Clinical Next-Generation Sequencing Pipeline Outperforms a Combined Approach Using Sanger Sequencing and Multiplex Ligation-Dependent Probe Amplification in Targeted Gene Panel Analysis

Advances in next-generation sequencing (NGS) have facilitated parallel analysis of multiple genes enabling the implementation of cost-effective, rapid, and high-throughput methods for the molecular diagnosis of multiple genetic conditions, including the identification of BRCA1 and BRCA2 mutations in high-risk patients for hereditary breast and ovarian cancer. We clinically validated a NGS pipeline designed to replace Sanger sequencing and multiplex ligation-dependent probe amplification analysis and to facilitate detection of sequence and copy number alterations in a single test focusing on a BRCA1/BRCA2 gene analysis panel. Our custom capture library covers 46 exons, including BRCA1 exons 2, 3, and 5 to 24 and BRCA2 exons 2 to 27, with 20 nucleotides of intronic regions both 5′ and 3′ of each exon. We analyzed 402 retrospective patients, with previous Sanger sequencing and multiplex ligation-dependent probe amplification results, and 240 clinical prospective patients. One-hundred eighty-three unique variants, including sequence and copy number variants, were detected in the retrospective (n = 95) and prospective (n = 88) cohorts. This standardized NGS pipeline demonstrated 100% sensitivity and 100% specificity, uniformity, and high-depth nucleotide coverage per sample (approximately 7000 reads per nucleotide). Subsequently, the NGS pipeline was applied to the analysis of larger gene panels, which have shown similar uniformity, sample-to-sample reproducibility in coverage distribution, and sensitivity and specificity for detection of sequence and copy number variants

GNPAT variant (D519G) is not associated with an elevated serum ferritin or iron removed by phlebotomy in patients referred for C282Y-linked hemochromatosis

Background. Previous studies in high and low expressors has demonstrated that a variant in the GNPAT gene (D519G, Rs11558492, chromosome 1, exon 11) has been associated with severe iron overload in C282Y homozygotes for hemochromatosis. In this study, a GNPATvariant was assessed prospectively in patients referred for HFE testing over a range of serum ferritin levels.Material and methods. Consecutive patients sent for HFE testing were studied for the GNPAT variant using a TaqMan kit assay (Life Technologies, Burlington, ON). Serum ferritin and iron removed by phlebotomy was compared in C282Y homozygotes with and without the GNPATvariant. The frequency of the GNPATvariant in referred patients was compared to a control population of voluntary blood donors without HFE mutations.Results. There were 533 patients that had GNPATanalysis. The allele frequency for the GNPATvariant in C282Y homozygotes (n = 75) was 0.226 and in wild type control patients (n = 458) was 0.213 (p = .07). Forty-eight percent (of the C282Y homozygotes were heterozygous (n = 28) or homozygous (n = 8) for the GNPATvariant. The mean (log)ferritin and iron removed did not significantly differ between C282Y homozygous with GNPAT homozygotes, GNPAT heterozygotes, and without the GNPATvariant (p = 0.84).Conclusions. C282Y homozygotes referred for HFE testing commonly have a GNPATvariant. This GNPATvariant does not appear be a co-modifying gene affecting expression of HFE related hemochromatosis in this population. The GNPATvariant does not predict the severity of iron overload