Application of Next-Generation–Sequencing in Hearing Loss Diagnosis - Next Generation Sequencing Technologies in Medical Genetics - page 66

hearing loss panels for 34 ARNSHL genes and 24 well-studied SNHL genes,
respectively (Schrauwen et al. 2013 ; Sivakumaran et al. 2013 ). The two NGS plat-
forms targeted all exons and fl anking intron regions of the hearing loss genes.
Schrauwen et al. presented an overall mean coverage depth in the target area of
1,585x and 95 % of the bases were covered at 30x, while the panel developed by
Sivakumaran et al. had a 96 % of the targeted bases covered at 40x. Sanger sequenc-
ing was used to verify the known variants. These two NGS panels both achieved
>99 % sensitivity and specifi city, indicating that the enrichment is a reliable plat-
form for mutation detection of hearing loss genes.
To detect the concordance between NGS panel and Sanger sequencing,
Sivakumaran et al. used the NextGENe software (SoftGenetics, LLC) which
detected a total of 394 variants in fi ve genes, GJB2 , CDH23 , MYO7A , EYA1 , and
OTOF , that had been sequenced by Sanger sequencing to confi rm the accuracy. The
results showed a >99.99 % concordance between NGS and Sanger sequencing by
evaluating more than 30,000 bp in the fi ve SNHL genes, except that only one C>T
substitution in MYO7A detected by NGS was not identifi ed by Sanger sequencing
due to a misalignment issue. Small indels were detected in the NGS data including
a 22-bp deletion in intron 27 of MYO7A . Since the acceptable false-positive and
false-negative rates are more stringent for clinical diagnostic use, the authors favor-
ably suggested to set >40x as coverage threshold at the target bases (Sivakumaran
et al. 2013 ).
In the panel Schrauwen et al. designed, all genes were selected from an ARNSHL
gene list and 24 patients with prelingual, moderate to profound hereditary NSHL in
autosomal recessive inheritance, and without GJB2 mutations were carefully
selected. Nine out of 24 patients (37.5 %) were confi dently diagnosed. Six patients
were found to have homozygous mutations and three patients had compound het-
erozygous mutations. The results also suggested a possible digenic fi nding in OTOF
and SLC26A4 genes in one patient. However, these two genes perform completely
different functions in the inner ear and proteins are expressed at different ear loca-
tions which weakened the evidence of digenic inheritance in this patient. The con-
vincing follow-up family study is an important step to confi rm the diagnosis
(Schrauwen et al. 2013 ).

7.6 Whole Exome Sequencing

Whole exome sequencing is emerging as a diagnostic tool for many inherited dis-
eases (Table 7.1 ; Chap. 8 ; Licastro et al. 2012 ; Schrauwen et al. 2013 ). However,
comparison between the performance of RDT deafness libraries to the Agilent
SureSelect 50 Mb Exome, Illumina TrueSeq 62 Mb exome, and Agilent SureSelect
exome kits only had 50-67 % of the targeted deafness gene exons covered at 20x
(Table 7.1 ). These results suggest that current exome sequencing does not provide
suffi cient target enrichment for hearing loss genes. However, improvement in cap-
ture technologies may increase the coverage in the near future.