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- Publisher
- American Society for Biochemistry and Molecular Biology
- Copyright
- Copyright © 2009 Elsevier Inc.
- ISSN
- 0022-2275
- eISSN
- 1539-7262
- DOI
- 10.1194/jlr.p900013-jlr200
- Publisher site
- See Article on Publisher Site
Abstract
patient-oriented and epidemiological research A nonsynonymous gene variant in the adiponutrin gene is associated with nonalcoholic fatty liver disease severity 1, ,§, §, † Silvia Sookoian, * ** Gustavo O. Castaño, ** Adriana L. Burgueño, Tomas Fernández † ,† 1,† Gianotti, María Soledad Rosselli, * and Carlos Jose Pirola Laboratory of Clinical and Molecular Hepatology,* and Laboratory of Molecular Genetics and Biology of the Metabolic Syndrome, Department of Molecular Genetics and Biology of Complex Diseases, Institute of Medical Research, University of Buenos Aires-CONICET, Ciudad Autónoma de Buenos Aires, Argentina; Research Council of GCBA, Ciudad Autónoma de Buenos Aires, Argentina; and Department of Medicine and Surgery,** Hospital Abel Zubizarreta, Ciudad Autónoma de Buenos Aires, Argentina Abstract We explored the role of the adiponutrin ( PNPLA3 ) Nonalcoholic fatty liver disease (NAFLD) is an emerg- nonsynonymous-rs738409 single nucleotide polymorphism ing epidemic disease with increasing prevalence world- (SNP) in genetic susceptibility to nonalcoholic fatty liver dis- wide. NAFLD not only affects the adult population (an ease (NAFLD) and whether this SNP contributes to the sever- estimated 20–40% of adults in Western countries have ex- ity of histological disease. Two hundred sixty-six individuals cess fat accumulation in the liver) ( 1 ), but also is the most were evaluated in a case-control association study, which in- common cause of pediatric liver disease ( 2 ). cluded 172 patients with features of NAFLD and 94 control The pathogenesis of NAFLD is multifactorial; as a com- subjects. The rs738409 G allele was signifi cantly associated plex disease, the disorder develops from the interplay be- with NAFLD ( P < 0.001; OR 2.8 95%, CI 1.5–5.2), indepen- tween genetic susceptibility and the environment. dent of age, sex, body mass index (BMI), and Homeostasis Model Assessment (HOMA) index. When we tested the hy- NAFLD refers to a wide spectrum of liver diseases, rang- pothesis of a relation between the SNP and the histological ing from fatty liver alone to nonalcoholic steatohepatitis spectrum of NAFLD, a signifi cant association was observed (NASH) with evidence of liver cell injury, a mixed infl am- [chi 2 19.9, degree of freedom (df): 2, P < 5 × 10 , adjusted matory lobular infi ltrate, and variable fi brosis ( 3 ). Patients for HOMA and BMI]. The degree of liver steatosis, as evalu- with simple steatosis usually have a benign prognosis for ated by liver biopsy, was signifi cantly associated with the liver disease. In contrast, up to 20% of patients with NASH rs738409 G allele. Patients with CC genotype showed a lower can progress to cirrhosis ( 4 ). steatosis score (14.9% ± 3.9) in comparison with the CG geno- While it is well known that environmental risk factors type (26.3% ± 3.5) and GG genotype (33.3% ± 4.0) ( P < 0.005). The proportion of the total variation attributed to infl uence the development and progression of NAFLD ( 5, rs738409 genotypes was 5.3% ( 0.23 ± 0.07; P < 0.002). 6 ), the contribution of the individual genetic variation to Our data suggest that the rs738409 G allele is associated not disease predisposition is still uncertain, despite the fact only with fat accumulation in the liver but also with liver in- that several genes in isolated studies have been suggested jury, possibly triggered by lipotoxicity. —Sookoian, S., G. O. as potential candidates either for NAFLD susceptibility or Castano, A. L. Burgueno, T. F. Gianotti, M. S. Rosselli, and C. disease progression ( 7–14 ). J. Pirola. A nonsynonymous gene variant in the adiponutrin Advances in genome analysis (including the devel- gene is associated with nonalcoholic fatty liver disease sever- opment of comprehensive sets of informative genetic ity. J. Lipid Res . 2009. 50: 2111–2116 . markers, improved physical mapping methods, and improvements in high throughput genotyping technology) Supplementary key words SNP � genetics � replication study � PN- PLA3 � fatty liver � nonalcoholic steatohepatitis � NASH Abbreviations: ADPN , adiponutrin; ALT, alanine aminotrans- ferase; AP, alkaline phosphatase; AST, aspartate aminotransferase; BMI, body mass index; df, degree of freedom; GGT, glutamyl-trans- This work was supported in part by grants UBACYT M055 (Universidad de Buenos Aires), PICT 05-25920 and PICT 2006-124 (Agencia Nacional de ferase; GWAS, genome-wide association studies; HOMA, Homeostasis Promoción Científi ca y Tecnológica) and Consejo de Investigación de la Ciudad Model Assessment; NAFLD, nonalcoholic fatty liver disease; NASH, Autonóma de Bs.As. SS, ALB and CJP belong to Consejo Nacional de Investiga- nonalcoholic steatohepatitis; PNPLA3 , patatin-like phospholipase ciones Científi cas (CONICET). domain containing 3 gene; SABP, systolic arterial blood pressure; Manuscript received 2 February 2009 and in revised form 16 March 2009 and in SNP, single nucleotide polymorphism; US, ultrasonographic. re-revised form 1 May 2009. To whom correspondence should be addressed. e-mail: [email protected] or pirola.carlos@lanari. Published, JLR Papers in Press, May 20, 2009 fmed.uba.ar DOI 10.1194/jlr.P900013-JLR200 Copyright © 2009 by the American Society for Biochemistry and Molecular Biology, Inc. This article is available online at http://www.jlr.org Journal of Lipid Research Volume 50, 2009 2111 This is an Open Access article under the CC BY license. drugs known to precipitate steatosis were excluded. In addition, have strongly contributed to the understanding of the patients with any of the following diseases were excluded from pathogenesis of complex diseases. In fact, genome-wide as- participation in this study: autoimmune liver disease, metabolic sociation studies (GWAS) using a dense map of single nu- liver disease, Wilson’s disease, and -1-antitrypsin defi ciency. cleotide polymorphisms (SNP) enable scientists to detect For the purpose of exploring the hypothesis of a relation be- common genetic variants that infl uence susceptibility to tween the rs738409 and NAFLD, we included in the analysis all complex diseases, illuminating both disease mechanisms the NAFLD patients (n = 172), and NAFLD was considered as a and the translation of this knowledge for diagnosis, prog- discrete trait. For the purpose of testing the hypothesis of a rela- nosis, and therapy. A remarkable example of the impact of tion between the rs738409 and the histological severity of NAFLD, we included in the analysis only those patients that had the use of GWAS in the understanding of the genetic archi- histopathologic evidence of fatty liver disease, either simple ste- tecture of human diseases is given by the recent three type atosis or NASH, on liver biopsy performed within this study (n = 2 diabetes GWAS as meta-analyzed by Zeggini et al. ( 15 ). 103) (details in the liver biopsy description). Although factors promoting NAFLD include obesity Control subjects were selected from patients attending our and type 2 diabetes, and NAFLD is now considered the hospital for check-up purposes whose sex matched the NAFLD hepatic manifestation of the metabolic syndrome ( 16 ), patients (63 females and 31 males). there have been no studies using this approach until re- In addition to the standard heath examination, all the control cently when researchers of the Dallas Heart Study per- individuals were subjected to liver US. They were included in the formed a GWAS for liver fat content in 2,111 individuals study if they did not have evidence of fatty change or biochemical abnormalities. Furthermore, control subjects were confi rmed from different ancestry groups ( 17 ). Interestingly, this not to have any of the features of the metabolic syndrome as de- study examined 9,222 nonsynonymous variants and fi ned by the National Cholesterol Education Program Adult showed strong evidence of association of NAFLD, evalu- Treatment Panel III ( 20 ) and did not abuse alcohol. ated by proton magnetic resonance spectroscopy, with the The case participants and the controls were selected during rs738409 G allele of the patatin-like phospholipase do- the same study period from the same population of patients at- main containing 3 gene ( PNPLA3 ), also known as adipo- tending our institution, and all of them shared the same demo- nutrin ( ADPN gene) ( 17 ). In addition, a signifi cant graphic characteristics (occupation, educational level, place of association was also observed between the G allele and the residence and ethnicity). elevation of alanine aminotransferase (ALT) levels, at least Physical, anthropometric, and biochemical evaluation in the Hispanic population. Another population-based Health examination included anthropometric measurements, GWAS of plasma liver-enzyme levels in the Caucasian pop- a questionnaire on health-related behaviors, and biochemical ulation also reported that the PNPLA3 locus is strongly as- determinations. sociated with ALT levels ( 18 ), supporting an infl ammatory Body mass index (BMI) was calculated as weight/height (kg/ effect of this gene on the liver. m ) and used as the index for relative weight. Waist and hip cir- While the functional impact of the reported SNP is still cumference were also assessed. Blood was drawn from fasting unknown, data from these two studies are very consistent subjects who had been supine for at least 30 min. Serum insulin, and point out the role of the PNPLA3 rs738409 [G/C], total cholesterol, HDL and LDL cholesterol, triglycerides, plasma also known as Met148Ile, in the susceptibility to NAFLD. glucose, and liver function tests were measured by standard clini- cal laboratory techniques. Homeostasis Model Assessment None of the above-mentioned studies examined the rela- ( HOMA) was used to evaluate an insulin resistance index and tion between PNPLA3 SNP and NAFLD severity. The main was calculated as fasting serum insulin ( U/ml) × fasting plasma reason seems to be the population-based nature of both glucose (mmol/l)/22.5. studies; none of them included patients with NAFLD diag- Glucose metabolism was evaluated only in NAFLD patients us- nosed by liver biopsy. ing the oral glucose tolerance test performed with 75 g of glucose We performed a hospital-based adult case-control asso- according to World Health Organization criteria. ciation study to replicate for the fi rst time the association Elevated blood pressure was defi ned as systolic arterial blood between the rs738409 and NAFLD susceptibility and to pressure (SABP) 130 mm Hg and/or DABP 85 mm Hg or being prescribed antihypertensive medications. further evaluate the association between the SNP and the Measurement of body fat content was performed using a bio- histological disease severity. electrical impedance method at 50 kHz and 500 µA (OMRON Body Fat Analyzer, model HBF-306, OMRON Healthcare). The body fat content was calculated by a formula that includes fi ve PATIENTS AND METHODS factors: electric resistance, height, weight, age, and gender. Body fat percentage was calculated as body fat mass/body weight (lbs.) We performed a cross-sectional study on NAFLD in a county × 100 as indicated by the manufacturer. hospital of the city of Buenos Aires. The study involved a total of Patients were defi ned to have abnormal liver enzymes (LFT) 266 unrelated individuals (80 males and 186 females), of which in the presence of at least one of: 1 ) elevated serum ALT and/ 172 patients had features of NAFLD and 94 were control or aspartate aminotransferase (AST), defi ned as >41 U/l, 2 ) subjects. -glutamyl-transferase (GGT) > 50 U/l, and 3 ) alkaline phos- The screening criteria was liver ultrasonographic (US) exami- phatase (AP) > 250 UI/l. nation indicative of fatty infi ltration ( 19 ), which was carried out All the investigations performed in this study were conducted by the same operator and performed in all the participants. in accordance with the guidelines of the 1975 Declaration of Hel- Secondary causes of steatosis, including alcohol abuse ( 30 sinki. Written consent from individuals was obtained in accor- g/d alcohol for men and 20 g for women), total parenteral nu- dance with the procedures approved by the ethical committee of trition, hepatitis B and hepatitis C virus infection, and the use of our institution. 2112 Journal of Lipid Research Volume 50, 2009 no further improvement in the fi tting model by adding up to four Liver biopsy and histopathological evaluation clusters (the ln of likelihood was maximum for K = 1). Indication of liver biopsy was based on previous recommenda- tions: benefi ts of obtaining liver for histology should be weighed Statistical analysis against the possible morbidity and mortality of the procedure Unless otherwise indicated, phenotypic quantitative data were ( 21 ). Hence, NAFLD patients were offered a percutaneous liver expressed as mean ± SD. For univariate analysis, and to avoid any biopsy if they showed either abnormal liver enzymes (AST, assumption about variable distribution and homoscedasticity, dif- ALT, AP, or GGT), or severe insulin resistance (HOMA value > 3) ferences between groups were assessed by the nonparametric ( 4, 22 ). Mann-Whitney test or Kruskal-Wallis for two or more groups, re- Sixty-nine patients that showed US features of mild liver steato- spectively. To test the association between genotypes and disease sis as well as persistently normal ALT, AST, AP, and GGT during severity, we used chi-square test and a regression analysis for an 12 months of follow-up were not included in the histological ordinal multinomial distribution (Probit as the Link function) evaluation. with disease severity as the dependent (response) variable coding Liver biopsy was performed with US guidance and modifi ed controls, simple steatosis, and NASH subjects as 0, 1 and 2, re- 1.4-mm diameter Menghini needles (Hepafi x) on an outpatient spectively; HOMA and BMI as continuous predictor variables; basis. Liver biopsy specimens were routinely fi xed in 40 g/L and, sex and genotypes as grouping variables. Moreover, logistic formaldehyde (pH 7.4) embedded in paraffi n and stained with regression analysis was included for the evaluation of the associa- hematoxylin and eosin, Masson trichrome, and silver impregna- tion between genotypes and histological disease severity. tion for reticular fi bers. The same liver pathologist, who was To assess the association between genotypes with NAFLD or blinded to patient details, read all the biopsies. All the biopsies quantitative traits such as ALT and AST, we used chi-square test were at least 2 cm in length and contained a minimum of 8 portal and logistic regression or multiple regression, adjusting for covari- tracts. The degree of steatosis was assessed according to the sys- ables such as age, HOMA, BMI, etc., respectively. tem developed by Brunt et al. ( 23 ) recently modifi ed by Kleiner We used the CSS/Statistica program package, StatSoft V 6.0 et al. ( 24 ) based on the percentage of hepatocytes containing (Tulsa, OK) for the analyses. macrovesicular fat droplets, as follows: grade 0, no steatosis; grade 1, <33% of hepatocytes containing macrovesicular fat droplets; grade 2, 33–66% of hepatocytes containing macrove- sicular fat droplets; and grade 3, >66% of hepatocytes containing RESULTS macrovesicular fat droplets. NASH was defi ned as steatosis plus Clinical features, anthropometric variables, and labora- mixed infl ammatory cell infi ltration, hepatocyte ballooning and necrosis, glycogen nuclei, Mallory’s hyaline, and any stage of fi - tory fi ndings at diagnosis in NAFLD patients and controls brosis, including absent fi brosis ( 3 ). The severity of necroinfl am- are shown in Table 1 . NAFLD patients were older and matory activity was expressed on a 3-point scale, as follows: grade showed most of the risk factors of the metabolic syndrome: 1 (mild), grade 2 (moderate), and grade 3 (severe) as described elevated BMI, waist-hip ratio, fasting glucose, and insulin by Brunt et al. ( 23 ). The severity of fi brosis was expressed on a and HOMA index; 46 individuals had type 2 diabetes, 22 of 5-point scale, as follows: 0 = none, 1 = perivenular and/or perisi- them received metformin. The rest of the patients were as- nusoidal fi brosis in zone 3, 2 = combined pericellular portal fi - signed to the conventional management of type 2 diabetes brosis, 3 = septal/bridging fi brosis, and 4 = cirrhosis. with a program of diet, weight loss, and physical activity. Based on histological fi ndings, 40 patients were assigned Genotype analysis to the simple steatosis group and 63 were included in the The genetic analyses were done on genomic DNA extracted NASH group. The histological features of patients who un- from white blood cells by a standard method as previously de- derwent liver biopsy are shown in Table 1 . scribed ( 25 ). In the controls, the frequencies of the G allele (Met148) Genotyping of the PNPLA3 rs738409 was performed by a high- and the C allele (Ile148) were 33.6% and 66.4%, respec- throughput genotyping method involving PCR amplifi cation of tively, and the distribution of the genotypes was in Hardy- genomic DNA with two-tailed allele-specifi c primers that intro- duce priming sites for universal energy transfer-labeled primers Weinberg equilibrium (data not shown). as previously described ( 26 ). Discrete trait analysis of NAFLD in the whole popula- To ensure genotyping quality, we included DNA samples as tion showed that the rs738409 G allele was associated with internal controls, hidden samples of known genotype, and nega- a 2-fold increase in the risk for NAFLD (OR: 2.04, 95% CI: tive controls (water). Genotypes with a signal below a negative 1.43–2.89; P < 0.00007). The association persisted even control were not scored. The analysis error was estimated by rep- after adjusting for covariates, because in the multivariate licating a blinded sample (always belonging to the same individ- logistic regression analysis, the rs738409 was strongly ual) eight times across the templates of the project. Of the 216 associated with NAFLD ( P < 0.001; OR 2.80 per G allele; genotypes for the blinded sample, we had only one not-matched genotype (0.46% error); the observed error rate is estimated to 95% CI 1.50–5.20) independent of age, sex, BMI, and be <0.5%. Overall genotype completion rate was 100%. HOMA index. The association was still signifi cant after ex- To explore possible stratifi cation in the population, we used cluding the type 2 diabetic patients ( P < 0.004; OR 2.75; a collection of 13 SNPs (rs6830727, rs12639788, rs1282807, 95% CI: 1.38–5.48), independent of age, sex, BMI, and rs1947745, rs7162312, rs12951674, rs7212346, rs1934869, HOMA index. rs9542666, rs11843545, rs9725124, rs2798659, and rs2199940) at In the analysis of the association of rs738409 and NA- different loci (located in chromosome 4, 15, 17, 13, 1, and 3) and FLD progression from simple steatosis to NASH, we ob- then analyzed the data with the Structure program Version 2 ( 27 ). served a signifi cant association between the rs738409 We found no evidence of stratifi cation in our sample, because the genotypes and histological disease severity ( Table 2 ). In cases and the controls showed similar Q values and were assigned with a similar distance to clusters by the program Structure with fact, we observed signifi cantly higher scores of disease Adiponutrin and fatty liver 2113 TABLE 1. Clinical and biochemical characteristics of the whole TABLE 2. PNPLA3 -rs738409 genotypes according to histological population according to disease status and histological features of features of NAFLD patients who underwent a percutaneous liver patients with NAFLD biopsy Allele Nominal Frequency Variables Control Subjects NAFLD Patients P -Value CC CG GG Difference OR Population Genotype Genotype Genotype P (95%CI) Number of subjects 94 172 — Female/male 63/31 123/49 NS 5 Patients with 10 18 12 0.02 2.095 (1.172- Age (years) 46.6 ± 11.3 55.3 ± 11.9 2 x 10 2) 7 simple steatosis 3.745) BMI (kg/m 24.9 ± 4.4 32.5 ± 5.7 1 x 10 7 (n = 40) Waist circumference (cm) 83.4 ± 16.0 103.7 ± 14.6 1 x 10 Patients with 822 33 SABP (mmHg) 116.1 ± 13.8 122.5 ± 14.7 0.02 NASH DABP (mmHg) 72.0 ± 9.0 76.3 ± 10.6 0.01 6 (n = 63) Body fat content (%) 29.6 ± 6.8 37.8 ± 7.3 1 x 10 Fasting plasma glucose 4.5 ± 0.6 5.7 ± 2.0 1 x 10 P -value stands for statistical signifi cance using Pearson’s goodness- (mmol/L) of-fi t chi-square (degree of freedom = 1). Fasting plasma insulin 48.1 ± 36.1 94.6 ± 72.4 1 x 10 (pmol/L) HOMA index 1.4 ± 1.2 3.5 ± 3.1 1 x 10 persisted after adjusting for HOMA and BMI as indepen- Total cholesterol 5.4 ± 1.0 5.5 ± 1.4 NS dent continuous predictor variables. As this result may (mmol/L) merely represent the association between NAFLD and the HDL cholesterol 1.4 ± 0.3 1.2 ± 0.5 0.02 (mmol/L) SNP, we performed a logistic regression analysis consider- LDL-cholesterol 3.3 ± 0.9 3.2 ± 1.4 NS ing the NASH group versus the simple steatosis one (in- (mmol/L) cluding only the 103 patients that had liver biopsy). We Triglycerides (mmol/L) 1.3 ± 0.8 1.9 ± 1.3 0.0001 Uric acid (µmol/L) 174 ± 7 258 ± 246 1 x 10 still observed a signifi cant association between the rs738409 ALT (U/L) 16.7 ± 9.4 46.5 ± 51.4 1 x 10 genotypes and the histological disease severity (OR 1.88 AST (U/L) 18.5 ± 9.2 35.8 ± 25.4 1 x 10 per G allele; 95% CI 1.03–3.43; P < 0.04). As both the GGT (U/L) 24.3 ± 23.1 54.1 ± 55.3 1 x 10 AP (U/L) 138.5 ± 56.7 225.5 ± 115.4 1 x 10 groups included type 2 diabetic patients, after adjusting Histological features of Simple steatosis NASH patients — for type 2 diabetes, we observed that the association still NAFLD patients patients persisted (OR 1.79; 95% CI 1.05–3.20; P < 0.05). Further- Number of subjects 40 63 — Degree of steatosis — more, after excluding from the analysis the type 2 diabetic 111 6 — patients, the association was signifi cant and persisted after 218 22 — adjusting for HOMA index (data not shown). 311 35 — Necroinfl ammatory The degree of liver steatosis (as evaluated by liver bi- activity opsy) was signifi cantly associated with the rs738409 G al- 1NA 26 — lele, as subjects with CC genotype showed lower steatosis 235 — scores (14.9% ± 3.9) in comparison with CG genotype 32 — Fibrosis stage (26.3% ± 3.5) and GG genotype (33.3% ± 4.0) ( P < 0.005). 0NA 31 — The proportion of the total variation accounted for the 119 — rs738409 genotypes, after correcting for age, sex, BMI, 21 — 311 — and HOMA, was 5.3% ( ± SE: 0.23 ± 0.07; P < 0.002). 41 — Finally, the rs738409 G allele at PNPLA3 was signifi cantly DABP: diastolic arterial blood pressure. Results are expressed as associated with serum ALT and AST levels. These associa- mean ± SD. Nominal P -value stands for statistical signifi cance using tions persisted after adjusting for age, BMI, HOMA, and Mann-Whitney Test. NS: non signifi cant. All measurements are in SI even plasma triglycerides ( Table 3 ). units. NA: not applicable. Degree of steatosis: grade 1, <33% of hepatocytes containing macrovesicular fat droplets; grade 2, 33%–66% of hepatocytes containing macrovesicular fat droplets; and grade 3, DISCUSSION >66% of hepatocytes containing macrovesicular fat droplets. Necroinfl ammatory activity: grade 1 (mild), grade 2 (moderate) and A large association study from the multiethnic popula- grade 3 (severe). Fibrosis stage: 0 = none, 1 = perivenular and/or perisinusoidal fi brosis in zone 3, 2 = combined pericellular portal tion-based Dallas Heart Study revealed an association be- fi brosis, 3 = septal/bridging fi brosis, and 4 = cirrhosis. tween a nonsynonymous variant (rs738409) in PNPLA3 gene and liver fat content, as measured and quantifi ed by severity in individuals with the GG genotype (2.12 ± 0.15) proton magnetic resonance ( 17 ). To our knowledge, we in comparison with those with the GC genotype (1.32 ± have performed for the fi rst time a replication study in a 0.15) and the CC genotype (0.70 ± 0.09; P < 0.00001). hospital-based population to evaluate not only the rela- To adjust for potential confounders, we used a regres- tionship between the SNP and the presence of fatty liver sion analysis for an ordinal multinomial distribution with but also a possible association with NAFLD severity, as de- Probit function by coding the histological grade that termined by liver biopsy. In the multivariate logistic re- ranged from healthy subjects to NASH patients: control gression analysis, we observed that the rs738409 was subjects, simple steatosis patients proven by liver biopsy signifi cantly associated with fatty liver, showing that carri- and NASH patients proven by liver biopsy as 0, 1 and ers of the G allele (Met148 variant) are, at least, 1.5-fold 2, respectively. Following this analysis, the association per G allele (OR 2.8; 95% CI 1.5–5.2) more likely to have [chi-square 19.9, degree of freedom (df ): 2; P < 5 x10 ] NAFLD in comparison with noncarriers, independent of 2114 Journal of Lipid Research Volume 50, 2009 TABLE 3. Multiple regression analysis of serum liver enzymes (ALT and AST) as dependent variable and PNPLA3 rs738409 genotypes, age, BMI, HOMA, and plasma triglycerides as independent ones ALT AST Variables ± SE B ± SE P -level ± SE B ± SE P -level rs738409 0.19 ± 0.09 13.99 ± 6.39 0.030 0.24 ± 0.09 8.60 ± 3.09 0.006 Age 0.06 ± 0.09 0.25 ± 0.41 0.53 0.05 ± 0.09 0.11 ± 0.20 0.58 BMI 0.08 ± 0.11 0.71 ± 1.03 0.49 0.05 ± 0.11 0.25 ± 0.50 0.62 HOMA 0.01 ± 0.10 0.11 ± 1.88 0.95 0.03 ± 0.10 0.24 ± 0.90 0.79 Plasma 0.04 ± 0.09 0.02 ± 0.04 0.62 0.09 ± 0.09 0.02 ± 0.02 0.29 triglycerides age, sex, BMI, and HOMA index. This association was also dictor of the impact of amino acid substitutions, PolyPhen observed after removing type 2 diabetic patients from the (http://genetics.bwh.harvard.edu/pph/). analysis. In addition, we observed a signifi cant association In conclusion, based on the knowledge about human between the histological spectrum of NAFLD (simple ste- genetic variation information provided by the HapMap atosis and NASH) and the rs738409 G allele, independent Project, GWAS are opening powerful insights on the of the effect of the BMI or insulin resistance. Furthermore, pathophysiology of the common diseases. In particular, carriers of G allele showed higher degrees of histological one of the major outcomes of these studies is the elucida- severity and higher liver steatosis scores; the presence of tion of important aspects of disease pathogenesis through each G allele increased the degree of liver steatosis by the discovery of novel genes or genomic regions previously 10%. This association with histological disease severity unrelated to a disease. Such is the case of the PNPLA3 as- persisted even after adjusting for type 2 diabetes. The lack sociated with the risk of NAFLD. However, while GWAS of association between the variant and type 2 diabetes ob- are becoming increasingly affordable, they are still costly served in our study suggests that the underlying mecha- and laborious and require optimal multistage designs that nisms by which the rs738409 operate are independent of include genotyping hundreds of thousands of markers in type 2 diabetes. thousands of subjects. Thus, while other genes infl uencing Moreover, the rs738409 G allele was signifi cantly and fatty liver disease predisposition and progression remain positively associated with serum levels of both ALT and to be identifi ed, the replication of the data generated by ex- AST, a fi nding that also replicates the results of a recently ploratory research strategies in small but well-characterized published GWAS on several loci infl uencing plasma levels populations should be encouraged. In this setting, studies of liver enzymes ( 18 ). of new outcomes such as disease severity are recom- Our results on patients carrying the 738409 G allele hav- mended. ing higher scores of liver steatosis on liver biopsy may be REFERENCES explained by a possible direct relation between a loss-of- function of the PNPLA3 and an increase in hepatic triglyc- 1 . Browning , J. D. , L. S. Szczepaniak , R. Dobbins , P. Nuremberg , eride content. Earlier data has shown that the greater the J. D. Horton , J. C. Cohen , S. M. Grundy , and H. H. Hobbs . 2004 . Prevalence of hepatic steatosis in an urban population in the liver fat accumulation, the more liver necrosis is induced United States: impact of ethnicity. Hepatology . 40 : 1387 – 1395 . by intracellular lipid toxicity ( 28 ). This may additionally 2 . Barshop , N. J. , C. B. Sirlin , J. B. Schwimmer , and J. E. Lavine . explain the signifi cant association we have observed be- 2008 . Review article: epidemiology, pathogenesis and potential treatments of paediatric non-alcoholic fatty liver disease. Aliment. tween the SNP and the histological disease severity and Pharmacol. Ther. 28 : 13 – 24 . serum liver enzymes. 3 . Neuschwander-Tetri , B. A. , and S. H. Caldwell . 2003 . Nonalcoholic Notwithstanding the diffi culties in replicating fi ndings steatohepatitis: summary of an AASLD Single Topic Conference. Hepatology . 37 : 1202 – 1219 . in genetic association studies, our results support the im- 4 . Sanyal , A. J. 2002 . AGA technical review on nonalcoholic fatty liver portance of the rs738409 in modifying not only hepatic disease. Gastroenterology . 123 : 1705 – 1725 . triglyceride content-explaining population differences in 5 . Suzuki , A. , K. Lindor , S. J. St , J. Lymp , F. Mendes , A. Muto , T. Okada , disease susceptibility, but also explaining interindividual and P. Angulo . 2005 . Effect of changes on body weight and lifestyle in nonalcoholic fatty liver disease. J. Hepatol. 43 : 1060 – 1066 . differences in infl ammatory response to liver lipotoxicity. 6 . Harrison , S. A. , and C. P. Day . 2007 . Benefi ts of lifestyle modifi ca- While no functional studies about the role of the variant tion in NAFLD. Gut . 56 : 1760 – 1769 . have been conducted, the fact that rs738409 is a nonsyn- 7 . Dong , H. , J. Wang , C. Li , A. Hirose , Y. Nozaki , M. Takahashi , M. Ono , N. Akisawa , S. Iwasaki , T. Saibara , et al . 2007 . The phosphati- onymous SNP supports a possible functional impact lead- dylethanolamine N-methyltransferase gene V175M single nucle- ing, for instance, to a loss-of-function of the protein, otide polymorphism confers the susceptibility to NASH in Japanese particularly as the amino acid switch (Met148Ile) is located population. J. Hepatol. 46 : 915 – 920 . 8 . Miele , L. , G. Beale , G. Patman , V. Nobili , J. Leathart , A. Grieco , in the lipolytic and lipogenic patatin domain of the pro- M. Abate , S. L. Friedman , G. Narla , E. Bugianesi , et al . 2008 . The tein. Although rs738409 SNP deserves biochemical investi- Kruppel-like factor 6 genotype is associated with fi brosis in nonal- gation, the Function Analysis and Selection Tool for Single coholic fatty liver disease. Gastroenterology . 135 : 282 – 291 . Nucleotide Polymorphisms resource ( 29 ) showed that it 9 . Namikawa , C. , Z. Shu-Ping , J. R. Vyselaar , Y. Nozaki , Y. Nemoto , M. Ono , N. Akisawa , T. Saibara , M. Hiroi , H. Enzan , et al . 2004 . has a high score risk for splicing regulation with protein Polymorphisms of microsomal triglyceride transfer protein gene structural damage, consequently abolishing the protein and manganese superoxide dismutase gene in non-alcoholic ste- domain. Similar results were observed using another pre- atohepatitis. J. Hepatol. 40 : 781 – 786 . Adiponutrin and fatty liver 2115 10 . Sookoian , S. , G. Castano , C. Gemma , T. F. Gianotti , and C. J. 20 . 2001 . Executive Summary of The Third Report of The National Pirola . 2007 . Common genetic variations in CLOCK transcription Cholesterol Education Program (NCEP) Expert Panel on factor are associated with nonalcoholic fatty liver disease. World J. Detection, Evaluation, And Treatment of High Blood Cholesterol Gastroenterol. 13 : 4242 – 4248 . In Adults (Adult Treatment Panel III). JAMA . 285 : 2486 – 2497 . 11 . Sookoian , S. , Castano , G. , Gianotti , T. F. , Gemma , C. , and Pirola , C. 21 . Grant , A. , and J. Neuberger . 1999 . Guidelines on the use of liver J. 2008 . Polymorphisms of MRP2 (ABCC2) are associated with sus- biopsy in clinical practice. British Society of Gastroenterology. Gut . ceptibility to nonalcoholic fatty liver disease. J. Nutr. Biochem. Epub 45 ( Suppl. 4 ): IV1 – IV11 . ahead of print. October 14, 2008. 22 . Fracanzani , A. L. , L. Valenti , E. Bugianesi , M. Andreoletti , A. Colli , 12 . Sookoian , S. , G. Castano , T. F. Gianotti , C. Gemma , M. S. Rosselli , E. Vanni , C. Bertelli , E. Fatta , D. Bignamini , G. Marchesini , et al . and C. J. Pirola . 2008 . Genetic variants in STAT3 are associated 2008 . Risk of severe liver disease in nonalcoholic fatty liver disease with nonalcoholic fatty liver disease. Cytokine . 44 : 201 – 206 . with normal aminotransferase levels: a role for insulin resistance 13 . Tokushige , K. , M. Takakura , N. Tsuchiya-Matsushita , M. Taniai , E. and diabetes. Hepatology . 48 : 792 – 798 . Hashimoto , and K. Shiratori . 2007 . Infl uence of TNF gene poly- 23 . Brunt , E. M. , C. G. Janney , A. M. Di Bisceglie , B. A. Neuschwander- morphisms in Japanese patients with NASH and simple steatosis. Tetri , and B. R. Bacon . 1999 . Nonalcoholic steatohepatitis: a J. Hepatol. 46 : 1104 – 1110 . proposal for grading and staging the histological lesions. Am. J. 14 . Wilfred de Alwis , N. M. , and C. P. Day . 2008 . Genes and nonalco- Gastroenterol. 94 : 2467 – 2474 . holic fatty liver disease. Curr. Diab. Rep. 8 : 156 – 163 . 24 . Kleiner , D. E. , E. M. Brunt , N. M. Van , C. Behling , M. J. Contos , 15 . Zeggini , E. , L. J. Scott , R. Saxena , B. F. Voight , J. L. Marchini , T. O. W. Cummings , L. D. Ferrell , Y. C. Liu , M. S. Torbenson , Hu , P. I. de Bakker , G. R. Abecasis , P. Almgren , G. Andersen , et al . A. Unalp-Arida , et al . 2005 . Design and validation of a histological 2008 . Meta-analysis of genome-wide association data and large- scoring system for nonalcoholic fatty liver disease. Hepatology . 41 : scale replication identifi es additional susceptibility loci for type 2 1313 – 1321 . diabetes. Nat. Genet. 40 : 638 – 645 . 25 . Kawasaki , E. S. 1990 . Sample preparation from blood, cells, 16 . Marchesini , G. , M. Brizi , G. Bianchi , S. Tomassetti , E. Bugianesi , M. and other fl uids. In PCR Protocols. A Guide to Methods and Lenzi , A. J. McCullough , S. Natale , G. Forlani , and N. Melchionda . Applications. M. A. Innis, D. H. Gelfand, J. J. Sninsky, and T. J. 2001 . Nonalcoholic fatty liver disease: a feature of the metabolic White, editors. Academic Press, Inc., San Diego. 146–152. syndrome. Diabetes . 50 : 1844 – 1850 . 26 . Myakishev , M. V. , Y. Khripin , S. Hu , and D. H. Hamer . 2001 . 17 . Romeo , S. , J. Kozlitina , C. Xing , A. Pertsemlidis , D. Cox , L. A. High-throughput SNP genotyping by allele-specifi c PCR with Pennacchio , E. Boerwinkle , J. C. Cohen , and H. H. Hobbs . 2008 . universal energy-transfer-labeled primers. Genome Res. 11 : Genetic variation in PNPLA3 confers susceptibility to nonalcoholic 163 – 169 . fatty liver disease. Nat. Genet. 40 : 1461 – 1465 . 27 . Pritchard , J. K. , M. Stephens , and P. Donnelly . 2000 . Inference of 18 . Yuan , X. , D. Waterworth , J. R. Perry , N. Lim , K. Song , J. C. population structure using multilocus genotype data. Genetics . 155 : Chambers , W. Zhang , P. Vollenweider , H. Stirnadel , T. Johnson , 945 – 959 . et al . 2008 . Population-based genome-wide association studies re- 28 . Wanless , I. R. , and K. Shiota . 2004 . The pathogenesis of nonalco- veal six loci infl uencing plasma levels of liver enzymes. Am. J. Hum. holic steatohepatitis and other fatty liver diseases: a four-step model Genet. 83 : 520 – 528 . including the role of lipid release and hepatic venular obstruction 19 . Mendler , M. H. , P. Bouillet , A. Le Sidaner , E. Lavoine , F. Labrousse , in the progression to cirrhosis. Semin. Liver Dis. 24 : 99 – 106 . D. Sautereau , and B. Pillegand . 1998 . Dual-energy CT in the di- 29 . Yuan , H. Y. , J. J. Chiou , W. H. Tseng , C. H. Liu , C. K. Liu , Y. J. Lin , agnosis and quantifi cation of fatty liver: limited clinical value in H. H. Wang , A. Yao , Y. T. Chen , and C. N. Hsu . 2006 . FASTSNP: an comparison to ultrasound scan and single-energy CT, with special always up-to-date and extendable service for SNP function analysis reference to iron overload. J. Hepatol. 28 : 785 – 794 . and prioritization. Nucleic Acids Res. 34 : W635 – W641 . 2116 Journal of Lipid Research Volume 50, 2009
Journal
Journal of Lipid Research – American Society for Biochemistry and Molecular Biology
Published: Oct 1, 2009
Keywords: SNP; genetics; replication study; PNPLA3; fatty liver; nonalcoholic steatohepatitis; NASH