Increased diagnostic yield in a cohort of hearing loss families using a comprehensive stepwise strategy of molecular testing

Abstract

Hearing loss is one of the most common sensory disorders in humans. This study proposes a stepwise strategy of deafness gene detection using multiplex PCR combined with high-throughput sequencing, Sanger sequencing, multiplex ligation-dependent probe amplification (MLPA), and whole-exome sequencing (WES) to explore its application in molecular diagnosis of hearing loss families. A total of 152 families with hearing loss were included in this study, the highest overall diagnosis rate was 73% (111/152). The diagnosis rate of multiplex PCR combined with high-throughput sequencing was 52.6% (80/152). One families was diagnosed by Sanger sequencing of GJB2 exon 1. Two families were diagnosed by MLPA analysis of the STRC gene. The diagnosis rate with additional contribution from WES was 18.4% (28/152). We identified 21 novel variants from 15 deafness genes by WES. Combining WES and deep clinical phenotyping, we diagnosed 11 patients with syndromic hearing loss (SHL). This study demonstrated improved diagnostic yield in a cohort of hearing loss families and confirmed the advantages of a stepwise strategy in the molecular diagnosis of hearing loss.

PKHD1L1, a gene involved in the stereocilia coat, causes autosomal recessive nonsyndromic hearing loss

“…This set comprises a total of 1450 hearing loss probands mainly from Henan Province, China, and it includes syndromic and nonsyndromic hearing loss. Most of the cohort was first tested by a lab-developed multiplex PCR kit covering the total coding sequencing of GJB2 , SLC26A4 , and MT-RNR1 [described in a previous study (Zeng et al 2022b )], as these are the most common causative genes in this population. If negative, exome sequencing was performed [the so-called step-wise approach as described previously (Zeng et al 2022b )].…”

“…Most of the cohort was first tested by a lab-developed multiplex PCR kit covering the total coding sequencing of GJB2 , SLC26A4 , and MT-RNR1 [described in a previous study (Zeng et al 2022b )], as these are the most common causative genes in this population. If negative, exome sequencing was performed [the so-called step-wise approach as described previously (Zeng et al 2022b )]. A fraction of patients chose exome sequencing as the first-tier test, while others with negative multiplex PCR were not tested by exome sequencing if DNA from both parents was not available.…”

PKHD1L1, A Gene Involved in the Stereocilia Coat, Causes Autosomal Recessive Nonsyndromic Hearing Loss

“…This set is comprised of a total of 1450 hearing loss probands and it includes syndromic and non-syndromic hearing loss. Most of the cohort was first tested by a lab-developed multiplex PCR kit covering the total coding sequencing of GJB2 , SLC26A4 , and MT-RNR1 (described in a previous study (Zeng et al 2022b)), as these are the most common causative genes in this population. If negative, exome sequencing was performed (the so-called step-wise approach as described previously (Zeng et al 2022b)).…”

“…Most of the cohort was first tested by a lab-developed multiplex PCR kit covering the total coding sequencing of GJB2 , SLC26A4 , and MT-RNR1 (described in a previous study (Zeng et al 2022b)), as these are the most common causative genes in this population. If negative, exome sequencing was performed (the so-called step-wise approach as described previously (Zeng et al 2022b)). A fraction of patients chose exome sequencing as the first-tier test, while others with negative multiplex PCR were not tested by exome sequencing if DNA from both parents was not available.…”

“…Family 4: Exome sequencing, bioinformatics analysis, and variant filtering for the proband were performed as previously described (Zeng et al 2022b). Copy number analysis was performed using DECoN (Fowler et al 2016) with default settings and the BAM files from the same enrichment panel and sequencing run as controls.…”