O angioedema hereditário (AEH) é uma doença autossômica dominante, caracterizada por episódios recorrentes de edema subcutâneo, do trato gastrointestinal e das vias aéreas superiores. Em sua forma clássica, o AEH é causado por deficiência do inibidor de C1 (C1- INH) e está associado a mutações no gene SERPING1. Recentemente, foi descrito o AEH com inibidor de C1 normal, resultante de mutações no gene F12, que codifica o Fator XII da coagulação (FXII), denominado AEH-FXII; e de mutações nos genes PLG e ANGPT1, que codificam Plasminogênio e Angiopoietina-1, caracterizando os tipos AEH-PLG e AEHANGPT1. O AEH com inibidor de C1 normal não cursa com diminuição de C1-INH quantitativo e/ou funcional, ou de C4, sendo o diagnóstico realizado pela presença de sintomas clínicos sugestivos e história familiar. Os objetivos do presente estudo foram: avaliar a discriminação alélica como método alternativo ao sequenciamento por método de Sanger, para o diagnóstico de AEH com inibidor de C1 normal por mutação c.983C>A no gene F12; determinar se a discriminação alélica seria uma técnica de melhor custo-benefício; e investigar outras mutações previamente descritas no exon 9 do gene F12, e nos genes PLG e ANGPT1 em pacientes com suspeita clínica de AEH com inibidor de C1 normal, e negativos para mutação c.983C>A em F12. Cento e oitenta e quatro indivíduos incluindo 51 pacientes- índice com suspeita clínica de AEH com inibidor de C1 normal, e seus familiares, foram previamente investigados por sequenciamento de Sanger. Esses indivíduos foram investigados por discriminação alélica, e a concordância entre os resultados para a mutação c.983C>A foi verificada por estatística Kappa. Custos e tempo de execução entre os dois métodos foram avaliados. Casos-índice negativos para a mutação c.983C>A no gene F12 foram avaliados para outras mutações no exon 9 do gene F12 e para mutações previamente descritas c.988A>G e c.807G>T em PLG e ANGPT1, respectivamente, usando sequenciamento por método de Sanger. Mutação no gene F12 foi identificada em 96 indivíduos (24 pacientes- índice e 72 familiares). Em todos esses pacientes foi encontrada a mutação c.983C>A. 88 indivíduos foram negativos para esta mutação. Os resultados foram 100% concordantes entre os dois métodos. Setenta e um dos 96 pacientes positivos para mutação c.983C>A eram do sexo feminino; 78,9% e 56% eram pacientes sintomáticos do sexo feminino e masculino, respectivamente. A discriminação alélica apresentou custo e tempo de execução 79,7% e 82,3% menores em comparação ao sequenciamento por Sanger, respectivamente. Outras mutações no gene F12, e mutações em PLG e ANGPT1 não foram encontradas. Entre os pacientes negativos para mutações associadas ao AEH com inibidor de C1 normal, 11 foram diagnosticados como AEH-desconhecido e 16 como angioedema idiopático adquirido não histaminérgico. Os resultados do presente estudo nos permitiram construir um algoritmo para diagnóstico de pacientes com AEH com inibidor de C1 normal. Em áreas onde a mutação c.983C>A em F12 é predominante entre pacientes com AEH-FXII, a discriminação alélica pode ser um método adequado para screening inicial. Em pacientes com resultados negativos, sequenciamento do exon 9 de F12 pelo método de Sanger estaria indicado. Pacientes remanescentes com resultados negativos seriam genotipados para mutações previamente descritas em PLG e ANGPT1. Estudos futuros em outros locais serão necessários para estabelecer se a discriminação alélica apresentaria melhor custo-benefício, com potencial para mais acessibilidade ao diagnóstico em pacientes portadores da doença no Brasil / Hereditary angioedema (HAE) is an autosomal dominant disease characterized by recurrent episodes of edema of subcutaneous tissue, gastrointestinal tract, and upper airways. In its classical form, HAE is caused by deficiency of C1 inhibitor (C1-INH) and it is associated with mutations in the SERPING1 gene. Recently, HAE with normal C1 inhibitor has been described, resulting from mutations in F12 gene, which encodes coagulation factor XII (FXII), designated as FXII-HAE; and from mutations in PLG and ANGPT1 genes, which encode Plasminogen and Angiopoietin-1, characterizing PLG-HAE and ANGPT1-HAE types. HAE with normal C1-INH does not present with decrease in quantitative and/or functional levels of C1-INH or C4, and diagnosis is carried out by presence of suggestive clinical symptoms and family history. Diagnosis can only be confirmed by genetic sequencing. The objectives of the present study were: to evaluate the allelic discrimination as an alternative method to sequencing by Sanger method for the diagnosis of HAE with normal C1-INH due to mutation c.983C>A in F12 gene; to determine whether allelic discrimination would be a better cost-effective technique; and to investigate presence of other mutations previously described in exon 9 of F12 gene and in PLG and ANGPT1 genes in patients with clinical suspicion of HAE with normal C1-INH who were negative for c.983C>A mutation in F12. One hundred and eighty-four individuals including 51 index patients with clinical suspicion of HAE with normal C1-INH and their relatives were previously investigated by Sanger sequencing. These individuals were investigated for allelic discrimination, and concordance of the results for the c.983C>A mutation was verified by Kappa statistics. Costs and execution time of the two methods were evaluated. Negative index cases for c.983C>A mutation in F12 gene were evaluated for other mutations in exon 9 of the F12 gene and for mutations previously described c.988A>G and c.807G>T in PLG and ANGPT1, respectively, using sequencing by Sanger method. Mutation in F12 gene was identified in 96 individuals (24 index patients and 72 relatives). In all these patients, the c.983C>A mutation was found. 88 subjects were negative for this mutation. The results were 100% concordant between the two methods. Seventy-one of the 96 patients positive for the c.983C>A mutation were female; 78.9% and 56% were symptomatic female and male patients, respectively. The allelic discrimination presented cost and execution time 79.7% and 82.3% lower in comparison to sequencing by Sanger, respectively. Other mutations in F12 gene, and mutations in PLG and ANGPT1 were not found. Among patients who were negative for mutations associated to HAE with normal C1-INH, 11 were diagnosed as unknown HAE and 16 as idiopathic nonhistaminergic acquired angioedema. The results of present study allowed us to construct an algorithm for diagnosis of patients with HAE with normal C1-INH. In areas where the c.983C>A mutation in F12 is predominant among patients with FXII-HAE, allelic discrimination may be a suitable method for initial screening. In patients with negative results, sequencing of F12 exon 9 by the Sanger method would be indicated. Remaining patients with negative results would be genotyped for mutations previously described in PLG and ANGPT1. Future studies at other sites will be needed to establish whether allelic discrimination would be more cost-effective, with potential for more accessibility to diagnosis in patients with the disease in Brazil

<p> </p><p>Genotyping can be used to link genetic variation among individuals to certain diseases or conditions. Some known disorders and states that are dependent on single nucleotide polymorphism (SNPs) are lactose intolerance, venous thrombosis, hereditary hemochromatosis and the difference in sensibility among people to metabolise drugs.</p><p>In this project a new kit, TaqManÒ Sample-to-SNP KitÔ <strong>for extraction of DNA and preparation of the extract for genotyping with real-time PCR and allelic discrimination, was evaluated. QIAamp® DNA Blood Biorobot® MDx Kit was used as the reference method.</strong></p><p>The purpose of the comparison was to find a method that makes DNA extraction from blood samples cheaper and faster, but with the same reliability as the reference procedure.</p><p>The results of the evaluation showed a complete agreement of the genotype results between the methods tested, which means that the new method was as reliable as the reference method. The costs of reagents and material would be reduced with 52% if the new method is adopted, that alone would result in a cost reduction of 144 000SEK a year with a sample volume of 650 samples/month. The time for DNA extraction would also be reduced with the new procedure.</p><p> </p>

Genotyping can be used to link genetic variation among individuals to certain diseases or conditions. Some known disorders and states that are dependent on single nucleotide polymorphism (SNPs) are lactose intolerance, venous thrombosis, hereditary hemochromatosis and the difference in sensibility among people to metabolise drugs. In this project a new kit, TaqManÒ Sample-to-SNP KitÔ for extraction of DNA and preparation of the extract for genotyping with real-time PCR and allelic discrimination, was evaluated. QIAamp® DNA Blood Biorobot® MDx Kit was used as the reference method. The purpose of the comparison was to find a method that makes DNA extraction from blood samples cheaper and faster, but with the same reliability as the reference procedure. The results of the evaluation showed a complete agreement of the genotype results between the methods tested, which means that the new method was as reliable as the reference method. The costs of reagents and material would be reduced with 52% if the new method is adopted, that alone would result in a cost reduction of 144 000SEK a year with a sample volume of 650 samples/month. The time for DNA extraction would also be reduced with the new procedure.

Equine herpesvirus-1 (EHV-1) can cause acute upper respiratory tract disease, abortion, neonatal death and neurological disease in horses. Rapid, accurate and timely diagnosis of EHV-1 infection in horses is important to curtail the spread of this pathogen. It has been reported that the neuropathogenic phenotype of EHV-1 can result from a single non-synonymous nucleotide substitution at position 2254 (A→G2254) in open reading frame 30 (ORF30). This was the basis for the development of an allelic discrimination, real-time PCR assay to distinguish between potential neuropathogenic and non-neuropathogenic EHV-1 strains. However, PCR analysis of a panel of EHV-1 abortion isolates revealed that other point mutations within ORF30 could produce false negative results with this previously described assay. Therefore, one of the objectives of this dissertation project was to develop a more sensitive and specific allelic discrimination real-time PCR assay for the detection of EHV-1. This was achieved by redesigning the primers and probes targeting ORF30. The new assay was ten times more sensitive than the original assay, with a lower detection limit of 10 infectious virus particles. While mutations within EHV-1’s genome can hinder diagnosis, they can also impact the virulence of the virus. Objective two, therefore, was to determine if sequential cell passage of T953 would induce sufficient attenuation of the EHV-1 genome to produce a low virulence phenotype. Two separate groups of 28 BALB/c mice were inoculated with either the parental strain or passage 135 (T953 P135) of EHV-1 strain T953. The animals were observed for fourteen days, euthanized and their tissues analyzed for the presence of EHV-1. At the conclusion of the fourteen day observation period, all of the mice infected with T953 P135 survived and regained their pre-inoculation body condition. Furthermore, there were significant differences in virus titer and viral DNA concentrations between T953 P135 and the parental strain, further confirming the attenuated phenotype of the virus. Taken together, data from this study clearly demonstrates that sequential cell culture passage of the neuropathogenic T953 strain of EHV-1 results in attenuation for young adult BALB/C mice.