Objectives: In autism and other neurobehavioral phenotypes, to determine the molecular heterogeneity and pathogenic effects if any for the 1.6 and 0.43-0.68 Mb duplications, to search for functionally significant point mutations in CHRNA7, and to evaluate the clinical significance of inversions within 15q13.3 and of any variation in the CHRFAM7A fusion genotype,
Methods: Array comparative genomic hybridization (CGH), MLPA, FISH and DNA sequencing are being used to compare samples from individuals with autism and other phenotypic abnormalities to controls.
Results: The deletions and duplications were observed at decreasing frequency from 0.43-0.68 Mb duplication, to 1.6 Mb deletion, 1.6 Mb duplication, and 0.68 Mb deletion in a series of ~10,000 pediatric samples submitted for clinically indicated array CGH studies. Phenotypes of mental retardation, developmental delay and/or autism were common in the deletion and duplication samples. Although the deletions are very likely pathogenic, any harmful effects of the duplications remain to be determined based in part on additional studies of control samples. MLPA provides reliable copy number information for exons 2 and 4 which are unique to CHRNA7 and for exons 5 and 10 which are common to CHRNA7 and the CHRFAM7A fusion gene. PCR assays were developed to detect the presence or absence of the 2-bp deletion in exon 6 of the fusion gene or the functional gene. DNA sequencing using a first step of long range PCR to distinguish exons 5-10 of the CHRNA7 gene detected two de novo missense mutations, many inherited missense mutations, and one nonsense mutation.
Conclusions: Loss-of-function mutations in CHRNA7 likely account for most of the phenotypic consequences seen with deletions of 15q13.3. It remains unknown whether the CHRFAM7A gene produces a stable fusion protein with any function, although its structure would be impacted by the 2-bp deletion in exon 6. Similarly it is unknown whether the various duplication mutations result in over-expression of the normal CHRNA7 protein or in some cases may produce transcripts subject to nonsense mediated decay and/or produce truncated proteins with dominant negative effects. In depth genotypying for 15q13.3 requires, array CGH or another general method for copy number assessment best supplemented with MLPA to determine copy number for CHRNA7 and CHRFAM7A.Tiling arrays show some heterogeneity of the common 0.68 Mb duplications suggesting the possibility of functional heterogeneity in this class of duplication. In addition, sequencing of CHRNA7 is important for detection of point mutations associated with autism and other neurobehavioral abnormalities.