HLA-G 3’UTR 14 bp Insertion Is Associated with a Decreased Risk of Developing Human African Trypanosomiasis in the Côte d’Ivoire Population

Human African trypanosomiasis (HAT), or sleeping sickness, caused by Trypanosoma brucei gambiense, is associated with diverse clinical outcomes. Host’s genetic factors involved in immunity are potential factors that can re-gulate infection. Genetic polymorphisms insertion homozygous genotype. Genetic polymorphisms in HLA-G may be associated with a variable risk to develop HAT. The 14 bp insertion appears to favour the occurrence of long-lasting T. b. gambiense latent infections.


Introduction
Human African trypanosomiasis (HAT), or sleeping sickness, caused by Trypanosoma brucei gambiense (T. b. gambiense) is classically described as a chronic infection characterized by an early hemolymphatic stage (stage 1) associated with nonspecific symptoms such as intermittent fevers and headaches, followed by a meningoencephalitic stage (stage 2) in which the parasite invades the central nervous system and causes neurological disorders and death if left untreated.
However, asymptomatic carriers and spontaneous cure without treatment have been described [1], underlining the phenomenon of human trypanotolerance/resistance [2] [3]. Host's genetic factors might be involved in the control of the infection and the progression of disease, as clearly shown for Trypanosoma congolense infections in experimental models [4] [5] and also in humans [6]- [11]. Genes coding immune system proteins such as the Human leucocyte antigen (HLA)-G are important candidates.
HLA-G is a non-classical major histocompatibility complex (MHC) class I gene. It is located on chromosome 6p21.3 and encompasses 4144 nucleotides distributed across eight exons and seven introns. HLA-G can generate four forms of membrane-bound proteins (HLA-G1, G2, G3, and G4) and three forms of soluble proteins (HLA-G5, G6, and G7) due to HLA-G alternative splicing.
HLA-G has a restricted distribution and is mainly expressed in trophoblastic cells, thymic epithelium, pancreas, cornea, nail matrix, and erythroid and endothelial precursors under normal physiological condition [12]. As an immune checkpoint molecule, HLA-G can bind its inhibitory receptors present on immune cells (NK, T, B and dendritic cells, monocytes/macrophage and neutrophils) and inhibit crucial functions of these effectors such as proliferation, differentiation, cytotoxicity, phagocytosis, reactive oxygen species and antibody production [13] [14] [15]. Exon 8 consists in a regulatory 3' untranslated region (3'UTR) that plays an important role in gene expression [16] Among the 17 variation sites that have been identified in 3'UTR, the 14 bp Insertion/Deletion polymorphism has been extensively studied in different pathologies including in-Open Journal of Genetics (HCMV), Human Papilloma Virus (HPV) [19] and more recently with HAT susceptibility in Democratic Republic of the Congo (DRC) [20]. In Côte d'Ivoire, only two studies have focussed on the role of human genetic variants in HAT susceptibility. The first one investigated the role of genetic polymorphisms in genes coding for cytokines (IL10 and TNFA) [6] and the second one investigated association between single markersin16 candidate genes (IL1, IL4, IL4R, IL6,  IL8, IL10, IL12, IL12R, TNFA, INFG, MIF, APOL1, HPR, CFH, HLA-A and HLA-G) and the disease [21]. However, the role played by the 14-bp Indel (rs16375) polymorphism was not investigated in these studies. In the present study, we investigated the association of the 14 bp Indel (rs16375) polymorphism from HLA-G regarding susceptibility/resistance to HAT.

Ethics Statement
All samples were collected within the framework of medical surveys and epidemiological surveillance activities supervised by the HAT National Control Program (HAT NCP). Samples were collected during routine screening and diagnostic procedures implemented by NCP. All participants were informed on the purpose of the study in their own language and signed an informed consent form. Approval from the Ivorian National Ethics Committee, No. 0308/MSLS/ CNER-P, January 30, 2012 was obtained for the study.

Study Population and Phenotype Definition
The samples consisted of 383 individuals, 119 cases, 221 controls and 43 seropositive individuals (latent infections). Blood samples were collected between 2012 and 2016 from study participants from the main HAT foci located in western-central Côte d'Ivoire (Bonon, Bouaflé, Zoukougbeu, Oumé, and Sinfra). Controls were matched with HAT cases and seropositive individuals (latent infections) on age, sex and ethnic group. For each individual, an aliquot of plasma was used to perform the immune trypanolysis test that detects Litat 1.3 and Litat 1.5 variable surface antigens specific for T.b. gambiense [22] Cases were defined as individuals with trypanosomes detected in either lymph node aspirate, blood, or cerebrospinal fluid (CSF) by microscopy, after concentrating parasites, if necessary [23]. Individuals with positive CATT [24] and trypanolysis test results (TL+) [22], in whom no trypanosomes were detected by microscopy were considered as seropositive individuals. Controls were defined as individuals living in an endemic area for which CATT and trypanolysis test are negative, with no suggestive signs or symptoms of HAT and without evidence of previous HAT infection. All individuals with end titers of 1/4 or greater were submitted to the microscopic examination of lymph node aspirates whenever swollen lymph nodes were present; 500 µl of the buffy coat (BC) was then examined by using the mini-anion exchange centrifugation test (mAECT) [23] to detect trypanosome parasites. Some HAT cases and seropositives included in the study were Open Journal of Genetics diagnosed during active HAT survey performed between 2009 and 2012 in collaboration with the National Control Programme (NCP), the Institut de Recherche pour le Développement (IRD) and Institut Pierre Richet (IPR) using serological and parasitological investigations. Study sites were revisited between 2012 and 2016 to consent and resample previously diagnosed and treated patients and seropositive individuals. All cases, seropositive and controls subjects included in this study were well defined. For each subject, the following covariates were collected: 1) age at diagnosis in years; 2) ethnic group, classified as native and non-native (natives originate from the HAT focus and non-natives are ethnic groups from northern Côte d'Ivoire, Mali and Burkina Faso areas where HAT was not present) and; 3) sex.
Collected samples were then frozen directly in the field at −20˚C and stored at −20˚C at Jean Lorougnon Guédé University in Daloa, Côte d'Ivoire until used.

HLA-G 14 bp Insertion/Deletion (Indel) Allele Genotyping
Genomic DNA was obtained from peripheral blood samples. Extraction was performed using Qiagen kits according to the manufacturer's instructions. DNA was stored at −20˚C until the time of analysis.
The HLA-G 14 bp Indel polymorphism in exon 8 (3'-UTR) of the HLA-G gene was identified by polymerase chain reaction (PCR) and performed as previously described [25]. The PCR primers were the following: forward primer/5'-GTG ATG GGC TGT TTA AAG TGT CAC C-3' and reverse primer/5'-GGA AGG AAT GCA GTT CAG CAT GA-3'. 20 ng of DNA sample was amplified in 25 μl of a reaction mixture containing 0.5 units of Taq DNA polymerase, dNTPs (2.5 μM each) (biotechrabbit, Germany), using an automated PCR thermal cycler (BIO RAD T100 TM Thermal Cycler). Thermal cycling was performed with an initial 94˚C for 5 min followed by 39 cycles at 94˚C for 40 s, annealing at 58˚C for 40 s and extension at 72˚C at 60 s and a final extension at 72˚C for 10 min. The amplified products were visualized by electrophoresis on a 2% agarose gel (120V for 1 hour) containing ethidium bromide (0.5 mg/ml). PCR products were either 224 or 210 bp, or both 224 and 210 bp, depending on the insertion/deletion of the 14 bp in exon 8. The number of 14 bp indel alleles was directly counted (Figure 1).

Statistical Analysis
All statistical analyses were performed using the Plink 1.9 and Rv 3.2.1 software. The Pearson Chi-square test was used to compare the frequencies of observed and expected genotypes under Hardy-Weinberg equilibrium. The allele and genotype frequencies were calculated by direct gene counting from the genotyping results. Association of HAT with recorded covariates (age and ethnic group) were analysed by multivariate analysis. Tests were performed under a recessive model to examine the association between HLA-G genotypes and alleles and the risk to develop HAT. Unadjusted odds ratios (OR) were reported with 95% confidence intervals (CI). A significant threshold of P value ≤ 0.05 was taken.

HLA-G Allele and Genotype Association with HAT
The distributions of allele and genotype frequencies of HLA-G 14 bp Indel polymorphism are reported in Table 2. The HLA-G Indel 14 bp polymorphism was in Hardy-Weinberg equilibrium in our population (P = 0.12).

HLA-G 14 bp Indel Polymorphism in HAT Patients, Controls and Seropositive Individuals
No significant differences were observed in the frequencies of 14 bp HLA-G alleles between HAT cases and controls. Nevertheless, the 14 bp insertion/insertion homozygote genotype was underrepresented in HAT cases (p = 0.012, OR = 0.27, 95% CI: 0.09 -0.8) ( Table 3). No significant difference was observed in both allelic and genotypic frequencies between seropositive and control groups (Table 3), whereas the 14 bp insertion/insertion genotype was also significantly associated with seropositivity in the Seropositive/HAT cases comparison (p = 0.043, OR = 0.26, 95% CI: 0.07 -0.99, Table 3) indicating that seropositive subjects carrying the insertion/insertion homozygote genotypes may be at lower risk of developing the disease. Estimation

Discussion
First described at the maternal-foetal interface [26] it is increasingly evident that  been extensively studied in a number of diseases and was shown to be associated with low HLAG mRNA expression levels [17] through its influence on mRNA stability and splicing patterns [18] [31]. Individuals homozygote for the 14-bp insertion had a lower risk of developing HAT in our study population as suggested by the fact that the 14-bp insertion/insertion genotype was significantly more frequent in both endemic controls (OR = 0.27; p = 0.012) and seropositive individuals suspected to harbour latent infections (OR = 0.26; p = 0.043).
Therefore, in our population, the HLAG 3'UTR 14 bp polymorphism does not appear to provide protection against infection itself but rather against infection progressing to severe disease. This result suggests that some HLAG polymorphisms may favour T. b. gambiense asymptomatic carriage and contribute to disease maintenance in HAT foci despite control measures targeted to HAT patients only [32]. As stated above the 14 bp insertion has mainly been associated with low HLAG mRNA expression levels. We add however no data to analyse HLAG expression in the framework of this study, we may, however, speculate that in our HAT patients high HLAG expression contributed to T. b. gambiense immune evasion, leading to parasite multiplication and disease progression, whereas in seropositive lower levels enable better control of infection. In agreement with this hypothesis, high levels of s-HLAG were reported recently from Guinea in the plasma of HAT patients. High s-HLAG plasmatic levels were also prognostic of subsequent disease development in seropositive subjects that were followed up in time after their initial diagnosis [33]. A different association was however described in the Democratic Republic of Congo where the HLAG UTR-2 haplotype (including the 14 bp insertion and a G at position +3196) was associated with increased susceptibility to HAT in a family-based association study [20]. The reasons for this discrepancy are yet unclear but may reflect para- consequently the interaction with HLA-G receptors and/or formation of HLA-G dimers that may more efficiently bind to HLA-G receptors. Thus, a particular allele and a particular molecule could provide susceptibility or protection against disease development [27].
In conclusion we have shown that the 3'UTR 14 bp insertion is associated with differential susceptibility to HAT in the population of Côte d'Ivoire. At the homozygous state this insertion appears to favour T. b. gambiense asymptomatic carriage and may thus favour disease persistence in endemic foci. A better characterization of HLA-G polymorphisms combined with the analysis of the gene expression levels will be required to better understand the biological mechanisms at play in the host-parasite interaction.