Background
Methods
Subjects
Samples and data collection
Genotyping of the selected polymorphisms
SNP No. | Gene polymorphism | Primer sequence | Amplicon (bp) | Restriction enzymes | Genotypes |
---|---|---|---|---|---|
SNP 1 | ESR1 IVS1–397 T/C (rs2234693) | 5′-CTG CCA CCC TAT CTG TAT CTT TTC CTA TTC TCC- 3′ (F) 5′-TCT TTC TCT GCC ACC CTG GCG TCG ATT ATC TGA- 3′ (R) | 1372 | PvuII | TT = 982 & 390 bp TC = 982, 390 & 1372 bp CC = 1372 bp |
SNP 2 | ESR1- IVS1–351 A/G (rs9340799) | 5′-CTG CCA CCC TAT CTG TAT CTT TTC CTA TTC TCC- 3′ (F) 5′-TCT TTC TCT GCC ACC CTG GCG TCG ATT ATC TGA- 3′ (R) | 1372 | XbaI | AA = 936 & 436 bp AG = 936, 436 & 1372 bp GG = 1372 bp |
SNP 3 | CETP-TaqIB (C277T) (rs708272) | 5′-CAC TAG CCC AGA GAG AGG AGT GCC-3′ (F) 5′-CTG AGC CCA GCC GCA CAC TAA C-3′ (R) | 535 | TaqI | B2B2 = 535 bp B1B2 = 535, 361 & 174 bp B1B1 = 361 & 174 bp |
SNP 4 | 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 | HinfI | CC = 198 bp CT = 198, 175 & 23 bp TT = 175 & 23 bp |
SNP 5 | 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 | BsrbI | GG = 233 & 77 bp GA = 310, 233 & 77 bp AA = 310 bp |
SNP 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 | HaeIII | AA = 211 bp AG = 211, 131 & 80 bp GG = 131 & 80 bp |
Statistical analysis
Results
Non-genetic factors
Parameters | Patients (N = 400) | Controls (N = 400) | Odds Ratio | p-value |
---|---|---|---|---|
BMI | 24.99 ± 5.75 | 23.21 ± 4.28 | – | < 0.0001 |
WHR | 0.99 ± 0.08 | 0.96 ± 0.13 | – | 0.008 |
Blood Pressure (mm Hg) | ||||
Systolic BP (SBP) | 142.90 ± 20.12 | 125.04 ± 8.30 | – | < 0.0001* |
Diastolic BP (DBP) | 89.17 ± 11.13 | 83.46 ± 8.48 | – | < 0.0001* |
Pulse Pressure (PP) | 53.73 ± 16.77 | 41.58 ± 8.12 | – | < 0.0001* |
Pulse Rate (PR) | 82.11 ± 12.23 | 74.41 ± 4.91 | – | < 0.0001* |
Total Cholesterol (TC) (mg/dl) | 172.75 ± 55.30 | 143.30 ± 27.87 | – | < 0.0001* |
Triglycerides (TG) (mg/dl) | 191.27 ± 68.39 | 166.57 ± 30.22 | – | < 0.0001* |
HDL- C (mg/dl) | 40.67 ± 7.71 | 44.88 ± 6.93 | – | < 0.0001* |
LDL-C (mg/dl) | 121.29 ± 61.56 | 112.42 ± 33.94 | – | 0.01* |
HDL/TC ratio | 0.259 ± 0.10 | 0.327 ± 0.91 | – | < 0.0001* |
Smoking | ||||
Smokers | 159 (39.75%) | 50 (12.5%) | 4.62 (3.23–6.60) | < 0.0001* |
Non-smokers | 241 (60.25%) | 350 (87.5%) | Ref. (1) | – |
Physical inactivity | ||||
Yes | 249 (62.25%) | 163 (40.75%) | 2.40 (1.80–3.18) | < 0.0001* |
No | 151 (37.75%) | 237 (59.25%) | Ref. (1) | – |
Dwelling | ||||
Urban | 215 (53.75%) | 158 (39.5%) | 0.00005* | |
Rural | 185 (46.25%) | 242 (60.5%) |
Genetic polymorphisms
Genotypic/Allelic frequencies | Cases (N = 400) | Controls (N = 400) |
---|---|---|
ESR1IVS1–397 T/C | ||
TT | 168 (42%) | 175 (43.75%) |
TC | 187 (46.75%) | 185 (46.25%) |
CC | 45 (11.25%) | 40 (10%) |
T (Major) | 0.65 | 0.67 |
C (Minor) | 0.35 | 0.33 |
χ2 | 0.65 | 0.5 |
p-value | 0.77 | 0.4 |
ESR1- IVS1–351 A/G | ||
AA | 105 (26.25%) | 133 (33.25%) |
AG | 196 (49%) | 202 (50.5%) |
GG | 99 (24.75%) | 65 (16.25%) |
A (Major) | 0.51 | 0.59 |
G (Minor) | 0.49 | 0.42 |
χ2 | 0.16 | 0.7 |
p-value | 0.64 | 0.4 |
CETP-(C277T) (TaqIB) | ||
B2B2 | 104 (26%) | 116 (29%) |
B1B2 | 215 (53.75%) | 212 (53%) |
B1B1 | 81 (20.25%) | 72 (18%) |
B2 (Major) | 0.53 | 0.56 |
B1 (Minor) | 0.47 | 0.45 |
χ2 | 2.47 | 0.1 |
p-value | 2.13 | 0.1 |
MTHFRC677T | ||
CC | 358 (89.5%) | 391 (97.75%) |
CT | 38 (9.5%) | 9 (2.25%) |
TT | 4 (1%) | 0 |
C (Major) | 0.94 | 0.98 |
T (Minor) | 0.06 | 0.02 |
χ2 | 6.1 | 0.01* |
p-value | 0.05 | 0.82 |
MTHFRG1793A | ||
GG | 311 (77.75%) | 330 (82.5%) |
GA | 85 (21.25%) | 70 (17.5%) |
AA | 4 (1%) | 0 |
G (Major) | 0.88 | 0.9 |
A (Minor) | 0.12 | 0.1 |
χ2 | 0.47 | 0.06 |
p-value | 3.68 | 0.5 |
MSA2756G | ||
AA | 251 (62.75%) | 294 (73.5%) |
AG | 139 (34.75%) | 103 (25.75%) |
GG | 10 (2.5%) | 3 (0.75%) |
A (Major) | 0.8 | 0.86 |
G (Minor) | 0.2 | 0.14 |
χ2 | 3.32 | 0.1 |
p-value | 3.54 | 0.06 |
MODEL | OR (95% CI) | p-value |
---|---|---|
ESR1 IVS1–397 T/C | ||
Co-dominant | ||
TC vs TT | 1.05 [0.79–1.41] | 0.7 |
CC vs TT | 1.17 [0.73–1.88] | 0.5 |
Dominant | ||
TC + CC vs TT | 1.07 [0.81–1.42] | 0.6 |
Recessive | ||
CC vs TC + TT | 3.39 [0.92–12.42] | 0.6 |
Allelic | ||
C vs T | 1.07 [0.87–1.31] | 0.5 |
ESR1- IVS1–351 A/G | ||
Co-dominant | ||
AG vs AA | 1.23 [0.89–1.70] | 0.2 |
GG vs AA | 1.93 [1.29–2.89] | 0.001* |
Dominant | ||
AG + GG vs AA | 1.40 [1.03–1.90] | 0.03* |
Recessive | ||
GG vs AG+ AA | 1.70 [1.20–2.40] | 0.003* |
Allelic | ||
G vs A | 1.37 [1.12–1.67] | 0.002* |
CETP-(C277T) (TaqIB) | ||
Co-dominant | ||
B1B2 vs B2B2 | 1.13 [0.82–1.57] | 0.4 |
B1B1 vs B2B2 | 1.25 [0.83–1.90] | 0.3 |
Dominant | ||
B1B2 + B1B1 vs B2B2 | 1.63 [0.85–1.59] | 0.3 |
Recessive | ||
B1B1 vs B1B2 + B2B2 | 1.16 [0.81–1.64] | 0.4 |
Allelic | ||
B1 vs B2 | 1.11 [0.91–1.35] | 0.3 |
MTHFR C677T | ||
Co-dominant | ||
CT vs CC | 4.61 [2.20–9.67] | 0.00001* |
TT vs CC | Not possible† | – |
Dominant | ||
CT + TT vs CC | Not possible† | – |
Recessive | ||
TT vs CT + CC | Not possible† | – |
Allelic | ||
T vs C | 5.36 [2.45–10.62] | < 0.0001* |
MTHFR G1793A | ||
Co-dominant | ||
GA vs GG | 1.29 [0.91–1.83] | 0.15 |
AA vs GG | Not possible† | – |
Dominant | ||
GA + AA vs GG | Not possible† | – |
Recessive | ||
AA vs GA+ GG | Not possible† | – |
Allelic | ||
A vs G | 1.37 [1.00–1.90] | 0.06 |
MS A2756G | ||
Co-dominant | ||
AG vs AA | 1.58 [1.16–2.14] | 0.003* |
GG vs AA | 3.90 [1.06–14.34] | 0.03* |
Dominant | ||
AG + GG vs AA | 1.64 [1.22–2.22] | 0.001* |
Recessive | ||
GG vs AG+ AA | 3.39 [0.93–12.42] | 0.1 |
Allelic | ||
G vs A | 1.57 [1.20–2.05] | 0.001* |
Variant ESR1 IVS1–397 T > C/ -351 A > G | Haplotype frequencies | OR (95% CI) | p-value† | |
---|---|---|---|---|
Patients (n = 400) | Controls (n = 400) | |||
C-A | 0.068 | 0.258 | 0.210 [0.153–0.289] | 2.22e-016* |
C-G | 0.278 | 0.073 | 4.906 [3.604–6.678] | 5.00e-015* |
T-A | 0.439 | 0.327 | 1.616 [1.318–1.980] | 3.61e-006* |
T-G | 0.215 | 0.342 | 0.525 [0.420–0.656] | 1.27e-008* |
Variant MTHFR C677T/ G1793A | ||||
C-A | 0.109 | 0.087 | 1.29 [0.93–1.79] | 0.1 |
C-G | 0.833 | 0.901 | 0.57 [0.42–0.77] | 0.0002 |
T-G | 0.050 | 0.011 | 4.71 [2.27–9.76] | 5.18e-006* |
T-A | 0.007 | 0.000 | – | – |
Gene –gene and gene –environment interaction
Interaction Model | TBA | CVC | p-value |
---|---|---|---|
Gene-Gene Interaction | |||
SNP 6 | 0.52 | 8/10 | 0.001 |
SNP 2, 5 | 0.5948 | 10/10 | < 0.0001* |
SNP 2, 3, 5 | 0.6574 | 9/10 | < 0.0001* |
SNP 1, 2, 3, 5 | 0.6563 | 6/10 | < 0.0001* |
SNP 1, 2, 3, 5, 6 | 0.7111 | 9/10 | < 0.0001* |
SNP 1, 2, 3, 4, 5, 6 | 0.7186 | 10/10 | < 0.0001* |
Gene-Environment Interaction | |||
A1 | 0.6363 | 10/10 | < 0.0001* |
SNP 5, A1 | 0.6347 | 6/10 | < 0.0001* |
SNP 2, 5, A1 | 0.705 | 10/10 | < 0.0001* |
SNP 1, 2, 5, A1 | 0.7153 | 10/10 | < 0.0001* |
SNP 1, 2, 3, 5, A1 | 0.7103 | 7/10 | < 0.0001* |
SNP 1, 2, 3, 5, 6, A1 | 0.7301 | 9/10 | < 0.0001* |
SNP 1, 2, 3, 5, 6, A1, A2 | 0.7379 | 7/10 | < 0.0001* |
SNP 1, 2, 3, 4, 5, 6, A1, A2 | 0.7403 | 10/10 | < 0.0001* |