Summary: American Red Cross-Northeast Region, Dedham, MA; 11 Department of Pathology, University of New Mexico, Albuquerque, NM; 12

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1 (2000) 25, Mcmilln Pulishers Ltd All rights reserved /00 $ The extent of HLA clss II llele level disprity in unrelted one mrrow trnsplnttion: nlysis of 1259 Ntionl Mrrow Donor Progrm donor recipient pirs CK Hurley 1, LA Bxter-Lowe 2,3, AB Begovich 4, M Fernndez-Vin 5, H Noreen 6, B Schmeckpeper 7,8, Z Awdeh 9, M Chopek 5,, M Slzr 10, TM Willims 11, EJ Yunis 12, D Kitjim 13, K Shipp 13, J Splett 13, T Winden 13, C Kollmn 13, D Johnson 14,JNg 1,15, RJ Hrtzmn 15 nd J Heglnd 13 1 Deprtment of Microiology nd Immunology nd Deprtment of Peditrics, Georgetown University, Wshington DC; 2 Center for Cncer Tretment nd Reserch, University of South Crolin, Columi, SC; 4 Roche Moleculr Systems, Almed, CA; 5 Americn Red Cross-Ntionl, Bltimore, MD; 6 Immunology/Histocomptiility Lortory, Firview-University Medicl Center, Minnepolis MN; 7 Immunogenetics Lortories, The Johns Hopkins University, Bltimore, MD; 9 CBR Lortories, Inc., Boston, MA; 10 Americn Red Cross-Northest Region, Dedhm, MA; 11 Deprtment of Pthology, University of New Mexico, Aluquerque, NM; 12 Deprtment of Cncer Immunology nd AIDS, Dn-Frer Cncer Institute, Boston, MA; 13 Ntionl Mrrow Donor Progrm, Minnepolis, MN; 14 Blood Centers of the Pcific-Irwin Center, Sn Frncisco, CA; nd 15 Nvl Medicl Reserch Center, Bethesd, MD, USA Summry: A comprehensive nlysis of the HLA-D region loci, DRB1, DRB3, DRB5, DQA1, DQB1, DPA1 nd DPB1, ws performed to determine llelic diversity nd underlying HLA disprity in 1259 one mrrow recipients nd their unrelted donors trnsplnted through the Ntionl Mrrow Donor Progrm. Although 43.0% of DRB1 lleles known to exist t the eginning of the study were found in this predominntly Cucsin trnsplnt popultion, few lleles predominted t ech locus. In recipients, 67.1% of DRB1 lleles identified were one or two of six common DRB1 lleles. Only 118 (9.4%) donor recipient pirs were mtched for ll lleles of DRB1, DQA1, DQB1, DPA1 nd DPB1. While 79.4% of the pirs were mtched for DRB1, only 13.2% were mtched for DPB1 lleles. Almost 66% of pirs differed y more thn one llele mismtch nd 59.0% differed t more thn one HLA-D locus. DQB1 ws mtched in 85.9% of DRB1-mtched pirs. In contrst, only 13.9% of the pirs mtched for DRB1, DQA1 nd DQB1 were lso mtched for DPA1 nd DPB1. This dtse, highlighting the underlying HLA disprity within the pirs, forms the foundtion of n ongoing study to estlish the reltionship etween HLA mtching nd successful outcome in unrelted llogeneic stem cell trnsplnt. (2000) 25, Keywords: one mrrow trnsplnttion; HLA mtching Correspondence: C Hurley, E404 Reserch Building, Georgetown University Medicl Center, 3970 Reservoir Rd NW, Wshington DC 20007, USA Current ddresses: 3 University of Cliforni, Sn Frncisco, CA, USA; 8 Americn Red Cross-Ntionl, Bltimore, MD, USA, Decesed Received 10 My 1999; ccepted 28 Septemer 1999 Trnsplnttion of llogeneic hemtopoietic stem cells hs ecome n ccepted tretment for mny hemtologic disorders. The optiml hemtopoietic stem cell donor is n HLA-identicl siling ecuse mtching of mjor histocomptiility ntigens significntly reduces the risks of trnsplnt-relted moridity nd mortlity. 1 5 Since pproximtely 65% of ptients do not hve well-mtched relted donor, significnt effort hs een expended in developing DNA-sed HLA typing methods to effectively identify n HLA mtched unrelted donor to chieve trnsplnt outcome similr to tht found with n HLA mtched siling donor. 6 The loci encoding the HLA ntigens re highly polymorphic nd the proility of finding two unrelted individuls who shre ll of their HLA lleles is quite low. 7 9 In the pst, tests used for identifiction of n optiml llogeneic mrrow donor included serologic typing of HLA-A, -B nd -DR ntigens s well s cellulr ssys of comptiility. It is now known tht mny of the serologiclly defined HLA specificities include severl sutypes, 10 nd mismtches etween lleles expressing the sme serologic specificity cn elicit deleterious immune responses. 11,12 Tody, DNA-sed identifiction of HLA-A, -B nd -DRB1 lleles hs replced the use of lterntive histocomptiility testing methods. 13,14 DNA-sed typing of dditionl HLA loci (ie HLA-DQB1, -DPB1 nd -C) is used y some centers to provide dditionl selection criteri. Since 1987, the US Ntionl Mrrow Donor Progrm (NMDP) 15 hs required minimum five of six ntigen-level mtch t HLA-A, -B nd -DR. It is now possile to retrospectively identify the lleles present in stored NMDP ptient nd donor smples collected since 1988 using n identicl level of typing resolution for ll of the pirs. This report summrizes the results of comprehensive DNAsed testing for HLA clss II lleles of 1259 unrelted donor recipient pirs trnsplnted through the NMDP etween 1988 nd 1994 to determine the extent of HLA-D region disprity found within these pirs.

2 386 Mterils nd methods HLA disprity in mrrow trnsplnttion Pired smples selected NMDP trnsplnt nd donor centers sumit to the NMDP Reserch Smple Repository pretrnsplnt whole lood smples from unrelted donors nd their recipients. Cells from 1259 trnsplnt pirs collected since 1988 were selected for study sed on smple vilility within the repository. The smples in the study represented 44.2% of ptients trnsplnted through the NMDP during tht time period (Tle 1). A is nlysis ws performed to determine if the trnsplnt pirs included in the study were representtive of ll trnsplnt pirs from tht time period. The study ws somewht ised for disese (P ), gender (P = 0.008), recipient rce/ethnic group (P = 0.04), yer of trnsplnt (P ), nd trnsplnt center (P ). While mny of the chrcteristics exhiiting is re not likely to ffect the conclusions of this study, differences in the ethnic mix of the ptient popultions served y trnsplnt centers included or not included in the study will likely ffect the llelic frequencies 8,16 nd the types of mismtches oserved in the trnsplnts under study. At the time of the trnsplnt, donors nd recipients were, Tle 1 Donor nd recipient chrcteristics Percentge of study popultion Percentge of totl trnsplnts in this study (No. in study/totl No. trnsplnts) Yer of trnsplnt (53/80) (78/177) (151/291) (241/455) (273/555) (275/680) (188/840) Diseses ALL (219/599) AML (182/518) CML (570/1149) Myelodysplstic disorders (96/229) Severe plstic nemi (49/175) Other mlignnt diseses c (49/135) Other non-mlignnt diseses d (85/214) Rce/Ethnic group % Donors (No.) % Recipients (No.) c Asin/Pcific Islnder 1.0 (12) 0.9 (11) Africn Americn 1.0 (13) 2.6 (32) Cucsin 87.8 (1105) 83.3 (1042) Hispnic 2.2 (28) 4.1 (51) Ntive Americn 0.7 (9) 0.7 (9) Other/Unknown/Decline 7.3 (92) 8.4 (105) Numers in ll chrts my not dd to 100% due to rounding. For exmple, in 1988, the NMDP fcilitted 80 trnsplnts. Donors nd recipients from 53 of these trnsplnts (66.3%) re included in this study. c Includes lymphom, other leukemis, nd plsm cell disorders. d Includes histiocytic disorders, inherited erythrocyte normlities, inherited immune system disorders, inherited metolic disorders, nd other non-mlignnt disorders. e Nine recipients received mrrow from two different donors. t minimum, chrcterized for HLA-A, HLA-B nd HLA- DR ntigens y vrious typing methods. In significnt numer of cses, the donor recipient pirs were lso evluted for comptiility using dditionl techniques such s MLC; however, this informtion ws not collected y the NMDP nd is not prt of this study. Finl donor selection ws determined y ech trnsplnt center sed on their own criteri nd the minimum criterion estlished y the NMDP (5/6 ntigen-level mtch t HLA-A, -B nd -DR). DNA-sed typing The DRB, DQA1, DQB1, DPA1, nd DPB1 lleles of ech smple were identified using locus-specific nd/or one or more group-specific mplifictions followed y hyridiztion with SSOP. DNA sequencing of DRB1 ws lso used s primry method to identify lleles in pproximtely 1000 of these smples. Additionl ssys using sequencespecific mplifiction, restriction frgment length polymorphism nlysis, nd direct DNA sequencing of mplified DNA were used s needed in specific cses to id in llele identifiction (Willims et l, in preprtion; Schmeckpeper et l, in preprtion). Ech smple ws typed t llele level y t lest two independent typing lortories in linded fshion. Discrepnt typings were reviewed nd, if required, further testing ws used to identify the correct llele. Interprettion of the typing results ws sed on the lleles descried in the 1994 WHO HLA nomenclture report. 17 Alleles differing only y silent sustitutions were not distinguished. Alleles descried (or deleted) since 1994, including new lleles identified in this study, 18 were incorported into the interprettion if novel proe hyridiztion pttern or nucleotide sequence ws otined. DRB4 llele typing is not included here nd will e descried in seprte report (Schmeckpeper et l, in preprtion). Smples with only single llele identified y hyridiztion or sequence nlysis were ssumed to e homozygous fter review of the complete typing for common hplotypic ssocitions (eg DRB1*04/*07/*09 with DRB4), heterozygosity t other loci, nd fter review of trnsplnt center typing. Sttisticl nlysis Sttisticl tests pplied to the dt re descried in the Results section. Results Donor nd recipient chrcteristics Clss II HLA-D region lleles in 1259 unrelted donorrecipient pired smples from one mrrow trnsplnts occurring from 1988 to 1994 were identified (Tle 1). These smples were sumitted from 62 trnsplnt centers in the United Sttes nd 18 trnsplnt centers in other countries. The mjority of the trnsplnt recipients (R) nd donors (D) were Cucsin (R: 83.3%, D: 87.8%). In most of the trnsplnts (76.6%), the ptient nd donor were from the sme rcil or ethnic group; 9.5% involved individuls

3 from different rcil or ethnic groups nd 13.9% involved ptient nd/or donor whose rcil or ethnic ckground ws not reported. Allele frequency Of the 107 DRB1 lleles descried in the 1994 WHO nomenclture report 17 (numer excludes lleles which differ only y silent sustitutions), 43 (40.2%) ppered in the recipient popultion nd 42 (39.3%) in the donor popultion (Tle 2). A similrly high percentge (52.8%) of the DPB1 lleles were found. In ddition, five new DPB1 lleles, DPB1*5901, *6801, *7001, *7101, *7301, were identified in this smple set. 18 A greter percentge of known lleles t other, less polymorphic loci ppered in the recipients or donors (DRB3: 4 oserved/4 totl known lleles; DRB5: 3/4; DQA1: 10/11; DQB1: 18/22; DPA1: 6/6). In generl, few lleles ccounted for the mjority of the types identified t most of the loci. For DRB1, 67 68% of the lleles found in these smples were ccounted for y one or two of the following six lleles, DRB1*0101, * 0301, *0401, *0701, *1301 nd *1501, ech present t frequencies of t lest 5%. DRB1*0301, *0701, *1501, ech represented 14 15% of identified DRB1 lleles. Of the 10 DQA1 lleles oserved in the trnsplnt popultion, 59 60% of the identified lleles were ccounted for y one or two of three lleles, DQA1*0102, *0201 nd *0501, present t frequencies greter thn 15%. Of the 18 DQB1 lleles oserved in the trnsplnt popultion, 78 79% of identified lleles were ccounted for y one or two of six lleles, DQB1*0201, *0202, *0301, *0302, *0501, *0602, present t frequencies greter thn 10%. A single llele, DPA1*0103, represented 81% of identified DPA1 lleles. Out of 33 DPB1 lleles oserved, 77 78% of identified lleles were ccounted for y one or two of four lleles, DPB1*0201, *0301, *0401, *0402, present t frequencies greter thn 9 10%. A single llele, DPB1*0401, represented 43 46% of identified DPB1 lleles. These oservtions reflect the reltively homogeneous nture of this predomintely Cucsin (eg R: 83.3%) trnsplnt popultion. The most frequent lleles found in the recipients nd donors re the lleles found in highest frequency in the US Cucsin popultion 16 which most likely incresed the vilility of n HLA mtch. 19 DRB1 mtching Almost 80% of the pirs were mtched for oth lleles t the DRB1 locus (Tle 3). The reltively high level of DRB1 mtching reflects the use of DR serologic nd/or DNA-sed typing in donor selection, the use of cellulr ssys to eliminte potentil donors with HLA-D region differences, nd the potentil is for selection of ptients with common HLA hplotypes (ie ptients with rre HLA hplotypes would hve een unlikely to find donors during the time period evluted, ). Of the 259 pirs with DRB1 mismtches, single llele mismtches were found in 218 (84.2%) pirs nd oth DRB1 lleles were mismtched in 41 (15.8%) pirs (Tle 3). Ten of the single llele mismtches involved DRB1 homozygous donors who shred n llele with the HLA disprity in mrrow trnsplnttion recipient (eg D: 1501, R: 1501,0103) nd 12 involved DRB1 homozygous recipients who shred n llele with the donor (eg D: 0301,1401, R: 0301). Homozygosity in one memer of the pir my ffect the impct of the mismtch on the type of immune response generted (rejection/grft-versus-host disese). 4 Aout 76% of the DRB1 mismtches involved lleles within n llele group (ie etween lleles likely encoding molecules with the sme serologic type) s defined y the first two numers of the llele nme (Figure 1). The remining mismtches (23.7%) involved different llele groups (ie etween lleles encoding molecules with different serologic types). The incresed numer of mismtches of DRB1* 04, *11 nd *13 compred to other DR groups reflects the frequency of these llele groups in the recipient popultion nd the diversity within ech of these llele groups. For exmple, DRB1*04 lleles represented 17.1% of the identified DRB1 lleles ut this llele group contined 19 known lleles (DRB1*0401 DRB1* ). Severl of these lleles pper t reltively high ( 1%) frequencies in the trnsplnt popultion (Tle 2). The typing of DR4 y serology would not hve identified these llele sutypes lthough the use of the MLC under optiml conditions 14 or, for some trnsplnts in more recent yers, high resolution DNA-sed testing should hve detected the mismtch. In contrst, lthough DRB1*07 ws present t high frequency (15.3%) in the trnsplnt popultion, the limited llelic diversity in this group 17 mens tht donors nd recipients mtched y serology for DR7 would lso hve een mtched t the llele level. Mtching t other clss II loci Trnsplnt centers re required to type for DR lthough some centers my hve typed nd mtched for other HLA- D region loci (eg DQ). The level of llele mtching for clss II loci other thn DRB1 vried from 87.2% mtching for the second DRB locus (DRB3,DRB4,DRB5) to 13.2% mtching t the DPB1 locus (Tle 3). The difference in the degree of mtch etween the closely linked loci, DPA1 (56.1%) nd DPB1 (13.2%), most likely reflects the reltively limited numer of DPA1 lleles (n = 6) compred to the extensive DPB1 diversity (n = 53) nd the high frequency (80.8%) of single DPA1 llele, DPA1*0103 (Tle 2), in this predominntly Cucsin popultion. Since DQ nd DP molecules re encoded y polymorphic A1 nd B1 genes, mismtches t A1 or B1 loci result in mismtch for the DQ or DP ntigen. Of the 1259 pirs, 24.4% crry mismtches t DQA1 nd/or DQB1 loci nd 87.6% crry mismtches t DPA1 nd/or DPB1 loci (Tle 3). Totl level of HLA-D region llelic disprity in trnsplnt pirs One hundred nd eighteen (9.4%) donor recipient pirs were mtched for ll lleles of DRB1, DQA1, DQB1, DPA1 nd DPB1 (Figure 2). All of the 118 pirs were mtched for the second DRB locus (ie DRB3, DRB5), if present. A chi-squre nlysis indicted tht no unique HLA chrcteristics were ssocited with the completely 387

4 388 Tle 2 Clss II llele frequencies HLA disprity in mrrow trnsplnttion DRB1* Donor frequency Recipient DQA1* Donor frequency Recipient DPA1* Donor frequency Recipient (%) frequency (%) (%) frequency (%) (%) frequency (%) DPB1* DQB1* DRB3* 3* * * * DRB5* 5* * * Tle summrizes the llele frequencies of 1259 recipients nd their donors. Frequency ws defined s the numer of times n llele ppers divided y the totl numer of lleles (ie 2518 DRB1 lleles totl in the recipient pool). Smples which ppered to e homozygous for prticulr llele were counted twice in this nlysis. Nine recipients received mrrow from two different donors nd these recipients were counted twice in the frequency tles. Not ll individuls crry DRB3 or DRB5 loci. The llele frequencies re clculted only for those hplotypes tht crry the locus sed on known ssocitions etween DRB1 nd DRB3 nd etween DRB1 nd DRB5 loci. If n individul expressed single llele t DRB3, for exmple, nd the DRB1 lleles were oth expected to e ssocited with DRB3 locus, the individul ws ssumed to crry two identicl DRB3 lleles. DRB4 lleles were not evluted in this report.

5 HLA disprity in mrrow trnsplnttion Tle 3 Summry of HLA-D region mtches Impct of mtching one locus on mtching t nery loci 389 Locus No. of Mtched t Mismtched t llele level pirs llele level typed (%) Totl 1 Allele 2 Alleles mismtches mismtched mismtched (%) (% of totl) (% of totl) DRB (79.4) 259 (20.6) 218 (84.2) 41 (15.8) DRB3/4/ c 1077 (87.2) d 158 (12.8) ND e ND DQA (83.6) 207 (16.4) 189 (91.3) 18 (8.7) DQB (76.6) 294 (23.4) 272 (92.5) 22 (7.5) DPA (56.1) 553 (43.9) 505 (91.3) 48 (8.7) DPB (13.2) 1093 (86.8) 687 (62.9) 406 (37.1) Both lleles t the locus re identicl in donor nd recipient. One or oth lleles re mismtched. c Of the 1259 pirs, only 1235 pirs hd t lest one DRB3, DRB4 or DRB5 llele in donor nd/or recipient. d Mtching ws evluted sed on the numer of loci nd the lleles present, for exmple, D: DRB3*0101, lnk ws considered mtched with R: DRB3*0101, DRB3*0101 ut mismtched with R: DRB3*0101,*0201 or R: DRB5:0101, lnk. DRB4 mtching ws evluted sed on the presence or sence of n mplified DRB4 llele. DRB4 disprity will e evluted in seprte report (Schmeckpeper et l, in preprtion). e ND = not determined. Since DRB3, DRB4, nd DRB5 lleles re not found on ll hplotypes, the nlysis of the numer of lleles mismtched ws not evluted. mtched pirs. The reminder of the pirs hd vrile degree of clss II mismtch differing from single llele mismtch t one locus (n = 309, 24.5%) to mismtches for two lleles t one locus (n = 90, 7.1%) to mismtches for nine lleles t ll five loci (n = 3, 0.2%) (Figure 2). No pir differed for 10 lleles t ll five loci, the mximum extent of vrition possile. Approximtely 66% of ll pirs demonstrted mismtch for two or more lleles t one or more loci including DRB1, DQA1, DQB1, DPA1 nd DPB1, nd 59.0% of ll pirs were mismtched t more thn one of these five loci. The mjority of the mismtching ws contriuted y the DP loci. Within the 309 pirs which differed only y single llele mismtch, most (280 pirs) differed only t the DPB1 locus nd just 12 pirs differed only t the DRB1 locus (Tle 4) (DRB3/DRB4/DRB5 mtching ws not included ecuse not ll hplotypes crry these loci). Becuse specific lleles t closely linked HLA loci tend to e ssocited t the popultion level (eg in linkge disequilirium), 9,20 the impct of mtching t one HLA clss II locus on the level of mtching for other clss II lleles ws evluted using chi-squre nlysis. Overll, the mtching t specific loci within the DR/DQ suregions ws significntly correlted with mtching t nother locus in tht region (Tle 5). Thus, 92.8% of smples mtched for DRB1 lleles were lso mtched for DQA1 lleles nd 85.9% were mtched for DQB1. Of the 965 pirs mtched for DQB1, 98.7% were lso mtched for DQA1. Overll, 85.1% of ll pirs were mtched for DRB1, DQA1 nd DQB1 loci. Since strong linkge disequilirium is oserved etween specific DRB1 nd DRB3/DRB4/DRB5 lleles nd since the second expressed DRB locus is less polymorphic thn the DRB1 locus, the level of mtching of the second DRB locus mong the DRB1 mtched pirs ws lso nlyzed. Mtching for DRB3 lleles ws high mong DRB3 positive, DRB1 mtched pirs; 87.5% were mtched. Likewise, mtching for DRB5 ws high mong DRB5 positive, DRB1 mtched pirs; 99.4% were mtched. Mtching of DRB1 nd DRB4 ws not evluted due to the lck of DRB4 llele level dt. DR/DQ mtching did not result in mtching t the DP loci (nd vice vers) supporting the previous oservtions tht recomintion etween the DR/DQ region nd DP loci occurs t rte of pproximtely 1% resulting in miniml linkge disequilirium etween DP nd the rest of the HLA-D region. 20,21 Since testing for DP differences ws not routine in the unrelted donor mtch process nd there is miniml linkge disequilirium, there ws not preferentil selection of donors mtched t these loci. Only 14.2% of pirs mtched for DRB1 lleles were mtched for DPB1 nd only 14.6% of the pirs mtched for DRB1, DQA1 nd DQB1 lleles were lso mtched for DPB1 lleles. Anlysis y odds rtios, however, did suggest tht mtching of DPA1 ws correlted with mtching t DPB1 nd vice vers. Of the 166 pirs mtched t DPB1, 94.0% were lso mtched for DPA1. 35 % Mismtches Donor llele Other Within *01 *03 *04 *07 *08 *09 *10 *011 *012 *013 *014 *015 *016 Recipient DRB1 llele Figure 1 Ctegories of DRB1 mismtches. Ctegories of the kinds of DRB1 mismtches in the trnsplnt pirs plotted s the percentge of the totl DRB1 mismtches. Homozygous lleles which differed etween donor nd recipient were counted twice. Donor lleles leled within involve mismtches of donor lleles within the sme llele group (eg R: 0401, D: 0402); other indictes mismtches etween llele groups (eg R: 0401, D: 0701).

6 HLA disprity in mrrow trnsplnttion No. loci mismtched % Pirs No. lleles mismtched Exmple of donor-recipient pir mismtched for 9 lleles t 5 loci Donor Recipient DRB DRB DQA DQA DQB DQB DPA DPA DPB DPB Figure 2 Disprity in donor recipient pirs. () Percentge of donor recipient pirs mismtched for none to 10 HLA-D region lleles t none to five loci (DRB1, DQA1, DQB1, DPA1, DPB1). Homozygous lleles which differed etween donor nd recipient were counted twice. Three pirs differed for nine lleles t ll five loci. () The HLA-D region lleles crried y one of the three pirs. Tle 4 (n = 309) Locus with mismtch Trnsplnt pirs differing only y single llele mismtch No. of pirs (% of totl) DRB1 12 (3.9) DQA1 1 (0.3) DQB1 10 (3.2) DPA1 6 (1.9) DPB1 280 (90.6) Tle 5 Impct of mtching on mtch t nery loci Pirs mtched t loci (No. ) Also mtched t locus (No.; % ) DRB1 (583 c ) DRB3 (510; 87.5) DRB1 (330) DRB5 (328; 99.4) DRB1 (1000) DQA1 (928; 92.8) DRB1 (1000) DQB1 (859; 85.9) DRB1 (1000) DPA1 (561; 56.1) DRB1 (1000) DPB1 (142; 14.2) DRB1-DQA1 (928) DQB1 (851; 91.7) DRB1-DQB1 (859) DQA1 (851; 99.1) DQB1 (965) DQA1 (952; 98.7) DRB1-DQA1-DQB1 (851) DPA1 (488; 57.3) DRB1-DQA1-DQB1 (851) DPB1 (124; 14.6) DRB1-DQA1-DQB1-DPA1 (488) DPB1 (118; 24.2) DPB1 (166) DPA1 (156; 94.0) The numer of pirs mtched t the indicted locus (loci). The reltive gene order within the mjor histocomptiility complex: telomere-dra- DRB3/4/5-DRB1-DQA1-DQB1-DPA1-DPB1-centromere. The numer nd percentge of pirs lso mtched for the indicted locus. c 583 pirs tht re mtched t DRB1 lso crry DRB3 lleles. Of these pirs, 510 or 87.5% were mtched for oth DRB1 nd DRB3 lleles. Common llele comintions of DR nd DQ The frequency with which specific DRB1 lleles were found with specific DQB1 lleles ws evluted for ll DRB1 lleles with frequency greter thn or equl to 1% Tle 6 Common DRB1/DQB1 ssocitions DRB1 No. individuls DQB1 Frequency of with DRB1 llele individuls with oth DRB1 nd DQB1 lleles (%) c (99.0) (100.0) (99.2) (58.9) d (49.7) (98.8) (82.7) d (77.7) (26.1) (98.0) (96.2) (94.9) (96.9) (96.8) (77.7) (21.8) (96.0) (96.3) DRB1 lleles present t 1% in the donor recipient popultion. DQB1 llele found in linkge disequilirium with the indicted DRB1 llele in Cucsins in other studies. 9,16,22 Since fmily studies were not performed, it is not known if the two lleles re encoded y the sme chromosome (cis vs trns). c For exmple, of the 392 individuls who crried DRB1*0101 llele, 388 or 99% lso crried DQB1*0501 llele. d It is likely tht some of these DQB1*0301 lleles re not on the sme hplotype s DRB1*0401 or DRB1*0407. For exmple, 19 DRB1*0401 individuls crry oth DQB1*0301 nd DQB1*0302 lleles. These individuls lso crry DRB1*1101 or DRB1*1104 lleles which re usully ssocited with DQB1*0301. of the lleles of the study popultion (Tle 6). For exmple, DRB1*0101 ws found in 392 individuls (15.6% of 2518 totl individuls); 388 (99.0%) of those individuls lso crried DQB1*0501 llele. While it is not known, in these individuls, if DQB1*0501 is encoded y the sme

7 chromosome crrying DRB1*0101 (ie in cis position), this frequent ssocition coupled with fmily segregtion dt from mny previous studies 9,16,22 suggest tht mtching for DRB1*0101 will result in mtch for DQB1*0501. The level of DQB1 mtching vried with the DRB1 llele. DRB1*0701 ws found ssocited with t lest two different DQB1 lleles, DQB1*0202 (77.7% of individuls crrying DRB1*0701) nd DQB1*0303 (26.1%). In this circumstnce, DQB1 typing would e required to select the donor with the est DQB1 mtch. A similr resoning pplies to DRB1*0401 *0407 nd *1302 which re lso frequently ssocited with more thn one DQB1 llele. Hplotype mtches Of the 1259 pirs, 821 (65.2%) were mtched for t lest one llele t every locus. Since fmily studies were not crried out to confirm segregtion of hplotypes, it is not known if these mtches might e equivlent to hploidenticl siling mtch. The predicted HLA hplotypes of ll of the donor recipient pirs will e descried in nother study (Begovich, in preprtion). Discussion One chllenge in the use of unrelted vs relted donors hs een the incresed potentil for HLA llele level mismtches. While fmily memers crry lleles of the HLA loci segregting within the fmily s linkge groups with limited possiility of recomintion, the outred humn popultion crries much more diverse collection of lleles on n rry of hplotypes. This diversity is not fully evident using serologic-sed testing ut is reveled using the high resolution, llele level DNA-sed methods employed in this study. This unrecognized disprity likely contriutes to the oserved differences in trnsplnt moridity nd mortlity rtes etween HLA-identicl siling trnsplnts nd HLA-mtched unrelted donors in mny studies. 4,23 26 In this study, the 1259 unrelted donor recipient pirs were derived primrily from the Cucsin US popultion nd exhiited few predominnt lleles t ech locus. Since limited informtion exists on llele frequencies in lrge popultions, it is not possile to compre frequencies of the trnsplnt popultion with, for exmple, the US popultion. In spite of the pprent homogeneity in the pirs studied, only 9.4% of pirs were mtched for five clss II loci (DRB1, DQA1, DQB1, DPA1, DPB1) nd the mjority of pirs (70.5%) differed y one to three lleles ffecting one to three of these loci. Pirs mtched for DRB1 nd oth DQ loci represented 67.6% of the trnsplnts evluted. The DPB1 locus dded the most extensive disprity mong the pirs; 86.8% of the pirs were mismtched. Retrospective llele level clss I typing of these smples is currently eing nlyzed; therefore, dditionl llelic mismtches t HLA-A, -B, -C loci re likely, further reducing the extent of HLA mtching (Hurley et l, in preprtion). Studies of the effect of HLA mtching should tke this extensive disprity t multiple loci into considertion in evlution of the role of single HLA loci on outcome since the inevitle mismtches t other loci will likely hve n impct on the HLA disprity in mrrow trnsplnttion conclusions 27,28 (Bxter-Lowe et l, in preprtion). Extension of this nlysis to trnsplnt pirs from more geneticlly diverse rcil or ethnic groups will likely identify n even greter level of disprity. While the methodology to identify the full extent of HLA diversity now exists in the form of DNA-sed techniques, the expense of extended HLA-D region typing mke this chrcteriztion imprcticl for trnsplnt center tht my hve to HLA type multiple donors within short period of time due to the ptient s condition. Although the dt re still limited, centers should focus their efforts on defining lleles t those loci which pper to e the most criticl for trnsplnt success. Mny trnsplnt centers specify DRB1 llele mtching in donor selection since studies on the impct of mtching t individul HLA-D loci suggest tht DRB1 identity reduces the risk of cute grft-versus-host disese nd improves survivl. 1,11 As result, the primry focus of HLA-D region typing is plced on identifying DRB1 lleles crried y donor nd recipient. A similr study 29 suggests tht DQB1 my lso encode trnsplnttion ntigen nd should e evluted in selecting mong severl HLA-A, -B, -DRB1 mtched donors. Strong linkge disequilirium etween DQB1 nd DRB1 lleles should enhnce the proility of finding DRB1 nd DQB1 mtched donor. Since the llorecognition process likely detects the DQ heterodimer, lleles t oth DQ loci must e mtched to insure DQ identity. The dt presented here show tht mtching t DRB1 nd DQA1 mtches DQB1 91.7% of the time, while mtching for DRB1 nd DQB1 mtches DQA1 99.1% of the time. This suggests tht typing cn e focused on DQB1 in preference to DQA1 to select donor who is potentilly mtched for DR nd oth DQ loci. The impct of mtching DRB3, DRB4 or DRB5 lleles is not known. The HLA molecules encoded y these loci do ply role in immune recognition 30,31 ut they re expressed t lower level thn DRB1-encoded molecules nd my e secondry llorective trget. Since lleles t these loci re lso in strong linkge disequilirium with DRB1, DRB1 mtched pirs shring lleles t other expressed DRB loci should not e difficult to identify. Typing of DRB3, DRB4 nd DRB5 loci is recommended to identify these mtches when selecting mong severl HLA-A, -B, -DRB1 mtched donors. The impct of DPB1 disprity is not yet known 35,36 (Bxter-Lowe et l, in preprtion). Finlly, selection of HLA clss I mtched donors is lso importnt. The importnce of mtching t the ntigen level for HLA-A nd -B, t minimum, nd, more recently, for HLA-A, -B nd -C lleles hs een demonstrted. 2,5,27,28,37 Becuse of the extensive HLA llele nd hplotype diversity in the popultion, otining llele level mtches for ll HLA loci is difficult. The existence of extended hplotypes improves mtching proilities for some individuls since these hplotypes re found in most rcil or ethnic groups. 8 This study of the HLA clss II lleles crried y 1259 unrelted donor recipient pirs is the lrgest, most comprehensive nlysis of HLA-D region disprity nd mtching in trnsplnt popultion to dte. The study encompsses ptients trnsplnted over 7 yers from 80 trnsplnt cen- 391

8 392 HLA disprity in mrrow trnsplnttion ters. The DNA-sed typing crried out in linded duplicte pproch, using constnt level of resolution nd some of the most powerful methodologies for llele identifiction, ensures the consistency nd reliility of this dt set. The focus on defining only those llelic vritions tht result in differences in mino cid sequence etween llelic products hs identified those mismtches which might stimulte llorecognition. In contrst, erlier studies 1,3,11,23,29,35 were often focused on one or few HLA-D region loci nd/or utilized less informtive typing methodologies (eg serology) so tht the underlying llelic disprity ws not identified. The informtion provided in this retrospective study forms the foundtion of dtse to evlute the role of HLA mtching on one mrrow trnsplnt outcome (Bxter-Lowe et l, in preprtion; Petersdorf et l, in preprtion), to develop strtegies for permissive mismtches, nd to enhnce HLA typing strtegies to select the optiml donor. These clss II dt will e complemented y typing dt from ongoing studies to identify the clss I lleles, HLA-A, -B nd -C, crried y the sme pirs (Hurley et l, in preprtion). The lrge numer of trnsplnt pirs eing chrcterized (1259) will improve the power of the sttisticl nlysis, limittion in mny previous outcome studies. Continuing high resolution llele level HLA typing of more recent trnsplnt pirs y the NMDP will continue to uild this dt set. Acknowledgements This reserch hs een supported y funding from the Office of Nvl Reserch N to Ntionl Mrrow Donor Progrm nd N J-4036 nd N to the CW Bill Young Mrrow Donor Recruitment nd Reserch Progrm. The views expressed in this rticle re those of the uthors nd do not reflect the officil policy or position of the Deprtment of the Nvy, the Deprtment of Defense, or the US government. We would like to cknowledge the ssistnce of the NMDP Trnsplnt nd Donors Centers in providing cells nd dt for this study, the DNA-sed registry typing lortories for their ssistnce in determining the preliminry DRB nd DQB1 typing of the smples, nd our lortory stff nd NMDP personnel for their ssistnce with the HLA typing nd dt nlysis. Dr Bo Dupont ws instrumentl in the originl design of this project nd oversight ws provided y the NMDP Histocomptiility Committee. This mnuscript is dedicted to the memory of our friend nd collegue, Michel Chopek, MD. Dr Chopek prticipted in the erly phses of the design, dt collection nd nlysis of this project. His enthusism nd commitment hve inspired us nd we re sddened tht he ws unle to see this work come to fruition. References 1 Devergie A, Apperley JF, Lopin M et l. Europen results of mtched unrelted donor one mrrow trnsplnttion for chronic myeloid leukemi. Impct of HLA clss II mtching. 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The predictive vlue of HLA-DR oligotyping for MLC responses. Trnsplnttion 1992; 53: Mickelson EM, Longton G, Ansetti C et l. Evlution of the mixed lymphocyte culture (MLC) ssy s method for selecting unrelted donors for mrrow trnsplnttion. Tissue Antigens 1996; 47: Perkins HA, Hnsen JA. The US Ntionl Mrrow Donor Progrm. Am J Ped Hemtol Oncol 1994; 16: Imnishi T, Akz T, Kimur A et l. Allele nd hplotype frequencies for HLA nd complement loci in vrious ethnic groups. In: Tsuji K, Aizw M, Sszuki T (eds). HLA 1991, vol. 1. Oxford University Press: New York, 1992, pp Bodmer JG, Mrsh SGE, Alert ED et l. Nomenclture for fctors of the HLA system, Hum Immunol 1994; 41: Noreen H, Steiner L, Dvidson M et l. Six new DPB1 lleles identified in study of 1302 unrelted one mrrow donorrecipient pirs. Tissue Antigens 1997; 49: Howrd MR, Gore SM, Hows JM et l. 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