Measuring the efficiency of purging by non-random mating in human populations.

Human populations harbour a high concentration of deleterious genetic variants. Here, we tested the hypothesis that non-random mating practices affect the distribution of these variants, through exposure in the homozygous state, leading to their purging from the population gene pool. To do so, we produced whole-genome sequencing data for two pairs of Asian populations exhibiting different alliance rules and rates of inbreeding, but with similar effective population sizes. The results show that populations with higher rates of inbred matings do not purge deleterious variants more efficiently. Purging therefore has a low efficiency in human populations, and different mating practices lead to a similar mutational load.Keywords: purifying selection, kinship, mate choice, inbreeding, genomics, deleterious variants, mutational load.

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.

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.

Splenic clearance of rigid erythrocytes as an inherited mechanism for splenomegaly and natural resistance to malaria.

BACKGROUND: In malaria-endemic areas, subjects from specific groups like Fulani have a peculiar protection against malaria, with high levels of IgM but also frequent anaemia and splenomegaly. The mechanisms underlying this phenotype remain elusive. METHODS: In a cohort study set up in Benin, West Africa, after a careful evaluation of malaria-related phenotypes, we measured the deformability of circulating erythrocytes in genetically distinct groups (including Fulani) living in sympatry, using ektacytometry and microsphiltration, a mimic of how the spleen clears rigid erythrocytes. Heritability of erythrocytes deformability was calculated, followed by a genome-wide association study (GWAS) of the same phenotype. FINDINGS: Compared to non-Fulani, Fulani displayed a higher deformability of circulating erythrocytes, pointing to an enhanced clearance of rigid erythrocytes by the spleen. This phenotype was observed in individuals displaying markers of Plasmodium falciparum infection. The heritability of this new trait was high, with a strong multigenic component. Five of the top 10 genes selected by a population structure-adjusted GWAS, expressed in the spleen, are potentially involved in splenic clearance of erythrocytes (CHERP, MB, PALLD, SPARC, PDE10A), through control of vascular tone, collagen synthesis and macrophage activity. INTERPRETATION: In specific ethnic groups, genetically-controlled processes likely enhance the innate retention of infected and uninfected erythrocytes in the spleen, explaining splenomegaly, anaemia, cryptic intrasplenic parasite loads, hyper-IgM, and partial protection against malaria. Beyond malaria-related phenotypes, inherited splenic hyper-filtration of erythrocytes may impact the pathogenesis of other hematologic diseases. FUNDING: ANR, National Geographic Society, IMEA, IRD, and Région Ile-de-France.

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.

High level of soluble human leukocyte antigen (HLA)-G at beginning of pregnancy as predictor of risk of malaria during infancy.

Placental malaria has been associated with an immune tolerance phenomenon and a higher susceptibility to malaria infection during infancy. HLA-G is involved in fetal maternal immune tolerance by inhibiting maternal immunity. During infections HLA-G can be involved in immune escape of pathogens by creating a tolerogenic environment. Recent studies have shown an association between the risk of malaria and HLA-G at both genetic and protein levels. Moreover, women with placental malaria have a higher probability of giving birth to children exhibiting high sHLA-G, independently of their own level during pregnancy. Our aim was to explore the association between the level of maternal soluble HLA-G and the risk of malaria infection in their newborns. Here, 400 pregnant women and their children were actively followed-up during 24 months. The results show a significant association between the level of sHLA-G at the first antenatal visit and the time to first malaria infection during infancy adjusted to the risk of exposure to vector bites (aHR = 1.02, 95%CI [1.01-1.03], p = 0.014). The level of sHLA-G is a significant predictor of the occurrence of malaria infection during infancy consistent with the hypothesis that mother sHLA-G could be a biomarker of malaria susceptibility in children.

The genetic diversity within the 1.4 kb HLA-G 5' upstream regulatory region moderately impacts on cellular microenvironment responses.

The HLA-G 5'URR extending 1.4 kb from the ATG presents a unique set of regulatory elements among HLA genes. Several variable sites have been described that coincide with or are close to these elements, thus HLA-G 5'URR polymorphism might influence the HLA-G expression level. We cloned the ten most frequent HLA-G 5'URR haplotypes to evaluate their activity on a luciferase reporter gene in HLA-G+ cell lines (JEG-3/choriocarcinoma and FON+/melanoma). We also investigated associations between the plasma HLA-G (sHLA-G) levels and the HLA-G 5'URR variability in 157 healthy individuals. Cell lines were transfected with pGL3-Basic vector constructions containing HLA-G 5'URR sequences. The G010101a (in JEG-3) and G010101b (in FON+) haplotypes exhibited higher promoter activity, whereas the G010101d (in JEG-3) and G010102a (in FON+) haplotypes exhibited lower promoter activity. In the presence of HLA-G inducers (interferon-ß and progesterone) or repressors (cyclopamine) HLA-G promoter activity was modulated, but certain haplotypes exhibited differential responses. No strict association was observed between plasma sHLA-G levels and the 5'URR haplotypes or genotypes; however, the G010101b haplotype was underrepresented among HLA-G-negative plasmas. Therefore, the HLA-G 5'URR polymorphism may have an impact on the modulation of HLA-G gene expression, but alone provides a limited predictive value for sHLA-G levels in vivo.

Inherited GINS1 deficiency underlies growth retardation along with neutropenia and NK cell deficiency.

Inborn errors of DNA repair or replication underlie a variety of clinical phenotypes. We studied 5 patients from 4 kindreds, all of whom displayed intrauterine growth retardation, chronic neutropenia, and NK cell deficiency. Four of the 5 patients also had postnatal growth retardation. The association of neutropenia and NK cell deficiency, which is unusual among primary immunodeficiencies and bone marrow failures, was due to a blockade in the bone marrow and was mildly symptomatic. We discovered compound heterozygous rare mutations in Go-Ichi-Ni-San (GINS) complex subunit 1 (GINS1, also known as PSF1) in the 5 patients. The GINS complex is essential for eukaryotic DNA replication, and homozygous null mutations of GINS component-encoding genes are embryonic lethal in mice. The patients' fibroblasts displayed impaired GINS complex assembly, basal replication stress, impaired checkpoint signaling, defective cell cycle control, and genomic instability, which was rescued by WT GINS1. The residual levels of GINS1 activity reached 3% to 16% in patients' cells, depending on their GINS1 genotype, and correlated with the severity of growth retardation and the in vitro cellular phenotype. The levels of GINS1 activity did not influence the immunological phenotype, which was uniform. Autosomal recessive, partial GINS1 deficiency impairs DNA replication and underlies intra-uterine (and postnatal) growth retardation, chronic neutropenia, and NK cell deficiency.

Soluble human leukocyte antigen -G during pregnancy and infancy in Benin: Mother/child resemblance and association with the risk of malaria infection and low birth weight.

Human leukocyte antigen (HLA) G is a tolerogenic molecule involved in the maternal-fetal immune tolerance phenomenon. Its expression during some infectious diseases leading to immune evasion has been established. A first study conducted in Benin has shown that the production of soluble HLA-G (sHLA-G) during the first months of life is strongly correlated with the maternal level at delivery and associated with low birth weight and malaria. However sHLA-G measurements during pregnancy were not available for mothers and furthermore, to date the evolution of sHLA-G in pregnancy is not documented in African populations. To extend these previous findings, between January 2010 and June 2013, 400 pregnant women of a malaria preventive trial and their newborns were followed up in Benin until the age of 2 years. Soluble HLA-G was measured 3 times during pregnancy and repeatedly during the 2 years follow-up to explore how sHLA-G evolved and the factors associated. During pregnancy, plasma levels of sHLA-G remained stable and increased significantly at delivery (p<0.001). Multigravid women seemed to have the highest levels (p = 0.039). In infants, the level was highest in cord blood and decreased before stabilizing after 18 months (p<0.001). For children, a high level of sHLA-G was associated with malaria infection during the follow-up (p = 0.02) and low birth weight (p = 0.06). The mean level of sHLA-G during infancy was strongly correlated with the mother's level during pregnancy (<0.001), and not only at delivery. Moreover, mothers with placental malaria infection had a higher probability of giving birth to a child with a high level of sHLA-g (p = 0.006). High sHLA-G levels during pregnancy might be associated with immune tolerance related to placental malaria. Further studies are needed but this study provides a first insight concerning the potential role of sHLA-G as a biomarker of weakness for newborns and infants.

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.

Inborn errors of the development of human natural killer cells.

PURPOSE OF REVIEW: Inborn errors of human natural killer (NK) cells may affect the development of these cells, their function, or both. There are two broad categories of genetic defects of NK cell development, depending on whether the deficiency is apparently specific to NK cells or clearly affects multiple hematopoietic lineages. We review here recent progress in the genetic dissection of these NK deficiencies (NKDs). RECENT FINDINGS: Patients with severe combined immunodeficiencies bearing mutations of adenosine deaminase, adenylate kinase 2, interleukin-2 receptor gamma chain, and Janus kinase 3 genes present NKDs and are prone to a broad range of infections. Patients with GATA binding protein 2 deficiency are susceptible to both mycobacterial and viral infections, and display NKDs and a lack of monocytes. Rare patients with mini chromosomal maintenance 4 deficiency display an apparently selective NKD associated with viral infections, but they also display various nonhematopoietic phenotypes, including adrenal insufficiency and growth retardation. SUMMARY: These studies have initiated a genetic dissection of the development of human NK cells. Further studies are warranted, including the search for genetic causes of NKD in particular. This research may lead to the discovery of molecules specifically controlling the development of NK cells and to improvements in our understanding of the hitherto elusive function of these cells in humans.

Human RHOH deficiency causes T cell defects and susceptibility to EV-HPV infections.

Epidermodysplasia verruciformis (EV) is a rare genetic disorder characterized by increased susceptibility to specific human papillomaviruses, the betapapillomaviruses. These EV-HPVs cause warts and increase the risk of skin carcinomas in otherwise healthy individuals. Inactivating mutations in epidermodysplasia verruciformis 1 (EVER1) or EVER2 have been identified in most, but not all, patients with autosomal recessive EV. We found that 2 young adult siblings presenting with T cell deficiency and various infectious diseases, including persistent EV-HPV infections, were homozygous for a mutation creating a stop codon in the ras homolog gene family member H (RHOH) gene. RHOH encodes an atypical Rho GTPase expressed predominantly in hematopoietic cells. Patients' circulating T cells contained predominantly effector memory T cells, which displayed impaired TCR signaling. Additionally, very few circulating T cells expressed the ß7 integrin subunit, which homes T cells to specific tissues. Similarly, Rhoh-null mice exhibited a severe overall T cell defect and abnormally small numbers of circulating ß7-positive cells. Expression of the WT, but not of the mutated RHOH, allele in Rhoh-/- hematopoietic stem cells corrected the T cell lymphopenia in mice after bone marrow transplantation. We conclude that RHOH deficiency leads to T cell defects and persistent EV-HPV infections, suggesting that T cells play a role in the pathogenesis of chronic EV-HPV infections.

Partial MCM4 deficiency in patients with growth retardation, adrenal insufficiency, and natural killer cell deficiency.

Natural killer (NK) cells are circulating cytotoxic lymphocytes that exert potent and nonredundant antiviral activity and antitumoral activity in the mouse; however, their function in host defense in humans remains unclear. Here, we investigated 6 related patients with autosomal recessive growth retardation, adrenal insufficiency, and a selective NK cell deficiency characterized by a lack of the CD56(dim) NK subset. Using linkage analysis and fine mapping, we identified the disease-causing gene, MCM4, which encodes a component of the MCM2-7 helicase complex required for DNA replication. A splice-site mutation in the patients produced a frameshift, but the mutation was hypomorphic due to the creation of two new translation initiation methionine codons downstream of the premature termination codon. The patients' fibroblasts exhibited genomic instability, which was rescued by expression of WT MCM4. These data indicate that the patients' growth retardation and adrenal insufficiency likely reflect the ubiquitous but heterogeneous impact of the MCM4 mutation in various tissues. In addition, the specific loss of the NK CD56(dim) subset in patients was associated with a lower rate of NK CD56(bright) cell proliferation, and the maturation of NK CD56(bright) cells toward an NK CD56(dim) phenotype was tightly dependent on MCM4-dependent cell division. Thus, partial MCM4 deficiency results in a genetic syndrome of growth retardation with adrenal insufficiency and selective NK deficiency.

Complementation of a pathogenic IFNGR2 misfolding mutation with modifiers of N-glycosylation.

Germline mutations may cause human disease by various mechanisms. Missense and other in-frame mutations may be deleterious because the mutant proteins are not correctly targeted, do not function correctly, or both. We studied a child with mycobacterial disease caused by homozygosity for a novel in-frame microinsertion in IFNGR2. In cells transfected with the mutant allele, most of the interferon gamma receptor 2 (IFN-gamma R2) protein was retained within the cell, and that expressed on the cell surface had an abnormally high molecular weight (MW). The misfolding mutation was not gain-of-glycosylation, as it created no new N-glycosylation site. The mutant IFNGR2 allele was null, as the patient's cells did not respond to IFN-gamma. Based on the well-established relationship between protein N-glycosylation and protein quality control processes, we tested 29 compounds affecting maturation by N-glycosylation in the secretory pathway. Remarkably, up to 13 of these compounds reduced the MW of surface-expressed mutant IFN-gamma R2 molecules and restored cellular responsiveness to IFN-gamma. Modifiers of N-glycosylation may therefore complement human cells carrying in-frame and misfolding, but not necessarily gain-of-glycosylation, mutations in genes encoding proteins subject to trafficking via the secretory pathway. Some of these compounds are available for clinical use, paving the way for clinical trials of chemical complementation for various human genetic traits.

Familial NK cell deficiency associated with impaired IL-2- and IL-15-dependent survival of lymphocytes.

We previously reported the clinical phenotype of two siblings with a novel inherited developmental and immunodeficiency syndrome consisting of severe intrauterine growth retardation and the impaired development of specific lymphoid lineages, including transient CD8 alphabeta T lymphopenia and a persistent lack of blood NK cells. We describe here the elucidation of a plausible underlying pathogenic mechanism, with a cellular phenotype of impaired survival of both fresh and herpesvirus saimiri-transformed T cells, in the surviving child. Clearly, NK cells could not be studied. However, peripheral blood T lymphocytes displayed excessive apoptosis ex vivo. Moreover, the survival rates of CD4 and CD8 alphabeta T cell blasts generated in vitro, and herpesvirus saimiri-transformed T cells cultured in vitro, were low, but not nil, following treatment with IL-2 and IL-15. In contrast, Fas-mediated activation-induced cell death was not enhanced, indicating a selective excess of cytokine deprivation-mediated apoptosis. In keeping with the known roles of IL-2 and IL-15 in the development of NK and CD8 T cells in the mouse model, these data suggest that an impaired, but not abolished, survival response to IL-2 and IL-15 accounts for the persistent lack of NK cells and the transient CD8 alphabeta T lymphopenia documented in vivo. Impaired cytokine-mediated lymphocyte survival is likely to be the pathogenic mechanism underlying this novel form of inherited and selective NK deficiency in humans.

A novel primary immunodeficiency with specific natural-killer cell deficiency maps to the centromeric region of chromosome 8.

We describe four children with a novel primary immunodeficiency consisting of specific natural-killer (NK) cell deficiency and susceptibility to viral diseases. One child developed an Epstein-Barr virus-driven lymphoproliferative disorder; two others developed severe respiratory illnesses of probable viral etiology. The four patients are related and belong to a large inbred kindred of Irish nomadic descent, which suggests autosomal recessive inheritance of this defect. A genomewide scan identified a single 12-Mb region on chromosome 8p11.23-q11.21 that was linked to this immunodeficiency (maximum LOD score 4.51). The mapping of the disease-causing genomic region paves the way for the identification of a novel pathway governing NK cell differentiation in humans.