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.

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.

Rapid and accurate G M1-gangliosidosis diagnosis using a parentage testing microsatellite

The G M1-gangliosidosis is an autosomal recessive lysosomal storage disease caused by structural defects of the beta-galactosidase gene (GLB1) which lead to a severe phenotypical impairment in homozygous individuals, whereas heterozygous carriers remain clinically normal. Currently employed DNA parentage tests include the analysis of microsatellites, which also have a diagnostic predictive value. The aim of this study was to provide a reliable tool for genotyping the canine GLB1 which can be effectively integrated in parentage testing investigations. For this purpose the association between the GLB1 gene and the AHT K253 microsatellite was analyzed in 30 Alaskan huskies (11 GLB1+/+, 17 GLB1+/- and 2 GLB1-/- dogs). The 143 bp AHT K253 microsatellite allele was identified only in GLB1+/- and GLB1-/- animals and was in strong linkage disequilibrium with the causative mutation for G M1-gangliosidosis, a 19 bp duplication within exon 15 of the GLB1 gene. The results of the present study revealed a 100% concordance between the previous established genotypes and those obtained after the analysis of the AHT K253 microsatellite. Thus, the genotype of the AHT K253 microsatellite, which is routinely determined during dog parentage testing, has a high predictive value for the G M1-gangliosidosis carrier status.

Isolation and genotyping of Toxoplasma gondii causing fatal systemic toxoplasmosis in an immunocompetent 10-year-old cat

A 10-year-old male, neutered domestic shorthair cat was presented with fever, anorexia, vomiting, and diarrhea. Serologic testing for Feline immunodeficiency virus and Feline leukemia virus were negative. Fine-needle aspirates of mesenteric lymph nodes revealed the presence of banana-shaped apicomplexan parasites. The cat died after 4 days of hospitalization. Postmortem polymerase chain reaction (PCR) analysis confirmed the presence of Toxoplasma gondii in all examined organs. Parasites were ex vivo isolated in outbred mice and subsequently transferred into cell culture. Genotyping, using genetic markers for SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico for PCR-restriction fragment length polymorphism, revealed infection with type II T. gondii displaying type II alleles at all loci except Apico, which exhibited a type I allele. This is the most frequently identified genotype among cats acting as definitive hosts in central Europe, but to the authors' knowledge, it has never been associated with systemic toxoplasmosis in an adult, immunocompetent cat.

Genotyping of Mycoplasma hyopneumoniae in wild boar lung samples

Mycoplasma hyopneumoniae is the etiologic agent of enzootic pneumonia (EP), an important cause of disease-associated losses in swine production and a role of wild boar in recurrent infections can be supposed. Genotypes of M. hyopneumoniae from wild boar are unknown but could indicate its role as a potential reservoir. Therefore, 34 lung samples being PCR-positive for M. hyopneumoniae from wild boar from the Geneva region in Switzerland were assayed by genotyping using the p146 and multi-locus sequence typing (MLST) approaches and compared to data from outbreak cases from domestic swine in Switzerland. Successful genotyping was dependent on a sufficiently high concentration of M. hyopneumoniae DNA in the samples as assessed by different real-time PCR assays. The p146 genotyping was more successful with 24 samples (70.5%) being typeable whereas only 6 samples (17.6%) could be genotyped using the MLST approach. Variability of genotypes was high but identical types were found in geographically related animals. Genotypes from wild boar showed phylogenetic relatedness to those from domestic pigs but no matching types could be identified. Results show that direct genotyping from wild boar lung samples is possible and provides a promising approach to investigate future EP outbreak related samples from wild boar. (C) 2011 Elsevier B.V. All rights reserved.

The impact of CpG island methylator phenotype and microsatellite instability on tumour budding in colorectal cancer

In colorectal cancer, tumour budding, a process likened to epithelial mesenchymal transition, is an adverse prognostic factor which is rarely found in tumours with high-level microsatellite instability (MSI-H). Cases with MSI-H or high-level CpG island methylator phenotype (CIMP-H) have similar histomorphological features, yet seemingly opposite prognosis. We hypothesized that tumour budding is related to CIMP, thus partially explaining this prognostic difference.

The genomic Echinococcus microsatellite EmsB sequences: from a molecular marker to the epidemiological tool

In the field of molecular and epidemiological parasitology, characterization of fast evolving genetic markers appears as an important challenge to consider the diversity and genetic structure of parasites. The study of respective populations can help us to understand their adaptive strategies to survive and perpetuate the species within different host populations, all trying to resist infection. In the past, the relative monomorphic features of Echinococcus multilocularis, the causative agent of alveolar echinococcosis and a severe human parasitic disease, did not stimulate studies dealing with the genetic variability of Echinococcus species or respective populations. A recently developed, characterized and validated original multilocus microsatellite, named EmsB, tandemly repeated in the genome, offered an additional opportunity for this line of investigation. We have compiled in this review new insights brought by this molecular tracker on the transmission activity of Echinococcus among different hosts and at different geographical scales.

A new DPYD genotyping assay for improving the safety of 5-fluorouracil therapy

Chemotherapeutic use of 5-fluorouracil (5FU) is compromised by 10-20% of patients developing severe toxicity. Recently described genetic variation in dihydropyrimidine dehydrogenase (DPYD) has been shown to be a major predictor of 5FU toxicity. Here, we describe a new genotyping assay for routine clinical use that covers all the major DPYD risk variants.

Genetic analysis of syndactyly in German Holstein cattle

Congenital syndactyly with a variable number of affected feet was observed in eight black and white German Holstein calves. Analysis of the pedigree data revealed that all affected individuals could be traced back to a single founder. The pedigree was consistent with monogenic autosomal recessive inheritance and variable expressivity. Bovine syndactyly or "mulefoot" has been previously shown to map on the telomeric end of bovine chromosome 15 and we performed PCR genotyping of microsatellite markers spanning 27 cM of this chromosomal region to test the new cases for genetic linkage with the phenotype. The haplotype segregation confirmed the suggested inheritance pattern of the mulefoot mutation in this family and markers RM004, BM848 and BMS820 showed significant linkage to the phenotype. The results confirmed the chromosomal location of the mulefoot gene in this pedigree. Furthermore the study demonstrated that although marker testing has been available for nearly a decade the use of mulefoot carriers in cattle breeding remains uncontrolled. The presented family provides a resource for positional cloning of the causative mutation.

Genotyping of human and porcine Yersinia enterocolitica, Yersinia intermedia, and Yersinia bercovieri strains from Switzerland by amplified fragment length polymorphism analysis

In this study, 231 strains of Yersinia enterocolitica, 25 strains of Y. intermedia, and 10 strains of Y. bercovieri from human and porcine sources (including reference strains) were analyzed using amplified fragment length polymorphism (AFLP), a whole-genome fingerprinting method for subtyping bacterial isolates. AFLP typing distinguished the different Yersinia species examined. Representatives of Y. enterocolitica biotypes 1A, 1B, 2, 3, and 4 belonged to biotype-related AFLP clusters and were clearly distinguished from each other. Y. enterocolitica biotypes 2, 3, and 4 appeared to be more closely related to each other (83% similarity) than to biotypes 1A (11%) and 1B (47%). Biotype 1A strains exhibited the greatest genetic heterogeneity of the biotypes studied. The biotype 1A genotypes were distributed among four major clusters, each containing strains from both human and porcine sources, confirming the zoonotic potential of this organism. The AFLP technique is a valuable genotypic method for identification and typing of Y. enterocolitica and other Yersinia spp.

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.

Rapid and reliable genotyping of polymorphic loci modifying correct splicing of CFTR pre-mRNA using mass spectrometry

We describe a fast and unambiguous method for haplotyping the (TG)mTn repeat in IVS8 and determining three other single nucleotide polymorphisms (SNPs) in exons 10, 14a and 24 in the cystic fibrosis transmembrane conductance regulator (CFTR) gene affecting correct splicing of the CFTR pre-mRNA using primer extension and mass spectrometry. The diagnostic products are generated by primer extension (PEX) reactions, which require a single detection primer complementary to a region downstream of a target strand's variable site. On addition of a polymerase and an appropriate mixture of dNTP's and 2', 3'-dideoxynucleotide triphosphates (ddNTP's), the primer is extended through the mutation region until the first ddNTP is incorporated and the mass of the extension products determines the composition of the variable site. Analysis of patient DNA assigned the correct and unambiguous haplotype for the (TG)mTn repeat in intron 8 of the CFTR gene. Additional crucial SNPs influencing correct splicing in exon 10, 14 and 24 can easily be detected by biplexing the assay to genotype allelic variants important for correct splicing of the CFTR pre-mRNA. Different PEX reactions with subsequent mass spectrometry generate sufficient data, to enable unambiguous and easy haplotyping of the (TG)mTn repeat in the CFTR gene. The method can be easily extended to the inclusion of additional SNPs of interest by biplexing some of the PEX reactions. All experimental steps required for PEX are amenable to the high degree of automation desirable for a high-throughput diagnostic setting, facilitating the work of clinicians involved in the diagnosis of non-classic cystic fibrosis.