Splice variant rs72613567 prevents worst histologic outcomes in patients with nonalcoholic fatty liver disease
2018; Elsevier BV; Volume: 60; Issue: 1 Linguagem: Inglês
10.1194/jlr.p089953
ISSN1539-7262
AutoresCarlos J. Pirola, Martín Garaycoechea, Diego Flichman, Marco Arrese, Julio San Martino, Carla Gazzi, Gustavo Castaño, Silvia Sookoian,
Tópico(s)Diet, Metabolism, and Disease
ResumoHydroxysteroid 17-#x03B2; dehydrogenase 13 (HSD17B13) is a lipid droplet-associated protein; its gene-encoding variants affect the chronic liver diseases, including nonalcoholic fatty liver disease (NAFLD). To estimate the effect of rs72613567, a splice variant with an adenine insertion (A-INS), on NAFLD susceptibility and severity, we performed a case-control study with 609 individuals. We investigated the effect of carrying the A-INS allele in 356 patients with biopsy-proven disease and explored the relationship between rs72613567 genotypes and the hepatic transcriptome. The A-INS allele protected against NAFLD [odds ratio (OR) per adenine allele = 0.667; 95#x0025; CI, 0.486−0.916; P = 0.012]; this effect was nonsignificant when logistic regression analysis included BMI. The A-INS allele protected against nonalcoholic steatohepatitis (NASH) (OR = 0.612; 95#x0025; CI, 0.388−0.964; P = 0.033), ballooning degeneration (OR = 0.474; 95#x0025; CI, 0.267−0.842; P = 0.01), lobular inflammation (OR = 0.475; 95#x0025; CI, 0.275−0.821; P = 0.007), and fibrosis (OR = 0.590; 95#x0025; CI, 0.361−0.965; P = 0.035). In patients carrying A-INS, HSD17B13 levels decreased proportionally to allele dosage. Whole-transcriptome genotype profiling showed overrepresented immune response-related pathways. Thus, the rs72613567 A-INS allele reduces the risk of NASH and progressive liver damage and may become a therapeutic target. Hydroxysteroid 17-#x03B2; dehydrogenase 13 (HSD17B13) is a lipid droplet-associated protein; its gene-encoding variants affect the chronic liver diseases, including nonalcoholic fatty liver disease (NAFLD). To estimate the effect of rs72613567, a splice variant with an adenine insertion (A-INS), on NAFLD susceptibility and severity, we performed a case-control study with 609 individuals. We investigated the effect of carrying the A-INS allele in 356 patients with biopsy-proven disease and explored the relationship between rs72613567 genotypes and the hepatic transcriptome. The A-INS allele protected against NAFLD [odds ratio (OR) per adenine allele = 0.667; 95#x0025; CI, 0.486−0.916; P = 0.012]; this effect was nonsignificant when logistic regression analysis included BMI. The A-INS allele protected against nonalcoholic steatohepatitis (NASH) (OR = 0.612; 95#x0025; CI, 0.388−0.964; P = 0.033), ballooning degeneration (OR = 0.474; 95#x0025; CI, 0.267−0.842; P = 0.01), lobular inflammation (OR = 0.475; 95#x0025; CI, 0.275−0.821; P = 0.007), and fibrosis (OR = 0.590; 95#x0025; CI, 0.361−0.965; P = 0.035). In patients carrying A-INS, HSD17B13 levels decreased proportionally to allele dosage. Whole-transcriptome genotype profiling showed overrepresented immune response-related pathways. Thus, the rs72613567 A-INS allele reduces the risk of NASH and progressive liver damage and may become a therapeutic target. Nonalcoholic fatty liver disease (NAFLD), which affects a high proportion of the global adult and child population, has emerged as the leading cause of end-stage liver disease (1Brunt E.M. Wong V.W. Nobili V. Day C.P. Sookoian S. Maher J.J. Bugianesi E. Sirlin C.B. Neuschwander-Tetri B.A. Rinella M.E. Nonalcoholic fatty liver disease.Nat. Rev. Dis. Primers. 2015; 1: 15080Crossref PubMed Scopus (474) Google Scholar, 2Friedman S.L. Neuschwander-Tetri B.A. Rinella M. Sanyal A.J. Mechanisms of NAFLD development and therapeutic strategies.Nat. Med. 2018; 24: 908-922Crossref PubMed Scopus (1524) Google Scholar). A growing body of evidence indicates that NAFLD is a heritable (3Speliotes E.K. Yerges-Armstrong L.M. Wu J. Hernaez R. Kim L.J. Palmer C.D. Gudnason V. Eiriksdottir G. Garcia M.E. Launer L.J. et al.Genome-wide association analysis identifies variants associated with nonalcoholic fatty liver disease that have distinct effects on metabolic traits.PLoS Genet. 2011; 7: e1001324Crossref PubMed Scopus (694) Google Scholar) and polygenic complex trait (4Sookoian S. Pirola C.J. Genetic predisposition in nonalcoholic fatty liver disease.Clin. Mol. Hepatol. 2017; 23: 1-12Crossref PubMed Scopus (115) Google Scholar). Variants located in genes that regulate lipid metabolism and lipid droplet (LD) biology, including patatin-like phospholipase domain containing 3 (PNPLA3)-rs738409 and transmembrane 6 superfamily member 2 transmembrane 6 superfamily member 2 (TM6SF2)-rs58542926, have been consistently replicated as major modifiers of the natural history of NAFLD (5Romeo S. Kozlitina J. Xing C. Pertsemlidis A. Cox D. Pennacchio L.A. Boerwinkle E. Cohen J.C. Hobbs H.H. Genetic variation in PNPLA3 confers susceptibility to nonalcoholic fatty liver disease.Nat. Genet. 2008; 40: 1461-1465Crossref PubMed Scopus (2233) Google Scholar, 8Pirola C.J. Sookoian S. The dual and opposite role of the TM6SF2-rs58542926 variant in protecting against cardiovascular disease and conferring risk for nonalcoholic fatty liver: A meta-analysis.Hepatology. 2015; 62: 1742-1756Crossref PubMed Scopus (110) Google Scholar), increasing the risk of developing severe histological forms of the disease, such as nonalcoholic steatohepatitis (NASH) and NASH-fibrosis. Of note, variants, more specifically SNPs, in the aforementioned genes have been discovered as a part of genome-wide (5Romeo S. Kozlitina J. Xing C. Pertsemlidis A. Cox D. Pennacchio L.A. Boerwinkle E. Cohen J.C. Hobbs H.H. Genetic variation in PNPLA3 confers susceptibility to nonalcoholic fatty liver disease.Nat. Genet. 2008; 40: 1461-1465Crossref PubMed Scopus (2233) Google Scholar) or exome-wide (7Kozlitina J. Smagris E. Stender S. Nordestgaard B.G. Zhou H.H. Tybjaerg-Hansen A. Vogt T.F. Hobbs H.H. Cohen J.C. Exome-wide association study identifies a TM6SF2 variant that confers susceptibility to nonalcoholic fatty liver disease.Nat. Genet. 2014; 46: 352-356Crossref PubMed Scopus (758) Google Scholar) association studies. Likewise, recent remarkable results from exome-sequence data coupled with electronic health records of 46,455 participants uncovered the association of the rs72613567 (−/adenine) deletion/insertion-variant in hydroxysteroid 17-#x03B2; dehydrogenase 13 (HSD17B13) with a reduced risk of chronic liver disease, including NASH (9Abul-Husn N.S. Cheng X. Li A.H. Xin Y. Schurmann C. Stevis P. Liu Y. Kozlitina J. Stender S. Wood G.C. et al.A protein-truncating HSD17B13 variant and protection from chronic liver disease.N. Engl. J. Med. 2018; 378: 1096-1106Crossref PubMed Scopus (403) Google Scholar). Research findings further indicate that the rs72613567 variant is reproducibly associated with decreased alanine aminotransferase (ALT) and aspartate aminotransferase (AST) serum levels (9Abul-Husn N.S. Cheng X. Li A.H. Xin Y. Schurmann C. Stevis P. Liu Y. Kozlitina J. Stender S. Wood G.C. et al.A protein-truncating HSD17B13 variant and protection from chronic liver disease.N. Engl. J. Med. 2018; 378: 1096-1106Crossref PubMed Scopus (403) Google Scholar). HSD17B13 encodes a protein that is involved in the regulation of lipid biosynthetic process (10Su W. Wang Y. Jia X. Wu W. Li L. Tian X. Li S. Wang C. Xu H. Cao J. et al.Comparative proteomic study reveals 17beta-HSD13 as a pathogenic protein in nonalcoholic fatty liver disease.Proc. Natl. Acad. Sci. USA. 2014; 111: 11437-11442Crossref PubMed Scopus (126) Google Scholar); molecular analysis (11Horiguchi Y. Araki M. Motojima K. 17beta-Hydroxysteroid dehydrogenase type 13 is a liver-specific lipid droplet-associated protein.Biochem. Biophys. Res. Commun. 2008; 370: 235-238Crossref PubMed Scopus (55) Google Scholar) and recent proteomic studies (10Su W. Wang Y. Jia X. Wu W. Li L. Tian X. Li S. Wang C. Xu H. Cao J. et al.Comparative proteomic study reveals 17beta-HSD13 as a pathogenic protein in nonalcoholic fatty liver disease.Proc. Natl. Acad. Sci. USA. 2014; 111: 11437-11442Crossref PubMed Scopus (126) Google Scholar) have identified HSD17B13 as a novel liver lipid droplet-associated protein. Findings yielded by recent functional studies demonstrate that the rs72613567, regarded as a splice region variant, is indeed a loss-of-function variant, which consists of an adenine insertion (A-INS) in the coding gene region (chr4:87310241, GRCh38.p7) adjacent to the donor splice site of intron 6 that results in a frame-shift and premature truncation of the HSD17B13 protein (9Abul-Husn N.S. Cheng X. Li A.H. Xin Y. Schurmann C. Stevis P. Liu Y. Kozlitina J. Stender S. Wood G.C. et al.A protein-truncating HSD17B13 variant and protection from chronic liver disease.N. Engl. J. Med. 2018; 378: 1096-1106Crossref PubMed Scopus (403) Google Scholar). Of note, the protective effect of the rs72613567 A-INS allele against NASH was recently observed in a sample of morbidly obese patients recruited from a bariatric cohort (9Abul-Husn N.S. Cheng X. Li A.H. Xin Y. Schurmann C. Stevis P. Liu Y. Kozlitina J. Stender S. Wood G.C. et al.A protein-truncating HSD17B13 variant and protection from chronic liver disease.N. Engl. J. Med. 2018; 378: 1096-1106Crossref PubMed Scopus (403) Google Scholar). As a part of the same study, the original description of the protective effect of the rs72613567-insertion variant on chronic liver damage was examined in a population of European Americans among whom the A-INS frequency is ∼24#x0025; (9Abul-Husn N.S. Cheng X. Li A.H. Xin Y. Schurmann C. Stevis P. Liu Y. Kozlitina J. Stender S. Wood G.C. et al.A protein-truncating HSD17B13 variant and protection from chronic liver disease.N. Engl. J. Med. 2018; 378: 1096-1106Crossref PubMed Scopus (403) Google Scholar). Therefore, it remains to be established whether the observed influential effect of the variant on the course of NAFLD can be generalized to other populations. Hence, we explored the association between rs72613567 and NAFLD disease severity, including a putative protecting effect on histological features associated with progressive damage. Furthermore, we aimed to explore the impact of carrying the rs72613567 A-INS allele in abundance and cell localization of the HSD17B13 protein in the liver of the affected patients. To gain further biological insight into the role of rs72613567, we also explored differentially expressed genes of the whole transcriptome according to the variant genotypes. We performed a case-control association study to estimate the effect of the rs72613567 variant on the susceptibility of NAFLD; this study involved a sample of 609 unrelated individuals, including 429 patients with NAFLD and 180 control subjects. Associations with NAFLD severity, including histological features known to be related to an aggressive course, were explored in a sample of 356 patients that showed the whole spectrum of the disease [nonalcoholic fatty liver (NAFL), NASH, and NASH-fibrosis], and who had histopathological confirmation of NAFLD. Patients and controls were selected from hospital-based settings, including patients diagnosed with NAFLD and metabolic syndrome (MetS) (Hospital Abel Zubizarreta, Buenos Aires, Argentina) and morbidly obese patients that underwent bariatric surgery (Hospital de Alta Complejidad en Red #x201C;El Cruce#x201D;, Buenos Aires, Argentina). Inclusion criteria were as follows: liver ultrasonographic examination was the initial screening criteria for selecting the cases and controls in the group of individuals included in the study of NAFLD and MetS, and it was performed in all the participants. Patients who presented fatty liver on liver ultrasonogram were indicated a liver biopsy if they showed either abnormal liver enzymes or severe insulin resistance [homeostatic model assessment of insulin resistance (HOMA-IR) value greater than 3] as explained earlier (6Sookoian S. Castano G.O. Burgueno A.L. Gianotti T.F. Rosselli M.S. Pirola C.J. A nonsynonymous gene variant in the adiponutrin gene is associated with nonalcoholic fatty liver disease severity.J. Lipid Res. 2009; 50: 2111-2116Abstract Full Text Full Text PDF PubMed Scopus (301) Google Scholar). A liver biopsy was performed in 268 patients who met the criteria; patients that showed ultrasonographic features of liver steatosis plus persistently normal liver enzymes during 12 months of follow-up were not included in the histological evaluation. All morbidly obese patients that were included in this study (n = 121) had histological confirmation of either NAFLD or normal liver by a liver biopsy that was performed during bariatric surgery. Exclusion criteria were as follows: secondary causes of steatosis, including alcohol abuse (≥30 g for men and ≥20 g for women, of alcohol daily), total parenteral nutrition, hepatitis B and hepatitis C virus infection, and the use of drugs known to precipitate steatosis were excluded. In addition, patients with any of the following diseases were excluded: autoimmune liver disease, metabolic liver disease, Wilson's disease, and α-1-antitrypsin deficiency. Control subjects that matched patients with NAFLD-MetS were selected from subjects attending our hospital for check-up purposes whose age and sex matched the NAFLD patients. In addition to the standard heath examination, all control individuals were subjected to a liver ultrasonographic examination. They were included in the study if they did not have evidence of fatty change or biochemical abnormalities. Furthermore, control subjects were confirmed not to have any of the features of MetS as defined by the National Cholesterol Education Program Adult Treatment Panel III and did not abuse alcohol. In the population of morbidly obese patients, control subjects were obese patients who also underwent bariatric surgery and had no features of NAFLD demonstrated in the liver biopsy. Liver biopsies from all patients as well as DNA samples from white blood cells from all the subjects included in this study were obtained with written informed consent in accordance with the Institutional Review Board-approved protocols. All the investigations performed in this study were conducted in accordance with the guidelines of the 1975 Declaration of Helsinki, as revised in 1993. The case participants and control subjects were consecutively selected during the same study period from the same population of patients attending the participant institutions of Argentina, and all of them shared the same demographic characteristics (occupation, educational level, place of residence, and ethnicity). Health examinations included anthropometric measurements, a questionnaire on health-related behaviors, and biochemical determinations (see the supplemental information). The disease severity was assessed by liver biopsy that was performed before any intervention with ultrasound guidance and a modified 1.4 mm-diameter Menghini needle (Hepafix, Braun, Germany) on an outpatient basis or during bariatric surgery. All liver biopsies were evaluated by the same pathologist. A portion of each liver biopsy specimen was routinely fixed in 40 g/l formaldehyde (pH 7.4), embedded in paraffin, and stained with hematoxylin and eosin, Masson's trichrome, and silver impregnation for reticular fibers. All the biopsies were at least 3 cm in length and contained a minimum of eight portal tracts. The degree of steatosis was assessed according to the system developed by Kleiner et al. (12Kleiner D.E. Brunt E.M. Van N.M. Behling C. Contos M.J. Cummings O.W. Ferrell L.D. Liu Y.C. Torbenson M.S. Unalp-Arida A. et al.Design and validation of a histological scoring system for nonalcoholic fatty liver disease.Hepatology. 2005; 41: 1313-1321Crossref PubMed Scopus (7163) Google Scholar), based on the percentage of hepatocytes containing macrovesicular fat droplets. NASH and NAFLD activity score (NAS) (12Kleiner D.E. Brunt E.M. Van N.M. Behling C. Contos M.J. Cummings O.W. Ferrell L.D. Liu Y.C. Torbenson M.S. Unalp-Arida A. et al.Design and validation of a histological scoring system for nonalcoholic fatty liver disease.Hepatology. 2005; 41: 1313-1321Crossref PubMed Scopus (7163) Google Scholar, 13Brunt E.M. Kleiner D.E. Wilson L.A. Belt P. Neuschwander-Tetri B.A. Nonalcoholic fatty liver disease (NAFLD) activity score and the histopathologic diagnosis in NAFLD: distinct clinicopathologic meanings.Hepatology. 2011; 53: 810-820Crossref PubMed Scopus (790) Google Scholar) were defined as reported previously, and a NAS threshold of 5 was used for further comparisons with variables of interest; NASH was defined as steatosis plus mixed inflammatory cell infiltration, hepatocyte ballooning and necrosis, Mallory's hyaline, and any stage of fibrosis, including absent fibrosis (12Kleiner D.E. Brunt E.M. Van N.M. Behling C. Contos M.J. Cummings O.W. Ferrell L.D. Liu Y.C. Torbenson M.S. Unalp-Arida A. et al.Design and validation of a histological scoring system for nonalcoholic fatty liver disease.Hepatology. 2005; 41: 1313-1321Crossref PubMed Scopus (7163) Google Scholar, 13Brunt E.M. Kleiner D.E. Wilson L.A. Belt P. Neuschwander-Tetri B.A. Nonalcoholic fatty liver disease (NAFLD) activity score and the histopathologic diagnosis in NAFLD: distinct clinicopathologic meanings.Hepatology. 2011; 53: 810-820Crossref PubMed Scopus (790) Google Scholar). Intra-acinar (lobular) inflammation was defined as the presence of cellular components of inflammation (polymorphonuclear leukocytes, lymphocytes and other mononuclear cells, eosinophils, and microgranulomas) located in sinusoidal spaces, surrounding Mallory's hyaline, or in hepatocellular necrosis (1Brunt E.M. Wong V.W. Nobili V. Day C.P. Sookoian S. Maher J.J. Bugianesi E. Sirlin C.B. Neuschwander-Tetri B.A. Rinella M.E. Nonalcoholic fatty liver disease.Nat. Rev. Dis. Primers. 2015; 1: 15080Crossref PubMed Scopus (474) Google Scholar, 13Brunt E.M. Kleiner D.E. Wilson L.A. Belt P. Neuschwander-Tetri B.A. Nonalcoholic fatty liver disease (NAFLD) activity score and the histopathologic diagnosis in NAFLD: distinct clinicopathologic meanings.Hepatology. 2011; 53: 810-820Crossref PubMed Scopus (790) Google Scholar). It was graded 0–3 and was defined as 0 (absent) = no foci; 1 = <2 foci per 200× field; 2 = 2–4 foci per 200× field; and 3 = >4 foci per 200× field. Ballooning was scored as: 0 = none; 1 = rare or few; and 2 = many (12Kleiner D.E. Brunt E.M. Van N.M. Behling C. Contos M.J. Cummings O.W. Ferrell L.D. Liu Y.C. Torbenson M.S. Unalp-Arida A. et al.Design and validation of a histological scoring system for nonalcoholic fatty liver disease.Hepatology. 2005; 41: 1313-1321Crossref PubMed Scopus (7163) Google Scholar, 13Brunt E.M. Kleiner D.E. Wilson L.A. Belt P. Neuschwander-Tetri B.A. Nonalcoholic fatty liver disease (NAFLD) activity score and the histopathologic diagnosis in NAFLD: distinct clinicopathologic meanings.Hepatology. 2011; 53: 810-820Crossref PubMed Scopus (790) Google Scholar). The severity of fibrosis was expressed on a four-point scale, as follows: 0 = none; 1 = perivenular and/or perisinusoidal fibrosis in zone 3; 2 = combined pericellular portal fibrosis; 3 = septal/bridging fibrosis; and 4 = cirrhosis (12Kleiner D.E. Brunt E.M. Van N.M. Behling C. Contos M.J. Cummings O.W. Ferrell L.D. Liu Y.C. Torbenson M.S. Unalp-Arida A. et al.Design and validation of a histological scoring system for nonalcoholic fatty liver disease.Hepatology. 2005; 41: 1313-1321Crossref PubMed Scopus (7163) Google Scholar, 13Brunt E.M. Kleiner D.E. Wilson L.A. Belt P. Neuschwander-Tetri B.A. Nonalcoholic fatty liver disease (NAFLD) activity score and the histopathologic diagnosis in NAFLD: distinct clinicopathologic meanings.Hepatology. 2011; 53: 810-820Crossref PubMed Scopus (790) Google Scholar). The genetic analyses were done on genomic DNA extracted from white blood cells (6Sookoian S. Castano G.O. Burgueno A.L. Gianotti T.F. Rosselli M.S. Pirola C.J. A nonsynonymous gene variant in the adiponutrin gene is associated with nonalcoholic fatty liver disease severity.J. Lipid Res. 2009; 50: 2111-2116Abstract Full Text Full Text PDF PubMed Scopus (301) Google Scholar). Genotyping of rs72613567 was performed using a custom TaqMan genotyping assay (see the supplemental information) (Applied Biosystems, Foster City, CA) according to manufacturer's instructions, and confirmed by DNA Sanger sequencing (Macrogen, Inc., South Korea) (see the supplemental information and supplemental Fig. S1). Genotyping of the PNPLA3 rs738409 was performed as previously described (6Sookoian S. Castano G.O. Burgueno A.L. Gianotti T.F. Rosselli M.S. Pirola C.J. A nonsynonymous gene variant in the adiponutrin gene is associated with nonalcoholic fatty liver disease severity.J. Lipid Res. 2009; 50: 2111-2116Abstract Full Text Full Text PDF PubMed Scopus (301) Google Scholar). To ensure genotyping quality, we included DNA samples as internal controls, hidden samples of known genotype, and negative controls (water). The overall genotype completion rate was 100#x0025;. To account for possible population stratification, we used a collection of 13 SNPs at different loci (located in chromosomes 4, 15, 17, 13, 1, and 3) and then analyzed the data with the Structure program version 2 (14Tian C. Gregersen P.K. Seldin M.F. Accounting for ancestry: population substructure and genome-wide association studies.Hum. Mol. Genet. 2008; 17: R143-R150Crossref PubMed Scopus (277) Google Scholar) as we explained elsewhere (6Sookoian S. Castano G.O. Burgueno A.L. Gianotti T.F. Rosselli M.S. Pirola C.J. A nonsynonymous gene variant in the adiponutrin gene is associated with nonalcoholic fatty liver disease severity.J. Lipid Res. 2009; 50: 2111-2116Abstract Full Text Full Text PDF PubMed Scopus (301) Google Scholar, 15Sookoian S. Castano G.O. Scian R. Mallardi P. Fernandez T.G. Burgueno A.L. San Martino J. Pirola C.J. Genetic variation in transmembrane 6 superfamily member 2 and the risk of nonalcoholic fatty liver disease and histological disease severity.Hepatology. 2015; 61: 515-525Crossref PubMed Scopus (157) Google Scholar, 16Sookoian 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.Hepatology. 2011; 53: 1883-1894Crossref PubMed Scopus (672) Google Scholar). We found no evidence of stratification in our sample because the cases and the controls showed similar Q values and the Structure program assigned a similar distance to clusters with no further improvement in the fitting model by adding up to four clusters (the ln of likelihood was maximum for K = 1). Using the CaTS power calculator for genetic association studies (17Skol A.D. Scott L.J. Abecasis G.R. Boehnke M. Joint analysis is more efficient than replication-based analysis for two-stage genome-wide association studies.Nat. Genet. 2006; 38: 209-213Crossref PubMed Scopus (1046) Google Scholar) and assuming a prevalence of NAFLD of 0.30, minor allele frequency A-INS 0.16 (the frequency found in our population, 0.84 for the risk allele), and an odds ratio (OR) of 1.8/0.56, our sample had 83#x0025; and 99#x0025;, for the additive and multiplicative genetic model, respectively. Four micrometer sections were mounted onto silane-coated glass slides to ensure section adhesion through subsequent staining procedures. Briefly, sections were deparaffinized, rehydrated, washed in PBS, and treated with 3#x0025; H2O2 in PBS for 20 min at room temperature to block endogenous peroxidase. Following microwave heat-induced epitope retrieval in 0.1 M citrate buffer at pH 6.0 for 20 min, the slides were incubated with a dilution of 1:150 of rabbit polyclonal antibody for human anti-HSD17B13 [HSD17B13 antibody (OAAN01691) size 50 UL, Aviva Systems Biology, San Diego, CA]. Immunostaining was performed using the VECTASTAIN Elite ABC kit (Vector Laboratories, Burlingame, CA) detection system. Subsequently, slides were immersed in a 0.05#x0025; 3,3′-diaminobenzidine solution in 0.1 M Tris buffer, pH 7.2, containing 0.01#x0025; H2O2. After a brown color developed, slides were removed and the reaction was stopped by immersion in PBS. Negative controls were carried out with rabbit serum diluted to the same concentration as the primary antibody. HSD17B13 immunostaining was evaluated in a blinded fashion regarding any of the histological and clinical characteristics of the patients. The extent of staining was scored according to its amount and intensity by a four-point scoring system as follows: 0 = no staining, 1 = positive staining in less than 20#x0025; of cells, 2 = 21–50#x0025; of positive cells, and 3 = positive staining in more than 50#x0025; of cells. Histological specimens were assessed by a LEICA DM 2000 (Leica, Germany) trinocular microscope equipped with a high-definition camera (Leica MC190 HD); all images were recorded using the Leica Application Suite software. Total RNA was prepared from fresh liver tissue using phenol extraction step method, with an additional DNase digestion. After extraction, RNA quantity was measured using the ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE), the ratio of absorbance 260 nm/280 nm being 1.9–2.0. For NAFLD patients carrying the rs72613567 (−/−) homozygous genotype (n = 8), one target for each sample was prepared. For the reference group [rs72613567 (A/A) homozygous genotype], pooled samples from five patients were used. The same proportion of NASH and NAFL patients were included per rs72613567-genotype group; selection of cases was based on the availability of RNA of sufficient quality and quantity to perform the microarray experiment. Samples were further processed using the SurePrint G3 Unrestricted Gene Expression 8x60K microarray (product number G4140-90050; Agilent, Santa Clara, CA) according to instructions provided by the manufacturer. This array allows the testing of 26,083 Entrez Genes (https://www.ncbi.nlm.nih.gov/gene) and 30,606 long noncoding RNAs. The cDNA (cRNA) synthesis involved a single amplification for two color labeling; probes from rs72613567 (−/−) tissue and from the reference tissue [rs72613567 (A/A)] were differentially labeled by the incorporation of cyanine (Cy)3 and Cy5, respectively. Feature extraction, quantification of the intensity of fluorescent images, and scanned images were processed and analyzed by Agilent SureScan microarray scanner bundle, Agilent Scan control software, and Agilent GeneSpring 14.5-GX software, respectively. We used fold change values generated by the software from the array data as the ratio of Lowess normalized background subtracted Cy5/Cy3 signals if they passed the built-in quality control analysis of the software executed according to the manufacturer's protocol for two-color experiments. Linearization of the data was undertaken by log2 transformation. In addition to the liver whole-genome expression exploration, we conducted gene set enrichment analysis to investigate the pathways and biological processes potentially associated with the differentially expressed genes. FunRich v.3.1.3 (http://www.funrich.org) software was used; only pathways that reached significant threshold were considered. Quantitative data are expressed as mean ± SD unless otherwise indicated. As a significant difference in SD was observed between the groups in most of the variables and the distribution was significantly skewed in most cases, we chose to be conservative and assessed the differences between the groups using nonparametric Mann-Whitney U or Kruskal-Wallis tests. The Cochran-Armitage test for trend was used in the categorical data analysis to assess the presence of association between the variant and the disease severity, as well as histological outcomes; chi-square (χ2), OR, and P value are informed. Ordinal variables were dichotomized as ballooning degeneration, lobular inflammation, and liver fibrosis: yes (1)/ no (0). In addition, we used generalized linear and nonlinear models (logistic regression and ANCOVA using binomial or ordinal multinomial distribution with logit and probit as link functions, respectively), adjusting for covariables, which were log-transformed. Then, adjustment for potential demographic, clinical, and genetic confounders (rs738409) was also performed. The CSS/Statistica program package version 6.0 (StatSoft, Tulsa, OK) was used in these analyses. Clinical, biochemical, and histological features of patients with histological confirmation of NAFLD are shown in Table 1. While the studied population was primarily comprised of patients with NAFLD and features of MetS, the cohort also included 88 morbidly obese persons who represented 24.7#x0025; of the total sample. The control group characteristics are shown in supplemental Table S1.TABLE 1Clinical and biochemical features of patients with NAFLD according to disease severityVariablesNAFLNASHPNumber of subjects (β)165 (46)191 (54)Female, #x0025;59.465NSAge, years49.5 ± 1250.5 ± 12NSBMI, kg/m238.3 ± 1336.5 ± 9.4NSArterial hypertension, #x0025;41.6#x0025;51.3#x0025;NSGlucose-related profileFasting plasma glucose, mg/dl100 ± 25129 ± 1101.0 × 10−8Fasting plasma insulin, μU/ml13.3 ± 8.819 ± 201.0 × 10−8HOMA-IR index3.34 ± 2.56.6 ± 161.0 × 10−8HbA1c6.3 ± 1.37.5 ± 4.4NSType 2 diabetes, #x0025;2951.30.000038Lipid profileTotal cholesterol, mg/dl199 ± 50204 ± 46NSHDL-cholesterol, mg/dl49.5 ± 2148 ± 14NSLDL-cholesterol, mg/dl125 ± 44125 ± 42NSTriglycerides, mg/dl157 ± 88187 ± 1150.059Uric acid, mg/dl5.6 ± 7.55.0 ± 2.0NSLiver phenotypeALT, U/L49 ± 5370 ± 551.0 × 10−8AST, U/L32 ± 1749 ± 331.0 × 10−8GGT, U/L67 ± 5788 ± 960.098AP, U/L183 ± 113202 ± 1150.098Histological features of NAFLDDegree of steatosis (0–3)1.85 ± 0.812.4 ± 0.750.000068Lobular inflammation (0–3)0.52 ± 0.611.33 ± 0.681.0 × 10−8Hepatocellular ballooning (0–2)0.09 ± 0.280.87 ± 0.601.0 × 10−8Fibrosis stage0.06 ± 0.331.6 ± 0.701.0 × 10−8NAS2.4 ± 1.204.5 ± 1.21.0 × 10−8Results are expressed as mean ± SD. P stands for statistical significance using Mann-Whitney U test, except for female/male proportion, arterial hypertension, and type 2 diabetes where P stands for statistical significance using the
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