Citizen science reveals unexpected continental-scale evolutionary change in a model organism

Organisms provide some of the most sensitive indicators of climate change and evolutionary responses are becoming apparent in species with short generation times. Large datasets on genetic polymorphism that can provide an historical benchmark against which to test for recent evolutionary responses are very rare, but an exception is found in the brown-lipped banded snail (Cepaea nemoralis). This species is sensitive to its thermal environment and exhibits several polymorphisms of shell colour and banding pattern affecting shell albedo in the majority of populations within its native range in Europe. We tested for evolutionary changes in shell albedo that might have been driven by the warming of the climate in Europe over the last half century by compiling an historical dataset for 6,515 native populations of C. nemoralis and comparing this with new data on nearly 3,000 populations. The new data were sampled mainly in 2009 through the Evolution MegaLab, a citizen science project that engaged thousands of volunteers in 15 countries throughout Europe in the biggest such exercise ever undertaken. A known geographic cline in the frequency of the colour phenotype with the highest albedo (yellow) was shown to have persisted and a difference in colour frequency between woodland and more open habitats was confirmed, but there was no general increase in the frequency of yellow shells. This may have been because snails adapted to a warming climate through behavioural thermoregulation. By contrast, we detected an unexpected decrease in the frequency of Unbanded shells and an increase in the Mid-banded morph. Neither of these evolutionary changes appears to be a direct response to climate change, indicating that the influence of other selective agents, possibly related to changing predation pressure and habitat change with effects on micro-climate.

Molecular characterization of the porcine deleted in malignant brain tumors 1 gene (DMBT1)

The human gene deleted in malignant brain tumors 1 (DMBT1) is considered to play a role in tumorigenesis and pathogen defense. It encodes a protein with multiple scavenger receptor cysteine-rich (SRCR) domains, which are involved in recognition and binding of a broad spectrum of bacterial pathogens. The SRCR domains are encoded by highly homologous repetitive exons, whose number in humans may vary from 8 to 13 due to genetic polymorphism. Here, we characterized the porcine DMBT1 gene on the mRNA and genomic level. We assembled a 4.5 kb porcine DMBT1 cDNA sequence from RT-PCR amplified seminal vesicle RNA. The porcine DMBT1 cDNA contains an open reading frame of 4050 nt. The transcript gives rise to a putative polypeptide of 1349 amino acids with a calculated mass of 147.9 kDa. Compared to human DMBT1, it contains only four N-terminal SRCR domains. Northern blotting revealed transcripts of approximately 4.7 kb in size in the tissues analyzed. Analysis of ESTs suggested the existence of secreted and transmembrane variants. The porcine DMBT1 gene spans about 54 kb on chromosome 14q28-q29. In contrast to the characterized cDNA, the genomic BAC clone only contained 3 exons coding for N-terminal SRCR domains. In different mammalian DMBT1 orthologs large interspecific differences in the number of SRCR exons and utilization of the transmembrane exon exist. Our data suggest that the porcine DMBT1 gene may share with the human DMBT1 gene additional intraspecific variations in the number of SRCR-coding exons.

Connexin37 protects against atherosclerosis by regulating monocyte adhesion

A genetic polymorphism in the human gene encoding connexin37 (CX37, encoded by GJA4, also known as CX37) has been reported as a potential prognostic marker for atherosclerosis. The expression of this gap-junction protein is altered in mouse and human atherosclerotic lesions: it disappears from the endothelium of advanced plaques but is detected in macrophages recruited to the lesions. The role of CX37 in atherogenesis, however, remains unknown. Here we have investigated the effect of deleting the mouse connexin37 (Cx37) gene (Gja4, also known as Cx37) on atherosclerosis in apolipoprotein E-deficient (Apoe(-/-)) mice, an animal model of this disease. We find that Gja4(-/-)Apoe(-/-) mice develop more aortic lesions than Gja4(+/+)Apoe(-/-) mice that express Cx37. Using in vivo adoptive transfer, we show that monocyte and macrophage recruitment is enhanced by eliminating expression of Cx37 in these leukocytes but not by eliminating its expression in the endothelium. We further show that Cx37 hemichannel activity in primary monocytes, macrophages and a macrophage cell line (H36.12j) inhibits leukocyte adhesion. This antiadhesive effect is mediated by release of ATP into the extracellular space. Thus, Cx37 hemichannels may control initiation of the development of atherosclerotic plaques by regulating monocyte adhesion. H36.12j macrophages expressing either of the two CX37 proteins encoded by a polymorphism in the human GJA4 gene show differential ATP-dependent adhesion. These results provide a potential mechanism by which a polymorphism in CX37 protects against atherosclerosis.

Taxonomic position and geographical distribution of the common sheep G1 and camel G6 strains of Echinococcus granulosus in three African countries

The taxonomic and phylogenetic status of Echinococcus granulosus strains are still controversial and under discussion. In the present study, we investigated the genetic polymorphism of E. granulosus isolates originating from three countries of Africa, including a region of Algeria, where the common G1 sheep and the camel G6 strains coexist sympatrically. Seventy-one hydatid cysts were collected from sheep, cattle, camels, and humans. Two mitochondrial markers (cox1 and nad1) were used for strain identification. Two nuclear markers (actII and hbx2) were used to study the possible occurrence of cross-fertilization. Despite the heterogeneity observed among the G1 isolates, they were all localized within one robust cluster. A second strong cluster was also observed containing all of the G6 isolates. Both strains appeared as two distinct groups, and no cases of interbreeding were found. Thus, the attribution of a species rank can be suggested. We also found the Tasmanian sheep G2 strain for the first time in Africa. Because of the slight variations observed between the common sheep and the Tasmanian sheep strains, further studies should be carried out to elucidate the epidemiological relevance of this genetic discrimination.

Influence of ABCB1, ABCC1, ABCC2, and ABCG2 haplotypes on the cellular exposure of nelfinavir in vivo

OBJECTIVES: The human immunodeficiency virus protease inhibitor nelfinavir is substrate of polyspecific drug transporters encoded by ABCB1 (P-glycoprotein), ABCC1 (MRP1) and ABCC2 (MRP2), and an inhibitor of BCRP, encoded by ABCG2. Genetic polymorphism in these genes may be associated with changes in transport function. METHODS: A comprehensive evaluation of single nucleotide polymorphisms (39 SNPs in ABCB1, 7 in ABCC1, 27 in ABCC2, and 16 in ABCG2), and inferred haplotypes was done to assess possible associations of genetic variants with cellular exposure of nelfinavir in vivo. Analysis used peripheral mononuclear cells from individuals receiving nelfinavir (n=28). Key results were re-examined in a larger sample size (n=129) contributing data on plasma drug levels. RESULTS AND CONCLUSIONS: There was no significant association between cellular nelfinavir area under the curve (AUC) and SNPs or haplotypes at ABCC1, ABCC2, ABCG2. There was an association with cellular exposure for two loci in strong linkage disequilibrium: ABCB1 3435C>T; AUCTT>AUCCT>AUCCC (ratio 2.1, 1.4, 1, Ptrend=0.01), and intron 26 +80T>C; AUCCC> AUCCT > AUCTT (ratio 2.4, 1.3, 1, Ptrend=0.006). Haplotypic analysis using tagging SNPs did not improve the single SNP association values.

Altered transmission of HOX and apoptotic SNPs identify a potential common pathway for clubfoot.

Clubfoot is a common birth defect that affects 135,000 newborns each year worldwide. It is characterized by equinus deformity of one or both feet and hypoplastic calf muscles. Despite numerous study approaches, the cause(s) remains poorly understood although a multifactorial etiology is generally accepted. We considered the HOXA and HOXD gene clusters and insulin-like growth factor binding protein 3 (IGFBP3) as candidate genes because of their important roles in limb and muscle morphogenesis. Twenty SNPs from the HOXA and HOXD gene clusters and 12 SNPs in IGFBP3 were genotyped in a sample composed of non-Hispanic white and Hispanic multiplex and simplex families (discovery samples) and a second sample of non-Hispanic white simplex trios (validation sample). Four SNPs (rs6668, rs2428431, rs3801776, and rs3779456) in the HOXA cluster demonstrated altered transmission in the discovery sample, but only rs3801776, located in the HOXA basal promoter region, showed altered transmission in both the discovery and validation samples (P = 0.004 and 0.028). Interestingly, HOXA9 is expressed in muscle during development. An SNP in IGFBP3, rs13223993, also showed altered transmission (P = 0.003) in the discovery sample. Gene-gene interactions were identified between variants in HOXA, HOXD, and IGFBP3 and with previously associated SNPs in mitochondrial-mediated apoptotic genes. The most significant interactions were found between CASP3 SNPS and variants in HOXA, HOXD, and IGFBP3. These results suggest a biologic model for clubfoot in which perturbation of HOX and apoptotic genes together affect muscle and limb development, which may cause the downstream failure of limb rotation into a plantar grade position.

Smoking, apolipoprotein E genotypes, and mortality (the Ludwigshafen RIsk and Cardiovascular Health study).

AIMS The genetic polymorphism of apolipoprotein E (APOE) has been suggested to modify the effect of smoking on the development of coronary artery disease (CAD) in apparently healthy persons. The interaction of these factors in persons undergoing coronary angiography is not known. METHODS AND RESULTS We analysed the association between the APOE-genotype, smoking, angiographic CAD, and mortality in 3263 participants of the LUdwigshafen RIsk and Cardiovascular Health study. APOE-genotypes were associated with CAD [ε22 or ε23: odds ratio (OR) 0.56, 95% confidence interval (CI) 0.43-0.71; ε24 or ε34 or ε44: OR 1.10, 95% CI 0.89-1.37 compared with ε33] and moderately with cardiovascular mortality [ε22 or ε23: hazard ratio (HR) 0.71, 95% CI 0.51-0.99; ε33: HR 0.92, 95% CI 0.75-1.14 compared with ε24 or ε34 or ε44]. HRs for total mortality were 1.39 (95% CI 0.39-0.1.67), 2.29 (95% CI 1.85-2.83), 2.07 (95% CI 1.64-2.62), and 2.95 (95% CI 2.10-4.17) in ex-smokers, current smokers, current smokers without, or current smokers with one ε4 allele, respectively, compared with never-smokers. Carrying ε4 increased mortality in current, but not in ex-smokers (HR 1.66, 95% CI 1.04-2.64 for interaction). These findings applied to cardiovascular mortality, were robust against adjustment for cardiovascular risk factors, and consistent across subgroups. No interaction of smoking and ε4 was seen regarding non-cardiovascular mortality. Smokers with ε4 had reduced average low-density lipoprotein (LDL) diameters, elevated oxidized LDL, and lipoprotein-associated phospholipase A2. CONCLUSION In persons undergoing coronary angiography, there is a significant interaction between APOE-genotype and smoking. The presence of the ε4 allele in current smokers increases cardiovascular and all-cause mortality.

A history and overview of phenotypic variability in CYP2D6 activity

CYP2D6 is a human cytochrome P450 that is responsible for the metabolism of a large number of drugs and chemicals. Interest in CYP2D6 has largely centered on the wide interindividual variability in its catalytic activity that stems from a common genetic polymorphism in the CYP2D6 gene. Two major phenotypes exist, extensive metabolizer (EM) and poor metabolizer (PM), together with the two less studied phenotypes of ultrarapid metabolizer (UM) and intermediate metabolizer. These phenotypes are the expression of an underlying allelomorphism in CYP2D6 and are also context dependent. Several drugs that are CYP2D6 substrates display polymorphic metabolism, that is, the existence in the population of multiple phenotypes, in particular EM and PM. The most notable drugs in this regard are debrisoquine and sparteine, although there are also data for a few others, in particular, dextromethorphan and metoprolol. Many nongenetic factors can alter the expression of CYP2D6 phenotypes, the most significant of which is the presence of other drugs. In this context, the EM phenotype may not be immutable, with potential conversion into a PM phenocopy, due to significantly impaired CYP2D6 metabolism in the presence of other CYP2D6 substrates and inhibitors. This phenotype interconversion generated great concern and helped drive the movement away from phenotyping based upon drug administration to genotyping of acquired DNA samples. However, ascertaining the presence of CYP2D6 alleles in a DNA sample does not determine the metabolism and pharmacokinetics of CYP2D6 substrates in that subject: it is a forecast, much like the weather forecast and, as we all know regarding the weather, the forecast can be inaccurate at times.

Detecting the signature of natural selection with microsatellites

Natural selection is one of the major factors in the evolution of all organisms. Detecting the signature of natural selection has been a central theme in evolutionary genetics. With the availability of microsatellite data, it is of interest to study how natural selection can be detected with microsatellites. ^ The overall aim of this research is to detect signatures of natural selection with data on genetic variation at microsatellite loci. The null hypothesis to be tested is the neutral mutation theory of molecular evolution, which states that different alleles at a locus have equivalent effects on fitness. Currently used tests of this hypothesis based on data on genetic polymorphism in natural populations presume that mutations at the loci follow the infinite allele/site models (IAM, ISM), in the sense that at each site at most only one mutation event is recorded, and each mutation leads to an allele not seen before in the population. Microsatellite loci, which are abundant in the genome, do not obey these mutation models, since the new alleles at such loci can be created either by contraction or expansion of tandem repeat sizes of core motifs. Since the current genome map is mainly composed of microsatellite loci and this class of loci is still most commonly studied in the context of human genome diversity, this research explores how the current test procedures for testing the neutral mutation hypothesis should be modified to take into account a generalized model of forward-backward stepwise mutations. In addition, recent literature also suggested that past demographic history of populations, presence of population substructure, and varying rates of mutations across loci all have confounding effects for detecting signatures of natural selection. ^ The effects of the stepwise mutation model and other confounding factors on detecting signature of natural selection are the main results of the research. ^

Antigenic variation in surface proteins of Trypanosoma cruzi (Chagas' disease)

Chagas' disease, a devastating illness in the Western Hemisphere, is caused by the protozoan parasite Trypanosoma cruzi. Transmission is via bloodsucking insect vectors, congenitally, or through blood transfusion and/or organ transplantation. A significant percentage of heart-related illnesses and deaths each year are attributable to the number of persons with Chagas' disease. Currently, there is no FDA-approved routine screening of the U.S. blood supply being conducted by blood banks. The only current commercial assays available for detection of Trypanosoma cruzi are based on South American isolates, which may differ antigenically from those found in the US. In this study, the assay used intact parasites as antigen in an ELISA-type assay. Therefore, serological differences presumably reflected variations in surface antigens. The basis of differential antibody binding to these antigens is unknown. In this study, biochemical characterization and genetic polymorphism analysis will be performed on three defined surface proteins of T. cruzi epimastigotes.^

Evidence suggesting that the odortypes of pregnant women are a compound of maternal and fetal odortypes.

Odortypes--namely, body odors that distinguish one individual from another on the basis of genetic polymorphism at the major histocompatibility complex and other loci--are a fundamental element in the social life and reproductive behavior of the mouse, including familial imprinting, mate choice, and control of early pregnancy. Odortypes are strongly represented in urine. During mouse pregnancy, an outcrossed mother's urine acquires fetal major histocompatibility complex odortypes of paternal origin, an observation that we took as the focus of a search for odortypes in humans, using a fully automated computer-programmed olfactometer in which trained rats are known to distinguish precisely the odortypes of another species. Five women provided urine samples before and after birth, which in each case appropriately trained rats were found to distinguish in the olfactometer. Whether this olfactory distinction of mothers' urine before and after birth reflects in part the odortype and hence genotype of the fetus, and not just the state of pregnancy per se, was tested in a second study in which each mother's postpartum urine was mixed either with urine from her own infant or with urine of a different, same-aged infant. Responses of trained rats were more positive with respect to the former (congruous) mixtures than to the latter (incongruous) mixtures, implying that, as in the mouse, human fetal odortypes of paternal genomic origin are represented in the odortype of the mother, doubtless by circulatory transfer of the pertinent odorants.

Molecular modelling of human CYP1B1 substrate interactions and investigation of allelic variant effects on metabolism

Molecular modelling of human CYP1B1 based on homology with the mammalian P450, CYP2C5, of known three-dimensional structure is reported. The enzyme model has been used to investigate the likely mode of binding for selected CYP1B1 substrates, particularly with regard to the possible effects of allelic variants of CYP1B1 on metabolism. In general, it appears that the CYP1B1 model is consistent with known substrate selectivity for the enzyme, and the sites of metabolism can be rationalized in terms of specific contacts with key amino acid residues within the CYP1B1 heme locus. Further-more, a mode of binding interaction for the inhibitor, a-naphthoflavone, is presented which accords with currently available information. The current paper shows that a combination of molecular modelling and experimental determinations on the substrate metabolism for CYP1B1 allelic variants can aid in the understanding of structure-function relationships within P450 enzymes. (C) 2003 Elsevier Science Ireland Ltd. All rights reserved.

Detection of fruit and the selection of primate visual pigments for color vision

Primates have X chromosome genes for cone photopigments with sensitivity maxima from 535 to 562 nm. Old World monkeys and apes (catarrhines) and the New World ( platyrrhine) genus Alouatta have separate genes for 535-nm ( medium wavelength; M) and 562-nm ( long wavelength; L) pigments. These pigments, together with a 425-nm ( short wavelength) pigment, permit trichromatic color vision. Other platyrrhines and prosimians have a single X chromosome gene but often with alleles for two or three M/L photopigments. Consequently, heterozygote females are trichromats, but males and homozygote females are dichromats. The criteria that affect the evolution of M/L alleles and maintain genetic polymorphism remain a puzzle, but selection for finding food may be important. We compare different types of color vision for detecting more than 100 plant species consumed by tamarins ( Saguinus spp.) in Peru. There is evidence that both frequency-dependent selection on homozygotes and heterozygote advantage favor M/L polymorphism and that trichromatic color vision is most advantageous in dim light. Also, whereas the 562-nm allele is present in all species, the occurrence of 535- to 556-nm alleles varies between species. This variation probably arises because trichromatic color vision favors widely separated pigments and equal frequencies of 535/543- and 562-nm alleles, whereas in dichromats, long-wavelength pigment alleles are fitter.

Molecular analysis of evolutionary relationships of Phaseolus dumosus with the gene pool of common bean

Valuable genetic variation for bean breeding programs is held within the common bean secondary gene pool which consists of Phaseolus albescens, P. coccineus, P. costaricensis, and P. dumosus. However, the use of close relatives for bean improvement is limited due to the lack of knowledge about genetic variation and genetic plasticity of many of these species. Characterisation and analysis of the genetic diversity is necessary among beans' wild relatives; in addition, conflicting phylogenies and relationships need to be understood and a hypothesis of a hybrid origin of P. dumosus needs to be tested. This thesis research was orientated to generate information about the patterns of relationships among the common bean secondary gene pool, with particular focus on the species Phaseolus dumosus. This species displays a set of characteristics of agronomic interest, not only for the direct improvement of common bean but also as a source of valuable genes for adaptation to climate change. Here I undertake the first comprehensive study of the genetic diversity of P. dumosus as ascertained from both nuclear and chloroplast genome markers. A germplasm collection of the ancestral forms of P. dumosus together with wild, landrace and cultivar representatives of all other species of the common bean secondary gene pool, were used to analyse genetic diversity, phylogenetic relationships and structure of P. dumosus. Data on molecular variation was generated from sequences of cpDNA loci accD-psaI spacer, trnT-trnL spacer, trnL intron and rps14-psaB spacer and from the nrDNA the ITS region. A whole genome DArT array was developed and used for the genotyping of P. dumosus and its closes relatives. 4208 polymorphic markers were generated in the DArT array and from those, 742 markers presented a call rate >95% and zero discordance. DArT markers revealed a moderate genetic polymorphism among P. dumosus samples (13% of polymorphic loci), while P. coccineus presented the highest level of polymorphism (88% of polymorphic loci). At the cpDNA one ancestral haplotype was detected among all samples of all species in the secondary genepool. The ITS region of P. dumosus revealed high homogeneity and polymorphism bias to P. coccineus genome. Phylogenetic reconstructions made with Maximum likelihood and Bayesian methods confirmed previously reported discrepancies among the nuclear and chloroplast genomes of P. dumosus. The outline of relationships by hybridization networks displayed a considerable number of interactions within and between species. This research provides compelling evidence that P. dumosus arose from hybridisation between P. vulgaris and P. coccineus and confirms that P. costaricensis has likely been involved in the genesis or backcrossing events (or both) in the history of P. dumosus. The classification of the specie P. persistentus was analysed based on cpDNA and ITS sequences, the results found this species to be highly related to P. vulgaris but not too similar to P. leptostachyus as previously proposed. This research demonstrates that wild types of the secondary genepool carry a significant genetic variation which makes this a valuable genetic resource for common bean improvement. The DArT array generated in this research is a valuable resource for breeding programs since it has the potential to be used in several approaches including genotyping, discovery of novel traits, mapping and marker-trait associations. Efforts should be made to search for potential populations of P. persistentus and to increase the collection of new populations of P. dumosus, P. albescens and P. costaricensis that may provide valuable traits for introgression into common bean and other Phaseolus crops.