Single-molecule optical mapping enables quantitative measurement of D4Z4 repeats in facioscapulohumeral muscular dystrophy (FSHD)

Abstract

PurposeFacioscapulohumeral muscular dystrophy (FSHD) is a common adult muscular dystrophy. Over 95% of FSHD cases are associated with contraction of the D4Z4 tandem repeat (~3.3 kb per unit) at 4q35 with a specific genomic configuration (haplotype) called 4qA. Molecular diagnosis of FSHD typically requires pulsed-field gel electrophoresis with Southern blotting. We aim to develop novel genomic and computational methods for characterising D4Z4 repeat numbers in FSHD.MethodsWe leveraged a single-molecule optical mapping platform that maps locations of restriction enzyme sites on high molecular weight (>150 kb) DNA molecules. We developed bioinformatics methods to address several challenges, including the differentiation of 4qA with 4qB alleles, the differentiation of 4q35 and 10q26 segmental duplications, the quantification of repeat numbers with different enzymes that may or may not have recognition sites within D4Z4 repeats. We evaluated the method on 25 human subjects (13 patients, 3 individual control subjects, 9 control subjects from 3 families) labelled by the Nb.BssSI and/or Nt.BspQI enzymes.ResultsWe demonstrated that the method gave a direct quantitative measurement of repeat numbers on D4Z4 repeats with 4qA allelic configuration and the levels of postzygotic mosaicism. Our method had high concordance with Southern blots from several cohorts on two platforms (Bionano Saphyr and Bionano Irys), but with improved quantification of repeat numbers.ConclusionWhile the study is limited by small sample size, our results demonstrated that single-molecule optical mapping is a viable approach for more refined analysis on genotype-phenotype relationships in FSHD, especially when postzygotic mosaicism is present.

Chromosome‐level genome assembly of a cyprinid fish Onychostoma macrolepis by integration of nanopore sequencing, Bionano and Hi‐C technology

“…Bionano Genomics has developed a complementary single molecule optical mapping technology, which contributes to long‐range ordering of sequence contigs. Due to its advantages in obtaining high repetitive sequence and detecting genome structure variation, Bionano technology has become a powerful tool for genome assembly (Dai et al., 2019; Lind et al., 2019). High‐throughput chromatin conformation capture (Hi‐C) technology enables the generation of genome‐wide 3D proximity maps and is an efficient and low‐cost strategy for sequence clustering, ordering, and orientation for pseudomolecule construction (Burton et al., 2013).…”

Rapid prenatal diagnosis of Facioscapulohumeral Muscular Dystrophy 1 by combined Bionano optical mapping and karyomapping

“…There are two methods of FSHD1 genetic diagnosis currently in use: Southern Blot and molecular combing. Recently, the Bionano optical mapping technique has been used for FSHD1 gene diagnosis . In the present study, we utilized Bionano optical mapping for prenatal diagnosis in a FSHD1 familiy with positive history.…”

“…Southern Blot is still the “gold standard” , but it is not so accurate because it detects the length of all D4Z4 repeats, and not their exact number. Research by Yi et al [] and Qian et al [] showed that Bionano optical mapping provided an accurate molecular diagnosis of FSHD, deriving haplotypes consistent with gold standard Southern blotting.…”

“…The genetic diagnosis of FSHD1 is very difficult, because of the special DNA structural features involved in the genetics of this disease: Recently, the Bionano optical mapping technique has been used for FSHD1 gene diagnosis 8,13 . In the present study, we utilized Bionano optical mapping for prenatal diagnosis in a FSHD1 familiy with positive history.…”

“…These deficiencies are tolerable in gene diagnosis but intolerable in prenatal diagnosis, so a simpler and more accurate method would be advantageous. In a recent approach shown by Dai Y et al , the commercial Bionano Saphyr single DNA molecule optical mapping system is well suited for the rapid diagnosis of FSHD1. This method produces ordered genome‐wide restriction maps by optical inspection and sizing of restriction fragments from single linearized DNA molecules .…”