Joo Wook Ahn

Rare deletions at the neurexin 3 locus in autism spectrum disorder.

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.

Male-Biased Autosomal Effect of 16p13.11 Copy Number Variation in Neurodevelopmental Disorders

Copy number variants (CNVs) at chromosome 16p13.11 have been associated with a range of neurodevelopmental disorders including autism, ADHD, intellectual disability and schizophrenia. Significant sex differences in prevalence, course and severity have been described for a number of these conditions but the biological and environmental factors underlying such sex-specific features remain unclear. We tested the burden and the possible sex-biased effect of CNVs at 16p13.11 in a sample of 10,397 individuals with a range of neurodevelopmental conditions, clinically referred for array comparative genomic hybridisation (aCGH); cases were compared with 11,277 controls. In order to identify candidate phenotype-associated genes, we performed an interval-based analysis and investigated the presence of ohnologs at 16p13.11; finally, we searched the DECIPHER database for previously identified 16p13.11 copy number variants. In the clinical referral series, we identified 46 cases with CNVs of variable size at 16p13.11, including 28 duplications and 18 deletions. Patients were referred for various phenotypes, including developmental delay, autism, speech delay, learning difficulties, behavioural problems, epilepsy, microcephaly and physical dysmorphisms. CNVs at 16p13.11 were also present in 17 controls. Association analysis revealed an excess of CNVs in cases compared with controls (OR = 2.59; p = 0.0005), and a sex-biased effect, with a significant enrichment of CNVs only in the male subgroup of cases (OR = 5.62; p = 0.0002), but not in females (OR = 1.19, p = 0.673). The same pattern of results was also observed in the DECIPHER sample. Interval-based analysis showed a significant enrichment of case CNVs containing interval II (OR = 2.59; p = 0.0005), located in the 0.83 Mb genomic region between 15.49–16.32 Mb, and encompassing the four ohnologs NDE1, MYH11, ABCC1 and ABCC6. Our data confirm that duplications and deletions at 16p13.11 represent incompletely penetrant pathogenic mutations that predispose to a range of neurodevelopmental disorders, and suggest a sex-limited effect on the penetrance of the pathological phenotypes at the 16p13.11 locus.

Validation and implementation of array comparative genomic hybridisation as a first line test in place of postnatal karyotyping for genome imbalance

BackgroundSeveral studies have demonstrated that array comparative genomic hybridisation (CGH) for genome-wide imbalance provides a substantial increase in diagnostic yield for patients traditionally referred for karyotyping by G-banded chromosome analysis. The purpose of this study was to demonstrate the feasibility of and strategies for, the use of array CGH in place of karyotyping for genome imbalance, and to report on the results of the implementation of this approach.ResultsFollowing a validation period, an oligoarray platform was chosen. In order to minimise costs and increase efficiency, a patient/patient hybridisation strategy was used, and analysis criteria were set to optimise detection of pathogenic imbalance. A customised database application with direct links to a number of online resources was developed to allow efficient management and tracking of patient samples and facilitate interpretation of results. Following introduction into our routine diagnostic service for patients with suspected genome imbalance, array CGH as a follow-on test for patients with normal karyotypes (n = 1245) and as a first-line test (n = 1169) gave imbalance detection rates of 26% and 22% respectively (excluding common, benign variants). At least 89% of the abnormalities detected by first line testing would not have been detected by standard karyotype analysis. The average reporting time for first-line tests was 25 days from receipt of sample.ConclusionsArray CGH can be used in a diagnostic service setting in place of G-banded chromosome analysis, providing a more comprehensive and objective test for patients with suspected genome imbalance. The increase in consumable costs can be minimised by employing appropriate hybridisation strategies; the use of robotics and a customised database application to process multiple samples reduces staffing costs and streamlines analysis, interpretation and reporting of results. Array CGH provides a substantially higher diagnostic yield than G-banded chromosome analysis, thereby alleviating the burden of further clinical investigations.

Detection of subtelomere imbalance using MLPA: validation, development of an analysis protocol, and application in a diagnostic centre

BackgroundCommercial MLPA kits (MRC-Holland) are available for detecting imbalance at the subtelomere regions of chromosomes; each kit consists of one probe for each subtelomere.MethodsFor validation of the kits, 208 patients were tested, of which 128 were known to be abnormal, corresponding to 8528 genomic regions overall. Validation samples included those with trisomy 13, 18 and 21, microscopically visible terminal deletions and duplications, sex chromosome abnormalities and submicroscopic abnormalities identified by multiprobe FISH. A robust and sensitive analysis system was developed to allow accurate interpretation of single probe results, which is essential as breakpoints may occur between MLPA probes.ResultsThe validation results showed that MLPA is a highly efficient technique for medium-throughput screening for subtelomere imbalance, with 95% confidence intervals for positive and negative predictive accuracies of 0.951-0.996 and 0.9996-1 respectively. A diagnostic testing strategy was established for subtelomere MLPA and any subsequent follow-up tests that may be required. The efficacy of this approach was demonstrated during 15 months of diagnostic testing when 455 patients were tested and 27 (5.9%) abnormal cases were detected.ConclusionThe development of a robust, medium-throughput analysis system for the interpretation of results from subtelomere assays will be of benefit to other Centres wishing to implement such an MLPA-based service.

Array CGH as a first line diagnostic test in place of karyotyping for postnatal referrals - results from four years’ clinical application for over 8,700 patients

BackgroundArray CGH is widely used in cytogenetics centres for postnatal constitutional genome analysis, and is now recommended as a first line test in place of G-banded chromosome analysis. At our centre, first line testing by oligonucleotide array CGH for all constitutional referrals for genome imbalance has been in place since June 2008, using a patient vs patient hybridisation strategy to minimise costs.FindingsOut of a total of 13,412 patients tested with array CGH, 8,794 (66%) had array CGH as the first line test. Referral indications for this first line group ranged from neonatal congenital anomalies through to adult neurodisabilities; 25% of these patients had CNVs either in known pathogenic regions or in other regions where imbalances have not been reported in the normal population. Of these CNVs, 46% were deletions or nullisomy, 53% were duplications or triplications, and mosaic imbalances made up the remainder; 87% were <5Mb and would likely not be detected by G-banded chromosome analysis. For cases with completed inheritance studies, 20% of imbalances were de novo.ConclusionsArray CGH is a robust and cost-effective alternative to traditional cytogenetic methodology; it provides a higher diagnostic detection rate than G-banded chromosome analysis, and adds to the sum of information and understanding of the role of genomic imbalance in disease. Use of novel hybridisation strategies can reduce costs, allowing more widespread testing.

CHD2 haploinsufficiency is associated with developmental delay, intellectual disability, epilepsy and neurobehavioural problems

BackgroundThe 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.MethodsWe conducted a multicentre study to identify and characterise the clinical features associated with haploinsufficiency of CHD2. Patients with deletions of this gene were identified from among broadly ascertained clinical cohorts undergoing genomic microarray analysis for developmental delay, congenital anomalies and/or autism spectrum disorder.ResultsDetailed clinical assessments by clinical geneticists showed recurrent clinical symptoms, including developmental delay, intellectual disability, epilepsy, behavioural problems and autism-like features without characteristic facial gestalt or brain malformations observed on magnetic resonance imaging scans. Parental analysis showed that the deletions affecting CHD2 were de novo in all four patients, and analysis of high-resolution microarray data derived from 26,826 unaffected controls showed no deletions of this gene.ConclusionsThe results of this study, in addition to our review of the literature, support a causative role of CHD2 haploinsufficiency in developmental delay, intellectual disability, epilepsy and behavioural problems, with phenotypic variability between individuals.

Phenotypic features in patients with 15q11.2(BP1‐BP2) deletion: Further delineation of an emerging syndrome

15q11.2 deletions flanked by BP1 and BP2 of the Prader‐Willi/Angelman syndrome region have recently been linked to a range of neurodevelopment disorders including intellectual disability, speech and language delay, motor delay, autism spectrum disorders, epilepsy, and schizophrenia. Array CGH analysis of 14,605 patients referred for diagnostic cytogenetic testing found that 83 patients (0.57%) carried the 15q11.2(BP1‐BP2) deletion. Phenotypic frequencies in the deleted cohort (n = 83) were compared with frequencies in the non‐deleted cohort (n = 14,522); developmental delay, motor delay, and speech and language delay were all more prevalent in the deleted cohort. Notably, motor delay was significantly more common (OR = 6.37). These data indicate that developmental delay, motor delay, and speech and language delay are common clinical features associated with this deletion, providing substantial evidence to support this CNV as a susceptibility locus for a spectrum of neurodevelopmental disorders.

MLPA for confirmation of array CGH results and determination of inheritance

BackgroundArray CGH has recently been introduced into our laboratory in place of karyotype analysis for patients with suspected genomic imbalance. Results require confirmation to check sample identity, and analysis of parental samples to determine inheritance and thus assess the clinical significance of the abnormality. Here we describe an MLPA-based strategy for the follow-up of abnormal aCGH results.ResultsIn the first 17 months of our MLPA-based aCGH follow-up service, 317 different custom MLPA probes for novel aCGH-detected abnormalities were developed for inheritance studies in 164 families. In addition, 110 samples were tested for confirmation of aCGH-detected abnormalities in common syndromic or subtelomeric regions using commercial MLPA kits. Overall, a total of 1215 samples have been tested by MLPA. A total of 72 de novo abnormalities were confirmed.ConclusionsConfirmation of aCGH-detected abnormalities and inheritance of these abnormalities are essential for accurate diagnosis and interpretation of aCGH results. The development of a new service utilising custom made MLPA probes and commercial MLPA kits for follow-up studies of array CGH results has been found to be efficient and flexible in our laboratory.

Parental Origin of Interstitial Duplications at 15q11.2-q13.3 in Schizophrenia and Neurodevelopmental Disorders

Duplications at 15q11.2-q13.3 overlapping the Prader-Willi/Angelman syndrome (PWS/AS) region have been associated with developmental delay (DD), autism spectrum disorder (ASD) and schizophrenia (SZ). Due to presence of imprinted genes within the region, the parental origin of these duplications may be key to the pathogenicity. Duplications of maternal origin are associated with disease, whereas the pathogenicity of paternal ones is unclear. To clarify the role of maternal and paternal duplications, we conducted the largest and most detailed study to date of parental origin of 15q11.2-q13.3 interstitial duplications in DD, ASD and SZ cohorts. We show, for the first time, that paternal duplications lead to an increased risk of developing DD/ASD/multiple congenital anomalies (MCA), but do not appear to increase risk for SZ. The importance of the epigenetic status of 15q11.2-q13.3 duplications was further underlined by analysis of a number of families, in which the duplication was paternally derived in the mother, who was unaffected, whereas her offspring, who inherited a maternally derived duplication, suffered from psychotic illness. Interestingly, the most consistent clinical characteristics of SZ patients with 15q11.2-q13.3 duplications were learning or developmental problems, found in 76% of carriers. Despite their lower pathogenicity, paternal duplications are less frequent in the general population with a general population prevalence of 0.0033% compared to 0.0069% for maternal duplications. This may be due to lower fecundity of male carriers and differential survival of embryos, something echoed in the findings that both types of duplications are de novo in just over 50% of cases. Isodicentric chromosome 15 (idic15) or interstitial triplications were not observed in SZ patients or in controls. Overall, this study refines the distinct roles of maternal and paternal interstitial duplications at 15q11.2-q13.3, underlining the critical importance of maternally expressed imprinted genes in the contribution of Copy Number Variants (CNVs) at this interval to the incidence of psychotic illness. This work will have tangible benefits for patients with 15q11.2-q13.3 duplications by aiding genetic counseling.

Microdeletions of ELP4 Are Associated with Language Impairment, Autism Spectrum Disorder, and Mental Retardation.

Copy‐number variations (CNVs) are important in the aetiology of neurodevelopmental disorders and show broad phenotypic manifestations. We compared the presence of small CNVs disrupting the ELP4‐PAX6 locus in 4,092 UK individuals with a range of neurodevelopmental conditions, clinically referred for array comparative genomic hybridization, with WTCCC controls (n = 4,783). The phenotypic analysis was then extended using the DECIPHER database. We followed up association using an autism patient cohort (n = 3,143) compared with six additional control groups (n = 6,469). In the clinical discovery series, we identified eight cases with ELP4 deletions, and one with a partial duplication of ELP4 and PAX6. These cases were referred for neurological phenotypes including language impairment, developmental delay, autism, and epilepsy. Six further cases with a primary diagnosis of autism spectrum disorder (ASD) and similar secondary phenotypes were identified with ELP4 deletions, as well as another six (out of nine) with neurodevelopmental phenotypes from DECIPHER. CNVs at ELP4 were only present in 1/11,252 controls. We found a significant excess of CNVs in discovery cases compared with controls, P = 7.5 × 10−3, as well as for autism, P = 2.7 × 10−3. Our results suggest that ELP4 deletions are highly likely to be pathogenic, predisposing to a range of neurodevelopmental phenotypes from ASD to language impairment and epilepsy.