Updated Structure of CNBP Repeat Expansions in Patients With Myotonic Dystrophy Type 2 and Its Implication for Standard Diagnostics

Myotonic dystrophy type 2 (DM2) is a multisystemic repeat disorder caused by the expansion of an unstable CCTG tetranucleotide repeat in the noncoding region of the gene. Standard diagnostic is based on Southern blot analysis or a unidirectional RP-PCR that amplifies the repeat from the downstream end. Our study reevaluated 80 patients (cohort 1) with clinical suspicion of DM2 but homozygous negative results using the standard diagnostic repeat-primed PCR (RP-PCR). Reanalysis was performed using a second RP-PCR that amplifies the repeat from the opposite direction. Individual samples were further analyzed by Oxford Nanopore Technology long-read sequencing, Sanger sequencing, and another RP-PCR. In addition, repeat expansions were further characterized in 168 patients with confirmed DM2 (cohort 2). We identified 5 of the 80 patients (cohort 1) with expanded repeats in and, as such, reclassified them as positive for DM2. The initial false-negative results were attributed to variants within the primer binding site of the standard RP-PCR in one patient and an additional novel (TCTG) repeat downstream to the known (CCTG) repeat in 4 other patients. By analyzing a cohort of 168 patients with confirmed DM2 (cohort 2), we found that the additional (TCTG) repeat is present in at least 84% of patients. Our study revealed the presence of an additional repeat (TCTG) in most of the patients living with DM2. Large expansions of this repeat likely hinder sufficient amplification of the disease causing (CCTG) repeat. Because the (TCTG) repeat is likely mosaic in length, (CCTG) repeat expansions are correctly detected in most patients. However, a few patients are at risk of a false-negative result using the standard RP-PCR, which had a false-negative rate of 0.7% (5/674) and a sensitivity of 97.3% in the cohort studied. Based on our findings, we propose (TG) (TCTG) (CCTG) (TCTG') as the updated model for the structure of repeat expansions and recommend adapting the diagnostic guidelines accordingly. The effect of the (TCTG) repeat on the phenotype remains to be determined but could be key for establishing a phenotype-genotype correlation for DM2 that remained elusive so far.