Hong Kong Med J 2018;24:Epub 2 Mar 2018
Genetic basis of channelopathies and cardiomyopathies in Hong Kong Chinese patients: a 10-year regional laboratory experience
Chloe M Mak1; Sammy PL Chen2; NS Mok3; WK Siu2; Hencher HC Lee2; CK Ching2; PT Tsui3; NC Fong4; YP Yuen2; WT Poon2; CY Law2; YK Chong2; YW Chan2; TC Yung5; Katherine YY Fan6; CW Lam7
1 Chemical Pathology Laboratory, Kowloon West Cluster Laboratory Genetic Service, Department of Pathology, Princess Margaret Hospital, Laichikok, Hong Kong
2 Department of Pathology, Princess Margaret Hospital, Laichikok, Hong Kong
3 Department of Medicine, Princess Margaret Hospital, Laichikok, Hong Kong
4 Department of Paediatrics and Adolescent Medicine, Princess Margaret Hospital, Laichikok, Hong Kong
5 Department of Paediatric Cardiology, Queen Mary Hospital, Pokfulam, Hong Kong
6 Department of Cardiac Medicine, Grantham Hospital, Wong Chuk Hang, Hong Kong
7 Department of Pathology, The University of Hong Kong, Pokfulam, Hong Kong
Corresponding author: Dr Chloe M Mak (firstname.lastname@example.org)
Introduction: Hereditary channelopathies and cardiomyopathies are potentially lethal and are clinically and genetically heterogeneous, involving at least 90 genes. Genetic testing can provide an accurate diagnosis, guide treatment, and enable cascade screening. The genetic basis among the Hong Kong Chinese population is largely unknown. We aimed to report on 28 unrelated patients with positive genetic findings detected from January 2006 to December 2015.
Methods: Sanger sequencing was performed for 28 unrelated patients with a clinical diagnosis of channelopathies or cardiomyopathies, testing for the following genes: KCNQ1, KCNH2, KCNE1, KCNE2, and SCN5A, for long QT syndrome; SCN5A for Brugada syndrome; RYR2 for catecholaminergic polymorphic ventricular tachycardia; MYH7 and MYBPC3 for hypertrophic cardiomyopathy; LMNA for dilated cardiomyopathy; and PKP2 and DSP for arrhythmogenic right ventricular dysplasia/cardiomyopathy.
Results: There were 17 males and 11 females; their mean age at diagnosis was 39 years (range, 1-80 years). The major clinical presentations included syncope, palpitations, and abnormal electrocardiography findings. A family history was present in 13 (46%) patients. There were 26 different heterozygous mutations detected, of which six were novel—two in SCN5A (NM_198056.2:c.429del and c.2024-11T>A), two in MYBPC3 (NM_000256.3:c.906-22G>A and c.2105_2106del), and two in LMNA (NM_170707.3:c.73C>A and c.1209_1213dup).
Conclusions: We have characterised the genetic heterogeneity in channelopathies and cardiomyopathies among Hong Kong Chinese patients in a 10-year case series. Correct interpretation of genetic findings is difficult and requires expertise and experience. Caution regarding issues of non-penetrance, variable expressivity, phenotype-genotype correlation, susceptibility risk, and digenic inheritance is necessary for genetic counselling and cascade screening.
New knowledge added by this study
- We characterised the genetic heterogeneity in channelopathies and cardiomyopathies among Hong Kong Chinese patients and described 26 mutations with six novel variants.
- This is the first case series of cardiac genetics in Hong Kong.
- This study provides genetic information for variant interpretation and insight into the clinical application of genetic testing for channelopathies and cardiomyopathies.
Cardiac genetics is evolving rapidly and many new insights have recently been achieved. Genetic causes are found in various potentially lethal channelopathies and cardiomyopathies including long and short QT syndrome (LQTS and SQTS), Brugada syndrome, catecholaminergic polymorphic ventricular tachycardia (CPVT), hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy (DCM), arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVD/C), Barth syndrome, and left ventricular non-compaction.1 Knowledge of genetics deepens the understanding of pathophysiology and remarkably changes the diagnosis, treatment, and genetic counselling for recurrence risk and family planning. This group is highly genetically heterogeneous (Table 12).
Table 1. Common channelopathy- and cardiomyopathy-associated genes2
The genetic basis of inherited cardiac diseases in the Hong Kong Chinese population is largely unknown. The Princess Margaret Hospital provides a comprehensive cardiac genetic service. We conducted this study to review the clinical and genetic findings of 28 unrelated positive cases encountered between January 2006 and December 2015.
Diagnosis of the cardiac conditions was based on clinical assessments by a cardiologist and practice guidelines.3 4 5 The patients were referred by cardiologists from various public hospitals for genetic analysis. Only patients with positive genetic findings are reported in this study. There were seven patients with LQTS, two with Brugada syndrome, two with CPVT, nine with HCM, four with DCM, and four with ARVD/C. Local ethics board approval was obtained. Peripheral blood samples were collected from the proband after informed consent was obtained. Genomic DNA was extracted using a QIAamp Blood Kit (Qiagen, Hilden, Germany). The coding exons and the flanking introns (10 bp) of each gene were amplified by polymerase chain reaction. The primer sequences and protocol are available on request. Sanger sequencing was performed in the following order and stopped once a positive finding was detected: KCNQ1, KCNH2, KCNE1, KCNE2, and SCN5A for LQTS; SCN5A for Brugada syndrome; RYR2 for CPVT; MYH7 and MYBPC3 for HCM; LMNA for DCM; and PKP2 and DSP for ARVD/C. The order was based on prevalence according to the literature and local experience. All coding exons were amplified for each gene except selected exons 3, 8, 14, 45, 46, 47, 49, 88, 89, 90, 93, 96, 97, 100, 101, and 103 for RYR2.6 The GenBank accession numbers are shown in Table 2. The pathogenicity of novel missense variants was analysed by Alamut Visual (Interactive Biosoftware, Rouen, France) with Polymorphism Phenotyping v2 (PolyPhen-2), Sorting Intolerant from Tolerant (SIFT), MutationTaster, and Assessing Pathogenicity Probability in Arrhythmia by Integrating Statistical Evidence (APPRAISE, https://cardiodb.org/APPRAISE/) and that of novel splicing variants by Splice Site Finder-like, MaxEntScan, NNSPLIC, GeneSplicer, and Human Splicing Finder, wherever appropriate. Splicing variants were considered to be damaging if there was a >10% lower score when compared with the wild-type prediction. Allele frequencies among populations were referred to the Exome Aggregation Consortium (ExAC; http://exac.broadinstitute.org/).
Over the 10-year period, more than 90 patients with channelopathies or cardiomyopathies were referred for genetic analysis. Among them, 28 unrelated patients had positive genetic results, comprising 17 males and 11 females. Their mean age at diagnosis was 39 years (range, 1-80 years). The major clinical presentations included syncope, palpitations, and abnormal electrocardiography (ECG) findings. Four patients were asymptomatic and were diagnosed following an incidental abnormal finding related to other medical issues. A family history was present in only 13 (46%) patients. All detected mutations were heterozygous, and 26 different heterozygous mutations were detected. These encompassed 11 missense, two nonsense, and five splicing mutations, as well as eight small insertions and deletions. There were six novel mutations—two in SCN5A (NM_198056.2:c.429del and c.2024-11T>A), two in MYBPC3 (NM_000256.3:c.906-22G>A and c.2105_2106del), and two in LMNA (NM_170707.3:c.73C>A and c.1209_1213dup) [Table 3]. All were considered pathogenic or likely pathogenic according to the Practice Guidelines for the Evaluation of Pathogenicity and the Reporting of Sequence Variants in Clinical Molecular Genetics by the Association for Clinical Genetic Science.7 Further clinical details and genotypes are shown in Table 3.
Table 3. Clinical and genetic findings of 28 Chinese patients with channelopathies and cardiomyopathies
There were seven patients with LQTS, two with Brugada syndrome, two with CPVT, nine with HCM, four with DCM, and four with ARVD/C. Three patients with LQTS had mutations in KCNQ1 (cases 1-3) and four had mutations in KCNH2 (cases 4-7). Two patients (cases 8 and 9) with Brugada syndrome had mutations in SCN5A, including two novel mutations. Two patients (cases 10 and 11) with CPVT had mutations in RYR2. Four patients with HCM (cases 12-15) had MYH7 mutations and five (cases 16-20) had MYBPC3 mutations, including two novel mutations. Four patients with DCM (cases 21-24) had LMNA mutations, including two novel mutations. Finally, three patients with ARVD/C had PKP2 mutations (cases 25-27) and one had a DSP mutation (case 28).
This is the first report of a cardiac genetic case series among Hong Kong Chinese patients with channelopathies and cardiomyopathies. A total of 28 patients are reported, and 26 different mutations and six novel mutations have been identified. Wide genetic diversity is observed, with no common mutation found. Hereditary channelopathies and cardiomyopathies are mainly inherited in an autosomal dominant manner. Mutations can be either inherited or de novo. Risk to proband sibling(s) and first-degree relatives depends on the genetic status of the parents. Offspring of the proband have a 50% risk of inheriting the mutation. Siblings of the proband have the same risk if the mutation is transmitted from either parent. Patients carrying a mutation of these sudden arrhythmia death syndromes show incomplete penetrance. In general, a mutation carrier will show symptoms/signs in 80% of those with CPVT, 20% to 50% of ARVD/C patients, 18% to 63% of LQTS patients, 80% to 94% of SQTS patients, and 80% of patients with Brugada syndrome who have abnormal ECG findings when challenged with a sodium channel blocker.8 No exact figure is available for HCM. The data could be more specific if a particular mutation was considered alongside clinical findings and family history. Pre-symptomatic testing of at-risk family members cannot be used to predict age of onset, severity, type of symptoms, or rate of progression. Detailed clinical, ECG, and genetic characterisation of affected and unaffected family members is helpful.
Long QT syndrome
Long QT syndrome is genetically heterogeneous, with at least 12 genes involved. Mutations in the four genes, KCNQ1, KCNH2, KCNE1, and KCNE2, are detected in 46%, 38%, 2%, and 1% of affected patients, respectively.8 A small proportion of patients (3%) have double heterozygous mutations in more than one disease loci.9 Specific arrhythmogenic triggers are associated with a particular subtype, such as exertion, swimming, and near-drowning for LQT1; auditory triggers and cardiac events occurring in the postpartum period for LQT2; and cardiac events during sleep or at rest for LQT3. Three patients had KCNQ1 mutations. Case 1 had recurrent syncope induced by exercise and swimming, but genetic testing confirmed LQTS type 1. Other patients had no specific provoking factor. LQTS type 2 caused by KCNH2 mutations accounts for about 38% of all LQTS.8 Four patients (cases 4-7) carried KCNH2 mutations and two (cases 4 and 6) presented with Torsades de pointes and one (case 7) had survived cardiac arrest requiring an implantable cardioverter defibrillator. Case 6 was the youngest patient, presenting at the age of 1 year. Genotype-guided treatment in LQTS is recommended and LQT1 responds best to beta-blockers.10 11
Brugada syndrome is characterised by cardiac conduction abnormalities (ST-segment abnormalities in leads V1-V3 on ECG and a high risk for ventricular arrhythmias) that can result in sudden death. The Shanghai Score System has been recently published for the diagnosis of Brugada syndrome.12 13 The prevalence of Brugada syndrome or its characteristic ECG pattern is reportedly higher among Asians, such as Japanese (0.14%-1.22%).14 15 16 17
Brugada syndrome is genetically heterogeneous and can be attributed to defects in at least 23 genes at the time of reporting.8 Mutations in SCN5A are detected in 11% to 14% of affected individuals in Japan and <10% in Taiwan where mutations in CACNA1C account for 1% to 7%.18 Approximately 65% to 70% of patients remain genetically undiagnosed. Expressivity is variable and penetrance is incomplete and low.
Conventionally, Brugada syndrome has been described as a monogenic disease that has autosomal dominant inheritance with incomplete penetrance; it is caused by rare genetic variants with a large effect size. Most individuals diagnosed with Brugada syndrome have an affected parent. The proportion of cases caused by a de-novo mutation is approximately 1%. Recent studies indicate that genetic inheritance is likely more complex, and models of an oligogenic disorder or susceptibility risk/genetic predisposition have been suggested.19 20 21 22
Among the two patients in this series, none had a positive family history. Symptoms were more non-specific, such as palpitation and syncope. It is noteworthy that convulsion can be a presentation of channelopathies (case 8). Clinical suspicion should be higher with more specific investigations, such as exercise-stress ECG and flecainide challenge tests, are required in order to reveal the real culprit. Sudden cardiac death can be the first presenting symptom in Brugada syndrome.
Two novel mutations are described in SCN5A: c.429del and c.2024-11T>A. The former is predicted to cause a frameshift and premature protein truncation. The latter is predicted to abolish the acceptor splice site and create a cryptic site upstream. At the time of reporting, both are absent from controls in the Exome Sequencing Project, 1000 Genomes Project, and ExAC. SCN5A mutations can cause either LQTS or Brugada syndrome.
Catecholaminergic polymorphic ventricular tachycardia
Catecholaminergic polymorphic ventricular tachycardia can present with syncope and sudden death during physical exertion or emotion, due to catecholamine-induced bidirectional ventricular tachycardia, polymorphic ventricular tachycardia or ventricular fibrillation. The reported mean age of onset is between 7 and 12 years.8 Exercise stress testing or an adrenaline provocation test may induce ventricular arrhythmia and enable a clinical diagnosis. About half of these cases are related to a dominantly inherited RYR2 gene mutation, with a small proportion (1%-2%) related to recessively inherited CASQ2 gene mutations. RYR2 is a large gene with 105 exons. Tier testing has been proposed by Medeiros-Domingo et al.6 First-tier RYR2 genetic testing of the 16 selected exons allows identification of about 65% of CPVT cases. There were two paediatric CPVT patients (cases 10 and 11) in our series, with two known disease-causing mutations detected, namely NM_001035.2(RYR2):c.11836G>A (p.Gly3946Ser)23 24 25 26 and c.14848G>A (p.Glu4950Lys).23 24 Both mutations were detected in first-tier screening.
Hypertrophic cardiomyopathy is the most prevalent hereditary cardiac disease, causing about one third of sudden cardiac deaths in young athletes. Its prevalence in China is approximately 1 in 1250.27 The clinical manifestations are markedly variable, ranging from asymptomatic to sudden cardiac death. Genetic testing provides an accurate diagnosis in the probands and enables screening of asymptomatic family members. Although the genetic background of HCM is heterogeneous, involving at least 30 genes, MYH7 and MYBPC3 are the most common and each accounts for approximately 40%.8
Nine patients with HCM are reported here: four had known MYH7 mutations and five had MYBPC3 mutations, including two novel mutations. NM_000256.3:c.906-22G>A was detected in case 16 and was a novel variant. Neither population frequency nor known pathogenicity have been reported. In-silico analysis showed creation of a novel acceptor site and insertion of 20 nucleotides into exon 10. This conceivably would lead to a frameshift and premature protein termination. Exon 10 of MYBPC3 is a microexon in which the stability of its original splicing site is easily disrupted by intronic variants. A similar mutation has been reported as c.906-36G>A.28 Nonetheless, cDNA analysis was not performed. NM_000256.3(MYBPC3):c.1223+1G>A at the critical canonical +1 splice site is also novel. In addition, other known disease-causing splicing mutations affecting the same nucleotide have been reported.26 29 30 Case 19 had two variants detected in MYBPC3 (c.2215G>A and c.3624del). The small deletion c.3624del is a mutation known to cause HCM in the Chinese population31 and predicted to cause a frameshift and premature termination of the protein. The missense variant c.2215G>A is as yet unreported and is predicted by in-silico analyses to cause an amino acid change from glutamate to lysine at codon 739 and probably damage. At the time of reporting, the variant is absent from controls in the Exome Sequencing Project, 1000 Genomes Project, and ExAC databases. This variant is considered to have uncertain significance. The mother of the patient in case 19 was available for testing. She was 48 years old at the time of genetic testing, asymptomatic, and heterozygous for c.3624del only. Hence, the two variants c.2215G>A and c.3624del of MYBPC3 were in-trans in the patient and elder brother of the patient in case 19. Both had a more severe form of HCM, with a younger onset.
Familial DCM is a group of genetically heterogeneous disorders. Laminopathy can manifest as several allelic disorders affecting muscle, nerve, adipose, and vascular tissues; one of them is cardiomyopathy, dilated 1A. We identified four patients with DCM, two of whom also had proximal muscle weakness. Two novel mutations in LMNA were detected (c.73C>A and c.1209_1213dup). NM_005572.3(LMNA):c.73C>A is a novel variant that is predicted to be deleterious by SIFT, probably causing damage according to PolyPhen-2 and disease-causing according to MutationTaster. Other missense mutations have been reported in the same amino acid codon.32 33 34 NM_005572.3(LMNA):c.1609-1G>A is predicted to significantly affect splicing by in-silico analysis. At the time of reporting, all variants are absent from controls in the Exome Sequencing Project, 1000 Genomes Project, and ExAC. In case 22 with NM_005572.3(LMNA):c.73C>A, one of the parents died of chronic heart failure in the fourth decade of life, and one sibling died of heart block and chronic heart failure with a diagnosis of muscular dystrophy at age 38 years. Nonetheless, there was no sample left for genotyping.
Arrhythmogenic right ventricular dysplasia/cardiomyopathy
Arrhythmogenic right ventricular dysplasia/cardiomyopathy is associated with fibrofatty replacement of cardiomyocytes, ventricular tachyarrhythmias, and sudden cardiac death. Although the right ventricle is primarily affected in this condition, left-dominant arrhythmogenic cardiomyopathy has also been described, and mutations have been identified in DSP as well as in other genes.35 Four patients are reported here, with three having mutations in PKP2 and one in DSP. Interestingly, the patient in case 26 presented at age 80 years with episodic palpitations. His ECG results showed paroxysmal ventricular tachycardia. He had a deletion in PKP2, c.1125_1132del (p.Phe376Alafs*8), resulting in a truncated incomplete protein product. Age of onset in patients with PKP2 mutations is older than that of the patient with DSP mutation. The latter patient (case 28) died at age 23 years, with sudden collapse as the first presentation.
Primary arrhythmogenic disorders including LQTS/SQTS, CPVT, Brugada syndrome, and cardiomyopathies account for about one third of sudden cardiac deaths in the young.36 Identification of a pathogenic variant can solve the diagnostic mystery, provide relief to the family, and enable family screening and counselling for other at-risk family members. In some developed countries, molecular autopsy is an essential part of a formal forensic investigation in unexplained sudden death.37 We support the implementation of molecular autopsy in routine autopsy investigation of sudden cardiac death victims. Our group has conducted the first local prospective study to determine the prevalence and types of sudden arrhythmia death syndrome underlying sudden cardiac death among local young victims through clinical and molecular autopsy of sudden cardiac death victims and clinical and genetic evaluation of their first-degree relatives (http://www.sadshk.org/en/medical_research.php). Such data can serve as the groundwork for the feasibility of implementation of such investigations in Hong Kong.
Genetic tests for cardiac conditions can aid diagnosis and guide treatment. Nonetheless, there are limitations that complicate the translational use of genetic results in patient care, such as incomplete penetrance, variable expressivity, and findings of variants of uncertain significance. In addition, since the genetic heterogeneity is large among cardiomyopathies and channelopathies and more genes are yet to be discovered, a negative genetic finding does not necessarily exclude a genetic basis of disease in patients.
Major limitations of the current study include its small sample size, incomplete family data for co-segregation study, and lack of functional study of novel variants. We observed a lower rate of use of genetic tests in early years that might have been due to insufficient awareness among clinicians about the clinical usefulness of such tests for channelopathies and cardiomyopathies. Clinical indications published in an expert consensus statement on the state of genetic testing for channelopathies and cardiomyopathies from the Heart Rhythm Society and European Heart Rhythm Association provide a good reference to determine when a genetic test should be requested.5 In our hospital, referral information can be accessed on http://kwcpath.home/genetics/ and more information about genetic service provision in public hospitals is available in the Hong Kong Hospital Authority Genetic Test Formulary (http://gtf.home/). A comprehensive system of cardiac genetics service is required for an efficient referral system, resource funding, training, and appropriate long-term follow-up.
We present the phenotypic and genotypic characteristics of 28 unrelated Hong Kong Chinese patients diagnosed across a 10-year period. For each disease entity, it was beyond our reach in the last decade to exhaustively screen for all known genes. We therefore focus on the most common ones when investigating cardiac genetics. Even so, genetic analysis can provide an accurate diagnosis and is of utmost importance for the management of patients and their families. It also allows for genetic counselling on the recurrence risk. Nonetheless, non-penetrance, variable expressivity, phenotype-genotype correlation, susceptibility risk, and digenic inheritance have been reported. Correct interpretation of genetic findings for careful genetic counselling requires professional expertise with relevant experience in both clinical medicine and molecular genetics. With the advent of next-generation sequencing, the diagnostic performance in this genetically heterogeneous group of channelopathies and cardiomyopathies will improve. Application of next-generation sequencing would be the mainstay diagnostic tool.
All authors have disclosed no conflicts of interest.
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