【作者】 王怡璐；

【导师】 惠汝太；

【作者基本信息】 北京协和医学院 ， 内科学， 2013， 博士

【摘要】 背景：50年来,肥厚型心肌病(hypertrophic cardiomyopathy, HCM)被认为是具有高猝死风险的家族性心脏病,其临床表型、自然病史以及基因突变谱有很大的异质性,而这种异质性源于遗传与环境因素的共同作用。多基因突变作为重要的遗传因素尽管已经常见报道于儿童或者具有严重临床表型的肥厚型心肌病患者,被推测与恶劣预后相关,但是尚没有其提示恶性临床预后的前瞻性研究报道。另外,作为HCM重要的心肌重塑表现,纤维化是近年来备受关注的与恶性预后相关的危险因素,大内皮素-1作为心肌重构的重要生物标记物是否能够影响和提示预后也是尚未解决的问题。方法：连续纳入552名无亲缘关系的HCM患者,应用多重靶向测序技术对已知的最常见HCM致病基因进行全外显子捕获测序,最终测序质量合格者529人。对于高通量测序所检出的突变致病性的判断遵循如下标准：在已公布的突变数据库或者已发表的文献中报道的与HCM相关的突变；新发现的突变应满足：在本研究纳入的312名健康对照中无此突变,并且生物信息学分析证明该突变引起高保守的氨基酸改变及蛋白结构改变,该突变在其家系中与HCM共分离,如果家系资料对于基因突变与疾病共分离分析不可用(家系过小或无其他家庭成员发病),一定要无义突变或者插入缺失突变,如果是剪切位点突变则需证实突变损伤了剪切位点。根据患者所携带的突变数量分三组：未检出突变组、单突变组及多突变组(携带突变数量≥2个)。通过历时4.7+3.2年的随访,获知心血管死亡等临床事件,并评估多突变与各类事件的相关性。并且我们将多突变组中的4名先证者扩大测序的范围至26个与HCM相关的基因,并对这四个家系的成员进行了详细的临床及遗传筛查,以观察多基因突变对于相同遗传背景者临床表型的影响。其中随机纳入245名患者在入组时进行了血浆大内皮素-1的检测,并进行了历时3(2-5)年的随访,以观察血浆大内皮素与预后的相关性。结果：我们在232名患者中发现了256个致病突变：38名(7.2%)患者携带了多突变,194名(36.7%)患者携带单突变。携带多突变的患者不论入组年龄、发病年龄还是确诊年龄都更年轻(p<10-6)、具有猝死危险因素患者的比例更高(p=0.002)。随访发现,42名患者发生了心血管死亡,5年、10年的心血管死亡生存率在多突变组分别为65.0%和55.7%,在单突变组分别为95.8%和88.3%,在未检出突变组分别为92.1%和86.5%(p<10-6)。与单突变组相比,多突变组心血管死亡危险显著增加(校正后的风险比[HR]为3.74；95%可信区间[CI]为2.51-12.231.84-7.58；p=0.0003)。并且与携带≤1个突变的患者相比,多突变携带者发生心力衰竭(校正后HR为3.53；95%CI为1.25-9.92；p=0.017)及猝死(校正后HR为3.57；95%CI为1.23-10.35；p=0.019)的危险更高。对于猝死危险预测,具有≥1个传统危险因素的患者较其他患者HR为2.74(95%CI为1.08-6.94；p=0.033),而具有≥1个传统危险因素并且携带多突变的患者较其他患者HR为5.52(95%CI为1.83-16.65；p=0.002)。以生存年龄为时长进行统计分析得到类似结果。在家系筛查中,以家系内基因突变数量不同的携带者之间的临床表型相比较,也提示携带突变数量越多发病年龄相对越早、症状更重,更易出现心衰症状。并且发现某些变异单独存在并不致病而同时共存时却能引起表型,其遗传模式符合隐性遗传。在进行血浆大内皮素-1检测的患者中发现,血浆大内皮素-1水平高的患者左房内径更大、心功能更差以及伴发房颤的患者比例更高。血浆大内皮素-1水平与N端B型钠尿肽的水平(r=0.291,p<0.001)以及钆延迟增强显像(r=0.222,p=0.016)显著相关。随访发现,高水平的血浆大内皮素水平与全因死亡、心血管死亡以及进展为慢性心力衰竭均有显著的相关性(p=0.020,0.044及0.032)。多因素校正后的回归模型显示,血浆大内皮素-1高水平组比低水平组全因死亡(HR=4.94,95%CI1.07-22.88；p=0.041)及进展为心衰(HR=4.10,95%CI1.32-12.75, p=0.015)的风险均显著增加。结论：作为遗传因素,多突变是HCM患者心血管死亡以及其他恶性临床事件的独立危险因素,其加入能够提高传统猝死危险因素模型对猝死风险的阳性预测价值。并且在家系内的筛查,家系中有多个患者时应当注意先证者的选择,或者必要时所有发病者均行全面的筛查,以便将整个家系中所携带的相关基因突变查全。并且应当重视单独存在并不致病而共存时却能引起表型的变异,它们可能以隐性遗传模式致病,在以往的基因筛查方式中易被遗漏。作为重要的生物标记物,高水平的血浆大内皮素-1是全因死亡以及进展为心衰的独立危险因素,可应用于HCM患者危险分层。并且血浆大内皮素-1作为生物标志物可能成为新的干预目标,并且打开一个观察HCM机制的新窗口。更多还原

【Abstract】 BACKGROUNDFor over50years, hypertrophic cardiomyopathy (HCM) has been recognized as a familial cardiac disease with highly visible risk for sudden death and disease progression, characterizedby heterogeneous phenotypic expression, natural history, and genetic profile. Although multiple mutations was often reported in HCM patients at childhood and with severe phenotype and thereby considered with worse prognosis, its predictive value for malignant clinical outcomes had not been well explored. And cardiac remodeling is one of major pathological process in hypertrophic cardiomyopathy (HCM). Endothelin-1has been linked to cardiac remodeling. Big endothelin-1is the precursor of endothelin-1. We tested if it can become the marker for predicting the prognosis of HCM..METHODSA total of529patients from a prospectively collected HCM cohort were genotyped by screening10disease genes with multiplexing targeted resequencing and were subsequently divided into3groups based on pathogenic mutation dosage:no mutation was detected (no mutation), single mutation and multiple mutation s (≥2mutations). Mutation was considered to be pathogenic if it was absent in312healthy controls, the variant residues are highly conserved throughout evolution and segregated with disease in family. If family co-segregation analysis was unavailable, one of the following additional criteria must be met:previously reported to be pathogenic, nonsense or indels mutations, in silico prediction of damaging effect, and destroyed existing splice site predicted by Human Splicing Finder.The incidence of cardiovascular death and other adverse outcomes were recorded through a follow-up of4.7±3.2years. Correlations of multiple mutations with clinical outcomes were assessed. The families of four probands with multiple mutations were clinically and genetically screened. Twenty-six HCM-related genes were comprehensively screened for mutations in the probands with targeted second generation sequencing, and the identified mutation was confirmed with bi-directional Sanger sequencing in all family members and healthy controls. And a total of245consecutive patients with HCM were enrolled from1999to2011and partitioned to low, middle and high level groups according to their plasma big endothelin-1levels. RESULTSWe identified232mutations from256patients:38(7.2%) with multiple mutations and194(36.7%) with single mutation. Patients with multiple mutations were younger (P<10-6) and higher frequencies of carrying≥1conventional sudden cardiac death (SCD) risk factors (P=0.002). During follow-up,42cardiovascular deaths occurred. The survival rates of free of cardiovascular death at5and10years of follow up were65.0%and55.7%for patients with multiple mutations,95.8%and88.3%for those with single mutation and92.1%and86.5%for those with no mutation, respectively (P<10-6). Multiple mutations significantly increased the risk of cardiovascular death compared with single mutation (adjusted hazard ratio [HR],3.74;95%confidence interval [CI],2.51-12.231; P=0.0003). Patients with multiple mutations had higher risk for heart failure (adjusted HR,3.53;95%CI,1.25-9.92; P=0.017) and SCD (adjusted HR,3.57;95%CI,1.23-10.35; P=0.019) than those with≤1mutation. The HR ratio for SCD prediction was increased from2.74(95%CI,1.08-6.94; P=0.033) by using≥1conventional risk factors to5.52(95%CI,1.83-16.65; P=0.002) by using the presence of≥1conventional risk factors and multiple mutations. Lifelong-time analysis also showed similar associations in various adverse clinical outcomes. In the4families with multiple mutations, compared to the members with single mutation, the members with multiple mutations were younger, with severer symptom and more susceptible to heart failure.We also found that at baseline, significant associations were found between high levels of big endothelin-1and left atrium size, heart function and atrial fibrillation. Big endothelin-1was positively correlated with N-terminal B-type natriuretic peptide (r=0.291, p<0.001) and late gadolinium enhancement (LGE) on magnetic resonance imaging (r=0.222, p=0.016). During a follow-up of3(range,2-5) years, big endothelin-1level was positively associated with the risks of all-cause mortality, cardiovascular death and progression to heart failure (p=0.020,0.044and0.032, respectively). After adjusting for multiple factors related to survival and cardiac function, the significance remained in the association of big endothelin-1with the risk of all-cause mortality (hazard ratio (HR)=4.94,95%confidence interval (CI)1.07-22.88; p=0.041) and progression to heart failure (HR=4.10,95%CI1.32-12.75, p=0.015).CONCLUSIONSMultiple mutations in HCM are an independent risk factor for cardiovascular death and other adverse clinical outcomes. The positive rate for SCD prediction can be improved by adding genetic analysis into conventional frisk stratification algorithm. In the screening of a family with more than one patient who presents various phenotypes, there should not be only one proband chosen for genetic screening, for recognize all mutations correlated with HCM in the family. Our study also showed that high level of plasma big endothelin-1predicted prognosis for patients with HCM and it can be added to the marker panel in stratifying HCM patients for giving treatment priority to those at high risk. It needs to further explore whether the biomarkers could be used to evaluate the efficacy of intervention and opens a new window to look at the pathogenesis of HCM.更多还原