BIOCHEMICAL AND MOLECULAR ASPECTS OF GLUCOSE-6-PHOSPHATE DEHYDROGENASE DEFICIENCY IN MAN

Abstract

The red cell Glucose-6-Phosphate Dehydrogenase (G6PD) is a highly polymorphic X-linked enzyme, with about 300 variants reported worldwide. This enzyme deficiency has a widespread prevalence in Southern Asia with a total of 78 variants reported, 30 of which are common variants with at least 13 polymorphic in nature. There have been no studies on the biochemical characterization of red cell G6PD in Singapore. In this study, about 10,000 cord blood samples from various hospitals in Singapore were screened for G6PD enzyme activity and electrophoretic mobility using starch gel electrophoresis in Tris-EDTA-Boric Acid (TEB) buffer (pH 8.6) system. About 200 variant samples (either deficient or electrophoretic or both) were obtained and of these, only 69 were partially purified using DEAE-Cellulose liquid chromatography before biochemical characterization was carried out to determine the level of red cell enzyme activity, relative electrophoretic mobilities, Km for G6P and NADP, relative rates of utilization of analogues of G6P and NADP (2dG6P, Gal6P and deamino NADP), heat stability and pH optimum. Wherever possible, a family study of this X-linked inheritance was also carried out. Of these 69 fully characterized variant samples, 58% belonged to Class 2 deficiency (less than 10% of normal activity), 16% to Class 3 (10-60%) and 26% to Class 4 with 60-150% activity. According to electrophoretic mobility, 28% were classified as fast (>100% of normal), 49% as normal (or 100%) and 17% as slow (<100% of normal) variants. Twenty-eight out of these 69 (or 41%) variant samples were from Chinese, 21 or 30% belonged to Malays and 20 or 29% were from Indians. Based on the above classification, a total of 21 (3 new and 18 reported) variants were identified from the 28 Chinese variant samples investigated, 11 (2 new. and 9 reported) variants were found from the 21 Malay variant samples while 12 (3 new and 9 reported) were identified from the 20 Indian variant samples. Hence, a total of 37 G6PD variants (7 new while 30 reported) have been identified from the 69 variant samples investigated. This G6PD locus is therefore tremendously heterogeneous in Singapore. It is possible that some of these variants were first introduced into this country in that early days of colonization as a result of population migration from other regions and the malaria endemic environment then helped to preserve these presumably protective mutants against falciparum malaria. Seven new variants were identified from these 69 variant samples investigated. One of these new variants, named G6PD Singapore, was found in a Malay family and 3 unrelated Indian females and was noticed to be similar to G6PD A in terms of its biochemical properties : both are non-deficient fast variants, with G6PD Singapore migrating at a faster rate at 140% as compared to 110% of G6PD A in TEB starch gel electrophoresis. G6PD A is found in about 20% of the African populations. Recent studies have identified a point mutation at nucleotide 376 (exon 5), with A -> G transition that creates an additional Fok I site, as the cause of this rapid anodal electrophoretic mobility characteristic of G6PD A. Another mutation at nucleotide 202 (exon 4), with G-> A transition that creates an additional Nla III site, was also reported in some G6PD A-, a deficient fast variant found in about 12% of Africans. This prompted us to study G6PD Singapore closely at the molecular level to determine its relationship with G6PD A and G6PD A-. DNAs of variant G6PD Singapore were subjected to Polymerase Chain Reaction using specific sets of primers to amplify exon 4 (109 bp fragment) and 5 (97 bp fragment for par-t of the exon and 301 bp fragment for the entire exon 5) of the gene. These amplified DNAs were then restricted with Fok I and Nla III for both 97 bp and 109 bp fragments, and Rsa I, Hae II, Hae III and Hha I for the 301 bp fragment. Since G6PD Singapore resembles closely to G6PD A in terms of biochemical properties, a similar point mutation at nucleotide 376 was suspected as in the A type variant. However, when subjected to restriction digestion, amplified DNAs of both the Malay mother and son as well as one of the three Indian samples showed neither cleavage at exon 5 (97 bp fragment) by Fok I nor at exon 4 (109 bp fragment) by Nla III. But, both Fok I and Nla III were able to digest the 109 bp and 97 bp fragments respectively in the normal G6PD B+. This implies the absence of point mutations A->G and G-> A at both nucleotide 376 and nucleotide 202 in G6PD Singapore respectively. The amplified 301 bp fragment from the Malay son, when restricted with Rsa I, Hae II, Hae III and Hha I enzymes, showed that only Hae III was able to digest it but not the other restriction enzymes as in the normal G6PD B+, indicating that exon 5 of G6PD Singapore is not normal G6PD B+ either. Our results, therefore, suggest that mutation(s) that results in the very fast anodal electrophoretic mobility of G6PD Singapore may also lie possibly on the exon 5, and that mutation at nucleotide 376 may not be the only transition that results in the faster-than-normal B+ anodal electrophoretic mobility. The very fast anodal electrophoretic migration of G6PD Singapore may be indeed genetically distinct from that of G6PD A as a result of mutation(s) elsewhere in the gene, possibly on exon 5.