[Show abstract][Hide abstract] ABSTRACT: Next-generation sequencing (NGS) technologies can be a boon to human mutation detection given their high throughput: consequently, many genes and samples may be simultaneously studied with high coverage for accurate detection of heterozygotes. In circumstances requiring the intensive study of a few genes, particularly in clinical applications, a rapid turn around is another desirable goal. To this end, we assessed the performance of the bench-top 454 GS Junior platform as an optimized solution for mutation detection by amplicon sequencing of three type 3 semaphorin genes SEMA3A, SEMA3C, and SEMA3D implicated in Hirschsprung disease (HSCR). We performed mutation detection on 39 PCR amplicons totaling 14,014 bp in 47 samples studied in pools of 12 samples. Each 10-hr run was able to generate ∼75,000 reads and ∼28 million high-quality bases at an average read length of 371 bp. The overall sequencing error was 0.26 changes per kb at a coverage depth of ≥20 reads. Altogether, 37 sequence variants were found in this study of which 10 were unique to HSCR patients. We identified five missense mutations in these three genes that may potentially be involved in the pathogenesis of HSCR and need to be studied in larger patient samples.
[Show abstract][Hide abstract] ABSTRACT: Hirschsprung disease (HSCR) is a neurocristopathy characterized by absence of intramural ganglion cells along variable lengths of the gastrointestinal tract. The HSCR phenotype is highly variable with respect to gender, length of aganglionosis, familiality and the presence of additional anomalies. By molecular genetic analysis, a minimum of 11 neuro-developmental genes (RET, GDNF, NRTN, SOX10, EDNRB, EDN3, ECE1, ZFHX1B, PHOX2B, KIAA1279, TCF4) are known to harbor rare, high-penetrance mutations that confer a large risk to the bearer. In addition, two other genes (RET, NRG1) harbor common, low-penetrance polymorphisms that contribute only partially to risk and can act as genetic modifiers. To broaden this search, we examined whether a set of 67 proven and candidate HSCR genes harbored additional modifier alleles. In this pilot study, we utilized a custom-designed array CGH with ∼33,000 test probes at an average resolution of ∼185 bp to detect gene-sized or smaller copy number variants (CNVs) within these 67 genes in 18 heterogeneous HSCR patients. Using stringent criteria, we identified CNVs at three loci (MAPK10, ZFHX1B, SOX2) that are novel, involve regulatory and coding sequences of neuro-developmental genes, and show association with HSCR in combination with other congenital anomalies. Additional CNVs are observed under relaxed criteria. Our research suggests a role for CNVs in HSCR and, importantly, emphasizes the role of variation in regulatory sequences. A much larger study will be necessary both for replication and for identifying the full spectrum of small CNV effects.