Development and Validation of a Next-Generation Sequencing Panel for Syndromic and Nonsyndromic Hearing Loss.
Alleles
Amino Acid Substitution
Chromosome Mapping
Computational Biology
/ methods
Genetic Association Studies
Genetic Predisposition to Disease
Genetic Testing
/ methods
Genotype
Hearing Loss
/ diagnosis
High-Throughput Nucleotide Sequencing
/ methods
Humans
Molecular Diagnostic Techniques
Reproducibility of Results
DFNA
DFNB
deafness
hearing loss
next generation sequencing
nonsyndromic hearing loss
syndromic hearing loss
Journal
The journal of applied laboratory medicine
ISSN: 2576-9456
Titre abrégé: J Appl Lab Med
Pays: England
ID NLM: 101693884
Informations de publication
Date de publication:
01 05 2020
01 05 2020
Historique:
received:
05
08
2019
accepted:
21
09
2019
entrez:
24
5
2020
pubmed:
24
5
2020
medline:
20
7
2021
Statut:
ppublish
Résumé
Deafness and hearing loss are common conditions that can be seen independently or as part of a syndrome and are often mediated by genetic causes. We sought to develop and validate a hereditary hearing loss panel (HHLP) to detect single nucleotide variants (SNVs), insertions and deletions (indels), and copy number variants (CNVs) in 166 genes related to nonsyndromic and syndromic hearing loss. We developed a custom-capture next-generation sequencing (NGS) reagent to detect all coding regions, ±10 flanking bp, for the 166 genes related to nonsyndromic and syndromic hearing loss. Our validation consisted of testing 52 samples to establish accuracy, reproducibility, and analytical sensitivity. In addition to NGS, supplementary methods, including multiplex ligation-dependent probe amplification, long-range PCR, and Sanger sequencing, were used to ensure coverage of regions that had high complexity or homology. We observed 100% positive and negative percentage agreement for detection of SNVs (n = 362), small indels (1-22 bp, n = 25), and CNVs (gains, n = 8; losses, n = 17). Finally, we showed that this assay was able to detect variants with a variant allele frequency ≥20% for SNVs and indels and ≥30% to 35% for CNVs. We validated an HHLP that detects SNVs, indels, and CNVs in 166 genes related to syndromic and nonsyndromic hearing loss. The results of this assay can be utilized to confirm a diagnosis of hearing loss and related syndromic disorders associated with known causal genes.
Sections du résumé
BACKGROUND
Deafness and hearing loss are common conditions that can be seen independently or as part of a syndrome and are often mediated by genetic causes. We sought to develop and validate a hereditary hearing loss panel (HHLP) to detect single nucleotide variants (SNVs), insertions and deletions (indels), and copy number variants (CNVs) in 166 genes related to nonsyndromic and syndromic hearing loss.
METHODS
We developed a custom-capture next-generation sequencing (NGS) reagent to detect all coding regions, ±10 flanking bp, for the 166 genes related to nonsyndromic and syndromic hearing loss. Our validation consisted of testing 52 samples to establish accuracy, reproducibility, and analytical sensitivity. In addition to NGS, supplementary methods, including multiplex ligation-dependent probe amplification, long-range PCR, and Sanger sequencing, were used to ensure coverage of regions that had high complexity or homology.
RESULTS
We observed 100% positive and negative percentage agreement for detection of SNVs (n = 362), small indels (1-22 bp, n = 25), and CNVs (gains, n = 8; losses, n = 17). Finally, we showed that this assay was able to detect variants with a variant allele frequency ≥20% for SNVs and indels and ≥30% to 35% for CNVs.
CONCLUSIONS
We validated an HHLP that detects SNVs, indels, and CNVs in 166 genes related to syndromic and nonsyndromic hearing loss. The results of this assay can be utilized to confirm a diagnosis of hearing loss and related syndromic disorders associated with known causal genes.
Identifiants
pubmed: 32445360
pii: 5813833
doi: 10.1093/jalm/jfaa021
doi:
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
467-479Informations de copyright
© American Association for Clinical Chemistry 2020. All rights reserved. For permissions, please email: journals.permissions@oup.com.