Phylogeography of hepatitis B virus: The role of Portugal in the early dissemination of HBV worldwide.

In Portugal, the genetic diversity, origin of HBV and the Portuguese role in the dissemination of HBV worldwide were never investigated. In this work, we studied the epidemic history and transmission dynamics of HBV genotypes that are endemic in Portugal. HBV pol gene was sequenced from 130 patients followed in Lisbon. HBV genotype A was the most prevalent (n = 54, 41.5%), followed by D (n = 44, 33.8%), and E (n = 32, 24.6%). Spatio-temporal evolutionary dynamics was reconstructed in BEAST using a Bayesian Markov Chain Monte Carlo method, with a GTR nucleotide substitution model, an uncorrelated lognormal relaxed molecular clock model, a Bayesian skyline plot, and a continuous diffusion model. HBV subgenotype D4 was the first to be introduced in Portugal around 1857 (HPD 95% 1699-1931) followed by D3 and A2 a few decades later. HBV genotype E and subgenotype A1 were introduced in Portugal later, almost simultaneously. Our results indicate a very important role of Portugal in the exportation of subgenotypes D4 and A2 to Brazil and Cape Verde, respectively, in the beginning of the XX century. This work clarifies the epidemiological history of HBV in Portugal and provides new insights in the early and global epidemic history of this virus.

Phenotype-Genotype Correlation in Colorectal Cancer: A Real-Life Study.

BACKGROUND AND AIMS: Colorectal cancer (CRC) is a heterogeneous disease with distinctive genetic pathways, such as chromosomal instability, microsatellite instability and methylator pathway. Our aim was to correlate clinical and genetic characteristics of CRC patients in order to understand clinical implications of tumour genotype. METHODS: Single-institution retrospective cohort of patients who underwent curative surgery for CRC, from 2012 to 2014. RAS and BRAF mutations were evaluated with the real-time PCR technique Idylla®. Mismatch repair deficiency (dMMR) was characterized by absence of MLH1, MSH6, MSH2 and/or PMS2 expression, evaluated by tissue microarrays. Overall survival (OS) and disease-free survival (DFS) were assessed using survival analysis. RESULTS: Overall, 242 patients were included (males 57.4%, age 69.3 ± 12.9 years; median follow-up 49 months). RAS-mutated tumours were associated with reduced DFS (p = 0.02) and OS (p = 0.045) in stage I-III CRC. BRAF-mutated tumours were more predominant in females and in the right colon, similarly to dMMR tumours. BRAF status did not influence OS (4 years)/DFS (3.5 years) in stage I-III disease. However, after relapse, length of survival was 3.5 months in BRAF-mutated tumours in contrast to 18.6 months in BRAF wild-type tumours (p = NS). No germline mutations in mismatch repair genes were so far identified in the patients with dMMR tumours. Molecular phenotype (RAS, BRAF and MMR) did not influence OS in metastatic patients. Our small sample size may be a limitation of the study. CONCLUSION: In our cohort, RAS-mutated tumours were associated with worse DFS and OS in early-stage CRC, whereas the remaining molecular variables had no prognostic influence.

INTRODUÇÃO: O cancro colo-rectal (CCR) é uma doença heterogénea, com vias genéticas distintas, nomeadamente instabilidade cromossómica, instabilidade de microssatélites e via metiladora. O nosso objetivo foi correlacionar as características clínicas e genéticas dos doentes com CCR e, deste modo, conhecer as implicações na prática clínica do genótipo tumoral. MÉTODOS: Estudo de coorte retrospectivo unicêntrico de doentes diagnosticados com CCR e submetidos a cirurgia com intuito curativo, entre 2012 e 2014. As mutações RAS e BRAF foram avaliadas pela técnica de real time PCR Idylla®. A deficiência de mismatch repair (MMR) foi avaliada pela técnica de tissue microarrays e definida pela ausência de expressão de MLH1, MSH6, MSH2 e/ou PMS2. A sobrevivência global (SG) e a sobrevivência livre de doença (SLD) foram avaliadas por análise de sobrevivência. RESULTADOS: No total, foram incluídos 242 doentes (homens 57.4%, idade 69.3 ± 12.9 anos, mediana de seguimento de 49 meses). Os tumores RAS-mutados associaram-se a menor SLD (p = 0.02) e SG (p = 0.045) em doentes com CCR estadio I­III. Os tumores BRAF-mutados foram mais frequentes em mulheres e nos tumores do cólon direito, assim como os tumores com deficiência para MMR. O status BRAF não influenciou a SG (4 anos)/SLD (3.5 anos) nos estadio I­III. Contudo, após a recidiva, o tempo de sobrevivência foi de 3.5 meses nos tumores BRAF-mutados, em comparação com 18.6 meses nos tumores sem esta mutação (p = NS). Não se identificaram mutações germinativas nos genes de mismatch repair nos doentes com tumores deficientes para estas proteinas (dMMR). O perfil molecular (RAS, BRAF e MMR) não influenciou a sobrevivência global dos doentes com metástases ao diagnóstico. O tamanho da amostra pode ser uma limitação do estudo. CONCLUSÃO: Na nossa coorte, os tumores RASmutados associaram-se a pior SLD e SG nos estádios precoces de CCR. Os restantes marcadores moleculares não influenciaram o prognóstico dos doentes.

Detection and quantification of EGFR T790M mutation in liquid biopsies by droplet digital PCR.

BACKGROUND: Liquid biopsy allows the identification of targetable cancer mutations in a minimally invasive manner. In patients with advanced non-small cell lung cancer (NSCLC), droplet digital PCR (ddPCR) is increasingly used to genotype the epidermal growth factor receptor (EGFR) gene in circulating cell-free DNA (cfDNA). However, the sensitivity of this method is still under debate. The aim of this study was to implement and assess the performance of a ddPCR assay for detecting the EGFR T790M mutation in liquid biopsies. METHODS: A ddPCR assay was optimized to detect the EGFR T790M mutation in plasma samples from 77 patients with NSCLC in progression. RESULTS: Our ddPCR assay enabled the detection and quantification of the EGFR T790M mutation at cfDNA allele frequency as low as 0.5%. The mutation was detected in 40 plasma samples, corresponding to a positivity rate of 52%. The number of mutant molecules per mL of plasma ranged from 1 to 6,000. A re-biopsy was analyzed for 12 patients that had a negative plasma test and the mutation was detected in 2 cases. A second liquid biopsy was performed for 6 patients and the mutation was detected in 3 cases. CONCLUSIONS: This study highlights the value of ddPCR to detect and quantify the EGFR T790M mutation in liquid biopsies in a real-world clinical setting. Our results suggest that repeated ddPCR tests in cfDNA may obviate tissue re-biopsy in patients unable to provide a tumor tissue sample suitable for molecular analysis.

Quantitative Genetics Validates Previous Genetic Variants and Identifies Novel Genetic Players Influencing Alzheimer's Disease Cerebrospinal Fluid Biomarkers.

Cerebrospinal fluid (CSF) biomarkers have been extensively investigated in the Alzheimer's disease (AD) field, and are now being applied in clinical practice. CSF amyloid-beta (Aß1-42), total tau (t-tau), and phosphorylated tau (p-tau) reflect disease pathology, and may be used as quantitative traits for genetic analyses, fostering the identification of new genetic factors and the proposal of novel biological pathways of the disease. In patients, the concentration of CSF Aß1-42 is decreased due to the accumulation of Aß1-42 in amyloid plaques in the brain, while t-tau and p-tau levels are increased, indicating the extent of neuronal damage. To better understand the biological mechanisms underlying the regulation of AD biomarkers, and its relation to AD, we examined the association between 36 selected single nucleotide polymorphisms (SNPs) and AD biomarkers Aß1-42, t-tau, and p-tau in CSF in a cohort of 672 samples (571 AD patients and 101 controls) collected within 10 European consortium centers.Our results highlighted five genes, APOE, LOC100129500, PVRL2, SNAR-I, and TOMM40, previously described as main players in the regulation of CSF biomarkers levels, further reinforcing a role for these in AD pathogenesis. Three new AD susceptibility loci, INPP5D, CD2AP, and CASS4, showed specific association with CSF tau biomarkers. The identification of genes that specifically influence tau biomarkers point out to mechanisms, independent of amyloid processing, but in turn related to tau biology that may open new venues to be explored for AD treatment.

Validation of a quantitative cerebrospinal fluid alpha-synuclein assay in a European-wide interlaboratory study.

Decreased levels of alpha-synuclein (aSyn) in cerebrospinal fluid (CSF) in Parkinson's disease and related synucleinopathies have been reported, however, not consistently in all cross-sectional studies. To test the performance of one recently released human-specific enzyme-linked immunosorbent assay (ELISA) for the quantification of aSyn in CSF, we carried out a round robin trial with 18 participating laboratories trained in CSF ELISA analyses within the BIOMARKAPD project in the EU Joint Program - Neurodegenerative Disease Research. CSF samples (homogeneous aliquots from pools) and ELISA kits (one lot) were provided centrally and data reported back to one laboratory for data analysis. Our study showed that although factors such as preanalytical sample handling and lot-to-lot variability were minimized by our study design, we identified high variation in absolute values of CSF aSyn even when the same samples and same lots of assays were applied. We further demonstrate that although absolute concentrations differ between laboratories the quantitative results are comparable. With further standardization this assay may become an attractive tool for comparing aSyn measurements in diverse settings. Recommendations for further validation experiments and improvement of the interlaboratory results obtained are given.

Genetic polymorphisms of proangiogenic factors seem to favor hepatocellular carcinoma development in alcoholic cirrhosis.

OBJECTIVE: Angiogenesis has been associated with hepatic cirrhosis and hepatocellular carcinoma (HCC). Alcohol promotes liver hypoxia, a trigger of angiogenesis. We aimed to evaluate whether the frequency of three polymorphisms in hypoxia-induced factor-1α (HIF-1α), vascular endothelial growth factor A (VEGFA), and KDR (encoding vascular endothelial growth factor receptor 2) genes was higher in alcoholics presenting liver disease (ALD) and ALD patients who developed HCC. MATERIALS AND METHODS: Functional HIF-1α 1744C/T, VEGFA 2578C/A, and KDR 1416A/T single-nucleotide polymorphisms were studied in 125 ALD patients and 88 heavy drinkers without liver disease (NLD). ALD patients were followed up to 9 years or until they died; 26 patients developed HCC. RESULTS: ALD patients were older than NLD (56±11 vs. 50±13, P<0.001), but drank less (215±164 vs. 331±293 g/day, P<0.001). No differences were found between HIF-1α, VEGFA, or KDR allelic frequencies or genotypes, isolated or simultaneously, between ALD and NLD. In ALD patients, those who developed HCC had a higher KDR 1416T allele frequency (36 vs. 15%, P=0.004; odds ratio 2.72; 95% confidence interval 1.35-5.46). There was also a progressive increase in genotypes with one or two T alleles in patients who developed HCC: AA 50 vs. 73%, AT 35 vs. 23%, and TT 15 vs. 4% (P=0.009). The simultaneous presence of KDR 1416T and VEGFA 2578A was associated with an increased risk of HCC (odds ratio 3.088; 95% confidence interval 1.20-7.96). CONCLUSION: Genetic polymorphisms in proangiogenic factors did not associate with the risk of ALD in heavy drinkers. However, KDR and VEFGA polymorphisms may confer an increased risk of HCC in patients with ALD.

Pachydermoperiostosis in an African patient caused by a Chinese/Japanese SLCO2A1 mutation-case report and review of literature.

OBJECTIVES: Pachydermoperiostosis is a rare clinical entity characterized by skin thickening of the forehead, eyelids, and hands, digital clubbing, and periostosis. Two genes have been associated, HPGD and recently SLCO2A1. We present a detailed clinical and genetic description of an African pachydermoperiostosis patient with a SLCO2A1 mutation. METHODS: Standard clinical and laboratory evaluation was carried out. Genetic screening was done with PCR followed by direct sequencing. We discuss the clinical features and known mutations of previously reported cases identified through a PubMed literature review. RESULTS: The clinical findings showed special features, including exuberant knee effusions and an extraordinary good response on surgery of the blepharoptosis. We found a splice site mutation in the SLCO2A1 gene in homozygous form: c.940+1G>A. This mutation was previously reported only in 1 Chinese and 3 Japanese cases and was considered as a founder mutation in Japan. Beside our case, only one other patient in the literature carried this mutation in homozygous condition, but with different main clinical symptoms. CONCLUSIONS: Our case demonstrates phenotypic heterogeneity of PDP even between homozygous carriers of the same mutation, suggesting further modifiers. Besides, it shows that this rare SLCO2A1 mutation is not exclusively present in East-Asia, but can occur in various ethnicities, with different origin, thus the incidence is probably underestimated.