Introduction
Nonalcoholic Fatty Liver Disease (NAFLD) is a clinicopathological syndrome characterized by excessive deposition of fat in liver cells, which is closely related to insulin resistance and genetic susceptibility and is caused by alcohol and other clear liver damage factors.1,2 The risk factors for NAFLD include a high-fat diet, a high-calorific diet, a sedentary lifestyle, insulin resistance, and metabolic syndrome,3–5 which are all risk factors for cardiovascular disease.2,6 Coronary artery disease (CAD) refers to coronary artery atherosclerosis caused by lumen stenosis or occlusion, resulting in myocardial ischemia, hypoxia, or necrosis caused by heart disease. Cardiovascular diseases have become the main cause of death globally, with more than 17.6 million deaths in 2016, and the number is expected to grow to more than 23.6 million by 2030.7 NAFLD and CAD are complex diseases resulting from the presence of susceptibility genes combined with environmental exposure.
MTHFR rs1801133 and rs1801131 are the most common genetic mutations of methylenetetrahydrofolate reductase (MTHFR).8 PolyPhen was used to predict the effect of the SNP site on proteins, the results showed that rs1801131 and rs1801133 may lead to impaired protein function, which may affect the function of MTHFR. MTHFR is the critical enzyme in folate 1-carbon and homocysteine (Hcy) metabolism.9,10 It has been reported that increased serum Hcy levels may affect intracellular fat metabolism and promote liver fat infiltration, leading to NAFLD.11 Studies by Xie Jun et al.10 showed that a history of high Hcy is an independent risk factor for cardiovascular and cerebrovascular diseases. Increased circulating levels of homocysteine accelerate atherosclerosis through several mechanisms.10,11 Some studies support the association of polymorphisms with susceptibility to NAFLD and CAD,8,12,13 while others do not.14–16
This study aims to explore the association between MTHFR gene rs1801131 and rs1801133 polymorphisms and the susceptibility of NAFLD and CAD.
Subjects and methods
Study subjects
This case-control study was approved by the Qingdao Hospital Ethics Committee (Approval NO. 2017-20), and was based on the principles of the Declaration of Helsinki and its appendices.17 All the subjects were informed and signed an informed agreement upon joining this study. From June 2018 to June 2019, a total of 556 patients from Qingdao Municipal Hospital participated in the study, including 103 NAFLD patients, 176 CAD patients, 94 patients with NAFLD complicated with CAD (NAFLD+CAD), and 183 healthy controls. The NAFLD patients were diagnosed according to the Guidelines of prevention and treatment of nonalcoholic fatty liver disease (2018),18 while the CAD patients were diagnosed according to the Guidelines for Diagnosis and Treatment of Stable Coronary Heart Disease.19 None of the patients with abnormal blood glucose content in this study was diagnosed with diabetes.
Biochemical analyses
Basic clinical information was collected such as sex, age, height, and weight. The body mass index (BMI) could be calculated by mass (kg)/height (m2). Fasting blood was taken from the subjects to test their biochemical parameters, such as alanine aminotransferase (ALT), aspartate aminotransferase, fasting plasma glucose (FPG), triglyceride (TG), total cholesterol (TC), high-density lipoprotein (HDL), low-density lipoprotein (LDL), gamma-glutamyl transpeptadase (GGT), alkaline phosphatase (ALP), total bilirubin.
Genomic DNA extraction and genotyping
Whole blood genomic DNA was extracted (blood genomic DNA extraction kit; Beijing Bomiao Biotechnology Co. Ltd, Beijing, China) and stored at 20°C. MTHFR rs1801133 and rs1801131 were genotyped by the polymerase chain reaction combined with sequencing, and specific steps were described in the references.20 Primer sequence of MTHFR was as follows: rs1801133, 5′-ACGTTGGATGCTTGAAGGAGAAGGTGTCTG-3′ and 5′-ACGTTGGATGACACGTTGGATGCTTCACAAAGCGGAAGAATG-3′; rs1801131, 5′-ACGTTGGATGTGAAGAGCAAGTCCCCCAAG-3′ and 5′-ACGTTGGATGCCGAGAGGTAAAGAACGAAG-3′. MTHFR rs1801131 showed that there were three genotypes: AA, CC, and AC, and MTHFR rs1801133 showed that there were three genotypes: TT, CC, and CT.
Statistical analysis
The data were analyzed using SPSS version 26.0. Pearson’s χ2 test was used to analyze the Hardy-Weinberg balance. Genotypes, allele frequencies, and other qualitative data comparisons were tested by Pearson’s χ2 test. After normality tests, continuous variables were expressed as mean ± standard deviation or median (interquartile range) for normal and abnormal distributed parameters, respectively. The measurement data were tested by the t-test and Wilcoxon rank sum test. The association between SNPs and the risk of NAFLD and CAD was estimated by computing odds ratios (ORs) and 95% confidence interval (95% CI). p < 0.05 was statistically significant.
Results
Demographic and clinical characteristics
The general clinical data and biochemical indicators were compared in Table 1. The NAFLD patients had higher BMI values and serum levels of FPG, ALT, GGT, TC, TG, and LDL than the healthy controls (all p < 0.05), with the two groups matched for gender (all p > 0.05); The CAD patients had higher BMI values and serum levels of FPG, ALT, GGT, and ALP than the healthy controls, besides, the serum level of TC, HDL, and LDL in CAD patients was significantly lower compared to the healthy controls (all p < 0.05), and the two groups were matched for gender and age (all p > 0.05); The NAFLD+CAD patients had higher BMI values and serum levels of FPG, ALT, GGT, and ALP than the healthy controls, besides, the serum level of HDL and LDL in CAD patients was significantly lower compared to the healthy controls (all p < 0.05).
Table 1Association of non-genetic variables in the study subjects
| Healthy controls (n = 183) | NAFLD (n = 103) | CAD (n = 176) | NAFLD+CAD (n = 94) | p0 |
|---|
| Male/Female | 104.00/79.00 | 69.00/34.00 | 116.00/60.00 | 68.00/26.00# | 0.055 |
| Age, y | 47.00 (40.00, 57.00) | 43.00 (38.00, 45.00)# | 66.00 (59.20, 75.75) | 63.00 (57.00, 68.00)# | <0.001 |
| BMI, kg/m2 | 23.60 ± 3.19 | 26.24 ± 2.56# | 24.59 ± 3.22# | 25.08 ± 2.67# | <0.001 |
| FPG, mmol/L | 4.57 (4.06, 5.05) | 4.85 (4.52, 5.21)# | 5.21 (4.55, 6.43)# | 5.42 (4.80, 6.07)# | <0.001 |
| ALT, U/L | 19.02 (13.36, 26.58) | 22.67 (18.30, 39.44)# | 21.85 (14.98, 32.22)# | 22.67 (15.36, 32.45)# | <0.001 |
| AST, U/L | 20.87 (18.84, 25.04) | 22.20 (18.77, 26.21) | 22.34 (17.08, 34.49) | 21.50 (16.80, 32.10) | 0.515 |
| GGT, U/L | 22.43 (16.45, 30.44) | 30.09 (20.19, 45.27)# | 27.35 (18.75, 41.58)# | 26.11 (18.25, 43.93)# | <0.001 |
| ALP, U/L | 69.31 (55.98, 83.91) | 67.36 (57.40, 79.17) | 82.71 (64.59, 107.38)# | 82.50 (70.99, 98.06)# | <0.001 |
| TC, mmol/L | 5.00 (4.20, 5.64) | 5.44 (4.96, 5.99)# | 4.48 (3.77, 5.35)# | 4.25 (3.83, 5.51) | <0.001 |
| TG, mmol/L | 1.21 (0.90, 1.94) | 1.49 (1.08, 2.20)# | 1.36 (0.99, 1.86) | 1.35 (0.94, 2.08) | 0.177 |
| HDL, mmol/L | 1.28 (1.07, 1.51) | 1.22 (1.08, 1.35) | 1.01 (0.85, 1.16)# | 1.05 (0.88, 1.19)# | <0.001 |
| LDL, mmol/L | 3.06 (2.64, 3.54) | 3.27 (2.82, 3.59)# | 2.69 (2.07, 3.30)# | 2.51 (2.14, 3.37)# | <0.001 |
Genotypes and alleles distributions of MTHFR rs1801131 and rs1801133
The distribution of MTHFR rs1801131 and rs1801133 polymorphisms in healthy controls was consistent with the Hardy-Weinberg equilibrium (rs1801131: χ2 = 0.094, p = 0.954; rs1801133: χ2 = 0.482, p = 0.786). There was no significant difference in the genotype distribution and allele frequency of rs1801131 among the four groups (NAFLD, CAD, NAFLD+CAD, and Healthy controls) (all p > 0.05) (Table 2).
Table 2Distributions of the MTHFR rs1801131 genotypes and alleles in the study groups
| NAFLD+CAD | NAFLD | CAD | Healthy controls | p0 | p1 | p2 | p3 |
|---|
| Genotypes |
| AA | 79 (73.8) | 80 (76.1) | 139 (77.7) | 138 (74.2) | 0.684 | 1.000 | 0.957 | 0.278 |
| CC | 2 (1.9) | 1 (1.0) | 0 (0) | 3 (1.6) | | | | |
| AC | 26 (24.3) | 24 (22.9) | 40 (22.3) | 45 (24.2) | | | | |
| Alleles |
| C | 30 (14.0) | 26 (12.4) | 40 (11.2) | 51 (13.7) | 0.695 | 0.917 | 0.650 | 0.300 |
| A | 184 (86.0) | 184 (87.6) | 318 (88.8) | 321 (86.3) | | | | |
The genotype distribution of rs1801133 was statistically different among the four groups (NAFLD, CAD, NAFLD+CAD, and Healthy controls) (p = 0.014), while the allele distribution was the same among the 4 groups (p = 0.139). Moreover, there were significant differences in the allele distribution of rs1801133 between the NAFLD+CAD and CAD groups (p2 = 0.021). The genotypes of the three groups (NAFLD, CAD, and Healthy controls) were statistically different from those of the NAFLD+CAD group (all p < 0.05) (Table 3).
Table 3Distributions of the MTHFR rs1801133 genotypes and alleles in the study groups
| NAFLD+CAD | NAFLD | CAD | Healthy controls | p0 | p1 | p2 | p3 |
|---|
| Genotypes |
| TT | 16 (16.0) | 35 (33.0) | 64 (36.2) | 63 (33.7) | 0.014 | 0.009 | 0.001 | 0.002 |
| CC | 13 (13.0) | 16 (15.1) | 23 (13.0) | 29 (15.5) | | | | |
| CT | 71 (71.0) | 55 (51.9) | 90 (50.8) | 95 (50.8) | | | | |
| Alleles |
| T | 103 (51.5) | 125 (59.0) | 218 (61.6) | 221 (59.1) | 0.139 | 0.128 | 0.021 | 0.081 |
| C | 97 (48.5) | 87 (41.0) | 136 (38.4) | 153 (40.9) | | | | |
Analysis of MTHFR rs1801133 genotype model
Analysis of the MTHFR rs1801133 genotypes model showed that the genotype distribution was statistically significant under the dominant model (TT vs CT+CC) and the co-dominant model (TT+CC vs CT) (all p < 0.05). After adjusting for age, BMI, and gender, there was no statistical significance between the NAFLD and NAFLD+CAD groups. (TT vs CT+CC: p1 = 0.074, TT+CC vs CT: p1 = 0.881), but there remained a statistical difference in other groups (all p < 0.05) (Table 4).
Table 4Comparison of MTHFR rs1801133 genotypic distribution under different gene models
| NAFLD | NAFLD+CAD | OR | 95%CI | p1 | CAD | NAFLD+CAD | OR | 95%CI | p2 | Healthy controls | NAFLD+CAD | OR | 95%CI | p3 |
|---|
| Recessive model |
| TT+CT | 90 | 87 | 1.190 | (0.541–2.619) | 0.666 | 154 | 87 | 1.000 | (0.482–2.072) | 0.999 | 158 | 87 | 1.228 | (0.607–2.485) | 0.567 |
| CC | 16 | 13 | | | | 23 | 13 | | | | 29 | 13 | | | |
| Dominant model |
| TT | 35 | 16 | 2.588 | (1.324–5.061) | 0.005 | 64 | 16 | 2.973 | (1.605–5.507) | 0.001 | 63 | 16 | 2.667 | (1.443–4.932) | 0.002 |
| CT+CC | 71 | 84 | | | | 113 | 84 | | | | 124 | 84 | | | |
| Dominant modela |
| TT | 35 | 16 | 1.391 | (0.198–9.786) | 0.740 | 64 | 16 | 3.192 | (1.678–6.071) | <0.001 | 63 | 16 | 3.423 | (1.623–7.222) | 0.001 |
| CT+CC | 71 | 84 | | | | 113 | 84 | | | | 124 | 84 | | | |
| Co-dominant model |
| TT+CC | 51 | 29 | 2.270 | (1.276–4.038) | 0.005 | 87 | 29 | 2.367 | (1.403–3.992) | 0.001 | 93 | 29 | 2.371 | (1.412–3.982) | 0.001 |
| CT | 55 | 71 | | | | 90 | 71 | | | | 95 | 71 | | | |
| Co-dominant modela |
| TT+CC | 51 | 29 | 0.880 | (0.165–4.691) | 0.881 | 87 | 29 | 2.468 | (1.433–4.251) | 0.001 | 93 | 29 | 2.584 | (1.372–4.867) | 0.003 |
| CT | 55 | 71 | | | | 90 | 71 | | | | 95 | 71 | | | |
Association of MTHFR rs1801131 and rs1801133 gene polymorphism with clinical parameters characteristics in all subjects
The clinical data of all participants were compared between carriers and non-carriers of the rs1801131 allele A, and the differences were not statistically significant (p > 0.05).
The clinical data of healthy controls, NAFLD, and CAD patients were compared between the homozygous (TT+CC) and heterozygous (CT) genotypes of the rs1801133, and the differences were not statistically significant (p > 0.05). In the NAFLD+CAD group, FPG levels of different genotypes were statistically different (Dominant model: p = 0.047, Co-dominant model: p = 0.002) (Tables 5 and 6).
Table 5Correlation analysis between rs1801133 genotypes and non-genetic variables in the NAFLD+CAD group under the dominant model
| TT | CC+CT | Statistics (t/z) | p |
|---|
| Age, y | 62.63 ± 7.80 | 61.79 ± 7.57 | 0.405 | 0.687 |
| BMI, kg/m2 | 25.67 ± 2.24 | 24.99 ± 2.66 | 0.958 | 0.341 |
| FPG, mmol/L | 5.13 (4.79, 5.41) | 5.58 (4.82, 6.52) | −1.984 | 0.047 |
| ALT, U/L | 22.50 (13.14, 39.48) | 22.71 (16.03, 32.77) | −0.188 | 0.851 |
| AST, U/L | 22.93 (17.29, 41.75) | 21.60 (16.92, 31.52) | −0.498 | 0.618 |
| GGT, U/L | 29.93 (19.48, 46.60) | 25.64 (18.12, 42.51) | −0.672 | 0.501 |
| ALP, U/L | 83.21 (76.90, 102.59) | 80.69 (69.09, 95.69) | −0.846 | 0.397 |
| TC, mmol/L | 4.25 (3.96, 5.57) | 4.22 (3.78, 5.46) | −0.155 | 0.877 |
| TG, mmol/L | 1.34 (1.00, 1.87) | 1.32 (0.95, 2.11) | −0.141 | 0.888 |
| HDL, mmol/L | 1.06 (0.98, 1.18) | 1.02 (0.85, 1.19) | −1.020 | 0.308 |
| LDL, mmol/L | 2.55 (2.21, 3.36) | 2.50 (2.02, 3.39) | −0.028 | 0.977 |
| TBIL, umol/L | 13.05 (9.93, 14.30) | 14.15 (10.73, 17.10) | −1.133 | 0.257 |
Table 6Correlation analysis between rs1801133 genotypes and non-genetic variables in the NAFLD+CAD group under the co-dominant model
| TT+CC | CT | Statistics (t/z) | p |
|---|
| Age, y | 62.17 ± 8.20 | 61.82 ± 7.36 | 0.212 | 0.833 |
| BMI, kg/m2 | 24.90 ± 2.41 | 25.18 ± 2.68 | −0.485 | 0.629 |
| FPG, mmol/L | 5.01 (4.57, 5.51) | 5.64 (4.89, 6.74) | −3.073 | 0.002 |
| ALT, U/L | 18.15 (14.94, 33.09) | 22.84 (16.63, 32.89) | −0.574 | 0.566 |
| AST, U/L | 21.52 (18.64, 54.64) | 21.88 (16.83, 28.83) | −0.593 | 0.554 |
| GGT, U/L | 25.44 (17.94, 36.64) | 27.09 (18.49, 44.92) | −0.479 | 0.632 |
| ALP, U/L | 82.40 (70.25, 98.11) | 83.72 (70.19, 96.39) | −0.406 | 0.684 |
| TC, mmol/L | 4.27 (3.75, 5.70) | 4.21 (3.79, 5.42) | −0.266 | 0.790 |
| TG, mmol/L | 1.37 (0.95, 2.06) | 1.31 (0.96, 2.13) | −0.562 | 0.574 |
| HDL, mmol/L | 1.03 (0.89, 1.16) | 1.04 (0.85, 1.20) | −0.167 | 0.867 |
| LDL, mmol/L | 2.61 (2.08, 3.35) | 2.46 (2.03, 3.40) | −0.034 | 0.973 |
| TBIL, umol/L | 13.00 (9.95, 14.75) | 14.30 (10.80, 17.70) | −1.516 | 0.130 |
Discussion
The findings of the present study provide a comprehensive understanding of the correlation between MTHFR rs1801131 and rs1801133 polymorphism in MTHFR and the susceptibility to NAFLD and CAD in China.
As mentioned in the introduction, MTHFR, as a key enzyme, is involved in the occurrence and development of NAFLD and CAD diseases by regulating Hcy metabolism. MTHFR polymorphisms may be closely related to NAFLD and CAD susceptibility. Although some studies have shown that the rs1801131 genotype is associated with CAD susceptibility,13–15 other studies showed that MTHFR rs1801131 polymorphism had no significant relationship with CAD.15,21,22 There are also inconsistent results in studies on the correlation between rs1801131 polymorphisms and NAFLD susceptibility.21,23,24 In the Turkish and Italian populations, rs1801131 polymorphism was significantly associated with NAFLD,23,24 while in the Chinese population, rs1801131 polymorphism was not associated with NAFLD.21 In this study, no correlation was found between rs1801131 polymorphism and NAFLD and CAD susceptibility (p > 0.05). Allele A frequencies in this study (86.3%) were consistent with the Chinese Beijing population (A 81.6%).25 This difference may be due to regional, lifestyle, and ethnic differences. Qingdao’s economy is relatively developed: the local people enjoy good nutrition and eat more seafood.
The correlation between rs1801133 polymorphism and NAFLD and CAD susceptibility is also controversial. For MTHFR rs1801133, T allele frequencies in this study (59.1%) were consistent with the Chinese Tianjin population (T 56%)26 and differed from the Chinese Beijing population (T 41.3%).25 Some studies showed that the T allele of rs1801133 gene polymorphism was a risk factor for CAD.25,27 while some Chinese studies showed that the CT genotype might be the susceptibility factor of CAD patients.21 A Meta-analysis conducted by Sun et al. revealed that MTHFR rs1801133 gene polymorphism was implicated in susceptibility to NAFLD.8 Literature shows that the genotype frequency of MTHFR rs1801133 varies greatly by race.28,29 Our study showed that MTHFR rs1801133 gene polymorphism was not associated with the risk of CAD or NAFLD, however, MTHFR rs1801133 polymorphism was associated with the risk of NAFLD complicated with CAD. There are no other studies on the correlation between polymorphism and susceptibility to NAFLD and CAD. According to our results, for healthy people, NAFLD, and CAD patients, rs1801133 polymorphism was associated with the risk of NAFLD combined with CAD disease. In this study, different gene models were used to analyze the genotype distribution of rs1801133 polymorphism. In the codominant model, the CT genotype of MTHFR rs1801133 was a risk factor for NAFLD combined with CAD, while in the dominant model, the CT+CC genotype was a risk factor for NAFLD combined with CAD. This is not completely consistent with other studies on NAFLD or CAD. Considering the complexity of the disease and the absence of relevant references, the rationality of the results of this study cannot be denied.
MTHFR rs1801133 could affect the total serum Hcy level, which might affect the risk of Type 2 diabetes (T2DM).30MTHFR rs1801133 polymorphism was found to be significantly associated with T2DM.31,32 Different meta-analyses showed a significant relationship between rs1801133 polymorphism and T2DM.33,34 Elevated FPG (≥7.0 mmol/L) is currently used to diagnose T2DM.9
In this study, the CT genotype and CC+CT genotype of MTHFR rs1801133 were associated with an increased FPG level in NAFLD+CAD patients (both p < 0.05). Given that rs1801133 polymorphisms were strongly associated with diabetes risk, it was reasonable to influence FPG levels in the patients with NAFLD complicated with CAD. This study has its limitations in that all samples were only collected in Qingdao, China, which has regional limitations. Compared with the south of China, the taste in food of the Qingdao area is heavy; People there like to eat pickled food, the dietary structure protein fat content is higher, and people generally eat more. Qingdao produces seafood, and the seafood intake is higher than in other areas. Also, the diagnosis of fatty liver relied on ultrasound examinations and liver biopsy was not performed.
Conclusion
In conclusion, the CT genotype and CC+CT genotype of MTHFR rs1801133 were the risk factors for NAFLD combined with CAD. The CT genotype of MTHFR rs1801133 was associated with the up-regulation of FPG levels in patients with NAFLD combined with CAD.
Abbreviations
- ALP:
alkaline phosphatase
- ALT:
alanine aminotransferase
- AST:
aspartate aminotransferase
- BMI:
body mass index
- CAD:
coronary artery disease
- FPG:
fasting plasma glucose
- GGT:
gamma-glutamyl transpeptadase
- Hcy:
homocysteine
- HDL:
high-density lipoprotein
- LDL:
low-density lipoprotein
- MTHFR:
methylenetetrahydrofolate reductase
- NAFLD:
nonalcoholic fatty liver disease
- OR:
odd ratio
- TC:
total cholesterol
- TG:
triglyceride
- T2DM:
Type 2 diabetes
- 95% CI:
95% confidence interval
Declarations
Acknowledgement
Not applicable.
Ethical statement
This case-control study was approved by the Qingdao Hospital Ethics Committee (Approval NO. 2017-20), and was based on the principles of the Declaration of Helsinki and its appendices. All the subjects were informed and signed an informed agreement upon joining this study.
Data sharing statement
The data used in support of the findings of this study are available from the corresponding author at [email protected] upon request.
Funding
The work was supported in part by a grant from the National Natural Science Foundation of China (32171277).
Conflict of interest
The authors have no conflict of interests related to this publication.
Authors’ contributions
Study concept and design (XYN and ZY); subjects collection (SH and LCM); acquisition and analysis of data (SH and ZZZ); drafting of the manuscript (SH and ZZZ); the revision of the manuscript (LSS, XYN, and ZY). Huan Song and Zhenzhen Zhao contributed equally to the article and are first authors, while Yongning Xin and Yong Zhou are corresponding authors. All authors have made a significant contribution to this study and have approved the final manuscript.