Pierre Darlu

Evolutionary implications of the frequent horizontal transfer of mismatch repair genes.

Mutation and subsequent recombination events create genetic diversity, which is subjected to natural selection. Bacterial mismatch repair (MMR) deficient mutants, exhibiting high mutation and homologous recombination rates, are frequently found in natural populations. Therefore, we have explored the possibility that MMR deficiency emerging in nature has left some imprint in the sequence of bacterial genomes. Comparative molecular phylogeny of MMR genes from natural Escherichia coli isolates shows that, compared to housekeeping genes, individual functional MMR genes exhibit high sequence mosaicism derived from diverse phylogenetic lineages. This apparent horizontal gene transfer correlates with hyperrecombination phenotype of MMR-deficient mutators. The sequence mosaicism of MMR genes may be a hallmark of a mechanism of adaptive evolution that involves modulation of mutation and recombination rates by recurrent losses and reacquisitions of MMR gene functions.

Revisiting Neandertal diversity with a 100,000 year old mtDNA sequence

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Worldwide distribution of NAT2 diversity: Implications for NAT2 evolutionary history

BackgroundThe N-acetyltransferase 2 (NAT2) gene plays a crucial role in the metabolism of many drugs and xenobiotics. As it represents a likely target of population-specific selection pressures, we fully sequenced the NAT2 coding region in 97 Mandenka individuals from Senegal, and compared these sequences to extant data on other African populations. The Mandenka data were further included in a worldwide dataset composed of 41 published population samples (6,727 individuals) from four continental regions that were adequately genotyped for all common NAT2 variants so as to provide further insights into the worldwide haplotype diversity and population structure at NAT2.ResultsThe sequencing analysis of the NAT2 gene in the Mandenka sample revealed twelve polymorphic sites in the coding exon (two of which are newly identified mutations, C345T and C638T), defining 16 haplotypes. High diversity and no molecular signal of departure from neutrality were observed in this West African sample. On the basis of the worldwide genotyping survey dataset, we found a strong genetic structure differentiating East Asians from both Europeans and sub-Saharan Africans. This pattern could result from region- or population-specific selective pressures acting at this locus, as further suggested in the HapMap data by extremely high values of FST for a few SNPs positions in the NAT2 coding exon (T341C, C481T and A803G) in comparison to the empirical distribution of FST values accross the whole 400-kb region of the NAT gene family.ConclusionPatterns of sequence variation at NAT2 are consistent with selective neutrality in all sub-Saharan African populations investigated, whereas the high level of population differentiation between Europeans and East Asians inferred from SNPs could suggest population-specific selective pressures acting at this locus, probably caused by differences in diet or exposure to other environmental signals.

Role of Intraspecies Recombination in the Spread of Pathogenicity Islands within the Escherichia coli Species

Horizontal gene transfer is a key step in the evolution of bacterial pathogens. Besides phages and plasmids, pathogenicity islands (PAIs) are subjected to horizontal transfer. The transfer mechanisms of PAIs within a certain bacterial species or between different species are still not well understood. This study is focused on the High-Pathogenicity Island (HPI), which is a PAI widely spread among extraintestinal pathogenic Escherichia coli and serves as a model for horizontal transfer of PAIs in general. We applied a phylogenetic approach using multilocus sequence typing on HPI-positive and -negative natural E. coli isolates representative of the species diversity to infer the mechanism of horizontal HPI transfer within the E. coli species. In each strain, the partial nucleotide sequences of 6 HPI–encoded genes and 6 housekeeping genes of the genomic backbone, as well as DNA fragments immediately upstream and downstream of the HPI were compared. This revealed that the HPI is not solely vertically transmitted, but that recombination of large DNA fragments beyond the HPI plays a major role in the spread of the HPI within E. coli species. In support of the results of the phylogenetic analyses, we experimentally demonstrated that HPI can be transferred between different E. coli strains by F-plasmid mediated mobilization. Sequencing of the chromosomal DNA regions immediately upstream and downstream of the HPI in the recipient strain indicated that the HPI was transferred and integrated together with HPI–flanking DNA regions of the donor strain. The results of this study demonstrate for the first time that conjugative transfer and homologous DNA recombination play a major role in horizontal transfer of a pathogenicity island within the species E. coli.

Decreasing the effects of horizontal gene transfer on bacterial phylogeny: the Escherichia coli case study.

Phylogenetic reconstructions of bacterial species from DNA sequences are hampered by the existence of horizontal gene transfer. One possible way to overcome the confounding influence of such movement of genes is to identify and remove sequences which are responsible for significant character incongruence when compared to a reference dataset free of horizontal transfer (e.g., multilocus enzyme electrophoresis, restriction fragment length polymorphism, or random amplified polymorphic DNA) using the incongruence length difference (ILD) test of Farris et al. [Cladistics 10 (1995) 315]. As obtaining this whole genome dataset prior to the reconstruction of a phylogeny is clearly troublesome, we have tested alternative approaches allowing the release from such reference dataset, designed for a species with modest level of horizontal gene transfer, i.e., Escherichia coli. Eleven different genes available or sequenced in this work were studied in a set of 30 E. coli reference (ECOR) strains. Either using ILD to test incongruence between each gene against the all remaining (in this case 10) genes in order to remove sequences responsible for significant incongruence, or using just a simultaneous analysis without removals, gave robust phylogenies with slight topological differences. The use of the ILD test remains a suitable method for estimating the level of horizontal gene transfer in bacterial species. Supertrees also had suitable properties to extract the phylogeny of strains, because the way they summarize taxonomic congruence clearly limits the impact of individual gene transfers on the global topology. Furthermore, this work allowed a significant improvement of the accuracy of the phylogeny within E. coli.

On the use of haplotype phylogeny to detect disease susceptibility loci.

BackgroundThe cladistic approach proposed by Templeton has been presented as promising for the study of the genetic factors involved in common diseases. This approach allows the joint study of multiple markers within a gene by considering haplotypes and grouping them in nested clades. The idea is to search for clades with an excess of cases as compared to the whole sample and to identify the mutations defining these clades as potential candidate disease susceptibility sites. However, the performance of this approach for the study of the genetic factors involved in complex diseases has never been studied.ResultsIn this paper, we propose a new method to perform such a cladistic analysis and we estimate its power through simulations. We show that under models where the susceptibility to the disease is caused by a single genetic variant, the cladistic test is neither really more powerful to detect an association nor really more efficient to localize the susceptibility site than an individual SNP testing. However, when two interacting sites are responsible for the disease, the cladistic analysis greatly improves the probability to find the two susceptibility sites. The impact of the linkage disequilibrium and of the tree characteristics on the efficiency of the cladistic analysis are also discussed. An application on a real data set concerning the CARD15 gene and Crohn disease shows that the method can successfully identify the three variant sites that are involved in the disease susceptibility.ConclusionThe use of phylogenies to group haplotypes is especially interesting to pinpoint the sites that are likely to be involved in disease susceptibility among the different markers identified within a gene.

Inferring haplotypes at the NAT2 locus: the computational approach

BackgroundNumerous studies have attempted to relate genetic polymorphisms within the N-acetyltransferase 2 gene (NAT2) to interindividual differences in response to drugs or in disease susceptibility. However, genotyping of individuals single-nucleotide polymorphisms (SNPs) alone may not always provide enough information to reach these goals. It is important to link SNPs in terms of haplotypes which carry more information about the genotype-phenotype relationship. Special analytical techniques have been designed to unequivocally determine the allocation of mutations to either DNA strand. However, molecular haplotyping methods are labour-intensive and expensive and do not appear to be good candidates for routine clinical applications. A cheap and relatively straightforward alternative is the use of computational algorithms. The objective of this study was to assess the performance of the computational approach in NAT2 haplotype reconstruction from phase-unknown genotype data, for population samples of various ethnic origin.ResultsWe empirically evaluated the effectiveness of four haplotyping algorithms in predicting haplotype phases at NAT2, by comparing the results with those directly obtained through molecular haplotyping. All computational methods provided remarkably accurate and reliable estimates for NAT2 haplotype frequencies and individual haplotype phases. The Bayesian algorithm implemented in the PHASE program performed the best.ConclusionThis investigation provides a solid basis for the confident and rational use of computational methods which appear to be a good alternative to infer haplotype phases in the particular case of the NAT2 gene, where there is near complete linkage disequilibrium between polymorphic markers.

Data-Mining Methods as Useful Tools for Predicting Individual Drug Response: Application to CYP2D6 Data

Objectives: Selecting a maximally informative subset of polymorphisms to predict a clinical outcome, such as drug response, requires appropriate search methods due to the increased dimensionality associated with looking at multiple genotypes. In this study, we investigated the ability of several pattern recognition methods to identify the most informative markers in the CYP2D6 gene for the prediction of CYP2D6 metabolizer status. Methods: Four data-mining tools were explored: decision trees, random forests, artificial neural networks, and the multifactor dimensionality reduction (MDR) method. Marker selection was performed separately in eight population samples of different ethnic origin to evaluate to what extent the most informative markers differ across ethnic groups. Results: Our results show that the number of polymorphisms required to predict CYP2D6 metabolic phenotype with a high accuracy can be dramatically reduced owing to the strong haplotype block structure observed at CYP2D6. MDR and neural networks provided nearly identical results and performed the best. Conclusion: Data-mining methods, such as MDR and neural networks, appear as promising tools to improve the efficiency of genotyping tests in pharmacogenetics with the ultimate goal of pre-screening patients for individual therapy selection with minimum genotyping effort.

Analyse cladistique numérique et analyse de parcimonie;l'exemple des Elephantidae*

Resume La confrontation des differentes hypotheses publiees ces dernieres annees au sujet de lorigine des Elephantidae a ete loccasion dutiliser un programme danalyse de parcimonie (le «PHYLIP package) et de discuter quelques points methodologiques inherents a laspect numerique des approches cladistiques informatisees. Dans letat actuel des connaissances, il apparait que la somme des convergences alliee a une information tres lacunaire sont un obstacle a lobtention dune solution parcimonieuse unique au probleme de la phylogenie des Elephantidae et des Elephantoidea de grade tetralophodonte. Quoique la logique interne des analyses de parcimonie puisse aboutir a des solutions fondees sur des combinaisons heteroclites de caracteres, ces analyses soulevent quelques points qui necessitent des recherches futures, notamment le controle de la monophylie des Stegodontidae. La monophylie des Elephantidae et les relations de parente a linterieur du groupe sont stables en letat actuel de linformation; en revanche, les relations de parente du groupe avec tel ou tel elephantoide de grade tetralophodonte restent incertaines.

aes, the gene encoding the esterase B in Escherichia coli, is a powerful phylogenetic marker of the species.

BackgroundPrevious studies have established a correlation between electrophoretic polymorphism of esterase B, and virulence and phylogeny of Escherichia coli. Strains belonging to the phylogenetic group B2 are more frequently implicated in extraintestinal infections and include esterase B2 variants, whereas phylogenetic groups A, B1 and D contain less virulent strains and include esterase B1 variants. We investigated esterase B as a marker of phylogeny and/or virulence, in a thorough analysis of the esterase B-encoding gene.ResultsWe identified the gene encoding esterase B as the acetyl-esterase gene (aes) using gene disruption. The analysis of aes nucleotide sequences in a panel of 78 reference strains, including the E. coli reference (ECOR) strains, demonstrated that the gene is under purifying selection. The phylogenetic tree reconstructed from aes sequences showed a strong correlation with the species phylogenetic history, based on multi-locus sequence typing using six housekeeping genes. The unambiguous distinction between variants B1 and B2 by electrophoresis was consistent with Aes amino-acid sequence analysis and protein modelling, which showed that substituted amino acids in the two esterase B variants occurred mostly at different sites on the protein surface. Studies in an experimental mouse model of septicaemia using mutant strains did not reveal a direct link between aes and extraintestinal virulence. Moreover, we did not find any genes in the chromosomal region of aes to be associated with virulence.ConclusionOur findings suggest that aes does not play a direct role in the virulence of E. coli extraintestinal infection. However, this gene acts as a powerful marker of phylogeny, illustrating the extensive divergence of B2 phylogenetic group strains from the rest of the species.