Vitamin D Level and Vitamin D Receptor Genetic Variation Were Involved in the Risk of Non-Alcoholic Fatty Liver Disease: A Case-Control Study

Background It has been demonstrated that vitamin D receptor (VDR), a key gene in the metabolism of vitamin D (VD), may affect the development of Non-alcoholic fatty liver disease (NAFLD) by regulating VD level and its biological effects. Objectives To investigate the effects of serum VD level, VDR variation, and a combination of VDR SNP and environmental behavior factor on the risk of NAFLD. Methods A total of 3023 subjects from a community in Nanjing were enrolled, including 1120 NAFLD cases and 1903 controls. Serum 25(OH)D3 levels were measured and eight single nucleotide polymorphisms (SNPs) in VDR gene were genotyped. Results Logistic regression analyses indicated that VD sufficiency and VD insufficiency were significantly associated with a low risk of NAFLD (all P<0.05; all P trend <0.05, in a locus-dosage manner). After adjusting for gender and age, VDR rs2228570-A and rs11168287-A alleles were all reduced the risk of NAFLD (all P FDR=0.136, in dominant model; P trend =0.039, combined effects in a locus-dosage manner). The protective effects of two favorable alleles were more evident among subjects ≤40 years, non-hypertension, non-hyperglycemia and non-low high density lipoprotein-cholesterol (all P<0.05). The area under the receiver operating curve of the combination of VDR SNP and exercise time for assessing NAFLD risk was slightly higher than that of only including exercise time or neither (all P<0.05). Conclusion High serum VD levels and VDR variants (rs2228570-A and rs11168287-A) might contribute to a low risk of NAFLD in Chinese Han population. The inclusion of VDR SNP and exercise time could improve the efficiency in assessment of NAFLD risk, which might provide a novel perspective for early screening and preventing NAFLD.


INTRODUCTION
Non-alcoholic fatty liver disease (NAFLD), as the most prevalent liver disease worldwide, comprises a wide disease spectrum ranging from steatosis to nonalcoholic steatohepatitis (NASH) and NASH-related cirrhosis (1,2). The reported prevalence reaches 10-30% in the United States, Europe and Asia (3,4). Most patients with NAFLD are asymptomatic, and only few may complain of nonspecific symptoms, like discomfort, fatigue, and vague right upper abdominal pain (5), but NAFLD can increase the risk of type 2 diabetes mellitus (T2DM) as well as cardiovascular disease (6), and NASH may develop into fibrosis, even causing cirrhosis and hepatocellular carcinoma (7). There are currently no approved therapies for NAFLD (8,9), and it is still necessary to explore the potential influencing factors affecting the development and progression of NAFLD.
Many factors are associated with NAFLD including insulin resistance (IR), metabolic syndrome and genetic variation (10,11). Studies have increasingly found that vitamin D (VD) can affect the development of NAFLD by regulating IR (12), immune inflammation (13), lipid metabolism and target gene expression (14). Vitamin D deficiency is implicated in NAFLD etiology (15,16), insulin resistance (17), visceral obesity, and metabolic syndrome (18). A meta-analysis found that compared to controls, patients with NAFLD had a lower level of VD (0.36 ng/mL) and were 1.26 times more likely to be VD-deficient (19). A population-based case-control study in China found that low serum VD was associated with advanced liver fibrosis in NAFLD patients (20). However, another systematic review and metaanalysis suggested that serum VD level might not change with NAFLD histologic severity (21). A randomized, double-blind, placebo-controlled trial in Italy suggested that although high-dose oral VD supplementation could increase the serum VD level, no effect was shown on hepatic steatosis or metabolic/cardiovascular parameters in T2DM patients with NAFLD (22). Thus, the relationship between VD and NAFLD was controversial.
VD must be transported, hydroxylated and finally combined with vitamin D receptor (VDR) to accomplish its biological roles (23). Serum VD may produce different biological effects due to genetic heterogeneity, which may help to explain why some people are prone to NAFLD, while others are not (even if they have metabolic syndrome components). Thus, exploring the influence of genetic variation on NAFLD related to VD metabolism is necessary. VDR, a key gene in VD metabolic pathway and a member of steroid/thyroid hormone receptor family (24), transcriptionally activates target genes after binding to vitamin D responsive elements localized in the promoter regions (25). These target genes mediate various NAFLDassociated processes, including lipid and glucose metabolism, cholesterol efflux and bile acid homeostasis, hepatic fibrogenesis, cellular differentiation, apoptosis and immune response (26,27).
Notably, NAFLD is considered a complex disease trait such that interactions between the environment and a susceptible genetic host background influence disease development and progression (2). Studying data suggests that genetic background may influence the response to lifestyle modification. For example, reduction in liver fat and liver enzyme levels in response to weight loss was larger in subjects homozygous for the PNPLA3 I148M variant compared to non-carriers (28,29). However, there is little information available on the association between environmental behaviors and VDR polymorphisms in the development and progression of NAFLD. Therefore, the aim of this study was to explore the effects of serum VD level, VDR variation, and a combination of VDR SNP and environmental behavior factor on the risk of NAFLD.

Participants and Study Design
A total of 3023 subjects from a community in Nanjing (Jiangsu, China) were enrolled in this case-control study during July to September 2018. The NAFLD cases were recruited from those diagnosed based on "Guideline of prevention and treatment for nonalcoholic fatty liver disease: a 2018 update". NAFLD can be diagnosed if the following items 1-4 coexist with the fifth or sixth item: (1) no drinking or history of overdose drinking (less than 210g/week ethanol for men and 140g/week for women in the past 12 months); (2) excluding drug hepatitis, hepatitis C virus genotype 3 infection, hepatolenticular degeneration and other specific diseases that could result in fatty liver; (3) serum levels of transaminase and g-glutamyl transpeptidase (g-GT) increase mildly to moderately (<5 times above the upper normal limit), usually presenting as an increase of alanine aminotransferase (ALT); (4) metabolic syndrome constituents such as visceral obesity, hyperglycemia, blood lipid disorder and hypertension; (5) the result of liver imaging study meets the imaging diagnostic criteria of diffuse fatty liver; (6) the histological findings of liver biopsy meet the pathological diagnostic criteria of fatty liver disease. The non-NAFLD controls were collected from the same community during the study period and randomly assigned to the control group. The constituent ratios of gender and age between cases and controls were considered similar, according the results of frequency-matching.
Excluded were: (1) less than 18 years old; (2) subjects with infection, acute or chronic gastrointestinal diseases, autoimmune diseases or malignant tumors; (3) subjects with history of other viral hepatitis, alcoholic liver disease or primary liver cancer; (4) subjects of excessive drinking (alcohol consumption ≥30g/d in males and ≥20g/d in females); (5) subjects who received a liver transplant within the previous year, or had complications of advanced liver disease (varicose veins, ascites, etc.); (6) subjects with drug-induced fatty hepatitis; (7) subjects with history of psychiatric disorders.
By reviewing the literature, we assumed the frequency of gene mutation in the general population was 20%, odds ratio (OR) was 1.5, two-sided test a was 0.05, and power of test (1-b) was 90%. Therefore, the minimum sample size was estimated by NCSS-PASS 11 software (Dawson edition; Kaysville, UT) to be 784. This study had a sample size large enough to guarantee the production of reliable results.
The current study protocol was in accordance with the Declaration of Helsinki, and was approved by the Institutional Ethics Review Committee of Nanjing Medical University (Nanjing, China). Written informed consent was obtained before blood test and genetic analysis.

Collection of Basic Data and Blood Samples
Self-designed questionnaires and an electronic medical record system were used to collect the demographic and clinical characteristics of all participants. 5-mL ethylene diamine tetraacetic acid (EDTA) anticoagulant venous blood was collected from each subject in the morning after an overnight fast. Within 2 hours, the serum and blood cells in each blood sample were separated and frozen at -80°C until further serological tests and genotyping assays.

Serum 25(OH)D 3 Level Detection
In two groups (gender and age matched at a ratio of 1:1, 336 subjects in the NAFLD group and 336 in the control group), the serum 25(OH)D 3 level was measured by enzyme-linked immunosorbent assay (ELISA) according to the manufacturer's instructions (Human 25-Dihydroxy vitamin D3 (25(OH)D3) ELISA Kit; Jin Yibai Biological Technology Co., Ltd.; Nanjing, China).

Statistical Analysis
All statistical analyses were processed using SPSS (version 22.0, SPSS Inc., Chicago, IL, USA) and MedCalc (Version 19.1, Ostend, Belgium). Distributions of demographic and clinical characteristics of two groups were compared using c 2 -test, student's t test or Mann-Whitney U test, wherever appropriate. Logistic regression analysis was used to calculate odds ratio (OR) and 95% confidence interval (95% CI) for quantifying the association of serum 25(OH)D 3 level with the risk of NAFLD. The correlation of serum 25(OH)D 3 level with VDR SNPs was assessed using general linear regression model adjusted for gender and age. The Hardy-Weinberg equilibrium (HWE) was tested using a goodness of-fit c 2 -test among the control subjects. The relationships between VDR SNPs and the risk of NAFLD were analyzed by dominant model (heterozygote + mutant homozygote VS. wild homozygote), recessive model (mutant homozygote VS. wild homozygote + heterozygote), and additive model (mutant homozygote VS. heterozygote VS. wild homozygote), respectively. For multiple SNPs comparisons, false discovery rate (FDR) correction was used and the P FDR value ≤ 0.25 was regarded as modest confidence that the correlation represented a positive result (30). Subgroup analysis was performed for positive SNPs, and Q test was performed to calculate the heterogeneity between subgroups. The area under the receiver operating curves (AUROCs) was performed to assess the predictive power of the combination of positive SNPs, environmental behavior factors and mainly related clinical factors for NAFLD risk. A two-tailed test with a P value < 0.05 was regarded as statistically significant in all analyses.

Associations Between VDR SNPs and Serum 25(OH)D 3 Level
Serum 25(OH)D 3 levels were Lg transformed into an approximately normal distribution, and SNPs were coded in an additive genetic model. The general linear regression analysis indicated that there were no significant associations between eight VDR SNPs and serum 25(OH)D 3 levels after adjusting for gender and age (all P>0.05) (Supplementary Table 1).

Association Between VDR SNPs and NAFLD Risk
The genotype distributions of the eight SNPs in both groups were shown in Table 3. After adjusting for gender and age, logistic  Table 2). In addition, the combined effects of VDR variants rs2228570 and rs11168287 on the risk of NAFLD were estimated by the number of favorable alleles from the two SNPs, as shown in Supplementary Table 3. The subjects were first divided into three groups with "0", "1-2" and "3-4" favorable alleles. Compared with those who had 0 favorable alleles, subjects with "3-4" favorable alleles had significantly decreased risk of NAFLD (adjusted OR=0.750, 95%CI=0.577-0.974, P=0.031) and the more favorable alleles, the lower NAFLD risk, suggesting a significant locus-dosage effect of the combined alleles on NAFLD risk (P trend =0.039). The subjects were then distributed into two groups with "0" and "1-4" favorable alleles, and we found that the presence of "1-4" alleles was related to a 0.798-fold lower risk of NAFLD (adjusted 95% CI=0.644-0.990, P=0.040).

DISCUSSION
In the current study, our findings suggested that high serum vitamin D level, VDR rs2228570-A and rs11168287-A were associated with a decreased risk of NAFLD, and the combination of VDR rs2228570 and exercise time was more effective in the risk assessment of NAFLD.
Accumulative evidence suggests that VD deficiency is highly prevalent among the general population in China (32), and low VD level is a risk factor of NAFLD (33). VD poses protective effects on many other metabolic-related diseases, such as obesity, hypertension, insulin resistance, type 2 diabetes, metabolic syndrome and cardiovascular disease (34)(35)(36). Consistently, our study manifested a high prevalence of NAFLD (59.2%) in the subjects with low serum 25(OH)D 3 level (<20ng/mL), and VD deficiency might increase the risk of NAFLD. The regulatory mechanism of VD level and VDR activity in the development of NAFDL has not been fully elucidated yet (37,38). Therefore, the present study provides a new insight into the association between VDR genetic mutation and NAFLD risk.
The VDR SNPs rs7975232, rs2228570 were reported to be associated with VD deficiency (39,40). However, other research showed no association of rs7975232, rs11568820, rs11574129 with serum 25(OH)D 3 in a Han Chinese population (41). A casecontrol study in Caucasian population found no correlation between VD level and rs2228570 genotype (42). In our study, no significant associations were found between eight VDR polymorphisms and 25(OH)D 3 level either. Possible explanations include geographical location, racial backgrounds and sunlight exposure (43).
Our study suggested that the SNPs of rs2228570-A and rs11168287-A in VDR gene exhibited an association with the decrease in NAFLD risk, with an evident gene-gene combined effect. Metabolic impairment and alteration of the glucoseinsulinhomeostasis are the primarily pathogenesis of NAFLD (44). Evidence indicated that the rs2228570 polymorphism of VDR gene was associated with the risk of fasting glucose in a Chinese Han population (45). It was found that the expression of VDR mRNA in the liver of obese individuals with biopsy-proven NAFLD was higher than that of non-NAFLD (46). Study reported that the combination of rs2228570 (FokI), rs1544410 (BsmI) and rs731236 (TaqI) was involved in the risk of T2DM in north Indians (47). However, another study showed that VDR-rs2225780 genetic variation was not associated with T2DM in a Caucasian population (42). The possible explanation might have various ethnic groups. The Jackson Heart Study indicated that VDR variants are associated with abdominal visceral adipose tissue volume and adiponectin concentrations, but not with BMI or WC in African Americans (48), a finding that is consistent with the stratified analysis of the combined effect of the two positive SNPs in this study. Although there is no research on rs11168287  polymorphisms and NAFLD, we found that rs11168287 is situated in the peak of H3k4me1 and near the peak of transcription levels by searching the UCSC genome browser (https://genome.ucsc. edu), which means that rs11168287 mutation might affect the serum VD level and its biological effects including insulin sensitivity, lipid metabolism as well as immune inflammation by dysregulating the transcription and expression of VDR gene, and ultimately affect the risk of NAFLD. Further study is warranted to identify the role and function of these VDR polymorphisms in VD metabolism and NAFLD risk. This study also demonstrated that age ≤40 years, exercise time ≥150 min/week, and rs2228570-A were independent protective factors of NAFLD; visceral obesity, hypertension, hypertriglyceridemia, Low HDL-C and ALT >40U/L were independent risk factors of NAFLD, which is consistent with the results of previous studies (49)(50)(51)(52). Based on these independent factors, we constructed combined factors to assess NAFLD risk. The results showed that the assessment efficiency of combined factor 3 (including age, visceral obesity, blood pressure, TG, HDL-C, ALT, exercise time and rs2228570) in NAFLD risk was higher than that of combined factor 2 (including age, visceral obesity, blood pressure, TG, HDL-C, ALT and exercise time) and combined factor 1 (including age, visceral obesity, blood pressure, TG, HDL-C and ALT). There is no research on the combination of genetic and environmental behavioral factors to evaluate the risk of NAFLD in Chinese population so far. A study of NAFLD risk in Italian obese children and adolescents found that combining genetic variants with clinical risk factors improved the predictability of NAFLD (53). For a disease related to heredity and environment, the predictive power of genetic-environmental factors is reasonably stronger than a single one. However, in our study, compared with the AUROC of combined factor 1 or combined factor 2, the AUROC of combined factor 3 only increased slightly, probably due to the weak effect of a single SNP on disease risk (54). In spite of this, we cannot ignore the effect of individual differences in genetic mutation on disease susceptibility. Once the risk of a particular genotype and environmental exposure combination is known, medical interventions, including medical surveillance, lifestyle advice, diet or drug treatment, could then be taken for high-risk groups or individuals to prevent disease (55,56).
There are a few limitations in our study. Firstly, based on the case-control study, we collected the data of the subjects and measured their VD level, which may not represent the average level of different seasons. A convenient and inexpensive ELISA method was used to detect 25(OH)D 3 levels in this study. Although it is not as accurate as liquid chromatography tandem mass spectrometry (LC-MS), it can reflect the level of 25(OH)D 3 to some extent (41,57). Secondly, only subjects from one community were enrolled, which may not be representative enough. In response to this problem, the frequency-matching of gender and age was used in the design stage, and multivariate analysis and stratified analysis were carried out to control the influence of the confounding factors to a certain extent. Finally, we only selected one key gene in the VD metabolic pathway, which may not be able to fully analyze the relationship between genetic factors and NAFLD risk. It is necessary to further explore the impact of polygenic polyloci and their combination with environmental factors on disease risk in a multicenter population of different races.
In conclusion, our results supported that high serum VD levels and VDR variants (rs2228570-A and rs11168287-A) might be involved in a low risk of NAFLD in the Chinese Han population, and a combination of VDR SNP and exercise time could improve the efficiency in assessment of NAFLD risk. These findings might provide new insight for risk evaluation of NAFLD and early screening of high-risk population.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding author.

ETHICS STATEMENT
The studies involving human participants were reviewed and approved by The Institutional Ethics Review Committee of Nanjing Medical University (Nanjing, China). The patients/ participants provided their written informed consent to participate in this study.