Mol Biol Rep DOI 10.1007/s11033-014-3688-2
HLA DQB1 alleles are related with nonalcoholic fatty liver disease Levent Doganay • Seyma Katrinli • Yasar Colak • Ebubekir Senates • Ebru Zemheri • Oguzhan Ozturk • Feruze Yilmaz Enc • Ilyas Tuncer • Gizem Dinler Doganay
Received: 21 September 2013 / Accepted: 20 August 2014 Ó Springer Science+Business Media Dordrecht 2014
Abstract Nonalcoholic fatty liver disease (NAFLD) is the leading cause of chronic liver disease. NAFLD is a complex disease and inflammation is a crucial component in the disease pathogenesis. Recent genome wide association studies in hepatology area highlighted significant relations with human leukocyte antigen (HLA) DQ region and certain liver diseases. The previous animal models also emphasized the involvement of adaptive immune system in the liver damage pathways. To investigate possible polymorphisms in the HLA region that can contribute to the immune response affecting the NAFLD, we enrolled 93 consecutive biopsy proven NAFLD patients and a control group consisted of 101 healthy people and genotyped HLA DQB1 alleles at high resolution by sequence specific primers-polymerase chain reaction. The mean NAFLD activity score (NAS) was 5.2 ± 1.2, fibrosis score was 0.9 ± 0.9, ALT was 77 ± 47.4 U/L, AST was
49.4 ± 26.3 U/L. Among 13 HLA DQB1 alleles analyzed in this study, DQB1*06:04 was observed significantly at a more frequent rate among the NAFLD patients compared to that of healthy controls (12.9 vs. 2 % v2 = 8.6, P = 0.003, Pc = 0.039, OR: 7.3 95 % CI 1.6–33.7). In addition, the frequency of DQB1*03:02 was significantly higher in the healthy control group than the NAFLD patients (24.8 vs. 7.5 %, v2 = 10.4, P = 0.001, Pc = 0.013, OR: 0.2, 95 % CI 0.1–0.6). NAFLD patients were grouped according to their fibrosis score and NAS. The distribution of DQB1 alleles over stratified NAFLD patients did not reveal any statistically significant relation. Taken together, immune repertoire of individuals may have an effect on NAFLD pathogenesis and therefore, in NAFLD, adaptive immunity pathways should be investigated. Keywords Nonalcoholic fatty liver disease (NAFLD) Nonalcoholic steatohepatitis (NASH) HLA DQ Liver Inflammation
Electronic supplementary material The online version of this article (doi:10.1007/s11033-014-3688-2) contains supplementary material, which is available to authorized users. L. Doganay Y. Colak F. Y. Enc I. Tuncer Department of Gastroenterology, Goztepe Teaching and Research Hospital, Medeniyet University, Istanbul, Turkey
E. Senates Department of Gastroenterology, School of Medicine, Dicle University, Diyarbakir, Turkey
L. Doganay O. Ozturk Department of Gastroenterology, Umraniye Teaching and Research Hospital, Istanbul, Turkey
E. Zemheri Department of Pathology, Goztepe Teaching and Research Hospital, Medeniyet University, Istanbul, Turkey
L. Doganay (&) Gastroenteroloji Klinigi, Umraniye Egitim ve Arastirma Hastanesi, Umraniye, 34764 Istanbul, Turkey e-mail: [email protected]
G. D. Doganay (&) Istanbul Teknik Universitesi, Ayazaga Kampusu, MOBGAM, 34469 Istanbul, Turkey e-mail: [email protected]
S. Katrinli G. D. Doganay Department of Molecular Biology and Genetics, Istanbul Technical University, Istanbul, Turkey
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Introduction Nonalcoholic fatty liver disease (NAFLD) is one of the most common causes of chronic liver diseases and it is a hepatic manifestation of metabolic syndrome [1]. The rising prevalence of NAFLD correlates with the epidemic of obesity. NAFLD shows a disease spectrum ranging from simple steatosis to nonalcoholic steatohepatitis (NASH), cirrhosis and hepatocellular carcinoma (HCC) [2]. Currently there are two hypotheses for the explanation of the progression of NAFLD from simple steatosis to more advanced forms and both of these put the inflammation as the key driving force for this progression [3, 4]. Present molecular studies mainly highlighted the role of innate immune system in this inflammation [4, 5]. However, there are animal model studies demonstrating the up-regulation of major histocompability complex (MHC) class II molecules in the liver damage pathways [6]. Moreover recent genome wide association studies in hepatology revealed significant association of single nucleotide polymorphisms in the human leukocyte antigen (HLA) DQ region with liver diseases, such as drug induced liver injury (rs312990 (P = 4.4 9 10-12) and rs92754407 (P = 4.8 9 10-14), fine mapping revealed DQB*06:02 allele association with both Lumiracoxib induced and Amoxicillin-Clavulanate induced liver injury, respectively) [7, 8], primary biliary cirrhosis (rs2856683, rs9275312, rs9275390, and rs7775228 (P values were in between 1.70 9 10-10 and 8.58 9 10-17), SNPs were associated with DQB1 gene region) [9], spontaneous resolution of hepatitis C viral disease (rs4273729 (P = 1.7 9 10-16), fine mapping revealed an association with DQB1*03:01) [10], chronic hepatitis B infection (rs28556718 (P = 5.9 9 10-28) and rs7453920 (P = 3.9 9 10-37), fine mapping revealed associations with DQB1*03:03 and DQB1*06:04 alleles) [11], HCC induced by hepatitis C (rs9275572 (P = 9.3 9 10-9), the SNP was associated with DQB1 gene region) [12], HCC and cirrhosis induced by hepatitis B virus (rs9275319 (P = 2.7 9 10-17), fine mapping revealed an association with DQB1*04:01) [13] and primary sclerosing cholangitis (many HLA alleles were associated with the disease, DQB*02:01 revealed significance at P = 2.4 9 10-187) [14]. Furthermore, our recent findings on the chronic hepatitis B disease demonstrated DQB1*05:01 and DQB1*05:03 alleles’ correlation to the chronic active disease state and treatment non-response, respectively [15, 16]. A recent pathway analysis with arrays including more than 300,000 single nucleotide polymorphisms in NAFLD patients demonstrated that antigen processing and presenting pathways were related with disease progression to NASH and HCC [17]. Human leukocyte antigens are essential elements of adaptive immunity and represents antigens to T cells [18].
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In liver; dendritic cells, kupffer cells, liver sinusoidal endothelial cells, and hepatocytes express MHC class II molecules that are coded by HLA genes [19–21]. According to our medline search there is no study genotyping HLA in biopsy proven NAFLD patients, this study aims to genotype at a high resolution HLA DQB1 alleles in biopsy proven NAFLD patients.
Methods Study participants In this case control study a total of 93 biopsy proven NAFLD patients (46 male and 47 female, mean age 42 ± 10.2 years) and a gender and age matched comparison group consisting of 101 healthy people (48 male 53 female, mean age 41 ± 9.5) were recruited. Patient and control inclusion and exclusion criteria were described before [22]. Patients were consecutively seen and followed up at hepatology outpatient clinic. Local ethics committee approved the study, and all participants gave informed consent. Patients with other hepatic diseases were excluded. None of the patients were on any estrogen, amiadrone, diltiazem, steroid, tamoxifen or herbal supplement treatment. The control group was consisted of people who were healthy on medical examination, had liver biochemistry tests in normal range and had normal liver on sonographic examination. Consuming alcohol greater than 20 g/day was an exclusion criteria for both patient and the control group. Clinical assessment All patients went through physical examination, anthropometric measurements, and biochemical tests. The diagnosis of NAFLD was done by histologically and on haematoxylin-eosin-stained liver specimens all patients had [5 % macrovesicular steatosis. Biopsies were obtained under ultrasonographic guidance by 16 G Hepafix needle. The minimum length of the biopsies was 2.5 cm. All biopsy specimens were stained with haematoxyin-eosin and Masson’s trichrome. A pathologist who was experienced in NAFLD and was blinded to all the study data examined the biopsies according to the National Institute of Diabetes and Digestive and Kidney Diseases NASH Clinical Research Network scoring system [23]. Polymorphism genotyping Genomic DNA was extracted from 200 lL of peripheral blood by using Invitrogen PureLink genomic DNA purification kit according to manufacturer’s protocol. The alleles of HLA-DQB1 were detected by sequence-specific
Mol Biol Rep Table 1 Characteristics of patients and the control group NAFLD (n = 93)
Control (n = 101)
P
46/47
48/53
Ns
Age (years)
42.7 ± 9.5
41 ± 9.5
Ns
Body mass index (kg/ m2) Metabolic syndrome
31.5 ± 5.1
24.9 ± 4.5
\0.001
60/33
0/101
\0.001
Gender (male/female)
Diabetes mellitus
22/70
0/101
\0.001
Triglyceride (mmol/l)
197 ± 145
102.3 ± 70.5
\0.001
LDL cholesterol (mmol/l)
133.4 ± 35.5
114.1 ± 29.1
\0.001
HDL cholesterol (mmol/l)
45.1 ± 8.3
50.2 ± 11
\0.001
ALT (U/l)
76.9 ± 47.2
18.2 ± 10.2
\0.001
AST (U/l)
49.3 ± 26.2
21.1 ± 7.1
\0.001
LDL low density lipoprotein, HDL high density lipoprotein, ALT alanine transferase, AST aspartate transaminase
SyberGreen in the presence of 100 bp DNA ladder (Fermentas) (Fig. 1). The allelic type was determined according to the presence or absence of the desired length PCR products. Statistical analysis The frequencies of HLA alleles were calculated by direct counting. Statistical analyses were done to evaluate the difference between NAFLD patients and control patients. During the HLA analysis, 2 9 2 tables and Chi square test were used. To evaluate the association of DQB1 gene with the disease, a dominant model where the 13 genotyped alleles represented in the tables as yes/no fashion was applied. An individual carries two alleles, so the sum of the percentages in the disease association table may not be one hundred. To reduce an overestimation, if only one allele was determined in an individual that allele was counted once rather than considering it as a homozygote. When the sample sizes were small or expected values in cells of Chi square table were \5, then, Fisher’s exact test was used. Parametric variables were analyzed by the student’s t test. Parametric variables were given as mean ± standard deviation. All P values were calculated double sided, and if the P value is below 0.05, it was considered as statistically significant unless there are multiple comparisons. Bonferroni’s correction was applied for multiple comparisons and corrected P values were given. All analyses were done in a computer with SPSS 21 (Chicago, IL).
primers–polymerase chain reaction (SSP–PCR) [24]. Primers sequences and PCR product sizes are listed in supplementary Table 1. Positive internal control primers concentration was adjusted as 0.8 fold of specific primers concentration, and then both were pooled into the reaction mix to eradicate pseudonegative possibilities. The internal control is a fragment of human growth hormone gene 1 (chr 17) with 439 bp [25]. The sequences of internal control primers are also shown in supplementary Table 1. PCR is performed in 25 lL reaction mixture containing 50–100 ng genomic DNA, 0.625 U DNA DreamTaq Green DNA polymerase, 10X DreamTaq Green buffer, 4 mM MgCl2 (Intron) and 0.2 mM of each dNTP (Intron), 0.5 lM primers, 0.4 lM internal control primers. PCR cycling parameters were as follows: predenaturation at 95 °C for 3 min, denaturation at 95 °C for 30 s, annealing at 65 °C for 30 s, extension at 72 °C for 50 s, repetition for 30 cycles and final extension at 72 °C for 7 min. After amplification, products were identified by ultraviolet light after electrophoresis in 2 % agarose gel stained by
Characteristics and baseline laboratory results of patients and control group are given in Table 1. These two groups were not different in terms of gender or age but body mass index (BMI), lipid profile, ALT, AST were significantly higher in the patient group. Mean NAFLD activity score (NAS) of the patients was 5.2 ± 1.2, the mean fibrosis score was 0.9 ± 0.9. Steatosis grade, NAS and fibrosis
Fig. 1 Electrophoretic figures of PCR product by 2 % agarose gel. Lane numbers present: 1-DQB1*03:01/04; 2-DQB1*05:01; 3DQB1*06:01; 4-DQB1*02:01 and DQB1*05:02; 5-DQB1*04:01; 6-
DQB1*04:02; 7-DQB1*05:03; 8-DQB1*02:01; 9-DQB1*02:01/ 03:02; 10-DQB1*03:03; 11-DQB1*03:02/03; 12-DQB1*03:02/03; 13-DQB1*06:04
Results
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Mol Biol Rep Table 2 Histologic scores of patients (n = 93)
Histologic variable
n (%)
genotypes over patients and controls did not reveal significant difference.
Steatosis grade 0
0
1
24 (25.8)
2
49 (52.6)
3
19 (20.4)
NAS 0–2
7 (7.5)
3–4
22 (23.7)
5–8
57 (61.2)
Fibrosis stage
NAS, NAFLD activity score
0
35 (37.6)
1
44 (47.3)
2
7 (7.5)
3
6 (6.5)
4
1 (1.1)
stage of the patients are given in Table 2. Among the genotyped 13 DQB1 alleles, DQB1*06:04 was observed significantly at a more frequent rate among the NAFLD patients compared to that of healthy controls (OR = 7.3, P = 0.003, Pc = 0.039). In addition, the frequency of DQB1*03:02 is significantly higher in the healthy control group than the NAFLD patients (OR = 0.3, P = 0.001, Pc = 0.013). The distribution of HLA DQB1 alleles over patients and control group is given in Table 3. The most frequent DQB1 genotypes in the whole study population were DQB1*03:01/03:04, DQB1*06:01 (n = 16, 8.2 %) and DQB1*03:01/03:04, DQB1*05:01 (n = 10, 5.1 %). The distribution of these most frequent
Table 3 The distribution of HLA alleles among patients and control group
DQB1 allele
Discussion The effect of immune system on NAFLD pathogenesis is an evolving issue. To date, studies indicated the impact of parts of innate immune system on NAFLD (e.g. kupffer cells [26, 27], natural killer cells [28], natural killer T cells [29, 30] and Toll like receptors [31, 32]). Although the elements of adaptive immune response are not thoroughly investigated, there is growing evidence suggesting the role of adaptive immune pathways in inflammation in NAFLD. Animal models exploring the liver damage pathways demonstrated the importance of MHC class II molecules. For instance; in the thioacetamide induced rat liver fibrosis model, MHC class II related pathway was shown to be induced [33]. In porcine serum induced rat model of fibrosis, MHC class II related genes were upregulated [6, 34]. In another animal model, it was demonstrated that liver fibrosis relied on the B lymphocyte presence [35]. In carbon tetrachloride induced steatohepatitis model in obese mice, expression of MHC II was increased in kuppfer cells [36]. Moreover in clinical studies it was shown that circulating CD4 positive lymphocytes and regulatory T cells were increased in NASH patients [37, 38]. In a biopsy study from pediatric NASH patients the profile liver infiltrating immune cells indicated the role of T helper 1 response besides innate immune pathway [39]. All these studies reveal the importance of MHC class II involvement in NAFLD from a mechanistic view; we think that there is a necessity to explore the MHC class II gene relation with the disease since these polymorphic sequences can be
NAFLD n: 93 (%)
Control group n: 101 (%)
V2
P
OR
CI 95 %
1
*05:01
16 (17.2)
15 (14.9)
0.2
0.66
1.1
0.5–2.6
2
*05:02
15 (16.1)
11 (10.9)
1.1
0.3
1.5
0.6–3.6
3
*05:03
10 (10.8)
20 (19.8)
3
0.08
0.49
0.2–1.1
4
*06:01
23 (24.7)
16 (15.8)
2.4
0.12
1.7
0.9–3.6
5
*06:02
0
0
NA
NA
NA
NA
6
*06:03/06:08
0
0
NA
NA
NA
NA
7
*06:04
12 (12.9)
2 (2)
8.6
0.003a
7.3
1.6–33.7 0.7–5.2
8
*02:01
12 (12.9)
7 (6.9)
1.96
0.16
2
NA not available
9
*03:01/03:04
34 (36.6)
42 (41.6)
0.5
0.5
0.8
0.5–1.4
a
10
*03:02
7 (7.5)
25 (24.8)
10.4
0.001b
0.2
0.1–0.6
11
*03:03
6 (6.5)
12 (11.9)
1.6
0.2
0.5
0.2–1.4
12
*04:02
3 (3.2)
6 (5.9)
c
0.5
0.5
0.1–2.1
13
*04:01
1 (1.1)
5 (5)
c
0.2
0.2
0.1–1.8
Pcorrected = 0.039 (after Bonferroni’s correction) b
Pcorrected = 0.013 (after Bonferroni’s correction) c
Fisher’s exact test is applied
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determinants of susceptibility or resistance, as observed for various diseases. Our study demonstrated that NAFLD patients had more frequently DQB1*06:04 allele (Pc = 0.039) compared to healthy control group, whereas control group showed more frequently DQB1*03:02 allele (Pc = 0.013) compared to that of the patients. In a previous study from Turkey, Celikbilek and his coworkers demonstrated that HLA B65 and DQ5 are the risk factors for NAFLD [40]. That study was consisted of 66 NAFLD patients diagnosed by ultrasound and 50 controls. Both patients and controls were selected from liver donor candidates and HLA subtypes were defined by serotyping. In our study, NAFLD was diagnosed by liver biopsy in consecutive patients and DQB1 alleles were genotyped at high resolution. Up until June 2014, according to our medline search this study is the first report revealing the association of genotyped HLA alleles in biopsy proven NAFLD patients. Antigen presenting cells (APC) present antigens to CD4 positive lymphocytes and these lymphocytes secrete numerous cytokines and promote adaptive immunity. In liver, dendritic cells, kupffer cells, liver sinusoidal endothelial cells, and hepatocytes present antigens to CD4 positive lymphocytes via MHC II molecules [19–21]. These APCs are involved in the pathogenesis of both chronic viral infections and autoimmune diseases of the liver [19, 41]. Additionally a novel pathway suggested to be involved in the pathogenesis of NAFLD is autophagy besides well-known ones such as proinflammatory mediators secreted by visceral adipose tissue [42, 43], gut derived products [31], and endoplasmic reticulum stress [44]. Dysregulation of autophagy affects both cellular lipid accumulation and inflammation in NAFLD [45, 46]. Autophagy in hepatocytes may activate innate immune pathways but also it is well shown that autophagy may induce self-antigen presentation via MHC II molecules [47, 48]. When our results are taken into account with the background knowledge of the growing evidence indicating the adaptive immune pathway involvement in the NAFLD pathogenesis, it might be considered as HLA DQ has a role in the junction between innate and adaptive immunity cross-talks. Interestingly the recent GWA studies in hepatology revealed association of HLA DQ region with many disease conditions. These studies investigated drug induced liver injury [7, 8], primary sclerosing cholangitis [14], primary biliary cirrhosis [9], hepatitis C clearance [10], liver damage in hepatitis C [49], hepatocellular cancer due to hepatitis C [12], hepatitis B seroclearance [11], hepatocellular cancer due to hepatitis B [13, 50], and all revealed association with single nucleotide polymorphisms in HLA DQ region. In this study we did not demonstrate any relation with fibrosis stage or NAS and DQB1 alleles (statistics not
shown). To search a relation, we classified patients according to NAS and fibrosis scores but statistics did not reveal any association with DQB1 alleles and histology. As we genotyped 13 different DQB1 types, the patient numbers in subgroups might be insufficient to demonstrate a significant relation. This study has some limitations. This is a cross sectional case control study, and it is not possible to conclude a direct causative relation between our findings and the disease. Replication studies to confirm HLA relation with the disease and molecular studies to investigate adaptive immunity pathway in NAFLD are necessary. For subgroup analysis (e.g. advanced fibrosis) the patient number should be higher. On the other hand, consecutive patient enrollment, very well-defined biopsy proven patient selection, and genotyping in high resolution are the strengths of this study. In conclusion the immune repertoire of an individual may affect NAFLD development. This study demonstrated that HLA DQB1*06:04 allele was associated with NALFD and DQB1*03:02 is a protective factor from NAFLD. Acknowledgments This study is supported by internal research funds of Istanbul Technical University.
References 1. Lazo M, Clark JM (2008) The epidemiology of nonalcoholic fatty liver disease: a global perspective. Semin Liver Dis 28(4): 339–350. doi:10.1055/s-0028-1091978 2. Cohen JC, Horton JD, Hobbs HH (2011) Human fatty liver disease: old questions and new insights. Science 332(6037): 1519–1523. doi:10.1126/science.1204265 3. Day CP, James OF (1998) Steatohepatitis: a tale of two ‘‘hits’’? Gastroenterology 114(4):842–845 4. Tilg H, Moschen AR (2010) Evolution of inflammation in nonalcoholic fatty liver disease: the multiple parallel hits hypothesis. Hepatology 52(5):1836–1846. doi:10.1002/hep.24001 5. Harmon RC, Tiniakos DG, Argo CK (2011) Inflammation in nonalcoholic steatohepatitis. Expert Rev Gastroenterol Hepatol 5(2):189–200. doi:10.1586/egh.11.21 6. Baba Y, Doi K (2004) MHC class II-related genes expression in porcine-serum-induced rat hepatic fibrosis. Exp Mol Pathol 77(3):214–221. doi:10.1016/j.yexmp.2004.08.001 7. Singer JB, Lewitzky S, Leroy E, Yang F, Zhao X, Klickstein L, Wright TM, Meyer J, Paulding CA (2010) A genome-wide study identifies HLA alleles associated with lumiracoxib-related liver injury. Nat Genet 42(8):711–714. doi:10.1038/ng.632 8. Lucena MI, Molokhia M, Shen Y, Urban TJ, Aithal GP, Andrade RJ, Day CP, Ruiz-Cabello F, Donaldson PT, Stephens C, Pirmohamed M, Romero-Gomez M, Navarro JM, Fontana RJ, Miller M, Groome M, Bondon-Guitton E, Conforti A, Stricker BH, Carvajal A, Ibanez L, Yue QY, Eichelbaum M, Floratos A, Pe’er I et al (2011) Susceptibility to amoxicillin-clavulanateinduced liver injury is influenced by multiple HLA class I and II alleles. Gastroenterology 141(1):338–347. doi:10.1053/j.gastro. 2011.04.001
123
Mol Biol Rep 9. Hirschfield GM, Liu X, Xu C, Lu Y, Xie G, Lu Y, Gu X, Walker EJ, Jing K, Juran BD, Mason AL, Myers RP, Peltekian KM, Ghent CN, Coltescu C, Atkinson EJ, Heathcote EJ, Lazaridis KN, Amos CI, Siminovitch KA (2009) Primary biliary cirrhosis associated with HLA, IL12A, and IL12RB2 variants. New Engl J Med 360(24):2544–2555. doi:10.1056/NEJMoa0810440 10. Duggal P, Thio CL, Wojcik GL, Goedert JJ, Mangia A, Latanich R, Kim AY, Lauer GM, Chung RT, Peters MG, Kirk GD, Mehta SH, Cox AL, Khakoo SI, Alric L, Cramp ME, Donfield SM, Edlin BR, Tobler LH, Busch MP, Alexander G, Rosen HR, Gao X, Abdel-Hamid M, Apps R et al (2013) Genome-wide association study of spontaneous resolution of hepatitis C virus infection: data from multiple cohorts. Ann Intern Med 158(4):235–245. doi:10. 7326/0003-4819-158-4-201302190-00003 11. Mbarek H, Ochi H, Urabe Y, Kumar V, Kubo M, Hosono N, Takahashi A, Kamatani Y, Miki D, Abe H, Tsunoda T, Kamatani N, Chayama K, Nakamura Y, Matsuda K (2011) A genome-wide association study of chronic hepatitis B identified novel risk locus in a Japanese population. Hum Mol Genet 20(19):3884–3892. doi:10.1093/hmg/ddr301 12. Kumar V, Kato N, Urabe Y, Takahashi A, Muroyama R, Hosono N, Otsuka M, Tateishi R, Omata M, Nakagawa H, Koike K, Kamatani N, Kubo M, Nakamura Y, Matsuda K (2011) Genomewide association study identifies a susceptibility locus for HCVinduced hepatocellular carcinoma. Nat Genet 43(5):455–458. doi:10.1038/ng.809 13. Jiang DK, Sun J, Cao G, Liu Y, Lin D, Gao YZ, Ren WH, Long XD, Zhang H, Ma XP, Wang Z, Jiang W, Chen TY, Gao Y, Sun LD, Long JR, Huang HX, Wang D, Yu H, Zhang P, Tang LS, Peng B, Cai H, Liu TT, Zhou P et al (2013) Genetic variants in STAT4 and HLA-DQ genes confer risk of hepatitis B virusrelated hepatocellular carcinoma. Nat Genet 45(1):72–75. doi:10. 1038/ng.2483 14. Liu JZ, Hov JR, Folseraas T, Ellinghaus E, Rushbrook SM, Doncheva NT, Andreassen OA, Weersma RK, Weismuller TJ, Eksteen B, Invernizzi P, Hirschfield GM, Gotthardt DN, Pares A, Ellinghaus D, Shah T, Juran BD, Milkiewicz P, Rust C, Schramm C, Muller T, Srivastava B, Dalekos G, Nothen MM, Herms S et al (2013) Dense genotyping of immune-related disease regions identifies nine new risk loci for primary sclerosing cholangitis. Nat Genet 45(6):670–675. doi:10.1038/ng.2616 15. Doganay L, Tuncer I, Katrinli S, Enc FY, Ozturk O, Colak Y, Ulasoglu C, Dinler G (2013) The effect of HLA-DQB1 alleles on virologic breakthroughs during chronic hepatitis B treatment with genetically low barrier drugs. Clin Res Hepatol Gastroenterol 37(4):359–364. doi:10.1016/j.clinre.2012.10.013 16. Doganay L, Fejzullahu A, Katrinli S, Yilmaz Enc F, Ozturk O, Colak Y, Ulasoglu C, Tuncer I, Dinler Doganay G (2014) Association of human leukocyte antigen DQB1 and DRB1 alleles with chronic hepatitis B. World J Gastroenterol : WJG 20(25):8179–8186. doi:10.3748/wjg.v20.i25.8179 17. Chen QR, Braun R, Hu Y, Yan C, Brunt EM, Meerzaman D, Sanyal AJ, Buetow K (2013) Multi-SNP Analysis of GWAS Data Identifies Pathways Associated with Nonalcoholic Fatty Liver Disease. PLoS One 8(7):e65982. doi:10.1371/journal.pone. 0065982 18. Germain RN (1994) MHC-dependent antigen processing and peptide presentation: providing ligands for T lymphocyte activation. Cell 76(2):287–299 19. Racanelli V, Rehermann B (2006) The liver as an immunological organ. Hepatology 43(2 Suppl 1):S54–S62. doi:10.1002/hep. 21060 20. Herkel J, Jagemann B, Wiegard C, Lazaro JF, Lueth S, Kanzler S, Blessing M, Schmitt E, Lohse AW (2003) MHC class IIexpressing hepatocytes function as antigen-presenting cells and
123
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
activate specific CD4 T lymphocyutes. Hepatology 37(5):1079–1085. doi:10.1053/jhep.2003.50191 Winau F, Hegasy G, Weiskirchen R, Weber S, Cassan C, Sieling PA, Modlin RL, Liblau RS, Gressner AM, Kaufmann SH (2007) Ito cells are liver-resident antigen-presenting cells for activating T cell responses. Immunity 26(1):117–129. doi:10.1016/j. immuni.2006.11.011 Colak Y, Karabay CY, Tuncer I, Kocabay G, Kalayci A, Senates E, Ozturk O, Doganay HL, Enc FY, Ulasoglu C, Kiziltas S (2012) Relation of epicardial adipose tissue and carotid intima-media thickness in patients with nonalcoholic fatty liver disease. Eur J Gastroenterol Hepatol 24(6):613–618. doi:10.1097/MEG. 0b013e3283513f19 Kleiner DE, Brunt EM, Van Natta M, Behling C, Contos MJ, Cummings OW, Ferrell LD, Liu YC, Torbenson MS, UnalpArida A, Yeh M, McCullough AJ, Sanyal AJ, Nonalcoholic Steatohepatitis Clinical Research N (2005) Design and validation of a histological scoring system for nonalcoholic fatty liver disease. Hepatology 41(6):1313–1321. doi:10.1002/hep.20701 Olerup O, Aldener A, Fogdell A (1993) HLA-DQB1 and -DQA1 typing by PCR amplification with sequence-specific primers (PCR–SSP) in 2 hours. Tissue Antigens 41(3):119–134. doi:10. 1111/j.1399-0039.1993.tb01991.x Xi-Lin Z, Te D, Jun-Hong L, Liang-Ping L, Xin-Hui G, Ji-Rong G, Chun-Yan G, Zhuo L, Ying L, Hui L (2006) Analysis of HLADQB1 gene polymorphisms in asymptomatic HBV carriers and chronic hepatitis B patients in the Chinese Han population. Int J Immunogenet 33(4):249–254. doi:10.1111/j.1744-313X.2006. 00607.x Rivera CA, Adegboyega P, van Rooijen N, Tagalicud A, Allman M, Wallace M (2007) Toll-like receptor-4 signaling and Kupffer cells play pivotal roles in the pathogenesis of non-alcoholic steatohepatitis. J Hepatol 47(4):571–579. doi:10.1016/j.jhep.2007. 04.019 Baffy G (2009) Kupffer cells in non-alcoholic fatty liver disease: the emerging view. J Hepatol 51(1):212–223. doi:10.1016/j.jhep. 2009.03.008 Kahraman A, Schlattjan M, Kocabayoglu P, Yildiz-Meziletoglu S, Schlensak M, Fingas CD, Wedemeyer I, Marquitan G, Gieseler RK, Baba HA, Gerken G, Canbay A (2010) Major histocompatibility complex class I-related chains A and B (MIC A/B): a novel role in nonalcoholic steatohepatitis. Hepatology 51(1):92–102. doi:10.1002/hep.23253 Tajiri K, Shimizu Y, Tsuneyama K, Sugiyama T (2009) Role of liver-infiltrating CD3 ? CD56 ? natural killer T cells in the pathogenesis of nonalcoholic fatty liver disease. Eur J Gastroenterol Hepatol 21(6):673–680. doi:10.1097/MEG.0b013e328 31bc3d6 Syn WK, Oo YH, Pereira TA, Karaca GF, Jung Y, Omenetti A, Witek RP, Choi SS, Guy CD, Fearing CM, Teaberry V, Pereira FE, Adams DH, Diehl AM (2010) Accumulation of natural killer T cells in progressive nonalcoholic fatty liver disease. Hepatology 51(6):1998–2007. doi:10.1002/hep.23599 Vijay-Kumar M, Aitken JD, Carvalho FA, Cullender TC, Mwangi S, Srinivasan S, Sitaraman SV, Knight R, Ley RE, Gewirtz AT (2010) Metabolic syndrome and altered gut microbiota in mice lacking Toll-like receptor 5. Science 328(5975):228–231. doi:10.1126/science.1179721 Miura K, Kodama Y, Inokuchi S, Schnabl B, Aoyama T, Ohnishi H, Olefsky JM, Brenner DA, Seki E (2010) Toll-like receptor 9 promotes steatohepatitis by induction of interleukin-1beta in mice. Gastroenterology 139 (1):323-334 e327. doi:10.1053/j.gas tro.2010.03.052 Ide M, Yamate J, Machida Y, Nakanishi M, Kuwamura M, Kotani T, Sawamoto O (2003) Emergence of different macrophage
Mol Biol Rep
34.
35.
36.
37.
38.
39.
40.
41.
42.
populations in hepatic fibrosis following thioacetamide-induced acute hepatocyte injury in rats. J Comp Pathol 128(1):41–51 Baba Y, Saeki K, Onodera T, Doi K (2005) Serological and immunohistochemical studies on porcine-serum-induced hepatic fibrosis in rats. Exp Mol Pathol 79(3):229–235. doi:10.1016/j. yexmp.2005.08.007 Novobrantseva TI, Majeau GR, Amatucci A, Kogan S, Brenner I, Casola S, Shlomchik MJ, Koteliansky V, Hochman PS, Ibraghimov A (2005) Attenuated liver fibrosis in the absence of B cells. J Clin Investig 115(11):3072–3082. doi:10.1172/JCI24798 Chatterjee S, Rana R, Corbett J, Kadiiska MB, Goldstein J, Mason RP (2012) P2X7 receptor-NADPH oxidase axis mediates protein radical formation and Kupffer cell activation in carbon tetrachloride-mediated steatohepatitis in obese mice. Free Radic Biol Med 52(9):1666–1679. doi:10.1016/j.freeradbiomed.2012. 02.010 Inzaugarat ME, Ferreyra Solari NE, Billordo LA, Abecasis R, Gadano AC, Chernavsky AC (2011) Altered phenotype and functionality of circulating immune cells characterize adult patients with nonalcoholic steatohepatitis. J Clin Immunol 31(6):1120–1130. doi:10.1007/s10875-011-9571-1 Soderberg C, Marmur J, Eckes K, Glaumann H, Sallberg M, Frelin L, Rosenberg P, Stal P, Hultcrantz R (2011) Microvesicular fat, inter cellular adhesion molecule-1 and regulatory T-lymphocytes are of importance for the inflammatory process in livers with non-alcoholic steatohepatitis. APMIS : acta pathologica, microbiologica, et immunologica Scandinavica 119(7):412–420. doi:10.1111/j.1600-0463.2011.02746.x Ferreyra Solari NE, Inzaugarat ME, Baz P, De Matteo E, Lezama C, Galoppo M, Galoppo C, Chernavsky AC (2012) The role of innate cells is coupled to a Th1-polarized immune response in pediatric nonalcoholic steatohepatitis. J Clin Immunol 32(3):611–621. doi:10.1007/s10875-011-9635-2 Celikbilek M, Selcuk H, Yilmaz U (2011) A new risk factor for the development of non-alcoholic fatty liver disease: hLA complex genes. Turk J Gastroenterol : Off J Turk Soc Gastroenterol 22(4):395–399 Tiegs G, Lohse AW (2010) Immune tolerance: what is unique about the liver. J Autoimmun 34(1):1–6. doi:10.1016/j.jaut.2009. 08.008 Ruhl CE, Everhart JE (2010) Trunk fat is associated with increased serum levels of alanine aminotransferase in the United
43.
44.
45.
46.
47.
48.
49.
50.
States. Gastroenterology 138 (4):1346-1356, 1356 e1341-1343. doi:10.1053/j.gastro.2009.12.053 van der Poorten D, Milner KL, Hui J, Hodge A, Trenell MI, Kench JG, London R, Peduto T, Chisholm DJ, George J (2008) Visceral fat: a key mediator of steatohepatitis in metabolic liver disease. Hepatology 48(2):449–457. doi:10.1002/hep.22350 Hotamisligil GS (2010) Endoplasmic reticulum stress and the inflammatory basis of metabolic disease. Cell 140(6):900–917. doi:10.1016/j.cell.2010.02.034 Amir M, Czaja MJ (2011) Autophagy in nonalcoholic steatohepatitis. Expert Rev Gastroenterol Hepatol 5(2):159–166. doi:10.1586/egh.11.4 Wang Y, Singh R, Massey AC, Kane SS, Kaushik S, Grant T, Xiang Y, Cuervo AM, Czaja MJ (2008) Loss of macroautophagy promotes or prevents fibroblast apoptosis depending on the death stimulus. J Biol Chem 283(8):4766–4777. doi:10.1074/jbc. M706666200 Suri A, Walters JJ, Rohrs HW, Gross ML, Unanue ER (2008) First signature of islet beta-cell-derived naturally processed peptides selected by diabetogenic class II MHC molecules. J Immunol 180(6):3849–3856 Dengjel J, Schoor O, Fischer R, Reich M, Kraus M, Muller M, Kreymborg K, Altenberend F, Brandenburg J, Kalbacher H, Brock R, Driessen C, Rammensee HG, Stevanovic S (2005) Autophagy promotes MHC class II presentation of peptides from intracellular source proteins. Proc Natl Acad Sci USA 102(22):7922–7927. doi:10.1073/pnas.0501190102 Urabe Y, Ochi H, Kato N, Kumar V, Takahashi A, Muroyama R, Hosono N, Otsuka M, Tateishi R, Lo PH, Tanikawa C, Omata M, Koike K, Miki D, Abe H, Kamatani N, Toyota J, Kumada H, Kubo M, Chayama K, Nakamura Y, Matsuda K (2013) A genome-wide association study of HCV-induced liver cirrhosis in the Japanese population identifies novel susceptibility loci at the MHC region. J Hepatol 58(5):875–882. doi:10.1016/j.jhep.2012. 12.024 Li S, Qian J, Yang Y, Zhao W, Dai J, Bei JX, Foo JN, McLaren PJ, Li Z, Yang J, Shen F, Liu L, Yang J, Li S, Pan S, Wang Y, Li W, Zhai X, Zhou B, Shi L, Chen X, Chu M, Yan Y, Wang J, Cheng S et al (2012) GWAS identifies novel susceptibility loci on 6p21.32 and 21q21.3 for hepatocellular carcinoma in chronic hepatitis B virus carriers. PLoS Genet 8(7):e1002791. doi:10. 1371/journal.pgen.1002791
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HLA DQB1 alleles are related with nonalcoholic fatty liver disease Levent Doganay • Seyma Katrinli • Yasar Colak • Ebubekir Senates • Ebru Zemheri • Oguzhan Ozturk • Feruze Yilmaz Enc • Ilyas Tuncer • Gizem Dinler Doganay
Received: 21 September 2013 / Accepted: 20 August 2014 Ó Springer Science+Business Media Dordrecht 2014
Abstract Nonalcoholic fatty liver disease (NAFLD) is the leading cause of chronic liver disease. NAFLD is a complex disease and inflammation is a crucial component in the disease pathogenesis. Recent genome wide association studies in hepatology area highlighted significant relations with human leukocyte antigen (HLA) DQ region and certain liver diseases. The previous animal models also emphasized the involvement of adaptive immune system in the liver damage pathways. To investigate possible polymorphisms in the HLA region that can contribute to the immune response affecting the NAFLD, we enrolled 93 consecutive biopsy proven NAFLD patients and a control group consisted of 101 healthy people and genotyped HLA DQB1 alleles at high resolution by sequence specific primers-polymerase chain reaction. The mean NAFLD activity score (NAS) was 5.2 ± 1.2, fibrosis score was 0.9 ± 0.9, ALT was 77 ± 47.4 U/L, AST was
49.4 ± 26.3 U/L. Among 13 HLA DQB1 alleles analyzed in this study, DQB1*06:04 was observed significantly at a more frequent rate among the NAFLD patients compared to that of healthy controls (12.9 vs. 2 % v2 = 8.6, P = 0.003, Pc = 0.039, OR: 7.3 95 % CI 1.6–33.7). In addition, the frequency of DQB1*03:02 was significantly higher in the healthy control group than the NAFLD patients (24.8 vs. 7.5 %, v2 = 10.4, P = 0.001, Pc = 0.013, OR: 0.2, 95 % CI 0.1–0.6). NAFLD patients were grouped according to their fibrosis score and NAS. The distribution of DQB1 alleles over stratified NAFLD patients did not reveal any statistically significant relation. Taken together, immune repertoire of individuals may have an effect on NAFLD pathogenesis and therefore, in NAFLD, adaptive immunity pathways should be investigated. Keywords Nonalcoholic fatty liver disease (NAFLD) Nonalcoholic steatohepatitis (NASH) HLA DQ Liver Inflammation
Electronic supplementary material The online version of this article (doi:10.1007/s11033-014-3688-2) contains supplementary material, which is available to authorized users. L. Doganay Y. Colak F. Y. Enc I. Tuncer Department of Gastroenterology, Goztepe Teaching and Research Hospital, Medeniyet University, Istanbul, Turkey
E. Senates Department of Gastroenterology, School of Medicine, Dicle University, Diyarbakir, Turkey
L. Doganay O. Ozturk Department of Gastroenterology, Umraniye Teaching and Research Hospital, Istanbul, Turkey
E. Zemheri Department of Pathology, Goztepe Teaching and Research Hospital, Medeniyet University, Istanbul, Turkey
L. Doganay (&) Gastroenteroloji Klinigi, Umraniye Egitim ve Arastirma Hastanesi, Umraniye, 34764 Istanbul, Turkey e-mail: [email protected]
G. D. Doganay (&) Istanbul Teknik Universitesi, Ayazaga Kampusu, MOBGAM, 34469 Istanbul, Turkey e-mail: [email protected]
S. Katrinli G. D. Doganay Department of Molecular Biology and Genetics, Istanbul Technical University, Istanbul, Turkey
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Introduction Nonalcoholic fatty liver disease (NAFLD) is one of the most common causes of chronic liver diseases and it is a hepatic manifestation of metabolic syndrome [1]. The rising prevalence of NAFLD correlates with the epidemic of obesity. NAFLD shows a disease spectrum ranging from simple steatosis to nonalcoholic steatohepatitis (NASH), cirrhosis and hepatocellular carcinoma (HCC) [2]. Currently there are two hypotheses for the explanation of the progression of NAFLD from simple steatosis to more advanced forms and both of these put the inflammation as the key driving force for this progression [3, 4]. Present molecular studies mainly highlighted the role of innate immune system in this inflammation [4, 5]. However, there are animal model studies demonstrating the up-regulation of major histocompability complex (MHC) class II molecules in the liver damage pathways [6]. Moreover recent genome wide association studies in hepatology revealed significant association of single nucleotide polymorphisms in the human leukocyte antigen (HLA) DQ region with liver diseases, such as drug induced liver injury (rs312990 (P = 4.4 9 10-12) and rs92754407 (P = 4.8 9 10-14), fine mapping revealed DQB*06:02 allele association with both Lumiracoxib induced and Amoxicillin-Clavulanate induced liver injury, respectively) [7, 8], primary biliary cirrhosis (rs2856683, rs9275312, rs9275390, and rs7775228 (P values were in between 1.70 9 10-10 and 8.58 9 10-17), SNPs were associated with DQB1 gene region) [9], spontaneous resolution of hepatitis C viral disease (rs4273729 (P = 1.7 9 10-16), fine mapping revealed an association with DQB1*03:01) [10], chronic hepatitis B infection (rs28556718 (P = 5.9 9 10-28) and rs7453920 (P = 3.9 9 10-37), fine mapping revealed associations with DQB1*03:03 and DQB1*06:04 alleles) [11], HCC induced by hepatitis C (rs9275572 (P = 9.3 9 10-9), the SNP was associated with DQB1 gene region) [12], HCC and cirrhosis induced by hepatitis B virus (rs9275319 (P = 2.7 9 10-17), fine mapping revealed an association with DQB1*04:01) [13] and primary sclerosing cholangitis (many HLA alleles were associated with the disease, DQB*02:01 revealed significance at P = 2.4 9 10-187) [14]. Furthermore, our recent findings on the chronic hepatitis B disease demonstrated DQB1*05:01 and DQB1*05:03 alleles’ correlation to the chronic active disease state and treatment non-response, respectively [15, 16]. A recent pathway analysis with arrays including more than 300,000 single nucleotide polymorphisms in NAFLD patients demonstrated that antigen processing and presenting pathways were related with disease progression to NASH and HCC [17]. Human leukocyte antigens are essential elements of adaptive immunity and represents antigens to T cells [18].
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In liver; dendritic cells, kupffer cells, liver sinusoidal endothelial cells, and hepatocytes express MHC class II molecules that are coded by HLA genes [19–21]. According to our medline search there is no study genotyping HLA in biopsy proven NAFLD patients, this study aims to genotype at a high resolution HLA DQB1 alleles in biopsy proven NAFLD patients.
Methods Study participants In this case control study a total of 93 biopsy proven NAFLD patients (46 male and 47 female, mean age 42 ± 10.2 years) and a gender and age matched comparison group consisting of 101 healthy people (48 male 53 female, mean age 41 ± 9.5) were recruited. Patient and control inclusion and exclusion criteria were described before [22]. Patients were consecutively seen and followed up at hepatology outpatient clinic. Local ethics committee approved the study, and all participants gave informed consent. Patients with other hepatic diseases were excluded. None of the patients were on any estrogen, amiadrone, diltiazem, steroid, tamoxifen or herbal supplement treatment. The control group was consisted of people who were healthy on medical examination, had liver biochemistry tests in normal range and had normal liver on sonographic examination. Consuming alcohol greater than 20 g/day was an exclusion criteria for both patient and the control group. Clinical assessment All patients went through physical examination, anthropometric measurements, and biochemical tests. The diagnosis of NAFLD was done by histologically and on haematoxylin-eosin-stained liver specimens all patients had [5 % macrovesicular steatosis. Biopsies were obtained under ultrasonographic guidance by 16 G Hepafix needle. The minimum length of the biopsies was 2.5 cm. All biopsy specimens were stained with haematoxyin-eosin and Masson’s trichrome. A pathologist who was experienced in NAFLD and was blinded to all the study data examined the biopsies according to the National Institute of Diabetes and Digestive and Kidney Diseases NASH Clinical Research Network scoring system [23]. Polymorphism genotyping Genomic DNA was extracted from 200 lL of peripheral blood by using Invitrogen PureLink genomic DNA purification kit according to manufacturer’s protocol. The alleles of HLA-DQB1 were detected by sequence-specific
Mol Biol Rep Table 1 Characteristics of patients and the control group NAFLD (n = 93)
Control (n = 101)
P
46/47
48/53
Ns
Age (years)
42.7 ± 9.5
41 ± 9.5
Ns
Body mass index (kg/ m2) Metabolic syndrome
31.5 ± 5.1
24.9 ± 4.5
\0.001
60/33
0/101
\0.001
Gender (male/female)
Diabetes mellitus
22/70
0/101
\0.001
Triglyceride (mmol/l)
197 ± 145
102.3 ± 70.5
\0.001
LDL cholesterol (mmol/l)
133.4 ± 35.5
114.1 ± 29.1
\0.001
HDL cholesterol (mmol/l)
45.1 ± 8.3
50.2 ± 11
\0.001
ALT (U/l)
76.9 ± 47.2
18.2 ± 10.2
\0.001
AST (U/l)
49.3 ± 26.2
21.1 ± 7.1
\0.001
LDL low density lipoprotein, HDL high density lipoprotein, ALT alanine transferase, AST aspartate transaminase
SyberGreen in the presence of 100 bp DNA ladder (Fermentas) (Fig. 1). The allelic type was determined according to the presence or absence of the desired length PCR products. Statistical analysis The frequencies of HLA alleles were calculated by direct counting. Statistical analyses were done to evaluate the difference between NAFLD patients and control patients. During the HLA analysis, 2 9 2 tables and Chi square test were used. To evaluate the association of DQB1 gene with the disease, a dominant model where the 13 genotyped alleles represented in the tables as yes/no fashion was applied. An individual carries two alleles, so the sum of the percentages in the disease association table may not be one hundred. To reduce an overestimation, if only one allele was determined in an individual that allele was counted once rather than considering it as a homozygote. When the sample sizes were small or expected values in cells of Chi square table were \5, then, Fisher’s exact test was used. Parametric variables were analyzed by the student’s t test. Parametric variables were given as mean ± standard deviation. All P values were calculated double sided, and if the P value is below 0.05, it was considered as statistically significant unless there are multiple comparisons. Bonferroni’s correction was applied for multiple comparisons and corrected P values were given. All analyses were done in a computer with SPSS 21 (Chicago, IL).
primers–polymerase chain reaction (SSP–PCR) [24]. Primers sequences and PCR product sizes are listed in supplementary Table 1. Positive internal control primers concentration was adjusted as 0.8 fold of specific primers concentration, and then both were pooled into the reaction mix to eradicate pseudonegative possibilities. The internal control is a fragment of human growth hormone gene 1 (chr 17) with 439 bp [25]. The sequences of internal control primers are also shown in supplementary Table 1. PCR is performed in 25 lL reaction mixture containing 50–100 ng genomic DNA, 0.625 U DNA DreamTaq Green DNA polymerase, 10X DreamTaq Green buffer, 4 mM MgCl2 (Intron) and 0.2 mM of each dNTP (Intron), 0.5 lM primers, 0.4 lM internal control primers. PCR cycling parameters were as follows: predenaturation at 95 °C for 3 min, denaturation at 95 °C for 30 s, annealing at 65 °C for 30 s, extension at 72 °C for 50 s, repetition for 30 cycles and final extension at 72 °C for 7 min. After amplification, products were identified by ultraviolet light after electrophoresis in 2 % agarose gel stained by
Characteristics and baseline laboratory results of patients and control group are given in Table 1. These two groups were not different in terms of gender or age but body mass index (BMI), lipid profile, ALT, AST were significantly higher in the patient group. Mean NAFLD activity score (NAS) of the patients was 5.2 ± 1.2, the mean fibrosis score was 0.9 ± 0.9. Steatosis grade, NAS and fibrosis
Fig. 1 Electrophoretic figures of PCR product by 2 % agarose gel. Lane numbers present: 1-DQB1*03:01/04; 2-DQB1*05:01; 3DQB1*06:01; 4-DQB1*02:01 and DQB1*05:02; 5-DQB1*04:01; 6-
DQB1*04:02; 7-DQB1*05:03; 8-DQB1*02:01; 9-DQB1*02:01/ 03:02; 10-DQB1*03:03; 11-DQB1*03:02/03; 12-DQB1*03:02/03; 13-DQB1*06:04
Results
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Mol Biol Rep Table 2 Histologic scores of patients (n = 93)
Histologic variable
n (%)
genotypes over patients and controls did not reveal significant difference.
Steatosis grade 0
0
1
24 (25.8)
2
49 (52.6)
3
19 (20.4)
NAS 0–2
7 (7.5)
3–4
22 (23.7)
5–8
57 (61.2)
Fibrosis stage
NAS, NAFLD activity score
0
35 (37.6)
1
44 (47.3)
2
7 (7.5)
3
6 (6.5)
4
1 (1.1)
stage of the patients are given in Table 2. Among the genotyped 13 DQB1 alleles, DQB1*06:04 was observed significantly at a more frequent rate among the NAFLD patients compared to that of healthy controls (OR = 7.3, P = 0.003, Pc = 0.039). In addition, the frequency of DQB1*03:02 is significantly higher in the healthy control group than the NAFLD patients (OR = 0.3, P = 0.001, Pc = 0.013). The distribution of HLA DQB1 alleles over patients and control group is given in Table 3. The most frequent DQB1 genotypes in the whole study population were DQB1*03:01/03:04, DQB1*06:01 (n = 16, 8.2 %) and DQB1*03:01/03:04, DQB1*05:01 (n = 10, 5.1 %). The distribution of these most frequent
Table 3 The distribution of HLA alleles among patients and control group
DQB1 allele
Discussion The effect of immune system on NAFLD pathogenesis is an evolving issue. To date, studies indicated the impact of parts of innate immune system on NAFLD (e.g. kupffer cells [26, 27], natural killer cells [28], natural killer T cells [29, 30] and Toll like receptors [31, 32]). Although the elements of adaptive immune response are not thoroughly investigated, there is growing evidence suggesting the role of adaptive immune pathways in inflammation in NAFLD. Animal models exploring the liver damage pathways demonstrated the importance of MHC class II molecules. For instance; in the thioacetamide induced rat liver fibrosis model, MHC class II related pathway was shown to be induced [33]. In porcine serum induced rat model of fibrosis, MHC class II related genes were upregulated [6, 34]. In another animal model, it was demonstrated that liver fibrosis relied on the B lymphocyte presence [35]. In carbon tetrachloride induced steatohepatitis model in obese mice, expression of MHC II was increased in kuppfer cells [36]. Moreover in clinical studies it was shown that circulating CD4 positive lymphocytes and regulatory T cells were increased in NASH patients [37, 38]. In a biopsy study from pediatric NASH patients the profile liver infiltrating immune cells indicated the role of T helper 1 response besides innate immune pathway [39]. All these studies reveal the importance of MHC class II involvement in NAFLD from a mechanistic view; we think that there is a necessity to explore the MHC class II gene relation with the disease since these polymorphic sequences can be
NAFLD n: 93 (%)
Control group n: 101 (%)
V2
P
OR
CI 95 %
1
*05:01
16 (17.2)
15 (14.9)
0.2
0.66
1.1
0.5–2.6
2
*05:02
15 (16.1)
11 (10.9)
1.1
0.3
1.5
0.6–3.6
3
*05:03
10 (10.8)
20 (19.8)
3
0.08
0.49
0.2–1.1
4
*06:01
23 (24.7)
16 (15.8)
2.4
0.12
1.7
0.9–3.6
5
*06:02
0
0
NA
NA
NA
NA
6
*06:03/06:08
0
0
NA
NA
NA
NA
7
*06:04
12 (12.9)
2 (2)
8.6
0.003a
7.3
1.6–33.7 0.7–5.2
8
*02:01
12 (12.9)
7 (6.9)
1.96
0.16
2
NA not available
9
*03:01/03:04
34 (36.6)
42 (41.6)
0.5
0.5
0.8
0.5–1.4
a
10
*03:02
7 (7.5)
25 (24.8)
10.4
0.001b
0.2
0.1–0.6
11
*03:03
6 (6.5)
12 (11.9)
1.6
0.2
0.5
0.2–1.4
12
*04:02
3 (3.2)
6 (5.9)
c
0.5
0.5
0.1–2.1
13
*04:01
1 (1.1)
5 (5)
c
0.2
0.2
0.1–1.8
Pcorrected = 0.039 (after Bonferroni’s correction) b
Pcorrected = 0.013 (after Bonferroni’s correction) c
Fisher’s exact test is applied
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determinants of susceptibility or resistance, as observed for various diseases. Our study demonstrated that NAFLD patients had more frequently DQB1*06:04 allele (Pc = 0.039) compared to healthy control group, whereas control group showed more frequently DQB1*03:02 allele (Pc = 0.013) compared to that of the patients. In a previous study from Turkey, Celikbilek and his coworkers demonstrated that HLA B65 and DQ5 are the risk factors for NAFLD [40]. That study was consisted of 66 NAFLD patients diagnosed by ultrasound and 50 controls. Both patients and controls were selected from liver donor candidates and HLA subtypes were defined by serotyping. In our study, NAFLD was diagnosed by liver biopsy in consecutive patients and DQB1 alleles were genotyped at high resolution. Up until June 2014, according to our medline search this study is the first report revealing the association of genotyped HLA alleles in biopsy proven NAFLD patients. Antigen presenting cells (APC) present antigens to CD4 positive lymphocytes and these lymphocytes secrete numerous cytokines and promote adaptive immunity. In liver, dendritic cells, kupffer cells, liver sinusoidal endothelial cells, and hepatocytes present antigens to CD4 positive lymphocytes via MHC II molecules [19–21]. These APCs are involved in the pathogenesis of both chronic viral infections and autoimmune diseases of the liver [19, 41]. Additionally a novel pathway suggested to be involved in the pathogenesis of NAFLD is autophagy besides well-known ones such as proinflammatory mediators secreted by visceral adipose tissue [42, 43], gut derived products [31], and endoplasmic reticulum stress [44]. Dysregulation of autophagy affects both cellular lipid accumulation and inflammation in NAFLD [45, 46]. Autophagy in hepatocytes may activate innate immune pathways but also it is well shown that autophagy may induce self-antigen presentation via MHC II molecules [47, 48]. When our results are taken into account with the background knowledge of the growing evidence indicating the adaptive immune pathway involvement in the NAFLD pathogenesis, it might be considered as HLA DQ has a role in the junction between innate and adaptive immunity cross-talks. Interestingly the recent GWA studies in hepatology revealed association of HLA DQ region with many disease conditions. These studies investigated drug induced liver injury [7, 8], primary sclerosing cholangitis [14], primary biliary cirrhosis [9], hepatitis C clearance [10], liver damage in hepatitis C [49], hepatocellular cancer due to hepatitis C [12], hepatitis B seroclearance [11], hepatocellular cancer due to hepatitis B [13, 50], and all revealed association with single nucleotide polymorphisms in HLA DQ region. In this study we did not demonstrate any relation with fibrosis stage or NAS and DQB1 alleles (statistics not
shown). To search a relation, we classified patients according to NAS and fibrosis scores but statistics did not reveal any association with DQB1 alleles and histology. As we genotyped 13 different DQB1 types, the patient numbers in subgroups might be insufficient to demonstrate a significant relation. This study has some limitations. This is a cross sectional case control study, and it is not possible to conclude a direct causative relation between our findings and the disease. Replication studies to confirm HLA relation with the disease and molecular studies to investigate adaptive immunity pathway in NAFLD are necessary. For subgroup analysis (e.g. advanced fibrosis) the patient number should be higher. On the other hand, consecutive patient enrollment, very well-defined biopsy proven patient selection, and genotyping in high resolution are the strengths of this study. In conclusion the immune repertoire of an individual may affect NAFLD development. This study demonstrated that HLA DQB1*06:04 allele was associated with NALFD and DQB1*03:02 is a protective factor from NAFLD. Acknowledgments This study is supported by internal research funds of Istanbul Technical University.
References 1. Lazo M, Clark JM (2008) The epidemiology of nonalcoholic fatty liver disease: a global perspective. Semin Liver Dis 28(4): 339–350. doi:10.1055/s-0028-1091978 2. Cohen JC, Horton JD, Hobbs HH (2011) Human fatty liver disease: old questions and new insights. Science 332(6037): 1519–1523. doi:10.1126/science.1204265 3. Day CP, James OF (1998) Steatohepatitis: a tale of two ‘‘hits’’? Gastroenterology 114(4):842–845 4. Tilg H, Moschen AR (2010) Evolution of inflammation in nonalcoholic fatty liver disease: the multiple parallel hits hypothesis. Hepatology 52(5):1836–1846. doi:10.1002/hep.24001 5. Harmon RC, Tiniakos DG, Argo CK (2011) Inflammation in nonalcoholic steatohepatitis. Expert Rev Gastroenterol Hepatol 5(2):189–200. doi:10.1586/egh.11.21 6. Baba Y, Doi K (2004) MHC class II-related genes expression in porcine-serum-induced rat hepatic fibrosis. Exp Mol Pathol 77(3):214–221. doi:10.1016/j.yexmp.2004.08.001 7. Singer JB, Lewitzky S, Leroy E, Yang F, Zhao X, Klickstein L, Wright TM, Meyer J, Paulding CA (2010) A genome-wide study identifies HLA alleles associated with lumiracoxib-related liver injury. Nat Genet 42(8):711–714. doi:10.1038/ng.632 8. Lucena MI, Molokhia M, Shen Y, Urban TJ, Aithal GP, Andrade RJ, Day CP, Ruiz-Cabello F, Donaldson PT, Stephens C, Pirmohamed M, Romero-Gomez M, Navarro JM, Fontana RJ, Miller M, Groome M, Bondon-Guitton E, Conforti A, Stricker BH, Carvajal A, Ibanez L, Yue QY, Eichelbaum M, Floratos A, Pe’er I et al (2011) Susceptibility to amoxicillin-clavulanateinduced liver injury is influenced by multiple HLA class I and II alleles. Gastroenterology 141(1):338–347. doi:10.1053/j.gastro. 2011.04.001
123
Mol Biol Rep 9. Hirschfield GM, Liu X, Xu C, Lu Y, Xie G, Lu Y, Gu X, Walker EJ, Jing K, Juran BD, Mason AL, Myers RP, Peltekian KM, Ghent CN, Coltescu C, Atkinson EJ, Heathcote EJ, Lazaridis KN, Amos CI, Siminovitch KA (2009) Primary biliary cirrhosis associated with HLA, IL12A, and IL12RB2 variants. New Engl J Med 360(24):2544–2555. doi:10.1056/NEJMoa0810440 10. Duggal P, Thio CL, Wojcik GL, Goedert JJ, Mangia A, Latanich R, Kim AY, Lauer GM, Chung RT, Peters MG, Kirk GD, Mehta SH, Cox AL, Khakoo SI, Alric L, Cramp ME, Donfield SM, Edlin BR, Tobler LH, Busch MP, Alexander G, Rosen HR, Gao X, Abdel-Hamid M, Apps R et al (2013) Genome-wide association study of spontaneous resolution of hepatitis C virus infection: data from multiple cohorts. Ann Intern Med 158(4):235–245. doi:10. 7326/0003-4819-158-4-201302190-00003 11. Mbarek H, Ochi H, Urabe Y, Kumar V, Kubo M, Hosono N, Takahashi A, Kamatani Y, Miki D, Abe H, Tsunoda T, Kamatani N, Chayama K, Nakamura Y, Matsuda K (2011) A genome-wide association study of chronic hepatitis B identified novel risk locus in a Japanese population. Hum Mol Genet 20(19):3884–3892. doi:10.1093/hmg/ddr301 12. Kumar V, Kato N, Urabe Y, Takahashi A, Muroyama R, Hosono N, Otsuka M, Tateishi R, Omata M, Nakagawa H, Koike K, Kamatani N, Kubo M, Nakamura Y, Matsuda K (2011) Genomewide association study identifies a susceptibility locus for HCVinduced hepatocellular carcinoma. Nat Genet 43(5):455–458. doi:10.1038/ng.809 13. Jiang DK, Sun J, Cao G, Liu Y, Lin D, Gao YZ, Ren WH, Long XD, Zhang H, Ma XP, Wang Z, Jiang W, Chen TY, Gao Y, Sun LD, Long JR, Huang HX, Wang D, Yu H, Zhang P, Tang LS, Peng B, Cai H, Liu TT, Zhou P et al (2013) Genetic variants in STAT4 and HLA-DQ genes confer risk of hepatitis B virusrelated hepatocellular carcinoma. Nat Genet 45(1):72–75. doi:10. 1038/ng.2483 14. Liu JZ, Hov JR, Folseraas T, Ellinghaus E, Rushbrook SM, Doncheva NT, Andreassen OA, Weersma RK, Weismuller TJ, Eksteen B, Invernizzi P, Hirschfield GM, Gotthardt DN, Pares A, Ellinghaus D, Shah T, Juran BD, Milkiewicz P, Rust C, Schramm C, Muller T, Srivastava B, Dalekos G, Nothen MM, Herms S et al (2013) Dense genotyping of immune-related disease regions identifies nine new risk loci for primary sclerosing cholangitis. Nat Genet 45(6):670–675. doi:10.1038/ng.2616 15. Doganay L, Tuncer I, Katrinli S, Enc FY, Ozturk O, Colak Y, Ulasoglu C, Dinler G (2013) The effect of HLA-DQB1 alleles on virologic breakthroughs during chronic hepatitis B treatment with genetically low barrier drugs. Clin Res Hepatol Gastroenterol 37(4):359–364. doi:10.1016/j.clinre.2012.10.013 16. Doganay L, Fejzullahu A, Katrinli S, Yilmaz Enc F, Ozturk O, Colak Y, Ulasoglu C, Tuncer I, Dinler Doganay G (2014) Association of human leukocyte antigen DQB1 and DRB1 alleles with chronic hepatitis B. World J Gastroenterol : WJG 20(25):8179–8186. doi:10.3748/wjg.v20.i25.8179 17. Chen QR, Braun R, Hu Y, Yan C, Brunt EM, Meerzaman D, Sanyal AJ, Buetow K (2013) Multi-SNP Analysis of GWAS Data Identifies Pathways Associated with Nonalcoholic Fatty Liver Disease. PLoS One 8(7):e65982. doi:10.1371/journal.pone. 0065982 18. Germain RN (1994) MHC-dependent antigen processing and peptide presentation: providing ligands for T lymphocyte activation. Cell 76(2):287–299 19. Racanelli V, Rehermann B (2006) The liver as an immunological organ. Hepatology 43(2 Suppl 1):S54–S62. doi:10.1002/hep. 21060 20. Herkel J, Jagemann B, Wiegard C, Lazaro JF, Lueth S, Kanzler S, Blessing M, Schmitt E, Lohse AW (2003) MHC class IIexpressing hepatocytes function as antigen-presenting cells and
123
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
activate specific CD4 T lymphocyutes. Hepatology 37(5):1079–1085. doi:10.1053/jhep.2003.50191 Winau F, Hegasy G, Weiskirchen R, Weber S, Cassan C, Sieling PA, Modlin RL, Liblau RS, Gressner AM, Kaufmann SH (2007) Ito cells are liver-resident antigen-presenting cells for activating T cell responses. Immunity 26(1):117–129. doi:10.1016/j. immuni.2006.11.011 Colak Y, Karabay CY, Tuncer I, Kocabay G, Kalayci A, Senates E, Ozturk O, Doganay HL, Enc FY, Ulasoglu C, Kiziltas S (2012) Relation of epicardial adipose tissue and carotid intima-media thickness in patients with nonalcoholic fatty liver disease. Eur J Gastroenterol Hepatol 24(6):613–618. doi:10.1097/MEG. 0b013e3283513f19 Kleiner DE, Brunt EM, Van Natta M, Behling C, Contos MJ, Cummings OW, Ferrell LD, Liu YC, Torbenson MS, UnalpArida A, Yeh M, McCullough AJ, Sanyal AJ, Nonalcoholic Steatohepatitis Clinical Research N (2005) Design and validation of a histological scoring system for nonalcoholic fatty liver disease. Hepatology 41(6):1313–1321. doi:10.1002/hep.20701 Olerup O, Aldener A, Fogdell A (1993) HLA-DQB1 and -DQA1 typing by PCR amplification with sequence-specific primers (PCR–SSP) in 2 hours. Tissue Antigens 41(3):119–134. doi:10. 1111/j.1399-0039.1993.tb01991.x Xi-Lin Z, Te D, Jun-Hong L, Liang-Ping L, Xin-Hui G, Ji-Rong G, Chun-Yan G, Zhuo L, Ying L, Hui L (2006) Analysis of HLADQB1 gene polymorphisms in asymptomatic HBV carriers and chronic hepatitis B patients in the Chinese Han population. Int J Immunogenet 33(4):249–254. doi:10.1111/j.1744-313X.2006. 00607.x Rivera CA, Adegboyega P, van Rooijen N, Tagalicud A, Allman M, Wallace M (2007) Toll-like receptor-4 signaling and Kupffer cells play pivotal roles in the pathogenesis of non-alcoholic steatohepatitis. J Hepatol 47(4):571–579. doi:10.1016/j.jhep.2007. 04.019 Baffy G (2009) Kupffer cells in non-alcoholic fatty liver disease: the emerging view. J Hepatol 51(1):212–223. doi:10.1016/j.jhep. 2009.03.008 Kahraman A, Schlattjan M, Kocabayoglu P, Yildiz-Meziletoglu S, Schlensak M, Fingas CD, Wedemeyer I, Marquitan G, Gieseler RK, Baba HA, Gerken G, Canbay A (2010) Major histocompatibility complex class I-related chains A and B (MIC A/B): a novel role in nonalcoholic steatohepatitis. Hepatology 51(1):92–102. doi:10.1002/hep.23253 Tajiri K, Shimizu Y, Tsuneyama K, Sugiyama T (2009) Role of liver-infiltrating CD3 ? CD56 ? natural killer T cells in the pathogenesis of nonalcoholic fatty liver disease. Eur J Gastroenterol Hepatol 21(6):673–680. doi:10.1097/MEG.0b013e328 31bc3d6 Syn WK, Oo YH, Pereira TA, Karaca GF, Jung Y, Omenetti A, Witek RP, Choi SS, Guy CD, Fearing CM, Teaberry V, Pereira FE, Adams DH, Diehl AM (2010) Accumulation of natural killer T cells in progressive nonalcoholic fatty liver disease. Hepatology 51(6):1998–2007. doi:10.1002/hep.23599 Vijay-Kumar M, Aitken JD, Carvalho FA, Cullender TC, Mwangi S, Srinivasan S, Sitaraman SV, Knight R, Ley RE, Gewirtz AT (2010) Metabolic syndrome and altered gut microbiota in mice lacking Toll-like receptor 5. Science 328(5975):228–231. doi:10.1126/science.1179721 Miura K, Kodama Y, Inokuchi S, Schnabl B, Aoyama T, Ohnishi H, Olefsky JM, Brenner DA, Seki E (2010) Toll-like receptor 9 promotes steatohepatitis by induction of interleukin-1beta in mice. Gastroenterology 139 (1):323-334 e327. doi:10.1053/j.gas tro.2010.03.052 Ide M, Yamate J, Machida Y, Nakanishi M, Kuwamura M, Kotani T, Sawamoto O (2003) Emergence of different macrophage
Mol Biol Rep
34.
35.
36.
37.
38.
39.
40.
41.
42.
populations in hepatic fibrosis following thioacetamide-induced acute hepatocyte injury in rats. J Comp Pathol 128(1):41–51 Baba Y, Saeki K, Onodera T, Doi K (2005) Serological and immunohistochemical studies on porcine-serum-induced hepatic fibrosis in rats. Exp Mol Pathol 79(3):229–235. doi:10.1016/j. yexmp.2005.08.007 Novobrantseva TI, Majeau GR, Amatucci A, Kogan S, Brenner I, Casola S, Shlomchik MJ, Koteliansky V, Hochman PS, Ibraghimov A (2005) Attenuated liver fibrosis in the absence of B cells. J Clin Investig 115(11):3072–3082. doi:10.1172/JCI24798 Chatterjee S, Rana R, Corbett J, Kadiiska MB, Goldstein J, Mason RP (2012) P2X7 receptor-NADPH oxidase axis mediates protein radical formation and Kupffer cell activation in carbon tetrachloride-mediated steatohepatitis in obese mice. Free Radic Biol Med 52(9):1666–1679. doi:10.1016/j.freeradbiomed.2012. 02.010 Inzaugarat ME, Ferreyra Solari NE, Billordo LA, Abecasis R, Gadano AC, Chernavsky AC (2011) Altered phenotype and functionality of circulating immune cells characterize adult patients with nonalcoholic steatohepatitis. J Clin Immunol 31(6):1120–1130. doi:10.1007/s10875-011-9571-1 Soderberg C, Marmur J, Eckes K, Glaumann H, Sallberg M, Frelin L, Rosenberg P, Stal P, Hultcrantz R (2011) Microvesicular fat, inter cellular adhesion molecule-1 and regulatory T-lymphocytes are of importance for the inflammatory process in livers with non-alcoholic steatohepatitis. APMIS : acta pathologica, microbiologica, et immunologica Scandinavica 119(7):412–420. doi:10.1111/j.1600-0463.2011.02746.x Ferreyra Solari NE, Inzaugarat ME, Baz P, De Matteo E, Lezama C, Galoppo M, Galoppo C, Chernavsky AC (2012) The role of innate cells is coupled to a Th1-polarized immune response in pediatric nonalcoholic steatohepatitis. J Clin Immunol 32(3):611–621. doi:10.1007/s10875-011-9635-2 Celikbilek M, Selcuk H, Yilmaz U (2011) A new risk factor for the development of non-alcoholic fatty liver disease: hLA complex genes. Turk J Gastroenterol : Off J Turk Soc Gastroenterol 22(4):395–399 Tiegs G, Lohse AW (2010) Immune tolerance: what is unique about the liver. J Autoimmun 34(1):1–6. doi:10.1016/j.jaut.2009. 08.008 Ruhl CE, Everhart JE (2010) Trunk fat is associated with increased serum levels of alanine aminotransferase in the United
43.
44.
45.
46.
47.
48.
49.
50.
States. Gastroenterology 138 (4):1346-1356, 1356 e1341-1343. doi:10.1053/j.gastro.2009.12.053 van der Poorten D, Milner KL, Hui J, Hodge A, Trenell MI, Kench JG, London R, Peduto T, Chisholm DJ, George J (2008) Visceral fat: a key mediator of steatohepatitis in metabolic liver disease. Hepatology 48(2):449–457. doi:10.1002/hep.22350 Hotamisligil GS (2010) Endoplasmic reticulum stress and the inflammatory basis of metabolic disease. Cell 140(6):900–917. doi:10.1016/j.cell.2010.02.034 Amir M, Czaja MJ (2011) Autophagy in nonalcoholic steatohepatitis. Expert Rev Gastroenterol Hepatol 5(2):159–166. doi:10.1586/egh.11.4 Wang Y, Singh R, Massey AC, Kane SS, Kaushik S, Grant T, Xiang Y, Cuervo AM, Czaja MJ (2008) Loss of macroautophagy promotes or prevents fibroblast apoptosis depending on the death stimulus. J Biol Chem 283(8):4766–4777. doi:10.1074/jbc. M706666200 Suri A, Walters JJ, Rohrs HW, Gross ML, Unanue ER (2008) First signature of islet beta-cell-derived naturally processed peptides selected by diabetogenic class II MHC molecules. J Immunol 180(6):3849–3856 Dengjel J, Schoor O, Fischer R, Reich M, Kraus M, Muller M, Kreymborg K, Altenberend F, Brandenburg J, Kalbacher H, Brock R, Driessen C, Rammensee HG, Stevanovic S (2005) Autophagy promotes MHC class II presentation of peptides from intracellular source proteins. Proc Natl Acad Sci USA 102(22):7922–7927. doi:10.1073/pnas.0501190102 Urabe Y, Ochi H, Kato N, Kumar V, Takahashi A, Muroyama R, Hosono N, Otsuka M, Tateishi R, Lo PH, Tanikawa C, Omata M, Koike K, Miki D, Abe H, Kamatani N, Toyota J, Kumada H, Kubo M, Chayama K, Nakamura Y, Matsuda K (2013) A genome-wide association study of HCV-induced liver cirrhosis in the Japanese population identifies novel susceptibility loci at the MHC region. J Hepatol 58(5):875–882. doi:10.1016/j.jhep.2012. 12.024 Li S, Qian J, Yang Y, Zhao W, Dai J, Bei JX, Foo JN, McLaren PJ, Li Z, Yang J, Shen F, Liu L, Yang J, Li S, Pan S, Wang Y, Li W, Zhai X, Zhou B, Shi L, Chen X, Chu M, Yan Y, Wang J, Cheng S et al (2012) GWAS identifies novel susceptibility loci on 6p21.32 and 21q21.3 for hepatocellular carcinoma in chronic hepatitis B virus carriers. PLoS Genet 8(7):e1002791. doi:10. 1371/journal.pgen.1002791
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