Frequency of PRRS live vaccine virus (European and North American genotype) in vaccinated and non-vaccinated pigs submitted for respiratory tract diagnostics in North-Western Germany

The frequency of PRRSV corresponding to live vaccines and wild-type was determined in 902 pigs from North-Western Germany submitted for post-mortem examination. Overall, 18.5% of the samples were positive for the EU wild-type virus. EU genotype vaccine virus was detected in 1.3% and the NA genotype vaccine virus in 8.9% of all samples. The detection of the EU vaccine was significantly higher in pigs vaccinated with the corresponding vaccine (OR=9.4). Pigs vaccinated with NA genotype had significantly higher detection chances for the corresponding vaccine virus when compared to non-vaccinated animals (OR=3.34) animals, however, NA vaccine was also frequently detected in non-vaccinated pigs. Concluding, the dynamics of NA genotype vaccine and EU wild-type virus corresponds with studies on PRRSV spread in endemically infected herds. The potential of spontaneous spread of the NA genotype vaccine should be considered in the planning of eradication programs.

Novel mitochondrial tRNA(Ile) m.4282A>G gene mutation leads to chronic progressive external ophthalmoplegia plus phenotype

BACKGROUND/AIM To investigate the underlying pathomechanism in a 33-year-old female Caucasian patient presenting with chronic progressive external ophthalmoplegia (CPEO) plus symptoms. METHODS Histochemical analysis of skeletal muscle and biochemical measurements of individual oxidative phosphorylation (OXPHOS) complexes. Genetic analysis of mitochondrial DNA in various tissues with subsequent investigation of single muscle fibres for correlation of mutational load. RESULTS The patient's skeletal muscle showed 20% of cytochrome c oxidase-negative fibres and 8% ragged-red fibres. Genetic analysis of the mitochondrial DNA revealed a novel point mutation in the mitochondrial tRNA(Ile) (MTTI) gene at position m.4282G>A. The heteroplasmy was determined in blood, buccal cells and muscle by restriction fragment length polymorphism (RFLP) combined with a last fluorescent cycle. The total mutational load was 38% in skeletal muscle, but was not detectable in blood or buccal cells of the patient. The phenotype segregated with the mutational load as determined by analysis of single cytochrome c oxidase-negative/positive fibres by laser capture microdissection and subsequent LFC-RFLP. CONCLUSIONS We describe a novel MTTI transition mutation at nucleotide position m.4282G>A associated with a CPEO plus phenotype. The novel variant at position m.4282G>A disrupts the middle bond of the D-stem of the tRNA(Ile) and is highly conserved. The conservation and phenotype-genotype segregation strongly suggest pathogenicity and is in good agreement with the MTTI gene being frequently associated with CPEO. This novel variant broadens the spectrum of MTTI mutations causing CPEO.

Carriage of the PNPLA3 rs738409 C >G polymorphism confers an increased risk of non-alcoholic fatty liver disease associated hepatocellular carcinoma.

BACKGROUND & AIMS Subtle inter-patient genetic variation and environmental factors combine to determine disease progression in non-alcoholic fatty liver disease (NAFLD). Carriage of the PNPLA3 rs738409 c.444C >G minor allele (encoding the I148M variant) has been robustly associated with advanced NAFLD. Although most hepatocellular carcinoma (HCC) is related to chronic viral hepatitis or alcoholic liver disease, the incidence of NAFLD-related HCC is increasing. We examined whether rs738409 C >G was associated with HCC-risk in patients with NAFLD. METHODS PNPLA3 rs738409 genotype was determined by allelic discrimination in 100 European Caucasians with NAFLD-related HCC and 275 controls with histologically characterised NAFLD. RESULTS Genotype frequencies were significantly different between NAFLD-HCC cases (CC=28, CG=43, GG=29) and NAFLD-controls (CC=125, CG=117, GG=33) (p=0.0001). In multivariate analysis adjusted for age, gender, diabetes, BMI, and presence of cirrhosis, carriage of each copy of the rs738409 minor (G) allele conferred an additive risk for HCC (adjusted OR 2.26 [95% CI 1.23-4.14], p=0.0082), with GG homozygotes exhibiting a 5-fold [1.47-17.29], p=0.01 increased risk over CC. When compared to the UK general population (1958 British Birth Cohort, n=1476), the risk-effect was more pronounced (GC vs. CC: unadjusted OR 2.52 [1.55-4.10], p=0.0002; GG vs. CC: OR 12.19 [6.89-21.58], p<0.0001). CONCLUSIONS Carriage of the PNPLA3 rs738409 C >G polymorphism is not only associated with greater risk of progressive steatohepatitis and fibrosis but also of HCC. If validated, these findings suggest that PNPLA3 genotyping has the potential to contribute to multi-factorial patient-risk stratification, identifying those to whom HCC surveillance may be targeted.

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.

Increasing the Glutathione Content in a Chilling-Sensitive Maize Genotype Using Safeners Increased Protection against Chilling-Induced Injury

With the aim of analyzing their protective function against chilling-induced injury, the pools of glutathione and its precursors, cysteine (Cys) and gamma -glutamyl-Cys, were increased in the chilling-sensitive maize (Zea mays) inbred line Penjalinan using a combination of two herbicide safeners. Compared with the controls, the greatest increase in the pool size of the three thiols was detected in the shoots and roots when both safeners were applied at a concentration of 5 muM. This combination increased the relative protection from chilling from 50% to 75%. It is interesting that this increase in the total glutathione (TG) level was accompanied by a rise in glutathione reductase (GR; EC 1.6.4.2) activity. When the most effective safener combination was applied simultaneously with increasing concentrations of buthionine sulfoximine, a specific inhibitor of glutathione synthesis, the total gamma -glutamyl-Cys and TG contents and GR activity were decreased to very low levels and relative protection was lowered from 75% to 44%. During chilling, the ratio of reduced to oxidized thiols first decreased independently of the treatments, but increased again to the initial value in safener-treated seedlings after 7 d at 5 degreesC. Taking all results together resulted in a linear relationship between TG and GR and a biphasic relationship between relative protection and GR or TG, thus demonstrating the relevance of the glutathione levels in protecting maize against chilling-induced injury.

Staphylococcus aureus genotype B and other genotypes isolated from cow milk in European countries

Staphylococcus aureus is globally one of the most important pathogens causing contagious mastitis in cattle. Previous studies, however, have demonstrated in Swiss cows that Staph. aureus isolated from bovine intramammary infection is genetically heterogeneous, with Staph. aureus genotype B (GTB) and GTC being the most prominent genotypes. In addition, Staph. aureus GTB was found to be contagious, whereas Staph. aureus GTC and all the remaining genotypes were involved in individual cow disease. The aim of this study was to subtype strains of Staph. aureus isolated from bovine mastitic milk and bulk tank milk to obtain a unified view of the presence of bovine staphylococcal subtypes in 12 European countries. A total of 456 strains of Staph. aureus were subjected to different typing methods: ribosomal spacer PCR, detection of enterotoxin genes, and detection of gene polymorphisms (lukE, coa). Major genotypes with their variants were combined into genotypic clusters (CL). This study revealed 5 major CL representing 76% of all strains and comprised CLB, CLC, CLF, CLI, and CLR. The clusters were characterized by the same genetic properties as the Swiss isolates, demonstrating high clonality of bovine Staph. aureus. Interestingly, CLB was situated in central Europe whereas the other CL were widely disseminated. The remaining 24% of the strains comprised 41 genotypes and variants, some of which (GTAM, GTBG) were restricted to certain countries; many others, however, were observed only once.

Open-label study of faldaprevir plus peginterferon and ribavirin in hepatitis C virus genotype 1-infected patients who failed placebo plus peginterferon and ribavirin.

Faldaprevir, a hepatitis C virus (HCV) NS3/4A protease inhibitor, was evaluated in HCV genotype 1-infected patients who failed peginterferon and ribavirin (PegIFN/RBV) treatment during one of three prior faldaprevir trials. Patients who received placebo plus PegIFN/RBV and had virological failure during a prior trial were enrolled and treated in two cohorts: prior relapsers (n = 43) and prior nonresponders (null responders, partial responders and patients with breakthrough; n = 75). Both cohorts received faldaprevir 240 mg once daily plus PegIFN/RBV for 24 weeks. Prior relapsers with early treatment success (ETS; HCV RNA <25 IU/mL detectable or undetectable at week 4 and <25 IU/mL undetectable at week 8) stopped treatment at week 24. Others received PegIFN/RBV through week 48. The primary efficacy endpoint was sustained virological response (HCV RNA <25 IU/mL undetectable) 12 weeks post treatment (SVR12). More prior nonresponders than prior relapsers had baseline HCV RNA ≥800 000 IU/mL (80% vs 58%) and a non-CC IL28B genotype (91% vs 70%). Rates of SVR12 (95% CI) were 95.3% (89.1, 100.0) among prior relapsers and 54.7% (43.4, 65.9) among prior nonresponders; corresponding ETS rates were 97.7% and 65.3%. Adverse events led to faldaprevir discontinuations in 3% of patients. The most common Division of AIDS Grade ≥2 adverse events were anaemia (13%), nausea (10%) and hyperbilirubinaemia (9%). In conclusion, faldaprevir plus PegIFN/RBV achieved clinically meaningful SVR12 rates in patients who failed PegIFN/RBV in a prior trial, with response rates higher among prior relapsers than among prior nonresponders. The adverse event profile was consistent with the known safety profile of faldaprevir.

Phenotype and genotype in 103 patients with tricho-rhino-phalangeal syndrome.

Tricho-rhino-phalangeal syndrome (TRPS) is characterized by craniofacial and skeletal abnormalities, and subdivided in TRPS I, caused by mutations in TRPS1, and TRPS II, caused by a contiguous gene deletion affecting (amongst others) TRPS1 and EXT1. We performed a collaborative international study to delineate phenotype, natural history, variability, and genotype-phenotype correlations in more detail. We gathered information on 103 cytogenetically or molecularly confirmed affected individuals. TRPS I was present in 85 individuals (22 missense mutations, 62 other mutations), TRPS II in 14, and in 5 it remained uncertain whether TRPS1 was partially or completely deleted. Main features defining the facial phenotype include fine and sparse hair, thick and broad eyebrows, especially the medial portion, a broad nasal ridge and tip, underdeveloped nasal alae, and a broad columella. The facial manifestations in patients with TRPS I and TRPS II do not show a significant difference. In the limbs the main findings are short hands and feet, hypermobility, and a tendency for isolated metacarpals and metatarsals to be shortened. Nails of fingers and toes are typically thin and dystrophic. The radiological hallmark are the cone-shaped epiphyses and in TRPS II multiple exostoses. Osteopenia is common in both, as is reduced linear growth, both prenatally and postnatally. Variability for all findings, also within a single family, can be marked. Morbidity mostly concerns joint problems, manifesting in increased or decreased mobility, pain and in a minority an increased fracture rate. The hips can be markedly affected at a (very) young age. Intellectual disability is uncommon in TRPS I and, if present, usually mild. In TRPS II intellectual disability is present in most but not all, and again typically mild to moderate in severity. Missense mutations are located exclusively in exon 6 and 7 of TRPS1. Other mutations are located anywhere in exons 4-7. Whole gene deletions are common but have variable breakpoints. Most of the phenotype in patients with TRPS II is explained by the deletion of TRPS1 and EXT1, but haploinsufficiency of RAD21 is also likely to contribute. Genotype-phenotype studies showed that mutations located in exon 6 may have somewhat more pronounced facial characteristics and more marked shortening of hands and feet compared to mutations located elsewhere in TRPS1, but numbers are too small to allow firm conclusions.

Phenotype-genotype studies in kuru: Implications for new variant Creutzfeldt–Jakob disease

The PRNP polymorphic (methionine/valine) codon 129 genotype influences the phenotypic features of transmissible spongiform encephalopathy. All tested cases of new variant Creutzfeldt–Jakob disease (nvCJD) have been homozygous for methionine, and it is conjectural whether different genotypes, if they appear, might have distinctive phenotypes and implications for the future “epidemic curve” of nvCJD. Genotype-phenotype studies of kuru, the only other orally transmitted transmissible spongiform encephalopathy, might be instructive in predicting the answers to these questions. We therefore extracted DNA from blood clots or sera from 92 kuru patients, and analyzed their codon 129 PRNP genotypes with respect to the age at onset and duration of illness and, in nine cases, to detailed clinical and neuropathology data. Homozygosity at codon 129 (particularly for methionine) was associated with an earlier age at onset and a shorter duration of illness than was heterozygosity, but other clinical characteristics were similar for all genotypes. In the nine neuropathologically examined cases, the presence of histologically recognizable plaques was limited to cases carrying at least one methionine allele (three homozygotes and one heterozygote). If nvCJD behaves like kuru, future cases (with longer incubation periods) may begin to occur in older individuals with heterozygous codon 129 genotypes and signal a maturing evolution of the nvCJD “epidemic.” The clinical phenotype of such cases should be similar to that of homozygous cases, but may have less (or at least less readily identified) amyloid plaque formation.

Quantitative analysis of senile plaques in Alzheimer disease: observation of log-normal size distribution and molecular epidemiology of differences associated with apolipoprotein E genotype and trisomy 21 (Down syndrome).

The discovery that the epsilon 4 allele of the apolipoprotein E (apoE) gene is a putative risk factor for Alzheimer disease (AD) in the general population has highlighted the role of genetic influences in this extremely common and disabling illness. It has long been recognized that another genetic abnormality, trisomy 21 (Down syndrome), is associated with early and severe development of AD neuropathological lesions. It remains a challenge, however, to understand how these facts relate to the pathological changes in the brains of AD patients. We used computerized image analysis to examine the size distribution of one of the characteristic neuropathological lesions in AD, deposits of A beta peptide in senile plaques (SPs). Surprisingly, we find that a log-normal distribution fits the SP size distribution quite well, motivating a porous model of SP morphogenesis. We then analyzed SP size distribution curves in genotypically defined subgroups of AD patients. The data demonstrate that both apoE epsilon 4/AD and trisomy 21/AD lead to increased amyloid deposition, but by apparently different mechanisms. The size distribution curve is shifted toward larger plaques in trisomy 21/AD, probably reflecting increased A beta production. In apoE epsilon 4/AD, the size distribution is unchanged but the number of SP is increased compared to apoE epsilon 3, suggesting increased probability of SP initiation. These results demonstrate that subgroups of AD patients defined on the basis of molecular characteristics have quantitatively different neuropathological phenotypes.

Alternativas de análise para experimentos G × E multiatributo

Geralmente, nos experimentos genótipo por ambiente (G × E) é comum observar o comportamento dos genótipos em relação a distintos atributos nos ambientes considerados. A análise deste tipo de experimentos tem sido abordada amplamente para o caso de um único atributo. Nesta tese são apresentadas algumas alternativas de análise considerando genótipos, ambientes e atributos simultaneamente. A primeira, é baseada no método de mistura de máxima verossimilhança de agrupamento - Mixclus e a análise de componentes principais de 3 modos - 3MPCA, que permitem a análise de tabelas de tripla entrada, estes dois métodos têm sido muito usados na área da psicologia e da química, mas pouco na agricultura. A segunda, é uma metodologia que combina, o modelo de efeitos aditivos com interação multiplicativa - AMMI, modelo eficiente para a análise de experimentos (G × E) com um atributo e a análise de procrustes generalizada, que permite comparar configurações de pontos e proporcionar uma medida numérica de quanto elas diferem. Finalmente, é apresentada uma alternativa para realizar imputação de dados nos experimentos (G × E), pois, uma situação muito frequente nestes experimentos, é a presença de dados faltantes. Conclui-se que as metodologias propostas constituem ferramentas úteis para a análise de experimentos (G × E) multiatributo.

HBV core sequence: definition of genotype-specific variability and correlation with geographical origin

There are eight genotypes and nine subtypes of HBV. Small differences in geographical origin are associated with sequence changes in the surface gene. Here, we compared core gene sequences from different genotypes and geographical regions. Specific combinations of 24 amino acid substitutions at nine residues allowed allocation of a sequence to a subtype. Six of these nine residues were located in different T cell epitopes depending on HBV geographical area and/or genotype. Thirty-seven nucleotide changes were associated uniquely with specific genotypes and subtypes. Unique amino acid and nucleotide variants were found in a majority of sequences from specific countries as well as within subtype ayw2 and adr. Specific nucleotide motifs were defined for Korean, Indian, Chinese, Italian and Pacific region isolates. Finally, we observed amino acid motifs that were common to either South-east Asian or Western populations, irrespective of subtype. We believe that HBV strains spread within constrained ethnic groups, result in selection pressures that define sequence variability within each subtype. It suggests that particular T cell epitopes are specific for geographical regions, and thus ethnic groups; this may affect the design of immunomodulatory therapies.