Risk factors for Hyperhomocysteinemia for Specic MTHFR C677T Genotypes and Gender in Chinese Population.

Background -- Our previous studies have identied that both gender and genotype of MTHFR C677T were independent variables for plasma homocysteine (Hcy) levels. Based on these results, we want to further explore their systemic information, in order to nd specic risk factors for each genetic group, which might be used as predictors or targeting markers for lowering Hcy levels. Patients and Methods --This cross section study was performed through November 2017 to July 2019. A total of 4534 adults aged 20-75y were selected for this study, and all of them underwent a physical examinations and MTHFR C677T genotyping. Results--The average of Hcy level was higher in TT genotype than CC and CT genotypes (P=0.000). Multiple linear regression analysis found that except the common protective factors (folate and Vit B12) and risk factor (Cr), each group has it specic risk factors for HHcy---female-CT (age, SBP and Hb), female-TT (SBP and AST); male-CC (age, AST and Hb), male-CT (age and AST) and male-TT (SBP, AST and Hb). Conclusion--The plasma Hcy level was inuenced by different risk factors for specic gender and genotype. These risk factors might be useful for prediction or prevention of HHcy in the future.

The aim of this study was to explore the risk factors for HHcy, which might be used as predictors or targeting markers for HHcy. Before this study, there have been several studies which also targeting for exploring risk factors for HHcy (12,14), but they were not strati ed by genders or genotypes (12,14). Our previous studies have found that both gender and genotype of MTHFR C677T were independent variables for plasma Hcy levels. (12,15). And food consumption would pose different in uence on Hcy levels for each gender and genotype (13). Based on these results, we supposed that each gender and genotype may have their speci c risk factors for HHcy.

Study Design and Ethics Approval
The Ethics Committee at PLA General Hospital approved the protocol of this cross-sectional, observational, non randomized study, which was designed in accordance with the principle of the Declaration of Helsinki (clinical trial ID: S2016-098-02). All subjects gave their informed consent to participate in the study. The study was carried out at the Health Management Institute of Chinese PLA General Hospital during the period of November 2017 to July 2019.
Written, informed consent was obtained from all patients to publish their innominate data.

Subjects
A total of 4770 participants who completed a health examination, were enrolled in this study. The inclusion criteria were: aged between 20 and 80, not taking folic acid supplements or using agents that affect vitamin B and folic acid metabolism, such as methotrexate and anticonvulsants, and being free from folic medicine for at least 6 months.
Among those participants, 34 participants with serious renal disease, 54 participants with hepatic disease, and 123 participants with hypothyroidism were excluded from the study. In addition, 25 participants were excluded for the inaccuracy of the results. In all, 1369 females and 3165 males were selected for studying in this cross section study.

Outcome Measures
Assessment of MTHFR C677T Genotype. Genetic polymorphisms MTHFR 677 C→T were detected using gene chip hybrid analysis. Genomic DNA was extracted from the whole blood of the participants using the QIAamp® DNA Mini Kit (CAT No. 51304, Germany). The PCR, hybridization, gene array detection and analysis were conducted strictly according to the manuals of the BaiO genotype detecting gene array kit and equipment (BaiO Technology Corp).
Assessment of Covariate. The patients were subjected to a health examination, including height and weight measurements and blood pressure. Fasting blood was extracted for detection. Plasma Hcy was analyzed by HPLC with uorometric detection (16,17). Folate concentration was measured using a dual count Solid Phase Boil Radio assay (Diagnostic Products, Los Angeles, CA). The vitamin (Vit) B12 was detected by liquid chromatography-tandem mass spectrometry (18). The blood glucose levels were measured using the hexokinase method; serum levels of bilirubin, Alanine aminotransferase (ALT) and Asparagine aminotransferase (AST) were measured using a BM Hitachi 711 Chemistry Analyzer. The total cholesterol (TC), high-density lipoprotein (HDL), low-density lipoprotein (LDH) and TG levels were measured with a colorimetric method (Cobas c 501 autoanalyzer, Roche Diagnostics, Germany). Serum Cr and UA were measured by a modi ed kinetic rate Jaffe reaction method using a Dade Dimension Chemistry Analyzer (Siemens). Cancer biomarkers of CEA and AFP were measured using electrochemiluminescence immunoassay. As the baseline of the Hcy level was remarkably different between sexes, HHcy was de ne distinctively for male ( ≧ 15 µmol/L) and female ( ≧ 10 µmol/L) (12,14).
Standard quality control procedures were performed each day with standard samples (CV < 10%).

Statistical Analysis
Participants were grouped by genders and genotypes. Comparison of the variables was assessed using an ANOVA or Chi-square test. Multiple linear regression analyses were performed between Hcy and the variables with stepwise methods. The standard coe cients for the variable were used to estimate the strength of the association. All tests were two-tailed, and P values less than 0.05 were considered to indicate signi cant differences. The statistical analyses were carried out using SPSS version 17.0 and SAS version 8.02 or 9.1.

Clinical Characteristics
There were 4534 individuals selected for this study, including 3165 males (CC = 724, CT = 1523 and TT = 918) and 1369 females (CC = 320, CT = 683 and TT = 366). There were signi cant difference between males and females in the clinical characteristics (see Supplement Table ).
Further analysis was performed in each gender group and strati ed by genotypes. For males, Hcy level was signi cantly different between 3 genotypes with the mean values of 12.30 ± 3.34 µmol, 12.94 ± 4.57 µmol and 19.37 ± 9.26 µmol for male-CC, male-CT and male-TT, respectively. And other factors were also identi ed with statistical signi cance, such as folate, Vit B12, HDL, TG and height (p < 0.05).

The Risk Factors for HHcy in Each Gender and Genotype
In the males, after adjustment of weight, height, BMI and other variables, we found that folate, VitB12 were negatively correlated with HHcy, while Cr were positively correlated with HHcy. And genotype (CC and CT) was a protective factor for HHcy (see Supplement Table II). After strati ed by genotypes, we found speci c positive risk factors for each genotype, even though they have some common negative factors, such as folate and VitB12. In male CC group, age, AST and Hb were positive risk factors for HHcy; In the male CT group, age and AST were positive risk factors for HHcy; In the male TT group, SBP, AST and Hb were positive risk factors for HHcy (see Table 2). In the females, we also found negative risk factors (genotype, folate and VitB12) and positive risk factor (Cr), after adjustment of weight, height, BMI and other variables (see Supplement Table II). We also found speci c risk factors after strati ed by genotype, though they share the negative factors of folate and VitB12. In the female CT group, one positive risk factor (Cr) was identi ed. In female CT group, 4 positive risk factor (age, SBP, Hb and Cr) were correlated with Hcy level. In female TT group, 3 positive risk factors (SBP, AST and Cr) were found to be correlated with Hcy level (see Table 3).

Discussion
HHcy has been proven to be independent risk factors for hypertension and stroke (19). People have found several factors that would cause HHcy, such as MTHFR C677T polymorphism, folate and vitamin B (20)(21)(22). At present, it is recommended that Hcy concentration should be lowered by supplement of folate and Vit B12 through food nutrition (23) or Synthetic medicine (24). However, this intervention did not show equal effects in all the populations-it is most effective in TT genotype, which carries the highest risk of developing HHcy, but with mild or modest effects in CC and CT genotype. Our previous studies have found that the correlation between Hcy and folate was stronger in male (CC, CT and TT) and female TT group, while the correlation of Hcy and Vit B12 was stronger in female-CC and female-CT groups (13). This give us a hint that different group might have speci c correctable factors that may improve the Hcy level (9).
In this study, we found that Cr was another common risk factor, which was positively correlated with HHcy in all the subjects. Our ndings are in accordance with previous report by Han,et al (25), which found that Cr was a common factor for HHcy in both healthy and hypertensive subjects. Another support for this relationship was that Hcy level could be increased in a dose-response effect by Guanidinoacetic acid (GAA), which is an intermediate in the biosynthesis of Cr (26).
Furthermore, we identi ed some speci c risk factors for each gender and genotype (see Table 4). This is quite unique from the other previous studies, which targeting the whole genotype population (12,25). First, we found that aging was just a risk factor for CC and CT genotypes, but not for TT genotype. Though former research has identi ed age as a positive risk factor for HHcy, yet it was not for all the genotypes. In fact, we observed that participants with TT genotype already had a high level of Hcy at the early age of 20 40y. Therefore, the intervention of decreasing Hcy should be performed at an early age for TT genotype, and it can be intervened later for CC and CT genotypes. SBP is also highly correlated with HHcy, and both SBP and HHcy are independent predictors for the stroke morbidity in hypertension population (27). Data analysis from the National Health and Nutrition Examination Survey (NHANES) showed that Hcy was positively associated with SBP, and this association was stronger in women than in men (28). However, it was controversial in the Hordaland study, which showed that the correlation of Hcy with SBP was really weak, even though positive (29). In this study, we discovered that correlation SBP was positively correlated with Hcy in TT and CT genotypes, but not in CC groups for both genders (see Table 4).This suggested that gene-related HHcy may play an important role for H-type hypertension. However, it does not mean that HHcy would not in uence SBP for CC group, only indicated that some other risk factors might outweigh this relationship.
AST was another positive risk factor for HHcy for male (CC, CT and TT) and female (TT). At present, the association between Hcy and AST were still controversial. Li et al. (30) have investigate the effect of MTHFR gene polymorphisms and serum Hcy and folate level on the hepatic functions in a Chinese hypertensive population, but they did not nd a signi cant correlation between Hcy and AST. However, an inverse correlation was found between Hcy and AST in hemodialysis patients, whose remethylation of Hcy was impaired (31). The inconformity of these studies might come from the selection criteria of the subjects. A strict cohort study may clarify their associations by a much longer observation.
We also found a positive correlation between Hcy and Hb in male (CC and TT) and female (CT). At present, few studies have focus on the association of Hcy and Hb. Schaffer A and his colleague (32) had reported that Hb was one of the positive risk factors for HHcy, but further studies are still needed to con rm this relationship and explore its mechanism.
The strength of this study is that we classi ed people into different gender and genotype groups, both of which are strong independent variables for HHcy. There are several bene cial from this classi cation. First, we exclude some confound bias among groups, such as age, which showed different in uential strength among groups. Second, we discovered some new variables that may in uence the Hcy level, even though they still need further prospective studies to con rm.
However, there are still some limitations for this study. First, there might be more risk factors to be discovered in addition to our ndings. Variables selected for this study was only the physical and laboratory examination. Other factors should be taken into consideration as they were proved to be important for Hcy metabolism, such as smoking, drinking, nutrition, physical exercise, et al. Second, the correlation between HHcy and the risk factors in this cross-section study cannot re ect the causal relationship. A well-designed cohort study or randomized clinical trial is needed to con rm the causality. Third, the result of this single-center study would be corroborated by multi-center collaborations, and it would be more authentic if the result can be veri ed in different centers.
In summary, we have found that except the common protective factors (folate and Vit B12) and risk factor (Cr), each gender and genotype group has it speci c risk factors for HHcy: female-CT (age, SBP and Hb), female-TT (SBP and AST) male-CC (age, AST and Hb), male-CT (age and AST) and male-TT (SBP, AST and Hb). These results might be useful for precise predicting or prevention of HHcy in the future.  Supplementary Files This is a list of supplementary les associated with this preprint. Click to download. supplementarytalbeIII.docx