Diagnosis of Inherited Neuromuscular Disorders by Next-Generation–Sequencing - Next Generation Sequencing Technologies in Medical Genetics - page 56

genotypes were unconfi rmed, the mutation detection rate might provide further
support for the accuracy of this method. Additionally, inclusion of noncoding
regions in the design permitted breakpoint junction mapping.
Lim et al. present a more practical approach by testing a relatively large number of
patients and demonstrating the detection of complex mutational spectra in a single
platform (Lim et al. 2011 ). This approach allows reaching a genetic diagnosis of
DMD/BMD in a shorter time as compared to the combined MLPA and Sanger
sequencing methods. Moreover, inclusion of other related genes might contribute to
reduction of cost for clinical use. The current approach has an advantage for the
genetic diagnosis of Duchenne muscular dystrophy and Becker muscular dystrophy
wherein a comprehensive mutational search may be feasible using a single platform.

6.4 Duchenne and Becker Muscular Dystrophy Diagnosis

by Next-Generation-Sequencing

Dystrophin is a large, rod-like cytoskeletal protein and located mainly in skeletal
muscles and cardiac muscles (Xie et al. 2012 ). This protein stabilizes and pro-
tects muscle fi bers and may play a role in signaling within cells. Mutations in the
DMD gene can alter the structure or function of dystrophin resulting in fatal,
X-linked disorder occurring at a frequency of about 1 in 3,500 new-born males
(Sironi et al. 2006 ). There are two identifi ed hot spots of DMD gene: the major
hot spot involves the region of exons 40-55, while the minor one is located
around intron 7 (Sironi et al. 2006 ). The high demand for low-cost and high-
throughput sequencing has urged the development and progress of high-through-
put NGS. With the need to obtain a genetic diagnosis more rapidly and to avoid
the gene-by-gene Sanger sequencing approach, NGS' position on the stage of
DNA sequencing and causative mutations detection is important (Sironi et al.
2006 ). To this end, a targeted NGS strategy was employed in detecting DMD
gene mutations, a large gene containing 79 exons with different mutation types
and regions (Xie et al. 2012 ).
Xie et al. carried out DMD mutation analysis by NGS, employing solid phase
capture followed by HiSeq 2000 sequencing, on a Chinese pedigree that included a
proband and a proband's cousin with the clinical diagnosis of Duchenne muscular
dystrophy (Xie et al. 2012 ) (Table 6.1 ). The proband and proband's cousin devel-
oped symptoms of muscle weakness, diffi culty walking, and large calves. Progressive
muscle weakness resulted in the inability to walk, breathing diffi culties, contrac-
tures of the ankles, knees, and hips, and scoliosis. For the DMD gene of the pro-
band, the average depth was 471X, the average depth for each base pair was 160X,
and the average median-depth across 79 exons was 168X. On average, 98.5 % of
base pairs with greater than 20X coverage were successfully detected. Two same
variants were detected in exon 70 of the proband and his cousin. A literature-
annotated disease nonsense mutation (c.10141C>T, NM_004006.1) that has been