【经验】基因检测技术在心脏病学实践中的应用

Matteo Vatta 教授教授

机构： 印第安纳大学医学院 介绍：

印第安纳大学医学院Matteo Vatta 教授

在过去几年内，由于被称作新一代测序（NGS）的高通量测序技术的出现，基因检测在心脏病学中的应用已获得了越来越多的关注以及展现出了不断增长的势头。然而，尽管基因检测的作用在很久以前就已经得到了证实，但其并未因此被纳入所有心脏病学实践的日常操作。

在此，我将讨论分子诊断在QT间期延长综合征（LQTS）患者中的应用。LQTS是进行基因检测与咨询的I类指征，这表示“有证据证明和/或人们广泛认同一项特定操作或治疗是有益、有用且有效的。”

作为一名临床分子遗传学者，我认为基因检测应在心脏病学医师进行了全面的临床评估后开始，之后通常是临床遗传学者进行会诊，在合适的情况下，遗传咨询师会给予预先测试。

尽管大量LQTS患者的基线ECG表现不同，提示比电流有所改变（如LQT1、LQT2、LQT3），但只能在进行了全面的临床基因检测后才能精确定位潜在的分子缺陷。目前所知，三个基因上的缺陷可导致约75%的LQTS病例发生，这三个基因（KCNQ1、KCNH2、和SCN5A）分别是编码两个钾离子通道α亚基的基因和一个钠离子通道α亚基的基因。此外还有10个基因（AKAP9、ANK2、CACNA1C、CAV3、KCNE1、KCNE2、KCNJ2、KCNJ5、SCN4B和SNTA1）与LQTS相关，累计覆盖了其他5-10%的病例。另外，大量重排影响了所有LQTS基因所致的的拷贝数目变异（CNV）解释了其他的少数病例，因而提示，仍有10%的病例其遗传学基础未知。

一旦确定了LQTS的诊断，基因检测则成为了标准的操作方式。众所周知，基因检测结果或有助于患者处理。与其他两个基因发生变异相比，KCNQ1携带有害变异的患者可能对β受体阻滞剂治疗的应答更好（KCNQ1>KCNH2>SCN5A）。此外，在SCN5A发生有害变异的症状性患者中，应考虑应用永久性起搏器和植入型复律除颤器（ICD）治疗。尽管QT间期延长和心律失常是LQTS患者的主要表现，他们还能表现出其他的临床特征，包括但不限于并指征、自闭症、面部畸形、脊柱侧弯和身材矮小，这可能提示了更为复杂的情况，如CACNA1C发生有害突变所致的Timothy综合征，或与KCNJ2变异相关的Andersen-Tawil综合征。此外，近期还确定LQTS与一些类型的癫痫之间存在相关性。在所有病例中，确定涉及其他医疗服务的非心脏特征、联合深入了解既往治疗史及家族史，这对于解读诊断性实验室检查和检测前、后LQTS患者的咨询服务有很大帮助。这对于诊断由与LQTS相关的某一基因库发生有害变异所致的重叠疾病尤其重要。因此，选择覆盖多数LQTS病例的基因组，可能包含有与上述更复杂的表现相关的基因，是最为理想的策略。

一旦决定进行特异性检测，则会出现三种可能的实验室检查结果：阴性结果、阳性结果以及不确定——检测到的变异临床意义不确定（VUS）。这一基本的分类方法解释了致病性的三种主要证据水平，如

1.检测到的变异类型（如剪接、无义突变、移码突变、错义突变、非移码突变、同义突变、大片段突变）及其在基因及蛋白质上的位置，联合所谓的潜在致病机制（如，单纯功能不全、获得功能、显性-阴性等）。

2.进行的遗传学研究（如在一个大规模的多代家族中或在多个小规模多代家族中大量LOD评分>3的关联数据，在这些情况下受累个体中变异与疾病同时存在，或突变在既往无家族史的先证者中首次出现）。

3.进行功能性研究（如，体外研究、分子模型研究、动物模型研究等），证实检测到的变异具有有害作用。

尽管LATS被认为是单基因孟德尔疾病，是由于上述所提及的一种基因发生了一种有害突变所致的，但约8%的患者具有两种或两种以上有害突变，导致了更加严重的临床表现。多数功能性研究证实的有害突变是先证者及其家人所特有的突变（“私人”突变）或罕见突变，而其他突变，如KCNE1 p.D85N，可发生于约0.8%的人口。这提示，一些更为常见的变异或许具有一种破坏性的作用，但他们或许不足以分离成一种明显的LQTS表型。

因此，目前有关基因检测的解读遭遇到了LQTS分子基础的复杂性，而且更甚于以前，一套全面的临床方式联合检测前后的遗传咨询对于评估每个突变的作用及在患者表型中的作用，以及检测先证者一级亲属的何种突变一确定风险家族成员而言都是必要的。

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The use of genetic testing in cardiology practice has gained increasing interest and momentum in the last several years thanks to the advent of massive parallel sequencing technology called Next-Generation Sequencing (NGS). However, even though the usefulness of genetic testing has been recognized for a long time, it has not made it into day-to-day operations in all cardiology practice yet.

Here I will discuss the molecular diagnostic approach to patients with long QT syndrome (LQTS), for which genetic testing and counseling is a Class I indication, meaning that “evidence and/or general agreement that a given procedure or treatment is beneficial, useful, and effective.”

As a clinical molecular geneticist, my approach to genetic testing begins after a comprehensive clinical evaluation by a cardiologist, usually followed by a clinical geneticist consult, when appropriate, and pretesting counseling by a genetic counselor.

While a significant number of patients with LQTS exhibit a baseline ECG with distinctive patterns suggesting alterations in specific currents (such as LQT1, LQT2, LQT3), the precise underlying molecular defect can be identified only after performing comprehensive clinical genetic tests. Currently, defects in three genes (KCNQ1, KCNH2, and SCN5A) coding for the alpha subunits of two potassium channels and one sodium channel, respectively, cause approximately 75% of all LQTS cases. However, an additional 10 genes (AKAP9, ANK2, CACNA1C, CAV3, KCNE1, KCNE2, KCNJ2, KCNJ5, SCN4B, and SNTA1) have been linked to LQTS, covering cumulatively an extra 5% to 10% of cases. Moreover, copy number variant (CNV) due to large rearrangements affecting all LQTS genes can explain an additional handful of cases, thus suggesting that the genetic basis of the remaining 10% of cases is still unknown.

Genetic testing for LQTS has become standard of care once the diagnosis has been made. It is well-known that genetic test results may support patient management. Individuals harboring a deleterious variant in KCNQ1 are more likely to respond well to beta-blocker therapy compared with variants in the other two genes (KCNQ1>KCNH2>SCN5A). Moreover, in symptomatic patients with deleterious variants in SCN5A, a permanent pacemaker and implantable cardioverter defibrillator (ICD) therapy should be considered. Although prolonged QTc interval and cardiac arrhythmias could represent the main findings in LQTS patients, they may also show evidence of additional clinical features such as, but not limited to, syndactyly, autism, facial dysmorphologies, scoliosis, and short stature, which could suggest more complex presentations, such as in Timothy syndrome caused by deleterious variants in CACNA1C, or Andersen–Tawil syndrome, associated with KCNJ2 variants. Moreover, it has been recently recognized that there is a link between LQTS and some forms of epilepsy. In all cases, recognizing non-cardiac features involving other medical services and detailing the clinical picture, along with in-depth past clinical and family history, greatly helps the interpretation of diagnostic laboratory tests and the pre- and post-test counseling of LQTS patients. This is particularly important provided the range of overlapping diseases caused by deleterious variants from a gene pool such as that associated with LQTS. Thus, opting for a panel of genes covering the majority of LQTS cases, and possibly including those genes associated with more complex presentations, as discussed above, represents the best strategy.

Once, the specific test has been decided, there are three possible outcomes from the laboratory tests: negative, positive, and uncertain, which occurs when a “variant of uncertain clinical significance” (VUS) is detected. This basic classification encompasses an interpretation based on three major levels of evidence of pathogenicity such as:

Type of variant detected (ie, splicing, nonsense, frameshift, missense, non-frameshift, synonymous, gross alterations) and its position in the gene and protein, along with what is known about the underlying pathogenic mechanism (ie, haplo-insufficiency, gain of function, dominant-negative, etc)

Genetic studies performed (ie, extensive linkage data with LOD score >3 in a large multigenerational family or in multiple small multigenerational families in which the variants co-segregate with the disease in affected individuals or when the variant occurs de novo in the index case with no previous family history)

Functional studies performed (ie, in vitro, cellular model, animal model, etc) demonstrating a deleterious effect of the detected variant

Although LQTS is regarded as a monogenic Mendelian disease, caused by one deleterious variant in one of the above-mentioned genes, up to 8% of patients harbor two or more deleterious variants, causing a more severe clinical presentation. Most functionally proven deleterious variants are unique to the proband and his/her family (“private” variant) or rare, but others, such as the KCNE1 p.D85N, can occur in approximately 0.8% of the general population. This suggests that some more common variants may have a known damaging effect, but they might not be sufficient in isolation to unveil an overt LQTS phenotype.

Therefore, currently the interpretation of genetic tests is subjected to the complexity of the molecular basis of LQTS, and, even more than before, a comprehensive clinical approach along with pre- and post-genetic counseling is necessary to assess each variant effect and weight in the patient phenotype, as well as which variant(s) to be tested for in the proband’s first-degree relatives to identify at-risk family members.

Copyright © 2015 Elsevier Inc. All rights reserved.

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