Hypertrophic cardiomyopathy (HCM) represents one of the most common inherited cardiac conditions. It is genetically and clinically heterogeneous and is most commonly caused by variants in sarcomere-encoding genes, including MYH7, which represents the second most common cause of familial HCM.¹ Missense variants in MYH7 are believed to cause HCM through gain-of-function actions: Variants produce an abnormal activated protein that incorporates into the sarcomere as a poison peptide.² Haploinsufficiency in MYH7 is not a recognized disease mechanism for HCM, and heterozygous variants that introduce premature termination codons (PTCs; ie, nonsense, frameshift, and splice variants) have not previously been demonstrated to be associated with this disease.¹

Here, we report the frameshift variant c.5769delG in MYH7 (ENST00000355349) that is associated with HCM in a large series of unrelated Egyptian patients with HCM assessed at the Aswan Heart Center. All patients gave written informed consent, and the study was reviewed and approved by the institutional research ethics committee (FWA00019142; Research Ethics Committee code 20130405MYFAHC_CMR_20130330). c.5769delG was found in 3.3% of patients (17 of 514) yet was absent from locally recruited ethnically matched controls (n=400, P_Fisher=5.0×10⁻⁵), the Genome Aggregation Database (n=125,748, P_Fisher=2.0×10⁻⁴¹), and the Iranome reference database (n=800, P_Fisher=9.98×10⁻⁸). The variant was identified in 1 individual from the Great Middle Eastern population study³ (n=993, P_Fisher=1.2×10⁻⁷), but the cardiac phenotype is not known for this subject. The variant reported here has not previously been seen in >6000 predominantly White patients with HCM.⁴

Detailed study of a large family showed the cosegregation of c.5769delG with HCM (logarithm of the odds score, 3.01), and no other rare protein-altering variants were found in other established HCM genes.⁴ The proband carrying the frameshift variant presented with the disease at 29 years of age, and all affected family members carried the variant (Figure [A], left). Proband and family members were clinically evaluated by cardiologists experienced in diagnosing and managing patients with HCM who were initially blinded to the genotype of each individual (Figure [A], left and Figure [B]). The diagnosis of HCM was established according to the 2014 European Society of Cardiology HCM guidelines: individuals with wall thickness ≥15 mm involving ≥1 segments of the left ventricle (using transthoracic echocardiography) that cannot be explained by loading conditions. In addition, first-degree family members of patients with confirmed HCM were diagnosed if the wall thickness of ≥1 left ventricular segments was ≥13 mm in the absence of an alternative explanation. In patients with borderline increased wall thickness (13–14 mm), other features suggestive of HCM were evaluated, including diastolic function, left atrial dimensions, systolic anterior motion of the mitral valve, dynamic left ventricular outflow tract obstruction, and late gadolinium enhancement on cardiac magnetic resonance imaging (Figure [B]). In total, 16 members of the family were evaluated, with 6 patients being diagnosed with HCM. Histopathological characterization of patients’ tissues shows high levels of interstitial fibrosis, elevated transforming growth factor-β1 expression, and a significant increase in myocyte diameter characteristic of HCM (Figure [C]).

Figure. A frameshift variant in MYH7 (c.5769delG) is associated with HCM. A, Left, MYH7 (c.5769delG) segregates with hypertrophic cardiomyopathy (HCM) in a large Egyptian family (limit of detection, 3.01). The proband is marked with an arrow. Ages of family members at diagnosis are shown. The variant segregated in all affected family members. Right, Clinical and genetic characteristics of the proband and affected family members depicted in the pedigree. Clinical history at presentation for patients with HCM. V.1: dyspnea, New York Heart Association (NYHA) class III, palpitations, and myectomy; IV.2: dyspnea, NYHA class II, and palpitations; IV.3: dyspnea, NYHA class II, and presyncope; IV.4: dyspnea, NYHA class III, and angina; IV.9: dyspnea, NYHA class II, paroxysmal nocturnal dyspnea, and myectomy; IV.10: dyspnea, NYHA class II, angina, palpitations, and myectomy; and IV.12: dyspnea, NYHA class III, angina, palpitations, and myectomy. Cardiac magnetic resonance (CMR) data were not available for V.2, V.4, IV.6, II.1, IV.2, and IV.3. B, Proband (ie, V.1 in pedigree) showing classic HCM features assessed by echocardiography and CMR. Top left and top right, Severe asymmetrical septal hypertrophy (basal interventricular septum measuring 36 mm) with systolic anterior motion of the mitral valve (bottom left) causing a peak systolic gradient of 50 mm Hg across the left ventricular outflow tract at rest (bottom right). C, Left, Histological characterization of HCM myectomy tissues (proband: V.1 and affected family members IV.9, IV.10, IV.12) compared with control myocardial tissues, stained with hematoxylin and eosin (H&E), picrosirius red (PSR), transforming growth factor-β1 (TGFβ1) immunohistochemistry, and wheat germ agglutinin (WGA). Scale bars are 20 µm. Right, WGA quantification in control subjects vs patients with HCM (P_Fisher<0.004, unpaired t test). D, RNA sequencing analysis of human heart tissue from the proband confirms the presence of both wild-type (WT; blue asterisk) and mutant (black asterisk) transcripts. MYH7 is on the reverse strand. E, Schematic of the MYH7 frameshift variant (MYH7fs) is predicted to lead to expression and translation of a truncated peptide. The cDNA sequence and predicted resulting amino acid sequence are shown for WT (top) and variant MYH7 (bottom) alleles. The frameshift variant (deletion of nucleotide C in exon 39) is predicted to result in an nonsense mediated decay–incompetent premature termination codon downstream of the last exon-exon junction. F, Left, Differentially expressed genes in RNA sequencing in left ventricular tissue from patients with MYH7fs, MYH7 rare missense variants (p.A355V, p.M690V, and p.D1208H), and control subjects. Each row and column in the heat map represent a differentially expressed gene and sample. Samples and genes are clustered according to the correlation among the genes and among the samples, respectively. The expression values are color-coded and scaled for each gene (ie, by row). Principal component (PC) analysis of gene signature of patients with HCM with the c.5769delG variant, patients with HCM with different rare missense MYH7 variants, and control subjects (inset). Right, Enrichment analysis of differentially expressed Gene Ontology terms in patients with HCM with the c.5769delG variant and controls. Enriched pathways (top) and diseases (bottom) that were downregulated in samples with MYH7 c.5769delG compared with controls.

RNA sequencing of myocardial tissue (obtained during surgical myectomy) from the proband carrying c.5769delG confirmed expression of the variant MYH7 transcript (Figure [D]). The predicted sequence of the variant MYH7 transcript shows that the PTC resides downstream of the last exon-exon junction (Figure [E]). PTCs in the last exon of a gene, or in the last 50 to 55 bp of the penultimate exon, generally escape nonsense mediated decay, a posttranscriptional mechanism that degrades mRNAs harboring a PTC.⁵ The translated protein sequence would produce an alternate c-terminal sequence just 4 amino acids shorter than the wild-type protein.

We then investigated the gene expression profiles of patients with HCM carrying the MYH7 frameshift variant (n=3), patients with HCM carrying different MYH7 variants (rare missense; n=3), and control subjects (n=4). The analysis identified 254 differentially expressed genes (Bonferroni-corrected P_Fisher<0.05) across the 3 groups with the MYH7 frameshift variant clustering with HCM (Figure [F], left). Principal component analysis shows distinct clustering of patients with HCM (both groups) compared with control subjects (Figure [F], left). Enrichment analysis for differentially expressed genes between patients with the MYH7 frameshift variant and control subjects showed pathways related to muscle contraction, cardiac conduction, and dilated/hypertrophic cardiomyopathy, consistent with the molecular changes characteristic of HCM (Figure [F], right).

In MYH7, only missense variants have previously been proven to cause HCM. Here, we provide the first evidence of an association with an nonsense mediated decay–incompetent frameshift variant, implicating a new class of genetic variants in disease. Although heterozygous null alleles, including nonsense mediated decay–competent PTCs, would not be considered pathogenic for HCM in isolation (but could be potent modifiers if found in trans with a disease allele or pathogenic if homozygous), distal PTCs may lead to expression of an abnormal protein that may be disease causing.

In conclusion, this study identifies a new class of pathogenic variants in HCM with important clinical implications and confirms the value of studying diverse populations.

Data, analytical methods, and study materials will be made available to other researchers through direct communication.

This study is part of the Egyptian Collaborative Cardiac Genomics Project. It was supported by the Science and Technology Development Fund government grant (Egypt), the Wellcome Trust (107469/Z/15/Z; 200990/A/16/Z), the Medical Research Council (United Kingdom), the NIHR Royal Brompton Cardiovascular Biomedical Research Unit, the NIHR Imperial College Biomedical Research Center, and a Health Innovation Challenge Fund award from the Wellcome Trust and Department of Health, United Kingdom (HICF-R6–373). Drs Allouba and Halawa are funded by Al Alfi Foundation to support their PhD degrees at Imperial College London and American University in Cairo, respectively. Dr Aguib is supported by Fondation Leducq (11 CVD-01). Dr Whiffin is supported by a Rosetrees and Stoneygate Imperial College Research Fellowship. Dr Walsh is supported by an Amsterdam Cardiovascular Science Fellowship.

Disclosures None.

*Y. Aguib, M. Allouba, and R. Walsh contributed equally.

†J.S. Ware, P.J.R. Barton, and M. Yacoub contributed equally.

For Sources of Funding and Disclosures, see page 757.

https://www.ahajournals.org/journal/circ

肥厚型心肌病 (HCM) 是最常见的遗传性心脏病之一。它在遗传和临床上具有异质性，最常见的原因是肌节编码基因的变异，包括MYH7，它代表了家族性 HCM 的第二大常见原因。¹ MYH7中的错义变体被认为通过功能获得作用引起 HCM：变体产生异常的活化蛋白，该蛋白作为毒肽结合到肌节中。² MYH7单倍体不足不是公认的 HCM 疾病机制，并且引入提前终止密码子（PTC；即无义、移码和剪接变体）的杂合变体以前没有被证明与这种疾病有关。¹

在这里，我们报告了MYH7 (ENST00000355349) 中的移码变体 c.5769delG，它与在阿斯旺心脏中心评估的大量不相关的埃及 HCM 患者中的 HCM 相关。所有患者均签署了书面知情同意书，该研究经机构研究伦理委员会审查和批准（FWA00019142；研究伦理委员会代码 20130405MYFAHC_CMR_20130330）。c.5769delG 在 3.3% 的患者（514 名中的 17 名）中发现，但在当地招募的种族匹配对照（n=400，P _Fisher =5.0×10 ^{- 5}）、基因组聚合数据库（n=125,748，P _Fisher =2.0×10 ^{- 41} )，以及 Iranome 参考数据库 (n=800,P_费舍尔=9.98×10 ^{- 8}）。在来自大中东人口研究³ (n=993, P _Fisher =1.2×10 ^-7 ) 的 1 个个体中发现了变异，但该受试者的心脏表型未知。这里报道的变异以前没有在 > 6000 名以白人为主的 HCM 患者中出现。⁴

对一个大家族的详细研究表明，c.5769delG 与 HCM 共分离（优势得分的对数，3.01），并且在其他已建立的 HCM 基因中未发现其他罕见的蛋白质改变变体。⁴携带移码变体的先证者在 29 岁时出现该病，所有受影响的家庭成员都携带该变体（图 [A]，左）。先证者和家庭成员由在诊断和管理 HCM 患者方面经验丰富的心脏病专家进行临床评估，这些患者最初对每个个体的基因型不知情（图 [A]、左图和图 [B]）。HCM 的诊断是根据 2014 年欧洲心脏病学会 HCM 指南确定的：个体壁厚 ≥15 mm，累及 ≥1 个左心室节段（使用经胸超声心动图），无法用负荷条件解释。此外，如果 ≥1 个左心室节段的壁厚≥13 mm，且没有其他解释，则诊断为确诊 HCM 患者的一级家庭成员。在壁厚临界增加（13-14 mm）的患者中，评估提示 HCM 的其他特征，包括舒张功能、左心房尺寸、二尖瓣收缩期前向运动、动态左心室流出道梗阻和晚期钆增强心脏磁共振成像（图 [B]）。总共评估了 16 名家庭成员，其中 6 名患者被诊断为 HCM。患者组织的组织病理学特征显示高水平的间质纤维化，转化生长因子-β1表达升高，

数字。 MYH7 (c.5769delG) 中的移码变体与 HCM 相关。A，左，MYH7 (c.5769delG) 在埃及大家庭中与肥厚型心肌病 (HCM) 分离（检测限，3.01）。先证者标有箭头。显示诊断时家庭成员的年龄。该变体在所有受影响的家庭成员中分离。正确的，谱系中描述的先证者和受影响的家庭成员的临床和遗传特征。HCM 患者就诊时的临床病史。V.1：呼吸困难、纽约心脏协会 (NYHA) III 级、心悸和肌切除术；IV.2：呼吸困难、NYHA II 级和心悸；IV.3：呼吸困难、NYHA II 级和先兆晕厥；IV.4：呼吸困难、NYHA III 级和心绞痛；IV.9：呼吸困难、NYHA II 级、阵发性夜间呼吸困难和肌切除术；IV.10：呼吸困难、NYHA II 级、心绞痛、心悸和肌切除术；IV.12：呼吸困难、NYHA III 级、心绞痛、心悸和肌切除术。V.2、V.4、IV.6、II.1、IV.2 和 IV.3 的心脏磁共振 (CMR) 数据不可用。B，先证者（即谱系中的 V.1）显示通过超声心动图和 CMR 评估的经典 HCM 特征。左上方和右上，严重不对称的室间隔肥大（基底室间隔测量 36 毫米），收缩期二尖瓣前移（左下）导致休息时左心室流出道的收缩压梯度峰值为 50 毫米汞柱（右下）。C，左，HCM 肌切除组织（先证者：V.1 和受影响的家庭成员 IV.9、IV.10、IV.12）与对照心肌组织相比的组织学特征，用苏木精和伊红 (H&E) 染色，天狼星红 ( PSR)、转化生长因子-β1 (TGFβ1) 免疫组化和小麦胚芽凝集素 (WGA)。比例尺为 20 µm。正确的，对照受试者与 HCM 患者的 WGA 量化（P _Fisher <0.004，非配对t检验）。D，来自先证者的人类心脏组织的 RNA 测序分析证实了野生型（WT；蓝色星号）和突变型（黑色星号）转录本的存在。MYH7在反向链上。E，MYH7移码变体 ( MYH7fs ) 的示意图预计会导致截短肽的表达和翻译。显示了 WT (顶部) 和变体MYH7 (底部)的 cDNA 序列和预测的氨基酸序列) 等位基因。移码变体（外显子 39 中核苷酸 C 的缺失）预计会导致最后一个外显子-外显子连接下游的无义介导的衰变-无能的过早终止密码子。F ,左, MYH7fs , MYH7患者左心室组织 RNA 测序中差异表达基因罕见的错义变体（p.A355V、p.M690V 和 p.D1208H）和对照受试者。热图中的每一行和每一列代表一个差异表达的基因和样本。样本和基因分别根据基因之间和样本之间的相关性进行聚类。对每个基因的表达值进行颜色编码和缩放（即，按行）。具有 c.5769delG 变体的 HCM 患者、具有不同罕见错义MYH7变体的 HCM 患者和对照受试者的基因特征的主成分 (PC) 分析（插图）。右图，富集分析具有 c.5769delG 变体和对照的 HCM 患者中差异表达的基因本体术语。丰富的途径（上）和疾病（下）)与对照相比，在MYH7 c.5769delG 的样本中下调。

携带 c.5769delG 的先证者的心肌组织 RNA 测序（在手术心肌切除术中获得）证实了MYH7转录本变异体的表达（图 [D]）。MYH7变异转录本的预测序列显示 PTC 位于最后一个外显子-外显子连接点的下游（图 [E]）。在基因的最后一个外显子或倒数第二个外显子的最后 50 至 55 bp 中的 PTC 通常会逃避无意义介导的衰变，这是一种降解含有 PTC 的 mRNA 的转录后机制。⁵翻译后的蛋白质序列将产生一个替代的 c 端序列，仅比野生型蛋白质短 4 个氨基酸。

然后，我们研究了携带MYH7移码变体（n=3）的 HCM 患者、携带不同MYH7变体（罕见错义；n=3）和对照受试者（n=4）的 HCM 患者的基因表达谱。该分析在 3 组中鉴定了 254 个差异表达基因（Bonferroni 校正的P _Fisher <0.05），其中MYH7移码变体与 HCM 聚类（图 [F]，左）。主成分分析显示 HCM 患者（两组）与对照组相比有明显的聚类（图 [F]，左）。MYH7患者之间差异表达基因的富集分析移码变异体和对照受试者显示出与肌肉收缩、心脏传导和扩张/肥厚型心肌病相关的通路，这与 HCM 的分子变化特征一致（图 [F]，右）。

在MYH7中，只有错义变体先前已被证明会导致 HCM。在这里，我们提供了与无意义介导的衰变无能移码变体相关的第一个证据，表明疾病中存在一类新的遗传变体。尽管杂合的无效等位基因，包括无义介导的衰变能力 PTC，在孤立的情况下不会被认为是 HCM 的致病性（但如果在反式中发现具有疾病等位基因或纯合的致病性，则可能是有效的修饰物），远端 PTC 可能导致表达可能导致疾病的异常蛋白质。

总之，本研究确定了一类新的 HCM 致病变异，具有重要的临床意义，并证实了研究不同人群的价值。

数据、分析方法和研究材料将通过直接交流提供给其他研究人员。

这项研究是埃及合作心脏基因组学项目的一部分。它得到了科学技术发展基金政府赠款（埃及）、威康信托基金（107469/Z/15/Z；200990/A/16/Z）、医学研究委员会（英国）、NIHR Royal Brompton 的支持心血管生物医学研究部、NIHR 帝国理工学院生物医学研究中心，以及英国威康信托基金会和卫生部颁发的健康创新挑战基金奖 (HICF-R6-373)。Allouba 博士和 Halawa 博士由 Al Alfi 基金会资助，分别支持他们在伦敦帝国理工学院和开罗美国大学攻读博士学位。Aguib 博士得到 Leducq 基金会 (11 CVD-01) 的支持。Whiffin 博士得到了 Rosetrees 和 Stoneygate 帝国学院研究奖学金的支持。

披露无。

*是。Aguib、M. Allouba 和 R. Walsh 做出了同样的贡献。

†JS Ware、PJR Barton 和 M. Yacoub 贡献相同。

有关资金来源和披露信息，请参见第 757 页。

https://www.ahajournals.org/journal/circ