肥厚型心肌病和假肥大型肌营养不良症家系的致病基因及临床研究

【作者】 刘亚欣；

【导师】 周宪梁；

【作者基本信息】 北京协和医学院 ， 内科学， 2014， 博士

【摘要】 研究背景肥厚型心肌病（hypertrophic cardiomyopathy, HCM）是由于单基因突变而导致的常染色体显性遗传性疾病,主要表现为左心室和（或）右心室及室间隔不对称肥厚、心室腔变小、心室顺应性降低。是青少年和运动员心源性猝死（Sudden cardiac death,SCD）最常见原因,可在任何年龄段发病,有显著的家族聚集性,家族性发病者占全部病例的50%,年死亡率约为1%-2%,具有发病年龄、发病程度和猝死风险等临床表型多样性的特点。研究发现：相关突变基因主要定位在β肌球蛋白重链基因、肌球蛋白结合蛋白C基因、心脏肌钙蛋白T基因等肌小节蛋白基因以及相关的线粒体基因与修饰基因上,超过900种不同的基因突变类型与HCM发生发展以及临床表型有关。目前在已经明确导致HCM的基因中发现至少1400个突变位点。指南中8个明确导致HCM的基因为p肌球蛋白重链（MYH7）、肌球蛋白结合蛋白c（MYBPC3）、肌钙蛋白T（TNNT2）、肌钙蛋白I（TNNI3）、a肌球蛋白（TPM1）、肌动蛋白（ACTC）、调节轻链（MYL2）和基本轻链基因（MYL3）。其中p肌球蛋白重链基因（MYH7）、肌球蛋白结合蛋白C基因（MYBPC3）及心脏肌钙蛋白T基因（TNT）是最常见的3个HCM致病基因,由这些基因突变引起的HCM约占75%。传统的Sanger法测序是遗传病分子诊断的金标准,但是将已知候选基因进行逐一排查耗时、费力及价格昂贵,特别是HCM基因比较多而且复杂。第二代高通量基因测序（Next generation squencing, NGS）以高通量、速度快、低成本为主要特征,可直接通过聚合酶或连接酶进行体外测序合成。运用NGS方法,不仅能检测到遗传性疾病的致病基因突变,而且能够发现新的基因或突变,全面地勾勒出基因组上的变异图谱。研究目的对家族性肥厚型心肌病患者进行第二代测序和家系验证,来分析家族性肥厚型心肌病患者的的致病基因及突变位点,分析基因型与临床表型及预后的关系。研究方法应用二代测序方法对家族性肥厚型心肌病家系的先证者进行基因检测,根据测序结果设计目标基因片段的引物,对患者及其家系成员进行第一代Sanger测序,即聚合酶链式反应扩增其功能区外显子片断,双脱氧末端终止法测序。分析该家系中所有患者的临床表型特点,并对其进行临床随访。研究结果1个肥厚型心肌病家系的先证者经第二代测序检测发现MYH7基因的18号外显子上c.1987G>A突变,导致精氨酸变为半胱氨酸,即Arg663Cys （R663C）。对其家系中其他成员进行基因筛查,全部进行p肌球蛋白重链（MYH7）18号外显子的目的基因片段的筛查,其中有4个家系成员同时有Arg663Cys突变,而正常对照组在同一位置未见异常。该家系患者临床表现为活动后胸闷伴晕厥或猝死,呈恶性表现,先证者进行了改良的Morrow外科手术,术后症状消失,临床随访2年,患者恢复良好,无活动后胸闷胸痛不适,超声心动图未发现进展性改变。另外1个肥厚型心肌病家系的先证者经第二代测序检测发现MYBPC3基因的14号外显子上新的移码突变c.1374delC,移码在465号氨基酸终止,终止密码（TGA）,即p.Arg458ArgfsX8（p.R458Rfs）,该突变为世界上首次报道。对其家系中其他成员进行Sanger测序,其中有3个家系成员同时有p.R458Rfs突变,而正常对照组在同一位置未见异常。该家系患者临床表现和预后有异质性,先证者及其弟弟表现为活动后胸闷,先证者的母亲和小姨已猝死,而其妹妹和女儿仅为突变携带者,无临床表现。结论p肌球蛋白重链基因的18号外显子突变Arg663Cys是家系1的致病突变。Arg663Cys所致肥厚型心肌病的临床症状出现早,多合并左室流出道的梗阻,容易出现晕厥、猝死等不良事件,预后较差,但是积极地进行手术治疗可缓解症状并能改善预后。家系2的致病突变是MYBPC3基因的14号外显子上新的移码突变c.1374delC突变,p.R458Rfs,该突变为世界上首次报道,该突变的表型有异质性,预后不同。研究背景肥厚型心肌病是一种以左室非对称性肥厚为特征的原发性心肌病,其主要并发症有心房血栓、心律失常、心衰和猝死,但合并左室血栓的肥厚型心肌病临床报道较少,本研究分析阜外心血管病医院10年间有左室血栓的肥厚型心肌病患者的临床资料,并探讨其临床特征、治疗及预后情况,为临床工作提供参考。研究目的探讨经心脏核磁证实合并有左心室血栓的肥厚型心肌病（Hypertrophic cardiomyopathy, HCM）患者的临床特征和预后情况。研究方法回顾性分析阜外心血管病医院2002年1月～2012年12月住院病人中经心脏核磁共振（cardiac magnetic resonance, CMR）证实有左室血栓的HCM患者的临床资料,并进行临床随访。研究结果经心脏核磁证实有左室血栓的HCM患者共6例（血栓组）,无左室血栓的HCM患者共284例（无血栓组）,血栓组的家族史比例、室性心律失常、左房内径、左室内径和室壁瘤明显高于无血栓组（p<0.05）,而左室射血分数和流出道梗阻比例明显低于无血栓组（p<0.05）。有左室血栓的患者接受华法令抗凝治疗,随访17～59月,未发现血栓栓塞,2例复查血栓消失,3例死亡,1例进行了全心脏移植。结论肥厚型心肌病合并左室血栓见于有室壁瘤或左室射血分数低下的患者,多合并室性心律失常,预后差；可发生栓塞事件,华法令可有效治疗左室血栓。背景：假肥大型肌营养不良症是由抗肌萎缩蛋白基因突变所致的一种X连锁隐性遗传神经肌肉疾病,又称为杜氏或贝克肌营养不良（Duchenne’s or Becker’s muscular dystrophy, DMD or BMD）,是以缓慢进行性加重的对称性肌无力和肌萎缩为特点的遗传性肌肉病变,其中DMD病情严重,预后差；BMD病情进展相对缓慢,可累及心脏。目的：总结假肥大型肌营养不良症伴心脏扩大患者家系的临床特征并进行基因诊断、分析和治疗及预后,为临床诊治提供借鉴。方法：对假肥大型肌营养不良症伴心脏扩大的先证者和家系成员进行血清肌酶、肌电图、心电图、心脏彩色超声检查及心脏核磁等临床检查,并采用Sanger直接测序的方法进行抗肌萎缩蛋白基因突变的检测,同时对100例无血缘关系的健康人群进行该位点的基因扩增测序,以排除所发现的突变为未知人群多态位点的可能。对确诊患者根据病情进行对症治疗并对其进行随访。结果：先证者符合BMD诊断,基因检测结果提示先证者携带dystrophin基因突变（c.4998₅000Del GCA,p.1667delAla,为首次报道）。家系成员中检测出3例患者携带以上突变,携带者均表现为四肢近端肌肉萎缩、无力,双侧腓肠肌假性肥大,血清肌酶的水平显著增高,心脏均受累,先证者表现为晚期心衰,并进行心脏移植手术,后恢复良好。结论：基因检测可为假肥大型肌营养不良症先证者及其家系成员进行明确诊断；对于假肥大型肌营养不良症伴心脏扩大引起心力衰竭的患者进行心脏移植术是治疗晚期患者可行有效的方法。更多还原

【Abstract】 BackgroundHypertrophic cardiomyopathy （HCM） is an autosomal-dominant monogenic form of cardiac disease caused by a mutation in one of the genes that encode a protein from the sarcomere, Z-disc, or intracellular calcium modulator and is characterized by unexplained left ventricular hypertrophy. It shows high variability in genetic heterogeneity and phenotypic characteristics. The genetic etiology responsible for HCM in many individuals remains unclear. Many causative genes have already been identified, including β-tropomyosin or myosin heavy chain7（MYH7gene）, cardiac myosin binding protein C （MYBPC3gene）, cardiac troponin T type2（TNNT2gene）, cardiac troponin I type3（TNNI3gene）, myosin light chain3（MYL3gene）, myosin light chain2regulatory （MYL2gene）, cardiac a-actin （ACTC gene） and, titin （TTN gene） and etc.Sanger sequencing is the traditional gold standard in the molecule diagnosis of genetic disease, but time-consuming, laborious and expensive to each investigate for the known candidate genes, especially in HCM. Next generation sequencing technology, with features of high through put, rapid sequencing and low cost, brings revolutionary changes in the fields of detecting gene mutations. The process of using this method to dectect mutations is simple. Researchers can combine whole genomere sequencing, target sequencing and transcriptome sequencing to dectect mutations in all-around, high accurate way.ObjectiveOur aims are to identify the disease-causing gene mutation with familial hypertrophic cardiomyopathy （FHCM） by next generation sequencing, and to analyze the correlation between their genotype and phenotype, prognosis.MethodsWe choose two Chinese pedigrees with HCM and extracted their DNA from whole blood. According to each of the21established HCM genes, the exons in the functional regions of the genes with mutation were amplified with PCR by targeted next-generation DNA sequencing. Proband were sequenced with the next generation DNA sequencing method. According to the results of sequencing, the probands were followed by Sanger sequencing, and the exon in the functional regions of the gene were amplified with PCR and the products were sequenced for their family members. The relation between the genotype and phenotype was analyzed in these families, and the products were directly sequenced.100normal subjects were recruited as control.ResultsOne missence mutation c.1987G>A （Arg663Cys） was identified in exon18of the beta myosin heavy chain gene in one family hypertrophic cardiomyopathy by next generation sequencing. Screening for of her family members by Sanger sequencing, and there are4family members also with mutation Arg663Cys in Exon18of the beta myosin heavy chain gene. The results of genetic test were normal in one hundred controls. The patients with mutation Arg663Cys clinically showed chest pain early, syncope, or sudden cardiac death. After undergoing Morrow operation, the proband had recovered well, and in the follow-up period for two years, she never feels chest pain and syncope.A novel frame shift mutation c.1374delC （p.R458Rfs） was identified in exon14of the cardiac myosin binding protein C gene in pedigree2. Screening for of her family members by Sanger sequencing, and there are3family members also with mutation R458Rfs in Exon14of MYBPC3gene. The results of genetic test were normal in one hundred controls. However, the members of the same HCM family with the R458Rfs mutation showed differences in phenotype and prognosis.ConclusionsThe mutation Arg663Cys in Exon18of beta myosin heavy chain gene is the disease-causing gene in family1by next generation sequencing. The symptoms of patients occur early associated with left ventricular outflow tract obstruction and show chest pain, syncope and sudden death. Their prognosis seems poor in this pedigree of HCM. However, Morrow operation treatment can relieve symptoms and improve prognosis if the patients can actively carry out surgery.A novel frame shift mutation c.1374delC （p.R458Rfs） was identified in exon14of the cardiac myosin binding protein C gene in pedigree2. The patients with this mutation clinically showed differences in phenotype and prognosis. The heterogeneity suggested that multiple factors may be involved in the pathogenesis of HCM. Objective:We aimed to retrospectively analyze the clinical features and prognosis of Hypertrophic cardiomyopathy patients with left ventricular thrombus.Methods:We retrospectively analyzed the data of HCM patients with left ventricular thrombus diagnosed by CMR in FuWai Hospital from January2002to December2012, and followed-up their survival states.Results:Left ventricular thrombosis was confirmed by MRI in6cases of HCM （thrombosis group）, and no left ventricular thrombosis in284cases of HCM （no thrombus group）, the ratio of family history of thrombosis group, ventricular arrhythmia, left atrial diameter, left ventricular diameter and ventricular aneurysm were significantly higher than those without thrombosis group （p<0.05）, and left ventricular ejection fraction and outflow tract obstruction was significantly lower than no thrombus group （p<0.05）.All patients received anticoagulation therapy with warfarin. During follow up （17-59） months, no thromboembolic events was recorded, and thrombus disappear in2patients.3cases died of heart failure and SCD. One patient received heart transplantation.Conclusion:Left ventricular thrombosis was seen in Hypertrophic cardiomyopathy patients with left ventricular aneurysm and lower left ventricular ejection fraction. Ventricular arrhythmia and poor prognosis were found in the majority of patients. Although embolic events occurred because of the left ventricular thrombus, warfarin can cure them. Background:Duchenne’s or Becker’s muscular dystrophy is a serious X-linked neuromuscular disorder. Mutations in the dystrophin gene on chromosome Xp21.1. have been reported to cause BMD. It primarily involved the skeletal muscle, characterized by weakness and muscle atrophy of hereditary muscle disease following as low progressive increase of symmetry. DMD is usually with serious condition and with a poor prognosis. BMD patients can usually walk and have a near normal life span, and there is a chance of them developing cardiac problems.Objective:To study the clinical characteristics and genetic analysis and management of Becker’s muscular dystrophy combined with cardiac involvement, and to expand our understanding of this disorder.Methods:The proband and the family members had genetic testing, and these subjects also had physical examination and received muscle biopsy and routine pathological checks and cardiac evaluation. One hundred control subjects without diagnostic features of BMD were also recruited. Genomic DNA was extracted from leukocytes of peripheral blood from the patients and the control subjects. We screened the dystrophin gene in the indexes, and also screened for the mutation in their families and100controls.Results:The proband was diagnosed with BMD by the identification of a mutation （c.4998₅000Del GCA, p.1667delAla） in the exon35of the dystrophin gene. Three cases were diagnosed with BMD by the identification of this mutation. All genetically affected subjects had a history of weakness of the quadriceps femoris as well as increased serum creatine kinase level at rest and cardiac involvement. After heart transplantation, the proband recovered well. Conclusion:Genetic analysis enables a precise diagnosis of BMD, and heart transplantation is an effective treatment for the patient with severe heart failure.更多还原