|Year : 2022 | Volume
| Issue : 2 | Page : 220-224
Human leukocyte antigen-A, B, and DRB1 diversity in renal transplant patients and donors: A single-center retrospective observational study
Vikash Chandra Mishra, Dinesh Chandra, Trupti Deshpande, Parvind Singh, Archana Anthwal, Vimarsh Raina
Department of Molecular Genetics, Chimera Transplant Research Foundation, New Delhi, India
|Date of Submission||17-Aug-2020|
|Date of Acceptance||25-Nov-2020|
|Date of Web Publication||30-Jun-2022|
Dr. Vikash Chandra Mishra
Department of Molecular Genetics, Chimera Transplant Research Foundation, South Extension Part-II, New Delhi
Source of Support: None, Conflict of Interest: None
Background: The understanding of transplant acceptance or rejection derives directly from knowing human leukocyte antigen (HLA) involved in the immune response. The HLA of the major histocompatibility complex contains a numerous family of genes located on the short arm of the chromosome number 6 and divided majorly into two classes, Class I (HLA-A, B, and C) and Class II (HLA-DR, DQ, and DP). The detection and detailed information of these histocompatibility genes are essential for the better outcome of organ transplants. Aims and Objectives: The aim of the present study is to analyze the frequencies of HLA-A, B, and DRB1 in chronic kidney disease (CKD) patients and their prospective donors as well as to see the prevalence of heterogenicity. Materials and Methods: A total of 264 patients diagnosed with CKD and 333 potential donors were studied retrospectively in the present study. All these cases were analyzed for HLA-A, B, and DRB1 loci typing by PCR-SSOP method based on the Luminex platform. Results: We identified 15 different alleles of HLA-A, 29 of HLA-B, and 13 of HLA-DRB1 amongst studied samples. Out of these identified HLA alleles, HLA-A*02, HLA-B*35, and HLA-DRB1*15 were more frequent as compared to others. The results showed significant heterogenicity in the identified HLA-A, B, and DRB1alleles. Conclusion: The result highlights the diversity of HLA-A, B, and DRB1 alleles and is valuable as a reference for organ transplantations. Further, these findings can also guide in better donor selection and hence improved transplant outcome. In addition, this information could be a starting point for the development of an indigenous transplant assay kit.
Keywords: Human leukocyte antigen, human leukocyte antigen-A, human leukocyte antigen-B, human leukocyte antigen-DRB1, human leukocyte antigen diversity
|How to cite this article:|
Mishra VC, Chandra D, Deshpande T, Singh P, Anthwal A, Raina V. Human leukocyte antigen-A, B, and DRB1 diversity in renal transplant patients and donors: A single-center retrospective observational study. Indian J Transplant 2022;16:220-4
|How to cite this URL:|
Mishra VC, Chandra D, Deshpande T, Singh P, Anthwal A, Raina V. Human leukocyte antigen-A, B, and DRB1 diversity in renal transplant patients and donors: A single-center retrospective observational study. Indian J Transplant [serial online] 2022 [cited 2022 Sep 25];16:220-4. Available from: https://www.ijtonline.in/text.asp?2022/16/2/220/349365
| Introduction|| |
Chronic kidney disease (CKD) is a rapidly increasing health issue, both in developed and developing countries. The most common and standard care of treatment for these patients is the renal transplant. The posttransplant outcome of renal patients depends on the compatibility of human leukocyte antigen (HLA) between the recipient and prospective donor. The HLA of the MHC contains a numerous family of genes located on the short arm of the chromosome number 6 (6p21.3) and divided majorly into two classes, Class I (HLA-A, B, and C) and Class II (HLA-DR, DQ, DP). Knowledge of these HLA genes is crucial for the outcome of organ transplants. The outcome of renal transplantation involves the information of the HLA as part of the acceptance or rejection of the allograft. In the present study, we carried out the molecular analysis of HLA-A, HLA-B, and HLA-DRB1 alleles by polymerase chain reaction (PCR) sequence-specific oligonucleotide probe (SSOP) among renal transplant patients and their prospective donors to estimate the prevalence of heterogenicity.
| Materials and Methods|| |
This is a single-center retrospective analysis of data performed on renal transplant patients (264) diagnosed with CKD and their potential live donors (333) whose samples were analyzed in our laboratory for HLA-A, B, and DRB1 loci typing between January 2015 and June 2018. The laboratory is accredited for ISO15189: 2012, and the required quality control and quality assurance, both internal and external are routinely undertaken. Only live donors and patients of CKD from Indian origin were included in this study. All these samples (597) were analyzed by PCR-SSOP method based on the Luminex platform. An analysis was performed to establish the frequency and diversity of each HLA-A, B, and DRB1 allele identified among these enrolled samples.
From each enrolled participant (patients and their donors), 3 ml of whole blood was collected in ethylenediaminetetraacetic acid-coated vials as per our center sample collection protocol.
DNA from each participant blood samples was extracted by commercial NucleoSpin® Blood DNA Extraction Kit from Macherey-Nagel (Germany). This is a solid-phase extraction method in which sample lysis was done using proteinase K and lysis buffer as per the recommended protocol, followed by adjusting conditions for DNA binding by column method. Finally, a silica membrane wash and dry technique was performed to purify the DNA followed by elution of highly pure DNA. DNA concentration was determined by measuring the intensity of absorbance with a spectrophotometer (Qubit®2.0, Invitrogen).
Human leukocyte antigen typing
HLA typing from all the extracted DNA samples was performed by PCR SSOP method following the recommended protocol provided in kit insert (LIFECODES®HLA-SSOTYPING, USA), which majorly includes DNA amplification, hybridization, and analysis. The method is based on the hybridization of labeled single-stranded PCR products to SSO probes and each of these probes preferentially hybridizes to a complementary region in the amplified DNA. Further, the amplified DNA is also hybridized to one or more consensus probes homologous to sequences present in all the alleles of a locus.
The data obtained were exported as a comma-separated values named as “OUTPUT. CSV” file once the run was complete. This generated OUTPUT. CSV was saved in a folder with the batch name and then opened with the MatchIT DNA software to analyze the report.
Statistical analysis of human leukocyte antigen-A, B and DRB1 Allele
HLA-A, B, and DRB1 allele frequencies were estimated by direct counting (n/2N × 100) method in Microsoft Excel 2010, where 'n' is the number of particular allele and 'N' is the total number of samples studied detailed statistical analysis.
Declaration of participant consent
The consent has been taken from each participant before collecting the sample and included in this study. All participants were aware that the names, initials, and photographs would not be published and all standard protocols will be followed to hide their identity.
It was a retrospective analysis and hence, EC clearance was not needed. The study was carried out as per the declaration of Helsinki. The study was performed according to the guidelines in Declaration of Helsinki.
| Results|| |
In the present study after detailed analysis, we have identified 15 different alleles of HLA-A, 29 of HLA-B, and 13 of DRB1 among the studied samples. The summary of all these identified HLA-A, B, and DRB1 alleles is represented in [Table 1].
|Table 1: Representing human leukocyte antigen-A, B, DRB1 alleles frequency of the studied samples (n=597)|
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Human leukocyte antigen-A allele frequency
Among the identified HLA alleles, HLA-A*02 (17.34%) was found to be more frequent. Subsequent to HLA-A*02, the most frequent among HLA-A alleles were A*24 (15.24%), A*11 (14.82%), A*01 (10.97%), and A*03 (9.46%). [Figure 1] shows the frequency of HLA-A allele frequency (%). Among the identified HLA-A alleles, 91.96% were heterozygous and homozygosity was seen only in 8.04%.
|Figure 1: Graphical representation of human leukocyte antigen-A alleles frequencies (%)|
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Human leukocyte antigen-B allele frequency
HLA-B*35 (12.23%) was found to be more frequent compared to others. Subsequently, most frequent alleles found were HLA-B*15 (9.80%), B*40 (9.63%), B*51 (8.46%), and B*44 (7.79%). The frequency (%) of the HLA-B allele is represented in [Figure 2]. Of the identified HLA-B allele, 89.11% were heterozygous and homozygosity was seen only in 10.89%.
|Figure 2: Graphical representation of human leukocyte antigen-B alleles frequencies (%)|
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Human leukocyte antigen-DRB1 allele frequency
The most frequent HLA-DRB1 allele was DRB1*15 (18.76%). Subsequent to HLA-DRB1*15, the most frequent among HLA-DRB1 allele were HLA-DRB1*07 (15.08%), DRB1*13 (10.39%), DRB1*03 (10.13%), and DRB1*11 (9.13%) [Figure 3] shows the frequency of HLA-DRB1 allele frequency (%). Among the identified HLA-DRB1 allele, 86.77% were heterozygous and homozygosity was seen only in 13.23%.
|Figure 3: Graphical representation of human leukocyte antigen-DRB1 alleles frequencies (%)|
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| Discussion|| |
Kidney transplantation is most successful for CKD patients. However, the outcome of this depends on HLA compatibility between recipient and donor. Most of the previously published reports on HLA frequency estimation from India have been done mainly to understand migration and population genetics. As of July 2020, the total number of 19,587 HLA Class I alleles and 7302 HLA Class II alleles have been recognized. Most of the HLA Class I alleles can be discriminated by their exon 2 and 3 sequences, and exon 2 for Class II alleles. Here, an attempt was made to analyze the diversity of HLA-A, B, and DRB1 in CKD patients along with their prospective live donors. The result highlights the diversity of HLA-A, B, and DRB1 pattern and is valuable as a reference for organ transplantations in the era of the virtual crossmatch.
Among the studied samples, the most frequent HLA alleles were HLA-A*02, HLA-B*35, and HLA-DRB1*15. It was also observed that the HLA-B allele was most polymorphic as compared to HLA-A (15 alleles) and DRB1 (13 alleles) with 29 alleles.
A similar study conducted by Chowdary et al. identified 17, 38, and 17 alleles for HLA-A, B, and DRB1 respectively. HLA-A*24, A*02, A*01, B*35, B*61, B*44, and DRB1*15 were in the list of most frequent HLA alleles in tertiary care from Telangana and Andhra Pradesh. Another study by Adiga et al. identified 32 HLA-A and 40 HLA-B alleles. HLA-A*24 (16%), A*33 (13.6%), A*02 (13.5%), A*11 (11.4%), A*01 (9.6%), B*40 (15%), B*07 (13.4%), B*35 (12.7%), B*51 (8.8%), and B*44 (7.6%) were in the list of most frequent allele. This showed some similarity with our results like HLA-A*02, A*11, HLA-B*35, DRB1*07 were found to be most frequent in our study. Kankonkar et al., identified HLA-A*02 (19.25%), A*11 (13.17%), A*24 (15.70%), A*33 (11.82%), B*35 (15.37%), B*40 (12.66%), DRB1*15 (19.25%) and DRB1*07 (12.83%) as the most common alleles in the population of Mumbai. HLA-A*02 (20%–28%), HLA-B*40 (5%–20%) and HLA-DRB1*15 (10%–18%) are the most frequent and common antigens seen in South and South-East Asian countries by Shankarkumar et al. A study published by Arisa-murillo et al. identified 19 alleles for HLA-A, 28 for HLA-B, and 15 HLA DRB1 in Colombian populations. Among HLA alleles identified HLA-A*24 (26.2%), A*02 (26%), B*35 (22.7%) and B*44 (11.1%), DRB1*04 (24%), DRB1*15 (11.3%) and DRB1*07 (11.1%) were more prevalent. In another study done by Tuladhar et al., to analyze the frequency of these antigens in the Nepali population revealed12 antigens for HLA-A, 15 for HLA-B, and 13 HLA-DRB1.Amongst them, most frequent were A*24 (17%), A*11 (34.5), B*33 (19%), B*35 (19%), DRB1*15 (33.5%) and DRB1*04 (7.32%).
More precise techniques of PCR-SSOP and sample size could be attributed to the presence or absence of these HLA alleles from one region to other. These observed differences among frequency of HLA antigen from one region to other showed the diversity of the HLA system which plays a major role in graft acceptance and rejection. 90% of kidneys for transplantation are obtained from living donors while only 10% come from deceased donors in India. So, there is a need of large population data of HLA polymorphism which may be helpful in the creation of organ sharing networks or registries of donors for Indian patients and it would results in more numbers of renal transplant especially the cadaveric transplant from deceased donors.
| Conclusion|| |
Our results showed the occurrence of a large variety of HLA-A, B, and DRB1 alleles in the studied samples. Considering the diverse Indian population and importance of HLA compatibility in renal transplant, the present study highlights on typification and importance of profiling of HLA alleles on a larger sample size which may help in creating the organ-sharing network and registries for the Indian population like the “United Network for Organ Sharing (UNOS).”
In addition, at present, all accessible assays for identification of Anti-HLA antibody in cases of renal transplant patients are based on HLA antigen frequency of Caucasian population and do not represent antigen predominantly found in Indian population. Hence, this information could be a starting point for the development of such an indigenous kit.
Finally, it is easy to find the best compatible HLA-matched organ transplant recipient and donors by knowing the frequency of HLA in a specific population and hence improve transplant outcomes to decrease the risk of antibody-mediated rejection.
The study was limited by small sample size and opens the need for further detailed analysis with bigger sample size.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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