Validation for Clinical Use of, and Initial Clinical Experience with, a Novel Approach to Population-Based Carrier Screening using High-Throughput, Next-Generation DNA Sequencing

Abstract

Traditional carrier screening assays are designed to look for only the most common mutations within a gene owing to cost considerations. Although this can yield high detection rates in specific populations for specific genes (such as cystic fibrosis in Caucasians), they are suboptimal for other ethnicities or for patients of mixed or unknown ethnic background. Next-generation DNA sequencing provides an opportunity to provide carrier screening using more comprehensive mutation panels that are limited primarily by information about the clinical impact of detected sequence changes. We describe a next-generation DNA sequencing-based assay capable of reliably screening patient samples in a timely and comprehensive manner. The analytic accuracy in a research setting has been documented. Here, we describe the additional studies performed to ensure the accuracy (analytic validity) and robustness of our assay for use in clinical practice and provide data from our experience offering this testing. Our clinical experience using this approach to screen 11,691 in vitro fertilization patients has identified 449 mutant alleles: 447 in carriers and 2 in an affected individual. In total, we found 87 distinct mutations in 14 different genes. Approximately one quarter of the mutations found are not included in traditional, limited, mutation panels, including 16 known mutations unique to our panel, and novel truncating mutations in several genes.

Feasibility of Low-Throughput Next Generation Sequencing for Germline DNA Screening

“…Overall, 99.39% of 2635 replicate variants were detected in all 3 replicates at a sensitivity of 99.42%, specificity of 99.99%, PPV of 99.86%, and NPV) of 99.94% from 7500 shared interrogations between NGS and array analysis. These frequencies are similar to reproducibility rates of 95.6%-98.8% (21,22 ), sensitivities of 95.9%-99.4% (21,23,24 ), and specificity of 100% (21 ) reported in studies using higher-throughput NGS formats, indicating an equivalence in analytical performance between modes. Notably, 11/16 (69%) replicate variants that were detected inconsistently had allele frequencies Յ0.15, strand biases ϾϪ35, or genotype quality scores Յ80 ( Fig.…”

Carrier screening by next‐generation sequencing: health benefits and cost effectiveness

“…NGS allows for the detection of many more mutations than traditional genotyping‐based carrier screens, while still detecting so‐called common mutations (Hallam et al. 2014; Perreault‐Micale et al. 2014).…”

“…This is consistent with a recent multiethnic study in which a quarter of the mutations detected by NGS are not included in traditional, limited, mutation panels (Hallam et al. 2014). Our model predicts that, for a large population, NGS will identify 10% or more mutation carriers than traditional genotyping, which is also consistent with the improvements calculated using data from the clinical database of 71,070 patients described in Table S7.…”

“…Specifically, Hallam et al. (2014) used high‐throughput NGS to screen 11,691 patients visiting assisted reproductive technology (ART) centers and identified 449 mutant alleles (447 in carriers and 2 in an affected individual), and, in total, 87 distinct pathogenic mutations in 14 different genes. Most importantly, about one quarter of the mutations found were not included in traditional genotyping panels, including 16 known pathogenic mutations unique to the NGS panel, and novel truncating mutations in several genes.…”

Changing trends in carrier screening for genetic disease in the United States

“…This approach identifies an increased number of carriers and significantly increases sensitivity. Hallam et al have also reported a NGS screening approach 22. Approximately 25% of the mutations they found would not have been detected with traditional targeted genotyping screening panels.…”