Prevalence Of Copy Number Variants Research Articles

Single-cell and single-nucleus RNA-sequencing from paired normal-adenocarcinoma lung samples provide both common and discordant biological insights.

Whether single-cell RNA-sequencing (scRNA-seq) captures the same biological information as single-nucleus RNA-sequencing (snRNA-seq) remains uncertain and likely to be context-dependent. Herein, a head-to-head comparison was performed in matched normal-adenocarcinoma human lung samples to assess biological insights derived from scRNA-seq versus snRNA-seq and better understand the cellular transition that occurs from normal to tumoral tissue. Here, the transcriptome of 160,621 cells/nuclei was obtained. In non-tumor lung, cell type proportions varied widely between scRNA-seq and snRNA-seq with a predominance of immune cells in the former (81.5%) and epithelial cells (69.9%) in the later. Similar results were observed in adenocarcinomas, in addition to an overall increase in cell type heterogeneity and a greater prevalence of copy number variants in cells of epithelial origin, which suggests malignant assignment. The cell type transition that occurs from normal lung tissue to adenocarcinoma was not always concordant whether cells or nuclei were examined. As expected, large differential expression of the whole-cell and nuclear transcriptome was observed, but cell-type specific changes of paired normal and tumor lung samples revealed a set of common genes in the cells and nuclei involved in cancer-related pathways. In addition, we showed that the ligand-receptor interactome landscape of lung adenocarcinoma was largely different whether cells or nuclei were evaluated. Immune cell depletion in fresh specimens partly mitigated the difference in cell type composition observed between cells and nuclei. However, the extra manipulations affected cell viability and amplified the transcriptional signatures associated with stress responses. In conclusion, research applications focussing on mapping the immune landscape of lung adenocarcinoma benefit from scRNA-seq in fresh samples, whereas snRNA-seq of frozen samples provide a low-cost alternative to profile more epithelial and cancer cells, and yield cell type proportions that more closely match tissue content.

Copy Number Variant Analysis of Spinocerebellar Ataxia Genes in a Cohort of Dutch Patients With Cerebellar Ataxia.

The spinocerebellar ataxias (SCAs) are a genetically heterogeneous group of neurodegenerative disorders generally caused by single nucleotide variants (SNVs) or indels in coding regions or by repeat expansions in coding and noncoding regions of SCA genes. Copy number variants (CNVs) have now also been reported for 3 genes-ITPR1, FGF14, and SPTBN2-but not all SCA genes have been screened for CNVs as the underlying cause of the disease in patients. In this study, we aim to assess the prevalence of CNVs encompassing 36 known SCA genes. A cohort of patients with cerebellar ataxia who were referred to the University Medical Center Groningen for SCA genetic diagnostics was selected for this study. Genome-wide single nucleotide polymorphism (SNP) genotyping was performed using the Infinium Global Screening Array. Following data processing, genotyping data were uploaded into NxClinical software to perform CNV analysis per patient and to visualize identified CNVs in 36 genes with allocated SCA symbols. The clinical relevance of detected CNVs was determined using evidence from studies based on PubMed literature searches for similar CNVs and phenotypic features. Of the 338 patients with cerebellar ataxia, we identified putative clinically relevant CNV deletions in 3 patients: an identical deletion encompassing ITPR1 in 2 patients, who turned out to be related, and a deletion involving PPP2R2B in another patient. Although the CNV deletion in ITPR1 was clearly the underlying cause of SCA15 in the 2 related patients, the clinical significance of the deletion in PPP2R2B remained unknown. We showed that CNVs detectable with the limited resolution of SNP array are a very rare cause of SCA. Nevertheless, we suggest adding CNV analysis alongside SNV analysis to SCA gene diagnostics using next-generation sequencing approaches, at least for ITPR1, to improve the genetic diagnostics for patients.

Increased Prevalence of Rare Copy Number Variants in Treatment-Resistant Psychosis.

It remains unknown why ~30% of patients with psychotic disorders fail to respond to treatment. Previous genomic investigations of treatment-resistant psychosis have been inconclusive, but some evidence suggests a possible link between rare disease-associated copy number variants (CNVs) and worse clinical outcomes in schizophrenia. Here, we identified schizophrenia-associated CNVs in patients with treatment-resistant psychotic symptoms and then compared the prevalence of these CNVs to previously published schizophrenia cases not selected for treatment resistance. CNVs were identified using chromosomal microarray (CMA) and whole exome sequencing (WES) in 509 patients with treatment-resistant psychosis (a lack of clinical response to ≥3 adequate antipsychotic medication trials over at least 5 years of psychiatric hospitalization). Prevalence of schizophrenia-associated CNVs in this sample was compared to that in a previously published large schizophrenia cohort study. Integrating CMA and WES data, we identified 47 cases (9.2%) with at least one CNV of known or possible neuropsychiatric risk. 4.7% (n = 24) carried a known neurodevelopmental risk CNV. The prevalence of well-replicated schizophrenia-associated CNVs was 4.1%, with duplications of the 16p11.2 and 15q11.2-q13.1 regions, and deletions of the 22q11.2 chromosomal region as the most frequent CNVs. Pairwise loci-based analysis identified duplications of 15q11.2-q13.1 to be independently associated with treatment resistance. These findings suggest that CNVs may uniquely impact clinical phenotypes beyond increasing risk for schizophrenia and may potentially serve as biological entry points for studying treatment resistance. Further investigation will be necessary to elucidate the spectrum of phenotypic characteristics observed in adult psychiatric patients with disease-associated CNVs.

22.1 Gene Copy Number Variation in Pediatric Mental Health

Rare copy number variants (CNVs) likely play an important role in common childhood neuropsychiatric disorders such as ADHD and OCD. These disorders represent the extremes of broadly distributed behavioral traits that are influenced by underlying variation in fundamental cognitive processes such as response inhibition. Trait-based approaches in a population sample can help reduce heterogeneity and improve the power to detect rare CNVs associated with these disorders. Currently, we do not know the prevalence of CNVs related to neuropsychiatric disorders in youth in the general population.

Analysis of copy number variation in men with non-obstructive azoospermia.

Recent findings demonstrate that single nucleotide variants can cause non-obstructive azoospermia (NOA). In contrast, copy number variants (CNVs) were only analysed in few studies in infertile men. Some have reported a higher prevalence of CNVs in infertile versus fertile men. This study aimed to elucidate if CNVs are associated with NOA. We performed array-based comparative genomic hybridisation (aCGH) in 37 men with meiotic arrest, 194 men with Sertoli cell-only phenotype, and 21 control men. We filtered our data for deletions affecting genes and prioritised the affected genes according to the literature search. Prevalence of CNVs was compared between all groups. Exome data of 2,030 men were screened to detect further genetic variants in prioritised genes. Modelling was performed for the protein encoded by the novel candidate gene TEKT5 and we stained for TEKT5 in human testicular tissue. We determined the cause of infertility in two individuals with homozygous deletions of SYCE1 and in one individual with a heterozygous deletion of SYCE1 combined with a likely pathogenic missense variant on the second allele. We detected heterozygous deletions affecting MLH3, EIF2B2, SLX4, CLPP and TEKT5, in one subject each. CNVs were not detected more frequently in infertile men compared with controls. While SYCE1 and MLH3 encode known meiosis-specific proteins, much less is known about the proteins encoded by the other identified candidate genes, warranting further analyses. We were able to identify the cause of infertility in one out of the 231 infertile men by aCGH and in two men by using exome sequencing data. As aCGH and exome sequencing are both expensive methods, combining both in a clinical routine is not an effective strategy. Instead, using CNV calling from exome data has recently become more precise, potentially making aCGH dispensable.

Identification of novel rare copy number variants associated with sporadic tetralogy of Fallot and clinical implications

Tetralogy of Fallot (TOF) is the most common cyanotic congenital heart disease. Highly penetrant copy number variants (CNVs) and genes related to the etiology of TOF likely exist with differences among populations. We aimed to identify CNV contributions to sporadic TOF cases in Han Chinese. Genomic DNA was extracted from peripheral blood in 605 subjects (303 sporadic TOF and 302 unaffected Han Chinese [Control] from cardiac centers in China) and analyzed by genome-wide association study (GWAS). The GWAS results were compared with existing Database of Genetic Variants. These CNVs were further validated by qPCR. Bioinformatics analyses were performed with protein-protein interaction (PPI) network and KEGG pathway enrichment. Across all chromosomes 119 novel "TOF-specific CNVs" were identified with prevalence of CNVs of 21.5% in chromosomes 1-20 and 37.0% including Chr21/22. In chromosomes 1-20, CNVs on 11q25 (encompasses genes ACAD8, B3GAT1, GLB1L2, GLB1L3, IGSF9B, JAM3, LOC100128239, LOC283177, MIR4697, MIR4697HG, NCAPD3, OPCML, SPATA19, THYN1, and VPS26B) and 14q32.33 (encompasses genes THYN1, OPCML, and NCAPD3) encompass genes most likely to be associated with TOF. Specific CNVs found on the chromosome 21 (6.3%) and 22(11.9%) were also identified in details. PPI network analysis identified the genes covering the specific CNVs related to TOF and the signaling pathways. This study for first time identified novel TOF-specific CNVs in the Han Chinese with higher frequency than in Caucasians and with 11q25 and 14q32.33 not reported in TOF of Caucasians. These novel CNVs identify new candidate genes for TOF and provide new insights into genetic basis of TOF.

Prevalence of copy number variants (CNVs) and rhGH treatment efficacy in an Italian cohort of children born small for gestational age (SGA) with persistent short stature associated with a complex clinical phenotype.

Multiple factors influence intrauterine growth and lead to low birth sizes. The impact of genetic alterations on both pre- and post-natal growth is still largely unknown. The aim of this study was to investigate the prevalence of CNVs in an Italian cohort of SGA children with persistent short stature and complex clinical phenotype. rhGH treatment efficacy was evaluated according to the different genotypes. Twenty-four SGA children (10F/14M) with persistent short stature associated with dysmorphic features and/or developmental delay underwent CNV evaluation. CNVs were present in 14/24 (58%) SGA children. Six patients had a microdeletion involving the following regions: 3q24q25.1, 8p21.2p12, 15q26, 19q13.11, 20q11.21q12, 22q11.2. In three females, the same microdeletion involving 17p13.3 region was identified. In two different patients, two microduplications involving 10q21.3 and Xp11.3 region were observed. A further female patient showed both an 11q12.1 and an Xq27.1 microduplication, inherited from her mother and from her father, respectively. In a boy, the presence of a 12p13.33 microdeletion and a 19q13.43 microduplication was found. GH treatment efficacy, expressed by height gain and height velocity in the first 12months of therapy, was similar in subjects with and without CNVs. These results show that pathogenic CNVs are common in SGA children with short stature associated with additional clinical features. Interestingly, the involvement of 17p13.3 region occurs with a relative high frequency, suggesting that genes located in this region could play a key role in pre- and post-natal growth. rhGH therapy has similar efficacy in the short term whether CNVs are present or not.

Contribution of Copy Number Variation in Idiopathic Hypogonadotropic Hypogonadism

Introduction: While the role of single nucleotide variants (SNVs) in causal genes for Idiopathic Hypogonadotropic hypogonadism (IHH) is known, the contribution of copy number variants (CNVs) to IHH has not been systematically studied. Here, we examined the prevalence of CNVs in a large IHH cohort and their associated phenotypic spectrum. Methods: Exome sequencing (ES) from 1,441 IHH probands was analyzed using the GATK-gCNV pipeline to identify CNVs that spanned known IHH genes with a site frequency of <1%. ES data was also analyzed for rare SNVs in all IHH genes. IHH subjects were evaluated for reproductive and non-reproductive phenotypes (kidney, eye, cardiovascular, midline/ facial, bone and neurological abnormalities). Results: (i) CNV prevalence in IHH: Three percent of the IHH probands (46/1441 probands, 38 males and 8 females) harbored CNVs in 19/36 high confidence IHH genes (26 deletions 20 duplications). The vast majority of CNVs disrupted either the ANOS1 (26%) or FGFR1 (17%) genes. Intriguingly, CHD7 (a gene that carries SNVs in ~12% of our IHH subjects) did not harbor any CNVs. (ii) Phenotypic analysis: We found that CNVs were more common in IHH subjects with anosmia (Kallmann syndrome, N=33) compared to normosmic IHH (N=13). More than half of the subjects with CNVs affecting IHH genes carried at least 1 non-reproductive feature (26/46 IHH subjects, 56%). A syndromic presentation with multiple non-reproductive phenotypes was more common in IHH subjects harboring multigenic CNVs (IHH gene & additional genes) compared to IHH subjects with single IHH gene CNV or SNV in a single IHH gene (61% vs. 12% or 18%, respectively; p 0.0006). Conclusions: CNVs in known IHH genes contribute to 3% of the IHH genetic architecture in our cohort. IHH subjects with larger multigenic CNVs displayed phenotypes consistent with contiguous gene syndromes. The absence of genic CNVs in genes frequently found to carry SNVs, such as CHD7, requires additional analysis to establish the biologic or mechanistic reasons for their rarity.

Chromosomal microarray analysis in fetuses with central nervous system anomalies: An 8-year long observational study from a tertiary care university hospital.

To evaluate the prevalence of DNA copy number variants (CNVs) detected with array comparative genomic hybridization (CGH) in fetuses with central nervous system (CNS) anomalies. Secondary objectives were to describe the prevalence of CNV in specific CNS abnormalities, in isolated defects or associated with other malformations or fetal growth restriction (FGR). Observational cohort study in 238 fetuses with CNS anomalies in which an array-CGH had been performed between January 2009 and December 2017. Pathogenic CNV and variants of unknown significance (VUS) were reported. Pathogenic CNVs were found in 16/238 cases (6.7%), VUS in 18/238 (7.6%), and normal result in 204/238 (85.7%) cases. Pathogenic CNVs were more frequent in posterior fossa anomalies (cerebellar hypoplasia 33%, megacisterna magna 20%), moderate ventriculomegaly (11%) and spina bifida (3.7%). Pathogenic CNVs and VUS were found in 7/182 (3.8%) and 14/182 (7.7%) cases of isolated anomalies, in 9/49 (18.4%) and 4/49 (8.2%) presenting another malformation, and in 0/7 and 0/7 cases with associated FGR (P = .001, P = .741, respectively). These results provide strong evidence toward performing array in fetuses with CNS anomalies, particular in cases of posterior fossa anomalies. The prevalence of pathogenic CNVs is higher in association with other malformations.

Detection of copy number variants with chromosomal microarray in 10377 pregnancies at a single laboratory.

IntroductionInvasive prenatal testing with chromosomal microarray analysis may be a relevant option for all pregnant women, but there is only moderate‐quality evidence for such an offer. We intended to study the prevalence of copy number variants (CNVs) in prenatal samples using a single SNP‐array platform stratified by indication.Material and methodsA cross‐sectional study was performed based on a cohort. From January 2015 to December 2017, a total of 10 377 prenatal samples were received for prenatal single nucleotide polymorphism (SNP)‐array in the laboratory of the Genetics Generation Advancement Corporation. Indications for chromosomal microarray analysis studies included the confirmation of an abnormal karyotype, ultrasound abnormalities, advanced maternal age and parental anxiety. CNVs and region of homozygosity identified by the SNP‐array were analyzed.ResultsOf 10 377 cases, 689 had ultrasound abnormalities and 9688 were ascertained to have other indications. The overall prevalence of CNVs was 2.1% (n = 223/10 377, 95% confidence interval [CI] 1.9‐2.4), but the prevalence was 4.4% (95% CI 3.0‐6.1) for cases referred with abnormal ultrasound findings and 2.0% (95% CI 1.7‐2.3) for other indications. Of the 223 CNVs detected, 42/10 377 were pathogenic (0.4%, 95% CI 0.3‐0.6), 84 were susceptibility CNV (0.8%, 95% CI 0.6‐1.0) and 97 were variants of uncertain significance (0.9%, 95% CI 0.8‐1.1). Using an SNP‐based platform allowed for the detection of paternal uniparental disomy of chromosome 14 in a fetus with ultrasound abnormality.ConclusionsWith an indication of advanced maternal age but normal ultrasound scans, the prevalence of pathogenic CNVs was 0.4% and that of susceptibility CNV 0.7%. As CNVs are independent of maternal age, the prevalence is likely the same for younger women. Thus, this study provides further evidence that chromosomal microarray analysis should be available for all women who wish to receive diagnostic testing, as this risk is above the cut‐off of 1:300 for Down syndrome, leading to the suggestion of invasive testing. A chromosomal microarray analysis based on SNP‐array platform is preferable, as it can also detect uniparental disomy in addition to copy number variants.

Evaluation of the Genetic Association Between Adult Obesity and Neuropsychiatric Disease.

Extreme obesity (EO) (BMI >50 kg/m2) is frequently associated with neuropsychiatric disease (NPD). As both EO and NPD are heritable central nervous system disorders, we assessed the prevalence of protein-truncating variants (PTVs) and copy number variants (CNVs) in genes/regions previously implicated in NPD in adults with EO (n = 149) referred for weight loss/bariatric surgery. We also assessed the prevalence of CNVs in patients referred to University College London Hospital (UCLH) with EO (n = 218) and obesity (O) (BMI 35-50 kg/m2; n = 374) and a Swedish cohort of participants from the community with predominantly O (n = 161). The prevalence of variants was compared with control subjects in the Exome Aggregation Consortium/Genome Aggregation Database. In the discovery cohort (high NPD prevalence: 77%), the cumulative PTV/CNV allele frequency (AF) was 7.7% vs. 2.6% in control subjects (odds ratio [OR] 3.1 [95% CI 2-4.1]; P < 0.0001). In the UCLH EO cohort (intermediate NPD prevalence: 47%), CNV AF (1.8% vs. 0.9% in control subjects; OR 1.95 [95% CI 0.96-3.93]; P = 0.06) was lower than the discovery cohort. CNV AF was not increased in the UCLH O cohort (0.8%). No CNVs were identified in the Swedish cohort with no NPD. These findings suggest that PTV/CNVs, in genes/regions previously associated with NPD, may contribute to NPD in patients with EO.

The Genomic Landscape of Sporadic Prolactinomas.

Somatic GNAS and USP8 mutations have been implicated in sporadic somatotrophinomas and corticotrophinomas, respectively. However, no genes are known to be recurrently mutated in sporadic prolactinomas. The prevalence of copy number variants (CNV), which is emerging as a mechanism of tumorigenesis in sporadic pituitary adenomas in general, is also unclear in prolactinomas. To characterize the genetic events underpinning sporadic prolactinomas, we performed whole exome sequencing of paired tumor and germline DNA from 12 prolactinoma patients. We observed recurrent large-scale CNV, most commonly in the form of copy number gains. We also identified sequence variants of interest in 15 genes. This included the DRD2, PRL, TMEM67, and MLH3 genes with plausible links to prolactinoma formation. Of the 15 genes of interest, CNV was seen at the gene locus in the corresponding tumor in 10 cases, and pituitary expression of eight genes was in the top 10% of tissues. However, none of our shortlisted somatic variants appeared to be classical driver mutations as no variant was found in more than one tumor. Future directions of research include mechanistic studies to investigate how CNV may contribute to prolactinoma formation, larger studies of relevant prolactinoma subsets according to clinical characteristics, and additional genetic investigations for aberrations not captured by whole exome sequencing.

SA6 - COPY NUMBER VARIANTS IN BRAIN-RELATED GENES ARE ASSOCIATED WITH NEUROPSYCHIATRIC TRAITS IN CHILDHOOD

Background Rare Copy Number Variants (CNVs) likely play an important role in common childhood neuropsychiatric disorders such as ADHD and OCD. These disorders represent the extremes of broadly distributed behavioural traits that are influenced by underlying variation in fundamental cognitive processes such as response inhibition. Using behavioural and cognitive trait-based approaches in population samples can help reduce heterogeneity and improve power to detect rare CNVs associated with these disorders. Currently we do not know the prevalence of CNVs related to neuropsychiatric disorders in youth in the general population. Using the Spit for Science sample (Toronto, Canada), we examined the association of rare CNVs with ADHD, OCD and response inhibition traits. Methods We collected DNA and quantitative trait measures from 16,718 youth (ages 6–17 years) at a local science museum. We measured ADHD traits using the Strengths and Weaknesses of ADHD and Normal Behavior (SWAN) scale, obsessive-compulsive traits using the Toronto Obsessive-Compulsive scale (TOCS) and response inhibition, a putative endophenotype for ADHD, using the Stop-Task. We genotyped unrelated Caucasians (n=5,320) using Illumina HumanCoreExome beadchips. CNVs were called using three algorithms: iPattern (ipn), PennCNV (pcnv), and QuantiSNP (qsnp) and a high confidence dataset was generated by retaining variants detected by two or more algorithms and at least 10 kb in size. We defined rare variants as those with less than 0.1% frequency against our population controls. Using linear regression, we examined whether CNV burden, overall and within psychiatrically relevant gene sets (e.g., brain expression, synaptic function), is associated with response inhibition, ADHD and OCD traits. Results After stringent quality control, 4815 (90.5%) of participants remained with a total of 9,490 rare CNVs (1.97/individual on average). We observed CNVs affecting 23 genomic loci previously implicated in neurodevelopmental conditions (e.g. 22q11.2) in 59 (1.2%) of participants. Other large CNVs affecting known psychiatric genes (e.g., ASTN2, NRXN1) were also identified. Significantly more deletions (>500 kb; p=0.04) and enrichment of deletions in genes involved in synapse structure, neuron projection and neurophenotypes in mice (p Discussion In a large pediatric general population sample, brain-related CNVs were associated with neuropsychiatric traits in children although the pattern of enrichment differed between ADHD, OCD and response inhibition. Our results replicate several previous CNV burden findings for ADHD and OCD suggesting a similar genetic architecture between traits and disorders. Our approach shows the feasibility of CNV analysis and quality of CNV data from the HumanCoreExome array in a large population based sample.

Genetic analysis of Mayer-Rokitansky-Kuster-Hauser syndrome in a large cohort of families

To study the genetic cause of Mayer-Rokitansky-Kuster-Hauser syndrome (MRKH). Although a few candidate genes and genomic domains for have been reported for MRKH, the genetic underpinnings remain largely unknown. Some of the top candidate genes are WNT4, HNF1B, and LHX1. The goals of this study were to: 1) determine the prevalence of WNT4, HNF1B, and LHX1 point mutations, as well as new copy number variants (CNVs) in people with MRKH; and 2) identify and characterize MRKH cohorts. Laboratory- and community-based study. Academic medical centers. A total of 147 MRKH probands and available family members. DNA sequencing of WNT4, HNF1B, and LHX1 in 100 MRKH patients, chromosomal microarray analysis in 31 North American MRKH patients, and characterization and sample collection of 147 North American and Turkish MRKH probands and their families. DNA sequence variants and CNVs; pedigree structural analysis. We report finding CNVs in 6/31 people (∼19%) with MRKH, but no point mutations or small indels in WNT4, HNF1B, or LHX1 in 100 MRKH patients. Our MRKH families included 43 quads, 26 trios, and 30 duos. Of our MRKH probands, 87/147 (59%) had MRKH type 1 and 60/147 (41%) had type 2 with additional anomalies. Although the prevalence of WNT4, HNF1B, and LHX1 point mutations is low in people with MRKH, the prevalence of CNVs was ∼19%. Further analysis of our large familial cohort of patients will facilitate gene discovery to better understand the complex etiology of MRKH.

A novel role for genomic copy number variants in female infertility

ObjectiveCopy number variants (CNVs) are genomic aberrations that alter the number of copies of genomic segments and therefore have the potential to alter dosage-sensitive genes and/or functional elements. Recent studies have shown that CNVs underlie various complex spectrum disorders and that the severity of the disorder is correlated with overall CNV burden. To investigate the role of CNVs in female infertility spectrum disorders, we have performed a survey examining CNVs in a cohort of patients with unexplained infertility.DesignRetrospective cohort of female patients with unexplained infertility.Materials and MethodsGenomic DNA extracted from blood samples was obtained from patients undergoing non-donor in-vitro fertilization (IVF) cycles at a large academic medical center. CNVs were detected using a custom NimbleGen Comparative Genomic Hybridization (CGH) Whole-Genome Tiling Array optimized for fertility-related loci. The resulting log2 ratios of the raw intensities were then normalized and segmented. The prevalence of CNVs among the infertile cohort was compared to the Children's Hospital of Philadelphia (CHOP) CNV database of healthy individuals.ResultsApproximately 30% of the >3000 CNVs detected were unique to the infertile patients (P<10-7, Binomial Test of Proportions). Moreover, CNVs specific to the infertile cohort appear to be overrepresented among regions of the genome predicted to play a role in mammalian fertility (P<10-16, t-test).ConclusionThe set of CNVs uniquely observed in our female unexplained infertility cohort represent putative genetic biomarkers that may predict a range of infertility conditions difficult to diagnose with existing clinical data. ObjectiveCopy number variants (CNVs) are genomic aberrations that alter the number of copies of genomic segments and therefore have the potential to alter dosage-sensitive genes and/or functional elements. Recent studies have shown that CNVs underlie various complex spectrum disorders and that the severity of the disorder is correlated with overall CNV burden. To investigate the role of CNVs in female infertility spectrum disorders, we have performed a survey examining CNVs in a cohort of patients with unexplained infertility. Copy number variants (CNVs) are genomic aberrations that alter the number of copies of genomic segments and therefore have the potential to alter dosage-sensitive genes and/or functional elements. Recent studies have shown that CNVs underlie various complex spectrum disorders and that the severity of the disorder is correlated with overall CNV burden. To investigate the role of CNVs in female infertility spectrum disorders, we have performed a survey examining CNVs in a cohort of patients with unexplained infertility. DesignRetrospective cohort of female patients with unexplained infertility. Retrospective cohort of female patients with unexplained infertility. Materials and MethodsGenomic DNA extracted from blood samples was obtained from patients undergoing non-donor in-vitro fertilization (IVF) cycles at a large academic medical center. CNVs were detected using a custom NimbleGen Comparative Genomic Hybridization (CGH) Whole-Genome Tiling Array optimized for fertility-related loci. The resulting log2 ratios of the raw intensities were then normalized and segmented. The prevalence of CNVs among the infertile cohort was compared to the Children's Hospital of Philadelphia (CHOP) CNV database of healthy individuals. Genomic DNA extracted from blood samples was obtained from patients undergoing non-donor in-vitro fertilization (IVF) cycles at a large academic medical center. CNVs were detected using a custom NimbleGen Comparative Genomic Hybridization (CGH) Whole-Genome Tiling Array optimized for fertility-related loci. The resulting log2 ratios of the raw intensities were then normalized and segmented. The prevalence of CNVs among the infertile cohort was compared to the Children's Hospital of Philadelphia (CHOP) CNV database of healthy individuals. ResultsApproximately 30% of the >3000 CNVs detected were unique to the infertile patients (P<10-7, Binomial Test of Proportions). Moreover, CNVs specific to the infertile cohort appear to be overrepresented among regions of the genome predicted to play a role in mammalian fertility (P<10-16, t-test). Approximately 30% of the >3000 CNVs detected were unique to the infertile patients (P<10-7, Binomial Test of Proportions). Moreover, CNVs specific to the infertile cohort appear to be overrepresented among regions of the genome predicted to play a role in mammalian fertility (P<10-16, t-test). ConclusionThe set of CNVs uniquely observed in our female unexplained infertility cohort represent putative genetic biomarkers that may predict a range of infertility conditions difficult to diagnose with existing clinical data. The set of CNVs uniquely observed in our female unexplained infertility cohort represent putative genetic biomarkers that may predict a range of infertility conditions difficult to diagnose with existing clinical data.

Array-based DNA copy number analysis for mullerian anomalies

Copy number variants (CNVs) have been found in Mayer-Rokitansky-Kuster-Hauser Syndrome (MRKH). It is unknown if CNVs are associated with other Mullerian anomalies (MA). We sought to determine the prevalence of CNVs in an adolescent cohort with any MA, and validate and characterize detected CNVs.

Rare copy number variants in neuropsychiatric disorders: Specific phenotype or not?

From a number of genome-wide association studies it was shown that de novo and/or rare copy number variants (CNVs) are found at an increased frequency in neuropsychiatric diseases. In this study we examined the prevalence of CNVs in six genomic regions (1q21.1, 2p16.3, 3q29, 15q11.2, 15q13.3, and 16p11.2) previously implicated in neuropsychiatric diseases. Hereto, a cohort of four neuropsychiatric disorders (schizophrenia, bipolar disorder, major depressive disorder, and intellectual disability) and control individuals from three different populations was used in combination with Multilpex Amplicon Quantifiaction (MAQ) assays, capable of high resolution (kb range) and custom-tailored CNV detection. Our results confirm the etiological candidacy of the six selected CNV regions for neuropsychiatric diseases. It is possible that CNVs in these regions can result in disturbed brain development and in this way lead to an increased susceptibility for different neuropsychiatric disorders, dependent on additional genetic and environmental factors. Our results also suggest that the neurodevelopmental component is larger in the etiology of schizophrenia and intellectual disability than in mood disorders. Finally, our data suggest that deletions are in general more pathogenic than duplications. Given the high frequency of the examined CNVs (1-2%) in patients of different neuropsychiatric disorders, screening of large cohorts with an affordable and feasible method like the MAQ assays used in this study is likely to result in important progress in unraveling the genetic factors leading to an increased susceptibility for several psychiatric disorders.

SYSTEMS NEUROSCIENCE UNDERLYING SCHIZOPHRENIA MODELS – ICOSR SYMPOSIUM

To study the genetic cause of Mayer-Rokitansky-Kuster-Hauser syndrome (MRKH). Although a few candidate genes and genomic domains for have been reported for MRKH, the genetic underpinnings remain largely unknown. Some of the top candidate genes are WNT4, HNF1B, and LHX1. The goals of this study were to: 1) determine the prevalence of WNT4, HNF1B, and LHX1 point mutations, as well as new copy number variants (CNVs) in people with MRKH; and 2) identify and characterize MRKH cohorts.Laboratory- and community-based study.Academic medical centers.A total of 147 MRKH probands and available family members.DNA sequencing of WNT4, HNF1B, and LHX1 in 100 MRKH patients, chromosomal microarray analysis in 31 North American MRKH patients, and characterization and sample collection of 147 North American and Turkish MRKH probands and their families.DNA sequence variants and CNVs; pedigree structural analysis.We report finding CNVs in 6/31 people (∼19%) with MRKH, but no point mutations or small indels in WNT4, HNF1B, or LHX1 in 100 MRKH patients. Our MRKH families included 43 quads, 26 trios, and 30 duos. Of our MRKH probands, 87/147 (59%) had MRKH type 1 and 60/147 (41%) had type 2 with additional anomalies.Although the prevalence of WNT4, HNF1B, and LHX1 point mutations is low in people with MRKH, the prevalence of CNVs was ∼19%. Further analysis of our large familial cohort of patients will facilitate gene discovery to better understand the complex etiology of MRKH.

Submicroscopic chromosome abnormalities associated with spina bifida (myelomeningocele)

Background Visible chromosome abnormalities have been commonly reported in patients with spina bifida (myelomeningocele), particularly when associated with other malformations. Newer technologies such as array comparative genomic hybridization (CGH) have made it possible to identify smaller (submicroscopic) DNA deletions/duplications, also referred to as copy number variants (CNVs). Submicroscopic CNVs have been identified as the cause of a variety of disorders, substantially increasing the diagnostic yield in patients with intellectual disability and multiple congenital malformations. Submicroscopic CNVs have not been commonly reported in patients with spina bifida, but given the high frequency of visible chromosome rearrangements, we hypothesize that submicroscopic CNVs also contribute to the genetic etiology of spina bifida. Materials and methods We reviewed all array CGH results at a single children’s hospital laboratory for which neural tube defect was included in the indication for the test. During 20072009, array CGH was performed by the laboratory on a total of 1988 patients. Results Fourteen patients with spina bifida were evaluated by array CGH during the study period. CNVs were identified in two patients: 1) A 14 year old male with thoracosacral myelomeningocele, diastematomyelia, shunted hydrocephalus, bilateral clubfeet, bilateral subependymal heterotopias, bilateral closed-lip schizencephaly, seizures and polyarticular juvenile rheumatoid arthritis, had normal high-resolution karyotype (550-650 band), but array CGH revealed a 1.3Mb interstitial deletion in16p13.11. Of note, the patient’s brother has Gorlin syndrome, but no neural tube defect, and carries the same deletion. Parents have not been tested. 16p13.11 deletion has been reported in patients with autism, intellectual disability, epilepsy, holoprosencephaly and other malformations, but also in a few phenotypically normal individuals. 2) A 14 year old female with thoracic myelomeningocele, shunted hydrocephalus, bilateral hearing loss and caudal regression with imperforate anus had a normal highresolution karyotype (600-650 band), but array CGH revealed a 0.4-2.1 Mb duplication in 15q13.3. Mother does not carry the duplication, father has not been tested and a 17 year old sister with learning disabilities and ADHD has not been tested. 15q13.3 duplication has been reported in patients with intellectual disability, autistic behaviors and dysmorphic features. Conclusions Newer technologies for detecting smaller chromosome abnormalities may identify the cause of spina bifida in a proportion of patients. We hypothesize that atypical presentation may be associated with increased risk of an underlying CNV. Larger studies are required to determine the prevalence of CNVs in patients with spina bifida and whether patients with CNVs display atypical clinical features.