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Linkage disequilibria between HLA-B and the rarer properdin factor B alleles, BfF1 and BfS1
Linkage Disequilibria between HLAoB and the Rarer Properdin Factor B Alleles, B f f l and B f s I G.J. Stewart, H.M. Cann., F.C. Grurnet, J. Hay, R.L. Kirk, and R. Payne
ABSTRACT: Phenotypie association and highly significant link,~ge disequilibria ha*'e been demonstrated ]or HLA-BI8 and Bf~l and HLA-BwSO and BfSl alleles among Caucasians from Australia and the United States (San Francisco Bay areal The HLA-BI8, BfFI association appears to be associated with HLA-Aw30. It is possible that BfSl arose as a mutation, after ,'he evolutionary splitting of HLA-Bw21. on a~ HLA-BwSO haplo(vpe, and that Bf~l arose on an HLA-Aw30, B 18 haplotype.
ABBREVIATIONS Bf properdin factor B LD linkage disequilibrium
IDDM
insulin dependent diabetes mellitus
INTRODUCTION The recent reports of an associauon between the rarer alleles of properdin factor B (Bf), B f f l and Bf:~l, and insulin-dependent diabetes mellitus (IDDM) [1,2,3], particularly B f f l in young diabetic children [4], have focused attention on the associations between the HLA system and these Bf alleles. B f r l has been reported to occur predominantly with H I A-B 18 in normal German [5 ], Australian [6], Newfoundland [7] and French [8] populations. In the last two studies, although the numbers were small, the association appeared to be with the haplotype HLA-Aw30, B1.8, and possibl/ as part of a full southern European haplotyl)e: HLA-Aw30, Cw5, B18, B f f l , DRw3 [8,3,9]. An association between B f f l and H L A - B I 8 has also been noted among pa.tients with I D D M [10], B f S l was found to be significantly associated with HLA-Bw21, B13, and B I 4 in the German study, and Bw21 in the Australian, but occurred only on A9,B12 haplotypes in Newfoundland. N o significant associations were detected
From the Departments of Medh'ine. Pediatrics. Genetics. and Patholog;. Stanford Unirersity School of Medicine. Stanford, California, the South Australian Red Cross Blood Transfusfi~n Sert'ice. Adelaide. Australia, and the Department of Human Biology. John Curtin School of Medical Research. Canberra. Australia. Address requestsfor reprints ~o: Dr. G.J. Stewart. Department of MedMne. Dit'ision of immunolo~. Stanford University Schoolof Mediciue, Stanfird, CA 94305, Received 1979.
Human Immunolo~./I, 31-~6 (1980) © El~evior North Holland, Inc.. 1980 52 Vanderbilt Ave., New York, NY 10017
31 0198-8859/80J01031-06$2.25
32
G.J. Stewart et al. in the French popuhtion although one of the two BfSl haplotypes also carried Bw2 I. Since each of the above studies detected relatively small numbers of HLAB18 and Bw21 positive individuals, the strength of the associations between these antigens and BfF1 and BfSl, respectively, has been difficult to assess. We therefore sought known HLA-B18 and Bw21 positive normal people in both Australian and American populations for the purpose of Bf typing. In addition to selecting individuals for Bf typing on the basis of their known HLA status, haplotype data derived from randomly selected Caucasian families, typed as part of an o:~going study at Stanford, were scrutinized for linkage disequilibria (LD) betweet, Bf~l, BfSl, and HLA. The HLA-Bw21,BfSl association was of particular interest since HLA-Bw21 can be split into Bw49(21) and BwS0 (21) on the basis of cytotoxicity with specific antisera and with anti-Bw4 and anti-Bw6 antisera, respective/y, while Bw49 and BwS0 share a common antigenic determinant in absorption studies [11 ]. Although the evolutionary basis of such "splits" is not fully understood, it has been suggested that they resulted from a recombination event between the polymorphic HLA-B locus and the HLA-Bw4/Bw6 locus [12]. An association between BfSl and both HLA-Bw49 and Bw50 would suggest that the BfSl mutation arose prior to the separation of the two antigens, while an association between the Bf allele and only one of the HLA-Bw21 "splits" would suggest that BfSl arose after that separation.
MATERIALS A N D M E T H O D S Twenty--two HLA-B18 positive and 21 HLA-Bw21 positive, unrelated, normal, healthy Caucasian individuals, previously HLA typed at the South Australian Red Cross, were selected for Bf phenotyping. Phenotype data for Bf was also collected on 21 HLA-BI8 positive and 29 HLA-Bw21 positive normal Caucasian individuals studied at Stanford. Most of the HLA-BI8 and more than half of the HLA-Bw21 positive U.S. people had been typed as part of a haplotype study on Caucasian families (see below). The remainder were Bf typed because of their known HLA (B18 or Bw21) status. In contrast to the highly selected phenotype data, 361 HLA,Bf haplotypes have been derived from randomly selected two-generation (and some three-generation) Caucasian families, including both parents and two or more offspring, residing, with few exceptions, in the San Francisco Bay area. Linkage disequilibria between HLA-B alldes and BfF1 and 8fSl were sought. Delta values were calculated as previously described [6]. Bf allotypes were detected by immunofncation following high-voltage dectrophoresis of serum or plasma in 1% agarose [13]. Typing for 15 HLA-A, 23 HLA-B, and 6 HLA-C antigens was performed by routine lymphocyte microcytotoxicity assay using multiple alloantisera. HLABw49 and BwS0 were defined by HLA-~w4 and Bw6 reactivity, respectively, and by multiple antisera, including specific reagents Simpkius (serum 409, Eighth International Histocompatibility workshop), Kendrick (serum 405), and Heiser (serum 075). The significance of all associations was tested by Fisher's exact test. RESULTS The phenotype date indicated that approximately one-third of HLA-B18 positive individuals will also be positive for BfFt (Table 1). Thus five of 22 (24%) HLA-B18 positive Australians were BfFI positive (four SF~, one FF~) as were
HLA and B f r l , BfSl TABLE 1
3~
P h e n o t y p e distribution o f Bft:t and BfS, a m o n g normal Caucasians selected for H L A - B 1 8 or Bw21 Bf phenotype
HLA
Population
Number tested
F, pos
St ix~s
-B 18
Australia U.S.A.
22 21
5 8
0 0
-Bw49 (21)
Australia U.S.A. Australia U.S.A.
12 19 9 10
0 0 0 0
2 0 5 8
-BwS0 (21 )
eight o f 21 ( 3 8 % ) normal U.S. individuals (four SF, and FF,). Overall 43 H I , A - B 1 8 positive p e o p l e w e r e p h e n o t y p e d for B f and 13 w e r e B f F , positive (30%). 'The Stanford haplotype data strongly indicated that B f r l is predominantly associated with H L A - B 1 8 (Table 2). Seven o f the 361 haplotypes contained B f ~ l and all seven also coded for H L A - B 1 8 , yielding a delta value o f 0.0183 (p = 5.2 x 10-~0). T w e n t y H L A - B 1 8 haplotypes were e n c o u n t e r e d and hence 7, or 3 5 % , also coded for B f ~ l . Six o f the seven H L A ° B 1 8 , B f ~ l haplotypes also carried H L A - A w 3 0 compared to o n e o f 12 F I L A - B 1 8 positive, B f ~ l negative haplotypes assignable for H L A - A (p = 0.002). An association b e t w e e n H L A - B 1 8 , B f ~ 1 and H L A - A w 3 0 was d.so supported by the p h e n o t y p ~ data. Thus three o f the five Australians and seven o f the eight A m e r i c a n s w h o w e r e positive for b o t h H L A - B 1 8 and B f F t w e r e also positive for H L A - A w 3 0 . Overall a m o n g 43 H L A - B 1 8 positive individuals, H L A - A w 3 0 was e n c o u n t e r e d in ten o f the 13 ( 7 7 % ) w h o w e r e also
TABLE 2
H a p l o t y p e f r e q u e n c i e s a~d linkage disequilibria for the rare B f alleles and HLAoB18, B w 4 9 and B w 5 0 in a U.S. Caucasian population"
Flaplotype
Haplotype frequency
Delta
p value
B 18, Bf s B 18, B f ~" B 18, B f s I Bl8, Bfrl
0.0305 0.0055 0 0.0194
-0.0132 -0.0043 -0.0009 0.0183
0.009 ns ns 5.2 x 10-*°
Bw49, BJs Bw49, B f ~ Bw49, B f s 1 Bw49, Bf~l
0.0249 0.0055 0
0.0009 0.0002 -0.0005 -0.0006
ns ns ns ns
BwSO,B/rs BwS0, Bfr BwS0,BfSl BwS0. Bfrl
0.0028 0 0.0166 0
-0.0125 -0.0034 0.0163 -0.0004
0.0004 ns 2.4 x 10-*z ns
0
"Ba~edon 361 haplotypes. ns equalsnot -Asnificam~p ->0.05). Allele frequencies in parenttl population: HLA-BI$ = 0.055; HLA-Bw49= 0.031; HLA-BwS0=0,019; Bfs = 0.790;B~" = 0.17,~;Bffl = 0.019~and B~I = 0.017.
34
G.J. Stewart et al. BfFt positive, compared to two of the 30 (7%) who were BfF~ negative (p = 8.1 x 10-6). The results of our HLA-Cw5 typing differed from an earlier report of a full haplotype association: HLA-Aw30, CwS, B18, Bf~'l (see above). Cw5 was not found on the HLA-Aw30, B18, B.t'FI haplotype in five of seven individuals carrying this haplotype. Both the phenotype and haplotype data indicated that BfSl is predominantly associated with the HLA-Bwg0 "split" of Bw21. Two of 12 Australian and non,e of 19 American HLA-Bw49 positive individuals were positive, on phenotype testing, for BfSI compared to five of nine Australians and eight of ten Americans positive for HLA-BwS0 (Table 1). While BfSt was not significantly associated with HLA-BwS0 (compared to Bw49) in the Australian group, the exclusive association in the American data was highly significant (p = I. 1 x 10-:'). Despite this difference, we combined results and the association was significant (2 of 31 compared to 13 of 19,p = 1 x 10-.~). The haplotype data yielded six haplotypes that coded for BfSl and all six also contained HLA-BwS0. Seven HLA-BwS0 haplotypes were encountered providing a delta value of 0.0163 for Bw50, BfSl (Table 2) with a p value of 2.4 x 10 -Iz. A significant/~ value (0.0004) was also noted for the negative association between HLA-BwS0 and Bf s (Table 2), an unavoidable result in view of the strong LD between BfSl and HLA-BwS0.
DISCUSSION The haplotype data clearly establish that strong linkage disequilibria (LD) exist in the normal American population between Bf~'l and HLA-B18 and BfSl and HLA-~, ~50. Other less striking HLA-B,Bf LD were encountered but only the data relevant to the rare Bf alleles have been presented in this brief paper. The results obtained from phenotyping a larger number of HLA-B18 or HLA-Bw21 positive individuals than were encountered in the haplotype study, added sapport to the associations. It is likely that Bf~l occurs predominantly in both Australian and American Caucasian populations on a HLA-Aw30,BI8 haplotype although the constant inclusion of Cw5 in the hapiotype (see [8] ) was not confirmed in the present study. The findings of BfFI on 35% (7 of 20) of HLA-B18 haplotypes and in 30°~ (13 of 43) of HLA-BI8 positive individuals compare well to previous reports of 33% of 11 haplotypes [7] and 27% of 17 haplotypes [8] but contrast with the results of Bender et ai. [5] who detected BfF1 on only 2 of 30 (7%) HLA-BI8 German haplotypes. Some discrepancy was encountered between the U.S. and Australian phenotype data for HLA-Bw21 and BfSt. The finding of 19 American HLABw49 positive individuals, all of whom were BfSt negative, and ten I-ILA-BwS0 positive people, eight of whom were BfSt positive (Table 1) indicates that the Bf allele is associated with Bw50 and not Bw49 (p -- 1.1 x 10-~). Although BfSt occurred in a higher proportion of HLA-Bwg0 than Bw49 positive Australians, the detection of two Bw49 positive, BfSt positive people was not expected. Difficulty with the typing may have accounted for the problem although the same monospecific antisera were used for each group. The results overall, however, establish that B/Sl i$ predominantly, if not exclusively, associated with the HLA-Bwg0 "split" of Bw21. No other data is currently available in the literature for comparison. It appears likely, therefore, that g~/'Sl first appeared on a HLA-Bwg0 haplotype after the evolutionary separation of BwS0 from Bw49. The detection of LD between I:ILA-BwS0 and BfS~ in non-Caucasian individuals would place that event prior to the separation of the
HLA and Bf~l, BfSl
~5
races. We are currently seeking HLA-bw21 non-Caucasian individuals but may be hampered by the apparent restriction o f the HI.,A-Bw21 "'split" to Caucasian populations (see [12] ). There are several possible explanations fi)r the persistence o f these extraordinary gametic associations in the normal pol?uladon. First, it is po~,;sible that the rare Bf alleles arose as recent mutations, Bf~-~ on a HLA-Aw30, B 18 haplotype and BfSl on a HLA-Bw50 haplotype, and that insufficient time has elapsed to allow recombination events to randomly distribme the Bf alleles. Alternatively, some advantage from natural selection may be iinvolved for these haplotypes. Certainly, the demonstration o f Bfrl in almost one-quarter o f childhood diabetics [1,2] suggests the existence of important genes (perhaps affecting the immune response to viral pathogens) in LD with the BfVl allele. If so, the advantage conferred on the general populatiion has been at the ,expense o f increasing susceptibility to IDDM. In addition~ the apparent preservation o f the full haplotype containing HLA-Aw30,B 18,Bf~l ~,nd possibly Cw5,DRw3 and an allele of C4 [9] raises the possibility o f selective interactions between the products of more than one gene locus within the H L A region. In this regard, the absence o f Cw5 from most of our observed H L A - A w 3 0 , B 1 8 , B f r l haplotypes is o f interest because a single recombination would not have been expected to remove Cw5 from the founder haplotype while leaving Aw30 intact. Clearly, further study o f this three-to-six-point association is warranted. Although attractive as a concept, it remains to be determined whether o r not natural selection (aided by the closeness o f Bf to HLA-B) has maintained these associations as does a mechanism for such selection
ACKNOWLEDGMENT "l~his work was supported by National Institutes of Health Grants GM20832, AI 11313, A114438, and HL21662. Dr. Stewart is a recipient of" an Applied Health Science Travelling Fellowship, National Health and Med ical Research Council of Australia- The authors wish re thank Dolly Ness and Betty V~n West for invaluable assistance in this study. REFERENCES 1. Raum D, Alper CA, Stein R, Gabbay KH: Genetic marker for insulin-dependent diabetes mellitus. Lancet 1:1208-1210, 1979. 2. Kirk RL, Theophilus J, Whitehouse S, Court J, Zimmet P: Genetic susceptibility to diabetes mellitus: The distribution of proper.tin factor B (Bf) and glyoxalase (GLO) phenotypes. Diabetes 28:949-951, 1979. 3. Deschamps l, Lestradet H, Marcelli-Barge A, Benajam A, Busson M, Hors J, Dausset J: Properdin factor B alleles as markers for insulin-dependent diabetes. Lancet 2:793 1979. 4. Kirk RL, Ser.~eantson SW, Theophilus J, Zi~met P, Whitehouse S, C,:mrt JM: Age relationship between insulin-dependent diabetes and rare alleles of properdin factor B. Lancet 2:537, 1979. 5. Bender K, Mayerova A, Frank R, Hiller C, W:ienker T: Haplotype analysis of the linkage group HLA-A: HLA-B: Bf and its: bearing on the interpretation of the linkage disequilibrium. Human Genetics 36:~91-196, 1977. 6. Stewart GJ, Basten A, Kirk RL: Strong link~age disequilibrium be,~ween HLA-Dw2 and BfS in multiple sclerosis and in the normal population. Tissue Antigens 14:86-97, 1979.
36
G.J. Stewart et al. 7. Larsin B, Arnason A, Barnard JM, Buehler SK, Edwards JH, Marshall WH: Bf types of HLA haplotyped individuals in an isolated Newfoundland population. Tissue Antigens 10:403-409, 1977. 8. Dausset J, Legrand L, Lepage V, Conm L, Marcelli-Barge A, Wildloecher I, Benajam A, Meo T, Degos L: A haplotype study of HLA complex with special reference to the HI.A-DR series and to Bf, C2, and glyoxalase 1 polymorphisms. Tissue Antigens 12:297-307, 1978. 9. de Mouzon A, Ohayon E, Ducos J, Hauptmann G: Bf and C4 markers for insulin-dependent diabetes in Basques. Lancet 2:1364, 1970. 10. Bertrams J, Baur MP, Gmneklee D, Gries FA: Association of BfFI, HLA-BI8 and insulin-dependent diabetes mellitus. Lancet 2:98, 1979. 11. Grumet FC, Colombe BW, Goodfellow P: Bw21 partition and crossreactivity of the components. Tissue Antigens 11:39-44, 1978. 12. Barnstable CJ, Snary D, Crumpton MJ, Bodmer WF: The structure and evolution of the HLA-Bw4 and Bw6 antigens. Tissue Antigens 13:334-341, 1979. 13. Alper CA, Boenisch T, Watson L: Genetic polymorphism in human glycine-rich beta-glycoprotein.J Exp Med 135:68-80, 1972.
ABSTRACT: Phenotypie association and highly significant link,~ge disequilibria ha*'e been demonstrated ]or HLA-BI8 and Bf~l and HLA-BwSO and BfSl alleles among Caucasians from Australia and the United States (San Francisco Bay areal The HLA-BI8, BfFI association appears to be associated with HLA-Aw30. It is possible that BfSl arose as a mutation, after ,'he evolutionary splitting of HLA-Bw21. on a~ HLA-BwSO haplo(vpe, and that Bf~l arose on an HLA-Aw30, B 18 haplotype.
ABBREVIATIONS Bf properdin factor B LD linkage disequilibrium
IDDM
insulin dependent diabetes mellitus
INTRODUCTION The recent reports of an associauon between the rarer alleles of properdin factor B (Bf), B f f l and Bf:~l, and insulin-dependent diabetes mellitus (IDDM) [1,2,3], particularly B f f l in young diabetic children [4], have focused attention on the associations between the HLA system and these Bf alleles. B f r l has been reported to occur predominantly with H I A-B 18 in normal German [5 ], Australian [6], Newfoundland [7] and French [8] populations. In the last two studies, although the numbers were small, the association appeared to be with the haplotype HLA-Aw30, B1.8, and possibl/ as part of a full southern European haplotyl)e: HLA-Aw30, Cw5, B18, B f f l , DRw3 [8,3,9]. An association between B f f l and H L A - B I 8 has also been noted among pa.tients with I D D M [10], B f S l was found to be significantly associated with HLA-Bw21, B13, and B I 4 in the German study, and Bw21 in the Australian, but occurred only on A9,B12 haplotypes in Newfoundland. N o significant associations were detected
From the Departments of Medh'ine. Pediatrics. Genetics. and Patholog;. Stanford Unirersity School of Medicine. Stanford, California, the South Australian Red Cross Blood Transfusfi~n Sert'ice. Adelaide. Australia, and the Department of Human Biology. John Curtin School of Medical Research. Canberra. Australia. Address requestsfor reprints ~o: Dr. G.J. Stewart. Department of MedMne. Dit'ision of immunolo~. Stanford University Schoolof Mediciue, Stanfird, CA 94305, Received 1979.
Human Immunolo~./I, 31-~6 (1980) © El~evior North Holland, Inc.. 1980 52 Vanderbilt Ave., New York, NY 10017
31 0198-8859/80J01031-06$2.25
32
G.J. Stewart et al. in the French popuhtion although one of the two BfSl haplotypes also carried Bw2 I. Since each of the above studies detected relatively small numbers of HLAB18 and Bw21 positive individuals, the strength of the associations between these antigens and BfF1 and BfSl, respectively, has been difficult to assess. We therefore sought known HLA-B18 and Bw21 positive normal people in both Australian and American populations for the purpose of Bf typing. In addition to selecting individuals for Bf typing on the basis of their known HLA status, haplotype data derived from randomly selected Caucasian families, typed as part of an o:~going study at Stanford, were scrutinized for linkage disequilibria (LD) betweet, Bf~l, BfSl, and HLA. The HLA-Bw21,BfSl association was of particular interest since HLA-Bw21 can be split into Bw49(21) and BwS0 (21) on the basis of cytotoxicity with specific antisera and with anti-Bw4 and anti-Bw6 antisera, respective/y, while Bw49 and BwS0 share a common antigenic determinant in absorption studies [11 ]. Although the evolutionary basis of such "splits" is not fully understood, it has been suggested that they resulted from a recombination event between the polymorphic HLA-B locus and the HLA-Bw4/Bw6 locus [12]. An association between BfSl and both HLA-Bw49 and Bw50 would suggest that the BfSl mutation arose prior to the separation of the two antigens, while an association between the Bf allele and only one of the HLA-Bw21 "splits" would suggest that BfSl arose after that separation.
MATERIALS A N D M E T H O D S Twenty--two HLA-B18 positive and 21 HLA-Bw21 positive, unrelated, normal, healthy Caucasian individuals, previously HLA typed at the South Australian Red Cross, were selected for Bf phenotyping. Phenotype data for Bf was also collected on 21 HLA-BI8 positive and 29 HLA-Bw21 positive normal Caucasian individuals studied at Stanford. Most of the HLA-BI8 and more than half of the HLA-Bw21 positive U.S. people had been typed as part of a haplotype study on Caucasian families (see below). The remainder were Bf typed because of their known HLA (B18 or Bw21) status. In contrast to the highly selected phenotype data, 361 HLA,Bf haplotypes have been derived from randomly selected two-generation (and some three-generation) Caucasian families, including both parents and two or more offspring, residing, with few exceptions, in the San Francisco Bay area. Linkage disequilibria between HLA-B alldes and BfF1 and 8fSl were sought. Delta values were calculated as previously described [6]. Bf allotypes were detected by immunofncation following high-voltage dectrophoresis of serum or plasma in 1% agarose [13]. Typing for 15 HLA-A, 23 HLA-B, and 6 HLA-C antigens was performed by routine lymphocyte microcytotoxicity assay using multiple alloantisera. HLABw49 and BwS0 were defined by HLA-~w4 and Bw6 reactivity, respectively, and by multiple antisera, including specific reagents Simpkius (serum 409, Eighth International Histocompatibility workshop), Kendrick (serum 405), and Heiser (serum 075). The significance of all associations was tested by Fisher's exact test. RESULTS The phenotype date indicated that approximately one-third of HLA-B18 positive individuals will also be positive for BfFt (Table 1). Thus five of 22 (24%) HLA-B18 positive Australians were BfFI positive (four SF~, one FF~) as were
HLA and B f r l , BfSl TABLE 1
3~
P h e n o t y p e distribution o f Bft:t and BfS, a m o n g normal Caucasians selected for H L A - B 1 8 or Bw21 Bf phenotype
HLA
Population
Number tested
F, pos
St ix~s
-B 18
Australia U.S.A.
22 21
5 8
0 0
-Bw49 (21)
Australia U.S.A. Australia U.S.A.
12 19 9 10
0 0 0 0
2 0 5 8
-BwS0 (21 )
eight o f 21 ( 3 8 % ) normal U.S. individuals (four SF, and FF,). Overall 43 H I , A - B 1 8 positive p e o p l e w e r e p h e n o t y p e d for B f and 13 w e r e B f F , positive (30%). 'The Stanford haplotype data strongly indicated that B f r l is predominantly associated with H L A - B 1 8 (Table 2). Seven o f the 361 haplotypes contained B f ~ l and all seven also coded for H L A - B 1 8 , yielding a delta value o f 0.0183 (p = 5.2 x 10-~0). T w e n t y H L A - B 1 8 haplotypes were e n c o u n t e r e d and hence 7, or 3 5 % , also coded for B f ~ l . Six o f the seven H L A ° B 1 8 , B f ~ l haplotypes also carried H L A - A w 3 0 compared to o n e o f 12 F I L A - B 1 8 positive, B f ~ l negative haplotypes assignable for H L A - A (p = 0.002). An association b e t w e e n H L A - B 1 8 , B f ~ 1 and H L A - A w 3 0 was d.so supported by the p h e n o t y p ~ data. Thus three o f the five Australians and seven o f the eight A m e r i c a n s w h o w e r e positive for b o t h H L A - B 1 8 and B f F t w e r e also positive for H L A - A w 3 0 . Overall a m o n g 43 H L A - B 1 8 positive individuals, H L A - A w 3 0 was e n c o u n t e r e d in ten o f the 13 ( 7 7 % ) w h o w e r e also
TABLE 2
H a p l o t y p e f r e q u e n c i e s a~d linkage disequilibria for the rare B f alleles and HLAoB18, B w 4 9 and B w 5 0 in a U.S. Caucasian population"
Flaplotype
Haplotype frequency
Delta
p value
B 18, Bf s B 18, B f ~" B 18, B f s I Bl8, Bfrl
0.0305 0.0055 0 0.0194
-0.0132 -0.0043 -0.0009 0.0183
0.009 ns ns 5.2 x 10-*°
Bw49, BJs Bw49, B f ~ Bw49, B f s 1 Bw49, Bf~l
0.0249 0.0055 0
0.0009 0.0002 -0.0005 -0.0006
ns ns ns ns
BwSO,B/rs BwS0, Bfr BwS0,BfSl BwS0. Bfrl
0.0028 0 0.0166 0
-0.0125 -0.0034 0.0163 -0.0004
0.0004 ns 2.4 x 10-*z ns
0
"Ba~edon 361 haplotypes. ns equalsnot -Asnificam~p ->0.05). Allele frequencies in parenttl population: HLA-BI$ = 0.055; HLA-Bw49= 0.031; HLA-BwS0=0,019; Bfs = 0.790;B~" = 0.17,~;Bffl = 0.019~and B~I = 0.017.
34
G.J. Stewart et al. BfFt positive, compared to two of the 30 (7%) who were BfF~ negative (p = 8.1 x 10-6). The results of our HLA-Cw5 typing differed from an earlier report of a full haplotype association: HLA-Aw30, CwS, B18, Bf~'l (see above). Cw5 was not found on the HLA-Aw30, B18, B.t'FI haplotype in five of seven individuals carrying this haplotype. Both the phenotype and haplotype data indicated that BfSl is predominantly associated with the HLA-Bwg0 "split" of Bw21. Two of 12 Australian and non,e of 19 American HLA-Bw49 positive individuals were positive, on phenotype testing, for BfSI compared to five of nine Australians and eight of ten Americans positive for HLA-BwS0 (Table 1). While BfSt was not significantly associated with HLA-BwS0 (compared to Bw49) in the Australian group, the exclusive association in the American data was highly significant (p = I. 1 x 10-:'). Despite this difference, we combined results and the association was significant (2 of 31 compared to 13 of 19,p = 1 x 10-.~). The haplotype data yielded six haplotypes that coded for BfSl and all six also contained HLA-BwS0. Seven HLA-BwS0 haplotypes were encountered providing a delta value of 0.0163 for Bw50, BfSl (Table 2) with a p value of 2.4 x 10 -Iz. A significant/~ value (0.0004) was also noted for the negative association between HLA-BwS0 and Bf s (Table 2), an unavoidable result in view of the strong LD between BfSl and HLA-BwS0.
DISCUSSION The haplotype data clearly establish that strong linkage disequilibria (LD) exist in the normal American population between Bf~'l and HLA-B18 and BfSl and HLA-~, ~50. Other less striking HLA-B,Bf LD were encountered but only the data relevant to the rare Bf alleles have been presented in this brief paper. The results obtained from phenotyping a larger number of HLA-B18 or HLA-Bw21 positive individuals than were encountered in the haplotype study, added sapport to the associations. It is likely that Bf~l occurs predominantly in both Australian and American Caucasian populations on a HLA-Aw30,BI8 haplotype although the constant inclusion of Cw5 in the hapiotype (see [8] ) was not confirmed in the present study. The findings of BfFI on 35% (7 of 20) of HLA-B18 haplotypes and in 30°~ (13 of 43) of HLA-BI8 positive individuals compare well to previous reports of 33% of 11 haplotypes [7] and 27% of 17 haplotypes [8] but contrast with the results of Bender et ai. [5] who detected BfF1 on only 2 of 30 (7%) HLA-BI8 German haplotypes. Some discrepancy was encountered between the U.S. and Australian phenotype data for HLA-Bw21 and BfSt. The finding of 19 American HLABw49 positive individuals, all of whom were BfSt negative, and ten I-ILA-BwS0 positive people, eight of whom were BfSt positive (Table 1) indicates that the Bf allele is associated with Bw50 and not Bw49 (p -- 1.1 x 10-~). Although BfSt occurred in a higher proportion of HLA-Bwg0 than Bw49 positive Australians, the detection of two Bw49 positive, BfSt positive people was not expected. Difficulty with the typing may have accounted for the problem although the same monospecific antisera were used for each group. The results overall, however, establish that B/Sl i$ predominantly, if not exclusively, associated with the HLA-Bwg0 "split" of Bw21. No other data is currently available in the literature for comparison. It appears likely, therefore, that g~/'Sl first appeared on a HLA-Bwg0 haplotype after the evolutionary separation of BwS0 from Bw49. The detection of LD between I:ILA-BwS0 and BfS~ in non-Caucasian individuals would place that event prior to the separation of the
HLA and Bf~l, BfSl
~5
races. We are currently seeking HLA-bw21 non-Caucasian individuals but may be hampered by the apparent restriction o f the HI.,A-Bw21 "'split" to Caucasian populations (see [12] ). There are several possible explanations fi)r the persistence o f these extraordinary gametic associations in the normal pol?uladon. First, it is po~,;sible that the rare Bf alleles arose as recent mutations, Bf~-~ on a HLA-Aw30, B 18 haplotype and BfSl on a HLA-Bw50 haplotype, and that insufficient time has elapsed to allow recombination events to randomly distribme the Bf alleles. Alternatively, some advantage from natural selection may be iinvolved for these haplotypes. Certainly, the demonstration o f Bfrl in almost one-quarter o f childhood diabetics [1,2] suggests the existence of important genes (perhaps affecting the immune response to viral pathogens) in LD with the BfVl allele. If so, the advantage conferred on the general populatiion has been at the ,expense o f increasing susceptibility to IDDM. In addition~ the apparent preservation o f the full haplotype containing HLA-Aw30,B 18,Bf~l ~,nd possibly Cw5,DRw3 and an allele of C4 [9] raises the possibility o f selective interactions between the products of more than one gene locus within the H L A region. In this regard, the absence o f Cw5 from most of our observed H L A - A w 3 0 , B 1 8 , B f r l haplotypes is o f interest because a single recombination would not have been expected to remove Cw5 from the founder haplotype while leaving Aw30 intact. Clearly, further study o f this three-to-six-point association is warranted. Although attractive as a concept, it remains to be determined whether o r not natural selection (aided by the closeness o f Bf to HLA-B) has maintained these associations as does a mechanism for such selection
ACKNOWLEDGMENT "l~his work was supported by National Institutes of Health Grants GM20832, AI 11313, A114438, and HL21662. Dr. Stewart is a recipient of" an Applied Health Science Travelling Fellowship, National Health and Med ical Research Council of Australia- The authors wish re thank Dolly Ness and Betty V~n West for invaluable assistance in this study. REFERENCES 1. Raum D, Alper CA, Stein R, Gabbay KH: Genetic marker for insulin-dependent diabetes mellitus. Lancet 1:1208-1210, 1979. 2. Kirk RL, Theophilus J, Whitehouse S, Court J, Zimmet P: Genetic susceptibility to diabetes mellitus: The distribution of proper.tin factor B (Bf) and glyoxalase (GLO) phenotypes. Diabetes 28:949-951, 1979. 3. Deschamps l, Lestradet H, Marcelli-Barge A, Benajam A, Busson M, Hors J, Dausset J: Properdin factor B alleles as markers for insulin-dependent diabetes. Lancet 2:793 1979. 4. Kirk RL, Ser.~eantson SW, Theophilus J, Zi~met P, Whitehouse S, C,:mrt JM: Age relationship between insulin-dependent diabetes and rare alleles of properdin factor B. Lancet 2:537, 1979. 5. Bender K, Mayerova A, Frank R, Hiller C, W:ienker T: Haplotype analysis of the linkage group HLA-A: HLA-B: Bf and its: bearing on the interpretation of the linkage disequilibrium. Human Genetics 36:~91-196, 1977. 6. Stewart GJ, Basten A, Kirk RL: Strong link~age disequilibrium be,~ween HLA-Dw2 and BfS in multiple sclerosis and in the normal population. Tissue Antigens 14:86-97, 1979.
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G.J. Stewart et al. 7. Larsin B, Arnason A, Barnard JM, Buehler SK, Edwards JH, Marshall WH: Bf types of HLA haplotyped individuals in an isolated Newfoundland population. Tissue Antigens 10:403-409, 1977. 8. Dausset J, Legrand L, Lepage V, Conm L, Marcelli-Barge A, Wildloecher I, Benajam A, Meo T, Degos L: A haplotype study of HLA complex with special reference to the HI.A-DR series and to Bf, C2, and glyoxalase 1 polymorphisms. Tissue Antigens 12:297-307, 1978. 9. de Mouzon A, Ohayon E, Ducos J, Hauptmann G: Bf and C4 markers for insulin-dependent diabetes in Basques. Lancet 2:1364, 1970. 10. Bertrams J, Baur MP, Gmneklee D, Gries FA: Association of BfFI, HLA-BI8 and insulin-dependent diabetes mellitus. Lancet 2:98, 1979. 11. Grumet FC, Colombe BW, Goodfellow P: Bw21 partition and crossreactivity of the components. Tissue Antigens 11:39-44, 1978. 12. Barnstable CJ, Snary D, Crumpton MJ, Bodmer WF: The structure and evolution of the HLA-Bw4 and Bw6 antigens. Tissue Antigens 13:334-341, 1979. 13. Alper CA, Boenisch T, Watson L: Genetic polymorphism in human glycine-rich beta-glycoprotein.J Exp Med 135:68-80, 1972.