儿童原发性限制型心肌病三例的临床特征及遗传分析

Clinical characteristics and genetic analysis of three pediatric patients with idiopathic restrictive cardiomyopathy

目的研究3例儿童原发性限制型心肌病（ restrictive cardiomyopathy, RCM）的临床特征及心肌病理改变，并进行心肌肌节蛋白基因突变分析，以期发现遗传致病基础。方法采集和分析3例RCM患儿及其父母的临床资料。同时选择100名健康儿童作为正常对照。病例1和病例2接受心肌活检，标本行常规组织学和电镜检查。聚合酶链式反应（PCR）扩增RCM患儿肌钙蛋白I、肌钙蛋白T、β肌球蛋白重链、肌动蛋白基因编码外显子及附近上下游序列，直接双向测序并进行分析。如发现突变，则进一步检测该突变在患儿父母及正常对照儿童中的分布情况。结果3例患儿病情呈进行性加重。病例1合并小型膜周部室间隔缺损，病例2合并室间隔中下段轻度肥厚。组织学检查提示病例1存在广泛的心肌细胞排列紊乱和轻度纤维化，电镜显示肌节数量减少，线粒体显著增大变形；病例2心肌细胞轻度肥厚和间质纤维化，电镜检查显示z线排列紊乱，间距不规则。遗传分析分别在病例1和病例3中发现了肌钙蛋白I基因R204H、R192H杂合突变，在病例2中发现了肌钙蛋白T基因100—101delNE杂合突变，所有突变所在蛋白序列均高度保守。所有突变只出现在患儿，而父母及100名正常对照儿童中均未检测到相同突变。结论3例RCM患儿确诊后病情均进展迅速。3例患儿进行遗传分析均发现了致病突变，其中2个为肌钙蛋白I基因突变，1个为肌钙蛋白T基因突变。研究结果有助于加深对儿童原发性RCM发病机制的认识。

Objective Restrictive cardiomyopathy （RCM） is rare in children, and little is known about the molecular basis of RCM. The aim of this study was to investigate the clinical and myopathological characteristics and to detect mutations on cardiac sarcomere protein genes in three idiopathic pediatric RCMs. Methods Detailed clinical characteristies and familiar history were obtained in three idiopathic pediatric RCMs. One hundred healthy pediatric individuals were recruited as controls. Histological evaluation was performed with heart tissue retrieved at catheterization in case-1 and case-2. The entire coding sequences of four cardiac sarcomere protein genes, including cardiac troponin T （TNNT2）, cardiac troponin I（ TNNI3 ）, 13-myosin heavy chain（ MYH7 ）, and α-actin （ACTC） were screened for mutations. Sequence variants were then tested in the family as well as in 100 healthy control DNAs. Results All three index cases were diagnosed as primary RCMs without family history, and their clinical conditions deteriorated rapidly. Case-1 was in combination with ventricular septal defect. Case-2 was in combination with mid- and inferoseptal hypertrophy. In case-l, myocardial biopsies displayed extensive anisomorphism and disarray of cardiomyocytes ; electron microscopy showed large stacks of severely dysmorphic megamitochondria and focal Z-disc streaming. In case-2, endomyocardial biopsy revealed moderate myoctyte hypertrophy with mild interstitial fibrosis ; transmission electron microscopy showed misalignment of Z-bands and unequal Z-Z band distances. Genetic analysis identified two heterozygous missense mutations in TNNI3, with R204H in case-1 and R192H in case-3 respectively. A de rtovo heterozygous deletion in TNNT2 （p. AsnlOO_Glul01del ） was identified in case-2. Sequence analysis shows that all three mutations are located in a position highly conserved across many species. The three mutations were negative for their parents and controls. Conclusion The clinical conditions in all three index cases are deteriorated rapidly after diagnosed as primary RCM. Three heterozygous mutations including two in TNN13 and one in TNNT2 gene are identified in the three RCMs respectively, which are considered as causative mutations. These findings provide new insights into the molecular etiology responsible for pediatric RCM.