Hearing loss is among the most etiologically heterogeneous disorders, with more than 400 genetic syndromes that include hearing loss as a feature, more than 120 genes associated with non-syndromic genetic hearing loss, and a number of non-genetic causes.5,12 With the advent of NGS technologies, hundreds of candidate HL genes can be analyzed simultaneously in a cost-effective manner. Even so, the diagnostic yield varies among different patient cohorts and depends on the detection methods used. The degree of hearing loss, the onset age of hearing loss, the existence of family history, the ethnic origin, and the number of genes contained in the NGS panel may affect the rate of genetic diagnosis.8 In the present study, two NGS panels were applied to explore molecular etiology in a Chinese hearing loss cohort, and 40 out of 94 patients has been genetically diagnosed, rendering an overall yield of 42.6% in the study population. This is an acceptable yield, comparing to the 10–83% diagnostic rates reported in previous reports,8,13−16 since a large number of hearing loss patients in our hospital choose a stepwise approach and be pre-excluded for mutations in common deafness genes GJB2, SLC26A4 and MT-RNR1 prior to NGS.10 Therefore, it is an additional diagnostic rate. It also explains why the proportions of GJB2 and SLC26A4 gene variations are comparatively low in the study cohort (Fig. 1).
In the study, 39 patients received genetics evaluations by using the custom-designed HL panel and 24 were diagnosed (diagnostic rate was 61.5%); 55 patients received genetic testing by using clinical exome sequencing and 16 were diagnosed (diagnostic rate was 29.1%). Compared with CES, the custom-designed HL panel has comparatively higher diagnostic yield, less expensive price, and similar turn-around time. Besides, in this study cohort, all the genes detected by CES were included in the custom-designed HL panel. Thus, for patients with clinical diagnosed NSHL or SHL, the custom-designed HL panel can be an effective diagnostic tool, facilitates genetic diagnosis and improves the management of patients. However, due to the comparatively small sample size, the diagnostic yields of the two NGS tools may not adequately represent their diagnostic capacities. Besides, before the performance evaluation, doctors’ choices between the two NGS tools were subjective, and bias can be induced in the process. CES may have better performance in cases when we cannot distinguish whether hearing loss is a feature of certain syndromes or in addition to multi-malformations, since it evaluate all exons in the genome for variations associated with clinical symptoms. Moreover, CES does not rely on a list of genes involved in a particular disease process, and may identify genetic defects that were not directly associated with hearing loss (not included in HL panels) but cause hearing loss through altering bone structure or affecting sound conduction. In one of our previous cases, the patient suffered from multiple enchondromatosis, atresia of the external auditory canal, abnormality of the middle ear and severe hearing loss on the right side, but normal hearing on the left side. CES detected an AD likely pathogenic variant in EXT1 gene, inherited from the patient’s mother who had normal hearing but pelvic exostoses. EXT1 participate in forming a heterooligomeric complex, which is an essential factor in a signal transduction cascade for regulation of chondrocyte differentiation, ossification, and apoptosis.17 Defects in EXT1 gene may affect its function and cause hearing loss through altering bone structure and affecting sound conduction in the patient.
In the present study, a number of novel variants have been detected and the pathogenicity of them has been estimated according to the ACMG guidelines. As shown in Table 3, six variants predicted to generate direct stop codons were classified as pathogenic or likely pathogenic. One variant located at a canonical splice site (+/–1 and +/–2 positions of an intron) in MYO15A gene was classified as pathogenic for which loss of function is reported as cause of the disease. Another two variants predicted to affect splice-sites (c.3866 + 5G > A in MYO15A gene and c.285 + 5G > C in MYO7A gene) were classified as variants of uncertain clinical significance (VUS). Missense variant c.8138T > G (p.Leu2713Arg) found in trans with another previously described likely pathogenic variant in MYO15A gene was not found in gnomAD exomes / genomes and predicted as damaging by multi computational programs in Varsome. The variants c.1606C > T (p.Arg536Trp) in CDH23 gene and c.5275A > C (p.Ser1759Arg) in MYO15A gene located at highly conserved regions, were predicted to alter the structure / function of the proteins by bioinformatics predictions and segregation analyses, but remain classified as VUS according to the ACMG guidelines. The in-frame insertion c.289_290insTGG (p.Gln97delinsLeuGlu) in MYH14 gene was found in a patient and his affected father with an AD pattern of inheritance. The variant was not found in healthy control population databases, and was predicted as conserved by bioinformatic tool of PhyloP100way. The detection and evaluation of novel variants is of importance in expanding the spectrum of variants associated with hearing loss.
Furthermore, NGS panels allow the simultaneous analysis of hundreds of genes, and sometimes pathogenic variants in different genes can be found in one patient.8 In the study cohort, seven cases harbored pathogenic variants in different genes or multi pathogenic variants in the same genes. The patient from Case 21980 carried homozygous c.235delC (p.Leu79CysfsTer3) variant in GJB2 gene in addition to the heterozygous c.5217delT (p.Val1740LeufsTer4) AR variant in PTPRQ gene. The patient from Case 23697 harbored homozygous c.109G > A (p.Val37Ile) variant in GJB2 gene in addition to exon 16 deletion in STRC gene. The patient from Case 25257 carried homozygous c.453 + 2T > C variant in TMC1 gene in addition to the heterozygous c.109G > A (p.Val37Ile) variant in GJB2 gene, was born to a consanguineous family with another hearing loss child. The patient from Case 65920 carried c.291T > G (p.Tyr97Ter) and c.285 + 5G > C variants in TMC1 gene in compound heterozygous state, in addition to the heterozygous c.109G > A (p.Val37Ile) variant in GJB2 gene. The patient from Case 51456 carried homozygous c.109G > A (p.Val37Ile) variant in GJB2 gene, in addition to a de novel variant c.4353G > C (p.Gly1451=) in CHD7 gene, which may be responsible for CHARGE syndrome the patient presented with. The patient from Case 39130 carried c.919-2A > G and c.589G > A (p.Gly197Arg) variants in SLC26A4 gene, which may be responsible for hearing loss and enlarged vestibular aqueduct (EVA) in the patient. Besides, the patient carried whole gene deletion (17p12) of PMP22, which may be the cause of hereditary neuropathy with liability to pressure (HNLP) in the patient. The patient from Case 4377 presented with hearing loss and EVA has been detected with three heterozygous LP variants in SLC26A4 gene, c.1547dupC (p.Ser517PhefsTer10) inherited from her father, c.563T > C (p.Ile188Thr) and c.1746delG (p.Ala584ArgfsTer2) from her healthy mother. These findings have important implications for reproductive genetic counseling.