DQ linkage disequilibrium of 231 German Caucasoid patients and their corresponding 821 potential unrelated stem cell transplants

HLA-DRB1,3,4,5 and -DQB1 Allele Frequencies and HLA-DR/DQ Linkage Disequilibrium of 231 German Caucasoid Patients and Their Corresponding 821 Potential Unrelated Stem Cell Transplants Andreas Johannes Knipper, Peter Hakenberg, Ju¨rgen Enczmann, Andreas Kuhro¨ber, Ulrich Kiesel, Gesine Ko¨gler, and Peter Wernet ABSTRACT: Allelic matching within the HLA-DRB1 and -DQB1 loci significantly improves the clinical outcome of hematopoietic stem cell transplantation. Consequently, allelic typing of these loci is strongly recommended for the unrelated stem cell donor selection. In this study, the HLA-DRB1,3,4,5 and -DQB1 alleles of 231 patients and their corresponding 821 nonrandom potential stem cell donors were determined to define compatible donor/recipient pairs. Highly accurate HLA typing data were achieved by PCR-SSOP and a combination of group specific PCR-SSP and subsequent sequencingbased typing of nearly the whole second exon of each locus. The alleles DRB1*07, *09, and *10 were analyzed by PCR-reverse dot blot hybridization instead of sequencing. Additionally, DRB1 homozygosity was verified by temperature gradient gel electrophoresis. The identified 2104 HLA-DRB1 and HLA-DQB1 alleles as well as data on HLA-DRB3, -DRB4, and -DRB5 alleles were applied

ABBREVIATIONS USCD unrelated stem cell donor TGGE temperature gradient gel electrophoresis SSOP sequence specific oligonucleotide probing SSP sequence specific primers

INTRODUCTION The HLA-DRB1 and -DQB1 genes are located within the HLA class II region on chromosome 6 which encom-

From the Institute for Transplantation Immunology and Cell Therapeutics (A.J.K., P.H., J.E., A.K., U.K., G.K., P.W.), Moleculargenetic Lab., Du¨sseldorf, Germany. Address reprint requests to: A. J. Knipper, Institute for Transplantation Immunology and Cell Therapeutics, Moleculargenetic Lab., Bldg. 14.80, Moorenstr. 5, 40225 Du¨sseldorf, Germany; Tel: (⫹49) 211 81-18646; Fax: (⫹49) 211 93 48 435; E-Mail: [email protected]. Received January 26, 1998; revised January 26, 2000; accepted January 27, 2000. Human Immunology 61, 605– 614 (2000) © American Society for Histocompatibility and Immunogenetics, 2000 Published by Elsevier Science Inc.

to a statistical program and absolute and relative delta values of DR/DQ linkages were calculated. The achieved data on the HLA-DRB1 allele distribution and on DR/DQ associations in terms of subtypes significantly ensure the typing reliability, since rare allele combinations will result in further investigations. Furthermore, detailed data on the DR/DQ allele associations allow estimations of the number of HLA-A, -B, and -DR matched unrelated stem cell donors necessary for the identification of DRB and DQB subtype identical donors. Human Immunology 61, 605– 614 (2000). © American Society for Histocompatibility and Immunogenetics, 2000. Published by Elsevier Science Inc. KEYWORDS: HLA-DRB1 allele frequency; HLADR/DQ linkage disequilibrium; delta value; sequencingbased typing

SBT BMT TMAC

sequencing-based typing bone marrow transplantation tetramethylammonium chloride

passes approximately 1.0 Mbp of DNA. Allele matching within these loci has been described to significantly improve the outcome of unrelated bone marrow transplantation [1, 2]. The polymorphism of the HLA-DR and -DQ genes is mainly based on the second exon, which encodes the aminoterminal extracellular domain of these antigens. Therefore, the amplification of these hypervariable second exons allow HLA-DRB and -DQB1 typing by different techniques, such as PCR-sequencespecific oligonucleotide probing (PCR-SSOP) [3–5], PCR with sequence-specific primers (PCR-SSP) [6 – 8], 0198-8859/00/$–see front matter PII S0198-8859(00)00114-2

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reverse dot blot hybridization [9], PCR-restriction fragment length polymorphism (PCR-RFLP) [10], and PCR-sequencing-based typing (PCR-SBT) [11–13]. The rate of recombination events between two loci is depending on their physical distance. However, HLADRB1 and -DQB1 genes tend to be inherited together more often than predicted from their close location. The observed linkage can be estimated by means of statistically determined delta values [14, 15]. In this study, sequencing-based typing techniques were used for the highly accurate determination of the HLA-DRB and -DQB1 polymorphisms of a non-random cohort of 1052 individuals. Reliable subtyping data on these alleles were generated and the allele distribution of this cohort was determined. Based on these data, the predictive values of HLA-DRB1/DQB1 and HLADRB1/B3/4/5 linkages were calculated. Because no pedigrees were available for the unrelated donor cohort, the HLA-DR/DQ association was determined by calculating the coefficient of linkage disequilibrium in terms of the haplotype frequencies p(DR) and p(DQ) [14]. A statistical assignment of HLA-DQB1 and -DRB3/4/5 alleles to distinct HLA-DRB1 subtypes was performed by addition of the two relative delta values of both possible HLA-DR/DQ combinations of each sample. Combinations with the highest value were considered as associated. The summary on HLA-DRB1 frequencies as well as data on DR/DQ associations presented here are of great importance in HLA typing as well as in the USCD search. MATERIALS AND METHODS Stem cell donors were considered for an unrelated transplant if the best HLA match available was incompatible for no more than one HLA-A, -B, or -DR minor mismatch. A HLA class I minor mismatch is defined as HLA-A or -B antigens belonging to the same serologically defined cross-reactive group (CREG). A HLA-DR minor mismatch is defined as any two different DRB1 alleles encoding the same serologically defined DR antigen (DR1, DR4, DR7, DR8, DR9, DR10, DR11, DR12, DR13, DR14, DR15, DR16, DR17, or DR18). Initial matching criteria for an unrelated cord blood transplant required the placental blood to share at least four HLA-A, -B, or -DR antigens with the potential recipient. In applying these rules to the USCD search for 231 patients, up to nine potential donors were selected for HLA-class II subtyping. For three patients, no USCD could be found in the BMDW (bone marrow donor worldwide) registry. A total of 1052 samples (231 patients and their corresponding 748 unrelated adult stem cell donors and 73 unrelated cord blood transplant spec-

A. J. Knipper et al.

imen) were analyzed within their HLA-DRB1, 3, 4, 5, and -DQB1 loci. To achieve a maximum HLA typing reliability, the following typing strategy was used. Generic PCR-SSO Typing of the HLA-DRB1/3/4/5 and -DQB1 Loci DNA extractions from frozen or fresh blood samples were performed following the Qia Amp protocol (QIAamp 96 Spin Blood Kit, Qiagen, Hilden, Germany). Two PCRs were performed to amplify the second exons of HLADRB (including existing HLA-DRB1, -3, -4, and -5 genes) and -DQB1 genes. Primer sets and cycle profiles have been listed elsewhere [11]. The amplified DNA fragments were alkaline denaturated and dotted onto 16 (DRB) and 18 (DQB1) nylon membranes (GeneScreen Plus, NEN, Boston, MA, USA) [3, 4]. Sixteen chemically biotinylated DRB oligonucleotide probes with high specificity to the most common alleles of the HLA families HLA-DRB1*01, *02, *03, *04, *11, *12, *13, *14, *07, *08, *09, *10, -DRB3, DRB4, and DRB5 (DRB5*0101) and 18 biotinylated HLA-DQB1 probes for DQB1*05/*06 subtyping, and *02AB, *03AD, *0302, *0303, and *04 typing were applied to separate membranes [16]. A TMAC hybridization protocol was used for parallel hybridization of all oligonucleotide probes using ECL as a non-radioactive detection system for hybridized probes (Amersham, Braunschweig, Germany) [17]. Group-Specific Amplification and Subsequent Sequencing-Based Typing Based on the PCR-SSOP results, HLA-DRB primers for the selective amplification of alleles or allelic groups were used in separate PCR reactions [6]. Distinct groupspecific 3⬘-primers were applied in the HLA-DQB1specific PCR [11]. The use of one biotinylated primer for each PCR reaction allowed the isolation of single strand DNA for the consecutive sequencing reaction. Both the solid-phase single-strand separation and the non-radioactive sequencing method have been described elsewhere [11, 12]. For HLA-DRB1*07, -DRB1*09, and -DRB1*10 and certain HLA-DRB3 and -DRB4 alleles no sequencing based typing was performed. Here, typing data were confirmed by reverse dot blot hybridization (HLA DRB Typing Kit, Dynal, Oslo, Norway). The analysis of the HLA-DRB and DQB1 subtypes was concentrated on the highly polymorphic second exon of each locus. However, the nucleotide differences between the alleles DQB1*0201 and 0202 or DRB4*0101 and *0103 are located within exon 3. This third exon was not analyzed and we used the WHO nomenclature together with the NMDP multiple allele code to specify HLA class II typing results.

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607

Typing Resolution and Ambiguous Heterozygous Combinations The combination of allele or group-specific amplification by PCR-SSP and subsequent sequencing-based typing result in a high degree of typing resolution. Concerning the HLA-class II allele update of the Anthony Nolan database of September 1998 all DQB1 alleles except the ambiguous heterozygous combination DQB1*0301/ *0302 ⫽ DQB1*03032/*0304 could be determined. This DQB1 ambiguity can be resolved by an additional PCR-SSP approach.

TABLE 1 Ambiguous heterozygous allele combinations of the DRB1 locus

Temperature Gradient Gel Electrophoresis for Confirming DRB1 Homozygosity One critical aspect of several HLA typing strategies is the definition of HLA homozygosity. Here, the temperature gradient gel electrophoresis (TGGE) was used as a reliable technique for confirming DRB1 homozygosity [18].

more than 12% of the generic HLA-DR antigens, while HLA-DRB1*14, *08, *16, *12, *09, and *10 appeared with a frequency of less than 4% (Fig. 1A). Differences between the patient pool and the USCD group were found for the HLA antigens DRB1*13, *11, *04, and

Determination of Linkage Disequilibrium An essential prerequisite for a proper analysis and interpretation of the obtained HLA typing data is to test the homogeneity of this non-random pool of individuals. Here, a chi square test was used, which confirmed the independence of the USCD pool compared to the patient pool (p ⫽ 0.1481). This result allowed further statistical analysis. To allocate DRB3,4,5 and DQB1 subtypes to distinct DRB1 alleles the coefficient of linkage disequilibrium (delta) was determined using the linkage analysis program of Ott [14]. An approximate value of the delta value was calculated by subtracting the product of both the haplotype frequencies of DRB1 and DQB1 from the likelihood of their coupled appearing [Delta ⫽ p(DRQ) ⫺ p(DRB) p(DQB)]. A strong DR/DQ association is characterized by a high delta value. The calculation of relative delta values (rel. D) allows a comparison of DR/DQ linkages [15].

FIGURE 1 Generic HLA-DR and DQ frequency within a pool of 231 patients and 821 unrelated potential stem cell donors. PAT ⫽ patients; PD ⫽ potential unrelated stem cell donors.

RESULTS Based on the typing strategy described, PCR-SSOP, PCR-SSP, PCR-SBT, and TGGE, accurate HLADRB1,3,4,5 and -DQB1 typing data on 231 patients and 821 potential stem cell transplants including unrelated marrow donors and cord blood specimens were obtained. Only three ambiguous heterozygous combinations appeared within the analyzed population (Table 1). Here, no additional analysis was done and preference was given to the more common combinations. In all other cases, DRB1 alleles were accurately defined by the typing strategy described. The generic distribution of 2104 HLA-DRB1 and -DQB1 alleles is illustrated in Figure 1. HLADRB1*13, *04, *15, *07, and *11 each represented

Common HLA-DRB1 alleles DRB1*1101/*1302 DRB1*1102/*1301 DRB1*1104/*1301

⫽ ⫽ ⫽

Rare HLA-DRB1 alleles

Total

DRB1*1120/*1314 DRB1*1116/*1322 DRB1*1116/*1311

3 1 3

No complete typing resolution was performed for seven allele combinations within the DRB1 locus. Here, no further analysis was performed and preference was given to the more common allelic combination.

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TABLE 2 DRB1 allele frequencies and DRB1/DQB1 associations of 2104 haplotypes

DR1 DRB1*0101 P(DQ I DRB1*0101) Delta rel. Delta DRB1*0102 P(DQ I DRB1*0102) Delta rel. Delta DRB1*0103 P(DQ I DRB1*0103) Delta rel. Delta DR15 DRB1*1501 P(DQ I DRB1*1501) Delta rel. Delta DRB1*15021 P(DQ I DRB1*1502) Delta rel. Delta DR16 DRB1*1601 P(DQ I DRB1*1601) Delta rel. Delta DRB1*1602 P(DQ I DRB1*1602) Delta rel. Delta DR3 DRB1*0301 P(DQ I DRB1*0301) Delta rel. Delta DR4 DRB1*0401 P(DQ I DRB1*0401) Delta rel. Delta DRB1*0402 P(DQ I DRB1*0402) Delta rel. Delta DRB1*0403 P(DQ I DRB1*0403) Delta rel. Delta DRB1*0404 P(DQ I DRB1*0404) Delta rel. Delta DRB1*0405 P(DQ I DRB1*0405) Delta rel. Delta DRB1*0407 P(DQ I DRB1*0407) Delta rel. Delta

N

P(sub)

200 163

0.815

31

0.155

6

0.03

289 278

0.9619

11

0.0381

42 41

0.9762

1

0.0238

230 230

1

302 154

0.5099

17

0.0563

13

0.0430

60

0.1987

13

0.0430

28

0.0927

DQB1* *0501 156 0.9571 0.0663 0.95 31 1 0.0133 1 6 1.0000 0.0026 0.9800 *0602 263 0.9460 0.1080 0.97 2 0.1818 0.0003 0.06 *0502 39 0.9286 0.0181 0.95 1 1 0.0005 0.95 *02AB 227 0.9870 0.0851 0.98 *0301 54 0.3506 0.0102 0.18 1 0.0588 ⫺0.0012 0.71

4 0.0667 ⫺0.0039 ⫺0.67 3 0.2308 0.0009 0.19 26 0.9286 0.0096 0.91

*0502 1 0.0061 ⫺0.0014 ⫺0.74

*0504 6 0.0368 0.0026 0.98

*0603 6 0.0216 ⫺0.0094 ⫺0.77 1 0.0909 0.0000 0.01 *0501 1 0.0238 ⫺0.0015 ⫺0.76

*0502 6 0.0216 ⫺0.0003 ⫺0.09 1 0.0909 0.0004 0.07 *0602 1 0.0238 ⫺0.0020 ⫺0.81

*0602 2 0.0087 ⫺0.0131 ⫺0.93 *0302 100 0.6494 0.0414 0.62 16 0.9412 0.0069 0.93 11 0.8462 0.0047 0.83 50 0.8333 0.0212 0.82 6 0.4615 0.0022 0.39 2 0.0714 ⫺0.0002 ⫺0.20

*06011 1 0.0036 0.0000 0.05 7 0.6364 0.0033 0.87

*0501 1 0.0036 ⫺0.0128 ⫺0.96

*0604 1 0.0036 ⫺0.0049 ⫺0.91

*0302 1 0.0043 ⫺0.0093 ⫺0.95 *0304

*0305

*0402

*0401

*02AB

1 0.0769 0.0005 0.14

1 0.0769 0.0005 0.95

1 0.0769 0.0005 0.95

3 0.2308 ⫺0.0006 ⫺0.43

6 0.1000 0.0022 0.09

(Continued)

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609

TABLE 2 (Continued)

DRB1*0408 P(DQ I DRB1*0408) Delta rel. Delta DRB1*0411 P(DQ I DRB1*0411) Delta rel. Delta DRB1*0413 P(DQ I DRB1*0413) Delta rel. Delta DR11 DRB1*1101 P(DQ I DRB1*1101) Delta rel. Delta DRB1*1102 P(DQ I DRB1*1102) Delta rel. Delta DRB1*1103 P(DQ I DRB1*1103) Delta rel. Delta DRB1*1104 P(DQ I DRB1*1104) Delta rel. Delta DRB1*1111 P(DQ I DRB1*1111) Delta rel. Delta DRB1*1112 P(DQ I DRB1*1112) Delta rel. Delta DRB1*1114 P(DQ I DRB1*1114) Delta rel. Delta DRB1*1127 P(DQ I DRB1*1127) Delta rel. Delta DRB1*11neu P(DQ I DRB1*11neu) Delta rel. Delta DR12 DRB1*1201 P(DQ I DRB1*1201) Delta rel. Delta DR13 DRB1*1301 P(DQ I DRB1*1301) Delta rel. Delta

N

P(sub)

15

0.0497

1

0.0033

1

0.0033

259 161

0.6216

6

0.0232

15

0.0579

71

0.2741

1

0.0039

2

0.0077

1

0.0039

1

0.0039

1

0.0039

29 29

1

314 189

0.6019

DQB1* 10 0.6667 0.0033 0.58

*0301 159 0.9876 0.0599 0.98 6 1 0.0023 0.98 14 0.9333 0.0052 0.92 69 0.9718 0.0259 0.97 1 1 0.0004 0.94 2 1 0.0007 0.94 1 1 0.0004 0.94 1 1 0.0004 0.94 1 1 0.0004 0.93 *0301 29 1 0.0110 1 *0603 185 0.9788 0.0796 0.98

5 0.3333 0.0024 0.72 1 1 0.0004 0.95 1 1 0.0000 0.98 *0302 1 0.0062 ⫺0.0064 ⫺0.93

*0602 1 0.0062 ⫺0.0094 ⫺0.95

*0603

*0304

*0502

1 0.0667 0.0003 0.04

*0604 1 0.0053 ⫺0.0032 ⫺0.87

*0607 1 0.0053 0.0004 0.95

1 0.0141 ⫺0.0026 ⫺0.85

1 0.0141 0.0004 0.11

*0301 1 0.0053 ⫺0.0179 ⫺0.97

*0402 1 0.0053 ⫺0.0017 ⫺0.78

*0609

(Continued)

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TABLE 2 DRB1 allele frequencies and DRB1/DQB1 associations of 2104 haplotypes

DRB1*1302 P(DQ I DRB1*1302) Delta rel. Delta DRB1*1303 P(DQ I DRB1*1303) Delta rel. Delta DRB1*1304 P(DQ I DRB1*1304) Delta rel. Delta DRB1*1305 P(DQ I DRB1*1305) Delta rel. Delta DRB1*1306 P(DQ I DRB1*1306) Delta rel. Delta DRB1*1308 P(DQ I DRB1*1308) Delta rel. Delta DR14 DRB1*1401 DRB1*1402 DRB1*1404 DRB1*1405 P(DQ I DR14) Delta rel. Delta DR7 DRB1*0701 P(DQ I DRB1*0701) Delta rel. Delta DRB1*0801 P(DQ I DRB1*0801) Delta rel. Delta DRB1*0802 P(DQ I DRB1*0802) Delta rel. Delta DRB1*0803 P(DQ I DRB1*0803) Delta rel. Delta DRB1*0804 P(DQ I DRB1*0804) Delta rel. Delta DRB1*0806 P(DQ I DRB1*0806) Delta rel. Delta DRB1*0810 P(DQ I DRB1*0806) Delta rel. Delta

N

P(sub)

89

0.2834

27

0.0860

1

0.0032

6

0.0191

1

0.0032

1

0.0032

68 61 1 4 2

1 0.0147 0.0588 0.0294

287 287

DQB1* 2 0.0225 ⫺0.0030 ⫺0.76

83 0.9326 0.0377 0.98

4 0.0449 0.0018 1 27 1 0.0102 1 1 1 0.0003 0.76 6 1 0.0023 0.98 1 1 0.0004 0.94 1 1 0.0004 0.94

*0503

1

0.6842

2

0.0351

8

0.1404

6

0.1053

1

0.0175

1

0.0175

1 0.0313 1 *02AB 209 0.7282 0.0709 0.66 38 0.9744 0.0176 0.98 2 1 0.0009 0.95

3 0.5 0.0014 0.48

*0303 77 0.2683 0.0310 0.87

*0301 1 0.0035 ⫺0.0274 ⫺0.98 1 0.0256 ⫺0.0019 ⫺0.80

7 0.875 0.0026 0.84 3 0.5 0.0001 0.06

1 0.125 0.0004 0.10

1 1 0.0004 0.94 1 1 0.0004 0.94 (Continued)

HLA-DRB1/3/4/5 and -DQ Subtyping Data and HLA-DR/DQ Linkage Disequilibrium

611

TABLE 2 (Continued)

DR9 DRB1*0901 P(DQ I DRB1*0901) Delta rel. Delta DR10 DRB1*1001 P(DQ I DRB1*1001) Delta rel. Delta

N

P(sub)

10 10

1

17 17

1

DQB1* *03032 10 1 0.0046 0.99 *0501 17 1 0.0073 1

N ⫽ total number of identified alleles; P(sub) ⫽ subtype frequency within the generic DR family; P(DQ I DRB1*0101) ⫽ the probability of typing a distinct DQB1 allele on condition that the DRB1 allele is the allele DRB1*0101.

*01 and were determined by chi square test as not significant deviations (p ⫽ 0.1481). According to the DR/DQ linkage disequilibrium, these differences were also visible for the associated DQ alleles (Fig. 1B). Also, 42.7% of the DQ alleles were represented by the DQ1 group (DQB1*05 and *06). The antigens DQB1*02 and DQB1*0301 each comprised nearly 21% of the DQB1 alleles. Forty-seven different DRB1 alleles were found in the analyzed caucasoid cohort (Table 2). In addition, the association of DRB1 alleles with 18 different DQB1 alleles was determined by calculating the absolute and relative delta values for the definition of DRB1/DRB3/ 4/5 and DRB1/DQB1 linkages. A total of 9.5% of all alleles were determined as HLA-DRB1*01. Within this DR family, DRB1*0101 had a frequency of 81%. DRB1*0101 was strongly associated with HLA-DQB1*0501 as demonstrated by the high relative delta value of 0.95. The probability of detecting the allele DQB1*0501 on condition that the DR1 typing result is DRB1*0101 was calculated to 96% [P(DQB1*0501 l DRB1*0101) ⫽ 0.957]. DRB1*1501/DQB1*0602 was the most frequent association within the DR15 family [P(DQ l DRB1*1501) ⫽ 0.946] with a relative delta value of 0.97, while DRB1*1502 was mainly associated with DQB1*0601 (rel. D ⫽ 0.87). The pairing DRB1*1601/ DQB1*0502 represent another DR/DQ association with a high relative delta value (rel. D ⫽ 0.95). The haplotype DRB1*1602/DQB1*0502 was rare within the European caucasoid population, but seem to be more frequent for American Caucasoids [16]. The probability of detecting DQB1*02AB on condition that the generic DR3 allele is DRB1*0301 was calculated to be 98% [P(DQB1*02AB l DRB1*0301) ⫽ 0.957]. This frequent association demonstrated a strong linkage disequilibrium (rel. D ⫽ 0.98). Nine different DRB1*04 alleles were detected within

the generic HLA-DR4 group. Seven out of these DR4 alleles have a frequency of more than 4% reflecting the heterogeneity of this generic group. DRB1*0401 was predominantly associated with DQB1*0302 (66%) or DQB1*0301 (33%). The alleles DRB1*0402, *0403 and *0404 showed high relative delta values with DQB1*0302 while the allele DRB1*0407 was mainly associated with DQB1*0301. DRB1*0405 was detected in combination with various DQB1 alleles. DQB1*0301 was strongly associated with DRB1*11 and *12. Within the DR13 family DRB1*1301/ DQB1*0603 and DRB1*1302/DQB1*0604 represented two frequent DR/DQ associations , while DRB1*1303 and *1305 were in linkage disequilibrium with DQB1*0301. The DRB1*14 group was found in association with DQB1*0503. There were two main DR/DQ associations for HLA-DRB1*07, namely DRB1*0701/DQB1*02AB (rel. D ⫽ 0.66) and DRB1*0701/DQB1*0303 (rel. D ⫽ 0.87). Within the DR8 family, DRB1*0801/DQB1*0402 (rel. D ⫽ 0.98) and DRB1*0803/DQB1*0301 (rel. D ⫽ 0.84) represented two stringent DR/DQ associations. The DR/DQ pairings DRB1*0901/DQB1*0303 and DRB1*1001/DQB1*0501 were found to be the only associations observed within these DR-families. 329 DR2 alleles associated with DRB5 were detected in the analyzed population. All DRB1*1501 alleles observed were found together with DRB5*0101 while DRB1*1502 was predominantly associated with DRB5*0102. DRB1*16 was only found in association with DRB5*02 (Table 3). A total of 900 haplotypes were included in the analysis of DRB1/DRB3 alleles (Table 3). DRB1*03 was associated mainly with DRB3*0101 and DRB3*02 alleles, but additionally 4 associations with DRB3*03 were found. The generic groups DR11 and DR14 showed a strong linkage disequilibrium with DRB3*02 subtypes. The high delta value of the pairing

612

A. J. Knipper et al.

TABLE 3 DRB1/DRB3 and DRB1/DRB5 associations of 1161 haplotypes DR2 DRB1*1501 P(DRB5 I DRB1*1501) Delta rel. Delta DRB1*1502 P(DRB5 I DRB1*1502) Delta rel. Delta DRB1*1601 P(DRB5 I DRB1*1601) Delta rel. Delta DRB1*1602 P(DRB5 I DRB1*1602) Delta rel. Delta DR3,5,6 DRB1*0301 P(DRB3 I DRB1*0301) Delta rel. Delta DRB1*11XX P(DRB3 I DRB1*0301) Delta rel. Delta DRB1*1201 P(DRB3 I DRB1*1201) Delta rel. Delta DRB1*1301 P(DRB3 I DRB1*1301) Delta rel. Delta DRB1*1302 P(DRB3 I DRB1*1302) Delta rel. Delta DRB1*1303 P(DRB3 I A69DRB1*1303) Delta rel. Delta DRB1*1304 P(DRB3 I DRB1*1304) Delta rel. Delta DRB1*1305 P(DRB3 I DRB1*1305) Delta rel. Delta DRB1*1306 P(DRB3 I DRB1*1306) Delta rel. Delta DRB1*1308 P(DRB3 I DRB1*1308) Delta rel. Delta

41

DRB5* *0101 278 1 0.1145 1 1 0.111 0.0007 0.16 —

1

—

N 278

9

230

DRB3* *0101 146 0.635 0.0541 0.57

259

29

189

89

27

1

6

1

1

1 0.034 ⫺0.0013 ⫺0.72 107 0.566 0.0397 0.51 1 0.011 ⫺0.0050 ⫺0.91 16 0.593 0.0065 0.59

*0102

8 0.889 0.0038 1 —

—

02XX 45 0.196 ⫺0.00235 ⫺0.09 243 0.938 0.0882 0.92 28 0.966 0.0104 0.95 77 0.407 0.0158 0.22

4 0.148 ⫺0.0013 ⫺0.51 1 1 0.0004 0.94 5 0.83333 0.0017 0.28 1 1 0.0004 0.94 1 1 0.0004 0.94

*02XX

—

41 1 0.019 1 1 1 0.0004 0.95 *0301 4 0.017 ⫺0.0028 ⫺0.6

present 35 0.152 0.0130 — 16 0.062 0.0033 nd

1 0.005 ⫺0.0034 ⫺0.88 86 0.966 0.0390 0.96

4 0.021 ⫺0.0009 nd 2 0.022 ⫺0.0005 nd 7 0.259 0.0029 nd

1 0.16667 0.0004 nd

N ⫽ total number of identified alleles; P(DRB5 I DRB1*1501) ⫽ the probability of finding a distinct DRB5 allele on condition that the DRB1 allele is the allele DRB1*1501; nd ⫽ not determined. For some samples only a medium resolution on DRB3 alleles was performed. A typing result like DRB3*02XX specifies the allele to belong to the DRB3*02 family.

HLA-DRB1/3/4/5 and -DQ Subtyping Data and HLA-DR/DQ Linkage Disequilibrium

DRB1*1302/DRB3*0301 reflect the strong association while DRB1*1301 was mainly associated with DRB3*0101. DRB4*01 was the only allelic group found to be associated with DRB1*04, *07, and *09. DISCUSSION The study presented here summarized the HLADRB1,3,4,5 and DQB1 subtyping data on 1052 individuals performed for the selection of compatible patient/ donor pairings. A maximum of typing accuracy was achieved by using PCR-SSOP, PCR-SSP, PCR-SBT, and TGGE typing techniques for the definition of each allele. Thus, the hypervariable region as well as conserved nucleotide areas of the second exon of each locus were inspected. According to the HLA-class II alleles up date from the Anthony Nolan database of September 1998, all DQB1 alleles except for the ambiguous heterozygous combination DQB1*0301/*0302 ⫽ DQB1*03032/ *0304 could be determined. This DQB1 ambiguity was resolved by an additional PCR-SSP approach. The analysis of the DRB1 locus revealed three ambiguous heterozygous combinations within the population analyzed (Table 1). Here, no additional analysis was done and preference was given to the more common combination. In all other cases, DRB1 alleles were accurately defined by the typing strategy described. The generic HLA distribution of the German caucasoid patient group (231 individuals) as well as the USCD group (821 individuals) are illustrated in Figure 1. Depending on the generic HLA haplotype frequencies of the patients, varying numbers of HLA-A, -B, and -DR typed potential USCDs were found to fulfill the matching criteria for further HLA-D subtyping analysis. Furthermore, different matching criteria were put on placental blood as a source of hematopoietic stem cells. In addition, the limited number of HLA haplotypes and the published association of distinct HLA-DR antigens with leukemia will influence the HLA frequencies of the patient pool [19, 20]. All these factors exert an influence on the generic HLA antigen frequencies of the USCD pool. To investigate the homogeneity of the cohort, HLA typing data were analyzed by chi square test. The achieved p value of 0.1481 confirmed the independence of the USCD pool compared to the patient pool. In addition, the generic DRB1 frequencies in a random cohort of the bone marrow donor registry Du¨sseldorf (1050 individuals) confirmed the homogeneity of the patient and the USCD cohort (data not shown). We are aware that this cohort cannot be defined as random. Thus, the achieved allele frequencies represent only a rough overview of the DRB1 distribution of the population analyzed. However, the achieved data can be used to performed statistics on DR/DQ associations.

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This study represents data on DRB1 allele frequencies and on DR/DQ associations from German caucasoid patients and their potential German, European, and even American stem cell donors. The HLA-DRB1 and -DQB1 allele distribution of this cohort was in concordance with published data on German allele frequencies [21]. Forty-seven different DRB1, 4 DRB3, 1 DRB4, 3 DRB5, and 18 DQB1 alleles were identified within the cohort (Table 2 and 3). Although 846 different DRB1/ DQB1 haplotype combinations were possible, only 88 different haplotypes were found. The different molecular organization of the haplotypes and the short physical distance between these genes are two reasons for the constraint recombination frequency observed [22]. DRB1/DQB1 associations were determined by calculating the absolute and relative delta values for each possible DR/DQ association. Only those pairs with the highest relative delta values were defined as associated. This strategy of linkage determination resulted in a slight shift towards the more frequent associations, but it is the only way of analyzing linkages, if no pedigree is available. The detected DR/DQ associations imply a Middle European (German) caucasoid origin of most donors. The resulting data on most samples were in concordance with family segregation studies [23]. Rare DRB1/DQB1 associations were analyzed for a second time and confirmed the pretyping results. The identified alleles represent the caucasoid HLA polymorphism a European molecular genetic tissue typing laboratory has to deal with. Based on these data, accurate PCR-SSOP typing strategies were developed. Data presented here on DQ typing elucidate, that approximately 45% of the DQ alleles were DQ1 (DQB1*05 and DQB1*06) (Fig. 1). The analysis of DQB1 subtyping data resulted in the development of a PCR-SSOP protocol for subtyping the DQB1 gene (data not shown). It is now employed as a routine typing method which significantly increase the accuracy of DRB1 typing. Thus, direct sequencing of DQB1 genes only needs to be employed for uncommon hybridization patterns. For stem cell donor search practice, the statistical evaluation of allele frequencies and associations presented here will allow predictions about the necessary number of generically matched USCDs, to identify a DR/DQ compatible donor for DRB1/DQB1 subtyped patients.

ACKNOWLEDGMENTS

The authors gratefully acknowledge S. Gross, I. Kubitzky, and G. Tillmann for their excellent technical assistance.

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