Identification of potential common genetic modifiers of neurofibromas: a genome-wide association study in 1333 patients with neurofibromatosis type 1.

BACKGROUND: Neurofibromatosis type 1 (NF1) is characterized by the highly variable and unpredictable development of benign peripheral nerve sheath tumours: cutaneous (cNFs), subcutaneous (scNFs) and plexiform (pNFs) neurofibromas. OBJECTIVES: To identify neurofibroma modifier genes, in order to develop a database of patients with NF1. METHODS: All patients were phenotypically evaluated by a medical practitioner using a standardized questionnaire and the causal NF1 variant identified. We enrolled 1333 patients with NF1 who were genotyped for > 7 million common variants. RESULTS: A genome-wide association case-only study identified a significant association with 9q21.33 in the pNF phenotype in the discovery cohort. Twelve, three and four regions suggestive of association at the P ≤ 1 × 10-6 threshold were identified for pNFs, cNFs and scNFs, respectively. Evidence of replication was observed for 4, 2 and 6 loci, including 168 candidate modifier protein-coding genes. Among the candidate modifier genes, some were implicated in the RAS-mitogen-activated protein kinase pathway, cell-cycle control and myelination. Using an original CRISPR/Cas9-based functional assay, we confirmed GAS1 and SPRED2 as pNF and scNF candidate modifiers, as their inactivation specifically affected NF1-mutant Schwann cell growth. CONCLUSIONS: Our study may shed new light on the pathogenesis of NF1-associated neurofibromas and will, hopefully, contribute to the development of personalized care for patients with this deleterious and life-threatening condition.

The limitations of kinship determinations using STR data in ill-defined populations.

The likelihood ratio (LR) method is commonly used to determine kinship in civil, criminal, or forensic cases. For the past 15 years, our research group has also applied LR to ancient STR data and obtained kinship results for collections of graves or necropolises. Although we were able to reconstruct large genealogies, some pairs of individuals showed ambiguous results. Second-degree relationships, half-sibling pairs for example, were often inconsistent with detected first-degree relationships, such as parent/child or brother/sister pairs. We therefore set about providing empirical estimations of the error rates for the LR method in living populations with STR allelic diversities comparable to that of the ancient populations we had previously studied. We collected biological samples in the field in North-Eastern Siberia and West Africa and studied more than 800 pairs of STR profiles from individuals with known relationships. Because commercial STR panels were constructed for specific regions (namely Europe and North America), their allelic makeup showed a significant deficit in diversity when compared to European populations, replicating a situation often faced in ancient DNA studies. We assessed the capacity of the LR method to confirm known relationships (effectiveness) and its capacity to detect those relationships (reliability). Concerns over the effectiveness of LR determinations are mostly an issue in forensic studies, while the reliability of the detection of kinship is an issue for the study of necropolises or other large gatherings of unidentified individuals, such as disaster victims or mass graves. We show that the application of LR to both test populations highlights specific issues (both false positives and false negatives) that prevent the confirmation of second-degree kinship or even full siblingship in small populations. Up to 29% of detected full sibling relationships were either overestimated half-sibling relationships or underestimated parent-offspring relationships. The error rate for detected half-sibling relationships was even higher, reaching 41%. Only parent-offspring pairs were reliably detected or confirmed. This implies that, in populations that are small, ill-defined, or for which the STR loci analyzed are inappropriate, an examiner might not be able to distinguish a pair of full siblings from a pair of half-siblings. Furthermore, half-sibling pairs might be overlooked altogether, an issue that is exacerbated by the common confusion, in many languages and cultures, between half-siblings and full siblings. Consequently, in the study of ancient populations, human remains of unknown origins, or poorly surveyed modern populations, we recommend a conservative approach to kinship determined by LR. Next-generation sequencing data should be used when possible, but the costs and technology involved might be prohibitive. Therefore, in potentially contentious situations or cases lacking sufficient external information, uniparental markers should be analyzed: ideally, complete mitochondrial genomes and Y-chromosome haplotypes (STR, SNP, and/or sequencing).

First genome-wide association study of non-severe malaria in two birth cohorts in Benin.

Recent research efforts to identify genes involved in malaria susceptibility using genome-wide approaches have focused on severe malaria. Here, we present the first GWAS on non-severe malaria designed to identify genetic variants involved in innate immunity or innate resistance mechanisms. Our study was performed on two cohorts of infants from southern Benin (525 and 250 individuals used as discovery and replication cohorts, respectively) closely followed from birth to 18-24 months of age, with an assessment of a space- and time-dependent environmental risk of exposure. Both the recurrence of mild malaria attacks and the recurrence of malaria infections as a whole (symptomatic and asymptomatic) were considered. Post-GWAS functional analyses were performed using positional, eQTL, and chromatin interaction mapping to identify the genes underlying association signals. Our study highlights a role of PTPRT, a tyrosine phosphatase receptor involved in STAT3 pathway, in the protection against both mild malaria attacks and malaria infections (p = 9.70 × 10-8 and p = 1.78 × 10-7, respectively, in the discovery cohort). Strong statistical support was also found for a role of MYLK4 (meta-analysis, p = 5.29 × 10-8 with malaria attacks), and for several other genes, whose biological functions are relevant in malaria infection. Results shows that GWAS on non-severe malaria can successfully identify new candidate genes and inform physiological mechanisms underlying natural protection against malaria.

Familial Aggregation and Heritability of Loa loa Microfilaremia.

Background: For a given prevalence of Loa loa microfilaremia, the proportion of people with high densities varies significantly between communities. We hypothesized that this variation is related to the existence of familial clusters of hypermicrofilaremic individuals that would be the consequence of a genetic predisposition to present high L. loa microfilarial densities. Methods: A familial study was performed in 10 villages in the Okola Health District of Cameroon. Intrafamilial correlation coefficients and heritability estimates were assessed for both the presence of L. loa microfilaremia and individual microfilarial densities by controlling for age, sex, Mansonella perstans coinfection, and household effects. Results: Pedigrees were constructed for 1126 individuals. A significant familial susceptibility to be microfilaremic for L. loa was found for first-degree relatives (ρ = 0.08, P < .05; heritability = 0.23). Regarding individual microfilarial densities, a significant familial aggregation was demonstrated (ρ = 0.36 for first-degree and 0.27 for second-degree relatives). For first-degree relatives, the highest coefficient was found between mothers and daughters (ρ = 0.57). The overall heritability estimate for L. loa microfilarial density was 0.24 (P = .003). Conclusions: A significant genetic component governs L. loa microfilarial density. This supports the hypothesis that a genetic predisposition to be hypermicrofilaremic exists, leading to the presence of familial clusters of individuals at risk for postivermectin severe adverse events. This finding should be taken into account while developing sampling strategies (including a household-level sampling) to identify villages where community-directed treatment with ivermectin cannot be applied.

Familial Aggregation and Heritability of Wuchereria bancrofti Infection.

BACKGROUND: The familial recurrence risk of lymphatic filariasis (LF) is unknown. This case study aimed to evaluate the familial susceptibility to infection with Wuchereria bancrofti and to microfilaremia in a village of the Republic of Congo. METHODS: The heritability and intrafamilial correlation coefficients were assessed for both W. bancrofti infection and microfilaremia by controlling for individual risk factors, environmental influence, and household effects. RESULTS: Pedigree charts were constructed for 829 individuals, including 143 individuals with a diagnosis of W. bancrofti circulating filarial antigens (CFAs) and 44 who also had microfilariae (MF). There was no intrafamilial correlation regarding CFA levels. However, the presence of MF (ρ = 0.45) and microfilarial density (ρ = 0.44) were significantly correlated among parent-offspring pairs. Heritability estimates for CFA positivity and intensity were 0.23 and 0.18, respectively. Heritability estimates were high for microfilarial positivity (h(2) = 0.74) and microfilarial density traits (h(2) = 0.81). CONCLUSIONS: Our study suggests that the acquisition of LF is mainly driven by environmental factors and habits and that genetic factors are moderately involved in the regulation of infection. By contrast, genetic factors play a major role in both the presence and intensity of microfilaremia.

Human Leukocyte Antigen-G: A Promising Prognostic Marker of Disease Progression to Improve the Control of Human African Trypanosomiasis.

BACKGROUND: Human African trypanosomiasis (HAT) caused by Trypanosoma brucei gambiense can be diagnosed in the early hemolymphatic stage (stage 1 [S1]) or meningoencephalitic stage (stage 2 [S2]). Importantly, individuals harbouring high and specific antibody responses to Tbg antigens but negative parasitology are also diagnosed in the field (seropositive [SERO]). Whereas some develop the disease in the months following their initial diagnosis (SERO/HAT), others remain parasitologically negative for long periods (SERO) and are apparently able to control infection. Human leucocyte antigen (HLA)-G, an immunosuppressive molecule, could play a critical role in this variability of progression between infection and disease. METHODS: Soluble HLA-G (sHLA-G) was measured in plasma for patients in the SERO (n = 65), SERO/HAT (n = 14), or HAT (n = 268) group and in cerebrospinal fluid for patients in S1 (n = 55), early S2 (n = 93), or late S2 (n = 110). Associations between these different statuses and the soluble level or genetic polymorphisms of HLA-G were explored. RESULTS: Plasma sHLA-G levels were significantly higher in HAT (P = 6 × 10-7) and SERO/HAT (P = .007) than SERO patients. No difference was observed between the SERO/HAT and HAT groups. Within the HAT group, specific haplotypes (HG010102 and HG0103) displayed increased frequencies in S1 (P = .013) and late S2 (P = .036), respectively. CONCLUSIONS: These results strongly suggest the involvement of HLA-G in HAT disease progression. Importantly, high plasma sHLA-G levels in SERO patients could be predictive of subsequent disease development and could represent a serological marker to help guide therapeutic decision making. Further studies are necessary to assess the predictive nature of HLA-G and to estimate both sensitivity and specificity.

Variation in NAT2 acetylation phenotypes is associated with differences in food-producing subsistence modes and ecoregions in Africa.

BACKGROUND: Dietary changes associated to shifts in subsistence strategies during human evolution may have induced new selective pressures on phenotypes, as currently held for lactase persistence. Similar hypotheses exist for arylamine N-acetyltransferase 2 (NAT2) mediated acetylation capacity, a well-known pharmacogenetic trait with wide inter-individual variation explained by polymorphisms in the NAT2 gene. The environmental causative factor (if any) driving its evolution is as yet unknown, but significant differences in prevalence of acetylation phenotypes are found between hunter-gatherer and food-producing populations, both in sub-Saharan Africa and worldwide, and between agriculturalists and pastoralists in Central Asia. These two subsistence strategies also prevail among sympatric populations of the African Sahel, but knowledge on NAT2 variation among African pastoral nomads was up to now very scarce. Here we addressed the hypothesis of different selective pressures associated to the agriculturalist or pastoralist lifestyles having acted on the evolution of NAT2 by sequencing the gene in 287 individuals from five pastoralist and one agriculturalist Sahelian populations. RESULTS: We show that the significant NAT2 genetic structure of African populations is mainly due to frequency differences of three major haplotypes, two of which are categorized as decreased function alleles (NAT2*5B and NAT2*6A), particularly common in populations living in arid environments, and one fast allele (NAT2*12A), more frequently detected in populations living in tropical humid environments. This genetic structure does associate more strongly with a classification of populations according to ecoregions than to subsistence strategies, mainly because most Sahelian and East African populations display little to no genetic differentiation between them, although both regions hold nomadic or semi-nomadic pastoralist and sedentary agriculturalist communities. Furthermore, we found significantly higher predicted proportions of slow acetylators in pastoralists than in agriculturalists, but also among food-producing populations living in the Sahelian and dry savanna zones than in those living in humid environments, irrespective of their mode of subsistence. CONCLUSION: Our results suggest a possible independent influence of both the dietary habits associated with subsistence modes and the chemical environment associated with climatic zones and biomes on the evolution of NAT2 diversity in sub-Saharan African populations.

NF1 single and multi-exons copy number variations in neurofibromatosis type 1.

Neurofibromatosis type 1 (NF1) is caused by dominant loss-of-function mutations of the tumor suppressor NF1 containing 57 constitutive coding exons. A huge number of different pathogenic NF1 alterations has been reported. The aim of the present study was to evaluate the usefulness of a multiplex ligation-dependent probe amplification (MLPA) approach in NF1 patients to detect single and multi-exon NF1 gene copy number variations. A genotype-phenotype correlation was then performed in NF1 patients carrying these types of genetic alterations. Among 565 NF1 index cases from the French NF1 cohort, single and multi-exon deletions/duplications screening identified NF1 partial deletions/duplications in 22 patients (~4%) using MLPA analysis. Eight single exon deletions, 11 multiple exons deletions, 1 complex rearrangement and 2 duplications were identified. All results were confirmed using a custom array-CGH. MLPA and custom array-CGH allowed the identification of rearrangements that were missed by cDNA/DNA sequencing or microsatellite analysis. We then performed a targeted next-generation sequencing of NF1 that allowed confirmation of all 22 rearrangements. No clear genotype-phenotype correlations were found for the most clinically significant disease features of NF1 in patients with single and multi-exons NF1 gene copy number changes.

Insights on the HLA-G evolutionary history provided by a nearby Alu insertion.

The AluyHG element belongs to the AluYb8 subfamily. It is a polymorphic insertion, located approximately 20 kb from the HLA-G 3'-untranslated region (3'-UTR), which has been used for evolution studies because it exhibits identity for descendants and it is still polymorphic in the human genome. To understand the evolutionary mechanisms acting on HLA-G, we evaluated the presence or absence of the AluyHG element, associating this variable site with others observed at HLA-G coding, 3'-UTR, or both regions in four distinct populations (Brazilian, French, Congolese, and Senegalese). The results were compared with the 1000Genomes Consortium data. The worldwide AluyHG frequencies showed an increment, starting lower in Africa and increasing following distance and time of human dispersion out of Africa. The same haplotype pattern was observed in all populations, indicating that most of the HLA-G haplotypes already detected were originated earlier in Africa, before Homo sapiens dispersion. The AluyHG insertion was associated with the G*01:01:01:01/UTR-1 haplotype, with rare recombinants. Despite its high frequency in worldwide populations, the G*01:01:01:01/UTR-1 haplotype should be very recent. The low frequency of recombinants indicates that the rate of recombination at the HLA-G gene is very low.

Differential protein expression profiles between Plasmodium falciparum parasites isolated from subjects presenting with pregnancy-associated malaria and uncomplicated malaria in Benin.

BACKGROUND: Plasmodium falciparum is responsible for severe malaria, including pregnancy-associated malaria (PAM). During intra-erythrocytic maturation, the infected erythrocyte (iE) membrane is modified by insertion of parasite-derived proteins, primarily consisting of variant surface antigens such as P. falciparum erythrocyte membrane protein-1. METHODS: To identify new PAM-specific parasite membrane proteins, we conducted a mass spectrometry-based proteomic study and compared the protein expression profiles of 10 PAM and 10 uncomplicated malaria (UM) samples. RESULTS: We focused on the 454/1139 membrane-associated and hypothetical proteins for comparative analysis. Using filter-based feature-selection methods combined with supervised data analysis, we identified a subset of 53 proteins that distinguished PAM and UM samples. Up to 19/20 samples were correctly assigned to their respective clinical group. A hierarchical clustering analysis of these 53 proteins based on the similarity of their expression profiles revealed 2 main clusters of 40 and 13 proteins that were under- or over-expressed, respectively, in PAM. CONCLUSIONS: VAR2CSA is identified and associated with PAM, validating our experimental approach. Other PAM-predictive proteins included PFI1785w, PF14_0018, PFB0115w, PFF0325c, and PFA_0410w. These proteomics data demonstrate the involvement of selected proteins in the pathophysiology of PAM, providing new insights for the definition of potential new targets for a vaccine against PAM.

NF1 molecular characterization and neurofibromatosis type I genotype-phenotype correlation: the French experience.

Neurofibromatosis type 1 (NF1) affects about one in 3,500 people in all ethnic groups. Most NF1 patients have private loss-of-function mutations scattered along the NF1 gene. Here, we present an original NF1 investigation strategy and report a comprehensive mutation analysis of 565 unrelated patients from the NF-France Network. A NF1 mutation was identified in 546 of the 565 patients, giving a mutation detection rate of 97%. The combined cDNA/DNA approach showed that a significant proportion of NF1 missense mutations (30%) were deleterious by affecting pre-mRNA splicing. Multiplex ligation-dependent probe amplification allowed the identification of restricted rearrangements that would have been missed if only sequencing or microsatellite analysis had been performed. In four unrelated families, we identified two distinct NF1 mutations within the same family. This fortuitous association points out the need to perform an exhaustive NF1 screening in the case of molecular discordant-related patients. A genotype-phenotype study was performed in patients harboring a truncating (N = 368), in-frame splicing (N = 36), or missense (N = 35) mutation. The association analysis of these mutation types with 12 common NF1 clinical features confirmed a weak contribution of the allelic heterogeneity of the NF1 mutation to the NF1 variable expressivity.

Rapid birth-and-death evolution of the xenobiotic metabolizing NAT gene family in vertebrates with evidence of adaptive selection.

BACKGROUND: The arylamine N-acetyltransferases (NATs) are a unique family of enzymes widely distributed in nature that play a crucial role in the detoxification of aromatic amine xenobiotics. Considering the temporal changes in the levels and toxicity of environmentally available chemicals, the metabolic function of NATs is likely to be under adaptive evolution to broaden or change substrate specificity over time, making NATs a promising subject for evolutionary analyses. In this study, we trace the molecular evolutionary history of the NAT gene family during the last ~450 million years of vertebrate evolution and define the likely role of gene duplication, gene conversion and positive selection in the evolutionary dynamics of this family. RESULTS: A phylogenetic analysis of 77 NAT sequences from 38 vertebrate species retrieved from public genomic databases shows that NATs are phylogenetically unstable genes, characterized by frequent gene duplications and losses even among closely related species, and that concerted evolution only played a minor role in the patterns of sequence divergence. Local signals of positive selection are detected in several lineages, probably reflecting response to changes in xenobiotic exposure. We then put a special emphasis on the study of the last ~85 million years of primate NAT evolution by determining the NAT homologous sequences in 13 additional primate species. Our phylogenetic analysis supports the view that the three human NAT genes emerged from a first duplication event in the common ancestor of Simiiformes, yielding NAT1 and an ancestral NAT gene which in turn, duplicated in the common ancestor of Catarrhini, giving rise to NAT2 and the NATP pseudogene. Our analysis suggests a main role of purifying selection in NAT1 protein evolution, whereas NAT2 was predicted to mostly evolve under positive selection to change its amino acid sequence over time. These findings are consistent with a differential role of the two human isoenzymes and support the involvement of NAT1 in endogenous metabolic pathways. CONCLUSIONS: This study provides unequivocal evidence that the NAT gene family has evolved under a dynamic process of birth-and-death evolution in vertebrates, consistent with previous observations made in fungi.

Association of IL-4 and IL-10 maternal haplotypes with immune responses to P. falciparum in mothers and newborns.

BACKGROUND: Particular cytokine gene polymorphisms are involved in the regulation of the antibody production. The consequences of already described IL-4, IL-10 and IL-13 gene polymorphisms on biological parameters and antibody levels were investigated among 576 mothers at delivery and their newborns in the context of P. falciparum placental malaria infection. METHODS: The study took place in the semi-rural area of Tori-Bossito, in south-west Benin, where malaria is meso-endemic. Six biallelic polymorphisms were determined by quantitative PCR using TaqMan® Pre-Designed SNP Genotyping Assays, in IL-4 (rs2243250, rs2070874), IL-10 (rs1800896, rs1800871, rs1800872) and IL-13 (rs1800925) genes. Antibody responses directed to P. falciparum MSP-1, MSP-2, MSP-3, GLURP-R0, GLURP-R2 and AMA-1 recombinant proteins were determined by ELISA. RESULTS: The maternal IL-4(-590)*T/IL-4(+33)*T haplotype (one or two copies) was associated with favorable maternal condition at delivery (high haemoglobin levels, absence of placental parasites) and one of its component, the IL-4(-590)TT genotype, was related to low IgG levels to MSP-1, MSP-2/3D7 and MSP-2/FC27. Inversely, the maternal IL-10(-1082)AA was positively associated with P. falciparum placenta infection at delivery. As a consequence, the IL-10(-819)*T allele (in CT and TT genotypes) as well as the IL-10(-1082)*A/IL-10(-819)*T/IL-10(-592)*A haplotype (one or two copies) in which it is included, were related to an increased risk for anaemia in newborns. The maternal IL-10(-1082)AA genotype was related to high IgG levels to MSP-2/3D7 and AMA-1 in mothers and newborns, respectively. The IL-13 gene polymorphism was only involved in the newborn's antibody response to AMA-1. CONCLUSION: These data revealed that IL-4 and IL-10 maternal gene polymorphisms are likely to play a role in the regulation of biological parameters in pregnant women at delivery (anaemia, P. falciparum placenta infection) and in newborns (anaemia). Moreover, IL-4, IL-10 and IL-13 maternal gene polymorphisms were related to IgG responses to MSP-1, MSP-2/3D7 and MSP-2/FC27 in mothers as well as to AMA-1 in newborns.

Combined polymorphisms involving the IgG heavy chain and Fc gamma receptors among Fulani and non-Fulani in Benin: implications for the natural protection of young Fulani against Plasmodium falciparum malaria infections.

A decreased susceptibility of Fulani populations to malaria infections has been shown in Africa. A previous longitudinal cohort study conducted in the Atacora region of northern Benin showed a high merozoite-phagocytosis capacity in young Fulani. Here, we explored the combined polymorphisms in the constant region of the IgG3 heavy chain (presence/absence of the G3m6 allotype) and in Fc gamma receptors (FcγRs) as potentially involved in the natural protection against malaria of young Fulani in Benin. An active malaria follow-up was conducted among individuals from Fulani, Bariba, Otamari and Gando ethnic groups living in sympatry in Atacora, over the full malaria transmission season. FcγRIIA 131R/H (rs1801274), FcγRIIC C/T (rs3933769) and FcγRIIIA 176F/V (rs396991) were determined using the TaqMan method; FcγRIIIB NA1/NA2 was assessed by polymerase chain reaction (PCR) using allele-specific primers and G3m6 using allotype by PCR-RFLP. Individual carriage of G3m6 (+) was associated with an increased risk of Pf malaria infection (logistic multivariate regression model (lmrm), OR = 2.25, 95% CI = 1.06;4.74, P = 0.034). Combined haplotype G3m6 (+) - FcγRIIA 131H - FcγRIIC T - FcγRIIIA 176F - FcγRIIIB NA2 was also associated with an increased risk of Pf malaria infection (lmrm, OR = 13.01, 95% CI = 1.69;99.76, P = 0.014). G3m6 (-), FcγRIIA 131R and FcγRIIIB NA1 were more prevalent in young Fulani (P = 0.002, P < 0.001 and P = 0.049, respectively), while no Fulani presented the combined G3m6 (+) - FcγRIIA 131H - FcγRIIC T - FcγRIIIA 176F - FcγRIIIB NA2 haplotype that was carried by a majority of infected children. Our results highlight the combined factors G3m6 - FcγR as potentially involved in the merozoite-phagocytosis capacity and in the natural protection of young Fulani individuals against P. falciparum malaria in Benin.

Evidence for Epistatic Interaction between HLA-G and LILRB1 in the Pathogenesis of Nonsegmental Vitiligo.

Vitiligo is the most frequent cause of depigmentation worldwide. Genetic association studies have discovered about 50 loci associated with disease, many with immunological functions. Among them is HLA-G, which modulates immunity by interacting with specific inhibitory receptors, mainly LILRB1 and LILRB2. Here we investigated the LILRB1 and LILRB2 association with vitiligo risk and evaluated the possible role of interactions between HLA-G and its receptors in this pathogenesis. We tested the association of the polymorphisms of HLA-G, LILRB1, and LILRB2 with vitiligo using logistic regression along with adjustment by ancestry. Further, methods based on the multifactor dimensionality reduction (MDR) approach (MDR v.3.0.2, GMDR v.0.9, and MB-MDR) were used to detect potential epistatic interactions between polymorphisms from the three genes. An interaction involving rs9380142 and rs2114511 polymorphisms was identified by all methods used. The polymorphism rs9380142 is an HLA-G 3'UTR variant (+3187) with a well-established role in mRNA stability. The polymorphism rs2114511 is located in the exonic region of LILRB1. Although no association involving this SNP has been reported, ChIP-Seq experiments have identified this position as an EBF1 binding site. These results highlight the role of an epistatic interaction between HLA-G and LILRB1 in vitiligo pathogenesis.

Susceptibility to malaria in fulani, Bariba, Otamari and gando individuals living in sympatry in Benin: Role of opsonizing antibodies to Plasmodium falciparum merozoites.

Objectives: Fulani in Africa are known to be less susceptible to Plasmodium falciparum (Pf) malaria. This study explored a potential involvement of antibody-mediated merozoite phagocytosis mechanism in this natural protection against malaria. Methods: Before the start of the malaria transmission season (MTS) in Benin, the functionality of antibodies against Pf merozoites was determined by the opsonic phagocytosis (OP) assay in plasma samples from Fulani, Bariba, Otamari and Gando groups. These individuals were actively followed-up for malaria detection from the beginning to the end of MTS. Anti-GLURP Immunoglobulin G antibody quantification, malaria Rapid Diagnostic Test (RDT) and spleen palpation were performed before and after MTS. Results: In Bariba, Otamari and Gando, but not in Fulani, plasma from adults promoted higher levels of OP than the children (P = 0.003; P = 0.012; P = 0.031 and P = 0.122). A high proportion of Fulani children had higher OP and anti-GLURP (P < 0.0001) antibody levels as compared to non-Fulani children; whereas this was not observed for Fulani adults (P = 0.223). High OP levels before MTS were significantly related to negative RDT after MTS (P = 0.011). Conclusion: Our results highlight the ability of opsonizing antibodies to potentially enhance natural protection of young Fulani individuals against Pf malaria in Benin.

ANRIL, a long, noncoding RNA, is an unexpected major hotspot in GWAS.

A large noncoding RNA called ANRIL (for antisense noncoding RNA in the INK4 locus) has been identified within the p15/CDKN2B-p16/CDKN2A-p14/ARF gene cluster. While the exact role of ANRIL awaited further elucidation, common disease genomewide association studies (GWAS) have surprisingly identified the ANRIL gene as a genetic susceptibility locus shared associated by coronary disease, intracranial aneurysm and also type 2 diabetes. Expression studies have confirmed the coregulation of p15/CDKN2B, p16/CDKN2A, p14/ARF, and ANRIL. Among the cluster, ANRIL expression showed the strongest association with the multiple phenotypes linked to the 9p21.3 region. More recent GWAS also identified ANRIL as a risk locus for gliomas and basal cell carcinomas in accordance with the princeps observation. Moreover, a mouse model has confirmed the pivotal role of ANRIL in regulation of CDKN2A/B expression through a cis-acting mechanism and its implication in proliferation and senescence. The implication of ANRIL in cellular aging has provided an attractive unifying hypothesis to explain its association with various susceptibility risk factors. ANRIL identification emphasizes the underestimated role of long noncoding RNAs. Many GWAS have identified trait-associated SNPs that felt in noncoding genomic regions. It is conceivable to anticipate that long, noncoding RNAs will map to many of these "gene deserts."

Multiple overlapping risk factors for childhood wheeze among children in Benin.

BACKGROUND: The African continent is currently facing an epidemiological transition characterized by a shift from communicable to non-communicable diseases. Prominent amongst the latter are allergies and asthma. In that context, wheeze has multiple potential contributory factors that could include some of the endemic helminth infections, as well as environmental exposures, such as household air pollution. We sought to determine the relative importance of these risk factors among children in Benin. METHODS: We included 964 children aged 6-14 years living in the commune of Comé, south-west Benin. All children were participants in the longitudinal monitoring cohort of the DeWorm3 trial designed to evaluate multiple rounds of community mass treatment with albendazole for interruption of the transmission of soil transmitted helminths (STH). We administered a standard ISAAC questionnaire to determine the presence of wheeze. In addition, we assessed exposure to household air pollution and to other potential allergy-inducing factors, dietary intake and anthropometry. Using STH infection status assessed at the pretreatment baseline timepoint, we used multivariate statistical modelling, controlling for covariates, to investigate associations between wheeze and the different factors measured. RESULTS: The prevalence of wheezing history was 5.2%, of current wheezing was 4.6% and of severe wheezing was 3.1%, while STH infections were found in 5.6% of children. These profiles did not vary as a function of either age or gender. Infection with Ascaris lumbricoides, but not hookworm species, was significantly associated with both current wheeze (adjusted Odds Ratio (aOR) = 4.3; 95% CI [1.5-12.0]) and severe wheeze (aOR = 9.2; 95% CI [3.1-27.8]). Significant positive associations with current wheeze, independent of each other and of STH infection status, were also found for (i) use of open cookstoves (aOR = 3.9; 95% CI [1.3-11.5]), (ii) use of palm cakes for fire lighting (aOR = 3.4; 95% CI [1.1-9.9]), (iii) contact with domestic animals and/or rodents (aOR = 2.5; 95% CI [1.1-6.0]), (iv) being overweight (aOR = 9.7; 95% CI [1.7-55.9]). Use of open cookstoves and being overweight were also independent risk factors for severe wheeze (aOR = 3.9; 95% CI [1.1-13.7]) and aOR = 10.3; 95% CI [1.8-60.0], respectively). CONCLUSIONS: Children infected with A. lumbricoides appear to be at elevated risk of wheeze. Deworming may be an important intervention to reduce these symptoms. Improving cooking methods to reduce household air pollution, modifying dietary habits to avoid overweight, and keeping animals out of the house are all additional measures that could also contribute to reducing childrens' risk of wheeze. Policymakers in LMIC should consider tailoring public health measures to reflect the importance of these different risk factors.

Genetic diversity of the LILRB1 and LILRB2 coding regions in an admixed Brazilian population sample.

Leukocyte immunoglobulin (Ig)-like receptors (LILR) LILRB1 and LILRB2 may play a pivotal role in maintaining self-tolerance and modulating the immune response through interaction with classical and nonclassical HLA molecules. Although both diversity and natural selection patterns over HLA genes have been extensively evaluated, little information is available concerning the genetic diversity and selection signatures on the LILRB1/2 regions. Therefore, we identified the LILRB1/2 genetic diversity using next-generation sequencing in a population sample from São Paulo State, Brazil. We identified 58 LILRB1 Single Nucleotide Variants (SNVs), which gave rise to 13 haplotypes, and 41 LILRB2 SNVs arranged into 11 haplotypes. Although we may not exclude as a possible effect of population structure, we found evidence of either positive or purifying selection on LILRB1/2 coding regions. Some residues in both proteins showed to be under the effect of positive selection, suggesting that amino acid replacements in these proteins resulted in beneficial functional changes. Finally, we have revealed that allelic variation (six and five amino acid exchanges in LILRB1 and LILRB2, respectively) affects the structure and/or stability of both molecules. Nonetheless, LILRB2 has shown higher average stability, with no D1/D2 residue affecting protein structure. Overall, our findings demonstrate that LILRB1 and LILRB2 are as polymorphic as HLA class Ib genes and provide strong evidence supporting the directional selection regime hypothesis.

Placental Malaria is Associated with Higher LILRB2 Expression in Monocyte Subsets and Lower Anti-Malarial IgG Antibodies During Infancy.

Background: Placental malaria (PM) is associated with a higher susceptibility of infants to Plasmodium falciparum (Pf) malaria. A hypothesis of immune tolerance has been suggested but no clear explanation has been provided so far. Our goal was to investigate the involvement of inhibitory receptors LILRB1 and LILRB2, known to drive immune evasion upon ligation with pathogen and/or host ligands, in PM-induced immune tolerance. Method: Infants of women with or without PM were enrolled in Allada, southern Benin, and followed-up for 24 months. Antibodies with specificity for five blood stage parasite antigens were quantified by ELISA, and the frequency of immune cell subsets was quantified by flow cytometry. LILRB1 or LILRB2 expression was assessed on cells collected at 18 and 24 months of age. Findings: Infants born to women with PM had a higher risk of developing symptomatic malaria than those born to women without PM (IRR=1.53, p=0.040), and such infants displayed a lower frequency of non-classical monocytes (OR=0.74, p=0.01) that overexpressed LILRB2 (OR=1.36, p=0.002). Moreover, infants born to women with PM had lower levels of cytophilic IgG and higher levels of IL-10 during active infection. Interpretation: Modulation of IgG and IL-10 levels could impair monocyte functions (opsonisation/phagocytosis) in infants born to women with PM, possibly contributing to their higher susceptibility to malaria. The long-lasting effect of PM on infants' monocytes was notable, raising questions about the capacity of ligands such as Rifins or HLA-I molecules to bind to LILRB1 and LILRB2 and to modulate immune responses, and about the reprogramming of neonatal monocytes/macrophages.