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Association of HLA alleles with hepatitis C infection in Maharashtra, western India.

Hepatitis C virus (HCV) is an important blood-borne pathogen that is eliminated from the host in approximately 15 per cent of acutely infected individuals but persists in the remaining 85 per cent (1) HCV is responsible for a wide spectrum of chronic liver lesions ranging from minimal to cirrhosis or hepatocellular carcinoma (HCC) and fatal outcome (2,3). Both virus-related factors such as viral heterogeneity and replicative activity (4) and the host determinants such as lack of efficient immune responses (5) are involved in the pathogenesis of chronic hepatitis. Further the liver damage in HCV infected patients is probably associated with direct cytopathic effects and immune mediated mechanisms (6). Though the exact basis for the differential clinical presentation of HCV infection is not fully understood, viral load and genotype (7) have been reported to influence the prognosis. The observation of different clinical presentations despite the same source of infection led to the recognition of the importance of host genetic factors in disease manifestations (8). In an Irish Cohort, of the 704 women infected with HCV from contaminated anti-D immune globulin, 390 (55%) became persistently infected (9). MHC class I and class II antigens are central to the host immune response and thus are ideal candidate genes to investigate for associations with HCV. Class I and Class II HLA are encoded by the most polymorphic genes that present antigens to CD8+ cytotoxic T cells and CD4+ helper T cells respectively. Polymorphisms in binding regions of these molecules determine antigenic specificities and the strength of the immune response to a given pathogen. Moreover during cellular immune response, HLA class I molecules may present HCV epitopes to cytotoxic T cells, resulting in a protective immune response. The human leukocyte antigen is a crucial genetic factor that initiates and regulates immune responses by presenting foreign or self-antigens to T lymphocytes. Certain HLA alleles have been shown to influence the outcome of chronic HCV infections (10,11). Various HLA alleles have been linked with either persistence or clearance of the virus. Several studies have aimed to identify the involvement of HLA with different outcomes of HCV infection, but the results have not been consistent. Moreover literature review revealed that the prevalence of HCV infection is significantly low in Indian population (12-14). So far, no data on HLA association with HCV infection have been reported from western India. Therefore, this study was undertaken to find out whether there is any genetic basis that involves HLA with HCV infection among anti-HCV positive individuals from the state of Maharashtra, western India.

Material & Methods

Study population: The study was carried out at NIV, Pune. A total of 43 anti-HCV positive individuals selected from different parts of Maharashtra over a period of three years (2003 and 2006) were included in this study. All these individuals were referred to NIV, Pune for diagnosis of hepatitis C infection by molecular and serological testing. All patients were tested for HLA A, B and C alleles but only 39 were studied for DQ and DR alleles. Of these 43, 33 were HCV RNA positives. Among these positives, 10 were on maintenance haemodialysis, 8 were voluntary blood donors and the remaining 15 were suffering from chronic liver diseases. Furthermore, patients suffering from chronic liver diseases had serum alanine aminotransferase (ALT) levels elevated leaving aside 8 of 15 patients suffering from chronic liver disease, none of the anti-HCV individuals were hospitalized. All of them were tested negative for both hepatitis B surface antigen (HbsAg) and anti-HIV antibodies. The control group consisted of 67 and 113 healthy unrelated normal Maharashtrian subjects for HLA A, B, C and DR, DQ respectively. Controls were negative for anti-HCV antibodies, HBsAg and anti-HIV antibodies.

Serological testing: All the samples were screened for the presence of anti-HCV antibodies using enzyme linked immunosorbent assay (ELISA, Ortho Clinical diagnostics, Inc. IIIrd generation, New Jersey, USA). Anti-HIV antibodies and HBsAg were screened by ELISA (Lab systems HIV EIA & Surase B-96, General Biological Corporation, Taiwan). Serum ALT was measured by a commercial kit (Span Diagnostics Ltd, India), all tests were done according to the manufactures protocol. HCV RNA was detected by reverse transcription nested polymerase chain reaction (RT-PCR) using primers located in the highly conserved 5' NCR region (15).

HLA typing: Genomic DNA was extracted from frozen peripheral blood mononuclear cells by Qiagen Blood mini kit (Germany). Molecular typing was carried out by polymerase chain reaction-sequence specific primer (PCR-SSP) method (Olerup SSP AB, GenoVision, Inc, Sweden) utilizing allele specific primers along with the control primers to identify the respective allele. The allele specific primers were provided in the Kit (Olerup SSP HLA-A-B-C SSP Combi Tray). The primer set contained 5' and 3' primers for grouping the HLA-A *0101 to *8001 alleles, 5' and 3' primers for grouping the B*0702 to *8302 alleles and 5' and 3' primers for grouping the Cw* 0102 to Cw* 1802 alleles. The amplified products were visualized under UV following agarose gel electrophoresis and the interpretations of the alleles were based on Helmberg SCORE programme (update V3.118 KIT software GenoVision, Inc, Sweden).

Statistical analysis: The phenotype frequencies, odds ratios (OR), probability value, Chi-square with Yates correction, aetiological and preventive fraction were estimated using the available data- base and computer programme16. Since each individual was tested for several HLA alleles and the same data were used for comparing frequency, it was possible that one of the alleles would by chance deviate significantly. To overcome this error, the P value was corrected by the use of Bonferroni inequality method (17), i.e. by multiplying with the number of alleles compared. The alleles were determined with the Helmberg SCORE V3.118T software supplied along with the kit.

Results

It was observed that among the HLA A locus, the frequencies of HLA A*03, A*26, A*32 and A*66 alleles increased, the increase of the first two alleles were highly significant (Table I). Similarly, A*11, A*24 and A*33 alleles decreased among patients compared to controls. Among the HLA B locus, the frequencies of alleles HLA B*08, B*15, B*55 and B*57 increased, increase in the allele frequency of B*15 was highly significant. Similarly, allele B*40 decreased. Among the HLA C locus, the frequencies of HLA Cw*01, Cw*08, CW*16 and Cw*18 alleles increased while Cw*06 decreased in HCV infected individuals compared to controls. Further among DRB1*16 and DQB1*03 alleles significantly increased while DRB1*01 and DQB1*05 decreased through not significantly. HLA II locus haplotype analysis revealed that HLA haplotype DRB1*11DQB1*03 was significantly associated among the patients (Table II). This haplotype was derived by direct counting.

Discussion

One of the striking features of HCV infection is the very high rate of development of chronicity. Approximately 15 per cent of infected patients successfully eliminate the virus, while others develop chronic infection with a wide spectrum of disease. Some will remain asymptomatic whereas others may have a more severe course leading to cirrhosis or hepatocellular carcinoma. There are evidences that immune mechanisms contribute to control the HCV infection. In the host immune reaction against viral infections HLA alleles play vital role in modulating the immune responses. Hence, this study was designed to examine the frequencies of HLA class I and class II genotype profiles in HCV infected western Indian individuals. The major findings of the present study were a significant increase among the allele frequencies of HLA A*03, A*32, HLA B*15, B*55, Cw*16, Cw*18, DRB1*03 and DQB1*03 in the western Indian patients population when compared with the controls from the same population and HLA II-locus haplotype DRB1*11-DQB1*03 was significantly increased among HCV infected individuals.

Yoon SK et al18 have reported that the frequencies of HLA-A3, HLA-B35 and HLA-B46 significantly increased in chronic HCV carriers compared with the controls in the Korean population. In an Egyptian population, Zekri et al (19) observed HLA class I and II alleles A28, A29, B14 and DR7 to be significantly encountered in HCV positive than negative cases. An association of HLA B 27 with spontaneous HCV clearance has also been reported (20).

Thio et al (11) have reported an association of Cw*0102 with HCV clearance in Caucasians and of A*2301 and Cw*04 with HCV persistence in both African-Americans and Caucasians. In Irish population McKiernan et al (21) have observed haplotypes A*03-B*07-DRB1*15-DQB1*06 and A*02-B27-Cw*01 DRB1*0101-DQB1*0501 to be associated with viral

clearance. We observed the allele A*32 among HCV antibody positive individuals from western India. An increased frequency of haplotype HLA A*11- Cw *04 in viraemic HCV patients was reported in a white population in Ireland (22). An association of B*15 allele with HCV infection was observed in our study. In a European population, Romero-Gomez et al (23) have reported the association of HLA--B*44 and sustained HCV response to ribavirin/interferon combination therapy. Among Irish population, McKierman et al (20) have reported increase frequency of B*08 and B*54 in those with chronic HCV infection when compared with those who cleared the infection. DRB1*08 has been reported to be associated with clearance of circulating HCV whereas DRB1*15 appears to predispose to progression of liver disease in Tunisian patients (24). In our study, it was interesting to note that among western Indians, the frequency of HLA DQB1*03 was higher in anti-HCV antibody positives than in controls, suggesting that the haplotype DRB1*11-DQB1*03 was generated because of the strong linkage disequilibrium of DRB1*11 with DQB1*03 and may or may not be related to the susceptibility to HCV infection. Earlier HLA DRB1*03-DQB1*03 haplotype association in western Indian systemic lupus erythromotosis (SLE) patients have been reported due to linkage disequilibrium (25). A trend with DRB1*11 alleles and less severe disease has already been reported (26).

Two studies from United Kingdom have reported higher frequency of DRB1*04, DQA1*03, DQB1*0301 to be associated with resistance to HCV infection (26,27). As found in our study, higher frequencies of DQB1*0301 and DRB1*1101 were also reported in patients with transient infection in a white population (26). The association of viral clearance with two alleles DRB1*01 and DQB1*03 was recently reported in a Brazilian population (28). DRB1*1101-DQB1*0301 haplotype seems to be associated with low hepatitis activity in a Chinese population (29). Harris et al (30) confirmed the previously reported associations between HCV clearance and two HLA types, i.e. DQB1*03 and DRB1*11. Strikingly, these associations were identified only among Caucasian, but not among African American patients. Our study showed the significant prevalence of haplotype HLA DRB*11-DQB1*03 in HCV antibody positive individuals.

An increased frequency of the haplotype DRB1*11-DQB1*03 in HCV antibody positive Indian population may have significance. Since, this observation is not due to the chance factor, involvement of a third factor could be ruled out. These results suggest that HLA association with HCV infection involves both class I and class II antigens. Taken together, our results suggest that HLA associations with the hepatitis C infection vary in relation to the ethnicity of the population studied. Nevertheless, differences in antigen frequency of selected HLA class I and class II alleles between normal subjects and in hepatitis C infected individuals from Maharashtra suggest that a susceptibility factor may contribute towards acquiring hepatitis C virus infection.

Acknowledgment

Authors acknowledge the financial support from Indian Council of Medical Research (ICMR), New Delhi.

Received November 21, 2007

References

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Reprint requests: Dr V.A. Arankalle, Scientist F and Head, Hepatitis Group, National Institute of Virology 130/1, Sus Road, Pashan, Pune 411 021, India e-mail: varankalle@yahoo.com

Anuradha S. Tripathy, U. Shankarkumar (*), Mandeep S. Chadha, Kanjashkya Ghosh (*) & Vidya A. Arankalle

National Institute of Virology (ICMR), Pune, & (*) National Institute of Immunohaematology (ICMR) Mumbai, India
Table I. HLA distribution in anti-HCV positive individuals and
controls from Maharashtra, India

HLA Anti-HCV Controls
 positives

 (n =43) (n =67)
 AF (%) AF (%) OR Ki2 EF PF

A*03 47.91 5.22 16.69 12.12 0.44
A*11 2.08 11.94 0.15 0.10
A*24 6.25 23.88 0.21 0.18
A*26 2.08 0.74 2.82 0.01
A*31 4.16 6.71 0.60
A*32 22.91 0.00 1474.00 0.21
A*33 2.08 8.95 0.21 0.06
A*66 6.25 1.49 4.40 0.04
A*68 6.25 4.47 1.42
B*07 10.41 9.70 1.08
B*08 6.25 2.23 2.91 0.03
B*15 43.75 5.22 14.11 10.42 0.39
B*27 4.16 2.98 1.41
B*40 8.33 23.88 0.28 0.17
B*44 8.33 17.16 0.43 0.09
B*51 8.33 4.47 1.93
B*55 8.33 0.74 12.09 1.81 0.07
B*57 2.08 0.00 134.00 0.01
Cw*01 6.25 1.49 4.40 0.04
Cw*02 4.16 3.73 1.12
Cw*03 4.16 10.44 0.37
Cw*04 16.66 16.41 1.01
Cw*06 6.25 19.40 0.27
Cw*07 12.50 22.38 0.49
Cw*08 2.08 0.00 134.00 0.01
Cw*12 8.33 5.22 1.64
Cw*14 2.08 2.98 0.69
Cw*15 14.58 12.68 1.17
Cw*16 14.58 2.23 7.45 0.12
Cw*18 6.25 0.00 402.00 0.05
DRB1*01 2.27 7.96 0.26
DRB1*03 11.36 3.09 4.01 0.08
DRB1*07 13.63 13.27 1.03
DRB1*08 2.27 2.65 0.85
DRB1*11 20.45 11.94 1.70
DRB1*12 4.54 0.88 5.30 0.03
DRB1*13 2.27 1.32 1.72
DRB1*15 18.18 10.61 1.87
DRB1*16 9.09 3.09 3.12 0.06
DQB1*02 15.90 15.04 1.06
DQB1*03 34.09 14.60 3.02 0.22
DQB1*05 6.81 8.84 0.75 0.02
DQB1*06 38.63 26.10 1.78

HLA
 P value

A*03 7.9E-12 **
A*11 0.040
A*24 0.007
A*26
A*31
A*32 1.8E-8 **
A*33
A*66
A*68
B*07
B*08
B*15 2.18E-10 **
B*27
B*40 0.020
B*44
B*51
B*55 0.005
B*57 0.090
Cw*01 0.080
Cw*02
Cw*03
Cw*04
Cw*06 0.030
Cw*07
Cw*08 0.090
Cw*12
Cw*14
Cw*15
Cw*16 0.001
Cw*18 0.003
DRB1*01
DRB1*03 0.010
DRB1*07
DRB1*08
DRB1*11
DRB1*12 0.060
DRB1*13
DRB1*15
DRB1*16 0.060
DQB1*02
DQB1*03 0.001
DQB1*05
DQB1*06

EF, aetiological fraction or attributable risk; PF, preventive
fraction; Ki2, Chi-square with Yates correction; OR, odds ratio;

** Very highly significant P value, obtained by multiplying with
the no. of alleles; AF, allele frequency

Table II. Significant haplotype associated with HCV infected
western Indians and corresponding controls

Haplotype HF HF OR K12 P EF

 Controls Patients
 n= 113 n=39

DRB1*11- 3.98 17.64 5.16 16.14 0.0001 * 0.13
DQB1*03

* Very highly significant P value; HF, Haplotype frequency in
percentage; OR, odds ratio; K12, Chi-square with gates
correction;

EF--aetiological fraction
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Title Annotation:human leukocyte antigen
Author:Tripathy, Anuradha S.; Shankarkumar, U.; Chadha, Mandeep S.; Ghosh, Kanjashkya; Arankalle, Vidya A.
Publication:Indian Journal of Medical Research
Article Type:Report
Geographic Code:9INDI
Date:Nov 1, 2009
Words:3578
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