Association of Common Variant Rs2281135 of PNPLA3 with Alcohol-Related Cirrhosis in Chinese Han Males

Research Article

Austin J Pathol Lab Med. 2021; 8(2): 1033.

Association of Common Variant Rs2281135 of PNPLA3 with Alcohol-Related Cirrhosis in Chinese Han Males

Chen H1#, Yang F2#, Zhang Y1#, Zhang Y3, Guo T4, Mao Y5, Liu C6, Cheng L6, Wang Y7, Li Y6* and Huang J1*

1Department of Clinical Laboratory, The First Hospital of Jilin University, Changchun, China

2Department of Pediatrics Outpatient Service, The First Hospital of Jilin University, Changchun, China

3Department of Clinical Laboratory, Beijing Tiantan Hospital, Capital Medical University, Beijing, China

4Department of Clinical Laboratory, Beijing Mentougou District Hospital, Beijing, China

5Department of Center of Clinical Laboratory Medicine, The Fifth Medical Center of the General Hospital of the Chinese People’s Liberation Army, Beijing, China

6Department of Clinical Laboratory, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China

7Department of Cancer Research Institute, The First Hospital of Jilin University, Changchun, China

#Co-First authors on this work

*Corresponding author: author: Jing Huang, Department of Clinical Laboratory, The First Hospital of Jilin University, Jilin, 130021, China

Yongzhe Li, Department of Clinical Laboratory, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, 100730, China

Received: April 27, 2021; Accepted: May 18, 2021; Published: May 25, 2021


Background and Aim: Alcoholic Liver Disease (ALD), caused by longterm heavy alcohol consumption, is influenced by genetic factors. Studies have illustrated the overlapping genetic mechanism in Nonalcoholic Fatty Liver Disease (NAFLD) and ALD. Recently, a number of Genome-Wide Association Studies (GWAS) have demonstrated several SNPs were strongly associated with NAFLD. The aim of present study is to evaluate the association between these NAFLD-associated SNPs and ALD in Chinese Han population.

Methods: Nine SNPs were selected and genotyped in a cohort of 507 patients with ALD and 645 healthy controls by using MassARRAY iPLEX system. Alleles and genotypes analysis of SNPs were performed in logistic regression. The association between SNP and the level of liver serum biomarkers was tested in chi-square test and linear regression model.

Results: Our data confirmed that rs2281135 A-allele in PNPLA3 and rs3761472 G-allele in SAMM50 were significantly associated with increased risk of ALD (P = 1.93×10-12, OR [95% CI] = 1.82 [1.54-2.15]; P = 2.08×10-16, OR [95% CI] = 1.06 [1.04-1.08], respectively). The genotypes of rs2281135 were associated with ALD in additive, dominant and recessive genetic model (P = 1.24×10-11, P = 1.46×10-7, P = 2.07×10-9, respectively). In addition, rs2281135 was found to be associated with serum elevated levels of ALT (P = 5.0×10-3), AST (P = 0.03), ALP (P = 0.02), GGT (P = 0.03) in patients with ALD.

Conclusions: The present study confirmed that PNPLA3 common variant rs2281135 was significantly associated with ALD in Chinese male Han population.

Keywords: Alcohol-associated liver disease; Nonalcoholic fatty liver disease; Single nucleotide polymorphism; PNPLA3; SAMM50


ALD: Alcoholic Liver Disease; AC: Alcoholic Cirrhosis; HCC: Hepatocellular Carcinoma; GWAS: Genome-Wide Association Study; SNP: Single-Nucleotide Polymorphism; PNPLA3: Patatin- Like Phospholipase Domain-Containing Protein 3; ALT: Alanine Aminotransferase; AST: Aspartate Aminotransferase; GGT: Gamma- Glutamyl Transpeptidase; NAFLD: Non-Alcoholic Liver Disease; ALP: Alkaline Phosphatase; ALB: Albumin; HWE: Hardy-Weinberg Equilibrium


Alcoholic Liver Disease (ALD) caused by long-term excessive alcohol consumption is a global public health issue and poses a significant burden on healthcare resources. ALD is a type of chronic liver disease that has a broad spectrum of progression, ranging from simple fatty liver to more severe forms of liver injury, including Alcoholic Hepatitis (AH), Alcoholic Cirrhosis (AC), even Hepatocellular Carcinoma (HCC) [1]. According to the Global Health Estimates (GHE) 2015 dataset by WHO, alcohol consumption was the second cause of deaths due to cirrhosis (20.8%) and liver cancer (29.8%) in the Asia-Pacific region, similar as in mainland China, 20.0% for all deaths due to cirrhosis and other chronic liver diseases and 32.5% for all deaths due to liver cancer, respectively [2]. The prevalence of ALD has increased rapidly in the developing countries, especially in China, but has decreased in Western countries [3].

The pathogenesis of ALD in humans is incompletely clear, genetic and environmental factors, including sex, ethnicity, obesity, drinking patterns and cigarette smoking et al. are associated with susceptibility to ALD [1]. Recently, a Genome-Wide Association Study (GWAS) [4] in population of European ancestry confirmed single-nucleotide polymorphism (SNP) rs738409 in patatin-like Phospholipase Domain-Containing Protein 3 (PNPLA3) was strongly associated with alcohol-associated cirrhosis (P = 1.54×10-48, OR [95%CI] = 2.19 [1.97- 2.43]), and identified Membrane-Bound O-Acyltransferase Domain- Containing Protein 7 (MBOAT7) gene polymorphisms (rs641738 and rs626283) and Transmembrane 6 Superfamily Member 2 (TM6SF2) gene polymorphisms (rs10401969 and rs58542926) as new risk loci for alcohol-associated cirrhosis. In our previous study, we tested the association between steatogenic genes (PNPLA3 rs738409, MBOAT7 rs626283 and rs641738, TM6SF2 rs58542926 and SUGP1 rs10401969) and ALD in Chinese Han population and validated that PNPLA3 rs738409 G-allele was associated with ALD (P = 1.25×10- 14, OR [95% CI] = 1.93[1.63-2.28]) and several biomarkers of liver including increased level of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma-glutamyl transpeptidase (GGT), and total bilirubin (TBil) in this cohort [5]. These SNPs also have been validated to be associated with non-alcoholic liver disease (NAFLD) [6-12], which demonstrated that ALD and NAFLD share overlapping pathogenic mechanism.

The study of NAFLD is faster than ALD, because of higher morbidity. GWAS is a powerful and broader approach to identify susceptibility genes for complicated human diseases. At present, a number of GWAS in NAFLD have been performed and identified multiple susceptibility loci associated with hepatic histology, increased level of serum AST and ALT or NAFLD activity score (NAS) in NAFLD [7,13-15]. Considering the overlap of genetic background in ALD and NAFLD and the association of these genetic variants with NAFLD, we hypothesized that these SNPs may be also associated with the prevalence of ALD in a Chinese Han population. Due to the different allele frequencies in Chinese Han population, we selected nine SNPs that Minor Allele Frequency (MAF) over 0.05 to investigate whether they were associated with ALD in Chinese Han population. Nine SNPs included rs2228603 in Neurocan (NCAN) gene, rs780094 in Glucokinase Regulatory Protein (GCKR) gene, rs2645424 in Farnesyl Diphosphate Farnesyl Transferase 1 (FDFT1) gene, rs1227756 in Collagen type XIII Alpha 1 (COL13A1) gene, rs6591182 in Latent Transforming Growth Factor-β Protein 3 (LTBP3) gene, rs6487679 in Pregnancy Zone Protein (PZP) gene, rs1421201 in solute carrier family 14 member 2 (SLC14A2), rs3761472 in sorting and assembly machinery component (SAMM50) gene and rs2281135 in PNPLA3 gene.

These SNPs have been verified to be associated with hepatic histology, increased level of serum AST and ALT or NAS in NAFLD. However, the role of these SNPs in development of ALD is currently unclear. The aim of this study was to test the potential association of nine variants with ALD in Chinese Han population.

Materials and Methods

Study population

We designed a multicenter case-control study, 507 individuals with alcohol-associated liver disease were collected from the first Hospital of Jilin University (Changchun, China), Peking Union Medical College Hospital (Beijing, China), the Fifth Medical Center of the General Hospital of the People’s Liberation Army (Beijing, China), Shengjing Hospital affiliated with China Medical University (Shenyang, China) and Hepatobiliary Hospital of Jilin (Changchun, China) from January 2016 to May 2017. All individuals were the outpatients or inpatients that diagnosed in five hospitals. The diagnostic criteria for ALD patients was based on the American Association for the Study of Liver Diseases (AASLD) practice guidelines (2010) [16], which revised by the Chinese Medical Association. The diagnosis of ALD was based on a combination of clinical features and characteristics, including history of alcohol consumption, ultrasonographic evidence of fatty liver or cirrhosis and supporting laboratory indicators abnormalities. Exclusion criteria for the study were as follows: 1) positivity to HBsAg, anti-HCV or HIV; 2) drug-induced liver disease, autoimmune liver disease, Wilson’s disease and other chronic liver disease. All subjects were unrelated Chinese Han male populations.

A total of 645 ethnicity-, age- and gender-matched healthy controls from the five hospitals were recruited during their routine physical examinations according to the following criteria: 1) had normal liver function, routine blood and urine results; 2) no history of HBV, HCV or HIV infection; 3) no evidence of liver disease in ultrasonography; and 4) no history of chronic disease.

All patients with ALD had a history of drinking for more than five years, the average alcohol intake is more than 40g per day for men or a large amount of alcohol consumption in the last two weeks averaging more than 80g per day. For healthy controls, the average alcohol consumption was less than 40g per day for men or did not had drinking habits in daily life. Clinical laboratory indicators were collected at baseline as following: ALT, AST, GGT, Alkaline Phosphatase (ALP), TBil, Albumin (ALB), Red Blood Cell Count (RBC), Mean Corpuscular Volume (MCV), Platelet (PLT) and Prothrombin Time (PT). The study was approved by The Ethics Committee of the First Hospital of Jilin University, and written informed consent was obtained from each subject.


2ml whole blood samples were collected into EDTA tubes from each individual by venous phlebotomy and stored at -80°C immediately after centrifuging. DNA was extracted from peripheral white blood cells of participants using Tiangen DNA extraction kit (Beijing, China) at the same time point, and then stored at -80°C until genotyped. Quality measurements were performed on all DNA samples. The nine SNPs (rs2228603, rs780094, rs2645424, rs1227756, rs6591182, rs6487679, rs1421201, rs3761472 and rs2281135) were genotyped using MassARRAY iPLEX system (Sequenom, USA). All the procedures were conducted following the manufacturer’s instructions. About 10-20ng DNA samples were amplified by Polymerase Chain Reaction (PCR) and the PCR products were performed to locus-specific single-base extension reactions. The final products were desalted and transferred to a 384-element SpectroCHIP array. Allele detection was carried out using matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOFMS). Then we used MassARRY Typer software v4.0 to analyze the mass spectrogram data.

Statistical analysis

To describe the study population, continuous traits were expressed as medians (and interquartile ranges) and categorical traits were expressed as numbers (and percentages). All statistical association analysis of genotype data was carried out using PLINK software v1.07 ( To account for multiple testing, we used the False Discovery Rate (Bonferroni) correction for P value. The Hardy-Weinberg equilibrium (HWE) was estimated separately in the case group and the control group. If P≥0.05, genotype frequencies of the population followed the HWE.

Differences in baseline characteristics between ALD patients (n=507) and controls (n=645) was tested by Student’s t’-test or Mann-Whitney U test and variant genotype frequencies were tested by Chi-square test. To explore the association between genotypes and ALD, we performed three model analyses (additive model, dominant model, and recessive model) by logistic regression analysis. The associations between genotype and serum biomarkers of liver were tested by two models. Model 1 was performed by Chi-square test using the traditionally cutoff of over normal reference interval as elevation in ALD group. And model 2 was performed by linear regression to investigate the association of genotypes and liver serum biomarkers level in all participants. All serum markers measurements were natural logarithmically transformed before performing regressions, due to non-normal distributions.

All P-values (adjusted by Bonferroni correction) less than 0.05 was considered statistically significant and were adjusted for age in regression models. Linkage Disequilibrium (LD) test was carried out using Haploview software v4.2. Statistical analyses were carried out using SPSS Statistics V.25 (IBM) and Prism V.8 (GraphPad).


Clinical characteristics of participants

The general characteristics of patients with ALD and controls are shown in Table 1. In present study, 507 ALD patients (median age 53 (47-59) years) and 645 healthy controls (median age 52 (45-59) years) were recruited from Chinese Han male population. All individuals were males, and age did not differ between two groups (P=0.21). Clinical characteristics comparisons between ALD group and control group, such as ALT, AST, ALP, GGT, ALB, RBC, MCV and PLT, were statistically significant (P<0.001).