Hum. Genet. 36, 191--196 (1977)

© by Springer-Verlag 1977

Haplotype Analysis of the Linkage Group HLA-A : HLA-B : Bf and Its Bearing on the Interpretation of the Linkage Disequilibrium

K. Bender 1 *, A. Ma~cerov~il, R. Frank 1, C. Hiller 2, and T. Wienker 1

11nstitut fiir Humangenetik und Anthropologie der Universit~it Freiburg, Albertstrage 11, D-7800 Freiburg, Federal Republic of Germany

-~ Institut fur Medizinische Mikrobiologie der Universit~it Hamburg, D-2000 Hamburg, Federal Republic of Germany

Summary. The analysis of 650 HLA-A: HLA-B : Bf three-factor haplotypes revealed significant associations only between alleles of the very closely linked genes HLA-A and HLA-B, and HLA-B and Bf, respectively. Most striking is the highly significant association of the rare Bfvariant F1 with HLA-B18 and of St with HLA-B 13, HLA-B 14, and HLA-Bw21. Only random allele distribu- tions were observed when considering the somewhat more distant genes HLA- A and Bf or the higher order interaction at, all three genes. From these findings it seems likely that the linkage disequilibrium within the M H C is not due to selective forces, but rather due to a short evolutionary period.

The complement system is comprised of at least 18 plasma proteins which are involved in 4 functional divisions: 2 different activation pathways, 1 amplification mechanism, and 1 common effector cascade (Mtiller-Eberhard, 1975). Inter- estingly enough, 3 genes controlling steps of the activation pathways, namely the C2 and the C4 loci of the classical and the Bf locus of the alternative complement sequence are located within or close to the major human histocompatibility complex (MHC) on chromosome 6 (Fu et al., 1974; Rittner et al., 1975; Allen, 1974). The possible implications of these linkage relationships have been discussed recently by Rittner (1976).

Among these 3 genes, the Bf locus is of special interest, since it is highly polymorphic with 2 common (Bf v and Bf s) and a number of rare alleles (Bf sl, Bf F1, Bf F1-6, Bf F°-55) in the Caucasoid population (Alper et al., 1972; Mauff et al., 1975; Hauptmann et al., 1976). Still other Bf variants have been described by Mauff et al. (1976) in South African Bantu tribes. The close linkage of the Bf and the HLA genes led to expect a linkage disequilibrium between certain HLA and Bf alleles. This indeed was found by Albert eta!. (1975) and Olaisen et al. (1975).

In this paper we present an analysis of 650 HLA-A : HLA-B : Bf three-factor haplotypes.

* To whom offprint requests should be sent

192 K. Bender et al.

Table 1. Observed three- fac tor hap lo types ( H L A - A : H L A - B : Bf) f r om 325 unre la ted persons

H L A - B Bf H L A - A

A1 A2 A3 A9 A10 A l l A28 A29 Aw30 Aw31 Aw32 Aw33 A w l 9 A"0"

B5 F 2 3 3 ! S 3 12 5 3 1

B7 F 2 3 2 S 4 19 27 13

B8 F 2 1 1 S 48 5 1 4 6 S1 1

B12 F 2 9 3 5 1 S 1 29 1 10 7

B13 S 2 8 2 1 S1 1 2 1

B14 F 1 S 1 2 3 1 S1

Bwl5 F 1 7 3 S 14 6 1 1

Bwl6 F S 1 5 6 8

B17 F 1 1 S 9 5 3 3

B18 F 1 1 S 4 I1 5 F1 2 1 S1

Bw21 S 2 2 1 1 S1 1 2 1

Bw22 F ! 1 1 S 1 2 1 4

B27 F S 2 6 3 3 6

Bw35 F 1 1 8 1 S 2 9 11 9

Bw37 F 1 2 S 2 1

Bw40 F 1 1 S 2 20 4 6 3

B"0" F 1 1 S 1 8 3 1 F1 1

6 4 2

3 1 l

1 4

1 6

1 1

1

1

1 1

1 1 1 1

3 1 13 3

3 2

2

5 4

1

1

1

1

1

1 1

1

1 3 2

1 1 1

1 3 1

1 1 1

Analysis of HLA-A : HLA-B : Bf Three-Factor Hyplotypes 193

Material and Methods

There were 325 healthy unrelated individuals from South West Germany tested for their HLA and Bf phenotypes and genotyped by family analyses.

HLA antigens were typed in peripheral blood lymphocytes using the lymphocytotoxic microtechnique of Kissmeyer-Nielsen and Kjerbye (1967). Bf phenotyping was performed by high-voltage agarosegel electrophoresis of serum samples followed by immunofixation ac- cording to Mauff et al. (1975) using anti-C3-activator (Behringwerke, Marburg, Germany) as specific anti-Bf reagent.

Results

The d i s t r ibu t ion of the 650 three- fac tor hap lo types as deduced by fami ly analyses is l isted in Table 1. The frequencies of the single H L A - A , HLA-B, and Bf alleles were de te rmined by gene-count ing and are given in Table 2. They are in good agreement with those f rom other studies of the Ge rman popu la t ion as referred by Bender et al. (1975) for the H L A and by Mauf f et al. (1975) for the Bf allele frequencies. As is to be expected, the most c o m m o n H L A two-fac tor com- b ina t ions (as for ins tance A I : B S , A2:B12 , A3 :B7 , A 2 : B w l 5 , A 2 : B w 4 0 or A3 :Bw35) are also involved in the most c o m m o n three-fac tor haplotypes .

The Bf s allele is inc luded in the 12 most f requent three-fac tor combina t ions . The somewha t rarer Bf F allele has its highest occurrence in the hap lo types A 2 : B 1 2 : F , A 3 : B w 3 5 : F , A 2 : B w l 5 : F and A 2 9 : B 1 2 : F .

One of the most in teres t ing quest ions on closely l inked po lymorph ic genes is, whether cer ta in gamet ic combina t ions exist more f requent ly than would be expec ted f rom the allele frequencies at the co r respond ing loci ( l inkage dis- equi l ibr ium). A first answer to this quest ion follows f rom the H L A - A : B f and H L A - B : B f two factor associa t ions (Table 3). The da t a show that the alleles of the more d is tan t loci H L A - A and Bf seem to be r a n d o m l y combined , whereas

Table 2. HLA-A, HLA-B, and Bf allele frequencies determined from 650 three-factor haplotypes by gene counting

Allele Frequency Allele Frequency

A1 0.1476 Bwl5 0.0569 A2 0.2938 Bwl6 0.0353 A3 0.1369 B17 0.0400 A9 0.1230 B18 0.0461 A10 0.0784 Bw21 0.0169 A 11 0.0523 Bw22 0.0184 A28 0.0446 B27 0.0446 Awl9 0.0938 Bw35 0.1031 A"0" 0.0292 Bw37 0.0107 B5 0.0723 Bw40 0.0734 B7 0.1200 B"0" 0.0292 B8 0.1092 BfF 0.1600 B12 0.1369 Bf s 0.8138 B13 0.0415 BfF1 0.0062 B 14 0.0446 BfS1 0.0200

194 K. Bender et al.

Table 3. HLA-A : Bf and HLA-B : Bftwo-factor haplotype frequencies (p)and the corresponding linkage disequilibrium parameters (D) from 650 haplotypes. Absence of interaction, i.e., signi- ficance of D, was tested by the exact method of Fisher and Yates basing upon the hypergeometric distribution (significance levels: *P~0.05 , **P_<0.01, ***P<0.01)

Haplotype p D Haplotype p D

HLA Bf HLA : Bf

A1 A1 A2 A2 A3 A3 A9 A9 AI0 A10 A l l A l l A28 A28 Awl9 Awl9 A"0" A"0" A2 A2 Awl9 B5 B5 B7 B7 B8 B8 B12 B12

F 0.0169 --0.006 B13 : S 0.0353 0.001 S 0.1276 0.007 B14 : F 0.0092 0.002 F 0.0415 --0.005 B14 : S 0.0307 --0.005 S 0.2430 0.004 Bwl5 : F 0.0184 0.009** F 0.0307 0.008 Bwl5 : S 0.0384 --0.008* S 0.1046 --0.006 Bwl6 : F 0.0015 0.004 F 0.0184 0.001 Bwl6 : S 0.0338 0.005 S 0.1000 0.000 B17 : F 0.0077 0.001 F 0.0107 --0.002 B17 : S 0.0323 --0.002 S 0.0661 0.002 B18 : F 0.0031 --0.004 F 0.0076 --0.001 B18 : S 0.0369 0.000 S 0.0446 0.002 Bw21 : S 0.0108 --0.003 F 0.0092 0.002 Bw22 : F 0.0046 0.001 S 0.0353 0.001 Bw22 : S 0.0138 --0.001 F 0.0183 0.003 B27 : F 0.0046 --0.003 S 0.0691 0.007 B27 : S 0.0400 0.003 F 0.0061 0.001 Bw35 : F 0.0230 0.006 S 0.0230 0.001 Bw35 : S 0.0800 --0.004 S1 0.0061 0.000 Bw37 : F 0.0046 0.003 F1 0.0031 0.001 Bw37 : S 0.0061 --0.003 S1 0.0061 0.004 Bw40 : F 0.0031 --0.009** F 0.0138 0.002 Bw40 : S 0.0707 0.010"* S 0.0584 0.000 B"0" : F 0.0061 0.001 F 0.0107 0.008* B"0" : S 0.0215 --0.002 S 0.1092 0.011" B13 : S1 0.0061 0.005** F 0.0061 0.011"* B14 : S1 0.0046 0.004* S 0.1015 0.013"* Bw21 : S1 0.0061 0.006*** F 0.0431 0.021"** B18 : F1 0.0046 0.004** S 0.0938 --0.018"*

t hose o f the m o r e a d j a c e n t genes H L A - B and Bf are s ign i f ican t ly assoc ia ted .

M o s t s t r ik ing is the h igh ly s ign i f i can t l i nkage d i s e q u i l i b r i u m b e t w e e n s o m e

i n f r e q u e n t H L A - B and ra re Bf alleles, as Bw21 :S1, B13: S1, B 1 4 : S 1 and B18: F1. T o d e t e r m i n e the t h r e e - f a c t o r l i nkage d i s e q u i l i b r i u m p a r a m e t e r [ D ( A , B, Bf)]

and the m u l t i p l e c o r r e l a t i o n coef f i c i en t o f t he al lele f r equenc ie s at the th ree loci

(ro) the d a t a were a r r a n g e d in a 2× 2 × 2 c o n t i n g e n c y tab le ( con t ingency c u b e ) f o r

e a c h t h r e e - f a c t o r c o m b i n a t i o n ; ca l cu l a t i ons were p e r f o r m e d a c c o r d i n g to fo r -

mu la s (5) a n d (7), r espec t ive ly , f r o m P i a z z a (1975). T h e hypo thes i s on absence o f

i n t e r a c t i o n in each c o n t i n g e n c y cube was tes ted by a Ch i - squa re m e t h o d f irs t

adv i sed by Bar t l e t t (1935) a n d c i ted f r o m P i a z z a (1975; f o r m u l a s 10, 11, and 12). O n l y a f r a c t i o n o f the d a t a in T a b l e 1 is a d e q u a t e fo r C h i - s q u a r e tests; we se lec ted those g a m e t i c c o m b i n a t i o n s fo r w h i c h the f r e q u e n c y in any cell o f the cor -

r e s p o n d i n g c o n t i n g e n c y cube is at least 3 (Tab le 4). Al l b u t one o f the 20

Analysis of HLA-A: HLA-B: Bf Three-Factor Hyplotypes 195

Table 4. Three-factor haplotype frequencies (p), linkage disequilibrium para- meters [D (A, B, Bt)] and multiple correlation coefficients (ro) of those haplotypes appropriate for test of independence by a Chi-square method (Ba~lett, 1935)

Haplotype p D (A, B, Bf) ro Z2

HLA-A : HLA-B : Bf

A2 : B5 : F 0.0046 0.0006 0.0000 0.14 A2 : B5 : S 0.0185 --0.0005 0.0000 0.00 A2 : B7 : F 0.0031 0.0010 0.0001 0.13 A2 : B7 : S 0.0292 --0.0009 --0.0001 0.72 A2 : B12 : F 0.0138 --0.0010 --0.0001 0.59 A2 : B12 : S 0.0446 0.0017 0.0001 1.06 A2 : Bwl5 : F 0.0108 0.0032 0.0001 0.38 A2 : Bwl5 : S 0.0215 --0.0027 --0.0001 0.20 A3 : B5 : F 0.0046 0.0017 0.0001 0.76 A3 : B5 : S 0.0077 --0.0018 --0.0001 1.10 A3 : B7 : F 0.0046 --0.0027 --0.0001 0.46 A3 : B7 : S 0.0415 0.0032 0.0001 0.27 A3 : Bw35 : F 0,0123 0.0058 0.0002 3.49 A3 : Bw35 : S 0,0169 0.0056 --0.0002 4.26 A9 : B12 : F 0.0077 0.0016 0.0001 0.29 A9 : B12 : S 0.0154 0.0012 --0.0001 0.08 A l l : Bw35 : F 0.0046 0.0004 0.0000 0.01 A l l : Bw35 : S 0.0200 0.0000 0.0000 0.08 A29 : B12 : F 0.0077 0.0044 0.0001 1.66 A29 : B12 : S 0.0061 --0.0042 --0.0001 2.02

h a p l o t y p e s ana ly sed a re r a n d o m l y ad jo ined . The e x c e p t i o n in wh ich the absence

o f i n t e r ac t i ons c o u l d n o t be c o n f i r m e d is A 3 : B w 3 5 : S , wh ich m i g h t be s ignif-

i can t ly u n d e r r e p r e s e n t e d (Z2=4 .26 ; df= l ; P = 0.039). H o w e v e r , one ou t o f

t w e n t y a s soc i a t i ons tes ted is e x p e c t e d to be s igni f ica t ive by chance a lone , i f a level

o f s ign i f i cance o f 5% is assessed (P iazza , 1975).

D i s c u s s i o n

T h e 650 h y p l o t y p e s u n d e r s tudy can be t a k e n as r ep re sen ta t ive fo r the G e r m a n

p o p u l a t i o n as seen f r o m the f r equenc ie s o f the s ingle H L A - A , H L A - B , and Bf

al leles a n d of the H L A - A : H L A - B hap lo types . A c c o r d i n g l y , the H L A - A : Bf and

H L A - B : B f t w o - l o c i a s soc i a t i ons as well as the H L A - A : H L A - B : B f three-

f a c t o r a s soc i a t i ons can be c o n s i d e r e d as r ep re sen ta t ive too.

F r o m the d a t a o f T a b l e 3 it can be seen tha t s ign i f ican t l i nkage d i sequ i l ib r i a

o c c u r on ly b e t w e e n the v e r y c lose ly l inked ( - 1 c M ) loci H L A - B and Bf as wel l as b e t w e e n HLA-A a n d H L A - B . C o n s i d e r i n g the d i s t ance be tween H L A - A and Bf

(which l ikely is twice t h a t o f H L A - B and Bf), su rpr i s ing ly no s ign i f ican t

a s s o c i a t i o n can be d e m o n s t r a t e d , The analys is o f the first and second o r d e r i n t e r ac t i ons b e t w e e n the th ree l inked loci d id n o t give c o n v i n c i n g ev idence for

ce r t a in ove r - o r u n d e r r e p r e s e n t e d game t i c a s soc ia t ions (Tab le 4). F r o m these d a t a

it seems tha t the p o s t u l a t e d se lec t ive forces wh ich shou ld have f a v o r e d ce r ta in

196 K. Bender et al.

assoc ia t ions by some kind of interact ive select ion might not be relevant. A poss ible e x p l a n a t i o n for the l inkage d i sequi l ib r ium between the very closely l inked genes could be tha t the evo lu t iona ry life span of the popu l a t i on s tudied is still too shor t to a l low for b r e a k up of all the founder hap lo types by recombi - nat ion.

In teres t ing are the highly s ignif icant associa t ions between Bw21 and Bf sl and be tween B18 and Bf F1. Poss ib ly S1 and F1 represent ra ther recent Bf-muta t ions ar isen in hap lo types involving Bw21 and B18, respectively.

Supported by the Deutsche Forschungsgemeinschaft Be 352/10.

References

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Received November 25, 1976