Hum. Genet. 52, 259--261 (1979) © by Springer-Verlag1979
Human Erythrocyte Carbonic Anhydrase Polymorphism in Kenya Alan Kendall* Department of Medicine, Kenyana National Hospital and University of Nairobi, Kenya, and McGill University, Montreal, Canada
Summary. In this study, 1736 Western Kenyans were examined for red cell carbonic anhydrase (CA) variants. No CA I variants were detected, but the CAII2 isozyme was found with a calculated gene frequency of 0.054. Introduction The human carbonic anhydrase isozymes CA I and CA II constitute a substantial fraction of the non-haemoglobin protein of the red cell. This facilitates their visualisafion along with haemoglobins when red cell haemolysates are subjected to electrophoresis. Thus, during haemoglobin screening programs and deliberate population surveys for the carbonic anhydrases, a number of genetic variants of these isozymes have been found (Tashian and Carter, 1976; Blake and Kirk, 1978). The discovery of such variants has not been clinically significant. Apart from their biochemical-physiological interest, these variants may prove of value as genetic markers in relation to population origins, movements and adaptations (Blake and Kirk, 1978). At the time of writing some 16 electrophoretic variants of CA I and 3 of CA II have been reported (Goriki et al., 1979; Tashian and Carter, 1976; Blake and Kirk, 1978; Ghosh, 1978). In only a third-of these has the mutant enzyme been characterised with respect to an amino-acid substitution. In each case a single base substitution in the structural gene might account for the anomalous protein. Until very recently the only CA II variant to be reported was that which has been variously called CAIIh, CA-H or CAII2 (Moore et al., 1971). This variant has been shown to have the substitution of aspartic acid for asparagine in position 253 of the CA II monomer (Lin and Deutsch, 1972). It has been found in polymorphic frequencies in Black Americans and West Africans (Moore et al., 1971; Carter, 1972; Welch, 1975). It has also been found in Central Africa in Zambia, but not in a small East African sample from Uganda and Ethiopia (Carter, 1972). Dr. A. G. Kendall, Division of Haematology, Royal Victoria Hospital, 687 Pine Ave., West Montreal, Que., Canada * P r e s e n t address:
0340-6717/79/0052/0259/$ 01.00
260
A. Kendall
Prior to 1970 detection of the heterozygous state for CA II2 was not feasible using standard electrophoretic techniques due to overlap of the CA I and CA II2 bands and the non-specificity of protein or esterase staining. This problem has been partially overcome by the use of an immunological technique by Moore et al. (1971) which differentiated the two enzymes. The subsequent development of an inexpensive method, using fluorogenic substrates, after electrophoresis, to differentiate the CA I and CA II isozymes, made possible mass population screening (Hopkinson et al., 1974). Modification of this technique for use with cellulose acetate electrophoresis was a useful simplification (Welch, 1975). Despite the obvious advantages of these improved techniques, practical considerations precluded their use in the present on site East African study, carried out between 1976 and 1978. A large group of western Kenyans, the subject of a haemoglobinopathy screening program, was also assessed for carbonic anhydrase variants. The non-specific techniques involved allow detection of heterozyg0sity for isozymes with non-overlapping electrophoretic mobility, or homozygosity for isozymes such as CA II2. Materials and Methods A total of 1736 Western Kenyans above the age of 5 years, belonging predominantly to the Luo and Luyia tribes, were the subjects of the study. Electrophoresis of red cell haemolysates was carried out on cellulose acetate strips in T.E.B. buffer, pH 8.4 (Helena Co., Beaumont, Texas, USA) and protein bands were stained with ponceau-S. An apparent absence of normal CA II was confirmed using the technique described by Carter (1972) using bromothymol blue indicator and CO2. In some instances the more sensitive protein stain, nigrosine, was used instead of ponceau-S. Results In this study no variants of CA I were found. In five unrelated individuals the electrophoretic pattern was that of the homozygous state for the CA II2 variant. Assuming a state of equilibrium, application of the Hardy-Weinberg calculation gives the following distribution of the CA II phenotypes: CA II homozygotes, 89.56%; CA I I / C A II2 phenotype, 10.16%; and CA II2 homozygotes, 0.29%. This gives a gene frequency for the CAII2 variant of approximately 0.054. Comment Although no CA I variants were detected in this East African population, the CA II2 variant isozyme appears to be present in polymorphic frequency. The gene frequency of 0.054 is roughly half that found in Black Americans and in other West African groups (Moore et al., 1971; Carter, 1972; Welch, 1975). The apparent ubiquitousness of the gene in the African continent is of interest. What relationship, if any, this bears to population movements remains to be determined.
Acknowledgments. I am grateful for the technical assistance of Mrs. N. Ouna of the University of Nairobi Department of Medicine.
CA Polymorphism in Kenya
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References Blake, N. M., Kirk, R. L.: Widespread distribution of variant forms of carbonic anhydrase in Australian Aboriginals. Med. J. Aust. 1, 183--185 (1978) Carter, N. D.: Carbonic anhydrase II polymorphism in Africa. Hum. Hered. 22, 539--541 (1972) Ghosh, A. K.: The distribution of genetic variants of glyoxalase I, esterase E, and carbonic anhydrase I and II in Indian populations. Indian J. Phys. Anthropol. Hum. Genet. (in press, 1978) Goriki, K., Tashian, R. E., Stroup, S. K., Yu, Y.-S. L., Henriksson, D. M.: Chemical characterization of a new Japanese variant of carbonic anhydrase I, CA I Nagasaki I (76 Arg-~Gln). Biochem. Genet. (in press, 1979) Hopkinson, D. A., Coppock, J. S., Mtihlemann, M. F., Edwards, Y. H.: The detection and differentiation of the products of the human carbonic anhydrase loci, CA~ and CAm using fluorogenic substrates. Ann. Hum. Genet. 38, 155--162 (1974) Lin, K.-T. D., Deutsch, H. F.: Human carbonic anhydrase. VIII. Isolation and characterization of a polymorphic form of the C type isozyme. J. Biol. Chem. 247, 3761--3766 (1972) Moore, M. J., Funakoshi, S., Deutsch, H. F.: Human carbonic anhydrases. VII. A new C type isozyme in erythrocytes of American Negroes. Biochem. Genet. 5, 497--504 (1971) Tashian, R. E., Carter, N. D.: Biochemical genetics of carbonic anhydrase. Adv. Hum. Genet. 7, 1--56 (1976) Welch, S.: Population and family studies on carbonic anhydrase II polymorphism in Gambia, West Africa. Humangenetik 27, 163--166 (1975)
Received April 12 / Revised June 26, 1979
Human Erythrocyte Carbonic Anhydrase Polymorphism in Kenya Alan Kendall* Department of Medicine, Kenyana National Hospital and University of Nairobi, Kenya, and McGill University, Montreal, Canada
Summary. In this study, 1736 Western Kenyans were examined for red cell carbonic anhydrase (CA) variants. No CA I variants were detected, but the CAII2 isozyme was found with a calculated gene frequency of 0.054. Introduction The human carbonic anhydrase isozymes CA I and CA II constitute a substantial fraction of the non-haemoglobin protein of the red cell. This facilitates their visualisafion along with haemoglobins when red cell haemolysates are subjected to electrophoresis. Thus, during haemoglobin screening programs and deliberate population surveys for the carbonic anhydrases, a number of genetic variants of these isozymes have been found (Tashian and Carter, 1976; Blake and Kirk, 1978). The discovery of such variants has not been clinically significant. Apart from their biochemical-physiological interest, these variants may prove of value as genetic markers in relation to population origins, movements and adaptations (Blake and Kirk, 1978). At the time of writing some 16 electrophoretic variants of CA I and 3 of CA II have been reported (Goriki et al., 1979; Tashian and Carter, 1976; Blake and Kirk, 1978; Ghosh, 1978). In only a third-of these has the mutant enzyme been characterised with respect to an amino-acid substitution. In each case a single base substitution in the structural gene might account for the anomalous protein. Until very recently the only CA II variant to be reported was that which has been variously called CAIIh, CA-H or CAII2 (Moore et al., 1971). This variant has been shown to have the substitution of aspartic acid for asparagine in position 253 of the CA II monomer (Lin and Deutsch, 1972). It has been found in polymorphic frequencies in Black Americans and West Africans (Moore et al., 1971; Carter, 1972; Welch, 1975). It has also been found in Central Africa in Zambia, but not in a small East African sample from Uganda and Ethiopia (Carter, 1972). Dr. A. G. Kendall, Division of Haematology, Royal Victoria Hospital, 687 Pine Ave., West Montreal, Que., Canada * P r e s e n t address:
0340-6717/79/0052/0259/$ 01.00
260
A. Kendall
Prior to 1970 detection of the heterozygous state for CA II2 was not feasible using standard electrophoretic techniques due to overlap of the CA I and CA II2 bands and the non-specificity of protein or esterase staining. This problem has been partially overcome by the use of an immunological technique by Moore et al. (1971) which differentiated the two enzymes. The subsequent development of an inexpensive method, using fluorogenic substrates, after electrophoresis, to differentiate the CA I and CA II isozymes, made possible mass population screening (Hopkinson et al., 1974). Modification of this technique for use with cellulose acetate electrophoresis was a useful simplification (Welch, 1975). Despite the obvious advantages of these improved techniques, practical considerations precluded their use in the present on site East African study, carried out between 1976 and 1978. A large group of western Kenyans, the subject of a haemoglobinopathy screening program, was also assessed for carbonic anhydrase variants. The non-specific techniques involved allow detection of heterozyg0sity for isozymes with non-overlapping electrophoretic mobility, or homozygosity for isozymes such as CA II2. Materials and Methods A total of 1736 Western Kenyans above the age of 5 years, belonging predominantly to the Luo and Luyia tribes, were the subjects of the study. Electrophoresis of red cell haemolysates was carried out on cellulose acetate strips in T.E.B. buffer, pH 8.4 (Helena Co., Beaumont, Texas, USA) and protein bands were stained with ponceau-S. An apparent absence of normal CA II was confirmed using the technique described by Carter (1972) using bromothymol blue indicator and CO2. In some instances the more sensitive protein stain, nigrosine, was used instead of ponceau-S. Results In this study no variants of CA I were found. In five unrelated individuals the electrophoretic pattern was that of the homozygous state for the CA II2 variant. Assuming a state of equilibrium, application of the Hardy-Weinberg calculation gives the following distribution of the CA II phenotypes: CA II homozygotes, 89.56%; CA I I / C A II2 phenotype, 10.16%; and CA II2 homozygotes, 0.29%. This gives a gene frequency for the CAII2 variant of approximately 0.054. Comment Although no CA I variants were detected in this East African population, the CA II2 variant isozyme appears to be present in polymorphic frequency. The gene frequency of 0.054 is roughly half that found in Black Americans and in other West African groups (Moore et al., 1971; Carter, 1972; Welch, 1975). The apparent ubiquitousness of the gene in the African continent is of interest. What relationship, if any, this bears to population movements remains to be determined.
Acknowledgments. I am grateful for the technical assistance of Mrs. N. Ouna of the University of Nairobi Department of Medicine.
CA Polymorphism in Kenya
261
References Blake, N. M., Kirk, R. L.: Widespread distribution of variant forms of carbonic anhydrase in Australian Aboriginals. Med. J. Aust. 1, 183--185 (1978) Carter, N. D.: Carbonic anhydrase II polymorphism in Africa. Hum. Hered. 22, 539--541 (1972) Ghosh, A. K.: The distribution of genetic variants of glyoxalase I, esterase E, and carbonic anhydrase I and II in Indian populations. Indian J. Phys. Anthropol. Hum. Genet. (in press, 1978) Goriki, K., Tashian, R. E., Stroup, S. K., Yu, Y.-S. L., Henriksson, D. M.: Chemical characterization of a new Japanese variant of carbonic anhydrase I, CA I Nagasaki I (76 Arg-~Gln). Biochem. Genet. (in press, 1979) Hopkinson, D. A., Coppock, J. S., Mtihlemann, M. F., Edwards, Y. H.: The detection and differentiation of the products of the human carbonic anhydrase loci, CA~ and CAm using fluorogenic substrates. Ann. Hum. Genet. 38, 155--162 (1974) Lin, K.-T. D., Deutsch, H. F.: Human carbonic anhydrase. VIII. Isolation and characterization of a polymorphic form of the C type isozyme. J. Biol. Chem. 247, 3761--3766 (1972) Moore, M. J., Funakoshi, S., Deutsch, H. F.: Human carbonic anhydrases. VII. A new C type isozyme in erythrocytes of American Negroes. Biochem. Genet. 5, 497--504 (1971) Tashian, R. E., Carter, N. D.: Biochemical genetics of carbonic anhydrase. Adv. Hum. Genet. 7, 1--56 (1976) Welch, S.: Population and family studies on carbonic anhydrase II polymorphism in Gambia, West Africa. Humangenetik 27, 163--166 (1975)
Received April 12 / Revised June 26, 1979
