Linkage Disequilibrium Between the HLA-DQA2 Alleles and the HLA-DR/DQ Complex Suvina Ratanachaiyavong, Jeffrey L. Bidwell, Elizabeth A. Bidwell, and Alan M. McGregor

ABSTRACT: A statisticallysignificantassociationwas

observed between alleles of the HLA-DQA2 and of the DIUDQ complex in a DNA-restriction fragmentlength polymorphismstudy of 219 membersfrom 21 multiplex familiesof patientswith hyperthyroidGraves'diseaseand ABBREVIATIONS DNA-RFLP DNA-restriction fragment length polymorphism IDDM insulin-dependent diabetes mellitus

773 unrelated individualsselected for homozygosityof the HLA-DQA2 alleles.This data provides evidencefor linkage disequilibriumrather than for a hot spot of recombinationwithin the HLA-DQsubregion.Human Immunology 30, 136-139 (1991)

LD MHC

linkage disequilibrium major histocompatibility complex

INTRODUCTION The human major histocompatibilit~ complex (MHC), consisting of three classes of genes (class I, II, and liD, spans over 3800 kb in the distal portion of band 6p21.3 on the short arm of chromosome 6. The MHC class I and II gene products are cell membrane glycoproteins whose functions are primarily concerned with the regulation of the immune response [1], whereas the MHC class III genes encode serum complement components C2, factor B, and C4 [2] whose functions seem likely to modulate the immune response [3]. More recently, genes encoding cytokines, tumor necrosis factors (TNF-a and TNF-/3), and members of the heat shock protein family (HSP-70) have also been mapped to this region [4, 5]. The association between certain HLA antigens, particularly class II (HLA-DR and -DQ), and a number of autoimmune disorders has been wellFrom the Department of Medicine, King's CollegeSchool of Medicine and Dentistry, BessemerRoad, London, and United Kingdom Transplant Service,SouthmeadRoad, Bristol, United Kingdom. Address reprint requeststo: Dr. Suvina Ratanachaiyavong, Department of Medicine, King's CollegeSchoolof Medicine and Dentistry, Bessemer Road, London, S£5 9PJ, UK. ReceivedFebruary 16, 1990; acceptedSeptember20, 1990.

1~6 0198-8859/91/$3.50

described [6]. More recently, following the availability of cDNA probes and recombinant DNA techniques, the human MHC has been thoroughly examined by many investigators. The human MHC class II region (HLA-D) can be subdivided into three main subregions: HLA-DR, -DQ, and -DP. Recently, two new loci lying between the DQ and DP subregions, namely DO (for DO/3) and DN (for DOn or DZ~), have been defined [7]. The DQ subregion consists of one pair of functionalgenes (DQA1 for DQa and DQB1 for DOff) and one pair of genes of as yet uncertain function (DQA2 for DX~ and DQB2 for DXfl) [8]. Linkage disequilibrium (LD) between the MHC genes is generally well-documented [9, 10] with the strongest LD appearing to exist between the HLA-DR and -DQ subregions [11,12]. A biallelic polymorphism, represented by 2.1- and 1.9-kb TaqI fragments detected by DQoe probe, has been described for the DQA2 gene [13]. The alleles have been designated DQA2-U and DQA2-L, respectively [14]. Apparent lack of association between specific DQA2 alleles and HLA-DR/DQ specificities, described in an earlier study [ 13], has led to the suggestion that a "recombination hot HumanImmunology30,136-139(1991) ©AmericanSocietyfor HistocompadbilityandImmunogenedcs,1991

LD of the HLA-DQA2 Alleles

spot" may exist between the DQB1 and DQA2 genes [15, 16]. The lack of association was also supported by more recent studies in French and Dutch populations [17, 18]. In this report we provide evidence suggesting that LD does exist between the DRB1-DQA1-DQB1 and the DQA2 subre#ons. METHODS Genomic DNA was prepared from whole blood samples taken from 219 members of 21 multiplex families of patients with Graves' disease and from 1501 unrelated individuals consisting of healthy control subjects and patients awaiting renal or bone marrow transplantation. DNA samples were first digested with Taql restr.iction endonuclease, size-fractionated by agarose gel electrophoresis, and transferred to membranes as previously described [19]. Filters were subsequently hybridized with locus-specific DIT~ (pRTV1) and full-length DQ~x (plIa-5) and DOff (plIfl-1) cDNA probes. When necessary, DNA samples were also subjected to MspI digest and hybridized with DQ~x probe to distinguish between DR7-DQw9 and DR9-DQw9 allogenotypes. The assignment of HLA specificitles was based on DNA-restriction fragment length polymorphism (DNA-RFLP) allogevotyping [12, 19]. RESULTS AND DISCUSSION The segregation of DNA-RFLP allogenotypes was observed within the Graves' families nor only between the HLA-DR,8-DQcx-DQfl DNA-RFLP patterns but also for the biallelic polymorphism of the DQA2 gene. No recombination occurred between the DRB1-DQA1DQB1 and DQA2 genes in 19 multiplex families with 160 informative meioses (two families were uninformative). The trend of association between either the DQA2-U or DQA2-L alleles and distinctive DNARFLP allogunotypes was observed in 158 extended haplogenotypes deduced from 219 members of 21 multiplex families. Similar trends of association were observed when analyses were performed in the larger population of 1501 unrelated individuals consisting of healthy subjects and patients awaiting renal or bone marrow transplantation. Due to the lack of family information, only 773 individuals were included in the analysis based on their homozygosity for either the DQA2-U or DQA2-L alleles. The HLA class I! complexes are not only highly polymorphic but also highly homologous, demonstrating complex interrelationships. Table 1 demonstrates the significance of the association between the biailelic polymorphism of the HLA-DQA2 gene and specific DNA-RFLP allogenotypes, and the HLA-DR/DQ serological and Dw cellular specificities.

137

In order to further substantiate these associations, the new HLA nomenclature defined by The WHO Nomenclature Committee for factors of the HLA system in August 1989 [19] has been incorporated. The total number of 1636 DQA2 alleles (684 DQA2-U and 952 DQA2-L) were derived from 773 unrelated individuals who were homozygous for either DQA2-U or DQA2-L alleles in conjunction with 90 haplogenotypes from the nonaffected members of the families (all haplogenotypes shared by affected members were excluded from the analysis). As shown in Table 1, all 16 of the more common allogenotypes (from the total of 30) showed statistically signhqcant associations with either the DQA2-U or DQA2-L alleles. S/milar trends of a s ~ i a tion were also observed for the remaining 14 of the tess frequent allogenotypes, though these did not reach statistical significance. These data suggest that LD does exist between the HLA-DQA2 and HLA-DR/DQ complex. However, the degree of LD does not appear to be as strong as the LD within the DRBI-DQAi-DQBI complex itself. There are two possible explanations for this. First, the lack of complete association with one or the other allele (DQA2-U or DQA2-L), in particular the DR34-DQ~3-DQf13a (DR4-DQw8) genotype, may indicate further heterogeneity of this genotype which is currently beyond the limit of detection by DNA-RFLP. For example, at least four different DRB1 alleles of the DRfl4 haplotype (DRBI*0401, *0402, *0403, and "0404) have the same DNA-RFLP allogenotype. Second, the weaker LD between the DQA2 and the DQB1-DQA1-DRB1 complex (as compared with the LD within the DRB1-DQA1-DQBI complex itself) may indicate that during genetic evolution, gene conversions which took place in the HLA-DR/DQ re#on were infrequently extended beyond the DRB 1-DQA 1DQB1 complex. It is interesting to note that all of the weLl-documented disease-associated DNA-RFLP aliogenotypes are associated with the DQA2-U allele. For example, DRfl15-DQalb-DO.~lb (DRwlS/Dw2-DQw6) in multiple sclerosis; DR~817t-DQ~2-DQ~2a (a subtype of DRw17) in insulin-dependent diabetes meLlitus (IDDM), tuyasthenia gravis, and Graves' disease; and Dl~4-DQ~3-DQ/83a (DR4-DQw8, TA10-ve/ IIB3+ve) in IDDM and rheumatoid arthritis. In contrast, the DRfl4-DQ~3-DQ~3b (DR4-DQw7, TA10+ve/llB3-ve) DNA-RFLP allogenotype, in Felty's syndrome is strongly associated with the DQA2-L allele (see Table 1). It is conceivable that the association of the DQA2-U allele previously described in IDDM [14] was in fact due to LD between the DQA2-U and the DR3 (DR~17~)/DR4 (DQ33a) haplotypes. In summary, though the DQA2 alleles have been

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The total number of both DQA2 alleles was derived from 773 unrelated individuals who are homozygoas fo~ either o f the two alleles and 90 nonaffected haplogenotypes deduced from 21 families of pasients with Gra,~es" disease. The significance of the association was ¢'~culated using l:isher's exact test with correction for the number of comparisons. "° = p < 0.02, "°° = p < 0.002, and " ' " = p < 0.0001. The corresponding HLA-DR/DQ serological and Dw cellular specificities were based on the assignment of specificities of homozygous typing cell (HTC) lines used in our study. The HLA t ~ e n c l a t u r e was based on ref. 20.

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TABLE 1 Association of DQA2-U and DQA2-L aileles with distinctive D N A - ~ ' L P aliogenotypes

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reported to segregate in families [18], LD between the DQA2 alleles and the D R / D Q complex has only been reported in one previous study of 256 haplotypes [21]. Our data, based on family study and analysis over 1600 haplotypes, confirms a significant LD between the biallelic polymorphisms of the DQA2 gene and the DRB ID Q A 1 - D Q B I complex. The lack of association suggested by earlier studies [13, 15-18] is most likely explicable on the basis of the heterogeneity of HLAD R / D Q serological specificities at the geaomic level. Based on the immunogenetic studies of many autoimmune disorders using D N A - R F L P of the M H C class lI region, we conclude that HLA D N A - R F L P allogenotyping can be used as a tool for (i) genotypic confirmation and subtyping of individuals who share the same HLA class II antigens at the phenotypic level (serological specificities), and (ii) identifying a subpopulation which is genetically more susceptible to developing a particular autoimmune disease, reflected by a particular combination of D N A - R F L P s which are in linkage disequilibrium.

ACKNOWLEDGMENT We would like to thank Dr. D. Larhammar of the Department of Cell Research, the Wallenberg Laboratory, University of Uppsala, Sweden, who kindly provided the pllc~-5 and pllB-I probes for our studies.

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8. Trowsdalej, Campbell RD: Physical map of huma~ HLA region, immunol Today 9:34, 1988. 9. Grange D, Tongio MM, Hauptmann G, Uring-L~-nbert B, North ML, Mayer S, Neugebauer M, Daur MP: Linkage between HLA-A, C, B Bf and DR alleles: Haplotype study of healthy families by fac¢orial correspondence analysis. [n Albert ED, Baur MP, Mayr WR (eds): His~ocompatibility Testing 1984. Berlin, Springer-Verlag, 1984, p 326. 10. Baur MP, Neugebauer M, Albert ED: Reference table for m'o locus haplotype frequencies for all MHC marker loci. In Albert ED, Baur MP, Mayr WR (eds): Histocompatibility Testing 1984. Berlin, Springer-Verlag, t984, p 677. 11. Schreuder GMT, Degos L: HLA-DR, DQ and Dw relationships. In Albert ED, Baur MP, Mayr WR (eds): Histocompatibility Testing 1984. Berlin, Springer-Verlag, 1984, p 234. 12. Bidwell 3L: DNA-RFLP analysis and genotyping of HLA-DR and DQ antigens. Immunol Today 9:18, 1988. 13. Spielman RS, Lee.L Bodmer WF, BodmerJG, Trowsdale J: Six HLA-D region ~x-ch~dngenes on human chromosome 6: Polymorphism and associations of DCc~-related sequences with DR types. Proc Nad Acad Sci USA 81:3461, 1984. 14. Festenstein H, Awad .1, Hitman GA, Cutbush S, Groves AV, Cassell P, Ollier W, Sachs JA: New HLA DNA polymorphisms associated with autolmmune diseases, Nature 322:64, 1986. 15. Bodmer JG, Bodmer WF: HistocompatibiliD" 1984. lmmunol Today 5:251, 1984. 16. Trowsdale.L YoungJAT, Kelly AP, Austin P.L Carson S. Munier H, So A, Erlich HA, Spielman RS, Bodmer JG, Bodmer WF: Structure sequence and polymorphism in the HLA-D region. Immunol Rev 85:5, 1985. 17. Le Gall I, Macadet A, Font M, Auffray C, StromingerJL, Lalouel L, Dausset .L Cohen D: Exuberrant restriction fragment lengch polymorphism associated with the DQ~ chain gene and the DXcx chain gene. Proc Natl Acad Sci USA 82:5433, 1985 18. Tilanus MG.L Van Eggermond MCJA, Fei H, D'AmaroJ, Schreuder GMTh, Giphart MJ: RFLP of the HLA-DQ region: A diallelic DX~x polymorphism, not linked to DR and DQ specificities. Tissue Antigens 30:128, 1987. 19. Bidwell JL, Jarrold EA: HLA-DR allogenotyping using exon-specific cDNA probes and application of rapid minigel methods. Molec Immuno123:1111, 1986. 20. Bodmer JG, Marsh SGE, Albert E: Nomenclature for factors of the HLA system 1989. Immunol Today 11:3, 1990. 21. Gao X, Stasmy P: Linkage study of the HLA-DX-c¢ alleles with DR and DQ specificities on 256 hap|otypes from DX-cx homozygous individuals. Hum [mmunol 23:98, 1988.