Role Of Copy Number Variations Research Articles

Tumour evolution inferred by single-cell sequencing

Genomic analysis provides insights into the role of copy number variation in disease, but most methods are not designed to resolve mixed populations of cells. In tumours, where genetic heterogeneity is common, very important information may be lost that would be useful for reconstructing evolutionary history. Here we show that with flow-sorted nuclei, whole genome amplification and next generation sequencing we can accurately quantify genomic copy number within an individual nucleus. We apply single-nucleus sequencing to investigate tumour population structure and evolution in two human breast cancer cases. Analysis of 100 single cells from a polygenomic tumour revealed three distinct clonal subpopulations that probably represent sequential clonal expansions. Additional analysis of 100 single cells from a monogenomic primary tumour and its liver metastasis indicated that a single clonal expansion formed the primary tumour and seeded the metastasis. In both primary tumours, we also identified an unexpectedly abundant subpopulation of genetically diverse 'pseudodiploid' cells that do not travel to the metastatic site. In contrast to gradual models of tumour progression, our data indicate that tumours grow by punctuated clonal expansions with few persistent intermediates.

Duplication of amyloid precursor protein (APP), but not prion protein (PRNP) gene is a significant cause of early onset dementia in a large UK series.

Amyloid precursor protein gene (APP) duplications have been identified in screens of selected probands with early onset familial Alzheimer's disease (FAD). A causal role for copy number variation (CNV) in the prion protein gene (PRNP) in prion dementias is not known. We aimed to determine the prevalence of copy number variation in APP and PRNP in a large referral series, test a screening method for detection of the same, and expand knowledge of clinical phenotype. We used a 3-tiered screening assay for APP and PRNP duplication (exonic real-time quantitative polymerase chain reaction [exon-qPCR], fluorescent microsatellite quantitative PCR [fm-q-PCR], and Illumina array [Illumina Inc., San Diego, CA, USA]) for analysis of a heterogeneous referral series comprising 1531 probands. Five of 1531 probands screened showed APP duplication, a similar prevalence to APP missense mutation. Real-time quantitative PCR and fluorescent microsatellite quantitative PCR were similar individually but are theoretically complementary; we used Illumina arrays as our reference assay. Two of 5 probands were from an autosomal dominant early onset Alzheimer's disease (familial Alzheimer's disease) pedigree. One extensive, noncontiguous duplication on chromosome 21 was consistent with an unbalanced translocation not including the Down's syndrome critical region. Seizures were prominent in the other typical APP duplications. A range of imaging, neuropsychological, cerebrospinal fluid, and pathological findings are reported that extend the known phenotype. APP but not PRNP duplication is a significant cause of early onset dementia in the UK. The recognized phenotype may be expanded to include the possibility of early seizures and apparently sporadic disease which, in part, may be due to different mutational mechanisms. The pros and cons of our screening method are discussed.

Genetic variation in the autophagy gene ULK1 and risk of Crohnʼs disease

The autophagy pathway has been linked with Crohn's disease (CD) through association of the ATG16L1 and IRGM genes with susceptibility for CD, and also to the Nod2 pathway, involved in CD. Our aim was to investigate polymorphisms in selected autophagy genes for their association with susceptibility to CD. We prioritized all known human homologs of yeast autophagy (Atg) genes according to their location in a known inflammatory bowel disease (IBD) locus or in a genomic region detected in a genome-wide association study (GWAS) or GWAS-meta-analysis. A total of 70 haplotype tagging single nucleotide polymorphisms (tSNPs) in 12 genes were genotyped in a cohort of CD patients (n = 947) and controls (n = 548). Transmission disequilibrium testing (TDT) was performed in an independent cohort of 335 parent-child CD-trios. The frequency of the T-allele of tSNP rs12303764 in ULK1 was significantly higher in CD (64%) versus controls (57%, corrected P-value 0.002). TDT demonstrated overtransmission of this allele to affected offspring (P = 0.02). Model-based multifactor dimensionality reduction (MB-MDR) interaction analysis confirmed a strong main effect for rs12303764. No interaction was found between ULK1 and CARD15, or between ULK1 genotypes and CD phenotypes. We report a genetic association with a tSNP in ULK1, an interesting candidate gene for IBD, given the role of ULK1 in autophagy initiation, and the interaction between Nod2 and autophagy pathways. To further clarify the role of ULK1 in CD, an in-depth investigation of the variation in the region and possible role for copy number variation in this region should be evaluated.

Investigation of the Role of Copy Number Variation In the Pathogenesis of Type 1 Von Willebrand Disease

AbstractAbstract 2218 Introduction:Type 1 von Willebrand disease (VWD) results from a partial quantitative deficiency of von Willebrand factor (VWF). Three multicentre studies were recently conducted to elucidate the molecular and clinical features of type 1 VWD; undertaken in the European Union (EU), Canada and the UK. All three successfully identified genetic alterations likely to cause type 1 VWD, but failed to identify a genetic cause in approximately 30–40% of patients. To date, relatively little is known regarding the extent to which large-scale deletions or duplications of VWF contribute to the pathogenesis of type 1 VWD due to the difficulty in identifying such events in heterozygous individuals. The aim of this initial investigation, on behalf of the TS Zimmerman Program for the Molecular and Clinical Biology of VWD (ZPMCB-VWD) and the EU study on VWD (EU-VWD), was to ascertain whether copy number variation (CNV) within VWF contributes to type 1 VWD phenotype in a cohort of index cases (IC) recruited by the EU study. Methods:Twenty-five mutation-negative IC and a further seven IC, in which the identified mutation did not explain the VWD phenotype, were investigated. Multiplex ligation-dependent probe amplification (MLPA), a well established technique based on sequence-specific probe hybridization to genomic DNA, was used to screen for CNV within VWF, utilizing a recently released VWF MLPA kit (P011-A1/P012-A1; MRC-Holland). In addition, five healthy control individuals (HC) were also screened. Fragment size analysis on an ABI 3730 DNA sequencer was followed by calculation of copy number ratios for each exon-specific amplicon in comparison with HC. Results:Analysis highlighted heterozygous CNV in 7/32 IC; five deletions and two apparent duplications. Regarding the deletions; three were the recently described in-frame deletion of exons 4 and 5 (p.Asp75_Gly178del; Sutherland et al., 2009), the other two patients both had a novel deletion of exons 32–34. Interestingly, the exon 32–34 deletion was also predicted to be in-frame (p.Arg1819_Cys1948delinsSer) and in both IC was associated with abnormal multimers (AbM) and low VWF:Ag (32 and 12 IU/dL respectively) and VWF:RCo (23 and 11 IU/dL respectively) levels. Deletions in all five cases segregated with disease phenotype. The two apparent duplications of exon 2 were both shown to be false positive results following further investigation with a new version of the MLPA kit (P011-B1/P012-B1). Conclusions:CNV of VWF has been shown to contribute to the pathogenesis of type 1 VWD. MLPA analysis of VWF has identified a previously described and a novel heterozygous deletion, both of which result in in-frame deletion of the VWF protein. The analysis also demonstrates that care must be taken when analyzing MLPA data to avoid false positive results. This adds to the mutations detected in the EU cohort, bringing the number to 113/150 IC (75%). Furthermore, MLPA analysis has increased the overall mutation detection rate in EU IC with AbM to 100%. These findings suggest that CNV is a significant contributor to mutation spectrum in type 1 VWD. Application of MLPA to the wider ZPMCB-VWD and EU-VWD patient cohorts will enable ascertainment of the contribution of large deletion/duplication events to all types of VWD pathogenesis. Disclosures:Vijzelaar: MRC-Holland b.v.: Employment.

Genome-Wide Detection of Copy Number Variation and Identification of Genes Associated with Risk of Multiple Myeloma

AbstractAbstract 4047 Background:Despite increasing efforts to characterize the role of copy number variants (CNVs) in MM, the genetic contribution to multiple myleoma (MM) has not been fully elucidated. Recent studies showed that chromosomal aberrations are detectable in MM and can be associated with susceptibility to MM. To gain insight into the incidence of the chromosomal aberrations in MM, we examined Korean MM genomes using high-resolution single-nucleotide polymorphism (SNP) array-based analysis. Patients and Methods:As part of a larger cohort study, 14 cases analyzed had been diagnosed with MM (9 male, 5 female). Median age at diagnosis was 58 years (range, 40≂f74). Of these patients, five patients had Ig Kappa type, five with IgG Lambda, two with light chain Kappa and two with light chain Lambda. 1,140,419 CNV markers were considered on these samples using Illumina HumanOmni1-Quad v1 BeadChip. Genome-wide CNV, genotyping of markers including 32119 non-synonymous SNPs, loss of heterozygosity (LOH) analyses were performed using the GenomeStudio v2010.1. Linkage disequilibrium was analyzed by Haploview 4.2. The gene set enrichment analysis was performed using GO software, Panther. Results:The average call rates were 99.9 %. The average number of CNVs per genome in this study (353.9) is much higher than that of CNVs called in the recent studies using lower-resolution SNP- or CNV arrays. The median size of CNVs was 1,902 (range 39 ≂f 2,263,901 bp). When we analyzed the number of CNVs per genome, there was no significant difference between MM patients of different subgroups. Interestingly copy number losses were 36.7 times more frequent than copy number gains. We defined CNV regions (CNVRs) by merging overlapping CNVs (30% of overlap threshold) detected in two or more genomes. In total 1271 CNVRs identified. When all CNVRs identified in the study were compared with the CNVRs in the DGV, 149 common (more than 2 incidences) CNVRs were novel, not found in DGV database. Like CNVs, CNVRs-losses were more frequent than CNVR-gains. Defined CNVRs encompassing 29.2Mb accounted for ≂f1% of the human genome. Total of 1029 NM numbered transcripts were located near or within the 1271 CNVRs. Through gene ontology (GO) analysis, putative target genes within the commonly gained or deleted region were categorized. Gene functions significantly enriched in the identified CNVRs include receptors for signal transduction pathways, transcription factors with nucleic acid binding proteins, defense/immunity molecules and regulatory molecule related functions involved in developmental processes. Hierarchical clustering of pooled datasets clearly distinguished IgG Kappa from Lambda subtypes. Genotype distributions for 32,110 non-synonymous SNPs in MM were also examined and compared to two lab-specific as well as 90 Korean HapMap samples as control reference. Conclusions:Power of High-resolution single-nucleotide polymorphism (SNP) array-based analysis allowed us a high incidence of gains and losses in MM patients. Many of those detectable legions were previously unidentified cryptic chromosomal aberrations. Although results reveals high degrees of heterogeneity in the genomic alterations detectable in MM, genes of the signal transduction pathway and defense/immunity processes were the most frequently altered targets whose deregulation may play a role in the pathogenesis of MM. CNVs/CNVRs identified in the study will be solid resources for investigating chromosomal aberrations in MM and its potential association with MM. Disclosures:No relevant conflicts of interest to declare.

Copy number variation in Parkinson's disease

A central theme of human genetic studies is to understand genomic variation and how this underlies the inherited basis of disease. Genomic variation can provide increased biological understanding of disease processes, which is necessary to develop future treatments. Recent technological advances have highlighted the role of copy number variants in normal and pathological phenotypic expression. These applications have been used in studies of Parkinson's disease, a common, late-onset, progressive neurodegenerative disorder. At present the main therapeutic approach is administration of symptom-alleviating drugs, which neither reverses the disease process nor halts its progression. However, the generation of in vivo model systems and development of novel disease intervention strategies for Parkinson's disease have come from research on monogenic forms of the disorder, including those caused by copy number variants. Here, we review the role of copy number variants and the mechanistic insights they have provided on the pathogenesis of Parkinson's disease.

The Human Genome Puzzle — the Role of Copy Number Variation in Somatic Mosaicism

The discovery of copy number variations (CNV) in the human genome opened new perspectives in the study of the genetic causes of inherited disorders and the etiology of common diseases. Differently patterned instances of somatic mosaicism in CNV regions have been shown to be present in monozygotic twins and throughout different tissues within an individual. A single-cell-level investigation of CNV in different human cell types led us to uncover mitotically derived genomic mosaicism, which is stable in different cell types of one individual. A unique study of immortalized B-lymphoblastoid cell lines obtained with 20 year interval from the same two subjects shows that mitotic changes in CNV regions may happen early during embryonic development and seem to occur only once, as levels of mosaicism remained stable. This finding has the potential to change our concept of dynamic human genome variation. We propose that further genomic studies should focus on the single-cell level, to understand better the etiology and physiology of aging and diseases mediated by somatic variations.

THIS MONTH IN Anesthesiology

The correlation between preoperative responses to experimental pain and clinical postoperative pain is investigated. See the accompanying Editorial View on page 1311 Adaptive support ventilation prevents diaphragmatic atrophy and maintains diaphragmatic contractile activity.Anesthesia and the patient with Restless Legs Syndrome are reviewed.Threshold volume and concentration of ropivacaine for interscalene block are investigated.Cellular effects of clinical usage of helium are reviewed.Although multiple perioperative guidelines acknowledge the prognostic value of symptomatic heart failure symptoms on postoperative outcome, the role of asymptomatic LV dysfunction remains unknown. This study used routine echocardiography in patients undergoing open or endovascular surgery to assess these potential prognostic values. Half of all patients had LV dysfunction, of which 80% were asymptomatic. In patients undergoing endovascular surgery (n = 356), only symptomatic heart failure was associated with 30-day cardiovascular events and long-term cardiovascular mortality. However, associations were seen with both asymptomatic systolic and isolated diastolic LV dysfunction in patients undergoing open vascular surgery (n = 649). This study demonstrated that asymptomatic LV dysfunction is predictive for 30-day and long-term cardiovascular outcome in open vascular surgery patients. Preoperative risk stratification should include heart failure symptoms and routine preoperative echocardiography should be considered for risk stratification in open vascular surgery patients. See the accompanying Editorial View on page 1303 Immune response and genetic predisposition play a role in the pathogenesis of sepsis. Specifically, human neutrophil peptides 1–3, endogenous cationic antimicrobial peptides, have been implicated in host defense against microbes. The genes encoding these human peptides (DEFA1/DEFA3 ) exhibit copy number variations. To evaluate the role of copy number variation on infection complication susceptibility, a case–control study was first performed in 179 patients with severe sepsis and 233 healthy blood donors, and then replicated in an independent cohort of 112 cases and 118 controls. Compared with controls, patients with sepsis were more likely to have high copy number (more than 8) of DEFA1/DEFA3 ; this held true after adjustment for age and gender. Results were replicated in a second age- and gender-matched case–control cohort. Furthermore, patients with large copy number had significantly lower plasma levels of human neutrophil peptides 1–3, tumor necrosis factor-α, interleukin-6, and interleukin-10 compared with patients with low copy number. A higher copy number of DEFA1/DEFA3 (more than 8 copies) was significantly associated with risk of severe sepsis. See the accompanying Editorial View on page 1307 Recently, irritant anesthetics were shown to activate peripheral nociceptors, which may contribute to their hyperalgesic actions and adverse effects in the airways. To test if this occurs via activation of the excitatory ion channel TRPA1, this study used models of isolated guinea pig bronchi to measure bronchoconstriction during exposure to anesthetics. TRPA1 was activated by exposure to isoflurane and desflurane, but not by the nonirritant volatile anesthetics sevoflurane and halothane. In contrast to nonirritants, isoflurane induced TRPA1-dependent constriction of isolated guinea pig bronchi in mice by a TRPA1-dependent mechanism. In addition to their beneficial effects, general anesthetics also produce excitatory effects such as airway irritation and bronchoconstriction. This study demonstrates that activation of TRPA1 may contribute to these adverse effects. See the accompanying Editorial View on page 1309

Genomic copy number variations in three Southeast Asian populations

Research on the role of copy number variations (CNVs) in the genetic risk of diseases in Asian populations has been hampered by a relative lack of reference CNV maps for Asian populations outside the East Asians. In this article, we report the population characteristics of CNVs in Chinese, Malay, and Asian Indian populations in Singapore. Using the Illumina Human 1M Beadchip array, we identify 1,174 CNV loci in these populations that corroborated with findings when the same samples were typed on the Affymetrix 6.0 platform. We identify 441 novel loci not previously reported in the Database of Genomic Variations (DGV). We observe a considerable number of loci that span all three populations and were previously unreported, as well as population-specific loci that are quite common in the respective populations. From this we observe the distribution of CNVs in the Asian Indian population to be considerably different from the Chinese and Malay populations. About half of the deletion loci and three-quarters of duplication loci overlap UCSC genes. Tens of loci show population differentiation and overlap with genes previously known to be associated with genetic risk of diseases. One of these loci is the CYP2A6 deletion, previously linked to reduced susceptibility to lung cancer.

Abstract 4720: Genome-wide copy number variation and breast cancer risk: Preliminary report

Abstract Background: DNA copy number variation (CNV) is a common phenomenon in human genome and some CNVs have been associated with susceptibility or resistance to disease. It has been suggested that gene copy number can be elevated in cancer cells. To evaluate potential role of CNV in breast cancer development, we conducted genome-wide association study of CNV in Seoul Breast Cancer Study. Methods: Cases were diagnosed with breast cancer and underwent curative surgery at two teaching hospitals located in Seoul, Korea, between 2001 and 2007 (n=2,386). Control subjects were selected from multi-center based cohort study in 2007 (n=2,386). The Affymetrix SNP assay 6.0 array, which includes 946,000 probes for the detection of copy number variation, was used. Nexus Copy Number version 4.1 was used for detecting CNVs (significance threshold=10−6; minimum number of probes per segment=4), and statistical analysis. After quality control step (i.e., robust variance sample QC), total 2,315 cases and 2,039 controls were compared for the difference of each CNV. In this study, we selected the CNVs that were different between cases and controls by at least 5% (P adjusted for multiple comparison <0.01). Adjacent CNVs were grouped into CNV regions ranging 10 kb to 300 kb. Results: Between case and controls, thirteen CNV regions including chr115q11.2, chr3q26.1, chr6q16.3, chr17q31.1 were significantly different for copy number. We note that those regions include genes such as UGT2B17 (UDP glucuronosyltransferase 2 family, polypeptide B18), OR4C11 (olfactory receptor, family 4, subfamily C, member 11), LRRC37A2 (leucine rich repeat containing 37, member A2), ARL17P1 (ADP-ribosylation factor-like 17 pseudogene 1), LOC644974, and the others. We are planning validation study for the regions by quantitative PCR to verify the association with breast cancer risk found in this study. Conclusion: Our results suggest that common CNVs may be associated with breast cancer in Korean women. However, further variation study is essential to support our observation and underlying biological mechanisms for the associations needs to be elucidated. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4720.

Gene copy number variation and common human disease

Variation in gene copy number is increasingly recognized as a common, heritable source of inter-individual differences in genomic sequence. The role of copy number variation is well established in the pathogenesis of rare genomic disorders. More recently, germline and somatic copy number variation have been shown to be important pathogenic factors in a range of common diseases, including infectious, autoimmune and neuropsychiatric diseases and cancer. In this review, we describe the range of methods available for measuring copy number variants (CNVs) in individuals and populations, including the limitations of presently available assays, and highlight some key examples of common diseases in which CNVs have been shown clearly to have a pathogenic role. Although there has been major progress in this field in the last 5 years, understanding the full contribution of CNVs to the genetic basis of common diseases will require further studies, with more accurate CNV assays and larger cohorts than have presently been completed.

Copy number variations and cancer susceptibility

DNA copy number variations (CNVs) comprise a recently discovered element of genetic variation that affects a greater cumulative fraction of the genome than single-nucleotide polymorphisms (SNPs). This review discusses current understanding of the characteristics of CNVs in the human genome and explores the emerging discoveries of both constitutional and somatic CNVs in an ever-expanding variety of human cancers. The advent of high-resolution SNP arrays has made it possible to identify CNVs. Characterization of widespread constitutional CNVs offers insight into their role in disease susceptibility, whereas somatic CNVs identify regions of the genome involved in disease phenotype. The role of CNVs in cancer has only emerged in the last 2 years, with constitutional CNVs originally being observed in the Li-Fraumeni cancer susceptibility syndrome, and more recently in neuroblastoma. It is not yet known how common or how functionally relevant CNVs will be to the process of carcinogenesis. Nonetheless, the inherent instability and structural variability that characterize cancer cell genomes make this form of genetic variation particularly intriguing to the study of cancer.

The role of copy number variation in schizophrenia

Recent developments in microarray technology have revealed the presence of many submicroscopic deletions and duplications in the human genome. Some of these have been found to increase the risk for neuropsychiatric disorders. Over the last 2 years, several large studies on schizophrenia have implicated large deletions and duplications that increase the risk of developing this disorder. It is now clear that rare deletions at 1q21.1, 15q13.3, 15q11.2 and 22q11.2, as well as duplications at 16p11.2 and 16p13.1, increase the risk of developing schizophrenia. They are found collectively in up to 3% of patients; therefore, they account for only a small proportion of the genetic causes of schizophrenia. In this paper I will review the evidence for these findings.

The Role of Copy Number Variation in Susceptibility to Amyotrophic Lateral Sclerosis: Genome-Wide Association Study and Comparison with Published Loci

BackgroundThe genetic contribution to sporadic amyotrophic lateral sclerosis (ALS) has not been fully elucidated. There are increasing efforts to characterise the role of copy number variants (CNVs) in human diseases; two previous studies concluded that CNVs may influence risk of sporadic ALS, with multiple rare CNVs more important than common CNVs. A little-explored issue surrounding genome-wide CNV association studies is that of post-calling filtering and merging of raw CNV calls. We undertook simulations to define filter thresholds and considered optimal ways of merging overlapping CNV calls for association testing, taking into consideration possibly overlapping or nested, but distinct, CNVs and boundary estimation uncertainty.Methodology and Principal FindingsIn this study we screened Illumina 300K SNP genotyping data from 730 ALS cases and 789 controls for copy number variation. Following quality control filters using thresholds defined by simulation, a total of 11321 CNV calls were made across 575 cases and 621 controls. Using region-based and gene-based association analyses, we identified several loci showing nominally significant association. However, the choice of criteria for combining calls for association testing has an impact on the ranking of the results by their significance. Several loci which were previously reported as being associated with ALS were identified here. However, of another 15 genes previously reported as exhibiting ALS-specific copy number variation, only four exhibited copy number variation in this study. Potentially interesting novel loci, including EEF1D, a translation elongation factor involved in the delivery of aminoacyl tRNAs to the ribosome (a process which has previously been implicated in genetic studies of spinal muscular atrophy) were identified but must be treated with caution due to concerns surrounding genomic location and platform suitability.Conclusions and SignificanceInterpretation of CNV association findings must take into account the effects of filtering and combining CNV calls when based on early genome-wide genotyping platforms and modest study sizes.

Inborn Genomic Variation as Predisposing Factors to Myeloid Malignancies.

Abstract 1590Poster Board I-616Single nucleotide polymorphism arrays (SNP-A) are increasingly accepted as a karyotyping tool, but with systematic application of this technology to study clonal somatic chromosomal lesion came a realization as to how widespread are copy number variations (CNV) and copy-number neutral runs of homozygosity (ROH) through the human genome. Recognition of recurrent CNVs and ROH is of importance for distinction of truly somatic lesions but clearly both ROH and CNVs themselves could constitute risk factors for development of MDS and AML prediction as sites frequently affected by these changes could constitute fragile sites for chromosomal breaks and recombination events. Alternatively these inherited genomic variants could affect expression of corresponding genes and alter regulatory elements. They can physically disrupt a gene, potentially creating new isoforms and can have a subtle effect on gene expression, either through variable penetrance of gene dosage effects or through interaction between the CNV/ROH and the genetic background. As all ROH is unlikely to be explained by autozygosity, meiotic errors or early embryonic mitotic events could be responsible. The role of CNVs in disease, in particular immune-related disorders, has become increasingly apparent, while acquired ROH is well-recognized as playing a role in carcinogenesis and malignant evolution. However, CNVs and inborn ROH as predisposing factors in myeloid malignancies has not been explored in depth. SNP-A (for example Affymetrix 6.0 arrays) offer an opportunity to systematically investigate not only somatic unbalanced translocations but also copy neutral loss of heterozygosity (CN-LOH), including ROH.To determine how CNVs and inborn ROH may affect predisposition to myeloid malignancies, we first studied an large cohort of control individuals (N=995) from internal and publicly-available sources. We identified 261 CNVs distributed across the entire genome; 15 were unique to our cohort and had not been previously reported. The remaining CNVs were verified against the Database of Genomic Variants (http://projects.tcag.ca/variation/) and their frequency was established. We also identified 153 non-clonal regions of ROH in the normal cohort (9.8%), distributed across all chromosomes and mapped frequently occurring ROH. No correlation was found between chromosome size and size of ROH on that chromosome. ROH could be divided into two groups: interstitial (N=147) and telomeric (N=6). Interstitial ROH ranged in size from 0.29-64.9Mb with a median of 7.2Mb; the rare telomeric germline ROH was 2.5-13.2Mb with a median of 5.8Mb. There were 30 recurrent regions of ROH; the most frequent (at 0.4%) were at 4q13.1-q13.3 and 5q15-q21.1. We concluded that interstitial ROH<24.5Mb and telomeric ROH<13.2Mb most likely reflected a germline event.Using this information as a reference we next investigated a large cohort of patients with MDS, MDS/MPN (N=395) and secondary AML (N=130). We identified several CNVs significantly overrepresented in each cohort. For MDS, gains at 3q26.1 (p<.0001), 4q13.2 (p<.0001), 12p11.1 (p<.0001), 15q11.2 (p=.015) 22q11.22 (p=.0008) and losses at 14q11.2 (p<.0001) and 1q21 (p<.0001) were significantly more frequent. In sAML, gain at 22q11.22 (p<.0001) and loss of 4q13.2, 7q34, 8p11.23 and 14q11.2 (all p<.0001) were more frequent than in normal controls. Regions of genomic structural instability, such as fragile sites or segmental duplications, are commonly associated with CNVs. The genomic architecture of each region was investigated with the UCSC Genome Browser (http://www.genome.ucsc.edu/cgi-bin/hgGateway). Overrepresented CNV were associated with regions harboring segmental duplications (1q21, 4q13.2, 12p11.1, 15q11.2, 22q11.22) or repetitive gene families (T cell receptor loci; 7p14.1, 7q34, 14q11.2). We also identified germline ROH in the patient cohorts. ROH at 11q14.1-q14.2 was more frequent in patients with MDS and sAML (6% MDS; 12% AML vs 0%, p=.02); these patients had high-risk disease and a normal karyotype or one chromosomal abnormality. In sAML, several regions were enriched in patients including 1q25.2-q25.3, 13q11q12.13 and 21q21.1 (all N=2; p=0.01).We conclude, genomic variations once thought to be pathologically silent is now recognized as potential predisposing pathogenic factor in disease; we have demonstrate that specific CNVs and ROH can be associated with myeloid malignancies. It is possible that the presence of CNV or LOH predisposes to somatic chromosomal aberrations and points towards fragile sites. DisclosuresMaciejewski:Taligen: Membership on an entity's Board of Directors or advisory committees; Genzyme: Research Funding; Celgene: Speakers Bureau; Eisai: Membership on an entity's Board of Directors or advisory committees.

Genetics of human neural tube defects

Neural tube defects (NTDs) are common, severe congenital malformations whose causation involves multiple genes and environmental factors. Although more than 200 genes are known to cause NTDs in mice, there has been rather limited progress in delineating the molecular basis underlying most human NTDs. Numerous genetic studies have been carried out to investigate candidate genes in cohorts of patients, with particular reference to those that participate in folate one-carbon metabolism. Although the homocysteine remethylation gene MTHFR has emerged as a risk factor in some human populations, few other consistent findings have resulted from this approach. Similarly, attention focused on the human homologues of mouse NTD genes has contributed only limited positive findings to date, although an emerging association between genes of the non-canonical Wnt (planar cell polarity) pathway and NTDs provides candidates for future studies. Priorities for the next phase of this research include: (i) larger studies that are sufficiently powered to detect significant associations with relatively minor risk factors; (ii) analysis of multiple candidate genes in groups of well-genotyped individuals to detect possible gene–gene interactions; (iii) use of high throughput genomic technology to evaluate the role of copy number variants and to detect ‘private’ and regulatory mutations, neither of which have been studied to date; (iv) detailed analysis of patient samples stratified by phenotype to enable, for example, hypothesis-driven testing of candidates genes in groups of NTDs with specific defects of folate metabolism, or in groups of fetuses with well-defined phenotypes such as craniorachischisis.

Genome-wide association studies in type 2 diabetes

Despite numerous candidate gene and linkage studies, the field of type 2 diabetes (T2D) genetics had until recently succeeded in identifying few genuine disease-susceptibility loci. The advent of genome-wide association (GWA) scans has transformed the situation, leading to an expansion in the number of established, robustly replicating T2D loci to almost 20. These novel findings offer unique insights into the pathogenesis of T2D and in the main point toward the etiologic importance of disorders of beta-cell development and function. All associated variants have common allele frequencies in the discovery populations, and exert modest to small effects on the risk of disease, characteristics that limit their prognostic and diagnostic potential. However, ongoing studies focusing on the role of copy number variation and targeting low-frequency polymorphisms should identify additional T2D susceptibility loci, some of which may have larger effect sizes and offer better individual prediction of disease risk.

Copy number variations and cancer

DNA copy number variations (CNVs) are an important component of genetic variation, affecting a greater fraction of the genome than single nucleotide polymorphisms (SNPs). The advent of high-resolution SNP arrays has made it possible to identify CNVs. Characterization of widespread constitutional (germline) CNVs has provided insight into their role in susceptibility to a wide spectrum of diseases, and somatic CNVs can be used to identify regions of the genome involved in disease phenotypes. The role of CNVs as risk factors for cancer is currently underappreciated. However, the genomic instability and structural dynamism that characterize cancer cells would seem to make this form of genetic variation particularly intriguing to study in cancer. Here, we provide a detailed overview of the current understanding of the CNVs that arise in the human genome and explore the emerging literature that reveals associations of both constitutional and somatic CNVs with a wide variety of human cancers.

Analysis of copy number variation (CNV) of the SPANXA-E gene family in infertile and fertile men

OBJECTIVE: The purpose of this study was to evaluate the role of copy number variation (CNVs) in male infertility by genotyping a candidate gene family, SPANXA-E, on the X chromosome in fertile and infertile men.DESIGN: An assay was developed to measure copy number variation (CNV) of SPANXB and its surrounding gene cluster using quantitative real time PCR (qPCR). This assay was used to compare CNV values of 100 infertile men (50 oligozoospermic and 50 azoospermic) and 67 normospermic men.MATERIALS AND METHODS: Assay validation was achieved via comparison to oligonucleotide-based CNV data from a set of eight individuals. The qPCR correlation coefficient for copy number of the SPANXB and SPANXA-E assays were 0.96 and 0.89, respectively, suggesting high assay validity. Copy number (CN) was estimated from qPCR results by comparing values for test individuals to a normal (single copy) female reference.RESULTS: SPANXB. CN was calculated for 96 infertile males and 51 fertile men. The average value for the infertile group was .52 (.03-1.96), and 47 (.01-1.7) for the fertile group. These values are consistent with the expected value of .5 indicating one X in a male in comparison to a female with two X chromosomes. The azoospermic and oligozoospermic group reported an average mean of .51, and .44 respectively. SPANXA-E Gene Cluster. CN was calculated for 96 infertile males and 44 fertile men. The average value for the infertile group was .49 (.05-2.6), and .44 (.35-1.34) for the fertile group. These values are consistent with the expected value of .5 indicating one X in a male in comparison to a female with two X chromosomes. The azoospermic group reported an average mean of .5, and the oligozoospermic group reported an average mean of .46.CONCLUSIONS: Initial evidence suggests no difference in CNV of SPANXB and SPANXA-E in infertile men compared to fertile men. The average means of groups stratified by diagnosis showed no difference when compared to the control group. The observed variation of the SPANXA-E gene cluster in these men suggests no correlation of CN with fertility status. Although SPANXA-E CN does not appear to be associated with male infertility, this area of genetic analysis remains a focus for complex diseases, such as male infertility, and further genome-wide CN analysis is warranted. OBJECTIVE: The purpose of this study was to evaluate the role of copy number variation (CNVs) in male infertility by genotyping a candidate gene family, SPANXA-E, on the X chromosome in fertile and infertile men. DESIGN: An assay was developed to measure copy number variation (CNV) of SPANXB and its surrounding gene cluster using quantitative real time PCR (qPCR). This assay was used to compare CNV values of 100 infertile men (50 oligozoospermic and 50 azoospermic) and 67 normospermic men. MATERIALS AND METHODS: Assay validation was achieved via comparison to oligonucleotide-based CNV data from a set of eight individuals. The qPCR correlation coefficient for copy number of the SPANXB and SPANXA-E assays were 0.96 and 0.89, respectively, suggesting high assay validity. Copy number (CN) was estimated from qPCR results by comparing values for test individuals to a normal (single copy) female reference. RESULTS: SPANXB. CN was calculated for 96 infertile males and 51 fertile men. The average value for the infertile group was .52 (.03-1.96), and 47 (.01-1.7) for the fertile group. These values are consistent with the expected value of .5 indicating one X in a male in comparison to a female with two X chromosomes. The azoospermic and oligozoospermic group reported an average mean of .51, and .44 respectively. SPANXA-E Gene Cluster. CN was calculated for 96 infertile males and 44 fertile men. The average value for the infertile group was .49 (.05-2.6), and .44 (.35-1.34) for the fertile group. These values are consistent with the expected value of .5 indicating one X in a male in comparison to a female with two X chromosomes. The azoospermic group reported an average mean of .5, and the oligozoospermic group reported an average mean of .46. CONCLUSIONS: Initial evidence suggests no difference in CNV of SPANXB and SPANXA-E in infertile men compared to fertile men. The average means of groups stratified by diagnosis showed no difference when compared to the control group. The observed variation of the SPANXA-E gene cluster in these men suggests no correlation of CN with fertility status. Although SPANXA-E CN does not appear to be associated with male infertility, this area of genetic analysis remains a focus for complex diseases, such as male infertility, and further genome-wide CN analysis is warranted.

Adaptive drool in the gene pool

A new study finds that copy number variation in the salivary amylase gene in humans is associated with amylase concentration in saliva and average starch consumption in populations. This provides a striking example of the role of copy number variants (CNVs) in adaptive evolution, and of diet in producing selective pressures.