Exon-Focused Microarray Analysis of Candidate Genes in Autism

Background: We are pursuing two hypotheses, first that de novo and recent mutations cause a major fraction of autism cases, and second that a different fraction of autism cases may be caused by epigenetic mutations (see Person et al. epigenetic abstract). Neither de novo genetic mutations nor epigenetic mutations (epimutations) would be detected by genome-wide linkage studies. The high heritability of autism as judged by high concordance in monozygotic (MZ) twins and the low concordance in dizygotic (DZ) twins is precisely what would be expected for an etiology based on de novo mutations and could fit as well for de novo epimutations occurring prior to MZ twinning. Much progress in understanding the etiology of autism is derived from the use of array technologies to detect genomic deletions and duplications followed by sequencing of candidate genes within these genomic regions. Objectives: The goal of this work is to discover novel genomic deletions and duplications causing autism with a particular focus on determination of exon copy number to identify small deletions that would implicate a single protein coding gene. Genes suspected of causing autism via haploinsufficiency would then be sequenced in other autism individuals. Methods: Customized Agilent arrays were designed to cover all the exons of tens to hundreds of autism candidate genes. Genes were selected based on literature support that they might cause autism (73 genes), on neurological or synaptic function (253 genes), or on an implied role in epigenetic regulation (~300 genes). A one-million oligonucleotide array to test copy number for all exons in the genome is in development. Further characterization of the pathogenic variations was performed utilizing the Agilent 244K Whole Human Genome CGH array. The study group included 98 patients from South Carolina Autism Project, 64 local patients, and 22 autistic individuals from AGRE collection. Results: These experiments identified several very small and apparently benign copy number variants (CNVs) within the NRXN3, CACNAIC, GIRK2, and many other genes. Multiple lymphoblast cell lines were trisomic for various chromosomes, three for chromosome 12, one for chromosomes 9 and 12, and one for chromosomes 9 and 14. These were interpreted to represent cell culture artifacts. We found 5 cases with pathogenic alterations. Two of these abnormalities were deletions: a 15q13.3 deletion encompassing the CHRNA7 gene and a paternally inherited 2-Mb deletion causing haploinsuffiency of the BDNF and LIN7C genes. In addition, there were three different duplications involving X-linked genes and the 15q11-q13 imprinted domain. The X-linked duplications were detected in males and included a maternally inherited 615-kb duplication encompassing the OCRL1 gene and a de novo duplication of the SYBL1 gene. Conclusions: These data provide additional support for the importance of copy number variants in the etiology of autism and suggest that exon-focused microarrays may be an effective method for detecting these chromosomal imbalances and identifying specific causative genes. The utilization of arrays that cover all exons in the genome is predicted to increase the detection of these chromosomal imbalances.