Next-generation sequencing of iron-metabolism related genes in Portuguese patients with iron overload: novel pathogenic genetic variants

Objective: In Southern European countries up to one-third of the patients with hereditary hemochromatosis (HH) do not present the common HFE risk genotype. In order to investigate the molecular basis of these cases we have designed a gene panel for rapid and simultaneous analysis of 6 HH-related genes (HFE, TFR2, HJV, HAMP, SLC40A1 and FTL) by next-generation sequencing (NGS). Materials and Methods: Eighty-eight iron overload Portuguese patients, negative for the common HFE mutations, were analysed. A TruSeq Custom Amplicon kit (TSCA, by Illumina) was designed in order to generate 97 amplicons covering exons, intron/exon junctions and UTRs of the mentioned genes with a cumulative target sequence of 12115bp. Amplicons were sequenced in the MiSeq instrument (IIlumina) using 250bp paired-end reads. Sequences were aligned against human genome reference hg19 using alignment and variant caller algorithms in the MiSeq reporter software. Novel variants were validated by Sanger sequencing and their pathogenic significance were assessed by in silico studies. Results: We found a total of 55 different genetic variants. These include novel pathogenic missense and splicing variants (in HFE and TFR2), a very rare variant in IRE of FTL, a variant that originates a novel translation initiation codon in the HAMP gene, among others. Conclusion: The merging of TSCA methodology and NGS technology appears to be an appropriate tool for simultaneous and fast analysis of HH-related genes in a large number of samples. However, establishing the clinical relevance of NGS-detected variants for HH development remains a hard-working task, requiring further functional studies.

Functionally characterization of the most common LDLR missense alterations found in Portuguese FH patients

Aims: Mutations in the LDLR gene are the major cause of familial hypercholesterolaemia (FH), which results in defective catabolism of LDL leading to premature coronary heart disease. Presently, more than 1700 different mutations in the LDLR gene have been described as causing FH but the majority of them remain without functional characterization. In the Portuguese Familial Hypercholesterolemia Study (PFHS), 123 LDLR alterations were found in 243 index patients and their relatives up to date. Until now, 70 of these alterations already have a final classification of pathogenic and 15 have been proved by in vitro studies to be non-pathogenic. The aim of the present work is to functionally characterize 16 LDLR missense alterations found in Portuguese FH patients and worldwide.

e_LIPID: Characterisation of the Lipid Profile in the Portuguese Population

AIM: Cardiovascular disease (CVD), particularly coronary heart disease and stroke, are the leading cause of morbidity and mortality worldwide. The common forms of CVD have a complex etiology in which interactions between multiple genetic and environmental factors play an important role. Dyslipidaemia is one of many independent cardiovascular risk factors that have been identified for CVD, and its correct identification is of great importance in order to implement specific interventions, especially for CVD prevention. The aim of this study was the construction of population specific lipid percentiles and the to present the characterization of the dyslipidaemia in the Portuguese population.

The effect of extreme cold temperatures on the risk of death in the two major Portuguese cities

It is well known that meteorological conditions influence the comfort and human health. Southern European countries, including Portugal, show the highest mortality rates during winter, but the effects of extreme cold temperatures in Portugal have never been estimated. The objective of this study was the estimation of the effect of extreme cold temperatures on the risk of death in Lisbon and Oporto, aiming the production of scientific evidence for the development of a real-time health warning system. Poisson regression models combined with distributed lag non-linear models were applied to assess the exposure-response relation and lag patterns of the association between minimum temperature and all-causes mortality and between minimum temperature and circulatory and respiratory system diseases mortality from 1992 to 2012, stratified by age, for the period from November to March. The analysis was adjusted for over dispersion and population size, for the confounding effect of influenza epidemics and controlled for long-term trend, seasonality and day of the week. Results showed that the effect of cold temperatures in mortality was not immediate, presenting a 1–2-day delay, reaching maximumincreased risk of death after 6–7 days and lasting up to 20–28 days. The overall effect was generally higher and more persistent in Lisbon than in Oporto, particularly for circulatory and respiratory mortality and for the elderly. Exposure to cold temperatures is an important public health problem for a relevant part of the Portuguese population, in particular in Lisbon.