PracticeUpdate: Neurology - Winter 2018

ICNMD 2018 25

" …unbiased next-generation sequencing methods (such as whole exome sequencing or whole genome sequencing) may represent the first step of a comprehensive diagnostic workflow for LGMD. "

Central to recognize similar individuals with the same genetic disease. The bioinformatic pipelinewas optimized, thanks to a collabora- tionwith the TelethonUndiagnosed Program. In addition, a subsequent approach for the still negative cases was developed. An “ultra exome” probe collection was designed to be used in connection with the 10x genomics. High-molecular-weight DNA fragments were partitioned into micelles, along with an adapter molecule and barcode sequence. Using this strategy, whole exome sequences are fully covered and phased and even small structural variations may be detected. Dr. Nigro concluded that unbiased next-generation sequencing methods (such as whole exome sequencing or whole genome sequencing) may represent the first step of a comprehensive diagnos- tic workflow for LGMD. This workflow includes traditional investigations such as muscle biopsies and electromyography and more specialized procedures such as ultrasound muscle imaging and magnetic resonance imaging.

subtype should be studied carefully and tailored to each clinical trial. Next-generation sequencing may be a first diagnostic step Vincenzo Nigro, MD, of the Università degli Studi della Campania “Luigi Vanvitelli,” Naples, Italy, explained that next-gener- ation sequencing has revolutionized the approach to genetic disorders, from sin- gle-gene testing to genomic studies. In particular, the care of individuals with het- erogeneous conditions having overlapping phenotypes, such as LGMD, has improved. Three next-generation sequencing strat- egies for DNA sequencing have been developed: • A targeted resequencing of specific regions/genes of interest captured by specific probes or amplified by poly- merase chain reaction • Whole exome sequencing targeting the entire set of coding regions (approxi- mately 1.5% of the human genome)

• Whole genome sequencing, which does not preselect DNA fragments Targeted approaches have included a strategy based on Haloplex selection (MotorPlex) and capture of 89 to >270 genes characterized by a megabase size of targeted regions. Using that original approach, approximately 50% of over 500 undiagnosed cases of LGMD were solved. However, further reduction of sequencing costs allow the opportunity to move toward whole exome sequencing. Whole exome sequencing is a unique test able to investigate both disease genes and novel candidate ones at the same time. Individuals with undiagnosed LGMD undergo standardized annotation using Phenotips. Selected cases are recruited for trio/quartet whole exome analysis. A complete whole exome enrichment with a target of approximately 69 Mb and cover- age of approximately 200x. Results are shared mainly using Phenome

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VOL. 3 • NO. 3 • 2018