Targeted gene panel use in 2249 neuromuscular patients: the Australasian referral center experience

Abstract

Objective To develop, test, and iterate a comprehensive neuromuscular targeted gene panel in a national referral center. Methods We designed two iterations of a comprehensive targeted gene panel for neuromuscular disorders. Version 1 included 336 genes, which was increased to 464 genes in Version 2. Both panels used TargetSeqTM probe‐based hybridization for target enrichment followed by Ion Torrent sequencing. Targeted high‐coverage sequencing and analysis was performed on 2249 neurology patients from Australia and New Zealand (1054 Version 1, 1195 Version 2) from 2012 to 2015. No selection criteria were used other than referral from a suitable medical specialist (e.g., neurologist or clinical geneticist). Patients were classified into 15 clinical categories based on the clinical diagnosis from the referring clinician. Results Six hundred and sixty‐five patients received a genetic diagnosis (30%). Diagnosed patients were significantly younger that undiagnosed patients (26.4 and 32.5 years, respectively; P = 4.6326E‐9). The diagnostic success varied markedly between disease categories. Pathogenic variants in 10 genes explained 38% of the disease burden. Unexpected phenotypic expansions were discovered in multiple cases. Triage of unsolved cases for research exome testing led to the discovery of six new disease genes. Interpretation A comprehensive targeted diagnostic panel was an effective method for neuromuscular disease diagnosis within the context of an Australasian referral center. Use of smaller disease‐specific panels would have precluded diagnosis in many patients and increased cost. Analysis through a centralized laboratory facilitated detection of recurrent, but under‐recognized pathogenic variants.

A Māori specific RFC1 pathogenic repeat configuration in CANVAS, likely due to a founder allele

“…For individuals where absence of a product upon flanking PCR suggested an expansion, we then performed repeat-primed PCR (RP-PCR), using primers specific for the AAAAG (reference allele), AAAGG (benign variant), and AAGGG (pathogenic) configurations. Because of Individual M2 V:1's unusually early clinical presentation, the Fulgent ataxia repeat expansion panel (ATN1, ATXN1, ATXN10, ATXN2, ATXN3, ATXN7, ATXN8, ATXN8OS, BEAN1, CACNA1A, FMR1, FXN, NOP56, PPP2R2B, TBP), and a broad neurogenetic gene panel (Beecroft et al, 2020) (genes are listed in Supplementary Table 1) were also performed to search for a possible other genetic condition explaining her early disease onset. In addition to these we performed functional studies to exclude Wilson's disease (copper studies), ataxia telangiectasia (alpha fetoprotein and ATM protein/kinase activity), vanishing white matter disease (transferrin isoforms) and white-cell enzyme testing.…”

Facilitations and Hurdles of Genetic Testing in Neuromuscular Disorders

“…GPS are frequently used in routine diagnostics since are cheaper then WES and WGS due to fewer genes targeted and require less data processing, analysis and storage. Since the analyzed region is smaller, deeper coverage is obtained, allowing a better detection of some copy number variations (CNVs) (e.g., PMP22 duplication/deletion [ 43 ]) and mosaicism, compared to WES [ 44 ]. In addition GPS do not reveal findings unrelated to the phenotype being investigated, avoiding incidental findings and ethical problems [ 44 ].…”

“…Although notable improvements in molecular analysis and bioinformatics are continually described, the technical limitations of short-read NGS are well known. Approximately 8.5% of the genome is extremely resistant to SNVs/small indels calling due to repetitive sequence or segmental duplications, causing poor variant detection in some clinically relevant genes [ 44 ]; this also have an effect in the detection of expansions or variants within NEB and TTN triplicated regions [ 43 ]. Moreover, in terms of capture efficiency, an important subset of GC-rich exons of coding genes is missed; accordingly, causative disease mutations present in these regions will be missed.…”

“…The use of short-read or long-read sequencing depends on the research or clinical application [ 89 ]; however, as the technology and bioinformatic tools continue to improve, long-read sequencing will likely become a regular feature in the rare disease genomics tools kit [ 43 ].…”

Heterozygous CAPN3 missense variants causing autosomal‐dominant calpainopathy in seven unrelated families

“…Where amplification across regions of the transcript were not successful, genomic DNA from muscle was isolated and primers, which included intron boundaries as described in Piluso et al [26] were used. Confirmatory sequencing was performed by gene panel at PathWest [27] and WES at the Broad Institute of MIT and Harvard. WES for patient F6, III:2, and familial variant confirmation sequencing for patient F6, II:1 and III:1 were performed by Blueprint Genetics.…”