Ancestral Haplotypes: Conserved Population MHC Haplotypes Mariapia A. Degli-Esposti, Anne L. Leaver, Frank T. Christiansen, Campbell S. Witt, Lawrence J. Abraham, and Roger L. Dawkins

ABSTRACT: We describe here a number of Caucasoid MHC haplotypes that extend from HLA-B to DR and that have been conserved en bloc. These haplotypes and recombinants between any two of them account for 73% of unselected haplotypes in our Caucasoid population. The existence of ancestral haplotypes implies conservation of large chromosomal segments. Irrespective of the mechanisms involved in preservation of ancestral haplo-

types, it is clear that these haplotypes carry several MHC genes, other than HLA, which may be relevant to antigen presentation, autoimmune responses, and transplantation rejection. In light of the existence of ancestral haplotypes, it is critical to evaluate MHC associations with disease and transplantation outcome in terms of associations with ancestral haplotypes rather than individual alleles. Human Immunology 34, 242-252 (1992)

ABBREVIATIONS AH ancestral haplotype Bf factor B EDTA ethylenediaminetetra-acetic acid HLA human leukocyte antigen IDDM Insulin-dependent diabetes mellitus

kb MHC PCR RFLP SLE

kilobase major histocompatibility complex polymerase chain reaction restriction fragment length polymorphism systemic lupus erythematosus

INTRODUCTION T h e initial definition o f a haplotype arose from the recognition that serologic patterns segregated within families [1]. Thus, allelic products o f closely linked genes were assumed to be inherited en bloc unless there was specific evidence o f recombination between the genes. It soon became obvious that haplotypes defined by one family study were often similar or identical to those found in other families, suggesting the possibility that there could be some r e m o t e ancestral relationship between different families and that haplotypes had been maintained en bloc o v e r many generations [2]. Alternatively, particular combinations of alleles could be found From the Department of Clinical Immunology, Royal Perth Hospital, and Sir Charles Gairdner Hospital and the University of Western Australia, Perth, Western Australia. Address reprint requests to Dr. R.L. Dawkins, Department of Clinical Immunology, Royal Perth Hospital, GPO Box X2213, Perth, Western Australia 6001. Received August 1991; acceptedJune 2, 1992. 242 0198-8859/92/$5.00

because of selection for combinations of products of genes separated by diverse and irrelevant intervening sequences. Pending distinction between these two possibilities, we chose to designate the combinations of alleles found in apparently unrelated subjects supratypes rather than haplotypes [3]. Interest in supratypes increased when it became clear that many o f those supratypes recognized from population studies were actually associated with autoimmune diseases [3]. By implication, these supratypes were markers for genes that regulated autoimmune responses. Therefore, we sought new methods for determining the physical structure responsible for diseaseassociated supratypes and also m o r e direct means of determining whether these reflected ancestral haplotypes that had been conserved en bloc from a very remote ancestor. W o r k by several groups has now provided unequivocal evidence for this interpretation [ 4 - 1 0 ] . It follows that the genes relevant to autoimmune diseases Human Immunology 34, 242-252 (1992) © American Society for Histocompatibility and lmmunogenetics, 1992

MHC Ancestral Haplotypes

could be anywhere within the megabase sequence that has been maintained over many thousands of generations. Mapping such genes might be possible using approaches that have been successful in inbred and recombinant strains of mice. On the present occasion, we have sought answers to the following questions: 1. Which of the previously described supratypes identify ancestral haplotypes? 2. Are rare supratypes recombinants of ancestral haplotypes? 3. Which HLA-B alleles are shared by more than a single ancestral haplotype? 4. Can haplospecific alleles or combinations of alleles be identified? 5. What proportion of unrelated haplotypes in the population is accounted for by ancestral haplotypes? To address these questions, we needed a large number of supratypes that could be assigned to nuclearhaplotypes by family study and that could be compared between unrelated individuals. To avoid the possibility of relatedness between the haplotypes in the database, we determined which supratypes are carried by three or more unrelated nuclear haplotypes, i.e., from different families. Having determined those supratypes that appeared to identify ancestral haplotypes, we analyzed the remaining haplotypes occurring three or more times to determine whether they would be consistent with historical recombination between two ancestral haplotypes. If multiple haplospecific markers are available, it should be possible to determine the regions where recombination has occurred. The haplotypes that cannot be explained by one historical recombination event may be either complex multiple recombinants or intrinsically rare combinations, perhaps resulting from racial admixture. This report shows that most common supratypes can be accounted for by ancestral haplotypes and their simple recombinants. A limited number of ancestral haplotypes and their telomeric (HLA-B-complotype) and centromeric (complotype-HLA-DR) recombinants are shown to account for 73% of unselected haplotypes in the Caucasoid population studied.

MATERIALS A N D M E T H O D S The data analyzed in this study include all haplotypes assigned from family studies performed by the Department of Clinical Immunology from 1980 to 1990. These studies were performed for transplantation purposes and to study the immunogenetics of autoimmune disorders.

243

HLA typing. HLA-A, C, B, DR, and DQ typing was performed using the standard microlymphotoxicity method. The sera used for this study were well characterized against cells typed in previous International Histocompatibility Workshops.

Complement allotyping, C4 allotypes were determined by immunofixation following electrophoresis of ethylenediaminetetra-acetic acid (EDTA) plasma treated with neuraminidase [11]. The number of C4A and C4B alleles was determined by comparing the relative densities of the C4A and C4B bands [12, 13]. Factor-B (Bf) typing was performed on plasma or serum samples using standard electrophoresis followed by immunofixation with antiserum to factor B [14].

Genotyping and computing. Genotypes were assigned by studying the segregation patterns of the HLA (A, Cw, B, DR, and DQ) and complement (Bf, C4A, and C4B) alleles in at least two generations within a nuclear family. The deduced haplotypes were stored in an IBMbased database (Database Manager). Each haplotype occurring in a family was recorded under the propositus' Unique Medical Record Number (UMRN). Each haplotype within one family was recorded only once with the appropriate haplotype code. For example, in a twogeneration family, the codes were a, b, c, and d, where a and b are the haplotypes carried by the father and c and d are the haplotypes carried by the mother. The following information was recorded for each haplotype: HLA-A, Cw, B, DR and DQ, Bf, C4A, and C4B, date of study, reason for study and ethnic extraction of family. Alleles were only assigned to a haplotype when segregation could be demonstrated unequivocally. Where an allele could not be assigned unequivocally to a haplotype, this was indicated by (a) two alleles being reported at one locus (e.g., C4A3 or C4AQ0) or (b) a "98" with an appropriate comment on the possible allele(s) present. When the observed segregation did not allow distinction between homozygosity for an allele as opposed to the presence of an unrecognized allele on one haplotype, this was indicated by a "99". Records with incomplete typing (i.e., not tested) at HLA-B, Bf, C4A, C4B, or DR were excluded from further analysis. A locally developed sorting program (Cobygram) was used to sort haplotypes based on similarity at HLA and/or C4. All haplotypes carrying combinations of alleles that occurred at least three times were analyzed further. The complete database included 836 haplotypes; seven haplotypes were excluded prior to analysis because of possible recombination within the nuclear family. The remaining 829 haplotypes from 219 unrelated families were studied (total data set 1). A subset of data set 1 was created by excluding all records where the

244

M.A. Degli-Esposti et al.

reason for study was the presence of an HLA-associated disease (ankylosing spondylitis, arthritis, complement deficiency, hemochromatosis, insullin-dependent diabetes mellitus (IDDM), IgA deficiency, myasthenia gravis, myositis, nephritis, rheumatoid arthritis, Sjogren's syndrome, systemic lupus erythematosus, or congenital adrenal hyperplasia) or where the reason for study was unknown. This data set, referred to as haplotype data set 2, included 348 haplotypes from 94 families. These 94 families were studied for the following reasons: bone marrow transplant, renal transplant, C3B deficiency, Ewing sarcoma, Hodgkin's disease, immune deficiency (not complement or IgA deficiency), melanoma, cryoglobulinemia, thalassemia major, or Wilson's disease. RESULTS Most supratypes are present on unrelated haplotypes. A number of supratypes have been identified following the analysis of HLA associations with autoimmune diseases. A list of 23 previously described supratypes [24, 15] and their disease associations is shown in Table 1. Using the Cobygram program, haplotypes were

sorted according to the HLA-B allele, followed by Bf, C4A, C4B, and HLA-DR. Haplotypes showing similarities at all of these loci were grouped together and ranked according to the frequency at which they occur in the data set. The analysis described above was performed for data sets 1 and 2. Most HLA-B alleles were found to occur with particular combinations of alleles at Bf, C4A, C4B, and DR. Thus, 49 supratypes were carried by three or more unrelated haplotypes (Table 2). Of the 23 previously described supratypes listed in Table 1, only two (B47, BfF, C4A1, C4BQ0, DR7 and B62, BfF, C4A3, C4B1, DR13) were found to occur less than three times in data set 1. The B47 supratype is rare and previously identified because of its association with congenital adrenal hyperplasia. The B62 supratype previously described in a renal transplant study [15] was not found in the current data set. Most common supratypes are ancestral haplotypes or simple recombinants of ancestral haplotypes. The 49 supratypes shown in Table 2 have been subclassified according to whether (a) their genomic structure in unrelated sub-

TABLE 1 List of Caucasoid supratypes and their disease associations HLA-

Central n o n - H L A

HLA-

A

Cw

B

C2

Bf

C4A

C4B

DR

Dw

DQw

Disease associations

Race

3 1 30 25 30 ?

7 7 6 ? 5 ? 4 4 4 5 4 6 3 6 3 3 3 3 3 ? 8 8 8

7 8 13 18 18 18 35 35 35 44 44 47 55 57 60 60 60 62 62 62 64 65 65

C C C Q0 C ? ? C ? C C C B C C C C C B C C C C

S S S S F1 S S F S S F F S S S S S S S F S S F

3 Q0 3 4 3 3 3 3 + 2 3 3 3 1 4 6 3 3 Q0 3 4 3 3 2 3

1 1 1 2 Q0 1 1 Q0 Q0 Q0 1 Q0 5 1 1 Q0 2 3 2 1 1 1 + 2 1

15 3 7 15 3 11 11 1 1 4 7 7 14 7 4 8 13 4 4 13 7 1 13

2 3 ? 2 3 ~ ? 1 1 4 17 ~ ? 11 ~ 8 19 4 4 ? 7 20 19

6 2 2 6 2 7 7 5 5 7 2 2 ~ 9 3 4 6 8 8 6 2 5 6

MS, CD, SLE, H e m o c h r G M G , I D D M , SLE RT C2 Def, SLE IDDM RT RT H I V rapid progression H I V rapid progression Felty's RA CD, IgA D e f CAH RT IgA Def, Psoriasis IDDM RT RT IDDM, RA IDDM, RA RT RT IgA Def, 2 1 O H D e f RT

C C C/M C C C C C C C C C C C/M/N C C C C C C C C C

3 11 2 29 3 ? 1 ? ? 2 2 ? ? ?

CAH, congenital adrenal hyperplasia; CD, celiac disease; Def, deficiency; GMG, generalized myasthenia gravis; Hemochr, hemochromatosis; IDDM, insulin-dependent diabetes mellitus; MS, multiple sclerosis; RA, rhematoid arthritis; SLE, systemic lupus erythematosus; and RT, renal transplant survival in Wilton et al. [15]. Supratypes are listed in numerical order according to their HLA-B allele. C, Caucasoid; M, Mongoloid; and N, Negroid.

MHC Ancestral Haplotypes

TABLE 2

245

List o f supratypes (occurring three or more times on a haplotype) sorted by their HLA*B allele

HLA-B

Bf

C4A

C4B

HLA-DR

n

5 51 51 7 7 7 7 8 13 14 14 14 14 18 18 18 21 21 49 22 27 27 27 27 35 35 35 35 35 37 39 39 40 40 60 60 44 44 44 44

S S S S S S S S S S S F S S F1 S F $1 S S S S S S F S S S S F F S S S S S s S S S F F S S S S S S S

3 3 3 3 3 3 3 Q0 3 2 2 3 3 4 3 3 3 2 3 4 3 4 3 3 3 ÷ 2 3 3 3 3 3 2 3 3 3 Q0 3 3 3 3 3 3 Q0 6 6 3 3 3 4 3

1 1 1 1 1 1 1 1 1 1÷2 1÷2 1 1 2 Q0 1 2 1 ÷ 1.5 1 5 1 2 1 1 Q0 Q0 1 1 1 1 Q0 1 1 1 2 Q0 1 Q0 1 1 1 1 1 1 3 1 2 2 1

2 5 7 1 2 4 6 3 7 1 5 6 7 2 3 5 4 7 5 6 1 1 2 4 1 1 4 5 6 6 1 2 4 6 6 8 2 4 4 5 7 7 6 7 4 4 4 4 5

7 8 3 3 53 8 7 104 10 10

44 44 57 57 62 62 62 62 62

4 6

5 7 11

7 3 4

4 8 5 3

37 6 8 3

7 53 3 4 10 3

93 7 26 4 12 19 3 3 20 14 5 3 4 3

jects has been studied and (b) they could be explained by a single historical recombination between two welldefined ancestral haplotypes (Table 3). For convenience column 1 of Table 3 lists previously described supratypes. The genomic structure of most of

these (boxed) has been studied previously, and at least three examples have been found to have identical gross genomic organization [4, 5, 7] and identical D N A structure at all sequences tested so far [ 8 - 1 0 , 16, 17]. Thus, we conclude that these supratypes are effectively identical by descent from a remote ancestor and can be designated ancestral haplotypes. We name ancestral haplotypes according to the B allele for convenience. When two or more ancestral haplotypes appear to carry the same B allele, we add sequential numbers to indicated the order of discovery (e.g., 18.1, 18.2, 18.3). Haplotypes in column 2 o f Table 3 carry a B allele or complotype (Bf, C4A, C4B) that has not been identified in the ancestral haplotypes listed in column 1. These might be rare ancestral haplotypes. Genomic characterization of these haplotypes is required and will progress when sufficient unrelated (nonconsanguineous) homozygous examples become available. The haplotypes listed in column 3 of Table 3 share some alleles with other ancestral haplotypes. These may represent additional ancestral haplotypes or multiple recombinants, i.e., recombinants in which two or more recombination events have occurred between portions of specific ancestral haplotypes. Additional polymorphic markers o f the relevant ancestral haplotypes will be required to distinguish between these two possibilities. It is interesting to note that all of these haplotypes carry the common BfS, C4A3, C4B1 complotype. The haplotypes listed in column 4 of Table 3 can be accounted for by a single historical recombination involving one o f the ancestral haplotypes listed in the first column. The B65(14), BfS, C4A2, C 4 B 1 ÷ 2 , DR5 and B57, BfS, C4A6, C4B1, DR6 haplotypes carry haplospecific markers of the 65.1 and 57.1 ancestral haplotypes, respectively. In these cases, because of the haplospecificity of the complotypes, recombination can be assumed with confidence. The remaining haplotypes in column 4 may be recombinants of the 7.1 ancestral haplotype, but other possibilities could be considered. The genomic organization o f these haplotypes must be analyzed. It should be noted from Table 2 that, as expected, intact ancestral haplotypes occur more frequently than their putative recombinants. Thus, we conclude that most of the common supratypes are either (a) ancestral haplotypes or (b) simple recombinants between these ancestral haplotypes. The combinations o f alleles that occurred less than 3 times may represent (a) very rare ancestral haplotypes or (b) complex recombinants and are not considered further here.

HLA-B alleles may be unique or shared by ancestral haplotypes. HLA-B8 is almost always carried by all or part of the 8.1 ancestral haplotype, viz. HLA-A1, B8, BfS,

246

M.A. Degli-Esposti et al.

TABLE 3

Haplotypes occurring three or more times can be subclassified Haplotypes that require further characterization

Possible ancestr~ haplotypes

Ancestral haplotypes

Probable recombinants of ancestral haplotypes Donor

HLA-B

Bf

C4A

C4B

HLA-DR

AH

HLA-B

Bf

C4 A

C4B

HLA-DR

HLA-B

Bf

C4A

C4B

HLA-DR

HLA-B

Bf

C4A

C4B

HLA-DR

AH

7

S

3

1

2

21

F

3

2

4

27

S

3

1

1

I'~---~---7--T;-2-I

5

65.1

8

S

Q0

1

3

49

S

3

1

5

35

S

3

1

4

~2_-'-_-r_-_-_~----__'C_q

6

5~.i

13

S

3

l

7

13.1

27

S

4

2

1

44

S

3

1

4

18.1

27

S

3

1

4

62

S

3

l

4

37

F

3

1

6

44

S

3

1

18

S

4

2

2

18

F1

3

Q0

3

5

L7,

18

S

3

1

5

18.3

39

F

2

Q0

l

62

S

3

1

5

C_r'__s___3__._~_A

35

S

3

l

5

35.1

44

F

Q0

1

7

35

S

3

1

6

]"~

35

F

3 + 2

Q0

1

51

S

3

1

5

40

S

3

1

6

62

S

3

2

4

51

S

3

l

7

35.3

s S

3 3

2v ~

1

1

7.1

4

71

6

7.1

1

2]

71

1

~

7,l

~

7.1

1

A !

35

S

3

Q0

1

44

S

3

QO

4

39

[~

44

F

3

1

7

44

[__!____3________2. . . . .

50

SI

2

1 + 1.5

7

50.1

55

S

4

5

6

55.1

57

S

6

1

7

60

S

3

1

4

60.1

60

S

3

Q0

8

60.2

60

S

Qo

2

6

62

S

3

3

4

62

S

4

2

4

64

S

3

1

7

65

S

2

1 +2

1

65

F

3

1

6

3

65.2

I ] Well-defined ancestral haplotypes: the gross and detailed D N A structure o f these haplotypes has been studied on three or more unrelated haplotypes. |'.~__"~ T h e p o r t i o n o f a haplotype that may be explained by recombination with a well-defined ancestral haplotype.

C4AQ0, C4B1, and DR3 [3]. For example, in the total data set, 118 haplotypes carry HLA-B8 (Fig. 1), and 104 (88%) of these carry the ancestral haplotype from HLA-B to DR (B8, BfS, C4AQ0, C4B1, DR3). In fact, 78 (75%) of 104 also carry HLA-A1. Of the 14 haplotypes with B8 but not DR3, six have the BfS, C4AQ0, C4B1 complotype, leaving only eight haplotypes (7%) with B8 alone. Interestingly, only a single haplotype with B8 and DR3 lacks the expected BfS, C4AQ0, C4B!. Therefore, if the 8.1 ancestral haplotype is assigned based on the presence of HLA-B8 and DR3, assignment will be correct in 99% of cases. The same sorting approach was used for all other HLA-B alleles. As shown in Fig. 2, 51% (21 of 41) of haplotypes carrying HLA-B 17 carry the 57.1 ancestral haplotype marked by HLA-B57, BfS, C4A6, C4B1, DR7. Six haplotypes (15%) carry the specific complotype (BfS, C4A6, C4B1), but not the relevant class II alleles. The remaining 14 haplotypes carry B17 with different complotypes and appear heterogeneous; two of these were classified as B58 rather than B57. It can be seen that the combination of B17 and DR7 is not sufficient to identify the 57.1 haplotype; in three instances, there are unexpected complotypes. In contrast to HLA-B8 and B57, some B alleles are

obviously shared by more than one ancestral haplotype. An example is HLA-B18, which is shared by three ancestral haplotypes: B18, BfS, C4A4, C4B2, DR2 (18.1) n -- 7; B18, BfF1, C4A3, C4BQ0, DR3 (18.2) n -- 13; and B18, BfS, C4A3, C4B1, DR5 (18.3) n -- 7 (Fig. 3). It should be noted that 18.1 and 18.2 have been recognized for many years because of their association with C2 deficiency and IDDM, respectively. Sharing of the HLA-B allele by more than one ancestral haplotype was observed for B18, B35, B44, B60, B62, and B65; most of these alleles are known to be heterogeneous by serology or other typing techniques. Recombinants between two ancestral haplotypes can be identified. If our designation of putative recombinants is correct, it should be possible to assign each component (telomeric and centromeric) to the ancestral haplotypes listed in Table 3. Examples of putative recombinant haplotypes are given in Fig. 4. As described previously, most HLA-B8 is carried as part of the 8.1 ancestral haplotype. Most haplotypes that carry B8, but no other identifiable portion of 8.1 (tail of B8), are explicable by recombination between 8.1 and another specific ancestral haplotype (Fig. 4). For example, haplotype A2062 contains B8 plus BfS, C4A3, C4B1, DR2, which is the

MHC Ancestral Haplotypes

UMRN C9029 A4292 A4202 R)O13 E034S H0013 E034S OO132 K0165 B5471 A04B K2071 D02,~ 04174 D0Se6 B5471 DO~ FSlm K0~e C0S~ AS3~0 J068S LO~5 D0188 FA071 IAISS O0S46 DS243 FO226 C00SS B~42 FS2S8 F4104S FSSSe BOZm C~2S K0460 AO4eO FS431 DS243 J9012 L400e A20e2 04187 LOS6S E4176 L421 SO 114176 R)012 COS2S 06042 D02nn G40~ A031S J0304 A4243 JS4S3 D0407 A0537 GOeSO EOS08 B2074 D54~ B2074 04174 E0~0 ¢01a4 FG482 J9012 D0331 JS413 O548O

A

gO on

gO

247

O

B

Elf

C4A

7 7 7 7 7 7 7 7 7

6 8 8 8 8 6 6 8 8 8 8 8 6 8 8 8 8 6 8 6 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 6 8 8 8 8 8 8

S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S .5 S $ S $ 5 $ S S S S S S ,5 S S S S S S S S S S S S S S S S S S S S S S S S S

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 9 0 0 0 0 0 0 0 0 0 O 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

gg

90 90

C4B

DR

3 3

3 9e

gO

0

3 3 3 S 3 $ 3 S 3 3 3 $ S S 8 3 $ S S 8 3 3 3 3 3 8 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 S 3 3 S 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3

DQ

2 2 go gO gO

0O444 C0160 L421 SO K0406 D0212 AS,lSO A0448 0O333

OS~I2 1(2057 020¢g A04.1 E4071 H0297 KgO~2 020¢0 JS4S3 06OO1 L0061 F0226 IM2M 0O132 E2~ AOS11 00O20 AGO12 L0184 L0184 o41ge FEH F0452 F314O A2O~ AOOm KOOCO E4071 L20~ EO560 05242 B42Sl

1 1 1 1 1 2 29 3 9 2 11 28 2S 2 2 2 2

8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 iS 8 8 8 8

Oe go

90

2 26 28 2 2 3 3 1 1 1 2 29 1 1 9 1 90 1 1 3 1 1 1 1 1 1 1 24

3 4 S 7 7

g0 90

1'

S S S S S S S S S S S S 90 S S S S S S S S S S S S $ S S S S S S S S 5 S S S S S S m S S S S F

;I 8

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 3 3 = 3 3 3 S 3

8 3 3 S 3 7 3 3 3 ~ 8 3 3 3 8 3 3 $

0

8 3 3 3 3 3 S 8 N 3 S 3 3 3 N $ 3

I" 4 1 1 2 4 2 4 4 5 7 7 7

~ N

~

N

90 90 9e

centromeric (complotype-DR) portion of the 7.1 ancestral haplotype (A3, B7, BfS, C4A3, C4B1, DR2). Haplotype B4251 is m o r e complicated and may be the result of multiple recombinations. W h e n the "tail" of the B57 haplotypes is examined, seven of 12 have a centromeric c o m p o n e n t that can be assigned to a second ancestral haplotype. It should be noted that the recombinant haplotypes referred to in this report are assumed to be the result of a remote recombination event, i.e., the recombination

FIGURE 1 The HLA-B8 allele is carried by one ancestral haplotype marked by A1, Cw7, B8, BfS, C4AQ0, C4B1, DR3. All the haplotypes in data set 1 carrying HLA-B8 are represented. These haplotypes have been sorted according to the frequency of complotype, DR, and A alleles. Haplotypes that carry alleles of 8.1 from HLA-A to DR are shown at the top of the figure, followed by haplotypes that extend from HLA-B to DR. Telomeric recombinants are shown at the bottom. The boxed areas schematically represent those portions of the 8.1 ancestral haplotype that are carried by unrelated B8containing haplotypes. Vertical lines approximately indicate the region where historical recombination has occurred. Alleles that may be carried by the 8.1 ancestral haplotype have been shaded (see DR3 on the B4251 haplotype).

has not occurred within the nuclear family available for examination, but in earlier generations.

Ancestral haplotypes carry haplospecific markers. As shown in Figs. 1 - 3 , unrelated subjects with the same ancestral haplotype share alleles at HLA-A, B, Bf, C4A, C4B, and DR. These alleles provide markers for ancestral haplotypes. All of the alleles carried by ancestral haplotypes, including others not included here, e.g., T N F , have been shown to be haplotypic, i.e., they have

248

M.A. Degli-Esposti et al.

UMRN

A

C

B

Bf

C4A

B0298 D9012 J9013 L9012 G4154 D0228 G6042 C0184 F4105 I:0681 B9012 JgO12 J9012 K9029 F5421 C4254 CO035 F5256 H0450 H0297 00212 J9012 H0276 C0452 G0169 H6046 L0554 F0881 A4278 C0336 E9013 G0251 D5438 F6001 C9013 A5224 H4293 E4218 G0606

1 1 1 1 1 1 1 1 I 1 29 2 2 2 28 2 32 3 3 26 2

6 6 6 6

17 57 57 57 57 57 17 17 17 17 57 57 57 17 57 17 17 17 17 57 57 57 57 17 17 17 57 17 17 17 57 17 17 17 57 17 17 17 17

S S S S S S S S S S S S S S S S S S S S S S S S S S S S S F S S S S S S F F S

6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 3 3 3 3 3 3 3 0 3 3 3 2

K5328 K9013

[,

6 6 6

2

1 1

24 24 11

i1 1 1 1

24 2 2 31 19 1 25 3 11 30

6

4

g9

58 58

1;' 17

17

17

17

S S

99

0 3

DR

C4B

2 0

7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 4 6 6 4 2 6 1 4

Most of the known HLA polymorphism is accounted for by only 22 ancestral haplotypes and their recombinations. An

DQ

estimate o f the frequency of ancestral haplotypes and their recombinants in a local Caucasoid population is shown in Table 5. This estimate was obtained from data set 2. This set includes 348 haplotypes from 94 unrelated families studied for reasons other than the presence of an HLA-associated disease. Since most, if not all, HLA associations with disease reflect associations with ancestral haplotypes, we analyzed the haplotypes in data set 2 in order to avoid inflating our estimate of the frequency of ancestral haplotypes in the population. The frequencies o f the 22 ancestral haplotypes listed in Table 2 (column 1) and their telomeric (HLA-B-complotype) and centromeric (complotype-DR) recombinants have been determined by simple counts. The 60.2 ancestral haplotype (B60, BfS, C4A3, C4BQ0, DR8)

3 99 99 99

2 4 4

0

6 98 5 5 5 99

1

3

6

FIGURE 2 The HLA-B57 allele is carried by one ancestral haplotype marked by A1, Cw6, B57(17), BfS, C4A6, C4B1, DR7. The 39 haplotypes carrying B57/B17 are shown. Two haplotypes with HLA-B58 are shown at the bottom. The haplotypes were sorted for complotypes, DR, and A alleles according to frequency. The boxed areas represent portions of the 57.1 ancestral haplotype. DR7 alleles not found with the BfS, C4A6, C4B1 complotype are shaded.

been found on all examples of a particular ancestral haplotype even when the donors are not known to be related by study o f nuclear or extended families. Thus, for example, all 8.1 ancestral haplotypes carry HLA-B8, BfS, C4AQ0, C4B 1 and DR3 as well as particular alleles at other loci. Some alleles or combinations o f alleles are haplospecific, being found uniquely on one particular ancestral haplotype. HLA-B8 and the BfS, C4AQ0, C4B1 complotype are examples o f such haplospecific markers. A preliminary list o f haplospecific markers carried by different ancestral haplotypes is shown in Table 4. These markers have been defined by serology (HLA-B), allotyping (Bf, C4A and C4B), and polymerase chain reaction (PCR) typing o f D N A with class II allele-specific oligonucleotide primers (DRB1, DRB3, DQA1, and DQB1). Haplospecific alleles may also be defined by restriction fragment length polymorphism (RFLP) typing and sequencing (data not shown).

FIGURE 3 The HLA-B18 allele is carried by three ancestral haplotypes. Haplotypes carrying HLA-B18 have been sorted on the basis of similarities in complotype, HLA-A, and DR. Blocks marked by the presence of the same HLA and complement alleles have then been arranged according to the frequency at which they occurred in data set 1. Three separate blocks can be identified, indicating that the HLA-B 18 antigen is carried by thre~ ancestral haplotypes. Each of the three blocks carries specific HLA-A, Bf, C4A, C4B (complotype), and DR alleles. UMRN B0053 1(4212 B0023 A0315 K0398 C0129 H9013 A0511 E4218 L0081 ,6.0315 L0081 C4219 D5243 H4184 L0589 K0165 00208 D0208 A0458 L6078 H9012 H6046 D4185 K9013 12054 J2034 K0009 J0346 L6013 L0184 C2046 L0554 C9013 C4219 E9013 J2034 J5453 C0381 D2103 B2040 J2034 J2034

A

C

B

Bf

C4A

C4B

DR

5

18 18 18 18 18 ] 18

F1 F1 F1 F1 F1 F1

3 3 3 3 3 3

0 0 0 0 0 0

I 18 18 18 18 18 18 18 18 18 18 18 18 18 18 ] 18 18 18 18 18 18 18 18 18 18 18 18 18 18

F1 F1 F1 F1 F1 F1 F1 FI F L S S S S S S S S S S S S S S S S S S S S

3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 98 0

00 0 0 0 0 0 1 2 2 2 2 2 2 2 2 I 1 I I 1 1 I I I I I I 1 I 0 0 0 0 2 2 I

3 3 3 3 3 3 3 3 3 3 3 3 3 3 2 2 2 2 2 2 2

5

24

5

11 2 1 24 2 3 24 19 10 25 25 25 25 25 19 25 24 24 24 2 2 1 3 3 1 11 3 26

5 5

99

2 7

9

4

2 2 28 2 2 32

0

2

99

2 3

4

99

18 18 lii 18 ~ 18 18 18 18

F F F S S S S

S $

0

0 0 0

0

98

1

12

F

DQ

99

99

99

8

5 5 5 5 5 0 5 0 5 0 98 99 4 99 6 4

7 6

0

6 1

4

99 0

MHC Ancestral Haplotypes

249

B8

HLA B

FIGURE 4 Most recombinants of the 8.1 and 57.1 ancestral haplotypes carry the complotype-DR portion of another ancestral haplotype. The haplotypes carrying HLA-B8 or B57(17) are shown on the left and right, respectively. The vertical height of the shaded areas is proportional to the frequency at which different portions of the 8.1 and 57.1 ancestral haplotypes occur. Haplotypes that carry HLA-B8 or B57, but not the appropriate complotypes and/or DR alleles, are shown individually. The composition of 7/8 haplotypes with B8 and 7/ 12 haplotypes with B57(17) can be explained by a single historical recombination with a second ancestral haplotype. The donor ancestral haplotypes are boxed and shown at the bottom of the figure. One haplotype with B8 (B4251) and 5 haplotypes with B57(17) cannot be explained by a single recombination event with another defined ancestral haplotype. These are probably multiple recombinants.

(n = 118)

BI

DR

I

HLA B

Oo~orAH

A2062 A0098 K0009 E4071 L2033

~ ~ I

eoms0

~

~ ! ]

05242 B4251

(n = 39)

B57

C4A C4B

IS IS I8 99] S IS

3 3 3 3 3

I 3 O 1 1

2 I 7.'~ 4 I 62.1 4 I 441 5 I ~8.3 7 1~3.~orS4.1

IS

3

i

71

IS F

6 3

1 1

7I 3

371

E9013 A4278 G0251 F6001 C0338 D5438 F0681 C9013 A5224 H4293 E4218 G0606 K6326 K9013

Bf

L

C4A C4B

DR

Donor AH

S

~i~l~i~i~!i

s

3 3

I o

2I 4I

;'.1 44.~

41 7 113.1 o r ~ l

:~i:11~::~:~!~!.:. !:i!i:i:i!~::,!::!i

F S

3 3

1 O

7 J

44.2

4 J

62.1

I I

35.3

7 8 98

~l~:i:iiii!i

F

3 0 1

S

0

1

5

S

3

1

6

5 S

58 58

Donor Ancestral Hsplotypes

17

Donor Ancestral Haplotypes

7 82

S S

3 3

1 3

2] 7,1 4 ] 62.1

I 44

F

3

1

71

44.2

I

S

3

1

4 I

62,1

44

S

3

O

4 ] 44.1

I 3s

s

3

o

11 35.3

18 13 64

S

3

1

5 I

S S

3 3

1 1

18.3 7 ] 13.1 7] 64.1

57

S

6

1

7]

was not represented among the haplotypes analysed. O f the 348 haplotypes studied, 133 (38%) were full HLAB - D R ancestral haplotypes and a further 122 haplotypes (35 %) were "simple" telomeric and centromeric recomo binants. Therefore, over 73% of the population haplotypes studied carry at least 400 kilobases (HLA-B-complotype -- 600 kb; and c o m p l o t y p e - D R = 400 kb) of one o f the ancestral haplotypes listed in Table 2 column 1. The remaining 2 7 % may include haplotypes with shorter fragments, as well as some rare ancestral haplotypes, which must be postulated to account for the rare alleles not carried by our current list of 22. DISCUSSION The present study c o m m e n c e d m o r e than a decade ago when we appreciated that autoimmune diseases were

62

57.1

associated with combinations o f alleles at several major histocompatibility complex (MHC) loci. Thus, we had reported that systemic lupus erythematosus (SLE) was associated with A1 and B8 if severe and with A2 and B7 if relatively mild [18]. It was obviously important to determine whether these associations reflected direct interactions between alleles at different H L A loci or whether H L A typing was providing markers for M H C haplotypes that might contain a variety of additional n o n - H L A genes [19]. At about this time, we appreciated that myasthenia gravis and SLE were associated with C4 deficiency [12, 20], undoubtedly reflecting the presence o f C4 null alleles on haplotypes containing HLA-A1, B8, and DR3. O t h e r putative haplotypes were found to be associated with different diseases so that in 1980 we established a database and developed software to examine combinations of alleles at diverse

250

TABLE 4

M.A. Degli-Esposti et al.

A n c e s t r a l h a p l o t y p e s carry a n u m b e r o f H L A and n o n - H L A haplospecific m a r k e r s Central non-HLA

Ancestral haplotype

HLA-B

C2

8.1

I-2_g_-3

13.1

rT~-'~ L_~J_...J

Bf

I s

C4A

QO

HLAC4B

DRB1

DRB3

DQA1

DQB1

1 I

18.1

"---t-Eli

18.2 35.2

37.1 44.1

r3'~-1,

47.1

I,.___,~

5o.1

,r'r~.q

57.1

r3'~-"l

Lo3oj_._. 02_0_2J F0-0Z - -

[_s07___ V -3

l ° 3 ° J. . . . £ ~ 3

r-s----:yIIIZZ2.

62.1 65.1

-- -1-

i . _ _ _ , J Haplospecificallelesor combinations of alleles. This haplospecific complotype is defined by a 6.4-kb Taql/PAT-A fragment.

M H C loci. W e q u i c k l y r e a l i z e d that th e m i n i m u m typing w o u l d have to i n c l u d e H L A - A , B, and D R as well as Bf, C 4 A , and C 4 B . T y p i n g f o r H L A - C , D Q , D P , and C2 a p p e a r e d to be less critical in defining c o m b i n a t i o n s associated w i t h disease. T h e p o t e n t i a l value o f typing the patient's relatives to

TABLE 5

assign h a p l o t y p e s was o b v i o u s , but, g i v e n the fact that we w e r e studying h u n d r e d s o f patients, o t h e r app r o a c h e s had to be c o n s i d e r e d . In 1982, w e c o n v e n e d a m e e t i n g that c o n s i d e r e d strategies and t e r m i n o l o g y that m i g h t be a p p r o p r i a t e [2]. It was r e c o g n i z e d t h a t the t e r m haplotype had b e e n

F r e q u e n c y o f ancestral h a p l o t y p e s and t h ei r " s i m p l e " r e c o m b i n a n t s in an A u s t r a l i a n p o p u l a t i o n : data set 2

Ancestral haplotype

HLA-B

Bf

C4A

C4B

HLA-DR

B-C4

7.1 8.1 13.1 18.1 18.2 18.3 35.1 35.2 35.3 44.1 44.2 50.1 55.1 57.1 60.1 60.3 62.1 62.2 64.1 65.1 65.2

7 8 13 18 18 18 35 35 35 44 44 50 55 57 60 60 62 62 64 65 65

S S S S F1 S S F S S F $1 S S S S S S S S F

3 0 3 4 3 3 3 3+ 2 3 3 3 1 4 6 3 0 3 4 3 2 3

1 1 1 2 0 1 1 0 0 0 1 1 + 1.5 5 1 1 2 3 2 1 1+ 2 1

2 3 7 2 3 5 5 1 1 4 7 7 6 7 4 6 4 4 7 1 6

13 1 0 0 0 2 5 0 1 4 2 1 1 2 4 1 1 2 1 0 0

Total

41

B-DR

C4-DR

Total

19 40 5 3 4 4 5 3 5 10 6 1 1 6 2 6 3 2 2 2 4

13 5 4 1 2 12 14 0 2 5 1 0 1 1 9 2 0 4 2 0 3

45 46 9 4 6 18 24 3 8 19 9 2 3 9 15 9 4 8 5 2 7

133

81

255/348

73.0%

MHC Ancestral Haplotypes

widely misused, but was appropriate in the context of the patterns that segregated within families. We therefore agreed to use the term supratype for combinations of alleles that could reflect haplotypes, but that were defined by the combination rather than the pattern of segregation within a family. At the time, it was theoretically possible that there could be an interaction between a B allele on one haplotype and a D R allele on another. Exhaustive family studies would be required to determine whether the two were necessarily in cis, reflecting the presence o f a haplotype. It soon became apparent that most supratypes of interest were actually carried on one haplotype--as expected, given that the component alleles were found to be in positive linkage disequilibrium in population studies. It was difficult to imagine how alleles could be maintained in positive linkage disequilibrium over many generations unless the haplotype was conserved en bloc [3]. From these considerations, we developed the concept of ancestral haplotypes, i.e., haplotypes of larger ancestral families. By definition, however, a haplotype could only be shown by demonstrating segregation within living relatives. Clearly, the difficulty was semantic rather than conceptual. Accordingly, in this study, we have used two terms: nuclear haplotypes defined by studying extant single nuclear families and ancestral haplotypes defined by comparing nuclear families and deducing haplotypes which are common to multiple and extended families i.e., which belong to a larger ancestral family. Other terminologies are possible; indeed, we suspect that our conserved haplotypes are the same at a sequence level as the extended haplotypes of Alper and colleagues [9, 21, 22]. At the sequence-level nuclear haplotypes must be identical (by descent). We speculated that ancestral haplotypes would also be identical but by remote descent. The database described in this report has enabled us to select examples of supratypes and to ask whether there is identity at the sequence level. The relevant molecular data have been reported elsewhere (see above), but, in every instance so far examined, disease-associated supratypes defined in terms of HLA-B, Bf, C4A, C4B, and D R have been found to be identical [4, 5, 7-10]. The purpose o f the present report is to demonstrate how a haplotype database can be useful in collecting the material for further studies o f ancestral haplotypes and their recombinants. We argue that Tables 2 and 3 provide a rather complete description o f the Caucasoid MHC. We recognize that we have ignored combinations (or supratypes) that occur with a frequency of ~ 3 / 8 2 9 or - 0 . 4 % , but for the time being we regard very rare supratypes as rare "mutants" in much the same way that a polymorphic allele is sometimes considered only when it attains a population frequency o f at least

251

1%. On the other hand, we have identified other combinations carried by many haplotypes in the population and we argue that at least most of these are ancestral haplotypes or recombinants between two of these. Thus, the basic pool of polymorphism within the M H C can probably be described in terms o f a relatively small number (perhaps 5 0 - 2 0 0 ) ancestral haplotypes and most individuals will be found to possess at least parts of one or more of these ancestral haplotypes. Further work is required to complete the analysis and to examine our conclusion in other population groups. There are several important implications of the present approach. The ability to recognize intact and recombinant ancestral haplotypes enables an approach to mapping M H C genes. Elsewhere we have determined that it is possible to map the M H C genes involved in Myasthenia Gravis [23], IDDM [24, 25], and perhaps AIDS [26]. Currently we are using the same approach in relation to transplantation. One of the more interesting implications of the present work relates to the mechanisms for the conservation of polymorphisms [27, 28]. In a sense, conservation and polymorphism are often contrasted and may appear to be incompatible, but, in fact, it is now certain that extremely polymorphic haplotypes have been maintained with minimal, if any, sequence variation through very many generations. Indeed, elsewhere we argue that polymorphism at the sequence level "freezes" the sequence by inhibiting recombination. Finally, it is apparent that similar studies are needed in other ethnic groups, and especially in situations where there is substantial racial admixture. Our preliminary data suggest that ancestral haplotypes are maintained if they exceed some threshold of frequency, but they fragment and perhaps even disappear if they fall below this threshold, as might occur after racial admixture. The high degree of conservation of ancestral haplotypes observed in relatively homogeneous populations may reflect reconstitution by preferential recombination within regions with identical genomic sequence. The maintenance of ancestral haplotypes may reflect the survival o f a distinct population group with a finite number of different ancestral haplotypes, but a restricted amount of racial admixture. ACKNOWLEDGMENTS Publication number 9109 of the Departments of Clinical Immunology, Royal Perth Hospital, Sir Charles Gairdner Hospital, and the University of Western Australia. We are grateful to Dr. B. Horton for his contribution in the initial stages of this project. We are especially thankful to Dr. M. Giphart for valuable discussions and criticisms. This work was supported by the National Health and Medical Research Council and the Immunogenetics Research Foundation.

252

M.A. Degli-Esposti et al.

REFERENCES 1. Ceppellini R, Curtoni ES, Mattiuz PL, Miggiano V, Scudeller G, Serra A: Genetics of leukocyte antigens: a family study of segregation and linkage. In Curtoni ES, Mattiuz PL, Tosi RM (eds): Histocompatibility Testing 1967. Copenhagen, Munksgaard, 1967. 2. Dawkins RL: Musculoskeletal disease and D-penicillamine: concepts and models. In Dawkins RL, Christiansen FT, Zilko PJ (eds): Immunogenetics in Rheumatology: Musculoskeletal Disease and D-Penicillamine. Amsterdam, Excerpta Medica, International Congress Series, 1982, p 1. 3. Dawkins RL, Christiansen FT, Kay PH, Garlepp MJ, McCluskey J, Hollingsworth PN, Zilko PJ: Disease associations with complotypes, supratypes and haplotypes. Immunol Rev 70:5, 1983. 4. Tokunaga K, Saueracker G, Kay PH, Christiansen FT, Anand R, Dawkins RL: Extensive deletions and insertions in different MHC supratypes detected by pulsed field gel electrophoresis. J Exp Med 168:933, 1988. 5. Dawkins RL, Kay PH, Martin E, Christiansen FT: The genomic structure of ancestral haplotypes revealed by pulsed field gel electrophoresis (PFGE): In Dupont B (ed): Immunology of HLA, vol 1. New York, SpringerVerlag, 1989, p 893. 6. Kambhu S, Falldorf P, LeeJS: Endogenous retroviral long terminal repeats within the HLA-DQ locus. Proc Natl Acad Sci USA 87:4927, 1990. 7. Zhang WJ, Degli-Esposti MA, Cobain TJ, Cameron PU, Christiansen FT, Dawkins RL: Differences in gene copy number carried by different MHC ancestral haplotypes: quantitation after physical separation of haplotypes by pulsed field gel electrophoresis. J Exp Med 171:2101, 1990. 8. Abraham LJ, ChinDu D, Zahedi K, Dawkins RL, Whitehead AS: Haplotypic polymorphisms of the TNFB gene. Immunogenetics 33:50, 1991. 9. Egea GE, Yunis I, Spies T, Strominger J, Awdeh ZL, Alper CA, Yunis EJ: Association of polymorphisms in the HLA-B region with extended haplotypes. Immunogenetics 33:4, 1991. 10. Wu X, Zhang WJ, Witt CS, Abraham LJ, Christiansen FT, Dawkins RL: Haplospecific polymorphism between HLA B and TNF. Hum Immunol 33:89, 1992. 11. Zhang WJ, Kay PH, Cobain TJ, Dawkins RL: C4 allotyping on plasma or serum: application to routine laboratories. Hum Immunol 21:165, 1988. 12. Christiansen FT, Dawkins RL, Uko G, McCluskey J, Kay PH, Zilko PJ: Complement allotyping in SLE: association with C4A null. Aust N Z J Med 13:483, 1983. 13. Kramer J, Gyodi E, Fust G: Usefulness of densitometry in typing of human complement component C4. Immunogenetics 29:121, 1989. 14. Alper CA, Boenisch T, Watson L: Genetic polymorphism

15.

16.

17.

18.

19.

20.

21.

22.

23.

24.

in human glycine-rich glycoprotein. J Exp Med 135:86, 1972. Wilton AN, Christiansen FT, Dawkins RL: Supratype matching improves renal transplant survival. Transplant Proc 17:2211, 1985. Martin E, Gin W, Kay PH, Christiansen FT, Dawkins RL: Complotypes and class III gene arrangements can be assigned from supratype specific restriction fragment length polymorphism. 10th Int Histocompatibility Workshop Newsl 13, 1987. Dawkins RL, Leaver A, Cameron PU, Martin E, Kay PH, Christiansen FT: Some disease-associated ancestral haplotypes carry a polymorphism of TNF. Hum Immunol 26:91, 1989. Rigby RJ, Dawkins RL, WetherallJD, Hawkins BR: HLA in systemic lupus erythematosus: influence on severity. Tissue Antigens 12:25, 1978. Hawkins 13R, Dawkins RL, Richmond J, Rigby RJ: Immunogenetic factors in systemic lupus erythematosus. Arth Rheum 22:94, 1979. Christiansen FT, Houliston, JB, Dawkins RL: HLA, antiDNA, and complement in myasthenia gravis. Muscle Nerve 1:467, 1978. Awdeh ZL, Raum LD, Yunis EJ, Alper CA: Extended HLA/complement allele haplotypes: evidence for T/tlike complex in man. Proc Natl Acad Sci USA 80:259, 1983. Alper CA, Awdeh ZL, Yunis EJ: Complotypes extended haplotypes, male segregation distortion and disease markers. Hum Immunol 15:366, 1986. Degli-Esposti MA, Andreas A, Christiansen FT, Albert E, Dawkins RL: An approach to the localisation of the susceptibility gene for generalised myasthenia gravis by mapping recombinant ancestral haplotypes Immunogenetics 35:355, 1992. Degli-Esposti MA, Abraham LJ, McCann V, Spies T, Christiansen FT, Dawkins RL: Ancestral haplotypes reveal the role of the central MHC in the immunogenetics of IDDM. Immunogenetics 1992 (in press).

25. Dawkins RL, Zhang WJ, Degli-Esposti MA, Abraham LJ, McCann V, Christiansen FT: Studies of haplotypes by pulsed field gel electrophoresis: In Tait B, Harrison L (eds): Bailliere's Clinical Endocrinology and Metabolism: The Genetics of Diabetes. London, Bailliere Tindall, 1991, p 285. 26. Mallal S, Cameron PU, French MAH, Dawkins RL: MHC genes and HIV infection. Lancet 335:1591, 1990. 27. Koop BF, Siemieniak D, SlightomJL, Goodman M, Dunbar J, Wright PC, Simons EL: Tarsius delta- and betaglobulin genes: conversions, evolution, and systematic implications. J Biol Chem 264:68, 1989. 28. Hughes AL: Testing for interlocus genetic exchange in the MHC: a reply to Andersson and co-workers. Immunogenetics 33:243, 1991.