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Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease with clinical and etiological heterogeneity and a complex genetic contribution. Clinical, neuropathological, and genetic evidence revealed that ALS and frontotemporal dementia (FTD) are in part of a single disease continuum. Genetic causes have been identified in sporadic (SALS) and familial patients (FALS) and the recurrent genetic factor underlying ALS and FTD is the C9orf72 hexanucleotide repeat expansion (HRE). However, in our population, the concomitance of ALS and FTD cannot be explained by C9orf72 HRE in many FALS and SALS cases. Our aim is to further understand the genetic basis of ALS in Portuguese patients. 34 patients with FALS or SALS-FTD, negative for C9orf72 HRE, were screened for rare variants in a panel of 29 relevant genes by next-generation sequencing. We detected 15 variants in 11 genes, one classified as pathogenic in TARDBP, two as likely pathogenic in TARDBP and PRPH, and the others as variants of unknown significance (VUS). Gene variants, including VUS, were found in 41.2% FALS patients and 40% SALS-FTD. In most patients, no potential pathogenic variants were found. Our results emphasize the need to enhance the efforts to unravel the genetic architecture of ALS-FTD. info:eu-repo/semantics/publishedVersion

Introduction: Autosomal dominant tubulo-interstitial kidney disease due to UMOD mutations (ADTKD-UMOD) is a rare condition associated with high variability in the age of end-stage kidney disease (ESKD). The minor allele of rs4293393, located in the promoter of the UMOD gene, is present in 19% of the population and downregulates uromodulin production by approximately 50% and might affect the age of ESKD. The goal of this study was to better understand the genetic and clinical characteristics of ADTKD-UMOD and to perform a Mendelian randomization study to determine if the minor allele of rs4293393 was associated with better kidney survival. Methods: An international group of collaborators collected clinical and genetic data on 722 affected individuals from 249 families with 125 mutations, including 28 new mutations. The median age of ESKD was 47 years. Men were at a much higher risk of progression to ESKD (hazard ratio 1.78, P < 0.001). Results: The allele frequency of the minor rs4293393 allele was only 11.6% versus the 19% expected (P < 0.01), resulting in Hardy-Weinberg disequilibrium and precluding a Mendelian randomization experiment. An in vitro score reflecting the severity of the trafficking defect of uromodulin mutants was found to be a promising predictor of the age of ESKD. Conclusion: We report the clinical characteristics associated with 125 UMOD mutations. Male gender and a new in vitro score predict age of ESKD. publishersversion published

Copyright: © 2015 Perpétuo et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, Introduction: Ankylosing Spondylitis (AS) is characterized by excessive local bone formation and concomitant systemic bone loss. Tumor necrosis factor (TNF) plays a central role in the inflammation of axial skeleton and enthesis of AS patients. Despite reduction of inflammation and systemic bone loss, AS patients treated with TNF inhibitors (TNFi) have ongoing local bone formation. The aim of this study was to assess the effect of TNFi in the differentiation and activity of osteoclasts (OC) in AS patients. Methods: 13 AS patients treated with TNFi were analyzed at baseline and after a minimum follow-up period of 6 months. 25 healthy donors were recruited as controls. Blood samples were collected to assess receptor activator of nuclear factor kappa-B ligand (RANKL) surface expression on circulating leukocytes and frequency and phenotype of monocyte subpopulations. Quantification of serum levels of bone turnover markers and cytokines, in vitro OC differentiation assay and qRT-PCR for OC specific genes were performed. Results: RANKL+ circulating lymphocytes (B and T cells) and IL-17A, IL-23 and TGF-β levels were decreased after TNFi treatment. We found no differences in the frequency of the different monocyte subpopulations, however, we found decreased expression of CCR2 and increased expression of CD62L after TNFi treatment. OC number was reduced in patients at baseline when compared to controls. OC specific gene expression was reduced in circulating OC precursors after TNFi treatment. However, when cultured in OC differentiating conditions, OC precursors from AS TNFi-treated patients showed increased activity as compared to baseline. Conclusion: In AS patients, TNFi treatment reduces systemic pro osteoclastogenic stimuli. However, OC precursors from AS patients exposed to TNFi therapy have increased in vitro activity in response to osteoclastogenic stimuli., This work was supported by Fundação para a Ciência e Tecnologia (http://www.fct.pt/index.phtml.en) PhD grant (SFRH/BD/70533/2010) and in part by a research grant from Investigator-Initiated Studies Program of Merck Sharp & Dohme Corp (http://www.merck.com/index.html - Merck_P08574). The opinions expressed in this paper are those of the authors and do not necessarily represent those of Merck Sharp & Dohme Corp.

Trabalho final de mestrado integrado em Medicina área científica de Otorrinolaringologia, apresentado á Faculdade de Medicina da universidade de Coimbra A meningite é uma das principais causas de surdez neurossensorial adquirida na infância e esta, por sua vez, é a principal sequela a longo prazo nos sobreviventes. Foi nosso propósito, com este estudo, avaliar a repercussão auditiva da meningite bacteriana em crianças. Assim, foram feitas avaliações otológica, neurológica e auditiva (impedancimetria, otoemissões acústicas, potenciais evocados auditivos e audiometria tonal, consoante a idade) de crianças com o diagnóstico de meningite bacteriana, admitidas no Departamento Pediátrico do Centro Hospitalar de Coimbra, entre 2000 e 2008. Foi identificada de surdez neurossensorial em 26% dos casos (em 13% bilateral). A Neisseria meningitidis foi o agente etiológico de meningite em 40% destes casos; em 20% não houve identificação de gérmen. Os outros agentes etiológicos identificados foram Streptococcus pneumoniae, Streptococcus do grupo B, Escherichia coli e Haemophilus influenzae, cada um deles identificado em 10% dos casos. As crianças com surdez apresentavam uma elevada incidência de sequelas neurológicas (40%). Em conclusão, a avaliação auditiva precoce após meningite é de extrema importância de forma a identificar lesões comprometedoras da função auditiva que possam interferir com a aquisição da linguagem. Um diagnóstico tardio pode comprometer o prognóstico e as opções terapêuticas Bacterial meningitis is an important and prevalent aetiology of acquired sensorineural hearing loss in paediatric population, and deafness is the main long term consequence in the survivors. Our main goal was to study audiological consequences in children victim of meningitis. This way, otologic, neurologic and auditory complete assessment of children admitted in Paediatric Department of Centro Hospitalar de Coimbra, with the diagnosis of meningitis, between 2000 e 2008, was conducted. Clinical otological and neurological examination, impedancimetry, otoacoustic emissions, auditory brainstem evoked potentials and tonal audiometry were performed, depending on age. The incidence of sensorineural hearing loss was twenty-six percent (13% bilateral). Neisseria meningitidis was the etiological agent responsible for 40% of these cases, and in 20% the agent was not identified. The other aetiological agents identified were Streptococcus pneumoniae, Streptococcus B, Escherichia coli and Haemophilus influenzae, each one responsible for 10% of the cases. Children with hearing impairment had a high incidence of neurological sequelae (40%). In conclusion, early auditory assessment after meningitis is of great importance in order to identify any ear damage that could determine restricted access to sound and lack of progress in development of auditory skills and speech. Later diagnosis could compromise outcomes and treatment options.

Ankylosing spondylitis (AS) is a chronic inflammatory rheumatic disease of the axial skeleton, characterised by systemic osteoporosis along with new local bone formation. Previous studies have shown that serum levels of TNF, IL-6 and IL-17 are increased in AS patients and may be implicated in the development of secondary osteoporosis, since these cytokines are able to induce osteoclast (OC) differentiation and, therefore, bone resorption. In this work we aimed to assess the effects of TNF-blocking therapy in the systemic inflammatory environment of AS patients with active disease as well as in OC differentiation and activity. Patients with AS starting TNF-blocking therapy were recruited for this study and blood was collected at baseline and 6 months after the first treatment administration. We performed ELISAs in the serum of these patients to assess cytokine levels and bone turnover markers. We also characterised RANKL surface expression in circulating neutrophils, B and T lymphocytes and monocyte subpopulation frequency and phenotype by flow cytometry. We cultured circulating monocytes from AS patients, before and after therapy, and from healthy controls under osteoclastogenic conditions. We performed two functional assays (TRAP staining and resorption pit assay) and analysed the expression of osteoclast specific genes. We found no differences (before and after treatment) in any of the circulating monocytes’ subpopulations regarding frequency or cell death assessed by annexin staining. Phenotype analysis only revealed differences in the classical monocyte subpopulation where there was a significant decrease in HLA-DR surface expression after treatment. The frequency of RANKL positive B lymphocytes was reduced after treatment. No changes were observed on RANKL expression in neutrophils or T lymphocytes after treatment. Before TNF-blocking treatment AS patients have increased levels of pro-inflammatory cytokines when compared with healthy subjects. After TNF-blocking therapy IL-17, TGF-β and osteoprotegerin were significantly decreased. Interestingly, we observed that after TNF-blocking therapy, gene expression was favouring osteoclastogenesis and that differentiated OCs have increased resorption activity. Our results suggest that in AS patients there might be a paradoxical effect of TNF blocking therapy that induces OC activity.