Vitamin D-Related Genetic Variations and Nonalcoholic Fatty Liver Disease: A Systematic Review

Background: Studies have demonstrated the link between vitamin-D-related genetic variations and nonskeletal outcomes. We aimed to identify all available data on the association of vitamin-D-related genetic variations with nonalcoholic fatty liver disease (NAFLD). Methods: Potentially eligible studies were identified from Embase and Medline databases from inception to June 2022 using a search strategy that comprised terms for “Vitamin D” and “NAFLD”. Eligible studies must report the association between vitamin D-related genetic variations and presence, severity or response to treatment of NAFLD. Data were extracted from each eligible study. Results: A total of 3495 articles were identified. After a systematic review, twelve studies were included. A total of 26 genetic variations were identified. Presence of NAFLD was associated with variations of GC (rs222054, rs222020, rs10011000, rs7041), VDR (rs2228570, rs11168287, rs10783219, rs4752), CYP24A1 (rs3787557, rs6068816, rs2296241, rs2248359) and CYP27B1 (rs4646536). Severity of NAFLD was associated with variations of GC (rs4588), VDR (rs2228570, rs4334089), CYP2R1 (rs10741657), DHCR7 (rs1544410, rs3829251, rs12785878) and CYP24A1 (rs3787557, rs6068816, rs6097809, rs6127119, rs2248359, rs3787554, rs4809960, rs6022999). Response to calcitriol treatment was associated with variation of VDR (rs10735810). Conclusions: Multiple vitamin D-related genetic variations were associated with NAFLD, indicating the role of vitamin D in the pathogenesis of NAFLD.


Introduction
Nonalcoholic fatty liver disease (NAFLD) is defined by hepatic fat accumulation in the absence of any secondary causes such as excessive alcohol consumption and hepatic viral diseases [1]. It is a spectrum of diseases that range from fatty acid accumulation in the liver, inflammation of the liver causing nonalcoholic steatohepatitis (NASH), advanced fibrosis, cirrhosis and hepatocellular carcinoma. NAFLD is the most common chronic liver disease in the world and soon to be the most common indication for liver transplantation [1,2]. Previous studies reported a global prevalence of NAFLD as high as one billion individuals, and 25% of these patients progress to NASH [3,4]. NAFLD is known to be a common comorbidity in obesity and metabolic syndrome given its reported prevalence of 65-85% among patients with obesity [5].
Vitamin D is a steroid hormone responsible for regulating calcium and phosphate metabolism. It is also known to exert multitudes of nonskeletal effects given that vitamin D receptor (VDR) is expressed in various types of tissues and cells, including the skin, skeletal muscle, adipose tissue, endocrine pancreas and immune cells, among others [6,7]. Humans get vitamin D from diets (vitamin D 2 from yeast and mushrooms and vitamin D 3 from animal products) and endogenous synthesis in the skin (vitamin D 3 from 7dehydrocholesterol exposure to UVB). Once entering the circulation, vitamin D (D 2 and D 3 ) gets converted into 25-hydroxyvitamin D [25(OH)D], the major circulating form of vitamin D, by the hepatic enzyme 25-hydroxylase. Then, 25(OH)D gets metabolized by the enzyme 25-hydroxyvitamin D-1α-hydroxylase in the kidney into the active form 1,25(OH) 2 D, which interacts with the intracellular VDR in the target tissues. The 1,25(OH)2D-activated VDR then interacts with the retinoid X receptor (RXR), which selectively recognizes the vitamin D-responsive elements in the promotor sites of the target genes resulting in changes in gene expression [8]. 1,25(OH) 2 D and 25(OH)D both get catabolized by the enzyme 24hydroxylase expressed in multiple tissues into inactive carboxylic acids, which are then excreted via the biliary system [6,7].
Vitamin D deficiency, indicated by low level of serum 25(OH)D of less than 20 ng/mL [12,13] has been shown to be associated with multiple chronic diseases, including cardiovascular disease, diabetes, autoimmune diseases and cancers [6,7,14]. Low level of 25(OH)D is also found to be associated with presence and severity of NAFLD in multiple studies [15,16]. Several underlying mechanisms of the association have been proposed. These include the observation that vitamin D plays a role in modulating the immune system and improving insulin sensitivity [17,18]. However, the causality of this association is still unclarified given that there is limited evidence from clinical trials on the impact of vitamin D supplementation on NAFLD prevention and treatment [19].
Interestingly, variations of genes involved in the vitamin D metabolism pathway described above have been shown to be associated with many conditions, such as coronary artery disease, osteoporosis, diabetes and autoimmune diseases, indicating the interaction between this pathway and health outcomes [20][21][22][23]. Additionally, a number of studies have reported the association between vitamin D-related genetic variations and NAFLD [24][25][26][27][28][29][30][31][32][33][34][35]. The objective of this systematic review was therefore to identify and summarize the results of all available studies that explored the association of vitamin D-related genetic polymorphisms with presence, severity and response to treatment of NAFLD.

Search Strategy
Two investigators (A.J., N.C.) independently searched records indexed in Embase and Medline from inception to June 2022. The search strategy included terms related to nonalcoholic fatty liver disease, vitamin D and genes involved in the vitamin D metabolic pathway, as shown in Supplemental Material S1. The PRISMA guideline for systematic review was followed, as indicated in Supplemental Material S2. No language restriction was applied.

Eligibility Criteria
Eligible studies must be observational studies that investigated the association between presence, severity or response to treatment of NAFLD and genetic variations in vitamin Drelated genes. These genes include DHCR7, CYP2R1, CYP2J2, CYP27A1, CYP3A4, CYP27B1, GC, VDR and CYP24A1.
Two investigators (A.J., B.P.) independently reviewed the titles and abstracts of retrieved records. Records that clearly did not fulfill the eligibility criteria based on type of article, study design or outcome of interest were excluded at this stage. Then, two investigators (A.J., B.P.) independently evaluated the full text of the remaining records for their final eligibility. The quality of each included study was assessed using the Newcastle-Ottawa quality assessment scale for the case-control study [36], which was performed by two investigators (AJ, BP). Different opinions in the eligibility and quality assessment of the records were resolved by discussion with the senior investigator (N.C.).

Data Extraction
Data from each eligible record were extracted using the standardized data collection form, which contained the following information: last name of the first author, country of the study, number of participants, evaluation of presence and/or severity of NAFLD, mean age of the participants, percentage of female participants and reported association of vitamin D-related genetic variations with outcomes.

Search Results
A total of 3495 records were identified from the electronic search. After removal of 523 duplicates, 2972 records underwent title and abstract review. A total of 2941 records were excluded at this stage as they clearly did not fulfill the eligibility criteria based on type of article, study design and outcome of interest, leaving 31 records for full-text review. A total of 19 records were further excluded at this stage since they did not report the outcome of interest. Finally, a total of 12 studies fulfilled the eligibility criteria [24][25][26][27][28][29][30][31][32][33][34][35].  [26][27][28], and the rest are from Australia [24], Germany, Iran [33], Japan [25] and the United States [34]. The average age of participants

Discussion
This is the first systematic review that explored the relationship between vitamin D-related genetic variations and NAFLD. Our systematic review revealed the association of presence, severity and response to treatment of NAFLD with variations of several genes in the vitamin D metabolic pathway, which include DHCR7, CYP2R1, CYP24A1, CYP27B1, GC and VDR. These findings support the notion that the vitamin D signaling pathway may play a significant role in the pathogenesis of NAFLD.
In fact, 1,25(OH) 2 D, the active form of vitamin D is known to have anti-inflammatory and antifibrogenic effects by inhibiting proinflammatory cytokines production (i.e., interleukin-1, interleukin-6 and tumor necrosis factor-alpha), enhancing anti-inflammatory cytokines production (i.e., interleukin-10 and adiponectin) and suppressing the function hepatic stellate cells [17,37,38]. These actions could therefore slow down the process of liver inflammation and fibrosis. In addition, 1,25(OH) 2 D was shown to enhance insulin receptor expression, thereby mitigating insulin resistance, which is known to be a major component of NAFLD pathogenesis [39,40].
It is worth noting that multiple observational studies revealed the association between low level of serum 25(OH)D and increased risk and severity of NAFLD [15]. Nevertheless, the causality and clinical significance of this association remain undetermined given that the association could be due to confounding effects (i.e., limited physical activity and high body mass index) as well as reverse causation since hepatic steatosis is shown to be associated with decreased activity of the 25-hydroxylase, which can result in decreased circulating 25(OH)D [15,16]. A few randomized controlled trials have shown that vitamin D supplementation can improve hepatic steatosis and insulin resistance and decrease biomarkers of inflammation in adults with NAFLD [41]. However, a mendelian randomization study by Wang et al. demonstrated no causal association between serum 25(OH)D concentration and presence of NAFLD [32].
Alteration of any steps of vitamin D metabolism could possibly affect levels and functions of vitamin D. For example, previous genome-wide association studies reported variations in the DHCR7(rs12785878), GC (rs2282679), CYP2R1(rs10741657), VDR (rs2228570, rs1544410, rs7975232, rs731236) and CYP24A1 (rs17216707) genes were associated with serum 25(OH)D level [42,43]. Another meta-analysis found that genetic variations of the GC (rs2282679, rs4588, rs1155563, rs7041) and CYP2R1 genes (rs10741657, rs10766197, rs2060793) were associated with vitamin D levels in more than 50% of the respective studies [44]. Therefore, variations in genes related to vitamin D metabolism may eventually affect the risks of NAFLD from multiple possible pathophysiologies mentioned above. It has been shown in an animal model that mice lacking VDR in the liver had increased hepatic steatosis and insulin resistance, as well as diminished the protective effect of vitamin D supplementation on NAFLD [45]. Mechanistic experiments demonstrated that the VDR interaction may improve lipid metabolism by interacting with the hepatocyte nuclear factor 4 α (HNF4α) [45]. Based on this observation, it is possible that genetic variations in the VDR may affect VDR expression in the liver, thereby modifying the risk of NAFLD. Data on animal models associated with other genes in the vitamin D metabolic pathway are, however, lacking.
Taken together, the relationship between vitamin D and pathogenesis and progression of NAFLD remains unclarified and may be complex. The observation that genetic variations of vitamin D-related genes were associated with presence, severity and response to treatment of NAFLD provides additional insights, as it may indicate interindividual differences in responsiveness to vitamin D. This can be supported by the observation by Barchetta et al. [46] in patients with NASH that vitamin D receptor expression on cholangiocytes was negatively correlated with steatosis severity, lobular inflammation and NAFLD score. Furthermore, the concept of individual responsiveness to vitamin D has been introduced based on findings from clinical trials that revealed differences in the degree of genome-wide expression in peripheral blood mononuclear cells as well as metabolomic profiles in response to vitamin D supplementation [47][48][49]. Future studies are warranted to further explain the impact of vitamin D-related genetic variations on metabolic outcomes.
The results of this systematic review may have some research implications as the reported genetic variations associated with NAFLD may be used as a reference for future animal models to further elucidate the link between vitamin D and NAFLD pathogenesis. In addition, these variations may be considered novel markers for determining individuals at risk for developing NAFLD. However, there are certain limitations of this systematic review that should be acknowledged. Most of the included studies are small in sample size, and many of them did not adjust for confounders as only five studies were performed in a large-scale cohort [24,31,32,34,35], and four studies conducted robust multivariate analysis [25,30,31,35]. Notably, none of the included studies considered calcium supplementation, a potential confounder that could affect NAFLD pathogenesis, in their multivariate analyses [50]. More importantly, none of the reported significant genetic variations were confirmed to be associated with outcomes in more than one study. All these could have jeopardized the reliability of the findings. Further studies are required to verify the findings of these studies.

Conclusions
This systematic review identified 26 vitamin D-related genetic variations in the DHCR7, CYP2R1, CYP24A1, CYP27B1, GC, and VDR genes to be associated with presence, severity or response to treatment of NAFLD. However, the confidence of these findings was relatively limited awaiting confirmation by future studies.

Conflicts of Interest:
The authors declare no conflict of interest.