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An analysis of association of human leukocyte antigen and liver disease patients in a tertiary care hospital, India
Simmi Mehra1, Aseem Kumar Tiwari1, Chhavi Rajvanshi1, Rajni Chauhan1, Randhir Sud2, Arvinder Singh Soin3
1 Department Of Transfusion Medicine, Molecular and Transplant Immunology Laboratory, Medanta - The Medicity, Gurgaon, Haryana, India
2 Department of Digestive and Hepatobiliary Sciences, Medanta - The Medicity, Gurgaon, Haryana, India
3 Department of Liver Transplant and Regenerative Medicine, Medanta - The Medicity, Gurgaon, Haryana, India
Department Of Transfusion Medicine, Molecular and Transplant Immunology Laboratory, Medanta - The Medicity, Gurgaon, Haryana
Source of Support: None, Conflict of Interest: None
Background and Objectives: In addition, the role of human leukocyte antigen (HLA) in transplantation, importance of HLA in disease susceptibility, and protection has been widely studied. The aim of the present study was to find out the HLA allelic frequency (HLA-A,-B,-DRB1) and its association with liver diseases. Methods: The present study was done for the duration of 4 years from 2013 to 2017 in a tertiary care center which primarily caters to north Indian patient population. A total of 429 patients were studied. The HLA genotyping was performed by molecular method using commercial available low resolution ABDR sequence-specific oligonucleotide probe-based method (Luminex platform). Results: There was an increased association of HLA-A*01 (n = 9, 18.6%), HLA-B*15, B*35, B*40, B*44 (n = 5, 11.6%), and HLA-DRB1*15 (n = 9, 20.9%) in 43 patients with hepatitis B virus (HBV)-related disease. There was an increased association of HLA-A*24 (n = 9, 20.7%), HLA-B*35, B*40 (n = 14, 15.2%), and HLA-DRB1*15 (n = 17, 18.5%) in 92 patients with alcohol-related disease. An increased association of HLA-A*11 (n = 30, 19.6%), HLA-B*35 (n = 24, 16.8%) and HLA-DRB1*15 (n = 28, 19.6%) in 143 patients with hepatitis C virus (HCV) related disease. Conclusion: This is possibly the first study in the north Indian patients having liver diseases related to HBV, HCV, and alcohol related with respect to A, B, and DR loci previous studies from India were conducted to see disease association of HLA class I and class II with respect to HCV only.
Keywords: Hepatitis B virus, hepatitis C virus, human leukocyte antigen
|How to cite this URL:|
Mehra S, Tiwari AK, Rajvanshi C, Chauhan R, Sud R, Soin AS. An analysis of association of human leukocyte antigen and liver disease patients in a tertiary care hospital, India. Glob J Transfus Med [Epub ahead of print] [cited 2021 Oct 16]. Available from: https://www.gjtmonline.com/preprintarticle.asp?id=317230
| Introduction|| |
In addition to the role of human leukocyte antigen (HLA) in transplantation, HLA and disease associations have been widely studied worldwide. HLA products are involved in immune regulation and cellular differentiation. HLA Class I and class II molecules expressed on cell surface presents the foreign or self-peptides (in case of autoimmune disease) to CD8+T cells and CD4+T cells, respectively.
One of the reasons to study the genetic association is for the disease susceptibility, risk assessment, immunological profiling, therapeutic decisions, and prognostication. Pathogenesis of diseases showing HLA association are related to the immune system, mostly autoimmune diseases.,,
There is paucity of data on HLA allelic frequency in liver diseases in the north Indian population. Therefore, we have compiled and analyzed this data in our patient population in the last 4 years.
Hepatitis B virus (HBV) infection remains one of the most common infectious diseases. According to the WHO fact sheets ~2 billion people who have been infected with HBV accounts for 1 million deaths each year. The variations in the natural history of HBV infection are mainly determined by (1) virological factors due to viral mutations, (2) immunological factors, (3) host genetic factors, and (4) environmental factor.
Hepatitis C virus (HCV) infection leads to a very high rate of the development of chronicity. There is evidence that immune mechanisms control the HCV infection.
Objectives of the study
The aim of the present study was to find out the HLA allelic frequency (HLA-A,-B,-DRB1), in North Indian patients (population) and its association with liver diseases. Further, the present study is aimed to compare our data with Western and other Indian literature.
| Materials And Methods|| |
The present study was done for the duration of 4 years from 2013 to 2017 in a 1250 bedded tertiary care center in India which primarily caters to the north Indian patient population. It was a retrospective and observational study. Patient registration includes residential address and patients from northern states (Rajasthan, Madhya Pradesh, Chhattisgarh, Jharkhand, Bihar, Uttar Pradesh, Uttaranchal, Himachal Pradesh, Punjab, and Haryana) northern union territories (Ladakh, Chandigarh, and Delhi) were included.
The required sample size for the study was estimated to be 203 for clinical significance by the following method:
n = n0/(1 + n0/N)
n0: Sample size pre-correction (rounded up)
pˆ: Sample proportion
z: Found by using a z-score table
MOE: Margin of error (to be divided to get a decimal)
n: Sample size
N: Population size
A total of 429 patients were studied, of which 321 (75%) were males with a mean age of 49.7 (18–68) and 108 (25%) females with a mean age of 44.55 (18–67).
The study population included all the liver disease patients including living donor liver transplant (LDLT). Patients who registered and provided consent for the study at the Department of Transfusion Medicine from 2013 to 20 were included in the study. The liver donor (n = 506) of all the LDLT patients was taken as control population. HLA typing of all the donors was done as well.
Inclusion and exclusion criteria
All liver disease patients suffering from HCV (n = 143, 33%), HBV (n = 43, 10%) or alcoholic liver diseases (n = 92, 21%) and their donors were included in the study. The patients having liver diseases related to HCC, Biliary Atresia, Chronic Cholecystitis, Biliary Cirrhosis, Nonalcoholic Fatty Liver Disease (NAFLD), Fulminant Hepatitis, Primary Sclerosing Cholangitis, Cirrhosis, Alcoholic and HCV, Autoimmune liver disease, Alcoholic and HBV, Fulminant Hepatitis and Chronic Cholecystitis, Nonalcoholic Steato-hepatitis (NASH), Drug Induced, HCV along with HBV were excluded from the study as the sample numbers were very low.
Human leukocyte antigen typing procedure
HLA class I and class II genotyping was performed using low resolution Sequence Specific Oligonucleotide Probe (SSOP) based method on Luminex platform. Genomic DNA was extracted from peripheral blood using spin-column based technology from Nucleospin blood kit (MACHEREY-NAGEL GmbH and Co. KG, Düren, Germany). The extracted DNA was quantified by the NanoDrop 2000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA) and the ratio of the absorbance at 260 and 280 nm (A260/280) between 1.8 and 2.0 was used to assess the purity of nucleic acids. SSOP method involves polymerase chain reaction (PCR) amplification, hybridization, streptavidin phycoerythrin (SA PE) reaction, and the measurement of the analytes. PCR primer (LIFECODES HLA-A SSO Typing Kit, LIFECODES HLA-B SSO Typing Kit, LIFECODES HLA-DRB1 SSO Typing Kit, Immucor, Inc., US) used were biotinylated at their 5 -ends. The amplicon sizes were 400–500 bp (exon 2) and 300–400 bp (exon 3) for the HLA-A, -B, and-C loci and 250–300 bp (exon 2) for the HLA-DRB1 locus. The 5 ′-biotin-labeled PCR amplicons were hybridized to the oligobeads in a 96-well plate by adding appropriate oligobead mixture using Applied Biosystems Veriti Thermal Cycler. After careful mixing with a pipette, the hybridized amplicons were labeled with SA–PE using Applied Biosystems Veriti Thermal Cycler. Then, the analyte were measured by the Luminex's xMAP® Technology (Immucor, Inc. USA). The median fluorescence intensity of PE was used to quantify the amount of DNA bound to the oligobeads. The measured data were read using dedicated software (MATCHT IT! DNA software) obtained from the Immucor, Inc., USA.
Type of blood samples
Two blood samples, 3 ml in each ethylenediaminetetraacetic acid tubes were taken from both patient and donor. Tubes were labeled with the unique donation identification number/bar-code for HLA typing.
The data of the patients were tabulated. The allele frequency was estimated by direct counting and calculated using the formula: N/2N; where n is the number of times that a particular allele was reported and N is the number of samples that were studied. The number of alleles for homozygous samples was counted as two. The relative risk (RR) was calculated using statistical software available online (MEDCALC). P < 0.05 was considered statistically significant. SPSS software version 24 (Statistical Package for the Social Sciences; IBM Bengaluru, Karnataka, India) was used for the analysis.
Patient consent was obtained for the diagnosis and treatment to the hospital. This was an observational study and no additional sample was drawn for the study. All investigations, treatment and monitoring were according to the current “standard of care.”
| Results|| |
As shown in [Table 1], the highest number of liver diseases were related to HCV (n = 143, 33%) followed by alcoholic (n = 92, 21%), HBV (n = 43, 10%), HCC (n = 33, 8%), Biliary Atresia (n = 23, 5%), Chronic Cholecystitis (n = 13, 3%), Biliary Cirrhosis (n = 12, 3%), NAFLD (n = 12, 3%), Fulminant Hepatitis (n = 11, 3%), Primary Sclerosing Cholangitis (n = 9, 2%), Cirrhosis (n = 8, 2%), Alcoholic and Hepatitis C (n = 7, 2%), Autoimmune liver disease (n = 7, 2%), Alcoholic and Hepatitis B (n = 4, 1%), Fulminant Hepatitis and Chronic Cholecystitis (n = 4, 1%), NASH (n = 4, 1%), Drug Induced (n = 3, 1%), Hepatitis C and B (n = 1, 0). In this study, we have only included HBV, HCV and alcoholic liver disease patients and the rest were excluded because of very low sample size.
|Table 1: Baseline characteristics of the patient|
undergoing liver transplant
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Human leukocyte antigen-B prevalence in liver disease patients
There was an increased association of HLA-A*01 (n = 9, 18.6%), HLA-A*02 (n = 8, 18.0%), HLA-A*11, A*24 (n = 7, 16.3%) and A*33 (n = 4, 93.3%) in 43 patients with HBV related disease [Table 2]. There was an increased association of HLA-A*24 (n = 9, 20.7%), HLA-A*02 (n = 18, 18.5%), HLA-A*01 (n = 16, 16.3%) and HLA-A*11 (n = 7, 12.0%) in 92 patients with alcohol related disease. There was an increased association of in HLA-A*11 (n = 30, 19.6%), HLA-A*24 (n = 25, 15.4%), HLA-A*02 (n = 22, 14.0%), and HLA-A*01 (n = 16, 9.8%) in 143 patients with HCV related disease.
|Table 2: Human leukocyte antigen‑A allele frequency distribution in hepatitis B virus, hepatitis C virus, and alcoholic liver disease patients|
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Human leukocyte antigen-B prevalence in liver diseases
There was an increased association of HLA-B*15, B*35, B*40, B*44 (n = 5, 11.6%), HLA-B*07, B*51, B*57 (n = 4, 9.3%) and HLA-B*48 (n = 3, 7.0%) in 43 patients with HBV related disease [Table 3]. There was an increased association of HLA-B*35, B*40 (n = 14, 15.2%) followed by HLA-B*15 (n = 13, 14.1%), HLA-B*07 (n = 7, 7.6%) and HLA-B*51, B*52 (n = 6, 6.5%) in 92 patients with alcohol related disease. There was an increased association of in HLA-B*35 (n = 24, 16.8%) followed by HLA-B*40 (n = 20, 14.0%), HLA-B*44 (n = 15, 10.5%) and HLA-B*15, B*51 (n = 11, 7.7%) in 143 patients with HCV-related disease.
|Table 3: Human leukocyte antigen‑B allele frequency distribution in hepatitis B virus, hepatitis C virus, and alcoholic liver disease patients|
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Human leukocyte antigen-DRB1 prevalence in liver diseases
There was an increased association of HLA-DRB1*15 (n = 9, 20.9%), HLA-DRB1*03 (n = 8, 18.9%) and HLA-DRB1*07 (n = 7, 16.3%) in 43 patients with HBV-related disease [Table 4]. There was an increased association of HLA-DRB1*15 (n = 17, 18.5%), HLA-DRB1*07 (n = 16, 17.4%), HLA-DRB1*11 (n = 14, 15.2%), and HLA-DRB1*04 (n = 10, 10.9%) in 92 patients with alcohol-related disease. There was an increased association in HLA-DRB1*15 (n = 28, 19.6%) followed by HLA-DRB1*07 (n = 24, 16.8%), HLA-DRB1*03 (n = 17, 11.9%), and HLA-DRB1*13 (n = 16, 11.2%) in 143 patients with HCV-related disease.
|Table 4: Human leukocyte antigen‑DRB1 allele frequency distribution in hepatitis B virus, hepatitis C virus, and alcoholic liver disease patients|
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| Discussion|| |
Human leukocyte antigen allele association with hepatitis B virus-related liver disease
HBV along with HCV accounts for 75% of liver diseases and is regarded as the major threat to public health globally. Rizzetto et al. mentioned that over two billion people are HBV infected during their lifetime and 350 million are estimated to be chronic carriers who can develop liver cirrhosis or HCC. The chronic HBV can cause mutagenesis and carcinogenesis, which can be due to HLA. Karan et al. showed that among class I and class II HLA alleles HLA B8, B13, DR7, DR13, and DQ3 appeared significantly more frequently in the patients with chronic HBV infection. We also found that DRB1*03 (P = 0.085, RR = 1.8), DRB1*07 (P = 0.880, RR = 1.1) frequencies were higher in HBV patients but were not significant. Furthermore, Almarri and Batchelor found HLA-DR7 was more frequent and there was a significant decrease in the frequency of HLA-DR2 in cases of, persistent HBV infection. Our study results are also concordant with the Chen et al. results who showed that the allele frequencies of HLA-DR3, A30, DQA1*0501 in patients with hepatitis B were markedly increased, suggesting that these alleles are associated with HBV. Thio et al. represented that four class I alleles were associated with viral persistence: B*08, B*44, Cw*0501, and Cw*1601 and among class II alleles, DQB1*0201 and DRB1*0301, were associated with viral persistence. In the present study, although the frequency of HLA-B*44 (P = 0.612, RR = 1.3) was highest among all but does not reach the significant level. Jiang et al. described that among Chinese patients HLA-DRB1*0301, HLA-DQA1*0501 and HLA-DQB1*0301 are closely related with susceptibility to HBV. Several studies carried out to find out the association of different HLA alleles to persistence and clearance of HBV. Amarapurpar et al. reported the negative association of DRB1*13XX to persistence of HBV and possible role of DRB1*11XX and DRB1*15XX in development of HBV hepatitis due to persistence of HBV among the Indian population. We also reported that that HLA-DRB1*15 (n = 9, 20.9%) was the highest among all alleles in HBV patients with P = 0.77 and RR = 1.1. In northern Chinese patients, Shen et al. suggested that the susceptibility to hepatitis B was strongly associated with HLA-DRB1*10 allele. However, in the present study, we have not found any significant increase of DRB1*13 which may suggest that it helps in the clearance of the infection. Our study results were also not correlating with Shen et al. study, in which DRB1*10 were significantly increased in HBV.
Human leukocyte antigen allele associations with hepatitis C virus-related liver disease
HCV infection leads to the development of hepatitis in about 70%–80% of cases and from these cases 20%–30% causes liver cirrhosis or HCC. Recent data show that HCV infection may be influenced by host genetic factors, especially HLA.
Tripathy et al. revealed HLA-A*03, A*32, HLA-B*15, B*55 and HLA-DRB1*03 association with HCV. The same also described that A*11, A*24, A*33, B*40, DRB1*01 and DQB1*05 alleles were decreasing among patients as compared to controls. Höhler et al. also found an association of HLA-DRB1*0301 with HCV. In our study HLA-B*15 (n = 11, 7.7%) and DRB1*03 (n = 17, 11.9%) were prevalent in HCV infected patients but again were not significant. Chowdhry et al. reported A*33, DRB1*03 and DRB1*10 frequencies were significantly high and A*02, A*31 and DRB1*15 were significantly low in HCV infected patients. However, A*03, B*15, B*55 and DRB1*11 frequencies were almost same as of normal controls and A*24, A*33 and B*40 were significantly high which are not concordant with Tripathy AS et al. studies. Corghi et al. found that there were higher frequencies of HLA-DRB1*13 (26.5% vs. 14.1%) and HLA-DQB1*02 (52.9% vs. 38.7%) in patients compared with controls but was not significant. We also found the same profile of DRB1*13 allele. Reported the association of HLA-DRB1*0405 and HLA-DQB1*0401 with liver disease due to HCV. However in our study, we have not found any significant prevalence of DRB1*04 in HCV. In a European population, Romero-Gómez et al. have reported the association of HLA-B*44 with HCV-infected patients and present study also released third-highest frequency of B*44. McKiernan et al. have reported increase frequency of B*08 and B*54 in those with HCV infection among Irish population. Moreover, we did not found the B*08 or B*54 prevalence in HCV infection.
Human leukocyte antigen allele associations with alcohol related liver disease
Alcoholism may have a precise genetic determination. Córsico et al. postulated that alcoholic group of patients had much higher frequency of HLA-B40 and HLA-DR4. In the Western part of Ireland HLA-A3, HLA-B5, and HLA-B40 was found in alcoholic cirrhosis, but the results were not statistically significant. In the present study, HLA-A*24 (20.7%), HLA-B*40 (15.2%) were found to have the maximum frequency in Alcohol-related liver disease patients.
An increased prevalence of HLA-B8 and an absence of HLA-A28 were found in alcoholic liver disease patients in Bell and Nordhagen study. However, H Bell has given an idea that HLA-BW40 may support that individual susceptibility to the development of alcoholic cirrhosis is genetically determined. Despite B*40, we also found B*15, A*02 and DRB1*15 increasing in these type of patients.
Limitations of the study
Many studies included in discussion were performed in general population.,,, The present study was conducted in hospital set-up which may not exactly represent the general population.
| Conclusion|| |
The 43 patients with HBV-related disease showed increased prevalence of HLA-A*01 (n = 9, 18.6%), HLA-B*15, B*35, B*40, B*44 (n = 5, 11.6%) and HLA-DRB1*15 (n = 9, 20.9%). There was an increased association of HLA-A*24 (n = 9, 20.7%), HLA-B*35, B*40 (n = 14, 15.2%) and HLA-DRB1*15 (n = 17, 18.5%) in 92 patients with alcohol-related disease. An increased association of HLA-A*11 (n = 30, 19.6%), HLA-B*35 (n = 24, 16.8%), and HLA-DRB1*15 (n = 28, 19.6%) in 143 patients with HCV-related disease. This is possibly the first study in the north Indian patients having liver diseases related to HBV, HCV, and alcohol-related with respect to A, B, and DR loci previous studies from India were conducted to see disease association of HLA class I and class II with respect to HCV only.
Research quality and ethics statement
The authors attest that this clinical investigation was approved by the Institutional Review Board (IRB approval letter number is 1046/2020 [Academic]). We also certify that we have not plagiarized the contents in this submission and have done a Plagiarism Check.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2], [Table 3], [Table 4]