Given that the prevalence of non-alcoholic fatty liver disease (NAFLD) covers higher than 25% of the population, it has become the most common chronic liver disease worldwide (1, 2). The onset of NAFLD is characterized by an imbalance in lipid metabolism, leading to the accumulation of lipid droplets in hepatocytes, a condition that progresses to non-alcoholic steatohepatitis, fibrosis, cirrhosis, and ultimately hepatocellular carcinoma (HCC) (3–5). Obesity, type 2 diabetes (T2DM), dyslipidemia, hypertension, and cardiovascular disorders, etc. were linked strongly to NAFLD (6, 7). Over the last decade, there has been growing interest on the role of single nucleotide polymorphisms (SNPs) in the progression of NAFLD. Multiple SNPs, including patatin-like phospholipase domain-containing protein 3 (PNPLA3, rs738409), transmembrane 6 superfamily member 2 (TM6SF2, rs58542926), etc., were found to be closely related to the incidence and progression of NAFLD (8, 9). Furthermore, they were also associated with reduced risk of atherosclerotic cardiovascular disease (ASCVD), which is the major cause of death among NAFLD patients (10–13). The effect of SNPs deepened our understanding of NAFLD and the relationship between NAFLD and cardio-metabolic disease.

Membrane bound O-acyltransferase domain containing 7 (MBOAT7) is a multi-transmembrane protein and an important member of the “Lands’ Cycle” of membrane phosphatidyl chain remodeling (14). In 2015, a genome-wide association study identified that the genetic variant near MBOAT7 (rs641738 C>T) was a risk factor for alcohol-related cirrhosis (15). Subsequently, studies conducted on NAFLD/NASH revealed that MBOAT7 rs641738 C> T was positively related to hepatic fat content and histological severity (16) only in European descent (17). Loss or variant of MBOAT7 reduced liver mRNA and protein expression and consequently led to changes in phosphorylated inositol species, which potentially contributed to liver injury (16, 18).

The clinical significance of MBOAT7 rs641738 C>T variant on NAFLD has been evaluated in various races and age groups; however, its impact on the elderly population remained unclear. In the current study, the relationship between MBOAT7 rs641738 variant with the risk of NAFLD and hepatic fibrosis in an elderly population. Meanwhile, the correlation of the rs641738 T allele with metabolic traits and ASCVD was also analyzed. Furthermore, we explored the effect of rs641738 variant on serum ANGPTL3 level in order to explain the link between MBOAT7 with ASCVD.

This was the first study on the significance of the MBOAT7 rs641738 variant in an elderly population-based cohort. We found that the MBOAT7 rs641738 T allele was associated with higher ALT and significant fibrosis mainly in elderly male NAFLD patients. Furthermore, the TT genotype was associated with a decreased risk of T2DM and MetS in NAFLD patients and with a decreased risk of ASCVD and obesity in the total population. In addition, serum ANGPTL3 levels were significantly lower in TT allele carriers compared with CC genotype carriers, suggesting that ANGPTL3 may be the key molecular link between MBOAT7 variant-driven NAFLD and ASCVD.

Age is an important influencing factor for NAFLD. The prevalence of NAFLD peaks between 40 and 50 years old in men and 60 and 69 years old in women and then slightly decrease in people older than 70 years (27). The severity of NAFLD was also affected by age. A Hongkong study found that being older than 50 years old was one of the risk factors for NAFLD-related cirrhosis (28). Miyaaki et al. reported that being older than 60 years old was a risk factor for severe fibrosis (29). Although the subjects in our study were not a population with the highest prevalence rate of NAFLD, they might have additional advantages: (1) the severity of NAFLD seemed to peak in elders; (2) metabolic-associated diseases, such as T2DM and ASCVD, were highly prevalent in the population and facilitate the observation of their correlation with MBOAT7 rs641738 variants. Therefore, the elderly seemed to be better subjects to evaluate the association of gene variants with metabolic disease. As shown in Table 1 , the prevalence rates of ASCVD and T2DM were as high as 35.28% and 41.58%.

NAFLD is a disease with high heterogeneity in which genetic predisposition plays an important role (30). Some studies have found several genes to be the major genetic determinants of NAFLD, including PNPLA3, TM6SF2, HSD17B13, and GCKR(31, 32). These gene variants combined with metabolic factors, such as obesity or diabetes, were proven to promote the progression of NAFLD by aggravating liver steatosis, inflammation, fibrosis, and hepatocarcinoma (30, 33). As mentioned above, MBOAT7 rs641738 C>T was an important inheritable factor in NAFLD incidence and progression (34–38). However, the relationships were mainly found in European descendants and not in Asians(Teo et al.; 17, 39–43). Only one study discovered that rs641738 C>T increased NAFLD occurrence in Chinese with an average age of 63 years old, and the frequency of the T allele was 43.42%. However, there was no relationship between the variant and fibrosis in the population (44). Different from the results, we found that the MBOAT7 rs641738 T allele promoted NAFLD inflammation and fibrosis but not NAFLD onset. As we all know, the effect of gene variants was not comparable in different ethnic groups due to the diversity of frequencies, penetrance, and relative risk. The frequency of the T allele in our cohort was 46.36%, which was higher than other Asian cohorts. In one Japanese cohort, two Korean cohorts, and one Taiwan children cohort, the T allele frequencies were 22.00% (39), 38.20% (43), 38.50% (40), and 19.60% (41), respectively. In European descents, the frequency was as high as 58.20% 16). Obviously, the higher prevalence of the variant facilitated the discovery of the effect of the variant.

The MBOAT7 gene encodes an acyltransferase that specifically esterifies arachidonic acid-CoA to lysophosphatidylinositol (LPI), thereby producing the main molecular species of phosphatidylinositol (PI) in the cell membrane (45). Thangapandi et al. (18) discovered that MBOAT7 deletion in hepatocytes can lead to the disruption of the intracellular PI side-chain remodeling pathway, which in turn promoted the pathological progression of liver fibrosis by upregulation of ECM genes in a high-fat, methionine-low, choline-deficient diet-fed mouse model of NAFLD. In addition, imbalance in LPI 18:0 levels have also been shown to be associated with the presence of fibrosis in human MBOAT7 rs641738 TT genotype carriers and in MBOAT7^Δhep mouse (18). The treatment of obese mice with exogenous LPI 18:1 promoted the expression of pro-fibrotic genes in the liver, and LPI lipids increased hepatic inflammation and fibrotic gene expression in an MBOAT7-dependent manner (46). Furthermore, circulating LPI levels have also been demonstrated to be significantly higher in patients with liver fibrosis compared to healthy individuals (46). The above-mentioned studies also demonstrated that MBOAT7-LPI-PI plays a key role in the development of fibrosis. NAFLD is an obvious sexual dimorphic disease (47, 48). The prevalence and severity of NAFLD are increasing in men more than in reproductive-age women. However, the incidence rate and severity of NAFLD were comparable or even worse than in men after menopause (49). However, NAFLD in female patients were all relatively mild compared to that in male patients in this study, characterized by lower ALT levels and less significant fibrosis. The effect of SNP is also different in male and female patients. A study showed that PNPLA3 rs738409 polymorphism was closely related to alcoholic liver disease in Chinese Han men (50). Gender differences in the MBOAT7 rs641738 variant have not been reported before. In our cohort, the associations of the T allele with ALT and fibrosis were only found in male NAFLD patients.

In our study, the association of the rs641738 variant with higher serum HDL was found but not TC and LDL-C. In a Spanish study, MBOAT7 variants were found to be associated with higher TC, LDL, and TG (51). Data from a genome-wide association study (852,409 participants) found that rs641738 variants were associated with higher TC only in Caucasian populations. A meta-analysis (17) showed that rs641738 variants were associated with lower TG and HDL only in Caucasian populations (17). We speculated that there were two reasons for the opposite results. First, the characteristics of the subjects were different in the studies. Second, there were more than 100 genes related to lipid metabolism. Thus, the relationship between a single gene and blood lipids was influenced by other factors, which were not taken into consideration collectively.

NAFLD was closely correlated with T2DM, and the relationship was recently proved to be influenced by SNPs. Moon et al. (52) discovered that a PNPLA3 polymorphism confers lower susceptibility to the incident of T2DM in NAFLD patients. In the NAFLD cohort of our study, the multivariant analysis showed that rs641738 T allele decreased the risk of T2DM. Such a correlation also resulted in the decrease in MetS in rs641738 T allele carriage. Previous research demonstrated that HDL reduce blood glucose levels through various mechanisms, such as promoting insulin secretion from pancreatic β (53) and enhancing glucose uptake into skeletal muscle by activating the AMPK signaling pathway (54). Clinical studies have also shown an association between lower levels of circulating HDL particles and T2DM, insulin resistance, and impaired glucose tolerance. (55). Femlak et al. found that low HDL-cholesterol (HDL-c) levels were an independent risk factor for type 2 diabetes and the development of microvascular complications of diabetes (56). In another study, the authors found a 50% decrease in both HDL₂ cholesterol and protein levels in obese individuals by comparing HDL concentrations in 68 age-matched obese individuals with controls (57). The above study showed that HDL levels were significantly and negatively associated with the progression of metabolic diseases. In this study, we found that TT genotype carriers had a significantly lower risk of MetS in the NAFLD population after adjusting for confounding factors, which may be associated with a high HDL level being found in this population. However, as the statistical significance of the T allele and T2DM tended towards borderline values, additional mechanistic research and prospective longitudinal studies are necessary to confirm these findings.

As aforementioned, several gene loci have been found to promote NAFLD progression and prevent ASCVD, such as PNPLA3 rs738409 and TM6SF2 rs58542926. MBOAT7 variants seemed to exhibit similar effects according to our findings. A similar result was reported in a prospective follow-up study carried out in China Changfeng society. They found that the MBOAT7 T allele decreased the mortality rate of cardiovascular disease together with PNPLA3 and TM6SF2 gene variants (44). Two other findings might support such a correlation in our study. One finding was that TT variants were associated with less obesity in the whole population; the other was its association with fewer MetS and T2DM in NAFLD patients. No other clinical study or meta-analysis came to the same conclusion as us and the Changfeng study (12, 58). Whether such an association also had ethnic differences needs to be further confirmed in more studies.

A lower level of serum ANGPTL3 has been shown to decrease the risk of ASCVD by regulating serum TG. In an analysis of 13,102 patients with cardiovascular disease, carriers with loss-of-function mutations in ANGPTL3 had an approximately 40% lower risk of cardiovascular disease than non-carriers (59). In addition, Thangapandi et al. demonstrated that ANGPTL3 mRNA levels increased significantly in the liver of MBOAT7 knockout mice (18). Therefore, we supposed that ANGPTL3 may be a key regulatory molecule linked to MBOAT7 rs641738-related NAFLD and ASCVD. The hypothesis that serum levels of ANGPTL3 were significantly decreased in the TT group compared to the CC and CT groups was verified. ANGPTL3 is mainly synthesized by hepatocytes and then cleaved by PCSK9 to generate an N-terminal coiled-coil region and a C-terminal fibrinogen-like domain, which could be detected in the serum. MBOAT7 is a protein located on the hepatocyte membrane. MBOAT7 rs641738 variant decreased protein level and may interfere with syntheses or secretion of ANGPTL3 protein. The underlying mechanism needs to be further elucidated through additional in vitro and in vivo studies.

Several limitations should be noted in our study. First, the sample size was relatively small and with less opportunity to get more results. Second, the analysis was designed for the NAFLD study, so the relationship with ASCVD needs confirmation in specially designed research. Third, only one SNP was analyzed in the study; the confounders such as PNPLA3 SNP were not adjusted.

In conclusion, the rs641738 variant near MBOAT7 was found to promote inflammation and fibrosis, particularly in male patients, and decrease the risk of T2DM and metabolic traits in NAFLD patients. In addition, it was related to decreased ASCVD risk in the whole population. These results will be meaningful to better understand the pathogenesis of NAFLD and identify distinct phenotypes of the disease, which will benefit for individualized management.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: https://figshare.com/, https://figshare.com/articles/dataset/MBOAT7_rs641738_C_T_is_associated_with_NAFLD_progression_and_decreased_ASCVD_risk_in_elder_Chinese_population/21506427.

The study protocol was approved by the individual Ethics Review Committee of Beijing Youan Hospital (IRB approval number [2020]-233). The patients/participants provided their written informed consent to participate in this study.

XX, HX and XL participated in the acquisition, analysis, and interpretation of data; prepared the manuscript; and had equal contribution to the study. SZ, GW, and LZ reviewed the literature and built the databases. ZC, and LQ contributed to the analysis and interpretation of the data and provided important scientific input. XD and YL statistically analyzed the data and wrote the first draft of the manuscript. JZ and YZ supervised the whole study. All authors collaboratively discussed key decisions throughout the course of the review, provided critical feedback on preliminary manuscript and interpretation of results. All authors contributed to the article and approved the submitted version.