A repeat-primed PCR assay for pentanucleotide repeat alleles in spinocerebellar ataxia type 37

Spinocerebellar ataxia 37 (SCA37) is caused by an (ATTTC)n insertion in a polymorphic ATTTT repeat in the non-coding region of DAB1. The non-pathogenic alleles have a configuration [(ATTTT)7-400], whereas pathogenic alleles have a complex structure of [(ATTTT)60-79(ATTTC)31-75(ATTTT)58-90]. Molecular diagnosis of SCA37 is laborious because about 7% of the pentanucleotide repeat alleles in DAB1 are larger than 30 units and, thus, fail to amplify with standard PCR conditions, resulting in apparently homoallelism or in complete lack of PCR amplification in several cases. The molecular test currently available requires long-range PCR and sequencing analysis for the detection and characterization of these large alleles. We developed a simple assay capable of rapidly detecting the presence or absence of large pentanucleotide repeat sizes. This assay is based on repeat-primed PCR followed by high-throughput capillary electrophoresis. Combining the standard PCR with RP-PCR allows completion of the diagnosis in more than 80% of individuals, minimizing the number of samples that require long-range PCR followed by Sanger sequencing analysis. This assay meets many of the requirements for pre-screening of large cohorts of affected individuals.This work was funded by Fundo Europeu de Desenvolvimento Regional-FEDER funds through the COMPETE 2020—Operational Programme for Competitiveness and Inter- nationalisation (POCI), Portugal 2020, and by funding from FCT— Fundação para a Ciência e a Tecnologia/Ministério da Ciência, Tec- nologia e Inovação, Portugal, in the framework of the project “Institute for Research and Innovation in Health Sciences” (POCI-01-0145- FEDER-007274); by Grant PTDC/SAU-GMG/098305/2008, from FCT, to I.S. J.R.L. was supported by scholarships from Grant PTDC/ GMG-SAU/098305/2008, FCT, PEst- C/SAU/LA0002/2013 and EMBO (ASTF494-2015). C.L.O. was supported by a scholarship from PEst-C/SAU/LA0002/2013. This work was also funded by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (FEDER), Portugal, that supports the Norte-01-0145-FEDER-000008—Porto Neurosciences and Neurologic Disease Research Initiative at I3S

Mutational mechanism for DAB1 (ATTTC) n insertion in SCA37: ATTTT repeat lengthening and nucleotide substitution

Dynamic mutations by microsatellite instability are the molecular basis of a growing number of neuromuscular and neurodegenerative diseases. Repetitive stretches in the human genome may drive pathogenicity, either by expansion above a given threshold, or by insertion of abnormal tracts in nonpathogenic polymorphic repetitive regions, as is the case in spinocerebellar ataxia type 37 (SCA37). We have recently established that this neurodegenerative disease is caused by an (ATTTC)n insertion within an (ATTTT)n in a noncoding region of DAB1. We now investigated the mutational mechanism that originated the (ATTTC)n insertion within an ancestral (ATTTT)n . Approximately 3% of nonpathogenic (ATTTT)n alleles are interspersed by AT-rich motifs, contrarily to mutant alleles that are composed of pure (ATTTT)n and (ATTTC)n stretches. Haplotype studies in unaffected chromosomes suggested that the primary mutational mechanism, leading to the (ATTTC)n insertion, was likely one or more T>C substitutions in an (ATTTT)n pure allele of approximately 200 repeats. Then, the (ATTTC)n expanded in size, originating a deleterious allele in DAB1 that leads to SCA37. This is likely the mutational mechanism in three similar (TTTCA)n insertions responsible for familial myoclonic epilepsy. Because (ATTTT)n tracts are frequent in the human genome, many loci could be at risk for this mutational process.We are grateful to the families and individuals who participated in this work. We thank Patricia Ribeiro for technical assistance. This study was financed by Fundo Europeu de Desenvolvimento Regional (FEDER), through the COMPETE 2020 Operational Pro- gram for Competitiveness and Internationalization (POCI) of Portugal 2020, and by the Fundacão para a Ciência e a Tecnologia (FCT) and Ministério da Ciência, Tecnologia e Ensino Superior (Portugal), in the framework of the project POCI-01-0145-FEDER-029255; (PTDC/MED-GEN/29255/2017) to I.S. J.R.L. and C.L.O. were sup- ported by scholarships from PEst-C/SAU/LA0002/2013. S.M. is funded by the project IF/00930/2013/ CP1184/CT0002 from FCT. This work was also funded by the Porto Neurosciences and Neurologic Disease Research Initiative at the Instituto de Investigação e Inovação em Saúde (Norte-01-0145-FEDER- 000008), supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTU- GAL 2020 Partnership Agreement with FEDER

Lack of HLA predominance and HLA shared epitopes in biliary Atresia

Biliary atresia (BA) is characterized by progressive inflammation and fibrosis of bile ducts. A theory of pathogenesis entails autoimmune-mediated injury targeting bile duct epithelia. One of the strongest genetic associations with autoimmunity is with HLA genes. In addition, apparently dissimilar HLA alleles may have similar antigen-binding sites, called shared epitopes, that overlap in their capacity to present antigens. In autoimmune disease, the incidence of the disease may be related to the presence of shared epitopes, not simply the HLA allelic association. Aim: To determine HLA allele frequency (high-resolution genotyping) and shared epitope associations in BA. Results: Analysis of every allele for HLA-A, -B, -C, -DRB1, -DPB1 and -DQB1 in 180 BA and 360 racially-matched controls did not identify any significant HLA association with BA. Furthermore, shared epitope analysis of greater than 10 million possible combinations of peptide sequences was not different between BA and controls. Conclusions: This study encompasses the largest HLA allele frequency analysis for BA in the United States and is the first study to perform shared epitope analysis. When controlling for multiple comparisons, no HLA allele or shared epitope association was identified in BA. Future studies of genetic links to BA that involve alterations of the immune response should include investigations into defects in regulatory T cells and non-HLA linked autoinflammatory diseases. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/2193-1801-2-42) contains supplementary material, which is available to authorized users

Levels of expression and immunogenicity of attenuated Salmonella enterica serovar typhimurium strains expressing Escherichia coli mutant heat-labile enterotoxin

The effects of heterologous gene dosage as well as Salmonella typhimurium strain variability on immune response toward both the heterologous antigen, the nontoxic mutant of the Escherichia coli heat-labile enterotoxin LTK63, and the carrier Salmonella strain have been analyzed, Effects of a single integration into the host DNA and different-copy-number episomal vectors were compared in S. typhimurium Delta cya Delta crp Delta asd strains of two different serotypes, UK-1 and SR-11, Expression of the enterotoxin in the different Salmonella isolates in vitro was found to vary considerably and, for the episomal vectors, to correlate with the plasmid copy number, LTK63-specific serum immunoglobulin G (IgG) and mucosal immunoglobulin A (IgA) antibodies were highest in mice immunized with the high-level-expression strain. High anti-LTK63 IgG and IgA titers were found to correspond to higher anti-Salmonella immunity, suggesting that LTK63 exerts an adjuvant effect on response to the carrier. Statistically significant differences in anti-LTK63 immune response were observed between groups of mice immunized with the attenuated Delta cya Delta crp UK-I and SR-II derivatives producing the antigen at the same rate, These data indicate that the same attenuation in S, typhimurium strains of different genetic backgrounds can influence significantly the immune response toward the heterologous antigen. Moreover, delivery of the LTK63 enterotoxin to the immune system by attenuated S. typhimurium strains is effective only when synthesis of the antigen is very high during the initial phase of invasion, while persistence of the S. typhimurium strain in deep tissues has only marginal influence.66122423

Validação conceitual das características definidoras de diagnósticos de enfermagem respiratórios em neonatos

OBJECTIVE:To develop and validate conceptual and operational definitions for the defining characteristics of the respiratory nursing diagnoses, ineffective breathing pattern, impaired gas exchange and impaired spontaneous ventilation, in newborns.METHODS:This was a methodological study of conceptual validation of the defining characteristics of three respiratory nursing diagnoses, by consensus analysis of a committee of five specialist nurses, and then a group of five non-nursing professionals, using the Delphi technique.RESULTS:After two rounds of evaluation, consensus was obtained that was equal to or greater than 80% on all of the definitions, which were then considered validated.CONCLUSION:The definitions developed for the defining characteristics of three nursing diagnoses were validated with a high level of consensus.OBJETIVO:Elaborar e validar definições conceituais e operacionais para as características definidoras dos diagnósticos de enfermagem respiratórios, Padrão Respiratório Ineficaz, Troca de Gases Prejudicada e Ventilação Espontânea Prejudicada em recém-nascidos.MÉTODOS:Estudo metodológico, de validação conceitual das características definidoras dos três diagnósticos de enfermagem respiratórios por meio da análise de consenso de um comitê de cinco enfermeiras especialistas e de cinco profissionais não enfermeiros, utilizando a técnica Delphi.RESULTADOS:Após duas rodadas de avaliação, obteve-se consenso igual ou superior a 80% na totalidade das definições, sendo consideradas validadas.CONCLUSÃO:As definições elaboradas para as características definidoras dos três diagnósticos de enfermagem foram validadas com elevado grau de consenso.Universidade Federal de São Paulo (UNIFESP) Escola Paulista de EnfermagemUNIFESP, EPESciEL

HEALTH AND PHYSIOLOGICAL QUALITY OF AROEIRA-PRETA ( Lithraea molleoides ) SEEDS EXPOSED TO METHODS OF OVERCOMING DORMANCY

O presente estudo teve como objetivo avaliar a qualidade fisiol\uf3gica e sanit\ue1ria de sementes de ( Lithraea molleoides (Vell.) Engl. comparando diferentes m\ue9todos de supera\ue7\ue3o da dorm\ueancia. Os m\ue9todos de supera\ue7\ue3o da dorm\ueancia utilizados foram: escarifica\ue7\ue3o \ue1cida por 10, 15, 20 e 25 minutos; imers\ue3o em \ue1gua quente, com temperatura de 70, 80 e 90\ub0C, at\ue9 resfriar por 24 horas, imers\ue3o em \ue1cido giber\ue9lico (GA3) na concentra\ue7\ue3o de 250 e 500 mg.L-1, por 24 e 48 horas; e imers\ue3o em nitrato de pot\ue1ssio (KNO3) por 24 e 48 horas. Foram realizadas avalia\ue7\uf5es de sanidade, germina\ue7\ue3o e comprimento m\ue9dio de pl\ue2ntulas. O delineamento experimental foi inteiramente casualizado, com quatro repeti\ue7\uf5es de 25 sementes por tratamento. Os dados em percentagem foram transformados segundo arco sen 1ax/100 e submetidos \ue0 an\ue1lise de vari\ue2ncia. A compara\ue7\ue3o das m\ue9dias foi realizada atrav\ue9s do teste de Tukey a 5 % de signific\ue2ncia. Foi realizada an\ue1lise de correla\ue7\ue3o simples entre sementes mortas do teste de germina\ue7\ue3o e os diferentes fungos identificados no teste de sanidade. No teste de sanidade, foram identificados com maior incid\ueancia os fungos Rhizoctonia spp., Penicillium spp., Aspergillus spp., Alternaria spp., Chaetomium spp., Epicoccum spp. De uma maneira geral, a utiliza\ue7\ue3o da \ue1gua quente controlou a incid\ueancia dos diferentes fungos e a utiliza\ue7\ue3o do \ue1cido giber\ue9lico proporcionou um aumento da incid\ueancia dos diferentes pat\uf3genos. A maior porcentagem de germina\ue7\ue3o foi observada quando se utilizou escarifica\ue7\ue3o \ue1cida por 20 minutos, imers\ue3o em \ue1gua quente a 70\ub0C, GA3 (250 mg L-1 por 48 horas) e KNO3 por 48 horas.This study aimed to evaluate the physiological and sanitary quality of seeds ( Lithraea molleoides (Vell.) Engl. comparing different methods to overcome dormancy. Methods of overcoming dormancy were used: acid scarification for 10, 15, 20 and 25 minutes soaking in hot water with temperatures of 70, 80 and 90\ub0C, for 24 hours until cool, soaking in gibberellic acid (GA3) in the concentration 250 and 500 mg.L-1 for 24 and 48 hours, and immersion in potassium nitrate (KNO3) for 24 and 48 hours. We evaluated health, germination and seedling length of the experimental design was completely randomized design with four replications of 25 seeds per treatment. The percentage data were transformed into the second arc sin 1ax/100 and subjected to analysis of variance. Comparison of means was performed using the Tukey test at 5% significance level. Analysis was a simple correlation between the test of dead seeds test and the different fungi in identified sanity. In the health test, the fungi which had the highest incidence were Rhizoctonia spp., Penicillium spp., Aspergillus spp., Alternaria spp., Chaetomium spp., Epicoccum spp. In general, the use of hot water controlled the incidence of different fungi and the use of gibberellic acid resulted in an increase in the incidence of different pathogens. The highest percentage of germination was observed when using acid scarification for 20 minutes, soaking in hot water at 70\ub0C, GA3(250 mg.L-1 for 48 hours) and KNO3 for 48 hours

A pentanucleotide ATTTC repeat insertion in the non-coding region of DAB1, mapping to SCA37, causes spinocerebellar ataxia.

Advances in human genetics in recent years have largely been driven by next-generation sequencing (NGS); however, the discovery of disease-related gene mutations has been biased toward the exome because the large and very repetitive regions that characterize the non-coding genome remain difficult to reach by that technology. For autosomal-dominant spinocerebellar ataxias (SCAs), 28 genes have been identified, but only five SCAs originate from non-coding mutations. Over half of SCA-affected families, however, remain without a genetic diagnosis. We used genome-wide linkage analysis, NGS, and repeat analysis to identify an (ATTTC)n insertion in a polymorphic ATTTT repeat in DAB1 in chromosomal region 1p32.2 as the cause of autosomal-dominant SCA; this region has been previously linked to SCA37. The non-pathogenic and pathogenic alleles have the configurations [(ATTTT)7-400] and [(ATTTT)60-79(ATTTC)31-75(ATTTT)58-90], respectively. (ATTTC)n insertions are present on a distinct haplotype and show an inverse correlation between size and age of onset. In the DAB1-oriented strand, (ATTTC)n is located in 5' UTR introns of cerebellar-specific transcripts arising mostly during human fetal brain development from the usage of alternative promoters, but it is maintained in the adult cerebellum. Overexpression of the transfected (ATTTC)58 insertion, but not (ATTTT)n, leads to abnormal nuclear RNA accumulation. Zebrafish embryos injected with RNA of the (AUUUC)58 insertion, but not (AUUUU)n, showed lethal developmental malformations. Together, these results establish an unstable repeat insertion in DAB1 as a cause of cerebellar degeneration; on the basis of the genetic and phenotypic evidence, we propose this mutation as the molecular basis for SCA37.We thank the families who participated in this study. We are grateful to Goncalo Abecasis, Miguel Costa, Tito Vieira, and Andre Torres for help with MERLIN analysis; Beatriz Sobrino, Jorge Amigo, and Pilar Cacheiro for next-generation sequencing analysis, performed at the Santiago de Compostela node of the Spanish National Genotyping Center; Nuno Santarem and Anabela Cordeiro-da-Silva for assistance with cloning; Antonio Amorim, Laura Vilarinho, and Paula Jorge for samples from the Portuguese population; and Paula Magalhaes from the Institute for Molecular and Cell Biology Cell Culture and Genotyping Core for DNA extraction. This work was financed by Fundo Europeu de Desenvolvimento Regional (FEDER) funds through the COMPETE 2020 Operational Program for Competitiveness and Internationalization (POCI) of Portugal 2020 and by Portuguese funds through the Fundacao para a Ciencia e a Tecnologia (FCT) and Ministerio da Ciencia, Tecnologia, e Inovacao in the framework of the project "Institute for Research and Innovation in Health Sciences" (POCI-01-0145-FEDER-007274); and by FCT grant PTDC/SAU-GMG/098305/2008 to I.S. A. I.S. was the recipient of an FCT scholarship (SFRH/BD/30702/2006). J.R.L. was supported by scholarships from PEst-C/SAU/LA0002/2013 and the European Molecular Biology Organization (ASTF494-2015). C.L.O. was supported by a scholarship from PEst-C/SAU/LA0002/2013. This work was also financed by the Porto Neurosciences and Neurologic Disease Research Initiative at the Instituto de Investigacao e Inovacao em Saude (Norte-01-0145-FEDER-000008), supported by Norte Portugal Regional Operational Programme (NORTE 2020) under the PORTUGAL 2020 Partnership Agreement through FEDER, and by the Fondo de Investigacion Sanitaria of the Instituto de Salud Carlos III (grant PI12/00742)