Diagnosis of Inherited Neuromuscular Disorders by Next-Generation–Sequencing - Next Generation Sequencing Technologies in Medical Genetics - page 57

reported as a DMD causing mutation was found in two patients, the proband and his
cousin. Therefore, the nonsense led to the generation of a truncated dystrophin lack-
ing the C-terminal domains. NGS was demonstrated to be an effective approach for
determining the genetic cause and reaching a molecular diagnosis for the patients in
the study.

6.5 Congenital Muscular Dystrophies Next-Generation-

Sequencing Panel

Congenital muscular dystrophy disorders (CMD) can be classifi ed into four major
groups, based on the affected genes and the location of their expressed protein: (1)
abnormalities of extracellular matrix proteins ( LAMA2 , COL6A1 , COL6A2 , and
COL6A3 ); (2) abnormalities of membrane receptors for the extracellular matrix
( FKTN , POMGNT1 , POMT1 , POMT2 , FKRP , LARGE , ITGA7 , and DAG1 ); (3)
abnormal endoplasmic reticulum protein ( SEPN1 ); and (4) intranuclear envelope
protein ( LMNA ; Wang et al. 2010 ). A specifi c diagnosis can be challenging because
muscle pathology may not yield a defi nitive diagnosis, limited access to and exper-
tise in using immunohistochemical stains, and phenotypic as well as genetic hetero-
geneity. Muscle biopsy and genetic test fi ndings must be interpreted in a clinical
context, yet the majority of diagnostic testing is not accompanied by a standard
clinical data set. Sequential gene sequencing by conventional Sanger sequencing
has been routinely used to diagnose this group of muscular dystrophies even though
it is a time-consuming and costly approach to resolve clinically heterogeneous
genetic disorders.
Valencia et al. investigated the application of NGS technologies for the molecu-
lar diagnosis of CMD (Table 6.1 ; Valencia et al. 2012 ). To this end, the authors
assessed the analytical sensitivity and specifi city of two different enrichment tech-
nologies, solution-based hybridization (SS), and microdroplet multiplex PCR target
enrichment (RainDance Technologies, RDT), in conjunction with NGS, to identify
mutations in 321 exons representing 12 different genes (65 kb target region) involved
with congenital muscular dystrophies (Valencia et al. 2012 ). In this study a wild-
type control, which had all 12 CMD genes Sanger sequenced, was included to serve
as a normal control reference, along with fi ve positive control samples, with previ-
ously known mutations, blinded to laboratory staff and six blinded samples, with
previously uncharacterized CMD genes, from patients presenting with clinical
features of CMD.
Valencia et al. reported that NGS results across several parameters, including
sequencing metrics and genotype concordance (Valencia et al. 2012 ). No statisti-
cally signifi cant differences between the two enrichment methods were observed
for average read numbers and percentage mapping to the genome. At 5X the
coverage was 96 % and 88 % for SS and RDT samples, respectively. Unfortunately,
regions with low sequence coverage typically had a high GC, making amplifi ca-
tion diffi cult. Moreover, the genotype concordance of SS and RDT was less than