EmsB, a tandem repeated multi-loci microsatellite, new tool to investigate the genetic diversity of Echinococcus multilocularis

In order to explore the genetic diversity within Echinococcus multilocularis (E. multilocularis), the cestode responsible for the alveolar echinococcosis (AE) in humans, a microsatellite, composed of (CA) and (GA) repeats and designated EmsB, was isolated and characterized in view of its nature and potential field application. PCR-amplification with specific primers exhibited a high degree of size polymorphism between E. multilocularis and Echinococcus granulosus sheep (G1) and camel (G6) strains. Fluorescent-PCR was subsequently performed on a panel of E. multilocularis isolates to assess intra-species polymorphism level. EmsB provided a multi-peak profile, characterized by tandemly repeated microsatellite sequences in the E. multilocularis genome. This "repetition of repeats" feature provided to EmsB a high discriminatory power in that eight clusters, supported by bootstrap p-values larger than 95%, could be defined among the tested E. multilocularis samples. We were able to differentiate not only the Alaskan from the European samples, but also to detect different European isolate clusters. In total, 25 genotypes were defined within 37 E. multilocularis samples. Despite its complexity, this tandem repeated multi-loci microsatellite possesses the three important features for a molecular marker, i.e. sensitivity, repetitiveness and discriminatory power. It will permit assessing the genetic polymorphism of E. multilocularis and to investigate its spatial distribution in detail.

A polymorphism within the equine CRISP3 gene is associated with stallion fertility in Hanoverian warmblood horses

Fertility of stallions is of high economic importance, especially for large breeding organisations and studs. Breeding schemes with respect to fertility traits and selection of stallions at an early stage may be improved by including molecular genetic markers associated with traits. The genes coding for equine cysteine-rich secretory proteins (CRISPs) are promising candidate genes because previous studies have shown that CRISPs play a role in the fertilising ability of male animals. We have previously characterised the three equine CRISP genes and identified a non-synonymous polymorphism in the CRISP1 gene. In this study, we report one non-synonymous polymorphism in the CRISP2 gene and four non-synonymous polymorphisms in the CRISP3 gene. All six CRISP polymorphisms were genotyped in 107 Hanoverian breeding stallions. Insemination records of stallions were used to analyse the association between CRISP polymorphisms and fertility traits. Three statistical models were used to evaluate the influence of single mutations, genotypes and haplotypes of the polymorphisms. The CRISP3 AJ459965:c.+622G>A SNP leading to the amino acid substitution E208K was significantly associated with the fertility of stallions. Stallions heterozygous for the CRISP3 c.+622G>A SNP had lower fertility than homozygous stallions (P = 0.0234). The pregnancy rate per cycle in these stallions was estimated to be approximately 7% lower than in stallions homozygous at this position.

Assessment of use of microsatellite polymorphism analysis for improving spatial distribution tracking of echinococcus multilocularis

Alveolar echinococcosis (AE)--caused by the cestode Echinococcus multilocularis--is a severe zoonotic disease found in temperate and arctic regions of the northern hemisphere. Even though the transmission patterns observed in different geographical areas are heterogeneous, the nuclear and mitochondrial targets usually used for the genotyping of E. multilocularis have shown only a marked genetic homogeneity in this species. We used microsatellite sequences, because of their high typing resolution, to explore the genetic diversity of E. multilocularis. Four microsatellite targets (EmsJ, EmsK, and EmsB, which were designed in our laboratory, and NAK1, selected from the literature) were tested on a panel of 76 E. multilocularis samples (larval and adult stages) obtained from Alaska, Canada, Europe, and Asia. Genetic diversity for each target was assessed by size polymorphism analysis. With the EmsJ and EmsK targets, two alleles were found for each locus, yielding two and three genotypes, respectively, discriminating European isolates from the other groups. With NAK1, five alleles were found, yielding seven genotypes, including those specific to Tibetan and Alaskan isolates. The EmsB target, a tandem repeated multilocus microsatellite, found 17 alleles showing a complex pattern. Hierarchical clustering analyses were performed with the EmsB findings, and 29 genotypes were identified. Due to its higher genetic polymorphism, EmsB exhibited a higher discriminatory power than the other targets. The complex EmsB pattern was able to discriminate isolates on a regional and sectoral level, while avoiding overdistinction. EmsB will be used to assess the putative emergence of E. multilocularis in Europe.

Distribution and genetic variability among Campylobacter spp. isolates from different animal species and humans in Switzerland

In Switzerland, a national database with 1028 Campylobacter isolates from poultry, pigs, cats, dogs, cattle, humans, zoo animals and water has been created. The database contains the genetic fingerprint and background information of each Campylobacter isolate. Dominant species could be identified in the different sources with a majority of Campylobacter jejuni in poultry (73%), humans (79%), cattle (95%), zoo animals (40%) and water (100%), of Campylobacter coli in pigs (72%), and of Campylobacter upsaliensis/helveticus in cats and dogs (55%). The comparison of three genotyping methods, amplified fragment length polymorphism (AFLP), pulsed field gel electrophoresis and restriction fragment length polymorphism, revealed that AFLP allows discrimination between the different Campylobacter species and is the most appropriate method to distinguish specific strains within the same species. Genotyping analysis demonstrated that the Campylobacter population is heterogeneous among the different sources and that no dominant clone is spread in the country. Genotyping and the resulting database are useful tools to trace back future Campylobacter infections.

Genetic polymorphisms of manganese-superoxide dismutase and glutathione-S-transferase in chronic alcoholic pancreatitis

Chronic alcohol consumption is a major risk factor for the development of chronic pancreatitis. However, chronic pancreatitis occurs only in a minority of heavy drinkers. This variability may be due to yet unidentified genetic factors. Several enzymes involved in the degradation of reactive oxidants and xenobiotics, such as glutathione-S-transferase P1 (GSTP1) and manganese-superoxide dismutase (MnSOD) reveal functional polymorphisms that affect the antioxidative capacity and may therefore modulate the development of chronic pancreatitis and long-term complications like endocrine and exocrine pancreatic insufficiency. Two functional polymorphisms of the MnSOD and the GSTP1 gene were assessed by polymerase chain reaction and restriction fragment length polymorphism in 165 patients with chronic alcoholic pancreatitis, 140 alcoholics without evidence of pancreatic disease and 160 healthy control subjects. The distribution of GSTP1 and MnSOD genotypes were in Hardy-Weinberg equilibrium in the total cohort. Genotype and allele frequencies for both genes were not statistically different between the three groups. Although genotype MnSOD Ala/Val was seemingly associated with the presence of exocrine pancreatic insufficiency, this subgroup was too small and the association statistically underpowered. None of the tested genotypes affected the development of endocrine pancreatic insufficiency. Polymorphisms of MnSOD and GSTP1 are not associated with chronic alcoholic pancreatitis. The present data emphasize the need for stringently designed candidate gene association studies with well-characterized cases and controls and sufficient statistical power to exclude chance observations.

Alcohol and colorectal cancer: the role of alcohol dehydrogenase 1C polymorphism

BACKGROUND: Chronic alcohol consumption is a risk factor for colorectal cancer. Animal experiments as well as genetic linkage studies in Japanese individuals with inactive acetaldehyde dehydrogenase leading to elevated acetaldehyde concentrations following ethanol ingestion support the hypothesis that acetaldehyde may be responsible for this carcinogenic effect of alcohol. In Caucasians, a polymorphism of alcohol dehydrogenase 1C (ADH1C) exists resulting in different acetaldehyde concentrations following ethanol oxidation. METHODS: To evaluate whether the association between alcohol consumption and colorectal tumor development is modified by ADH1C polymorphism, we recruited 173 individuals with colorectal tumors diagnosed by colonoscopy and 788 control individuals without colorectal tumors. Genotyping was performed using genomic DNA extracted from whole blood followed by polymerase chain reaction. RESULTS: Genotype ADH1C*1/1 was more frequent in patients with alcohol-associated colorectal neoplasia compared to patients without cancers in the multivariate model controlling for age, gender, and alcohol intake (odds ratio = 1.674, 95% confidence interval = 1.110-2.524, 2-sided p from Wald test = 0.0139). In addition, the joint test of the genetic effect and interaction between ADH1C genotype and alcohol intake (2-sided p = 0.0007) indicated that the difference in ADH1C*1 polymorphisms between controls and colorectal neoplasia is strongly influenced by the alcohol consumption and that only individuals drinking more than 30 g ethanol per day with the genotype ADH1C*1/1 had an increased risk for colorectal tumors. CONCLUSIONS: These data identify ADH1C homozygosity as a genetic risk marker for colorectal tumors in individuals consuming more than 30 g alcohol per day and emphasize the role of acetaldehyde as a carcinogenic agent in alcohol-related colorectal carcinogenesis.

Evaluation of the transforming growth factor beta1 codon 25 (Arg-->Pro) polymorphism in alcoholic liver disease

INTRODUCTION: Liver cirrhosis develops only in a minority of heavy drinkers. Genetic factors may account for some variation in the progression of fibrosis in alcoholic liver disease (ALD). Transforming growth factor beta 1 (TGFbeta1) is a key profibrogenic cytokine in fibrosis and its gene contains several polymorphic sites. A single nucleotide polymorphism at codon 25 has been suggested to affect fibrosis progression in patients with chronic hepatitis C virus infection, fatty liver disease, and hereditary hemochromatosis. Its contribution to the progression of ALD has not been investigated sufficiently so far. PATIENTS AND METHODS: One-hundred-and-fifty-one heavy drinkers without apparent ALD, 149 individuals with alcoholic cirrhosis, and 220 alcoholic cirrhotics who underwent liver transplantation (LTX) were genotyped for TGFbeta1 codon 25 variants. RESULTS: Univariate analysis suggested that genotypes Arg/Pro or Pro/Pro are associated with decompensated liver cirrhosis requiring LTX. However, after adjusting for patients' age these genotypes did not confer a significant risk for cirrhosis requiring LTX. CONCLUSION: TGFbeta1 codon 25 genotypes Arg/Pro or Pro/Pro are not associated with alcoholic liver cirrhosis. Our study emphasizes the need for adequate statistical methods and accurate study design when evaluating the contribution of genetic variants to the course of chronic liver diseases.

Do common genetic variants in endotoxin signaling pathway contribute to predisposition to alcoholic liver cirrhosis?

BACKGROUND: Tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta), produced by endotoxin-activated Kupffer cells, play a key role in the pathogenesis of alcoholic liver cirrhosis (ALC). Alleles TNFA -238A, IL1B -31T and variant IL1RN*2 of repeat polymorphism in the gene encoding the IL-1 receptor antagonist increase production of TNF-alpha and IL-1beta, respectively. Alleles CD14 -159T, TLR4 c.896G and TLR4 c.1196T modify activation of Kupffer cells by endotoxin. We confirmed the published associations between these common variants and genetic predisposition to ALC by means of a large case-control association study conducted on two Central European populations. METHODS: The study population comprised a Czech sample of 198 ALC patients and 370 controls (MONICA project), and a German sample of 173 ALC patients and 331 controls (KORA-Augsburg), and 109 heavy drinkers without liver disease. RESULTS: Single locus analysis revealed no significant difference between patients and controls in all tested loci. Diplotype [IL1RN 2/ 2; IL1B -31T+] was associated with increased risk of ALC in the pilot study, but not in the validation samples. CONCLUSIONS: Although cytokine mediated immune reactions play a role in the pathogenesis of ALC, hereditary susceptibility caused by variants in the corresponding genes is low in Central European populations.

Genetic diversity and ecological success of Staphylococcus aureus strains colonizing humans

The genetic determinants and phenotypic traits which make a Staphylococcus aureus strain a successful colonizer are largely unknown. The genetic diversity and population structure of 133 S. aureus isolates from healthy, generally risk-free adult carriers were investigated using four different typing methods: multilocus sequence typing (MLST), amplified fragment length polymorphism analysis (AFLP), double-locus sequence typing (DLST), and spa typing were compared. Carriage isolates displayed great genetic diversity which could only be revealed fully by DLST. Results of AFLP and MLST were highly concordant in the delineation of genotypic clusters of closely related isolates, roughly equivalent to clonal complexes. spa typing and DLST provided considerably less phylogenetic information. The resolution of spa typing was similar to that of AFLP and inferior to that of DLST. AFLP proved to be the most universal method, combining a phylogeny-building capacity similar to that of MLST with a much higher resolution. However, it had a lower reproducibility than sequencing-based MLST, DLST, and spa typing. We found two cases of methicillin-resistant S. aureus colonization, both of which were most likely associated with employment at a health service. Of 21 genotypic clusters detected, 2 were most prevalent: cluster 45 and cluster 30 each colonized 24% of the carrier population. The number of bacteria found in nasal samples varied significantly among the clusters, but the most prevalent clusters were not particularly numerous in the nasal samples. We did not find much evidence that genotypic clusters were associated with different carrier characteristics, such as age, sex, medical conditions, or antibiotic use. This may provide empirical support for the idea that genetic clusters in bacteria are maintained in the absence of adaptation to different niches. Alternatively, carrier characteristics other than those evaluated here or factors other than human hosts may exert selective pressure maintaining genotypic clusters.

The EmsB tandemly repeated multilocus microsatellite: a new tool to investigate genetic diversity of Echinococcus granulosus sensu lato

Cystic echinococcosis (CE) is a widespread and severe zoonotic disease caused by infection with the larval stage of the eucestode Echinococcus granulosus sensu lato. The polymorphism exhibited by nuclear and mitochondrial markers conventionally used for the genotyping of different parasite species and strains does not reach the level necessary for the identification of genetic variants linked to restricted geographical areas. EmsB is a tandemly repeated multilocus microsatellite that proved its usefulness for the study of genetic polymorphisms within the species E. multilocularis, the causative agent of alveolar echinococcosis. In the present study, EmsB was used to characterize E. granulosus sensu lato samples collected from different host species (sheep, cattle, dromedaries, dogs, and human patients) originating from six different countries (Algeria, Mauritania, Romania, Serbia, Brazil, and the People's Republic of China). The conventional mitochondrial cox1 and nad1 markers identified genotypes G1, G3, G5, G6, and G7, which are clustered into three groups corresponding to the species E. granulosus sensu stricto, E. ortleppi, and E. canadensis. With the same samples, EmsB provided a higher degree of genetic discrimination and identified variations that correlated with the relatively small-scale geographic origins of the samples. In addition, one of the Brazilian single hydatid cysts presented a hybrid genotypic profile that suggested genetic exchanges between E. granulosus sensu stricto and E. ortleppi. In summary, the EmsB microsatellite exhibits an interesting potential for the elaboration of a detailed map of the distribution of genetic variants and therefore for the determination and tracking of the source of CE.

Genotyping of Francisella tularensis strains by pulsed-field gel electrophoresis, amplified fragment length polymorphism fingerprinting, and 16S rRNA gene sequencing

We evaluated three molecular methods for identification of Francisella strains: pulsed-field gel electrophoresis (PFGE), amplified fragment length polymorphism (AFLP) analysis, and 16S rRNA gene sequencing. The analysis was performed with 54 Francisella tularensis subsp. holarctica, 5 F. tularensis subsp. tularensis, 2 F. tularensis subsp. novicida, and 1 F. philomiragia strains. On the basis of the combination of results obtained by PFGE with the restriction enzymes XhoI and BamHI, PFGE revealed seven pulsotypes, which allowed us to discriminate the strains to the subspecies level and which even allowed us to discriminate among some isolates of F. tularensis subsp. holarctica. The AFLP analysis technique produced some degree of discrimination among F. tularensis subsp. holarctica strains (one primary cluster with three major subclusters and minor variations within subclusters) when EcoRI-C and MseI-A, EcoRI-T and MseI-T, EcoRI-A and MseI-C, and EcoRI-0 and MseI-CA were used as primers. The degree of similarity among the strains was about 94%. The percent similarities of the AFLP profiles of this subspecies compared to those of F. tularensis subsp. tularensis, F. tularensis subsp. novicida, and F. philomiragia were less than 90%, about 72%, and less than 24%, respectively, thus permitting easy differentiation of this subspecies. 16S rRNA gene sequencing revealed 100% similarity for all F. tularensis subsp. holarctica isolates compared in this study. These results suggest that although limited genetic heterogeneity among F. tularensis subsp. holarctica isolates was observed, PFGE and AFLP analysis appear to be promising tools for the diagnosis of infections caused by different subspecies of F. tularensis and suitable techniques for the differentiation of individual strains.

Angiotensin I-converting enzyme-gene-polymorphism: relationship to albumin excretion and blood pressure in pediatric patients with type-I-diabetes mellitus

The purpose of this study was the evaluation of a predictive genetic marker for nephropathy and hypertension in patients with type-I-diabetes mellitus (IDDM). The study was performed on 247 pediatric patients with IDDM. The mean age was 15.5 years (range 3.1-29.3), the mean duration of diabetes was 7.6 years (range 0.1-25.7). Age-related blood pressure and nocturnal albumin excretion rate were compared with the insertion/deletion-(I/D) polymorphism of the angiotensin-I converting enzyme gene. The genotype distribution did not differ significantly between IDDM patients (ID 48%, D 28%, I 24%) and the control group (ID 44%, D 37%, I 19%). Neither in the entire group, nor in patients with IDDM for more than 5 years, was a correlation found bet-ween allele distribution and albumin excretion rate. No correlation was found between genotype and blood pressure. When patients with a chronological age above 12 years were analysed separately, the genotype distribution between the groups with normal and elevated blood pressure showed no significant difference. The previously reported association of the I/D-polymorphism with nephropathy could not be confirmed in this study. The development of microalbuminuria, nephropathy and hypertension will be followed in our pediatric patients.

Origins of Shared Genetic Variation in African Cichlids

Cichlid fishes have evolved tremendous morphological and behavioral diversity in the waters of East Africa. Within each of the Great Lakes Tanganyika, Malawi, and Victoria, the phenomena of hybridization and retention of ancestral polymorphism explain allele sharing across species. Here, we explore the sharing of single nucleotide polymorphisms (SNPs) between the major East African cichlid assemblages. A set of approximately 200 genic and nongenic SNPs was ascertained in five Lake Malawi species and genotyped in a diverse collection of 160 species from across Africa. We observed segregating polymorphism outside of the Malawi lineage for more than 50% of these loci; this holds similarly for genic versus nongenic SNPs, as well as for SNPs at putative CpG versus non-CpG sites. Bayesian and principal component analyses of genetic structure in the data demonstrate that the Lake Malawi endemic flock is not monophyletic and that river species have likely contributed significantly to Malawi genomes. Coalescent simulations support the hypothesis that river cichlids have transported polymorphism between lake assemblages. We observed strong genetic differentiation between Malawi lineages for approximately 8% of loci, with contributions from both genic and nongenic SNPs. Notably, more than half of these outlier loci between Malawi groups are polymorphic outside of the lake. Cichlid fishes have evolved diversity in Lake Malawi as new mutations combined with standing genetic variation shared across East Africa.

A test of genetic association among male nuptial coloration, female mating preference, and male aggression bias within a polymorphic population of cichlid fish

Both inter- and intrasexual selection have been implicated in the origin and maintenance of species-rich taxa with diverse sexual traits. Simultaneous disruptive selection by female mate choice and male-male competition can, in theory, lead to speciation without geographical isolation if both act on the same male trait. Female mate choice can generate discontinuities in gene flow, while male-male competition can generate negative frequency-dependent selection stabilizing the male trait polymorphism. Speciation may be facilitated when mating preference and/or aggression bias are physically linked to the trait they operate on. We tested for genetic associations among female mating preference, male aggression bias and male coloration in the Lake Victoria cichlid Pundamilia. We crossed females from a phenotypically variable population with males from both extreme ends of the phenotype distribution in the same population (blue or red). Male offspring of a red sire were significantly redder than males of a blue sire, indicating that intra-population variation in male coloration is heritable. We tested mating preferences of female offspring and aggression biases of male offspring using binary choice tests. There was no evidence for associations at the family level between female mating preferences and coloration of sires, but dam identity had a significant effect on female mate preference. Sons of the red sire directed significantly more aggression to red than blue males, whereas sons of the blue sire did not show any bias. There was a positive correlation among individuals between male aggression bias and body coloration, possibly due to pleiotropy or physical linkage, which could facilitate the maintenance of color polymorphism.

Nuclear markers reveal unexpected genetic variation and a Congolese-Nilotic origin of the Lake Victoria cichlid species flock

Phylogenetic analyses based on mitochondrial (mt) DNA have indicated that the cichlid species flock of the Lake Victoria region is derived from a single ancestral species found in East African rivers, closely related to the ancestor of the Lake Malawi cichlid species flock. The Lake Victoria flock contains ten times less mtDNA variation than the Lake Malawi radiation, consistent with current estimates of the ages of the lakes. We present results of a phylogenetic investigation using nuclear (amplified fragment length polymorphism) markers and a wider coverage of riverine haplochromines. We demonstrate that the Lake Victoria–Edward flock is derived from the morphologically and ecologically diverse cichlid genus Thoracochromis from the Congo and Nile, rather than from the phenotypically conservative East African Astatotilapia. This implies that the ability to express much of the morphological diversity found in the species flock may by far pre–date the origin of the flock. Our data indicate that the nuclear diversity of the Lake Victoria–Edward species flock is similar to that of the Lake Malawi flock, indicating that the genetic diversity is considerably older than the 15 000 years that have passed since the lake began to refill. Most of this variation is manifested in trans–species polymorphisms, indicating very recent cladogenesis from a genetically very diverse founder stock. Our data do not confirm strict monophyly of either of the species flocks, but raise the possibility that these flocks have arisen from hybrid swarms.