Article ID: CJ-21-0077
Background: Dilated cardiomyopathy (DCM) is an important cause of heart failure and cardiac transplantation. This study determined the prevalence of DCM-associated genes and evaluated the genotype-phenotype correlation in Vietnamese patients.
Methods and Results: This study analyzed 58 genes from 230 patients. The study cohort consisted of 64.3% men; age at diagnosis 47.9±13.7 years; familial (10.9%) and sporadic DCM (82.2%). The diagnostic yield was 23.5%, 44.0% in familial and 19.6% in sporadic DCM. TTN truncating variants (TTNtv) were predominant (46.4%), followed by TPM1, DSP, LMNA, MYBPC3, MYH6, MYH7, DES, TNNT2, ACTC1, ACTN2, BAG3, DMD, FKTN, PLN, TBX5, RBM20, TCAP (2–6%). Familial DCM, genotype-positive and TTNtv-positive patients were younger than those with genotype-negative and sporadic DCM. Genotype-positive patients displayed a decreased systolic blood pressure and left ventricular wall thickness compared to genotype-negative patients. Genotype-positive patients, particularly those with TTNtv, had a family history of DCM, higher left atrial volume index and body mass index, and lower right ventricle-fractional area change than genotype-negative patients. Genotype-positive patients reached the combined outcomes more frequently and at a younger age than genotype-negative patients. Major cardiac events occurred more frequently in patients positive with genes other than TTNtv.
Conclusions: The study findings provided an overview of Vietnamese DCM patients’ genetic profile and suggested that management of environmental factors may be beneficial for DCM patients.
Dilated cardiomyopathy (DCM) was characterized by left or biventricular dilatation and systolic dysfunction in the absence of secondary causes such as coronary artery disease.1 With a prevalence of 1 in 250 in the population, DCM is a common cause of heart failure and the leading indication for cardiac transplantation.2 DCM can be attributed to genetic and non-genetic causes, with approximately 40% of DCM cases having a genetic cause.3 Rare variants in multiple genes encoding cardiac sarcomeric, cytoskeletal, desmosomal, nuclear lamina, mitochondrial and ion flux-handling proteins have been linked to disease manifestations.4 The relationship between mutations in DCM-related genes and abnormalities of cardiac morphology and functions were investigated mostly in Western populations; data from Asian countries were scarce. With next-generation sequencing, multiple genes can be analyzed simultaneously, making genetic testing a powerful tool for disease management as for hypertrophic cardiomyopathy (HCM). However, the clinical utility of DCM genetic testing still needs to be established. Furthermore, a focused panel comprising the most prevalent DCM-associated genes in the Vietnamese population would enable a cost-effective DCM genetic testing program in Vietnam.
Editorial p ????
Our study aimed to determine the prevalence of rare variants from 58 DCM-related genes in 230 well-phenotyped DCM Vietnamese patients, and to analyze genotype-phenotype correlations in the study cohort.
A total of 230 unrelated patients admitted to the Heart Institute and Tam Duc Heart Hospital between September 2019 and August 2020 were enrolled in this study. All patients provided informed written consent and received genetic counseling prior to genetic testing. The study was approved by ethics committees of the 2 participating hospitals according to local regulations and followed the Declaration of Helsinki on human experimentation.
Diagnosis of DCM was issued based on left ventricular end-diastolic (LVED) volumes or diameters >2 SD from normal according to normograms (Z scores >2 SD) corrected by body surface area (BSA) and age, or BSA and gender and left ventricular ejection fraction (LVEF) ≤50%, not explained by abnormal loading conditions or coronary artery disease, valvular heart diseases, congenital heart lesions, and other systemic diseases.1 Before enrollment in the study, all patients were subjected to a physical examination, chest radiography, electrocardiography (ECG), echocardiography, 24-h ambulatory ECG monitoring, coronary artery angiography or coronary multislice computed tomography angiography. Patient information included family and personal history of DCM, and family history of sudden cardiac death (SCD). Familial DCM was assigned when confirmed disease and confirmed or probable disease was observed in the proband and in at least one relative.5
Targeted Next-Generation Sequencing and Bioinformatics Data ProcessingWe used the Illumina TruSight Cardio panel and selected 58 genes (Supplementary Table 1) either with ≥1 variant reported as pathogenic (P) for DCM in the Human Gene Mutation Database or having a DCM phenotype number in online mendelian inheritance in man (OMIM) for inclusion in the analysis.
DNA was extracted from peripheral blood using a QIAamp DNA Blood Mini Kit (Qiagen). Target enrichment was performed with the TruSight Rapid Capture kit (Illumina). Captured libraries were sequenced with 2 × 150 bp reads on a MiSeq/Miniseq platform (Illumina). Sequence reads were mapped onto the human reference genome, hg38, using the burrows-wheeler alignment (BWA) tool.6 The Genome Analysis ToolKit was used for variants (single nucleotide polymorphisms (SNPs) and indels) calling.7 Analytic validation of the gene panel used has been submitted elsewhere with a sensitivity of 100.0% for SNPs and 75.0% for indels; a specificity of 100.0% and 83.3% for SNPs and indels, respectively. Identified variants were annotated using Annotate variation (ANNOVAR).8
Variant InterpretationVariants with at least 20 × coverage were analyzed using Alamut Visual (Interactive Biosoftware) and interpreted according to recent the American college of medical genetics and genomics (ACMG) guidelines.9 Synonymous variants, intronic variants outside of the flanking regions, and variants with a minor allelic frequency (MAF) ≥0.1% in the Genome Aggregation (gnomAD) databases were excluded.10 The 1,000 Genomes database, including data from 99 Vietnamese subjects, was used to check for the presence of all P and likely pathogenic (LP) variants identified.11 For disease-specific refinement of the ACMG guidelines, we adopted CardioClassifier, a disease- and gene-specific computational decision support tool, which defines more specific thresholds for inherited cardiac disorders. According to CardioClassifier, the maximum credible population allele frequency for any DCM causative variant was set at 0.0056%; therefore, in this study, variants with a frequency less and greater than 0.0056% were categorized as PM2 and BS1, respectively.12 Various in silico prediction programs, including SIFT, PolyPhen-2, AlignGVGD, MutationTaster, Mutation Assessor, CADD, and REVEL were used to analyze missense variants.13,14 The analysis of intronic changes was performed with MaxEntScan, and Splice Site Finder-like;15 and GERP++ was used to explore nucleotide-specific estimates of evolutionary constraint.16 TTN missense variants were classified as benign variants.17 All detected P and LP variants were confirmed by Sanger sequencing. Patients harboring P/LP variants were classified as genotype positive. Non-carriers of P/LP variants were considered genotype negative.
Statistical AnalysisNormally distributed, continuous variables were expressed as mean±SD and non-parametric as median (interquartile range). Categorical variables were depicted using numbers (proportions). Independent sample’s t-test combined with Levene’s tests was used for comparison between the groups for all continuous variables, and Mann-Whitney U-tests were used for non-parametric variables. Categorical variables were compared using the chi-squared test or Fisher’s exact test. The Cox proportional hazard model was used for event-free survival analyses by comparing patients with and without mutation, TTNtv mutation and no mutation, and TTNtv mutation and mutation in other genes. Event-free survival was adjusted for gender, hypertension, and arrhythmia. The events used included death from any cause, heart transplantation, non-fatal stroke, life-threatening arrhythmia requiring implantable cardioverter-defibrillator (ICD) implant. Two-sided probability values were considered significant at P<0.05. All statistical analyses were performed with SPSS version 25.0 (SPSS Inc., Chicago, IL, USA).
Our study cohort consisted of 230 unrelated DCM patients of Vietnamese origin; 148 males (64.3%), mean age 51.3±14.0 years, mean age at diagnosis 47.9±13.7 years; 25 with familial DCM (10.9%) and 189 with sporadic DCM (82.2%). The mean LVEF and LV ED diameter index (LVEDDi) were 28.3±7.5 and 40.6±5.2, respectively. Patients characteristics stratified by genetic status are summarized in Table 1.
Characteristics | All (n=230) |
Mutation- positive (n=54) |
No mutation (n=176) |
P valuea | TTNtv- positive (n=26) |
P valueb |
Other gene- positive (n=28) |
P valuec | Familial DCM (n=25) |
Sporadic DCM (n=189) |
P valued |
---|---|---|---|---|---|---|---|---|---|---|---|
Patient age, years | 51.3±14.0 | 47.0±12.7 | 52.6±14.1 | 0.010* | 48.2±14.0 | 0.135 | 46.0±11.5 | 0.539 | 45.6±13.3 | 52.2±14.1 | 0.028* |
Age of diagnosis, years | 47.8±13.7 | 43.0±11.4 | 49.3±14.0 | 0.003* | 43.1±12.8 | 0.033* | 42.9±10.3 | 0.963 | 41.7±12.5 | 48.7±13.8 | 0.017* |
BMI, kg/m2 | 23.9±4.3 | 24.6±4.1 | 23.6±4.4 | 0.042* | 25.8±4.1 | 0.007* | 23.5±3.8 | 0.051 | 23.8±2.9 | 23.8±4.4 | 0.591 |
23.0 (5.4) | 24.4 (4.8) | 22.8 (5.1) | 25.7 (5.3) | 23.2 (4.5) | 24.0 (5.3) | 22.9 (5.3) | |||||
SBP, mmHg | 116.8±17.1 | 111.5±15.5 | 118.4±17.2 | 0.009* | 113.7±16.3 | 0.194 | 109.4±14.7 | 0.313 | 115.6±16.9 | 116.7±17.4 | 0.771 |
NYHA class at onset | 2.1±0.4 | 2.1±0.5 | 2.1±0.4 | 0.315 | 2.1±0.6 | 0.645 | 2.2±0.4 | 0.642 | 2.1±0.4 | 2.1±0.4 | 0.817 |
LVEF, % | 28.2±7.3 | 27.2±7.1 | 28.5±7.4 | 0.234 | 27.1±7.3 | 0.368 | 27.2±7.0 | 0.969 | 27.1±6.1 | 28.2±7.5 | 0.501 |
LV maximal wall thickness, mm | 10.5±2.1 | 10.0±1.9 | 10.6±2.2 | 0.046* | 10.0±1.8 | 0.138 | 10.0±2.0 | 0.972 | 9.7±1.4 | 10.5±2.1 | 0.024* |
LVEDD index, mm/m2 | 40.6±5.2 | 39.6±4.6 | 40.9±5.4 | 0.122 | 38.8±3.3 | 0.008* | 40.4±5.6 | 0.207 | 39.9±4.4 | 40.9±5.0 | 0.365 |
LAV index, mL/m2 | 49.0±28.2 | 55.7±21.1 | 47.0±29.8 | 0.001* | 58.4±19.4 | 0.001* | 53.4±22.7 | 0.322 | 42.2±21.2 | 49.5±29.5 | 0.181 |
39.0 (29.0) | 58.0 (38.0) | 38.0 (21.0) | 59.0 (33.3) | 45.0 (42.0) | 36.5 (22.7) | 39.0 (29.5) | |||||
RV-TAPSE, mm | 18.5±4.1 | 17.7±5.0 | 18.7±3.8 | 0.134 | 18.6±6.1 | 0.894 | 16.8±3.3 | 0.218 | 17.4±3.7 | 18.6±4.1 | 0.189 |
RV-FAC, % | 35.3±11.9 | 30.8±13.1 | 36.7±11.2 | 0.005* | 31.9±12.8 | 0.052 | 29.7±13.6 | 0.560 | 32.2±10.7 | 35.7±12.1 | 0.218 |
Patient gender, male | 148 (64.3) | 40 (74.1) | 108 (61.4) | 0.105 | 23 (88.5) | 0.007* | 17 (60.7) | 0.030* | 18 (72.0) | 118 (62.4) | 0.387 |
Family history of SD | 27 (11.7) | 11 (20.4) | 16 (9.1) | 0.134 | 6 (23.1) | 0.188 | 5 (17.9) | 0.741 | 11 (44.0) | 12 (6.3) | <0.001* |
Family history of DCM | 25 (10.9) | 11 (20.4) | 14 (8.0) | 0.024* | 6 (23.1) | 0.003* | 5 (17.9) | 0.246 | – | – | – |
Hypertension | 55 (23.9) | 8 (14.8) | 47 (26.7) | 0.136 | 4 (15.4) | 0.421 | 4 (14.3) | 1.000 | 6 (24.0) | 46 (24.3) | 0.934 |
Palpitation | 67 (29.1) | 19 (35.2) | 48 (27.3) | 0.095 | 10 (38.5) | 0.014* | 9 (32.1) | 0.484 | 8 (32.0) | 53 (28.0) | 0.865 |
Dyspnea | 222 (96.5) | 52 (96.3) | 170 (96.6) | 0.166 | 25 (96.2) | 0.022* | 27 (96.4) | 0.367 | 25 (100) | 182 (96.3) | 0.620 |
Syncope | 9 (3.9) | 3 (5.6) | 6 (3.4) | 0.148 | 1 (3.8) | 0.033* | 2 (7.1) | 0.511 | 1 (4.0) | 7 (3.7) | 0.934 |
Left bundle branch block | 134 (58.3) | 31 (57.4) | 103 (58.5) | 0.714 | 18 (69.2) | 0.533 | 13 (46.4) | 0.107 | 9 (36.0) | 115 (60.8) | 0.045* |
Atrial fibrillation | 19 (8.3) | 8 (14.8) | 11 (6.3) | 0.104 | 5 (19.2) | 0.065 | 3 (10.7) | 0.460 | 1 (4.0) | 16 (8.5) | 0.640 |
Arrhythmia | 58 (25.2) | 19 (35.2) | 39 (22.2) | 0.123 | 8 (30.8) | 0.553 | 11 (39.3) | 0.577 | 3 (12.0) | 52 (27.5) | 0.206 |
Mutation | – | – | – | – | – | – | – | – | 11 (44.0) | 37 (19.6) | 0.010* |
Data are presented as mean±SD (normal distribution) or median (interquartile range) (non‐parametric distribution) or n (%). *Statistically significant difference between compared groups. aMutation-positive and no-mutation. bTTNtv-positive and no-mutation. cTTNtv-positive and other gene-positive. dFamilial DCM and sporadic DCM. BMI, body mass index; DCM, dilated cardiomyopathy; FAC, fractional area change; LAV, Left atrial volume; LV, left ventricular; LVEDD, left ventricular end diastolic diameter; LVEF, left ventricular ejection fraction; NYHA, New York Heart Association functional class; RV, right ventricle; RV-FAC, right ventricle-fractional area change; SBP, systolic blood pressure; SD, sudden death; TAPSE, tricuspid annular plane systolic excursion; –, no information.
The analysis of 58 DCM-related genes in 230 patients revealed a total of 56 variants classified as P/LP in 54 patients. The overall diagnostic yield was 23.5%; higher in familial than in sporadic DCM (44.0% vs. 19.6%). Among P variants identified, 17 were missense, 13 were nonsense, 7 were splice-site, 16 were frameshift indels and 3 were in-frame indels. Thirty (53.6%) disease-causing variants were known variants and 26 (46.4%) were novel. Among 56 P variants identified in this study, 4 were previously described in HCM Vietnamese patients. All DCM-associated variants are listed in Table 2.
Gene | Transcript | Nucleotide change |
Coding effect | Zygosity | MAF (%) gnomAD, ALL |
MAF (%) gnomAD, EAS |
MAF (%) 1,000G, KHV |
ACMG class |
Patient no. |
ACMG criteria | References |
---|---|---|---|---|---|---|---|---|---|---|---|
ACTC1 | NM_005159.4 | c.941G>A | p.Arg314His | Het | 0.0014 | 0 | NA | LP | 169 | PS3 PS4 PM2 PP3 PP5 | Olson et al (1998)51 |
BAG3 | NM_004281.3 | c.974delC | p.Pro325Glnfs*17 | Het | 0.0000 | 0 | NA | P | 004 | PVS1 PM2 PM5 PP3 PP5 | – |
DES | NM_001927.3 | c.83C>T | p.(Ser28Phe) | Het | 0.0000 | 0 | NA | LP | 129 | PM1 PM2 PP2 PP3 | – |
DES | NM_001927.3 | c.193G>A | p.(Gly65Ser) | Het | 0.0025 | 0 | 0 | LP | 197 | PS4-M PM1 PM2 PP2 BP4 | Walsh et al (2017)52 |
DMD | NM_004006.2 | c.31+1G>T | – | Het | 0.0000 | 0 | NA | P | 161 | PVS1 PS4-M PM2 PP3 PP5 |
Milasin et al (1996)53; Feng et al (2002)54; Cho et al (2017)55 |
DSP | NM_004415.2 | c.748C>T | p.(Gln250*) | Het | 0.0000 | 0 | NA | P | 085 | PVS1 PM2 PP3 | – |
DSP | NM_004415.2 | c.2848dup | p.(Ile950Asnfs*3) | Het | 0.0000 | 0 | NA | P | 160 | PVS1 PS4-M PM2 PP3 PP5 |
Pugh et al (2014)56; Walsh et al (2017)52 |
DSP | NM_004415.2 | c.5428del | p.Gln1810ArgfsTer8 | Het | 0.0000 | 0 | NA | LP | 263 | PVS1 PM2 | – |
FKTN | NM_001351497.1 | c.538C>T | p.(Arg180*) | Het | 0.0008 | 0 | NA | P | 147 | PVS1 PS4 PM2 PP3 PP5 | Lévesque et al (2016)57 |
FKTN | NM_001351497.1 | c.-2-2A>G | – | Het | 0.0086 | 0.0099 | 0 | P | 147 | PVS1 PM2 PM3 PP3 | – |
LAMA2 | NM_000426.3 | c.7525_7528dup | p.(Ser2510Thrfs*3) | Het | 0.0000 | 0 | NA | LP | 182 | PM1 PM2 PM4 | – |
LAMP2 | NM_002294.2 | c.139C>T | (p.Gln47Ter) | Het | 0.0000 | 0 | NA | P | 016 | PVS1 PM1 PM2 PP3 | – |
LAMP2 | NM_002294.2 | c.35_52del | p.(Ser12_Val17del) | Het | 0.0000 | 0 | NA | LP | 082 | PVS1 PM2 | – |
LMNA | NM_170707.3 | c.566G>A | p.(Arg189Gln) | Het | 0.0032 | 0 | NA | LP | 087 | PM1 PM2 PM5 PP3 PP3 | – |
LMNA | NM_170707.3 | c.3G>A | – | Het | 0.0000 | 0 | NA | P | 143 | PVS1 PS4-M PM1 PM2 PP3 PP5 |
– |
MYBPC3 | NM_000256.3 | c.2308G>A | p.(Asp770Asn) | Het | 0.0016 | 0 | NA | LP | 035 | PVS1 PS4-M PM1 PM2 PP3 PP5 |
Van Driest et al (2004)58 |
MYBPC3 | NM_000256.3 | c.1504C>T | p.(Arg502Trp) | Het | 0.0046 | 0 | NA | LP | 149 | PM1 PM2 PM5 PP3 BP5 | Richard et al (2003)49 |
MYH6 | NM_002471.3 | c.2040del | p.(Lys681Argfs*5) | Het | 0.0000 | 0 | NA | LP | 037, 065 | PVS1 PM2 | – |
MYH7 | NM_000257.3 | c.4823G>A | p.(Arg1608His) | Het | 0.0008 | 0 | NA | LP | 036 | PM1 PM2 PM5 PP2 PP3 | – |
MYH7 | NM_000257.3 | c.2155C>T | p.(Arg719Trp) | Het | 0.0032 | 0 | NA | P | 116 | PS3 PM1 PM2 PM5 PP2 PP5 |
Tesson et al (1998)59 |
MYH7 | NM_000257.3 | c.2207T>C | p.(Ile736Thr) | Het | 0.0000 | 0 | NA | P | 149 | PS3 PM1 PM2 PP2 PP3 PP5 |
Minoche et al (2019)60 |
PLN | NM_002667.4 | c.40_42del | p.(Arg14del) | Het | 0.0007 | 0 | NA | P | 006 | PS3 PS4-M PM2 PM4 PP1 PP5 |
van der Zwaag et al (2012)61 |
RBM20 | NM_001134363.2 | c.1907G>A | p.(Arg636His) | Het | 0.0000 | 0 | NA | P | 200 | PS4 PM1 PM2 PM5 PP3 PP5 |
Brauch et al (2009)44 |
SDHA | NM_004168.3 | c.1352G>A | p.(Arg451His) | Het | 0.0004 | 0 | NA | LP | 215 | PS3 PM2 PM5 PP3 BP1 | Toledo et al (2017)62 |
SGCB | NM_000232.4 | c.1A>G | – | Het | 0.0000 | 0 | 0 | P | 131 | PVS1 PS4-M PM2 PP3 PP5 |
Semplicini et al (2015)63 |
TBX5 | NM_000192.3 | c.652C>G | p.Gln218Glu | Het | 0.0000 | 0 | NA | LP | 222 | PM1 PM2 PP2 PP3 | – |
TCAP | NM_003673.3 | c.472C>T | p.(Arg158Cys) | Het | 0.0000 | 0 | NA | LP | 081 | PM2 PM5 PP2 PP3 | Hirtle-Lewis et al (2013)64 |
TNNT2 | NM_001001430.2 | c.620_622del | p.(Lys210del) | Het | 0.0000 | 0 | NA | P | 043 | PS3 PS4 PM2 PP5 | Kamisago et al (2000)65 |
TNNT2 | NM_001001430.2 | c.518G>A | p.(Arg173Gln) | Het | 0.0000 | 0 | NA | P | 259 | PS4 PM2 PM5 PP1 PP2 PP3 |
Van Acker et al (2010)66; Lakdawala et al (2012)67; Fernlund et al (2017)68 |
TPM1 | NM_001018005.1 | c.644C>T | p.(Ser215Leu) | Het | 0.0004 | 0 | NA | P | 194 | PS3 PM1 PM2 PP2 PP3 PP5 |
Cecconi et al (2016)69 |
TPM1 | NM_001018005.1 | c.598G>C | p.(Val200Leu) | Het | 0.0000 | 0 | NA | LP | 208 | PM1 PM2 PP2 PP3 | – |
TPM1 | NM_001018005.1 | c.842T>C | p.(Met281Thr) | Het | 0.0004 | 0 | NA | LP | 271 | PS4-M PM2 PP2 PP3 | Van Driest et al (2003)48; Dorsch et al (2021)70 |
TTN | NM_001267550.2 | c.40688_40689insT | p.(Arg13565Lysfs*7) | Het | 0.0000 | 0 | NA | LP | 001 | PVS1 PM2 | – |
TTN | NM_001267550.2 | c.104974_104995dup | p.(Leu34999Glnfs*16) | Het | 0.0000 | 0 | NA | P | 009 | PVS1 PM2 PP3 | – |
TTN | NM_001267550.2 | c.13898_13899del | p.(Lys4633Argfs*7) | Het | 0.0000 | 0 | NA | LP | 013 | PVS1 PM2 | – |
TTN | NM_001267550.2 | c.49669A>T | p.(Lys16557*) | Het | 0.0000 | 0 | NA | P | 025 | PVS1 PM2 PP3 | – |
TTN | NM_001267550.2 | c.68302A>T | p.(Lys22768*) | Het | 0.0000 | 0 | NA | P | 026 | PVS1 PM2 PP3 | – |
TTN | NM_001267550.2 | c.86116C>T | p.(Arg28706*) | Het | 0.0004 | 0 | NA | P | 040 | PVS1 PS3 PM2 PP3 PP5 | – |
TTN | NM_001267550.2 | c.71706del | p.(Ile23902Metfs*33) | Het | 0.0000 | 0 | NA | LP | 055 | PVS1 PM2 | – |
TTN | NM_001267550.2 | c.42205C>T | p.(Arg14069*) | Het | 0.0000 | 0 | NA | P | 057 | PVS1 PM2 PP3 | – |
TTN | NM_001267550.2 | c.92683C>T | p.Arg30895Ter | Het | 0.0004 | 0 | NA | P | 066 | PVS1 PM2 PP3 | Roberts et al (2015)37 |
TTN | NM_001267550.2 | c.59926+1G>A | – | Het | 0.0004 | 0 | NA | P | 088 | PVS1 PS4 PM2 PP3 PP5 | Herman et al (2012)71 |
TTN | NM_001267550.2 | c.52307_52310dup | p.(Glu17437Aspfs*2) | Het | 0.0000 | 0 | NA | P | 107 | PVS1 PM2 PP5 | – |
TTN | NM_001267550.2 | c.94754T>G | p.(Leu31585*) | Het | 0.0000 | 0 | NA | P | 109 | PVS1 PM2 PP3 | – |
TTN | NM_001267550.2 | c.54809del | p.(Ile18270Asnfs*22) | Het | 0.0000 | 0 | NA | P | 141 | PVS1 PM2 PP3 | – |
TTN | NM_001267550.2 | c.41608+1G>T | – | Het | 0.0000 | 0 | NA | P | 144 | PVS1 PM2 PP3 | – |
TTN | NM_001267550.2 | c.59460G>A | p.(Trp19820*) | Het | 0.0032 | 0 | NA | P | 145 | PVS1 PM2 PP3 PP5 | – |
TTN | NM_001267550.2 | c.3073dup | p.(Ser1025Lysfs*15) | Het | 0.0000 | 0 | NA | P | 166 | PVS1 PM2 PP3 | – |
TTN | NM_001267550.2 | c.58240_58244del | p.(Pro19414Alafs*2) | Het | 0.0000 | 0 | NA | P | 177 | PVS1 PM2 PP3 | – |
TTN | NM_001267550.2 | c.47692C>T | p.(Arg15898*) | Het | 0.0005 | 0 | NA | P | 190 | PVS1 PS4-M PM2 PP3 PP5 |
Roberts et al (2015)37 |
TTN | NM_001267550.2 | c.87459_87460dup | p.(Ser29154Lysfs*3) | Het | 0.0000 | 0 | NA | LP | 207 | PVS1 PM2 | – |
TTN | NM_001267550.2 | c.67637-1G>C | – | Het | 0.0000 | 0 | NA | P | 002, 140 | PVS1 PM2 PP3 PP5 | – |
TTN | NM_001267550.2 | c.59535del | p.(Asn19846Metfs*12) | Het | 0.0000 | 0 | NA | LP | 087, 246 | PVS1 PM2 | – |
TTN | NM_001267550.2 | c.58870C>T | p.(Arg19624*) | Het | 0.0000 | 0 | NA | P | 120, 213 | PVS1 PM2 PP3 PP4 | – |
TTN | NM_001267550.2 | c.81274C>T | p.(Gln27092*) | Het | 0.0000 | 0 | NA | P | 251 | PVS1 PM2 PP3 | |
TTN | NM_001267550.2 | c.60359_60371del | p.(Lys20120Thrfs*19) | Het | 0.0000 | 0 | NA | LP | 270 | PVS1 PM2 | – |
ACMG, the American college of medical genetics and genomics; EAS, East Asia; Het, heterozygous; KHV, Kinh in Ho Chi Minh City, Vietnam; MAF, minor allele frequency; NA, not applicable. ACMG class: LP, likely pathogenic; P, pathogenic. ACMG criteria: _M, moderate; _P, supporting; _S, strong; PM, moderate pathogenicity evidence; PP, supporting pathogenicity evidence; PS, strong pathogenicity evidence; PVS, very strong pathogenicity evidence; –, no information.
The distribution of disease-causing mutations are presented in Figure 1. TTN gene mutations were predominant, found in 26/230 patients (11.3%). TPM1, DSP, LMNA, MYBPC3, MYH6, MYH7, DES, TNNT2, ACTC1, ACTN2, BAG3, DMD, FKTN, PLN, TBX5, RBM20, TCAP, contributed 2–6% each to the overall genotype-positive status. TTN truncating variants (TTNtv) accounted for 24% (6/25) and 7.9% (15/189) of familial and sporadic DCM, respectively. TTNtv were mostly found in the A band of the protein (17/23) (Supplementary Figure). The frequency of P variants in genes other than TTN was not sufficient for statistical analysis. Among 43 relatives of 21/56 genotype-positive index patients who accepted to participate in this study, 27 (62.8%) harbored the same mutation found in the proband.
Distribution of pathogenic and likely pathogenic variants in Vietnamese dilated cardiomyopathy (DCM) patients.
The RBM20 P variant, c.1907G>A, identified in one proband was found in 5 of 8 of his relatives. SCD was recorded for this proband; his mother and uncle who were diagnosed with DCM, all at a young age (Figure 2).
Pedigree of the family with dilated cardiomyopathy (DCM) due to RBM20 c.1907G>A (p.Arg636His). Index patients are marked with an arrow. d, died; dx, diagnosis; SCD, sudden cardiac death; y, years old.
To establish genotype-phenotype correlations, we compared clinical characteristics of genotype-positive and genotype-negative patients. Similar comparisons were made between patients harboring a TTNtv and genotype-negative patients. Familial and sporadic DCM patients were also analyzed for possible distinctive clinical manifestations. Correlation analyses are presented in Table 1.
In this study, familial DCM, genotype-positive and TTNtv-positive patients were younger than those with genotype-negative and sporadic DCM. Male gender was markedly associated with TTNtv-positive status (23/88.5%). Genotype-positive patients, particularly those with TTNtv had a higher BMI compared to genotype-negative patients. Genotype-positive, especially those with a TTNtv-positive status, was associated with a family history of DCM, whereas family history of SCD was significantly enriched in familial DCM. Genotype-positive patients displayed a significant slight decrease in systolic blood pressure (P=0.009). These patients also exhibited a decreased LV wall thickness compared to genotype-negative patients (P=0.013). A similar result, though not significant, was observed in TTNtv-positive patients (P=0.077), and familial DCM cases (P=0.057). Compared to genotype-negative patients, the right ventricle-fractional area change (RV-FAC) value was lower in genotype-positive and TTNtv-positive patients, although the difference was not significant for the latter group (P=0.052). Left atrial volume index (LAVi) was higher in genotype-positive and TTNtv-positive patients than in genotype-negative patients (P=0.001). Symptoms such as palpitation, dyspnea, and syncope were mostly observed in TTNtv-positive cases. Left bundle branch block was enriched in sporadic DCM compared to familial DCM. Interestingly, we noted no LV dilatation and no difference in LVEF values among all patient groups. Higher, though not significant, rates of atrial fibrillation and lower LVEDDi were observed in TTNtv-positive compared to genotype-negative patients (P=0.076) and in familial compared to sporadic DCM (P=0.078).
We defined major composite outcomes as death from any cause, heart transplantation, non-fatal stroke, life-threatening arrhythmia requiring ICD implant for the analysis of event-free survival curves. Event-free survival was measured from time of birth, and adjusted for gender, hypertension, and arrhythmia. Patients who did not have the outcome of interest were censored at the time of their last recorded follow up in this study. Genotype-positive patients reached the combined outcomes more frequently and at a younger age than genotype-negative patients (HR=3.4; 95% CI: 1.5–8.0; P=0.005) (Figure 3A). Major cardiac events occurred more frequently in patients with mutations in genes other than TTNtv (HR=9.2; 95% CI: 1.1–74.2; P=0.038) (Figure 3B). However, no difference in survival rate was observed between TTNtv-positive and genotype-negative patients (HR=0.7; 95% CI: 0.1–5.0; P=0.679) (Figure 3C).
Survival curves show freedom from composite outcomes (death from any cause, heart transplantation, non-fatal stroke, life-threatening arrhythmia requiring implantable cardioverter-defibrillator implant). (A) Survival curve for patients with and without detected disease-causing mutation. (B,C) Comparison between patients with TTN tv vs. other gene mutation, and TTN tv vs. no mutation, respectively. Event-free survival is measured from time of birth, and adjusted for gender, hypertension, and arrhythmia. CI, confidence interval; HR, hazard ratio.
To the best of our knowledge, this is the first time disease-causing variants in DCM-associated genes were analyzed in Vietnamese patients diagnosed with DCM.
Mutation PrevalenceThe overall diagnostic yield in our study was 23.5% (56/230); higher in familial (44%) than in sporadic DCM (19.6%), in line with previous observations.18,19 Our diagnostic yield was lower than that of some previous studies, but comparable to others that showed a diagnostic yield of 17–26%.18,19 A common point between these studies and ours was the use of a very stringent variant classification, whereas other reports were based on a less strict variant interpretation system. For example, a study in Han-Chinese DCM patients reported a diagnostic yield of 34.7%; however, the authors used less strict criteria for variant interpretation.20 Furthermore, the ratio of familial DCM in our study (10.8%) was much lower than that found in other published studies. P variants in our study cohort were found in genes that have robust evidence for DCM association including TTN, DSP, TPM1, LMNA, MYH7, TNNT2, BAG3, ACTC1, and RBM20.19 These P variants were not found in the 1,000 Genomes database and were determined using the strict ACMG guidelines combined with CardioClassifier, the computational tool specific for inherited cardiac conditions.
The number of P mutations found in a single patient may affect the clinical severity.21 In this study, 1 proband with compound mutations in MYBPC3 and 1 with combined mutations in MYBPC3 and MYH7 were identified, a rate much lower than previously described.22 Our finding was consistent with the predominant autosomal dominant inheritance of most DCM genes.23
Genotype-Phenotype CorrelationGenotype-phenotype correlations in DCM was a question for debate. Some studies showed no difference in term of clinical manifestations between patients with and without a mutation.20,24 In contrast, genotype-positive status associated with more adverse outcomes were reported for other study populations.19,25 Our genotype-positive and TTNtv-positive patients had a family history of DCM and an age at diagnosis significantly younger than genotype-negative patients, which is in agreement with previous research.18 However, some abnormal cardiac features characterizing DCM such as LVEF, LVEDDi, arrhythmia, and especially atrial fibrillation displayed no difference between genotype-positive and genotype-negative probands, in contrast with previous studies.19,25 The absence or very low prevalence of P variants in genes predominantly associated with arrhythmic DCM such as SCN5A, LMNA and RBM20 in our study cohort may partly explain these findings. Systolic blood pressure together with ejection fraction were the 2 predictors for long-term survival in DCM patients.26 Our genotype-positive patients displayed a slight though significant decrease in systolic blood pressure, even though it was still within the normal range. LV wall thinning and LV dilatation were factors that triggered LV remodelling.27 In this study, genotype-positive patients displayed a marked decreased of LV wall thickness compared with genotype-negative patients, but no difference in LV dilatation was observed between the 2 groups. In DCM patients with reduced LVEF, LAV was a powerful predictive marker and increased LAV conferred an increased risk of cardiac death.28,29 Findings that were replicated in this study with genotype-positive probands showed a marked increase in LAVi. RV systolic function was considered as a prognostic predictor of outcomes in DCM patients.30,31 In this study, right ventricle-tricuspid annular plane systolic excursion (RV-TAPSE) and RV-FAC were lower in genotype-positive patients compared to genotype-negative patients, but only RV-FAC showed a significant difference. This observation, in line with our other results, showed a correlation between genotype-positive status and patients’ adverse outcomes, as previously reported.30,31
Familial DCM was characterized by younger age and age at diagnosis, family history of SCD and was inversely correlated with the presence of left bundle branch block; findings that are in accordance with previous reports.19,32 Diagnostic yield in familial DCM was higher than in sporadic DCM, as expected. A tendency for lower LVEDDi (P=0.078) and LV wall thickness (P=0.057) was observed in familial vs. sporadic DCM, although this was not significant and possibly due to insufficient data.
DCM is a complex disorder caused by genetic and environmental factors that combine to drive disease onset and outcomes. A significant though slight difference in BMI value was recorded between genotype-positive and genotype-negative patients. An association between high BMI and cardiomyopathies, in particular DCM, was observed in a large follow-up study in Sweden.33 High BMI exacerbates genetic cardiac dysfunction, resulting in earlier onset of the disease. A prospective study showed that the risk of developing DCM in adulthood significantly increased with even mildly elevated body weight in late adolescence.34
TTNtv-Positive StatusTTNtv accounted for 11.3% of the study cohort, and 48% of all genotype-positive patients; 24% of familial cases and 7.9% of sporadic cases; these rates were similar to those determined in other published cohorts.18,35 A significant male predominance was observed in our TTNtv patients, which is also in agreement with previous reports.35,36 Clinical impacts of TTNtv depend on variant location in the entire gene. Variants situated in the A-band and the proximal or terminal part of the I-band were associated with higher cardiac expression and disease penetrance than those located in the Z-band and M-band regions.37 The high percentage of TTNtv found in the A-band of our study cohort (73.9%) was in accordance with these findings.
TTNtv that are highly expressed in the heart (hiPSI TTNtv) were associated with severe cardiac phenotypes, largely driven by DCM in individuals with European ancestry.36,38,39 In contrast, no difference in clinical manifestations or only a mild form of DCM were reported between TTNtv-positive and -negative patients.20,40 A clear family history of DCM and significant increase of LAVi characterized our TTNtv-positive probands. They also manifested several heart conditions such as palpitations, dyspnea and syncope, indicators of morbidity and mortality risks when associated with cardiac structural diseases.41 TTNtv-positive status created metabolic stress signaling, which can be accentuated by further increases in metabolic stressors, reflecting the age and BMI-induced onset of the disease.42 Our TTNtv-positive probands exhibited a younger age and a significantly higher BMI than that of genotype-negative patients. However, despite all the unfavorable characteristics including male sex, family history of DCM, increased LAVi, and cardiac manifestations, probands positive with TTNtv in this study displayed a higher survival rate than those with mutations in other genes, finding in agreement with previous reports on the treatable nature and milder manifestations of TTN-positive status compared to LMNA, SCN5A, and RBM20 genes.24
RBM20, a component of the RNA splicing machinery, regulates the splicing of at least 30 cardiac genes, including TTN.43 The missense P RBM20 variant c.1907G>A identified in this study was situated in the RS domain and considered as a mutational hotspot.44 The RBM20-positive case exhibited a marked family history of DCM and SCD. These findings confirmed the early onset, rapidly progressive nature and high mortality associated with RBM20 P variants.44
Overlapping DCM/HCMDifferent mutations in the same gene cause either HCM or DCM, but no mutation can lead to both diseases.45 Nevertheless, an overlapping spectrum of disease-causing mutations or DCM, HCM, arrhythmogenic right ventricular cardiomyopathy (ARVC) and channelopathies was also recognized.22,46 In this study, 5 P variants identified in 4 DCM patients were previously reported as associated with other cardiomyopathies in previous studies, including a Vietnamese HCM study cohort.47–49 Misdiagnosis of end-stage HCM was nearly excluded from our study because no long-standing history of HCM was recorded in any of these probands.4 Furthermore, LV wall thickness of these patients (8–12.5 mm) was much lower than that reported for Vietnamese HCM patients (mean 22.5±4.8 mm)47 (Supplementary Table 2).
Study LimitationsFirst, our study cohort was subjected to a selection bias because only patients with clear clinical symptoms were recruited from the 2 biggest heart hospitals in South Vietnam. Furthermore, due to limited awareness and capacity of access to health care of the general population, a certain number of DCM cases, especially those with milder manifestations, was probably missed. Second, the total number of analyzed cases was not sufficient to assess the penetrance of all identified variants. In cardiomyopathies including DCM, environmental epigenetic factors and common genetic variants also contributed to the manifestations of gene mutations. These factors were not considered in our study. Finally, the TruSight Cardio did not include FLNC, a gene with a strong association with DCM.
Determination of the most clinically relevant DCM genes and variants could provide evidence to increase the clinical utility through reducing the uncertainty associated with large number of variants of uncertain significance (VUS). Our findings, though, did not provide concluding results, but gave an overview of Vietnamese DCM patients’ genetic profile. Further studies are required for more elaborated analyses. Previous reports showed that a clear improvement in all aspects of patients’ quality of life can be obtained through early diagnosis leading to better risk stratification and follow up, and/or personalized therapy based on identification of etiological assessment.32,50 Our findings suggested that management of environmental factors may be beneficial for DCM patients, especially TTNtv-positive ones. Data should be taken into consideration for genetic counseling of patients and families.
This study could not have been carried out without the efficient cooperation from cardiologists in the participating institutions, namely Diem Trang Kieu Tran, Thi Lan Vo, Kim Tuyen Le, Van Phuoc Nguyen, Thuy Lan Dang Le, Chi Thanh Nguyen, and Mai Phuong Ngoc Nguyen.
This work was supported by a research grant (54/2018/HĐ-QKHCN) from the Department of Science and Technology – Ho Chi Minh City, Vietnam.
The authors declare no conflicts of interest.
The study was approved by the ethics committees of the 2 participating hospitals according to local regulations (reference numbers: 1759/VT-HDDD, 19.18/QD-NC-TD).
Please find supplementary file(s);
http://dx.doi.org/10.1253/circj.CJ-21-0077