Frequency of glucose-6-phosphate dehydrogenase deficiency in relation to altitude: a malaria hypothesis

Bulletin of the World Health Organization, 58 (4): 659-662 (1980) Frequency of glucose-6-phosphate dehydrogenase deficiency in relation to altitude: a malaria hypothesis M. TZONEVA,1 A. G. BULANOV,2 M. MAVRUDIEVA,3 S. LALCHEV,3 D. TONCHEVA,3 & D. TANEV3 Genetic markers have recently beenfound to bemuch morepolymorphic than expected. Such extensive human polymorphisms may bepartly explained by a number ofgenetic and environmentalfactors, including infectious diseases. Malaria, which was very widespread in thepast and stillposesaproblem in many countries today, is a good candidatefor research. The association between malaria and glucose-6-phosphate dehydrogenase (G6PD) deficiency is well-known, but moreshouldbe done to determine the mechanisms responsible for thispositive correlation and to confirm that malaria is a strong selectivefactorfor many other genotypes also. The present paper refers to a WHOproject on genetic markers and susceptibility to infectious diseases, which isconcernedmainly with G6PDdeficiencyandthe following genetic markers: haemoglobinopathies, including the P-thalassaemia trait and ABO, Rh, MN, Duffy, secretory types (Ss), and human leukocyte antigens (HLA). Since malaria was eradicated in Bulgaria manyyears ago, human populationsfrom this country, living at different altitudes above sea-level, were used as a modelfor analysis ofthe malaria hypothesis. The data for G6PD deficiency confirm that malaria was a selective factor in lowland areas where malaria infection was more frequent in the past. It is, moreover, apparent that in addition to malaria some otherfactors also play a selective role. Genetic polymorphisms have considerable varia- bility in different human populations and in different races and ethnic groups (1). The most striking feature is that nearly all human gene loci are polymorphic. One of the two theories developed to explain such genetic diversity is that of natural selection. Admit- tedly, there are some arguments in support of the adaptive (selective) character of genetic polymor- phisms, but more studies are needed to determine the real effect of different factors, especially diseases, on human populations. Special attention should be given to infectious diseases (2) since, because they were widespread in the past and still are in many areas, they are a primary factor in shaping the human gene pool. At the present time, it is accepted that haemoglobin S plays an adaptive role in the case of Plasmodium falciparum infection (3, 4). A certain amount ofinfor- mation exists on the possible adaptive effect of glucose-6-phosphate dehydrogenase (G6PD) de- ficiency on malaria (5, 6), but many questions still remain unanswered. ' Head, Department of Medical Genetics, Medico-Biological Institute, Medical Academy, Sofia 1040, Bulgaria. 2 Human Genetics Unit, World Health Organization, Geneva, Switzerland. 3Scientist, Department of Medical Genetics, Medico-Biological Institute, Medical Academy, Sofia, Bulgaria. In the past, malaria was endemic in Bulgaria. For example, in 1946 when antimalaria programmes were started on a wide scale, morbidity from this infection had reached 20.7 per 1000. Malaria was most frequent in lowland, river areas but was unheard of in areas situated above 1000 m above sea-level.a Our earlier investigations showed the existence in some areas of a high frequency of G6PD deficiency-from 2.86/o to 7.66%o (7). Twelve percent of newborns were found to have severe hyperbilirubinaemia due to G6PD deficiency (8). In addition, many cases of favism and haemolytic anaemia of unclear etiology were reported. More than 10 variants oftheG6PD deficient gene were identified, two of them for the first time ever (7). These findings led to the initiation in Bulgaria of a WHO project on genetic markers and suscepti- bility to infectious diseases. Our research followed two main paths of investi- gation: (1) demographic analysis of human popu- lations living at different altitudes in regions with different morbidity of malaria in the past; (2) testing of populations for G6PD deficiency, haemoglobino- pathies, including the,-thalassaemia trait, and the a PULL, J. H. Rapport non publie sur une mission effectu& en Bulganie en vue de I'attestation de 1'eradication du paludisme, Copenhagen. WHO Regional Office for Europe 1965 (Report No. EURO-232, BULGARIE-9). 3986 -659- M. TZONEVA ET AL. following immunogenetic markers-ABO, Rh, MN, Duffy, secretory types (Ss), human leukocyte antigens (HLA). In this paper, data are presented on the frequency of G6PD deficiency according to altitude and its correlation with the P-thalassaemia trait. Various population groups selected from areas of different altitude are used as a model for analysis of the malaria hypothesis. MATERIALS AND METHODS A total of 9962 individuals, mainly aged 18 years and over, were tested for G6PD deficiency in five areas of Bulgaria: Plovdiv, Blagoevgrad, Smolyan, Vratsa, and Vidin. These areas had each been partially affected by malaria in the past. For the detection of G6PD deficiency, including identification of heterozygotes, we applied the methods of Brewer et al. (9), Toenz & Rossi (10), and also fluorescent screening (11). Of the 9962 indi- viduals, 1496 were tested for the P-thalassaemia trait. The quantity of haemoglobin A2 was determined by electrophoresis on acetate folia, and alkaline-resistant haemoglobin F was estimated by Singer's method (12), as modified by Betke et al. (13), and by the cyto- logical method of Pembrey et al. (14). Table 1. Relation between altitude and frequency of G6PD deficiency Group and Number of Total number altitude individuals with of individuals Frequency in metres G6PD deficiency tested for G6PD (%) 1:0-199 205 5739 3.57 II: 200-999 136 3944 3.45 111:1000+ 3 279 1.08 Totals 344 9962 3.45 For the purpose of analysis, the individuals were divided into three groups according to the altitude above sea-level of the towns and villages: Group I = 0-199 m; Group II = 200-999 m; Group III = 1000 m and over. Statistical analysis was done by the x2 method. RESULTS AND DISCUSSION The results are presented in Tables I and 2, where all the G6PD-deficient individuals are considered to- gether without being classified into hemizygotes, homozygotes, and heterozygotes. Table I shows a sig- nificant difference in the prevalence of G6PD de- ficiency in human populations living at an altitude of over 1000 m (x,1 = 7.87; 4V1 = 7.13; 0.001 <P<0.01), where it is three times lower than in those living below 1000 m. Of the 1496 individuals tested for both G6PD deficiency and the P-thalassaemia trait, 54 (3.61 07o) were found to have G6PD deficiency and 6 (0.40%7o) were carriers of the P-thalassaemia trait as well (see Table 2). The percentage of G6PD-deficient and thalassaemic persons in Group II (0.94%o) was higher than in the other two groups. The same tendency is found among individuals with G6PD deficiency, where the frequency of double carriers in group II is 33.30o. The data in Tables 1 and 2 suggest that several different genetic and environmental factors play a role in the accumulation of G6PD deficiency and of G6PD deficiency with the P-thalassaemia trait. Malaria can be considered as one example of a selective factor in lowland areas (below 1000 m) where this infection was prevalent in the past. The higher number of G6PD- deficient individuals in lowland areas (3.57%7o and 3.45%o) and the lower number in highland areas (1.0807o) confirms the malaria hypothesis. It is worth- while comparing our study with those carried out by Siniscalco et al. (6) and Piazza et al. (15) in Sardinia, Table 2. Relation between altitude and double carriers of G6PD deficiency and the P-thalassaemia trait IndivdualwithG6PDdeficency Individuals with G6PD deficiencyIndividuals ith defi ienc and the P-thalassaemia trait Total number of Group and individuals tested Percentage of all altitude for G6PD and the Number % Number % individuals with in metres P-thalassaemia trait G6PD deficiency 1: 0-199 886 38 4.29 1 0.11 2.63 II: 200-999 532 15 2.82 5 0.94 33.33 III: 1000+ 78 1 1.28 0 0 0 Totals 1496 54 3.61 6 0.40 11.11 660 G6PD DEFICIENCY AND MALARIA 661 where similar health problems of G6PD deficiency were encountered. They concluded that the G6PD- deficient gene was frequent in the lowlands and absent or decreased in the highland villages. When considering our data on the correlation between the frequency of double carriers of G6PD deficiency and the P-thalassaemia trait in relation to altitude, it is important to remember that in persons with G6PD deficiency the P-thalassaemia gene provides some protection against favism (16). The Vicia fava bean which provokes favism in persons with G6PD deficiency is grown for human consump- tion in the lowland areas (below 1000 m) but not in the highlands. This provides a logical explanation for the high frequency of the (3-thalassaemia trait among G6PD-deficient individuals (33.3%) in lowland areas of Bulgaria. It is very likely that in human populations living at less than 1000 m above sea-level the P-thalassaemia trait protects G6PD-deficient persons against favism and therefore plays an adaptive role. ACKNOWLEDGEMENTS The authors are grateful to Mr J. P. Bansal and Mme P. Roze for their valuable help with the computer programming and to Miss M. Eddison for technical assistance. RDSUMt FREQUENCE DU DEFICIT EN GLUCOSE-6-PHOSPHATE-DESHYDROGENASE EN FONCTION DE L'ALTITUDE: HYPOTHESE RELATIVE AU PALUDISME Une etude a Wtt effectuee sur les populations humaines de cinq regions de la Bulgarie (Plovdiv, Blagoevgrad, Smolyan, Vratsa, Vidin), qui avaient toutes ett partiellement impalu- dees dans le passe. II y avait eu differents taux d'infection paludeenne, celle-ci ayant sevi particulierement dans les regions basses, alors que les regions situ&es a 1000 metres ou plus au-dessus du niveau de la mer etaient totalement indemnes de la maladie. Les populations humaines choisies pour I'etude ont ett divis&es en trois groupes selon l'altitude: groupe I, 0-199 m; groupe II, 200-999 m; et groupe III, 1000 m et plus. Au total, on a recherche le d6ficit en G6PD chez 9962 individus et 1496 d'entre eux ont 6galement 6t6 eprouves en ce qui concerne le trait P-thalass6mique. II a &tt observe que la frequence du d6ficit en G6PD etait plus faible parmi les populations vivant dans les zones elevees, A 1000 metres ou plus au-dessus du niveau de la mer; les pourcentages de sujets deficients en G6PD, dans les plaines, etaient de 3,57% (groupe I) et 3,45% (groupe II), alors que pour les rtgions d'altitude le chiffre 6tait de 1,08% (groupe III). Cela confirme que le paludisme peut etre consider6 comme un exemple de facteur selectif dans les regions au-dessous de 1000 metres, ou cette affection sevissait dans le passe. La frequence des porteurs doubles de deficit en G6PD et du trait ,B-thalassemique 6tait en moyenne de 0,401., mais elle atteignait jusqu'a 0,94% dans le groupe II. La frtquence elevee de tels cas peut s'expliquer par le r6le protecteur que joue le gene ,-thalass6mique contre le favisme chez les personnes atteintes de deficit en G6PD dans les zones ob Viciafava est cultive en vue de la consommation humaine. REFERENCES 1. MOURANT, A. E. ET AL. The distribution of the human blood groups and other polymorphisms, London, Oxford University Press, 1976, p. 1055. 2. HALDANE, J. B. S. Natural selection in man. Progress in medical genetics, 1: 27-37, (1961). 3. ALLISON, A. C. Protection afforded by sickle-cell trait against subtertial malarial infection. British medical journal, 1: 290-294 (1954). 4. WHO Technical Report Series, No. 338, 1972 (Haemo- globinopathies and allied disorders: Report of a WHO Scientific Group). 5. LuzzArro, L. Genetic factors in malaria. Bulletin of the World Health Organization, 50: 195-202 (1974). 6. SINISCALCO, M. ET AL. Population genetics of haemo- globin variants, thalassaemia and glucose-6-phosphate dehydrogenase deficiency. Bulletin ofthe WorldHealth Organization, 34: 379-393 (1966). 7. TZONEVA, M. ET AL. [Incidence and polymorphism of glucose-6-phosphate dehydrogenase deficiency in some Bulgarian districts.] (in Bulgarian) Savremenna medicina, 29(10-11): 12-16 (1978). 8. TZONEVA, M. ET AL. Neonatal hyperbilirubinemia and glucose-6-phosphate dehydrogenase deficiency. Pediatria, No. 4, pp. 279-284 (1975). 662 M. TZONEVA ET AL. 9. BREWER, G. J. ET AL. Methemoglobin reduction test: new, simple, in vitro test for identifying primaquine sensitivity. Bulletin of the World Health Organization, 22: 633-640 (1960). 10. TOENZ, 0. & Rossi, E. Morphological demonstration of two red cell populations in human females hetero- zygous for glucose-6-phosphate dehydrogenase deficiency. Nature (London), 202: 606-607 (1964). 11. BEUTLER, E. & MITCHELL, M. Special modifications of the fluorescent screening method for glucose-6-phos- phate dehydrogenase deficiency. Blood, 32: 816-818 (1968). 12. SINGER, K. ET AL. Studies on abnormal haemoglobins. 1. Their demonstration in sickle cell anemia and other hematologic disorders by means of alkali denaturation. Blood, 6: 413-428 (1951). 13. BETKE, K. ET AL. Estimation of small percentages of foetal haemoglobin. Nature (London), 184: 1877-1878 (1959). 14. PEMBREY, M. ET AL. Reliable routine estimation of small amounts of foetal haemoglobin by alkali denaturation. Journal of clinical pathology, 25: 738-740 (1972). 15. PIAZZA, A. ET AL. HL-A variation in four Sardinian villages under differential selective pressure by malaria. Histocompatibility Testing 1972, Copenhagen, Munks- gaard, 1972, pp. 73-84. 16. BOTTINI, E. ET AL. On the relation between malaria and G6PD deficiency. Journal of medical genetics, 15: 363-365 (1978).