| dc.description.abstract | A study of human populations has provided fascinating insights into modes of genetic inheritance. The populations, made up of different endogamous groups on the basis of socioeconomic, religious and ethnic differences, have intermingled sometimes deliberately and often unintentionally. Genetic markers have provided clues to these processes. Environmental factors have played a major role in the selection and perpetuation of certain genetic traits. A major fascination of human genetics has been the study of inbreeding. Although several societies have frowned upon the practice of inbreeding, it is practised by a fairly large percentage of human populations. Although inbreeding has decreased or almost disappeared from Western societies, it is still practised in many parts of Asia and Africa.
The South Indian population has a long tradition of inbreeding, especially in the states of Karnataka, Andhra Pradesh, Tamil Nadu and parts of Kerala. The accepted marital unions are uncle–niece, I cousins and II cousins (Fig. 1). A large scale study carried out in this laboratory showed that inbreeding is still a prevalent practice in Bangalore, as more than 30% of the 1,07,000 newborn children examined were born to consanguineously related couples (Table 1).
Despite the presence of this high proportion of consanguineous couples, there was no alarmingly high incidence of autosomal recessive disorders of amino acid metabolism or increased mortality or morbidity compared to outbred populations. However, in a study of sick children, it was seen that inbreeding had an effect on the expression of autosomal recessive genes. These studies pointed out that the predicted “cleansing of the gene pool” had not occurred and genetic disorders were still prevalent in the population.
It was also suggested that the matrilineal inbreeding practised by this population could have increased the homozygosity at the X linked loci. The high proportion of males in the inbreeding populations could also affect the expression of X linked disorders. G6PD, a well characterized enzyme coded by a gene on the X chromosome, could be a good marker to test these hypotheses.
G6PD, which catalyses the reduction of NADP, has several functions in the red cell. One of them is the maintenance of membrane integrity. Loss of this function results in fragility of red cells and anemia. Decreased levels of the enzyme have been correlated with increased selection against malaria in populations, as the parasite uses this enzyme during one stage of its life cycle. Due to these multiple functions, several polymorphic forms of the enzyme have been recorded (Table 2).
The major forms of the enzyme are the A and B types. The variant A form is present in African populations, while the B form in Mediterranean populations is associated with favism. The polymorphic forms were identified by changes in electrophoretic mobility and RFLP patterns (Tables 2–4). More than 400 variants differing in mobility, reflecting alterations in structure, kinetic properties, and gene sequence changes have been reported.
The gene for G6PD was identified on the X chromosome by linkage studies and later located at Xq28 by cloning. The gene consists of 13 exons coding for a 1.2 kb mRNA. Methylation was identified as a regulator of its expression. The hypothesis that part of the N terminus was coded by an autosome was short lived.
The aim of the present investigation was to determine whether inbreeding in the South Indian population has resulted in an increased incidence of G6PD deficiency. It was also of interest to determine the incidence of deficiency in view of the population’s historical exposure to malaria. Identifying the nature of the deficiency and changes in phenotype was another important aim.
The materials and methods used-enzyme purification from normal and deficient blood samples, CAM electrophoresis, starch gel, PAGE, SDS PAGE, N terminal sequencing (Figs. 4, 5), kinetic analysis, Western and Southern blotting, RNA Dot blot, ELISA, etc.-are briefly described.
Blood spots from 5140 newborns were used to determine G6PD levels by CAM electrophoresis (Fig. 7). Samples were selected from those collected for amino acidopathy screening (Fig. 2). There was no sampling bias, as indicated by similar consanguinity profiles (Table 6) and sex ratios.
Approximately 1500 samples were screened each year (Fig. 6). The proportions of Muslims and Christians and the consanguinity profiles were similar to the general population (Tables 1, 8).
Among the 5140 neonates screened, 405 were deficient based on enzyme activity <3 IU, representing 7.8% of the total (Table 7).
The consanguinity profile of the G6PD deficient group (Table 8) was similar to the general newborn population (Table 1). The F values were 0.037 for normal, 0.038 for deficient, and 0.029 for general newborns. The reported F value for the South Indian population is 0.032. This suggests that consanguinity may not have played a major role in the higher incidence of G6PD deficiency observed.
Higher incidence in Middle East Muslims and Kurdish Jews is known. Since Muslim groups in India originated largely from conversions rather than migration, the religious profile of deficiency was examined. No significant increase was seen among Muslims (Tables 9 and 10). Consanguinity did not influence deficiency within religious groups.
A major finding was the high proportion of females classified as deficient based on enzyme analysis (Table 7). This would suggest that the phenotype does not truly reflect genotype.
Careful experimentation ruled out inhibitors. Mixing lysates (Table 11) and partially purifying both normal and variant enzyme preparations (Fig. 9, Table 12) confirmed this.
As recommended by WHO, detailed kinetic studies were carried out with partially purified enzymes. The Km values for G6P and NADP, Ki for NADPH (Figs. 16, 17), and substrate specificity with 2dG6P were not significantly altered between normal and deficient enzymes. No kinetic differences existed between males and females either (Tables 13 and 14). However, the values were decreased, suggesting lower enzyme amounts or catalytic efficiency. Dosage compensation appeared intact.
Electrophoretic mobility and SDS PAGE patterns were identical for normal and variant enzymes (Figs. 7, 22, 27). pH optimum (8.2) was unchanged.
The enzyme from normal males and females was purified to homogeneity using DEAE Sephadex and ADP Sepharose (Table 15). Attempts to purify the variant enzyme failed, likely due to instability.
Antibodies to the purified enzyme were raised in rabbits (Fig. 22) and used to quantify protein levels by ELISA and Western blot (Figs. 28, 29).
Western blotting (Figs. 31, 32) showed that deficient individuals had ~2.25 fold lower G6PD protein. When equal activity was loaded, deficient samples required ~2.5 fold more protein to give equivalent activity. These results indicate decreased enzyme content and catalytic efficiency.
mRNA analysis (Table 19, Fig. 33) showed identical G6PD mRNA levels in normal and deficient samples, indicating that reduced protein levels arise from decreased enzyme stability, not reduced transcription.
Family studies (25 families) showed classical X linked inheritance in some (Figs. 34, 36, 37), incomplete penetrance in others (Figs. 38, 40), and unusual pedigrees (Figs. 41–44).
Southern blotting using PstI and PvuII revealed patterns different from African reports (Fig. 35), suggesting population specific mutations.
In conclusion, G6PD deficiency occurs at ~8% frequency in this South Indian population. The high incidence is not related to consanguinity or religion. The deficiency results from reduced enzyme stability and catalytic efficiency, not from altered Km values or mRNA levels. | |
