DE NOVO POINT MUTATIONS and Copy Number Variations CNVs

De novo copy number variants associated with intellectual disability have a paternal origin and age bias

2011-10-05T08:54:00.000-07:00

J Med Genet. 2011 Oct 3. [Epub ahead of print]
De novo copy number variants associated with intellectual disability have a paternal origin and age bias.
Hehir-Kwa JY, Rodríguez-Santiago B, Vissers LE, de Leeuw N, Pfundt R, Buitelaar JK, Pérez-Jurado LA, Veltman JA.
Source1Department of Human Genetics, Nijmegen Centre for Molecular Life Sciences and Institute for Genetic and Metabolic Disorders, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands.

Abstract
BackgroundDe novo mutations and structural rearrangements are a common cause of intellectual disability (ID) and other disorders with reduced or null reproductive fitness. Insight into the genomic and environmental factors predisposing to the generation of these de novo events is therefore of significant clinical importance.MethodsThis study used information from single nucleotide polymorphism microarrays to determine the parent-of-origin of 118 rare de novo copy number variations (CNVs) detected in a cohort of 3443 patients with ID.ResultsThe large majority of these CNVs (76%, p=1.14×10(-8)) originated on the paternal allele. This paternal bias was independent of CNV length and CNV type. Interestingly, the paternal bias was less pronounced for CNVs flanked by segmental duplications (64%), suggesting that molecular mechanisms involved in the formation of rare de novo CNVs may be dependent on the parent-of-origin. In addition, a significantly increased paternal age was only observed for those CNVs which were not flanked by segmental duplications (p=0.02).ConclusionThis indicates that rare de novo CNVs are increasingly being generated with advanced paternal age by replication based mechanisms during spermatogenesis.

PMID:21969336[PubMed - as supplied by publisher]

2010-03-19T15:27:00.001-07:00

Activating mutations in FGFR3 and HRAS reveal a shared genetic origin for congenital disorders and testicular tumors

2009-10-26T12:10:00.000-07:00

Letter abstract
Nature Genetics Published online: 25 October 2009 doi:10.1038/ng.470
Activating mutations in FGFR3 and HRAS reveal a shared genetic origin for congenital disorders and testicular tumors
Anne Goriely1, Ruth M S Hansen1, Indira B Taylor1, Inge A Olesen2, Grete Krag Jacobsen3, Simon J McGowan4, Susanne P Pfeifer5, Gilean A T McVean5, Ewa Rajpert-De Meyts2 & Andrew O M Wilkie1
Abstract
Genes mutated in congenital malformation syndromes are frequently implicated in oncogenesis1, 2, but the causative germline and somatic mutations occur in separate cells at different times of an organism's life. Here we unify these processes to a single cellular event for mutations arising in male germ cells that show a paternal age effect3. Screening of 30 spermatocytic seminomas4, 5 for oncogenic mutations in 17 genes identified 2 mutations in FGFR3 (both 1948A>G, encoding K650E, which causes thanatophoric dysplasia in the germline)6 and 5 mutations in HRAS. Massively parallel sequencing of sperm DNA showed that levels of the FGFR3 mutation increase with paternal age and that the mutation spectrum at the Lys650 codon is similar to that observed in bladder cancer7, 8. Most spermatocytic seminomas show increased immunoreactivity for FGFR3 and/or HRAS. We propose that paternal age-effect mutations activate a common 'selfish' pathway supporting proliferation in the testis, leading to diverse phenotypes in the next generation including fetal lethality, congenital syndromes and cancer predisposition.Top of page
Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford, UK.
Department of Growth & Reproduction, Copenhagen University Hospital (Rigshospitalet), Copenhagen, Denmark.
Department of Pathology, Copenhagen University Hospital (Rigshospitalet), Copenhagen, Denmark.
Computational Biology Research Group, Oxford, UK.
Department of Statistics, University of Oxford, Oxford, UK.
Correspondence to: Andrew O M Wilkie1 e-mail: awilkie@hammer.imm.ox.ac.uk

Some Diseases More Common In Children Of Older Fathers: Testicular Tumors May Explain Why

2009-10-26T12:02:00.000-07:00

Some Diseases More Common In Children Of Older Fathers: Testicular Tumors May Explain Why

A rare form of testicular tumour has provided scientists with new insights into how genetic changes (mutations) arise in our children. The research, funded by the Wellcome Trust and the Danish Cancer Society, could explain why certain diseases are more common in the children of older fathers. Mutations can occur in different cells of the body and at different times during life. Some, such as those which occur in 'germ cells' (those which create sperm or eggs), cause changes which affect the offspring; those which occur in other cells can lead to tumours, but are not inherited. In work published in Nature Genetics, researchers at the University of Oxford and Copenhagen University Hospital describe a surprising link between certain severe childhood genetic disorders and rare testicular tumours occurring in older men: the germ cells that make the mutant gene-carrying sperm seem to be the same cells that produce the tumour. Although the original mutations occur only rarely in the sperm-producing cells, they encourage the mutant cells to divide and multiply. When the cell divides, it copies the mutation to each daughter cell, and the clump of mutant sperm-producing cells expands over time. Hence, the number of sperm carrying this mutation also increases as men get older, raising the risk to older fathers of having affected children. Professor Andrew Wilkie from the University of Oxford, who led the study, explains: "We think most men develop these tiny clumps of mutant cells in their testicles as they age. They are rather like moles in the skin, usually harmless in themselves. But by being located in the testicle, they also make sperm - causing children to be born with a variety of serious conditions. We call them 'selfish' because the mutations benefit the germ cell but are harmful to offspring." The work helps to explain the origins of several serious conditions that affect childhood growth and development. These include achondroplasia and Apert, Noonan and Costello syndromes, as well as some conditions causing stillbirth. The research links these conditions to a single pathway controlling cell multiplication, and will be valuable to doctors explaining to parents why the disorder has arisen, and informing them about the risks of it occurring again: in most cases, future children are unlikely to be affected. The findings may also help explain one of the mysteries of genetics: why scientists have yet to account for much of the genetic component of common diseases. Common diseases tend to be caused by the interaction of many genes, but despite powerful genome-wide association scans to search for these genes, relatively few have been uncovered. Several of these diseases, including breast cancer, autism and schizophrenia, seem to be more frequent in the offspring of older fathers, but the reasons are unknown. Professor Wilkie suggests that similar - but milder - mutations might contribute to these diseases. "What we have seen so far may just be the tip of a large iceberg of mildly harmful mutations being introduced into our genome," he explains. "These mutations would be too weak and too rare to be picked up by our current technology, but their sheer number would have a cumulative effect, leading to disease." Further research is needed to find other genes that are affected by this process. However, DNA sequencing technology has recently undergone a step change in capacity, enabling more sequence to be obtained in one day than was possible in a whole year just a decade ago. As the sequencing data emerge over the next decade, we should discover just how vulnerable we are to men's selfish mutation factories. Source: Craig Brierley Wellcome Trust

Sensitive and accurate detection of copy number variants using read depth of coverage.

2009-09-05T06:43:00.000-07:00

1: Genome Res. 2009 Sep;19(9):1586-92. Epub 2009 Aug 5.
Links
Sensitive and accurate detection of copy number variants using read depth of coverage.
Yoon S, Xuan Z, Makarov V, Ye K, Sebat J.
Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724, USA;
Methods for the direct detection of copy number variation (CNV) genome-wide have become effective instruments for identifying genetic risk factors for disease. The application of next-generation sequencing platforms to genetic studies promises to improve sensitivity to detect CNVs as well as inversions, indels, and SNPs. New computational approaches are needed to systematically detect these variants from genome sequence data. Existing sequence-based approaches for CNV detection are primarily based on paired-end read mapping (PEM) as reported previously by Tuzun et al. and Korbel et al. Due to limitations of the PEM approach, some classes of CNVs are difficult to ascertain, including large insertions and variants located within complex genomic regions. To overcome these limitations, we developed a method for CNV detection using read depth of coverage. Event-wise testing (EWT) is a method based on significance testing. In contrast to standard segmentation algorithms that typically operate by performing likelihood evaluation for every point in the genome, EWT works on intervals of data points, rapidly searching for specific classes of events. Overall false-positive rate is controlled by testing the significance of each possible event and adjusting for multiple testing. Deletions and duplications detected in an individual genome by EWT are examined across multiple genomes to identify polymorphism between individuals. We estimated error rates using simulations based on real data, and we applied EWT to the analysis of chromosome 1 from paired-end shotgun sequence data (30x) on five individuals. Our results suggest that analysis of read depth is an effective approach for the detection of CNVs, and it captures structural variants that are refractory to established PEM-based methods.

Unlike schizophrenia, the risk of BPAD seems to be associated with both paternal and maternal ages.

2009-07-25T18:52:00.000-07:00

1: Psychol Med. 2009 Jul 23:1-9. [Epub ahead of print]
Menezes PR, Lewis G, Rasmussen F, Zammit S, Sipos A, Harrison GL, Tynelius P, Gunnell D.Paternal and maternal ages at conception and risk of bipolar affective disorder in their offspring.

Department of Preventive Medicine, University of Sao Paulo, Brazil.
BACKGROUND: A consistent association between paternal age and their offspring's risk of schizophrenia has been observed, with no independent association with maternal age. The relationship of paternal and maternal ages with risk of bipolar affective disorders (BPAD) in the offspring is less clear. The present study aimed at testing the hypothesis that paternal age is associated with their offspring's risk of BPAD, whereas maternal age is not.MethodThis population-based cohort study was conducted with individuals born in Sweden during 1973-1980 and still resident there at age 16 years. Outcome was first hospital admission with a diagnosis of BPAD. Hazard ratios (HRs) were calculated using Cox's proportional hazard regression. RESULTS: After adjustment for all potential confounding variables except maternal age, the HR for risk of BPAD for each 10-year increase in paternal age was 1.28 [95% confidence interval (CI) 1.11-1.48], but this fell to 1.20 (95% CI 0.97-1.48) after adjusting for maternal age. A similar result was found for maternal age and risk of BPAD [HR 1.30 (95% CI 1.08-1.56) before adjustment for paternal age, HR 1.12 (95% CI 0.86-1.45) after adjustment]. The HR associated with having either parent aged 30 years or over was 1.26 (95% CI 1.01-1.57) and it was 1.45 (95% CI 1.16-1.81) if both parents were >30 years. CONCLUSIONS: Unlike schizophrenia, the risk of BPAD seems to be associated with both paternal and maternal ages.
PMID: 19627644 [PubMed - as supplied by publisher]
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Genetic Copy Variations and Disease

2009-06-05T12:49:00.000-07:00

From the June 2009 Scientific American Magazine | 0 comments

Genetic Copy Variations and Disease
A new sense for how variable numbers of genes cause disease
By Melinda Wenner

Scientists published the first draft of the human genome nearly a decade ago, but the hunt for disease genes is far from over. Most researchers have focused on single changes in DNA base pairs (AT and CG) that cause fatal diseases, such as cystic fibrosis. Such mutations among the genome’s three billion base pairs don’t tell the whole story, however. Recently geneticists have taken a closer look at a genetic aberration previously considered rare: copy number variation (CNV). The genes may be perfectly normal, yet there is a shortage or surplus of DNA sequences that may play a role in diseases that defy straightforward genetic patterns, such as autism, schizophrenia and Crohn’s disease, the causes of which have stumped researchers for decades.

American geneticist Calvin Bridges discovered copy number variation in 1936, when he noticed that flies that inherit a duplicate copy of a gene called Bar develop very small eyes. Two decades later a French researcher studying human chromosomes under a microscope identified CNV as the cause of Down syndrome: sufferers inherit an extra copy of chromosome 21. By all appearances, CNV was rare and always a direct cause of disease....

Note: This article was originally published with the title, "Too Little, Too Much".

ABOUT THE AUTHOR(S)
Melinda Wenner, based in New York City, described in the May issue how "quorum-sensing" bacteria could help beat antibiotic-resistant germs.

The architecture of our genomes is anything but basic

2009-04-11T07:54:00.000-07:00

Shared Differences
The architecture of our genomes is anything but basic

By Tina Hesman Saey April 25th, 2009; Vol.175 #9 (p. 16) Text Size Enlarge
SHARED DIFFERENCESView Larger Version | Glowing strands of DNA from five people highlight differences among humans in the number of copies of amylase genes, which encode enzymes that break down sugars. Red and green probes bind to regions hosting the genes, and each DNA strand has a different number of the genes on the short arm of chromosome 1. From the American Journal of Human Genetics, January 2008Whether you like it or not, you’re a little different. If it makes you feel any better, so is everybody else. In fact, everybody is far more different than anybody had imagined.

2009-03-23T18:05:00.001-07:00

Public release date: 23-Mar-2009
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Contact: Charlotte Webber
charlotte.webber@biomedcentral.com
44-782-531-7342
BioMed Central

Genomic variations in African-American and white populations
Deletions, duplications or rearrangements of genomic regions in the human genomes produce differences in gene copy numbers, referred to as copy number variations (CNV). Those variations account for a substantial portion of human genetic diversity, and in a few cases, have been associated with behavioural traits or increased susceptibility to disease. A study published today in the open access journal BMC Genetics, describes a CNV map of the African American genome, and compares frequencies of CNVs between African American and white American/European populations.

Joseph P McElroy and colleagues from the Department of Neurology, University of California at San Francisco, recruited African Americans from 28 States and used their genomes to draw CNV comparisons with the White dataset. "To the best of our knowledge, this is the first detailed map of copy number variations in African Americans. Understanding the distributions of CNVs in a population is a first step to addressing their role in disease".

The authors employed an array of over 500,000 sequences whose position in the human genome is already known due to single nucleotide polymorphisms. They first analysed the interaction of 50 blood samples of healthy African American females with this gene chip platform, and then used the results as a reference to assess copy number variation in samples from a further 385 African Americans, and an additional set of samples from 435 White individuals. In total, 1362 CNVs were detected in African Americans and 1972 in the White cohort. Across most of the genome, the frequency of CNVs did not differ greatly between the two populations. However, there were two duplications, one on chromosome 15, and one on chromosome 17, whose frequency varied markedly between the two groups.

The research team discovered that the duplication in chromosome 17 (region 17q21) is present in 45% of White but only in 8% of African American individuals. Another independent study has implicated the same region in mental retardation caused by a deletion due to duplication. Among the deleted genes, two of them, CRHR1 (corticitropin releasing hormone receptor 1) and MAPT (microtubule-associated protein tau), were previously associated with some neurological disorders. These two genes are not contained within the 17q21 region of CNV duplication, but map very close to it.

According to McElroy, "It would be good to know if the CNV duplication of the region might have an effect on the expression of these genes, which in turn could result in neurological disease. It is also interesting to find out whether the type of mental retardation associated with this locus is more common in Whites than in Africans or African Americans. If this is true, then it might be one of the first reported diseases with differing ethnic frequencies due to CNVs."

###

Rosetta Biosoftware Announces the Syllego System Version 3.5 with Support for Copy Number Variation

2009-03-16T08:01:00.000-07:00

March 16, 2009 08:00 AM Eastern Daylight Time
Rosetta Biosoftware Announces the Syllego System Version 3.5 with Support for Copy Number Variation
New Software Release Mitigates Data Challenges, Improves Research Productivity for Integrative Genomic Studies
Cambridge Healthtech Institute's Comprehending Copy Number Variation Conference
SEATTLE--(BUSINESS WIRE)--Rosetta Biosoftware, a global leader in life science informatics solutions, today announced the availability of copy number variation (CNV) data management and analysis capabilities in the latest release of the Syllego™ system. Syllego version 3.5 also offers scientists the ability to co-analyze gene expression (GE), CNV and genotyping data through enhanced statistical and visualization features and a data exchange gateway to the Rosetta Resolver® system. The new software release adds CNV to the Syllego system's current support for genome-wide association (GWA), linkage and eQTL studies, improving research productivity and expanding the software system's effectiveness in addressing the informatics challenges of scientists engaged in integrative genomic research.
In recent years, the existence of copy number as a common form of genetic variation, and its ability to influence phenotype, have been well established. Copy number variation is the subject of an increasing set of investigations to determine the extent and nature of its role in complex diseases. These studies look beyond single nucleotide polymorphisms (SNPs) in an effort to create a more complete picture of the associations between genetic variation and disease-related phenotypes. The need to manage, integrate and derive complicated dependencies between diverse data types – copy number, SNP, gene expression and phenotypic – from both public and proprietary sources is central to the success of this research. Syllego version 3.5 mitigates these challenges by providing
a common repository for commercial and custom copy number data and associated phenotypic data
a platform to integrate public copy number variation, genome data with proprietary data and analyze the composite data set
integrative analysis of genotyping, copy number variation and gene expression datasets
an open analysis platform that allows researchers to instantly use new public or proprietary analysis methods in easy-to-use interfaces
geneticists, biologists, informaticians and IT specialists with a single software solution to perform data management, data analysis, store results, and visualize results
"With the addition of CNV support, scientists can now use the Syllego system to interrogate complex data and find answers they've not been able to find before," said Yelena Shevelenko, General Manager, Rosetta Biosoftware. "We continue to execute on our strategy to deliver innovative technology to life scientists as they transform our understanding of biology and shape a new future for healthcare."
The Syllego system version 3.5 will be showcased at Cambridge Healthtech Institute's Comprehending Copy Number Variation Conference in San Diego on March 16, 2009; and during a Webinar hosted by Rosetta Biosoftware on March 25, 2009 at 9:00 AM Pacific Daylight Time. For more information, visit www.rosettabio.com/company/events.
About the Syllego System
The Syllego system is Rosetta Biosoftware’s practical solution for genetic data management and analysis. The Syllego system includes the Affymetrix GeneChip®-compatible designation and is part of the Illumina® Connect program. For more information on the Syllego system, please visit www.rosettabio.com/syllego.
About Rosetta Biosoftware
Rosetta Biosoftware is a leading provider of informatics solutions for life science research. Its comprehensive software solutions, including the Rosetta Resolver, Rosetta Elucidator, and Syllego systems, empower life scientists with advanced, scalable, and easy-to-use analysis platforms that accelerate discovery research. Rosetta Biosoftware is a business unit of Rosetta Inpharmatics LLC, a wholly-owned subsidiary of Merck & Co., Inc. More information about Rosetta Biosoftware is available at www.rosettabio.com.
Forward-Looking Statements
This press release contains "forward-looking statements" as that term is defined in the Private Securities Litigation Reform Act of 1995. These statements involve risks and uncertainties, which may cause results to differ materially from those set forth in the statements. The forward-looking statements may include statements regarding product development, product potential, or financial performance. No forward-looking statement can be guaranteed, and actual results may differ materially from those projected. Neither Rosetta Inpharmatics nor Merck & Co., Inc. undertakes any obligation to publicly update any forward-looking statement, whether as a result of new information, future events, or otherwise. Forward-looking statements in this press release should be evaluated together with the many uncertainties that affect the business of Merck & Co., Inc. including, among others, the extent to which Rosetta Inpharmatics' technology platform can be used in drug discovery programs, uncertainty of market acceptance of Rosetta Inpharmatics' technologies, ability to compete against existing technologies, and those mentioned in the cautionary statements in Item 1 of Merck’s Form 10-K for the year ended Dec. 31, 2008, and in its periodic reports on Form 10-Q and Form 8-K (if any) which are incorporated by reference.
Rosetta Resolver, Resolver, Elucidator, and Syllego are trademarks of Rosetta Inpharmatics LLC.

Are CNVs a Product of Paternal Age?

2009-03-13T15:46:00.000-07:00

1: PLoS Med. 2009 Mar 10;6(3):e40. [Epub ahead of print] Links
Advanced Paternal Age Is Associated with Impaired Neurocognitive Outcomes during Infancy and Childhood.Saha S, Barnett AG, Foldi C, Burne TH, Eyles DW, Buka SL, McGrath JJ.
BACKGROUND: Advanced paternal age (APA) is associated with an increased risk of neurodevelopmental disorders such as autism and schizophrenia, as well as with dyslexia and reduced intelligence. The aim of this study was to examine the relationship between paternal age and performance on neurocognitive measures during infancy and childhood. METHODS AND FINDINGS: A sample of singleton children (n = 33,437) was drawn from the US Collaborative Perinatal Project. The outcome measures were assessed at 8 mo, 4 y, and 7 y (Bayley scales, Stanford Binet Intelligence Scale, Graham-Ernhart Block Sort Test, Wechsler Intelligence Scale for Children, Wide Range Achievement Test). The main analyses examined the relationship between neurocognitive measures and paternal or maternal age when adjusted for potential confounding factors. Advanced paternal age showed significant associations with poorer scores on all of the neurocognitive measures apart from the Bayley Motor score. The findings were broadly consistent in direction and effect size at all three ages. In contrast, advanced maternal age was generally associated with better scores on these same measures. CONCLUSIONS: The offspring of older fathers show subtle impairments on tests of neurocognitive ability during infancy and childhood. In light of secular trends related to delayed fatherhood, the clinical implications and the mechanisms underlying these findings warrant closer scrutiny.

PMID: 19278291 [PubMed - as supplied by publisher]

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Children Of Older Dads Do Less Well In Intelligence Tests

2009-03-11T07:38:00.001-07:00

Pediatrics / Children's Health News

Children Of Older Dads Do Less Well In Intelligence Tests Featured ArticleMain Category: Pediatrics / Children's HealthAlso Included In: Fertility; Men's healthArticle Date: 11 Mar 2009 -
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0 postsResearchers from Australia looking at data on US families found that young children of older fathers performed less well in a range of cognitive intelligence tests up to the age of 7 years, but were unable to say whether those children were able to catch up when they got older.The study was the work of Sukanta Saha from the Queensland Centre for Mental Health Research, The Park Centre for Mental Health, Richlands, Australia, and colleagues from other research centres in Australia, and was published online in the open access journal PLoS Medicine.There is good evidence that specific disorders are linked with older fathers, but the link between children's general intelligence and fathers' age is not very clear, said an editorial comment accompanying the article. One study has shown a link between lower intelligence and very young and older fathers, so the authors wanted to look at the issue more closely and see if there was a link between age of fathers and children's ability on intelligence tests. They also wanted to see if there was evidence to support another finding that older mothers tend to have more intelligent children.For the study the researchers looked at records from the US Collaborative Perinatal Project, and analyzed data on 33,437 children who had undergone tests of cognitive ability at 8 months, 4 years and 7 years. The tests measured children's ability to think and reason, assessing things like concentration, memory, learning, understanding, speaking, and reading. Some tests also assessed physical co-ordination or "motor skills".The tests included the Wechsler Intelligence Scale for Children, Bayley scales, the Stanford Binet Intelligence Scale, the Graham-Ernhart Block Sort Test, and the Wide Range Achievement Test.Saha and colleagues analyzed the data using two models. In the first model they took into account physical factors such as the age of the parents. In the second model they added social factors such as parents' education and income, both factors that are known to affect intelligence. The researchers also grouped the children according to maternal age, and within each group looked for links between the lowest scores and paternal age.They found that the older the father, the poorer the results (the exception was the Bayley Motor score), but there was no such link with the age of the mother; in fact maternal age was "was generally associated with better scores on these same measures", they wrote.Saha and colleagues also wrote that the "findings were broadly consistent in direction and effect size at all three ages". They concluded:"The offspring of older fathers show subtle impairments on tests of neurocognitive ability during infancy and childhood.""In light of secular trends related to delayed fatherhood, the clinical implications and the mechanisms underlying these findings warrant closer scrutiny," they added.The sudy is the first to show that older fatherhood gives rise to children that perform less well on intelligence tests when young, but it can't say whether these children catch up with their peers after the age of 7.The editorial comment also cautioned that the results could be biased because some of the records did not have any information on the father's age.The last few decades have seen an increasing trend in the developing world toward couples waiting until their late thirties to have children. And while we have known for some time that older mothers are more likely to give birth to children with disabilities like Down's syndrome, it wasn't until recently that we discovered older fatherhood might also bring risks.We now know that older fatherhood is linked to miscarriages, birth deformities, cancer, and brain development disorders such as autism and schizophrenia, as well as dyslexia and reduced intelligence.Rates of autism have gone up in recent decades, but nobody knows why; some suspect it is genetic, others that it could be damage to sperm, which increases as a man gets older. While women's eggs are formed in the female fetus before she is born and stay like that until they are fertilized and develop into a baby, a man's sperm keeps dividing throughout his life, increasing the chance of mutation.Some studies suggest that children of older mothers benefit because they are nurtured more at home. If that is the case, then it appears that children of older fathers don't experience this benefit.But this study cannot answer these questions, it can only point to the need for more research, and perhaps emphasize the importance of doing so, especially as the trend toward older fatherhood appears likely to continue."Advanced Paternal Age Is Associated with Impaired Neurocognitive Outcomes during Infancy and Childhood."Saha S, Barnett AG, Foldi C, Burne TH, Eyles DW, Buka SL and McGrath JJ.PLoS Medicine Vol. 6, No. 3, e40. Published online March 10, 2009.doi:10.1371/journal.pmed.1000040

Why is the Paternal Age Effect and Genetic Disorders Kept Out of the Public Mind

2009-02-20T08:34:00.000-08:00

Why are the wealthy corporate monied families in America funding the research at genome labs?
http://www.hemophiliatoday.co.cc/why-are-the-wealthy-corporate-monied-families-in-america-funding-the-research-at-genome-labs/
Alex asked: Are genetic disease and disorders caused by older paternal age and will there never be cures or for Alzheimer’s, diabetes, MS, hemophilia, autism, schizophrenia,cancers because in non-familial cases they are basic degradations of the human genome caused by genetic copy number variations?

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IN QUESTION Abnormal gene expression may be tied to in vitro techniques.

2009-02-16T21:49:00.000-08:00

Picture Emerging on Genetic Risks of IVF

IN QUESTION Abnormal gene expression may be tied to in vitro techniques.

By GINA KOLATA
Published: February 16, 2009
Over the past 30 years, in vitro fertilization has been reassuringly safe. Millions of healthy children have been born and developed normally. And the first IVF baby, Louise Brown, born in England on July 25, 1978, now has her own child, 2-year-old Cameron, conceived without the technique.
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Health Guide: In Vitro Fertilization IVF
But researchers have always wondered whether there might be subtle changes in an embryo that is grown for several days in a petri dish, as IVF embryos are — and, if so, whether would there be any consequences.
Now, with new epidemiological studies and new techniques that allow scientists to probe the genes of embryo cells, some tentative answers are starting to emerge.
The issues have nothing to do with the chances that a woman will have twins, triplets or even, as just happened in California, octuplets. Instead, they involve questions of whether there are changes in gene expression or in developmental patterns, which may or may not be obvious at birth.
For example, some studies indicate that there may be some abnormal patterns of gene expression associated with IVF and a possible increase in rare but devastating genetic disorders that appear to be directly linked to those unusual gene expression patterns. There also appears to be an increased risk of premature birth and of babies with low birth weight for their gestational age.
In November, the Centers for Disease Control and Prevention published a paper reporting that babies conceived with IVF, or with a technique in which sperm are injected directly into eggs, have a slightly increased risk of several birth defects, including a hole between the two chambers of the heart, a cleft lip or palate, an improperly developed esophagus and a malformed rectum. The study involved 9,584 babies with birth defects and 4,792 babies without. Among the mothers of babies without birth defects, 1.1 percent had used IVF or related methods, compared with 2.4 percent of mothers of babies with birth defects.
The findings are considered preliminary, and researchers say they believe IVF does not carry excessive risks. There is a 3 percent chance that any given baby will have a birth defect.
But the real question — what is the chance that an IVF baby will have a birth defect? — has not been definitively answered. That would require a large, rigorous study that followed these babies. The C.D.C. study provides comparative risks but not absolute risks.
Yet even though the risks appear to be small, researchers who are studying the molecular biology of embryos grown in petri dishes say they would like a better understanding of what happens, so they can improve the procedure and allow couples to make more informed decisions.
“There is a growing consensus in the clinical community that there are risks,” said Richard M. Schultz, associate dean for the natural sciences at the University of Pennsylvania. “It is now incumbent on us to figure out what are the risks and whether we can do things to minimize the risks.”
And although the questions are well known, the discussion has been largely confined to scientists, said Dr. Elizabeth Ginsburg, president of the Society for Assisted Reproductive Technology.
Dr. Ginsburg, who is the medical director of in vitro fertilization at Brigham and Women’s Hospital in Boston, says her center’s consent forms mention that there might be an increased risk for certain rare genetic disorders. But, she says, none of her patients have been dissuaded.
Richard G. Rawlins, who directs the in vitro fertilization and assisted reproduction laboratories at the Rush Centers for Advanced Reproductive Care in Chicago, said that when he spoke to patients he never heard questions about growing embryos in the laboratory and the possible consequences.
“I have never had a patient ask me anything” about it, he said, adding, “For that matter, not many doctors have ever asked, either.”
Dr. Andrew Feinberg, a professor of medicine and genetics at Johns Hopkins, became concerned about the lack of information about IVF eight years ago when he and a colleague, Dr. Michael R. DeBaun, were studying changes in gene expression that can lead to cancer.
Their focus was on children with Beckwith-Wiedemann syndrome, characterized by a 15 percent risk of childhood cancers of the kidney, liver or muscle; an overgrowth of cells in the kidney and other tissues; and other possible abnormalities, among them a large tongue, abdominal-wall defects and low levels of blood sugar in infancy.
The syndrome, Dr. Feinberg and Dr. DeBaun found, was often caused by changes in the expression of a cluster of genes, and those changes also are found in colon and lung cancers. Children with those gene alterations had a 50 percent risk of the childhood cancers. The normal risk is less than 1 in 10,000.
The two investigators recruited children with the disorder, following them and studying them in their clinic. Then, several mothers in the study who had had IVF asked the researchers: Was it possible that the fertility treatments had caused Beckwith-Wiedemann syndrome?
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Autism and Schizophrenia and As Fathers Age on a Populations level

2009-02-16T21:23:00.000-08:00

Autism and Schizophrenia and As Fathers Age on a Populations level
at：2009-02-17 08:56:45 Click: 21
Autism and Schizophrenia and As Fathers Age on a Populations levelat：2009-02-04 01:06:45 Click: 23 Schizophrenia Risk and the Paternal Germ LineBy Dolores MalaspinaDolores Malaspina Paternal age at conception is a robust risk factor for schizophrenia. Possible mechanisms include de novo point mutations or defective epigenetic regulation of paternal genes. The predisposing genetic events appear to occur probabilistically (stochastically) in proportion to advancing paternal age, but might also be induced by toxic exposures, nutritional deficiencies, suboptimal DNA repair enzymes, or other factors that influence the fidelity of genetic information in the constantly replicating male germ line. We propose that de novo genetic alterations in the paternal germ line cause an independent and common variant of schizophrenia. Seminal findingsWe initially examined the relationship between paternal age and the risk for schizophrenia because it is well established that paternal age is the major source of de novo mutations in the human population, and most schizophrenia cases have no family history of psychosis. In 2001, we demonstrated a monotonic increase in the risk of schizophrenia as paternal age advanced in the rich database of the Jerusalem Perinatal Cohort. Compared with the offspring of fathers aged 20-24 years, in well-controlled analyses, each decade of paternal age multiplied the risk for schizophrenia by 1.4 (95 percent confidence interval: 1.2-1.7), so that the relative risk (RR) for offspring of fathers aged 45+ was 3.0 (1.6-5.5), with 1/46 of these offspring developing schizophrenia. There were no comparable maternal age effects (Malaspina et al., 2001). Epidemiological evidenceThis finding has now been replicated in numerous cohorts from diverse populations (Sipos et al., 2004; El-Saadi et al., 2004; Zammit et al., 2003; Byrne et al., 2003; Dalman and Allenbeck, 2002; Brown et al., 2002; Tsuchiya et al., 2005). By and large, each study shows a tripling of the risk for schizophrenia for the offspring of the oldest group of fathers, in comparison to the risk in a reference group of younger fathers. There is also a "dosage effect" of increasing paternal age; risk is roughly doubled for the offspring of men in their forties and is tripled for paternal age >50 years. These studies are methodologically sound, and most of them have employed prospective exposure data and validated psychiatric diagnoses. Together they demonstrate that the paternal age effect is not explained by other factors, including family history, maternal age, parental education and social ability, family social integration, social class, birth order, birth weight, and birth complications. Furthermore, the paternal age effect is specific for schizophrenia versus other adult onset psychiatric disorders. This is not the case for any other known schizophrenia risk factor, including many of the putative susceptibility genes (Craddock et al., 2006). There have been no failures to replicate the paternal age effect, nor its approximate magnitude, in any adequately powered study. The data support the hypothesis that paternal age increases schizophrenia risk through a de novo genetic mechanism. The remarkable uniformity of the results across different cultures lends further coherence to the conclusion that this robust relationship is likely to reflect an innate human biological phenomenon that progresses over aging in the male germ line, which is independent of regional environmental, infectious, or other routes. Indeed, the consistency of these data is unparalleled in schizophrenia research, with the exception of the increase in risk to the relatives of schizophrenia probands (i.e., 10 percent for a sibling). Yet, while having an affected first-degree relative confers a relatively higher risk for illness than having a father >50 years (~10 percent versus ~2 percent), paternal age explains a far greater portion of the population attributable risk for schizophrenia. This is because a family history is infrequent among schizophrenia cases, whereas paternal age explained 26.6 percent of the schizophrenia cases in our Jerusalem cohort. If we had only considered the risk in the cases with paternal age >30 years, our risk would be equivalent to that reported by Sipos et al. (2004) in the Swedish study (15.5 percent). When paternal ages >25 years are considered, the calculated risk is much higher. Although the increment in risk for fathers age 26 through 30 years is small (~14 percent), this group is very large, which accounts for the magnitude of their contribution to the overall risk. The actual percentage of cases with paternal germ line-derived schizophrenia in a given population will depend on the demographics of paternal childbearing age, among other factors. With an upswing in paternal age, these cases would be expected to become more prevalent. Biological plausibilityWe used several approaches to examine the biological plausibility of paternal age as a risk factor for schizophrenia. First, we established a translational animal model using inbred mice. Previously it had been reported that the offspring of aged male rodents had less spontaneous activity and worse learning capacity than those of mature rodents, despite having no noticeable physical anomalies (Auroux et al., 1983). Our model carefully compared behavioral performance between the progeny of 18-24-month-old sires with that of 4-month-old sires. We replicated Auroux's findings, demonstrating significantly decreased learning in an active avoidance test, less exploration in the open field, and a number of other behavioral decrements in the offspring of older sires (Bradley-Moore et al., 2002). Next, we examined if parental age was related to intelligence in healthy adolescents. We reasoned that if de novo genetic changes can cause schizophrenia, there might be effects of later paternal age on cognitive function, since cognitive problems are intertwined with core aspects of schizophrenia. For this study, we cross-linked data from the Jerusalem birth cohort with the neuropsychological data from the Israeli draft board (Malaspina et al., 2005a). We found that maternal and paternal age had independent effects on IQ scores, each accounting for ~2 percent of the total variance. Older paternal age was exclusively associated with a decrement in nonverbal (performance) intelligence IQ, without effects on verbal ability, suggestive of a specific effect on cognitive processing. In controlled analyses, maternal age showed an inverted U-shaped association with both verbal and performance IQ, suggestive of a generalized effect. Finally, we examined if paternal age was related to the risk for autism in our cohort. We found very strong effects of advancing paternal age on the risk for autism and related pervasive developmental disorders (Reichenberg et al., in press). Compared to the offspring of fathers aged 30 years or younger, the risk was tripled for offspring of fathers in their forties and was increased fivefold when paternal age was >50 years. Together, these studies provide strong and convergent support for the hypothesis that later paternal age can influence neural functioning. The translational animal model offers the opportunity to identify candidate genes and epigenetic mechanisms that may explain the association of cognitive functioning with advancing paternal age. A variant of schizophreniaA persistent question is whether the association of paternal age and schizophrenia could be explained by psychiatric problems in the parents that could both hinder their childbearing and be inherited by their offspring. If this were so, then cases with affected parents would have older paternal ages. This has not been demonstrated. To the contrary, we found that paternal age was 4.7 years older for sporadic than familial cases from our research unit at New York State Psychiatric Institute (Malaspina et al., 2002). In addition, epidemiological studies show that advancing paternal age is unrelated to the risk for familial schizophrenia (Byrne et al., 2003; Sipos et al., 2004). For example, Sipos found that each subsequent decade of paternal age increased the RR for sporadic schizophrenia by 1.60 (1.32 to 1.92), with no significant effect for familial cases (RR = 0.91, 0.44 to 1.89). The effect of late paternal age in sporadic cases was impressive. The offspring of the oldest fathers had a 5.85-fold risk for sporadic schizophrenia (Sipos et al., 2004); relative risks over 5.0 are very likely to reflect a true causal relationship (Breslow and Day, 1980). It is possible that the genetic events that occur in the paternal germ line are affecting the same genes that influence the risk in familial cases. However, there is evidence that this is not the case. First, a number of the loci linked to familial schizophrenia are also associated with bipolar disorder (Craddock et al., 2006), whereas advancing paternal age is specific for schizophrenia (Malaspina et al., 2001). Next, a few genetic studies that separately examined familial and sporadic cases found that the "at-risk haplotypes" linked to familial schizophrenia were unassociated with sporadic cases, including dystrobrevin-binding protein (Van Den Bogaert et al., 2003) and neuregulin (Williams et al., 2003). Segregating sporadic cases from the analyses actually strengthened the magnitude of the genetic association in the familial cases, consistent with etiological heterogeneity between familial and sporadic groups. Finally, the phenotype of cases with no family history and later paternal age are distinct from familial cases in many studies. For example, only sporadic cases showed a significant improvement in negative symptoms between a "medication-free" and an "antipsychotic treatment" condition (Malaspina et al., 2000), and sporadic cases have significantly more disruptions in their smooth pursuit eye movement quality than familial cases (Malaspina et al., 1998). A recent study also showed differences between the groups in resting regional cerebral blood flow (rCBF) patterns, in comparison with healthy subjects. The sporadic group of cases had greater hypofrontality, with increased medial temporal lobe activity (frontotemporal imbalance), while the familial group evidenced left lateralized temperoparietal hypoperfusion along with widespread rCBF changes in cortico-striato-thalamo-cortical regions (Malaspina et al., 2005b). Other data linking paternal age with frontal pathology in schizophrenia include a proton magnetic resonance spectroscopy study that demonstrated a significant association between prefrontal cortex neuronal integrity (NAA) and paternal age in sporadic cases only, with no significant NAA decrement in the familial schizophrenia group (Kegeles et al., 2005). These findings support the hypothesis that schizophrenia subgroups may have distinct neural underpinnings and that the important changes in some sporadic (paternal germ line) cases may particularly impact on prefrontal cortical functioning. Genetic mechanismSeveral genetic mechanisms might explain the relationship between paternal age and the risk for schizophrenia (see Malaspina, 2001). It could be due to de novo point mutations arising in one or several schizophrenia susceptibility loci. Paternal age is known to be the principal source of new mutations in mammals, likely explained by the constant cell replication cycles that occur in spermatogenesis (James Crow, 2000). Following puberty, spermatogonia undergo some 23 divisions per year. At ages 20 and 40, a man's germ cell precursors will have undergone about 200 and 660 such divisions, respectively. During a man's life, the spermatogonia are vulnerable to DNA damage, and mutations may accumulate in clones of spermatogonia as men age. In contrast, the numbers of such divisions in female germ cells is usually 24, all but the last occurring during fetal life. Trinucleotide repeat expansions could also underlie the paternal age effect. Repeat expansions have been demonstrated in several neuropsychiatric disorders, including myotonic dystrophy, fragile X syndrome, spinocerebellar ataxias, and Huntington disease. The sex of the transmitting parent is frequently a major factor influencing anticipation, with many disorders showing greater trinucleotide repeat expansion with paternal inheritance (Lindblad and Schalling, 1999; Schols et al., 2004; Duyao et al., 1993). Larger numbers of repeat expansions could be related to chance molecular events during the many cell divisions that occur during spermatogenesis. Later paternal age might confer a risk for schizophrenia if it was associated with errors in the "imprinting" patterns of paternally inherited alleles. Imprinting is a form of gene regulation in which gene expression in the offspring depends on whether the allele was inherited from the male or female parent. Imprinted genes that are only expressed if paternally inherited alleles are reciprocally silenced at the maternal allele, and vice versa. Imprinting occurs during gametogenesis after the methylation patterns from the previous generation are "erased" and new parent of origin specific methylation patterns are established. Errors in erasure or reestablishment of these imprint patterns may lead to defective gene expression profiles in the offspring. The enzymes responsible for methylating DNA are the DNA methyltransferases, or DNMTs. These enzymes methylate cytosine residues in CpG dinucleotides, usually in the promoter region of genes, typically to reduce the expression of the mRNA. The methylation may become inefficient for a variety of reasons; one possibility is reduced DNA methylation activity in spermatogenesis, since DNMT levels diminish as paternal age increases (Benoit and Trasler, 1994; La Salle et al., 2004). Another possible mechanism is that this declining DNMT activity could be epigenetically transmitted to the offspring of older fathers. There are a number of different DNMTs that differ in whether they initiate or sustain methylation, and which are active at different ages and in different tissues. Human imprinted genes have a critical role in the growth of the placenta, fetus, and central nervous system, in behavioral development, and in adult body size. It is an appealing hypothesis that loss of normal imprinting of genes critical to neurodevelopment may play a role in schizophrenia. Indeed, one of the most consistently identified molecular abnormalities in schizophrenia has been theorized to result from abnormal epigenetic mechanisms (Veldic et al., 2004), that is, the reduced GABA and reelin expression in prefrontal GABAergic interneurons. An overexpression of DNMT in these GABAergic interneurons, hypermethylating the reelin and GAD67 promoter regions, might be responsible for reducing their mRNA transcripts and expression levels. These decrements could functionally impair the role of GABAergic interneurons in regulating the activity and firing of pyramidal neurons, thereby causing cognitive dysfunction. Later paternal age could be related to the abnormal regulation or expression of DNMT activity in specific cells. ConclusionThese findings suggest exciting new directions for research into the etiology of schizophrenia. If there is a unitary etiopathology for paternal age-related schizophrenia, then it is likely to be the most common form of the condition in the population and in treatment settings, since genetic linkage and association studies indicate that familial cases are likely to demonstrate significant allelic heterogeneity and varying epistatic effects. Schizophrenia is commonly considered to result from the interplay between genetic susceptibility and environmental exposures, particularly those that occur during fetal development and in adolescence. The data linking paternal age to the risk for schizophrenia indicate that we should expand this event horizon to consider the effects of environmental exposures over the lifespan of the father. The mutational stigmata of an exposure may remain in a spermatogonial cell, and be manifest in the clones of spermatozoa that it will subsequently generate over a man's reproductive life. References:Auroux M. Decrease of learning capacity in offspring with increasing paternal age in the rat. Teratology. 1983 Apr;27(2):141-8. Abstract Benoit G, Trasler JM. Developmental expression of DNA methyltransferase messenger ribonucleic acid, protein, and enzyme activity in the mouse testis. Biol Reprod. 1994 50:1312-9. Abstract Bradley-Moore M, Abner R, Edwards T, Lira J, Lira A, Mullen T, Paul S, Malaspina D, Brunner D, Gingrich JA. Modeling The Effect Of Advanced Paternal Age On Progeny Behavior In Mice. Developmental Psychobiology, abstract, 2002; (41)3, 230. Breslow, N. E. and Day, N. E. (1980). The analysis of case-control data. In Statistical Methods in Cancer Research , Volume 1. Lyon: World Health Organization. Brown AS, Schaefer CA, Wyatt RJ, Begg MD, Goetz R, Bresnahan MA, Harkavy-Friedman J, Gorman JM, Malaspina D, Susser ES. Paternal age and risk of schizophrenia in adult offspring. Am J Psychiatry. 2002 Sep;159(9):1528-33. Abstract Byrne M, Agerbo E, Ewald H, Eaton WW, Mortensen PB. Parental age and risk of schizophrenia: a case-control study. Arch Gen Psychiatry. 2003 Jul;60(7):673-8. Abstract Crow JF (1997). The high spontaneous mutation rate: is it a health risk? Proc Natl Acad Sci USA 94:8380-8386. Craddock N, O'Donovan MC, Owen MJ. Genes for schizophrenia and bipolar disorder? Implications for psychiatric nosology. Schizophr Bull. 2006 Jan;32(1):9-16. Abstract Dalman C, Allebeck P. Paternal age and schizophrenia: further support for an association. Am J Psychiatry. 2002 Sep;159(9):1591-2. Abstract Duyao M, Ambrose C, Myers R, Novelletto A, Persichetti F, Frontali M, Folstein S, Ross C, Franz M, Abbott M, et al. Trinucleotide repeat length instability and age of onset in Huntington's disease. Nat Genet. 1993 Aug;4(4):387-92. Abstract El-Saadi O, Pedersen CB, McNeil TF, Saha S, Welham J, O'Callaghan E, Cantor-Graae E, Chant D, Mortensen PB, McGrath J. Paternal and maternal age as risk factors for psychosis: findings from Denmark, Sweden and Australia.Schizophr Res. 2004 Apr 1;67(2-3):227-36. Abstract Kegeles LS, Shungu DC, Mao X, Goetz R, Mikell CB, Abi-Dargham A, Laurelle M, Malaspina D. Relationship of age and paternal age to neuronal functional integrity in the prefrontal cortex in schizophrenia determined by proton magnetic resonance spectroscopy. Schizophrenia Bulletin, 31:443; 2005. La Salle S, Mertineit C, Taketo T, Moens PB, Bestor TH, Trasler JM. Windows for sex-specific methylation marked by DNA methyltransferase expression profiles in mouse germ cells. Dev Biol. 2004 268:403-15. Abstract Lindblad K, Schalling M. Expanded repeat sequences and disease. Semin Neurol. 1999;19(3):289-99. Abstract Malaspina D, Friedman JH, Kaufmann C, Bruder G, Amador X, Strauss D, Clark S, Yale S, Lukens E, Thorning H, Goetz R, Gorman J. Psychobiological heterogeneity of familial and sporadic schizophrenia. Biol Psychiatry. 1998 Apr 1;43(7):489-96. Abstract Malaspina D, Goetz RR, Yale S, Berman A, Friedman JH, Tremeau F, Printz D, Amador X, Johnson J, Brown A, Gorman JM. Relation of familial schizophrenia to negative symptoms but not to the deficit syndrome. Am J Psychiatry. 2000 Jun;157(6):994-1003. Abstract Malaspina D, Harlap S, Fennig S, Heiman D, Nahon D, Feldman D, Susser ES. Advancing paternal age and the risk of schizophrenia. Arch Gen Psychiatry. 2001 Apr;58(4):361-7. Abstract Malaspina D. Paternal factors and schizophrenia risk: de novo mutations and imprinting. Schizophr Bull. 2001;27(3):379-93. Review. Abstract Malaspina D, Corcoran C, Fahim C, Berman A, Harkavy-Friedman J, Yale S, Goetz D, Goetz R, Harlap S, Gorman J. Paternal age and sporadic schizophrenia: evidence for de novo mutations. Am J Med Genet. 2002 Apr 8;114(3):299-303. Abstract Malaspina D, Harkavy-Friedman J, Corcoran C, Mujica-Parodi L, Printz D, Gorman JM, Van Heertum R. Resting neural activity distinguishes subgroups of schizophrenia patients. Biol Psychiatry. 2005 (a) Dec 15;56(12):931-7. Abstract Malaspina D, Reichenberg A, Weiser M, Fennig S, Davidson M, Harlap S, Wolitzky R, Rabinowitz J, Susser E, Knobler HY. Paternal age and intelligence: implications for age-related genomic changes in male germ cells. Psychiatr Genet. 2005 (b) Jun;15(2):117-25. Abstract Reichenberg A, Gross R, Weiser M, Bresnahan M, Silverman J, Harlap, Rabinowitz J, Shulman L, Malaspina D, Lubin G, Knobler HY, Davidson M, Susser E: Advancing paternal age and Autism. Archives of General Psychiatry. Schols L, Bauer P, Schmidt T, Schulte T, Riess O. Autosomal dominant cerebellar ataxias: clinical features, genetics, and pathogenesis. Lancet Neurol. 2004 May;3(5):291-304. Abstract Sipos A, Rasmussen F, Harrison G, Tynelius P, Lewis G, Leon DA, Gunnell D. Paternal age and schizophrenia: a population based cohort study. BMJ. 2004 Nov 6;329(7474):1070. Epub 2004 Oct 22. Abstract Tsuchiya KJ, Takagai S, Kawai M, Matsumoto H, Nakamura K, Minabe Y, Mori N, Takei N. Advanced paternal age associated with an elevated risk for schizophrenia in offspring in a Japanese population. Schizophr Res. 2005 Jul 15;76(2-3):337-42. Epub 2005 Apr 21. Abstract Van Den Bogaert A, Schumacher J, Schulze TG, Otte AC, Ohlraun S, Kovalenko S, Becker T, Freudenberg J, Jonsson EG, Mattila-Evenden M, Sedvall GC, Czerski PM, Kapelski P, Hauser J, Maier W, Rietschel M, Propping P, Nothen MM, Cichon S. The DTNBP1 (dysbindin) gene contributes to schizophrenia, depending on family history of the disease. Am J Hum Genet. 2003 Dec;73(6):1438-43. Abstract Veldic M, Caruncho HJ, Liu WS, Davis J, Satta R, Grayson DR, Guidotti A, Costa E. DNA-methyltransferase 1 mRNA is selectively overexpressed in telencephalic GABAergic interneurons of schizophrenia brains. Proc Natl Acad Sci U S A. 2004 Jan 6;101(1):348-53. Abstract Williams NM, Preece A, Spurlock G, Norton N, Williams HJ, Zammit S, O'Donovan MC, Owen MJ. Support for genetic variation in neuregulin 1 and susceptibility to schizophrenia. Mol Psychiatry. 2003 May;8(5):485-7. Abstract Zammit S, Allebeck P, Dalman C, Lundberg I, Hemmingson T, Owen MJ, Lewis G. Paternal age and risk for schizophrenia. Br J Psychiatry. 2003 Nov;183:405-8. Abstract

Men Must Contend With a Biological Clock, Too Older males face higher risk of fathering children with medical problems, research finds ( CNVs)

2009-02-14T13:41:00.000-08:00

Men Must Contend With a Biological Clock, Too Older males face higher risk of fathering children with medical problems, research finds
By Kathleen DohenyHealthDay Reporter
SATURDAY, Feb. 14 (HealthDay News) -- It wasn't all that long ago that any suggestion that a man had a "biological clock" like a woman, and should father children sooner rather than later, would have been given short scientific shrift.
Not anymore. Today, a growing body of evidence suggests that as men get older, fertility can and does decline, while the chances of fathering a child with serious birth defects and medical problems increase.
Some studies have linked higher rates of serious health problems such as autism and schizophrenia in children born to men as young as their mid-40s.
And doctors and researchers are busy trying to figure out how men who choose to delay fatherhood -- either by choice or necessity, such as a lack of a partner -- can offset the effects of their biological clocks as those clocks wind down.
Interestingly, problems with reduced fertility can start long before middle age, said Dr. Harry Fisch, one of the pioneers in the field in male fertility and director of the Columbia University College of Physicians and Surgeons' Male Reproductive Center, in New York City.
"We know after age 30, testosterone levels decline about 1 percent per year," said Fisch, author of the book The Male Biological Clock.
Research done at the University of Washington has found that "as men age, DNA damage occurs to their sperm," said Dr. Narendra P. Singh, a research associate professor in the department of bioengineering, who co-authored a study on the subject.
Several other studies point to problems in the offspring of older fathers, as well as older men experiencing fertility problems.
For instance, Fisch and his colleagues found that if a woman and a man were both older than age 35 at the time of conception, the father's age played a significant role in the prevalence of Down syndrome. And this effect was most detectable if the woman was 40 or older -- the incidence of Down syndrome was about 50 percent attributable to the sperm.
Other researchers have found that children born to fathers 45 or older are more likely to have poor social skills, and that children born to men 55 and older are more likely to have bipolar disorder than those born to men 20 to 24 years of age at the time of conception.
On other fronts, researchers at Mount Sinai School of Medicine in New York City found that children of men aged 40 or older were about six times more likely to have autism. Still another study found that the children of fathers who were 50 or older when they were born were almost three times more likely to be diagnosed with schizophrenia....
But Fisch did say, "The sooner, the better."...

Genome Study Points to New Culprit for Schizophrenia

2009-02-06T17:27:00.000-08:00

Genome Study Points to New Culprit for Schizophrenia
February 6 (HealthDay News) -- Large, rare structural changes in DNA called copy number variants may play a role in schizophrenia, according to U.S. researchers, who said their findings support a sharp change of direction in genetics research on schizophrenia.

Over the past two decades, researchers have identified dozens of genes and single nucleotide polymorphisms (SNPs or "snips") that could be linked to schizophrenia. But this new study dismisses all of them.

"The literature is replete with dozens of genes and SNPs identified as associated with schizophrenia. But we systematically retested all the leading candidates and concluded that most, if not all of them, are false positives," study lead author Anna Need, a postdoctoral associate at the Center for Human Genome Variation at the Duke Institute for Genome Sciences & Policy, said in a Duke University news release.

Most of the previous studies were too small to properly assess the role of SNPs in schizophrenia, Need said.

She and her colleagues analyzed the genomes of schizophrenia patients and healthy people for SNPs and copy number variants (CNVs). None of the previously identified SNPs appeared significant in schizophrenia, but the researchers identified several CNVs they believe may be associated with the psychiatric disorder.

CNVs are common and usually appear as deletions or duplications of significant stretches of DNA. But the largest deletions -- those over 2 million bases long -- appear only in people with schizophrenia, Need said.

The study was published Feb. 6 in the journal PLoS Genetics.

"What this means is that if we are going to make real headway in assessing genetic links to schizophrenia, we will have to sequence the entire genome of each schizophrenia patient," Need said. "That is a tremendous amount of work, but it is the only way we will be able to find these extremely rare variations."

SOURCE: Duke University, news release, Feb. 5, 2009

Copy Number Variation, Genetic Interactions and Disease

2009-01-30T19:29:00.000-08:00

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Copy Number Variation, Genetic Interactions and Disease

30 January 2009. Understanding how genetic variation contributes to human disease has become a major thrust of modern science. For example, considerable effort currently goes into genomewide association studies, which aim to identify markers that associate with a given disease, such as Alzheimer’s. But the effort is not paying off too well just yet. When it comes to teasing apart complex genetic traits driven by multiple genetic variations, many strategies fall woefully short. Likewise, the contribution to human disease of copy number variation, where duplication or deletions of whole sections of chromosomes can add or subtract complete genes, remains poorly understood. Two recent papers address these challenges—and reveal some surprises. In the January 21 American Journal of Human Genetics online, researchers led by Howard Hughes Investigator Evan Eichler at the University of Washington, Seattle, report that single copy number variants (CNV) may predispose individuals to a broad range of neurologic diseases, including schizophrenia, autism, and forms of mental retardation, suggesting that these variants may interact with other genetic and environmental factors to yield particular pathologies. And in the January 23 Science, researchers led by Barak Cohen at the University of Washington School of Medicine, St. Louis, Missouri, describe how different genetic variations can interact to influence expression of specific phenotypes in the model organism brewer’s yeast. Such epistatic interactions are thought to play a major role in human diseases, such as Alzheimer’s and schizophrenia, but little headway has been made in deciphering those relationships.
Eichler and colleagues used array-based analysis of DNA samples from about 2,500 healthy people to estimate copy number variation in the human population as a whole. This study stands out for its large sample size, which enables it to give an overview of this type of genetic idiosyncrasy in the population at large. First author Andy Itsara and colleagues report that most individuals (extrapolated to 65 to 85 percent of the general population) harbor a CNV that is at least 100 kb of DNA long. Much larger (>500 kb) variants occur in 5 to 10 percent of individuals, while at least 1 percent of the population carries a CNV exceeding 1 Mb. CNVs longer than 100 kb are rare, while those topping 500 kb tend to be found in only one individual. These findings are in keeping with the idea that huge CNVs are bad for your health. Counted from the other side, any given CNV is present in the population at a frequency of 0.2 to 1.0 percent.

Previous analysis of a sample set designed to measure human genome diversity on a global scale (see Cann et al., 2002) suggested that certain world populations carry more than their fair share of CNVs—20 to 30 per person compared to the average of seven to nine (see Jakobsson et al., 2008). Itsara and colleagues examined the same sample set, and while they confirmed that two of those three populations, Melanesian and Papuan, did have a higher prevalence (11.9, and 10.3 CNV per individual) than other populations, the difference was marginal. The third group, the Kalash, had fewer CNVs than average. “Deeper population screens to assess the distribution of large and rare CNVs in the human population are clearly warranted, because although such variants may segregate within specific populations because of genetic drift, others may contribute disproportionately to disease susceptibility or alternatively be adaptive within those populations,” write the authors.

CNVs are clearly linked to disease. Triplication of the entire chromosome 21, for example, gives rise to Down syndrome, which is accompanied by a much greater risk for dementia. Duplication of the amyloid precursor protein gene on chromosome 21 leads to early-onset, familial AD (see ARF related news story), while duplication or triplication of the α-synuclein gene causes familial Parkinson disease (see ARF related news story and Ibanez et al., 2009). Abnormally high CNVs have also been linked to schizophrenia and autism (see related story on Schizophrenia Research Forum).

To assess the impact of CNVs on some neurologic diseases, Itsara and colleagues combined their data with those from nine genomewide association studies of schizophrenia, autism, and mental retardation, assembling CNV data on 6,860 affected individuals and 5,674 controls. Their analysis recovered known associations (e.g., deletions at chromosome 22q11 in some schizophrenia patients) and also revealed new, unexpected relationships.

The Seattle geneticists found that a chromosome 17p11.2 microdeletion normally associated with a disease called heredity neuropathy with liability to pressure palsies (HNPP) is also deleted in patients with schizophrenia and autism. At a locus on chromosome 16p12 that is predicted to be a risk candidate for schizophrenia (see Stone et al., 2008), Itsara and colleagues found a deletion in one autistic patient and no deletions in any controls, again suggesting some overlap between autism and schizophrenia risk factors. And their data suggest that at chromosome 3q29, where a microdeletion leads to a syndrome that includes mental retardation and other neurologic abnormalities (see Willatt et al., 2005), deletions again increase the risk for schizophrenia. The findings tie clinically separate disorders together through the same CNV, leading the authors to suggest that these loci render their carriers generally vulnerable to mental illness such that the specific manifestation in a particular person depends on genetic modifier or environmental effects.

Exactly such genetic modification is what Barak Cohen and colleagues tried to come to grips with in their yeast study, which focused on a widely variable trait—sporulation. In Saccharomyces cerevisiae sporulation is a complex, heritable trait. It is subject to environmental influence, and believed to fall under different selection pressure in different environments, such as the oak grove and the oak barrel. Yeast from the former sporulate at near 100 percent efficiency, but those from the latter, perhaps not surprisingly, are much less competent. Sporulation serves as a model for complex traits, including susceptibility to disease and resistance to pharmacological intervention, exhibited by humans. Many individual genetic risk factors for late-onset Alzheimer disease have been identified, for example (see AlzRisk database ), but it is not clear how any of them interact with each other to increase or even decrease susceptibility.

First author Justin Gerke and colleagues identified four nucleotide changes among three transcription factors that explain the natural variation in yeast sporulation efficiency. The researchers crossed two parent strains, one from the North American oak and one from a California wine barrel, and looked for quantitative trait loci in the offspring that match their sporulation efficiency.

The researchers identified five loci that accounted for most of the variation among the different offspring. Three of these loci, all of which turned out to harbor transcription factors, had large effects. By sequencing the different strains, the researchers found four nucleotide substitutions that account for almost 80 percent of the sporulation variation: a single nucleotide change in the regulatory region of RME1, a transcription factor that can suppress sporulation in certain cell types; two non-synonymous substitutions in the coding region of IME1, a master regulator that initiates sporulation; and a single coding change in RSF1, transcriptional activator of mitochondrial genes essential for respiration. By replacing nucleotides one, two, three, and four at a time, Gerke and colleagues found that all four alleles interact to alter the phenotype.

“Knowing how individual genetic polymorphisms combine to produce phenotypic change could strengthen evolutionary theory and advance applications such as personalized medicine,” write the authors. In general, epistatic interactions between genes are poorly understood and this study highlights the effect that even single nucleotides can have on a given trait. “This emphasizes the need to incorporate genetic interactions into models that seek to accurately predict phenotype from genotype,” write the authors, adding that “if prevalent, genetic interactions between nucleotides will be a major hurdle in the endeavor to connect genetic and phenotypic variation in humans.”—Tom Fagan.

References:
Itsara A, Cooper GM, Baker C, Girirajan S, Li J, Absher D, Krauss RM, Myers RM, Ridker PM, Chasman DI, Mefford H, Ying P, Nickerson DA, Eichler EE. Population analysis of large copy number variants. Am. J. Hum. Genet. 2009, Jan 21. Abstract

Gerke J, Lorenz K, Cohen B. Genetic interactions between transcription factors cause natural variation in yeast. Science. 2009, Jan. 23; 323:498-501. Abstract

New study backs parent age-autism link

2008-12-12T17:08:00.000-08:00

New study backs parent age-autism link
Last Updated: 2008-12-12 9:12:21 -0400 (Reuters Health)

By Anne Harding

NEW YORK (Reuters Health) - Advanced parental age does indeed appear to boost autism risk in children, and the risk is seen with both mothers and fathers, new research shows.

"What we found was that actually it's both parents age, and when you control for one parent's age you still see the effect of the other parent's age, and vice versa," Dr. Maureen Durkin of the University of Wisconsin School of Medicine and Public Health in Madison, the lead researcher of the study reported in the American Journal of Epidemiology, told Reuters Health.

The findings may offer clues to understanding the causes of autism and why it's on the rise, but they shouldn't be used to guide family planning decisions, Durkin said. Even though the oldest child born to two older parents is three times as likely to be autistic than a middle or youngest child with younger parents, she explained, there's still a 97 percent chance that the higher-risk child will be perfectly fine. "The vast majority of children don't develop autism," she emphasized.

Several studies have suggested links between a father's age or the age of both parents and a child's likelihood of having autism. The current study included twice as many autism cases as any other research on this issue to date, which made it possible to tease out the effects of both maternal and paternal age.

The researchers looked at 253,347 children born in 1994 at 10 sites included in the Centers for Disease Control and Prevention's Autism and Developmental Disabilities Monitoring Network. There were 1,251 children who met standard criteria for an autism spectrum disorder at age 8 for whom information on both parents' age was available.

Alzheimer's link to older fathers

2008-11-21T19:00:00.000-08:00

Alzheimer's link to older fathers
Independent, The (London), Sep 17, 1998 by Charles Arthur Technology Editor
E-mail Print Link CHILDREN BORN to fathers who are approaching middle age have a higher than average risk of developing Alzheimer's disease in later life, a study suggests.

A retrospective investigation of 206 people who have the degenerative illness, but no history of it occurring in the family, revealed a statistically significant link with the age of their father when they were born.

Some genes are known to contibute to the chance of developing Alzheimer's, but the new study, carried out by Lars Bertram at the Technical University of Munich, suggests that simply having an older father - average age 35.7 - can be a risk factor even in the absence of those genes. For those where there was a family history of Alzheimer's, the average age of the father was 31.3 years.

Though the sample is comparatively small, it is in line with the knowledge that ageing is associated with genetic damage to the sperm, which carry the father's genetic contribution to the child. That might eventually lead to Alzheimer's in the offspring. "There's an accumulation of environmental factors which somehow alter the genome of the father," Dr Bertram told New Scientist magazine.

Similar effects are already known to occur in women, where mothers over 35 have a far higher chance of giving birth to babies with Down's syndrome, which is caused by a genetic defect in the embryo. People with Down's syndrome are also more likely eventually to develop Alzheimer's.

Copyright 1998 Newspaper Publishing PLC
Provided by ProQuest Information and Learning Company. All rights Reserved.

Older parents, epigenomics and psychiatric illness

2008-11-11T07:36:00.000-08:00

Older parents, epigenomics and psychiatric illness
2008-11-11 — Dave Bath
Nature’s British Journal of Pharmacology has (for free!) an editorial that is getting my nose twitching, and pushes me to speed up a Balneus post that has been brewing for a while. The growing literature on the diseases of children caused by advanced parental age suggests that the societal pattern of people building their careers before having children needs to be reviewed by social policy makers.
"Epigenetic biomarkers in psychiatric disorders" British Journal of Pharmacology (2008) 155, 795–796; doi:10.1038/bjp.2008.254; published online 23 June 2008 (also as PDF is yet another paper stressing the importance of epigenetics in pathogenesis, and introduces a new word, "epigenomics" that relates to testing and markers.
Basically, the older the person (male or female) when conceiving a child, the more likely something epigenetic has gone awry and will cause problems.
Another relatively recent paper highlighted the relationship between advanced parental age and schizophrenia: "Aberrant Epigenetic Regulation Could Explain the Relationship of Paternal Age to Schizophrenia" Schizophrenia Bulletin doi:10.1093/schbul/sbm093 (advance publication 2007-08-21) contains the following:
In 2001, Malaspina et al showed that the incidence of schizophrenia increased progressively with increasing paternal age, the risk being 2-fold and 3-fold for offspring of fathers aged 45–49 and 50 or more years, compared with those of fathers aged less than 25 years.
It’s not just schizophrenia: autism, cognitive and learning difficulties, longevity … the list gets longer every year.
It’s a far cry from what we were taught at uni in the seventies: that old ova stuck in meiosis for 40 years accumulated damage (leading to increased incidence of trisomy 21 or Down’s Syndrome), but because spermatogenesis was continuous, older males didn’t cause such problems.
This raises questions about how social policy affects societal health perhaps more serious than the "diabesity" epidemic, as obesity is more easily treated than something caused at the time of conception (even before).
The easy recommendation is for ladies: ignore the flattery and bank balances of older men!
For males, it’s worthwhile trying to settle down earlier, do the parenting bit with your career on hold.
For politicians, this means that education patterns and work/life balance policies need some attention - unless we want each generation of teenagers to be nuttier than than the previous one.
Someone in Canberra should be crunching the numbers between the census details on parental age and epidemiology, taking into account greater diagnostic capabilities across the years.
I’m much relieved that at 48, my grandson is approaching 2, not only because of this research, but because I’ve got just enough energy to keep up with him for a couple of days (I stay with my daughter and grandson every second weekend on average). I’d be much less fun for him if my joints were any creakier!
Posted in Biology and Health, Politics, Society

Harry Fisch's advice? "If my son or daughter was to ask, I'd tell them to have kids early -- and that's before 30."

2008-11-08T09:32:00.000-08:00

Yo, dude, check your bio clock -- now
New studies warn that it isn't just women who become less fertile as they age
Sarah Treleaven , The Ottawa Citizen
Recently, I've had a lot of conversations about baby-making with my male friends.

"I worry that I might be too selfish to ever have children," said my friend Joe, 29, somewhat pensively over gin and cucumber cocktails. Ditto for Colin, who just broke up with a woman he loves because she wants to have kids in the next few years and, at 35, he just doesn't feel ready yet. Kids or no, they both feel like they have all the time in the world to decide.

I, on the other hand, just turned 30 and have been making a lot of jokes about needing an apartment with a second bedroom for my soon-to-be-frozen eggs.

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Font:****Lots of women wring their hands about having a baby. Not only do we have to worry about our plummeting fertility (which begins to tank in our mid-20s), but we also have to worry about job retention and advancement once those kids (come biology, adoption or surrogacy) eventually appear. And it's the physical limitations of the female ability to procreate that have placed such a heavy emphasis on the reproductive biological clock, shaping the way many women live, work and even date.

But evidence is increasingly emerging that men, too, have a reproductive biological clock -- and that it ticks much more loudly than most of us have thought. Even as stories occasionally emerge about septuagenarian and octogenarian men becoming proud papas -- author Saul Bellow, for example, fathered a child at 84 -- several recent studies are challenging the conventional wisdom that men have an invincible ability to procreate.

A French study released in July found that women's pregnancy rates drop and miscarriages increase when the mother is over 35 and the father is over 40. Another study suggests that a man's fertility begins to decrease as early as his 20s. Researchers from the University of California at Berkeley and the Lawrence Livermore National Laboratory tested men between the ages of 22 and 80, and found that semen volume and sperm motility were both significantly compromised by aging.

Additionally, the increased odds for older fathers producing genetic abnormalities have been well documented, and studies have demonstrated that fathers over 40 are six times more likely to produce an autistic child than fathers under 30.

The numbers related to schizophrenia are similarly compelling. A study utilizing health databases in Jerusalem found that fathers over 40 were twice as likely to produce schizophrenic children as fathers who were under 25; for fathers over 50, the odds tripled when compared to fathers who were under 25.

Dr. Harry Fisch, director of the Male Reproductive Center at New York-Presbyterian Hospital/Columbia University Medical Center and the author of The Male Biological Clock, says that he's been ringing the alarm bell for years.

"There's a female biological clock; we all agree on the decline in fertility, more genetic problems and a decline in estrogen.

"The same thing happens in men -- a little bit differently, but essentially the same," Fisch says. "Why is it important? Well, demographically more men and women are waiting until they're over 30 to have a baby."

Study sheds light on genetic differences that cause a childhood eye disease

2008-10-31T09:58:00.000-07:00

Public release date: 31-Oct-2008

Contact: Lindsay Elleker
lindsay.elleker@ualberta.ca
780-492-0647
University of Alberta Faculty of Medicine & Dentistry

Study sheds light on genetic differences that cause a childhood eye disease

Medical researchers at the University of Alberta have unlocked part of the mystery underlying a childhood eye disease. New research shows how children with some types of glaucoma end up with missing or extra pieces of DNA.

The missing or extra bits of DNA are called copy number variations (CNVs). The U of A research team had previously shown how they play a major role in causing some types of pediatric glaucoma – a disease that can lead to blindness. In their current study, published in Human Molecular Genetics, the authors describe how the CNVs that cause childhood glaucomas are formed.

Using genetic samples from patients living with pediatric glaucoma, the research team studied the locations where extra or missing pieces of DNA begin and end. Close examination of these break points allowed the team to determine how these copy number variations occur.

"Our findings broaden the mechanisms known to cause copy number variations, which improves our understanding not only of pediatric glaucoma, but also of the growing number of genetic diseases linked to copy number variations, including heart disease and psoriasis. We're really only looking at the tip of the iceberg in terms of how CNVs cause disease." said Dr. Ordan Lehmann, an associate professor with the Faculty of Medicine & Dentistry at the University of Alberta and an ophthalmologist with Alberta Health Services. "These findings will also help us to improve the detection of pediatric glaucoma and, by allowing earlier diagnosis, will help lead to earlier treatment of this condition."

###
The research team has received funding from the Emerging Research Teams Grant Program, which was created by the Faculty of Medicine & Dentistry and Alberta Health Services to provide startup money to promising research groups.

The study was undertaken in collaboration with researchers at the University of Chicago

Overlap Found Between Autism, Schizophrenia-Spectrum Disorders

2008-10-03T12:29:00.000-07:00

Psychiatr News October 3, 2008
Volume 43, Number 19, page 20
© 2008 American Psychiatric Association

Articles by Arehart-Treichel, J.

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Clinical & Research News

Overlap Found Between Autism, Schizophrenia-Spectrum Disorders
Joan Arehart-Treichel
Some patients may have traits of both autism and schizophrenia because the autism-spectrum and schizophrenia-spectrum disorders share some of the same susceptibility genes.

Although autism and schizophrenia are now generally recognized as two separate illnesses, there is reason to believe that autistic traits and schizophrenia traits co-occur in some individuals.

For instance, some children with autism disorder have been found to develop schizophrenia later in life, the negative symptoms of schizophrenia have been found to co-vary with autistic traits in certain schizophrenia subjects, and a link between autistic traits and schizophrenia traits was found in a sample of college students.

Now certain individuals with schizotypal personality disorder—considered the mildest schizophrenia-spectrum illness—have been found to possess an unusual preponderance of autistic traits. The results of the study, which was led by Michelle Esterberg, M.P.H., of Emory University, were published in the September Schizophrenia Research.

The study included 121 adolescent subjects—35 with schizotypal personality disorder; 38 with other types of personality disorders (antisocial, avoidant, borderline, narcissistic, obsessive-compulsive, paranoid, or schizoid); and 48 with no personality disorders. The subjects were evaluated for various autistic characteristics, and the results for each group were then compared.

The schizotypal group scored significantly higher than the other two groups on a number of autistic traits. They included being socially anxious, having no close friends, using a limited number of facial expressions, not showing affection, being unaware of social cues, having circumscribed or unusual interests, and being resistant to change. Furthermore, the schizotypal group scored especially high on deficits in the social-functioning domain.

"The present findings indicate significant ... overlap between autism-spectrum and schizophrenia-spectrum disorders," Esterberg and her colleagues concluded.

Why might autistic traits and schizophrenia traits coexist in certain persons? Esterberg and her group suspect that it is because the autism-spectrum disorders and the schizophrenia-spectrum disorders share some of the same susceptibility genes or because some of the susceptibility genes contributing to each spectrum are occasionally inherited together.

For instance, individuals who lack genes on a particular stretch of chromosome 22—called the 22q11 chromosomal deletion—are known to be at heightened risk for both the autistic-spectrum and schizophrenia-spectrum disorders, they pointed out, suggesting that some genes located in this stretch are complicit in both disorders (Psychiatric News, September 19).

But one point they are quite sure about, as are many other investigators, is that autism and schizophrenia are not identical illnesses. One reason is because 10 of their schizotypal subjects, as well as two other subjects from the "other personality disorder" category, developed schizophrenia during a three-year follow-up period. Yet the researchers could find no link between having autistic traits and subsequently developing schizophrenia.

The study was funded by the National Institute of Mental Health.

An abstract of "Childhood and Current Autistic Features in Adolescents With Schizotypal Personality Disorder" can be accessed at by clicking on "Browse A-Z," "S," and then "Schizophrenia Research."

Children whose fathers were over 33 were 1.8 times more likely to have autism than those fathers were under 29.

2008-09-30T17:11:00.000-07:00

Father age link to autism in children
Older fathers are almost twice as likely to have autistic children as younger men, research has found.

By Rebecca Smith, Medical Editor
Last Updated: 12:31AM BST 01 Oct 2008

A small study of children with autism spectrum disorder, the umbrella term for a range of similar conditions, found they were more likely to have been fathered by men over the age of 33.

There was no link with the condition and the mother's age, the Japanese study found.

The research involved 84 children with high-functioning autism spectrum disorders, meaning they had the social impairments of the condition but had normal intelligence, and 208 children without the disorder.

Children whose fathers were over 33 were 1.8 times more likely to have autism than those fathers were under 29. Men who fathered children between the age of 29 and 32 were 30 per cent more likely to have an autistic child.

The research is published in the British Journal of Psychiatry.

This is the first study to explore the effect of paternal age on the risk of high-functioning autistic spectrum disorder. Its findings correspond with previous studies which have shown a link between older fathers and a low IQ in children.

Benet Middleton, director of communications at The National Autistic Society, said: "The causes of autism are still being investigated. Many experts believe that the pattern of behaviour from which autism is diagnosed may not result from a single cause. Autism affects around one in 100 people in the UK and does not solely affect children of older parents.

"Members of the NAS are made up of parents of children from a variety of ages and backgrounds; in addition there is evidence to suggest that complex genetic factors are responsible for some forms of autism."

Some experts have argued that the measles, mumps and rubella vaccination is linked to the development of autism but this has been widely discredited and other studies have failed to find any link.

Analysis of copy number variation using quantitative interspecies competitive PCR

2008-09-26T09:20:00.000-07:00

Nucleic Acids Research Advance Access originally published online on August 12, 2008
Nucleic Acids Research 2008 36(17):e112; doi:10.1093/nar/gkn495

Nucleic Acids Research, 2008, Vol. 36, No. 17 e112
© 2008 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Methods Online

Analysis of copy number variation using quantitative interspecies competitive PCR
Nigel M. Williams1,*, Hywel Williams1, Elisa Majounie1, Nadine Norton1, Beate Glaser1, Huw R. Morris2, Michael J. Owen1 and Michael C. O’Donovan1
1Department of Psychological Medicine, Wales School of Medicine, Cardiff University, Heath Park, Cardiff CF14 4XN and 2Department of Neurology, Ophthalmology and Audiological Medicine, Wales School of Medicine, Cardiff University, Cardiff, UK

*To whom correspondence should be addressed. Tel: +44(0)2920 687070; Fax: +44(0)2920 687068; Email: williamsnm@cf.ac.uk

Received May 23, 2008. Revised July 17, 2008. Accepted July 17, 2008.

Over recent years small submicroscopic DNA copy-number variants (CNVs) have been highlighted as an important source of variation in the human genome, human phenotypic diversity and disease susceptibility. Consequently, there is a pressing need for the development of methods that allow the efficient, accurate and cheap measurement of genomic copy number polymorphisms in clinical cohorts. We have developed a simple competitive PCR based method to determine DNA copy number which uses the entire genome of a single chimpanzee as a competitor thus eliminating the requirement for competitive sequences to be synthesized for each assay. This results in the requirement for only a single reference sample for all assays and dramatically increases the potential for large numbers of loci to be analysed in multiplex. In this study we establish proof of concept by accurately detecting previously characterized mutations at the PARK2 locus and then demonstrating the potential of quantitative interspecies competitive PCR (qicPCR) to accurately genotype CNVs in association studies by analysing chromosome 22q11 deletions in a sample of previously characterized patients and normal controls.

Paternal Age a major cause of new disorders in the population

2008-09-14T07:12:00.001-07:00

Am J Med Genet A. 2008 Sep 15;146A(18):2385-9. Links
The population-based prevalence of achondroplasia and thanatophoric dysplasia in selected regions of the US.Waller DK, Correa A, Vo TM, Wang Y, Hobbs C, Langlois PH, Pearson K, Romitti PA, Shaw GM, Hecht JT.
Houston Health Science Center, The University of Texas, Houston, Texas 77030, USA. kim.waller@uth.tmc.edu

There have been no large population-based studies of the prevalence of achondroplasia and thanatophroic dysplasia in the United States. This study compared data from seven population-based birth defects monitoring programs in the United States. We also present data on the association between older paternal age and these birth defects, which has been described in earlier studies. The prevalence of achondroplasia ranged from 0.36 to 0.60 per 10,000 livebirths (1/27,780-1/16,670 livebirths). The prevalence of thanatophoric dysplasia ranged from 0.21 to 0.30 per 10,000 livebirths (1/33,330-1/47,620 livebirths). In Texas, fathers that were 25-29, 30-34, 35-39, and > or =40 years of age had significantly increased rates of de novo achondroplasia among their offspring compared with younger fathers. The adjusted prevalence odds ratios were 2.8 (95% CI; 1.2, 6.7), 2.8 (95% CI; 1.0, 7.6), 4.9 (95% CI; 1.7, 14.3), and 5.0 (95% CI; 1.5, 16.1), respectively. Using the same age categories, the crude prevalence odds ratios for de novo cases of thanatophoric dysplasia in Texas were 5.8 (95% CI; 1.7, 9.8), 3.9 (95% CI; 1.1, 6.7), 6.1 (95% CI; 1.6, 10.6), and 10.2 (95% CI; 2.6, 17.8), respectively. These data suggest that thanatophoric dysplasia is one-third to one-half as frequent as achondroplasia. The differences in the prevalence of these conditions across monitoring programs were consistent with random fluctuation. Birth defects monitoring programs may be a good source of ascertainment for population-based studies of achondroplasia and thanatophoric dysplasia, provided that diagnoses are confirmed by review of medical records. Copyright 2008 Wiley-Liss, Inc.

PMID: 18698630 [PubMed - indexed for MEDLINE]

Related ArticlesEffect of paternal age in achondroplasia, thanatophoric dysplasia, and osteogenesis imperfecta. [Am J Med Genet. 1995] The birth prevalence rates for the skeletal dysplasias. [J Med Genet. 1986] Thanatophoric dysplasia: an autosomal dominant condition? [Am J Med Genet. 1988] Association of paternal age with prevalence of selected birth defects. [Birth Defects Res A Clin Mol Teratol. 2007] Prevalence of spina bifida at birth--United States, 1983-1990: a comparison of two surveillance systems. [MMWR CDC Surveill Summ. 1996] » See all Related Articles...

Bipolar disorder: What you need to know

2008-09-05T23:16:00.000-07:00

Bipolar disorder: What you need to know
By NIH
Sep 3, 2008 - 8:08:23 AM

Older men may be at risk of having children with bipolar disorder, according to a study published in the Sep. 2008 issue of Archives of General Psychiatry.

Emma M. Frans, M.Med.Sc., of the Karolinska Institutet, Stockholm, Sweden, and colleagues came to the conclusion after they compared 13,428 patients in Swedish registers with a diagnosis of bipolar disorder with those with sex and age matched, but without the condition.

They found the older an individual's father, the more likely he or she was to develop bipolar disorder. An increase risk was observed among children who were fathered by men age 29 or older.

"After controlling for parity [number of children], maternal age, socioeconomic status and family history of psychotic disorders, the offspring of men 55 years and older were 1.37 times more likely to be diagnosed as having bipolar disorder than the offspring of men aged 20 to 24 years," the authors wrote.

The ages of older mothers also had an effect on the risk, which was not as significant as the ages of older fathers. There was no association between the mother's age and early bipolar disorder (diagnosed before the age of 20). But the association existed for the father's age.

The researchers explained that de novo mutations occur in germ cell replications while women's eggs do not have as many replications and mutations may not be as common in eggs. Because of this, maternal age does not affect the risk of bipolar disorder in children as much as the father's age.

Few risk factors have been identified for bipolar disorder.

Older paternal age has been linked in previous studies to higher risk of complex neurodevelopmental disorders, including schizophrenia and autism, according to the press release by JAMA and Archives Journals.

Bipolar risk rises with father's age

2008-09-01T09:09:00.000-07:00

Bipolar risk rises with father's age

Adam Cresswell, Health editor September 02, 2008
CHILDREN of older fathers are more likely to have bipolar disorder - a discovery that could explain the increasing numbers of people diagnosed with the condition.
Compared with the offspring of fathers aged 20 to 24, people whose fathers were aged 55 or over at the time of their birth are 37 per cent more likely to be diagnosed with bipolar disorder.
Children of fathers aged 30-34 had an 11 per cent increased risk of bipolar, while children of fathers aged 40-44 had a 15 per cent increased risk.
Having an older mother also increased the risk, but the effect was far less pronounced.
The research is based on nearly 13,500 Swedish people with bipolar disorder, a severe mood disorder that causes repeated peaks of euphoria and hyperactivity followed by troughs of depression.
The authors of the study, published in the US journal Archives of General Psychiatry, said paternal age was already known to be linked to other developmental disorders such as schizophrenia and autism. They suggested the findings might reflect the increased risk of DNA mutations in sperm cells, which, unlike a woman's eggs, undergo hundreds of replication cycles in which errors can occur.
Australian psychiatrist Gordon Parker, executive director of the Black Dog Institute, said the findings were important, and might explain why diagnoses of bipolar disorder had been rising.
In 1992, according to the Australian Bureau of Statistics, 35 per cent of fathers of children aged 0-14 were under 35. This fell to 26 per cent by 2003. The proportion of fathers aged 45 and over rose from 19 per cent in 1992 to 25 per cent in 2003.
However, Professor Parker said as the existing risk of bipolar disorder was thought to be between 4 and 6 per cent, the effect of the increases remained slight.
"I would hate to see any concern in the community that people shouldn't have babies because they have bipolar disorder in their family," he said.
"If you have bipolar disorder in your family, you are more likely to end up in Who's Who, because high intelligence and creativity is over-represented in families with bipolar disorder."

A Brilliant Explanation of the Male Biological Clock

2008-07-15T21:49:00.000-07:00

Male Biological Clock

CNVs vs SNPs: Understanding Human Structural Variation in Disease

2008-07-12T14:40:00.000-07:00

Advancing paternal age causes copy number variations Science Webinar: CNVs vs SNPs July 12, 2008
Posted by Bertalan Meskó in Webinar, science.
trackback
Science Magazine will organize a webinar about copy number variations and single nucleotide polymorphisms.

CNVs vs SNPs: Understanding Human Structural Variation in Disease

July 16, 2008 at 12 noon Eastern Time (9 a.m. Pacific, 4 p.m. GMT)

Genetic variation—differences in both the coding and noncoding portions of our DNA—is what makes each human being a unique individual. It also can determine our unique susceptibility to disease. Exhaustive analysis of human single nucleotide polymorphisms (SNPs) has led to the identification of interesting SNP markers for certain disorders. But these small changes are not the whole picture. Copy number variations (CNVs)—gain or loss of segments of genomic DNA relative to a reference—have also been shown to be associated with several complex and common disorders. Using array-based comparative genomic hybridization (CGH) techniques, CNVs at multiple loci can be assessed simultaneously allowing for their identification and characterization. CNV microarrays allow exploration of the genome for sources of variability beyond SNPs that could explain the strong genetic component of several of these disorders. Now, advances in microarray probe density have provided more comprehensive coverage of CNVs, enabling more in depth genotyping research.

You can register here!

Speakers:

Charles Lee, Ph.D., FACMG; Department of Pathology, Brigham and Women’s Hospital, Boston MA
Lars Feuk, Ph.D., The Centre for Applied Genomics; The Hospital for Sick Children, Toronto, Ontario
Alexandra I Blakemore, Ph.D., Division of Medicine, Imperial College London, United Kingdom
The moderator will be Sean Sanders, Ph.D., Commercial Editor, Science/AAAS.

CCR5 haplotypes HHE and HHG*2 strongly influence the risk of SLE

2008-07-11T18:02:00.000-07:00

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Published Online First: 30 October 2007. doi:10.1136/ard.2007.078048
Annals of the Rheumatic Diseases 2008;67:1076-1083
Copyright © 2008 BMJ Publishing Group Ltd & European League Against Rheumatism
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CCL3L1 gene-containing segmental duplications and polymorphisms in CCR5 affect risk of systemic lupus erythaematosus
M Mamtani 1, B Rovin 2, R Brey 3, J F Camargo 1, H Kulkarni 1, M Herrera 1, P Correa 4, S Holliday 5, J-M Anaya 4,6, S K Ahuja 1,7
1 The Veterans Administration Center for AIDS and HIV-1 Infection, South Texas Veterans Health Care System and Department of Medicine, University of Texas Health Science Center at San Antonio, Texas, USA
2 The Ohio State University College of Medicine and Public Health and Davis Heart and Lung Research Institute, Columbus, Ohio, USA
3 Department of Neurology, University of Texas Health Science Center at San Antonio, Texas, USA
4 Cellular Biology and Immunogenetics Unit, Corporacion para Investigaciones Biologicas, Medellin, Colombia
5 Psychology Service, South Texas Veterans Health Care System, San Antonio, Texas, USA
6 School of Medicine, Universidad del Rosario, Bogota, Colombia
7 Departments of Microbiology and Immunology, and Biochemistry, University of Texas Health Science Center, San Antonio, Texas, USA

Correspondence to:
S K Ahuja, Veterans Administration Center for AIDS and HIV infection, University of Texas Health Science Center, 7703 Floyd Curl Drive, Room 5.009R, San Antonio, Texas, 78229-7870, USA; ahujas@uthscsa.edu

Objectives: There is an enrichment of immune response genes that are subject to copy number variations (CNVs). However, there is limited understanding of their impact on susceptibility to human diseases. CC chemokine ligand 3 like-1 (CCL3L1) is a potent ligand for the HIV coreceptor, CC chemokine receptor 5 (CCR5), and we have demonstrated previously an association between CCL3L1-gene containing segmental duplications and polymorphisms in CCR5 and HIV/AIDS susceptibility. Here, we determined the association between these genetic variations and risk of developing systemic lupus erythaematosus (SLE), differential recruitment of CD3+ and CD68+ leukocytes to the kidney, clinical severity of SLE reflected by autoantibody titres and the risk of renal complications in SLE.

Methods: We genotyped 1084 subjects (469 cases of SLE and 615 matched controls with no autoimmune disease) from three geographically distinct cohorts for variations in CCL3L1 and CCR5.

Results: Deviation from the average copy number of CCL3L1 found in European populations increased the risk of SLE and modified the SLE-influencing effects of CCR5 haplotypes. The CCR5 human haplogroup (HH)E and CCR5-32-bearing HHG*2 haplotypes were associated with an increased risk of developing SLE. An individual’s CCL3L1–CCR5 genotype strongly predicted the overall risk of SLE, high autoantibody titres, and lupus nephritis as well as the differential recruitment of leukocytes in subjects with lupus nephritis. The CCR5 HHE/HHG*2 genotype was associated with the maximal risk of developing SLE.

Conclusion: CCR5 haplotypes HHE and HHG*2 strongly influence the risk of SLE. The copy number of CCL3L1 influences risk of SLE and modifies the SLE-influencing effects associated with CCR5 genotypes. These findings implicate a key role of the CCL3L1–CCR5 axis in the pathogenesis of SLE.

Additionally the risk of autism, epilepsy or schizophrenia also increased in these kids, which led to accidental deaths as well,

2008-07-08T09:48:00.000-07:00

Man's Ability to Have Kids is Dependent on His Age

Written by Theresa Maher
Monday, 07 July 2008
MONDAY, July 7, (News Locale) - Contrary to popular perception, a man is not able to have kids anytime he wishes. New research out of France indicates male fertility is also dependent on age and men who delay fatherhood may have a tough time conceiving later on.
It is believed that unlike women, men have no biological clock and can father children throughout their life. In fact it is not uncommon to see celebrities having babies well after they have crossed their 50s.

Now researchers at the Eylau Centre for Assisted Reproduction in Paris have revealed men who delay fatherhood have a less chance of impregnating their partners.

The study of more than 20,000 couples who sought fertility help at the center found men over the age of 35 are almost a third less likely to conceive as compared to their younger counterparts. Furthermore men over the age of 40 had poor quality of sperm, which could lead to frequent miscarriages in their partners. In fact the risk of miscarriage if the father was over the age of 40 was 75 percent.

Researchers believe the DNA in sperm starts to decay with age and this may be the cause of fertility issues in older men.

The details of the study were presented at the European Society of Human Reproduction and Embryology conference.

An earlier study by Danish researchers had revealed kids born to older fathers were more likely to die before they entered adulthood when compared to kids born to younger fathers. This incidence was attributed to the declining quality of sperm due to ageing.

The scientists found that congenital defects like heart and spine problems were the main cause of death in these children.

Additionally the risk of autism, epilepsy or schizophrenia also increased in these kids, which led to accidental deaths as well, the researchers had reported in the European Journal of Epidemiology.

Consumers must be aware that the mother's age has always been associated with pregnancy complications. The above study provides evidence that a father's age may also have a say in conception.

'Miscarriage rate rises with age of father' CNVs?

2008-07-06T19:15:00.000-07:00

'Miscarriage rate rises with age of father'

By Steve Connor, Science Editor
Monday, 7 July 2008

Women with older partners may be at higher risk of suffering miscarriages irrespective of their own age, according to a study that has linked the increased chance of a failed pregnancy with men over the age of 40.

copy number variants related to advanced paternal age may also contribute to the differential trajectory of brain development associated with autism

2008-06-27T18:40:00.000-07:00

and schizophrenia.
1: Behav Brain Sci. 2008 Jun;31(3):264-265.
Animal models may help fractionate shared and discrete pathways underpinning schizophrenia and autism.Burne TH, Eyles DW, McGrath JJ.
Queensland Centre for Mental Health Research, The Queensland Brain Institute, The University of Queensland, St Lucia, Brisbane, 4072, Australia. t.burne@uq.edu.au http://www.qbi.uq.edu.au eyles@uq.edu.au http://www.qbi.uq.edu.au john_mcgrath@qcmhr.uq.edu.au http://www.qbi.uq.edu.au.

Crespi & Badcock (C&B) present an appealing and parsimonious synthesis arguing that schizophrenia and autism are differentially regulated by maternal versus paternal genomic imprinting, respectively. We argue that animal models related to schizophrenia and autism provide a useful platform to explore the mechanisms outlined by C&B. We also note that schizophrenia and autism share certain risk factors such as advanced paternal age. Apart from genomic imprinting, copy number variants related to advanced paternal age may also contribute to the differential trajectory of brain development associated with autism and schizophrenia.

PMID: 18578908 [PubMed - as supplied by publisher]

IVF And De novo point mutations, copy number variations etc. etc.Paternal Age Sperm mutations

2008-06-16T13:48:00.000-07:00

"Furthermore, genetic damage to the spermatozoa of aging males is thought to contribute to the etiology of more complex polygenic conditions such as autism, spontaneous schizophrenia and epilepsy" RJ Aitken
Expert calls for vigilance on IVF technology
By Anna Salleh for ABC Science Online

Posted Sat Jun 14, 2008 10:46am AEST

A 3D ultrasound showing a foetus inside the womb. (Getty Images)
As humans become more dependent on reproductive technologies, an Australian reproductive biologist says we must remain vigilant to avoid the spread of genetic defects.

The warning comes in an editorial by Professor John Aitken, of the University of Newcastle, in the current issue of Expert Review of Obstetrics and Gynecology.

"People shouldn't be too confident that just because the baby looks normal there is no damage there that won't appear later in life," he said.

"People underestimate how much genetic damage they're passing onto the embryos."

Professor Aitkin says one in every 35 babies born in Australia are a result of IVF.

"In some countries it's more like one in 20 and there are models that predict it will be one in 10 before too long," he said.

Professor Aitken says because IVF allows infertile men to reproduce, the more we use it the more it will be needed in the future.

"So we better make sure it's safe because a large proportion of the population will be generated in this way," he said.

Ageing sperm

Professor Aitken says a number of factors are known, or suspected, to cause genetic damage to sperm that do not necessarily cause defects obvious at birth.

For example, Professor Aitken says the sperm of ageing males is thought to contribute to conditions such as autism, schizophrenia and epilepsy.

He says there is strong evidence linking sperm DNA damage to smoking, which can lead to the development of childhood cancers.

Epigenetic changes to sperm DNA that can affect fertility through several generations have also been reported.

For example, several recent papers have shown that infertile men have a dramatically altered DNA methylation profile.

Screening and monitoring

Professor Aitken says genetic problems mean it is important that reproductive clinics do a good job at screening sperm samples for genetic damage.

He is presenting the latest evidence on one screening technique he is developing with biotech company nuGEN at the Australian Research Council's Graeme Clark Research Outcomes Forum in Canberra next week.

But Professor Aitken says long-term monitoring of children born through IVF and other reproductive technologies is also essential, because such techniques can not pick up epigenetic damage.

"There are all kinds of things that can and could still go wrong," he said.

While he says IVF children are being monitored, he is concerned about complacency among clinics who celebrate their ability to produce normal looking babies from sperm with high levels of DNA damage.

IVF defended

Professor Michael Chapman of the Fertility Society of Australia, who also works for IVF Australia, says genetic damage is considered by IVF clinics.

"They're concerns that are shared within the IVF profession," he said.

Professor Chapman says one rare epigenetic disease has shown up in IVF children, at a rate of one in 1,500 versus one in 5,000 in the general population.

But he says Professor Aitken's "provocative" article overstates the problem since in the 20 years that IVF has been around, few long-term problems have arisen, despite thousands of children being monitored.

"I'm sure that if something starts to turn up, it will jump out at us," he said.

Sandra Hill, chief executive officer of ACCESS Australia, a group led by patients seeking IVF treatment, is confident that IVF is well-monitored, and she agrees this should continue.

But she says many of the concerns raised by Professor Aitken also apply to natural conception and she thinks the use of IVF should not be singled out.

She says it could be useful to educate men in general about the concerns raised by Professor Aitken - especially the need for men to have children before they get too old.

Professor Aitken says this may be so, but IVF still presents a unique challenge.

"With IVF you are facilitating the fertilisation of eggs with sperm that would otherwise be unsuccessful," he said.

Professor Aitken also says the rate of birth defects in IVF children are up to twice that of normally-conceived children, although he expects that to improve as techniques improve.

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Professor R. John Aitken ScD, FRSE
Director, ARC Centre of Excellence in Biotechnology and Development,
Professor of Biological Sciences,
School of Environmental and Life Sciences,
University of Newcastle
Callaghan
NSW 2308.

E mail: jaitken@mail.newcastle.edu.au
Tel: (+61 2) 4921 6143
Mobile: 0414 667 878
Fax: (+61 2) 4921 6308

John Aitken is currently Director of ARC Centre of Excellence in Biotechnology and Development and Professor of Biological Sciences at the University of Newcastle, NSW. His research interests focus on the cell biology of male germ cells, particularly the cell biology of human spermatozoa and the mechanisms regulating the formation and recruitment of primordial follicles within the ovary.

Qualifications: BSc (Special Hons) University of London

MSc University of Wales

PhD University of Cambridge

ScD University of Cambridge

Fellow of the Royal Society of Edinburgh

Positions:

1982-1987 Senior Scientist, Medical Research Council Reproductive Biology Unit, University of Edinburgh.

1987-1998 Special Appointment, Professorial Grade, Medical Research Council Reproductive Biology Unit, University of Edinburgh

1992- Honorary Professorship, Department of Obstetrics and Gynaecology, University of Edinburgh

1998- Professor of Biological Sciences, Faculty of Science and IT, University of Newcastle, NSW.

1998 Director of the Centre for Life Sciences, University of Newcastle, NSW.

2003 Director of the ARC Centre of Excellence in Biotechnology and Development

Honours:

1984 Honorary Fellow of the Faculty of Medicine,University of Edinburgh.

1985 Ayerst Lecturer, Pacific Coast Fertility Society,Caesar's Palace, Las Vegas.

1985 Ortho-McMaster Lecturer, McMaster University Medical Centre, Hamilton, Canada.

1985 Convener, Chairman and Editor, WHO symposium on The Zona-free Hamster Oocyte Penetration test in the Diagnosis of Male Infertility, Boston, Massachusetts, USA.

1986 The Walpole Prize, Society for the Study of Fertility, United Kingdom

1987 The Walpole Prize, Society for the Study of Fertility, United Kingdom

1989 1989 University of Catania Prize, Scientific Committee, Faculty of Medicine.

University of Catania, Italy.

1990 The Puvan Memorial Lecture. Opening Address of the 27th Malaysian Congress of Obstetrics and Gynaecology.

1990 The Jennifer Hallum Memorial Lecture. Royal College of Obstetricians and Gynaecologists,

1992 Honorary Professorship, Department of Obstetrics and Gynaecology, Faculty of Medicine, University of Edinburgh.

1994 Opening Address Thaddeus Mann Symposium. Seventh International Congress of Spermatology, Cairns, Australia.

1994 American Fertility Society State-of-the-Art Lecture. Annual Meeting, San Antonio, Texas,

1995 Elected a Fellow of the Royal Society of Edinburgh

1997 Plenary Lecture European Society of Human Reproduction and Embryology Congress 1997. Edinburgh Conference Centre.

1997 The Bruce Stewart Memorial Lecture 1997 American Urology Society Lecture, American Society for Reproductive Medicine, Cincinnati, OH, USA.

1998 The 1998 Amoroso Lecture. The human spermatozoon-a cell in crisis? Society for the Study of Fertility, Annual Meeting, University of Glasgow, UK

1998 Society for Male Reproduction/Urology Prize Paper. 16th World Congress on Fertility and Sterility/54th Annual Meeting of the American Society for Reproductive Medicine, San Francisco

1999 M.J. Edwards Lecture. Australian Birth Defects Society. University of Sydney.

2000 Best Poster Award. Combined meeting of the Society for Free Radical Research on Oxidants, Antioxidants and Nutrition and ComBio 2000. Wellington, New Zealand.

2002 Plenary Lecture, World Congress on Human Reproduction, Montreal, Canada

2003 Lloyd Cox memorial Lecture, University of Adelaide.

2004 The Founders Lecture, Society for the Study of Reproduction, Annual Meeting, Sydney.

Relevant Employment History:

1987-98 Special Appointment -Professorial Level

MRC Reproductive Biology Unit, University of Edinburgh,

1992-present Honorary Professorship, Faculty of Medicine, University of Edinburgh.

1982-1987 Senior Scientist

MRC Reproductive Biology Unit, University of Edinburgh.

1977-1982 Research Scientist Grade 1

MRC Reproductive Biology Unit, University of Edinburgh.

1976-1977 Chargé de Recherche

Faculte de Medicine, Universitie de Bordeaux.

1975-1976 Consultant Scientist

Human Reproduction Programme, World Health Organisation, Geneva.

1973-1975 MRC Post-Doctoral Fellowship

Department of Genetics, University of Edinburgh.

Publications and Presentations:
More than 350 peer review publications and more than 300 presentations at national and international meetings. Examples:

Angell, R.R., Aitken, R.J., Van Look, P.F.A., Lumsden, M.A. & Templeton, A.A. (1983) Chromosome abnormalities in human embryos after in vitro fertilization. Nature, 303, 336-338.

Aitken, R.J. & Clarkson, J.S. (1987) Cellular basis of defective sperm function and its association with the genesis of reactive oxygen species by human spermatozoa. Journal of Reproduction and Fertility, 83, 459-469.

This article was awarded the Walpole Memorial Prize and Lecture by the Society for the Study of Fertility

Henderson, C.J., Hulme, M.J. & Aitken, R.J. (1988) Contraceptive potential of antibodies to the zona pellucida. Journal of Reproduction and Fertility, 8, 325-343.

This article was awarded the Walpole Memorial Prize and Lecture by the Society for the Study of Fertility

Nasr-Esfahani, M, Aitken, R.J. & Johnson, M.H. (1990) The measurement of H2O2 levels in preimplantation embryos from blocking and non-blocking strains of mice. Development, 109, 501-507.

Aitken, R. J., M. Paterson, H. Fisher, D.W. Buckingham & Van Duin, M. (1995) Redox regulation of tyrosine phosphorylation in human spermatozoa is involved in the control of human sperm function. Journal of Cell Science, 108, 2017-2025.

Aitken, R. J., Buckingham, D.W. & Irvine, D.S. (1996) The extragenomic action of progesterone on human spermatozoa: evidence for a ubiquitous response that is rapidly down-regulated. Endocrinology 137, 3999-4009.

Twigg, J., Fulton, N., Gomez, E, Irvine D. S. & Aitken, R. J. (1998) Analysis of the impact of intracellular reactive oxygen species generation on the structural and functional integrity of human spermatozoa: lipid peroxidation, DNA fragmentation and effectiveness of antioxidants. Human Reproduction 13, 1429-1437. Featured as an ‘Outstanding Article’ by the journal

Aitken, R. J., Harkiss, D., Knox, W., Paterson, M. and Irvine, D. S. (1998) A novel signal transduction cascade in capacitating human spermatozoa characterized by a redox-regulated, cAMP-mediated induction of tyrosine phosphorylation. Journal of Cell Science 111, 645-656.

Aitken, R. J. (1999) The human spermatozoon: a cell in crisis? The Amoroso Lecture. Journal of Reproduction and Fertility 115, 1-7.

Aitken, R.J. & Marshall Graves, J. A. (2002) The Y chromosome, oxidative stress and the future of sex. Nature 415, 963.

Ecroyd, H., Asquith, K., Jones, R.C. & Aitken R.J. (2004) The development of signal transduction pathways during epididymal maturation is calcium dependent. Developmental Biology 268, 53-63.

Baker, M.A., Hetherington, L., Ecroyd, H. & Aitken, R.J. (2004) Analysis of the mechanism by which calcium negatively regulates the tyrosine phosphorylation cascade associated with sperm capacitation. Journal of Cell Science 117, 211-222.

Asquith, K. L., Baleato, R. M. McLaughlin, E. A., Nixon B. & Aitken R.J. (2004) Analysis of the mechanisms by which tyrosine phosphorylation regulates sperm-zona recognition in the mouse, a chaperone-mediated event? Journal of Cell Science 117, 3645-3657.

Aitken, R.J., Koopman, P. & Lewis S. E. (2004). Seeds of concern. Nature. 432, 48-52.
Full Text
Expert Review of Obstetrics & Gynecology
May 2008, Vol. 3, No. 3, Pages 267-271
(doi:10.1586/17474108.3.3.267)

Just how safe is assisted reproductive technology for treating male factor infertility?
R John Aitken
Sections: ChooseINTRODUCTIONThe male factorParental ageEnvironmental pollutionErrors of endogenous meta...Electromagnetic radiation...Epigenetic damageIs ART a dangerous form o...References

INTRODUCTION ChooseTop of pageINTRODUCTION The male factorParental ageEnvironmental pollutionErrors of endogenous meta...Electromagnetic radiation...Epigenetic damageIs ART a dangerous form o...References

Assisted reproductive technology (ART) has been responsible for the birth of over 3 million babies since the delivery of Louise Brown in the UK 28 years ago. Currently, one in 80–100 children born in the USA, one in 50 born in Sweden, one in 40 born in Australia and one in 24 born in Denmark are the product of this form of treatment. In 2003, more than 100,000 in vitro fertilization (IVF) cycles were reported from 399 clinics in the USA, resulting in the birth of more than 48,000 babies [1,101]. Worldwide, this figure has now exceeded 200,000 births per annum [2] and is continuing to rise. Indeed, it is a biological certainty that the more ART is used in one generation, the more it will be needed in the next. Given the cost of this form of treatment, and the fact that children born as a consequence of ART stand a 30–40% increased risk of birth defects [3], the current widespread use of assisted conception may constitute the beginnings of a serious public health problem.

The male factor ChooseTop of pageINTRODUCTIONThe male factor Parental ageEnvironmental pollutionErrors of endogenous meta...Electromagnetic radiation...Epigenetic damageIs ART a dangerous form o...References

There is general agreement that the two major reasons for patient referral to assisted conception programs are increased maternal age and male subfertility. The former can be easily reversed by public awareness and a change in social attitudes to family planning. However, the latter is a more intractable problem, as we have little or no understanding about the origins of this pathology. As a result, rational treatment or prevention of male infertility is all but impossible.

The importance of the male factor in human infertility has been highlighted by recent analyses of population trends in Denmark. This population has witnessed a steady decline in fertility rates in recent years, which is being addressed by increasing reliance on ART [4]. At the present time, 21% of young Danish men exhibit semen quality (in terms of sperm count and morphology) that falls below the internationally accepted thresholds of normality set by the WHO [5]. Moreover, it has been suggested that this situation is getting worse with the passage of time and, according to a recent publication [6]:

‘we may now have reached a level where semen quality of a significant segment of men in the population is so poor that it may contribute to the current widespread use of assisted reproduction’.

Although Denmark affords a particularly striking example of secular trends in male reproduction, semen quality in human males is notoriously poor. Indeed, it is a feature of the human condition, with at least one in 20 men in developed countries suffering from some level of infertility [7]. Most men produce sufficient numbers of spermatozoa to fertilize an egg in vivo; however, the gametes they generate have lost their biological potential for fertilization and the support of normal embryonic development. An important characteristic of these defective spermatozoa is a high level of DNA damage, which is, in turn, correlated with poor fertility, high rates of miscarriage and an increased incidence of disease in the offspring, including childhood cancer [8].

The use of such DNA-damaged spermatozoa in ART is thought to be a major contributor to the increased incidence of birth defects and other diseases seen in children conceived in vitro. Specifically, it has been proposed that the DNA damage brought into the fertilized egg by the spermatozoon may increase the mutational load carried by the embryo as a consequence of the aberrant or incomplete repair of this damage in the interval between fertilization and initiation of the first cleavage division [9,10]. Experimental verification of this relationship between DNA damage in the fertilizing sperm and embryo development has recently been secured in an animal model [11]. In these studies, intracytoplasmic sperm injection (ICSI) was performed in mice with fresh or DNA-damaged spermatozoa. Use of the latter was associated with poor preimplantation development and a reduction in the number of live births. Postnatal examination of the progeny revealed that the use of DNA-damaged spermatozoa in ICSI was associated with behavioral defects (increased anxiety, lack of habituation pattern, deficit in short-term spatial memory and age-dependent hypolocomotion in an open field test), the appearance of mesenchyme tumors, premature aging and a shortened lifespan. These results are supported by clinical data [8] and have profound implications for the safety of ICSI, which must frequently involve the use of DNA-damaged spermatozoa [12]. Currently, both the nature of this genetic damage and its origins are a matter of intense investigation. In terms of etiology, the ensuing paragraphs summarize data suggesting that paternal age, environmental toxicants, errors of endogenous metabolism and exposure to electromagnetic radiation are all potential contributors to DNA damage in the male germ line.

Parental age ChooseTop of pageINTRODUCTIONThe male factorParental age Environmental pollutionErrors of endogenous meta...Electromagnetic radiation...Epigenetic damageIs ART a dangerous form o...References

As men age they do not lose their capacity to generate spermatozoa; however, the quality of these gametes deteriorates. This change can be visualized as an age-dependent increase in DNA fragmentation in spermatozoa [13,14]. Paternal age is also widely recognized as a key factor in the etiology of dominant genetic diseases, such as Apert syndrome or achondroplasia [15]. Furthermore, genetic damage to the spermatozoa of aging males is thought to contribute to the etiology of more complex polygenic conditions such as autism, spontaneous schizophrenia and epilepsy [8]. Since older men tend to be married to older women it is significant that as oocytes age in the ovary, they suffer the depletion of several key genes involved in protection against oxidative stress and the maintenance of DNA integrity, including genes with a probable role in DNA repair [16]. Thus, age-related changes to the integrity of DNA in the spermatozoa are compounded by age-related declines in the oocytes’ capacity for DNA stabilization and repair. In combination, these factors could well make a significant contribution to the elevated incidence of birth defects associated with assisted conception therapy. Whichever way you look at it, aging and reproduction are incompatible bedfellows.

Environmental pollution ChooseTop of pageINTRODUCTIONThe male factorParental ageEnvironmental pollution Errors of endogenous meta...Electromagnetic radiation...Epigenetic damageIs ART a dangerous form o...References

An impact of environmental pollutants on DNA integrity in spermatozoa has been known for some time. For example, men who smoke heavily produce spermatozoa suffering from high levels of oxidative DNA damage. This does not necessarily impair the capacity of these cells for fertilization, however, it does impact upon the subsequent ability of the fertilized egg to develop normally. As a result, the children of heavy smokers stand a four- to fivefold increased chance of developing childhood cancer: a fact that is not often appreciated in the antismoking debate [9].

Recently, exposure of mice to particulate air pollution in an urban/industrial location has also been shown to induce high levels of DNA damage in spermatozoa [17]. Analyses of young men exposed to high levels of air pollution as a result of excessive coal combustion during Eastern European winters have substantiated these results in a clinical context [18]. Similarly, toxicological studies have demonstrated elevated levels of DNA damage in human spermatozoa, which are linked to the presence of metabolites of insecticides or persistent organochlorine pollutants in blood or urine [19,20]. Exposure to environmental endocrine disruptors, such as nonylphenol [21], as well as heavy metals [22], have also been demonstrated to induce oxidative DNA damage in human spermatozoa. Further resolution of the kinds of environmental pollutant that might be damaging to human spermatozoa is clearly needed. Elucidation of the significance of enzyme polymorphisms in defining an individual’s susceptibility to toxicant exposure is also required, as exemplified by a recent study demonstrating that men who are homozygous null for glutathione-S-transferase M1 are more likely to respond to air pollution with high levels of DNA damage in their spermatozoa than men possessing this isoform [23].

Errors of endogenous metabolism ChooseTop of pageINTRODUCTIONThe male factorParental ageEnvironmental pollutionErrors of endogenous meta... Electromagnetic radiation...Epigenetic damageIs ART a dangerous form o...References

Induction of DNA fragmentation in human spermatozoa is not solely due to exposure to environmental toxins, it can also result from errors of endogenous metabolism. An extremely important observation in this context is a recent preliminary report indicating that young male patients suffering from diabetes mellitus exhibit high levels of DNA damage in their spermatozoa [24]. These results have been confirmed in animal studies demonstrating that the experimental induction of diabetes in male mice is associated with oxidative stress and a postmeiotic genotoxic effect reflected in high rates of embryonic resorption in mated females [25]. Our laboratory has also demonstrated that endogenously generated estrogens, particularly catechol estrogens, can have a profound effect on DNA integrity in human spermatozoa, as a consequence of their inherent redox cycling activity [26]. Such studies reinforce the generally held view that most endogenously generated DNA damage in human spermatozoa is a consequence of oxidative stress [8,28]. If this is the case, then any ion (lead or cadmium), organic compound (phthalate ester), enzyme (NADPH oxidase), organelle (mitochondria) or cell (neutrophil), capable of generating reactive oxygen species in the vicinity of human spermatozoa is potentially capable of contributing to DNA damage in the male germ line [8–10,22,28]. In addition to oxidative damage, it is possible that in some patient’s sperm DNA is cleaved by the sequential action of topoisomerase IIB and an uncharacterized nuclease in a process analogous to apoptosis in somatic cells [29]. The relative significance of nuclease- and free radical-mediated mechanisms in the cleavage of sperm DNA, is a key issue that awaits resolution.

Electromagnetic radiation ChooseTop of pageINTRODUCTIONThe male factorParental ageEnvironmental pollutionErrors of endogenous meta...Electromagnetic radiation... Epigenetic damageIs ART a dangerous form o...References

Various forms of electromagnetic radiation are also known to have a detrimental effect on DNA integrity in the male germ line. A classic example is heat. The scrotum is designed to maintain the testes and epididymis slightly below core body temperature. It has been known for some time that elevated testicular temperature impairs spematogenesis. However, recent data have also indicated the ability of mild scrotal heat stress (42°C for 30 min) to induce DNA damage in epididymal mouse spermatozoa [30]. Radiofrequency electromagnetic radiation has also been demonstrated to induce DNA damage in epididymal sperm in animal models [31] and there are some reports of mobile phone exposure having a detrimental effect on semen quality in men [32]. Thus, any practice that elevates testicular temperature, such as wearing clothes or a sedentary occupation, or exacerbated exposure to other forms of electromagnetic radiation, such as excessive mobile phone use, are possible contributors to DNA damage in human spermatozoa.

Epigenetic damage ChooseTop of pageINTRODUCTIONThe male factorParental ageEnvironmental pollutionErrors of endogenous meta...Electromagnetic radiation...Epigenetic damage Is ART a dangerous form o...References

In some couples, the damage brought into the oocyte by the fertilizing spermatozoon may be epigenetic rather than genetic. These epigenetic factors are reviewed in an article in this edition of Expert Review of Obstetrics and Gynecology [31] and include: a functional centrosome to regulate cell division in the embryo; an appropriate pattern of chromatin remodeling; an appropriate population of mRNA and miRNA species that are carried into the zygote by the fertilizing spermatozoon and may play a role in the regulation of early embryonic development; and an appropriate pattern of DNA methylation. There are several recent papers indicating that the DNA methylation profile is dramatically altered in the spermatozoa of infertile men and we already know that the incidence of imprinting defects is elevated in children born as a result of ART [31,32]. The importance of epigenetic defects in the male germ line has recently been highlighted by analyses of vinclozolin, a fungicide used in the wine-making industry [33]. Transient embryonic exposure to vinclozolin in utero resulted in the birth of male offspring exhibiting a spermatogenenic defect. This defect was epigenetic in origin and was vertically transmitted through at least four generations.

Is ART a dangerous form of therapy? ChooseTop of pageINTRODUCTIONThe male factorParental ageEnvironmental pollutionErrors of endogenous meta...Electromagnetic radiation...Epigenetic damageIs ART a dangerous form o... References

Given the evidence that both IVF and ICSI are associated with a significant increase in birth defects, should ART be regarded as safe? On one hand, there is no denying that ART, and particularly ICSI, is an effective form of treatment for infertility. After 12 months approximately 90% of couples submitting to this form of therapy walk away with a baby. Furthermore, even though the risk of birth defects is significantly elevated following ART, the incidence is still relatively rare and should decrease as the field moves towards single-embryo transfers, thereby eliminating complications created by multiple births. Moreover, several clinical groups have trumpeted their ability to successfully perform ART in couples where the male partner’s spermatozoa exhibit high levels of DNA damage, without any obvious consequences as far as the health and wellbeing of the offspring are concerned [34]. These and other data tell us that even if DNA-damaged spermatozoa are used for assisted human conception, the risk of generating a visible phenotypic change in the offspring is extremely low.

However, we should also recognize that the absence of a pathological phenotype in the vast majority of children born as a result of ART, does not mean that the genome has not been damaged, or that the damage will not emerge in some future generation, as a result of mechanisms such as haploid insufficiency, the expression of X-linked defects in male offspring or the future creation of double-recessive combinations. It also does not mean that we will not find defects if we look hard enough. The controversial discovery of fertility-threatening Y-chromosome deletions in the offspring of genotypically normal males as a consequence of ART, is an example of a condition that may take 25–30 years to surface even though the mutation was probably created shortly after fertilization [35].

Clearly, we must continue to be vigilant in our long-term monitoring of the health and wellbeing of children produced by ART. Given recent advances in our understanding of epigenetic defects in the spermatozoa of infertile male patients, we should also extend this surveillance to the DNA methylation profiles of children born as a result of assisted conception. It is also incumbent upon embryologists to optimize the quality of the gametes that are used for ART, particularly where ICSI is involved. The development of prophylactic antioxidant therapies [36], improved culture conditions [37], novel gamete selection technologies [38] and noninvasive methods for the assessment of embryo quality [39] will all contribute to the future evolution of ART as a safe, effective means of treating human infertility.

Financial & competing interests disclosure
Aitken is a Consultant for NuSep. The author has no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

No writing assistance was utilized in the production of this manuscript.

References ChooseTop of pageINTRODUCTIONThe male factorParental ageEnvironmental pollutionErrors of endogenous meta...Electromagnetic radiation...Epigenetic damageIs ART a dangerous form o...References <<

Papers of special note have been highlighted as: • of interest •• of considerable interest

1 Van Voorhis‌ BJ. Clinical practice. in vitro fertilization. N. Engl. J. Med. 356, 379–386 (2007). [CrossRef] [Medline]
2 Adamson‌ GD, de Mouzon J, Lancaster P, Nygren KG, Sullivan E, Zegers-Hochschild F; International Committee for Monitoring Assisted Reproductive Technology. World collaborative report on in vitro fertilization, 2000. Fertil. Steril. 85, 1586–1622 (2006). [CrossRef] [Medline]
3 Hansen‌ M, Bower C, Milne E, de Klerk N, Kurinczuk JJ. Assisted reproductive technologies and the risk of birth defects – a systematic review. Hum. Reprod. 20, 328–338 (2005)
•• Meta-analysis suggesting a 30–40% increased risk of birth defects associated with assisted reproductive technology (ART).

[CrossRef] [Medline]
4 Jensen‌ TK, Sobotka T, Hansen MA, Pedersen AT, Lutz W, Skakkebæk NE. Declining trends in conception rates in recent birth cohorts of native Danish women: a possible role of deteriorating male reproductive health. Int. J. Androl. 31(2), 81–92 (2007).
• Recent review highlighting the declining fertility rates typical of the Danish population.

[CrossRef] [Medline]
5 Jorgensen‌ N, Carlsen E, Nermoen I et al. East–West gradient in semen quality in the Nordic–Baltic area: a study of men from the general population in Denmark, Norway, Estonia and Finland. Hum. Reprod. 17, 2199–2208 (2002). [CrossRef] [Medline]
6 Andersen‌ AN, Erb K. Register data on assisted reproductive technology (ART) in Europe including a detailed description of ART in Denmark. Int. J. Androl. 29, 12–16 (2006). [CrossRef]
7 McLachlan‌ RI, de Kretser DM. Male infertility: the case for continued research. Med. J. Aust. 174, 116–117 (2001). [Medline]
8 Aitken‌ RJ, De Iuliis GN. Origins and consequences of DNA damage in male germ cells. Reprod. Biomed. Online 14, 727–733 (2007)
•• Recent review of the causes and consequences of DNA damage in the male germ line.

[Medline]
9 Aitken‌ RJ, Koopman P, Lewis SE. Seeds of concern. Nature 432, 48–52 (2004).
• Review of potential environmental impacts on DNA damage in the germ line.

[CrossRef] [Medline]
10 Aitken‌ RJ, Krausz C. Oxidative stress, DNA damage and the Y chromosome. Reproduction 122, 497–506 (2001). [CrossRef] [Medline]
11 Fernández-Gonzalez‌ R, Moreira P, Pérez-Crespo M et al. Long-term effects of mouse intracytoplasmic sperm injection with DNA-fragmented sperm on health and behavior of adult offspring. Biol. Reprod. 78(4), 761–72 (2008).
•• Important recent paper providing experimental evidence that the performance of intracytoplasmic sperm injection (ICSI) with DNA-damaged spermatozoa can have long-lasting impacts on the health and wellbeing of the offspring.

[CrossRef] [Medline]
12 Irvine‌ DS, Twigg JP, Gordon EL, Fulton N, Milne PA, Aitken RJ. DNA integrity in human spermatozoa: relationships with semen quality. J. Androl. 21, 33–44 (2000). [Medline]
13 Schmid‌ TE, Eskenazi B, Baumgartner A et al. The effects of male age on sperm DNA damage in healthy non-smokers. Hum. Reprod. 22, 180–187 (2007). [CrossRef] [Medline]
14 Singh‌ NP, Muller CH, Berger RE. Effects of age on DNA double-strand breaks and apoptosis in human sperm. Fertil. Steril. 80, 1420–1430 (2003). [CrossRef] [Medline]
15 Crow‌ JF. The origins, patterns and implications of human spontaneous mutation. Nat. Rev. Genet. 1, 40–47 (2000). [CrossRef] [Medline]
16 Hamatani‌ T, Falco G, Carter MG et al. Age-associated alteration of gene expression patterns in mouse oocytes. Hum. Mol. Genet. 13, 2263–2278 (2004). [CrossRef] [Medline]
17 Yauk‌ C, Polyzos A, Rowan-Carroll A et al. Germ-line mutations, DNA damage, and global hypermethylation in mice exposed to particulate air pollution in an urban/industrial location. Proc. Natl Acad. Sci. USA 105, 605–610 (2008).
•• Important recent paper clearly demonstrating the impact that air pollution has on the epigenetic programming and integrity of sperm DNA.

[CrossRef] [Medline]
18 Rubes‌ J, Selevan SG, Evenson DP et al. Episodic air pollution is associated with increased DNA fragmentation in human sperm without other changes in semen quality. Hum. Reprod. 20, 2776–2783 (2005). [CrossRef] [Medline]
19 Rignell-Hydbom‌ A, Rylander L, Giwercman A et al. Exposure to PCBs and p,p´-DDE and human sperm chromatin integrity. Environ. Health Perspect. 113, 175–179 (2005). [Medline]
20 Meeker‌ JD, Singh NP, Ryan L et al. Urinary levels of insecticide metabolites and DNA damage in human sperm. Hum. Reprod. 19, 2573–2580 (2004). [CrossRef] [Medline]
21 Anderson‌ D, Schmid TE, Baumgartner A, Cemeli-Carratala E, Brinkworth MH, Wood JM. Oestrogenic compounds and oxidative stress (in human sperm and lymphocytes in the COMET assay). Mutat. Res. 544, 173–178 (2003). [CrossRef] [Medline]
22 Xu‌ DX, Shen HM, Zhu QX et al. The associations among semen quality, oxidative DNA damage in human spermatozoa and concentrations of cadmium, lead and selenium in seminal plasma. Mutat. Res. 534, 155–163 (2003). [Medline]
23 Rubes‌ J, Selevan SG, Sram RJ, Evenson DP, Perreault SD. GSTM1 genotype influences the susceptibility of men to sperm DNA damage associated with exposure to air pollution. Mutat. Res. 625, 20–28 (2007). [Medline]
24 Agbaje‌ IM, Rogers DA, McVicar CM et al. Insulin dependant diabetes mellitus: implications for male reproductive function. Hum. Reprod. 22, 1871–1877 (2007).
• Sentinel paper indicating that diabetic patients possess high levels of DNA damage in their spermatozoa.

[CrossRef] [Medline]
25 Shrilatha‌ B, Muralidhara. Early oxidative stress in testis and epididymal sperm in streptozotocin-induced diabetic mice: its progression and genotoxic consequences. Reprod. Toxicol. 23, 578–587 (2007). [CrossRef] [Medline]
26 Bennetts‌ LE, De Iuliis GN, Nixon B et al. Analysis of the impact of estrogenic compounds on DNA integrity in the male germ line. Mutat. Res. (2007) (In Press).
27 Wang‌ X, Sharma RK, Sikka SC, Thomas AJ Jr, Falcone T, Agarwal A. Oxidative stress is associated with increased apoptosis leading to spermatozoa DNA damage in patients with male factor infertility. Fertil. Steril. 80, 531–535 (2003). [CrossRef] [Medline]
28 Aitken‌ RJ, Baker HW. Seminal leukocytes: passengers, terrorists or good samaritans? Hum. Reprod. 10, 1736–1739 (1995).
•• Discussion of the significance of leukocytic infiltration in the origins of oxidative stress in the male reproductive tract.

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29 Shaman‌ JA, Yamauchi Y, Ward WS. Sperm DNA fragmentation: awakening the sleeping genome. Biochem. Soc. Trans. 35, 626–628 (2007). [CrossRef] [Medline]
30 Banks‌ S, King SA, Irvine DS, Saunders PT. Impact of a mild scrotal heat stress on DNA integrity in murine spermatozoa. Reproduction 129, 505–514 (2005). [CrossRef] [Medline]
31 Carrell‌ DT. Paternal genetic and epigenetic influences on IVF outcome. Expert Rev. Obstet. Gynecol. 3(3), 359–367 (2008). [Abstract]
32 Houshdaran‌ S, Cortessis VK, Siegmund K, Yang A, Laird PW, Sokol RZ. Widespread epigenetic abnormalities suggest a broad DNA methylation erasure defect in abnormal human sperm. PLoS ONE 2, e1289 (2007). [CrossRef]
33 Anway‌ MD, Cupp AS, Uzumcu M, Skinner MK. Epigenetic transgenerational actions of endocrine disruptors and male fertility. Science 308, 1466–1469 (2005). [CrossRef] [Medline]
34 Gandini‌ L, Lombardo F, Paoli D et al. Full-term pregnancies achieved with ICSI despite high levels of sperm chromatin damage. Hum. Reprod. 19, 1409–1417 (2004). [CrossRef] [Medline]
35 Feng‌ C, Wang LQ, Dong MY, Huang HF. Assisted reproductive technology may increase clinical mutation detection in male offspring. Fertil. Steril. (2008) (Epub ahead of print).
• Recent publication indicating that the treatment of male infertility patients with ART is associated with the de novo appearance of Y chromosome deletions in the offspring.

36 Greco‌ E, Iacobelli M, Rienzi L, Ubaldi F, Ferrero S, Tesarik J. Reduction of the incidence of sperm DNA fragmentation by oral antioxidant treatment. J. Androl. 26, 349–353 (2005). [CrossRef] [Medline]
37 Friedler‌ S, Schachter M, Strassburger D, Esther K, Ron El R, Raziel A. A randomized clinical trial comparing recombinant hyaluronan/recombinant albumin versus human tubal fluid for cleavage stage embryo transfer in patients with multiple IVF-embryo transfer failure. Hum. Reprod. 22, 2444–2448 (2007). [CrossRef] [Medline]
38 Ainsworth‌ C, Nixon B, Jansen RP, Aitken RJ. First recorded pregnancy and normal birth after ICSI using electrophoretically isolated spermatozoa. Hum. Reprod. 22, 197–200 (2007). [CrossRef] [Medline]
39 Patrizio‌ P, Fragouli E, Bianchi V, Borini A, Wells D. Molecular methods for selection of the ideal oocyte. Reprod. Biomed. Online 15, 346–353 (2007). [Medline]
Website 101 Australian Babies. 4102.0. Australian Social Trends. Australian Bureau of Statistics (2007). www.abs.gov.au/AUSSTATS

Affiliations
R John Aitken
Laureate Professor of Biological Sciences, Faculty of Science and IT, University of Newcastle, Callaghan, NSW 2308, Australia. john.aitken@newcastle.edu.au
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Genetic clock ticks for men Is this where copy number variations arise?

2008-06-12T22:46:00.000-07:00

Genetic clock ticks for men Les Sheffield

June 12, 2008 12:00am
MOST men would have been surprised to read that overseas researchers had found the death rate of young adults was higher if they had been born to older fathers.

This is no surprise to me. It has been scientifically established that genetic changes occur more often in the sperm of older fathers than younger fathers.

As men age there is a higher chance of changes in the genes in the sperm.

These changes can cause genetic conditions in their offspring, such as birth defects, autism and schizophrenia.

Their partners can also have an increased risk of miscarriages.

The presumed reason for the increase occurrence of all of these conditions is that they are all due to a new genetic change in the sperm of the older father.

Genetic changes are occurring all the time. Sometimes they have a beneficial effect, such as making the individual stronger, taller or smarter.

This is part of the concept of "survival of the fittest".

Sometimes, when the gene change is in a non-coding part of the genome, they have no effect. At other times, they can be harmful.

The problem is that these harmful effects are extremely varied because they can affect any one of the 20,000 or so human genes.

For example, they often change the structure of the body. One example is dwarfism, where the arms and legs are short due to a genetic change. The commonest type of dwarfism is achondroplasia.

An individual with this condition will have a 50 per cent risk of having an affected child themselves.

Indeed, about 20 per cent of the parents of achondroplastic babies have one of the parents with this condition, but the remaining 80 per cent do not.

If you look at the parents of babies with achondroplasia, who do not have the condition themselves, you find their average age is older than other people having babies in the population.

Significantly, statistics show it is the father's age which is important and not the mother's.

Achondroplasia is rare and it is only one of the many genes that can go wrong. Collectively, any of the 20,000 genes can change and this causes an increase in risk from about the age of 40.

The risk in men for any single gene change is one in 200 at age of 40, 20 at age 50 and rises steeply after that.

This increase in risk with paternal age is no surprise to me, but it is a surprise to practically everyone else.

The increase risk for older mothers for Down syndrome is well-known.

As part of my work as a clinical geneticist, I see couples every week who come to ask about the risk of having babies because of the age of the mother.

We talk about this and often, as the male partner is also older, we talk about the risk of his age. Most of the partners are quite surprised and even taken aback with this news.

In today's society, delaying pregnancy until later is often done for career and other purposes but usually only the age of the mother is taken into account in planning when to start a family. Why is the increased risk in relation to a father's age not widely known?

There are many possible reasons. Some of the information - such as increased death rates of adults - is new.

But information about single gene changes, such as achondroplasia, has been around for many years.

I think the real reason for the lack of knowledge is the conditions that can be caused are varied and can't really be prevented by a screening program like the one offered for Down syndrome.

In fact, most of the conditions, such as achondroplasia, can't even be picked up by the normal ultrasound scan for abnormalities done at 18-20 weeks of a pregnancy.

So, if you're a male, the only way not to be exposed to this increased risk of genetic defects in your offspring is to plan your children early and regard the increasing risks of the woman in her late 30s and early 40s as also applying to you.

In other words, stop your child bearing at the same sort of age that women stop child bearing. This may not be what older men want to hear, but they need to seek information about what the risks actually are before making child-bearing decisions.

We hear about the positive sides of parenthood in some older celebrity fathers but the story last week about the increase in death rates of the offspring brings out the hidden risks associated with fathering children at an older age.

Associate Professor Les Sheffield is a clinical geneticist with the Victorian Clinical Genetics Services

New Mutations Linked to Sporadic Schizophrenia Advancing Paternal Age

2008-05-30T11:06:00.000-07:00

Medical News: Schizophrenia

New Mutations Linked to Sporadic Schizophrenia
By Michael Smith, North American Correspondent, MedPage Today
Published: May 30, 2008
Reviewed by Dori F. Zaleznik, MD; Associate Clinical Professor of Medicine, Harvard Medical School, Boston. Earn CME/CE credit
for reading medical news

NEW YORK, May 30 -- About 10% of sporadic cases of schizophrenia appear to arise from spontaneous genetic mutations not inherited from either parent, researchers here said.

So-called copy number variants were seen in 15 of 152 patients with the disease but not in their unaffected parents, according to Maria Karayiorgou, M.D., of Columbia, and colleagues.

In contrast, such uninherited variants were only seen in two of 159 unaffected controls, Dr. Karayiorgou and colleagues reported online in Nature Genetics.

"We now know the cause of around 10% of the cases of sporadic schizophrenia," Dr. Karayiorgou added. Action Points
--------------------------------------------------------------------------------

Explain to interested patients that schizophrenia affects about 1% of the population, with 40% being inherited while the other 60% appeared sporadically in people without a family history of the disease.

Note that this study suggests that new genetic mutations -- copy number variants -- are significantly associated with sporadic cases of the disease and may account for about 10% of cases.
The findings mean that "schizophrenia is not as much of a 'big black box' as it used to be," Dr. Karayiorgou said.

Abnormal deletions or duplications of genetic material "are increasingly being implicated in schizophrenia and autism," said Thomas Insel, M.D., director of the National Institute of Mental Health, which was one of the supporters of the research.

"Now we have a dramatic demonstration that genetic vulnerabilities for these illnesses may stem from both hereditary and non-hereditary processes," Dr. Insel said in a statement.

The research "holds promise for improved treatments -- and perhaps someday even prevention -- of developmental brain disorders," he added.

The researchers carried out a whole-genome scan of 1,077 volunteers from a genetically homogenous Afrikaner population in South Africa, including 152 patients with sporadic schizophrenia, 159 controls, 48 people with familial schizophrenia, and both biological parents in all cases.

In the first step, the researchers used microarray technologies to identify copy number variation -- either gains or losses from the parental genome -- among the sporadic cases and the controls.

They identified 19 copy number variations, including 11 gains and eight losses, among the 15 cases and two controls.

The variants were roughly eight times as common among the cases as among the unaffected controls, Dr. Karayiorgou and colleagues said, and the association with schizophrenia was significant at P=0.00078, using a two-tailed Fisher's exact test.

Among the 48 volunteers with familial schizophrenia, there were no spontaneous copy number variations, implying that the association is "primarily confined to the sporadic cases," the researchers said.

On the other hand, when the researchers looked at rare inherited copy number mutations among the sporadic cases and controls, they found little difference. Thirty percent of the cases carried such a mutation compared with 20% of the controls.

Dr. Karayiorgou and co-workers have previously shown that loss of genes on chromosome 22, in the region 22q11.2, is responsible for introducing sporadic cases of schizophrenia into the population.

In the current study, three of the copy number variations found among sporadic cases occurred in that region, the researchers said, and all involved loss of function.

One possibility is that the identification of the mutated genes will allow researchers to discern the brain-development pathways that are involved in disease onset, "so that in the future we can look at better ways of treating this devastating disease," she said.

The study was supported by the National Institute of Mental Health and the Lieber Center for Schizophrenia Research at Columbia University Medical Center. The microarray experiments were carried out in the Vanderbilt Microarray Shared Resource, which is supported by the Vanderbilt Ingram Cancer Center, the Vanderbilt Digestive Disease Center, and the Vanderbilt Vision Center. Dr. Karayiorgou did not report any potential conflicts.

Primary source: Nature Genetics
Source reference:
Xu B, et al "Strong association of de novo copy number mutations with sporadic schizophrenia" Nature Genetics 2008; DOI:10.1038/ng.162.

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It is also recognized that paternal age is increased among affected children."

2008-05-03T15:52:00.000-07:00

1: Nat Rev Genet. 2008 May;9(5):341-55. Links
Advances in autism genetics: on the threshold of a new neurobiology.Abrahams BS, Geschwind DH.
Neurology Department, and Semel Institute for Neuroscience and Behaviour, David Geffen School of Medicine, University of California at Los Angeles, Los Angeles, California 90095-1769 USA. brett.abrahams@gmail.com

Autism is a heterogeneous syndrome defined by impairments in three core domains: social interaction, language and range of interests. Recent work has led to the identification of several autism susceptibility genes and an increased appreciation of the contribution of de novo and inherited copy number variation. Promising strategies are also being applied to identify common genetic risk variants. Systems biology approaches, including array-based expression profiling, are poised to provide additional insights into this group of disorders, in which heterogeneity, both genetic and phenotypic, is emerging as a dominant theme.

PMID: 18414403 [PubMed - in process]

We conclude that the absence of oligomer-dependent ligand interactions of DISC1 can be associated with sporadic mental disease of mixed phenotypes.

2008-04-09T21:43:00.000-07:00

The Journal of Neuroscience, April 9, 2008, 28(15):3839-3845; doi:10.1523/JNEUROSCI.5389-07.2008

Neurobiology of Disease
Insolubility of Disrupted-in-Schizophrenia 1 Disrupts Oligomer-Dependent Interactions with Nuclear Distribution Element 1 and Is Associated with Sporadic Mental Disease

S. Rutger Leliveld,1,2 Verian Bader,1 Philipp Hendriks,1 Ingrid Prikulis,1 Gustavo Sajnani,3 Jesús R. Requena,3 and Carsten Korth1

1Department of Neuropathology, Heinrich Heine University of Düsseldorf, 40225 Düsseldorf, Germany, 2Department of Molecular Biophysics-II, Forschungszentrum Jülich, 52425 Jülich, Germany, and 3Department of Medicine, University of Santiago de Compostela, 15782 Santiago de Compostela, Spain

Correspondence should be addressed to Dr. Carsten Korth, Department of Neuropathology, Heinrich Heine University of Düsseldorf, Moorenstrasse 5, 40225 Düsseldorf, Germany. Email: ckorth@uni-duesseldorf.de

Disrupted-in-schizophrenia 1 (DISC1) and other genes have been identified recently as potential molecular players in chronic psychiatric diseases such as affective disorders and schizophrenia. A molecular mechanism of how these genes may be linked to the majority of sporadic cases of these diseases remains unclear. The chronic nature and irreversibility of clinical symptoms in a subgroup of these diseases prompted us to investigate whether proteins corresponding to candidate genes displayed subtle features of protein aggregation. Here, we show that in postmortem brain samples of a distinct group of patients with phenotypes of affective disorders or schizophrenia, but not healthy controls, significant fractions of DISC1 could be identified as cold Sarkosyl-insoluble protein aggregates. A loss-of-function phenotype could be demonstrated for insoluble DISC1 through abolished binding to a key DISC1 ligand, nuclear distribution element 1 (NDEL1): in human neuroblastoma cells, DISC1 formed expression-dependent, detergent-resistant aggregates that failed to interact with endogenous NDEL1. Recombinant (r) NDEL1 expressed in Escherichia coli selectively bound an octamer of an rDISC1 fragment but not dimers or high molecular weight multimers, suggesting an oligomerization optimum for molecular interactions of DISC1 with NDEL1. For DISC1-related sporadic psychiatric disease, we propose a mechanism whereby impaired cellular control over self-association of DISC1 leads to excessive multimerization and subsequent formation of detergent-resistant aggregates, culminating in loss of ligand binding, here exemplified by NDEL1. We conclude that the absence of oligomer-dependent ligand interactions of DISC1 can be associated with sporadic mental disease of mixed phenotypes.

Key words: psychiatric disease; depression; bipolar disorder; multimerization; protein conformational disease; protein aggregation

Autism Connected To Gene Central To Neuron Formation, Study Shows

2008-03-19T22:27:00.000-07:00

Autism Connected To Gene Central To Neuron Formation, Study Shows
ScienceDaily (Mar. 20, 2008) — Eli Hatchwell, M.D., Ph.D., Associate Professor of Pathology at Stony Brook University Medical Center, and colleagues have found that a disruption of the Contactin 4 gene on chromosome 3 may be linked to autism spectrum disorder (ASD). What causes ASD, a developmental disorder of the central nervous system, is largely unknown. Dr. Hatchwell’s finding suggests that mutations affecting Contactin 4 may be relevant to ASD pathogenesis, and thus a potential biomarker for some individuals with the disorder.

According to the Centers for Disease Control and Prevention, the prevalence of ASD in the United States may be as high as 1 in 150 children. The disorder is divided into five subtypes, including autism proper. Pathogenesis of ASD may be environmental and/or biological. Experts suspect that many genes may play a role in the etiology of ASD.

“Given the prevalence of ASD, a clearer understanding of its etiology is necessary for both diagnostic and therapeutic purposes,” says Dr. Hatchwell, also Director of Stony Brook University’s Genomics Core Facility and Geneticist at the Cody Center for Autism and Developmental Disabilities at SBU. “Our study implicates Contactin 4 as a candidate gene in ASD, a finding that significantly contributes to our understanding of the biological basis of autism.”

A total of 92 patients with ASD from the Cody Center participated in the genetics study. The participants came from 81 families. Genomic DNA was analyzed from all subjects and, where relevant, from their biological parents. More than 500 normal control patients were included in the analysis.

A whole genome analysis of the 92 subjects revealed that three subjects had chromosome 3 copy number variations that disrupted the same gene, Contactin 4. A deletion was detected in two subjects (siblings), and a duplication was found in a third, unrelated, individual. Subsequent array analysis of parental DNA indicated that both variations were paternally inherited, specifically inherited from fathers without a history of ASD.

According to Dr. Hatchwell, when mutations are found that explain just one percent of a given ASD population, the results are significant, as ASD likely has a multitude of genetic causes. For example, a recent study reported in the New England Journal of Medicine showed that copy number variations of chromosome 16p11.2 accounted for one percent of all cases of the syndrome. Dr. Hatchwell explains that the genetic analysis with the Cody Center patients, detailed in the article entitled “Disruption of Contactin 4 in 3 Subjects with Autism Spectrum Disorder,” is highly significant in that two of 81 families (2.5 percent) presented with a disruption of Contactin 4.

The mutations found in Dr. Hatchwell’s study directly interrupt Contactin 4. The gene codes for an axon-associated cell adhesion molecule that is expressed in the brain and is known to be important in axonal development.

Dr. Hatchwell’s multidisciplinary research team is planning to analyze Contactin 4 in large numbers of patients with ASD and normal controls, in order to identify mutations that might be involved in the pathogenesis of ASD in a subset of affected individuals.

Details of the study are reported in the early online edition of the Journal of Medical Genetics. Dr. Hatchwell’s co-authors from Stony Brook University include: Jasmin Roohi, B.A., Department of Genetics; John C. Pomeroy, M.D., David H. Tegay, D.O., and Carla DeVincent, Ph.D., of the Department of Pediatrics; Lance E. Palmer, Ph.D., Department of Microbiology. Other authors include: Cristina Montagna, Ph.D., Department of Pathology and Molecular Genetics, Albert Einstein College of Medicine; Susan L. Christian, Ph.D., Department of Human Genetics, University of Chicago; and Norma Nowak, Ph.D., Department of Cancer Prevention and Population Sciences, University of Buffalo.

The study was supported in part by grants from the Cody Center for Autism and Developmental Disabilities, National Alliance for Autism Research, National Institute of Neurological Diseases and Stroke, the National Cancer Institute, and the General Clinical Research Center at SBUMC.

Adapted from materials provided by Stony Brook University Medical Center.

CNVs in Contactin 4 and 2.5 percent of Autism

2008-03-19T08:56:00.000-07:00

http://news.aol.com/story/_a/gene-for-brain-connections-linked-with/n20080318150209990016

Gene for brain connections linked with autism
By Maggie Fox,
Reuters
Posted: 2008-03-18 15:02:04
WASHINGTON (Reuters) - A gene that helps the brain make connections may underlie a significant number of autism cases, researchers in the United States reported on Tuesday.Disruptions in the gene, called contactin 4, stop the gene from working properly and appear to stop the brain from making proper networks, the researchers reported in the Journal of Medical Genetics.These disruptions, in which the child has either three copies of the gene or just one copy when two copies is normal, could account for up to 2.5 percent of autism cases, said Dr. Eli Hatchwell of Stony Brook University Medical Center in New York, who led the study."That is a significant number," said Hatchwell.

Generally the mistake that people make is they are looking for one unifying cause for autism, and there is no such thing and there never will be," Hatchwell said in a telephone interview.

Hatchwell's team tested 92 patients from 81 families with autism spectrum disorder and compared them to 560 people without autism.They did a whole genome analysis, looking at the entire DNA map, and found three of the patients had deletions or duplications of DNA that disrupted contactin 4.They were all inherited from fathers without a history of autism, which can cause severe social and developmental delays and even mental retardation.

Both genes have been affected by CNVs in patients with autism and mental retardation, but neither has been previously implicated in SZ

2008-03-03T21:44:00.000-08:00

1: Hum Mol Genet. 2008 Feb 1;17(3):458-65. Epub 2007 Nov 6. Links
Comparative genome hybridization suggests a role for NRXN1 and APBA2 in schizophrenia.Kirov G, Gumus D, Chen W, Norton N, Georgieva L, Sari M, O'Donovan MC, Erdogan F, Owen MJ, Ropers HH, Ullmann R.
Department of Psychological Medicine, Cardiff University, Henry Wellcome Building, Heath Park, Cardiff CF14 4XN, UK.

Copy number variations (CNVs) account for a substantial proportion of human genomic variation, and have been shown to cause neurodevelopmental disorders. We sought to determine the relevance of CNVs to the aetiology of schizophrenia (SZ). Whole-genome, high-resolution, tiling path BAC array comparative genomic hybridization (array CGH) was employed to test DNA from 93 individuals with DSM-IV SZ. Common DNA copy number changes that are unlikely to be directly pathogenic in SZ were filtered out by comparison to a reference dataset of 372 control individuals analyzed in our laboratory, and a screen against the Database of Genomic Variants. The remaining aberrations were validated with Affymetrix 250K SNP arrays or 244K Agilent oligo-arrays and tested for inheritance from the parents. A total of 13 aberrations satisfied our criteria. Two of them are very likely to be pathogenic. The first one is a deletion at 2p16.3 that was present in an affected sibling and disrupts NRXN1. The second one is a de novo duplication at 15q13.1 spanning APBA2. The proteins of these two genes interact directly and play a role in synaptic development and function. Both genes have been affected by CNVs in patients with autism and mental retardation, but neither has been previously implicated in SZ.

PMID: 17989066 [PubMed

Structural variation of chromosomes in autism spectrum disorder

2008-03-03T21:31:00.000-08:00

http://www.ncbi.nlm.nih.gov/pubmed/18252227?ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum

1: Am J Hum Genet. 2008 Feb;82(2):477-88. Epub 2008 Jan 17. Links
Structural variation of chromosomes in autism spectrum disorder.Marshall CR, Noor A, Vincent JB, Lionel AC, Feuk L, Skaug J, Shago M, Moessner R, Pinto D, Ren Y, Thiruvahindrapduram B, Fiebig A, Schreiber S, Friedman J, Ketelaars CE, Vos YJ, Ficicioglu C, Kirkpatrick S, Nicolson R, Sloman L, Summers A, Gibbons CA, Teebi A, Chitayat D, Weksberg R, Thompson A, Vardy C, Crosbie V, Luscombe S, Baatjes R, Zwaigenbaum L, Roberts W, Fernandez B, Szatmari P, Scherer SW.
The Centre for Applied Genomics, The Hospital for Sick Children, Department of Molecular and Medical Genetics, University of Toronto, Toronto, Ontario M5G 1L7, Canada.

Structural variation (copy number variation [CNV] including deletion and duplication, translocation, inversion) of chromosomes has been identified in some individuals with autism spectrum disorder (ASD), but the full etiologic role is unknown. We performed genome-wide assessment for structural abnormalities in 427 unrelated ASD cases via single-nucleotide polymorphism microarrays and karyotyping. With microarrays, we discovered 277 unbalanced CNVs in 44% of ASD families not present in 500 controls (and re-examined in another 1152 controls). Karyotyping detected additional balanced changes. Although most variants were inherited, we found a total of 27 cases with de novo alterations, and in three (11%) of these individuals, two or more new variants were observed. De novo CNVs were found in approximately 7% and approximately 2% of idiopathic families having one child, or two or more ASD siblings, respectively. We also detected 13 loci with recurrent/overlapping CNV in unrelated cases, and at these sites, deletions and duplications affecting the same gene(s) in different individuals and sometimes in asymptomatic carriers were also found. Notwithstanding complexities, our results further implicate the SHANK3-NLGN4-NRXN1 postsynaptic density genes and also identify novel loci at DPP6-DPP10-PCDH9 (synapse complex), ANKRD11, DPYD, PTCHD1, 15q24, among others, for a role in ASD susceptibility. Our most compelling result discovered CNV at 16p11.2 (p = 0.002) (with characteristics of a genomic disorder) at approximately 1% frequency. Some of the ASD regions were also common to mental retardation loci. Structural variants were found in sufficiently high frequency influencing ASD to suggest that cytogenetic and microarray analyses be considered in routine clinical workup.

CNVs will be found in non-familial autism, schizophrenia, alzheimer's in offspring of older father's, I predict

2008-02-20T17:13:00.000-08:00

Researchers release most detailed global study of genetic variation

A schematic of worldwide human genetic variation, with colors representing different genetic types. The figure illustrates the great amout of genetic variation in Africa. Illustration by Martin Soave/University of Michigan

University of Michigan scientists and their colleagues at the National Institute on Aging have produced the largest and most detailed worldwide study of human genetic variation, a treasure trove offering new insights into early migrations out of Africa and across the globe.

Like astronomers who build ever-larger telescopes to peer deeper into space, population geneticists like U-M's Noah Rosenberg are using the latest genetic tools to probe DNA molecules in unprecedented detail, uncovering new clues to humanity's origins.

The latest study characterizes more than 500,000 DNA markers in the human genome and examines variations across 29 populations on five continents.

"Our study is one of the first in a new wave of extremely high-resolution genome scans of population genetic variation," said Rosenberg, an assistant research professor at U-M's Life Sciences Institute and co-senior author of the study, to be published in the Feb. 21 edition of Nature.

"Now that we have the technology to look at thousands, or even hundreds of thousands, of genetic markers, we can infer human population relationships and ancient migrations at a finer level of resolution than has previously been possible."

The new study, led by Rosenberg and National Institute on Aging colleague Andrew Singleton, produced genetic data nearly 100 times more detailed than previous worldwide assessments of human populations. It shows that:

• A recently discovered type of human genetic variation, known as a copy-number variant or CNV, is a reliable addition to the toolkit of population geneticists and should speed the discovery of disease-related genes. Rosenberg and his colleagues discovered 507 previously unknown CNVs, which are large chunks of DNA—up to 1,000,000 consecutive "letters" of the genetic alphabet—that are either repeated or deleted entirely from a person's genome. Various diseases can be triggered by an abnormal gain or loss in the number of gene copies.

• It's sometimes possible to trace a person's ancestry to an individual population within a geographic region. While previous studies have found that broad-scale geographic ancestry could be successfully traced, the new results indicate "it's becoming increasingly possible to use genomics to refine the geographic position of an individual's ancestors with more and more precision," Rosenberg said.

• Human genetic diversity decreases as distance from Africa—the cradle of humanity—increases. People of African descent are more genetically diverse than Middle Easterners, who are more diverse than Asians and Europeans. Native Americans possess the least-diverse genomes. As a result, searching for disease-causing genes should require the fewest number of genetic markers among Native Americans and the greatest number of markers among Africans.

The results are being made available on publicly shared databases.

"I hope the study will be an invaluable resource for understanding genomic variability and investigating genetic association with disease," said the NIA's Singleton.

The researchers analyzed DNA from 485 people. They examined three types of genetic variation: single-nucleotide polymorphisms, or SNPs; haplotypes; and CNVs.

If the human genome is viewed as a 3-billion-letter book of life, then SNPs represent single-letter spelling changes, haplotype variations equate to word changes, and CNVs are wholesale deletions or duplications of full pages.

The patterns revealed by the new study support the idea that humans originated in Africa, then spread into the Middle East, followed by Europe and Asia, the Pacific Islands, and finally to the Americas.

The results also bolster the notion of "serial founder effects," meaning that as people began migrating eastward from East Africa about 100,000 years ago, each successive wave of migrants carried a subset of the genetic variation held by previous groups.

"Diversity has been eroded through the migration process," Rosenberg said.

In addition to his position at the Life Sciences Institute, Rosenberg is an assistant professor of human genetics at the Medical School; an assistant professor of biostatistics at the School of Public Health; an assistant professor of ecology and evolutionary biology at the College of Literature, Science, and the Arts; and an assistant research professor of bioinformatics at the Medical School's Center for Computational Medicine and Biology.

"This data set is so rich. It provides a much more comprehensive, cross-sectional snapshot of the human genome than previous studies," said Paul Scheet, a post-doctoral researcher in the U-M Department of Biostatistics and one of the lead authors.

"The next step for these studies is to sequence whole genomes," said Mattias Jakobsson, a post-doctoral researcher at the U-M Center for Computational Medicine and Biology and another lead author. "You would take 500 individuals, and you would just completely sequence everything, and then you'd have almost every important variant that's out there."

Source: University of Michigan

CNVs amd phenotypic disorders

2008-02-16T22:21:00.001-08:00

> Perspective > Full Text
Perspective

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Nature Genetics 39, S37 - S42 (2007)doi:10.1038/ng2080

Copy-number variation and association studies of human disease
Steven A McCarroll1 & David M Altshuler1,2

The authors are in the Program in Medical and Population Genetics, Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA; and Department of Molecular Biology and the Center for Human Genetic Research, Massachusetts General Hospital, Boston, Massachusetts, USA. e-mail: smccarro@broad.mit.edu
David Altshuler is also in the Department of Medicine at Massachusetts General Hospital and the Departments of Genetics and Medicine, Harvard Medical School, Boston, Massachusetts, USA.

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AbstractThe central goal of human genetics is to understand the inherited basis of human variation in phenotypes, elucidating human physiology, evolution and disease. Rare mutations have been found underlying two thousand mendelian diseases; more recently, it has become possible to assess systematically the contribution of common SNPs to complex disease. The known role of copy-number alterations in sporadic genomic disorders, combined with emerging information about inherited copy-number variation, indicate the importance of systematically assessing copy-number variants (CNVs), including common copy-number polymorphisms (CNPs), in disease. Here we discuss evidence that CNVs affect phenotypes, directions for basic knowledge to support clinical study of CNVs, the challenge of genotyping CNPs in clinical cohorts, the use of SNPs as markers for CNPs and statistical challenges in testing CNVs for association with disease. Critical needs are high-resolution maps of common CNPs and techniques that accurately determine the allelic state of affected individuals.

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IntroductionEmpirical evidence that CNVs are associated with phenotypes
The first evidence that copy-number alterations can influence human phenotypes came from sporadic diseases, termed 'genomic disorders', caused by de novo structural alterations1. The number of genomic disorders has grown, with several dozen reported to date2. In addition to such sporadic diseases, inherited CNVs have been found to underlie mendelian diseases in several families3, 4, 5. Nonetheless, CNVs have been implicated in only a few percent of the 2,000 or more mendelian diseases so far explained at a molecular level.

Little is known about the genetic basis of common, complex phenotypes, and thus it would be premature to predict the relative proportion of complex disease explained by SNPs and CNVs. In principle, complex disease might be more susceptible to 'soft' forms of variation — such as variation in noncoding sequences and copy number — which alter gene dose without abolishing gene function. Common CNPs have been reported to be associated with several complex disease phenotypes, including HIV acquisition and progression6, lupus glomerulonephritis7 and three systemic autoimmune diseases: systemic lupus erythematosus, microscopic polyangiitis and Wegener's granulomatosis8, 9. A recent study of gene expression variation as a model complex phenotype measured the fraction of gene expression 'traits' that could be associated with either SNPs or CNVs; in this study, SNP genotypes and CNV measurements were associated with 83% and 18% of those gene expression traits for which statistically significant associations were found10. This may still underestimate the role of CNVs, given the greater completeness and accuracy with which SNPs can be queried at present.

Technical issues in assessing CNVs for a role in disease
The power to discover a relationship between DNA variation and phenotype is limited by the sensitivity and accuracy with which that DNA variation is measured in each individual. (Accuracy in measuring phenotype, environment and behavior are equally or more important; these challenges, which are not specific to CNV studies, are beyond the scope of this review.) To the extent that the precise allelic state of any DNA variant is not well measured, power declines. But although this issue has been the subject of extensive discussion in the literature on SNP association studies11, little has been written about the extent to which current attempts to measure copy number provide precise and accurate measures of the underlying DNA variation in each individual.

Insufficient data have been collected at a sequence level to estimate the correlation between quantitative measures of CNVs by existing techniques and the true allelic state of each CNV in each individual. However, there are reasons to believe that the correlation is low — much lower, for example, than that offered by current SNP genotyping products for the underlying SNP variation in the genome12. This difference is due fundamentally to the greater challenge of measuring multibase, often multiallelic variants compared with single-base, diallelic SNPs. The specific challenge of genotyping CNVs is discussed in a later section.

Enabling core knowledge about CNVs for association studies
An indispensable starting point for association studies is basic knowledge about the genetic variations that are present in the human population — their alleles, their frequencies, their precise locations. SNP databases developed through a series of phases: first, rapid growth in methods to detect the locations of putative SNPs; second, calibration and standardization of discovery methods to maximize sensitivity and minimize false positives13; third, accurate genotyping of large numbers of SNPs to validate (or invalidate) SNPs and characterize their properties14, 15, 16; and fourth, assessment of the sensitivity of the resulting map in comparison to systematic resequencing data16. The resulting resource enables researchers and technology companies to design assays for any given SNP of interest (or a genome-wide collection) and to assess the relationship between any given SNP and others that may or may not have been typed.

Pioneering genome-wide surveys of CNVs17, 18, 19, 20, 21, 22, 23, 24, 25, 26 and databases holding the results of those studies27 are an important initial step toward a comprehensive database of CNVs, but much work lies ahead. Most reported CNV locations actually correspond to the locations of CNV-containing regions (CNVRs), generally the genomic coordinates of a BAC probe, set of oligonucleotide probes, or fosmid from which a variant was discovered. A reported CNVR is consistent with a large number of potential variants (Fig. 1); of importance for the design of assays, seldom is it known which precise locus or gene within the CNVR is actually affected. An important step toward enabling knowledge of CNP locations is an ongoing effort to sequence fosmid clones containing structurally variant haplotypes28. Until the locations of CNVs within reported CNVRs are known, researchers interested in studying a reported CNV in clinical samples must first perform experiments to find the CNV(s) within the reported CNVR. Ultimately, the utility of CNV databases will be enhanced by data on the validation state of each putative CNV (to avoid wasting resources on false positive CNVs) and on the frequency of each allele in different populations (to estimate statistical power when designing association studies), and by a map of the linkage disequilibrium (or lack thereof) with nearby SNPs that may be easier to genotype or may already have been assessed.

Figure 1: Many potential CNV locations are consistent with the coordinates of a reported CNV-containing region (CNVR).
An investigator will typically have to perform extensive experiments to find the CNV within a CNVR. An ongoing project to map structural variants at the sequence level28 will provide important enabling knowledge for clinical genetic studies.

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Genotyping CNVs in disease association studies
Disease association studies rely on accurate genotypes. Most CNV studies to date have been discovery studies (generating lists of regions that contain CNVs) rather than association studies (assessing the correlation between phenotype and genotype). The underlying problems in CNV discovery and CNV genotyping are different. A discovery study begins with a null hypothesis that no variation exists at a locus and assesses whether the evidence for variation exceeds a genome-wide significance threshold; a high false-negative rate (failure to discover variants) is tolerated in order to preserve a low false-positive rate29. An association study tests a null hypothesis that variation is not associated with phenotype; all forms of misclassification (both over- and underascertainment of altered copy-number levels) are problematic, and all levels of copy number must be distinguished. This is a much more exacting requirement: for example, of some 1,500 CNVs that were identified in one recent genome-wide survey, only 70 common, diallelic CNVs yielded genotypes of the quality that could be used for linkage disequilibrium analysis26. Indeed, of the thousands of CNV-containing regions that have been identified in the literature to date, only a few percent have been genotyped in available reference samples (Fig. 2).

Figure 2: Although the number of reported copy-number-variable regions has increased dramatically, only a few percent of CNVs have been successfully genotyped.
An important step in association study of any CNV will be the development of a genotyping assay that accurately determines the allelic state of every individual in a cohort.

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The development of assays for accurately typing CNVs in clinical cohorts has required enormous effort in CNV-disease association studies to date6, 7, 8, 9 and is one of the most pressing needs in CNV research. Although it is not yet clear what technology will be used, it is critical that any assay be reproducible in other labs. Standards for publishing a CNV-disease association should include genotypes for a publicly available set of reference samples, which can be used by other labs to develop additional assays and to assess the original assay.

Copy-number measurements versus copy-number genotypes
At any given nucleotide, biological copy numbers are integers. The more precise and localized a measurement of copy number, the more its distribution in a population shows a discrete distribution reflecting the underlying integer distribution of copy numbers (for example, 0, 1, 2; or 2, 3, 4; or even 2, 3, 4, 5, 6) (Fig. 3). Frequently, though, copy-number measurements seem to be continuously distributed across a population (Fig. 3c). The factors which cause imprecision in copy-number measurement can be divided into two categories. Measurement imprecision refers to the noise inherent in making any measurement. Spatial imprecision occurs when an assay aggregates information across a large region into a single measurement.

Figure 3: Using copy-number measurements and copy-number genotypes in association studies.
(a) A common CNP containing a gene encoding pyruvate dehydrogenase phosphatase regulatory protein (PDPR; blue arrow) interrogated by a BAC probe (Chr16tp-9C8, red rectangle) on a BAC array-CGH platform26, and by a series of oligonucleotide probes (vertical line segments) on an oligonucleotide platform (Affymetrix GenomeWide 5.0). (b–d) Copy-number measurements for the two platforms across the same set of samples (HapMap CEU sample of individuals with European ancestry) are correlated (b), confirming that they interrogate the same CNP. Measurements on the BAC array-CGH platform26 show a continuous distribution (c), whereas measurements on the oligonucleotide platform show a discrete distribution (d). (e,f) Association analysis using raw intensity measurements. PDPR gene expression is found to be associated with PDPR copy number using raw measurements of copy number on both platforms. Colors indicate the discrete genotype 'calls' on each platform (not used in this analysis, but used in the analysis in panels g,h). The association in e was discovered by Stranger et al. (2007)10. (g,h) Association analysis using discrete genotype 'calls'. Where raw measurements show a continuous distribution (c,e,g), hardening of raw measurements into discrete 'call' loses information that was present in the original measurements, with the result that association with phenotype is no longer detected. Where raw measurements show a discrete distribution (d,f,h), conversion of raw measurements into genotypes can increase the correlation with phenotype, though the primary benefit may simply be greater clarity about the distribution of genetic variation and its relationship to phenotype.

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Surveys of copy-number variation have further summarized copy-number measurements into discrete values of 'gain' or 'loss' in each sample; although these assessments are sometimes referred to as 'genotypes', inspection of the underlying data often shows that these discrete distinctions are not reflected in the underlying distribution of measurements (Fig. 3c,e). Summarizing raw copy-number measurements into such 'calls' may lose information present in the original measurements, and is of uncertain relationship to the true genotype (Fig. 3e,g).

Until approaches for genome-wide CNP genotyping mature, a placeholder strategy may be to rely on raw hybridization measurements as an approximation to an unknown, underlying genotype. This approach was used in a recent study of CNPs and gene expression10 that used copy number data from an immediately preceding study26; the analysis dispensed with the CNV 'calls' from the previous study, instead using the raw hybridization measurements for association analysis10, 26 (Fig. 3). The paucity of effective CNP genotypes means that techniques and algorithms for making genotype calls are a critical need in CNP disease research; until such approaches mature, raw measurements may be the preferred basis for a preliminary association analysis.

Using SNPs as markers for CNPs
Given the technical challenges in finding and typing CNPs, and the early stage of basic knowledge about their locations and molecular structures, an appealing strategy might be to rely on more-easily-typed SNPs to serve as markers by linkage disequilibrium for common variants throughout the genome. Linkage disequilibrium–based approaches utilize the observation that the human recombination rate is (i) low relative to the typical age of alleles in the human population and (ii) clustered into hotspots across the genome30. These features mean that ancestral variants (whatever their molecular nature) segregate in the population on haplotypes, are correlated with one another and thus can be 'tagged' by a reduced set of SNPs31. Because such linkage disequilibrium–based approaches require neither a priori identification of all variants nor technology for typing every variant individually, they might address the limitations of current knowledge and genotyping technology in the CNP field.

To assess a specific CNP through linkage disequilibrium, one would genotype the CNP in the HapMap (or other reference) samples and assess whether nearby SNPs were able to capture the CNP through linkage disequilibrium; if so, one would then type those SNPs in affected cohorts as a proxy for the CNP. To analyze a genomic region, one would select a dense set of SNPs sufficient to capture almost all common, ancestral polymorphisms through linkage disequilibrium11 and test them for association with disease. On a genome-wide scale, one would presumably use commercial whole-genome SNP genotyping products. In all cases, positive association (if found) could be due to a CNV or to anything else in linkage disequilibrium with the associated SNP — possibilities that would be assessed by directed resequencing, copy-number analysis and additional genotyping in following up any initial association.

The performance of linkage disequilibrium–based approaches will depend on the strength and generality of linkage disequilibrium between CNPs and SNPs. Using available SNP data and PCR-based genotyping of deletion polymorphisms, initial studies found that deletion polymorphisms are generally ancestral and are tagged by SNPs22, 23. A subsequent study of the linkage-disequilibrium properties of CNPs in the genome's segmental-duplication-rich regions found that copy-number measurements from such CNPs were less well captured by HapMap SNPs24; a more recent study of 70 genotyped CNPs found that the CNPs showed appreciable linkage disequilibrium with SNPs, but were less well tagged than frequency-matched SNPs were26. The extent of linkage disequilibrium between SNPs and CNPs remains unclear, for two reasons. First, assessing linkage disequilibrium around CNPs requires accurate genotyping of a large and representative collection of CNPs in samples with dense SNP genotypes — and yet accurate genotypes exist for only a small and nonrandom collection of CNPs (Fig. 2). Second, regions rich in segmental duplications contain almost half of all reported CNPs19, 24, 26, but contain a density of validated SNPs (that could serve as potential tags) much lower than that of the rest of the genome24.

Integrated association studies for SNPs and CNPs
Many genome-wide SNP association studies, each involving hundreds to thousands of affected individuals, are underway. The raw intensity data generated during SNP genotyping can be mined for copy-number information32, 33, 34, 35, making such studies a potential source of data for CNP-disease association studies. However, several factors limit the utility of previous generations of SNP arrays for this purpose. Most important is coverage: because common CNPs cause SNP genotyping assays to fail Hardy-Weinberg and mendelian inheritance checks, genomic regions harboring common CNPs had been filtered out (partially or completely) of commercial whole-genome SNP array platforms during the selection of high-performance SNP assays. Another limitation is technical: because SNP assays are optimized for allelic discrimination rather than copy-number measurement, the copy-number measurements they provide are noisy, with the result that only large variants are typically detected. Commercial SNP arrays are used to find the large copy-number alterations typical of cancer32, 33, 34, 35, but have not to date been used to perform association studies for germline CNPs, and seem to detect many more rare CNVs than common CNPs26.

Ideally, every DNA sample would be simultaneously queried for SNPs and CNVs in a single, integrated analysis. We have been working with collaborators to develop hybrid oligonucleotide arrays that contain both SNP allele-discrimination probes and dedicated 'copy-number probes' — probes whose sequences have been optimized for copy-number quantification by (i) designing them to nonpolymorphic sequences, (ii) selecting sequence features predictive of technical efficacy and (iii) empirically assessing responsiveness in screening experiments (Fig. 3d,f,h). Such hybrid arrays (or some other technological solution) offer the potential for integrated association studies in which SNP and copy-number variation are considered together. Moreover, as databases of CNPs and SNPs become ever more complete, the content of such arrays should similarly approach completeness.

Testing the disease association of common CNPs
Once an accurate and complete set of CNV measurements is obtained in a sample, there are few unprecedented statistical challenges to the assessment of association with disease. As with SNPs, a key dividing line is whether the statistical test involves common variants or a collection of individually rare events.

For common CNPs, statistical tests will involve a straightforward comparison of allele frequencies (or of diploid genotype frequencies): between affected individuals and controls in a population cohort; between transmitted and untransmitted chromosomes in families with affected offspring; or between affected and unaffected siblings. Most successfully genotyped CNPs seem to be diallelic, showing 2 or 3 diploid copy-number classes and therefore most likely representing two underlying alleles23, 26. Such variants are readily incorporated into current frameworks for SNP association testing; in fact, the copy-number classes could be subjected to the same quality-control tests (mendelian inheritance, Hardy-Weinberg equilibrium) used to ensure the quality of SNP genotypes. Such CNPs could for practical reasons be recoded as SNP genotypes (for example, 'AA' for zero copies, 'AC' for one copy, 'CC' for two copies) and thereby benefit from the software and analytical approaches already available for SNP-based analyses, including correcting for population stratification (discussed below) and scrutinizing a genome-wide study for P-value inflation.

Some CNPs seem to involve more than three copy-number classes, and therefore more than two copy-number alleles (Fig. 3). Nineteen such loci were identified in a recent genome-wide CNV survey26. A related class of CNPs appears to harbor both deletion and duplication alleles24, 26. Notably, the common CNPs reported to be associated with HIV progression and autoimmune phenotypes are multiallelic6, 7, 8, 9. For the population-based analyses in those studies, researchers used a variety of techniques to test for disease association, including (i) reducing the copy-number genotype to a binary class (for example, >4 versus <4 copies), then performing a chi-squared analysis on the distribution of disease status between these two groups6; (ii) a logistic regression analysis, with copy number as an explanatory variable and age and gender as covariates7; and (iii) nonparametric tests of the null hypothesis that affected individuals and controls were drawn from the same distribution of copy numbers7. Family-based analyses — which are favored by many researchers because they are more robust to population stratification (discussed below) — will also need to be generalized to address multiallelic CNPs and continuously distributed copy-number measurements.

Testing the disease association of rare CNVs
For rare variants, association analysis is more challenging, as it is less constrained: there are many potential ways to group a collection of unique events, and thus more degrees of freedom. When copy-number ascertainment was limited to large, microscopically visible (and therefore usually functional) variants, such variants were generally assumed to be causative (although the specific gene involved is, conversely, very imprecisely localized). The new ability to detect smaller, submicroscopic CNVs — hundreds of which may be present in any one individual, and the vast majority of which are benign — requires statistically well founded assessment of their association with disease.

As submicroscopic CNVs cannot be assumed to have functional consequence, it is critical to search for them in affected individuals and controls with equal rigor, and to use a statistical framework to determine whether rearrangements are truly more common in the affected. It is critical that CNVs not be discovered in a set of cases and then the specific variants that were found queried in controls; such an approach is subject to 'ascertainment bias' and is statistically unsound. Given the existence of hundreds of rare CNVs with apparent frequencies of less than one percent, even in a well designed study it will frequently occur that a CNV is present (for example) in 3/200 cases and 0/200 controls. Such results are expected to occur by chance in a genome-wide search, and so do not necessarily imply a causal effect. (The observation of three independent, de novo structural mutations at the same locus in a disease cohort might be highly significant, because the rate of sporadic structural mutation seems to be much lower than the rate of CNV inheritance; such sporadic genomic disorders are discussed in an accompanying Perspective2).

It is natural to also consider the hypothesis that distinct CNVs at the same genomic locus may similarly influence disease risk in different individuals. An important precedent for such reasoning is the argument that diverse sequence variants in candidate genes are more frequently found in affected individuals than in controls36, 37. In the case of rare coding SNPs, a framework is typically used in which nonsynonymous SNPs are examined based on their a priori likelihood of functionality. In the case of CNVs, similar paradigms may be useful: for example, pooling just those CNVs confirmed as affecting a candidate gene's coding sequence and nearby highly conserved elements. Although defining the right a priori criteria is not straightforward, the need for such criteria is: there is a great danger in (and long history of) post hoc explanations that can be invoked to support nonsignificant findings in discovery research.

Systematic biases can lead to false association
Years of SNP association studies — the vast majority of which proved irreproducible — have led to increased awareness of the factors that cause artifactual associations between genetic variants and phenotypes. CNP association studies are equally susceptible to these artifacts, which include population stratification, technical artifacts attributable to variability in the quality of DNA samples, and the general problem (inherent in all genome-wide studies) of distinguishing true signals from a genome-wide distribution of statistical sampling fluctuations.

Many phenotypes are associated with continental ancestry, and many CNPs (like many SNPs) vary in their frequencies across populations24, 25, 26. In disease association studies, such variants can be associated with disease owing to the confounding effect of ancestry (known as population stratification). Even in a study of individuals of European ancestry, variants that differ in frequency between northern and southern Europeans (such as the lactase persistence allele) can be artifactually associated with phenotypes (such as stature) that differ between northern and southern Europeans38. Methods to correct for stratification have been developed39, 40 and require the investigator to obtain extensive genetic information beyond the locus in question; it would seem reasonable to require such analyses in any CNP-based genome-wide association study, as in any SNP-based study. Family-based designs are another way to prevent stratification.

Disease association studies often utilize DNA that has been collected at a variety of clinical sites, extracted by different techniques, and prepared or assayed at different times. To the extent that DNA samples from affected individuals and controls differ, systematic technical bias can be introduced between the two groups. Some SNP assays are sensitive to DNA quality in ways that bias toward a particular result in lower-quality samples and can thereby lead to artifactual associations with disease41. This observation seems certain to apply to CNV studies as well. For example, the sensitivity of array comparative genomic hybridization (CGH) for detecting variants has been shown to vary from sample to sample based on variation in DNA and hybridization quality29. To the extent that altered copy numbers are undercalled in lower-quality DNA samples and hybridizations, heterogeneity in DNA preparation could lead to artifactual associations.

Although such biases may be sporadic and infrequent in focused, single-locus candidate-gene association studies, they are pervasive in genome-wide studies. This is because such studies involve looking for effects in the tails of a P-value distribution, where artifacts inevitably collect. As genome-wide CNP-disease association studies begin to be performed, it will be critical to seek out any systematic bias that distinguishes how DNA samples from affected individuals and controls are treated throughout the process of research. An important assessment is the extent to which the genome-wide distribution of P values conforms to the expected uniform distribution.

Perhaps the greatest cause of false association in the SNP literature has been the use of statistical thresholds inadequate to distinguish true associations from false positives. This is particularly problematic because of the low prior probability that any given variant (SNP or CNP) truly influences the trait of interest (at least, to an extent measurable with the sample size, technical approach and statistical framework employed)42. Just as in SNP association studies, it seems unlikely that an association of a CNP with disease that displays a P value of 0.05 will prove reproducible. On the other hand, a robustly significant P value (given the lower prior probability intrinsic in a genome-wide search), perhaps combined with functional data, surely can result in a compelling finding. Insistence upon the highest standards for proof in the early days of a field will save the community the consternation of irreproducible findings muddying the literature.

Opportunities
The coming years are likely to be tremendously exciting, as initial observations of common human common copy-number variation mature into an understanding that is crisp in molecular detail, complete in knowledge of location and frequency, and conducive to discoveries in the pathogenesis and genetic epidemiology of human disease. Perhaps the greatest impediments to such a future would be not the discovery of too few CNP-disease associations in the next year or two, but an insufficient investment in a truly effective set of tools and databases for their study, coupled with overly enthusiastic (but not quite reproducible) early claims of association with disease. With the proper focus and standards, CNP research will yield important insights, elucidating not only human genetic variation, but biological pathways and the mechanisms of human disease.

Competing interests statement: The authors declare no competing financial interests.

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Nature Genetics 39, S37 - S42 (2007)doi:10.1038/ng2080

Copy-number variation and association studies of human disease
Steven A McCarroll1 & David M Altshuler1,2

The authors are in the Program in Medical and Population Genetics, Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA; and Department of Molecular Biology and the Center for Human Genetic Research, Massachusetts General Hospital, Boston, Massachusetts, USA. e-mail: smccarro@broad.mit.edu
David Altshuler is also in the Department of Medicine at Massachusetts General Hospital and the Departments of Genetics and Medicine, Harvard Medical School, Boston, Massachusetts, USA.

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AbstractThe central goal of human genetics is to understand the inherited basis of human variation in phenotypes, elucidating human physiology, evolution and disease. Rare mutations have been found underlying two thousand mendelian diseases; more recently, it has become possible to assess systematically the contribution of common SNPs to complex disease. The known role of copy-number alterations in sporadic genomic disorders, combined with emerging information about inherited copy-number variation, indicate the importance of systematically assessing copy-number variants (CNVs), including common copy-number polymorphisms (CNPs), in disease. Here we discuss evidence that CNVs affect phenotypes, directions for basic knowledge to support clinical study of CNVs, the challenge of genotyping CNPs in clinical cohorts, the use of SNPs as markers for CNPs and statistical challenges in testing CNVs for association with disease. Critical needs are high-resolution maps of common CNPs and techniques that accurately determine the allelic state of affected individuals.

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IntroductionEmpirical evidence that CNVs are associated with phenotypes
The first evidence that copy-number alterations can influence human phenotypes came from sporadic diseases, termed 'genomic disorders', caused by de novo structural alterations1. The number of genomic disorders has grown, with several dozen reported to date2. In addition to such sporadic diseases, inherited CNVs have been found to underlie mendelian diseases in several families3, 4, 5. Nonetheless, CNVs have been implicated in only a few percent of the 2,000 or more mendelian diseases so far explained at a molecular level.

Little is known about the genetic basis of common, complex phenotypes, and thus it would be premature to predict the relative proportion of complex disease explained by SNPs and CNVs. In principle, complex disease might be more susceptible to 'soft' forms of variation — such as variation in noncoding sequences and copy number — which alter gene dose without abolishing gene function. Common CNPs have been reported to be associated with several complex disease phenotypes, including HIV acquisition and progression6, lupus glomerulonephritis7 and three systemic autoimmune diseases: systemic lupus erythematosus, microscopic polyangiitis and Wegener's granulomatosis8, 9. A recent study of gene expression variation as a model complex phenotype measured the fraction of gene expression 'traits' that could be associated with either SNPs or CNVs; in this study, SNP genotypes and CNV measurements were associated with 83% and 18% of those gene expression traits for which statistically significant associations were found10. This may still underestimate the role of CNVs, given the greater completeness and accuracy with which SNPs can be queried at present.

Technical issues in assessing CNVs for a role in disease
The power to discover a relationship between DNA variation and phenotype is limited by the sensitivity and accuracy with which that DNA variation is measured in each individual. (Accuracy in measuring phenotype, environment and behavior are equally or more important; these challenges, which are not specific to CNV studies, are beyond the scope of this review.) To the extent that the precise allelic state of any DNA variant is not well measured, power declines. But although this issue has been the subject of extensive discussion in the literature on SNP association studies11, little has been written about the extent to which current attempts to measure copy number provide precise and accurate measures of the underlying DNA variation in each individual.

Insufficient data have been collected at a sequence level to estimate the correlation between quantitative measures of CNVs by existing techniques and the true allelic state of each CNV in each individual. However, there are reasons to believe that the correlation is low — much lower, for example, than that offered by current SNP genotyping products for the underlying SNP variation in the genome12. This difference is due fundamentally to the greater challenge of measuring multibase, often multiallelic variants compared with single-base, diallelic SNPs. The specific challenge of genotyping CNVs is discussed in a later section.

Enabling core knowledge about CNVs for association studies
An indispensable starting point for association studies is basic knowledge about the genetic variations that are present in the human population — their alleles, their frequencies, their precise locations. SNP databases developed through a series of phases: first, rapid growth in methods to detect the locations of putative SNPs; second, calibration and standardization of discovery methods to maximize sensitivity and minimize false positives13; third, accurate genotyping of large numbers of SNPs to validate (or invalidate) SNPs and characterize their properties14, 15, 16; and fourth, assessment of the sensitivity of the resulting map in comparison to systematic resequencing data16. The resulting resource enables researchers and technology companies to design assays for any given SNP of interest (or a genome-wide collection) and to assess the relationship between any given SNP and others that may or may not have been typed.

Pioneering genome-wide surveys of CNVs17, 18, 19, 20, 21, 22, 23, 24, 25, 26 and databases holding the results of those studies27 are an important initial step toward a comprehensive database of CNVs, but much work lies ahead. Most reported CNV locations actually correspond to the locations of CNV-containing regions (CNVRs), generally the genomic coordinates of a BAC probe, set of oligonucleotide probes, or fosmid from which a variant was discovered. A reported CNVR is consistent with a large number of potential variants (Fig. 1); of importance for the design of assays, seldom is it known which precise locus or gene within the CNVR is actually affected. An important step toward enabling knowledge of CNP locations is an ongoing effort to sequence fosmid clones containing structurally variant haplotypes28. Until the locations of CNVs within reported CNVRs are known, researchers interested in studying a reported CNV in clinical samples must first perform experiments to find the CNV(s) within the reported CNVR. Ultimately, the utility of CNV databases will be enhanced by data on the validation state of each putative CNV (to avoid wasting resources on false positive CNVs) and on the frequency of each allele in different populations (to estimate statistical power when designing association studies), and by a map of the linkage disequilibrium (or lack thereof) with nearby SNPs that may be easier to genotype or may already have been assessed.

Figure 1: Many potential CNV locations are consistent with the coordinates of a reported CNV-containing region (CNVR).
An investigator will typically have to perform extensive experiments to find the CNV within a CNVR. An ongoing project to map structural variants at the sequence level28 will provide important enabling knowledge for clinical genetic studies.

Full size image (49 KB)

Genotyping CNVs in disease association studies
Disease association studies rely on accurate genotypes. Most CNV studies to date have been discovery studies (generating lists of regions that contain CNVs) rather than association studies (assessing the correlation between phenotype and genotype). The underlying problems in CNV discovery and CNV genotyping are different. A discovery study begins with a null hypothesis that no variation exists at a locus and assesses whether the evidence for variation exceeds a genome-wide significance threshold; a high false-negative rate (failure to discover variants) is tolerated in order to preserve a low false-positive rate29. An association study tests a null hypothesis that variation is not associated with phenotype; all forms of misclassification (both over- and underascertainment of altered copy-number levels) are problematic, and all levels of copy number must be distinguished. This is a much more exacting requirement: for example, of some 1,500 CNVs that were identified in one recent genome-wide survey, only 70 common, diallelic CNVs yielded genotypes of the quality that could be used for linkage disequilibrium analysis26. Indeed, of the thousands of CNV-containing regions that have been identified in the literature to date, only a few percent have been genotyped in available reference samples (Fig. 2).

Figure 2: Although the number of reported copy-number-variable regions has increased dramatically, only a few percent of CNVs have been successfully genotyped.
An important step in association study of any CNV will be the development of a genotyping assay that accurately determines the allelic state of every individual in a cohort.

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The development of assays for accurately typing CNVs in clinical cohorts has required enormous effort in CNV-disease association studies to date6, 7, 8, 9 and is one of the most pressing needs in CNV research. Although it is not yet clear what technology will be used, it is critical that any assay be reproducible in other labs. Standards for publishing a CNV-disease association should include genotypes for a publicly available set of reference samples, which can be used by other labs to develop additional assays and to assess the original assay.

Copy-number measurements versus copy-number genotypes
At any given nucleotide, biological copy numbers are integers. The more precise and localized a measurement of copy number, the more its distribution in a population shows a discrete distribution reflecting the underlying integer distribution of copy numbers (for example, 0, 1, 2; or 2, 3, 4; or even 2, 3, 4, 5, 6) (Fig. 3). Frequently, though, copy-number measurements seem to be continuously distributed across a population (Fig. 3c). The factors which cause imprecision in copy-number measurement can be divided into two categories. Measurement imprecision refers to the noise inherent in making any measurement. Spatial imprecision occurs when an assay aggregates information across a large region into a single measurement.

Figure 3: Using copy-number measurements and copy-number genotypes in association studies.
(a) A common CNP containing a gene encoding pyruvate dehydrogenase phosphatase regulatory protein (PDPR; blue arrow) interrogated by a BAC probe (Chr16tp-9C8, red rectangle) on a BAC array-CGH platform26, and by a series of oligonucleotide probes (vertical line segments) on an oligonucleotide platform (Affymetrix GenomeWide 5.0). (b–d) Copy-number measurements for the two platforms across the same set of samples (HapMap CEU sample of individuals with European ancestry) are correlated (b), confirming that they interrogate the same CNP. Measurements on the BAC array-CGH platform26 show a continuous distribution (c), whereas measurements on the oligonucleotide platform show a discrete distribution (d). (e,f) Association analysis using raw intensity measurements. PDPR gene expression is found to be associated with PDPR copy number using raw measurements of copy number on both platforms. Colors indicate the discrete genotype 'calls' on each platform (not used in this analysis, but used in the analysis in panels g,h). The association in e was discovered by Stranger et al. (2007)10. (g,h) Association analysis using discrete genotype 'calls'. Where raw measurements show a continuous distribution (c,e,g), hardening of raw measurements into discrete 'call' loses information that was present in the original measurements, with the result that association with phenotype is no longer detected. Where raw measurements show a discrete distribution (d,f,h), conversion of raw measurements into genotypes can increase the correlation with phenotype, though the primary benefit may simply be greater clarity about the distribution of genetic variation and its relationship to phenotype.

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Surveys of copy-number variation have further summarized copy-number measurements into discrete values of 'gain' or 'loss' in each sample; although these assessments are sometimes referred to as 'genotypes', inspection of the underlying data often shows that these discrete distinctions are not reflected in the underlying distribution of measurements (Fig. 3c,e). Summarizing raw copy-number measurements into such 'calls' may lose information present in the original measurements, and is of uncertain relationship to the true genotype (Fig. 3e,g).

Until approaches for genome-wide CNP genotyping mature, a placeholder strategy may be to rely on raw hybridization measurements as an approximation to an unknown, underlying genotype. This approach was used in a recent study of CNPs and gene expression10 that used copy number data from an immediately preceding study26; the analysis dispensed with the CNV 'calls' from the previous study, instead using the raw hybridization measurements for association analysis10, 26 (Fig. 3). The paucity of effective CNP genotypes means that techniques and algorithms for making genotype calls are a critical need in CNP disease research; until such approaches mature, raw measurements may be the preferred basis for a preliminary association analysis.

Using SNPs as markers for CNPs
Given the technical challenges in finding and typing CNPs, and the early stage of basic knowledge about their locations and molecular structures, an appealing strategy might be to rely on more-easily-typed SNPs to serve as markers by linkage disequilibrium for common variants throughout the genome. Linkage disequilibrium–based approaches utilize the observation that the human recombination rate is (i) low relative to the typical age of alleles in the human population and (ii) clustered into hotspots across the genome30. These features mean that ancestral variants (whatever their molecular nature) segregate in the population on haplotypes, are correlated with one another and thus can be 'tagged' by a reduced set of SNPs31. Because such linkage disequilibrium–based approaches require neither a priori identification of all variants nor technology for typing every variant individually, they might address the limitations of current knowledge and genotyping technology in the CNP field.

To assess a specific CNP through linkage disequilibrium, one would genotype the CNP in the HapMap (or other reference) samples and assess whether nearby SNPs were able to capture the CNP through linkage disequilibrium; if so, one would then type those SNPs in affected cohorts as a proxy for the CNP. To analyze a genomic region, one would select a dense set of SNPs sufficient to capture almost all common, ancestral polymorphisms through linkage disequilibrium11 and test them for association with disease. On a genome-wide scale, one would presumably use commercial whole-genome SNP genotyping products. In all cases, positive association (if found) could be due to a CNV or to anything else in linkage disequilibrium with the associated SNP — possibilities that would be assessed by directed resequencing, copy-number analysis and additional genotyping in following up any initial association.

The performance of linkage disequilibrium–based approaches will depend on the strength and generality of linkage disequilibrium between CNPs and SNPs. Using available SNP data and PCR-based genotyping of deletion polymorphisms, initial studies found that deletion polymorphisms are generally ancestral and are tagged by SNPs22, 23. A subsequent study of the linkage-disequilibrium properties of CNPs in the genome's segmental-duplication-rich regions found that copy-number measurements from such CNPs were less well captured by HapMap SNPs24; a more recent study of 70 genotyped CNPs found that the CNPs showed appreciable linkage disequilibrium with SNPs, but were less well tagged than frequency-matched SNPs were26. The extent of linkage disequilibrium between SNPs and CNPs remains unclear, for two reasons. First, assessing linkage disequilibrium around CNPs requires accurate genotyping of a large and representative collection of CNPs in samples with dense SNP genotypes — and yet accurate genotypes exist for only a small and nonrandom collection of CNPs (Fig. 2). Second, regions rich in segmental duplications contain almost half of all reported CNPs19, 24, 26, but contain a density of validated SNPs (that could serve as potential tags) much lower than that of the rest of the genome24.

Integrated association studies for SNPs and CNPs
Many genome-wide SNP association studies, each involving hundreds to thousands of affected individuals, are underway. The raw intensity data generated during SNP genotyping can be mined for copy-number information32, 33, 34, 35, making such studies a potential source of data for CNP-disease association studies. However, several factors limit the utility of previous generations of SNP arrays for this purpose. Most important is coverage: because common CNPs cause SNP genotyping assays to fail Hardy-Weinberg and mendelian inheritance checks, genomic regions harboring common CNPs had been filtered out (partially or completely) of commercial whole-genome SNP array platforms during the selection of high-performance SNP assays. Another limitation is technical: because SNP assays are optimized for allelic discrimination rather than copy-number measurement, the copy-number measurements they provide are noisy, with the result that only large variants are typically detected. Commercial SNP arrays are used to find the large copy-number alterations typical of cancer32, 33, 34, 35, but have not to date been used to perform association studies for germline CNPs, and seem to detect many more rare CNVs than common CNPs26.

Ideally, every DNA sample would be simultaneously queried for SNPs and CNVs in a single, integrated analysis. We have been working with collaborators to develop hybrid oligonucleotide arrays that contain both SNP allele-discrimination probes and dedicated 'copy-number probes' — probes whose sequences have been optimized for copy-number quantification by (i) designing them to nonpolymorphic sequences, (ii) selecting sequence features predictive of technical efficacy and (iii) empirically assessing responsiveness in screening experiments (Fig. 3d,f,h). Such hybrid arrays (or some other technological solution) offer the potential for integrated association studies in which SNP and copy-number variation are considered together. Moreover, as databases of CNPs and SNPs become ever more complete, the content of such arrays should similarly approach completeness.

Testing the disease association of common CNPs
Once an accurate and complete set of CNV measurements is obtained in a sample, there are few unprecedented statistical challenges to the assessment of association with disease. As with SNPs, a key dividing line is whether the statistical test involves common variants or a collection of individually rare events.

For common CNPs, statistical tests will involve a straightforward comparison of allele frequencies (or of diploid genotype frequencies): between affected individuals and controls in a population cohort; between transmitted and untransmitted chromosomes in families with affected offspring; or between affected and unaffected siblings. Most successfully genotyped CNPs seem to be diallelic, showing 2 or 3 diploid copy-number classes and therefore most likely representing two underlying alleles23, 26. Such variants are readily incorporated into current frameworks for SNP association testing; in fact, the copy-number classes could be subjected to the same quality-control tests (mendelian inheritance, Hardy-Weinberg equilibrium) used to ensure the quality of SNP genotypes. Such CNPs could for practical reasons be recoded as SNP genotypes (for example, 'AA' for zero copies, 'AC' for one copy, 'CC' for two copies) and thereby benefit from the software and analytical approaches already available for SNP-based analyses, including correcting for population stratification (discussed below) and scrutinizing a genome-wide study for P-value inflation.

Some CNPs seem to involve more than three copy-number classes, and therefore more than two copy-number alleles (Fig. 3). Nineteen such loci were identified in a recent genome-wide CNV survey26. A related class of CNPs appears to harbor both deletion and duplication alleles24, 26. Notably, the common CNPs reported to be associated with HIV progression and autoimmune phenotypes are multiallelic6, 7, 8, 9. For the population-based analyses in those studies, researchers used a variety of techniques to test for disease association, including (i) reducing the copy-number genotype to a binary class (for example, >4 versus <4 copies), then performing a chi-squared analysis on the distribution of disease status between these two groups6; (ii) a logistic regression analysis, with copy number as an explanatory variable and age and gender as covariates7; and (iii) nonparametric tests of the null hypothesis that affected individuals and controls were drawn from the same distribution of copy numbers7. Family-based analyses — which are favored by many researchers because they are more robust to population stratification (discussed below) — will also need to be generalized to address multiallelic CNPs and continuously distributed copy-number measurements.

Testing the disease association of rare CNVs
For rare variants, association analysis is more challenging, as it is less constrained: there are many potential ways to group a collection of unique events, and thus more degrees of freedom. When copy-number ascertainment was limited to large, microscopically visible (and therefore usually functional) variants, such variants were generally assumed to be causative (although the specific gene involved is, conversely, very imprecisely localized). The new ability to detect smaller, submicroscopic CNVs — hundreds of which may be present in any one individual, and the vast majority of which are benign — requires statistically well founded assessment of their association with disease.

As submicroscopic CNVs cannot be assumed to have functional consequence, it is critical to search for them in affected individuals and controls with equal rigor, and to use a statistical framework to determine whether rearrangements are truly more common in the affected. It is critical that CNVs not be discovered in a set of cases and then the specific variants that were found queried in controls; such an approach is subject to 'ascertainment bias' and is statistically unsound. Given the existence of hundreds of rare CNVs with apparent frequencies of less than one percent, even in a well designed study it will frequently occur that a CNV is present (for example) in 3/200 cases and 0/200 controls. Such results are expected to occur by chance in a genome-wide search, and so do not necessarily imply a causal effect. (The observation of three independent, de novo structural mutations at the same locus in a disease cohort might be highly significant, because the rate of sporadic structural mutation seems to be much lower than the rate of CNV inheritance; such sporadic genomic disorders are discussed in an accompanying Perspective2).

It is natural to also consider the hypothesis that distinct CNVs at the same genomic locus may similarly influence disease risk in different individuals. An important precedent for such reasoning is the argument that diverse sequence variants in candidate genes are more frequently found in affected individuals than in controls36, 37. In the case of rare coding SNPs, a framework is typically used in which nonsynonymous SNPs are examined based on their a priori likelihood of functionality. In the case of CNVs, similar paradigms may be useful: for example, pooling just those CNVs confirmed as affecting a candidate gene's coding sequence and nearby highly conserved elements. Although defining the right a priori criteria is not straightforward, the need for such criteria is: there is a great danger in (and long history of) post hoc explanations that can be invoked to support nonsignificant findings in discovery research.

Systematic biases can lead to false association
Years of SNP association studies — the vast majority of which proved irreproducible — have led to increased awareness of the factors that cause artifactual associations between genetic variants and phenotypes. CNP association studies are equally susceptible to these artifacts, which include population stratification, technical artifacts attributable to variability in the quality of DNA samples, and the general problem (inherent in all genome-wide studies) of distinguishing true signals from a genome-wide distribution of statistical sampling fluctuations.

Many phenotypes are associated with continental ancestry, and many CNPs (like many SNPs) vary in their frequencies across populations24, 25, 26. In disease association studies, such variants can be associated with disease owing to the confounding effect of ancestry (known as population stratification). Even in a study of individuals of European ancestry, variants that differ in frequency between northern and southern Europeans (such as the lactase persistence allele) can be artifactually associated with phenotypes (such as stature) that differ between northern and southern Europeans38. Methods to correct for stratification have been developed39, 40 and require the investigator to obtain extensive genetic information beyond the locus in question; it would seem reasonable to require such analyses in any CNP-based genome-wide association study, as in any SNP-based study. Family-based designs are another way to prevent stratification.

Disease association studies often utilize DNA that has been collected at a variety of clinical sites, extracted by different techniques, and prepared or assayed at different times. To the extent that DNA samples from affected individuals and controls differ, systematic technical bias can be introduced between the two groups. Some SNP assays are sensitive to DNA quality in ways that bias toward a particular result in lower-quality samples and can thereby lead to artifactual associations with disease41. This observation seems certain to apply to CNV studies as well. For example, the sensitivity of array comparative genomic hybridization (CGH) for detecting variants has been shown to vary from sample to sample based on variation in DNA and hybridization quality29. To the extent that altered copy numbers are undercalled in lower-quality DNA samples and hybridizations, heterogeneity in DNA preparation could lead to artifactual associations.

Although such biases may be sporadic and infrequent in focused, single-locus candidate-gene association studies, they are pervasive in genome-wide studies. This is because such studies involve looking for effects in the tails of a P-value distribution, where artifacts inevitably collect. As genome-wide CNP-disease association studies begin to be performed, it will be critical to seek out any systematic bias that distinguishes how DNA samples from affected individuals and controls are treated throughout the process of research. An important assessment is the extent to which the genome-wide distribution of P values conforms to the expected uniform distribution.

Perhaps the greatest cause of false association in the SNP literature has been the use of statistical thresholds inadequate to distinguish true associations from false positives. This is particularly problematic because of the low prior probability that any given variant (SNP or CNP) truly influences the trait of interest (at least, to an extent measurable with the sample size, technical approach and statistical framework employed)42. Just as in SNP association studies, it seems unlikely that an association of a CNP with disease that displays a P value of 0.05 will prove reproducible. On the other hand, a robustly significant P value (given the lower prior probability intrinsic in a genome-wide search), perhaps combined with functional data, surely can result in a compelling finding. Insistence upon the highest standards for proof in the early days of a field will save the community the consternation of irreproducible findings muddying the literature.

Opportunities
The coming years are likely to be tremendously exciting, as initial observations of common human common copy-number variation mature into an understanding that is crisp in molecular detail, complete in knowledge of location and frequency, and conducive to discoveries in the pathogenesis and genetic epidemiology of human disease. Perhaps the greatest impediments to such a future would be not the discovery of too few CNP-disease associations in the next year or two, but an insufficient investment in a truly effective set of tools and databases for their study, coupled with overly enthusiastic (but not quite reproducible) early claims of association with disease. With the proper focus and standards, CNP research will yield important insights, elucidating not only human genetic variation, but biological pathways and the mechanisms of human disease.

Competing interests statement: The authors declare no competing financial interests.

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Nature Genetics 39, S37 - S42 (2007)doi:10.1038/ng2080

Copy-number variation and association studies of human disease
Steven A McCarroll1 & David M Altshuler1,2

The authors are in the Program in Medical and Population Genetics, Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA; and Department of Molecular Biology and the Center for Human Genetic Research, Massachusetts General Hospital, Boston, Massachusetts, USA. e-mail: smccarro@broad.mit.edu
David Altshuler is also in the Department of Medicine at Massachusetts General Hospital and the Departments of Genetics and Medicine, Harvard Medical School, Boston, Massachusetts, USA.

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AbstractThe central goal of human genetics is to understand the inherited basis of human variation in phenotypes, elucidating human physiology, evolution and disease. Rare mutations have been found underlying two thousand mendelian diseases; more recently, it has become possible to assess systematically the contribution of common SNPs to complex disease. The known role of copy-number alterations in sporadic genomic disorders, combined with emerging information about inherited copy-number variation, indicate the importance of systematically assessing copy-number variants (CNVs), including common copy-number polymorphisms (CNPs), in disease. Here we discuss evidence that CNVs affect phenotypes, directions for basic knowledge to support clinical study of CNVs, the challenge of genotyping CNPs in clinical cohorts, the use of SNPs as markers for CNPs and statistical challenges in testing CNVs for association with disease. Critical needs are high-resolution maps of common CNPs and techniques that accurately determine the allelic state of affected individuals.

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IntroductionEmpirical evidence that CNVs are associated with phenotypes
The first evidence that copy-number alterations can influence human phenotypes came from sporadic diseases, termed 'genomic disorders', caused by de novo structural alterations1. The number of genomic disorders has grown, with several dozen reported to date2. In addition to such sporadic diseases, inherited CNVs have been found to underlie mendelian diseases in several families3, 4, 5. Nonetheless, CNVs have been implicated in only a few percent of the 2,000 or more mendelian diseases so far explained at a molecular level.

Little is known about the genetic basis of common, complex phenotypes, and thus it would be premature to predict the relative proportion of complex disease explained by SNPs and CNVs. In principle, complex disease might be more susceptible to 'soft' forms of variation — such as variation in noncoding sequences and copy number — which alter gene dose without abolishing gene function. Common CNPs have been reported to be associated with several complex disease phenotypes, including HIV acquisition and progression6, lupus glomerulonephritis7 and three systemic autoimmune diseases: systemic lupus erythematosus, microscopic polyangiitis and Wegener's granulomatosis8, 9. A recent study of gene expression variation as a model complex phenotype measured the fraction of gene expression 'traits' that could be associated with either SNPs or CNVs; in this study, SNP genotypes and CNV measurements were associated with 83% and 18% of those gene expression traits for which statistically significant associations were found10. This may still underestimate the role of CNVs, given the greater completeness and accuracy with which SNPs can be queried at present.

Technical issues in assessing CNVs for a role in disease
The power to discover a relationship between DNA variation and phenotype is limited by the sensitivity and accuracy with which that DNA variation is measured in each individual. (Accuracy in measuring phenotype, environment and behavior are equally or more important; these challenges, which are not specific to CNV studies, are beyond the scope of this review.) To the extent that the precise allelic state of any DNA variant is not well measured, power declines. But although this issue has been the subject of extensive discussion in the literature on SNP association studies11, little has been written about the extent to which current attempts to measure copy number provide precise and accurate measures of the underlying DNA variation in each individual.

Insufficient data have been collected at a sequence level to estimate the correlation between quantitative measures of CNVs by existing techniques and the true allelic state of each CNV in each individual. However, there are reasons to believe that the correlation is low — much lower, for example, than that offered by current SNP genotyping products for the underlying SNP variation in the genome12. This difference is due fundamentally to the greater challenge of measuring multibase, often multiallelic variants compared with single-base, diallelic SNPs. The specific challenge of genotyping CNVs is discussed in a later section.

Enabling core knowledge about CNVs for association studies
An indispensable starting point for association studies is basic knowledge about the genetic variations that are present in the human population — their alleles, their frequencies, their precise locations. SNP databases developed through a series of phases: first, rapid growth in methods to detect the locations of putative SNPs; second, calibration and standardization of discovery methods to maximize sensitivity and minimize false positives13; third, accurate genotyping of large numbers of SNPs to validate (or invalidate) SNPs and characterize their properties14, 15, 16; and fourth, assessment of the sensitivity of the resulting map in comparison to systematic resequencing data16. The resulting resource enables researchers and technology companies to design assays for any given SNP of interest (or a genome-wide collection) and to assess the relationship between any given SNP and others that may or may not have been typed.

Pioneering genome-wide surveys of CNVs17, 18, 19, 20, 21, 22, 23, 24, 25, 26 and databases holding the results of those studies27 are an important initial step toward a comprehensive database of CNVs, but much work lies ahead. Most reported CNV locations actually correspond to the locations of CNV-containing regions (CNVRs), generally the genomic coordinates of a BAC probe, set of oligonucleotide probes, or fosmid from which a variant was discovered. A reported CNVR is consistent with a large number of potential variants (Fig. 1); of importance for the design of assays, seldom is it known which precise locus or gene within the CNVR is actually affected. An important step toward enabling knowledge of CNP locations is an ongoing effort to sequence fosmid clones containing structurally variant haplotypes28. Until the locations of CNVs within reported CNVRs are known, researchers interested in studying a reported CNV in clinical samples must first perform experiments to find the CNV(s) within the reported CNVR. Ultimately, the utility of CNV databases will be enhanced by data on the validation state of each putative CNV (to avoid wasting resources on false positive CNVs) and on the frequency of each allele in different populations (to estimate statistical power when designing association studies), and by a map of the linkage disequilibrium (or lack thereof) with nearby SNPs that may be easier to genotype or may already have been assessed.

Figure 1: Many potential CNV locations are consistent with the coordinates of a reported CNV-containing region (CNVR).
An investigator will typically have to perform extensive experiments to find the CNV within a CNVR. An ongoing project to map structural variants at the sequence level28 will provide important enabling knowledge for clinical genetic studies.

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Genotyping CNVs in disease association studies
Disease association studies rely on accurate genotypes. Most CNV studies to date have been discovery studies (generating lists of regions that contain CNVs) rather than association studies (assessing the correlation between phenotype and genotype). The underlying problems in CNV discovery and CNV genotyping are different. A discovery study begins with a null hypothesis that no variation exists at a locus and assesses whether the evidence for variation exceeds a genome-wide significance threshold; a high false-negative rate (failure to discover variants) is tolerated in order to preserve a low false-positive rate29. An association study tests a null hypothesis that variation is not associated with phenotype; all forms of misclassification (both over- and underascertainment of altered copy-number levels) are problematic, and all levels of copy number must be distinguished. This is a much more exacting requirement: for example, of some 1,500 CNVs that were identified in one recent genome-wide survey, only 70 common, diallelic CNVs yielded genotypes of the quality that could be used for linkage disequilibrium analysis26. Indeed, of the thousands of CNV-containing regions that have been identified in the literature to date, only a few percent have been genotyped in available reference samples (Fig. 2).

Figure 2: Although the number of reported copy-number-variable regions has increased dramatically, only a few percent of CNVs have been successfully genotyped.
An important step in association study of any CNV will be the development of a genotyping assay that accurately determines the allelic state of every individual in a cohort.

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The development of assays for accurately typing CNVs in clinical cohorts has required enormous effort in CNV-disease association studies to date6, 7, 8, 9 and is one of the most pressing needs in CNV research. Although it is not yet clear what technology will be used, it is critical that any assay be reproducible in other labs. Standards for publishing a CNV-disease association should include genotypes for a publicly available set of reference samples, which can be used by other labs to develop additional assays and to assess the original assay.

Copy-number measurements versus copy-number genotypes
At any given nucleotide, biological copy numbers are integers. The more precise and localized a measurement of copy number, the more its distribution in a population shows a discrete distribution reflecting the underlying integer distribution of copy numbers (for example, 0, 1, 2; or 2, 3, 4; or even 2, 3, 4, 5, 6) (Fig. 3). Frequently, though, copy-number measurements seem to be continuously distributed across a population (Fig. 3c). The factors which cause imprecision in copy-number measurement can be divided into two categories. Measurement imprecision refers to the noise inherent in making any measurement. Spatial imprecision occurs when an assay aggregates information across a large region into a single measurement.

Figure 3: Using copy-number measurements and copy-number genotypes in association studies.
(a) A common CNP containing a gene encoding pyruvate dehydrogenase phosphatase regulatory protein (PDPR; blue arrow) interrogated by a BAC probe (Chr16tp-9C8, red rectangle) on a BAC array-CGH platform26, and by a series of oligonucleotide probes (vertical line segments) on an oligonucleotide platform (Affymetrix GenomeWide 5.0). (b–d) Copy-number measurements for the two platforms across the same set of samples (HapMap CEU sample of individuals with European ancestry) are correlated (b), confirming that they interrogate the same CNP. Measurements on the BAC array-CGH platform26 show a continuous distribution (c), whereas measurements on the oligonucleotide platform show a discrete distribution (d). (e,f) Association analysis using raw intensity measurements. PDPR gene expression is found to be associated with PDPR copy number using raw measurements of copy number on both platforms. Colors indicate the discrete genotype 'calls' on each platform (not used in this analysis, but used in the analysis in panels g,h). The association in e was discovered by Stranger et al. (2007)10. (g,h) Association analysis using discrete genotype 'calls'. Where raw measurements show a continuous distribution (c,e,g), hardening of raw measurements into discrete 'call' loses information that was present in the original measurements, with the result that association with phenotype is no longer detected. Where raw measurements show a discrete distribution (d,f,h), conversion of raw measurements into genotypes can increase the correlation with phenotype, though the primary benefit may simply be greater clarity about the distribution of genetic variation and its relationship to phenotype.

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Surveys of copy-number variation have further summarized copy-number measurements into discrete values of 'gain' or 'loss' in each sample; although these assessments are sometimes referred to as 'genotypes', inspection of the underlying data often shows that these discrete distinctions are not reflected in the underlying distribution of measurements (Fig. 3c,e). Summarizing raw copy-number measurements into such 'calls' may lose information present in the original measurements, and is of uncertain relationship to the true genotype (Fig. 3e,g).

Until approaches for genome-wide CNP genotyping mature, a placeholder strategy may be to rely on raw hybridization measurements as an approximation to an unknown, underlying genotype. This approach was used in a recent study of CNPs and gene expression10 that used copy number data from an immediately preceding study26; the analysis dispensed with the CNV 'calls' from the previous study, instead using the raw hybridization measurements for association analysis10, 26 (Fig. 3). The paucity of effective CNP genotypes means that techniques and algorithms for making genotype calls are a critical need in CNP disease research; until such approaches mature, raw measurements may be the preferred basis for a preliminary association analysis.

Using SNPs as markers for CNPs
Given the technical challenges in finding and typing CNPs, and the early stage of basic knowledge about their locations and molecular structures, an appealing strategy might be to rely on more-easily-typed SNPs to serve as markers by linkage disequilibrium for common variants throughout the genome. Linkage disequilibrium–based approaches utilize the observation that the human recombination rate is (i) low relative to the typical age of alleles in the human population and (ii) clustered into hotspots across the genome30. These features mean that ancestral variants (whatever their molecular nature) segregate in the population on haplotypes, are correlated with one another and thus can be 'tagged' by a reduced set of SNPs31. Because such linkage disequilibrium–based approaches require neither a priori identification of all variants nor technology for typing every variant individually, they might address the limitations of current knowledge and genotyping technology in the CNP field.

To assess a specific CNP through linkage disequilibrium, one would genotype the CNP in the HapMap (or other reference) samples and assess whether nearby SNPs were able to capture the CNP through linkage disequilibrium; if so, one would then type those SNPs in affected cohorts as a proxy for the CNP. To analyze a genomic region, one would select a dense set of SNPs sufficient to capture almost all common, ancestral polymorphisms through linkage disequilibrium11 and test them for association with disease. On a genome-wide scale, one would presumably use commercial whole-genome SNP genotyping products. In all cases, positive association (if found) could be due to a CNV or to anything else in linkage disequilibrium with the associated SNP — possibilities that would be assessed by directed resequencing, copy-number analysis and additional genotyping in following up any initial association.

The performance of linkage disequilibrium–based approaches will depend on the strength and generality of linkage disequilibrium between CNPs and SNPs. Using available SNP data and PCR-based genotyping of deletion polymorphisms, initial studies found that deletion polymorphisms are generally ancestral and are tagged by SNPs22, 23. A subsequent study of the linkage-disequilibrium properties of CNPs in the genome's segmental-duplication-rich regions found that copy-number measurements from such CNPs were less well captured by HapMap SNPs24; a more recent study of 70 genotyped CNPs found that the CNPs showed appreciable linkage disequilibrium with SNPs, but were less well tagged than frequency-matched SNPs were26. The extent of linkage disequilibrium between SNPs and CNPs remains unclear, for two reasons. First, assessing linkage disequilibrium around CNPs requires accurate genotyping of a large and representative collection of CNPs in samples with dense SNP genotypes — and yet accurate genotypes exist for only a small and nonrandom collection of CNPs (Fig. 2). Second, regions rich in segmental duplications contain almost half of all reported CNPs19, 24, 26, but contain a density of validated SNPs (that could serve as potential tags) much lower than that of the rest of the genome24.

Integrated association studies for SNPs and CNPs
Many genome-wide SNP association studies, each involving hundreds to thousands of affected individuals, are underway. The raw intensity data generated during SNP genotyping can be mined for copy-number information32, 33, 34, 35, making such studies a potential source of data for CNP-disease association studies. However, several factors limit the utility of previous generations of SNP arrays for this purpose. Most important is coverage: because common CNPs cause SNP genotyping assays to fail Hardy-Weinberg and mendelian inheritance checks, genomic regions harboring common CNPs had been filtered out (partially or completely) of commercial whole-genome SNP array platforms during the selection of high-performance SNP assays. Another limitation is technical: because SNP assays are optimized for allelic discrimination rather than copy-number measurement, the copy-number measurements they provide are noisy, with the result that only large variants are typically detected. Commercial SNP arrays are used to find the large copy-number alterations typical of cancer32, 33, 34, 35, but have not to date been used to perform association studies for germline CNPs, and seem to detect many more rare CNVs than common CNPs26.

Ideally, every DNA sample would be simultaneously queried for SNPs and CNVs in a single, integrated analysis. We have been working with collaborators to develop hybrid oligonucleotide arrays that contain both SNP allele-discrimination probes and dedicated 'copy-number probes' — probes whose sequences have been optimized for copy-number quantification by (i) designing them to nonpolymorphic sequences, (ii) selecting sequence features predictive of technical efficacy and (iii) empirically assessing responsiveness in screening experiments (Fig. 3d,f,h). Such hybrid arrays (or some other technological solution) offer the potential for integrated association studies in which SNP and copy-number variation are considered together. Moreover, as databases of CNPs and SNPs become ever more complete, the content of such arrays should similarly approach completeness.

Testing the disease association of common CNPs
Once an accurate and complete set of CNV measurements is obtained in a sample, there are few unprecedented statistical challenges to the assessment of association with disease. As with SNPs, a key dividing line is whether the statistical test involves common variants or a collection of individually rare events.

For common CNPs, statistical tests will involve a straightforward comparison of allele frequencies (or of diploid genotype frequencies): between affected individuals and controls in a population cohort; between transmitted and untransmitted chromosomes in families with affected offspring; or between affected and unaffected siblings. Most successfully genotyped CNPs seem to be diallelic, showing 2 or 3 diploid copy-number classes and therefore most likely representing two underlying alleles23, 26. Such variants are readily incorporated into current frameworks for SNP association testing; in fact, the copy-number classes could be subjected to the same quality-control tests (mendelian inheritance, Hardy-Weinberg equilibrium) used to ensure the quality of SNP genotypes. Such CNPs could for practical reasons be recoded as SNP genotypes (for example, 'AA' for zero copies, 'AC' for one copy, 'CC' for two copies) and thereby benefit from the software and analytical approaches already available for SNP-based analyses, including correcting for population stratification (discussed below) and scrutinizing a genome-wide study for P-value inflation.

Some CNPs seem to involve more than three copy-number classes, and therefore more than two copy-number alleles (Fig. 3). Nineteen such loci were identified in a recent genome-wide CNV survey26. A related class of CNPs appears to harbor both deletion and duplication alleles24, 26. Notably, the common CNPs reported to be associated with HIV progression and autoimmune phenotypes are multiallelic6, 7, 8, 9. For the population-based analyses in those studies, researchers used a variety of techniques to test for disease association, including (i) reducing the copy-number genotype to a binary class (for example, >4 versus <4 copies), then performing a chi-squared analysis on the distribution of disease status between these two groups6; (ii) a logistic regression analysis, with copy number as an explanatory variable and age and gender as covariates7; and (iii) nonparametric tests of the null hypothesis that affected individuals and controls were drawn from the same distribution of copy numbers7. Family-based analyses — which are favored by many researchers because they are more robust to population stratification (discussed below) — will also need to be generalized to address multiallelic CNPs and continuously distributed copy-number measurements.

Testing the disease association of rare CNVs
For rare variants, association analysis is more challenging, as it is less constrained: there are many potential ways to group a collection of unique events, and thus more degrees of freedom. When copy-number ascertainment was limited to large, microscopically visible (and therefore usually functional) variants, such variants were generally assumed to be causative (although the specific gene involved is, conversely, very imprecisely localized). The new ability to detect smaller, submicroscopic CNVs — hundreds of which may be present in any one individual, and the vast majority of which are benign — requires statistically well founded assessment of their association with disease.

As submicroscopic CNVs cannot be assumed to have functional consequence, it is critical to search for them in affected individuals and controls with equal rigor, and to use a statistical framework to determine whether rearrangements are truly more common in the affected. It is critical that CNVs not be discovered in a set of cases and then the specific variants that were found queried in controls; such an approach is subject to 'ascertainment bias' and is statistically unsound. Given the existence of hundreds of rare CNVs with apparent frequencies of less than one percent, even in a well designed study it will frequently occur that a CNV is present (for example) in 3/200 cases and 0/200 controls. Such results are expected to occur by chance in a genome-wide search, and so do not necessarily imply a causal effect. (The observation of three independent, de novo structural mutations at the same locus in a disease cohort might be highly significant, because the rate of sporadic structural mutation seems to be much lower than the rate of CNV inheritance; such sporadic genomic disorders are discussed in an accompanying Perspective2).

It is natural to also consider the hypothesis that distinct CNVs at the same genomic locus may similarly influence disease risk in different individuals. An important precedent for such reasoning is the argument that diverse sequence variants in candidate genes are more frequently found in affected individuals than in controls36, 37. In the case of rare coding SNPs, a framework is typically used in which nonsynonymous SNPs are examined based on their a priori likelihood of functionality. In the case of CNVs, similar paradigms may be useful: for example, pooling just those CNVs confirmed as affecting a candidate gene's coding sequence and nearby highly conserved elements. Although defining the right a priori criteria is not straightforward, the need for such criteria is: there is a great danger in (and long history of) post hoc explanations that can be invoked to support nonsignificant findings in discovery research.

Systematic biases can lead to false association
Years of SNP association studies — the vast majority of which proved irreproducible — have led to increased awareness of the factors that cause artifactual associations between genetic variants and phenotypes. CNP association studies are equally susceptible to these artifacts, which include population stratification, technical artifacts attributable to variability in the quality of DNA samples, and the general problem (inherent in all genome-wide studies) of distinguishing true signals from a genome-wide distribution of statistical sampling fluctuations.

Many phenotypes are associated with continental ancestry, and many CNPs (like many SNPs) vary in their frequencies across populations24, 25, 26. In disease association studies, such variants can be associated with disease owing to the confounding effect of ancestry (known as population stratification). Even in a study of individuals of European ancestry, variants that differ in frequency between northern and southern Europeans (such as the lactase persistence allele) can be artifactually associated with phenotypes (such as stature) that differ between northern and southern Europeans38. Methods to correct for stratification have been developed39, 40 and require the investigator to obtain extensive genetic information beyond the locus in question; it would seem reasonable to require such analyses in any CNP-based genome-wide association study, as in any SNP-based study. Family-based designs are another way to prevent stratification.

Disease association studies often utilize DNA that has been collected at a variety of clinical sites, extracted by different techniques, and prepared or assayed at different times. To the extent that DNA samples from affected individuals and controls differ, systematic technical bias can be introduced between the two groups. Some SNP assays are sensitive to DNA quality in ways that bias toward a particular result in lower-quality samples and can thereby lead to artifactual associations with disease41. This observation seems certain to apply to CNV studies as well. For example, the sensitivity of array comparative genomic hybridization (CGH) for detecting variants has been shown to vary from sample to sample based on variation in DNA and hybridization quality29. To the extent that altered copy numbers are undercalled in lower-quality DNA samples and hybridizations, heterogeneity in DNA preparation could lead to artifactual associations.

Although such biases may be sporadic and infrequent in focused, single-locus candidate-gene association studies, they are pervasive in genome-wide studies. This is because such studies involve looking for effects in the tails of a P-value distribution, where artifacts inevitably collect. As genome-wide CNP-disease association studies begin to be performed, it will be critical to seek out any systematic bias that distinguishes how DNA samples from affected individuals and controls are treated throughout the process of research. An important assessment is the extent to which the genome-wide distribution of P values conforms to the expected uniform distribution.

Perhaps the greatest cause of false association in the SNP literature has been the use of statistical thresholds inadequate to distinguish true associations from false positives. This is particularly problematic because of the low prior probability that any given variant (SNP or CNP) truly influences the trait of interest (at least, to an extent measurable with the sample size, technical approach and statistical framework employed)42. Just as in SNP association studies, it seems unlikely that an association of a CNP with disease that displays a P value of 0.05 will prove reproducible. On the other hand, a robustly significant P value (given the lower prior probability intrinsic in a genome-wide search), perhaps combined with functional data, surely can result in a compelling finding. Insistence upon the highest standards for proof in the early days of a field will save the community the consternation of irreproducible findings muddying the literature.

Opportunities
The coming years are likely to be tremendously exciting, as initial observations of common human common copy-number variation mature into an understanding that is crisp in molecular detail, complete in knowledge of location and frequency, and conducive to discoveries in the pathogenesis and genetic epidemiology of human disease. Perhaps the greatest impediments to such a future would be not the discovery of too few CNP-disease associations in the next year or two, but an insufficient investment in a truly effective set of tools and databases for their study, coupled with overly enthusiastic (but not quite reproducible) early claims of association with disease. With the proper focus and standards, CNP research will yield important insights, elucidating not only human genetic variation, but biological pathways and the mechanisms of human disease.

Competing interests statement: The authors declare no competing financial interests.

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