(20) Optical Genome Mapping and Long Read Sequencing Identifies a Novel 27kb Dystrophin Gene Inversion in a Patient with Duchenne’s Muscular Dystrophy

Background: Genetic testing is the current standard approach to diagnose patients with suspected Muscular Dystrophy. Current testing uses targeted sequencing panels or comprehensive genome sequencing, analyzing candidate genes like Dystrophin for deletions, duplications, and mutations, identifying a pathogenic variant in 95-99% of DMD cases.
We report a case of a male patient who presented to clinic at 5 years of age with a history of delayed motor milestones, frequent falls, and difficulty with stairs due to generalized muscle weakness. Labs showed elevated CK (13,041) with chronically elevated transaminases. On exam, patient showed a positive Gower’s sign and round calves. He had neuromuscular scoliosis and cognitive impairment requiring an IEP.
At age 6, a neuromuscular panel found multiple non-significant VUSs. A subsequent muscle biopsy (western blot/IHC) showed severely reduced dystrophin rod domain labeling. Follow-up comprehensive DMD short-read sequencing (del/dup analysis) at age 11 was again negative.

Objectives: Due to the negative genetic testing results, we used two novel DNA analysis methods: Optical Genome Mapping (OGM, Bionano Inc.), a high-res cytogenetic method, and Long Read (LR) sequencing (ONT, Oxford Nanopore Technologies), for DNA variant analysis and detection of structural variants missed by short-read methods.

Design/Methods: For LR sequencing, we performed WGS at a targeted 30x coverage with 23kb average read lengths; structural variants were called with Sniffles2 and viewed in IGV. OGM achieved 252x average coverage with 335kb average fragment sizes. De-novo assembly used Bionano Access/Solve. Structural variants were identified against hg38.

Results: Both methods independently identified a novel 27kb inversion encompassing exons 68-73 (ChrX:31,171,362-31,202,982) which we classified as pathogenic. LR sequencing did not identify further DNA variants.

Conclusions: In summary, we describe a case of a patient with suspected DMD where current standard genetic testing did not identify a disease variant. Smaller inversions pose a unique challenge for current short-read sequencing. Both LR sequencing and OGM identified a pathogenic 27kb inversion in the Dystrophin gene with well-defined breakpoints demonstrating the clear advantages over current short-read technologies. We recommend that in patients with suspected DMD, OGM and LRS should be considered as methods to comprehensively assess DNA and structural variants in advance of more invasive procedures.