CFTR haplotype distribution in the Brazilian Western Amazonian region

Two hundred twenty-one individuals from four groups located around the Brazilian town of Porto Velho, Rond?nia, were studied in relation to four sites located within or near the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The allele frequencies, when considered individually, do not depart markedly from frequencies obtained from other populations of mainly European descent. However, when haplotypes were estimated, two of the groups departed markedly from other Brazilian and non-Brazilian samples. This finding is probably related to the complex multiethnic origin of these groups.     

Worldwide genetic relationships among Francisella tularensis isolates determined by multiple-locus variable-number tandem repeat analysis

The intracellular bacterium Francisella tularensis is the causative agent of tularemia and poses a serious threat as an agent of bioterrorism. We have developed a highly effective molecular subtyping system from 25 variable-number tandem repeat (VNTR) loci. In our study, multiple-locus VNTR analysis (MLVA) was used to analyze genetic relationships and potential population structure within a global collection of 192 F. tularensis isolates, including representatives from each of the four subspecies. The VNTR loci displayed between 2 and 31 alleles with Nei's diversity values between 0.05 and 0.95. Neighbor-joining cluster analysis of VNTR data revealed 120 genotypes among the 192 F. tularensis isolates, including accurate subspecies identification. F. tularensis subsp. tularensis (type A) isolates showed great diversity at VNTR loci, while F. tularensis subsp. holarctica (type B) isolates showed much lower levels despite a much broader geographical prevalence. The resolution of two distinct clades within F. tularensis subsp. tularensis (designated A.I and A.II) revealed a previously unrecognized genetic division within this highly virulent subspecies. F. tularensis subsp. holarctica appears to have recently spread globally across continents from a single origin, while F. tularensis subsp. tularensis has a long and complex evolutionary history almost exclusively in North America. The sole non-North American type A isolates (Slovakian) were closely related to the SCHU S4 strain. Significant linkage disequilibrium was detected among VNTR loci of F. tularensis consistent with a clonal population structure. Overall, this work greatly augments the study of tularemia ecology and epidemiology, while providing a framework for future forensic analysis of F. tularensis isolates.     

A molecular genetic analysis of the mutations in the exons of the CFTR gene in cystic fibrosis patients in Ukraine

The results of DNA analysis of major mutations in CFTR gene in 260 families from Ukraine with high risk of cystic fibrosis are presented. The perspectives of molecular diagnosis as a main tool for cystic fibrosis prevention are discussed.     

Identification of a region of strong discrimination in the pore of CFTR

The variety of methods used to identify the structural determinants of anion selectivity in the cystic fibrosis transmembrane conductance regulator Cl(-) channel has made it difficult to assemble the data into a coherent framework that describes the three-dimensional structure of the pore. Here, we compare the relative importance of sites previously studied and identify new sites that contribute strongly to anion selectivity. We studied Cl(-) and substitute anions in oocytes expressing wild-type cystic fibrosis transmembrane conductance regulator or 12-pore-domain mutants to determine relative permeability and relative conductance for 9 monovalent anions and 1 divalent anion. The data indicate that the region of strong discrimination resides between T338 and S341 in transmembrane 6, where mutations affected selectivity between Cl(-) and both large and small anions. Mutations further toward the extracellular end of the pore only strongly affected selectivity between Cl(-) and larger anions. Only mutations at S341 affected selectivity between monovalent and divalent anions. The data are consistent with a narrowing of the pore between the extracellular end and a constriction near the middle of the pore.     

Worldwide genetic differentiation in the common fouling barnacle,Amphibalanus amphitrite

Amphibalanus amphitrite is a common fouling barnacle distributed globally in tropical and subtropical waters. In the present study, the genetic (mitochondrial cytochrome oxidase subunit I) and morphological differentiation in A. amphitrite from 25 localities around the world were investigated. The results revealed three clades within A. amphitrite with a genetic divergence of ~ 4% among clades, whereas there were no diagnostic morphological differences among clades. Clade 1 is widely distributed in both temperate and tropical waters, whereas Clade 3 is currently restricted to the tropical region. The deep divergence among clades suggests historical isolation within A. amphitrite; thus, the present geographical overlaps are possibly a result of the combined effects of rising sea level and human-mediated dispersals. This study highlights the genetic differentiation that exists in a common, widely distributed fouling organism with great dispersal potential; future antifouling research should take into account the choice of lineages.     

Refined genetic analysis of the region II che mutants in Salmonella typhimurium

Summary From a detailed complementation analysis of the region II che mutants of Salmonella typhimurium, we have located five che genes, cheA, cheW, cheR, cheB, and cheY. We have shown that corrections are required in the previous assignment of the mutations in four strains: both SL2514 and SL2515 which have been reported to be cheY mutants are cheR mutants, SL2539 is not a cheA but a cheW mutant, and ST171 which has been reported to be a cheZ mutant is a double mutant with defects in both cheA and cheB. Since ST171 is the only cheZ mutant so far isolated, the idea that the cheZ gene might play an essential role in chemotaxis in S. typhimurium as in Escherichia coli has lost its experimental basis. Furthermore, a number of deletion mutants in region II resulting from the excision of Tn10 have been isolated and analysed. From these experiments, we propose that the gene order in region II is flaK-flaE-motA-motB-cheA-cheW-cheR-cheB-cheY-flaM-flaC, which is identical with that in E. coli.     

Worldwide genetic structure in 37 genes important in telomere biology

Telomeres form the ends of eukaryotic chromosomes and are vital in maintaining genetic integrity. Telomere dysfunction is associated with cancer and several chronic diseases. Patterns of genetic variation across individuals can provide keys to further understanding the evolutionary history of genes. We investigated patterns of differentiation and population structure of 37 telomere maintenance genes among 53 worldwide populations. Data from 898 unrelated individuals were obtained from the genome-wide scan of the Human Genome Diversity Panel (HGDP) and from 270 unrelated individuals from the International HapMap Project at 716 single-nucleotide polymorphism (SNP) loci. We additionally compared this gene set to HGDP data at 1396 SNPs in 174 innate immunity genes. The majority of the telomere biology genes had low to moderate haplotype diversity (45-85%), high ancestral allele frequencies (>60%) and low differentiation (FST<0.10). Heterozygosity and differentiation were significantly lower in telomere biology genes compared with the innate immunity genes. There was evidence of evolutionary selection in ACD, TERF2IP, NOLA2, POT1 and TNKS in this data set, which was consistent in HapMap 3. TERT had higher than expected levels of haplotype diversity, likely attributable to a lack of linkage disequilibrium, and a potential cancer-associated SNP in this gene, rs2736100, varied substantially in genotype frequency across major continental regions. It is possible that the genes under selection could influence telomere biology diseases. As a group, there appears to be less diversity and differentiation in telomere biology genes than in genes with different functions, possibly due to their critical role in telomere maintenance and chromosomal stability.     

Worldwide genetic relationships of pigs as inferred from X chromosome SNPs

The phylogeography of the porcine X chromosome has not been studied despite the unique characteristics of this chromosome. Here, we genotyped 59 single nucleotide polymorphisms (SNPs) in 312 pigs from around the world, representing 39 domestic breeds and wild boars in 30 countries. Overall, widespread commercial breeds showed the highest heterozygosity values, followed by African and American populations. Structuring, as inferred from F_ST and analysis of molecular variance, was consistently larger in the non‐pseudoautosomal (NPAR) than in the pseudoautosomal regions (PAR). Our results show that genetic relationships between populations can vary widely between the NPAR and the PAR, underscoring the fact that their genetic trajectories can be quite different. NPAR showed an increased commercial‐like genetic component relative to the PAR, probably because human selection processes to obtain individuals with high productive parameters were mediated by introgressing boars rather than sows.     

Analysis of the complete coding region of the CFTR gene in ten Algerian cystic fibrosis families

The spectrum of cystic fibrosis (CF) mutations in the North African population remains poorly known. In order to offer an effective diagnostic service and to determine accurate risk estimates, we decided to identify the CF mutations in 10 Algerian CF families. We carried out a chemical-clamp denaturing gradient gel electrophoresis analysis of the CFTR gene and automated direct DNA sequencing. We identified 5 mutations and we characterized 60% of the CF chromosomes. Taking advantage of the homogeneity of the sample, we report clinical features of homozygous CF patients.     

Worldwide genetic variability of the Duffy binding protein: insights into Plasmodium vivax vaccine development

The dependence of Plasmodium vivax on invasion mediated by Duffy binding protein (DBP) makes this protein a prime candidate for development of a vaccine. However, the development of a DBP-based vaccine might be hampered by the high variability of the protein ligand (DBP(II)), known to bias the immune response toward a specific DBP variant. Here, the hypothesis being investigated is that the analysis of the worldwide DBP(II) sequences will allow us to determine the minimum number of haplotypes (MNH) to be included in a DBP-based vaccine of broad coverage. For that, all DBP(II) sequences available were compiled and MNH was based on the most frequent nonsynonymous single nucleotide polymorphisms, the majority mapped on B and T cell epitopes. A preliminary analysis of DBP(II) genetic diversity from eight malaria-endemic countries estimated that a number between two to six DBP haplotypes (17 in total) would target at least 50% of parasite population circulating in each endemic region. Aiming to avoid region-specific haplotypes, we next analyzed the MNH that broadly cover worldwide parasite population. The results demonstrated that seven haplotypes would be required to cover around 60% of DBP(II) sequences available. Trying to validate these selected haplotypes per country, we found that five out of the eight countries will be covered by the MNH (67% of parasite populations, range 48-84%). In addition, to identify related subgroups of DBP(II) sequences we used a Bayesian clustering algorithm. The algorithm grouped all DBP(II) sequences in six populations that were independent of geographic origin, with ancestral populations present in different proportions in each country. In conclusion, in this first attempt to undertake a global analysis about DBP(II) variability, the results suggest that the development of DBP-based vaccine should consider multi-haplotype strategies; otherwise a putative P. vivax vaccine may not target some parasite populations.     

Extent of the selectivity filter conferred by the sixth transmembrane region in the CFTR chloride channel pore

Point mutations within the pore region of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl^? channel have previously been shown to alter the selectivity of the channel between different anions, suggesting that part of the pore may form an anion 'selectivity filter'. However, the full extent of this selectivity filter region and the location of anion binding sites in the pore are currently unclear. As a result, comparisons between CFTR and other classes of Cl^? channel of known structure are difficult. We compare here the effects of point mutations at each of eight consecutive amino acid residues (arginine 334-serine 341) in the crucial sixth transmembrane region (TM6) of CFTR. Anion selectivity was determined using patch-clamp recording from inside-out membrane patches excised from transiently transfected mammalian cell lines. The results suggest that selectivity is predominantly controlled by a single site involving adjacent residues phenylalanine 337 and threonine 338, and that the selectivity conferred by this 'filter' region is modified by anion binding to flanking sites involving the more extracellular arginine 334 and the more intracellular serine 341. Other residues within this part of the pore play only minor roles in controlling anion permeability and conductance. Our results support a model in which specific TM6 residues make important contributions to a single, localized anion selectivity filter in the CFTR pore, and also contribute to multiple anion binding sites both within and on either side of the filter region.     

CFTR regulatory region interacts with NBD1 predominantly via multiple transient helices

The regulatory (R) region of the cystic fibrosis transmembrane conductance regulator (CFTR) is intrinsically disordered and must be phosphorylated at multiple sites for full CFTR channel activity, with no one specific phosphorylation site required. In addition, nucleotide binding and hydrolysis at the nucleotide-binding domains (NBDs) of CFTR are required for channel gating. We report NMR studies in the absence and presence of NBD1 that provide structural details for the isolated R region and its interaction with NBD1 at residue-level resolution. Several sites in the R region with measured fractional helical propensity mediate interactions with NBD1. Phosphorylation reduces the helicity of many R-region sites and reduces their NBD1 interactions. This evidence for a dynamic complex with NBD1 that transiently engages different sites of the R region suggests a structural explanation for the dependence of CFTR activity on multiple PKA phosphorylation sites.     

Extent of the selectivity filter conferred by the sixth transmembrane region in the CFTR chloride channel pore

Point mutations within the pore region of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel have previously been shown to alter the selectivity of the channel between different anions, suggesting that part of the pore may form an anion 'selectivity filter'. However, the full extent of this selectivity filter region and the location of anion binding sites in the pore are currently unclear. As a result, comparisons between CFTR and other classes of Cl(-) channel of known structure are difficult. We compare here the effects of point mutations at each of eight consecutive amino acid residues (arginine 334-serine 341) in the crucial sixth transmembrane region (TM6) of CFTR. Anion selectivity was determined using patch-clamp recording from inside-out membrane patches excised from transiently transfected mammalian cell lines. The results suggest that selectivity is predominantly controlled by a single site involving adjacent residues phenylalanine 337 and threonine 338, and that the selectivity conferred by this 'filter' region is modified by anion binding to flanking sites involving the more extracellular arginine 334 and the more intracellular serine 341. Other residues within this part of the pore play only minor roles in controlling anion permeability and conductance. Our results support a model in which specific TM6 residues make important contributions to a single, localized anion selectivity filter in the CFTR pore, and also contribute to multiple anion binding sites both within and on either side of the filter region.