第一部分   肥厚型心肌病拟表型基因突变谱及表型关联研究

背景及目的：  肥厚型心肌病（HCM）拟表型是由于调控心脏代谢或清除代谢产物的蛋白质发生基因突变而引起一类与HCM有同样表型的疾病，包括法布雷病（GLA）、Danon病（LAMP2）、淀粉样变心肌病（TTR）和糖原贮积心肌病（PRKAG2）等。HCM拟表型所导致的左室肥厚在临床影像检查中无法同HCM相区分，常被误诊为HCM而无法得到有效治疗。本研究旨在明确HCM患者队列中拟表型基因变异图谱，建立HCM拟表型的基因型-表型关联。

方法：  研究对象为1999年到2010年期间，阜外医院入组529例HCM患者。HCM患者的诊断标准为超声心动图或心脏核磁检查显示左心室壁最大厚度≥15mm ，并排除原发性高血压、主动脉畸形等可能引起心肌肥厚的疾病。同时设立307名经常规体格检查、心电图和超声心动图检查未见明显异常的个体作为健康对照。所有患者和健康对照均通过panel二代测序筛查GLA、LAMP2、TTR和PRKAG2基因罕见变异。罕见变异排除标准为：1）同义突变；2）1000 Genomes Project 和Exome Varian Server中最小等位基因频率≥0.5%。所有患者均进行定期随访，随访事件为心血管病死亡。

结果：我们在19个患者（3.6%）中发现了16个HCM拟表型基因的罕见变异，同时在3个健康对照（1.0%）中发现3个罕见变异, 肥厚型患者中拟表型基因罕见变异的比例显著高于正常对照组(P=0.024)。患者中筛查到的罕见变异中2个为GLA突变，2个TTR突变，4个LAMP2突变以及8个PRKAG2突变，而对照组中筛查到的3个罕见变异均为PRKAG2突变。这19个罕见变异中16个为错义突变，2个移码突变和1个剪接位点变异。携带肥厚型心肌病拟表型罕见变异的患者发病年龄明显早于未携带罕见变异患者（35.9±13.7岁VS 43.5±14.8岁, P=0.028）。而且携带肥厚型心肌病拟表型变异的患者合并家族成员心肌肥厚比例更高（52% VS 23.5%, P=0.01）。所有患者随访4.7± 3.2年，共有42名(7.9%)患者出现心血管死亡，其中19名患者发生猝死，20名患者死于心衰以及3名患者死于血管栓塞。携带HCM拟表型罕见基因患者心血管死亡率同非携带者相比无显著差异(HR=0.58, 95CI: 0.08-4.18, P=0.58)。

结论：  本研究是国际上第一次在大样本HCM患者人群中进行的拟表型基因全面筛查。我们发现约4%的HCM患者为HCM拟表型疾病，这类患者发病年龄更早，但临床结局与其他患者无明显差异。

关键词：   肥厚型心肌病；拟表型；基因检测；基因型-表型关联

第二部分  肥厚型心肌病的遗传阻断研究

背景及目的：肥厚型心肌病（HCM）是青年人心源性猝死的主要原因，许多HCM患者最终会恶化为心力衰竭。HCM是一种常染色体显性遗传疾病，主要由肌小节蛋白编码基因突变导致。HCM往往呈家族性聚集发病，给患者家庭和社会造成沉重负担。目前HCM的治疗主要是根据临床症状进行针对性治疗，尚无彻底治愈的药物或手段。而随着辅助生殖技术的进步，通过产前或胚胎植入前诊断，进行选择性生育，可以有效阻断疾病在家族中的世代传递。本研究旨在通过产前羊水检测胎儿是否携带HCM致病突变，进而实现阻断致病基因突变在患者家族中遗传的目的。

方法：入组有生育要求的适龄HCM患者，所有患者及配偶均签署知情同意书。二代测序检测肌小节蛋白编码基因MYH7、MYBPC3、TNNT2、MYL2、MYL3、TNNI3和ACTC1，明确肥厚型心肌病患者的致病突变。女性患者或患者配偶怀孕16-20周时进行羊水穿刺，提取纯化羊水DNA。通过Sanger测序和短片段重复序列（STR）检测方法明确胎儿是否携带肥厚型心肌病致病突变。STR标记物的选择标准为: 1)同致病基因连锁；2）长度在100-500bp；3）在人群中的杂合度≥0.7；4）位置尽量靠近致病基因。合成STR引物携带荧光标记，经毛细管电泳检测确定胎儿是否携带致病基因连锁的STR。胎儿出生后，抽取脐带血进行sanger测序验证产前诊断结果。

结果：共有4例HCM患者接受产前诊断，致病基因均为人β心肌肌球蛋白重链（MYH7）。筛选出6对与MYH7连锁的STR：D14S50、D14S283、D14S990、D14S972、D14S64 和D14S264进行检测。胎儿父母及羊水DNA经一代测序和STR检测均显示4例胎儿均未携带肥厚型心肌病致病突变，胎儿脐带血检测结果与产前诊断一致。第一例患者为51岁女性，其弟弟及妹妹均诊断为HCM。家系调查表明MYH7 Arg663Ser突变与HCM呈共分离，确定Arg663Ser突变为致病突变。STR检测确定同Arg663Ser突变连锁的D14S50长度为173bp，胎儿D14S50长度为171/177bp；同Arg663Ser突变连锁的D14S990长度为151bp，胎儿D14S990长度为141/147bp。羊水DNA Sanger测序及STR检测确定胎儿未携带Arg663Ser突变。第二例患者为46岁男性于42岁时因心衰行心脏移植术，患者女儿同样确诊为HCM。家系调查表明MYH7 Arg249Gln突变同HCM表型呈共分离。STR检测确定同Arg249Gln突变连锁的D14S283长度为137bp，胎儿D14S283长度为133/141bp；同Arg249Gln突变连锁的D14S263长度为222bp，胎儿D14S263长度为226/228bp。Sanger测序和STR检测均确定胎儿未携带Arg249Gln突变。第三例患者为29岁男性，因心衰植入起搏器，其母亲因HCM于49岁时发生心衰后死亡。家系调查确定MYH7 Arg453His为致病突变。STR检测确定同Arg453His突变连锁的D14S50长度为169bp，胎儿D14S50长度为171/175bp；同Arg453His突变连锁的D14S283长度为145bp，胎儿D14S283长度为129/129bp。Sanger测序和STR检测均确定胎儿未携带Arg453His突变。第四例患者为31岁男性HCM患者，其叔父同样确诊为HCM，家系调查确定MYH7 Ala797Thr突变为致病突变。STR标记物检测结果示同MYH7 Ala797Thr突变连锁的D14S264长度为226bp，胎儿的D14S264长度为222/222bp；Ala797Thr突变连锁的D14S990长度为131bp，胎儿的D14S990长度为143/143bp。Sanger测序和STR检测均确定胎儿未携带Ala797Thr突变。

结论：产前诊断可以明确诊断胎儿是否携带HCM致病突变，实现选择性生育，阻断HCM在家族遗传，降低HCM患病率。

关键词：肥厚型心肌病；产前诊断；短片段重复序列检测

Part I：Screening of the Phenocopy Genes and Genotype-Phenotype Correlation in A Large Chohort of Hypertrophic Cardiomyopathy 

Background and objectives: Some monogenic phenocopy diseases causing left ventricular hypertrophic mimic hypertrophic cardiomyopathy (HCM) in cardiac imaging findings.The disease genes of HCM phenocopies mainly encodeproteins that function in cardiac metabolism or clearance of cellular byproducts, including Fabry disease (GLA)、transthyretinrelated amyloid cardiomyopathy(TTR)、Danon disease(LAMP2) and glycogen-storage cardiomyopathy(PRKAG2). HCM phenocopies were used to misdiagnosied as HCM and minght be treated inappropriately. The aim of the present study  was to determine the variant profile of phenocopy genes and analyze the genotype-phenotype correlation in a large Chinese cohort of patients diagnosed as HCM clinically.

Methods: A cohort of 529 Chinese HCM patients, diagnosed by the maxium left ventricular wall thickness ≥15mm presented by echocardiography and/or cardiac magnetic resonance were recruited between 1999 and 2010 in Fuwai hospital. Every patients was followed up regularly, with cardiovascular deaths as the primary endpoint. A total of 307 individuals without any cardiac disease in physical examination, electrogram and echocardiogram were also recruited as healthy controls. The the coding exons and their flanking intronic regions of phenocopy genes, including GLA、LAMP2、TTR and PRKAG2, were sequenced by targeted panes resequencing. The phenotype-genotype analysis of baseline characteristics and clinical outcomes during follow-up were performed after exclusing the following variants:1)synonymous mutations；2）variants with a minor allele frequency ≥0.5% in 1000 Genomes Project or Exome Varian Server. 

Results: We identified 16 rare variants in phenocopy genes in 19 patients with HCM (3.6%), and 3 rare variants in 3 healthy controls (1.0%). The frequency of rare variants in phenocopy genes was significantly higher in patines than in healthy controls (P=0.024). The identified variants include 16 missense, 2 frameshift and 1 splicing mutations. Among the 16 variants identified in patients, 2 located in GLA, 2 in TTR, 4 in LAMP2 and 8 in PRKAG2. Whereas, all the 3 variants identied in healthy controls were located in PRKAG2.The onset age of patients with rare variants was 35.9±13.7 years old, significantly younger than patients without rare variants (43.5±14.8, P=0.028). Furthermore, patients with rare variants were more likely having family history of unexplained cardiac hypertrophy (52.0% vs. 23.5%, P=0.01). Duing the follow-up of 4.7± 3.2 years, 42(7.9%) patients occurred cardiovascular mortality, including 19 sudeen cardiac deaths, 20 heat failure-related and 3 stroke-realated deaths. The risk of cardiovascular mortality was comparable between HCM patients with and without rare variants in phenocopy genes (HR=0.58, 95CI: 0.08-4.18, P=0.584).

Conclusions:The present study comprehensively screened the phenocopy genes in a larege cohort of patients with HCM for the first time in the world. Our results indicated that phenocopy disease might accourt for a small but significant proportion of patients with clinically diagnosed HCM patients.

Key Words: Hypertrophy Cardiomyopathy; phenocopy disease; genetic testing；genotype-phenotype correlation

Part II：The Prenatal Diagnosis of Hypertrophic Cardiomyopathy 

Background and objectives: Hypertrophic cardiomyopathy(HCM) is the most common cause of sudden cardiac death in youth, and many HCM patients would suffer from heart failure. HCM is an autosomal dominant Mendelian disorder mainly caused by mutations in sarcomeric genes, creating heavy burden for family and society. Symptomatic treatments are mainly used to treat HCM, meaning there is no way to cure this disease. With the development of assisted reproductive technique, we could block the transmission of the disease within the family by selective fertility after prenatal diagnosis or preimplantation genetic diagnosis. The aim of study is to diagnose whether the fetuses carried the pathogenic variants of HCM though amniotic fluid test, and to block the transmission of the disease within family.

Methods: HCM patients at reproductive age were recruited. Informed consent was obtained from all patients and their spouses. Pathogenic variants were identified by familial linkage analysis and multiplex targeted sequencing for sarcomeric genes, including MYH7、MYBPC3、TNNT2、MYL2、MYL3、TNNI3 and ACTC1. Female patients or the spouse of male patients underwent amniocentesis after 16-20 weeks of gestation. Genomic DNA was extracted from amniotic fluid. Sanger sequencing and short tandem repeat(STR) test were used for validate whether fetuses carried pathogenic variants. The criterions for STR markers included: 1）linked with pathogenic gene；2）length between 100 to 500bp；3）heterozygosity of STR markers ≥ 0.7 among population；4）located close to the pathogenic gene. DNA from amniotic fluid was tested though capillary electrophoresis, with the use of fluorescent-labeled primers of STR markers. DNA from Umbilical blood was sequenced to confirm the result of prenatal diagnosis.

Results: The prenatal diagnosis was performed in 4 families, and the pathogenic gene of all patients was MYH7. Six STR markers linked with MYH7 were selected for testing: D14S50, D14S283, D14S990, D14S972, D14S64 and D14S264. Sanger sequencing and STR test of both parents and amniotic fluid revealed that these 4 fetuses didn’t carry pathogenic variants. The results of umbilical blood sequencing were consistent with STR test. The first HCM patient was a woman of 51 years old whose brother and sister were diagnosed with HCM. Strong cosegregation of Arg663Ser with clinical status were observed among the family, suggesting that Arg663Ser as the pathogenic variant. The length of D14S50 linked with Arg663Ser mutation was 173bp, while the length of D14S50 of the fetus were 171bp and 177bp. And the length of D14S990 linked with Arg663Ser was 151bp, while the length of D14S990 of the fetus were 141bp and 147bp. Sanger sequencing and STR test confirmed that the fetus didn’t carry Arg663Ser. The second one was 46-year-old male HCM patients, who received cardiac transplantation for heart failure at 42 years old. His daughter was also diagnosed with HCM, and Arg249Gln was identified as the pathogenic variant with complete cosegregation among the family. The length of D14S283 linked with Arg249Gln was 137bp, while the length of D14S283 of the fetus were 133bp and 141bp. And the length of D14S263 linked with Arg249Gln was 222bp, while the length of D14S263 of the fetus were 226bp and 228bp. Sanger sequencing and STR test showed that the fetus didn’t carry Arg249Gln. The third patient was a 29-year-old male HCM patient, with implanted pacemaker for heart failure. His mother died of heart failure at 49 years old. Cosegregation of Arg453His was found among the family, suggesting Arg453Hisas the pathogenic variant. The length of D14S50 linked with Arg453Hiswas 169bp, while the length of D14S50 of the fetus were 171bp and 175bp. And the length of D14S283 linked with Arg453His was 145bp, while the length of D14S283 of the fetus were 129bp and 129bp.Sanger sequencing and STR test confirmed the result the fetus didn’t carry Arg453His. The last patient was a male HCM patients of 31-years old, whose uncle was diagnosed with HCM. Ala797Thr wasidentifiedas the cause of HCM in this family. The length of D14S263 linked withAla797Thr was 226bp, while the length of D14S263 of the fetus were 222bp and 222bp. And the length of D14S990 linked with Ala797Thr was 131bp, while the length of D14S990 of the fetus were 143bp and 143bp. Sanger sequencing and STR test indicated that the fetus didn’t carry Ala797Thr

Conclusions:prenatal dianosis could be used to indentify whether fetuses carried the pathogenic variants of HCM or not.

Key words:hypertrophic cardiomyopathy; prenatal diagnosis; preimplantation genetic diagnosis; short tandem repeat test