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.

Indoor air quality in Portuguese schools: levels and sources of pollutants

Indoor air quality (IAQ) parameters in 73 primary classrooms in Porto were examined for the purpose of assessing levels of volatile organic compounds (VOCs), aldehydes, particulate matter, ventilation rates and bioaerosols within and between schools, and potential sources. Levels of VOCs, aldehydes, PM2.5 , PM10 , bacteria and fungi, carbon dioxide (CO2 ), carbon monoxide, temperature and relative humidity were measured indoors and outdoors and a walkthrough survey was performed concurrently. Ventilation rates were derived from CO2 and occupancy data. Concentrations of CO2 exceeding 1000 ppm were often encountered, indicating poor ventilation. Most VOCs had low concentrations (median of individual species <5 μg/m(3) ) and were below the respective WHO guidelines. Concentrations of particulate matter and culturable bacteria were frequently higher than guidelines/reference values. The variability of VOCs, aldehydes, bioaerosol concentrations, and CO2 levels between schools exceeded the variability within schools. These findings indicate that IAQ problems may persist in classrooms where pollutant sources exist and classrooms are poorly ventilated; source control strategies (related to building location, occupant behavior, maintenance/cleaning activities) are deemed to be the most reliable for the prevention of adverse health consequences in children in schools.

Accessing Planktothrix species diversity and associated toxins using quantitative real-time PCR in natural waters

Tese de Doutoramento em Biologia apresentada à Faculdade de Ciências da Universidade do Porto, 2015.