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HLA-DR2-associated DRB1 and DRB5 alleles and haplotypes in Koreans
HLA-DR2-Associated DRB1 and DRB5 Alleles and Haplotypes in Koreans Eun Young Song, Su Jin Kang, Young-Joon Lee, and Myoung Hee Park ABSTRACT: There are considerable racial differences in the distribution of HLA-DR2-associated DRB1 and DRB5 alleles and the characteristics of linkage disequilibrium between these alleles. In this study, the frequencies of DR2-associated DRB1 and DRB5 alleles and related haplotypes were analyzed in 186 DR2-positive individuals out of 800 normal Koreans registered for unrelated bone marrow donors. HLA class I antigen typing was performed by the serological method and DRB1 and DRB5 genotyping by the PCR-single strand conformational polymorphism method. Only 3 alleles were detected for DR2-associated DRB1 and DRB5 genes, respectively: DRB1*1501 (gene frequency 8.0%), *1502 (3.2%), *1602 (0.9%); DRB5*0101 (8.0%), *0102 (3.2%), and *0202 (0.9%). DRB1-DRB5 haplotype analysis showed an exclusive association between these alleles: DRB1*1501-DRB5*0101 (haplotype frequency 8.0%),
ABBREVIATIONS HLA human leukocyte antigen GF gene frequency HF haplotype frequency
INTRODUCTION HLA class II polymorphism is functionally important in the control of immune response, transplantation immunobiology, and susceptibility to autoimmune and infectious diseases. HLA-DR2 is of special interest for that DR2 occurs with relatively high frequencies in many populations and is known to be positively or negatively associated with several diseases such as narcolepsy, multiple sclerosis, Goodpasture’s syndrome, systemic lupus
From the Department of Clinical Pathology (E.Y.S., Y.-J.L., M.H.P.), Seoul National University College of Medicine, Seoul, Korea; the Clinical Research Institute (S.J.K.), Seoul National University Hospital, Seoul, Korea. Address reprint requests to: Myoung Hee Park, M.D., Department of Clinical Pathology, Seoul National University Hospital, 28 Yongon-dong, Chongno-gu, Seoul 110-744, Korea; Tel: ⫹82-2-760-3388; Fax: ⫹822-3672-3337; E-mail: [email protected]. Received March 2, 2000; accepted June 1, 2000. Human Immunology 61, 937–941 (2000) © American Society for Histocompatibility and Immunogenetics, 2000 Published by Elsevier Science Inc.
DRB1*1502-DRB5*0102 (3.2%), and DRB1*1602DRB5*0202 (0.9%). The 5 most common DR2-associated A-B-DRB1 haplotypes occurring at frequencies of 肁0.5% were A24-B52-DRB1*1502 (1.8%), A2-B62DRB1*1501, A2-B54-DRB1*1501, A26-B61DRB1*1501, and A24-B51-DRB1*1501. The remarkable homogeneity in the haplotypic associations between DR2-associated DRB1 and DRB5 alleles in Koreans would be advantageous for organ transplantation compared with other ethnic groups showing considerable heterogeneity in the distribution of DRB1-DRB5 haplotypes. Human Immunology 61, 937–941 (2000). © American Society for Histocompatibility and Immunogenetics, 2000. Published by Elsevier Science Inc. KEYWORDS: HLA-DR2; HLA-DRB1; HLA-DRB5; haplotype; Korean
PCR SSCP
polymerase chain reaction single strand conformational polymorphism
erythematosus, and insulin-dependent diabetes mellitus [1–5]. Molecular analyses have identified two DR2-associated genes, the DRB1 and DRB5, each encoding a DR chain that bears the serologic specificity of DR2 and DR51, respectively. With reporting of an ever-increasing number of new alleles, the current nomenclature for factors of the HLA system enlists 20 different DR2associated DRB1 alleles and 14 different DRB5 alleles [6]. There is a strong association between the DRB1 and DRB5 loci in DR2 haplotypes [7]. The associations of DRB5*0101 with DRB1*1501, DRB5*0102 with DRB1*1502, and DRB5*02 with DRB1*1601 and *1602 are frequently observed in Caucasoids but there are considerable racial differences in the distribution of DR2 haplotypes [8 –10]. Although heterogeneity of DR2 haplotypes has been studied in many populations 0198-8859/00/$–see front matter PII S0198-8859(00)00155-5
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TABLE 1 Gene and haplotype frequencies of HLA-DRB1 (DR2) and DRB5 in Koreans (n ⫽ 800) Allele
Na
GFb (%)
PFc (%)
Haplotype
HFd (%)
LDe
DRB1*1501 DRB1*1502 DRB1*1602
123 52 14
8.0 3.2 0.9
15.4 6.5 1.8
DRB1*1501-DRB5*0101 DRB1*1502-DRB5*0102 DRB1*1602-DRB5*0202
8.0 3.2 0.9
7.36 3.10 0.89
DRB5*0101 DRB5*0102 DRB5*0202
123 52 14
8.0 3.2 0.9
15.4 6.5 1.8
a
N: number of individuals positive for each allele. GF: gene (allele) frequency. c PF: phenotype frequency (PF ⫽ N/n, n: total number of individuals tested). d HF: haplotype frequency. e LD: linkage disequilibrium value (⫻100). b
[8 –13], little is known in Koreans. In this study, we have analyzed HLA-DRB1 and DRB5 alleles and haplotypes in 186 DR2-positive unrelated Koreans.
were derived from DR2-associated DRB1 allele frequencies.
DRB1 and DRB5 Genotyping Genomic DNA was extracted from anticoagulated peripheral blood by the salting out method [14]. DRB1 and DRB5 genes were typed using the PCR-single strand conformational polymorphism (SSCP) method. HLA-DRB1 and DRB5 genes were amplified using group-specific primers and SSCP analysis was performed according to the methods described previously [15, 16] with minor modifications.
RESULTS All 186 DR2-positive individuals included in this study were typed as DRB1*15 and/or DRB1*16 by DRB1 genotyping. In this group, 12 individuals were DR2 homozygous, contributing a total of 198 DR2-carrying haplotypes. The distribution of DR15/16 alleles showed that the most frequent allele was DRB1*1501 (gene frequency 8.0%) followed by *1502 (3.2%) and *1602 (0.9%) (Table 1). No other alleles were detected in our population. Molecular analysis of the DRB5 locus also showed the presence of 3 alleles: DRB5*0101 (8.0%), *0102 (3.2%) and *0202 (0.9%). Haplotypic associations between these DRB1 and DRB5 alleles were exclusive with very high linkage disequilibrium (LD) values. All DRB1*1501 alleles identified were exclusively associated with DRB5*0101, whereas DRB1*1502 was associated only with DRB5*0102 and DRB1*1602 only with DRB5*0202 (Chi square value of 1600 for each haplotype). The composition and frequencies of HLA A-BDRB1*15/16 haplotypes in the Korean population are shown in Table 2. The 5 most common DR2-associated haplotypes in Koreans were A24-B52-DRB1*1502 (haplotype frequency 1.8%), A2-B62-DRB1*1501 (1.1%), A2-B54-DRB1*1501 (0.8%), A26-B61-DRB1*1501 (0.7%), and A24-B51-DRB1*1501 (0.5%).
Statistical Analysis The frequencies of HLA-A, B, and DRB1 alleles and A-B-DRB1 haplotypes were estimated by the maximum likelihood method using a computer program developed in the 11th International Histocompatibility Workshop [17]. There was an exclusive association between DR2associated DRB1 and DRB5 alleles, and thus the frequencies of DRB5 alleles and DRB1-DRB5 haplotypes
DISCUSSION In this study, we have analyzed the polymorphism and distribution of HLA-DR2 alleles and related DRB1DRB5 and A-B-DRB1 haplotypes on a relatively large number of individuals (n ⫽ 800). The predominance of DRB1*1501 and the absence of DRB1*1503 and DRB1*1601 alleles in our population are similar to the
MATERIALS AND METHODS Population Studied The study was conducted on 186 DR2-positive (including 12 DR2 homozygous) individuals out of 800 normal Koreans registered for unrelated bone marrow donors. Serological typing of HLA class I (A, B, C) antigens was performed using a commercial kit designed for Oriental populations (OT-72, One Lambda, Canoga Park, CA, USA). HLA-DR generic typing was performed by reverse line blot hybridization method using the AMPLICOR威 HLA DRB Test (Roche Diagnostic System, Branchburg, NJ, USA). DRB1 genotyping was performed for 800 individuals and DRB5 genotyping for 186 DR2-positive individuals.
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TABLE 2 Haplotype frequencies of HLA-A, B and DRB1 (DR2) in Koreans (n ⫽ 800) Haplotype
HFa (%)
LDb
A24 B52 DRB1*1502 A2 B62 DRB1*1501 A2 B54 DRB1*1501 A26 B61 DRB1*1501 A24 B51 DRB1*1501 A2 B38 DRB1*1502 A2 B48 DRB1*1501 A2 B52 DRB1*1502 A26 B62 DRB1*1501 A2 B51 DRB1*1501
1.83 1.07 0.77 0.74 0.50 0.44 0.42 0.34 0.32 0.31
1.81 0.81 0.64 0.68 0.30 0.43 0.34 0.31 0.25 0.05
a b
HF: haplotype frequency. LD: linkage disequilibrium value (⫻100).
rarity of these alleles in other Asian populations [18, 19]. Distribution of DR2 subtypes is significantly different among different ethnic groups. DRB1*1501 is predominant (⬎50% of DR2 subtypes) in most Caucasians and Asian populations [10, 18, 19], and DRB1*1502 is predominant in Japanese [18, 19]. DRB1*1503 is predominant in African Americans and is rare in other populations [10, 18]. DRB1*1601 is predominant in Romanian, Sardinian and Spanish Gypsy [18] and comprises more than 10% of DR2 subtypes in Caucasoid Americans [10], but is rare in populations of AsiaOceania [8, 9, 18, 19]. DRB1*1602 comprises more than 20% of DR2 subtypes in Southern Chinese, such as Singapore Chinese and Xiamen Chinese [8, 10]. There is a strong association between DR2-associated TABLE
DRB1 alleles and DRB5 alleles [7]. However, substantial heterogeneity is present in the distribution of DR2associatied DRB1-DRB5 haplotypes among different ethnic populations [8 –13]. Analysis of the DRB1-DRB5 haplotypes in the present study of Koreans showed an exclusive association between DRB1 and DRB5 alleles: DRB1*1501-DRB5*0101 (65.7%), DRB1*1502DRB5*0102 (27.3%), and DRB1*1602-DRB5*0202 (7.1%). The distribution of DRB1-DRB5 haplotypes in this study is similar to that of Northern Chinese [8] and Asian Indians [11] (Table 3), and probably that of Japanese (data not available). It is of interest that DRB1*1602 is predominantly associated with DRB5*02 in Northeast Asians, whereas associated with DRB5*0101 in Southern Chinese such as Singapore Chinese and Xiamen Chinese [8, 10]. Significant heterogeneity in the distribution of DRB1-DRB5 haplotypes is observed in Chinese Americans, Caucasoid Americans, and African Americans, revealing 11 to 12 different haplotypes in each population. The heterogeneity of DR2 haplotypes is considered to be generated via multiple gene conversion events together with point mutations and reciprocal recombinations [10]. The remarkable homogeneity in the linkage between DR2-associated DRB1 and DRB5 alleles in the Korean population would be advantageous in matching unrelated donor recipient pairs for marrow transplantation, compared with other populations showing heterogeneity in the distribution of DRB1-DRB5 haplotypes. This finding of exclusive association between DR2-associated DRB1 and DRB5 alleles in our population could also be
3 Relative frequencies of DRB1 (DR2)-DRB5 haplotypes in Koreans and six other ethnic groups
DRB1-DRB5a *1501-*0101 *1502-*0101 *1502-*0102 *1503-*0101 *1503-blank *1601-*0101 *1601-*02 *1602-*0101 *1602-*02 *1602-*0203 Others
Koreansb n ⫽ 198 (3)c
Asian Indiansd n ⫽ 65 (3)
Northern Chinesee n ⫽ 76 (5)
Southern Chinesee n ⫽ 50 (5)
Chinese Americansf n ⫽ 298 (11)
Caucasoid Americansf n ⫽ 527 (11)
African Americansf n ⫽ 380 (12)
65.7
60.0
50.0
27.3
33.8
38.2
50.0 2.0 16.0
53.4 5.7 19.8
78.0 1.7 5.3 1.0
6.2
1.3 1.3 9.2
2.4 1.3 9.7 5.0 1.3 1.3
1.3 9.5 0.2 1.9
17.1 0.8 1.1 64.8 2.4 0.3 0.5 1.1 11.1
1.1
1.0
7.1
28.0 4.0g
Haplotypes observed at frequencies of ⬎1% in one or more ethnic groups are listed. Present study. c n: number of DR2-positive haplotypes studied (number of different haplotypes observed). d Mehra et al. [11]. e Gao and Serjeantson [8]. f Lee et al. [10]. g DRB1*1602-DRB5*new: probably *1602-*0203 haplotype observed in Chinese Americans (1.3%) and Xiamen Chinese (1.5%) by Lee et al. [10]. a
b
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TABLE 4 Comparison of HLA-A, B, DRB1(DR2) haplotype frequencies among different ethnic groups in Northeast Asia
A-B-DRB1*a 2-61-1501 2-BL-1501 24-52-1502 11-52-1502
Koreansb n ⫽ 800 HF ⱖ 0.3%
Japanesec n ⫽ 195 HF ⱖ 0.5%
Hanc n ⫽ 57 HF ⱖ 0.9%
Hoton-Mongolianc n ⫽ 85 HF ⱖ 0.6%
Manc n ⫽ 172 HF ⱖ 0.6% 2.9
2.3 1.8
7.2 6.5
A-B-DRB1(DR2) haplotypes with haplotype frequencies (HF) of ⱖ2% in some of the 4 different ethnic groups in Northeast Asia are listed. Present study, HF ⱖ 0.3% are shown. c Tanaka et al. [19], number (n) of individuals studied and the minimum level of HF listed are shown. a
b
applied to determining DR2 subtypes in low resolution HLA-DRB DNA typing. We used the AMPLICOR威 HLA DRB Test (Roche Diagnostic System), based on reverse SSO hybridization method using 29 probes for DRB1, DRB3, DRB4, and DRB5 genes. This HLADRB typing kit can not distinguish DR2 subtypes. However, applying the finding of DRB1-DRB5 associations in Koreans, 97% of DR2-positive cases had expectable DR15/DR16 subtypes at serological level of resolution, which turned out to be correct by DRB1 and DRB5 genotyping (unpublished data). HLA-A, B, DRB1 match between donor and recipient is important in organ transplantations, especially in unrelated marrow transplantation. Table 4 shows common HLA-DR2-associated A-B-DRB1 haplotypes (frequency 肁 2%) in 4 different ethnic groups in Northeast Asia [19] in comparison with the result of the present study of Koreans. None of the common DR2-associated haplotypes in Han, Hoton-Mongolian and Man populations are shared with Japanese or Koreans. The most frequent DR2-associated A-B-DRB1 haplotype in Koreans, A24B52-DRB1*1502 (1.8%), is shared with Japanese occurring at high frequency (7.2%). Analysis of A-B-DRB1 haplotypes in Koreans (n ⫽ 800) revealed 5 DR2-associated haplotypes occurring at frequency of 肁0.5% (Table 3), and that in Japanese (n ⫽ 1216) revealed only 2 DR2-associated haploytpes occurring at frequency of 肁0.5% [20]. Koreans show a higher degree of polymorphism in the distribution of DR2-associated A-B-DRB1 haplotypes compared with Japanese and only the most common one (A24-B52-DRB1*1502) in both populations are shared. In the present study, we have analyzed the distribution of DR2-associated DRB1 and DRB5 alleles and related haplotypes in 800 Koreans. Koreans showed remarkable homogeneity in the haplotypic associations between DRB1 and DRB5 alleles. The results obtained in this study will be useful as basic data on Koreans for anthropology, disease association studies and organ transplantations.
REFERENCES 1. Juji T, Satake M, Honda Y, Doi Y: HLA antigens in Japanese patients with narcolepsy. All the patients were DR2 positive. Tissue Antigens 24:316, 1984. 2. Olerup O, Hillert J: HLA class II-associated genetic susceptibility in multiple sclerosis: a critical evaluation. Tissue Antigens 38:1, 1991. 3. Burns AP, Fisher M, Li P, Pusey CD, Rees AJ: Molecular analysis of HLA class II genes in Goodpasture’s disease. QJM 88:93, 1995. 4. Hartung K, Baur MP, Coldewey R, Fricke M, Kalden JR, Lakomek HJ, Peter HH, Schendel D, Schneider PM, Seuchter SA, Strangel W, Deicher HRG: Major histocompatibility complex haplotypes and complement C4 alleles in systemic lupus erythematosus. Results of a multicenter study. J Clin Invest 90:1346, 1992. 5. Ilonen J, Herva E, Tiilikainen A, Akerblom HK, Koivukangas T, Kouvalainen K: HLA-Dw2 as a marker of resistance against juvenile diabetes mellitus. Tissue Antigens 11:144, 1978. 6. Bodmer JG, Marsh SGE, Albert ED, Bodmer WF, Bontrop RE, Dupont B, Erlich HA, Hansen JA, Mach B, Mayr WR, Parham P, Petersdorf EW, Sasazuki T, Schreuder GMT, Strominger JL, Svejgaard A, Terasaki PI: Nomenclature for factors of the HLA system, 1998. Tissue Antigens 53:407, 1999. 7. Tiercy JM, Jeannet M, Mach B: Oligonucleotide typing analysis for the linkage disequilibrium between the polymorphic DRB1 and DRB5 loci in DR2 haplotypes. Tissue Antigens 37:161, 1991. 8. Gao X, Serjeantson SW: Heterogeneity in HLA-DR2related DR, DQ haplotypes in eight populations of AsiaOceania. Immunogenetics 34:401, 1991. 9. Trejaut J, Bhatia K, Greville WD, Hu KR, Duraisamy G, Nuchprayoon C, Donald J, Aziz A, Dunckley H: HLADR2 haplotypic diversity in populations of South-East Asia, northern China, Melanesia and Australian aborigines using PCR-RFLP for DRB1, DRB5, DQA1 and DQB1.
HLA-DR2 Alleles and Haplotypes in Koreans
10.
11.
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15.
A novel DRB1 allele: DRB1*16022. Eur J Immunogenet 23:437, 1996. Lee A, Huang R, Yan L, Shaw CK, Zeng S, Lin PY, Lee TD: Heterogeneity of HLA-DR2 haplotypes in Caucasoid Americans, African Americans, Chinese Americans, Native Americans and Xiamen Chinese. Eur J Immunogenet 26:275, 1999. Mehra NK, Verduijn W, Drabbels TJ, Singh SPN, Giphart MJ: Analysis of HLA-DR2-associated polymorphisms by oligonucleotide hybridization in an Asian Indian population. Hum Immunol 32:246, 1991. Fan L, Chandanayingyong D, Smith AG, Hansen JA: The Dai minority population of Southwest China: heterogeneity of DR2-associated HLA-DRB1, DRB5, DQA1, and DQB1 haplotypes. Hum Immunol 45:143, 1996. Constantinidou N, Chaidaroglou A, Van den BergLoonen EM, Koniavitou K: Polymorphism and distribution of HLA-DR2 alleles and haplotypes in a Greek population. Tissue Antigens 52:153, 1998. Miller SA, Dykes DD, Polesky HF: A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res 16:1215, 1988. Bannai M, Tokunaga K, Lin L, Kuwata S, Mazda T, Amaki I, Fujisawa K, Juji T: Discrimination of human HLA-DRB1 alleles by PCR-SSCP (single-strand confor-
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mation polymorphism) method. Eur J Immunogenet 21:1, 1994. Takeuchi F, Nakano K, Yamada H, Hong GH, Nabeta H, Yoshida A, Matsuta K, Bannai M, Tokunaga K, Ito K: Association of HLA-DR with progressive systemic sclerosis in Japanese. J Rheumatol 21:857, 1994. Imanishi T, Akaza T, Kimura A, Tokunaga K, Gojobori T: Estimation of allele and haplotype frequencies for HLA and complement loci. In Tsuji K, Aizawa M, Saszuki T (eds): HLA 1991. vol 1. New York, Oxford University Press, 1992. Imanishi T, Akaza T, Kimura A, Tokunaga K, Gojobori T: Allele and haplotype frequencies for HLA and complement loci in various ethnic groups. In Tsuji K, Aizawa M, Saszuki T (eds): HLA 1991. vol. 1. New York, Oxford University Press, 1992. Tanaka H, Tokunaga K, Inoko H, Tsuji K, Chimge NO, Jia GJ, Park MH, Han H, Nose Y, Nagao N, Saitou S, Akaza T, Juji T: Distribution of HLA-A, B, and DRB1 alleles and haplotypes in Northeast Asia. In Charron D (ed): Genetic diversity of HLA: functional and medical implication. vol I. Paris, EDK Publisher, 1997. Akaza T, Imanishi T, Fujiwara K, Tokunaga K, Juji T, Yashiki S, Sonoda S: HLA allele and haplotype frequencies in Japanese. Transplant Now 7:87, 1994.
ABBREVIATIONS HLA human leukocyte antigen GF gene frequency HF haplotype frequency
INTRODUCTION HLA class II polymorphism is functionally important in the control of immune response, transplantation immunobiology, and susceptibility to autoimmune and infectious diseases. HLA-DR2 is of special interest for that DR2 occurs with relatively high frequencies in many populations and is known to be positively or negatively associated with several diseases such as narcolepsy, multiple sclerosis, Goodpasture’s syndrome, systemic lupus
From the Department of Clinical Pathology (E.Y.S., Y.-J.L., M.H.P.), Seoul National University College of Medicine, Seoul, Korea; the Clinical Research Institute (S.J.K.), Seoul National University Hospital, Seoul, Korea. Address reprint requests to: Myoung Hee Park, M.D., Department of Clinical Pathology, Seoul National University Hospital, 28 Yongon-dong, Chongno-gu, Seoul 110-744, Korea; Tel: ⫹82-2-760-3388; Fax: ⫹822-3672-3337; E-mail: [email protected]. Received March 2, 2000; accepted June 1, 2000. Human Immunology 61, 937–941 (2000) © American Society for Histocompatibility and Immunogenetics, 2000 Published by Elsevier Science Inc.
DRB1*1502-DRB5*0102 (3.2%), and DRB1*1602DRB5*0202 (0.9%). The 5 most common DR2-associated A-B-DRB1 haplotypes occurring at frequencies of 肁0.5% were A24-B52-DRB1*1502 (1.8%), A2-B62DRB1*1501, A2-B54-DRB1*1501, A26-B61DRB1*1501, and A24-B51-DRB1*1501. The remarkable homogeneity in the haplotypic associations between DR2-associated DRB1 and DRB5 alleles in Koreans would be advantageous for organ transplantation compared with other ethnic groups showing considerable heterogeneity in the distribution of DRB1-DRB5 haplotypes. Human Immunology 61, 937–941 (2000). © American Society for Histocompatibility and Immunogenetics, 2000. Published by Elsevier Science Inc. KEYWORDS: HLA-DR2; HLA-DRB1; HLA-DRB5; haplotype; Korean
PCR SSCP
polymerase chain reaction single strand conformational polymorphism
erythematosus, and insulin-dependent diabetes mellitus [1–5]. Molecular analyses have identified two DR2-associated genes, the DRB1 and DRB5, each encoding a DR chain that bears the serologic specificity of DR2 and DR51, respectively. With reporting of an ever-increasing number of new alleles, the current nomenclature for factors of the HLA system enlists 20 different DR2associated DRB1 alleles and 14 different DRB5 alleles [6]. There is a strong association between the DRB1 and DRB5 loci in DR2 haplotypes [7]. The associations of DRB5*0101 with DRB1*1501, DRB5*0102 with DRB1*1502, and DRB5*02 with DRB1*1601 and *1602 are frequently observed in Caucasoids but there are considerable racial differences in the distribution of DR2 haplotypes [8 –10]. Although heterogeneity of DR2 haplotypes has been studied in many populations 0198-8859/00/$–see front matter PII S0198-8859(00)00155-5
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TABLE 1 Gene and haplotype frequencies of HLA-DRB1 (DR2) and DRB5 in Koreans (n ⫽ 800) Allele
Na
GFb (%)
PFc (%)
Haplotype
HFd (%)
LDe
DRB1*1501 DRB1*1502 DRB1*1602
123 52 14
8.0 3.2 0.9
15.4 6.5 1.8
DRB1*1501-DRB5*0101 DRB1*1502-DRB5*0102 DRB1*1602-DRB5*0202
8.0 3.2 0.9
7.36 3.10 0.89
DRB5*0101 DRB5*0102 DRB5*0202
123 52 14
8.0 3.2 0.9
15.4 6.5 1.8
a
N: number of individuals positive for each allele. GF: gene (allele) frequency. c PF: phenotype frequency (PF ⫽ N/n, n: total number of individuals tested). d HF: haplotype frequency. e LD: linkage disequilibrium value (⫻100). b
[8 –13], little is known in Koreans. In this study, we have analyzed HLA-DRB1 and DRB5 alleles and haplotypes in 186 DR2-positive unrelated Koreans.
were derived from DR2-associated DRB1 allele frequencies.
DRB1 and DRB5 Genotyping Genomic DNA was extracted from anticoagulated peripheral blood by the salting out method [14]. DRB1 and DRB5 genes were typed using the PCR-single strand conformational polymorphism (SSCP) method. HLA-DRB1 and DRB5 genes were amplified using group-specific primers and SSCP analysis was performed according to the methods described previously [15, 16] with minor modifications.
RESULTS All 186 DR2-positive individuals included in this study were typed as DRB1*15 and/or DRB1*16 by DRB1 genotyping. In this group, 12 individuals were DR2 homozygous, contributing a total of 198 DR2-carrying haplotypes. The distribution of DR15/16 alleles showed that the most frequent allele was DRB1*1501 (gene frequency 8.0%) followed by *1502 (3.2%) and *1602 (0.9%) (Table 1). No other alleles were detected in our population. Molecular analysis of the DRB5 locus also showed the presence of 3 alleles: DRB5*0101 (8.0%), *0102 (3.2%) and *0202 (0.9%). Haplotypic associations between these DRB1 and DRB5 alleles were exclusive with very high linkage disequilibrium (LD) values. All DRB1*1501 alleles identified were exclusively associated with DRB5*0101, whereas DRB1*1502 was associated only with DRB5*0102 and DRB1*1602 only with DRB5*0202 (Chi square value of 1600 for each haplotype). The composition and frequencies of HLA A-BDRB1*15/16 haplotypes in the Korean population are shown in Table 2. The 5 most common DR2-associated haplotypes in Koreans were A24-B52-DRB1*1502 (haplotype frequency 1.8%), A2-B62-DRB1*1501 (1.1%), A2-B54-DRB1*1501 (0.8%), A26-B61-DRB1*1501 (0.7%), and A24-B51-DRB1*1501 (0.5%).
Statistical Analysis The frequencies of HLA-A, B, and DRB1 alleles and A-B-DRB1 haplotypes were estimated by the maximum likelihood method using a computer program developed in the 11th International Histocompatibility Workshop [17]. There was an exclusive association between DR2associated DRB1 and DRB5 alleles, and thus the frequencies of DRB5 alleles and DRB1-DRB5 haplotypes
DISCUSSION In this study, we have analyzed the polymorphism and distribution of HLA-DR2 alleles and related DRB1DRB5 and A-B-DRB1 haplotypes on a relatively large number of individuals (n ⫽ 800). The predominance of DRB1*1501 and the absence of DRB1*1503 and DRB1*1601 alleles in our population are similar to the
MATERIALS AND METHODS Population Studied The study was conducted on 186 DR2-positive (including 12 DR2 homozygous) individuals out of 800 normal Koreans registered for unrelated bone marrow donors. Serological typing of HLA class I (A, B, C) antigens was performed using a commercial kit designed for Oriental populations (OT-72, One Lambda, Canoga Park, CA, USA). HLA-DR generic typing was performed by reverse line blot hybridization method using the AMPLICOR威 HLA DRB Test (Roche Diagnostic System, Branchburg, NJ, USA). DRB1 genotyping was performed for 800 individuals and DRB5 genotyping for 186 DR2-positive individuals.
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TABLE 2 Haplotype frequencies of HLA-A, B and DRB1 (DR2) in Koreans (n ⫽ 800) Haplotype
HFa (%)
LDb
A24 B52 DRB1*1502 A2 B62 DRB1*1501 A2 B54 DRB1*1501 A26 B61 DRB1*1501 A24 B51 DRB1*1501 A2 B38 DRB1*1502 A2 B48 DRB1*1501 A2 B52 DRB1*1502 A26 B62 DRB1*1501 A2 B51 DRB1*1501
1.83 1.07 0.77 0.74 0.50 0.44 0.42 0.34 0.32 0.31
1.81 0.81 0.64 0.68 0.30 0.43 0.34 0.31 0.25 0.05
a b
HF: haplotype frequency. LD: linkage disequilibrium value (⫻100).
rarity of these alleles in other Asian populations [18, 19]. Distribution of DR2 subtypes is significantly different among different ethnic groups. DRB1*1501 is predominant (⬎50% of DR2 subtypes) in most Caucasians and Asian populations [10, 18, 19], and DRB1*1502 is predominant in Japanese [18, 19]. DRB1*1503 is predominant in African Americans and is rare in other populations [10, 18]. DRB1*1601 is predominant in Romanian, Sardinian and Spanish Gypsy [18] and comprises more than 10% of DR2 subtypes in Caucasoid Americans [10], but is rare in populations of AsiaOceania [8, 9, 18, 19]. DRB1*1602 comprises more than 20% of DR2 subtypes in Southern Chinese, such as Singapore Chinese and Xiamen Chinese [8, 10]. There is a strong association between DR2-associated TABLE
DRB1 alleles and DRB5 alleles [7]. However, substantial heterogeneity is present in the distribution of DR2associatied DRB1-DRB5 haplotypes among different ethnic populations [8 –13]. Analysis of the DRB1-DRB5 haplotypes in the present study of Koreans showed an exclusive association between DRB1 and DRB5 alleles: DRB1*1501-DRB5*0101 (65.7%), DRB1*1502DRB5*0102 (27.3%), and DRB1*1602-DRB5*0202 (7.1%). The distribution of DRB1-DRB5 haplotypes in this study is similar to that of Northern Chinese [8] and Asian Indians [11] (Table 3), and probably that of Japanese (data not available). It is of interest that DRB1*1602 is predominantly associated with DRB5*02 in Northeast Asians, whereas associated with DRB5*0101 in Southern Chinese such as Singapore Chinese and Xiamen Chinese [8, 10]. Significant heterogeneity in the distribution of DRB1-DRB5 haplotypes is observed in Chinese Americans, Caucasoid Americans, and African Americans, revealing 11 to 12 different haplotypes in each population. The heterogeneity of DR2 haplotypes is considered to be generated via multiple gene conversion events together with point mutations and reciprocal recombinations [10]. The remarkable homogeneity in the linkage between DR2-associated DRB1 and DRB5 alleles in the Korean population would be advantageous in matching unrelated donor recipient pairs for marrow transplantation, compared with other populations showing heterogeneity in the distribution of DRB1-DRB5 haplotypes. This finding of exclusive association between DR2-associated DRB1 and DRB5 alleles in our population could also be
3 Relative frequencies of DRB1 (DR2)-DRB5 haplotypes in Koreans and six other ethnic groups
DRB1-DRB5a *1501-*0101 *1502-*0101 *1502-*0102 *1503-*0101 *1503-blank *1601-*0101 *1601-*02 *1602-*0101 *1602-*02 *1602-*0203 Others
Koreansb n ⫽ 198 (3)c
Asian Indiansd n ⫽ 65 (3)
Northern Chinesee n ⫽ 76 (5)
Southern Chinesee n ⫽ 50 (5)
Chinese Americansf n ⫽ 298 (11)
Caucasoid Americansf n ⫽ 527 (11)
African Americansf n ⫽ 380 (12)
65.7
60.0
50.0
27.3
33.8
38.2
50.0 2.0 16.0
53.4 5.7 19.8
78.0 1.7 5.3 1.0
6.2
1.3 1.3 9.2
2.4 1.3 9.7 5.0 1.3 1.3
1.3 9.5 0.2 1.9
17.1 0.8 1.1 64.8 2.4 0.3 0.5 1.1 11.1
1.1
1.0
7.1
28.0 4.0g
Haplotypes observed at frequencies of ⬎1% in one or more ethnic groups are listed. Present study. c n: number of DR2-positive haplotypes studied (number of different haplotypes observed). d Mehra et al. [11]. e Gao and Serjeantson [8]. f Lee et al. [10]. g DRB1*1602-DRB5*new: probably *1602-*0203 haplotype observed in Chinese Americans (1.3%) and Xiamen Chinese (1.5%) by Lee et al. [10]. a
b
940
E. Y. Song et al.
TABLE 4 Comparison of HLA-A, B, DRB1(DR2) haplotype frequencies among different ethnic groups in Northeast Asia
A-B-DRB1*a 2-61-1501 2-BL-1501 24-52-1502 11-52-1502
Koreansb n ⫽ 800 HF ⱖ 0.3%
Japanesec n ⫽ 195 HF ⱖ 0.5%
Hanc n ⫽ 57 HF ⱖ 0.9%
Hoton-Mongolianc n ⫽ 85 HF ⱖ 0.6%
Manc n ⫽ 172 HF ⱖ 0.6% 2.9
2.3 1.8
7.2 6.5
A-B-DRB1(DR2) haplotypes with haplotype frequencies (HF) of ⱖ2% in some of the 4 different ethnic groups in Northeast Asia are listed. Present study, HF ⱖ 0.3% are shown. c Tanaka et al. [19], number (n) of individuals studied and the minimum level of HF listed are shown. a
b
applied to determining DR2 subtypes in low resolution HLA-DRB DNA typing. We used the AMPLICOR威 HLA DRB Test (Roche Diagnostic System), based on reverse SSO hybridization method using 29 probes for DRB1, DRB3, DRB4, and DRB5 genes. This HLADRB typing kit can not distinguish DR2 subtypes. However, applying the finding of DRB1-DRB5 associations in Koreans, 97% of DR2-positive cases had expectable DR15/DR16 subtypes at serological level of resolution, which turned out to be correct by DRB1 and DRB5 genotyping (unpublished data). HLA-A, B, DRB1 match between donor and recipient is important in organ transplantations, especially in unrelated marrow transplantation. Table 4 shows common HLA-DR2-associated A-B-DRB1 haplotypes (frequency 肁 2%) in 4 different ethnic groups in Northeast Asia [19] in comparison with the result of the present study of Koreans. None of the common DR2-associated haplotypes in Han, Hoton-Mongolian and Man populations are shared with Japanese or Koreans. The most frequent DR2-associated A-B-DRB1 haplotype in Koreans, A24B52-DRB1*1502 (1.8%), is shared with Japanese occurring at high frequency (7.2%). Analysis of A-B-DRB1 haplotypes in Koreans (n ⫽ 800) revealed 5 DR2-associated haplotypes occurring at frequency of 肁0.5% (Table 3), and that in Japanese (n ⫽ 1216) revealed only 2 DR2-associated haploytpes occurring at frequency of 肁0.5% [20]. Koreans show a higher degree of polymorphism in the distribution of DR2-associated A-B-DRB1 haplotypes compared with Japanese and only the most common one (A24-B52-DRB1*1502) in both populations are shared. In the present study, we have analyzed the distribution of DR2-associated DRB1 and DRB5 alleles and related haplotypes in 800 Koreans. Koreans showed remarkable homogeneity in the haplotypic associations between DRB1 and DRB5 alleles. The results obtained in this study will be useful as basic data on Koreans for anthropology, disease association studies and organ transplantations.
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