Molecular basis of G6PD deficiency Dr Narazah Mohd Yusoff Director, Human Genome Center, USM

Introduction G6PD deficiency – one of the most common inherited disorders, 400 million people affected –tropical and subtropical countries Epidemiological and in vitro studies - selection advantage during Plasmodium falciparum infection Most affected individuals asymptomatic, however risk of acute haemolysis

Genetics 1989 - more than 400 variants of G6PD Different biochemical forms of the enzyme exhibited, distinguished by different electrophoretic mobility, kinetic properties. Variants divided 5 classes according to the residual enzyme activity based (WHO). Mediterranean and African (A-) variants - by far the most clinically significant. Enzyme activity scarcely detectable in the Mediterranean type but close to normal in the African variant.

Genetics Thus, latter variant is considered to be the less severe form G6PD deficiency More recently, the dev.- new gene cloning techniques and complementary DNA sequencing techniques - identify the precise mutation of variants Many variants previously thought to be unique have proved to be identical.

Genetics Majority of the variants - from a single point-mutation resulting in amino acid substitution in gene encoding for G6PD located at the Xq28 region on the tip of the long arm of the X- chromosome.

The G6PD gene

TABLE 1 Molecular data on human G6PD DNA Size of gene (in kilobases) 18.5 Number of exons 13 Number of introns 12 mRNA Size (in nucleotides) 2269 Protein Number of amino acids 515 Molecular weight (in Daltons) 59,265 Subunits per molecule of active enzyme 2 or 4

Genetics G6PD deficiency - genetically heterogeneous condition. This explains why G6PD deficiency is predominantly a male syndrome. Males possess only one copy of the gene, thus they are either normal or G6PD deficient However, females - can be either normal, heterozygous or homozygous It is often quite difficult to differentiate between them based on the phenotypic expression alone

Genetics Heterozygous females with G6PD deficiency - two red blood cells populations, one normal and one that enzyme deficient. One copy of the gene governs the synthesis of the normal G6PD and the other makes the variant. In the early embryonic stage of life one of the two X-chromosomes is inactivated, hence each cell afterwards synthesise only either the normal or the deficient enzyme

Genetics This phenomenon - insight into why female heterozygotes appear to have a greater resistance against P.falciparum malaria than do deficient male hemizygotes. The growth of malarial parasites - impaired upon first passage from normal to G6PD deficient RBC. Through subsequent passages, they could adapt and grow normally though by inducing their own production of G6PD to protect it against oxidative damage.

Genetics In heterozygous G6PD deficient females however, such adaptation is not possible due to the presence of a mosaic of deficient and normal red blood cells, thus the protection against malaria

G6PD and its resistance to malaria Interesting - the incidence of G6PD deficiency - relatively high in places where P. falciparum malaria has been a life-threatening factor for centuries, as in the Mediterranean but rare elsewhere. This distribution - typical of other main genetically controlled traits, e.g. sickle cell anaemia, thalassaemia, HbE and persistent foetal haemoglobin (HbF). Apparently, - a positive biological selective pressure for the emergence of these mutations

G6PD and its resistance to malaria Several attempts -made to associate other conditions with G6PD deficiency based on the study of genetic locations. Many other genes located on X-chromosome besides the G6PD gene. By examining genes located close to the Xq28 region, a possible linkage with the expression of G6PD enzyme can be postulated.

Genetics E.g the proximity of genes for red and green pigments to Xq28 has lead to the suggestion of a linkage between G6PD deficiency and congenital colour blindness by Yucel. A study was carried out in Turkey to test the hypothesis. It was found that that none of the colour blind subjects were G6PD deficient and vice versa.

G6PD and its resistance to malaria Based on the results obtained Yucel concluded that there was a linkage disequilibrium between congenital colour blindness and G6PD deficiency, which may be due to crossing over of the closely situated genes during meiosis Nevertheless, this does not preclude conclusively the possibility of a linkage between G6PD deficiency and other clinically significant conditions.

G6PD and its resistance to malaria The answer - the malarial parasites which show a surprisingly high susceptibility to oxidative stress require glutathione and the products of the G6PD oxidative shunt for optimal growth. Not unlike the sickle cell trait which utilises an independent mechanism of protection against malaria, G6PD deficiency creates an inhospitable environment for the malarial parasites and discourage lodging of the protozoa in the red blood cells

Summary G6PD deficiency – commonest enzymopathy Varied clinical manifestations Genetic heterogeneity Malaria protection Public health problem