Table_3_TNNT2 Missplicing in Skeletal Muscle as a Cardiac Biomarker in Myotonic Dystrophy Type 1 but Not in Myotonic Dystrophy Type 2.xls

Cardiac involvement is one of the most important manifestations of the multisystemic phenotype of patients affected by myotonic dystrophy (DM) and represents the second cause of premature death. Molecular mechanisms responsible for DM cardiac defects are still unclear; however, missplicing of the cardiac isoform of troponin T (TNNT2) and of the cardiac sodium channel (SCN5A) genes might contribute to the reduced myocardial function and conduction abnormalities seen in DM patients. Since, in DM skeletal muscle, the TNNT2 gene shows the same aberrant splicing pattern observed in cardiac muscle, the principal aim of this work was to verify if the TNNT2 aberrant fetal isoform expression could be secondary to myopathic changes or could reflect the DM cardiac phenotype. Analysis of alternative splicing of TNNT2 and of several genes involved in DM pathology has been performed on muscle biopsies from patients affected by DM type 1 (DM1) or type 2 (DM2) with or without cardiac involvement. Our analysis shows that missplicing of muscle-specific genes is higher in DM1 and DM2 than in regenerating control muscles, indicating that these missplicing could be effectively important in DM skeletal muscle pathology. When considering the TNNT2 gene, missplicing appears to be more evident in DM1 than in DM2 muscles since, in DM2, the TNNT2 fetal isoform appears to be less expressed than the adult isoform. This evidence does not seem to be related to less severe muscle histopathological alterations that appear to be similar in DM1 and DM2 muscles. These results seem to indicate that the more severe TNNT2 missplicing observed in DM1 could not be related only to myopathic changes but could reflect the more severe general phenotype compared to DM2, including cardiac problems that appear to be more severe and frequent in DM1 than in DM2 patients. Moreover, TNNT2 missplicing significantly correlates with the QRS cardiac parameter in DM1 but not in DM2 patients, indicating that this splicing event has good potential to function as a biomarker of DM1 severity and it should be considered in pharmacological clinical trials to monitor the possible effects of different therapeutic approaches on skeletal muscle tissues.

心脏受累是肌强直性营养不良（myotonic dystrophy, DM）患者多系统表型的核心表现之一，亦是其过早死亡的第二大诱因。目前，DM心脏缺陷的分子机制尚未完全阐明；不过，心肌肌钙蛋白T（cardiac isoform of troponin T, TNNT2）与心脏钠通道（cardiac sodium channel, SCN5A）基因的异常剪接，可能与DM患者出现的心肌功能减退及传导异常密切相关。鉴于在DM骨骼肌中，TNNT2基因呈现与心肌组织一致的异常剪接模式，本研究的首要目标为验证：TNNT2异常胎儿型异构体的表达，究竟是继发于肌病性改变，还是能够直接反映DM的心脏表型。本研究对伴或不伴心脏受累的1型DM（DM1）与2型DM（DM2）患者的肌肉活检样本，开展了TNNT2及多种参与DM病理过程的基因的可变剪接分析。分析结果显示，DM1与DM2患者的肌肉特异性基因异常剪接程度均高于再生对照肌肉，提示这类异常剪接或在DM骨骼肌病理进程中发挥重要作用。针对TNNT2基因的分析表明，其异常剪接在DM1肌肉组织中较DM2更为显著：DM2患者的TNNT2胎儿型异构体表达水平低于成年型异构体。这一现象似乎与肌肉组织病理改变的严重程度无关——DM1与DM2肌肉的病理改变程度相近。上述结果提示，DM1中更为显著的TNNT2异常剪接，或许并非仅由肌病性改变所致，而是能够反映DM1较DM2更为严重的整体表型，包括DM1患者较DM2更为严重且高发的心脏受累问题。此外，在DM1患者中，TNNT2异常剪接与QRS心脏参数呈显著相关，但在DM2患者中未观察到此关联。这表明该剪接事件具备成为DM1病情严重程度生物标志物的良好潜力，亦可在药物临床试验中用于评估不同治疗方案对骨骼肌组织的潜在疗效。