Application of Next-Generation–Sequencing in Hearing Loss Diagnosis - Next Generation Sequencing Technologies in Medical Genetics - page 65

detect 246 genes responsible for either human or mouse deafness. With a 95 % of
coverage of the targeted bases at 10x, pathogenic mutations were identifi ed in six of
the 11 probands and their families in CDH23 , MYO15A , TECTA , TMC1 , and WFS1
(Brownstein et al. 2011 ).
Even though successes have been shown, hybridization capture-based enrich-
ment has restrictions in the capture of GC-rich or repetitive elements as well as gene
family members that share sequence homology. The presence of repetitive or high
GC content sequences results in incomplete selection, selection bias, and uneven
capture effi ciency. This may result in reduced sensitivity and specifi city that are
highly required in diagnostic testing (De Keulenaer et al. 2012 ; Schrauwen et al.
2013 ). To address these disadvantages, PCR amplifi cation-based enrichment has
been employed in several hearing loss studies followed by NGS.

7.5.1 PCR-Based Hearing Loss Panels

A European group designed a primer library including 646 specifi c primer pairs for
exons and most of the UTR of 15 ARNSHL genes, using semiautomated conven-
tional PCR. All amplicons were pooled in an equal molar concentration and ana-
lyzed using Roche 454 NGS technology (Table 7.1 ; De Keulenaer et al. 2012 ). This
platform generated the coverage of 95 % targeted bases at 30x depth. Among fi ve
patients with congenital genetic deafness, causative mutations were identifi ed in
four patients. Among these, two novel mutations in CDH23 and OTOF were found
in three patients that were also characterized as interesting regions in a previous
linkage study. Similarly, Licastro et al. used a long-PCR-based enrichment and
NGS to develop a diagnostic panel for Usher syndrome genes (Licastro et al. 2012 ).
Molecular diagnosis in Usher syndrome is hindered by signifi cant genetic heteroge-
neity, the large size of some of the Usher genes, numerous polymorphic variants in
genes such as MYO7A and USH2A , and digenic inheritance which was also pro-
posed in some Usher syndrome cases (Bonnet et al. 2011 ). At least 11 loci and nine
causative genes have been reported associated with three subtypes of Usher syn-
drome. Current diagnostic strategies for Usher syndrome include Sanger sequenc-
ing of Usher genes, which is a demanding procedure in terms of both cost and time,
or microarrays-based genotyping method that only detects the previously reported
mutation. This study showed this NGS platform had 94 % coverage of target bases
at 25x. Eleven pathogenic mutations in MYO7A , CLRN1 , GPR98 , USH2A , and
PCDH15 were identifi ed in ten out of the 12 USHER patients, while genetic causa-
tion of two patients still stayed negative, indicating a positive diagnostic rate of
84 % in this study.
The main advantage of microdroplet PCR-based technology, such as RainDance,
is able to combine high-throughput automation and highly sensitive, specifi c, and
uniform amplifi cation using target specifi c primers (Tewhey et al. 2009 ). Recently,
two research groups used a similar strategy, applying RDT microdroplet PCR
enrichment and sequencing on the Illumina HiSeq 2000 sequencer, to develop NGS