Next-generation sequencing of hereditary hemochromatosis-related genes: novel likely pathogenic variants found in the Portuguese population

Hereditary hemochromatosis (HH) is an autosomal recessive disorder characterized by excessive iron absorption resulting in pathologically increased body iron stores. It is typically associated with common HFE gene mutation (p.Cys282Tyr and p.His63Asp). However, in Southern European populations up to one third of HH patients do not carry the risk genotypes. This study aimed to explore the use of next-generation sequencing (NGS) technology to analyse a panel of iron metabolism-related genes (HFE, TFR2, HJV, HAMP, SLC40A1, and FTL) in 87 non-classic HH Portuguese patients. A total of 1241 genetic alterations were detected corresponding to 53 different variants, 13 of which were not described in the available public databases. Among them, five were predicted to be potentially pathogenic: three novel mutations in TFR2 [two missense (p.Leu750Pro and p.Ala777Val) and one intronic splicing mutation (c.967-1GNC)], one missense mutation in HFE (p.Tyr230Cys), and one mutation in the 5′-UTR of HAMP gene(c.-25GNA). The results reported here illustrate the usefulness of NGS for targeted iron metabolism-related gene panels, as a likely cost-effective approach for molecular genetics diagnosis of non-classic HH patients. Simultaneously, it has contributed to the knowledge of the pathophysiology of those rare iron metabolism-related disorders.

Hemorheological alterations in sickle cell anemia and their clinical consequences: the role of genetic modulators

Sickle cell anemia (SCA) is an autosomal recessive chronic hemolytic anemia, caused by homozygosity for the HBB:c.20A>T mutation. The disease presents with high clinical heterogeneity, stroke being the most devastating manifestation. This study aimed to identify genetic modulators of severe hemolysis and stroke risk in children with SCA, as well as understand their consequences at the hemorheological level. Sixty-six children with SCA were categorised according to their degree of cerebral vasculopathy (Stroke/Risk/Control). Relevant data were collected from patients’ medical records. Several polymorphic regions in genes related to vascular cell adhesion and tonus were characterized by molecular methodologies. Data analyses were performed using R software. Several in silico tools (e.g. TFBind, MatInspector) were applied to investigate the main variant consequences. Some genetic variants in vascular adhesion molecule-1 gene promoter and endothelial nitric oxide synthase gene were associated with higher levels of hemolysis and stroke events. They modify important transcription factor binding sites or disturb the corresponding protein structure/function. Our findings emphasize the relevance of the genetic variants in modulating the degree of hemolysis and development of cerebral vasculopathy due to their effect on gene expression, modification of protein biological activities related with erythrocyte/endothelial interactions and consequent hemorheological abnormalities in SCA.

Occupational exposure to environmental tobacco smoke in hospitality venues: are genetic- or proteomics-based biomarkers predictive of respiratory diseases?

Environmental tobacco smoke (ETS) is recognized as an occupational hazard in the hospitality industry. Although Portuguese legislation banned smoking in most indoor public spaces, it is still allowed in some restaurants/bars, representing a potential risk to the workers’ health, particularly for chronic respiratory diseases. The aims of this work were to characterize biomarkers of early genetic effects and to disclose proteomic signatures associated to occupational exposure to ETS and with potential to predict respiratory diseases development. A detailed lifestyle survey and clinical evaluation (including spirometry) were performed in 81 workers from Lisbon restaurants. ETS exposure was assessed through the level of PM 2.5 in indoor air and the urinary level of cotinine. The plasma samples were immunodepleted and analysed by 2D-SDSPAGE followed by in-gel digestion and LC-MS/MS. DNA lesions and chromosome damage were analysed innlymphocytes and in exfoliated buccal cells from 19 cigarette smokers, 29 involuntary smokers, and 33 non-smokers not exposed to tobacco smoke. Also, the DNA repair capacity was evaluated using an ex vivo challenge comet assay with an alkylating agent (EMS). All workers were considered healthy and recorded normal lung function. Interestingly, following 2D-DIGE-MS (MALDI-TOF/TOF), 61 plasma proteins were found differentially expressed in ETS-exposed subjects, including 38 involved in metabolism, acute-phase respiratory inflammation, and immune or vascular functions. On the other hand, the involuntary smokers showed neither an increased level of DNA/chromosome damage on lymphocytes nor an increased number of micronuclei in buccal cells, when compared to non-exposed non-smokers. Noteworthy, lymphocytes challenge with EMS resulted in a significantly lower level of DNA breaks in ETS-exposed as compared to non-exposed workers (P<0.0001) suggestive of an adaptive response elicited by the previous exposure to low levels of ETS. Overall, changes in proteome may be promising early biomarkers of exposure to ETS. Likewise, alterations of the DNA repair competence observed upon ETS exposure deserves to be further understood. Work supported by Fundação Calouste Gulbenkian, ACSS and FCT/Polyannual Funding Program.

Estudo mutacional em tumor de Wilms por sequenciação de nova geração

O Tumor de Wilms (TW) é o tumor renal mais comum da infância, com uma incidência de 1 em ~10000 crianças. Esta patologia é de etiologia genética complexa e diversificada. No entanto, cerca de um terço dos doentes apresenta mutações somáticas associadas aos genes WT1, CTNNB1, TP53 e/ou AMER1. Assim, foi desenvolvido um painel de amplicões destes 4 genes para a identificação de mutações num grupo de doentes portugueses com TW, através de uma metodologia baseada na sequenciação de nova geração. As bibliotecas de DNA foram preparadas a partir de amostras de sangue periférico e tumor de 36 doentes com TW e sequenciadas no MiSeq. Foram identificadas alterações somáticas em 7 dos 36 (19,4%) doentes estudados. Conclui-se que a sequenciação de um painel de genes é um método rápido para a deteção de mutações somáticas quando desenhado com cuidado de forma a serem evitados problemas de perda de cobertura.