Polymorphic variants p.66L>R/H (g.7081T>G/A; rs10127939) and p.176F>V (g.10872T>G; rs396991) in FCGR3A (CD16A) have been associated with defects in cytotoxic function of natural killer (NK) cells in humans. Genotyping of these variants in genomic DNA has been ambiguous because of high degree of homology between FCGR3A and FCGR3B. We designed a strategy to genotype these polymorphisms and to evaluate their effects on NK cells' cytotoxic activity. One hundred and fifteen individuals from different geographical regions of Colombia were included. Specific primers were designed to amplify FCGR3A exons 4 and 5 encompassing g.7081T>G/A and g.10872T>G by long-range and nested polymerase chain reaction and sequencing. The binding of different monoclonal antibodies to CD16A and NK antibody-dependent cellular cytotoxicity (ADCC) were evaluated. We demonstrate that amplifying and sequencing FCGR3A allows genotyping of g.7081T>G/A and g.10872T>G without interference from FCGR3B. Allele frequencies in our population were as follows: 7081T = 0.895, 7081G = 0.065, 7081 A = 0.039, 10872T = 0.673, and 10872G = 0.326. We also observed linkage disequilibrium between variants 7081T and 10872G. Interestingly, 176FF variant affected the reactivity of MEM154 monoclonal antibody against CD16A, but it did not affect ADCC. Our studies aimed to determine whether clinical association exists between these polymorphisms and NK cell function defects in patients with compatible phenotypes.
Although phlebotomine sand flies breeding sites have been identified and recorded by several studies, the microhabitats exploited by these insects remain little-known and hard to find. In this context, the difficulty of finding immature stages, and the limited number of taxonomic studies to identify immature stages of phlebotomine sand flies, are considered the major obstacles when attempting a complete inventory of Lutzomyia species. The objective of this study is to validate Cytochrome Oxidase I (Barcode region) as a marker for the identification of immature stages of Lutzomyia species recovered from natural breeding sites in Colombia. Among 142 collected sand flies, 18 immature individuals that did not complete their life cycle were identified to species level through sequencing of the COI gene. Values of K2P genetic distance between 0.002–0.031 allowed the identification of larvae at species level. The bootstrap support values (96%) in the Neighbor-Joining dendrogram were consistent for the majority of the established MOTUS of Lutzomyia atroclavata, Lutzomyia micropyga, Lutzomyia serrana, Lutzomyia cayennensis, Lutzomyia rangeliana, Lutzomyia shannoni and some species of the genus Brumptomyia. The COI gene is validated as a marker for the identification of immature stages of the genus Lutzomyia.
Familial Hemophagocytic Lymphohistiocytosis type 2 (FHL2) results from mutations in PRF1. We described two unrelated individuals who presented with FHL, in whom severely impaired NK cytotoxicity and decrease perforin expression was observed. DNA sequencing of PRF1 demonstrated that both were not only heterozygous for the p.54R > C/91A > V haplotype but also presented with the novel variant p.47G > V at the perforin protein. Perforin mRNA was found to be increased in a individual with that genotype. A carrier of the novel variant also demonstrated altered perforin mRNA and protein expression. Phylogenetic analysis and multiple alignments with perforin orthologous demonstrated a high level of conservation at Gly47. PolyPhen-2 and PROVEAN predicted p.47G > V to be "probably damaging" and "deleterious", respectively. A thermodynamic analysis showed that this variant was highly stabilizing, decreasing the protein internal energy. The ab initio perforin molecular modeling indicated that Gly47 is buried inside the hydrophobic core of the MACPF domain, which is crucial for the lytic pore formation and protein oligomerization. After the in silico induction of the p.47G > V mutation, Val47 increased the interactions with the surrounding amino acids due to its size and physical properties, avoiding a proper conformational change of the domain. To our knowledge, this is the first description supporting that p.47G > V is a pathogenic variant that in conjunction with p.54R > C/91A > V might result in the clinical phenotype of FHL2.
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