Wendlandt Martin, Erdmann Hannes, Rost Simone, Lucas Morghan C, Becker Kerstin, Kleinle Stephanie, Timmer Manuela, Bier Andrea, Wunderlich Gilbert, Wenninger Stephan, Walter Maggie C, Neuhann Teresa, Schoser Benedikt, Holinski-Feder Elke, Abicht Angela
MGZ-Medical Genetics Center, Munich.
Institute of Medical Biochemistry and Molecular Biology, University Medicine of Greifswald.
Neurol Genet. 2024 Dec 18;11(1):e200220. doi: 10.1212/NXG.0000000000200220. eCollection 2025 Feb.
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.
2型强直性肌营养不良症（DM2）是一种多系统重复序列疾病，由基因非编码区不稳定的CCTG四核苷酸重复序列扩增引起。标准诊断基于Southern印迹分析或从下游末端扩增重复序列的单向RP-PCR。
我们的研究重新评估了80例临床怀疑患有DM2但使用标准诊断重复引物PCR（RP-PCR）结果为纯合阴性的患者（队列1）。使用从相反方向扩增重复序列的第二种RP-PCR进行重新分析。个体样本通过牛津纳米孔技术长读长测序、桑格测序和另一种RP-PCR进一步分析。此外，在168例确诊为DM2的患者（队列2）中进一步对重复序列扩增进行了特征分析。
我们在80例患者（队列1）中鉴定出5例重复序列扩增，因此将他们重新分类为DM2阳性。最初的假阴性结果归因于1例患者标准RP-PCR引物结合位点内存在变异，以及另外4例患者在已知（CCTG）重复序列下游存在一个额外的新型（TCTG）重复序列。通过分析168例确诊为DM2的患者队列（队列2），我们发现至少84%的患者存在额外的（TCTG）重复序列。
我们的研究揭示了大多数DM2患者中存在额外的重复序列（TCTG）。该重复序列的大量扩增可能阻碍致病（CCTG）重复序列的充分扩增。由于（TCTG）重复序列的长度可能是嵌合的，大多数患者中致病（CCTG）重复序列的扩增能够被正确检测到。然而，少数患者使用标准RP-PCR有出现假阴性结果的风险，在本研究队列中其假阴性率为0.7%（5/674），灵敏度为97.3%。基于我们的研究结果，我们提出（TG）（TCTG）（CCTG）（TCTG'）作为重复序列扩增结构的更新模型，并建议相应地调整诊断指南。（TCTG）重复序列对表型的影响仍有待确定，但可能是建立迄今仍难以捉摸的DM2表型-基因型相关性的关键。
