The identification of germline variants that predispose to cancer is important to further our understanding of tumorigenesis, guide patient management, prevent disease in unaffected relatives, and inform best practice for health care. We describe a kindred with multiple gastrointestinal malignancies where a novel MSH6 germline susceptibility variant was identified by exome sequencing after eluding serial routine testing in multiple affected members. This case fosters discussion of our current understanding of DNA mismatch repair deficiency, the management of Lynch Syndrome, and the emerging role of next generation sequencing in laboratory medicine to identify rare pathogenic germline variants in a comprehensive, unbiased fashion.
We describe a method for pooling and sequencing DNA from a large number of individual samples while preserving information regarding sample identity. DNA from 576 individuals was arranged into four 12 row by 12 column matrices and then pooled by row and by column resulting in 96 total pools with 12 individuals in each pool. Pooling of DNA was carried out in a two-dimensional fashion, such that DNA from each individual is present in exactly one row pool and exactly one column pool. By considering the variants observed in the rows and columns of a matrix we are able to trace rare variants back to the specific individuals that carry them. The pooled DNA samples were enriched over a 250 kb region previously identified by GWAS to significantly predispose individuals to lung cancer. All 96 pools (12 row and 12 column pools from 4 matrices) were barcoded and sequenced on an Illumina HiSeq 2000 instrument with an average depth of coverage greater than 4,000×. Verification based on Ion PGM sequencing confirmed the presence of 91.4% of confidently classified SNVs assayed. In this way, each individual sample is sequenced in multiple pools providing more accurate variant calling than a single pool or a multiplexed approach. This provides a powerful method for rare variant detection in regions of interest at a reduced cost to the researcher.
Cisplatin is a widely used and effective chemotherapeutic agent, although its use is restricted by the high incidence of irreversible ototoxicity associated with it. In children, cisplatin ototoxicity is a serious and pervasive problem, affecting more than 60% of those receiving cisplatin and compromising language and cognitive development. Candidate gene studies have previously reported associations of cisplatin ototoxicity with genetic variants in the genes encoding glutathione S-transferases and megalin. We report association analyses for 220 drug-metabolism genes in genetic susceptibility to cisplatin-induced hearing loss in children. We genotyped 1,949 SNPs in these candidate genes in an initial cohort of 54 children treated in pediatric oncology units, with replication in a second cohort of 112 children recruited through a national surveillance network for adverse drug reactions in Canada. We identified genetic variants in TPMT (rs12201199, P value = 0.00022, OR = 17.0, 95% CI 2.3-125.9) and COMT (rs9332377, P value = 0.00018, OR = 5.5, 95% CI 1.9-15.9) associated with cisplatin-induced hearing loss in children.
Human respiratory syncytial virus (HRSV) is one of the major etiologic agents of respiratory tract infections among children worldwide.
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