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HLA-B∗44 allele frequencies and haplotypic associations in Koreans
HLA-B*44 Allele Frequencies and Haplotypic Associations in Koreans Eun Young Song, Dong Hee Whang, Mina Hur, Su Jin Kang, Kyou Sup Han, and Myoung Hee Park ABSTRACT: We have investigated the frequencies of HLA-B*44 alleles and their haplotypic associations with HLA-A, -C, and -DRB1 loci in 450 healthy unrelated Koreans, including 213 parents from 107 families. All 79 samples (17.6%) typed as B44 by serology were analyzed for B*44 alleles using polymerase chain reaction (PCR) single-strand conformation polymorphism (SSCP) method. A total of three different B*44 alleles were detected: B*44031 (allele frequency 4.7%), B*44032 (3.1%), and B*4402 (1.3%). Three characteristic haplotypes revealing strong linkage disequilibrium were A*3303-Cw*1403-B*44031-DRB1*1302 (3.6%), A*3303-Cw*07-B*44032-DRB1*0701 (2.8%), and A*3201-Cw*05-B*4402-DRB1*0405 (0.4%). In addition, a strong association was observed for B*4402 with
ABBREVIATIONS AF allele frequency HF haplotype frequency PCR polymerase chain reaction RFLP restriction fragment length polymorphism
INTRODUCTION HLA-B44 is one of the most frequently found HLA-B specificities in many Caucasian and Asian populations with allele frequencies of approximately 5%–15% [1]. Although serologic HLA-B44 specificity is encoded by at least 25 different alleles (B*4402-B*4424) identified to date [2], B*4402 and B*4403 are by far the two predominant alleles found in all populations studied [3–10]. B*4403 alleles are subdivided into B*44031 and
From the Department of Clinical Pathology (E.Y.S., D.H.W., M.H., K.S.H., M.H.P.), Seoul National University College of Medicine, and Clinical Research Institute (S.J.K., M.H.P.), Seoul National University Hospital, Seoul, Korea. Address reprint requests to: Dr. Myoung Hee Park, Department of Clinical Pathology, Seoul National University College of Medicine, 28 Yongon-dong, Chongno-gu, Seoul 110-744, Korea; Tel: ⫹82 (2) 7603388; Fax: ⫹82 (2) 3672-3337; E-mail: [email protected]. Received November 15, 2000; revised February 12, 2001; accepted June 15, 2001. Human Immunology 63, 1142–1147 (2001) © American Society for Histocompatibility and Immunogenetics, 2001 Published by Elsevier Science Inc.
A*0301. The B*4403-bearing haplotypes of Koreans appear to be relatively common in Asian populations, whereas the B*4402-bearing haplotypes share some similarity to those of Caucasians. HLA-B44 alleles demonstrate a limited allelic diversity and comprise distinctive extended haplotypes in the Korean population. It is suggested that the frequencies of B44 subtype mismatches among ABDRB1-matched unrelated donor-recipient pairs would be low in this population. Human Immunology 63, 1142–1147 (2001). © American Society for Histocompatibility and Immunogenetics, 2001. Published by Elsevier Science Inc. KEYWORDS: allele frequency; HLA-B*44; HLA-B44 subtype; haplotype; Korean
SSCP SSO SSP
single strand conformation polymorphism sequence specific oligonucleotide sequence specific primer
B*44032 by a silent substitution of T for C at nucleotide 369 [11]. Previous studies suggested that the frequencies of B44 subtypes are quite different among various ethnic groups, and these B44 subtypes seem to be in close linkage disequilibrium with different HLA alleles [3–10, 12]. Despite the well-known ethnic differences in the distribution of B44 subtypes in Caucasians, the distribution and haplotypic association of B44 alleles with other HLA loci have not been well studied in Asian populations. The clinical importance of HLA-B44 polymorphism has been highlighted by a report on the generation of cytotoxic T lymphocytes (CTL) directed against B44 subtype mismatch in bone marrow allograft rejection [13, 14]. Identification of conserved and uncommon B44 haplotypes among different populations would be useful for unrelated bone marrow donor searches. This report summarizes the B44 allelic diversity and haplotypic associations in Koreans. 0198-8859/01/$–see front matter PII S0198-8859(01)00304-4
HLA-B*44 Alleles and Haplotypes in Koreans
MATERIALS AND METHODS Population Studied A total of 450 healthy unrelated Koreans from two groups of individuals were studied. One group consisted of 213 parents from 107 families, as previously reported [15], and the other group consisted of 237 volunteer bone marrow donors enrolled in the Korean Marrow Donor Program (KMDP). For family samples, HLA-A, -B, -C, and -DR antigens were typed by the standard serologic method previously reported [15]. For KMDP donors, HLA-A, -B, -C serologic typing was performed using a commercial kit designed for Oriental populations (OT-72; One Lambda, Canoga Park, CA, USA) and HLA-DRB1 generic DNA typing was performed using the Amplicor威 HLA DRB kit (Roche Diagnostic System, Branchburg, NJ, USA). Molecular Typing of HLA-B44, -Cw, -DRB1, and -DQB1 Alleles HLA-B44 allele typing was performed for all of the samples serologically typed as B44-positive (n ⫽ 79) using two-step polymerase chain reaction (PCR) and single-strand conformation polymorphism (SSCP) analysis. First, PCR was performed to amplify the fragment encompassing exons 2 and 3 using dimorphic primers (5BIN1-CG and 3BIN3-37) as previously described [16]. Second, PCR was performed separately using BEX2-1 and BEX2-FC primers for exon 2, and BEX3-1 and BINT3-1C primers for exon 3 [17, 18]. Exon 2 and 3 fragments, amplified in the second PCR, were separately analyzed using the SSCP method described previously for DRB1 alleles [19] and for B22 alleles [18] with minor modifications. Electrophoresis was carried out in a 10% polyacrylamide gel, and the electrophoretic temperature was 22 °C for exon 2 and 30 °C for exon 3. Each of different B*44 alleles (B*4402, *44031, and *44032), which was discriminated in the SSCP analysis, was confirmed by direct sequencing of randomly selected two samples from each of the three different B*44 subtypes. Absence of other rare B*44 alleles was also verified by reverse SSO typing using the Dynal RELI™ SSO HLA-B Test (56 probes; Dynal Biotech, United Kingdom), which can differentiate B*4402, *44031/44032/4407, *4404, *4405, *4406, *4408, *4409, *4410, and *4411 alleles. Molecular typing of HLA-Cw alleles was performed using the Dynal RELI™ SSO HLA-Cw Test (36 probes; Dynal Biotech) on all 450 samples. HLADRB1 allele typing was performed using the PCR-SSCP method [19]. HLA-DQB1 allele typing was performed for family samples using a combination of the PCRRFLP and PCR-SSCP method as previously described [20].
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Molecular Typing of HLA-A Alleles Haplotype analysis revealed strong associations between HLA-B44 subtypes and A3, A32, or A33 alleles. The majority (71/79) of the B44-positive individuals was positive for A33 (n ⫽ 63), A3 (n ⫽ 6), or A32 (n ⫽ 4); these HLA-A specificities were further analyzed for exons 2 and 3 at allelic level. A33 genotyping was performed by the sequence-specific primer (SSP) method. A*3301/ 3/4 (⫹A*23/*24/*29/*30/*31) PCR was done using A137 and A90c primers as previously described [21]. A*3301 PCR was done with A137 and A33-1 (GTTCCCGTTCTCCAGGTG) primers, and A*3304 PCR was done with A137 and A33-4 (CGCCGCGGACCAGGAGCT) primers. A*03 genotpying was performed by the PCR-SSCP method. Two-step PCRs were performed. The first PCR was perfomed to amplify the fragment encompassing exons 2 and 3 of A*03 alleles, using AI1-221 and A90c primers [21, 22], and then a second PCR was perfomed separately using AI1-221 and ASEQ3 primers for exon 2, and ASEQ5 and A90c primers for exon 3 [21, 22]. Exon 2 and 3 fragments, amplified in the second PCR, were separately analyzed using the SSCP method (10% polyacrylamide gel, 22°C). A*32 alleles were amplified using 5⬘FR-279 and A90c primers [21, 22], and were analyzed by direct sequencing. All of the A33- and A3-positive samples were typed as A*3303 and A*0301, and two randomly selected samples from each allelic type were further confirmed by direct sequencing. Statistical Analysis Allele and haplotype (two-, three-, and four-locus) frequencies were estimated by the maximum likelihood method using a computer program developed for the 11th International Histocompatibility Workshop [23]. Linkage disequilibrium and Chi-square values were calculated by the same method. RESULTS The distribution of B*44 alleles and related two-locus haplotypes with haplotype frequencies (HF) of 肁 0.3% and significant associations (2 肁 10.83, p ⬍ 0.001) are illustrated in Table 1. Three- and four-locus haplotypes carrying B*44 alleles with frequencies of 肁 0.3% are also depicted in Table 2. Three different B*44 alleles were detected in 450 Koreans: B*44031 (allele frequency 4.7%), B*44032 (3.1%), and B*4402 (1.3%). B*44031 was strongly associated with A*3303, Cw*1403, and DRB1*1302 alleles; these alleles comprised the most common B*44-bearing extended haplotype: A*3303Cw*1403-B*44031-DRB*1302 (HF: 3.6%). B*44032 was strongly associated with A*3303, Cw*07, and DRB1*0701; these alleles comprised the second most
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TABLE 1 Two-locus haplotypes carrying HLA-B*44 with significant positive linkage disequilibrium in Koreans Two-locus haplotypea B*44
AF (%)
B*4402
1.3
B*44031
4.7
B*44032
3.1
HF (%)
LD
2
0.6 0.4 1.3 0.6 4.2 4.7 3.9 2.9 3.0 3.0
0.5 0.4 1.3 0.4 3.5 4.4 3.6 2.4 2.6 2.8
135.5 177.5 896.9 17.7 186.9 866.2 362.4 137.0 219.8 359.6
A*0301 A*3201 Cw*05 DRB1*0405 A*3303 Cw*1403 DRB1*1302 A*3303 Cw*07 DRB1*0701
Two-locus haplotypes with haplotype frequency 肁 0.3% and Chi-square value 肁 10.83 (p ⬍ 0.001) are listed. Abbreviations: AF ⫽ allele frequency; HF ⫽ haplotype frequency; LD ⫽ linkage disequilibrium value (⫻ 100). a
common haplotype: A*3303-Cw*07-B*44032DRB1*0701 (2.8%). B*4402 was strongly associated with A*3201, Cw*05, and less strongly with DRB1*0405; these alleles comprised a distinctive haplotype: A*3201-Cw*05-B*4402-DRB1*0405 (0.4%). B*4402 also revealed a strong association with A*0301, and this haplotype was associated with Cw*05, however, it was not extended to distinctive DRB1 association. These haplotypic associations estimated by the maximum likelihood method were well correlating with those of the segregated haplotypes observed in 213 parents from 107 families by the direct counting method. A total of 46 B44-bearing A-B-DR haplotypes were identified in 44 B44-positive individuals. A total of 13 different B44-bearing A-B-DR haplotypes were identified: A*3303-B*44031-DRB1*1302 (n ⫽ 22) and A*3303B*44032-DRB1*0701 (n ⫽ 13) comprised 76% of the total 46 haplotypes, and each of the remaining 11 different haplotypes occurred only once. For the B44-bearing extended haplotype, DQB1 data was available for the family samples. The two predominant B44-bearing haplotypes in family samples revealed
very strong associations with DQB1 alleles. The A*3303-Cw*1403-B*44031-DRB1*1302 haplotype was almost exclusively (21/22) associated with DQB1*0604, and A*3303-Cw*07-B*44032-DRB1*0701 was exclusively (13/13) associated with DQB1*02. In Koreans, DRB1*1302 is associated with either DQB1*0604 or *0609 (about 3:1 ratio, unpublished data). However, B*44031-DRB1*1302 haplotype was associated mainly with DQB1*0604 and not with DQB1*0609, which is similar to that observed in Japanese for Cw*1403B*4403-DRB1*1302-DQB1*0604 haplotype [10]. DISCUSSION B*44 allele frequencies from this study of 450 Koreans were compared with those of other ethnic groups and depicted in Table 3. In Koreans, B*4403 was the predominant subtype with a subtype frequency of 86% (B*44031, 52%; B*44032, 34%), and B*4402 comprised only a small portion (14%). Other rare B44 subtypes encountered in other populations were not observed in Koreans. Generally, in European and North
TABLE 2 Three- and four-locus haplotypes carrying HLA-B*44 alleles in Koreans Haplotypea A* 3201 3201 3303 3303 3303 3303
Cw* 05 1403 07
B*
DRB1*
HF (%)
LD
4402 4402 44031 44031 44032 44032
0405 0405 1302 1302 0701 0701
0.4 0.4 3.6 3.6 2.8 2.8
0.4 0.4 3.5 3.6 2.7 2.8
Three- and four-locus haplotypes with haplotype frequency 肁 0.3% are listed. Abbreviations: HF ⫽ haplotype frequency; LD ⫽ linkage disequilibrium value (⫻ 100). a
HLA-B*44 Alleles and Haplotypes in Koreans
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TABLE 3 Comparison of B*44 allele frequencies (per 10,000) in Koreans with other ethnic groups
B*4402 B*44031 B*44032 B*4404 B*4405 B*4406 Othersc
Koreansa (n ⫽ 450)
Asianb (n ⫽ 3599)
Africanb (n ⫽ 253)
Eur. Caub (n ⫽ 29,204)
N. Am. Caub (n ⫽ 417)
125.7 473.7 305.8
90.5 143.5 111.4 20.4 20.4 20.4 20.4
102.2 646.9 155.5 39.6 39.6 39.6 89.1
863.3 279.4 87.6 22.8 57.1 33.2 40.6
907.0 98.6 98.6 36.1 36.1 36.1 98.6
Abbreviations: Eur. Cau ⫽ European Caucasian; N. Am. Cau ⫽ North American Caucasian. The highest allele frequency in each ethnic group is shown in boldface. a Present study. b Allele frequencies according to Gjertson and Lee [12]. c B*4407 and B*4409.
American Caucasians, the most common B44 subtype is B*4402 [3, 6, 9, 12]. However, there are some ethnic differences and B*4403 is more frequent than B*4402 in Southern European Caucasians, such as Spanish and Italian populations [7, 8]. The distribution of B44 subtypes has not been well studied in Asian and African populations, and limited data suggest that B*4403 is predominant in these populations, with B*44031 occurring at a higher frequency than B*44032 [12]. Besides marked differences in the distribution of B44 subtypes, there are also marked differences in their haplotypic associations among different ethnic groups. Characteristic B*44-bearing A*-B* haplotypes in this study (HF 肁 0.3%) and the two most common B*44-bearing haplotypes in each four other ethnic groups [12] are compared in Table 4. In Koreans and other Asian populations, the two most common haplotypes are A*3303B*44031 and A*3303-B*44032. Although B*44031 is the predominant B44 subtype in Africans, the haplotypic association is quite different, revealing predominant as-
sociation with A*0301 and A*2301. The two B*4402bearing haplotypes of Koreans, A*0301-B*4402 and A*3201-B*4402, are quite rare in Asian and African populations. It is of interest to note that these haplotypes are present in Caucasians at similar frequencies as observed in Koreans, although the predominant B*4402bearing haplotype is A*0201-B*4402 in both European and North American Caucasians. As for the B*44-bearing extended haplotypes, two characteristic haplotypes were observed in Koreans that reveal strong linkage disequilibrium: A*3303Cw*1403-B*44031-DRB1*1302 (3.6%) and A*3303Cw*07-B*44032-DRB1*0701 (2.8%). These two haplotypes appear to be relatively common in Asian populations, although the latter is rare in Japanese [1, 10]. A33-B44-DR6 (1.5%) and A33-B44-DR7 (1.3%) are the second and the fifth most common haplotypes represented in the NMDP donor file of Asian ethnic group [24]. In Caucasians A2-B44-DR4 (2.2%) and A29-B44-DR7 (1.5%) are the third and the fifth most
TABLE 4 Comparison of B*44-bearing A*-B* haplotype frequencies (per 10,000) in Koreans with other ethnic groups A*-B*a 0201-4402 0301-4402 3201-4402 0301-44031 2301-44031 2902-44031 3303-44031 3303-44032
Koreans (n ⫽ 450) 55.4 44.4
419.1 287.3
Asianb (n ⫽ 3599)
Africanb (n ⫽ 253)
Eur. Caub (n ⫽ 29,204)
N. Am. Caub (n ⫽ 417)
3.4 7.9 0.2 0.4 0.2 0.1 71.3 51.4
6.5 5.4 9.5 62.4 77.9 7.3 7.4 1.8
451.3 63.4 41.7 6.6 16.3 136.4 0.2 0.1
393.4 61.2 13.2 8.0 3.6 8.5 0.2 0.2
Abbreviations: Eur. Cau ⫽ European Caucasian; N. Am. Cau ⫽ North American Caucasian. a Two-locus (A*-B*) haplotypes with haplotype frequency (per 10,000) 肁 30.0 and Chi-square value 肁 10.83 (p ⬍ 0.001) from the present study of Koreans and the two most common haplotypes in each of four other ethnic groups are listed. The two most common haplotypes in each ethnic group are depicted in boldface. b Haplotype frequencies according to Gjertson and Lee [12].
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common haplotypes represented in the NMDP donor file [24]. In the present study, analysis of 107 families revealed that the diversity of B44-bearing A-B-DR haplotypes is much lower in Koreans compared with that of Caucasians. A total of 13 different haplotypes were identified among 46 B44-bearing haplotypes, and the two most common haplotypes comprised 76% of the total haplotypes. In comparison, in Spanish and Italian families, the numbers of different A-B-DR haplotypes among B44-bearing haplotypes were 19/31 and 37/85, respectively, and the two most common haplotypes comprised only 25%–32% of the total haplotypes [7, 8]. The distinct epitopes that define the B44 molecules can be recognized by T lymphocytes and cause strong allogeneic responses [14, 25]. It has been reported that some of the serologically matched donor-recipient pairs in unrelated bone marrow transplantation mismatch for B44 subtypes [8, 14, 26]. The strong linkage disequilibrium between HLA-B*44 alleles and other HLA alleles is the key for the low frequency of HLA-B44 mismatches between patients and their serologically matched unrelated donors [8]. When a patient presents unusual haplotypes, B44 subtype mismatches could possibly occur if donor selection is based on A, B serology. The identification of conserved and unusual B44 haplotypes in the present study of Koreans would have important clinical implications for matching donor-recipient pairs in unrelated bone marrow transplantation. In summary, B*4403 is the predominant B44 subtype (86%) in Koreans, and B*4402 (14%) accounts for the rest of the B44 alleles. Two characteristic B*4403-bearing haplotypes of Koreans (A*3303-Cw*1403B*44031-DRB1*1302 and A*3303-Cw*07-B*44032DRB1*0701) appear to be relatively common in Asian populations, whereas the B*4402-bearing haplotypes share some similarity to those of Caucasians. The limited diversity in the distribution of B*44 alleles and associated haplotypes in Koreans suggests that the frequencies of B44 subtype mismatches among ABDRB1-matched unrelated donor-recipient pairs would be low in this population. ACKNOWLEDGMENT
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This study was supported by grant No. 03-98-060-2 from the SNUH Research Fund. 12.
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ABBREVIATIONS AF allele frequency HF haplotype frequency PCR polymerase chain reaction RFLP restriction fragment length polymorphism
INTRODUCTION HLA-B44 is one of the most frequently found HLA-B specificities in many Caucasian and Asian populations with allele frequencies of approximately 5%–15% [1]. Although serologic HLA-B44 specificity is encoded by at least 25 different alleles (B*4402-B*4424) identified to date [2], B*4402 and B*4403 are by far the two predominant alleles found in all populations studied [3–10]. B*4403 alleles are subdivided into B*44031 and
From the Department of Clinical Pathology (E.Y.S., D.H.W., M.H., K.S.H., M.H.P.), Seoul National University College of Medicine, and Clinical Research Institute (S.J.K., M.H.P.), Seoul National University Hospital, Seoul, Korea. Address reprint requests to: Dr. Myoung Hee Park, Department of Clinical Pathology, Seoul National University College of Medicine, 28 Yongon-dong, Chongno-gu, Seoul 110-744, Korea; Tel: ⫹82 (2) 7603388; Fax: ⫹82 (2) 3672-3337; E-mail: [email protected]. Received November 15, 2000; revised February 12, 2001; accepted June 15, 2001. Human Immunology 63, 1142–1147 (2001) © American Society for Histocompatibility and Immunogenetics, 2001 Published by Elsevier Science Inc.
A*0301. The B*4403-bearing haplotypes of Koreans appear to be relatively common in Asian populations, whereas the B*4402-bearing haplotypes share some similarity to those of Caucasians. HLA-B44 alleles demonstrate a limited allelic diversity and comprise distinctive extended haplotypes in the Korean population. It is suggested that the frequencies of B44 subtype mismatches among ABDRB1-matched unrelated donor-recipient pairs would be low in this population. Human Immunology 63, 1142–1147 (2001). © American Society for Histocompatibility and Immunogenetics, 2001. Published by Elsevier Science Inc. KEYWORDS: allele frequency; HLA-B*44; HLA-B44 subtype; haplotype; Korean
SSCP SSO SSP
single strand conformation polymorphism sequence specific oligonucleotide sequence specific primer
B*44032 by a silent substitution of T for C at nucleotide 369 [11]. Previous studies suggested that the frequencies of B44 subtypes are quite different among various ethnic groups, and these B44 subtypes seem to be in close linkage disequilibrium with different HLA alleles [3–10, 12]. Despite the well-known ethnic differences in the distribution of B44 subtypes in Caucasians, the distribution and haplotypic association of B44 alleles with other HLA loci have not been well studied in Asian populations. The clinical importance of HLA-B44 polymorphism has been highlighted by a report on the generation of cytotoxic T lymphocytes (CTL) directed against B44 subtype mismatch in bone marrow allograft rejection [13, 14]. Identification of conserved and uncommon B44 haplotypes among different populations would be useful for unrelated bone marrow donor searches. This report summarizes the B44 allelic diversity and haplotypic associations in Koreans. 0198-8859/01/$–see front matter PII S0198-8859(01)00304-4
HLA-B*44 Alleles and Haplotypes in Koreans
MATERIALS AND METHODS Population Studied A total of 450 healthy unrelated Koreans from two groups of individuals were studied. One group consisted of 213 parents from 107 families, as previously reported [15], and the other group consisted of 237 volunteer bone marrow donors enrolled in the Korean Marrow Donor Program (KMDP). For family samples, HLA-A, -B, -C, and -DR antigens were typed by the standard serologic method previously reported [15]. For KMDP donors, HLA-A, -B, -C serologic typing was performed using a commercial kit designed for Oriental populations (OT-72; One Lambda, Canoga Park, CA, USA) and HLA-DRB1 generic DNA typing was performed using the Amplicor威 HLA DRB kit (Roche Diagnostic System, Branchburg, NJ, USA). Molecular Typing of HLA-B44, -Cw, -DRB1, and -DQB1 Alleles HLA-B44 allele typing was performed for all of the samples serologically typed as B44-positive (n ⫽ 79) using two-step polymerase chain reaction (PCR) and single-strand conformation polymorphism (SSCP) analysis. First, PCR was performed to amplify the fragment encompassing exons 2 and 3 using dimorphic primers (5BIN1-CG and 3BIN3-37) as previously described [16]. Second, PCR was performed separately using BEX2-1 and BEX2-FC primers for exon 2, and BEX3-1 and BINT3-1C primers for exon 3 [17, 18]. Exon 2 and 3 fragments, amplified in the second PCR, were separately analyzed using the SSCP method described previously for DRB1 alleles [19] and for B22 alleles [18] with minor modifications. Electrophoresis was carried out in a 10% polyacrylamide gel, and the electrophoretic temperature was 22 °C for exon 2 and 30 °C for exon 3. Each of different B*44 alleles (B*4402, *44031, and *44032), which was discriminated in the SSCP analysis, was confirmed by direct sequencing of randomly selected two samples from each of the three different B*44 subtypes. Absence of other rare B*44 alleles was also verified by reverse SSO typing using the Dynal RELI™ SSO HLA-B Test (56 probes; Dynal Biotech, United Kingdom), which can differentiate B*4402, *44031/44032/4407, *4404, *4405, *4406, *4408, *4409, *4410, and *4411 alleles. Molecular typing of HLA-Cw alleles was performed using the Dynal RELI™ SSO HLA-Cw Test (36 probes; Dynal Biotech) on all 450 samples. HLADRB1 allele typing was performed using the PCR-SSCP method [19]. HLA-DQB1 allele typing was performed for family samples using a combination of the PCRRFLP and PCR-SSCP method as previously described [20].
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Molecular Typing of HLA-A Alleles Haplotype analysis revealed strong associations between HLA-B44 subtypes and A3, A32, or A33 alleles. The majority (71/79) of the B44-positive individuals was positive for A33 (n ⫽ 63), A3 (n ⫽ 6), or A32 (n ⫽ 4); these HLA-A specificities were further analyzed for exons 2 and 3 at allelic level. A33 genotyping was performed by the sequence-specific primer (SSP) method. A*3301/ 3/4 (⫹A*23/*24/*29/*30/*31) PCR was done using A137 and A90c primers as previously described [21]. A*3301 PCR was done with A137 and A33-1 (GTTCCCGTTCTCCAGGTG) primers, and A*3304 PCR was done with A137 and A33-4 (CGCCGCGGACCAGGAGCT) primers. A*03 genotpying was performed by the PCR-SSCP method. Two-step PCRs were performed. The first PCR was perfomed to amplify the fragment encompassing exons 2 and 3 of A*03 alleles, using AI1-221 and A90c primers [21, 22], and then a second PCR was perfomed separately using AI1-221 and ASEQ3 primers for exon 2, and ASEQ5 and A90c primers for exon 3 [21, 22]. Exon 2 and 3 fragments, amplified in the second PCR, were separately analyzed using the SSCP method (10% polyacrylamide gel, 22°C). A*32 alleles were amplified using 5⬘FR-279 and A90c primers [21, 22], and were analyzed by direct sequencing. All of the A33- and A3-positive samples were typed as A*3303 and A*0301, and two randomly selected samples from each allelic type were further confirmed by direct sequencing. Statistical Analysis Allele and haplotype (two-, three-, and four-locus) frequencies were estimated by the maximum likelihood method using a computer program developed for the 11th International Histocompatibility Workshop [23]. Linkage disequilibrium and Chi-square values were calculated by the same method. RESULTS The distribution of B*44 alleles and related two-locus haplotypes with haplotype frequencies (HF) of 肁 0.3% and significant associations (2 肁 10.83, p ⬍ 0.001) are illustrated in Table 1. Three- and four-locus haplotypes carrying B*44 alleles with frequencies of 肁 0.3% are also depicted in Table 2. Three different B*44 alleles were detected in 450 Koreans: B*44031 (allele frequency 4.7%), B*44032 (3.1%), and B*4402 (1.3%). B*44031 was strongly associated with A*3303, Cw*1403, and DRB1*1302 alleles; these alleles comprised the most common B*44-bearing extended haplotype: A*3303Cw*1403-B*44031-DRB*1302 (HF: 3.6%). B*44032 was strongly associated with A*3303, Cw*07, and DRB1*0701; these alleles comprised the second most
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TABLE 1 Two-locus haplotypes carrying HLA-B*44 with significant positive linkage disequilibrium in Koreans Two-locus haplotypea B*44
AF (%)
B*4402
1.3
B*44031
4.7
B*44032
3.1
HF (%)
LD
2
0.6 0.4 1.3 0.6 4.2 4.7 3.9 2.9 3.0 3.0
0.5 0.4 1.3 0.4 3.5 4.4 3.6 2.4 2.6 2.8
135.5 177.5 896.9 17.7 186.9 866.2 362.4 137.0 219.8 359.6
A*0301 A*3201 Cw*05 DRB1*0405 A*3303 Cw*1403 DRB1*1302 A*3303 Cw*07 DRB1*0701
Two-locus haplotypes with haplotype frequency 肁 0.3% and Chi-square value 肁 10.83 (p ⬍ 0.001) are listed. Abbreviations: AF ⫽ allele frequency; HF ⫽ haplotype frequency; LD ⫽ linkage disequilibrium value (⫻ 100). a
common haplotype: A*3303-Cw*07-B*44032DRB1*0701 (2.8%). B*4402 was strongly associated with A*3201, Cw*05, and less strongly with DRB1*0405; these alleles comprised a distinctive haplotype: A*3201-Cw*05-B*4402-DRB1*0405 (0.4%). B*4402 also revealed a strong association with A*0301, and this haplotype was associated with Cw*05, however, it was not extended to distinctive DRB1 association. These haplotypic associations estimated by the maximum likelihood method were well correlating with those of the segregated haplotypes observed in 213 parents from 107 families by the direct counting method. A total of 46 B44-bearing A-B-DR haplotypes were identified in 44 B44-positive individuals. A total of 13 different B44-bearing A-B-DR haplotypes were identified: A*3303-B*44031-DRB1*1302 (n ⫽ 22) and A*3303B*44032-DRB1*0701 (n ⫽ 13) comprised 76% of the total 46 haplotypes, and each of the remaining 11 different haplotypes occurred only once. For the B44-bearing extended haplotype, DQB1 data was available for the family samples. The two predominant B44-bearing haplotypes in family samples revealed
very strong associations with DQB1 alleles. The A*3303-Cw*1403-B*44031-DRB1*1302 haplotype was almost exclusively (21/22) associated with DQB1*0604, and A*3303-Cw*07-B*44032-DRB1*0701 was exclusively (13/13) associated with DQB1*02. In Koreans, DRB1*1302 is associated with either DQB1*0604 or *0609 (about 3:1 ratio, unpublished data). However, B*44031-DRB1*1302 haplotype was associated mainly with DQB1*0604 and not with DQB1*0609, which is similar to that observed in Japanese for Cw*1403B*4403-DRB1*1302-DQB1*0604 haplotype [10]. DISCUSSION B*44 allele frequencies from this study of 450 Koreans were compared with those of other ethnic groups and depicted in Table 3. In Koreans, B*4403 was the predominant subtype with a subtype frequency of 86% (B*44031, 52%; B*44032, 34%), and B*4402 comprised only a small portion (14%). Other rare B44 subtypes encountered in other populations were not observed in Koreans. Generally, in European and North
TABLE 2 Three- and four-locus haplotypes carrying HLA-B*44 alleles in Koreans Haplotypea A* 3201 3201 3303 3303 3303 3303
Cw* 05 1403 07
B*
DRB1*
HF (%)
LD
4402 4402 44031 44031 44032 44032
0405 0405 1302 1302 0701 0701
0.4 0.4 3.6 3.6 2.8 2.8
0.4 0.4 3.5 3.6 2.7 2.8
Three- and four-locus haplotypes with haplotype frequency 肁 0.3% are listed. Abbreviations: HF ⫽ haplotype frequency; LD ⫽ linkage disequilibrium value (⫻ 100). a
HLA-B*44 Alleles and Haplotypes in Koreans
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TABLE 3 Comparison of B*44 allele frequencies (per 10,000) in Koreans with other ethnic groups
B*4402 B*44031 B*44032 B*4404 B*4405 B*4406 Othersc
Koreansa (n ⫽ 450)
Asianb (n ⫽ 3599)
Africanb (n ⫽ 253)
Eur. Caub (n ⫽ 29,204)
N. Am. Caub (n ⫽ 417)
125.7 473.7 305.8
90.5 143.5 111.4 20.4 20.4 20.4 20.4
102.2 646.9 155.5 39.6 39.6 39.6 89.1
863.3 279.4 87.6 22.8 57.1 33.2 40.6
907.0 98.6 98.6 36.1 36.1 36.1 98.6
Abbreviations: Eur. Cau ⫽ European Caucasian; N. Am. Cau ⫽ North American Caucasian. The highest allele frequency in each ethnic group is shown in boldface. a Present study. b Allele frequencies according to Gjertson and Lee [12]. c B*4407 and B*4409.
American Caucasians, the most common B44 subtype is B*4402 [3, 6, 9, 12]. However, there are some ethnic differences and B*4403 is more frequent than B*4402 in Southern European Caucasians, such as Spanish and Italian populations [7, 8]. The distribution of B44 subtypes has not been well studied in Asian and African populations, and limited data suggest that B*4403 is predominant in these populations, with B*44031 occurring at a higher frequency than B*44032 [12]. Besides marked differences in the distribution of B44 subtypes, there are also marked differences in their haplotypic associations among different ethnic groups. Characteristic B*44-bearing A*-B* haplotypes in this study (HF 肁 0.3%) and the two most common B*44-bearing haplotypes in each four other ethnic groups [12] are compared in Table 4. In Koreans and other Asian populations, the two most common haplotypes are A*3303B*44031 and A*3303-B*44032. Although B*44031 is the predominant B44 subtype in Africans, the haplotypic association is quite different, revealing predominant as-
sociation with A*0301 and A*2301. The two B*4402bearing haplotypes of Koreans, A*0301-B*4402 and A*3201-B*4402, are quite rare in Asian and African populations. It is of interest to note that these haplotypes are present in Caucasians at similar frequencies as observed in Koreans, although the predominant B*4402bearing haplotype is A*0201-B*4402 in both European and North American Caucasians. As for the B*44-bearing extended haplotypes, two characteristic haplotypes were observed in Koreans that reveal strong linkage disequilibrium: A*3303Cw*1403-B*44031-DRB1*1302 (3.6%) and A*3303Cw*07-B*44032-DRB1*0701 (2.8%). These two haplotypes appear to be relatively common in Asian populations, although the latter is rare in Japanese [1, 10]. A33-B44-DR6 (1.5%) and A33-B44-DR7 (1.3%) are the second and the fifth most common haplotypes represented in the NMDP donor file of Asian ethnic group [24]. In Caucasians A2-B44-DR4 (2.2%) and A29-B44-DR7 (1.5%) are the third and the fifth most
TABLE 4 Comparison of B*44-bearing A*-B* haplotype frequencies (per 10,000) in Koreans with other ethnic groups A*-B*a 0201-4402 0301-4402 3201-4402 0301-44031 2301-44031 2902-44031 3303-44031 3303-44032
Koreans (n ⫽ 450) 55.4 44.4
419.1 287.3
Asianb (n ⫽ 3599)
Africanb (n ⫽ 253)
Eur. Caub (n ⫽ 29,204)
N. Am. Caub (n ⫽ 417)
3.4 7.9 0.2 0.4 0.2 0.1 71.3 51.4
6.5 5.4 9.5 62.4 77.9 7.3 7.4 1.8
451.3 63.4 41.7 6.6 16.3 136.4 0.2 0.1
393.4 61.2 13.2 8.0 3.6 8.5 0.2 0.2
Abbreviations: Eur. Cau ⫽ European Caucasian; N. Am. Cau ⫽ North American Caucasian. a Two-locus (A*-B*) haplotypes with haplotype frequency (per 10,000) 肁 30.0 and Chi-square value 肁 10.83 (p ⬍ 0.001) from the present study of Koreans and the two most common haplotypes in each of four other ethnic groups are listed. The two most common haplotypes in each ethnic group are depicted in boldface. b Haplotype frequencies according to Gjertson and Lee [12].
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common haplotypes represented in the NMDP donor file [24]. In the present study, analysis of 107 families revealed that the diversity of B44-bearing A-B-DR haplotypes is much lower in Koreans compared with that of Caucasians. A total of 13 different haplotypes were identified among 46 B44-bearing haplotypes, and the two most common haplotypes comprised 76% of the total haplotypes. In comparison, in Spanish and Italian families, the numbers of different A-B-DR haplotypes among B44-bearing haplotypes were 19/31 and 37/85, respectively, and the two most common haplotypes comprised only 25%–32% of the total haplotypes [7, 8]. The distinct epitopes that define the B44 molecules can be recognized by T lymphocytes and cause strong allogeneic responses [14, 25]. It has been reported that some of the serologically matched donor-recipient pairs in unrelated bone marrow transplantation mismatch for B44 subtypes [8, 14, 26]. The strong linkage disequilibrium between HLA-B*44 alleles and other HLA alleles is the key for the low frequency of HLA-B44 mismatches between patients and their serologically matched unrelated donors [8]. When a patient presents unusual haplotypes, B44 subtype mismatches could possibly occur if donor selection is based on A, B serology. The identification of conserved and unusual B44 haplotypes in the present study of Koreans would have important clinical implications for matching donor-recipient pairs in unrelated bone marrow transplantation. In summary, B*4403 is the predominant B44 subtype (86%) in Koreans, and B*4402 (14%) accounts for the rest of the B44 alleles. Two characteristic B*4403-bearing haplotypes of Koreans (A*3303-Cw*1403B*44031-DRB1*1302 and A*3303-Cw*07-B*44032DRB1*0701) appear to be relatively common in Asian populations, whereas the B*4402-bearing haplotypes share some similarity to those of Caucasians. The limited diversity in the distribution of B*44 alleles and associated haplotypes in Koreans suggests that the frequencies of B44 subtype mismatches among ABDRB1-matched unrelated donor-recipient pairs would be low in this population. ACKNOWLEDGMENT
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This study was supported by grant No. 03-98-060-2 from the SNUH Research Fund. 12.
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