Yu Q T, Ifegwu J, Marian A J, Mares A, Hill R, Perryman M B, Bachinski L L, Roberts R
Baylor College of Medicine, Houston, TX 77030.
Circulation. 1993 Feb;87(2):406-12. doi: 10.1161/01.cir.87.2.406.
The beta-myosin heavy chain (beta-MHC) gene has been identified as a major locus for familial hypertrophic cardiomyopathy (FHCM). We recently showed that one of the common mutations associated with FHCM is expressed in the cardiac muscle messenger RNA (mRNA) of an affected individual. Since beta-MHC is a major sarcomeric protein of cardiac and skeletal muscle, studies were performed to determine whether the mutation is also expressed in skeletal muscle.
Biopsies were obtained of skeletal muscle (biceps brachii) from a proband with FHCM known to have the missense mutation in exon 13 of the beta-MHC gene. RNA was extracted from skeletal muscle and lymphocytes by the RNAzol method. First-strand complementary DNA was synthesized by reverse transcription using an antisense primer to exon 16. Polymerase chain reaction (PCR) was performed using primers to exons 12 and 14 to amplify the segment encompassing exon 13. The PCR products were digested with Ddel restriction endonuclease. Undigested PCR product in the control and the proband was 321 base-pairs (bp). Ddel digestion of the PCR product from normal skeletal and lymphocytes showed two DNA fragments of 181 and 140 bp as expected, whereas digestion of the PCR product from the proband's skeletal muscle and lymphocytes showed four DNA fragments of 181, 149, 140, and 32 bp due to the mutation in exon 13. This indicates that the mutation in affected individuals is also expressed in the mRNA of skeletal muscle and lymphocytes.
To our knowledge, this is the first documentation of a beta-MHC gene mutation expressed in skeletal muscle. This finding is provocative. Does it impair skeletal muscle function? If so, how? If not, why not? Is the impairment, or lack of it, a clue to the molecular defect of cardiac muscle? Furthermore, skeletal muscle provides a readily accessible source of mRNA for expression studies and for purification of the beta-MHC protein, which is probably essential to future investigation designed to unravel the molecular basis of this disorder.
β-肌球蛋白重链（β-MHC）基因已被确定为家族性肥厚型心肌病（FHCM）的一个主要基因座。我们最近发现，与FHCM相关的常见突变之一在一名受影响个体的心肌信使核糖核酸（mRNA）中表达。由于β-MHC是心肌和骨骼肌的一种主要肌节蛋白，因此开展了研究以确定该突变是否也在骨骼肌中表达。
从一名已知β-MHC基因第13外显子存在错义突变的FHCM先证者身上获取骨骼肌（肱二头肌）活检样本。采用RNAzol法从骨骼肌和淋巴细胞中提取RNA。使用针对第16外显子的反义引物通过逆转录合成第一链互补DNA。使用针对第12和14外显子的引物进行聚合酶链反应（PCR），以扩增包含第13外显子的片段。PCR产物用Ddel限制性内切酶消化。对照和先证者中未消化的PCR产物为321个碱基对（bp）。正常骨骼肌和淋巴细胞的PCR产物经Ddel消化后显示出预期的181和140 bp的两个DNA片段，而先证者骨骼肌和淋巴细胞的PCR产物消化后显示出181、149、140和32 bp的四个DNA片段，这是由于第13外显子中的突变所致。这表明受影响个体中的突变也在骨骼肌和淋巴细胞的mRNA中表达。
据我们所知，这是首次记录到β-MHC基因突变在骨骼肌中表达。这一发现颇具启发性。它是否会损害骨骼肌功能？如果是，如何损害？如果不是，为何不会？这种损害或缺乏是否是心肌分子缺陷的线索？此外，骨骼肌为表达研究和β-MHC蛋白的纯化提供了易于获取的mRNA来源，这可能对未来旨在阐明该疾病分子基础的研究至关重要。
