第一部分 FHOD3基因突变在肥厚型心肌病危险分层中的价值研究

研究背景：

肥厚型心肌病（hypertrophic cardiomyopathy，HCM）发病率达到1/500-1/200，该疾病绝大多数情况为单基因遗传，在此类疾病中发病率颇高，以不明原因的左心室肥厚（left ventricular hypertrophy，LVH）为最典型的病理特征。多年来，心肌肥厚领域不断深入的研究逐渐发现，FHOD3（formin homology 2 domain-containing 3）基因及其编码蛋白与肥厚型心肌病的发生有重要关联，近年来的研究中也有证据支持其可能是一种肥厚型心肌病的致病基因。然而，FHOD3上不同变异的致病性仍有待全面评估。本研究通过对较大体量的肥厚型心肌病队列和对照队列中FHOD3变异谱的分析，深入探讨其与肥厚型心肌病发病和预后情况的相关性。

研究方法：

本研究纳入1000例肥厚型心肌病患者和761例非肥厚型心肌病对照，入组后行全面检查并采血、提取保存DNA样本，而后进行2.6±1.6年门诊或电话随访，末次随访时间为2018年12月。HCM组和对照组均进行全外显子组测序，对罕见变异进行gene-based关联分析，对检出的所有变异根据人类基因组变异学会推荐数据、以FHOD3基因的最长转录本（NM_001281740.1）为基础进行注释，再根据美国医学遗传学和基因组学学院的标准，构建FHOD3变异分类的改良分类方案，同时将致病性、可能致病性和未知意义状态的FHOD3突变定义为本次研究的候选突变。在以上工作的基础上，本研究进一步根据FHOD3基因上变异发生情况，将患者分为FHOD3-HCM和非FHOD3-HCM两组，对组间的临床特征和终点事件风险进行对比分析，对带有FHOD3候选变异的肥厚型心肌病患者及其所有家庭成员的DNA样本使用Sanger毛细管法测序进行测试、完成家系分析，以进一步确认各FHOD3候选变异的致病性。

研究结果：

本研究共鉴定出37个FHOD3基因的候选变异，包括25个错义突变和2个截断变异。具体而言，在33例（3.3%）HCM组患者中检测到了27个候选变异，在12个（1.6%）对照组中共计发现了12个变异，呈现显著差异（OR=2.13；P<0.05）。以上变异中，6个变异聚集在FHOD3蛋白的一个心脏亚型独有的结构域（amino acids 400-574）中，且仅在9例HCM患者中测到。在进一步的家系分析中， 4名患者检测到新发现的截断变异c.1286+2delT，该变异依据指南判定为可能致病类别。对该变异进一步进行连锁分析，得出其综合LOD（logarithm of the odds）值为1.44，根据公认的标准，本研究认定其为这些患者肥厚型心肌病发病的原因。此外，与不携带FHOD3候选变异的HCM患者相比，携带 FHOD3候选变异的HCM患者发生心血管死亡和心脏性猝死的风险显著为高（校正后HR [95% CI]= 3.71 [1.32-8.59]；P=0.016；校正后HR [95% CI]= 3.02 [1.09-6.85]；P=0.035）。同时，在携带肌小节蛋白编码基因致病性变异的患者中，得到的结果与整体人群类似，携带FHOD3候选变异的HCM患者，其终点事件发生率同样显著高于不携带的患者。

研究结论：本研究确认截断变异c.1286+2delT为4名患者的病因变异，进一步证实了FHOD3基因对于肥厚型心肌病的致病性。同时，HCM患者携带FHOD3候选变异将增加心血管死亡和全因死亡的风险， FHOD3候选变异可能具有纳入肥厚型心肌病危险分层体系、改善患者治疗管理的价值。

第二部分 心力衰竭分型在肥厚型心肌病危险分层中的价值研究

研究背景：

肥厚型心肌病（hypertrophic cardiomyopathy，HCM）发病率达到1/500-1/200，以不明原因的左心室肥厚（left ventricular hypertrophy，LVH）为最典型的病理特征，而心力衰竭（heart failure，HF）则是HCM患者病程发展的重要过程。心力衰竭主要有两个重要分型，射血分数保留型心力衰竭（preserved ejection fraction heart failure，HFpEF）是心力衰竭的主要形式，且与不良生存质量和过早死亡有显著关联。BNP及其等效物NT-proBNP，在心衰的临床诊断和危险分层中已有现行标准，但目前高钠尿肽浓度的界值仍然存在争议。本研究旨在探讨肥厚型心肌病患者HFpEF的特点和预后，并评估哪一种NT-proBNP界值更适宜用于肥厚型心肌病患者HFpEF的诊断，有助于完善肥厚型心肌病的危险分层。

研究方法：

本项前瞻性队列研究共纳入1178例具有NT-proBNP结果的肥厚型心肌病患者。根据美国心脏协会（AHA，HFpEF定义为LVEF≥50%，有HF症状或体征，NT-proBNP≥800pg/mL）和欧洲心脏病学会（ESC，NT-proBNP浓度＞125 pg/mL）指南所述的不同标准，患者被分为HFpEF和无心力衰竭（非HF）两组。终点事件定义为全因死亡、心血管死亡和心脏性猝死（sudden cardiac death，SCD）。研究采用K-M法及对数秩检验、Cox回归分析、ROC曲线、敏感性分析等方法进行分析研究。同时通过基因检测对HCM患者的遗传特征进行描述和分析。

研究结果：

在1178例肥厚型心肌病患者中，根据AHA标准，513例（43.5%）被确定患有HFpEF, 使用ESC指南中标准的诊断HFpEF，则得到761例（64.6%）HFpEF患者。在总量为5827人年的随访中，79名患者发生了全因死亡（每100人年1.4例），其中60例为心血管死亡，包含29例SCD事件。采用AHA标准，HFpEF患者的全因死亡和心血管死亡发生率分别为1.92/100人年和1.51/100人年，而非HF患者的全因死亡和心血管死亡发生率仅为0.96/100人年和0.70/100人年。与非心衰患者相比，HFpEF患者的最大室壁厚度（P＜0.001）、最大左室流出道梯度（P＜0.001）、心房颤动比例（P＜0.001）、全因死亡（对数秩检验，P=0.002）和心血管死亡（对数秩检验，P=0.005）显著增加。多因素Cox分析显示，HFpEF患者的全因死亡风险（HR [95% CI]=1.80 [1.11-2.90]；P=0.017）和心血管死亡风险（HR [95% CI]=1.82 [1.05-3.18]；P=0.033）几乎是非心衰患者的两倍，且当NT-proBNP＞1780pg/ml，得到的约登指数最高，亦即ROC曲线性能最优。而在ESC标准下绝大多数分析未见两组之间有显著差异。同时，基因检测结果发现在HCM相关的八个最主要的肌小节基因上，HFpEF组的突变阳性患者比例显著高于非HF组（36.6% vs 30.3%，P=0.041），可能提示基因型与HFpEF之间的内在关联，有待在后续研究进一步发掘。

研究结论：

对心力衰竭进行分型，显示HCM患者的HFpEF患病率较高，而HFpEF患者的疾病严重程度和死亡率显著高于非HF患者，HFpEF与全因死亡和心血管死亡风险升高独立相关。因此，HCM患者可能需要适当且更积极的HF治疗，使用AHA标准对HCM患者进行HFpEF诊断可以为患者危险分层提供指导。

PART1: Value of Formin Homology 2 Domain-Containing 3 Gene variants in Risk Stratification of Hypertrophic Cardiomyopathy

Background

Hypertrophic cardiomyopathy (HCM) is the most common monogenic cardiac disorder characterized by unexplained left ventricular hypertrophy (LVH), affecting 1/500-1/200 individuals in the general population. The FHOD3 (formin homology 2 domain-containing 3) gene has recently been identified as a probably causative gene of hypertrophic cardiomyopathy (HCM). However, the pathogenicity of FHOD3 variants remains to be evaluated. This study analyzed the spectrum of FHOD3 variants in a large HCM and control cohort, and explored its correlation with the disease.

Methods

The genetic analysis of FHOD3 was performed using the whole exome sequencing  (WES) data from 1000 patients with HCM and 761 controls without HCM. After the patients were enrolled, clinical evaluation was performed in all patients, and DNA samples were collected at the same time. Follow-up was performed for 2.6±1.6 years until December 2018. WES performed in both HCM group and control group, and gene-based association analysis was performed for rare variants. All detected variants were annotated based on the longest transcript of FHOD3 gene (NM_001281740.1) according to the data recommended by  the Human Genome Variation Society, and then a modified classification scheme, based on the criteria of American College of Medical Genetics and Genomics, was constructed for FHOD3 variant classification. On the basis of the above work, according to the occurrence of FHOD3 gene mutation, the patients were further divided into FHOD3-HCM and non FHOD3-HCM，and the clinical characteristics and risks between the two groups were compared. Patients with FHOD3 candidate mutations of FHOD3-HCM group were sequenced by Sanger capillary method to complete family analysis, together with all their family members, to further confirm the pathogenicity of each FHOD3 candidate variant.

Results

A total of 37 FHOD3 candidate variants were identified, including 25 missense variants and 2 truncating variants. In detail, there were 27 candidate variants detected in 33 (3.3%) patients with HCM, which was significantly higher than in the 12 controls (3.3% versus 1.6%; odds ratio, 2.13; P<0.05). On the basis of familial segregation, we identified one truncating variant (c.1286+2delT) as a causal variant in 4 patients. Furthermore, this FHOD3 candidate variant experienced significantly more risk of cardiovascular death and all-cause death (adjusted hazard ratio [HR], 3.71; 95%CI, 1.32-8.59; P=0.016; and adjusted HR, 3.02; 95% CI, 1.09-6.85; P=0.035, respectively). And among the HCM patients with pathogenic related variants on sarcomere genes, the results were similar to the overall population.

Conclusions

Our study suggests that FHOD3 is a causal gene for hypertrophic cardiomyopathy. And that the presence of FHOD3 candidate variants is an independent risk for cardiovascular death and all-cause death in HCM. Carrying FHOD3 candidate variants may increase the risk of cardiovascular death and all-cause death. FHOD3 candidate variants probably has the value of incorporating into the risk stratification system of hypertrophic cardiomyopathy and improving patient management.

PART2: Classification of Heart Failure Helps Risk Stratification for Hypertrophic Cardiomyopathy

Background

Hypertrophic cardiomyopathy (HCM) is the most common monogenic cardiac disorder characterized by unexplained left ventricular hypertrophy (LVH), affecting 1/500-1/200 individuals in the general population. Heart failure (HF) is a major cause of mortality in patients with HCM. There are two main types of heart failure (HF), heart failure with preserved ejection fraction (HFpEF) is the dominant form. As established biomarkers for the diagnosis and prognosis of heart failure, neither BNP or NT-proBNP have reached a recognized critical value. We here aimed to investigate the characteristics and prognosis of HFpEF in patients with HCM, and analyse which criteria for the cut off value for high natriuretic peptides perform better in HCM patients.

Methods

This was a prospective cohort study and patients with HCM with available NT-proBNP results were enrolled. Patients were categorized into HFpEF [defined as LVEF≥50%, with symptoms or signs of HF, and N-terminal pro-brain natriuretic peptide≥800 pg/mL according to American Heart Association (AHA) criteria] and without heart failure (non-HF). The outcomes of interest were all-cause death, cardiovascular death, and sudden cardiac death (SCD).

Results

Of 1178 included patients with HCM, 513 (43.5%) were identified as having HFpEF according to AHA criteria. Over a total follow-up of 5827 patient years, all-cause death occurred in 79 patients (1.4 per 100 patient years). The incidence of all-cause death and cardiovascular death in patients with HFpEF were 1.92 and 1.51 per 100 patient-years, whereas only 0.96 and 0.70 per 100 patient years in non-HF patients. Compared with non-HF patients, patients with HFpEF had significantly larger maximal wall thickness (P<0.001), higher maximal left ventricular outflow tract gradient (P<0.001), higher proportion of atrial fibrillation (P<0.001), higher incidence of all-cause death (log-rank test, P=0.002), and cardiovascular death (log-rank test, P=0.005). Multivariable Cox analysis showed that patients with HFpEF had a nearly two-fold higher risk of all-cause death (adjusted HR=1.80, 95% CI=1.11-2.90; P=0.017) and cardiovascular death (adjusted HR =1.82, 95% CI 1.05-3.18; P=0.033) than non-HF patients.

Conclusions

Patients with HCM have a high prevalence of HFpEF and those with HFpEF present greater disease severity and higher mortality than non-HF patients, and thus may require an appropriate and more aggressive treatment for HF management. Identification of patients with HFpEF using AHA criteria can provide guidance on patient risk stratification for patients with HCM.