HLA-A∗28 allele frequencies in the five major U.S. ethnic groups

HLA-A*28 Allele Frequencies in the Five Major U.S. Ethnic Groups Eileen Hsu, Mian Bei, Rebecca Slack, Robert J. Hartzman, Jennifer Ng, and Carolyn Katovich Hurley ABSTRACT: The frequency of each A*28 allele was determined by PCR-SSOP typing in 5 major U.S. ethnic populations: Caucasians, African Americans, Asians/Pacific Islanders, Hispanics, and Native Americans. The percent of serologically defined A28-positive individuals in the 5 populations ranged from 2.7–17.9%. Fifty-nine individuals who were previously serologically typed as A28, A68 or A69 were randomly chosen for allele-level typing from each ethnic group from a database of 82,979 consecutively typed unrelated individuals. The most common A*28 allele for Caucasians, Asians/Pacific Islanders, Hispanics, and Native Americans was A*68012, while

INTRODUCTION HLA matching of donor and recipient is essential for optimal outcome in hematopoietic stem cell transplantation. Frequency studies have shown that the distribution of HLA alleles can vary greatly among different racial and ethnic populations in unrelated stem cell registries [1–3] and can impact donor searches [4]. A patient is more likely to find an HLA matched donor within the same ethnic group, but some groups, such as African Americans, have a broader distribution of HLA alleles making it difficult for a patient to find a match [4]. A further complication in donor searches arises from the limitations of the methodology used to define HLA types of registry donors. Serologic typing lacks the ability to distinguish each HLA allelic product and crossreactivity of alloantisera creates the problem of inconsisFrom the Departments of Microbiology and Immunology (E.H., M.B., C.K.H.), Biostatistics (R.S.), and Pediatrics (J.N.), Georgetown University Medical Center, Washington, District of Columbia; Naval Medical Research Institute (R.J.H., J.N.), Bethesda, Maryland, USA. Address reprint requests to: Carolyn Katovich Hurley, Ph.D., E404 Research Building, Georgetown University Medical Center, 3970 Reservoir Road NW, Washington, DC 20007, USA; Fax: 11 202-687-7505; E-Mail: [email protected] Received 27 August 1998; accepted 13 October 1998. Human Immunology 60, 159 –167 (1999) © American Society for Histocompatibility and Immunogenetics, 1999 Published by Elsevier Science Inc.

A*6802 was found in the majority of African Americans. Only four and three A*28 alleles were seen in Caucasians and African Americans, respectively, while five to six A*28 alleles were seen in the other population groups. The A*6804 and A*6806 alleles were not observed in any of the five ethnic groups. Human Immunology 60, 159 –167 (1999). © American Society for Histocompatibility and Immunogenetics, 1999. Published by Elsevier Science Inc. KEYWORDS: HLA-A*28; SSOP typing; subtyping; allele frequency

tent serologic assignments [5–7]. Recently, DNA-based typing has been used to define HLA types because of its increased accuracy, specificity and reliability [8]. The ability of DNA typing methods to resolve HLA types to the allelic level now allows a transplant center to select an HLA allele-matched donor, potentially decreasing the probability of graft versus host disease and graft rejection and increasing survival [9 –11]. DNA-based allelic level typing can be performed for HLA-A subtypes that are difficult to define by serology, such as HLA-A28, which is subdivided into its serologic splits (A68 and A69) (Fig. 1) only 10 –15% of the time [2]. A previous study had shown that HLA-A28 is found in significant portions of the African American and Hispanic populations (9 –10%) and in smaller portions of Caucasian, Asian/Pacific Islander and Native American groups (2–5%) in the U.S. registry [2]. To date, there are nine identified alleles in the A*28 family, eight A68 alleles (A*68011, A*68012, A*6802, A*68031, A*68032, A*6804, A*6805, A*6806) and one A69 allele (A*6901) (Fig. 1) [12–14]. The allelic products differ from each other by 1– 8 amino acid residues in the a1 and a2 domains. Two pairs of alleles (A*68011, A*68012 and A*68031, A*68032) differ in nucleotide 0198-8859/99/$–see front matter PII S0198-8859(98)00105-0

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FIGURE 1 The HLA-A alleles specifying the broad antigen A28 can be further divided into two groups that are defined by the split antigens A68 and A69. Currently, nine A*28 alleles have been identified [12–14].

sequences but share the same amino acid sequence (Fig. 2). In this study, we determined the frequencies of eight of the nine HLA-A*28 alleles in five different U.S. ethnic populations. The alleles, A*68031 and A*68032, which differ by a silent nucleotide substitution in exon 2 were not distinguished in this study. MATERIALS AND METHODS Unrelated individuals from many geographic locations throughout the United States were serologically typed using standard microcytotoxicity assays with commercial and local trays. The ethnic background(s) of each individual was self-defined. Individuals who selected multiple ethnic backgrounds were classified into a single ethnic group for this study. In order to have a 95% probability of finding a rare allele that occurs in 5% of the A28 positive population, 59 samples per ethnic group were randomly chosen from a database of individuals previously serologically typed as A28, A68 or A69. A randomization list was created using the random numFIGURE 2 The exon 2 and 3 nucleotide sequence differences among the nine A*28 alleles. GenBank accession numbers for each allele: A*68011, X03070, X03071; A*68012, L06425; A*6802, U03861; A*68031, U43336; A*68032, U89947, A*6804, U41844; A*6805, U43335; A*6806, U91627, U91628; A*6901, X03158, X03159 [12–14].

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ber generator for a uniform distribution in GAUSS (GAUSS 3.2 Mathematical and Statistical System, Aptech System, Maple Valley, WA). Since there were only 57 A28 positive Asian/Pacific Islander samples available, all were used in the study. Genomic DNA was extracted from whole blood or peripheral blood lymphocytes using the QIAamp Blood Kit (QIAGEN, Santa Clarita, CA) or Pel-Freez DNA Isolation Kit (Brown Deer, WI). The HLA-A alleles were amplified with generic A-locus primers (5AIn1-46/ 3AIn3-62) and identified using SSOP as previously described [15, 16] (Table 1). HLA-A alleles were identified using an HLA typing software program which compared the hybridization patterns of the samples to known alleles [17]. Samples whose initial SSOP hybridization results could not resolve the A*28 allele were further analyzed by separation of HLA-A alleles. In some cases, the A*28 allele was amplified specifically from genomic DNA using A*28-specific primers (Table 2). PCR reaction mixtures with the primer pairs 5AEx2/3AIn3-62 and 5AIn1-A/AL#H contained 1.0 mM MgCl2. For 5AEx2/ 3AIn3-62, 30 amplification cycles were run at 94°C for 22 s, 63°C 50 s, and 72°C 30 sec. For 5AIn-A/AL#H, 30 cycles were run at 94°C for 22 s, 64°C 50 s, and 72°C 30 s. PCR reactions with the primer pair 5AIn-A/ 3AIn3-62 were performed under the same conditions as the generic A locus amplification [15]. Amplified HLA-A locus DNA from samples with alleles that could not be separated by PCR was ligated into the pCR™II vector (Invitrogen, Carlsbad, CA) and cloned in NovaBlue Competent Cells (Novagen, Madison, WI). SSOP typing determined the identity of the cloned alleles. For some samples, SSOP hybridization experiments exhibited unclear SSOP reactivity patterns. The PCRamplified HLA-A alleles in these samples were sequenced using an ABI Prism™ Dye Terminator Cycle Sequencing Ready Reaction Kit (Perkin-Elmer, Forster City, CA) on an ABI Prism 377 automatic sequencer. The sequencing primers 5AIn1-A, 5AIn1-G (59 GGG GCG CAG GAC CCG GGG), 3AIn3-62, Internal 2F (59

HLA-A*28 Frequencies in 5 US Ethnic Groups

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TABLE 1 SSOP used for HLA-A typinga

TTA CCC GGT TTC ATT TTC AG), and Internal 2R (59 GGA TCT CGG ACC CGG AG) were used. The frequency distributions of the A*28 alleles were tested using chi-square tests for a difference among the 5 ethnic groups. The test was considered significant if the p-value was less than 0.05. In order to control for the ten tests comparing each pair of ethnic groups, a Bonferroni correction was used. Each test of overall frequency for two ethnic groups was significant if the p-value was less than 0.005 (0.05/10) [18]. Comparisons were made for specific allele frequencies that contributed large values to the significant chi-square tests. Each comparison was considered significant if the p-value was less than 0.006 (0.05/8; testing for 8 of 9 possible A*28 alleles). Any specific allele frequencies appearing different between two similar ethnic groups were tested, but were only considered significant if the p-value was less than 0.0006 (0.05/80; 10 combinations of ethnic groups, 8 alleles in each).

Name

Sequence (59-39)

8FFTb,c 43Rc 56R 62EG 62G 62LQ 62RN 70Hc 70Qb,c 73I 74Hc 76AN 76VDb,c 77S 80IALRc 114EH 114Q 114R 131R 142IK 142TK 144Q 149T 150V 151R 152E 152W 156Lb,c 156Q 156W 161D 163Ec 163R 163Tc 166DG 9YTc 10Tc 12Mc

ACGGATGTGAAGAAATAC ATCCTCCGGCTCGCG GAGAGGCCTGAGTAT CGAGGAGACAGGGAAA GACGGGGAGACACGG TGGGACCTGCAGACA GACCGGAACACACGG AAGGCCCACTCACAGA TCTGTGACTGGGCCTT TCACAGATTGACCGAG ACTCGGTGAGTCTGTG GACCGAGCGAACCTG CCCAGGTCCACTCGG GAGAGCCTGCGGATC CGGATCGCGCTCCGCTAC TATGAACAGCACGCC TACCAGCAGGACGCC GGGTACCGGCAGGAC CGCTCTTGGACCGCG CAGATCACCAAGCGCA ACCACCAAGCACAAG AGATCACCCAGCGCA TGGGAGACGGCCCAT AGGCGGTCCATGCG GCGGCCCGTGTGGCG GCCCATGAGGCGGAG GCCCGTTGGGCGGAG GGCTCTCAACTGCTCC GAGCAGCAGAGAGCC GAGCAGTGGAGAGCC CTGGATGGCACGTGC GAGGGCGAGTGCGT GAGGGCCGGTGCGTG GAGGGCACGTGCGT GTGGACGGGCTCCG ACACCTCCGTGTCCC CTACACTTCCGTGTCCC CTACACCTCCATGTCCCG

a

All probes described in Oh et al. [16] except where indicated. Probe for non-coding strand. c Designed for this and other studies. b

RESULTS AND DISCUSSION Study Population The database used in this study consisted of 82,979 consecutive unrelated individuals serologically typed between December, 1990 and May, 1996. Individuals were grouped into five self-reported ethnic groups: 74.3% were Caucasian, 10.0% African American, 2.7% Asian/ Pacific Islander, 5.9% Hispanic, and 7.1% Native American (Fig. 3). The overall ethnic distribution appeared similar to the total U.S. population [19] although Native Americans appeared higher in the study population (7.1% vs. 0.7%). Individuals were typed for HLA-A by serology and the approximate frequency of individuals carrying each HLA-A broad antigen type in each ethnic group was calculated (Table 3). Within the database, the serologic type A28 (including A68 and A69) occurred with a frequency of 7.8% in Caucasians, 17.9% in African Americans, 2.7% in Asians/Pacific Islanders, 16.2% in Hispanics, and 8.9% in Native Americans.

TABLE 2 A*28 specific amplification Ambiguous combinations

Forward primer (59-39 sequence)

Reverse primer (59-39 sequence)

A*02subseta/A*6803 or A*02subset/A*6805 A*3002/A*6802 or A*3004/A*6901 A*10subsetb/A*68011 or A*10subset/A*68012

5AEx2c (CAC TCC TCG CCC CCA GGC TCC) 5AIn1-A (GGG GCG CAG GAC CCG GGA) 5AIn1-A

3AIn3-62d (GTC CCA ATT GTC TCC CCT CCT T) 3AIn3-62d

a

A*0201, A*0204, A*0207, A*0215N, A*0217, A*0218, or A*0220. A*2601, A*3401, A*3402, or A*6601. c Primer designed for this study. d Primer described by Cereb et al. [15]. b

AL#Hc (GTC CAA GAG CGC AGG CCT TCT)

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FIGURE 3 A comparison of the ethnic distribution in 82,979 consecutively typed unrelated individuals and in the total U.S. population [19]. Other includes other ethnic designations and individuals whose ethnic group is not available.

The distribution of the A*28 alleles was determined in a random sample of these individuals typed as A28, A68 or A69. In order to have the 95% probability of finding a rare allele that occurs in 5% of the A*28positive population, 59 samples were chosen for each ethnic group. One African American, 1 Hispanic, and 4 Native Americans were randomly chosen twice and counted twice in the frequency estimation. Since there were only 57 available Asian/Pacific Islander individuals TABLE 3 Frequency (%) of individuals carrying HLA-A specificities in different ethnic groups in a U.S. populationa,b African Asian/Pacific Native HLA-A Caucasian American Islander Hispanic American antigen n 5 61,655 n 5 8,288 n 5 2,275 n 5 4,879 n 5 5,882 A1 A2 A3 A9 A10 A11 A19 A28 A36 A43 A80c AXd

30.4 49.6 26.8 19.7 10.5 11.8 24.9 7.8 0.1 — — 0.6

11.1 34.6 16.6 24.5 14.0 2.8 50.8 17.9 3.8 — 0.7 2.1

9.5 36.0 8.2 47.6 21.1 28.6 25.2 2.7 0.1 — — 1.5

15.4 46.9 17.1 29.3 10.6 9.5 36.0 16.2 0.6 — 0.2 1.3

29.1 49.7 25.8 19.6 9.8 11.1 26.0 8.9 0.3 — 0.1 0.9

Individuals in this study (n 5 82,979) were unrelated. HLA-A types defined by split antigens were included in the respective broad antigen specificities. An individual carrying two different splits of a broad type (e.g., A68,A69) was counted once. c Since alloantisera detecting A80 has only recently become available, the frequency of A80 is likely underestimated. d Undefined specificity. a b

who were typed as A28, all samples were used in this study. SSOP Typing HLA-A alleles were amplified from genomic DNA using generic A locus primers and were probed with 38 SSOPs (Table 1). The probes were selected to give allele level typing of most A*28 alleles and antigen level typing of non-A28 alleles. Some heterozygous combinations could not be distinguished using the 38 probe panel. For example, an individual with the HLA-A type A*3002, A*6802 exhibited the same hybridization pattern as an individual with A*3004, A*6901. In these cases, the alleles were separated by allele-specific PCR or cloning to identify the A*28 allele. New primers were designed to selectively amplify the A*28 allele in many of these instances. The A*28 group does not have a unique sequence located within exons 2 and 3 that can serve as a primer-binding site for only A*28 alleles. Therefore, primer sets were designed to specifically amplify a set of HLA-A alleles that included A*28 alleles, but specifically not the other allele present in the sample (Table 2). For samples containing allelic combinations that could not be separated by allele-specific amplification (such as A*24, A*68 combinations), cloning was used to separate the HLA-A alleles. The alleles, A*68031 and A*68032, which differ by a silent nucleotide substitution in exon 2 were not distinguished in this study. Serologic Assignments of A28, A68 and A69 As previously observed [20, 21], the serologic assignments most frequently given to cells carrying A*6801,

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TABLE 4 The distribution of broad and split serologic assignments for A*28 allelesa Serologic Assignment N (%)

Allele A*68011

A*68012

A*6802

Broad antigen 8 (42%) 9 (53%) 7 (44%) 4 (33%) 11 (65%) 24 (75%) 1 (33%) 14 (61%) 13 (56.5%) 16 (67%) 3 (43%) 24 (63%) 9 (69%) 6 (50%) 12 (92%)

A*6803 2 (29%) 1 (50%) A*6805 A*6901

Correct split antigen 11 (58%) 7 (41%) 9 (56%) 8 (67%) 6 (35%) 8 (25%) 2 (67%) 9 (39%) 10 (43.5%) 8 (33%) 4 (57%) 13 (34%) 4 (31%) 6 (50%) 1 (8%) 1 (100%) 5 (71%) 1 (50%) 1 (100%)

ID# Incorrect split antigen 1 (6%)

1 (3%)

1 (100%) 2 (50%)

1 (100%) 1 (25%) 1 (100%)

TABLE 5 HLA-A28 discrepancies between serologic and DNA typing

1 (25%)

Race

09393257b

AFRa

Raceb

09366725b

ASI

CAU AFR ASI HIS NAT CAU AFR ASI HIS NAT CAU AFR ASI HIS NAT ASI HIS NAT HIS CAU ASI HIS NAT

19640176b

ASI

19511443b,c

HIS

19704634b

ASI

09318346b

NAT

09366691b,c

NAT

14119036

AFR

14313233

AFR

14189971

HIS

a

Homozygous individuals were counted once. A*28 allelic products that were serologically mistyped as non-A28 antigens were not evaluated. b CAU 5 Caucasian, AFR 5 African American, ASI 5 Asian/Pacific Islander, HIS 5 Hispanic, NAT 5 Native American.

A*6802, A*6803 or A*6805 alleles were A68 or A28; the majority of the assignments for cells carrying A*6901 were A69 or A28 (Table 4). Overall, 58.2% and 40.8% of A*28 alleles were assigned the broad antigen (A28) and the correct split antigen (A68 or A69), respectively. Since the presence of the second HLA molecule may affect A28/A68/A69 serologic typing results, there may be variations between different ethnic groups in the frequency of broad and split antigen identifications. DNA-based typing analysis also revealed some discrepancies with serologic typing in 25 samples. There were a total of 7 samples that were serologically typed as A28 but identified as A*28-negative by DNA typing (Table 5). There were no A*28-negative Caucasians found, but at least one A*28-negative individual was found in the other four ethnic groups. Among individuals serologically assigned as A28, the frequency of A*28-negative individuals was 1.7% in African Americans, 5.3% in Asians/Pacific Islanders, 3.2% in Hispanics, and 5.0% in Native Americans. Three samples had

Serologic assignment

DNA assignment

A28 A74 A28 A2 A28 A2 A28 A2 A68 A11 A28 A3 A28 A24(A9) A69 A2 A69 A1 A68 A30

A*02 A*74 A*02 A*02 A*02 A*33(A19) A*11 A*33(A19) A*03 A*24(A9) A*6802 A*02 A*68011 A*0101 A*6901 A*3004

a CAU 5 Caucasian, AFR 5 African American, ASI 5 Asian/Pacific Islander, HIS 5 Hispanic, NAT 5 Native American. b A*28-negative sample that was serologically mistyped as A28. c This sample was chosen twice in the random selection. Therefore, it was counted twice in the calculation of frequency of A*28-negative individuals.

incorrectly assigned A28 splits (e.g., A69 instead of A68) (Tables 4, 5). Fourteen other samples had misassignments involving the non-A28 antigen (e.g., misses of antigens like A74 or incorrect assignment of antigens in cross-reactive groups such as A30/A31) and are not described in this study. The misassignments of A28 may be due to the lack of specific antibody reagents and reference cells for serologic typing of non-Caucasians and the cross-reactivity of the accompanying non-A28 antigen [5, 22]. The ambiguity becomes more serious for cells that exhibit only a single specificity, since homozygotes often exhibit cross-reactivity with sera detecting closely related HLA-A molecules through a gene dosage effect [23]. In 4 of 11 discrepancies for A28, an A*02 allelic product was mistaken for an A*28-encoded molecule. A comparison of HLA-A*02 and A*28 allelic products shows only a few differences in the a1 and a2 domains [12], and the A2 and A28 alloantisera are known to cross-react [24]. In 2 samples, A*33 alleles were mistyped as A28 or A68. The alloantisera cross-reactivity can be explained by the observation that A28 shares the 1C public epitope with the A33 family [25]. Additionally, A28, A2 and A24(9) antigens share the 2C public epitope and are grouped in the same CREG [25]. This similarity coupled with homozygosity at the HLA-A locus would explain why one

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TABLE 6 A*28 allele frequencies and tests of overall significance of A*28 frequency distribution tests in Caucasians (CAU), African Americans (AFR), Asians/Pacific Islanders (ASI), Hispanics (HIS), and Native Americans (NAT)

Allele

Caucasian n 5 60

African American n 5 59

Asian/Pacific Islander n 5 54

Hispanic n 5 60

Native American n 5 57

Allele

N (%)

N (%)

N (%)

N (%)

N (%)

A*68011 A*68012 A*6802 A*6803 A*6804 A*6805 A*6806 A*6901

20 (33) 32 (53) 7 (12) — — — — 1 (2)

18 (31) 3 (5) 38 (64) — — — — —

16 (30) 23 (43) 13 (24) 1 (2) — — — 1 (2)

12 (20) 24 (40) 12 (20) 7 (12) — 1 (2) — 4 (7)

17 (30) 24 (42) 13 (23) 2 (4) — — — 1 (2)

Ethnic group

p-value

p-value

p-value

p-value

CAU AFR ASI HIS

,0.001*

0.731 ,0.001*

0.047 ,0.001* 0.366

0.646 ,0.001* .0.999 0.493

* Significant at the level specified for each group of tests.

sample that contained an apparent homozygous A*24 was mistyped as A28. It is also likely that some A*28 allelic products were mistyped as A2, A9, or A33, but this was not evaluated in this study. Distribution of A*28 Alleles The distribution of A*28 alleles was calculated by including all haplotypes that encoded an A*28 allele. A*28-negative individuals were not counted. For example, if an individual had two different A*28 alleles (e.g., A*68011, A*68012), both alleles were counted. Presumed homozygous alleles were counted twice in the allele frequency calculation (e.g., an individual typed by serology and DNA as only A68/A*68011 was assumed to carry two copies, A*68011, A*68011). Individuals whose SSOP typing result did not match their A28 serologic type were not included. The group of 59 Caucasians carried 60 A*28 alleles, 58 A*28-positive African Americans carried 59 A*28 alleles, 54 A*28-positive Asians/Pacific Islanders carried 54 A*28 alleles, 57 Hispanics carried 60 A*28 alleles, and 56 Native Americans carried 57 A*28 alleles. The distribution of A*28 alleles was calculated separately for each ethnic group (Table 6, Fig. 4). A*68012 was the most frequent allele in Caucasians, Asians/Pacific Islanders, Hispanics and Native Americans (53%, 43%, 40%, 42%, respectively), while A*68011 was the second most frequent allele in these groups (33%, 30%, 20%, 30%, respectively). Taken together, these two alleles that encode the same polypep-

tide account for 86%, 73%, 60%, and 72% of all A*28 alleles in these populations, respectively. In contrast, the most frequent allele in African Americans was A*6802 (64%). A*68011 and A*68012 occurred at lower frequencies (31% and 5%, respectively) in that population. African Americans were the least diverse group in terms of the number (3) of alleles present. Caucasians also did not show extensive diversity in A*28 alleles since only 4 alleles of the 9 known A*28 alleles were seen. The greatest number of alleles seen in one group was 6 alleles in Hispanics. Asians/Pacific Islanders and Native Americans carried 5 A*28 alleles. A*6804 and A*6806 were not seen in any of the individuals tested, suggesting that these alleles are present in less than 5% of the A*28positive populations or the products of these alleles do not encode antigens serologically typed as A28. The two alleles were originally described in an African American [21] and a Hispanic individual [14], respectively. The serologic type associated with A*6804 is A68 [21]; however, the serologic type associated with A*6806 is not known [14]. The overall distribution of alleles between the ethnic groups was tested and found to be significantly different in at least one ethnic group (p , 0.001). The African American population differed from all other populations, but no other combination of ethnic groups was significantly different (Table 6). This difference is due primarily to a smaller proportion of A*68012 in African Americans (5% compared to 40 –53% in other ethnic groups) and a larger proportion of A*6802 (64% compared to

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165

FIGURE 4 The frequency of A*28 alleles in 5 ethnic groups from a U.S. population. There were 60 A*28 alleles in 59 Caucasians, 59 alleles in 58 African Americans, 54 alleles in 54 Asians/Pacific Islanders, 60 alleles in 57 Hispanics, and 57 alleles in 56 Native Americans.

12–24%) (Tables 6,7). A*6802 has been shown to be prevalent in African Americans as well as in Mediterranean populations [20, 26, 27]. The frequency of A*6803 in Caucasians and Hispanics appeared different, but this difference was not significant at the necessary significance level of 0.0006. Because the alleles, A*68011 and A*68012, differ only at the nucleotide level and are predicted to encode for the same HLA polypeptide, only two distinct HLAA28 (or -A68) molecules were expressed in the African Americans studied. The low number of A*28 alleles seen in African Americans in this study was unexpected. Previous observations suggested that African Americans are a diverse population with a broader distribution of HLA phenotypes [4, 28]. This diversity has been explained by the origin of African Americans as descendants of highly diverse African populations and admixture with Europeans and other non-African populations. If other A*28 alleles are present, their frequency is likely

to be less than 5% of the A28 positive alleles. The missing alleles, A*6803-A*6806 and A*6901, are less frequent in other populations which may explain why these alleles have not been introduced at a high frequency through admixture. Perhaps a small number of A*28 alleles originated from the African founder population and the A*28 family diversified in populations after they migrated out of Africa. Another theory may be that selective forces in Africa acted specifically on a large pool of A*28 alleles and favored the inheritance of certain A*28 alleles while selection did not occur in populations that migrated out of Africa. Since the Hispanic population group is defined on the basis of linguistics and culture, it encompasses individuals with Caucasian, African American, and Native American origins. As predicted by this population structure, Hispanics had the highest degree of diversity in A*28 alleles. The recently identified alleles, A*6803 and A*6805, were initially observed in Hispanics [21] and

TABLE 7 Specific A*28 allele distribution tests in Caucasians (CAU), African American (AFR), Asians/Pacific Islanders (ASI), Hispanics (HIS), and Native Americans (NAT) CAU

AFR A*68012 A*6802 CAU A*6803

AFR

N (%)

p-value

N (%)

32 (53) 7 (12) 0 (0)

,0.001* ,0.001*

3 (5) 38 (64)

ASI

p-value

a ND 5 not done * Significant at the level specified above for each group of tests.

NDa

HIS

NAT

N (%)

p-value

N (%)

p-value

N (%)

p-value

23 (43) 12 (24)

,0.001* ,0.001* ND

24 (40) 12 (20) 7 (12)

,0.001* ,0.001* 0.0064

24 (42) 13 (23)

,0.001* ,0.001* ND

166

were found to occur at higher frequencies in Hispanics in this study. CONCLUSIONS The use of the SSOP methodology to identify alleles has its limitations. Since the selection of probes used to define alleles is based on the alleles known at the time, it is possible that as yet unidentified A*28 alleles will be missed. This is demonstrated in an earlier studies of A*28 allele frequency [20, 26] in which only 3 alleles, A*68011, A*6802, and A*6901, were considered in the interpretation of the typing. While the presence of an additional allele (termed A68.3) was suggested in these earlier studies by the loss of probe hybridization around codon 10, the set of probes utilized would have not distinguished A*68011 from A*6804 and A*68012 from A*68031, A*68032, A*6805, and A*6806. This study has focused on the alleles in the five major U.S. ethnic populations. Because individuals were categorized into broad self-defined ethnic groups, the definitions of each group were not based on consistent or established biological criteria [2]. Furthermore, the heterogeneity of each population should be considered when analyzing data on allele frequencies. For example, the diversity in the A*28 allele family in Asians/Pacific Islanders may reflect the combination of several genetically distinct groups including Korean, Chinese and Filipino populations [29]. Despite this complication and the limitations of SSOP typing described above, information on allele frequencies will be useful in the clinical setting of hematopoietic stem cell transplantation. The data from this study might be used to calculate the probability of finding an A68 or A69 antigen match for A28 typed donors or even an A*28 allele matched donor for a transplant patient. Although it is unclear whether matching at the allelic level of resolution for HLA class I will provide a better outcome in transplants than matching at the serologic level, previous observations suggest that T cells can detect differences between serologically identical allelic products and may affect transplant outcome [30, 31]. Furthermore, allele frequency studies such as this one will be important in developing strategies for registry size and composition to allow for selection of optimally matched stem cell donors. ACKNOWLEDGMENTS

We thank Noriko Steiner for the primer sequences and Susan Mowry for the DNA extractions. This research is supported by funding from the Office of Naval Research N00014-94-10049 and NCI P30-CA-51008 (Lombardi Cancer Center Shared Resources). The views expressed in this article are those of the authors and do not reflect the official policy or position of the Department of Navy, Department of Defense, or the U.S. Government.

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