Allele-Specific Droplet Digital PCR Combined with a Next-Generation Sequencing-Based Algorithm for Diagnostic Copy Number Analysis in Genes with High Homology: Proof of Concept Using Stereocilin.

Clin Chem
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Keywords
Abstract

BACKGROUND: Copy number variants (CNVs) can substantially contribute to the pathogenic variant spectrum in several disease genes. The detection of this type of variant is complicated in genes with high homology to other genomic sequences, yet such genomics regions are more likely to lead to CNVs, making it critical to address detection in these settings.

METHODS: We developed a copy number analysis approach for high homology genes/regions that consisted of next-generation sequencing (NGS)-based dosage analysis accompanied by allele-specific droplet digital PCR (ddPCR) confirmatory testing. We applied this approach to copy number analysis in , a gene with 98.9% homology to a nonfunctional pseudogene, , and characterized its accuracy in detecting different copy number states by use of known samples.

RESULTS: Using a cohort of 517 patients with hearing loss, we prospectively demonstrated the clinical utility of the approach, which contributed 30 of the 122 total positives (6%) to the diagnostic yield, increasing the overall yield from 17.6% to 23.6%. Positive genotypes included homozygous (n = 15) or compound heterozygous (n = 8) deletions, or heterozygous deletions with pathogenic sequence variants (n = 7). Finally, this approach limited ddPCR testing to cases with NGS copy number findings, thus markedly reducing the number of costly and laborious, albeit specific, ddPCR tests.

CONCLUSIONS: NGS-based CNV detection followed by allele-specific ddPCR confirmatory testing is a reliable and affordable approach for copy number analysis in medically relevant genes with homology issues.

Year of Publication
2018
Journal
Clin Chem
Volume
64
Issue
4
Pages
705-714
Date Published
2018 04
ISSN
1530-8561
DOI
10.1373/clinchem.2017.280685
PubMed ID
29339441
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