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Iterative Reanalysis of Hypertrophic Cardiomyopathy Exome Data Reveals Causative Pathogenic Mitochondrial DNA Variants
Circulation: Genomic and Precision Medicine ( IF 7.4 ) Pub Date : 2021-05-10 , DOI: 10.1161/circgen.121.003388
Luis R Lopes 1, 2 , David Murphy 3 , Enrico Bugiardini 4 , Reem Salem 5 , Joanna Jager 1 , Marta Futema 1 , Mohammed Majid Akhtar 1, 2 , Konstantinos Savvatis 1, 2, 6 , Cathy Woodward 7 , Alan M Pittman 8 , Michael G Hanna 4 , Petros Syrris 1 , Robert D S Pitceathly 4 , Perry M Elliott 1, 2
Affiliation  

Mitochondrial cytopathies caused by mitochondrial DNA (mtDNA) mutations have an estimated prevalence of 1/5000 adults.1 Cardiac manifestations are common (up to 40%) and include hypertrophic cardiomyopathy (HCM). Some mtDNA mutations (eg,m.3243A>G,m.8344A>G,m.4300A>G) are well-recognized causes of cardiomyopathy and may occur as part of a multi-organ syndrome, such as Mitochondrial Encephalomyopathy, Lactic Acidosis, and Stroke-like episodes, or as the only manifestation.


Up to 60% of HCM probands have no detectable causal mutations, while the prevalence of pathogenic mtDNA variants in large HCM cohorts has not previously been determined. We hypothesized that the presence of mtDNA mutations might account for a proportion of genotype-negative cases and applied a workflow to reliably identify mtDNA variants from whole-exome sequencing data.


The study population and clinical evaluation have been previously described.2 All patients provided written informed consent, and the study had ethics committee approval (15/LO/0549). DNA extraction, library preparation, whole-exome sequencing, variant calling, and annotation were previously reported.2,3 Mitochondrial variants with read-depth ≥10× and heteroplasmy level ≥10% were chosen for validation and confirmation using whole mtDNA next-generation sequencing. Data that support the findings are available upon request.


The cohort comprised 770 unrelated HCM patients (67% males, age 49.3±15.9 years at diagnosis); 33% had candidate variants in sarcomeric genes robustly associated with HCM. Six hundred fifty-nine samples passed quality control with called mitochondrial variants (mean depth 20.2).


The MT-TL1 m.3243A>G mutation, a well-recognized cause of HCM, was detected at heteroplasmic levels in 2 probands (0.4% of sarcomere-negatives) in whom a primary mitochondrial disease diagnosis had not previously been suspected. A third proband was homoplasmic for MT-ND1 m.3460G>A, a pathogenic variant associated with Leber hereditary optic neuropathy. The patients did not harbor any other candidate variants in nuclear-encoded HCM genes.


Proband 1 was a female who presented at 35 years due to breathlessness and chest pain. Past medical history included well-controlled hypertension diagnosed at 19 years, bilateral deafness attributed to parotiditis, repeat miscarriages (five), and gestational diabetes. Family history was unremarkable. ECG showed left ventricular hypertrophy and T wave inversion; echocardiography revealed symmetrical/concentric left ventricular hypertrophy maximum LV wall thickness 16 mm; cardiac magnetic resonance showed extensive fibrosis with subepicardial distribution (Figure [A]). Cardiopulmonary exercise test revealed a low peak oxygen consumption of 18 mL/min per kg (53% predicted) and anaerobic threshold of 33%. The m.3243A>G mutation was detected at 37% load in blood.


Figure. ECG and cardiac magnetic resonance (CMR) images of the described probands.A, ECG and cardiac magnetic resonance (CMR) images (from left to right, 4 chamber, short axis and 2 chamber views; upper row end-diastolic cine images, lower row late gadolinium enhancement images) for proband 1, harboring the m.3243A>G mutation in MT-TL1. ECG shows T-wave inversion V2 to V6, DI, DII, and aVL. CMR shows concentric hypertrophy and extensive fibrosis with a subepicardial distribution at basal lateral wall and mid-apical anterior, lateral, and inferior walls. B, CMR images (from left to right, 4 chamber and mid short-axis views; upper row end-diastolic cine images, lower row late gadolinium enhancement images), for poband 2 harboring the m.3243A>G mutation in MT-TL1, showing extensive circumferential mid-myocardial/subepicardial enhancement and localized inferior septum hypertrophy (14 mm); relative wall thickness was 0.46, indicative of concentric remodeling. C, ECG and CMR images (from left to right, 4 chamber and 2 chamber views; upper row end-diastolic cine images and lower row late gadolinium enhancement images) for proband 3, homoplasmic for the pathogenic m.3460G>A variant in ND1. ECG shows left ventricular hypertrophy and deep T-wave inversion V3 to V6, DI, DII, aVL, and aVF. CMR shows septal and apical left ventricular hypertrophy (LVH) and extensive patchy enhancement, mainly in the anterolateral wall and septum.


Proband 2 was a male who presented at 61 years with heart failure and atrial fibrillation. Past medical history included hypertension (well-controlled on medication), diabetes complicated by retinopathy, and multiple strokes. Family history was uninformative. ECG showed atrial fibrillation and left bundle branch block. Echocardiography revealed severe LV systolic dysfunction and septal hypertrophy(14 mm); relative wall thickness 0.46 (concentric remodeling). Cardiac magnetic resonance showed extensive circumferential mid-myocardial/subepicardial enhancement (Figure [B]) with an LV ejection fraction 33%. Creatine kinase was mildly increased at 340 IU/L. A dual-chamber implantable cardioverter defibrillator with resynchronization therapy was implanted at 72 years and an appropriate shock occurred 3 months thereafter. He died at 74 years due to decompensated heart failure. The m.3243A>G mutation was identified at 11% load.


Proband 3 was a male who presented at 39 years with chest pain and breathlessness. ECG showed marked left ventricular hypertrophy, inferolateral T-wave inversion(Figure [C]). Cardiac magnetic resonance revealed septal-apical left ventricular hypertrophy with maximum LV wall thickness 26 mm and extensive patchy enhancement, in the anterolateral wall and septum(Figure [C]). Nonsustained ventricular tachycardia was detected, and an implantable cardioverter-defibrillator was implanted. During follow-up, Leber hereditary optic neuropathy was diagnosed in a maternal aunt and cousin; his mother was known to carry the mutation with no clinical manifestations. He had an ophthalmologic assessment with no features of optic neuropathy. The m.3460G>A was detected at homoplasmic levels in blood.


In retrospect, both patients harboring the m.3243A>G mutation had features consistent with a nosnsarcomeric cause. Proband 1 had multiple miscarriages, gestational diabetes, and hearing loss, in addition to limited performance on the cardiopulmonary exercise test; proband 2 exhibited systolic dysfunction, diabetes, and multiple strokes. Finally, the distribution and extent of fibrosis was unusual for sarcomeric HCM but is consistent with one other case series describing cardiac magnetic resonance findings in patients with mitochondrial mutations.4


Sequencing off-target captured mtDNA from exome data has been described previously and refined in-house.3 This methodology had never been applied to screen a HCM cohort for pathogenic mtDNA variants. A previous study using whole-genome sequencing detected the pathogenic m.4300A>G variant in 1/46 genotype-negative HCM patients.5 The coverage achieved with whole-genome sequencing is higher, but most clinical and research cohorts are studied using whole-exome sequencing.


HCM caused by mtDNA mutations is characterized by ventricular arrhythmia, conduction disease, and evolution to systolic dysfunction. A thorough assessment for extra-cardiac manifestations is crucial if mitochondrial disease is suspected. The detection of pathogenic mtDNA variants has significant impact for the genetic counseling and management of the proband and their relatives.


Iterative reanalysis of whole-exome sequencing data for mtDNA mutations increases the yield of genetic testing in HCM and should, therefore, be considered in genetically undiagnosed HCM cohorts.


The University College London Hospitals/University College London Queen Square Institute of Neurology sequencing facility receives a proportion of funding from the Department of Health’s National Institute for Health Research Biomedical Research Centres funding scheme. The clinical and diagnostic Rare Mitochondrial Disorders Service in London is funded by the UK National Health Service Highly Specialised Commissioners. Dr Lopes is funded by an Medical Research Council UK Clinical Academic Research Partnership Award. D. Murphy is funded by the National Institute for Health Research University College London Hospitals Biomedical Research Centre. Dr Pitceathly is supported by a Medical Research Council Clinician Scientist Fellowship (MR/S002065/1). Drs Pitceathly and Hanna receive funding from a Medical Research Council strategic award to establish an International Centre for Genomic Medicine in Neuromuscular Diseases (MR/S005021/1). Dr Futema is funded by the Fondation Leducq Transatlantic Networks of Excellence Program grant (No. 14 CVD03).


Disclosures None.


*R.D.S. Pitceathly and P.M. Elliott contributed equally.


For Sources of Funding and Disclosures, see page 382.




中文翻译:

肥厚型心肌病外显子组数据的迭代再分析揭示了致病性线粒体 DNA 变异

由线粒体 DNA (mtDNA) 突变引起的线粒体细胞病估计患病率为 1/5000 成年人。1心脏表现很常见(高达 40%),包括肥厚型心肌病 (HCM)。一些 mtDNA 突变(例如,m.3243A>G,m.8344A>G,m.4300A>G)是公认的心肌病原因,可能作为多器官综合征的一部分发生,例如线粒体脑肌病、乳酸性酸中毒和中风样发作,或作为唯一表现。


高达 60% 的 HCM 先证者没有可检测到的因果突变,而大型 HCM 队列中致病性 mtDNA 变异的流行率之前尚未确定。我们假设 mtDNA 突变的存在可能占基因型阴性病例的一部分,并应用工作流程从全外显子组测序数据中可靠地识别 mtDNA 变体。


先前已经描述了研究人群和临床评估。2所有患者均提供书面知情同意书,该研究已获得伦理委员会批准 (15/LO/0549)。DNA 提取、文库制备、全外显子组测序、变异调用和注释之前已被报道。2,3选择读取深度≥10×、异质性水平≥10% 的线粒体变体使用全 mtDNA 下一代测序进行验证和确认。支持调查结果的数据可应要求提供。


该队列包括 770 名无关的 HCM 患者(67% 为男性,诊断时年龄为 49.3±15.9 岁);33% 的肌节基因的候选变异与 HCM 密切相关。659 个样本通过了称为线粒体变体的质量控制(平均深度 20.2)。


MT-TL1 m.3243A>G 突变是公认的 HCM 原因,在 2 名先证者(0.4% 的肌节阴性)的异质水平上检测到,这些先证者以前没有怀疑原发性线粒体疾病的诊断。第三个先证者是MT-ND1 m.3460G>A 的同质者,这是一种与 Leber 遗传性视神经病变相关的致病性变异。患者在核编码的 HCM 基因中没有任何其他候选变体。


先证者 1 是一名 35 岁的女性,因呼吸困难和胸痛而就诊。既往病史包括 19 岁时诊断出的控制良好的高血压、归因于腮腺炎的双侧耳聋、反复流产(5 次)和妊娠糖尿病。家族史无异常。心电图显示左心室肥厚和T波倒置;超声心动图显示对称/同心左心室肥厚最大 LV 壁厚 16 mm;心脏磁共振显示广泛的纤维化,分布在心外膜下(图 [A])。心肺运动测试显示低峰值耗氧量为每公斤 18 毫升/分钟(53% 的预测值)和 33% 的无氧阈值。m.3243A>G 突变在血液中检测到 37% 的负荷。


数字。 所述先证者的心电图和心脏磁共振 (CMR) 图像。A,先证者 1 的心电图和心脏磁共振 (CMR) 图像(从左到右,4 腔,短轴和 2 腔视图;上排舒张末期电影图像,下排晚期钆增强图像),包含 m。 MT-TL1 中的 3243A>G 突变。ECG 显示 T 波倒置 V2 至 V6、DI、DII 和 aVL。CMR 显示向心性肥大和广泛的纤维化,在基底侧壁和心尖中部前壁、外侧壁和下壁分布于心外膜下。B CMR图像(从左到右, 4 个腔室和中间短轴视图;上排舒张末期电影图像,下排晚期钆增强图像),用于在MT-TL1中携带 m.3243A>G 突变的 poband 2 ,显示广泛的周向心肌中部/心外膜下增强和局部下隔膜肥大(14 mm) ; 相对壁厚为0.46,表明同心重塑。先证者 3的C、ECG 和 CMR 图像(从左到右,4 室和 2 室视图;上排舒张末期电影图像和下排延迟钆增强图像),同质为致病性 m.3460G> ND1中的变异. 心电图显示左心室肥大和深部 T 波倒置 V3 至 V6、DI、DII、aVL 和 aVF。CMR 显示室间隔和心尖部左心室肥厚 (LVH) 和广泛的斑片状强化,主要位于前外侧壁和室间隔。


先证者 2 是一名 61 岁时出现心力衰竭和心房颤动的男性。既往病史包括高血压(药物控制良好)、糖尿病合并视网膜病变和多次中风。家族史没有提供任何信息。心电图显示心房颤动和左束支传导阻滞。超声心动图显示严重的 LV 收缩功能障碍和室间隔肥大(14 毫米);相对壁厚 0.46(同心重塑)。心脏磁共振显示广泛的周向心肌中部/心外膜下增强(图 [B]),左室射血分数为 33%。肌酸激酶在 340 IU/L 时轻度升高。72 岁时植入了带再同步治疗的双腔植入式心脏复律除颤器,3 个月后发生适当的休克。他因失代偿性心力衰竭去世,享年 74 岁。m.3243A>G 突变在 11% 负载下被鉴定。


先证者 3 是一名男性,39 岁时出现胸痛和呼吸困难。心电图显示左心室明显肥大,下外侧T波倒置(图[C])。心脏磁共振显示室间隔心尖肥大,左室壁最大厚度为 26 mm,前外侧壁和室间隔广泛斑片状强化(图 [C])。检测到非持续性室性心动过速,并植入了植入式心脏复律除颤器。随访期间,一位姨妈和表弟被诊断为 Leber 遗传性视神经病变;众所周知,他的母亲携带了没有临床表现的突变。他进行了眼科评估,没有视神经病变的特征。m.3460G>A 在血液中的同质水平上被检测到。


回想起来,这两名携带 m.3243A>G 突变的患者都具有与非肌节病原因一致的特征。先证者 1 有多次流产、妊娠糖尿病和听力损失,此外在心肺运动试验中表现有限;先证者 2 表现出收缩功能障碍、糖尿病和多发性中风。最后,肌节 HCM 的纤维化分布和程度不常见,但与描述线粒体突变患者心脏磁共振结果的另一系列病例一致。4


先前已经描述了从外显子组数据中对捕获的脱靶 mtDNA 进行测序,并在内部进行了改进。3这种方法从未用于筛选 HCM 队列中的致病性 mtDNA 变异。先前使用全基因组测序的研究在 1/46 基因型阴性 HCM 患者中检测到致病性 m.4300A>G 变异。5全基因组测序的覆盖率更高,但大多数临床和研究队列使用全外显子组测序进行研究。


由 mtDNA 突变引起的 HCM 的特征是室性心律失常、传导疾病和演变为收缩功能障碍。如果怀疑有线粒体疾病,对心脏外表现进行全面评估至关重要。致病性mtDNA变异的检测对先证者及其亲属的遗传咨询和管理具有重要影响。


对 mtDNA 突变的全外显子组测序数据进行迭代再分析提高了 HCM 中基因检测的产量,因此应在未确诊的 HCM 队列中考虑。


伦敦大学学院医院/伦敦大学学院皇后广场神经病学研究所测序设施从卫生部的国家健康研究所生物医学研究中心资助计划中获得一定比例的资金。伦敦的临床和诊断罕见线粒体疾病服务由英国国家卫生服务高度专业委员会资助。Lopes 博士由英国医学研究委员会临床学术研究合作奖资助。D. Murphy 由国家健康研究所伦敦大学学院医院生物医学研究中心资助。Pitceathly 博士得到了医学研究委员会临床医生科学家奖学金 (MR/S002065/1) 的支持。Pitceathly 博士和 Hanna 博士从医学研究委员会战略奖获得资金,以建立国际神经肌肉疾病基因组医学中心 (MR/S005021/1)。Futema 博士由基金会 Leducq 跨大西洋卓越网络计划赠款(编号 14 CVD03)资助。


披露无。


*RDS Pitceathly 和 PM Elliott 贡献相同。


有关资金来源和披露信息,请参见第 382 页。


更新日期:2021-06-15
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