Biallelic Loss of Function Variants in Myocardial Zonula Adherens Protein Gene (MYZAP) Cause a Severe Recessive Form of Dilated Cardiomyopathy,Circulation: Heart Failure

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Biallelic Loss of Function Variants in Myocardial Zonula Adherens Protein Gene (MYZAP) Cause a Severe Recessive Form of Dilated Cardiomyopathy
Circulation: Heart Failure ( IF 9.7 ) Pub Date : 2024-03-04 , DOI: 10.1161/circheartfailure.123.011226
Juan Pablo Ochoa _{1,

2,

3} , Laura Lalaguna ₃ , Jesús G. Mirelis ₁ , Fernando Dominguez _{1,

2,

3} , Esther Gonzalez-Lopez _{1,

2,

3} , Clara Salas _{1,

2} , Gaston Roustan ₁ , Kathryn A. McGurk _{4,

5} , Sean L. Zheng _{4,

5,

6} , Paul J.R. Barton _{4,

5,

6} , James S. Ware _{4,

5,

6} , María Victoria Gómez-Gaviro _{7,

8} , Enrique Lara-Pezzi _{2,

3} , Pablo Garcia-Pavia _{1,

2,

3,

9}

Affiliation

Hospital Universitario Puerta de Hierro Majadahonda, IDIPHISA, Madrid, Spain (J.P.O., J.G.M., F.D., E.G.-L., C.S., G.R., P.G.-P.).
CIBERCV, Instituto de Salud Carlos III, Madrid, Spain (J.P.O., F.D., E.G.-L., C.S., E.L.-P., P.G.-P.).
Centro Nacional de Investigaciones Cardiovasculares, Madrid, Spain (J.P.O., L.L., F.D., E.G.-L., E.L.-P., P.G.-P.).
National Heart and Lung Institute (K.A.M, S.L.Z., P.J.R.B., J.S.W.), Imperial College London, United Kingdom.
MRC London Institute of Medical Sciences (K.A.M., S.L.Z., P.J.R.B., J.S.W.), Imperial College London, United Kingdom.
Royal Brompton and Harefield Hospitals, Guy’s and St Thomas’ Hospital NHS Trust, London, UK (S.L.Z., P.J.R.B., J.S.W.).
Instituto de Investigación Sanitaria Gregorio Marañón, Madrid, Spain (M.V.G.-G.).
Centro de Investigación Biomédica en Red de Salud Mental, Madrid, Spain (M.V.G.-G.).
Universidad Francisco de Vitoria, Pozuelo de Alarcón, Spain (P.G.-P.).

Dilated cardiomyopathy (DCM) is the most frequent cause of heart failure in the young and the leading cause of transplantation. Almost half of the cases have a familial (hereditary) component, but even in familial cases, the diagnostic yield of genetic testing is lower than <40%.¹Myocardial zonula adherens protein (MYZAP) gene encodes a protein widely expressed in cardiac tissue, being an emerging candidate to become a DCM-associated gene.

We evaluated a family from Spanish origin (European ancestry) in which 2 sisters were affected by a severe form of DCM with healthy unaffected parents. The study was approved by the Hospital Universitario Puerta de Hierro ethics committee and patients provided informed consent. Data are available from the corresponding author upon reasonable request.

The proband was a 35-year-old female with dyspnea (New York Heart Association II) and palpitations after her first pregnancy. Echocardiography showed mild dilatation of the left ventricle (LV) with systolic dysfunction (LV ejection fraction, 44%) and frequent ventricular ectopic beats (1000/24 h) were detected on Holter ECG. Cardiac magnetic resonance showed biventricular midventricular fibrosis predominantly located in the LV. Familial work-up was performed. Both the 62-year-old father and 60-year-old mother had normal cardiac studies. However, her asymptomatic 32-year-old sister showed LV dilatation with LV ejection fraction of 35% and a similar pattern of late gadolinium enhancement on cardiac magnetic resonance (Figure [A]). Nonsustained VT episodes were detected, and an implantable cardioverter defibrillator was implanted. During follow-up she received an appropriate shock while sleeping due to sustained VT. None of the affected patients had extracardiac features. Initial genetic analysis of desmosomal genes and a next generation sequencing cardiomyopathy panel (126 genes) were negative.

Figure. Clinical and functional evaluations performed in the family. A, Family pedigree and cardiac magnetic resonance imaging of the index case (upper) showing LV dilatation (LVEDV=168 mL) and extensive areas of midventricular fibrosis predominantly in the left ventricle. Her affected sister (lower) had similar findings on magnetic resonance imaging (LVEDV=260 mL). B, MYZAP immunostaining in skin samples from affected siblings and unaffected parents. Skin biopsies were immunostained with anti-MYZAP antibody (white). Nuclei were stained with DAPI (blue). Images were acquired with a 20× objective. Magnifications of the selected areas (yellow squares) were acquired with a 40× objective. C, Western blot analysis of MYZAP in HL-1 cells transfected with MYZAP-WT, MYZAP-p.Arg117*, and MYZAP-c.933+1G>A. MYZAP-c.933+1G>A shows significantly lower expression level of MYZAP compared with MYZAP-WT and MYZAP-p.Arg117*. D, Immunofluorescence analysis of localization of MYZAP in transfected HL-1 cells showing that MYZAP-WT and MYZAP-c.933+1G>A are observed predominantly as perinuclear aggregations and in the cytoplasmatic membrane, while MYZAP-p.Arg117* delocalizes being distributed in the cytoplasm but not in any particular structure at cell level. Bar, 20 µm. DAPI indicates 4′,6-diamidino-2-phenylindole; LVEDV, left ventricle end diastolic volume; VINC, vinculin; and WT, wild-type.

Exome sequencing was performed in all family members. Assuming a recessive model, we identified MYZAP as the only gene with 2 rare candidate loss-of-function (LoF) variants in both siblings. The first was a nonsense variant (NM_001018100.5 c.349C>T) predicting a premature termination codon (p.Arg117*) in exon 4. The second, NM_001018100.5 c.933+1G>A, affected the first nucleotide of the splice donor site of intron 8, predicting the loss of the donor site. Each parent carried 1 variant (Figure [A]), confirming that they were present in compound heterozygosis (different alleles) in the affected daughters.

We hypothesized that only biallelic LoF variants in MYZAP would be associated with DCM. We performed immunostaining of skin samples with anti-MYZAP antibodies in the family to evaluate if there was protein expression as it had been described in pemphigus patients.² A similar distribution of the MYZAP staining pattern was observed in all individuals, which was preferentially located in perivascular areas, but with a weaker expression in samples from carriers of biallelic variants (Figure B).

Cardiac muscle cell lines (HL-1) were transfected with GFP (green fluorescent protein)-tagged expression vectors for MYZAP: peGFP-C1-MYZAP-WT, peGFP-C1-MYZAP-p.Arg117*, and peGFP-C1-MYZAP-c.933+1G>A. Western blot analysis showed a significantly lower expression of MYZAP c.933+1G>A compared with MYZAP-WT and MYZAP-p.Arg117* (Figure [C]). Immunofluorescence to evaluate the subcellular protein localization showed that MYZAP-WT and MYZAP-c.933+1G were predominantly localized in the plasma membrane and as perinuclear aggregates, whereas MYZAP-p.Arg117* was distributed randomly throughout the cytoplasm, without reaching intercellular junctions (Figure [D]). These experiments confirm that neither of the 2 alleles are viable: one of them produces a protein that is degraded and the other produces a dysfunctional protein that is delocalized in the cytoplasm. These results, in which no functional protein was produced in affected carriers, support previous findings of functional studies in zebrafish³ and knock-out mice⁴ that develop DCM with decreased contractile function, and increased mortality, being MYZAP localized in the intercalated discs.

Finally, to evaluate the relevance of MYZAP variants in humans, we obtained data from 200 571 participants from the UK Biobank and found 29 distinct rare LoF variants in 98 individuals. There were no biallelic LoF carriers and none of the LoF heterozygous individuals were diagnosed with cardiomyopathy. Furthermore, none of 1446 patients with cardiomyopathies studied by whole genome sequencing in the 100 000 Genomes Project England had biallelic LoF variants in MYZAP. Cardiomyopathy was not more prevalent in carriers of heterozygous MYZAP LoF variants (2/26; 7.7%) compared with individuals without MYZAP variants (1420/15 552; 8.4%; χ² test: odds ratio, 0.91 [95% CI, 0.15–3.14]).

In this research, we describe and demonstrate the association between biallelic LoF variants in MYZAP and a severe form of DCM, characterized by profuse biventricular fibrosis and ventricular arrhythmias. This phenotype is coincident with 2 homozygous LoF cases recently reported.⁵ Unlike the previous report, affected individuals in our family exhibited LoF in compound heterozygosity, providing further support to MYZAP as a recessive DCM-associated gene and suggesting that the relevance of MYZAP variants in DCM may be higher than previously anticipated. This could be particularly important in populations with high rates of consanguinity. Furthermore, by analyzing large population cohorts we show that although LoF variants in single heterozygosity in MYZAP are observed at low frequency in the general population, they are not associated with cardiac disease in the heterozygous state. Based on our findings, we propose MYZAP as a gene to be considered for evaluation in patients with DCM, particularly among gene-elusive patients with arrhythmogenic DCM whose parents do not show cardiac disease.

For the purpose of open access, the author has applied a Creative Commons Attribution (CC BY) license to any Author Accepted Manuscript version arising.

This study has been funded by grants from the Spanish Society of Cardiology (basic research grant 2021) to Dr Ochoa, Spanish Ministry of Science PLEC2022-009235 MCIN/AEI/10.13039/501100011033 co-funded by the European Union NextGenerationEU/PRTR to Drs Lara-Pezzi, Gómez-Gaviro, and Garcia-Pavia, and PID2021-124629OB-I00 and TED2021-129774B-C22 to Dr Lara-Pezzi, and from Instituto de Salud Carlos III (ISCIII) DTS22/00030 co-funded by the European Union to Dr Gómez-Gaviro. Drs Lara-Pezzi, Ware, and Garcia-Pavia are funded by the Pathfinder Cardiogenomics Programme of the European Innovation Council of the European Union (DCM-NEXT project; project 101115416). The CNIC is supported by the ISCIII, the Ministerio de Ciencia e Innovación (MCIN) and the Pro CNIC Foundation and is a Severo Ochoa Center of Excellence (grant CEX2020-001041-S funded by MICIN/AEI/10.13039/501100011033). Drs Ware, McGurk, and Barton have been funded by the Medical Research Council (United Kingdom), Sir Jules Thorn Charitable Trust (21JTA), Wellcome Trust (107469/Z/15/Z), British Heart Foundation (RE/18/4/34215, FS/IPBSRF/22/27059), and the National Institute for Health and Care Research (NIHR) Imperial College Biomedical Research Centre. Part of this research was made possible through access to the data and findings generated by the 100 000 Genomes Project. The 100 000 Genomes Project is managed by Genomics England Limited (a wholly owned company of the Department of Health and Social Care). The 100 000 Genomes Project is funded by the NIHR and National Heart Service (NHS) England. The Wellcome Trust, Cancer Research UK and the Medical Research Council have also funded research infrastructure. The 100 000 Genomes Project uses data provided by patients and collected by the NHS as part of their care and support. The UK Biobank recruited 500 000 participants aged 40 to 69 years across the UK between 2006 and 2010 (National Research Ethics Service, 11/NW/0382; 10.1371/journal.pmed.1001779). This study was conducted under terms of access approval number 47602. Written informed consent was provided.

Nonstandard Abbreviations and Acronyms

DCM	dilated cardiomyopathy
GFP	green fluorescent protein
LoF	loss of function
LV	left ventricle
MYZAP	myocardial zonula adherens protein

dilated cardiomyopathy

green fluorescent protein

loss of function

left ventricle

myocardial zonula adherens protein

Disclosures Dr Ochoa is an employee of Health in Code. Dr Ware has consulted for MyoKardia Inc, Foresite Labs, and Pfizer. The other authors reports no conflicts.

For Sources of Funding and Disclosures, see page 280.

中文翻译：

心肌粘连带蛋白基因 (MYZAP) 的双等位基因功能丧失变异导致严重隐性扩张型心肌病

扩张型心肌病（DCM）是年轻人心力衰竭的最常见原因，也是移植的主要原因。几乎一半的病例具有家族（遗传）成分，但即使在家族病例中，基因检测的诊断率也低于<40%。¹心肌小带粘附蛋白( MYZAP ) 基因编码一种在心脏组织中广泛表达的蛋白质，是 DCM 相关基因的新兴候选基因。

我们评估了一个西班牙血统（欧洲血统）的家庭，其中 2 名姐妹患有严重的扩张型心肌病，父母健康且未受影响。该研究得到了耶罗门大学医院伦理委员会的批准，并且患者提供了知情同意书。数据可根据合理要求从相应作者处获得。

先证者是一名 35 岁女性，在第一次怀孕后出现呼吸困难（纽约心脏协会 II）和心悸。超声心动图显示左心室 (LV) 轻度扩张，伴有收缩功能障碍（左心室射血分数，44%），动态心电图检测到频繁的室性异位搏动（1000/24 小时）。心脏磁共振显示双心室心室中段纤维化主要位于左心室。进行了家庭检查。 62 岁的父亲和 60 岁的母亲的心脏检查均正常。然而，她无症状的 32 岁姐姐在心脏磁共振上显示左心室扩张，左心室射血分数为 35%，且有类似的晚期钆增强模式（图 [A]）。检测到非持续性室性心动过速发作，并植入植入式心律转复除颤器。在随访期间，由于持续性室性心动过速，她在睡觉时接受了适当的电击。受影响的患者均未出现心外特征。桥粒基因的初始遗传分析和下一代测序心肌病组（126 个基因）均为阴性。

数字。 在家庭中进行临床和功能评估。 A，索引病例的家族谱系和心脏磁共振成像（上）显示左心室扩张（LVEDV=168 mL）和主要在左心室的广泛心室中纤维化区域。她受影响的妹妹（下）在磁共振成像中也有类似的发现（LVEDV=260 mL）。B，受影响兄弟姐妹和未受影响父母皮肤样本中的 MYZAP 免疫染色。皮肤活检用抗 MYZAP 抗体（白色）进行免疫染色。细胞核用 DAPI（蓝色）染色。图像是用 20 倍物镜采集的。使用 40 倍物镜获取所选区域（黄色方块）的放大倍数。C，用 MYZAP-WT、MYZAP-p.Arg117* 和 MYZAP-c.933+1G>A 转染的 HL-1 细胞中 MYZAP 的蛋白质印迹分析。与MYZAP-WT和MYZAP-p.Arg117*相比，MYZAP-c.933+1G>A显示出显着较低的MYZAP表达水平。D，转染的 HL-1 细胞中 MYZAP 定位的免疫荧光分析表明，MYZAP-WT 和 MYZAP-c.933+1G>A 主要作为核周聚集体和在细胞质膜中观察到，而 MYZAP-p.Arg117* 则离域分布在细胞质中，但不分布在细胞水平的任何特定结构中。条形，20 µm。 DAPI表示4',6-二脒基-2-苯基吲哚； LVEDV，左心室舒张末期容积； VINC，纽蛋白；和WT，野生型。

对所有家庭成员进行了外显子组测序。假设是隐性模型，我们确定MYZAP是兄弟姐妹中唯一具有 2 个罕见候选功能丧失 (LoF) 变异的基因。第一个是无义变体 (NM_001018100.5 c.349C>T)，预测外显子 4 中的过早终止密码子 (p.Arg117*)。第二个是 NM_001018100.5 c.933+1G>A，影响内含子8的剪接供体位点，预测供体位点的丢失。每个父母都携带 1 个变异（图 [A]），证实它们在受影响的女儿中存在复合杂合（不同的等位基因）。

我们假设只有MYZAP中的双等位基因 LoF 变体才会与 DCM 相关。我们用抗 MYZAP 抗体对该家族的皮肤样本进行了免疫染色，以评估是否存在天疱疮患者所描述的蛋白质表达。²在所有个体中均观察到 MYZAP 染色模式的相似分布，其优先位于血管周围区域，但在双等位基因变异携带者的样本中表达较弱（图 B）。

心肌细胞系 (HL-1) 用 MYZAP 的 GFP（绿色荧光蛋白）标记表达载体转染：peGFP-C1-MYZAP-WT、peGFP-C1-MYZAP-p.Arg117* 和 peGFP-C1-MYZAP -c.933+1G>A。蛋白质印迹分析显示，与 MYZAP-WT 和 MYZAP-p.Arg117* 相比，MYZAP c.933+1G>A 的表达显着降低（图 [C]）。评估亚细胞蛋白定位的免疫荧光显示 MYZAP-WT 和 MYZAP-c.933+1G 主要定位于质膜并作为核周聚集体，而 MYZAP-p.Arg117* 随机分布在整个细胞质中，没有到达细胞间连接（图[D]）。这些实验证实这两个等位基因均不可行：其中一个产生一种被降解的蛋白质，另一个产生一种在细胞质中离域的功能失调的蛋白质。这些结果（其中受影响的携带者中没有产生功能性蛋白质）支持了先前在斑马鱼³和基因敲除小鼠⁴中进行的功能研究的发现，这些小鼠由于 MYZAP 位于闰盘中而发展为 DCM，收缩功能下降，死亡率增加。

最后，为了评估MYZAP变异在人类中的相关性，我们从英国生物银行的 200 571 名参与者中获取了数据，并在 98 名个体中发现了 29 种不同的罕见 LoF 变异。没有双等位基因 LoF 携带者，并且没有 LoF 杂合子个体被诊断患有心肌病。此外，在英格兰 10 万基因组计划中，通过全基因组测序研究的 1446 名心肌病患者中，没有一个患者的MYZAP具有双等位基因 LoF 变异。与没有 MYZAP 变异的个体（1420/15 552；8.4%；χ ²检验：比值比，0.91 [95% CI，0.15- 3.14]）。

在这项研究中，我们描述并证明了MYZAP中的双等位基因 LoF 变异与严重 DCM 之间的关联，其特征是严重的双心室纤维化和室性心律失常。该表型与最近报道的 2 例纯合 LoF 病例一致。⁵与之前的报告不同，我们家族中受影响的个体在复合杂合性中表现出 LoF，这为MYZAP作为隐性 DCM 相关基因提供了进一步的支持，并表明MYZAP变异在 DCM 中的相关性可能高于之前的预期。这对于近亲结婚率高的人群尤其重要。此外，通过分析大型人群队列，我们发现虽然MYZAP中单一杂合性的 LoF 变异在普通人群中观察到的频率较低，但它们与杂合状态下的心脏病无关。根据我们的研究结果，我们建议将MYZAP作为 DCM 患者评估的一个基因，特别是在父母未表现出心脏病、基因难以捉摸的致心律失常性 DCM 患者中。

出于开放获取的目的，作者已对任何作者接受的手稿版本应用了知识共享署名 (CC BY) 许可。

这项研究由西班牙心脏病学会向 Ochoa 博士提供的资助（2021 年基础研究资助）、西班牙科学部 PLEC2022-009235 MCIN/AEI/10.13039/501100011033 共同资助，由欧盟 NextGenerationEU/PRTR 共同资助给 Lara 博士-Pezzi、Gómez-Gaviro 和 Garcia-Pavia，以及 Lara-Pezzi 博士的 PID2021-124629OB-I00 和 TED2021-129774B-C22，以及由欧盟共同资助的 Instituto de Salud Carlos III (ISCIII) DTS22/00030致戈麦斯-加维罗博士。 Lara-Pezzi、Ware 和 Garcia-Pavia 博士由欧盟欧洲创新理事会探路者心脏基因组学计划（DCM-NEXT 项目；项目 101115416）资助。 CNIC 得到 ISCIII、Ministryio de Ciencia e Innovación (MCIN) 和 Pro CNIC 基金会的支持，是一个 Severo Ochoa 卓越中心（由 MICIN/AEI/10.13039/501100011033 资助的赠款 CEX2020-001041-S）。 Ware、McGurk 和 Barton 博士得到了医学研究理事会（英国）、Jules Thorn 爵士慈善信托基金 (21JTA)、Wellcome Trust (107469/Z/15/Z)、英国心脏基金会 (RE/18/4) 的资助/34215、FS/IPBSRF/22/27059）以及国家健康与护理研究所 (NIHR) 帝国理工学院生物医学研究中心。这项研究的一部分是通过访问十万基因组计划产生的数据和研究结果而得以实现的。 100 000 基因组计划由 Genomics England Limited（卫生和社会保障部的全资公司）管理。 100 000 基因组计划由 NIHR 和英国国家心脏服务中心 (NHS) 资助。威康信托基金会、英国癌症研究中心和医学研究委员会也资助了研究基础设施。 100 000 基因组计划使用患者提供的数据以及 NHS 收集的数据作为他们护理和支持的一部分。 2006 年至 2010 年间，英国生物银行在英国招募了 50 万名年龄在 40 岁至 69 岁之间的参与者（国家研究道德服务中心，11/NW/0382；10.1371/journal.pmed.1001779）。本研究是根据准入批准号 47602 的条款进行的。提供了书面知情同意书。