G6PD Deficiency, Distribution and Variants in Saudi Arabia: An Overview

Introduction

Glucose-6-phosphate dehydrogenase (G6PD; EC 1.1.1.49) is the first enzyme of the hexose monophosphate shunt and catalyzes the conversion of glucose-6-phosphate to 6-phosphogluconolactone, with the concomitant reduction of NADP⁺ to NADPH.^1–3 The gene for G6PD is located on the long arm of the X-chromosome and is inherited as an X-linked recessive trait.⁴ The G6PD gene is highly polymorphic, and over 300 variants resulting from single-point mutations are known to exist in the different populations of the world.⁵ Several variants have significantly reduced activity and result in a condition referred to as “G6PD deficiency.”^3–6 The state is inherited as an X-linked recessive state, and presents one of the most frequently encountered red-cell enzymopathies. The major interest in the G6PD-deficient state results from the associated hemolytic anemia resulting from oxidative stress.⁶

The first report of G6PD deficiency in Saudi Arabia dates back over three decades when, in 1965, Gelpi⁷ reported the presence of this state in different villages in the Eastern Province of Saudi Arabia. In the following years, reports of G6PD deficiency in other provinces of Saudi Arabia were published,^8–21 and showed the occurrence of G6PD deficiency at a high though variable frequency. We conducted a nationwide study in different regions in an attempt to investigate the distribution pattern in different parts of the Kingdom. This paper summarizes our findings on the distribution and molecular aspects of G6PD deficiency in Saudis.

Materials and Methods

The study group included randomly selected males and females living in different provinces of Saudi Arabia, with ages ranging from 2–70 years. There were 13,796 males and 13,613 females, from whom 5.0 mL blood samples were collected by venipuncture in acid-citrate dextrose (ACD) tubes. The whole blood was used to determine the hematological parameters using Coulter Counter ZF6, with a hemoglobinometer attachment. The blood was centrifuged to separate the plasma from the cells. The red cells taken from the bottom of the tube were hemolyzed with cold distilled water and the fresh hemolysate was used to determine the G6PD activity spectrophotometrically, using kits from Boehringer Mannheim Diagnostica. The G6PD unit was defined as millimoles of substrate converted to product per minute at the specified pH and temperature. The activity was expressed as mU/10⁹ erythrocytes.⁵

Another sample of the fresh hemolysate was subjected to cellulose acetate electrophoresis using Titan III Plates (Helena Cat. No. 3023) and Supra Heme buffer at pH 8.6 for 20 min. at 350V (Helena Cat. No. 5802). Visualization of G6PD band was carried out by specific staining for G6PD using G6PD staining reagent (Helena Cat. No. 5620) for 20 min. at 20°C. After color development, the plates were fixed for 2–3 min. in 7.5% trichloroacetic acid, washed with 5% acetic acid, dried in air and stored. Scanning was carried our before fixing, using a Quick Scan Densitometer (Helena).

Results

A wide range of G6PD activity was encountered in the male and female samples. The normal reference range of G6PD was used as 60–130 mU/10⁹ erythrocytes, with a mean of 95 for Saudi males, and 60–140 mU/10⁹ erythrocytes with a mean of 100 mU/10⁹ erythrocytes for Saudi females.²³ Individuals with severe G6PD deficiency were considered as those with G6PD activity <20% of the lower normal. Of the total 13,796 males, 1249 were deficient and among the 13,613 females screened, 558 were deficient, giving a frequency of G6PD deficiency of 0.0905 and 0.041, respectively, in the males and females. The samples from different provinces were separated and the frequency of G6PD deficiency was calculated in each province. The results are presented in Table 1. The Eastern Province had the highest frequency of G6PD deficiency, while the Northern Province had the lowest. Within each province the samples were further separated on the basis of different areas and frequency of G6PD deficiency calculated. Table 2 presents the results and shows significantly variable frequency in different areas. The phenotyping showed the normal enzyme as G6PD-B⁺ and four of its variants: 1) G6PD-Mediterranean, which was most severely deficient, with activity ranging from 10.5±4.6 mU/10⁹ erythrocytes. It had a similar mobility to G6PD-B⁺, but in most cases it was very faintly visible or invisible: 2) G6PD-A⁺ moved faster and had almost normal activity (81.3±15 mU/10⁹ erythrocytes); 3) G6PD-A⁻ moved at the same place/pace as G6PD-A⁺ but had low activity (22.6±10.6 mU/10⁹ erythrocytes); and 4) a variant with the same mobility as G6PD-B⁺, but activity ranging between 20%–60% of normal (37.5±8.1 mU/10⁹ erythrocytes) was also identified. The frequency of the different variants in the various provinces and total frequency are presented in Table 3. In all provinces, the G6PD-B⁺ was the normal enzyme, and the most frequently encountered severely deficient variant was G6PD-Med, while G6PD-A⁺ and A⁻ occurred at low frequency.

Discussion

This study is a comprehensive investigation conducted over almost 10 years to determine the frequency of G6PD deficiency and its variants in Saudis. The activity of G6PD was determined spectrophotometrically using mature red cells obtained from the bottom of the tube containing the red-cell sediment after centrifugation. This step is essential in order to avoid using reticulocytes and young red cells, which are usually rich in G6PD levels, and hence can mask G6PD deficiency. All samples with G6PD activity less than 15 mU/10⁹ RBC were classified as severely deficient. On electrophoresis, either no G6PD band was seen for these samples or a very faint band was observed. Partially deficient variants had between 12–35 mU/10⁹ RBC.

The data generated during this study showed that G6PD deficiency occurs in all provinces of Saudi Arabia, though at a variable frequency. In the Eastern Province, both males and females have the highest frequency of G6PD deficiency, followed by those in the Southwestern Province. The number of deficient females was more than the number calculated using Hardy-Weinberg equilibrium. This is believed to be due to a high rate of consanguinity in Saudis,²⁴ or a higher inactivation of normal X-chromosome in heterozygous females, thus increasing the number of deficient females and disturbing the Hardy-Weinberg equilibrium.

The normal G6PD, as in every population of the world investigated to date, is G6PD-B⁺ in the Saudis in each region, but significant interregional variations are obvious in its frequency.²⁵ In addition, the frequency of G6PD-B⁺ differs in males and females. The other normal variant found in Saudis is G6PD-A⁺, an African variant. This is a variant with a higher mobility than G6PD-B⁺, but the activity is about 90% of the normal. It occurs in Saudis but at a nonpolymorphic level in most areas. G6PD-A⁻, a deficient African variant with the same mobility as G6PD-A⁺, is also found in Saudis, but at a lower prevalence.

G6PD-Mediterranean was the most frequently encountered deficient variant in all areas of Saudi Arabia. This is the variant frequently encountered in the countries around the Mediterranean and produces a severe deficiency, resulting in hemolytic anemia under oxidative stress. It is also the variant producing favism. A variant with the same mobility as G6PD-B⁺, but activity between 20%–60%, is encountered at a high frequency in some areas and needs to be further characterized.

The frequency of G6PD deficiency was correlated with past or present history of malaria endemicity.²⁶ In each area of Saudi Arabia where malaria had been encountered in the past or was presently endemic, the frequency of G6PD deficiency was high. This strongly supports the malaria hypothesis.^27–31

In conclusion, G6PD deficiency occurs frequently in several areas of Saudi Arabia, and G6PD-Mediterranean is the most frequently encountered variant producing severe G6PD deficiency. The frequency correlates with malaria endemicity, and thus it may provide a natural protection against malaria, as is the situation in several populations of the world where sickle cell gene (HbS), α- and β-thalassemias and G6PD deficiency provide a strong inborn resistance against malaria.