A Rare Duplication on Chromosome 16p11.2 Is Identified in Patients with Psychosis in Alzheimer's Disease

Epidemiological and genetic studies suggest that schizophrenia and autism may share genetic links. Besides common single nucleotide polymorphisms, recent data suggest that some rare copy number variants (CNVs) are risk factors for both disorders. Because we have previously found that schizophrenia and psychosis in Alzheimer''s disease (AD+P) share some genetic risk, we investigated whether CNVs reported in schizophrenia and autism are also linked to AD+P. We searched for CNVs associated with AD+P in 7 recurrent CNV regions that have been previously identified across autism and schizophrenia, using the Illumina HumanOmni1-Quad BeadChip. A chromosome 16p11.2 duplication CNV (chr16: 29,554,843-30,105,652) was identified in 2 of 440 AD+P subjects, but not in 136 AD subjects without psychosis, or in 593 AD subjects with intermediate psychosis status, or in 855 non-AD individuals. The frequency of this duplication CNV in AD+P (0.46%) was similar to that reported previously in schizophrenia (0.46%). This duplication CNV was further validated using the NanoString nCounter CNV Custom CodeSets. The 16p11.2 duplication has been associated with developmental delay, intellectual disability, behavioral problems, autism, schizophrenia (SCZ), and bipolar disorder. These two AD+P patients had no personal of, nor any identified family history of, SCZ, bipolar disorder and autism. To the best of our knowledge, our case report is the first suggestion that 16p11.2 duplication is also linked to AD+P. Although rare, this CNV may have an important role in the development of psychosis.     

Erasure and reestablishment of random allelic expression imbalance after epigenetic reprogramming

Clonal level random allelic expression imbalance and random monoallelic expression provides cellular heterogeneity within tissues by modulating allelic dosage. Although such expression patterns have been observed in multiple cell types, little is known about when in development these stochastic allelic choices are made. We examine allelic expression patterns in human neural progenitor cells before and after epigenetic reprogramming to induced pluripotency, observing that loci previously characterized by random allelic expression imbalance (0.63% of expressed genes) are generally reset to a biallelic state in induced pluripotent stem cells (iPSCs). We subsequently neuralized the iPSCs and profiled isolated clonal neural stem cells, observing that significant random allelic expression imbalance is reestablished at 0.65% of expressed genes, including novel loci not found to show allelic expression imbalance in the original parental neural progenitor cells. Allelic expression imbalance was associated with altered DNA methylation across promoter regulatory regions, with clones characterized by skewed allelic expression being hypermethylated compared to their biallelic sister clones. Our results suggest that random allelic expression imbalance is established during lineage commitment and is associated with increased DNA methylation at the gene promoter.     

Accurately Assessing the Risk of Schizophrenia Conferred by Rare Copy-Number Variation Affecting Genes with Brain Function

Investigators have linked rare copy number variation (CNVs) to neuropsychiatric diseases, such as schizophrenia. One hypothesis is that CNV events cause disease by affecting genes with specific brain functions. Under these circumstances, we expect that CNV events in cases should impact brain-function genes more frequently than those events in controls. Previous publications have applied “pathway” analyses to genes within neuropsychiatric case CNVs to show enrichment for brain-functions. While such analyses have been suggestive, they often have not rigorously compared the rates of CNVs impacting genes with brain function in cases to controls, and therefore do not address important confounders such as the large size of brain genes and overall differences in rates and sizes of CNVs. To demonstrate the potential impact of confounders, we genotyped rare CNV events in 2,415 unaffected controls with Affymetrix 6.0; we then applied standard pathway analyses using four sets of brain-function genes and observed an apparently highly significant enrichment for each set. The enrichment is simply driven by the large size of brain-function genes. Instead, we propose a case-control statistical test, cnv-enrichment-test, to compare the rate of CNVs impacting specific gene sets in cases versus controls. With simulations, we demonstrate that cnv-enrichment-test is robust to case-control differences in CNV size, CNV rate, and systematic differences in gene size. Finally, we apply cnv-enrichment-test to rare CNV events published by the International Schizophrenia Consortium (ISC). This approach reveals nominal evidence of case-association in neuronal-activity and the learning gene sets, but not the other two examined gene sets. The neuronal-activity genes have been associated in a separate set of schizophrenia cases and controls; however, testing in independent samples is necessary to definitively confirm this association. Our method is implemented in the PLINK software package.     

Schizophrenia: genome, interrupted

Structural chromosomal variation is increasingly recognized as an important contributor to human diseases, particularly those of neurodevelopment, such as autism. A current paper makes a significant advance to schizophrenia genetics by establishing an association with rare copy number variants (CNV), which are over-represented in neurodevelopmental genes.     

GALC deletions increase the risk of primary open-angle glaucoma: the role of Mendelian variants in complex disease

DNA copy number variants (CNVs) have been reported in many human diseases including autism and schizophrenia. Primary Open Angle Glaucoma (POAG) is a complex adult-onset disorder characterized by progressive optic neuropathy and vision loss. Previous studies have identified rare CNVs in POAG; however, their low frequencies prevented formal association testing. We present here the association between POAG risk and a heterozygous deletion in the galactosylceramidase gene (GALC). This CNV was initially identified in a dataset containing 71 Caucasian POAG cases and 478 ethnically matched controls obtained from dbGAP (study accession phs000126.v1.p1.) (p = 0.017, fisher's exact test). It was validated with array comparative genomic hybridization (arrayCGH) and realtime PCR, and replicated in an independent POAG dataset containing 959 cases and 1852 controls (p = 0.021, OR (odds ratio) = 3.5, 95% CI -1.1-12.0). Evidence for association was strengthened when the discovery and replication datasets were combined (p = 0.002; OR = 5.0, 95% CI 1.6-16.4). Several deletions with different endpoints were identified by array CGH of POAG patients. Homozygous deletions that eliminate GALC enzymatic activity cause Krabbe disease, a recessive Mendelian disorder of childhood displaying bilateral optic neuropathy and vision loss. Our findings suggest that heterozygous deletions that reduce GALC activity are a novel mechanism increasing risk of POAG. This is the first report of a statistically-significant association of a CNV with POAG risk, contributing to a growing body of evidence that CNVs play an important role in complex, inherited disorders. Our findings suggest an attractive biomarker and potential therapeutic target for patients with this form of POAG.     

Disruption of Contactin 4 in two subjects with autism in Chinese population

Autism is a heterogeneous childhood neurodevelopmental disorder that is characterised by deficits in verbal communication, impaired social interactions, restricted interests and repetitive behaviours. Using an Illumina HumanCNV370-Quad BeadChip, we identified two Han Chinese individuals with autism and large duplications (~1.6 Mb and ~2.4 Mb) disrupting the same CNTN4 gene. CNTN4 encodes a protein that functions as a cell-adhesion molecule and may play an essential role in the formation of axon connections in the developing nervous system. The disruption of this gene has been reported to be the cause of the 3p deletion syndrome and also a possible susceptibility factor for autism spectrum disorders (ASDs). Our results suggest that rare copy number variations (CNVs) in CNTN4 may also influence autism susceptibility in Asian populations. Interestingly, a comparison of the clinical phenotypes between the two subjects revealed that the subject with the 2.4 Mb CNV (involving several other genes) presented with a more severe phenotype than the subject with the 1.6 Mb CNV (disrupting only CNTN4 and CNTN6). This suggests that other genes in the nearby region may contribute to the pathogenesis.     

Neuronal cell adhesion genes

The major mental disorders, schizophrenia and bipolar disorder are substantially heritable. Recent genomic studies have identified a small number of common and rare risk genes contributing to both disorders and support epidemiological evidence that genetic susceptibility overlaps between them. Prompted by the question of whether risk genes cluster in specific molecular pathways or implicate discrete mechanisms we and others have developed hypothesis-free methods of investigating genome-wide association datasets at a pathway-level. The application of our method to the 212 experimentally-derived pathways in the Kyoto Encycolpaedia of Genes and Genomes (KEGG) database identified significant association between the cell adhesion molecule (CAM) pathway and both schizophrenia and bipolar disorder susceptibility across three GWAS datasets. Interestingly, a similar approach applied to an autistic spectrum disorders (ASDs) sample identified a similar pathway and involved many of the same genes. Disruption of a number of these genes (including NRXN1, CNTNAP2 and CASK) are known to cause diverse neurodevelopmental brain disorder phenotypes including schizophenia, autism, learning disability and specific language disorder. Taken together these studies bring the CAM pathway sharply into focus for more comprehensive DNA sequencing to identify the critical genes, and investigate their relationships and interaction with environmental risk factors in the expression of many seemingly different neurodevelopmental disorders.     

An Inherited Small Microdeletion at 15q13.3 in a Patient with Early- Onset Obsessive-Compulsive Disorder

Copy number variations (CNVs) have been previously associated with several different neurodevelopmental psychiatric disorders, such as autism, schizophrenia, and attention deficit hyperactivity disorder (ADHD). The present study consisted of a pilot genome-wide screen for CNVs in a cohort of 16 patients with early-onset obsessive-compulsive disorder (OCD) and 12 mentally healthy individuals, using array-based comparative genomic hybridization (aCGH) on 44K arrays. A small rare paternal inherited microdeletion (∼64 kb) was identified in chromosome 15q13.3 of one male patient with very early onset OCD. The father did not have OCD. The deletion encompassed part of the FMN1 gene, which is involved with the glutamatergic system. This finding supports the hypothesis of a complex network of several genes expressed in the brain contributing for the genetic risk of OCD, and also supports the glutamatergic involvement in OCD, which has been previously reported in the literature.     

Synergistic Interactions between Drosophila Orthologues of Genes Spanned by De Novo Human CNVs Support Multiple-Hit Models of Autism

Autism spectrum disorders (ASDs) are highly heritable and characterised by deficits in social interaction and communication, as well as restricted and repetitive behaviours. Although a number of highly penetrant ASD gene variants have been identified, there is growing evidence to support a causal role for combinatorial effects arising from the contributions of multiple loci. By examining synaptic and circadian neurological phenotypes resulting from the dosage variants of unique human:fly orthologues in Drosophila, we observe numerous synergistic interactions between pairs of informatically-identified candidate genes whose orthologues are jointly affected by large de novo copy number variants (CNVs). These CNVs were found in the genomes of individuals with autism, including a patient carrying a 22q11.2 deletion. We first demonstrate that dosage alterations of the unique Drosophila orthologues of candidate genes from de novo CNVs that harbour only a single candidate gene display neurological defects similar to those previously reported in Drosophila models of ASD-associated variants. We then considered pairwise dosage changes within the set of orthologues of candidate genes that were affected by the same single human de novo CNV. For three of four CNVs with complete orthologous relationships, we observed significant synergistic effects following the simultaneous dosage change of gene pairs drawn from a single CNV. The phenotypic variation observed at the Drosophila synapse that results from these interacting genetic variants supports a concordant phenotypic outcome across all interacting gene pairs following the direction of human gene copy number change. We observe both specificity and transitivity between interactors, both within and between CNV candidate gene sets, supporting shared and distinct genetic aetiologies. We then show that different interactions affect divergent synaptic processes, demonstrating distinct molecular aetiologies. Our study illustrates mechanisms through which synergistic effects resulting from large structural variation can contribute to human disease.     

A New Method for Detecting Associations with Rare Copy-Number Variants

Copy number variants (CNVs) play an important role in the etiology of many diseases such as cancers and psychiatric disorders. Due to a modest marginal effect size or the rarity of the CNVs, collapsing rare CNVs together and collectively evaluating their effect serves as a key approach to evaluating the collective effect of rare CNVs on disease risk. While a plethora of powerful collapsing methods are available for sequence variants (e.g., SNPs) in association analysis, these methods cannot be directly applied to rare CNVs due to the CNV-specific challenges, i.e., the multi-faceted nature of CNV polymorphisms (e.g., CNVs vary in size, type, dosage, and details of gene disruption), and etiological heterogeneity (e.g., heterogeneous effects of duplications and deletions that occur within a locus or in different loci). Existing CNV collapsing analysis methods (a.k.a. the burden test) tend to have suboptimal performance due to the fact that these methods often ignore heterogeneity and evaluate only the marginal effects of a CNV feature. We introduce CCRET, a random effects test for collapsing rare CNVs when searching for disease associations. CCRET is applicable to variants measured on a multi-categorical scale, collectively modeling the effects of multiple CNV features, and is robust to etiological heterogeneity. Multiple confounders can be simultaneously corrected. To evaluate the performance of CCRET, we conducted extensive simulations and analyzed large-scale schizophrenia datasets. We show that CCRET has powerful and robust performance under multiple types of etiological heterogeneity, and has performance comparable to or better than existing methods when there is no heterogeneity.     

The application of gene co-expression network reconstruction based on CNVs and gene expression microarray data in breast cancer

Copy number variations (CNVs) are one type of the human genetic variations and are pervasive in the human genome. It has been confirmed that they can play a causal role in complex diseases. Previous studies of CNVs focused more on identifying the disease-specific CNV regions or candidate genes on these CNV regions, but less on the synergistic actions between genes on CNV regions and other genes. Our research combined the CNVs with related gene co-expression to reconstruct gene co-expression network by using single nucleotide polymorphism microarray datasets and gene microarray datasets of breast cancer, and then extracted the modules which connected densely inside and analyzed the functions of modules. Interestingly, all of these modules’ functions were related to breast cancer according to our enrichment analysis, and most of the genes in these modules have been reported to be involved in breast cancer. Our findings suggested that integrating CNVs and gene co-expressed relations was an available way to analyze the roles of CNV genes and their synergistic genes in breast cancer, and provided a novel insight into the pathological mechanism of breast cancer.     

Deletion 17q12 is a recurrent copy number variant that confers high risk of autism and schizophrenia

Autism spectrum disorders (ASD) and schizophrenia are neurodevelopmental disorders for which recent evidence indicates an important etiologic role for rare copy number variants (CNVs) and suggests common genetic mechanisms. We performed cytogenomic array analysis in a discovery sample of patients with neurodevelopmental disorders referred for clinical testing. We detected a recurrent 1.4 Mb deletion at 17q12, which harbors HNF1B, the gene responsible for renal cysts and diabetes syndrome (RCAD), in 18/15,749 patients, including several with ASD, but 0/4,519 controls. We identified additional shared phenotypic features among nine patients available for clinical assessment, including macrocephaly, characteristic facial features, renal anomalies, and neurocognitive impairments. In a large follow-up sample, the same deletion was identified in 2/1,182 ASD/neurocognitive impairment and in 4/6,340 schizophrenia patients, but in 0/47,929 controls (corrected p = 7.37 × 10⁻⁵). These data demonstrate that deletion 17q12 is a recurrent, pathogenic CNV that confers a very high risk for ASD and schizophrenia and show that one or more of the 15 genes in the deleted interval is dosage sensitive and essential for normal brain development and function. In addition, the phenotypic features of patients with this CNV are consistent with a contiguous gene syndrome that extends beyond RCAD, which is caused by HNF1B mutations only.     

Genome arrays for the detection of copy number variations in idiopathic mental retardation, idiopathic generalized epilepsy and neuropsychiatric disorders: lessons for diagnostic workflow and research

We review the contributions and limitations of genome-wide array-based identification of copy number variants (CNVs) in the clinical diagnostic evaluation of patients with mental retardation (MR) and other brain-related disorders. In unselected MR referrals a causative genomic gain or loss is detected in 14-18% of cases. Usually, such CNVs arise de novo, are not found in healthy subjects, and have a major impact on the phenotype by altering the dosage of multiple genes. This high diagnostic yield justifies array-based segmental aneuploidy screening as the initial genetic test in these patients. This also pertains to patients with autism (expected yield about 5-10% in nonsyndromic and 10-20% in syndromic patients) and schizophrenia (at least 5% yield). CNV studies in idiopathic generalized epilepsy, attention-deficit hyperactivity disorder, major depressive disorder and Tourette syndrome indicate that patients have, on average, a larger CNV burden as compared to controls. Collectively, the CNV studies suggest that a wide spectrum of disease-susceptibility variants exists, most of which are rare (<0.1%) and of variable and usually small effect. Notwithstanding, a rare CNV can have a major impact on the phenotype. Exome sequencing in MR and autism patients revealed de novo mutations in protein coding genes in 60 and 20% of cases, respectively. Therefore, it is likely that arrays will be supplanted by next-generation sequencing methods as the initial and perhaps ultimate diagnostic tool in patients with brain-related disorders, revealing both CNVs and mutations in a single test.     

Accurate Distinction of Pathogenic from Benign CNVs in Mental Retardation

Copy number variants (CNVs) have recently been recognized as a common form of genomic variation in humans. Hundreds of CNVs can be detected in any individual genome using genomic microarrays or whole genome sequencing technology, but their phenotypic consequences are still poorly understood. Rare CNVs have been reported as a frequent cause of neurological disorders such as mental retardation (MR), schizophrenia and autism, prompting widespread implementation of CNV screening in diagnostics. In previous studies we have shown that, in contrast to benign CNVs, MR-associated CNVs are significantly enriched in genes whose mouse orthologues, when disrupted, result in a nervous system phenotype. In this study we developed and validated a novel computational method for differentiating between benign and MR-associated CNVs using structural and functional genomic features to annotate each CNV. In total 13 genomic features were included in the final version of a Naïve Bayesian Tree classifier, with LINE density and mouse knock-out phenotypes contributing most to the classifier''s accuracy. After demonstrating that our method (called GECCO) perfectly classifies CNVs causing known MR-associated syndromes, we show that it achieves high accuracy (94%) and negative predictive value (99%) on a blinded test set of more than 1,200 CNVs from a large cohort of individuals with MR. These results indicate that this classification method will be of value for objectively prioritizing CNVs in clinical research and diagnostics.     

CNV Analysis in Tourette Syndrome Implicates Large Genomic Rearrangements in COL8A1 and NRXN1

Tourette syndrome (TS) is a neuropsychiatric disorder with a strong genetic component. However, the genetic architecture of TS remains uncertain. Copy number variation (CNV) has been shown to contribute to the genetic make-up of several neurodevelopmental conditions, including schizophrenia and autism. Here we describe CNV calls using SNP chip genotype data from an initial sample of 210 TS cases and 285 controls ascertained in two Latin American populations. After extensive quality control, we found that cases (N = 179) have a significant excess (P = 0.006) of large CNV (>500 kb) calls compared to controls (N = 234). Amongst 24 large CNVs seen only in the cases, we observed four duplications of the COL8A1 gene region. We also found two cases with ∼400kb deletions involving NRXN1, a gene previously implicated in neurodevelopmental disorders, including TS. Follow-up using multiplex ligation-dependent probe amplification (and including 53 more TS cases) validated the CNV calls and identified additional patients with rearrangements in COL8A1 and NRXN1, but none in controls. Examination of available parents indicates that two out of three NRXN1 deletions detected in the TS cases are de-novo mutations. Our results are consistent with the proposal that rare CNVs play a role in TS aetiology and suggest a possible role for rearrangements in the COL8A1 and NRXN1 gene regions.     

Convergence of Genes and Cellular Pathways Dysregulated in Autism Spectrum Disorders

Rare copy-number variation (CNV) is an important source of risk for autism spectrum disorders (ASDs). We analyzed 2,446 ASD-affected families and confirmed an excess of genic deletions and duplications in affected versus control groups (1.41-fold, p = 1.0 × 10⁻⁵) and an increase in affected subjects carrying exonic pathogenic CNVs overlapping known loci associated with dominant or X-linked ASD and intellectual disability (odds ratio = 12.62, p = 2.7 × 10⁻¹⁵, ∼3% of ASD subjects). Pathogenic CNVs, often showing variable expressivity, included rare de novo and inherited events at 36 loci, implicating ASD-associated genes (CHD2, HDAC4, and GDI1) previously linked to other neurodevelopmental disorders, as well as other genes such as SETD5, MIR137, and HDAC9. Consistent with hypothesized gender-specific modulators, females with ASD were more likely to have highly penetrant CNVs (p = 0.017) and were also overrepresented among subjects with fragile X syndrome protein targets (p = 0.02). Genes affected by de novo CNVs and/or loss-of-function single-nucleotide variants converged on networks related to neuronal signaling and development, synapse function, and chromatin regulation.