Exploring association between MBL2 gene polymorphisms and the occurrence of clinical blackwater fever through a case–control study in Congolese children

This study aimed to test the association between MBL2 polymorphisms and Blackwater fever, one of the most severe complications of malaria, and provide the first distribution data for MBL2 haplotypes in Congolese individuals. This is a case–control study conducted over 2 years in 4 medical institutions across Kinshasa, namely University Hospitals of Kinshasa, Kimbanseke Hospital, Bondeko Hospital and General Provincial Hospital of Kinshasa. Sampling methods and case definition are published elsewhere [12]. Altogether, 43 cases and 86 controls were enrolled. Ages for cases and controls ranged from 2 to 15 years.

The medical history was obtained from parents, with particular attention to demographic data, including disease history and medications taken before BWF episode. Clinical data were recorded in a customized pre-tested clinical form. Malaria was confirmed by the presence of parasites on blood thick and film.

Twenty mL of fresh urine were collected from each participant. The presence of haemoglobin in urine was first detected by urinary dip strip (Medi test Combi9, MacheryEur, Paris, France) and then confirmed by spectrometer (Thermo Genesis 10 BIO, New York, USA) using protocol of 3,3′ dimethyl benzidine reagent [38]. The results of urine dip stick were read as either negative (yellow colour) or positive (change in blue colour) 1+, 2+, 3+, which corresponded approximately to haemoglobin concentrations of respectively 0.061 ± 0.0166 mg/L, 0.3986 ± 0.2612 mg/dL and 0.5679 ± 0.27688 mg/L as quantified using the spectrometer.

Human MBL2 gene was assessed from genomic DNA. Eight drops of blood were collected on FTA card^® WB 120067 (GE Healthcare, Amersham, UK) and stored in a fridge until the transfer to the Institute of Tropical Medicine of Nagasaki University in Japan for DNA testing in the Department of Immunogenetics according to a previously described protocol [39].

DNA samples from 43 cases and 86 controls were examined. The promoter region and exon 1 of MBL2 gene were PCR-amplified and Sanger sequenced. Prior to Sanger sequencing, PCR products were verified by gel electrophoresis to confirm the presence of expected band and exclude unexpected inserts. The PCR mixture contained 17.5 μL of ultra-pure water, 2.5 μL PCR 10 × buffer, 4 μL of dNTPs (2 µmol), 0.4 μL (2 units) of Taq polymerase and 0.8 μL of each primer (2.5 µmol). A disc containing between 5 and 20 ng of DNA was punched from the FTA card and added into the PCR reaction tube. In order to identify technical contaminations, a tube a No DNA template was also included in each run. This consisted of a punch from an unspotted FTA card. After an initial denaturation step of 5 min at 95 °C, 35 amplification cycles were applied including rapid denaturation at 95 °C for 1 min, annealing at 65 °C for 1 min and elongation at 72 °C for 1 min. The reaction ended with a final elongation step at 72 °C for 5 min. PCR product was sequenced by dideoxy termination sequencing using Big-Dye^® Terminator version 1.1. Sequencing product was analysed on a 3730 DNA ANALYSER, version 3.0, from HITACHI. Haplotypes were double- and triple-checked using visual inspection of sequencing traces.

Alleles were designated as suggested by Antonarakis et al. [7] for the 3 variants in the Exon 1. The MBL2*B, MBL2*C and other variants alleles were identified as described by Sumiya et al., Lipscombe et al. and Madsen et al. [3‐5, 9].

Alleles and genotypes frequencies were obtained by direct scoring of electropherogram. Data were recorded using the software Epi Info 7. All analyses were carried out using SPSS 18.0. All records were crosschecked with the original data sheets before the analysis. A non-conditional model was used. This was a binary logistic regression including covariates, anti-malaria drugs, MBL2 gene polymorphism, G6PD and parasitaemia. Multivariate logistic regression analysis was used to evaluate associations between MBL2 haplotypes/genotypes/alleles and the BWF. Odds ratio and confidence intervals were calculated. All tests were two-sided, and the level of significance was set at p < 0.05.

MBL2*A allele was the most common allele within the 2 groups compared to each of the derived alleles individually. However, when considered together, null alleles (allele 0) were more frequent among patients with UM compared to those with BWF, with allele frequencies of 0.39 and 0.233, respectively. 0 includes B, C and D alleles (Table 3). MBL2*C was the most frequent in both groups. Bellamy et al. [46] reported also a higher frequency of the MBL2*C in in the population of The Gambia. Compared to the other null alleles, the MBL2*C has been demonstrated to be extremely common in sub-Saharan Africans with a population frequency of 0.30, whereas the MBL2*B was predominant in Europeans, in Asians and in indigenous people of South America with population frequencies of 0.13, 0.20 and 0.50, respectively [3, 5, 46]. None of the alleles observed in the study population presented a significant preferential distribution between the 2 groups.

It has been hypothesized that *B, *C and *D alleles are positively selected in order to reduce susceptibility or mortality due to certain infectious diseases [5, 24, 34]. This study did not identify the MBL2*D allele within the 2 groups. This allele has been detected with frequencies up to 0.05 in the northeast of Africa, in Europe and India [3, 10]. Hence, the absence of the MBL2*D may simply indicate a low admixture with European and Indian in the Congolese population examined in this study [47‐49].

The present study revealed 9 haplotypes, including 3 high MBL expression haplotypes (Table 4) and 6 low expression haplotypes. The LYQA haplotype was the most prevalent haplotype both in BWF and UM group with, respectively, 45.5 and 39.5%, followed by MBL2⃰ LYQC in UM population with 23.3%. In Gabon, Boldt et al. defined 14 new haplotypes and reported that MBL2*LYQC, MBL2*LYQA and MBL2*LYPA were the most prevalent haplotypes in the children population [55]. A new haplotype HYPC only described in Zimbabwe individuals was observed in 2 patients: one BWF and one UM. A study in India reported that the MBL2*LYPA haplotypes confers protection, whereas MBL2*LXPA increases the malaria risk. These findings in Indian populations demonstrate that MBL2 functional variants are strongly associated with malaria and infection severity [10]. However, no significant association was find between BWF and a particular haplotype.

Lower parasitaemia was observed in BWF patients. Considering that quinine intake offers a strong clearance of parasite, low parasitaemia observed in BWF may be secondary to the quinine intake that triggers BWF occurrence. In that prospect, the time between quinine intake and the occurrence of BWF may influence parasitaemia. However, this timing remains unclear since reported time-lapses range from 12 h to multiple days after treatment [56‐58]. Another reason for low parasitaemia in BWF could be the activity of the immune system in AA individuals. The active immune response would provide a good clearance of parasite and accessorily cause BWF. Further studies may be needed to investigate this hypothesis.