Cytogenet. Cell Genet. 22: 651-654 (1978)
Complement C5: immunofixation electrophoresis, quantitative variants, and nonlinkage to HLA L.R. WiiiTKAMP, S. R osenfeld, and E. J ohnston Department of Psychiatry, Division of Genetics, Department of Pediatrics, and Department of Medicine, Division of Immunology, University of Rochester School of Medicine and Dentistry, Rochester, N.Y.
The absence of close linkage between the loci for complement C5 and HLA has been previously reported in a single female-informative kindred.1 The information has special relevance in view of the fact that some loci for quantitative or qualitative variants for proteins in the complement series are closely linked to HLA (C2, C4) and others are not (C3, C6, C7). Here we report a technique for C5 immunofixation electrophoresis and a second family segregating for a C5 quantitative variant. Agarose-gel electrophoresis was run for 2 lk h at 15 volts/cm on a 10-cm (20-cm wide) glass plate. The gel and electrode buffers were pH 8.6 barbital at an ionic strength of 0.075 with 0.002 M calcium lactate added. Immunofixation after electro phoresis was carried out as previously described,2 using a rabbit anti-human C5 and Coomassie blue stain. A single protein band appears 4 cm from the origin. The bands shown in fig. 1 were obtained using 3 ul of serum. More intense patterns could be obtained using 8 «1 of serum.
+
__ LJÜ __ ________ _ _ _
1 2 3 4 5 6
Fig. 1. C5 immunofixation electrophoresis. The arrow points to a single band of C5 protein. Samples 1, 2, and 3 are from ran domly selected black Americans. (Sample 2 is from H069-002.) Samples 4, 5, and 6 are from C5 heterozygotes II-2, III-4, and 111-5 from the pedigree of Rosenfeld et aid
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Supported in part by The National Foundation grant 1-443, NIH grants GM 19962 and AI 12568, NIMH grant 9-K02-MH00124, and a grant from the Monroe County chapter of the Arthritis Foundation.
652
W eitkamp et al.
Unassigned loci and general linkage studies
Fig. 2. Pedigree of family H069 showing the inheritance of C5 deficiency in relation to that for the HLA region. Squares indicate males, circles females. Erythrocyte glyoxalase (GLO), properdin factor B (Bf), and HLA types are given below the pedigree symbols in the most probable order of their linkage relationships on chromosome 6 and according to their probable phase of linkage.
Table I. C5 values in family H069.
001 002 003 004 005 006 007 008 009 013 014 015 016 017
Radial diffusion (ug/ml)
Hemolytic units/m lX 10-3
Genotype
69, 61 36, 32 126, 126 51, 50 54, 56 48, 50 54, 46 69, 82 54, 75 32 100, 110 69, 67 54, 50 92, 90
96 82, 60 226 73 56 108 92 153, 147 128, 95 102 217,199 98 78 171
heterozygote heterozygote normal heterozygote heterozygote heterozygote heterozygote uncertain heterozygote heterozygote normal heterozygote heterozygote normal
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No qualitative variations from a single precipitin band were seen in 33 unrelated black and 47 unrelated white Americans. However, one black individual (H069-002), who was noted to have a decreased quantity of C5 protein on electrophoresis (sample 2, fig. 1), had a decreased quantity
W eitkamp et al.
Unassigned loci and general linkage studies
653
Table II. Lod scores for the linkage relationships of C5 with 20 marker systems. Chromosome
1
Locus
Rh PGM, AMY Fy
2 6
ACP, GLO HLA
9
ABO
16
uHp
20 Other
ADA GPT MNSs Js Jk Le Se Cp Hb C3 Pr
Family»
D078 H069 H069 H069 D078 H069 H069 D078 H069 D078 H069 D078 H069 D078 H069 H069 D078 H069 D078 H069 D078 H069 D078 H069 D078 H069 D078 D078 H069 H069 H069
Phase-known offspring NR
R
0 0 1 2 3 0 1 0 0 4 0 0 0 0 0 2 0 0 6 0 0 0 0 0 0 0 3 7 1 2 3
0 0 2 1 5 0 2 0 0 5 0 0 0 0 0 1 0 0 3 0 0 0 0 0 0 0 1 5 2 1 0
Lod scores at & = 0.05
0.10
0.20
0.30
0.40
-0 .2 6 0.37 -3 .6 2 -2 .6 1 -5 .6 1 -2 .1 6 -3 .8 9 -2 .8 9 0.37 -5 .2 6 -0 .9 1 -0 .6 3 -0 .9 1 1.07 -1 .4 4 -1 .3 5 0.07 -0 .5 6 -2 .7 7 -0 .9 1 -1 .2 7 -0 .0 5 -1 .4 4 0.81 -1 .9 1 -0 .5 3 -0 .2 5 -5 .4 2 -1 .7 2 -0 .4 4 -0 .3 5
-0 .2 0 0.53 -2 .2 6 -1 .5 2 -3 .6 2 -1 .3 3 -2 .4 7 -1 .7 7 0.53 -2 .9 5 -0 .4 2 -0 .1 7 -0 .4 2 0.93 -0 .8 9 -0 .6 1 0.24 -0 .4 6 -1 .4 5 -0 .4 2 -0 .7 4 -0 .0 4 -0 .8 9 0.72 -1 .1 2 -0 .4 0 0.00 -3 .0 6 -1 .1 4 -0 .1 9 0.09
-0 .1 1 0.53 -1 .0 4 -0 .5 7 -1 .7 6 -0 .5 8 -1 .1 7 -0 .7 8 0.53 -1 .0 6 -0 .0 6 0.13 -0 .0 6 0.65 -0 .3 9 -0 .0 6 0.26 -0 .1 3 -0 .3 6 -0 .0 6 -0 .2 9 -0 .0 3 -0 .3 9 0.52 -0 .4 5 -0 .1 9 0.18 -1 .0 8 -0 .5 9 0.01 0.36
-0 .0 5 0.36 -0 .4 6 -0 .1 6 -0 .8 2 -0 .2 3 -0 .5 3 -0 .3 0 0.36 -0 .2 5 0.03 0.15 0.03 0.36 -0 .1 5 0.09 0.16 -0 .0 4 0.06 0.03 -0 .1 0 -0 .0 1 -0 .1 5 0.30 -0 .1 6 -0 .0 7 0.20 -0 .2 8 -0 .3 0 0.07 0.35
-0 .0 1 0.13 -0 .1 5 0.01 -0 .2 8 -0 .0 5 -0 .1 7 -0 .0 7 0.13 -0 .0 6 0.02 0.06 0.02 0.11 -0 .0 4 0.08 0.05 -0 .0 1 0.15 0.11 -0 .0 2 0.00 -0 .0 4 0.09 -0 .0 4 -0 .0 1 0.14 0.02 -0 .1 2 0.06 0.22
and activity of C5 protein by radial diffusion and hemolytic assay. A study of the family confirmed inheritance for a C5 deficiency gene in a second American black family (fig. 2 and table I). A summary of the linkage relationships of the C5 locus with 20 in-
Downloaded by: Nagoya University 133.6.82.173 - 1/15/2019 5:22:22 AM
» Data on family D078, almost entirely female-informative, were previously published.' Family H069 is male-informative only.
654
W eitkamp et al.
Unassigned loci and general linkage studies
formative marker systems is shown in table II. There is no evidence for linkage with any of the markers studied. The new H069 family contributes little additional information to the previously reported exclusion of linkage between C5 and HLA at 6 X 0 .1 5 in females (family D078). However, in the new family there is one recombinant between C5 and C3 among three phase-known male-informative offspring. The recombinant event (in H069-016) is probably not attributable to nonpaternity since there was no exclusion in a number of marker systems and the uncommon C5 deficiency gene was inherited from her father. Nor is the recombinant individual one in whom there is a borderline C5 phenotype. Although one recombination in three opportunities does not constitute much evidence, it is sufficient to make improbable very tight linkage, such as exists for C2, C4, Bf, and HLA-B. Also, linkage of C5 with the C3-linked Lewis (Le) locus is excluded for 0 < 0.07. Thus, C5 joins C6123 in being tightly linked to neither HLA or C3.
Downloaded by: Nagoya University 133.6.82.173 - 1/15/2019 5:22:22 AM
1 Rosenfeld, S.I.; W eitkamp, L.R., and Ward, F.: Hereditary C5 deficiency in man: genetic linkage studies. J. Immunol. 119: 604-608 (1977). 2 A lper, C.A. and J ohnson, A.M.: Immunofixation electrophoresis: a technique for the study of protein polymorphism. Vox Sang. 17: 445-452 (1969). 3 O lving, J.H.; O laisen, B.; T eisberg, P.; G edde-Dahl, T., J r ., and T horsby, E.: Nonlinkage between C6 and chromosome 6 markers. Hum. Genet. 37: 125-129 (1977).
Complement C5: immunofixation electrophoresis, quantitative variants, and nonlinkage to HLA L.R. WiiiTKAMP, S. R osenfeld, and E. J ohnston Department of Psychiatry, Division of Genetics, Department of Pediatrics, and Department of Medicine, Division of Immunology, University of Rochester School of Medicine and Dentistry, Rochester, N.Y.
The absence of close linkage between the loci for complement C5 and HLA has been previously reported in a single female-informative kindred.1 The information has special relevance in view of the fact that some loci for quantitative or qualitative variants for proteins in the complement series are closely linked to HLA (C2, C4) and others are not (C3, C6, C7). Here we report a technique for C5 immunofixation electrophoresis and a second family segregating for a C5 quantitative variant. Agarose-gel electrophoresis was run for 2 lk h at 15 volts/cm on a 10-cm (20-cm wide) glass plate. The gel and electrode buffers were pH 8.6 barbital at an ionic strength of 0.075 with 0.002 M calcium lactate added. Immunofixation after electro phoresis was carried out as previously described,2 using a rabbit anti-human C5 and Coomassie blue stain. A single protein band appears 4 cm from the origin. The bands shown in fig. 1 were obtained using 3 ul of serum. More intense patterns could be obtained using 8 «1 of serum.
+
__ LJÜ __ ________ _ _ _
1 2 3 4 5 6
Fig. 1. C5 immunofixation electrophoresis. The arrow points to a single band of C5 protein. Samples 1, 2, and 3 are from ran domly selected black Americans. (Sample 2 is from H069-002.) Samples 4, 5, and 6 are from C5 heterozygotes II-2, III-4, and 111-5 from the pedigree of Rosenfeld et aid
Downloaded by: Nagoya University 133.6.82.173 - 1/15/2019 5:22:22 AM
Supported in part by The National Foundation grant 1-443, NIH grants GM 19962 and AI 12568, NIMH grant 9-K02-MH00124, and a grant from the Monroe County chapter of the Arthritis Foundation.
652
W eitkamp et al.
Unassigned loci and general linkage studies
Fig. 2. Pedigree of family H069 showing the inheritance of C5 deficiency in relation to that for the HLA region. Squares indicate males, circles females. Erythrocyte glyoxalase (GLO), properdin factor B (Bf), and HLA types are given below the pedigree symbols in the most probable order of their linkage relationships on chromosome 6 and according to their probable phase of linkage.
Table I. C5 values in family H069.
001 002 003 004 005 006 007 008 009 013 014 015 016 017
Radial diffusion (ug/ml)
Hemolytic units/m lX 10-3
Genotype
69, 61 36, 32 126, 126 51, 50 54, 56 48, 50 54, 46 69, 82 54, 75 32 100, 110 69, 67 54, 50 92, 90
96 82, 60 226 73 56 108 92 153, 147 128, 95 102 217,199 98 78 171
heterozygote heterozygote normal heterozygote heterozygote heterozygote heterozygote uncertain heterozygote heterozygote normal heterozygote heterozygote normal
Downloaded by: Nagoya University 133.6.82.173 - 1/15/2019 5:22:22 AM
No qualitative variations from a single precipitin band were seen in 33 unrelated black and 47 unrelated white Americans. However, one black individual (H069-002), who was noted to have a decreased quantity of C5 protein on electrophoresis (sample 2, fig. 1), had a decreased quantity
W eitkamp et al.
Unassigned loci and general linkage studies
653
Table II. Lod scores for the linkage relationships of C5 with 20 marker systems. Chromosome
1
Locus
Rh PGM, AMY Fy
2 6
ACP, GLO HLA
9
ABO
16
uHp
20 Other
ADA GPT MNSs Js Jk Le Se Cp Hb C3 Pr
Family»
D078 H069 H069 H069 D078 H069 H069 D078 H069 D078 H069 D078 H069 D078 H069 H069 D078 H069 D078 H069 D078 H069 D078 H069 D078 H069 D078 D078 H069 H069 H069
Phase-known offspring NR
R
0 0 1 2 3 0 1 0 0 4 0 0 0 0 0 2 0 0 6 0 0 0 0 0 0 0 3 7 1 2 3
0 0 2 1 5 0 2 0 0 5 0 0 0 0 0 1 0 0 3 0 0 0 0 0 0 0 1 5 2 1 0
Lod scores at & = 0.05
0.10
0.20
0.30
0.40
-0 .2 6 0.37 -3 .6 2 -2 .6 1 -5 .6 1 -2 .1 6 -3 .8 9 -2 .8 9 0.37 -5 .2 6 -0 .9 1 -0 .6 3 -0 .9 1 1.07 -1 .4 4 -1 .3 5 0.07 -0 .5 6 -2 .7 7 -0 .9 1 -1 .2 7 -0 .0 5 -1 .4 4 0.81 -1 .9 1 -0 .5 3 -0 .2 5 -5 .4 2 -1 .7 2 -0 .4 4 -0 .3 5
-0 .2 0 0.53 -2 .2 6 -1 .5 2 -3 .6 2 -1 .3 3 -2 .4 7 -1 .7 7 0.53 -2 .9 5 -0 .4 2 -0 .1 7 -0 .4 2 0.93 -0 .8 9 -0 .6 1 0.24 -0 .4 6 -1 .4 5 -0 .4 2 -0 .7 4 -0 .0 4 -0 .8 9 0.72 -1 .1 2 -0 .4 0 0.00 -3 .0 6 -1 .1 4 -0 .1 9 0.09
-0 .1 1 0.53 -1 .0 4 -0 .5 7 -1 .7 6 -0 .5 8 -1 .1 7 -0 .7 8 0.53 -1 .0 6 -0 .0 6 0.13 -0 .0 6 0.65 -0 .3 9 -0 .0 6 0.26 -0 .1 3 -0 .3 6 -0 .0 6 -0 .2 9 -0 .0 3 -0 .3 9 0.52 -0 .4 5 -0 .1 9 0.18 -1 .0 8 -0 .5 9 0.01 0.36
-0 .0 5 0.36 -0 .4 6 -0 .1 6 -0 .8 2 -0 .2 3 -0 .5 3 -0 .3 0 0.36 -0 .2 5 0.03 0.15 0.03 0.36 -0 .1 5 0.09 0.16 -0 .0 4 0.06 0.03 -0 .1 0 -0 .0 1 -0 .1 5 0.30 -0 .1 6 -0 .0 7 0.20 -0 .2 8 -0 .3 0 0.07 0.35
-0 .0 1 0.13 -0 .1 5 0.01 -0 .2 8 -0 .0 5 -0 .1 7 -0 .0 7 0.13 -0 .0 6 0.02 0.06 0.02 0.11 -0 .0 4 0.08 0.05 -0 .0 1 0.15 0.11 -0 .0 2 0.00 -0 .0 4 0.09 -0 .0 4 -0 .0 1 0.14 0.02 -0 .1 2 0.06 0.22
and activity of C5 protein by radial diffusion and hemolytic assay. A study of the family confirmed inheritance for a C5 deficiency gene in a second American black family (fig. 2 and table I). A summary of the linkage relationships of the C5 locus with 20 in-
Downloaded by: Nagoya University 133.6.82.173 - 1/15/2019 5:22:22 AM
» Data on family D078, almost entirely female-informative, were previously published.' Family H069 is male-informative only.
654
W eitkamp et al.
Unassigned loci and general linkage studies
formative marker systems is shown in table II. There is no evidence for linkage with any of the markers studied. The new H069 family contributes little additional information to the previously reported exclusion of linkage between C5 and HLA at 6 X 0 .1 5 in females (family D078). However, in the new family there is one recombinant between C5 and C3 among three phase-known male-informative offspring. The recombinant event (in H069-016) is probably not attributable to nonpaternity since there was no exclusion in a number of marker systems and the uncommon C5 deficiency gene was inherited from her father. Nor is the recombinant individual one in whom there is a borderline C5 phenotype. Although one recombination in three opportunities does not constitute much evidence, it is sufficient to make improbable very tight linkage, such as exists for C2, C4, Bf, and HLA-B. Also, linkage of C5 with the C3-linked Lewis (Le) locus is excluded for 0 < 0.07. Thus, C5 joins C6123 in being tightly linked to neither HLA or C3.
Downloaded by: Nagoya University 133.6.82.173 - 1/15/2019 5:22:22 AM
1 Rosenfeld, S.I.; W eitkamp, L.R., and Ward, F.: Hereditary C5 deficiency in man: genetic linkage studies. J. Immunol. 119: 604-608 (1977). 2 A lper, C.A. and J ohnson, A.M.: Immunofixation electrophoresis: a technique for the study of protein polymorphism. Vox Sang. 17: 445-452 (1969). 3 O lving, J.H.; O laisen, B.; T eisberg, P.; G edde-Dahl, T., J r ., and T horsby, E.: Nonlinkage between C6 and chromosome 6 markers. Hum. Genet. 37: 125-129 (1977).
