Recurrent copy number variations as risk factors for autism spectrum disorders: analysis of the clinical implications

Chromosomal microarray analysis (CMA) is currently considered a first‐tier diagnostic assay for the investigation of autism spectrum disorders (ASD), developmental delay and intellectual disability of unknown etiology. High‐resolution arrays were utilized for the identification of copy number variations (CNVs) in 195 ASD patients of Greek origin (126 males, 69 females). CMA resulted in the detection of 65 CNVs, excluding the known polymorphic copy number polymorphisms also found in the Database of Genomic Variants, for 51/195 patients (26.1%). Parental DNA testing in 20/51 patients revealed that 17 CNVs were de novo, 6 paternal and 3 of maternal origin. The majority of the 65 CNVs were deletions (66.1%), of which 5 on the X‐chromosome while the duplications, of which 7 on the X‐chromosome, were rarer (22/65, 33.8%). Fifty‐one CNVs from a total of 65, reported for our cohort of ASD patients, were of diagnostic significance and well described in the literature while 14 CNVs (8 losses, 6 gains) were characterized as variants of unknown significance and need further investigation. Among the 51 patients, 39 carried one CNV, 10 carried two CNVs and 2 carried three CNVs. The use of CMA, its clinical validity and utility was assessed.     

Application of array comparative genomic hybridization in 102 patients with epilepsy and additional neurodevelopmental disorders

Copy-number variants (CNVs) collectively represent an important cause of neurodevelopmental disorders such as developmental delay (DD)/intellectual disability (ID), autism, and epilepsy. In contrast to DD/ID, for which the application of microarray techniques enables detection of pathogenic CNVs in ～10-20% of patients, there are only few studies of the role of CNVs in epilepsy and genetic etiology in the vast majority of cases remains unknown. We have applied whole-genome exon-targeted oligonucleotide array comparative genomic hybridization (array CGH) to a cohort of 102 patients with various types of epilepsy with or without additional neurodevelopmental abnormalities. Chromosomal microarray analysis revealed 24 non-polymorphic CNVs in 23 patients, among which 10 CNVs are known to be clinically relevant. Two rare deletions in 2q24.1q24.3, including KCNJ3 and 9q21.13 are novel pathogenic genetic loci and 12 CNVs are of unknown clinical significance. Our results further support the notion that rare CNVs can cause different types of epilepsy, emphasize the efficiency of detecting novel candidate genes by whole-genome array CGH, and suggest that the clinical application of array CGH should be extended to patients with unexplained epilepsies. ? 2012 Wiley Periodicals, Inc.     

Chromosomal microaberrations in patients with epilepsy,intellectual disability,and congenital anomalies

Epilepsy is a common finding in patients with chromosomal macro‐ and micro‐rearrangements but only few aberrations show a constant pattern of seizures. DNA array‐based studies have reported causative copy number variations (CNVs) in 5–30% of patients with epilepsy with or without co‐morbidities. The interpretation of many of the detected CNVs remains challenging. In order to identify CNVs carrying epilepsy‐related genes we investigated 43 children with various patterns of epileptic seizures, intellectual disability (ID), and minor dysmorphism, using the Illumina® Infinium Human1M‐DuoV1 array. In three patients we found likely causative de novo CNVs, i.e. deletions in 1q41q42.12 (3.4 Mb) and 19p13.2 (834 kb), and a mosaic two‐segment duplication in 17p13.2 (218 kb) and 17p13.1 (422 kb). In six additional patients there were aberrations (a deletion in one and duplications in five patients) with uncertain clinical consequences. In total, the finding of causative chromosomal micro‐rearrangements in 3 out of 43 patients (7%) and potentially causative CNVs in 6 additional patients (14%) with epilepsy and ID but without major malformations confirms the power of DNA arrays for the detection of new disease‐related genetic regions.     

Copy number variants in people with autism spectrum disorders and co-morbid psychosis

The genetic association between autism spectrum disorder (ASD) and psychotic disorders such as schizophrenia is complicated and mirrors the clinical overlap between these conditions to some degree. However, no studies to date have examined the genetics of individuals dually diagnosed with both ASD and psychosis. In this study, we present findings of copy number variants (CNVs) from a study of 116 well-characterised individuals with this dual diagnosis. DNA was extracted and arrayed using the Affymetrix CytoScan HD 2.8M array or the Affymetrix Cytogenetics arrays and compared with existing samples from the Database of Genomic Variants and the Simons Simplex Collection of CNVs from individuals with ASD and their families. Twenty-seven novel CNVs ≥20k base pairs were identified in the sample, most occurring in only a single individual, although two were found in two female participants. Forty-nine rare CNVs (<1.5% rate in general population) were also found at significantly higher frequencies than expected. The findings may provide evidence for areas of further study in the understanding of the development of both ASD and psychosis due to the number of affected genetic regions that have not previously been linked to these conditions.     

Array CGH in patients with developmental delay or intellectual disability: are there phenotypic clues to pathogenic copy number variants?

Shoukier M, Klein N, Auber B, Wickert J, Schröder J, Zoll B, Burfeind P, Bartels I, Alsat EA, Lingen M, Grzmil P, Schulze S, Keyser J, Weise D, Borchers M, Hobbiebrunken E, Röbl M, Gärtner J, Brockmann K, Zirn B. Array CGH in patients with developmental delay or intellectual disability: are there phenotypic clues to pathogenic copy number variants? Array comparative genomic hybridization (array CGH) is now widely adopted as a first‐tier clinical diagnostic test in individuals with unexplained developmental delay/intellectual disability (DD/ID) and congenital anomalies. Our study aimed at enlarging the phenotypic spectrum associated with clinically relevant copy number variants (CNVs) as well as delineating clinical criteria, which may help separating patients with pathogenic CNVs from those without pathogenic CNVs. We performed a retrospective review of clinical and array CGH data of 342 children with unexplained DD/ID. The phenotypic features of patients with clinically significant CNV were compared with those without pathogenic CNVs. Array CGH detected pathogenic CNVs in 13.2% of the patients. Congenital anomalies, especially heart defects, as well as primary microcephaly, short stature and failure to thrive were clearly more frequent in children with pathogenic CNVs compared with children with normal array CGH results. Thus, we assume that in patients with unexplained DD/ID, array CGH will more probably detect a significant CNV if any of these features is part of the patient's phenotype.     

Copy number variation and association analysis of SHANK3 as a candidate gene for autism in the IMGSAC collection

SHANK3 is located on chromosome 22q13.3 and encodes a scaffold protein that is found in excitatory synapses opposite the pre-synaptic active zone. SHANK3 is a binding partner of neuroligins, some of whose genes contain mutations in a small subset of individuals with autism. In individuals with autism spectrum disorders (ASDs), several studies have found SHANK3 to be disrupted by deletions ranging from hundreds of kilobases to megabases, suggesting that 1% of individuals with ASDs may have these chromosomal aberrations. To further analyse the involvement of SHANK3 in ASD, we screened the International Molecular Genetic Study of Autism Consortium (IMGSAC) multiplex family sample, 330 families, for SNP association and copy number variants (CNVs) in SHANK3. A collection of 76 IMGSAC Italian probands from singleton families was also examined by multiplex ligation-dependent probe amplification for CNVs. No CNVs or SNP associations were found within the sample set, although sequencing of the gene was not performed. Our data suggest that SHANK3 deletions may be limited to lower functioning individuals with autism.     

Copy number variation characteristics in subpopulations of patients with autism spectrum disorders

Autism spectrum disorders (ASDs) are a heterogeneous group of disorders with a complex genetic etiology. We used high‐resolution whole genome array‐based comparative genomic hybridization (array‐CGH) to screen 223 ASD patients for gene dose alterations associated with susceptibility for autism. Clinically significant copy number variations (CNVs) were identified in 18 individuals (8%), of which 9 cases (4%) had de novo aberrations. In addition, 20 individuals (9%) were shown to have CNVs of unclear clinical relevance. Among these, 13 cases carried rare but inherited CNVs that may increase the risk for developing ASDs, while parental samples were unavailable in the remaining seven cases. Classification of all patients into different phenotypic and inheritance pattern groups indicated the presence of different CNV patterns in different patient groups. Clinically relevant CNVs were more common in syndromic cases compared to non‐syndromic cases. Rare inherited CNVs were present in a higher proportion of ASD cases having first‐ or second‐degree relatives with an ASD‐related neuropsychiatric phenotype in comparison with cases without reported heredity (P = 0.0096). We conclude that rare CNVs, encompassing potential candidate regions for ASDs, increase the susceptibility for the development of ASDs and related neuropsychiatric disorders giving us further insight into the complex genetics underlying ASDs. © 2010 Wiley‐Liss, Inc.     

Identification of single gene deletions at 15q13.3: further evidence that CHRNA7 causes the 15q13.3 microdeletion syndrome phenotype

The 15q13.3 microdeletion syndrome (OMIM #612001) is characterized by a wide range of phenotypic features, including intellectual disability, seizures, autism, and psychiatric conditions. This deletion is inherited in approximately 75% of cases and has been found in mildly affected and normal parents, consistent with variable expressivity and incomplete penetrance. The common deletion is approximately 2 Mb and contains several genes; however, the gene(s) responsible for the resulting clinical features have not been clearly defined. Recently, four probands were reported with small deletions including only the CHRNA7 gene. These patients showed a wide range of phenotypic features similar to those associated with the larger 15q13.3 microdeletion. To further correlate genotype and phenotype, we queried our database of >15,000 patients tested in the Mayo Clinic Cytogenetics Laboratory from 2008 to 2011 and identified 19 individuals (10 probands and 9 family members) with isolated heterozygous CHRNA7 gene deletions. All but two infants displayed multiple features consistent with 15q13.3 microdeletion syndrome. We also identified the first de novo deletion confined to CHRNA7 as well as the second known case with homozygous deletion of CHRNA7 only. These results provide further evidence implicating CHRNA7 as the gene responsible for the clinical findings associated with 15q13.3 microdeletion.     

Three patients with Schaaf–Yang syndrome exhibiting arthrogryposis and endocrinological abnormalities

MAGEL2 is the paternally expressed gene within Prader–Willi syndrome critical region at 15q11.2. We encountered three individuals in whom truncating mutations of MAGEL2 were identified. Patients 1 and 2, siblings born to healthy, non‐consanguineous Japanese parents, showed generalized hypotonia, lethargy, severe respiratory difficulty, poor feeding, and multiple anomalies including arthrogryposis soon after birth. We carried out whole‐exome sequencing, which detected a MAGEL2 mutation (c.1912C>T, p.Gln638*, heterozygous). The patients’ father was heterozygous for the mutation. Patient 3 was a female infant, showed respiratory difficulty reflecting pulmonary hypoplasia, generalized hypotonia, feeding difficulty and multiple anomalies soon after birth. Targeted next‐generation sequencing detected a novel heterozygous mutation in MAGEL2 (c.3131C>A, p.Ser1044*). This mutation was not found in the parents. MAGEL2 mutations, first reported to be the cause of the Prader–Willi like syndrome with autism by Schaaf et al. (2013) Nature Genetics, 45: 1405–1408 show the wide range of phenotypic spectrum from lethal arthrogryposis multiplex congenital to autism spectrum disorder (ASD) and mild intellectual disability (ID). Our results indicate that MAGEL2 mutations cause multiple congenital anomalies and intellectual disability accompanied by arthrogryposis multiplex congenita and various endocrinologic abnormalities, supporting that the view that clinical phenotypes of MAGEL2 mutations are variable.

     

Further evidence of GABRA4 and TOP3B as autism susceptibility genes

Chromosomal copy number variants (CNVs) are known contributors to neurodevelopmental conditions such as autism spectrum disorder (ASD). Both array comparative genomic hybridization and next-generation sequencing techniques have led to an increased detection of small CNVs and the identification of many candidate susceptibility genes for ASD. We report familial inheritance of two CNVs that include genes with known involvement in neurodevelopment. These CNVs are found in various combinations among four siblings with autism spectrum disorder, as well as in their neurodevelopmentally normal parents. We describe a 2.4 Mb duplication of 4p12 to 4p11 that includes GABRA4 (OMIM: 137141) and other GABA receptor genes, as well as a 246 kb deletion at 22q11.22 involving the TOP3B gene (OMIM: 603582). The maternally inherited 4p duplication was detected in three siblings, two of whom also had the paternally inherited 22q11.22 deletion. The fourth sibling only had the 22q11.22 deletion. These CNVs have rarely been reported in the literature. Upon review, a single publication was found describing a similar 4p duplication in three generations of a family with neurodevelopmental and neuropsychiatric disorders, as well as in an unrelated patient with autism (Polan et al., 2014). TOP3B falls within the distal 22q11.22 microdeletion syndrome and has been associated with schizophrenia, neurodevelopmental disorders including epilepsy, and cardiac defects. The identification of this family contributes to the understanding of specific genetic contributors to neurodevelopmental disorders and an emerging phenotype associated with proximal 4p duplication.     

Genotype–phenotype analysis of 18q12.1-q12.2 copy number variation in autism

Autism Spectrum Disorders (ASD) are complex neurodevelopmental conditions characterized by delays in social interactions and communication as well as displays of restrictive/repetitive interests. DNA copy number variants have been identified as a genomic susceptibility factor in ASDs and imply significant genetic heterogeneity. We report a 7-year-old female with ADOS-G and ADI-R confirmed autistic disorder harbouring a de novo 4 Mb duplication (18q12.1). Our subject displays severely deficient expressive language, stereotypic and repetitive behaviours, mild intellectual disability (ID), focal epilepsy, short stature and absence of significant dysmorphic features. Search of the PubMed literature and DECIPHER database identified 4 additional cases involving 18q12.1 associated with autism and/or ID that overlap our case: one duplication, two deletions and one balanced translocation. Notably, autism and ID are seen with genomic gain or loss at 18q12.1, plus epilepsy and short stature in duplication cases, and hypotonia and tall stature in deletion cases. No consistent dysmorphic features were noted amongst the reviewed cases. We review prospective ASD/ID candidate genes integral to 18q12.1, including those coding for the desmocollin/desmoglein cluster, ring finger proteins 125 and 138, trafficking protein particle complex 8 and dystrobrevin-alpha. The collective clinical and molecular features common to microduplication 18q12.1 suggest that dosage-sensitive, position or contiguous gene effects may be associated in the etiopathogenesis of this autism-ID-epilepsy syndrome.     

De novo variants in KLF7 are a potential novel cause of developmental delay/intellectual disability,neuromuscular and psychiatric symptoms

Due to small numbers of reported patients with pathogenic variants in single genes, the phenotypic spectrum associated with genes causing neurodevelopmental disorders such as intellectual disability (ID) and autism spectrum disorder is expanding. Among these genes is KLF7 (Krüppel‐like factor 7), which is located at 2q33.3 and has been implicated in several developmental processes. KLF7 has been proposed to be a candidate gene for the phenotype of autism features seen in patients with a 2q33.3q34 deletion. Herein, we report 4 unrelated individuals with de novo KLF7 missense variants who share similar clinical features of developmental delay/ID, hypotonia, feeding/swallowing issues, psychiatric features and neuromuscular symptoms, and add to the knowledge about the phenotypic spectrum associated with KLF7 haploinsufficiency.     

Epilepsy with cognitive deficit and autism spectrum disorders: Prospective diagnosis by array CGH

The clinical significance of chromosomal microdeletions and microduplications was predicted based on their gene content, de novo or familial inheritance and accumulated knowledge recorded on public databases. A patient group comprised of 247 cases with epilepsy and its common co‐morbidities of developmental delay, intellectual disability, autism spectrum disorders, and congenital abnormalities was reviewed prospectively in a diagnostic setting using a standardized oligo‐array CGH platform. Seventy‐three (29.6%) had copy number variations (CNVs) and of these 73 cases, 27 (37.0%) had CNVs that were likely causative. These 27 cases comprised 10.9% of the 247 cases reviewed. The range of pathogenic CNVs associated with seizures was consistent with the existence of many genetic determinants for epilepsy. © 2012 Wiley Periodicals, Inc.     

Detection of copy-number variation in AUTS2 gene by targeted exonic array CGH in patients with developmental delay and autistic spectrum disorders

Small genomic rearrangements and copy-number variations (CNVs) involving a single gene have been associated recently with many neurocognitive phenotypes, including intellectual disability (ID), behavioral abnormalities, and autistic spectrum disorders (ASDs). Such small CNVs in the Autism susceptibility candidate 2 (AUTS2) gene have been shown to be associated with seizures, ID, and ASDs. We report four patients with small CNVs ranging in size between 133–319 kb that disrupt AUTS2. Two patients have duplications involving single exons, whereas two have deletions that removed multiple exons. All patients had developmental delay, whereas two patients had a diagnosis of ASDs. The CNVs were detected by an exon-targeted array CGH with dense oligonucleotide coverage in exons of genes known or hypothesized to be causative of multiple human phenotypes. Our report further shows that disruption of AUTS2 results in a variety of neurobehavioral phenotypes. More importantly, it demonstrates the utility of targeted exon array as a highly sensitive clinical diagnostic tool for the detection of small genomic rearrangements in the clinically relevant regions of the human genome.     

High‐resolution molecular karyotyping in patients with developmental delay and/or multiple congenital anomalies in a clinical setting

Wincent J, Anderlid B‐M, Lagerberg M, Nordenskjöld M, Schoumans J. High‐resolution molecular karyotyping in patients with developmental delay and/or multiple congenital anomalies in a clinical setting. Microarray‐based comparative genomic hybridization (array‐CGH) enables genomewide investigation of copy‐number changes at high resolution and has recently been implemented as a clinical diagnostic tool. In this study we evaluate the usefulness of high‐resolution arrays as a diagnostic tool in our laboratory and investigate the diagnostic yield in the first 160 patients who were clinically referred for investigation of developmental delay (DD)/multiple congenital anomalies (MCA). During this period both 38K BAC‐arrays and 244K oligonucleotide‐arrays were used. Copy‐number variations (CNVs) not previously reported as normal variants were detected in 22.5% of cases. In 13.1% the aberrations were considered causal to the phenotype and in 9.4% the clinical significance was uncertain. There was no difference in diagnostic yield between patients with mild, moderate or severe DD. Although the effective resolution of the 244K oligonucleotide‐arrays was higher than the 38K BAC‐array, the diagnostic yield of both platforms was approximately equal and no causal aberrations <300 kb were detected in this patient cohort. We experienced that increasing the resolution of a whole genome screen in the diagnostic setting has its drawback of detecting an increased number of CNVs with uncertain contribution to a phenotype. Based on our experiences, array‐CGH is recommended as the first‐step analysis in the genetic evaluation of patients with DD and/or MCA.     

Genomic architecture of aggression: Rare copy number variants in intermittent explosive disorder

Copy number variants (CNVs) are known to be associated with complex neuropsychiatric disorders (e.g., schizophrenia and autism) but have not been explored in the isolated features of aggressive behaviors such as intermittent explosive disorder (IED). IED is characterized by recurrent episodes of aggression in which individuals act impulsively and grossly out of proportion from the involved stressors. Previous studies have identified genetic variants in the serotonergic pathway that play a role in susceptibility to this behavior, but additional contributors have not been identified. Therefore, to further delineate possible genetic influences, we investigated CNVs in individuals diagnosed with IED and/or personality disorder (PD). We carried out array comparative genomic hybridization on 113 samples of individuals with isolated features of IED (n = 90) or PD (n = 23). We detected a recurrent 1.35‐Mbp deletion on chromosome 1q21.1 in one IED subject and a novel ～350‐kbp deletion on chromosome 16q22.3q23.1 in another IED subject. While five recent reports have suggested the involvement of an ～1.6‐Mbp 15q13.3 deletion in individuals with behavioral problems, particularly aggression, we report an absence of such events in our study of individuals specifically selected for aggression. We did, however, detect a smaller ～430‐kbp 15q13.3 duplication containing CHRNA7 in one individual with PD. While these results suggest a possible role for rare CNVs in identifying genes underlying IED or PD, further studies on a large number of well‐characterized individuals are necessary. © 2011 Wiley‐Liss, Inc.     

Clinical utility of the X‐chromosome array

Previous studies have limited the use of specific X‐chromosome array designed platforms to the evaluation of patients with intellectual disability. In this retrospective analysis, we reviewed the clinical utility of an X‐chromosome array in a variety of scenarios. We divided patients according to the indication for the test into four defined categories: (1) autism spectrum disorders and/or developmental delay and/or intellectual disability (ASDs/DD/ID) with known family history of neurocognitive disorders; (2) ASDs/DD/ID without known family history of neurocognitive disorders; (3) breakpoint definition of an abnormality detected by a different cytogenetic test; and (4) evaluation of suspected or known X‐linked conditions. A total of 59 studies were ordered with 27 copy number variants detected in 25 patients (25/59 = 42%). The findings were deemed pathogenic/likely pathogenic (16/59 = 27%), benign (4/59 = 7%) or uncertain (7/59 = 12%). We place particular emphasis on the utility of this test for the diagnostic evaluation of families affected with X‐linked conditions and how it compares to whole genome arrays in this setting. In conclusion, the X‐chromosome array frequently detects genomic alterations of the X chromosome and it has advantages when evaluating some specific X‐linked conditions. However, careful interpretation and correlation with clinical findings is needed to determine the significance of such changes. When the X‐chromosome array was used to confirm a suspected X‐linked condition, it had a yield of 63% (12/19) and was useful in the evaluation and risk assessment of patients and families. © 2012 Wiley Periodicals, Inc.     

Assessment of patient clinical descriptions and pathogenic variants from gene panel sequences in the CAGI‐5 intellectual disability challenge

The Critical Assessment of Genome Interpretation‐5 intellectual disability challenge asked to use computational methods to predict patient clinical phenotypes and the causal variant(s) based on an analysis of their gene panel sequence data. Sequence data for 74 genes associated with intellectual disability (ID) and/or autism spectrum disorders (ASD) from a cohort of 150 patients with a range of neurodevelopmental manifestations (i.e. ID, autism, epilepsy, microcephaly, macrocephaly, hypotonia, ataxia) have been made available for this challenge. For each patient, predictors had to report the causative variants and which of the seven phenotypes were present. Since neurodevelopmental disorders are characterized by strong comorbidity, tested individuals often present more than one pathological condition. Considering the overall clinical manifestation of each patient, the correct phenotype has been predicted by at least one group for 93 individuals (62%). ID and ASD were the best predicted among the seven phenotypic traits. Also, causative or potentially pathogenic variants were predicted correctly by at least one group. However, the prediction of the correct causative variant seems to be insufficient to predict the correct phenotype. In some cases, the correct prediction has been supported by rare or common variants in genes different from the causative one.     

Complex autism spectrum disorder in a patient with a 17q12 microduplication

Autism spectrum disorders (ASDs) are phenotypically complex developmental neuropsychiatric disorders affecting approximately 0.6% of the population. About 30-70% of affected children are also considered to have intellectual disability (ID). The underlying genetic causes of ASDs are diverse with a defined etiology in 16-20%. Array comparative genomic hybridization (aCGH) has proven useful in identifying sub-microscopic chromosome aberrations in a subset of patients, some of which have been shown to be recurrent. One such aberration is the 1.4 Mb microdeletion at chromosome 17q12, which has been reported to be associated with renal disease, growth restriction, diabetes, cognitive impairment, seizures, and in some cases an ASD. Patients with the reciprocal chromosome 17q12 microduplication typically have also been identified with ID and in some cases seizures and behavioral abnormalities. Here we report a patient with a de novo, 1.4 Mb microduplication diagnosed with significant ID involving complex deficits and autism. To our knowledge, this is the first report of a patient with the 17q12 microduplication and a complex ASD phenotype.