Screening of mutations by targeted next generation sequencing

For family 1 a targeted next generation sequencing panel comprising 10 ion channel genes and 245 primary myopathies/muscular dystrophies-related genes (Suppl. Table ¹) was employed to screen the causal mutations. Genomic DNA from peripheral blood was extracted with High Pure PCR Template Preparation Kit (Roche, Basel,CH) according to the manufacturer’s instructions. DNA fragments were enriched by solution-based hybridization capture and followed by sequencing with an Illumina Miseq platform (Illumina, San Diego, CA, USA) with the 2 × 300 bp paired-end read module. The hybridization capture procedure was performed with the SureSelect Library Prep Kit (Agilent, Santa Clara, CA, USA). For further verification of the candidate mutations, we performed the Sanger sequencing with the DNA samples extracted from the patient and the parents. PCR was performed with GoldStar Taq DNA Polymerase (CWbiotech) according to standard protocol. PCR products were sequenced on an ABI3730xl DNA Analyzer (Applied Biosystems). Public databases including dbSNP138, 1000 Genome project, Exome Sequencing Project, ExAC, ClinicVar and HGMD were used to screen variants. Prediction of functional effect was evaluated by PolyPhen-2 and SIFT scores. To detect CNV (Copy number variant), sequencing depth of each region covered by the probes was calculated according to the alignment files. ExomeDepth Package was also used to find potential CNVs.

For family 2 genetic analysis of ion channel genes SCN4A, CACNA1S, KCNJ2 and CLCN1 was performed at the Neurogenetics Unit, National Hospital for Neurology and Neurosurgery as provided by the Channelopathy Highly Specialized National Service for rare disease. Samples underwent Next-Generation Sequencing on an Illumina HiSeq following enrichment with an Illumina custom Nextera Rapid Capture panel (Illumina, Inc., San Diego, CA).