Diagnosis of Inherited Neuromuscular Disorders by Next-Generation–Sequencing - Next Generation Sequencing Technologies in Medical Genetics - page 55

assessment decreases following WGS or WES due to lower coverage (Wheeler
et al. 2008 ). Sequencing more genes at a lower coverage leads to an increased risk
of false negative and an increased number of false positive variants that are time-
consuming to validate. Therefore, the major reasons for use of the NMD panel
strategy as a routine approach in genetic diagnostic laboratories are the reproduc-
ibility, detection sensitivity, and it applications to examine mutations in genetically
heterogeneous disorders.

6.3 Next-Generation-Sequencing Panel for Duchenne

and Becker, Congenital, and Limb Girdle Muscular
Dystrophies

Duchenne muscular dystrophy and Becker muscular dystrophies are the most com-
mon forms of childhood muscular dystrophy (Emery 1991 ). Genetic testing of the
DMD gene is commonly used to confi rm the diagnosis. Defi ning the mutational
spectrum is important for genetic counseling, prenatal diagnosis, and selecting the
patients eligible for future mutation-specifi c treatments. The mutational spectrum
can be approximated as follows: a large deletion in about 60 % of patients, a large
duplication in about 10 % of patients, and small mutations confi ned mostly to cod-
ing exons in about 30 % of patients (Lim et al. 2011 ). In most laboratories, methods
for detecting large deletions/duplications and methods for detecting small muta-
tions are conducted separately (Bennett et al. 2001 ; Flanigan et al. 2003 ; Lalic et al.
2005 ; Mendell et al. 2001 ; Prior and Bridgeman 2005 ).
A study reports the application of NGS in the genetic diagnosis development of
Duchenne muscular dystrophy or Becker and related muscular dystrophies to
address diagnostic issues related to complex mutational spectrum, large size of the
DMD gene, and the costly requirement of two or more analytical methods to account
for known point, small indels, and large indel mutations (Table 6.1 ; Lim et al. 2011 ).
The study subjects were 25 patients: 16 with defi cient dystrophin expression with-
out a large deletion/duplication and nine with a known large deletion/duplication.
Technically, Lim et al. used a custom solution-based target enrichment kit for DMD
and other muscular dystrophy-related genes and Illumina Genome Analyzer as a
sequencing platform (Lim et al. 2011 ). The mean coverage of the DMD gene was
107X and exonic regions had at least the read depth of 8X. Signifi cantly, small
mutations were detected in most patients (15 out of 16) without a large deletion/
duplication.
For the development of one platform for comprehensive DMD mutation analysis,
Lim et al. used these 16 patients as the standard and accurately predicted the deleted
or duplicated exons in the nine patients with known mutations. Pathogenic muta-
tions were observed in 15 of 16 patients, and the mutation detection rate and distri-
bution of mutation by types (12 nonsense, two small deletions causing frameshifts,
and one splicing mutation) were similar to the results of other studies using different
methods. Because 16 patients in this study were selected randomly and their