M.J. Way

Homozygosity for rs738409:G in PNPLA3 is associated with increased mortality following an episode of severe alcoholic hepatitis

BACKGROUND & AIMS Carriage of rs738409:G in PNPLA3 is associated with an increased risk of developing alcohol-related cirrhosis and has a significant negative effect on survival. Short-term mortality in patients with severe alcoholic hepatitis is high; drinking behaviour is a major determinant of outcome in survivors. The aim of this study was to determine whether carriage of rs738409:G has an additional detrimental effect on survival in this patient group. METHODS Genotyping was undertaken in 898 cases with severe alcoholic hepatitis, recruited through the UK Steroids or Pentoxifylline for Alcoholic Hepatitis (STOPAH) trial, and 1188 White British/Irish alcohol dependent controls with no liver injury, recruited via University College London. Subsequent drinking behaviour was classified, in cases surviving ≥90days, as abstinent or drinking. The relationship between rs738409 genotype, drinking behaviour and survival was explored. RESULTS The frequency of rs738409:G was significantly higher in cases than controls (29.5% vs. 18.9%; p=2.15×10-15; odds ratio 1.80 [95% confidence interval (CI) 1.55-2.08]). Case-mortality at days 28, 90 and 450 was 16%, 25% and 41% respectively. There was no association between rs738409:G and 28-day mortality. Mortality in the 90 to 450-day period was higher in survivors who subsequently resumed drinking (hazard ratio [HR] 2.77, 95% CI 1.79-4.29; p<0.0001) and in individuals homozygous for rs738409:G (HR 1.69, 95% CI 1.02-2.81, p=0.04). CONCLUSION Homozygosity for rs738409:G in PNPLA3 confers significant additional risk of medium-term mortality in patients with severe alcoholic hepatitis. Rs738409 genotype may be taken into account when considering treatment options for these patients. LAY SUMMARY Individuals misusing alcohol who carry a particular variant of the gene PNPLA3 are more at risk of developing severe alcoholic hepatitis, a condition with a poor chance of survival. The longer-term outcome in people with this condition who survive the initial illness is strongly influenced by their ability to remain abstinent from alcohol. However, carriers of this gene variant are less likely to survive even if they are able to stop drinking completely. Knowing if someone carries this gene variant could influence the way in which they are managed. Clinical trial numbers: EudraCT reference number: 2009-013897-42; ISRCTN reference number: ISRCTN88782125. CLINICAL TRIAL NUMBERS EudraCT reference number: 2009-013897-42; ISRCTN reference number: ISRCTN88782125.

Phenotypic heterogeneity in study populations may significantly confound the results of genetic association studies on alcohol dependence.

Background The interpretation of genetic studies on alcohol dependence may be confounded by the co-occurrence of substance dependence, psychiatric disorders and alcohol-related comorbidities, for example, cirrhosis. Significant single-marker and haplotypic associations between polymorphisms in the zinc finger gene, ZNF699, and alcohol dependence were reported in the Irish Affected Sib Pair Study of Alcohol Dependence population, one-third of whom had co-occurring substance dependence while 80% had identified psychiatric comorbidity. The aim of this study was to explore variant ZNF699 associations with alcohol dependence while exercising controls for potential confounders. Methods The study population was comprised of 1449 alcohol-dependent cases and 1283 population controls; all were of British or Irish ancestry. None of the cases had a history of dependence on other substances, and the frequency of comorbid depression was low. A separate, ancestry-matched cohort of 196 opioid-dependent cases was also included. Genotyping for the four previously identified SNPs of interest in ZNF699 was performed using K-Biosciences Competitive Allele Specific PCR. Results No single-marker associations were found between polymorphisms in ZNF699 and alcohol dependence per se. A significant allelic association was found between rs7254880 in ZNF699 and alcohol-related cirrhosis (n=292), using cases with no biopsy evidence of liver disease (n=314) as controls (P=0.013). Significant allelic associations were also found between rs12460279 (P=0.028), rs7252865 (P=0.012) and rs10854142 (P=0.016) in ZNF699 and opioid dependence. Conclusion Phenotypic variation in study populations may contribute towards the nonreplication of genetic association studies on alcohol dependence; controls for recognised confounding variables should be exercised whenever possible.

Genetic variation in GABRβ1 and the risk for developing alcohol dependence

Associations between the &ggr;-aminobutyric acid type-A receptors (GABAA) and alcohol dependence risk have been reported, although the receptor subunit driving the association is unclear. Recent work in mice has highlighted a possible role for variants in the Gabr &bgr;1 subunit (Gabr&bgr;1) in alcohol dependence risk, although this gene does not contain any common nonsynonymous variants in humans. However, the GABAA receptor is a heteropentamer so multiple potential variants within the gene complex could generate the alcohol dependence phenotype. The association between GABR&bgr;1 variants and alcohol dependence risk was explored in a British and Irish population of alcohol-dependent cases (n=450) and ancestrally-matched controls screened to exclude current or historical alcohol misuse (n=555). Twelve common single nucleotide polymorphisms (SNPs) and a rare nonsynonymous variant, rs41311286, were directly genotyped; imputation was then performed across the whole gene. No allelic association was observed between alcohol dependence risk and any of the directly genotyped or imputed SNPs. However, post-hoc testing for genotypic association identified five common intronic SNPs that showed modest evidence for association after correction for multiple testing; two, rs76112682 and rs141719901, were in complete linkage disequilibrium [Pcorrected=0.02, odds ratio (95% confidence interval)=5.9 (1.7–2.06)]. These findings provide limited support for an association between GABR&bgr;1 and the risk for developing alcohol dependence; further testing in expanded cohorts may be warranted.

Evidence for genetic susceptibility to the alcohol dependence syndrome from the thiamine transporter 2 gene solute carrier SLC19A3.

The risk for developing the alcohol dependence syndrome (ADS) has a substantial genetic component. The human thiamine transporter protein 2 (hTHTR2) is encoded by the SLC19A3 gene, which is on chromosome 2q37. hTHTR2 is responsible for the cellular uptake of thiamine (B1), a water-soluble essential vitamin that plays a fundamental and ubiquitous role in carbohydrate metabolism. This gene was also found to be associated with biotin-responsive basal ganglia disease, an autosomal recessive metabolic disorder characterized by encephalopathy and ophthalmoplegia (Ozand et al., 1998; Zeng et al., 2005). Homozygous or compound heterozygous mutations in SLC19A3 cause two distinct clinical phenotypes: biotin-responsive basal ganglia disease and Wernicke’s-like encephalopathy. Biotin and/or thiamine are effective therapies for both diseases (Yamada et al., 2010). A missense mutation in exon 5 of the SLC19A3 was found in 18 cases of biotin/thiamine-responsive basal ganglion disease presenting with subacute encephalopathy and extrapyramidal signs (Alfadhel et al., 2013). Kono et al. (2009) described two Japanese brothers, who were both compound heterozygotes for the K44E and E320Q mutations in SLC19A3, who developed a syndrome of thiamine-responsive diplopia, ophthalmoplegia and ataxia, similar to Wernicke’s encephalopathy, despite normal serum thiamine levels (Kono et al., 2009). Yamada et al. (2010) reported a pathogenic homozygous mutation (c.958G>C, [p.E320Q]) in SLC19A3 in four patients from a single family. They report a wide variety of neurological signs in SLC19A3 mutation carriers. Our previous unpublished research found that four markers in the SLC19A3 gene showed significant allelic association with Wernicke–Korsakoff syndrome (WKS) in a sample of 120 cases when compared with normal controls. In the present study, the entire SLC19A3 gene was screened for DNA variation in a WKS subset (n=120) of a UK ADS case–control sample comprised of 1032 alcohol-dependent cases and 1022 controls. High resolution melting curve analysis, which is based on the melting characteristic of double-stranded DNA, was carried out using a LightCycler 480 Real-Time PCR System (Roche, Burgess Hill, UK). Genetic variation was validated with Sanger DNA sequencing. Thirteen single nucleotide variants were identified through high resolution melting analysis. Two exon 3 variants that were predicted to cause amino acid substitutions, 2:228563818T/C and rs148144444, were selected for genotyping in the entire ADS case–control sample using an allele-specific fluorescent PCR method (KasPar; LGC Genomics, Hoddesdon, UK). Statistical analysis was carried out on the previously unreported 2:228563818T/C change of a T to C substitution at position 228 563 818 on chromosome 2. This variant causes an R250G amino acid substitution in the largest cytoplasmic domain of the protein and it is, therefore, likely to affect post-translational function. rs148144444 causes the amino acid change G141S which is likely to exert an effect on protein phosphorylation and conformation because of the introduction of the aliphatic chain of serine. Neither the cases nor the controls in the present study had the SLC19A3 disease susceptibility variants that have been reported previously (Zeng et al., 2005; Kono et al., 2009). The minor allele of 2:228563818T/C was detected in five ADS cases, but was absent in the control samples (P=0.033). The minor allele of rs148144444 was detected in five ADS cases and in four controls and was not associated with ADS. Neither of these variants was present in the 120 WKS cases in our ADS sample. Our data suggest that genetic variation in the SLC19A3 thiamine transporter at 2:228563818T/C may make a modest contribution towards the genetic susceptibility to ADS.

A TWO-STAGE GENOME-WIDE ASSOCIATION STUDY IDENTIFIES TM6SF2 AND MBOAT7 AS RISK LOCI FOR ALCOHOL-RELATED CIRRHOSIS

Material and Methods: Patients were randomized 1:1:1 to receive either SOF+RBV for 16 or 24 weeks or SOF+PEG/RBV for 12 weeks and stratified by HCV genotype and cirrhosis status. All patients received SOF 400mg daily and RBV 1000–1200mg in a divided daily dose. PEG was administered as 180mg weekly injection. The primary end point was sustained virologic response 12 weeks after treatment (SVR12). Results: Of 592 patients randomized and treated, 92% had GT3 HCV, 67% were male, 84% white, 53% treatment experienced, 62% had non-CC IL28B genotypes, and 37% had cirrhosis. GT2 treatmentexperienced patients with cirrhosis had high SVR12 rates in all treatment groups: 87% of those receiving SOF+RBV for 16 weeks, 100% of those receiving SOF+RBV for 24 weeks, and 94% of those receiving SOF+PEG/RBV for 12 weeks. Among GT3 patients, SVR12 rates were highest in those receiving SOF+PEG/RBV for 12 weeks (93%) as compared to SOF+RBV for 24 (84%, p 0.008) or 16 weeks (71%, p < 0.001) (Table). The most common adverse events in all arms were fatigue, headache, insomnia, and nausea. Overall, 6 (1%) patients discontinued treatment due to adverse events; one of them was treated with SOF+PEG/RBV.

Genetic variants in ALDH1B1 and alcohol dependence risk in a British and Irish population: A bioinformatic and genetic study.

Alcohol is metabolized in the liver via the enzymes alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH). Polymorphisms in the genes encoding these enzymes, which are common in East Asian populations, can alter enzyme kinetics and hence the risk of alcohol dependence and its sequelae. One of the most important genetic variants, in this regards, is the single nucleotide polymorphism (SNP) rs671 in ALDH2, the gene encoding the primary acetaldehyde metabolizing enzyme ALDH2. However, the protective allele of rs671 is absent in most Europeans although ALDH1B1, which shares significant sequence homology with ALDH2, contains several, potentially functional, missense SNPs that do occur in European populations. The aims of this study were: (i) to use bioinformatic techniques to characterize the possible effects of selected variants in ALDH1B1 on protein structure and function; and, (ii) to genotype three missense and one stop-gain, protein-altering, non-synonymous SNPs in 1478 alcohol dependent cases and 1254 controls of matched British and Irish ancestry. No significant allelic associations were observed between the three missense SNPs and alcohol dependence risk. The minor allele frequency of rs142427338 (Gln378Ter) was higher in alcohol dependent cases than in controls (allelic P = 0.19, OR = 2.98, [0.62–14.37]) but as this SNP is very rare the study was likely underpowered to detect an association with alcohol dependence risk. This potential association will needs to be further evaluated in other large, independent European populations.

OC-031 A Genome-Wide Association Study Identifies PNPLA3 and SLC38A4 as Risk Loci for Alcoholic Hepatitis

Introduction The clinical spectrum of alcohol-related liver disease varies widely but the majority of patients with evolving disease are asymptomatic. However, a small minority of patients develop severe alcoholic hepatitis (SAH), a clinical syndrome manifest as profound hepatocellular failure, often superimposed on cirrhosis. As only a relative minority of patients develop SAH genetic determination of susceptibility has been proposed. A genome-wide association study (GWAS) approach was used to identify potential risk loci for SAH. Methods Cases with SAH, of self-reported white British ancestry, were recruited prospectively through the Steroids or Pentoxifylline for Alcoholic Hepatitis trial (n = 860). Ancestry-matched controls with alcohol dependence but without alcohol-related liver injury were recruited from the Centre for Hepatology, Royal Free Hospital, London (n = 1191). A two-stage GWAS was performed. DNA samples from an exploratory cohort (332 cases, 318 controls) were genotyped on Illumina CoreExome BeadChips (Illumina, San Diego, USA) at the Wellcome Trust Sanger Institute, Cambridge, UK. The most significantly associated SNPs were genotyped in DNA samples from an independent replication cohort (528 cases, 873 controls) using KASPar chemistry (LGC Genomics, Hoddesdon, UK). Data were analysed in PLINK1.9. Results The variant rs738409 in patatin-like phospholipase domain-containing protein 3 (PNPLA3) was associated at genome-wide significance level (PTHRESHOLD <5x10−8; PEXPLORATORY =1.619x10−8, OR 2.21 [95% CI 1.68–2.91]). Ten additional, independent loci demonstrated suggestive association (PTHRESHOLD <1.1x10−5). Replication genotyping for the lead markers at each locus demonstrated disease association for rs738409 in PNPLA3 (PREPLICATION = 3.47x10−8; PMETA = 8.62x10−15; OR 1.87) and Solute Carrier Family 38, Member 4 (SLC38A4) (PREPLICATION = 0.029; PMETA = 4.13x10−5; OR 1.32).Abstract OC031 Figure 1 Conclusion This first analysis of data from a GWAS of severe alcoholic hepatitis confirms significant risk association with PNPLA3, consistent with the phenotypic overlap with alcohol-related cirrhosis. It also identified SLC38A4 as a potential, novel risk locus for development of alcoholic hepatitis per se; this gene is predominantly expressed in the liver and is involved in amino acid transport. Disclosure of Interest None Declared

PWE-033 Promoter Region Variations in The Glutaminase Gene as A Risk Factor for The Development Hepatic Encephalopathy in Patients with Cirrhosis: Abstract PWE-033 Table 1

Introduction Hepatic encephalopathy (HE) is the commonest complication of cirrhosis but its development is not invariable; thus approximately 20% of individuals remain free of neuropsychiatric complications and patients with minimal HE do not invariably develop overt HE. It is possible that genetic factors, most likely resulting in deregulation of ammonia metabolism, might determine an individual’s susceptibility to develop this complication. In 2010, Romero-Gomez and colleagues,1 identified a functional microsatellite consisting of GCA repeats near the promoter region of the glutaminase gene, GLS1, and proposed that patients with cirrhosis who were homozygous for the long microsatellite allele were more likely to develop overt HE. The aim of this study was to further investigate the association between GLS1 promoter microsatellite variants and HE. Methods The study population comprised 110 patients with cirrhosis, of British/Irish ancestry, in whom neuropsychiatic status had remained stable during monitoring for a minimum of 5 years, except in those treated for overt HE. Patients were classified using clinical, neurophysiological and neuropsychometric variables as: unimpaired (51; 46%) or as having minimal (24; 22%) or overt (35; 32%) HE. The control population comprised 325 ancestrally-matched healthy controls. DNA was genotyped using gel electrophoresis and verified by Sanger sequencing. Alleles were stratified as long (≥14 GCA repeats) or short (<14 GCA repeats). Genotype distributions and allele frequencies were compared between groups. Results There were no significant differences in allele frequencies or genotype distributions between cases and controls. The proportion of long-short heterozygotes was significantly higher in the unimpaired patients (62.7%) than in those with either minimal HE (37.5%) or overt HE (37.1%) (p = 0.029; Table).Abstract PWE-033 Table 1 Genotype Controls(n = 325) All Patients(n = 110) Unimpaired(n = 51) Minimal HE(n = 24) Overt HE(n = 35) Intergroupsignificance(p) n (%) Long-long 103 (31.7) 39 (35.5) 15 (29.4) 9 (37.5) 15 (42.9) 0.428 Long-short 163 (50.2) 54 (49.1) 32 (62.7) 9 (37.5) 13 (37.1) 0.029 Short-short 59 (18.2) 17 (15.5) 4 (7.8) 6 (25.0) 7 (20.0) 0.106 Conclusion There was no evidence that the long-long genotype is a risk factor for HE in the present study. However, there was evidence of possible heterozygote advantage resulting in significant protection against the development of this syndrome. More studies are needed to confirm or refute these findings. Reference 1 Romero-Gomez M, et al. Variations in the promoter region of the glutaminase gene and the development of hepatic encephalopathy in patients with cirrhosis: a cohort study. Ann Intern Med 2010;153:281–8. Disclosure of Interest None Declared