Journal ofImmunogenetics (1983) 10,6 1-67.

C6 L I N K A G E S T U D I E S

K . B E N D E R , S. B I S S B O R T , A . M A Y E R O V A , G . M A U F F * A N D

T. F. W I E N K E R

Institut f ur Humangenetik und Anthropologie der Universitat Freiburg, Freiburg, Germany and *Hygiene-Institut der Universitat Koln, Koln, Germany

(Received 7 June 198 I )

S U M M A R Y

No evidence was found for close linkage between the human C6 gene (6th component of complement) and 28 marker loci, using the LIPED 3 computer program of Ott (1974). But the data allow the exclusion of the C6 locus from large parts of the autosomes 1,2,4,6,8, 9, 13, 14, 16, 19, and 20.

I N T R O D U C T I O N

The compIement system is comprised of at least 18 plasma proteins (MulIer-Eberhard, 1975). They are involved in four functional divisions (two different activation pathways, one amplification mechanism and one common effector cascade). So far the genes of only four complement components could be assigned to their chromosomes: C2, C4 and Bf to chromosome 6 (Fu et ul., 1974; Rittner et al., 1975; Allen, 1974) and C3 to chromosome 19 (Whitehead et al., 1981). The location within the major histocompatibility complex (MHC) of just three genes prompted an intensive search on whether also other complement genes might be situated there. For at least nine genes, however, linkage relationships with the MHC could be excluded in man: CIq (Berkel et al., 1979), C l r (Day et al., 1975), CIINH (Blumenthal et al., 1978), C3 (Gedde-Dahl et al., 1974), C.5 (Rosenfeld et al., 1977), C6 (Olving et al., 1977), C7 (Hobart ef al., 1977), C8 (Jersild et af., 1976), and C9 (Lint et al., 1980). Mapping of the complement genes (but also that of most other serum proteins) will mainly be restricted upon linkage detection with already assigned marker loci, because the respective gene products are only spuriously or not expressed in fibroblasts to allow the use of somatic cell hybridization methods. Linkage studies, however, require the existence of genetic polymorphism. The C6 gene fulfills this essential by the occurrence of two frequent and a number of rare alleles (Hobart et al., 1975; Olving el al., 1977; Rittner et al., 1979; Kiihnl & Kreckel, 1980; Kunstmann et al., 1980). Two extensive reports on C6 linkage analyses were already given (Hobart e f al., 1977; Olving et al., 1979). No measurable linkages were found between C6 and altogether 37 marker loci in both investigations. The number of informative families for some comparisons was unavoidably small, so that the analysis of further independent material was desirable.

Correspondence: Prof. Dr Klaus Bender, lnstitut fur Humangenetik und Anthropologie der Universitat Freiburg, Albertstr. 1 1, Freiburg, Germany.

0305-18 I1/83/0200-0061%02.00 0 1983 Blackwell Scientific Publications 61

62 K. Bender et al. M A T E R I A L A N D M E T H O D S

The family material comprised of 40 two-generation families and six large pedigrees resulting in 66 matings with 331 children as listed in Table 1. Most were collected because of large sibships, but two pedigrees also because they are segregating for an autosomally inherited rare disease (muscular dystrophy, facio-scapulo-humeral type, McKusick No. 15890, ‘MDF’, and van der Woude syndrome, McKusick No., 11930 ‘VWS’). Except for two matings, in which one or both parents were of Turkish or Egyptian origin, all individuals were German.

TABLE 1. Segregation ratios for C, allotypes -~~ ~

Mating No. of No. of tY Pe matings children A AB B A A , AB, A M BA, BE, BM

A x A A x AB A x B AB x AB B x B B x AB A x A, A x AB, A x A,B, B x AA, B x A,B AB x AB, AB x BM AB x BB, Incomplete cases

Total

9 21

5 9 2 5 1 1 1 1 1 1 1 1 I

66

42 42 98 48 50 28 28 40 8 23 9 15 15 41 18 23

6 3 3 3 2 1 5 4 1 3 3 I 3 4 5 1 1 2 1 1 1 2 1 1

35 19 13 2 1

331 123 136 53 I 4 0 4 2 2

Allele frequencies (based on 106 unrelated parents and by omitting the foreign ones): C6A = 0.606; C6’= 0.349; CfjA’ = 0.020; C6B2 = 0.020; CCiM = 0.005.

C6 typing was done in serum samples (stored at -8OOC) by the technique of isoelectric focusing in polyacrylamide gel, pH range 5-8, with the LKB multiphor equipment for 3 hr at a maximum of 1400 volts. The detection of C6 was achieved by a haemolytic assay using an overlay consisting of 0.6% agarose, sensitized sheep erythrocytes and serum from C6-deficient rabbits (for details see Kunstmann et at., 1980). The other investigated blood groups, serum proteins, enzyme markers as well as the HLA phenotypes were tested using standard methods.

Linkage analysis has been performed by a generally accepted computer programme (LIPED 3) kindly provided by its author (J. Ott, 1974). LIPED 3 is Fortran written and has been modifled to our facilities and needs; however, the subroutines computing the likelihood of a pedigree according to the Elston-Stewart algorithm (Elston & Stewart, 197 1) remained unchanged. The programme has been directed to compute discrete points of the three-dimensional bivariate likelihood surface (z (em,,,, Bfema,J) for each pedigree, arranged in a two-dimensional square matrix. These matrices have been summed up over all pedigrees yielding the maximum likelihood joint estimate of the peak lods. As has been pointed out and discussed by Conneally & Rivas (1980) this exact procedure

C6 linkage studies TABLE 2. Linkage relations of C6

63

Lod scores at recombination fraction, 0

Locus Sex Matings Offspring 0.05 0.1 0.2 0.3 0.4

1. ABO

2. ACP,

3. ADA

4. AK

5. c o

6. C3

7. ESD

8. Fy

9. GALT

10. GC

11. GLO

12. Gm

13. GPT

14. HLAIBf

15. Hp

16. Jk

17. Km

M F Joint M F Joint M F Joint M F Joint M F Joint M F Joint M F Joint M F Joint M F Joint M F Joint M F Joint M F Joint M F Joint M F Joint M F Joint M F Joint M F Joint

26

22

4

4

3

24

10

22

10

29

21

25

18

41

24

24

9

132

113

28

21

21

126

60

118

54

155

127

123

106

207

116

121

42

-6.21 -7.73

-17.14 -1.89

-11.11 -14.31

+0.02 -0.72 -2.68 -0.46 -4.72 -5.20 -0.63 -1.44 -2.07 -7.13

-10.57 -16.28 -7.95 -3.29

-11.28 -4.06 -8.58

-12.67 -9.44 -5.33

-14.72 -8.57

-12.75 -23.57 -12.22

-6.70 -20.11 -9.39 -9.46

-20.86 -8.48 -7.64

-18.05 -30.00 -19.31 -52.36 -2.45

-10.80 -15.51 -1.90 -6.05

-11.72 -3.56 -1.18 -5.12

-3.69 -4.16 -9.40 -0.38 -6.60 -7.36 +0.83 -0.44 -1.30 -0.23 -2.86 -3.10 -0.17 -0.89 -1.06 -3.68 -6.60 -9.32 -4.69 -1.55 -6.26 -1.68 -5.17 -6.77 -5.28 -3.43 -8.52 -4.73 -7.32

-13.28 -7.25 -3.85

-11.42 -5.47 -5.27

-11.93 -4.64 -4.72

-10-23 -18.24 -9.88

-29.84 -1.38 -5.73 -8.16 -0.94 -3.29 -6.10 -2.03 -0.75 -2.95

- 1.45 -1.23 -3.09 +0.49 -2.79 -2.26 +0.79 -0.19 -0.3 1 -0.06 -1.21 -1.28 +O. 13 -0.39 -0.26 -0.95 -3.06 -3.75 -1.85 -0.24 -2.10 +0.02 -2.16 -2.08 -1.86 -1.54 -3.33 -1.16 -2.68 -4.71 -2.99 -1.50 -4.45 -2.19 -1.97 -4.59 -1.58 -2.10 -3.86 -7.97 -3.64

-11.29 -0.48 -1.54 -2.29 -0.20 -1.06 -1.81 -0.74 -0.33 -1.13

-0.48 -0.20 -0.76 +0.46 -1.18 -0.69 +0*46 -0.08 -0.02 -0.01 -0.46 -0.47 +0.15 -0.15

0.00 -0.04 -1.38 -1.41 -0.63 +o. 12 -0.51 +0.42 -0.81 -0.37 -0.53 -0.65 -1.16 -0.45 -0.78 -1.33 -1.16 -0.56 -1.70 -0.83 -0.65 -1.59 -0.39 -0.89 -1.34 -3.19 -0.46 -3.87 -0.11 -0.01 -0.17 +0.04 -0.27 -0.36 -0.23 -0.15 -0.40

-0.06 -0.00 -0.06 +0.16 -0.4 1 -0.25 +0.07 -0.02 +0.03

0.00 -0.11 -0.11 10.06 -0.04 +0.02 +o. 10 -0.49 -0.39 -0.12 +o. 11 -0.02 +0.31 -0.18 +0.13 -0.05 -0.19 -0.25 -0.06 -0.09 -0.15 -0.33 -0.16 -0.49 -0.23 -0.10 -0.35 -0.20 -0.28 -0.31 -0.90 +0.02 -0.97, +0.02 t0-34 +0.35 +0.06 -0.06 -0.01 -0.03 -0.07 -0.11

K. Bender et al. Table 2 (cont.)

Lod scores at recombination fraction, 6

Locus Sex Matings Offspring 0.05 0. I 0.2 0.3 0.4

18. KeN

19. Lu

20. M N S s

21. P

22. PGD

23. PGM,

24. PGM,

25. Pi

26. PLG

27. RHESUS

28. M D F

M F Joint M F Joint M F Joint M Joint M F Joint M F Joint M F Joint M F Joint M F Joint M F Joint M F Joint

2

2

32

5

2

18

13

22

19

21

2

9

9

170

24

8

104

17

I20

86

I29

8

-1.90 - - I .90 - 1.93 -

-1.93 -11.55 -12.79 -26.3 I

-0.72 -2.11 +0.81 - 1.44 -0.63 -4.56

-10.98 -16.59

-5.66 -2.63 -8.23 -5.82

-13.31 -20.55

-3.66 -2.04 -5.94 -8.6 1 -7.86

-18.88 -0.42 -0.11 -0.50

-1.09 -

- I .09 -1.13 -

-1.13 -6.64 -7.13 - 14.42 -0.44 -1.14 +0.72 -0.88 -0.16 -2.53 -6.58 -9.69 -3.26 -1.35 -4.2 I -2.82 -8.17

-11.00 -2.09 -0.83 -3.07 -4.67 -4.76

-10.58 -0.22 -0.09 -0.23

-0.39 -

-0.39 -0.45 -

-0.45 -2.47 -2.37 -4.95 -0.19 -0.39 t0 .52 -0.39 +0.13 -0.94 -2.50 -3.90 -1.24 -0- 3 3 -1.28 -0.55 -3.67 -4.30 -0.80 -0.05 -0.81 - 1.49 -1.99 -3.79 -0.07 -0.05 -0.12

-0.09

-0.09 -0.15

-

- -0.15 -0.77 -0.50 -1.30 -0.08 -0.1 1 +0.30 -0.15 +0.15 -0.36 -1.06 -1.50 -0.42 +0.02 -0.32 +0.08 - 1.60 -1.52 -0.21 +0.21 -0.08 -0.35 -0.14 -1.16 -0.02 -0.01 -0.03

+0.02

+0.02 -0.03

-

- -0.03 -0.15 +o. 10 -0.05 -0.02 -0.02 +0.09 -0.04 +0.05 -0.15 -0.29 -0.44 -0.09 t0.07 -0.01 +0.09 -0.54 -0.45 -0.06 +0.10 +0.04 -0.04 -0. I7 -0.22

0.00 -0.01 -0.01

complicates the presentation of data. To avoid complications associated with approximate factorizations for recombination fractions in both sexes (Morton, 1978), we have reduced the bivariate lod tables to a three-row representation given in Table 2.

R E S U L T S A N D D I S C U S S I O N

Formal genetics of the C6 polvmorphism The 40 two-generation families and the six larger pedigrees resulted in 66 matings with

altogether 33 1 children. All pairings showed the expected C6 segregation patterns and ratios (Table 1). The allele frequencies based on 106 unrelated German parents (omitting the three foreign persons) correspond well to those repcrted in other European populations

C6 linkage studies 6 5 (tabulated by Kuhnl & Kreckel, 1980). Thereby the regular formal genetic model of the C6 polymorphism is demonstrated once more.

C6 linkage studies The linkage relations between the C6 locus and 28 marker loci are given in Table 2. The

data base of each linkage test is given by the total of informative matings and offspring involved 'in the particular two-locus combination. Many families gave information on recombination in one sex only (double backcrosses), as contrasted to others, in which no exact factorization of the likelihood is possible (double intercrosses and untested ancestors, Morton, 1978). The informational contribution of the last mentioned situations is rather small, but varying, however. Therefore, the bivariate lod tables (z (emale, Oremale)) have been reduced to three lines (Table 2): the first line (M), in which recombination is attributed to the male only, assuming absence of linkage in the female (z (Omale, Ofemale = 0.5)); the second line (F) for the inverse situation (z (eremale, Omale = 0.05)); and the

........ h ....... .- ................ ................ ...............

.....

............... .....- .............

........ .......... - ........

0 0.10 0.20 0.30 0.40 8 Chromosome I ' ' ' ' ' ' ' -

1

ADA Unarsi nod

Jk K L e P ESA, Tf PTC EBSW

E,

2

4

6

0

9

11

13

14

15

16

19

20 ................ ..................... ................ ........ ....................... -. ...... ........

................ . ...................... m

................ ...............

............................... ................ ............... MNSr

Gc PLG

HLA/Bf G L O PGM3

........ ........

GPT

GALT ABO A K

H b B ESD

P i Grn

P I

H P

c3 L u SO

66 K. Bender et al. third line (Joint), giving the lod scores for recombination fractions equal in both (z (0 male = 0 female)), which is the diagonal of the lod table.

In accordance with the foregoing reports there is no convincing evidence for close linkage between C6 and any of the tested marker genes, also not for GALT, Lu, PLG and M D F included here for the first time. But weak positive lod scores resulted from the male meioses as to C6:ADA, C6:ACP, C6:Hp (as already observed by Olving el al., 1979), C6:PGD (as already found by Hobart et af., 1977) and C6:Fy (all three reports). Further studies should concentrate also on these comparisons.

A failing of conclusive linkage relations is, however, also of some gene mapping value. It will allow the localization exclusion of the gene from a number of chromosome regions (Cook et al., 1980), so that further studies can be concentrated on the remaining ones. By compilation of our data with those of Hobart et af. (1977) and Olving et a!. (1979) the C6 gene can be excluded (lod score (-2.00) from large parts of the genome, especially from big pieces of the autosomes 1, 2, 4 , 6 , 8 , 9, 13, 14, 16, and 19 (Fig. 1).

A C K N O W L E D G M E N T S

The authors appreciate the kind help of Dr Donald Raum (Center for Blood Research, Boston, USA) at the onset of their family studies, and the generous gift of C6 deficient rabbits from Prof. Dr Ursula Rother and Prof. Dr Klaus Rother (Institut f u r Immunologie der Universitat Heidelberg). For excellent technical assistance we thank Mrs Klara Burckhardt. Mrs Annemarie KIein and Mr Gerhard Kunstmann.

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