NCM 2021: Nanopore sequencing increases resolution of DMPK hypermethylation in myotonic dystrophy type 1 patients

Mytonic dystrophy type 1 (DM1) is a multisystemic disorder with many characteristics, the main ones being muscle degeneration, weakness, and myotonia. It is the most common form of muscular dystrophy in adults. The genetic background of DM1 is the expansion of a CTG repeat in the 3’ UTR of the DMPK gene on chromosome 19. This region alters the transcription of DMPK and its neighbouring genes. The result is a ‘toxic gain-of-function mRNA’ that leads to splicing, and deregulation. The CTG repeat is unstable in both meiosis and mitosis so it can expand over generations and during the lifespan of an individual. The CpG sites around the repeat may become hypermethylated, and there is a correlation between methylation and phenotype. Ulf’s aim was to see if long-read sequencing using Oxford Nanopore technology could give more detailed information on the CTG repeat and the surrounding methylation, compared to traditional methods. Four patients with hypermethylation and four controls were compared using microarray and nanopore sequencing, using the MinION and Cas9 Sequencing Kit. Very good coverage was seen in samples 1 and 3 but lower in samples 2 and 4 which was suggested to be due to the age of these samples (several years old). STRique was used to study the repeat length and enabled identification of the progenitor allele, which had the shortest repeat length, modal repeat length, and somatic mosaicism. Methylation was detected using three different programs: Oxford Nanopore’s Megalodon, Nanopolish, and DeepSignal, and the results showed very good correlation. There was also good correlation between the array and sequencing platforms; however, nanopore sequencing offered higher resolution, as there was data from all CpG sites, and the ability to simultaneously analyse CTG repeats with methylation status on a single molecule level. Ulf found that ‘hypermethylation can be ascribed to the expanded allele’.