Glucose-6-phosphate dehydrogenase deficiency allelic variants and their prevalence in malaria patients in Eritrea

Introduction Glucose 6-phosphate dehydrogenase (G6PD) deficiency is the most common enzymopathy with a relatively high frequency in malaria-endemic regions. In Eritrea, there is scanty knowledge of G6PD deficiency. The aim of the study was to characterize and determine the prevalence of four common G6PD allelic variants. Methods Three hundred and fourteen dried blood spot samples from unrelated microscopically diagnosed malaria patient Eritrean ethnic groups living in five zobas (regions) of Eritrea were analysed by PCR-RFLP method to identify the G6PD B, G6PD A (A376G), G6PD A-(G202A), and G6PD Mediterranean (C563T) variants. To confirm the RFLP results, samples positive for A376G but negative for G202A variants were subjected to Sanger sequencing and a subset of PCR products (exon 5) directly sequenced to identify A376G and other mutations. Results For G6PD genotyping, G6PD B was detected in 87.5% and A376G detected in 12.5% of malaria patients, whereas G202A and C563T were absent. Bivariate Statistical analysis showed a statistically significant association between G6PD genotypes and zoba (P < 0.004 < 0.05). Sequencing revealed the expected A376G variant. In exon 5, four common (A376G) mutations, three uncommon mutations rs782669677 (535G→A) and one potentially new mutation (451G→C), relative to the reference, mRNA NM_001042351 were detected. Bioinformatic analysis of these mutations' potential functional impact suggests minimal effect on protein function. Conclusion This is the first report indicating that G6PD B and G6PD A genotypes are prevalent in Eritrea. Similar findings were reported in neighboring countries. Further studies including phenotype analysis are needed to corroborate the observed results.


Introduction
Malaria remains to be a public health concern with 429,000 reported associated deaths in 2015 in spite of the effort to control and eventually eliminate it has recorded huge success in the last decades worldwide [1]. Malaria is a public health concern in Eritrea although the country has scored tremendous achievements in its control over the past 15 years through intensive control interventions [2,3].
Eritrea is located in the horn of Africa, where 67% of its populations live in malaria risk areas [4]. Plasmodium falciparum is responsible for 67% of malaria cases and Plasmodium vivax for 30%; there is a small percentage of cases due to mixed infections [5]. Eritrea has an estimated population of 5.19 million people [6] with nine officially recognized ethnic groups (Afar, Bilen, Hidareb, Kunama, Nara, Rashida, Saho, Tigre and Tigrigna). Tigrigna is the largest ethnic group followed by the Tigre group, whereas Rashaida is the smallest ethnic group in the country. Administratively, the country is divided into six zobas (regions): Maekel, Anseba, Gash Barka, Debub, Semienawi Keyih Bahri (Northern Red Sea) and Debubawi Keyih Bahri (Southern Red Sea). Among these zobas, Debub, Semienawi Keyih Bahri, Anseba and Gash Barka are endemic to malaria. Malaria in the country is mostly seasonal and unstable. The disease is transmitted through the Anopheles arabiensis primary vector, which is difficult to control using conventional interventions method due to its facultative indoor and/or outdoor feeding and resting behavior [7]. G6PD is a cytosolic enzyme present in all cells [8] and functions in the body's defense against oxidant damage [9]. In red blood cells (RBCs), G6PD is the only source of NADPH cofactor, which is needed for generating a reduced glutathione (GSH), the major antioxidant defense. G6PD gene encoding the G6PD enzyme is located on X chromosome and is highly polymorphic with more than 400 variants, described based on biochemical diagnosis [10]. G6PD deficiency is the most prevalent enzyme deficiency in humans [11] present in over 400 million people worldwide [12] with an estimated allelic frequency of 8% across malaria endemic countries [13]. As X-linked disorder, the transmission pattern results in G6PD deficiency hemizygous males or homozygous females, whereas heterozygous females can be either normal or deficient in G6PD activity because of mosaicism [14].
Although most individuals with G6PD deficiency are clinically asymptomatic, this defect causes neonatal jaundice, mild hemolytic anemia and chronic non-spherocytic hemolytic anemia triggered by infections, specific foods (fava beans), or drugs [15]. On the other hand, G6PD deficiency has protective power against malaria infection although whether this advantage is exclusive to heterozygous female individuals, or it is shared by both sexes equally has been controversial [16,17]. A case-control study performed on the coast of Kenya showed that only girls who were heterozygotes for the G6PD c.202T (G202A) variants were significantly protected from severe and complicated Plasmodium falciparum malaria [18]. Recent studies also revealed an increasing levels of G6PD deficiency associated with decreasing risk of cerebral malaria, but with increased risk of severe malarial anaemia [19]. To date, more than 400 G6PD variants have been identified, of which 186 variants are associated with G6PD deficiency by decreasing the activity or stability of G6PD [9,20,21].
However, in sub-Saharan Africa three variants occur with polymorphic frequencies (> 0.1%): G6PD*B, G6PD*A and G6PD*A-. G6PD*B is the wild type and the most common variant in Africa and worldwide.
G6PD*A has a single A→G substitution at nucleotide number 376. It is a normal variant with about 90% of the G6PD*B enzyme activity [22]. G6PD*A-is a deficient variant with about 8-20% of the wild type enzyme activity and caused by the A376G and G202A mutations [23].
G6PD Mediterranean variant is caused by the C563T mutation with less than 10% enzyme activity and found in Italy, Cyprus and in the Middle East [9]. In Eritrea, there is scant knowledge of G6PD deficiency. The Eritrean Government through its Ministry of Health is currently preparing pilot activities towards total elimination of malaria by 2030. Despite this, oxidative drugs and G6PD deficiency screenings are not part of the national policy for the treatment of Plasmodium falciparum and Plasmodium vivax infections in Eritrea. This study aimed at molecular characterization and determination of the prevalence of common G6PD variants, namely, B, A, Aand Mediterranean, in microscopically diagnosed malaria patient Eritrean ethnic groups.

Methods
Study sites and study population: this cross-sectional study was undertaken at 10 study sites located in five zobas of Eritrea ( Figure   1) between August and October 2016. Study sites were selected based on practical accessibility, on the availability of malaria cases and geography. The number of study participants enrolled from each study site was determined using probability proportional to size (PPS).
To limit double sampling of genetically related individuals, a single individual per family was recruited. Individuals born from parents of two different ethnic groups were excluded from this study.

Microscopic examination of malaria parasite: outpatients
visiting health stations/centres/hospitals and those clinically suspected to have malaria were diagnosed for Plasmodium species by microscopy. Thick and thin blood smears and dried filter paper blood spots (DBS) were made from a drop of finger-prick blood. After the slides were air-dried, the thin smear fixed in methanol and both smears were stained with 10% Giemsa for 15 minutes. The stained slides were subsequently air-dried and viewed under 100X oil immersion microscope (Olympus microscope CX21, Tokyo, Japan). An experienced microscopist read the slides. Species identification was done by visualizing thin blood smear. Parasitaemia was determined as the percent of malaria parasites observed per 200 white blood cells [24].   showing positive for A375G, but negative for G202A variants were further analyzed by DNA sequencing in one direction [26]. A subset of PCR products of exon 5 of the G6PD gene was also directly sequenced to identify A376G and other mutations. PCR products of G6PD exons, 3+4 and 5 were amplified using forward primers previously described in Tishkoff et al. [25]. PCR thermal cycling protocol was performed as previously described in Promega Madison, USA. PCR products were purified using ExoSap enzymes (USB, Affymetrix, USA) and subjected to cycle sequencing using Big Dye Phylogenetic analysis: phylogenetic analysis was performed using the nucleotide sequences obtained from G6PD exon 5 PCR products.
The nucleotide sequences were first manually edited by removing ambiguous sequences at the ends and generating reverse complements, which were then aligned using a Geneious version 11.1 [27]. Human G6PD exon 5 sequence was extracted from

Results
To characterize and determine the prevalence of common G6PD  [28]. The prevalence of G6PD A variant by sex was 11.9% in females and 13.1% in males in this study, but these difference are not statistically significant (P > 0.05). The higher prevalence of G6PD deficiency in male is consistent with other studies conducted in Afghanistan [29] and Solomon Islands [30]. The male predominance of G6PD deficiency can be attributed to the X-linked inheritance in G6PD gene [9]. The prevalence of the A376G variant in terms of frequency among the ethnic groups ranged from 0.00% to 21.4%.
Similar results were reported from a study done by G6PD fluorescent spot test among unrelated males of Karen (4.1%) and Burman (12.9%) ethnic groups in a malaria endemic region on the Thailand-Myanmar border [31] and in five ethnic groups in Pakistan [32]. In this study, higher G6PD A (A376G) variant prevalence was found to be associated with individuals residing in zoba Debub (28.3%) followed by zoba Gash Barka (12.4%) and these differences were statistically significant (P < 0.004 < 0.05) which is in agreement with previous findings in Ethiopia and Nepal [33,34]. Notably, the 8.3% of malaria patients with the A376G variant recorded in zoba Maekel, which is considered malaria free zone of Eritrea. These individuals who are mainly Tigrigna and Tigre ethnic groups resided and/or temporarily travelled to different zobas, and they had come for their medical treatment. The frequency of G6PD did differ by zoba (region) due to differing ethnic make-up of each zoba. As conclusion, the frequency of G6PD deficiency differed across the world depending on the ethnic groups and regions [13][14][15]35].

Considerable evidences showed that in African populations the G6PD
A-(G202A/A376G) mutations is exclusively the cause of G6PD deficiency. Few studies reported G6PD A-(202GA) frequency estimates considerably lower than those generally found in sub-Saharan Africa [10] and confirmed by a recent geostatistical modelbased map that predicted a 1.0% prevalence [15]. The prevalence G202A deficient variant was zero (0.0%) in this study, which is in line with the study findings in Ethiopia [28,36] and Northern Sudan [37].
In contrast, the frequency of G6PD Adeficient variant was higher in Uganda (20.41%) and in Mali (7.9%) respectively [38,39]. The absence of G6PD Amutation in this study could be due to geographical location and the molecular heterogeneity of G6PD deficiency among different populations [40]. G6PD Mediterranean variant is primarily found in populations within the Mediterranean region [41,42]. The prevalence of the G6PD Mediterranean (C563T) mutation in terms of frequency was zero (0.00%) in this study, which is in accordance with studies done in Angola [43] and in Ethiopia [28].
In contrast, Mediterranean mutation is the second most common protein function [28].

Conclusion
This study is the first report on molecular characterization and the  G6PD deficiency is the most common in malaria endemic areas and has a protective power against malaria.