Diagnostic use of Massively Parallel Sequencing in Neuromuscular Diseases: Towards an Integrated Diagnosis

Abstract

Massively parallel sequencing is revolutionizing the genetic testing in diagnosis laboratories, replacing gene-by-gene investigations with a "gene panel" strategy. This new approach is particularly promising for the diagnosis of neuromuscular disorders affecting children as well as adults, which is constrained by strong clinical and genetic heterogeneity. While it leads to a strong improvement in molecular diagnosis, this new approach is dramatically changing the whole diagnosis process, establishing new decision trees and requiring integrated strategies between clinicians and laboratories. To have an overview of the implementation and benefit of these novel sequencing strategies for the diagnosis of neuromuscular disorders, we surveyed the current literature on the application of targeted genes panel sequencing, exome sequencing and genome sequencing. We highlight advantages and disadvantages of these different strategies in a diagnosis setting, discuss about unresolved cases, and point potential validation approaches and outcomes of massively parallel sequencing. It appears important to integrate such novel strategies with clinical, histopathological and imaging investigations, for a faster and more accurate diagnosis and patient care, and to foster research projects and clinical trials.

Keywords: Neuromuscular disease; dystrophy; exome sequencing; genetic testing; genome sequencing; mutation; myasthenia; myopathy; neuropathy; next generation sequencing; targeted genes panel sequencing.

Fig.1

Coverage and depth in MPS. a) Targeted Sequencing (TS) and ExomeSequencing (ES) target exons of a panel of genes or of most genesrespectively. Genome Sequencing (GS) targets the genome and inparticular exons and introns of all genes. TS may be used for thestudy of genomic regions. b) Each region of interest istheoretically sequenced several times by overlapping sequencefragments called reads. When considering a sequence of 10 bases(in blue), in case 7 out of the 10 bases are covered by 5different reads, the coverage of this region is 70% at 5X depth.A potential single nucleotide variant (SNV; C to G transition) isshown at the heterozygous state.

Fig.2

Workflow of informatic analysis following MPS. Each step is monitored with quality scores. Sequence reads are used for mapping to a reference genome, variant calling, filtering and ranking. This unique pipeline is nevertheless prone to end in different conclusions depending on the sequencing platform, algorithms and softwares used, as well as the training and expertise of the users.

Fig.3

Close integration of clinical, biochemical, medical imaging, histopathological and genetic data for an integrated diagnosis. The whole process implies a great expertise in each disease/gene and in particular may require an expert molecular laboratory for final interpretation of sequence variants. The diagnosis may result in specific patient management including potential existing therapies, and should allow in any case genetic counselling within the family (carrier, predictive, prenatal or pre-implantation testing). Inclusion of data into molecular and clinical databases is a pre-requisite for patients recruitment in clinical trials and to allow development of research projects on the disease.