Effect of MBOAT7 variant on hepatitis B and C infections in Moroccan patients

The outcomes of HBV and HCV infections are associated both with viral and host genetic factors. Here, we explore the role of a genetic variation located in membrane-bound O-acyltransferase domain-containing protein 7 (MBOAT7) gene on spontaneous clearance of HBV and HCV infections and on liver fibrosis. We genotyped MBOAT7 rs641738 polymorphism in 971 consecutive Moroccan subjects, including 288 patients with chronic hepatitis C (CHC), 98 cases with spontaneous clearance of HCV, 268 patients with chronic hepatitis B (CHB), 126 spontaneously cleared HBV infections and 191 healthy controls. MBOAT7 rs641738 variant is not associated with spontaneous clearance of HBV (OR = 0.67, 95% CI: 0.39–1.14; p = 0.131) and HCV infections (OR = 1.33, 95% CI: 0.79–2.23; p = 0.278). Furthermore, multivariable logistic regression analysis adjusted for biologically relevant covariates and potential confounders associated with the risk of liver disease progression revealed that MBOAT7 rs641738 is not associated either with fibrosis progression in CHC group (OR = 1.12; 95% CI: 0.55–2.28; p = 0.761) or with chronic progressive state in CHB patients (OR = 0.81; 95% CI: 0.41–1.61; p = 0.547). We conclude that the variant MBOAT7 rs641738 genotype is not associated with spontaneous clearance of HBV and HCV infections or with the progression of liver disease in chronic hepatitis B or C in a genetic context of Mediterranean patients.

Hepatitis B virus (HBV) and hepatitis C virus (HCV) infections are major causes of acute and chronic liver disease, resulting in an estimated 1.4 million deaths annually 1,2 . Worldwide, it is estimated that 248 million people are living with chronic HBV infection (CHB), and that 110 million persons are HCV-antibody positive among whom 80 million suffer from a bona fide chronic infection (CHC). Thus, the burden of persistent infections with HBV or HCV remains disproportionately high in low-and middle-income countries, particularly in Asia and Africa 1 . Persons with chronic HBV or HCV infections are at high risk to develop progressive hepatic fibrosis and subsequent cirrhosis, two conditions associated with a higher risk of hepatocellular carcinoma (HCC). A recent genome-wide association study in individuals of European descent (with subsequent validation in two independent European cohorts) identified a novel single-nucleotide polymorphism (SNP; rs641738) in the membrane bound O-acyltransferase domain containing 7 gene (MBOAT7), as associated with alcoholic cirrhosis 3 . Furthermore, the rs641738 MBOAT7 SNP was subsequently found to influence histological liver damage in nonalcoholic fatty liver disease (NAFLD), hepatitis C and hepatitis B [4][5][6][7][8][9][10] . Interestingly, previous studies have shown that the rs641738 T allele is associated with lower MBOAT7 mRNA levels and protein expression, as well as reduced arachidonoyl-phosphatidylinositol/total phosphatidylinositol ratios coupled with higher values of oleyl-or linoleyl-phosphatidylinositol/total phosphatidylinositol ratios. These disturbances suggest that the rs641738 T allele may modulate the inflammation process independently of the etiology of the liver disease by down-regulating the MBOAT7 expression and protein synthesis [4][5][6] . Despite the wealth of data about the putative role of MBOAT7 variation in the progression of liver diseases, no data assessing the relationship between the MBOAT7 rs641738 genetic variant and earlier steps of the disease ie. spontaneous clearance of HBV and Serological and molecular analyses. Sera were tested for HBsAg, anti-HCV (Axsym/Architect, Abbott Diagnostics, Wiesbaden-Delkenheim, Germany) and anti-HIV (Genscreen Ag/Ab HIV Ultra, Biorad, Marnes La Coquette, France). Plasma HCV-RNA was measured by qPCR using COBAS AmpliPrep/COBAS TaqMan (Roche Diagnostics, Germany). HCV RNA level below the detection threshold (50IU/mL) were scored as negative for HCV RNA. ALT, AST, gamma-glutamyl transpeptidase (GGT), bilirubin, total cholesterol, high density lipoprotein (HDL), low density lipoprotein (LDL) and triglycerides were collected at time of blood sampling by two separate interviews. The study was conformed to the ethical guidelines of the 1975 Declaration of Helsinki and approved by the ethics committee of the Faculty of Medicine of Casablanca. All participants of this study gave their informed consent.
DNA isolation and MBOAT7 rs641738 Genotyping. Genomic DNA was isolated from the peripheral blood mononuclear cells as described previously 12,13 . Genotyping for MBOAT7 rs641738 was undertaken using the TaqMan SNP genotyping allelic discrimination method (Applied Biosystems, Foster City, CA, USA). All genotyping was blinded to clinical variables and SNP determination was scored by two independent investigators (R.E and H.C) in order to ensure correct typing results.
Statistical analysis. For descriptive statistics, continuous variables are shown as median and range.
Categorical variables are presented as number and proportion. Hardy-Weinberg equilibrium was performed by a χ 2 test with 1 degree of freedom. Comparisons of continuous variables between groups were conducted using ANOVA. We examined five potential genetic models that might explain the effect of MBOAT7 rs641738 on spontaneous clearance outcomes: co-dominant; dominant; recessive; over dominant; and additive. We investigated which model was the most appropriate by calculating the Akaike information criterion (AIC) values 14 . The lowest AIC is indicative of the best fit. Multivariable logistic regression analysis were adjusted for biologically relevant covariates and potential confounders associated with the risk of liver disease progression in chronic hepatitis C patients (age, sex, viral load, ALT, serum cholesterol, HDL, LDL, triglycerides, GGT and MBOAT7 rs641738 genotype) and (age, sex, viral load, ALT, AST and MBOAT7 rs641738 genotype) in CHB patients. All statistical   procedures were performed with R software for Windows and the effect of genetic polymorphism on spontaneous clearance was examined with the SNPassoc R package (https://www.r-project.org). P values less than 0.05 were considered statistically significant. All statistical tests were two-sided.

Results
Clinical and biochemical characteristics of the patients. Association between the MBOAT7 rs641738 polymorphism and spontaneous clearance or disease progression of HBV infection. First, we analyzed the effect of the rs641738 SNP on HBV infection outcomes. The recessive model for the minor allele (T) best fitted the data and was the most appropriate, as it had the lowest Akaike Information Criterion (AIC) value. The distribution of MBOAT7 rs641738 genotypes did not differ with the status of HBV-infection (Supplementary Table 1). Next, we evaluated whether the functional impact of MBOAT7 rs641738 polymorphism was related to disease progression in HBV infection by comparing the genotype frequencies between the HBV chronic infection patients and chronic hepatitis B group (HBeAg negative chronic hepatitis, LC and HCC). No significant differences were detected between the two groups regarding MBOAT7 rs641738 polymorphism. However, a significant association was found male sex, age, ALT, AST and viral load were associated with risk of progressive liver disease (Table 3).

Association between the MBOAT7 rs641738 polymorphism and spontaneous clearance or disease progression of HCV infection. Regarding HCV infection, the dominant model for the minor allele
(T) best fitted the data. The distribution of MBOAT7 rs641738 genotypes did not differ between HCV-persistent and HCV-cleared groups (Supplementary Table 2).   Next, we tested subsequently the association of rs641738 with fibrosis stage by multiple logistic regression analysis adjusted for age, gender, and viral load. CHC patients were stratified according to fibrosis as absent/ mild (F0-F2) or significant (F3-F4). The result indicated that age, male sex, cholesterol, GGT, ALT and AST were associated with advanced fibrosis (p < 0.05). On the other hand, we observed an absence of impact of rs641738 TT genotype on fibrosis progression (OR = 1.12; 95% CI: 0.55-2.28; p = 0.761; Table 4).
Biochemical and metabolic characteristics according to rs641738 genotype. No significant associations were found with any available biochemical features such as liver enzymes (Fig. 2), platelet counts, lipid profile, anthropometric traits or HCV-RNA according to rs641738 genotype in CHC group (Fig. 3). Furthermore, no significant results were found for the features analyzed in CHB patients (Supplementary Fig. 1).

Discussion
CHB and CHC are characterized by liver inflammation that promotes, on the long term, fibrosis, cirrhosis and HCC 15,16 . It is known that genetic variation of the host, viral and environmental factors are modulating disease progression 17,18 . In the present study, we investigated the role of rs641738 C > T genetic variant in MBOAT7 in Moroccan patients with a broad spectrum of liver disease. In Moroccan population, the MAF frequency was 0.44 which is in line with the frequencies reported in Caucasians population but lower than those reported Asian populations 6,8 . To our knowledge, this study is the first description of the relationship between MBOAT7 rs641738 polymorphism and clearance of HBV and HCV. The major finding of the present study was the lack of association of rs641738 C > T genetic variant in the MBOAT7 gene and spontaneous resolution of HBV and HCV infections.
The MBOAT7 rs641738 polymorphism was first identified as a predisposing locus for cirrhosis in alcoholic liver disease 3 . In the setting of CHB and CHC, the immune system initially attempts to eradicate the HBV and HCV, while it probably promotes progressive liver damage and fibrosis in the same time. In the present study, we found that MBOAT7 rs641738 is not associated with progression in both CHB and CHC. Our data are, thus, at odd with previous studies showing that MBOAT7 rs641738 variant accelerates the progression to fibrosis in CHC and CHB of Caucasian patients 6,8 . In addition, the MBOAT7 rs641738 SNP variant was shown to predispose Caucasian subjects to liver fibrosis subjects with excessive alcohol intake 3 and to progression of nonalcoholic fatty liver disease (NAFLD) 4,5 . In contrast, a Korean study did not reveal any association between MBOAT7 rs641738 SNP and the histologic severity of NAFLD 19 . The discrepancy between results found in studies conducted on subjects with different anthropological background may be related to allele frequency differences between populations, or to the genetic architecture of the populations studied and its inherent set of epistasis 4,8 . We speculate that the effect of MOBAT7 rs641738 variant on the development of liver disease progression may be influenced by ethnicity 4,8,10 . Moreover, concerning HBV infection, we compared patients with HBV infection without hepatitis versus patients with infection and hepatitis. This could explain at least in part the different findings of this study compared to previous study 8 . Nowadays, the functional mechanisms of MBOAT7 action in liver diseases remain unknown. MBOAT transfers arachidonic acid to lysophosphatidylinositol to produce arachidonic acid-containing phosphatidylinositol 20 . Moreover, previous study showed that MBOAT7 is independently associated with inflammation, oxidative stress, macrophage activation and the transition to early fibrosis 6 .
Our data did not reveal any significant associations with clinical characteristics such as liver enzymes, lipid profile, HCV-RNA, or HBV-DNA. This result seems to be in line with previous findings 6,8,10 . However, a higher ALT levels is observed in individuals carrying the T allele 8,21 .
As a conclusion, the MBOAT7 rs641738 variant is not associated with spontaneous resolution of HBV and HCV infections. Furthermore, our data show that MBOAT7 rs641738 polymorphism does not predispose infected patients to progress to an advanced stage. Our study has some limitations, including missing data on the MBOAT expression in HCV/HBV-infected liver biopsies. Moreover, we are fully aware that the limitation of the current study is the relatively small size of the cohort examined. Further surveys warrant the recruitment of a larger cohort and future analyses of additional polymorphisms in MBOAT7 gene and Transmembrane 6 superfamily 2 (TM6SF2) E167K variant will help to clarify their role in HCV/HBV infections and progression. Overall, further studies elucidating the mechanistic function and on large and others cohorts, notably North African patients with NAFLD, are warranted to definitively refute or confirm an effect of MOBAT7 rs641738 variant on liver diseases progression.