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Saudi Journal of Kidney Diseases and Transplantation
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ORIGINAL ARTICLE  
Year : 2012  |  Volume : 23  |  Issue : 3  |  Page : 467-470
HLA-C polymorphisms in two cohorts of donors for bone marrow transplantation


1 King Abdullah International Medical Research Centre, King Abdulaziz Medical City, Riyadh, Kingdom of Saudi Arabia
2 Department of Pathology and Laboratory Medicine, King Abdulaziz Medical City, Riyadh, Kingdom of Saudi Arabia
3 Istanbul University, Istanbul Medical Faculty, Department of Physiology, Istanbul, Turkey
4 Department of Pathology and Laboratory Medicine, King Abdulaziz Medical City; College of Medicine, King Saud bin Abdulaziz University for Health Sciences, National Guard Health Affairs, Riyadh, Kingdom of Saudi Arabia

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Date of Web Publication7-May-2012
 

   Abstract 

The typing for HLA-C in transplantation was rather neglected in the past. However, several recent studies have emphasized its role in transplantation and its association with the outcome. Serological typing of HLA-C could identify only a limited number of HLA-C antigens, resulting in a number of HLA-C blanks. This was mainly due to the low expression of surface HLA-C and the small number of available specific anti-sera. Performing molecular methods has identified new HLA-C alleles and filled the blank of most serological typed antigens. In this study, we compared serological and molecular typing of HLA-C in two cohorts of healthy Saudis. Our serological typing method identified HLA-C1-7 with different frequencies, 23.5% of the alleles were not identified and thus defined as blank. Using the SSP molecular method, all samples were typed and all alleles were defined. Both methods showed that C*07 and C*06 have the highest frequency in the Saudi population. Our study emphasizes the importance of molecular methods in identifying all possible HLA-C alleles.

How to cite this article:
Jawdat D, Al Saleh S, Sutton P, Al Anazi H, Shubaili A, Uyar F A, Hajeer AH. HLA-C polymorphisms in two cohorts of donors for bone marrow transplantation. Saudi J Kidney Dis Transpl 2012;23:467-70

How to cite this URL:
Jawdat D, Al Saleh S, Sutton P, Al Anazi H, Shubaili A, Uyar F A, Hajeer AH. HLA-C polymorphisms in two cohorts of donors for bone marrow transplantation. Saudi J Kidney Dis Transpl [serial online] 2012 [cited 2020 Jul 2];23:467-70. Available from: http://www.sjkdt.org/text.asp?2012/23/3/467/95705

   Introduction Top


The outcome of unrelated donor transplantation is highly influenced by the HLA matching between the donor and the recipient. HLA-C is a class I gene locus, yet its importance in transplantation has been less validated compared with HLA-A and HLA-B. However, a number of studies have linked HLA-C to graft rejection and emphasized on its importance in transplantation. For example, HLA-C matching has been shown to be important in bone marrow transplantation (BMT) and in the prevention of graft-versus-host disease. [1] In addition, HLA-C matching has been shown to play a role in renal and liver transplantation outcome. [2],[3] HLA-C has also been associated with a number of diseases such as multiple sclerosis, [4] Crohn's disease, [5] psoriasis [6] and familial leukemia. [7]

Approximately ten different HLA-C antigens were identified using serologic methods, while a high frequency of HLA-C phenotypes is serologically undefined resulting in a large percentage of HLA-C blank phenotypes. This is mainly due to HLA-C's low surface expression and the few specific anti-sera available. However, using molecular methods such as DNA sequence analysis has filled up many blanks of the HLA-C phenotypes and identified more than 150 different HLA-C alleles. The aim of this study was to compare serological and molecular typing methods for HLA-C in the Saudi population.


   Materials and Methods Top


Our study was performed retrospectively on two cohorts of Saudi subjects. These comprised of one group of individuals typed by serology (1997-2001) and another group typed by molecular methods (2002-2008). We have selected the first donor from the family studies for bone marrow transplantation (BMT) which were carried out at our laboratory, King Abdulaziz Medical City, Riyadh. Results of HLA-C typing by serology (381 individuals) were compared with results from recent years after the introduction of SSP molecular method (385 individuals). This study was approved by the local Research and IRB committees.

Serology Typing

Ten milliliters of ACD, (acid-citrate dextrose) anti-coagulated blood was collected from all individuals. Lymphocytes were separated from peripheral blood by density gradient centrifugation. [8] HLA-C antigens were typed by the standard NIH (National Institute of Health) microlymphocytotoxicity test following the manufacturer's instructions (Bio-test, Dreieich, Germany). Typing trays used contained antibodies to HLA-C 1-7. Briefly, 1 μL of a 2 × 10 6 /mL suspension of either T or B lymphocytes were added to Class I tray (T cells) or Class II tray (B cells), and was incubated for ½ h at room temperature. Then, 5 μL of rabbit complement was added and again incubated for 1 h. Reactions were read using an inverted ultraviolet (UV) microscope after addition of 1 μL of ethidium bromide/acridine orange stain.

Molecular Typing

Five milliliters of peripheral blood was collected in ethylene diammine tetraacetate. DNA was prepared from blood leukocytes using a salt-out procedure. White cells were separated using Ficoll Hypaque after lysis of erythrcytes in red cell lysis buffer and protein digestion in proteinase K solution. DNA was extracted by precipitation of proteins using a saturating salt solution using the QIAamp DNA Blood Mini Kit (Qiagen, Hilden, Germany).

All individuals were DNA typed for HLA-C using polymerase chain reaction-sequence specific primer (PCR-SSP). The HLA-C primers were obtained from the Dynal HLA-C SSP low-resolution standard kit (Deutsche Dynal AG, Hamburg, Germany). PCR was performed according to the manufacturer's recommendations. Reaction products were separated by gel electrophoresis (1.5% agarose in TBE buffer) for 10 min at 150 V. Ethidium bromide was added subsequently and the gel was examined under an UV transilluminator. The results were compared with the interpretation table supplied by Dynal.


   Data Analysis Top


The maximum-likelihood estimates of allele frequencies were computed using an expectation-maximisation algorithm by the ARLEQUIN software. [9] Allele frequencies of the HLA-C gene were compared with those of Ollier et al 1985. [10] Conformity to the Hardy-Weinberg equilibrium (HWE) was tested for the allele frequencies by using PyPop statistical software. [11] Alleles are said to be in HWE if the observed homozygote and heterozygote frequencies did not differ significantly from the expected frequencies (P >0.05). The frequencies of each HLA-C allele were compared between serology and molecular biology techniques, and P-values were calculated using Chi-square tests (or Fisher's exact tests, when appropriate), by means of the statistics program Epi Info Version 6.


   Results Top


A comparison of the serology and molecular typing methods for the Saudi population is presented in [Table 1]. Both methods identified C*01-*07. However, only the molecular method identified C*08, C*12, C*14, C*15, C*16, C*17 and C*18. Serology failed to identify 23.5% of the HLA-C alleles. This is attributed to the alleles not detected by serology (C*08, *12, *14-*18). The sum of the allele frequencies of those alleles detected by molecular technique only (C*08, *12, *14-*18) was 28.9%, which was not significantly different from the sum of the alleles not identified by serology (23.5%), X 2 3.3, P = 0.1.
Table 1: HLA-C allele distribution in Saudi individuals using serology typing results from our laboratory and those from Ollier et al10 and using molecular typing methodology from our laboratory.

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Distribution of the allele frequencies of HLA-C showed that C*07 and C*06 were the most common alleles in the Saudi population by both serology and PCR-SSP, with frequencies of 27.8% and 21.8% by serology and 26.5% and 18.1% by PCR, respectively. We also compared our serological typing results with another study by Ollier et al [10] on HLA polymermorphism in a Saudi population [Table 1]. The data in that study were similar to our results. Ollier et al found that C*07 and C*06 had the highest frequency among the Saudi population, with a frequency of 24.6% and 25.2%, respectively. In addition 21% of their alleles were typed as blank. [10]

The HLA-C allele frequency of this population, as identified by molecular biology, does not conform with HWE. This is evident by the over-representation of the homozygous genotypes [Table 2]. It is interesting to note that there are 41 individuals who are *07/*07 while the expected is 27 individuals; 13 individuals were *15/*15 genotype while the expected is five individuals.
Table 2: HWE in the HLA-C alleles using Pypop software.

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   Discussion Top


In this study, we identified HLA-C allele frequency on two cohorts totalling 766 Saudis, using two different methods; serological and DNA-based typing. The discrepancy between the two methods has been described previously. [12] The discrepancy is due to the low surface expression of the HLA-C antigen and the insufficient anti-sera available and, thus, it would be difficult to evaluate the clinical importance of HLA-C in transplantation and to associate any specific alleles with certain diseases. It is important to emphasize the advantage of using the molecular method for HLA typing compared with serology because it was only by PCR-SSP that we were able to identify all the HLA-C alleles within the samples. We found that HLA-C*7 and C*6 are the most frequent alleles in this Saudi population, which is also similar to the study by Ollier. [10] We also found that only 23% of the serologically typed alleles were blank compared with 50% seen in other populations. [12],[13] This is reflected by the fact that HLA-C*01 to C*07 comprise the majority of the allele frequency by serology and molecular typing methods.

When we analyzed the molecular typing data, we found that HLA-C alleles are not in linkage disequilibrium, a phenomenon that was also noticed by Ollier et al. [9] In their study, Ollier et al suggested that this deviation from HWE was due to the higher than expected C1/C5 genotype, although their results of the other genes were in HWE. The reason behind this phenomenon needs further investigation.

In this study, we noticed the presence of higher than expected homozygous genotypes. Nevertheless, our study concludes highlighting the importance of molecular typing techniques for HLA-C to evaluate its role in transplantation and disease association studies.

 
   References Top

1.Bishara A, Amar A, Brautbar C, Condiotti R, Lazarovitz V, Nagler A. The putative role of HLA-C recognition in graft versus host disease (GVHD) and graft rejection after unrelated bone marrow transplantation (BMT). Exp Hematol 1995;23:1667.  Back to cited text no. 1
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2.Moya-Quiles MR, Muro M, Torio A, et al. Human leukocyte antigen-C in short- and longterm liver graft acceptance. Liver Transpl 2003;9:218.  Back to cited text no. 2
    
3.Frohn C, Fricke L, Puchta JC, Kirchner H. The effect of HLA-C matching on acute renal transplant rejection. Nephrol Dial Transplant 2001;16:355.  Back to cited text no. 3
[PUBMED]  [FULLTEXT]  
4.Hao Q, Saida T, Kawakami H, et al. HLAs and genes in Japanese patients with multiple sclerosis: evidence for increased frequencies of HLA-C3, HLA-DR2, and HLA-DQB1*0602. Hum Immunol 1992;35:116.  Back to cited text no. 4
[PUBMED]  [FULLTEXT]  
5.Purrmann J, Bertrams J, Cleveland S, Gemsa R, Berges W, Strohmeyer G. Association of Crohn's disease with HLA phenotype B44, C5. Z Gastroenterol 1988;26:658.  Back to cited text no. 5
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6.Asahina A, Kuwata S, Tokunaga K, Juji T, Nakagawa H. Study of aspartate at residue 9 of HLA-C molecules in Japanese patients with psoriasis vulgaris. J Dermatol Sci 1996;13: 125.  Back to cited text no. 6
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7.Kuruvilla J, Gupta V, Gill KS, Lipton JH. Association of familial leukemia with HLA C3: is it real? Leuk Lymphoma 2003;44:309.  Back to cited text no. 7
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8.Boyum A. Isolation and removal of lymphocytes from bone marrow of rats and guinea-pigs. Scand J Clin Lab Invest Suppl 1968;97:91.  Back to cited text no. 8
    
9.Excoffier L, Laval G, Schneider S. Arlequin (version 3.0): An integrated software package for population genetics data analysis. Evol Bioinform Online 2005;1:47.  Back to cited text no. 9
    
10.Ollier W, Doyle P, Alonso A, et al. HLA polymorphisms in Saudi Arabs. Tissue Antigens 1985;25:87.  Back to cited text no. 10
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11.Lancaster AK, Single RM, Solberg OD, Nelson MP, Thomson G. PyPop update--a software pipeline for large-scale multilocus population genomics. Tissue Antigens 2007;69 Suppl 1:192.  Back to cited text no. 11
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12.Mytilineos J, Christ U, Lempert M, Opelz G. Comparison of typing results by serology and polymerase chain reaction with sequence-specific primers for HLA-C in 650 individuals. Tissue Antigens 1997;50:395.  Back to cited text no. 12
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13.Bunce M, Barnardo MC, Procter J, Marsh SG, Vilches C, Welsh KI. High resolution HLA-C typing by PCR-SSP: identification of allelic frequencies and linkage disequilibria in 604 unrelated random UK Caucasoids and a comparison with serology. Tissue Antigens 1997; 50:100.  Back to cited text no. 13
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Correspondence Address:
Ali H Hajeer
Department of Pathology and Laboratory Medicine, King Abdulaziz Medical City, Riyadh 11426
Kingdom of Saudi Arabia
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