Methodology

Method: Optical Mapping, Southern Blot; DNA sequencing

Details:  Peripheral blood leukocytes (or cultured prenatal cells) have high molecular weight DNA extracted. FSHD analysis via Bionano DLS labeling, Saphyr instrumentation, and EnFOCUS FSHD specific pipeline/de novo assembly pipeline is based on optical mapping data collected on the Saphyr Genome Imaging instrument. Based on specific labeling and mapping of ultra-high molecular weight DNA in nanochannel arrays, optical mapping provides a high-resolution analysis of the D4Z4 repeat array. The molecules aligning to regions of interest are extracted and assembled. The resulting consensus maps are used for the Bionano EnFocus FSHD Analysis. The D4Z4 repeat regions in chromosomes 4 and 10 are sized, and the permissive and non-permissive haplotypes (4qA and 4qB, respectively) are determined. Additional structural variants and copy number gains and losses are noted in the proximity of the D4Z4 repeat array on chromosome 4 and of the SMCHD1 gene on chromosome 18.

Additional testing for FSHD2 involves determining the methylation status of the D4Z4 repeats, via Southern blot analysis, that uses isolated DNA. In patients with a permissive 4qA allele and an appropriate level of hypomethylation, mutation analysis of the SMCHD1 gene is performed on a next generation sequencing platform.