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

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 CNBP 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 CNBP 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)_n repeat downstream to the known (CCTG)_n repeat in 4 other patients. By analyzing a cohort of 168 patients with confirmed DM2 (cohort 2), we found that the additional (TCTG)_n repeat is present in at least 84% of patients.

Our study revealed the presence of an additional repeat (TCTG)_n in most of the patients living with DM2. Large expansions of this repeat likely hinder sufficient amplification of the disease causing (CCTG)_n repeat. Because the (TCTG)_n repeat is likely mosaic in length, (CCTG)_n 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)_v(TCTG)_w(CCTG)_n(TCTG')_m as the updated model for the structure of CNBP repeat expansions and recommend adapting the diagnostic guidelines accordingly. The effect of the (TCTG)_n 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.