Diagnosis of Inherited Neuromuscular Disorders by Next-Generation–Sequencing - Next Generation Sequencing Technologies in Medical Genetics - page 53

6.2 A Broad Neuromuscular Diseases Panel

A study reported the development of an effi cient screening strategy for heterogeneous
neuromuscular disease genes using two groups of patients; with or without known
mutations (Vasli et al. 2012 ; Table 6.1 ). Solution-based targeted enrichment, Agilent
SureSelect (SS) custom kit, of 267 known NMD genes (1.6 Mb target region) fol-
lowed by NGS on an Illumina Genome Analyzer IIx was performed. After align-
ment with the human reference genome, the mean coverage of the targeted exons
was 138X and the percentage of nucleotides with at least 10X coverage was 94 %.
Most of the exons were captured (>97 %), but 168 exons had low coverage sequences
due to high GC content. Mapping, variant calling, fi ltering, and ranking were used
to prioritize variants. On average, 1,162 variants and 152 indels were identifi ed, of
which 341 were not reported as SNPs. In the NMD panel, on average 125 variants
affecting splice sites or predicted to change the amino acid sequence were described.
Further prioritization was based on VaRank, clinical phenotype, and segregation.
The challenge of NGS data analysis is the identifi cation of the pathogenic
changes among the large list of variants (Vasli et al. 2012 ). Signifi cantly, the custom
VaRank scoring program blindly ranked the known mutations and implicated genes
fi rst in the list based on disease class and inheritance mode. Importantly, clinical,
histological, and molecular data were necessary for matching the genetic data with

Table 6.1 Capture and sequencing technologies that have been applied in the mutation detection
of muscular dystrophies

Target
interval
(kbp)

Muscular dystrophy
type(s)

No. of
genes

No. of
exons

Enrichment
platform NGS platform Reference

Inherited
neuromuscular
disorders

267 4,604 1,600 Solution-based

capture a

Illumina's
Genome
Analyzer IIx

Vasli et al.
( 2012 )

Duchenne muscular
dystrophy

1 79 30 Solid phase

capture b

HiSeq 2000 Xie et al.

( 2012 )

Congenital muscular
dystrophies

12 321 65 RainDance c ,

Solution-based
capture a

SOLiD 3 Valencia

et al.
( 2012 )

Congenital muscular
dystrophies

12 321 65 RainDance c SOLiD 3 Valencia

et al.
( 2013 )

Duchenne muscular
dystrophy,
congenital
muscular
dystrophies, limb
girdle muscular
dystrophies

26 747 1,069 Solution-based

capture a

Illumina's
Genome
Analyzer IIx

Lim et al.
( 2011 )

a Solution-based capture, Agilent's hybridization-based SureSelect
b Solid phase capture, NimbleGen's hybridization-based microarray capture
c RainDance, highly multiplex PCR amplifi cation