Introduction
Methodology
Study population and area
Blood collection and DNA isolation
Genotyping
Gene polymorphism | Primer sequence | Amplicon (bp) | PCR conditions | Restriction enzymes | Genotypes | Reference |
---|---|---|---|---|---|---|
MTHFR C677T (rs1801133) | 5’-TGA AGG AGA AGG TGT CTG CGG GA-3’ (F) 5’-AGG ACG GTG CGG TGA GAG TG-3’ (R) | 198 | Pre-Denaturation: 94 °C/ 2 min Denaturation: 94 °C/ 30 s Annealing: 62 °C/ 60 s Extension: 72 °C/ 30 s Final Extension: 72 °C/ 7 min. (40 cycles) | HinfI | CC = 198 bp CT = 198, 175 & 23 bp TT = 175 & 23 bp Figure 1 | McBride et al., 2004 [36] |
MTHFR G1793A (rs2274976) | 5’-CTC TGT GTG TGT GTG CAT GTG TGC G-3’ (F) 5’-GGG ACA GGA GTG GCT CCA ACG CAG G-3’ (R) | 310 | Pre-Denaturation: 94 °C/ 1 min Denaturation: 94 °C/ 1 min Annealing: 67 °C/ 1 min Extension: 72 °C/ 1 min Final Extension: 72 °C/ 7 min. (40 cycles) | BsrbI | GG = 233 & 77 bp GA = 310, 233 & 77 bp AA = 310 bp Figure 2 | Rady et al., 2002 [6] |
MS A2756G (rs185087) | 5’- TGT TCC AGA CAG TTA GAT GAA AAT C-3’ (F) 5’- GAT CCA AAG CCT TTT ACA CTC CTC-3’ (R) | 211 | Pre-Denaturation: 95 °C/ 4 min Denaturation: 95 °C/ 1 min Annealing: 61 °C/ 1.5 min Extension: 72 °C/ 1 min Final Extension: 72 °C/ 7 min. (35 cycles) | HaeIII | AA = 211 bp AG = 211, 131 & 80 bp GG = 131 & 80 bp Figure 3 | Sahiner et al., 2014 [25] |
Statistical analyses
Meta-analysis
Literature search
Inclusion and exclusion criteria
Studies included | Studies excluded |
---|---|
● Studies with Case–control designs ● Report of the association between the MTHFR C677T, MTHFR G1793A and MS A2756G polymorphism and the risk of CHD ● Studies that included Pediatric participants ● Studies that follow Hardy Weinberg equilibrium (HWE) ● Studies with sufficient data ● Studies in English language | ● Case reports ● Meta analysis and review articles ● Studies without control group ● Studies with abstract only ● Studies that include maternal/ paternal cases only ● Studies without detailed genotype data ● Studies that are associated with other diseases like CVD’s, thrombosis, coronary artery defects etc |
Data extraction and quality assessment
Study | Age Group/Mean age of cases | Mean age of controls | Diagnostic criteria | Source of controls | Country/ Region | Ethnicity | Genotyping Method | Cases | Controls | NOS | HWE | ||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
CC | CT | TT | Total | CC | CT | TT | Total | ||||||||||
Junker et al., 2001 [9] | 0–16 | Age matched | Echocardiography excluding DS or Chromosomal abnormality | HB | Germany | Caucasian | PCR–RFLP | 51 | 42 | 21 | 114 | 129 | 78 | 21 | 228 | 9 | 0.0751 |
Lee et al., 2005 [10] | Children | - | Confirmed CHD patients for cardiac catheterization | HB (cord blood from healthy foetuses) | Taiwan | Asian | DHPLC | 110 | 89 | 14 | 213 | 114 | 68 | 13 | 195 | 9 | 0.5128 |
Li et al., 2005 [11] | Children | Age matched | Registered patients of birth defects confirmed for CHD | HB | China | Asian | PCR–RFLP | 30 | 95 | 58 | 183 | 22 | 57 | 24 | 103 | 9 | 0.2766 |
Shaw et al., 2005 [12] | 0–1 year & foetuses with CHD | Age matched | Conotruncal heart cases confirmed by Echocardiography | PB | America | Caucasian | DIRECT SEQUENCING | 69 | 68 | 16 | 153 | 180 | 202 | 52 | 434 | 9 | 0.6836 |
Zhu et al., 2006 [13] | 6.2 yrs | 8.4 yrs | Confirmed CHD by Echocardiography | PB | China | Asian | PCR–RFLP | 3 | 7 | 12 | 22 | 22 | 57 | 24 | 103 | 9 | 0.2766 |
Zhu et al., 2006 [13] | 6.2 yrs | 8.4 yrs | Confirmed CHD by Echocardiography | PB | China | Asian | PCR–RFLP | 4 | 15 | 15 | 34 | 22 | 57 | 24 | 103 | 9 | 0.2766 |
van Beynum et al., 2006 [4] | 3.4 yrs | 9.4 yrs | Echocardiography excluding NTD, cleft palate/lip, detected genetic abnormalities, known syndromes, and Vacterl- association | PB | Caucasian | Caucasian | PCR–RFLP | 79 | 66 | 20 | 165 | 98 | 104 | 18 | 220 | 8 | 0.1842 |
Galdieri et al., 2007 [14] | 0–11 yrs | - | Isolated cardiopathies (not associated with genetic syndromes or other malformations) confirmed by echocardiogram or cardiac catheterization | HB | Brazil | Caucasian | DIRECT SEQUENCING | 30 | 21 | 7 | 58 | 18 | 14 | 6 | 38 | 9 | 0.2631 |
van Driel et al., 2008 [15] | 16.8 months | 16.7 months | Confirmed CHD by echocardiography and/or cardiac catheterization and/or surgery | PB | European | Caucasian | Real time PCR, RFLP | 99 | 103 | 27 | 229 | 119 | 107 | 25 | 251 | 9 | 0.8951 |
Xu et al., 2010 [16] | 6.50 yrs | 6.69 yrs | Non-syndromic CHD cases confirmed by echocardiography | HB | China | Asian | 162 | 244 | 96 | 502 | 151 | 261 | 115 | 527 | 0.9115 | ||
Kuehl et al., 2010 [17] | Infants before one year of age | Age matched | Confirmed CHD by echocardiography and/or cardiac catheterization and/or surgery | PB | America | Caucasian | DIRECT SEQENCING | 12 | 33 | 10 | 55 | 134 | 134 | 32 | 300 | 7 | 0.8611 |
Oberman-Borst et al., 2011 [18] | 17 months | 17.3 months | Confirmed CHD by echocardiography and/or cardiac catheterization and/or surgery | PB | Netherlands | Caucasian | DIRECT SEQUENCING | 64 | 66 | 9 | 139 | 92 | 76 | 15 | 183 | 8 | 0.9 |
Kotby et al., 2012 [19] | 31.5 months | 32.7 months | Conotruncal heart defects excluding syndrome CHD | PB | Egypt | Caucasian | PCR–RFLP | 12 | 14 | 4 | 30 | 20 | 8 | 2 | 30 | 8 | 0.3613 |
Gong et al., 2012 [20] | 2.27 yrs | 1.58 yrs | Non-syndromic CHD cases confirmed by echocardiography and /or surgery | HB | Chinese Han population | Asian | MALDI-ToF–MS | 45 | 123 | 76 | 244 | 43 | 72 | 21 | 136 | 8 | 0.3088 |
El-Abd et al., 2012 [21] | Neonates | Neonates | Confirmed CHD except congenital heart disease associated with chromosomal anomalies and genetic syndromes, pre-mature infants (< 37 weeks gestation) and maternal diabetes, malabsorption, wasting syndromes, or any condition associated with folate deficiency | HB | Egypt | Caucasian | PCR–RFLP | 7 | 12 | 7 | 26 | 13 | 5 | 0 | 18 | 9 | 0.4938 |
Wang et al., 2013 [22] | - | - | Confirmed CHD by echocardiography | HB | China | Asian | SNaPShot genotyping, sequencing | 59 | 76 | 25 | 160 | 53 | 100 | 35 | 188 | 9 | 0.3124 |
Kocakap et al., 2014 [23] | 3.7 yrs | 8.7 yrs | Patients w ith echocardiographically proven conotruncal heart defect | HB | Turkey | Caucasian | HRM, PCR–RFLP, Sequencing | 40 | 33 | 2 | 75 | 43 | 44 | 8 | 95 | 9 | 0.4841 |
Chao et al., 2014 [24] | 46.7 yrs | 50.9yrs | Patients undergoing PDA ligation except patients diagnosed with diseases due to chromosomal defect or those born prematurely | HB | Taiwan | Asian | PCR–RFLP | 10 | 5 | 2 | 17 | 19 | 12 | 3 | 34 | 8 | 0.5863 |
Mohamad et al., 2014 [8] | Paediatric cases | > 21 years | Non-syndromic CHD patients confirmed by echocardiography | PB | Malaysians | Asian | PCR–RFLP | 118 | 32 | 0 | 150 | 131 | 19 | 0 | 150 | 7 | 0.4076 |
Sahiner et al., 2014 [25] | 7.63 yrs | - | Non-syndromic CHD patients confirmed by echocardiography | HB | Turkey | Caucasian | PCR–RFLP | 69 | 53 | 14 | 136 | 47 | 39 | 7 | 93 | 9 | 0.7791 |
Li et al., 2015 [26] | - | - | Clinically confirmed CHD patients by echocardiography | HB | China | Asian | DIRECT SEQUENCING | 31 | 78 | 41 | 150 | 59 | 66 | 25 | 150 | 9 | 0.3756 |
Shi et al., 2015 [27] | - | - | Clinically confirmed CHD patients by echocardiography | PB | China | Asian | PCR–RFLP | 55 | 68 | 30 | 153 | 70 | 101 | 45 | 216 | 8 | 0.4437 |
Wang et al., 2016 [28] | 1.46 yrs | 3.08 yrs | Non-syndromic CHD patients confirmed by echocardiogram or cardiac catheterization | HB | Chinese Han population | Asian | Taq-Man allelic discrimination assay | 14 | 73 | 60 | 147 | 49 | 84 | 35 | 168 | 9 | 0.9278 |
Noori et al., 2017 [29] | 4.2 yrs | 4.9 yrs | confirmed CHD patients by echocardiography, cardiac catherization and surgical procedures | HB | Iran | Asian | Tetra-ARMS PCR | 95 | 51 | 7 | 153 | 100 | 46 | 1 | 147 | 9 | 0.0781 |
Wang et al., 2018 [30] | - | - | Conotruncal heart defects CHD patients by echocardiography | HB | China | Asian | DIRECT SEQUENCING | 8 | 48 | 36 | 92 | 70 | 117 | 50 | 237 | 7 | 0.9316 |
Present study 2020 | 23.24 months | 59.26 months | Non-syndromic CHD patients confirmed by echocardiography and surgical procedures | HB | Indian | Asian | PCR–RFLP | 44 | 4 | 2 | 50 | 90 | 9 | 1 | 100 | 8 | 0.1796 |
Statistical analysis for meta-analysis
Trial sequential analysis (TSA)
Results
Case–control study
Type of CHD | No. of Cases (N = 50) | Percentage (%) |
---|---|---|
Ventricular septal defect (VSD) | 17 | 34% |
Atrial septal defect (ASD) | 13 | 26% |
Tetralogy of fallot (TOF) | 7 | 26% |
Patent ductus arteriosus (PDA) | 4 | 8% |
Endocardial cushion defect | 3 | 6% |
ASD with PDA | 2 | 4% |
VSD with peripheral arterial hypertension | 2 | 4% |
VSD with AV-canal defect | 1 | 2% |
Endocardial cushion defect along with dextrocardia | 1 | 2% |
Category | Genotypes/Alleles (%) | χ2 | p-value | ||||
---|---|---|---|---|---|---|---|
MTHFR (C677T) polymorphism | |||||||
CC (Wild) | CT (Hetero) | TT (Risk) | C (Wild) | T (Risk) | |||
CHD Cases (n = 50) | 44 (88%) | 4 (8%) | 2 (4%) | 0.92 | 0.08 | 10.42 | 0.001* |
Controls (n = 100) | 90 (90%) | 9 (9%) | 1 (1%) | 0.95 | 0.05 | 1.8 | 0.18 |
MTHFR (G1793A) polymorphism | |||||||
GG (Wild) | GA (Hetero) | AA (Risk) | G (Wild) | A (Risk) | |||
CHD Cases (n = 50) | 29 (58%) | 19 (38%) | 2 (4%) | 0.77 | 0.23 | 0.27 | 0.61 |
Controls (n = 100) | 90 (90%) | 10 (10%) | 0 | 0.95 | 0.05 | 0.28 | 0.60 |
MS (A2756G) gene polymorphism | |||||||
AA (Wild) | AG (Hetero) | GG (Risk) | A (Wild) | G (Risk) | |||
CHD Cases (n = 50) | 30 (60%) | 18 (36%) | 2 (4%) | 0.78 | 0.22 | 0.12 | 0.73 |
Controls (n = 100) | 73 (73%) | 26 (26%) | 1 (1%) | 0.86 | 0.14 | 0.64 | 0.43 |
MODEL | OR (95% CI) | p-value |
---|---|---|
MTHFR C677T polymorphism | ||
Co-dominant | ||
CT vs CC | 0.91 [0.27–3.12] | 0.879 |
TT vs CC | 4.09 [0.36–46.35] | 0.22 |
Dominant | ||
CT + TT vs CC | 1.23 [0.42–3.59] | 0.71 |
Recessive | ||
TT vs CT + CC | 4.12[0.36–46.63] | 0.234 |
Allelic | ||
T vs C | 1.49 [0.58–3.84] | 0.40 |
MTHFR G1793A polymorphism | ||
Co-dominant | ||
GA vs GG | 5.90 [2.46–14.11] | 0.00002b |
AA vs GG | Not possiblea | - |
Dominant | ||
GA + AA vs GG | 6.52 [2.75–15.43] | < 0.0001b |
Recessive | ||
AA vs GA + GG | Not possiblea | - |
Allelic | ||
A vs G | 5.68 [2.58–12.48] | < 0.0001b |
MS A2756G polymorphism | ||
Co-dominant | ||
AG vs AA | 1.68 [0.81–3.52] | 0.163 |
GG vs AA | 4.87 [0.43–55.71] | 0.20 |
Dominant | ||
AG + GG vs AA | 1.80 [0.88–3.69] | 0.11 |
Recessive | ||
GG vs AG + AA | 4.12[0.36–46.63] | 0.2 |
Allelic | ||
G vs A | 1.73 [0.93–3.22] | 0.08 |
Variant MTHFR C677T/ G1793A | CHD Cases (n = 50) | Controls (n = 100) | OR (95% CI) | p-value† |
---|---|---|---|---|
C-A | 0.230 | 0.050 | 5.67 [2.58–12.48] | 2.71e‐006a |
C-G | 0.690 | 0.895 | 0.26 [0.14–0.48] | 1.00e‐005a |
T-G | 0.080 | 0.055 | 1.49 [0.58–3.84] | 0.40 |
T-A | 0.000 | 0.000 | - | - |
Meta-analysis
Genetic Model | Number of studies | Test of association | Heterogeneity | Egger's test p- value | ||||
---|---|---|---|---|---|---|---|---|
OR | 95% CI | p-value | Model | p-value | I^2 | |||
Overall | ||||||||
Allele contrast (T vs. C) | 26 | 1.33 | 1.14–1.55 | 0.0002 | Random | 0.0001 | 0.7554 | 0.0259 |
Recessive model (TT vs. TC + CC) | 25a | 1.49 | 1.83–1.87 | 0.0007 | Random | 0.0001 | 0.5828 | 0.1945 |
Dominant model (TT + TC vs. CC) | 26 | 1.38 | 1.14- 1.69 | 0.001 | Random | 0.0001 | 0.696 | 0.0068 |
Homozygous model (TT vs CC) | 25a | 1.75 | 1.26–2.44 | 0.001 | Random | 0.0001 | 0.7286 | 0.0699 |
Heterozygous model (TT vs CT) | 25a | 1.34 | 1.11–1.60 | 0.002 | Random | 0.02 | 0.5157 | 0.6033 |
Caucasians | ||||||||
Allele contrast (T vs. C) | 11 | 1.21 | 0.97–1.50 | 0.1 | Random | 0.0006 | 0.6755 | 0.1529 |
Recessive model (TT vs. TC + CC) | 11 | 1.27 | 0.99–1.63 | 0.06 | Fixed | 0.1662 | 0.2933 | 0.8658 |
Dominant model (TT + TC vs. CC | 11 | 1.24 | 0.95- 1.62 | 0.1 | Random | 0.003 | 0.6234 | 0.0657 |
Homozygous model (TT vs CC) | 11 | 1.37 | 0.91- 2.07 | 0.1 | Random | 0.0237 | 0.5157 | 0.6033 |
Heterozygous model (TT vs CT) | 11 | 1.78 | 0.91- 1.53 | 0.2 | Fixed | 0.5288 | 0 | 0.8349 |
Asians | ||||||||
Allele contrast (T vs. C) | 15 | 1.42 | 1.15- 1.76 | 0.001 | Random | 0.0001 | 0.7988 | 0.0765 |
Recessive model (TT vs. TC + CC) | 14a | 1.67 | 1.21–2.31 | 0.002 | Random | 0.0001 | 0.6958 | 0.1205 |
Dominant model (TT + TC vs. CC | 15 | 1.50 | 1.12- 2.01 | 0.02 | Random | 0.0001 | 0.7438 | 0.0599 |
Homozygous model (TT vs CC) | 14a | 2.12 | 1.30–3.47 | 0.003 | Random | 0.0001 | 0.8067 | 0.08 |
Heterozygous model (TT vs CT) | 14a | 1.46 | 1.13–1.89 | 0.003 | Random | 0.03 | 0.4697 | 0.0834 |
Genetic Model | Number of studies | Test of association | Heterogeneity | Egger's test p- value | |||||
---|---|---|---|---|---|---|---|---|---|
OR | 95% CI | p-value | Model | p-value | I^2 | ||||
Overall | |||||||||
Allele contrast (G vs. A) | 6 | 1.05 | 0.88–1.26 | 0.6 | Fixed | 0.3 | 0.1993 | 0.4631 | |
Recessive model (GG vs. AG + AA) | 6 | 1.11 | 0.47–2.64 | 0.8 | Random | 0.07 | 0.5136 | 0.5171 | |
Dominant model (GG + AG vs. AA) | 6 | 1.08 | 0.86–1.35 | 0.5 | Fixed | 0.6 | 0 | 0.7422 | |
Homozygous model (GG vs AA) | 6 | 0.95 | 0.57–1.56 | 0.8 | Fixed | 0.1 | 0.4122 | 0.4344 | |
Heterozygous model (GG vs AG) | 6 | 1.10 | 0.45–2.72 | 0.8 | Random | 0.06 | 0.5204 | 0.5685 | |
Caucasians | |||||||||
Allele contrast (G vs. A) | 3 | 0.95 | 0.75–1.19 | 0.6 | Fixed | 0.5 | 0 | 0.9501 | |
Recessive model (GG vs. AG + AA) | 3 | 0.86 | 0.30–2.47 | 0.8 | Random | 0.03 | 0.7067 | 0.9516 | |
Dominant model (GG + AG vs. AA) | 3 | 0.96 | 0.71–1.31` | 0.8 | Fixed | 0.92 | 0 | 0.0379 | |
Homozygous model (GG vs AA) | 3 | 0.84 | 0.34–2.06 | 0.7 | Random | 0.1 | 0.5605 | 0.9324 | |
Heterozygous model (GG vs AG) | 3 | 0.87 | 0.26–2.91 | 0.82 | Random | 0.02 | 0.7476 | 0.9915 | |
Asians | |||||||||
Allele contrast (G vs. A) | 3 | 1.25 | 0.93–1.69 | 0.1 | Fixed | 0.3 | 0.2455 | 0.6974 | |
Recessive model (GG vs. AG + AA) | 3 | 2.26 | 0.51–9.94 | 0.3 | Fixed | 0.4 | 0.0104 | 0.5599 | |
Dominant model (GG + AG vs. AA) | 3 | 1.24 | 0.89–1.73 | 0.21 | Fixed | 0.3 | 0.0785 | 0.5501 | |
Homozygous model (GG vs AA) | 3 | 2.42 | 0.55–10.69 | 0.2 | Fixed | 0.3 | 0.1005 | 0.577 | |
Heterozygous model (GG vs AG) | 3 | 1.95 | 0.43–8.78 | 0.4 | Fixed | 0.5 | 0 | 0.4763 |