The identification of germline variants that predispose to cancer is important to further our understanding of tumorigenesis, guide patient management, prevent disease in unaffected relatives, and inform best practice for health care. We describe a kindred with multiple gastrointestinal malignancies where a novel MSH6 germline susceptibility variant was identified by exome sequencing after eluding serial routine testing in multiple affected members. This case fosters discussion of our current understanding of DNA mismatch repair deficiency, the management of Lynch Syndrome, and the emerging role of next generation sequencing in laboratory medicine to identify rare pathogenic germline variants in a comprehensive, unbiased fashion.
Synaptic function is central to brain function. Understanding the synapse is aided by studies of patients lacking individual synaptic proteins. Common neurological diseases are genetically complex. Their understanding is likewise simplified by studies of less common monogenic forms. We detail the disease caused by absence of the synaptic protein CNKSR2 in 8 patients ranging from 6 to 62 years old. The disease is characterized by intellectual disability, attention problems, and abrupt lifelong language loss following a brief early childhood epilepsy with continuous spike-waves in sleep. This study describes the phenotype of CNKSR2 deficiency and its involvement in systems underlying common neurological disorders. Ann Neurol 2014;76:758-764.
The genetic architecture of autism spectrum disorder involves the interplay of common and rare variants and their impact on hundreds of genes. Using exome sequencing, here we show that analysis of rare coding variation in 3,871 autism cases and 9,937 ancestry-matched or parental controls implicates 22 autosomal genes at a false discovery rate (FDR) < 0.05, plus a set of 107 autosomal genes strongly enriched for those likely to affect risk (FDR < 0.30). These 107 genes, which show unusual evolutionary constraint against mutations, incur de novo loss-of-function mutations in over 5% of autistic subjects. Many of the genes implicated encode proteins for synaptic formation, transcriptional regulation and chromatin-remodelling pathways. These include voltage-gated ion channels regulating the propagation of action potentials, pacemaking and excitability-transcription coupling, as well as histone-modifying enzymes and chromatin remodellers-most prominently those that mediate post-translational lysine methylation/demethylation modifications of histones.
A universal challenge in genetic studies of autism spectrum disorders (ASDs) is determining whether a given DNA sequence alteration will manifest as disease. Among different population controls, we observed, for specific exons, an inverse correlation between exon expression level in brain and burden of rare missense mutations. For genes that harbor de novo mutations predicted to be deleterious, we found that specific critical exons were significantly enriched in individuals with ASD relative to their siblings without ASD (P < 1.13 × 10(-38); odds ratio (OR) = 2.40). Furthermore, our analysis of genes with high exonic expression in brain and low burden of rare mutations demonstrated enrichment for known ASD-associated genes (P < 3.40 × 10(-11); OR = 6.08) and ASD-relevant fragile-X protein targets (P < 2.91 × 10(-157); OR = 9.52). Our results suggest that brain-expressed exons under purifying selection should be prioritized in genotype-phenotype studies for ASD and related neurodevelopmental conditions.
The chromodomain helicase DNA binding domain (CHD) proteins modulate gene expression via their ability to remodel chromatin structure and influence histone acetylation. Recent studies have shown that CHD2 protein plays a critical role in embryonic development, tumor suppression and survival. Like other genes encoding members of the CHD family, pathogenic mutations in the CHD2 gene are expected to be implicated in human disease. In fact, there is emerging evidence suggesting that CHD2 might contribute to a broad spectrum of neurodevelopmental disorders. Despite growing evidence, a description of the full phenotypic spectrum of this condition is lacking.
PRPS1 codes for the enzyme phosphoribosyl pyrophosphate synthetase-1 (PRS-1). The spectrum of PRPS1-related disorders associated with reduced activity includes Arts syndrome, Charcot-Marie-Tooth disease-5 (CMTX5) and X-linked non-syndromic sensorineural deafness (DFN2). We describe a novel phenotype associated with decreased PRS-1 function in two affected male siblings. Using whole exome and Sanger sequencing techniques, we identified a novel missense mutation in PRPS1. The clinical phenotype in our patients is characterized by high prenatal maternal ?-fetoprotein, intrauterine growth restriction, dysmorphic facial features, severe intellectual disability and spastic quadraparesis. Additional phenotypic features include macular coloboma-like lesions with retinal dystrophy, severe short stature and diabetes insipidus. Exome sequencing of the two affected male siblings identified a shared putative pathogenic mutation c.586C>T p.(Arg196Trp) in the PRPS1 gene that was maternally inherited. Follow-up testing showed normal levels of hypoxanthine in urine samples and uric acid levels in blood serum. The PRS activity was significantly reduced in erythrocytes of the two patients. Nucleotide analysis in erythrocytes revealed abnormally low guanosine triphosphate and guanosine diphosphate. This presentation is the most severe form of PRPS1-deficiency syndrome described to date and expands the spectrum of PRPS1-related disorders.European Journal of Human Genetics advance online publication, 25 June 2014; doi:10.1038/ejhg.2014.112.
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.
There is tremendous potential for genome sequencing to improve clinical diagnosis and care once it becomes routinely accessible, but this will require formalizing research methods into clinical best practices in the areas of sequence data generation, analysis, interpretation and reporting. The CLARITY Challenge was designed to spur convergence in methods for diagnosing genetic disease starting from clinical case history and genome sequencing data. DNA samples were obtained from three families with heritable genetic disorders and genomic sequence data were donated by sequencing platform vendors. The challenge was to analyze and interpret these data with the goals of identifying disease-causing variants and reporting the findings in a clinically useful format. Participating contestant groups were solicited broadly, and an independent panel of judges evaluated their performance.
Inherited monogenic disease has an enormous impact on the well-being of children and their families. Over half of the children living with one of these conditions are without a molecular diagnosis because of the rarity of the disease, the marked clinical heterogeneity, and the reality that there are thousands of rare diseases for which causative mutations have yet to be identified. It is in this context that in 2010 a Canadian consortium was formed to rapidly identify mutations causing a wide spectrum of pediatric-onset rare diseases by using whole-exome sequencing. The FORGE (Finding of Rare Disease Genes) Canada Consortium brought together clinicians and scientists from 21 genetics centers and three science and technology innovation centers from across Canada. From nation-wide requests for proposals, 264 disorders were selected for study from the 371 submitted; disease-causing variants (including in 67 genes not previously associated with human disease; 41 of these have been genetically or functionally validated, and 26 are currently under study) were identified for 146 disorders over a 2-year period. Here, we present our experience with four strategies employed for gene discovery and discuss FORGE's impact in a number of realms, from clinical diagnostics to the broadening of the phenotypic spectrum of many diseases to the biological insight gained into both disease states and normal human development. Lastly, on the basis of this experience, we discuss the way forward for rare-disease genetic discovery both in Canada and internationally.
Validating the potential pathogenicity of copy number variants (CNVs) identified in genome-wide studies of autism spectrum disorders (ASD) requires detailed assessment of case/control frequencies, inheritance patterns, clinical correlations, and functional impact. Here, we characterize a small recurrent duplication in the annexin A1 (ANXA1) gene, identified by the Autism Genome Project (AGP) study.
Obsessive-compulsive disorder (OCD) and Tourette syndrome (TS) are heritable neurodevelopmental disorders with a partially shared genetic etiology. This study represents the first genome-wide investigation of large (>500 kb), rare (<1%) copy number variants (CNVs) in OCD and the largest genome-wide CNV analysis in TS to date.
The emerging 3q13.31 microdeletion syndrome appears to encompass diverse neurodevelopmental conditions. However, the 3q13.31 deletion is rare and few adult cases have yet been reported. We examined a cohort with schizophrenia (n?=?459) and adult control subjects (n?=?26,826) using high-resolution microarray technology for deletions and duplications at the 3q13.31 locus.
Peninsular Malaysia is a strategic region which might have played an important role in the initial peopling and subsequent human migrations in Asia. However, the genetic diversity and history of human populations--especially indigenous populations--inhabiting this area remain poorly understood. Here, we conducted a genome-wide study using over 900,000 single nucleotide polymorphisms (SNPs) in four major Malaysian ethnic groups (MEGs; Malay, Proto-Malay, Senoi and Negrito), and made comparisons of 17 world-wide populations. Our data revealed that Peninsular Malaysia has greater genetic diversity corresponding to its role as a contact zone of both early and recent human migrations in Asia. However, each single Orang Asli (indigenous) group was less diverse with a smaller effective population size (N(e)) than a European or an East Asian population, indicating a substantial isolation of some duration for these groups. All four MEGs were genetically more similar to Asian populations than to other continental groups, and the divergence time between MEGs and East Asian populations (12,000--6,000 years ago) was also much shorter than that between East Asians and Europeans. Thus, Malaysian Orang Asli groups, despite their significantly different features, may share a common origin with the other Asian groups. Nevertheless, we identified traces of recent gene flow from non-Asians to MEGs. Finally, natural selection signatures were detected in a batch of genes associated with immune response, human height, skin pigmentation, hair and facial morphology and blood pressure in MEGs. Notable examples include SYN3 which is associated with human height in all Orang Asli groups, a height-related gene (PNPT1) and two blood pressure-related genes (CDH13 and PAX5) in Negritos. We conclude that a long isolation period, subsequent gene flow and local adaptations have jointly shaped the genetic architectures of MEGs, and this study provides insight into the peopling and human migration history in Southeast Asia.
Autism or autism spectrum disorder (ASD) is a range of neurodevelopmental disorders starting in early childhood and is characterized by impairments in communication and reciprocal social interaction and presence of restricted and repetitive patterns of behavior. The contribution of genetic factors to autism is clear in twin and family studies. It is apparent that, overall, ASD is a complex non-Mendelian disorder. Recent studies suggest that copy number variations (CNVs) play a significant role in the etiology of ASD. For the current work, we recruited 245 family members from 73 ASD families from Styria, Austria. The DNA from probands was genotyped with Affymetrix single nucleotide polymorphism (SNP) 6.0 microarrays to screen for CNVs in their genomes. Analysis of the microarray data was performed using three different algorithms, and a list of stringent calls was compared to existing CNV data from over 2,357 controls of European ancestry. For stringent calls not present in controls, quantitative real-time PCR (qRT-PCR) was used to validate the CNVs in the probands and in their family members. Twenty-two CNVs were validated from this set (five of which are apparently de novo), many of which appear likely to disrupt genes that may be considered as good candidates for neuropsychiatric disorders, including DLG2, S100B, ARX, DIP2A, HPCAL1, and GPHN. Several others disrupt genes that have previously been implicated in autism, such as BDNF, AUTS2, DPP6, and C18orf22, and our data add to the growing evidence of their involvement in ASD.
To determine the causative genetic lesion in 3 adult siblings with a slowly progressive, juvenile-onset phenotype comprising cerebellar atrophy and ataxia, intellectual decline, hearing loss, hypogonadism, hyperreflexia, a demyelinating sensorimotor neuropathy, and (in 2 of 3 probands) supratentorial white matter changes, in whom numerous prior investigations were nondiagnostic.
Purpose:Recurrent 15q13.3 deletions are enriched in multiple neurodevelopmental conditions including intellectual disability, autism, epilepsy, and schizophrenia. However, the 15q13.3 microdeletion syndrome remains ill-defined.Methods:We systematically compiled all cases of 15q13.3 deletion published before 2014. We also examined three locally available cohorts to identify new adults with 15q13.3 deletions.Results:We identified a total of 246 cases (133 children, 113 adults) with deletions overlapping or within the 15q13.3 (breakpoint (BP)4-BP5) region, including seven novel adult cases from local cohorts. No BP4-BP5 deletions were identified in 23,838 adult controls. Where known, 15q13.3 deletions were typically inherited (85.4%) and disproportionately of maternal origin (P < 0.0001). Overall, 198 cases (121 children, 77 adults; 80.5%) had at least one neuropsychiatric diagnosis. Accounting for ascertainment, developmental disability/intellectual disability was present in 57.7%, epilepsy/seizures in 28.0%, speech problems in 15.9%, autism spectrum disorder in 10.9%, schizophrenia in 10.2%, mood disorder in 10.2%, and attention deficit hyperactivity disorder in 6.5%. By contrast, major congenital malformations, including congenital heart disease (2.4%), were uncommon. Placenta previa occurred in the pregnancies of four cases.Conclusion:The 15q13.3 microdeletion syndrome is predominantly characterized by neuropsychiatric expression. There are implications for pre- and postnatal detection, genetic counseling, and anticipatory care.Genet Med advance online publication 31 July 2014Genetics in Medicine (2014); doi:10.1038/gim.2014.83.
MicroRNAs (miRNAs) are key regulators of gene expression in the human genome and may contribute to risk for neuropsychiatric disorders. miRNAs play an acknowledged role in the strongest of genetic risk factors for schizophrenia, 22q11.2 deletions. We hypothesized that in schizophrenia there would be an enrichment of other rare copy number variants (CNVs) that overlap miRNAs.
New genomic disorders associated with large, rare, recurrent copy number variations (CNVs) are being discovered at a rapid pace. Detailed phenotyping and family studies are rare, however, as are data on adult phenotypic expression. Duplications at 2q13 were recently identified as risk factors for developmental delay/autism and reported in the prenatal setting, yet few individuals (all children) have been extensively phenotyped. During a genome-wide CNV study of schizophrenia, we identified two unrelated probands with 2q13 duplications. In this study, detailed phenotyping and genotyping using high-resolution microarrays was performed for 12 individuals across their two families. 2q13 duplications were present in six adults, and co-segregated with clinically significant later-onset neuropsychiatric disorders. Convergent lines of evidence implicated GABAminergic dysfunction. Analysis of the genic content revealed promising candidates for neuropsychiatric disease, including BCL2L11, ANAPC1, and MERTK. Intrafamilial genetic heterogeneity and "second hits" in one family may have been the consequence of assortative mating. Clinical genetic testing for the 2q13 duplication and the associated genetic counseling was well received. In summary, large rare 2q13 duplications appear to be associated with variable adult neuropsychiatric and other expression. The findings represent progress toward clinical translation of research results in schizophrenia. There are implications for other emerging genomic disorders where there is interest in lifelong expression.
Copy number variation (CNV) has been recognized as a major contributor to human genome diversity. It plays an important role in determining phenotypes and has been associated with a number of common and complex diseases. However CNV data from diverse populations is still limited. Here we report the first investigation of CNV in the indigenous populations from Peninsular Malaysia. We genotyped 34 Negrito genomes from Peninsular Malaysia using the Affymetrix SNP 6.0 microarray and identified 48 putative novel CNVs, consisting of 24 gains and 24 losses, of which 5 were identified in at least 2 unrelated samples. These CNVs appear unique to the Negrito population and were absent in the DGV, HapMap3 and Singapore Genome Variation Project (SGVP) datasets. Analysis of gene ontology revealed that genes within these CNVs were enriched in the immune system (GO:0002376), response to stimulus mechanisms (GO:0050896), the metabolic pathways (GO:0001852), as well as regulation of transcription (GO:0006355). Copy number gains in CNV regions (CNVRs) enriched with genes were significantly higher than the losses (P value <0.001). In view of the small population size, relative isolation and semi-nomadic lifestyles of this community, we speculate that these CNVs may be attributed to recent local adaptation of Negritos from Peninsular Malaysia.
Rare copy number variants (CNVs) disrupting ASTN2 or both ASTN2 and TRIM32 have been reported at 9q33.1 by genome-wide studies in a few individuals with neurodevelopmental disorders. The vertebrate-specific astrotactins, ASTN2 and its paralog ASTN1, have key roles in glial-guided neuronal migration during brain development. To determine the prevalence of astrotactin mutations and delineate their associated phenotypic spectrum, we screened ASTN2/TRIM32 and ASTN1 (1q25.2) for exonic CNVs in clinical microarray data from 89,985 individuals across 10 sites, including 64,114 neurodevelopmental disorder subjects. In this clinical dataset, we identified 46 deletions and 12 duplications affecting ASTN2. Deletions of ASTN1 were much rarer. Deletions near the 3 terminus of ASTN2, which would disrupt all transcript isoforms (a subset of these deletions also included TRIM32), were significantly enriched in the neurodevelopmental disorder subjects (p=0.002) compared with 44,085 population-based controls. Frequent phenotypes observed in individuals with such deletions included Autism Spectrum Disorders (ASD), Attention Deficit Hyperactivity Disorder (ADHD), speech delay, anxiety and Obsessive Compulsive Disorder (OCD). The 3-terminal ASTN2 deletions were significantly enriched compared with controls in males with neurodevelopmental disorders, but not in females. Upon quantifying ASTN2 human brain RNA, we observed shorter isoforms expressed from an alternative transcription start site of recent evolutionary origin near the 3 end. Spatiotemporal expression profiling in the human brain revealed consistently high ASTN1 expression while ASTN2 expression peaked in the early embryonic neocortex and postnatal cerebellar cortex. Our findings shed new light on the role of the astrotactins in psychopathology and their interplay in human neurodevelopment.
Osteoporosis is a systemic skeletal disease characterised by reduced bone mineral density and increased susceptibility to fracture; these traits are highly heritable. Both common and rare copy number variants (CNVs) potentially affect the function of genes and may influence disease risk.
Individually rare, large copy number variants (CNVs) contribute to genetic vulnerability for schizophrenia. Unresolved questions remain, however, regarding the anticipated yield of clinical microarray testing in schizophrenia. Using high-resolution genome-wide microarrays and rigorous methods, we investigated rare CNVs in a prospectively recruited community-based cohort of 459 unrelated adults with schizophrenia and estimated the minimum prevalence of clinically significant CNVs that would be detectable on a clinical microarray. A blinded review by two independent clinical cytogenetic laboratory directors of all large (>500 kb) rare CNVs in cases and well-matched controls showed that those deemed to be clinically significant were highly enriched in schizophrenia (16.4-fold increase, P < 0.0001). In a single community catchment area, the prevalence of individuals with these CNVs was 8.1%. Rare 1.7 Mb CNVs at 2q13 were found to be significantly associated with schizophrenia for the first time, compared with the prevalence in 23 838 population-based controls (42.9-fold increase, P = 0.0002). Additional novel findings that will facilitate the future clinical interpretation of smaller CNVs in schizophrenia include: (i) a greater proportion of individuals with two or more rare exonic CNVs >10 kb in size (1.5-fold increase, P = 0.0109) in schizophrenia; (ii) the systematic discovery of new candidate genes for schizophrenia; and, (iii) functional gene enrichment mapping highlighting a differential impact in schizophrenia of rare exonic deletions involving diverse functions, including neurodevelopmental and synaptic processes (4.7-fold increase, P = 0.0060). These findings suggest consideration of a potential role for clinical microarray testing in schizophrenia, as is now the suggested standard of care for related developmental disorders like autism.
Autism Spectrum Disorders (ASD) are highly heritable and characterised by impairments in social interaction and communication, and restricted and repetitive behaviours. Considering four sets of de novo copy number variants (CNVs) identified in 181 individuals with autism and exploiting mouse functional genomics and known protein-protein interactions, we identified a large and significantly interconnected interaction network. This network contains 187 genes affected by CNVs drawn from 45% of the patients we considered and 22 genes previously implicated in ASD, of which 192 form a single interconnected cluster. On average, those patients with copy number changed genes from this network possess changes in 3 network genes, suggesting that epistasis mediated through the network is extensive. Correspondingly, genes that are highly connected within the network, and thus whose copy number change is predicted by the network to be more phenotypically consequential, are significantly enriched among patients that possess only a single ASD-associated network copy number changed gene (p?=?0.002). Strikingly, deleted or disrupted genes from the network are significantly enriched in GO-annotated positive regulators (2.3-fold enrichment, corrected p?=?2×10(-5)), whereas duplicated genes are significantly enriched in GO-annotated negative regulators (2.2-fold enrichment, corrected p?=?0.005). The direction of copy change is highly informative in the context of the network, providing the means through which perturbations arising from distinct deletions or duplications can yield a common outcome. These findings reveal an extensive ASD-associated molecular network, whose topology indicates ASD-relevant mutational deleteriousness and that mechanistically details how convergent aetiologies can result extensively from CNVs affecting pathways causally implicated in ASD.
Autism Spectrum Disorder (ASD) demonstrates high heritability and familial clustering, yet the genetic causes remain only partially understood as a result of extensive clinical and genomic heterogeneity. Whole-genome sequencing (WGS) shows promise as a tool for identifying ASD risk genes as well as unreported mutations in known loci, but an assessment of its full utility in an ASD group has not been performed. We used WGS to examine 32 families with ASD to detect de novo or rare inherited genetic variants predicted to be deleterious (loss-of-function and damaging missense mutations). Among ASD probands, we identified deleterious de novo mutations in six of 32 (19%) families and X-linked or autosomal inherited alterations in ten of 32 (31%) families (some had combinations of mutations). The proportion of families identified with such putative mutations was larger than has been previously reported; this yield was in part due to the comprehensive and uniform coverage afforded by WGS. Deleterious variants were found in four unrecognized, nine known, and eight candidate ASD risk genes. Examples include CAPRIN1 and AFF2 (both linked to FMR1, which is involved in fragile X syndrome), VIP (involved in social-cognitive deficits), and other genes such as SCN2A and KCNQ2 (linked to epilepsy), NRXN1, and CHD7, which causes ASD-associated CHARGE syndrome. Taken together, these results suggest that WGS and thorough bioinformatic analyses for de novo and rare inherited mutations will improve the detection of genetic variants likely to be associated with ASD or its accompanying clinical symptoms.
The GPHN gene codes for gephyrin, a key scaffolding protein in the neuronal postsynaptic membrane, responsible for the clustering and localization of glycine and GABA receptors at inhibitory synapses. Gephyrin has well-established functional links with several synaptic proteins that have been implicated in genetic risk for neurodevelopmental disorders such as autism spectrum disorder (ASD), schizophrenia and epilepsy including the neuroligins (NLGN2, NLGN4), the neurexins (NRXN1, NRXN2, NRXN3) and collybistin (ARHGEF9). Moreover, temporal lobe epilepsy has been linked to abnormally spliced GPHN mRNA lacking exons encoding the G-domain of the gephyrin protein, potentially arising due to cellular stress associated with epileptogenesis such as temperature and alkalosis. Here, we present clinical and genomic characterization of six unrelated subjects, with a range of neurodevelopmental diagnoses including ASD, schizophrenia or seizures, who possess rare de novo or inherited hemizygous microdeletions overlapping exons of GPHN at chromosome 14q23.3. The region of common overlap across the deletions encompasses exons 3-5, corresponding to the G-domain of the gephyrin protein. These findings, together with previous reports of homozygous GPHN mutations in connection with autosomal recessive molybdenum cofactor deficiency, will aid in clinical genetic interpretation of the GPHN mutation spectrum. Our data also add to the accumulating evidence implicating neuronal synaptic gene products as key molecular factors underlying the etiologies of a diverse range of neurodevelopmental conditions.
The contribution of copy-number variation (CNV) to disease has been highlighted with the widespread adoption of array-based comparative genomic hybridisation (aCGH) and microarray technology. Contiguous gene deletions involving ANKRD11 in 16q24.3 are associated with autism spectrum disorder (ASD) and intellectual disability (ID), while 16q24.1 deletions affecting FOXF1 are associated with congenital renal malformations, alveolar capillary dysplasia, and various other abnormalities. The disease associations of deletions in the intervening region, 16q24.2, have only been defined to a limited extent.
Genomic rearrangements involving AUTS2 (7q11.22) are associated with autism and intellectual disability (ID), although evidence for causality is limited. By combining the results of diagnostic testing of 49,684 individuals, we identified 24 microdeletions that affect at least one exon of AUTS2, as well as one translocation and one inversion each with a breakpoint within the AUTS2 locus. Comparison of 17 well-characterized individuals enabled identification of a variable syndromic phenotype including ID, autism, short stature, microcephaly, cerebral palsy, and facial dysmorphisms. The dysmorphic features were more pronounced in persons with 3AUTS2 deletions. This part of the gene is shown to encode a C-terminal isoform (with an alternative transcription start site) expressed in the human brain. Consistent with our genetic data, suppression of auts2 in zebrafish embryos caused microcephaly that could be rescued by either the full-length or the C-terminal isoform of AUTS2. Our observations demonstrate a causal role of AUTS2 in neurocognitive disorders, establish a hitherto unappreciated syndromic phenotype at this locus, and show how transcriptional complexity can underpin human pathology. The zebrafish model provides a valuable tool for investigating the etiology of AUTS2 syndrome and facilitating gene-function analysis in the future.
Metaphyseal dysplasia with maxillary hypoplasia and brachydactyly (MDMHB) is an autosomal-dominant bone dysplasia characterized by metaphyseal flaring of long bones, enlargement of the medial halves of the clavicles, maxillary hypoplasia, variable brachydactyly, and dystrophic teeth. We performed genome-wide SNP genotyping in five affected and four unaffected members of an extended family with MDMHB. Analysis for copy-number variations revealed that a 105 kb duplication within RUNX2 segregated with the MDMHB phenotype in a region with maximum linkage. Real-time PCR for copy-number variation in genomic DNA in eight samples, as well as sequence analysis of fibroblast cDNA from one subject with MDMHB confirmed that affected family members were heterozygous for the presence of an intragenic duplication encompassing exons 3 to 5 of RUNX2. These three exons code for the Q/A domain and the functionally essential DNA-binding runt domain of RUNX2. Transfection studies with murine Runx2 cDNA showed that cellular levels of mutated RUNX2 were markedly higher than those of wild-type RUNX2, suggesting that the RUNX2 duplication found in individuals with MDMHB leads to a gain of function. Until now, only loss-of-function mutations have been detected in RUNX2; the present report associates an apparent gain-of-function alteration of RUNX2 function with a distinct rare disease.
There is increasing evidence that the phenotypic effects of genomic sequence variants are best understood in terms of variant haplotypes rather than as isolated polymorphisms. Haplotype analysis is also critically important for uncovering population histories and for the study of evolutionary genetics. Although the sequencing of individual human genomes to reveal personal collections of sequence variants is now well established, there has been slower progress in the phasing of these variants into pairs of haplotypes along each pair of chromosomes. Here, we have developed a distinct approach to haplotyping that can yield chromosome-length haplotypes, including the vast majority of heterozygous single-nucleotide polymorphisms (SNPs) in an individual human genome. This approach exploits the haploid nature of sperm cells and employs a combination of genotyping and low-coverage sequencing on a short-read platform. In addition to generating chromosome-length haplotypes, the approach can directly identify recombination events (averaging 1.1 per chromosome) with a median resolution of <100 kb.
We report on two patients with developmental delay, hypotonia, and autistic features associated with duplications of chromosome region 2q23.1-2q23.2 detected by chromosome microarray analysis. The duplications include one OMIM Morbid Map gene, MBD5, as well as seven known RefSeq genes (ACVR2A, ORC4L, EPC2, KIF5C, MIR1978, LYPD6B, and LYPD6). MBD5 lies in the minimum area of overlap of the 2q23.1 microdeletion syndrome. This report provides the first detailed clinical examination of two individuals with a duplication of this region and suggests that brain development and cognitive function may be affected by an increased dosage of the genes involved.
The three members of the human neurexin gene family, neurexin 1 (NRXN1), neurexin 2 (NRXN2), and neurexin 3 (NRXN3), encode neuronal adhesion proteins that have important roles in synapse development and function. In autism spectrum disorder (ASD), as well as in other neurodevelopmental conditions, rare exonic copy-number variants and/or point mutations have been identified in the NRXN1 and NRXN2 loci. We present clinical characterization of four index cases who have been diagnosed with ASD and who possess rare inherited or de novo microdeletions at 14q24.3-31.1, a region that overlaps exons of the alpha and/or beta isoforms of NRXN3. NRXN3 deletions were found in one father with subclinical autism and in a carrier mother and father without formal ASD diagnoses, indicating issues of penetrance and expressivity at this locus. Notwithstanding these clinical complexities, this report on ASD-affected individuals who harbor NRXN3 exonic deletions advances the understanding of the genetic etiology of autism, further enabling molecular diagnoses.
Attention deficit hyperactivity disorder (ADHD) is a common and persistent condition characterized by developmentally atypical and impairing inattention, hyperactivity, and impulsiveness. We identified de novo and rare copy number variations (CNVs) in 248 unrelated ADHD patients using million-feature genotyping arrays. We found de novo CNVs in 3 of 173 (1.7%) ADHD patients for whom we had DNA from both parents. These CNVs affected brain-expressed genes: DCLK2, SORCS1, SORCS3, and MACROD2. We also detected rare inherited CNVs in 19 of 248 (7.7%) ADHD probands, which were absent in 2357 controls and which either overlapped previously implicated ADHD loci (for example, DRD5 and 15q13 microduplication) or identified new candidate susceptibility genes (ASTN2, CPLX2, ZBBX, and PTPRN2). Among these de novo and rare inherited CNVs, there were also examples of genes (ASTN2, GABRG1, and CNTN5) previously implicated by rare CNVs in other neurodevelopmental conditions including autism spectrum disorder (ASD). To further explore the overlap of risks in ADHD and ASD, we used the same microarrays to test for rare CNVs in an independent, newly collected cohort of 349 unrelated individuals with a primary diagnosis of ASD. Deletions of the neuronal ASTN2 and the ASTN2-intronic TRIM32 genes yielded the strongest association with ADHD and ASD, but numerous other shared candidate genes (such as CHCHD3, MACROD2, and the 16p11.2 region) were also revealed. Our results provide support for a role for rare CNVs in ADHD risk and reinforce evidence for the existence of common underlying susceptibility genes for ADHD, ASD, and other neuropsychiatric disorders.
Autism spectrum disorder (ASD) is a highly heritable disorder of complex and heterogeneous aetiology. It is primarily characterized by altered cognitive ability including impaired language and communication skills and fundamental deficits in social reciprocity. Despite some notable successes in neuropsychiatric genetics, overall, the high heritability of ASD (~90%) remains poorly explained by common genetic risk variants. However, recent studies suggest that rare genomic variation, in particular copy number variation, may account for a significant proportion of the genetic basis of ASD. We present a large scale analysis to identify candidate genes which may contain low-frequency recessive variation contributing to ASD while taking into account the potential contribution of population differences to the genetic heterogeneity of ASD. Our strategy, homozygous haplotype (HH) mapping, aims to detect homozygous segments of identical haplotype structure that are shared at a higher frequency amongst ASD patients compared to parental controls. The analysis was performed on 1,402 Autism Genome Project trios genotyped for 1 million single nucleotide polymorphisms (SNPs). We identified 25 known and 1,218 novel ASD candidate genes in the discovery analysis including CADM2, ABHD14A, CHRFAM7A, GRIK2, GRM3, EPHA3, FGF10, KCND2, PDZK1, IMMP2L and FOXP2. Furthermore, 10 of the previously reported ASD genes and 300 of the novel candidates identified in the discovery analysis were replicated in an independent sample of 1,182 trios. Our results demonstrate that regions of HH are significantly enriched for previously reported ASD candidate genes and the observed association is independent of gene size (odds ratio 2.10). Our findings highlight the applicability of HH mapping in complex disorders such as ASD and offer an alternative approach to the analysis of genome-wide association data.
Copy number variations (CNVs) can contribute to variable degrees of fitness and/or disease predisposition. Recent studies show that at least 1% of any given genome is copy number variable when compared to the human reference sequence assembly. Homozygous deletions (or CNV nulls) that are found in the normal population are of particular interest because they may serve to define non-essential genes in human biology.
There are four known pericentromeric euchromatic variants of chromosome 9 in the literature that are increasingly being observed in diagnostic cytogenetic laboratories. These variants pose diagnostic and counselling dilemmas, especially in prenatal settings, as distinction of a pathogenic alteration from a euchromatic variant is difficult. The molecular characterisation of three of these four variants has been reported. In this study, the genomic structure of the fourth variant, an additional G-positive band at 9q13-q21, is characterised.
Both obesity and being underweight have been associated with increased mortality. Underweight, defined as a body mass index (BMI) ??18.5?kg?per?m(2) in adults and ??-2 standard deviations from the mean in children, is the main sign of a series of heterogeneous clinical conditions including failure to thrive, feeding and eating disorder and/or anorexia nervosa. In contrast to obesity, few genetic variants underlying these clinical conditions have been reported. We previously showed that hemizygosity of a ?600-kilobase (kb) region on the short arm of chromosome 16 causes a highly penetrant form of obesity that is often associated with hyperphagia and intellectual disabilities. Here we show that the corresponding reciprocal duplication is associated with being underweight. We identified 138 duplication carriers (including 132 novel cases and 108 unrelated carriers) from individuals clinically referred for developmental or intellectual disabilities (DD/ID) or psychiatric disorders, or recruited from population-based cohorts. These carriers show significantly reduced postnatal weight and BMI. Half of the boys younger than five years are underweight with a probable diagnosis of failure to thrive, whereas adult duplication carriers have an 8.3-fold increased risk of being clinically underweight. We observe a trend towards increased severity in males, as well as a depletion of male carriers among non-medically ascertained cases. These features are associated with an unusually high frequency of selective and restrictive eating behaviours and a significant reduction in head circumference. Each of the observed phenotypes is the converse of one reported in carriers of deletions at this locus. The phenotypes correlate with changes in transcript levels for genes mapping within the duplication but not in flanking regions. The reciprocal impact of these 16p11.2 copy-number variants indicates that severe obesity and being underweight could have mirror aetiologies, possibly through contrasting effects on energy balance.
We report on a female patient with a de novo interstitial deletion of chromosome region 2q23.1-23.3 identified by array-CGH. She had significant global delay with developmental regression at age 6 years. She developed seizures at age 3 years with progressive difficulties with balance, loss of fine motor skills and aggressive behavior. She had short stature, microcephaly, and distinct facial features. Her speech was dysarthric, and she demonstrated repetitive hand movements. In this article, we compare the clinical features of our patient with previously reported cases with a 2q23.1 deletion.
The Autism Tissue Program (ATP), a science program of Autism Speaks, provides researchers with access to well-characterized postmortem brain tissues. Researchers access these tissues through a peer-reviewed, project-based approval process, and obtain related clinical information from a secure, online informatics portal. However, few of these samples have DNA banked from other sources (such as a blood sample from the same individual), hindering genotype-phenotype correlation and interpretation of gene expression data derived from the banked brain tissue. Here, we describe an initiative to extract DNA from Brodmann Area 19, and genotype these samples using both the Affymetrix Genome-Wide Human SNP Array 6.0 and the Illumina Human1M-Duo DNA Analysis BeadChip genome-wide microarray technologies. We additionally verify reported gender, and infer ethnic background from the single nucleotide polymorphism data. We have also used a rigorous, multiple algorithm approach to identify genomic copy number variation (CNV) from these array data. Following an initial proof of principle study using two samples, 52 experimental samples, consisting of 27 subjects with confirmed or suspected autism and related disorders, 5 subjects with cytogenetically visible duplications of 15q, 2 with epilepsy and 18 age-matched normal controls were processed, yielding high-quality genotype data in all cases. The genotype and CNV data are provided via the ATP informatics portal as a resource for the autism research community.
Dup(X)(p11.22-p11.23) has been shown to be associated with intellectual disability (ID, also referred to as mental retardation). Here, we characterize a 4.64 Mb de novo duplication of the same Xp11.22-p11.23 ID region in a female, but for this reference case the diagnosis was Autism Spectrum Disorder (ASD). Besides ASD, she also had very persistent trichotillomania, anxiety symptoms and some non-specific dysmorphic features. We report the detailed clinical features, as well as refine the rearrangement breakpoints of this disease-associated copy number variation region, which encompasses more than 50 genes. We propose that in addition to ID, the phenotypic spectrum associated with dup(X)(p11.22-p11.23) can include ASD, language impairment, and/or other primary psychiatric disorders.
Autism is a common neurodevelopmental disorder with a complex mode of inheritance. It is one of the most highly heritable of the complex disorders, although the underlying genetic factors remain largely unknown. Here, we report mutations in the X-chromosome PTCHD1 (patched-related) gene in seven families with autism spectrum disorder (ASD) and in three families with intellectual disability. A 167-kilobase microdeletion spanning exon 1 was found in two brothers, one with ASD and the other with a learning disability and ASD features; a 90-kilobase microdeletion spanning the entire gene was found in three males with intellectual disability in a second family. In 900 probands with ASD and 208 male probands with intellectual disability, we identified seven different missense changes (in eight male probands) that were inherited from unaffected mothers and not found in controls. Two of the ASD individuals with missense changes also carried a de novo deletion at another ASD susceptibility locus (DPYD and DPP6), suggesting complex genetic contributions. In additional males with ASD, we identified deletions in the 5 flanking region of PTCHD1 that disrupted a complex noncoding RNA and potential regulatory elements; equivalent changes were not found in male control individuals. Thus, our systematic screen of PTCHD1 and its 5 flanking regions suggests that this locus is involved in ~1% of individuals with ASD and intellectual disability.
Accumulating data indicate that there is significant genetic heterogeneity underlying the etiology in individuals diagnosed with autism spectrum disorder (ASD). Some rare and highly-penetrant gene variants and copy number variation (CNV) regions including NLGN3, NLGN4, NRXN1, SHANK2, SHANK3, PTCHD1, 1q21.1, maternally-inherited duplication of 15q11-q13, 16p11.2, amongst others, have been identified to be involved in ASD. Genome-wide association studies have identified other apparently low risk loci and in some other cases, ASD arises as a co-morbid phenotype with other medical genetic conditions (e.g. fragile X). The progress studying the genetics of ASD has largely been accomplished using genomic analyses of germline-derived DNA. Here, we used gene and miRNA expression profiling using cell-line derived total RNA to evaluate possible transcripts and networks of molecules involved in ASD. Our analysis identified several novel dysregulated genes and miRNAs in ASD compared with controls, including HEY1, SOX9, miR-486 and miR-181b. All of these are involved in nervous system development and function and some others, for example, are involved in NOTCH signaling networks (e.g. HEY1). Further, we found significant enrichment in molecules associated with neurological disorders such as Rett syndrome and those associated with nervous system development and function including long-term potentiation. Our data will provide a valuable resource for discovery purposes and for comparison to other gene expression-based, genome-wide DNA studies and other functional data.
Although autism spectrum disorders (ASDs) have a substantial genetic basis, most of the known genetic risk has been traced to rare variants, principally copy number variants (CNVs). To identify common risk variation, the Autism Genome Project (AGP) Consortium genotyped 1558 rigorously defined ASD families for 1 million single-nucleotide polymorphisms (SNPs) and analyzed these SNP genotypes for association with ASD. In one of four primary association analyses, the association signal for marker rs4141463, located within MACROD2, crossed the genome-wide association significance threshold of P < 5 × 10(-8). When a smaller replication sample was analyzed, the risk allele at rs4141463 was again over-transmitted; yet, consistent with the winners curse, its effect size in the replication sample was much smaller; and, for the combined samples, the association signal barely fell below the P < 5 × 10(-8) threshold. Exploratory analyses of phenotypic subtypes yielded no significant associations after correction for multiple testing. They did, however, yield strong signals within several genes, KIAA0564, PLD5, POU6F2, ST8SIA2 and TAF1C.
Recent advances in microarray technology are helping to identify more genetic anomalies associated with tetralogy of Fallot and other congenital heart defects. We report on a 24-year-old woman with a syndromic form of tetralogy of Fallot who was found to have a novel de novo deletion of the proximal long arm of chromosome 13. History of developmental delay and learning difficulties, mild dysmorphic facial features, and anal atresia prompted genetic investigations. A review of the literature on deletions that overlap this region showed that several were associated with major congenital heart defects. The results suggest that the 13q13.1-q13.2 region may harbour a gene or genes important in cardiac development.
The autism spectrum disorders (ASDs) are a group of conditions characterized by impairments in reciprocal social interaction and communication, and the presence of restricted and repetitive behaviours. Individuals with an ASD vary greatly in cognitive development, which can range from above average to intellectual disability. Although ASDs are known to be highly heritable ( approximately 90%), the underlying genetic determinants are still largely unknown. Here we analysed the genome-wide characteristics of rare (<1% frequency) copy number variation in ASD using dense genotyping arrays. When comparing 996 ASD individuals of European ancestry to 1,287 matched controls, cases were found to carry a higher global burden of rare, genic copy number variants (CNVs) (1.19 fold, P = 0.012), especially so for loci previously implicated in either ASD and/or intellectual disability (1.69 fold, P = 3.4 x 10(-4)). Among the CNVs there were numerous de novo and inherited events, sometimes in combination in a given family, implicating many novel ASD genes such as SHANK2, SYNGAP1, DLGAP2 and the X-linked DDX53-PTCHD1 locus. We also discovered an enrichment of CNVs disrupting functional gene sets involved in cellular proliferation, projection and motility, and GTPase/Ras signalling. Our results reveal many new genetic and functional targets in ASD that may lead to final connected pathways.
Using microarrays, we identified de novo copy number variations in the SHANK2 synaptic scaffolding gene in two unrelated individuals with autism-spectrum disorder (ASD) and mental retardation. DNA sequencing of SHANK2 in 396 individuals with ASD, 184 individuals with mental retardation and 659 unaffected individuals (controls) revealed additional variants that were specific to ASD and mental retardation cases, including a de novo nonsense mutation and seven rare inherited changes. Our findings further link common genes between ASD and intellectual disability.
The clinical use of array comparative genomic hybridization in the evaluation of patients with multiple congenital anomalies and/or mental retardation has recently led to the discovery of a number of novel microdeletion and microduplication syndromes. We present four male patients with overlapping molecularly defined de novo microdeletions of 16q24.3. The clinical features observed in these patients include facial dysmorphisms comprising prominent forehead, large ears, smooth philtrum, pointed chin and wide mouth, variable cognitive impairment, autism spectrum disorder, structural anomalies of the brain, seizures and neonatal thrombocytopenia. Although deletions vary in size, the common region of overlap is only 90 kb and comprises two known genes, Ankyrin Repeat Domain 11 (ANKRD11) (MIM 611192) and Zinc Finger 778 (ZNF778), and is located approximately 10 kb distally to Cadherin 15 (CDH15) (MIM 114019). This region is not found as a copy number variation in controls. We propose that these patients represent a novel and distinctive microdeletion syndrome, characterized by autism spectrum disorder, variable cognitive impairment, facial dysmorphisms and brain abnormalities. We suggest that haploinsufficiency of ANKRD11 and/or ZNF778 contribute to this phenotype and speculate that further investigation of non-deletion patients who have features suggestive of this 16q24.3 microdeletion syndrome might uncover other mutations in one or both of these genes.
Recurrent microdeletions and microduplications of approximately 555 kb at 16p11.2 confer susceptibility to autism spectrum disorder (ASD) in up to 1% of ASD patients. No physical or behavioural features have been identified that distinguish these individuals as having a distinct ASD subtype, but clinical data are limited.
Members of the Na+/Ca2+ exchanger (NCX) family are important regulators of cytosolic Ca2+ in myriad tissues and are highly conserved across a wide range of species. Three distinct NCX genes and numerous splice variants exist in mammals, many of which have been characterized in a variety of heterologous expression systems. Recently, however, we discovered a fourth NCX gene (NCX4), which is found exclusively in teleost, amphibian, and reptilian genomes. Zebrafish (Danio rerio) NCX4a encodes for a protein of 939 amino acids and shows a high degree of identity with known NCXs. Although knockdown of NCX4a activity in zebrafish embryos has been shown to alter left-right patterning, it has not been demonstrated that NCX4a functions as a NCX. In this study, we 1) demonstrated, for the first time, that this gene encodes for a novel NCX; 2) characterized the tissue distribution of zebrafish NCX4a; and 3) evaluated its kinetic and transport properties. While ubiquitously expressed, the highest levels of NCX4a expression occurred in the brain and eyes. NCX4a exhibits modest levels of Na+-dependent inactivation and requires much higher levels of regulatory Ca2+ to activate outward exchange currents. NCX4a also exhibited extremely fast recovery from Na+-dependent inactivation of outward currents, faster than any previously characterized wild-type exchanger. While this result suggests that the Na+-dependent inactive state of NCX4a is far less stable than in other NCX family members, this exchanger was still strongly inhibited by 2 microM exchanger inhibitory peptide. We demonstrated that a new putative member of the NCX gene family, NCX4a, encodes for a NCX with unique functional properties. These data will be useful in understanding the role that NCX4a plays in embryological development as well as in the adult, where it is expressed ubiquitously.
Autism is a complex childhood neurodevelopmental disorder with a strong genetic basis. Microdeletion or duplication of a approximately 500-700-kb genomic rearrangement on 16p11.2 that contains 24 genes represents the second most frequent chromosomal disorder associated with autism. The role of common and rare 16p11.2 sequence variants in autism etiology is unknown.
The identification of rare inherited and de novo copy number variations (CNVs) in human subjects has proven a productive approach to highlight risk genes for autism spectrum disorder (ASD). A variety of microarrays are available to detect CNVs, including single-nucleotide polymorphism (SNP) arrays and comparative genomic hybridization (CGH) arrays. Here, we examine a cohort of 696 unrelated ASD cases using a high-resolution one-million feature CGH microarray, the majority of which were previously genotyped with SNP arrays. Our objective was to discover new CNVs in ASD cases that were not detected by SNP microarray analysis and to delineate novel ASD risk loci via combined analysis of CGH and SNP array data sets on the ASD cohort and CGH data on an additional 1000 control samples. Of the 615 ASD cases analyzed on both SNP and CGH arrays, we found that 13,572 of 21,346 (64%) of the CNVs were exclusively detected by the CGH array. Several of the CGH-specific CNVs are rare in population frequency and impact previously reported ASD genes (e.g., NRXN1, GRM8, DPYD), as well as novel ASD candidate genes (e.g., CIB2, DAPP1, SAE1), and all were inherited except for a de novo CNV in the GPHN gene. A functional enrichment test of gene-sets in ASD cases over controls revealed nucleotide metabolism as a potential novel pathway involved in ASD, which includes several candidate genes for follow-up (e.g., DPYD, UPB1, UPP1, TYMP). Finally, this extensively phenotyped and genotyped ASD clinical cohort serves as an invaluable resource for the next step of genome sequencing for complete genetic variation detection.
CONTEXT Brain-derived neurotrophic factor (BDNF) is suspected of being a causative factor in psychiatric disorders based on case reports or studies involving large structural anomalies. OBJECTIVE To determine the involvement of BDNF in human psychopathology. DESIGN Case-control study. SETTING Microarray-based comparative genomic hybridization data from 7 molecular diagnostic centers including 38 550 affected subjects and 28 705 unaffected subjects. PATIENTS Subjects referred to diagnostic screening centers for microarray-based comparative genomic hybridization for physical or cognitive impairment. MAIN OUTCOME MEASURES Genomic copy number gains and losses. RESULTS We report 5 individuals with psychopathology and genomic deletion of a critical region including BDNF. The defined critical region was never disrupted in control subjects or diagnostic cases without developmental abnormalities. CONCLUSION Hemizygosity of the BDNF region contributes to variable psychiatric phenotypes including anxiety, behavioral, and mood disorders.
Rare, recurrent chromosome 1q21.1 duplications have been associated with developmental delay, congenital anomalies, and macrocephaly in children. Data on adult clinical expression would help to inform genetic counseling.
Left-sided congenital heart disease (CHD) encompasses a spectrum of malformations that range from bicuspid aortic valve to hypoplastic left heart syndrome. It contributes significantly to infant mortality and has serious implications in adult cardiology. Although left-sided CHD is known to be highly heritable, the underlying genetic determinants are largely unidentified. In this study, we sought to determine the impact of structural genomic variation on left-sided CHD and compared multiplex families (464 individuals with 174 affecteds (37.5%) in 59 multiplex families and 8 trios) to 1,582 well-phenotyped controls. 73 unique inherited or de novo CNVs in 54 individuals were identified in the left-sided CHD cohort. After stringent filtering, our gene inventory reveals 25 new candidates for LS-CHD pathogenesis, such as SMC1A, MFAP4, and CTHRC1, and overlaps with several known syndromic loci. Conservative estimation examining the overlap of the prioritized gene content with CNVs present only in affected individuals in our cohort implies a strong effect for unique CNVs in at least 10% of left-sided CHD cases. Enrichment testing of gene content in all identified CNVs showed a significant association with angiogenesis. In this first family-based CNV study of left-sided CHD, we found that both co-segregating and de novo events associate with disease in a complex fashion at structural genomic level. Often viewed as an anatomically circumscript disease, a subset of left-sided CHD may in fact reflect more general genetic perturbations of angiogenesis and/or vascular biology.
Structural genetic changes, especially copy number variants (CNVs), represent a major source of genetic variation contributing to human disease. Tetralogy of Fallot (TOF) is the most common form of cyanotic congenital heart disease, but to date little is known about the role of CNVs in the etiology of TOF. Using high-resolution genome-wide microarrays and stringent calling methods, we investigated rare CNVs in a prospectively recruited cohort of 433 unrelated adults with TOF and/or pulmonary atresia at a single centre. We excluded those with recognized syndromes, including 22q11.2 deletion syndrome. We identified candidate genes for TOF based on converging evidence between rare CNVs that overlapped the same gene in unrelated individuals and from pathway analyses comparing rare CNVs in TOF cases to those in epidemiologic controls. Even after excluding the 53 (10.7%) subjects with 22q11.2 deletions, we found that adults with TOF had a greater burden of large rare genic CNVs compared to controls (8.82% vs. 4.33%, p?=?0.0117). Six loci showed evidence for recurrence in TOF or related congenital heart disease, including typical 1q21.1 duplications in four (1.18%) of 340 Caucasian probands. The rare CNVs implicated novel candidate genes of interest for TOF, including PLXNA2, a gene involved in semaphorin signaling. Independent pathway analyses highlighted developmental processes as potential contributors to the pathogenesis of TOF. These results indicate that individually rare CNVs are collectively significant contributors to the genetic burden of TOF. Further, the data provide new evidence for dosage sensitive genes in PLXNA2-semaphorin signaling and related developmental processes in human cardiovascular development, consistent with previous animal models.
Progressive myoclonus epilepsies are severe, intractable, and neurodegenerative. They afflict patients of all ages, but more commonly adolescents, and comprise the main differential diagnosis of common juvenile myoclonic epilepsy. Genetic or minimally invasive pathologic diagnoses are available for many but not all teenage-onset progressive myoclonus epilepsies. We describe a multiplex family with autosomal recessive teenage-onset progressive myoclonus epilepsy that had remained undiagnosed despite extensive genetic and pathologic testing. We describe whole exome sequencing combined with homozygosity mapping to identify the disease gene directly and diagnose the family. The affected gene is CLN6, previously known to underlie variant late-infantile and adult-onset neuronal ceroid lipofuscinoses. Combined with other recent work, our results add CLN6 to the genetic mutations causing teenage-onset progressive myoclonus epilepsy, expand the group of teenage-onset progressive myoclonus epilepsy patients who can be diagnosed by genetic testing, and extend the clinical spectrum of CLN6 mutations to include teenage-onset progressive myoclonus epilepsy. This work also exemplifies the potentiality of next-generation sequencing in the genetic identification and diagnosis of patients with neurologic diseases of unknown cause.
Medulloblastoma, the most common malignant paediatric brain tumour, is currently treated with nonspecific cytotoxic therapies including surgery, whole-brain radiation, and aggressive chemotherapy. As medulloblastoma exhibits marked intertumoural heterogeneity, with at least four distinct molecular variants, previous attempts to identify targets for therapy have been underpowered because of small samples sizes. Here we report somatic copy number aberrations (SCNAs) in 1,087 unique medulloblastomas. SCNAs are common in medulloblastoma, and are predominantly subgroup-enriched. The most common region of focal copy number gain is a tandem duplication of SNCAIP, a gene associated with Parkinsons disease, which is exquisitely restricted to Group 4?. Recurrent translocations of PVT1, including PVT1-MYC and PVT1-NDRG1, that arise through chromothripsis are restricted to Group 3. Numerous targetable SCNAs, including recurrent events targeting TGF-? signalling in Group 3, and NF-?B signalling in Group 4, suggest future avenues for rational, targeted therapy.
Adenocarcinoma of the pancreas is a significant cause of cancer mortality, and up to 10 % of cases appear to be familial. Heritable genomic copy number variants (CNVs) can modulate gene expression and predispose to disease. Here, we identify candidate predisposition genes for familial pancreatic cancer (FPC) by analyzing germline losses or gains present in one or more high-risk patients and absent in a large control group. A total of 120 FPC cases and 1,194 controls were genotyped on the Affymetrix 500K array, and 36 cases and 2,357 controls were genotyped on the Affymetrix 6.0 array. Detection of CNVs was performed by multiple computational algorithms and partially validated by quantitative PCR. We found no significant difference in the germline CNV profiles of cases and controls. A total of 93 non-redundant FPC-specific CNVs (53 losses and 40 gains) were identified in 50 cases, each CNV present in a single individual. FPC-specific CNVs overlapped the coding region of 88 RefSeq genes. Several of these genes have been reported to be differentially expressed and/or affected by copy number alterations in pancreatic adenocarcinoma. Further investigation in high-risk subjects may elucidate the role of one or more of these genes in genetic predisposition to pancreatic cancer.
Hirschsprung disease (HSCR) is a congenital disorder characterized by aganglionosis of the distal intestine. To assess the contribution of copy number variants (CNVs) to HSCR, we analysed the data generated from our previous genome-wide association study on HSCR patients, whereby we identified NRG1 as a new HSCR susceptibility locus. Analysis of 129 Chinese patients and 331 ethnically matched controls showed that HSCR patients have a greater burden of rare CNVs (p = 1.50 × 10(-5)), particularly for those encompassing genes (p = 5.00 × 10(-6)). Our study identified 246 rare-genic CNVs exclusive to patients. Among those, we detected a NRG3 deletion (p = 1.64 × 10(-3)). Subsequent follow-up (96 additional patients and 220 controls) on NRG3 revealed 9 deletions (combined p = 3.36 × 10(-5)) and 2 de novo duplications among patients and two deletions among controls. Importantly, NRG3 is a paralog of NRG1. Stratification of patients by presence/absence of HSCR-associated syndromes showed that while syndromic-HSCR patients carried significantly longer CNVs than the non-syndromic or controls (p = 1.50 × 10(-5)), non-syndromic patients were enriched in CNV number when compared to controls (p = 4.00 × 10(-6)) or the syndromic counterpart. Our results suggest a role for NRG3 in HSCR etiology and provide insights into the relative contribution of structural variants in both syndromic and non-syndromic HSCR. This would be the first genome-wide catalog of copy number variants identified in HSCR.
Recent studies have highlighted the involvement of rare (<1% frequency) copy-number variations and point mutations in the genetic etiology of autism spectrum disorder (ASD); these variants particularly affect genes involved in the neuronal synaptic complex. The SHANK gene family consists of three members (SHANK1, SHANK2, and SHANK3), which encode scaffolding proteins required for the proper formation and function of neuronal synapses. Although SHANK2 and SHANK3 mutations have been implicated in ASD and intellectual disability, the involvement of SHANK1 is unknown. Here, we assess microarray data from 1,158 Canadian and 456 European individuals with ASD to discover microdeletions at the SHANK1 locus on chromosome 19. We identify a hemizygous SHANK1 deletion that segregates in a four-generation family in which male carriers--but not female carriers--have ASD with higher functioning. A de novo SHANK1 deletion was also detected in an unrelated male individual with ASD with higher functioning, and no equivalent SHANK1 mutations were found in >15,000 controls (p = 0.009). The discovery of apparent reduced penetrance of ASD in females bearing inherited autosomal SHANK1 deletions provides a possible contributory model for the male gender bias in autism. The data are also informative for clinical-genetics interpretations of both inherited and sporadic forms of ASD involving SHANK1.
Attention deficit hyperactivity disorder (ADHD) is a common, highly heritable psychiatric disorder. Because of its multifactorial etiology, however, identifying the genes involved has been difficult. The authors followed up on recent findings suggesting that rare copy number variants (CNVs) may be important for ADHD etiology.
There now exist multiple lines of evidence pointing to a significant genetic component underlying the aetiology of autism spectrum disorders (ASDs). The advent of methodologies for scanning the human genome at high resolution, coupled with the recognition of copy number variation (CNV) as a prevalent source of genomic variation, has led to new strategies in the identification of clinically relevant loci. Balanced genomic changes, such as translocations and inversions, also contribute to ASD, but current studies have shown that screening with microarrays has up to fivefold increase in diagnostic yield. Recent work by our group and others has shown unbalanced genomic alterations that are likely pathogenic in upwards of 10% of cases, highlighting an important role for CNVs in the genetic aetiology of ASD. A trend in our empirical data has shifted focus for discovery of candidate loci towards individually rare but highly penetrant CNVs instead of looking for common variants of low penetrance. This strategy has proven largely successful in identifying ASD-susceptibility candidate loci, including gains and losses at 16p11.2, SHANK2, NRXN1, and PTCHD1. Another emerging and intriguing trend is the identification of the same genes implicated by rare CNVs across neurodevelopmental disorders, including schizophrenia, attention deficit hyperactivity disorder, and intellectual disability. These observations indicate that similar pathways may be involved in phenotypically distinct outcomes. Although interrogation of the genome at high resolution has led to these novel discoveries, it has also made cataloguing, characterization, and clinical interpretation of the increasing amount of CNV data difficult. Herein, we describe the history of genomic structural variation in ASD and how CNV discovery has been used to pinpoint novel ASD-susceptibility loci. We also discuss the overlap of CNVs across neurodevelopmental disorders and comment on the current challenges of understanding the relationship between CNVs and associated phenotypes in a clinical context.
Ataxia demonstrates substantial phenotypic and genetic heterogeneity. We set out to determine the diagnostic yield of exome sequencing in pediatric patients with ataxia without a molecular diagnosis after standard-of-care assessment in Canada. FORGE (Finding Of Rare disease GEnes) Canada is a nation-wide project focused on identifying novel disease genes for rare pediatric diseases using whole-exome sequencing (WES). We retrospectively selected all FORGE Canada projects that included cerebellar ataxia as a feature. We identified 28 such families and a molecular diagnosis was made in 13; a success rate of 46%. In 11 families we identified mutations in genes associated with known neurological syndromes and in two we identified novel disease genes. Exome analysis of sib pairs and/or patients born to consanguineous parents was more likely to be successful (9/13) than simplex cases (4/15). Our data suggests that exome sequencing is an effective first line test for pediatric patients with ataxia where a specific single gene is not immediately suspected to be causative.
Related JoVE Video
Journal of Visualized Experiments
What is Visualize?
JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.
How does it work?
We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.
Video X seems to be unrelated to Abstract Y...
In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.