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Interaction Between Alcohol Consumption and PNPLA3 Variant in the Prevalence of Hepatic Steatosis in the US Population

  • Mariana Lazo
    Correspondence
    Reprint requests Address requests for reprints to: Mariana Lazo, PhD, ScM, MD, Drexel University, 3600 Market Street, Suite 750, Philadelphia, Pennsylvania 19104.
    Affiliations
    Department of Community Health and Prevention, Dornsife School of Public Health, Drexel University, Philadelphia, Pennsylvania

    Urban Health Collaborative, Dornsife School of Public Health, Drexel University, Philadelphia, Pennsylvania

    Division of General Internal Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland
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  • Usama Bilal
    Affiliations
    Urban Health Collaborative, Dornsife School of Public Health, Drexel University, Philadelphia, Pennsylvania

    Department of Epidemiology and Biostatistics, Dornsife School of Public Health, Drexel University, Philadelphia, Pennsylvania
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  • Mack C. Mitchell
    Affiliations
    Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, Texas
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  • James Potter
    Affiliations
    Division of Gastroenterology & Hepatology, Johns Hopkins University School of Medicine, Baltimore, Maryland
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  • Ruben Hernaez
    Affiliations
    Section of Gastroenterology, Michael E. DeBakey Veterans Affairs Medical Center, Houston, Texas

    Center for Innovations in Quality, Effectiveness and Safety, Michael E. DeBakey Veterans Affairs Medical Center, Houston, Texas

    Section of Gastroenterology and Hepatology, Department of Medicine, Baylor College of Medicine, Houston, Texas
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  • Jeanne M. Clark
    Affiliations
    Division of General Internal Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland

    Division of Gastroenterology & Hepatology, Johns Hopkins University School of Medicine, Baltimore, Maryland
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Published:August 31, 2020DOI:https://doi.org/10.1016/j.cgh.2020.08.054

      Background & aims

      To our knowledge, the interaction between alcohol consumption and PNPLA3 genotype on hepatic steatosis has not been explored in a representative sample. To examine the interaction between alcohol consumption and PNPLA3 genotype on hepatic steatosis in the US adult population.

      Methods

      Cross-sectional study of 4,674 adult participants of the Third National Health and Nutrition Examination Survey, Phase 2 (1991-1994) with data on PNPLA3 genotype, self-reported alcohol consumption, ultrasound-defined hepatic steatosis and socio-demographic characteristics.

      Results

      In 1991-1994 in the U.S. population, the weighted allele frequency of the G (risk) allele of the rs738409 at PNPLA3 was 25.4%. We confirmed both a J shaped association between alcohol consumption and hepatic steatosis among those with the CC genotype of PNPLA3, and a higher prevalence of hepatic steatosis among those with PNPLA3 gene G variant. We found evidence of an interaction of PNPLA3 G allele presence on the association between moderate alcohol consumption and hepatic steatosis on both the multiplicative (relative prevalence ratio [RPR]=1.95, 95% confidence interval [CI] 1.04-3.65) and additive scales (relative excess risk due to interaction=0.49, 95% CI 0.13-0.85). Compared to never drinkers, moderate alcohol drinking was associated with a 48% decreased risk of hepatic steatosis only among those without PNPLA3 G allele (PR=0.52, 95% CI 0.26-1.05), with no association among those with at least one copy of the PNPLA3 G allele (PR=1.02, 95% CI 0.68-1.54).

      Conclusions

      Our results suggest that a highly common and strong genetic susceptibility to liver disease is modifiable by the level of alcohol consumption. Keeping alcohol consumption low may offset genetic predisposition to liver disease.

      Keywords

      Abbreviations used in this paper:

      ALD (alcohol-related fatty liver disease), BMI (body mass index), CI (confidence interval), EAF (effect allele frequency), NAFLD (nonalcoholic fatty liver disease), NHANES III (Third National Health and Nutrition Examination Survey), PR (prevalence ratio), RERI (relative excess risk due to interaction), RPR (relative prevalence ratio)

       Background

      PNPLA3 variant (I148M) is of significant relevance given the strong association with fatty liver disease and because of the high prevalence in the population, especially among some racial and ethnic minorities. The empirical evidence of the role of alcohol consumption modulating this genetic vulnerability is extremely limited.

       Findings

      In this cross-sectional study of a U.S. nationally representative sample, we found evidence of an interaction of PNPLA3 G variant on the association between alcohol consumption on hepatic steatosis in which we observed a dose-response association between alcohol consumption and hepatic steatosis among those with the GG genotype, and the “J-shaped” association suggesting potential beneficial effect of moderate drinking only observed among those without G variant copies (CC genotype).

       Implications for patient care

      Our results suggest that strategies to reduce alcohol consumption may be an effective measure to offset genetic risk of liver disease. Furthermore, our results of a dose-response association of alcohol consumption and hepatic steatosis among individuals with PNPLA3 GG genotype help support recent guidelines emphasizing that there is no safe drinking level for patients with nonalcoholic fatty liver disease.
      The burden of liver diseases in the United States and other countries is substantial.
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      Currently, there are no Food and Drug Administration–approved treatments available for these conditions; therefore, prevention is key. Both obesity and alcohol consumption are highly prevalent modifiable risk factors in the Western hemisphere. However, even in the context of heavy alcohol consumption or obesity, some individuals do not develop liver disease. These findings are not surprising, given the multifactorial nature of the disease, and support the potential importance of interactions between genetic and environmental factors in explaining the variation of the occurrence of liver disease.
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      In 2008, a very strong association between a common genetic variation in the PNPLA3 gene, the G allele at rs738409, and hepatic steatosis was described.
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      We and others have documented that in the U.S. adult population the prevalence of the PNPLA3 risk variant varies considerably by race and ethnicity, with the highest prevalence among Hispanics (EAF, 0.54), intermediate prevalence among non-Hispanic Whites (EAF, 0.25), and the lowest prevalence among non-Hispanic Blacks (EAF 0.14), mirroring the observed racial and ethnic disparities in NAFLD.
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      • Pirola C.J.
      Meta-analysis of the influence of I148M variant of patatin-like phospholipase domain containing 3 gene (PNPLA3) on the susceptibility and histological severity of nonalcoholic fatty liver disease.
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      Whether risk factors, including alcohol consumption and obesity, modify the effects of the PNPLA3 genotype in the population is incompletely understood. Existing evidence supports an interaction between PNPLA3 genotype and adiposity on hepatic steatosis, by which body mass index (BMI) amplifies the effect of the G allele.
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      • Nordestgaard B.G.
      • et al.
      Adiposity amplifies the genetic risk of fatty liver disease conferred by multiple loci.
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      The interaction of rs738409, obesity, and alcohol: a population-based autopsy study.
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      • et al.
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      The GG phenotype confers little risk of hepatic steatosis among lean individuals but has a strong effect in individuals with obesity.
      • Stender S.
      • Kozlitina J.
      • Nordestgaard B.G.
      • et al.
      Adiposity amplifies the genetic risk of fatty liver disease conferred by multiple loci.
      However, to our knowledge, there has been no study of the interaction between alcohol consumption and PNPLA3 genotype on the occurrence of hepatic steatosis, in a large representative sample, with 1 prior small study suggesting an interaction.
      • Dunn W.
      • Zeng Z.
      • O'Neil M.
      • et al.
      The interaction of rs738409, obesity, and alcohol: a population-based autopsy study.
      The objective of our study is to examine the role of alcohol consumption modulating this genetic vulnerability of PNPLA3 genotype on hepatic steatosis in a large representative sample of the United States.

      Materials and Methods

       Study Population

      We used data from participants of the Third National Health and Nutrition Examination Survey (NHANES III), phase 2, a cross-sectional survey of the U.S. civilian population conducted in 1988–1994 by the National Center of Health Statistics. NHANES III was divided into 2 phases: phase 1 was conducted from 1988 to 1990 and phase 2 was conducted from 1991 to 1994. Genetic data are available for phase 2 participants only. We limited our analyses to participants who were 20–74 years of age (eligible for ultrasound to determine hepatic steatosis), with PNPLA3 genotype information, hepatic steatosis assessment, and information regarding alcohol consumption. The total analytical sample was 4674 (unweighted). The NHANES III was approved by the institutional review board of the National Center of Health Statistics.

       Exposures

      Self-reported past and current alcohol consumption was categorized into 4 mutually exclusive groups: never, former, current low-moderate alcohol consumption (≤1 and ≤2 drinks/d in women and men, respectively), and current heavy alcohol consumption (>1 and >2 drinks/d in women and men, respectively). These cut points of alcohol consumption are consistent with the literature on fatty liver disease to distinguish “alcoholic” and “nonalcoholic” nature.
      • Chalasani N.
      • Younossi Z.
      • Lavine J.E.
      • et al.
      The diagnosis and management of nonalcoholic fatty liver disease: practice guidance from the American Association for the Study of Liver Diseases.
      Genotyping of rs738409 (PNPLA3) was performed using the iPLEX Sequenom platform (CD Genomics, Shirley, NY).

       Outcome

      The protocol used to ascertain the presence of hepatic steatosis using gallbladder ultrasounds in NHANES III can be found elsewhere.
      Third National Health and Nutrition Examination Survey
      Hepatic Steatosis Assessment Procedure Manual.
      For the current analyses, to maximize specificity and accuracy of the outcome, we defined hepatic steatosis as the presence of severe hepatic steatosis by ultrasound.
      Additional details regarding methods are described in the Supplementary Material.

       Statistical Methods

      We characterized the study population by PNPLA3 genotype (CC, CG, GG) using descriptive statistics. We used robust Poisson regression to estimate adjusted prevalence estimates of hepatic steatosis by alcohol consumption categories and PNPLA3 genotype and adjusted prevalence ratios (PRs) and 95% confidence intervals (CIs). We adjusted for the following potential confounders: race and ethnicity, sex, age, education, and income; a priori, we did not adjust for BMI, given that the PNPLA3 genotype is not associated with BMI.
      • Romeo S.
      • Kozlitina J.
      • Xing C.
      • et al.
      Genetic variation in PNPLA3 confers susceptibility to nonalcoholic fatty liver disease.
      For simplicity and given the scope of the current manuscript, results for former drinkers are only shown in the Supplementary Material.
      We examined the interaction between alcohol consumption categories and PNPLA3 genotype in both the multiplicative scale (using the relative prevalence ratio [RPR]) and the additive scale (using the relative excess risk due to interaction [RERI]). Additional details about these measures can be found in the Supplementary Material.
      For the main analyses, we present the results using never drinkers and those with no copy of the allele as the reference group. In sensitivity analyses, we present the results using moderate drinkers with no copy of allele, the group with the lowest risk, as the reference group.

      Results

      In 1991–1994 in the United States, 57.3% of the U.S. population had the CC genotype, 34.8% had the CG genotype, and 7.9% had the GG genotype, for a weighted allele frequency of the G allele of the rs738409 at PNPLA3 in the overall U.S. population of 25.4%. There were notable racial and ethnic differences in the distribution of the PNPLA3 genotypes, with much higher prevalence of the GG genotype among Mexican Americans and lowest prevalence among non-Hispanic Blacks. Importantly, there were no differences in BMI, diabetes prevalence, and mean number of alcoholic drinks per week by PNPLA3 genotype (Table 1).
      Table 1Characteristics of the U.S. Adult Population (20–74 years of age) by PNPLA3 Genotype (Third National Health and Nutrition Examination Survey, Phase 2 [1991–1994])
      OverallCCCGGG
      Unweighted population4674 (100)2402 (57.3)1700 (34.8)572 (7.9)
      Age, y42.49 (41.39–43.59)42.85 (41.48–44.21)42.44 (40.89–43.98)40.14 (38.48–41.80)
      Female51.551.551.551.1
      Race/ethnicity
      Non-Hispanic White81.781.983.174.6
      Non-Hispanic Black12.115.78.62.4
      Mexican American6.22.58.423.0
      Education
      <12 y18.818.918.221.0
      12 y35.333.138.936.3
      >12 y45.848.042.942.7
      Metabolic parameters
      BMI, kg/m226.95 (26.61–27.30)26.83 (26.47–27.19)27.27 (26.75–27.79)26.48 (25.92–27.03)
      BMI category
      Normal40.441.937.741.3
      Overweight32.931.3033.641.2
      Obese25.024.8027.216.9
      Glucose, mg/dL97.28 (95.51–99.05)96.94 (94.82–99.07)98.06 (96.13–100.00)96.28 (94.51–98.06)
      Diabetes6.66.47.16.3
      Triglycerides, mg/dl108 [76–165]106 [76–160]108 [78–168]108 [73–170]
      Liver-related measures
      Severe hepatic steatosis8.57.59.112.8
      ALT, IU/L15 [11–21]14 [10–20]15 [11–22]17 [11–26]
      AST, IU/L19 [16–24]19 [16–23]19 [16–24]20 [17–25]
      GGT, IU/L19 [13–29]19 [13–28]19 [13–29]18 [13–33]
      Alcohol consumption
      Never10.59.712.19.2
      Former33.432.634.534.8
      Low-moderate (≤7 [women] and ≤14 [men] drinks/wk)47.247.846.247.2
      Heavy (≥7 [women] and ≥14 [men] drinks/wk)8.99.97.38.8
      Drinks per week3.0 [1.0–8.0]3.0 [1.0–8.0]3.0 [0.9–6.0]3.0 [0.9–8.0]
      NOTE. Values are n (%), mean (95% confidence interval), %, or median [interquartile range].
      ALT, alanine aminotransferase; AST, aspartate aminotransferase; BMI, body mass index; GGT, γ-glutamyltransferase.

       Main Effects of Alcohol and PNPLA3 Genotype on Severe Hepatic Steatosis

      Compared with never drinkers, low-moderate drinkers had lower prevalence of severe hepatic steatosis (6.7% vs 8.8%), whereas those with heavy drinking had higher prevalence (11.4%) (Figure 1). The corresponding PRs and 95% CIs for the whole population and by race and ethnicity are shown in the Supplementary Table 1. Compared with the CC genotype, those individuals with CG and GG genotypes had a higher prevalence of hepatic steatosis: 6.6% vs 8.0% and 11.2%, respectively (Figure 2). The corresponding PRs and 95% CIs for the whole population and by race and ethnicity are shown in the Supplementary Table 2.
      Figure thumbnail gr1
      Figure 1Adjusted prevalence (95% CI) of severe hepatic steatosis by alcohol consumption categories, overall and stratified by race and ethnicity (Third National Health and Nutrition Examination Survey, Phase 2 [1991–1994]).
      Figure thumbnail gr2
      Figure 2Adjusted prevalence (95% CI) of severe hepatic steatosis across PNPLA3 genotypes, overall and by race and ethnicity (Third National Health and Nutrition Examination Survey, Phase 2 [1991–1994]).

       Interaction Between Alcohol With PNPLA3 Genotype on Severe Hepatic Steatosis

      At the population level and after adjusting for sociodemographic characteristics, the lowest prevalence of severe hepatic steatosis was among individuals with no copies of the PNPLA3 G variant (CC) and with low-moderate alcohol consumption (5.3%; 95% CI, 2.0%–8.6%), with the highest prevalence observed among those with heavy drinking and homozygous GG (17.3%; 95% CI, 0%–36.4%).
      Furthermore, when we compared the shape of the association between alcohol consumption given the PNPLA genotype, we observed how the “J-shaped” association between alcohol levels and hepatic steatosis disappears in the presence of the G variant. Among those heterozygous individuals (CG), low-moderate drinkers no longer had a lower prevalence of hepatic steatosis compared with never drinkers (8.0% vs 7.9% for moderate and never drinkers, respectively), and among those with 2 copies (GG), low-moderate drinkers and never drinkers had equally high prevalence of severe hepatic steatosis (12.1% vs 6% for low-moderate and never drinkers, respectively). For heavy drinkers, the prevalence of hepatic steatosis increased monotonically with increases in copies of the G variant, and at any given PNPLA3 genotype, heavy drinkers had the highest prevalence (Figure 3).
      Figure thumbnail gr3
      Figure 3Adjusted prevalence (95% CI) of severe hepatic steatosis by alcohol consumption and PNPLA3 genotypes (Third National Health and Nutrition Examination Survey, Phase 2 [1991–1994]).

       Additive and Multiplicative Interaction Metrics

      We found evidence of an interaction of PNPLA3 G allele presence on the association between low-moderate alcohol consumption and severe hepatic steatosis on both the multiplicative (RPR, 1.95; 95% CI, 1.04–3.65; P = .048) and additive (RERI, 0.49; 95% CI, 0.13–0.85) scales. Compared with never drinkers, low-moderate alcohol drinking was associated with a 48% decreased risk of hepatic steatosis only among those without PNPLA3 G allele (PR, 0.52; 95% CI, 0.26–1.05), with no association among those with at least 1 copy of the PNPLA3 G allele (PR, 1.02; 95% CI, 0.68–1.54).
      For heavy alcohol drinkers compared with never drinkers, heavy alcohol drinking was associated with no increased risk of hepatic steatosis among those without any PNPLA3 G allele (PR, 0.98; 95% CI, 0.38–2.53), but among those with at least 1 copy of the PNPLA3 G, there was a nonsignificant but increased risk of hepatic steatosis (PR, 1.29; 95% CI, 0.66–2.53). The results of the interaction metrics were generally consistent with the ones observed for low-moderate drinking but did not reach statistical significance (RPR, 1.71; 95% CI, 0.65–4.51; P = .29; RERI, 0.54; 95% CI, –0.38 to 1.46) (Table 2). In sensitivity analyses, changing the reference to low-moderate drinkers with no PNPLA3 G allele, the results were consistent (Supplementary Table 3).
      Table 2aPR for the Interaction Between Alcohol Consumption and PNPLA3 Genotype and Severe Hepatic Steatosis (Third National Health and Nutrition Examination Survey, Phase 2 [1991–1994])
      PNPLA3 G alleleNeverLow-Moderate
      Low-moderate alcohol consumption: ≤1 and ≤2 drinks/d for women and men, respectively.
      Heavy
      Heavy alcohol consumption: >1 and 2 drinks/d in women and men, respectively.
      Stratified PR
      01.0 (reference)0.52 (0.26 to 1.05)0.98 (0.38 to 2.53)
      +1 copy1.0 (reference)1.02 (0.68 to 1.54)1.67 (0.93 to 3.00)
      Multiplicative interaction metric
      RPR
      The null hypothesis is that RPR, 1. An RPR > 1/RPR < 1 indicate positive/negative interaction on the multiplicative scale.
      1.0 (reference)1.95 (1.04 to 3.65)
      Calculated as: PR++-PR+--PR-++1. The null hypothesis is that RERI, 0. RERI > 0/RERI < 0 indicate positive/negative interaction on the additive scale.
      1.71 (0.65 to 4.52)
      Single referent PR
      01.0 (reference)0.52 (0.26 to 1.05)0.98 (0.38 to 2.53)
      +1 copy0.77 (0.45 to 1.32)0.79 (0.43 to 1.44)1.29 (0.66 to 2.53)
      Additive interaction metrics
      RERI
      Calculated as: PR++-PR+--PR-++1. The null hypothesis is that RERI, 0. RERI > 0/RERI < 0 indicate positive/negative interaction on the additive scale.
      0 (reference)0.49 (0.13 to 0.85)
      Calculated as: PR++-PR+--PR-++1. The null hypothesis is that RERI, 0. RERI > 0/RERI < 0 indicate positive/negative interaction on the additive scale.
      0.54 (–0.38 to 1.46)
      NOTE. Values are aPR (95% confidence interval). Data were adjusted for race and ethnicity, sex, age, age squared, education, and income.
      aPR, adjusted prevalence ratio; PR, prevalence ratio; RERI, relative excess risk due to interaction; RPR, relative prevalence ratio.
      a Low-moderate alcohol consumption: ≤1 and ≤2 drinks/d for women and men, respectively.
      b Heavy alcohol consumption: >1 and 2 drinks/d in women and men, respectively.
      c The null hypothesis is that RPR, 1. An RPR > 1/RPR < 1 indicate positive/negative interaction on the multiplicative scale.
      d Calculated as: PR++-PR+--PR-++1. The null hypothesis is that RERI, 0. RERI > 0/RERI < 0 indicate positive/negative interaction on the additive scale.

      Discussion

      In a representative sample of the adults in the U.S. civilian population, we found a significant interaction between the PNPLA3 gene G variant and alcohol consumption on hepatic steatosis in which we observed a dose-response association between alcohol consumption and hepatic steatosis among those with the GG genotype, and a “J-shaped” association suggesting potential beneficial effect of moderate drinking among those without G variant copies (CC genotype). These results imply that abstinence from alcohol may offset the genetic risk of fatty liver disease. Furthermore, the dose-response association of alcohol consumption and hepatic steatosis among individuals with the PNPLA3 GG genotype helps support recent guidelines, emphasizing that there is no safe drinking level for patients with NAFLD.
      From a clinical point of view, these findings are important given that moderate alcohol consumption is widespread worldwide, yet there is substantial and increasing controversy regarding the health effects of alcohol consumption.
      World Health Organization
      Global Status Report on Alcohol and Health.
      Current guidelines for patients with NAFLD concede a potential benefit of moderate drinking and give equivocal recommendations.
      • Chalasani N.
      • Younossi Z.
      • Lavine J.E.
      • et al.
      The diagnosis and management of nonalcoholic fatty liver disease: practice guidance from the American Association for the Study of Liver Diseases.
      ,
      EASL-EASD-EASO Clinical Practice Guidelines for the management of non-alcoholic fatty liver disease.
      Our results, although observational in nature, suggest that any potential beneficial effect of moderate alcohol consumption on fatty liver disease may be limited to those with the CC genotype (just over half the population) and therefore emphasize the role of keeping alcohol consumption low as an effective and modifiable way to protect against liver damage. Indeed, our findings help to further support the recommendations included in the recently published “Diagnosis and Treatment of Alcohol-Related Liver Disease: 2019 Practice Guidance from the American Association for the Study of Liver Diseases” that concludes “Patients with ALD or other liver diseases, in particular NAFLD, NASH, viral hepatitis, and hemochromatosis, should be counseled that there is no safe level of drinking, and that they should abstain.”
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      • Im G.Y.
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      Diagnosis and Treatment of Alcohol-Associated Liver Diseases: 2019 Practice Guidance From the American Association for the Study of Liver Diseases.
      In addition, the findings are of significant medical and public health relevance, as the PNPLA3 risk variant is highly common and strongly associated with liver-related outcomes; carriers of at least 1 copy of the risk alleles have a 2-fold risk of liver disease, and carriers of at least 2 copies have a greater than 3-fold risk.
      • Sookoian S.
      • Pirola C.J.
      Meta-analysis of the influence of I148M variant of patatin-like phospholipase domain containing 3 gene (PNPLA3) on the susceptibility and histological severity of nonalcoholic fatty liver disease.
      ,
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      PNPLA3 gene polymorphism is associated with predisposition to and severity of alcoholic liver disease.
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      PNPLA3 rs738409 polymorphism associated with hepatic steatosis and advanced fibrosis in patients with chronic hepatitis C virus: a meta-analysis.
      In that context, findings related to gene and environment interactions are important. Our findings extend prior studies observing interaction between PNPLA3 variant and adiposity,
      • Stender S.
      • Kozlitina J.
      • Nordestgaard B.G.
      • et al.
      Adiposity amplifies the genetic risk of fatty liver disease conferred by multiple loci.
      PNPLA3 variant and adiposity and alcohol,
      • Dunn W.
      • Zeng Z.
      • O'Neil M.
      • et al.
      The interaction of rs738409, obesity, and alcohol: a population-based autopsy study.
      and PNPLA3 variant and chronic hepatitis C and alcohol.
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      • et al.
      Distinct, alcohol-modulated effects of PNPLA3 genotype on progression of chronic hepatitis C.
      Together, these results imply that even though the effect of PNPLA3 genetic variant on liver disease is strong, this effect may be modulated by environmental factors. These findings emphasize the potential benefits of interventions to lower alcohol consumption and for weight loss at the population level to offset genetic risk for liver disease. Future research is needed to determine to what extent interventions to lower alcohol consumption and for weight loss based on PNPLA3 genotype screening are more efficacious than those without genetic testing.
      Moreover, given that the PNPLA3 risk variant has marked racial and ethnic differences (49% of individuals with Hispanic ancestry and 23% among individuals with European ancestry),
      • Romeo S.
      • Kozlitina J.
      • Xing C.
      • et al.
      Genetic variation in PNPLA3 confers susceptibility to nonalcoholic fatty liver disease.
      our findings have substantial relevance for reducing racial and ethnic disparities in liver disease among Hispanics, who have experienced large and sustained disparities in liver-related morbidity and mortality.
      • Rich N.E.
      • Oji S.
      • Mufti A.R.
      • et al.
      Racial and ethnic disparities in nonalcoholic fatty liver disease prevalence, severity, and outcomes in the United States: a systematic review and meta-analysis.
      From a biological perspective, the results of this study extend our current understanding of the mechanisms by which PNPLA3 mutation lead to hepatic steatosis and ethanol leads to triglycerides accumulation. It has been suggested that the presence of the PNPLA3 is not sufficient to cause hepatic steatosis,
      • Basantani M.K.
      • Sitnick M.T.
      • Cai L.
      • et al.
      Pnpla3/Adiponutrin deficiency in mice does not contribute to fatty liver disease or metabolic syndrome.
      and that overexpression of PNPLA3 is also required.
      • Li J.Z.
      • Huang Y.
      • Karaman R.
      • et al.
      Chronic overexpression of PNPLA3I148M in mouse liver causes hepatic steatosis.
      The lipid droplets coated by mutant PNPLA3 protein resists ubiquitylation and prevents degradation of triglycerides.
      • BasuRay S.
      • Smagris E.
      • Cohen J.C.
      • et al.
      The PNPLA3 variant associated with fatty liver disease (I148M) accumulates on lipid droplets by evading ubiquitylation.
      ,
      • BasuRay S.
      • Wang Y.
      • Smagris E.
      • et al.
      Accumulation of PNPLA3 on lipid droplets is the basis of associated hepatic steatosis.
      Based on animal studies, there is evidence that ethanol increases triglyceride synthesis independently of insulin action, for example through SREBP-1 expression,
      • You M.
      • Fischer M.
      • Deeg M.A.
      • et al.
      Ethanol induces fatty acid synthesis pathways by activation of sterol regulatory element-binding protein (SREBP).
      which, in turn, can increase expression of PNPLA3.
      • Huang Y.
      • He S.
      • Li J.Z.
      • et al.
      A feed-forward loop amplifies nutritional regulation of PNPLA3.
      In vivo studies in mice and in vitro experiments have shown that diets rich in either carbohydrates or alcohol induce the expression of PNPLA3.
      • Huang Y.
      • He S.
      • Li J.Z.
      • et al.
      A feed-forward loop amplifies nutritional regulation of PNPLA3.
      ,
      • Restrepo R.J.
      • Lim R.W.
      • Korthuis R.J.
      • et al.
      Binge alcohol alters PNPLA3 levels in liver through epigenetic mechanism involving histone H3 acetylation.
      The extent to which these factors induce the expression of PNPLA3 in humans is an area that deserves further attention. Moreover, recent evidence from in vivo studies demonstrated the role of downregulation of the PNPLA3 mutant allele in decreasing hepatic steatosis and inflammation,
      • Linden D.
      • Ahnmark A.
      • Pingitore P.
      • et al.
      Pnpla3 silencing with antisense oligonucleotides ameliorates nonalcoholic steatohepatitis and fibrosis in Pnpla3 I148M knock-in mice.
      implying that factors that target this gene variant may be considered as part of the therapeutic armamentarium for treating fatty liver.
      While this study had a number of strengths, such as its large size and representation of the U.S. adult population, there are also limitations to keep in mind when interpreting the results. These data were collected over 20 years ago. However, this is the only nationally representative study in the United States with data on PNPLA3 genotype, alcohol consumption, and hepatic steatosis. The prevalence estimates of both hepatic steatosis and the PNPLA3 G allele in the population are likely underestimated, given the steep increase in NAFLD and increases in the Hispanic population.
      • Lazo M.
      • Hernaez R.
      • Eberhardt M.S.
      • et al.
      Prevalence of nonalcoholic fatty liver disease in the United States: the Third National Health and Nutrition Examination Survey, 1988–1994.
      However, given that the mechanistic effects of alcohol or the PNPLA3 gene should not be dependent on their distribution in the population, the main inferences of this study should not be affected. This study had an observational cross-sectional design, limiting our ability to draw inferences about causality regarding the effect of alcohol consumption on liver steatosis. However, our study aim was to examine the role of alcohol consumption modulating the genetic vulnerability of PNPLA3 genotype on hepatic steatosis and the main exposure was a genetic variant and was not susceptible to confounding. Moreover, because we focused on a single self-reported measure of alcohol consumption, and heavy alcohol consumption is associated with higher morbidity and mortality, individuals with susceptibility to alcohol damage may have been selected out of our sample. This measure may also be subject to recall bias and misclassification. In both cases, the extent to which these biases affect our estimate of the interaction is likely to be toward the null. We used ultrasound to determine hepatic steatosis. While this is not the gold standard, it has shown to be a reliable and accurate tool for the detection of moderate-severe fatty liver and is widely used in large epidemiological studies.
      • Hernaez R.
      • Lazo M.
      • Bonekamp S.
      • et al.
      Diagnostic accuracy and reliability of ultrasonography for the detection of fatty liver: a meta-analysis.
      To maximize validity, we focused in the more extreme phenotype (severe steatosis) as the outcome. Future studies using liver biopsy, ultrasound elastography, magnetic resonance measures of hepatic steatosis and clinical outcomes, and alcohol consumption biomarkers are needed to allow for a more accurate estimation of the effect of the interaction, as well as the characterization of the interaction with liver fibrosis, a more advanced manifestation of fatty liver disease. Last, other genetic determinants of fatty liver such as TM6SF2 and MBOAT7 were not available in NHANES III and could not be examined.

      Conclusion

      Our results suggest that strategies to reduce alcohol consumption may be an effective measure to offset genetic risk of liver disease. Furthermore, our results of a dose-response association of alcohol consumption and hepatic steatosis among individuals with PNPLA3 GG genotype help support recent guidelines, emphasizing that there is no safe drinking level for patients with NAFLD.

      Materials and Methods

       Third National Health and Nutrition Examination Survey Phase 2

      By design, the Third National Health and Nutrition Examination Survey (NHANES III)was divided into 2 phases: phase 1 was conducted from 1988 to 1990 and phase 2 was conducted from 1991 to 1994. Genetic data are available for phase 2 participants only and constitute the sampling frame for the current analyses. Each phase can be analyzed separately and using the appropriate sampling weights, and results are generalizable to the entire U.S. civilian noninstitutionalized population.

       Exposures

      Genotyping of rs738409 (PNPLA3) was performed using the iPLEX Sequenom platform (CG Genomics, Shirley, NY)and using strict quality control criteria, including testing for deviations from the Hardy-Weinberg Equilibrium.
      Third National Health and Nutrition Examination Survey
      Hepatic Steatosis Assessment Procedure Manual.
      Participants were asked about lifetime consumption of alcoholic beverage (yes/no), past 12 months consumption of alcohol (yes/no), and among current drinkers, the frequency per week and number of alcoholic drinks per day on a drinking day over the past 12 months. We calculated average alcohol consumption per day multiplying the number of drinking days and the number of drinks per day, on average, on a drinking day.

       Outcome

      Gallbladder ultrasounds of NHANES III were performed on participants 20–74 years of age during the physical exam using a Toshiba SSA-90A machine (Toshiba, Tustin, CA). The protocol used to ascertain the presence of hepatic steatosis using these ultrasounds can be found elsewhere.
      Third National Health and Nutrition Examination Survey
      Hepatic Steatosis Assessment Procedure Manual.
      For the current analyses, to maximize specificity and accuracy of the outcome, we defined hepatic steatosis as the presence of severe hepatic steatosis by ultrasound.

       Other variables

      Questionnaires were used to obtain data on sex, age, race or ethnicity, education, income, and prevalent medical conditions. Standardized measurements of height and weight were obtained and used to calculate body mass index. Serum biochemistries were performed using the Hitachi 737 automated multichannel chemistry analyzer (Boehringer Mannheim Diagnostics, Indianapolis, IN). The following liver tests were assayed: aspartate aminotransferase, alanine aminotranferase, and γ-glutamyltransferase.

       Statistical methods

      We examined the interaction between alcohol consumption categories and PNPLA3 genotype in both the multiplicative scale and the additive scale. In the multiplicative scale, we estimated relative prevalence ratio (RPR) as a measure of departure of the effect expected of the product of the two exposures considered separately. For the additive scale, we used relative excess risk due to interaction (RERI) as a measure of departure of the sum of the individual effects on a relative scale. From a public health perspective, an interaction in the additive scale is important, as it provides a measure of the excess and deficit in the number of cases attributable to the 2 exposures in combination compared with what we would expect from the sum of the cases associated with each exposure.
      The RPR and the RERI were estimated as follows. From the interaction model:
      log(PSHS=1)=β0+β1Moderate+β2Heavy+β3Alleles+β4ModerateAlleles+β5HeavyAlleles+βcCc


      where moderate is an indicator variable for the individual being a low-moderate drinker, heavy is an indicator variable for the individual being a heavy drinker, alleles is an ordinal variable with the number of risk alleles, and C is a vector of the confounders described previously. The RPR is estimated as:
      RPRmoderate=eβ4andRPRheavy=eβ5


      While the RERI was estimated as:
      RERImoderate=eβ1+β3+β4eβ1eβ3+1andRERIheavy=eβ2+β3+β5eβ2eβ3+1


      For the RPR and the RERI, 95% confidence intervals were estimated using the delta method.
      • Hosmer D.W.
      • Lemeshow S.
      Confidence interval estimation of interaction.
      For the main analyses, we present the results using never drinkers and those with no copy of the allele as the reference group. In sensitivity analyses, we present the results using moderate drinkers with no copy of allele, the group with the lowest risk, as the reference group, to test the robustness of our results to the controversy around the use and interpretation of RERI for protective factors (e.g. moderate drinking).
      EASL-EASD-EASO Clinical Practice Guidelines for the management of non-alcoholic fatty liver disease.
      All analyses incorporated the sampling weights to obtain unbiased estimates from the complex sampling design. The standard errors for all estimates were obtained using the Taylor series (linearization).
      Analyses were conducted using STATA 13 (StataCorp, College Station, TX).
      Supplemental Table 1Adjusted Prevalence Ratios for Severe Hepatic Steatosis Comparing Alcohol Consumption Categories, Overall and by Race/Ethnicity (Third National Health and Nutrition Examination Survey, Phase 2)
      Overall (Non-Hispanic White, Non-Hispanic Black, Mexican American)
      Never1 (reference)
      Former0.80 (0.54–1.18)
      Low-moderate0.76 (0.49–1.19)
      Heavy1.30 (0.73–2.32)
      Non-Hispanic White
      Never1 (reference)
      Former0.78 (0.51–1.20)
      Low-moderate0.81 (0.47–1.39)
      Heavy1.42 (0.77–2.62)
      Non-Hispanic Black
      Never1 (reference)
      Former0.88 (0.44–1.76)
      Low-moderate0.52 (0.28–0.94)
      Heavy1.06 (0.40–2.85)
      Mexican-American
      Never1 (reference)
      Former1.02 (0.62–1.68)
      Low-moderate0.96 (0.54–1.71)
      Heavy1.42 (0.90–2.24)
      Values are adjusted prevalence ratio (95% confidence interval). Values were adjusted for race/ethnicity, sex, age, age squared, education, income, and PNPLA3 genotype
      Supplement Table 2Adjusted Prevalence Ratios for Severe Hepatic Steatosis by PNPLA3 Genotypes, Overall and by Race/Ethnicity (Third National Health and Nutrition Examination Survey. Phase 2)
      Overall (Non-Hispanic White, Non-Hispanic Black, Mexican American)
      CC1 (reference)
      CG1.21 (0.76–1.92)
      GG1.70 (0.83–3.49)
      Non-Hispanic White
      CC1 (reference)
      CG1.21 (0.70–2.08)
      GG1.59 (0.64–3.93)
      Non-Hispanic Black
      CC1 (reference)
      CG1.18 (0.87–1.61)
      GG1.70 (0.70–4.10)
      Mexican-American
      CC1 (reference)
      CG1.53 (0.89–2.63)
      GG2.52 (1.35–4.71)
      Values are adjusted prevalence ratio (95% confidence interval). Values were adjusted for race/ethnicity, sex, age, age squared, education, income, and PNPLA3 genotype.
      Supplement Table 3Adjusted Prevalence Ratios for the Interaction Between Alcohol Consumption and PNPLA3 Genotype and Severe Hepatic Steatosis (Third National Health and Nutrition Examination Survey. Phase 2)
      PNPLA3 G AlleleNeverLow-Moderate
      Low-moderate alcohol consumption: ≤1 and ≤2 drinks/d for women and men, respectively.
      Heavy
      Heavy alcohol consumption: >1 and 2 drinks/d in women and men, respectively.
      Stratified PR
      01.911.0 (reference)1.87
      +1 copy0.981.0 (reference)1.64
      Multiplicative interaction metric
      RPR
      The null hypothesis is that RP R = 1. An RPR > 1 indicates positive interaction on the multiplicative scale and RPR < 1 indicated negative interaction on the multiplicative scale.
      0.511.0 (reference)0.87
      Single referent PR
      01.911.0 (reference)1.87
      +1 copy1.471.502.46
      Additive interaction metrics
      RERI
      Calculated as: PR ++-PR +- -PR -++1. The null hypothesis is that RERI = 0. A RERI > 0 indicates positive interaction on the additive scale and RERI < 0 indicates negative interaction on the additive scale.
      –0.94
      Calculated as: PR ++-PR +- -PR -++1. The null hypothesis is that RERI = 0. A RERI > 0 indicates positive interaction on the additive scale and RERI < 0 indicates negative interaction on the additive scale.
      0 (reference)0.09
      Adjusted for race/ethnicity, sex, age, age squared, education, and income.
      aPR, adjusted prevalence ratio; PR, prevalence ratio; RERI, relative excess risk due to interaction; RPR, relative prevalence ratio.
      a Low-moderate alcohol consumption: ≤1 and ≤2 drinks/d for women and men, respectively.
      b Heavy alcohol consumption: >1 and 2 drinks/d in women and men, respectively.
      c The null hypothesis is that RP R = 1. An RPR > 1 indicates positive interaction on the multiplicative scale and RPR < 1 indicated negative interaction on the multiplicative scale.
      d Calculated as: PR ++-PR +- -PR -++1. The null hypothesis is that RERI = 0. A RERI > 0 indicates positive interaction on the additive scale and RERI < 0 indicates negative interaction on the additive scale.

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