Human genetic determinants of severe malaria in The Gambia

Projects:

Locations: The Gambia (GM)

 About this study

Genetic factors play an important role in resistance to malaria infection but many of the genes responsible remain unknown. It has been estimated that host genetic factors contribute 25% of total malaria protection (Mackinnon MJ et al, 2005) but only 2% of this total can be attributed to HbS; a polymorphism which confers a well-documented protective effect against severe malaria. This raises the question: What other genetic variants have evolved that contribute to malaria protection or risk? In The Gambia, we have been building a DNA resource since 1996 with the aim of investigating the human genetic determinants of severe malaria.

Summary

An unmatched case-control study was conducted between 1996 and 2009 with the aim of investigating the human genetic determinants of severe malaria in The Gambia.Severe malaria cases (children aged between 4 months and 15-years) were recruited from the Western Health Region of the Gambia, the main referral hospital in Banjul, the MRC wards in Fajara, and some governmental health facilities. Clinical data and DNA were collected from both cases and controls. In about 50% of the cases, we collected DNA samples from both parents to allow a family-based (TDT) analysis. Controls were collected to represent the population of the Gambia; blood was collected from umbilical cords at the maternity units of the main hospitals. As with the cases, we also collected parental samples (mothers only) in about 50% of the samples.

Clinical data and DNA samples were contributed to the MalariaGEN Consortial Project 1 (CP1) along with those of 11 other case-control studies from a total of 11 malaria-endemic countries. As part of the sample handling process, baseline genotyping data was generated for a number of malaria–associated single nucleotide polymorphisms (SNPs) and the appropriate data has been returned to each site for site-specific analysis. A total of 69 SNPs at candidate genes (selection based on previous reports of association with severe malaria or on their likely biological role in malaria infection/disease) will be included in our analysis. We will investigate the association between each SNP and several malaria phenotypes including severe malaria, cerebral malaria, severe malarial anaemia, respiratory distress and hyperparasitaemia.

Study site description

The Medical Research Council (MRC) Unit in The Gambia recruited severe malaria cases, healthy controls and parents from the western part of the country, including the Kanifing Municipal Council (KMC) with a population of 397,327, and the West Coast Region, with a population of 527,753. This catchment had a total population of 956,396 in 2009 (2009 population projected from 2003 Population Census). Some cases and their parents were also referred from the North Bank West Region which has a total population of 92,822.  The four largest ethnic groups in The Gambia are the Mandinka, Jola, Fula and Wollof. About half the inhabitants of the Western Health Region (WHR) are farmers, relying on their agricultural produce for subsistence. The GDP per capita for the country in 2009 was USD $436 (World Bank, accessed 12 Dec 2011).

The WHR is served by twelve government health facilities (in and out-patient) and four hospitals. In addition, there are 12 government-operated clinics (out-patient only) in the region. The MRC Clinical Services Department, Ahmadiyya Hospital and several private clinics in the study area also provide health care to the inhabitants in this region.

Transmission of malaria in The Gambia is seasonal. The long dry season lasts from November-June with a short rainy season from July-October with an average annual rainfall of 1,015 mm. Morbidity and mortality from malaria follows this pattern; both occur more frequently during the rainy season with a peak in October. P. falciparum is the dominant species, being responsible for all severe disease and for over 95% of clinical attacks. Few cases of clinical malaria are caused by P. malariae. The main vector species in the WHR are Anopheles gambiae, A. melas and A. arabiensis.

Methods

An unmatched case-control study, including parents of cases and mothers of controls was conducted between 1996 and 2009 recruiting patients admitted to the Edward Francis Small Teaching Hospital (EFSTH), formerly the Royal Victoria Teaching Hospital (RVTH), and the Medical Research Council (MRC) wards. The EFSTH is located in the capital, Banjul, and is The Gambia's main referral hospital. Patients come from all over the country, with the majority from the western region. The MRC wards are located in the main research site of the MRC in Fajara.

Cases consist of children (aged 4 months-15 years) with severe malaria. Severe malaria was defined by the presence of asexual parasitemia and any of the following: cerebral malaria (Blantyre coma score of ≤2), severe anaemia (haemoglobin <6 g/dl or hematocrit <18%), respiratory distress (respiratory rate >40 with 2 of the following: nasal flaring, intercostal and subcostal recession, and grunting), repeated generalised convulsions, hypoglycaemia (blood glucose <2.2mmol/l or <40mg/dl), renal failure (urine output <12ml/kg/day), haemoglobinuria, jaundice, prostration, hyperparasitaemia (≥500 parasites/HPF), hyperpyrexia, circulatory collapse, cold extremities, rapid pulses, systolic blood pressure below 50mmHg.

Controls consist of cord blood samples recruited from various labour wards, primarily in the western division of The Gambia, between 1999 and 2007. Blood samples were collected from the parents of a number of severe malaria cases, and mouth swab samples from mothers of a number of controls

A standardised case report form (CRF), was created for the Consortial Project 1 and used by all sites to collect standardised clinical data. The data collected in Gambia (and all other sites) was uploaded onto secure web-based software developed by MalariaGEN. Here, the integrity of the data was checked and data was standardised and amalgamated.

Genomic DNA was extracted at the MRC Unit in The Gambia from whole blood using the Nucleon™ BACC2 Genomic DNA extraction kit® (Gen-Probe Life Sciences Ltd., Manchester, UK) using manufacturer’s instructions. Aliquots of the DNA samples were shipped to the MalariaGEN Resource Centre in Oxford for further processing and quality control for quantity, quality (by genotyping) and confirming appropriate clinical data was available, prior to selection for the genome-wide experiment. Baseline genotype data for 69 malaria-associated SNPs was generated for all contributing samples; briefly, samples underwent a primer-extension pre-amplification (PEP) step (Xu K et al, 1993; Zhang L et al, 1992) prior to genotyping on the Sequenom® MassArray® platform. Following curation, the genotype data were returned to The Gambia to allow local analyses.

Table 1: Breakdown of samples
Number Gender: n (%) Age in years: n (%) Ethnicity: n (%)
Malaria cases: 2,801 Male: 1429 (51)

Female: 1296 (46)

Not recorded: 87 (3)

<1: 236 (8)

1-2: 405 (14)

2-5: 1336 (48)

5-15: 813 (29)

Not recorded: 11 (<1)

Fula: 344 (12)

Jola: 432 (15)

Mandinka:  900 (32)

Wollof: 335 (12)

Other: 780 (28)

Not recorded: 10 (<1)

Healthy controls: 4472 Male: 1979 (44)

Female: 2315 (51)

Not recorded: 221 (5)

<1: 3801 (85)

1-2: 0 (0)

2-5: 2 (<1)

5-15: 17 (<1)

>15: 211 (5)

Not recorded: 441 (10)

Fula: 784 (18)

Jola: 527 (12)

Mandinka:  1325 (30)

Wollof: 597 (13)

Other: 519 (12)

Not recorded: 359 (8)

Ethics

The study was approved by The Gambia Government/Medical Research Council Joint Ethics Committee (proposal numbers: 670/630 and 1029v2).

Informed consent was obtained from parents or guardians of cases and mothers for the population controls (cord bloods). Population controls were collected by consenting women attending maternity units to give birth. Further controls were collected from The Gambia Biobank with appropriate ethics permissions.

Additional contributors

  • Abdoulie Camara, Medical Research Council Unit, The Gambia
  • Abubacar Sadiq, Medical Research Council Unit, The Gambia
  • Abdou Bah, Royal Victoria Teaching Hospital, The Gambia
  • Aja Abie Khan, Department of State for Health and Social Welfare, The Gambia
  • Amie Jobarteh, Royal Victoria Teaching Hospital, The Gambia
  • Anthony Mendy, Medical Research Council Unit, The Gambia
  • Augustine Ebonyi, Royal Victoria Teaching Hospital, The Gambia
  • Bakary Danso, Medical Research Council Unit, The Gambia
  • Bintou Taal, Royal Victoria Teaching Hospital, The Gambia
  • Climent Casals-Pascual, Medical Research Council Unit, The Gambia
  • Emmanuel Onykwelu, Royal Victoria Teaching Hospital, The Gambia
  • Fatoumatta Sisay-Joof, Medical Research Council Unit, The Gambia
  • Haddy Kanyi, Medical Research Council Unit, The Gambia
  • Haddy Njie, Royal Victoria Teaching Hospital, The Gambia
  • Herbert Obu, Royal Victoria Teaching Hospital, The Gambia
  • Horeja Saine, Royal Victoria Teaching Hospital, The Gambia
  • Idrissa Sambou, Medical Research Council Unit, The Gambia
  • Ismaela Abubakar, Medical Research Council Unit, The Gambia
  • Jalimory Njie, Royal Victoria Teaching Hospital, The Gambia
  • Janet Fullah, Royal Victoria Teaching Hospital, The Gambia
  • Jula Jaiteh, Royal Victoria Teaching Hospital, The Gambia
  • Kebba Jammeh, Royal Victoria Teaching Hospital, The Gambia
  • Kumba Sabally-Ceesay, Department of State for Health and Social Welfare, The Gambia
  • Lamin Manneh, Medical Research Council Unit, The Gambia
  • Landing Camara, Royal Victoria Teaching Hospital, The Gambia
  • Lawrence Yamoah, Medical Research Council Unit, The Gambia
  • Madi Njie, Medical Research Council Unit, The Gambia
  • Malick Njie, Royal Victoria Teaching Hospital, The Gambia
  • Margaret Pinder, Medical Research Council Unit, The Gambia
  • Mariatou Jallow, Royal Victoria Teaching Hospital, The Gambia
  • Mohammed Aiyegbo, Royal Victoria Teaching Hospital, The Gambia
  • Momodou Jasseh, Medical Research Council Unit, The Gambia
  • Momodou Lamin Keita, Royal Victoria Teaching Hospital, The Gambia
  • Momodou Saidy-Khan, Medical Research Council Unit, The Gambia
  • Ndey Ceesay, Royal Victoria Teaching Hospital, The Gambia
  • Oba Rasheed, Royal Victoria Teaching Hospital, The Gambia
  • Pa Lamin Ceesay, Royal Victoria Teaching Hospital, The Gambia
  • Pamela Esangbedo, Royal Victoria Teaching Hospital, The Gambia
  • Ramou Cole-Ceesay, Department of State for Health and Social Welfare, The Gambia
  • Rasaq Olaosebikan, Royal Victoria Teaching Hospital, The Gambia
  • Simon Correa, Medical Research Council Unit, The Gambia
  • Sophie Njie, Royal Victoria Teaching Hospital, The Gambia
  • Stanley Usen, Medical Research Council Unit, The Gambia
  • Yaya Dibba, Medical Research Council Unit, The Gambia and Royal Victoria Teaching Hospital, The Gambia

Acknowledgements

We thank the Gambian children and their parents and guardians who made this study possible. We extend our appreciation and gratitude to the doctors especially Mariatou Jallow, Herbert Obu, Oba Rasheed, Augustine Ebonyi, Emmanuel Onykwelu and nurses and matrons particularly Haddy Njie, Ndey Ceesay, Landing Camara and Horeja Saine, and the laboratory technicians Janet Fullah Riddles and Abdou Bah and the Record Office staff especially Amie Jobarteh, Jalimory Njie and Nyakassi Bojang at the Royal Victoria Teaching Hospital, Banjul. We also thank the nurses particularly Sophie Njie and Jula Jaiteh at various government health facilities in the Western Health Region and staff of Regional Health Office especially Aja Abie Khan, Kumba Sabally-Ceesay and Alhagie Sankareh who assisted with this work. Our sincere gratitude and appreciation to Ramou Cole-Ceesay and the staff of the Reproductive and Child Health Unit and the Ministry of Health of The Gambia. We thank the study team- Yaya Dibba, Anthony Mendy, Abdoulie Camara, Momodou Saidy-Khan, Idrissa Sambou, Simon Correa, Mohammed Aiyegbo, Bintou Taal, Kebba Jammeh, Modou Lamin Keita, Madi Njie, Bakary Danso, Pa Lamin Ceesay, Ismaela Abubakar and Haddy Kanyi for their invaluable support, and Abubacar Sadiq, Rasaq Olaosebikan, Momodou Jasseh, Climent Casals-Pascual and staff of the Medical Council Unit for their contributions.

MalariaGEN’s primary funding is from the Wellcome Trust and from the Bill & Melinda Gates Foundation, through the Foundation for the National Institutes of Health as part of the Grand Challenges in Global Health initiative. The Wellcome Trust (Sanger Institute core funding) and the Medical Research Council provide additional support for genotyping, bioinformatics and analysis.