Validation of Optical Genome Mapping for the Molecular Diagnosis of Facioscapulohumeral Muscular Dystrophy

Abstract

The molecular diagnosis of facioscapulohumeral muscular dystrophy (FSHD) relies on detecting contractions of the unique D4Z4 repeat array at the chromosome 4q35 locus in the presence of a permissive 4q35A haplotype. Long, intact DNA molecules are required for accurate sizing of D4Z4 repeats. We validated the use of optical genome mapping to determine size and haplotype of D4Z4 alleles for FSHD analysis. The cohort included 36 unique DNA specimens from fresh blood samples or archived agarose plugs. High-molecular- weight DNA underwent sequence-specific labeling followed by separation and image analysis with data collection on the Saphyr system. D4Z4 allele sizes were calculated and haplotypes determined from the labeling patterns. Each specimen had previous diagnostic testing using restriction enzyme digests with EcoRI, EcoRI/BlnI, XapI, or HindIII, followed by pulsed field gel electrophoresis and Southern blot analysis with appropriate probes. Optical genome mapping detected 4q35 and 10q26 alleles ranging from 1 to 79 D4Z4 repeats and showed strong correlation with Southern blot allele sizing (R2 = 0.95) and haplotyping (133 of 134; 99.4% haplotype match). Analysis of inter-assay and intra-assay runs showed high reproducibility (0.03 to 0.94 %CV). Subsequent optical genome mapping for routine clinical testing from 315 clinical FSHD cases compared favorably with historical result trends. Optical genome mapping is an accurate and highly reproducible method for chromosomal abnormalities associated with FSHD.

Copyright © 2021 Association for Molecular Pathology and American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Figures

Figure 1

Linear regression shows that repeat array allele size according to optical genome mapping correlates well with the size determined by Southern blot analysis. D4Z4 repeat array sizes in kilobase pairs for optical genome mapping and Southern blot analysis from 1 to 79 repeats is shown with the trend line. Both the slope of the curve and the correlation (R2) approach 1.0 at 0.977 and 0.9541, respectively.

Figure 2

Optical genome mapping direct label patterns that differentiate the 4q35 A and B haplotypes adjacent to the D4Z4 repeat array (D4F104S1 region, nucleotides 4384 to 4858) (https://www.ncbi.nlm.nih.gov/genbank; GenBank accession number AF117653). Fluorescent labels [dark blue vertical lines are shown as predicted by the reference sequence (green bar) or as identified from results of optical genome mapping (blue bars)]. The genome reference sequence (hg38) contains sequence for both A and B haplotypes. The number of D4Z4 repeats is determined by measuring the distance from the most proximal and most distal fluorescent labels.

Figure 3

Optical genome mapping direct label pattern for chromosome 18q. The summary alignment of the labeled molecules (two bottom blue bands labeled Allele 1 and Allele 2) is shown aligned to the reference for chromosome 18 (Ref 18 band, centromere region shown in green) with the chromosome banding pattern at the top (white, gray, red, and black bars). A deletion of 18p11 to the telomere end from one chromosome is identified by the presence of only one pattern of labeled molecules and an absence of aligned molecules for one chromosome (Allele 2) consistent with the loss of approximately 15 Mb. The copy number window also highlights the loss (green line in the peach-colored bar).