Prevalence of thrombophilia-associated genetic risk factors in blood donors of a regional hospital in southern Brazil

ABSTRACT Introduction: Thromboembolic events occur due to an imbalance in the hemostasis and some factors associated with this condition can be inherited. In order to evaluate the frequency of genotypes considered to be common hereditary risk factors for thrombophilia associated with venous thrombosis (g.1691G>A and g.20210G>A) and hyperhomocysteinemia (g.677C>T and g.1298A>C), samples from voluntary healthy blood donors at the Hospital de Clínicas de Porto Alegre were tested. Methods: We examined 325 blood samples from blood donors collected from October 2017 to July 2018. Blood was collected on filter paper and the DNA was extracted for single nucleotide polymorphisms (SNPs) analysis using the qualitative real time polymerase chain reaction. Results: The calculated frequencies of each genetic variant in heterozygosity were 4% for the FV gene (g.1691G> A), 4% for the F2 gene (g.20210G> A) and 42% and 39% for methylenetetrahydrofolate reductase (MTHFR), g.677C>T and g.1298A>C, respectively. Only the genetic variants of MTHFR were found in homozygosity, with frequencies of 14% and 6% (g.677C>T and g.1298A>C), respectively. Discussion: Altogether, these results describe the frequencies of genetic variants associated with venous thrombosis and hyperhomocysteinemia in the analyzed group and are important to enhance our current knowledge about the genetic profiles of Brazilian blood donors.

Prevalence of thrombophilia and thrombotic events in patients with Fabry disease in a reference center for lysosomal disorders in Southern Brazil

Introduction: Venous thromboembolism (VTE) is a multifactorial genetic disorder that occurs in approximately one in a thousand adults per year. Because there is no laboratory test or clinical marker useful for predicting which patients with Fabry disease may develop thrombotic events, this study aimed to determine whether there is a hereditary predisposition to hypercoagulation in these patients. Methods: The prevalence of p.R506Q mutation in the factor V gene and of c.G20210A mutation in Factor II (prothrombin) gene was evaluated in 39 patients with Fabry disease from Southern Brazil and correlated with clinical findings. The DNA analysis was performed by real-time polymerase chain reaction on genomic DNA using TaqMan probes. Results: In this group of patients, the frequency of mutation in the prothrombin gene was 1.28%, whereas no patient showed mutation in the factor V gene; additionally, there was no correlation between these mutations and the incidence of thrombotic events. Conclusion: Hereditary thrombophilia due to mutations in factor V and prothrombin genes does not seem to be related to thrombotic events in Fabry patients in our cohort, although studies in larger cohorts and the inclusion of additional factors may be required to determine if a correlation exists.

Análise comparativa entre as metodologias de PCR metilação-específica (MSP), southern blot (SB) e FISH utilizadas no diagnóstico genético molecular de pacientes com suspeita clínica das síndromes de Prader-Willi ou Angelman / Comparative analysis of methylation-specific PCR (MSP), southern blot (SB) and FISH in molecular genetic diagnosis of patients with clinical picture suggestive of Prader-Willi or Angelman syndromes

Introdução: Prader-Willi (SPW) e Angelman (SA) são síndromes clinicamente distintas, causadas pela perda de expressão de genes na região cromossômica 15q11.2-q13, de origem paterna ou materna, respectivamente. Ambas compartilham os mesmos métodos diagnósticos. Nossos objetivos foram: a) analisar por PCR metilação-específica (MSP) pacientes com suspeita clínica de SPW/SA; b) comparar resultados de diferentes metodologias de diagnóstico molecular; c) aplicar a técnica MSP na rotina assistencial de pacientes encaminhados ao Serviço de Genética Médica/Hospital de Clínicas de Porto Alegre (SGM/HCPA). Métodos: Foram analisados 123 pacientes com suspeita clínica de SPW (n = 71) ou SA (n = 52) por MSP. Desses, 79 possuíam análise prévia por hibridação in situ fluorescente (FISH) e/ou Southern blot (SB). Resultados: Foram detectados 21 casos positivos – 15 de SPW (12,19%) e 6 de SA (4,88%). Nove pacientes tiveram etiologia molecular determinada, sendo sete com diagnóstico de SPW (quatro dissomias uniparentais – UPD15 materna – e três deleções na região 15q11-13) e dois com diagnóstico de SA (um com UPD15 paterna e um com deleção na região 15q11-13). Foram observados resultados equivalentes entre MSP e SB e resultados discrepantes entre MSP e FISH (n = 4). Foram padronizados dois protocolos de MSP para confirmação dos resultados e controle interno de qualidade. Conclusão: O perfil de detecção de cada técnica varia de acordo com o mecanismo etiológico presente. A análise por MSP detecta alterações no padrão de metilação geradas por deleção, UPD e defeitos de imprinting, sem identificar o mecanismo etiológico responsável...

Introduction: Prader-Willi (PWS) and Angelman (AS) are clinically different syndromes caused by loss of expression of genes located on the chromosome 15q11.2-q13, of paternal or maternal origin, respectively. Both syndromes have the same diagnostic methods. The aims of the present study were: a) to perform a molecular analysis of 123 patients with clinical findings suggestive of PWS or AS using methylation-specific PCR (MSP); b) to compare the results obtained using different molecular diagnostic methodologies; c) to standardize MSP to be used in the routine care of patients at Medical Genetics Service/Hospital de Clínicas de Porto Alegre (SGM/HCPA). Methods: 123 patients with clinical findings suggestive of PWS (n = 71) or AS (n = 52) were analyzed by MSP. 79 had undergone previous laboratory analysis by fluorescence in situ hybridization (FISH) and/or Southern blot (SB). Results: MSP detected 21 positive cases – 15 PWS (12,19%) and 6 AS (4,88%). Molecular etiology was determined in 9 patients only – 7 were diagnosed with PWS (4 had uniparental disomy – maternal UPD15 – and 3 had deletions at 15q11-13) and 2 were diagnosed with AS (1 of paternal UPD15 and 1 deletion at 15q11-13). Comparing both methodologies, it was possible to observe concordant results between MSP and SB and discordant results between MSP and FISH (n = 4). We standardized two MSP methods in order to confirm the results and for internal quality control. Conclusion: The resulting profile of each technique varies according to the existing etiological mechanism. The methylation analysis by MSP technique detects changes on methylation pattern caused by deletion, UPD and imprinting defects, but it does not identify the responsible etiologic mechanism...

The multidimensional evaluation and treatment of anxiety in children and adolescents: rationale, design, methods and preliminary findings / Avaliação multidimensional e tratamento da ansiedade em crianças e adolescentes: marco teórico, desenho, métodos e resultados preliminares

OBJECTIVE: This study aims to describe the design, methods and sample characteristics of the Multidimensional Evaluation and Treatment of Anxiety in Children and Adolescents - the PROTAIA Project. METHOD: Students between 10 and 17 years old from all six schools belonging to the catchment area of the Primary Care Unit of Hospital de Clínicas de Porto Alegre were included in the project. It comprises five phases: (1) a community screening phase; (2) a psychiatric diagnostic phase; (3) a multidimensional assessment phase evaluating environmental, neuropsychological, nutritional, and biological factors; (4) a treatment phase, and (5) a translational phase. RESULTS: A total of 2,457 subjects from the community were screened for anxiety disorders. From those who attended the diagnostic interview, we identified 138 individuals with at least one anxiety disorder (apart from specific phobia) and 102 individuals without any anxiety disorder. Among the anxiety cases, generalized anxiety disorder (n = 95; 68.8 percent), social anxiety disorder (n = 57; 41.3 percent) and separation anxiety disorder (n = 49; 35.5 percent) were the most frequent disorders. CONCLUSION: The PROTAIA Project is a promising research project that can contribute to the knowledge of the relationship between anxiety disorders and anxiety-related phenotypes with several genetic and environmental risk factors.

OBJETIVO: o objetivo deste estudo é descrever o desenho, os métodos e as características amostrais da Avaliação Multidimensional e Tratamento da Ansiedade em Crianças e Adolescentes - Projeto PROTAIA. MÉTODO: Escolares entre 10 e 17 anos de todas as escolas pertencentes à área de abrangência da unidade de atenção primária do Hospital de Clínicas de Porto Alegre foram incluídos no projeto. O projeto compreende cinco fases: 1) triagem comunitária; 2) diagnóstico psiquiátrico; 3) avaliação multidimensional, incluindo fatores ambientais, neuropsicológicos, nutricionais e marcadores biológicos; 4) tratamento; e 5) fase translacional. RESULTADOS: Um total de 2.457 sujeitos foram triados para transtornos de ansiedade na comunidade. Dos indivíduos que compareceram à avaliação diagnóstica, 138 foram detectados com ao menos um transtorno de ansiedade (excluindo fobia específica) e 102 indivíduos sem nenhum transtorno de ansiedade. Dentre os casos de ansiedade, o transtorno de ansiedade generalizada (n = 95; 68,8 por cento), transtorno de ansiedade social (n = 57; 41,3 por cento) e o transtorno de ansiedade de separação (n = 49; 35,5 por cento) foram os mais frequentes. CONCLUSÃO: O projeto PROTAIA é um projeto de pesquisa promissor que pode contribuir para o entendimento da relação entre transtornos de ansiedade e fenótipos relacionados à ansiedade com vários fatores de risco, tanto genéticos quanto ambientais.

Detection of human bocavirus and human metapneumovirus by real-time PCR from patients with respiratory symptoms in Southern Brazil

The introduction of newer molecular methods has led to the discovery of new respiratory viruses, such as human metapneumovirus (hMPV) and human bocavirus (hBoV), in respiratory tract specimens. We have studied the occurrence of hMPV and hBoV in the Porto Alegre (PA) metropolitan area, one of the southernmost cities of Brazil, evaluating children with suspected lower respiratory tract infection from May 2007-June 2008. A real-time polymerase chain reaction method was used for amplification and detection of hMPV and hBoV and to evaluate coinfections with respiratory syncytial virus (RSV), influenza A and B, parainfluenza 1, 2 and 3, human rhinovirus and human adenovirus. Of the 455 nasopharyngeal aspirates tested, hMPV was detected in 14.5 percent of samples and hBoV in 13.2 percent. A unique causative viral agent was identified in 46.2 percent samples and the coinfection rate was 43.7 percent. For hBoV, 98.3 percent of all positive samples were from patients with mixed infections. Similarly, 84.8 percent of all hMPV-positive results were also observed in mixed infections. Both hBoV and hMPV usually appeared with RSV. In summary, this is the first confirmation that hMPV and hBoV circulate in PA; this provides evidence of frequent involvement of both viruses in children with clinical signs of acute viral respiratory tract infection, although they mainly appeared as coinfection agents.

Mucopolysaccharidoses in northern Brazil: Targeted mutation screening and urinary glycosaminoglycan excretion in patients undergoing enzyme replacement therapy

Mucopolysaccharidoses (MPS) are rare lysosomal disorders caused by the deficiency of specific lysosomal enzymes responsible for glycosaminoglycan (GAG) degradation. Enzyme Replacement Therapy (ERT) has been shown to reduce accumulation and urinary excretion of GAG, and to improve some of the patients' clinical signs. We studied biochemical and molecular characteristics of nine MPS patients (two MPS I, four MPS II and three MPS VI) undergoing ERT in northern Brazil. The responsiveness of ERT was evaluated through urinary GAG excretion measurements. Patients were screened for eight common MPS mutations, using PCR, restriction enzyme tests and direct sequencing. Two MPS I patients had the previously reported mutation p.P533R. In the MPS II patients, mutation analysis identified the mutation p.R468W, and in the MPS VI patients, polymorphisms p.V358M and p.V376M were also found. After 48 weeks of ERT, biochemical analysis showed a significantly decreased total urinary GAG excretion in patients with MPS I (p < 0.01) and MPS VI (p < 0.01). Our findings demonstrate the effect of ERT on urinary GAG excretion and suggest the adoption of a screening strategy for genotyping MPS patients living far from the main reference centers.

Prenatal diagnosis of fetal chromosomal abnormalities: report of an 18-year experience in a Brazilian public hospital

The study of the fetal karyotype became an important tool for the fetal diagnosis of genetic diseases in the 1970s. Although application of this test has remained very restricted in Brazil, we had 905 referrals for prenatal fetal karyotyping between 1989 and 2007. In 879 cases, a fetal karyotype was obtained. We detected 74 abnormal karyotypes (8.4%), the majority being found when the prior indication was fetal malformation. When obtaining amniotic fluid or chorionic villus samples was difficult, alternative fetal materials (urine, cystic hygroma, cystic lung, intreperitoneal and cerebrospinal fluids) were collected and we had success in obtaining karyotypes in all 13 cases. Although, the option of terminating abnormal pregnancies does not legally exist in Brazil, the information gained in assessing the prognosis of on-going pregnancies or estimating recurrence risks justifies prenatal diagnosis of chromosome abnormalities. We conclude that, in keeping with the policy in most other countries, prenatal cytogenetic analysis is strongly recommended in high-risk pregnancies for fetal abnormalities. However, the unique aspect of this type of study is not its rarity in world terms, but its rarity in Brazil. This argues that Brazilian health policy on prenatal diagnosis requires reforming to make it much more widely available within the public health care sector.