Eli Hatchwell

Novel Submicroscopic Chromosomal Abnormalities Detected in Autism Spectrum Disorder

BACKGROUND One genetic mechanism known to be associated with autism spectrum disorders (ASD) is chromosomal abnormalities. The identification of copy number variants (CNV), i.e., microdeletions and microduplications that are undetectable at the level of traditional cytogenetic analysis, allows the potential association of submicroscopic chromosomal imbalances and human disease. METHODS We performed array comparative genomic hybridization (aCGH) utilizing a 19K whole genome tiling path bacterial artificial chromosome (BAC) microarray on 397 unrelated subjects with autism spectrum disorder. Common CNV were excluded using a control group comprised of 372 individuals from the National Institute of Mental Health (NIMH) Genetics Initiative Control samples. Confirmation studies were performed on all remaining CNV using fluorescence in situ hybridization (FISH), microsatellite analysis, and/or quantitative polymerase chain reaction (PCR) analysis. RESULTS A total of 51 CNV were confirmed in 46 ASD subjects. Three maternal interstitial duplications of 15q11-q13 known to be associated with ASD were identified. The other 48 CNV ranged in size from 189 kilobase (kb) to 5.5 megabase (Mb) and contained from 0 to approximately 40 National Center for Biotechnology Information (NCBI) Reference Sequence (RefSeq) genes. Seven CNV were de novo and 44 were inherited. CONCLUSIONS Fifty-one autism-specific CNV were identified in 46 of 397 ASD patients using a 19K BAC microarray for an overall rate of 11.6%. These microdeletions and microduplications cause gene dosage imbalance in 272 genes, many of which could be considered as candidate genes for autism.

High-resolution genome-wide cytosine methylation profiling with simultaneous copy number analysis and optimization for limited cell numbers

Many genome-wide assays involve the generation of a subset (or representation) of the genome following restriction enzyme digestion. The use of enzymes sensitive to cytosine methylation allows high-throughput analysis of this epigenetic regulatory process. We show that the use of a dual-adapter approach allows us to generate genomic representations that includes fragments of <200 bp in size, previously not possible when using the standard approach of using a single adapter. By expanding the representation to smaller fragments using HpaII or MspI, we increase the representation by these isoschizomers to more than 1.32 million loci in the human genome, representing 98.5% of CpG islands and 91.1% of refSeq promoters. This advance allows the development of a new, high-resolution version of our HpaII-tiny fragment Enrichment by Ligation-mediated PCR (HELP) assay to study cytosine methylation. We also show that the MspI representation generates information about copy-number variation, that the assay can be used on as little as 10 ng of DNA and that massively parallel sequencing can be used as an alternative to microarrays to read the output of the assay, making this a powerful discovery platform for studies of genomic and epigenomic abnormalities.

Disruption of contactin 4 in three subjects with autism spectrum disorder

Background: Autism spectrum disorder (ASD) is a developmental disorder of the central nervous system of largely unknown aetiology. The prevalence of the syndrome underscores the need for biological markers and a clearer understanding of pathogenesis. For these reasons, a genetic study of idiopathic ASD was undertaken. Methods and results: Array based comparative genomic hybridisation identified a paternally inherited chromosome 3 copy number variation (CNV) in three subjects: a deletion in two siblings and a duplication in a third, unrelated individual. These variations were fluorescence in situ hybridisation (FISH) validated and the end points further delineated using a custom fine tiling oligonucleotide array. Polymerase chain reaction (PCR) products unique to the rearrangements were amplified and sequence analysis revealed the variations to have resulted from Alu Y mediated unequal recombinations interrupting contactin 4 (CNTN4). Conclusion: CNTN4 plays an essential role in the formation, maintenance, and plasticity of neuronal networks. Disruption of this gene is known to cause developmental delay and mental retardation. This report suggests that mutations affecting CNTN4 function may be relevant to ASD pathogenesis.

A discovery resource of rare copy number variations in individuals with autism spectrum disorder.

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.

Genetic variation in brain‐derived neurotrophic factor and human fear conditioning

Brain‐derived neurotrophic factor (BDNF) has been implicated in hippocampal‐dependent learning processes, and carriers of the Met allele of the Val66Met BDNF genotype are characterized by reduced hippocampal structure and function. Recent nonhuman animal work suggests that BDNF is also crucial for amygdala‐dependent associative learning. The present study sought to examine fear conditioning as a function of the BDNF polymorphism. Fifty‐seven participants were genotyped for the BDNF polymorphism and took part in a differential‐conditioning paradigm. Participants were shocked following a particular conditioned stimulus (CS+) and were also presented with stimuli that ranged in perceptual similarity to the CS+ (20, 40 or 60% smaller or larger than the CS+). The eye blink component of the startle response was measured to quantify fear conditioning; post‐task shock likelihood ratings for each stimulus were also obtained. All participants reported that shock likelihood varied with perceptual similarity to the CS+ and showed potentiated startle in response to CS ± 20% stimuli. However, only the Val/Val group had potentiated startle responses to the CS+. Met allele carrying individuals were characterized by deficient fear conditioning – evidenced by an attenuated startle response to CS+ stimuli. Variation in the BDNF genotype appears related to abnormal fear conditioning, consistent with nonhuman animal work on the importance of BDNF in amygdala‐dependent associative learning. The relation between genetic variation in BDNF and amygdala‐dependent associative learning deficits is discussed in terms of potential mechanisms of risk for psychopathology.

Haploinsufficiency of MBD5 associated with a syndrome involving microcephaly, intellectual disabilities, severe speech impairment, and seizures

Microdeletion of chromosome 2q23.1 results in a novel syndrome previously reported in five individuals. Many of the del(2)(q23.1) cases were thought to have other syndromes such as Angelman, Prader–Willi, or Smith–Magenis because of certain overlapping clinical features. We report two new cases of the 2q23.1 microdeletion syndrome, describe the syndrome phenotype, define the minimal critical region, and analyze the expression of critical region genes toward identification of the causative gene(s) for the disorder. Individuals with del(2)(q23.1) have severe developmental and cognitive delays, minimal speech, seizures, microcephaly, mild craniofacial dysmorphism, behavioral disorders, and short stature. The deletions encompassing 2q23.1 range from >4 Mb to <200 kb, as identified by oligonucleotide and BAC whole-genome array comparative hybridization. The minimal critical region includes a single gene, MBD5, deleted in all cases, whereas all but one case also include deletion of EPC2. Quantitative real-time PCR of patient lymphoblasts/lymphocytes showed an ∼50% reduced expression of MBD5 and EPC2 compared with controls. With similar phenotypes among the 2q23.1 deletion patients, the idea of one or more common genes causing the pathological defect seen in these patients becomes evident. As all five previous cases and the two cases in this report share one common gene, MBD5, we strongly suspect that haploinsufficiency of MBD5 causes most of the features observed in this syndrome.

Characterization of the chromosome 1q41q42.12 region, and the candidate gene DISP1, in patients with CDH.

Cytogenetic and molecular cytogenetic studies demonstrate association between congenital diaphragmatic hernia (CDH) and chromosome 1q41q42 deletions. In this study, we screened a large CDH cohort (N = 179) for microdeletions in this interval by the multiplex ligation‐dependent probe amplification (MLPA) technique, and also sequenced two candidate genes located therein, dispatched 1 (DISP1) and homo sapiens H2.0‐like homeobox (HLX). MLPA analysis verified deletions of this region in two cases, an unreported patient with a 46,XY,del(1)(q41q42.13) karyotype and a previously reported patient with a Fryns syndrome phenotype [Kantarci et al., 2006 ]. HLX sequencing showed a novel but maternally inherited single nucleotide variant (c.27C>G) in a patient with isolated CDH, while DISP1 sequencing revealed a mosaic de novo heterozygous substitution (c.4412C>G; p.Ala1471Gly) in a male with a left‐sided Bochdalek hernia plus multiple other anomalies. Pyrosequencing demonstrated the mutant allele was present in 43%, 12%, and 4.5% of the patients lymphoblastoid, peripheral blood lymphocytes, and saliva cells, respectively. We examined Disp1 expression at day E11.5 of mouse diaphragm formation and confirmed its presence in the pleuroperitoneal fold, as well as the nearby lung which also expresses Sonic hedgehog (Shh). Our report describes the first de novo DISP1 point mutation in a patient with complex CDH. Combining this finding with Disp1 embryonic mouse diaphragm and lung tissue expression, as well as previously reported human chromosome 1q41q42 aberrations in patients with CDH, suggests that DISP1 may warrant further consideration as a CDH candidate gene.

X inactivation analysis in a female with hypomelanosis of Ito associated with a balanced X;17 translocation: evidence for functional disomy of Xp.

X inactivation analysis was performed on normal and hypopigmented skin samples obtained from a female with hypomelanosis of Ito associated with a balanced whole arm X;17 translocation. Severe skewing of X inactivation resulting in inactivity of the intact X was found in blood and cultures of both types of skin, but analysis of DNA prepared directly from hypopigmented skin showed significant inactivation of the translocated X, inconsistent with the usual mechanism of phenotypic expression in X;autosome translocations. In addition, dual colour FISH analysis using centromere specific probes for chromosomes X and 17 showed that the breakpoints on both chromosomes lie within the alphoid arrays, making interruption of a locus on either chromosome unlikely. While partial variable monosomy of loci on chromosome 17p cannot be excluded as contributing to the phenotype in this patient, it is argued that the major likely factor is partial functional disomy of sequences on Xp in cell lineages that have failed to inactivate the intact X chromosome.

Human MLPA Probe Design (H-MAPD): a probe design tool for both electrophoresis-based and bead-coupled human multiplex ligation-dependent probe amplification assays

BackgroundMultiplex ligation-dependent probe amplification (MLPA) is an efficient and reliable technique for gene dosage analysis. Currently MLPA can be conducted on two platforms: traditional electrophoresis-based, and FlexMAP bead-coupled. Since its introduction in 2002, MLPA has been rapidly adopted in both clinical and research situations. However, MLPA probe design is a time consuming process requiring many steps that address multiple criteria. There exist only one or two commercial software packages for traditional electrophoresis-based MLPA probe design. To our knowledge, no software is yet available that performs bead-coupled MLPA probe design.ResultsWe have developed H-MAPD, a web-based tool that automates the generation and selection of probes for human genomic MLPA. The software performs physical-chemical property tests using UNAFold software, and uniqueness tests using the UCSC genome browser. H-MAPD supports both traditional electrophoresis-based assays, as well as FlexMAP bead-coupled MLPA.ConclusionH-MAPD greatly reduces the efforts for human genomic MLPA probe design. The software is written in Perl-CGI, hosted on a Linux server, and is freely available to non-commercial users.

Brief Report: Glutamate Transporter Gene (SLC1A1) Single Nucleotide Polymorphism (rs301430) and Repetitive Behaviors and Anxiety in Children with Autism Spectrum Disorder

Investigated association of single nucleotide polymorphism (SNP) rs301430 in glutamate transporter gene (SLC1A1) with severity of repetitive behaviors (obsessive–compulsive behaviors, tics) and anxiety in children with autism spectrum disorder (ASD). Mothers and/or teachers completed a validated DSM-IV-referenced rating scale for 67 children with autism spectrum disorder. Although analyses were not significant for repetitive behaviors, youths homozygous for the high expressing C allele had more severe anxiety than carriers of the T allele. Allelic variation in SLC1A1 may be a biomarker for or modifier of anxiety symptom severity in children with ASD, but study findings are best conceptualized as tentative pending replication with larger independent samples.