Malaria Journal
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Lwanira et al. Malar J (2017) 16:322 DOI 10.1186/s12936-017-1970-1 Malaria Journal RESEARCH Open Access Prevalence of polymorphisms in glucose‑6‑phosphate dehydrogenase, sickle haemoglobin and nitric oxide synthase genes and their relationship with incidence of uncomplicated malaria in Iganga, Uganda Catherine Nassozi Lwanira1†, Fred Kironde2*† , Mark Kaddumukasa3 and Göte Swedberg4 Abstract Background: Host genetics play an important role in Plasmodium falciparum malaria susceptibility. However, information on host genetic factors and their relationships with malaria in the vaccine trial site of Iganga, Uganda is limited. The main objective of this study was to determine the prevalence of selected host genetic markers and their relationship to malaria incidence in the vaccine trial site of Iganga, Uganda. In a 1-year longitudinal cohort study, 423 children aged below 9 years were recruited and their malaria episodes were investigated. Host genetic polymor- phisms were assessed by PCR–RFLP, haemoglobin electrophoresis and DNA sequencing. Using a multivariate negative binomial regression model, estimates of the impact of human genetic polymorphisms on malaria incidence were performed. In all statistical tests, a P value of <0.05 was considered as signifcant. Results: The prevalences of sickle cell haemoglobin trait, G6PD c.202 G>A (rs 1050828) and NOS2 954 G>C (rs 1800482) variants were 26.6, 22.7 and 17.3%, respectively. Inducible nitric oxide synthase 2 (NOS2 −954 G>C; rs 1800482) heterozygosity was associated with lower incidence of malaria in all age groups {Adjusted− incident rates ratio (aIRR) 0.59; 95% CI [0.386–0.887]; P 0.012)}. About 4% of study subjects had co-existence of sickle cell Hb trait and G6PD defciency. Sickle cell Hb =heterozygotes (Hb AS) aged less than 1 year experienced signifcantly more malaria episodes annually than children with normal haemoglobin (Hb AA) {aIRR 1.98; 95% CI [1.240–3.175]; P 0.004}. There was no signifcant infuence of the sickle cell trait on malaria incidence= among older children of 1–9= years. Conclusions: Mutation (NOS2 954 G>C; rs 1800482) of nitric oxide synthase 2 gene promoter was associated with a lower incidence of acute malaria.− The normal haemoglobin (wild genotype; HbAA) was associated with reduced malaria incidence rates during the frst year of life. More understanding of the interplay between host genetics and malaria susceptibility is required. Keywords: Human gene polymorphisms, Plasmodium falciparum malaria, Incidence *Correspondence: faskironde@gmail.com †Catherine Nassozi Lwanira and Fred Kironde are joint-frst authors 2 Habib Medical School, Faculty of Health Sciences, Islamic University in Uganda (IUIU), Kampala Campus, Kampala, Uganda Full list of author information is available at the end of the article © The Author(s) 2017. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Lwanira et al. Malar J (2017) 16:322 Page 2 of 9 Background polymorphism (rs 9282799) with reduced malaria inci- Te infuence of human genetic factors on progression dence rates [20]. In other studies, NOS2 −954 G>C and severity of Plasmodium falciparum malaria has been polymorphism heterozygosity was associated with protec- extensively studied and a number of host genes have been tion against severe malaria [22], yet else where, no rela- suggested to confer specifc protection. However, the tion between NOS2 polymorphisms and susceptibility to sickle cell trait and glucose-6-phosphate dehydrogenase malaria [27] or asymptomatic malaria [28] was observed. (G6PD) defciency stand out among other host genetic In the present study, the associations between the inci- markers reported in genome wide association and mul- dence of uncomplicated malaria and HBB, G6PD and ticentre studies [1–3]. Various studies in African coun- NOS2 gene polymorphisms were determined in a 1 year tries have reported that sickle cell trait (HbAS) protects follow up study. Understanding the prevalence of these against severe malaria in 70–90% cases [4–6] and pre- genetic factors and their impact on malaria incidence is vents up to 75% of hospital admissions due to malaria important in providing baseline data of value in malaria [7]. In other studies, HbAS has been associated with vaccine trials and other malaria control interventions. reduced malaria incidence rates [6–9] and delayed onset of malaria [10]. More recently, larger association studies Methods have also consistently linked the sickle cell trait with pro- Participant recruitment tection against uncomplicated and severe malaria [3, 11]. A baseline survey was carried out and eligible children G6PD defciency afects over 400 million people glob- were enrolled into a cohort which was followed up for ally, 15-30% of whom are found in sub Saharan Africa 1 year. At the study site in Iganga, malaria transmission [12, 13]. More than 160 G6PD genetic variants have been largely occurs throughout the year [29, 30], but with identifed including G6PD B (wild type), G6PD A (non- minor transmission peaks being observed following the defcient type) and G6PD A− (African defcient type); major rains which usually occur from April to June and however the most common and more frequently associ- from September to December. In this project, the study ated with G6PD defciency in sub Saharan Africa is the children were recruited during November and followed 376G/202A haplotype [13]. While the protective efect up for 1 year. Te inclusion criteria, detailed enrolment of the HbAS against malaria seems to be clearer, associa- and follow up are described in an earlier publication [31]. tions with G6PD defciency vary widely in the diferent Study personnel sought for verbal community consent studies, with protection observed in females [14, 15], in to participate in a brief demographic survey, and writ- males [16], in both [17] and no protection at all [18, 19]. ten informed consent of each child’s guardian was subse- More recently, increasing levels of G6PD defciency were quently obtained before enrolment into the study. Using shown to protect against cerebral malaria, but increas- a standardized questionnaire, demographics and malaria ing the risk of severe malaria anaemia in both males and indicator information were collected. females in multicentre investigations [3, 13]. Notably, many of the earlier studies were largely case–control and Active case detection and determination of malaria examined efects of G6PD defciency on the risk of severe incidence malaria but not the incidence of uncomplicated malaria. After the baseline survey, parents or guardians were Also, these studies were done in other populations out- instructed to bring their children to the study malaria side Uganda. Only two longitudinal studies that were clinic based at Iganga Hospital whenever the children done in Uganda [20] and Gabon [21] found an increase in felt unwell. In addition, study children were actively fol- the malaria incidence rates among female heterozygotes. lowed up by two home visits at convenient times of day, Other host polymorphisms within the inducible nitric on Mondays and Tursdays, every week. A standardized oxide synthase 2 (NOS2) gene promoter also appear to be questionnaire was administered for collecting infor- protective against severe malaria [22] and uncomplicated mation regarding any illnesses that had occurred since malaria [20]. Particularly, a single nucleotide polymor- the last visit, use of health care facilities and medica- phism (NOS2 −954 G>C; rs 1800482) is believed to mod- tions used. At each visit, the tympanic temperature was ify NOS2 transcription and increases nitric oxide activity recorded using a digital thermometer. When fever (tym- [22]. In previous studies, increased levels of nitric oxide panic temperature of ≥37.5 °C) or history of recent fever were shown to be important in parasite clearance [22, 23] (within the last 24 h) was observed or reported for any and protection against P. falciparum infection [24–26], study child, a rapid diagnostic test (RDT, OPTIMAL ®) yet few studies have investigated the relationship between and microscopy of a stained blood smear were per- NOS2 polymorphisms and the incidence of uncomplicated formed to determine the presence of malaria parasites. malaria. One particular study from Uganda associated Malaria was defned as i) the child being ill or having the NOS2 −954 G>C, but not the −1173 C>T promoter any symptom of malaria illness and ii) the child having Lwanira et al. Malar J (2017) 16:322 Page 3 of 9 fever (tympanic temperature ≥37.5 °C) or a history of following the manufacturer’s protocol. Assessment of fever (within the past 24 h) plus iii) the child having any host gene polymorphism was performed by amplifying P. falciparum parasitaemia. Sick children found with specifc gene fragments with polymerase chain reaction malaria parasitaemia were administered artemisinin (PCR) followed by endonuclease restriction fragment combination therapy (ACT) at the study clinic following length polymorphism (RFLP) as described