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Introduction: Hemophilia B is an X-linked bleeding disorder caused by molecular defects in the Factor IX gene (F9), leading to either deficiency or functional abnormality of Factor IX. Actual data indicate a high heterogeneity of variants in F9. Over 1000 different variants have been reported, including pathogenic single nucleotide variants (SNPs), indels and complex variants. Materials and Methods: 86 index patients and 313 relatives were studied. F9 variant analysis was performed from total genomic DNA by PCR followed either by SSCP and DNA sequencing or direct DNA sequencing. When no variant was detected by sequencing, F9 analysis by MLPA was performed. Segregation studies were performed in each family. Results: Overall, 52 different F9 variants have been identified, including 49 SNPs or small indels, a gross duplication (exons 2-6) and two deletions of the entire gene. Ten of the variants had been firstly reported by us and three are novel: c.391+5G>T; c.432T>G, p.(Phe144Leu) and c.749C>A, p.(Ala250Glu). This approach allowed establishing the carrier state of over 300 women and 12 prenatal diagnoses were performed. Conclusions: The spectrum of F9 variants identified in the Portuguese population significantly overlaps that observed in other populations. Identification of F9 gene variants in patients allows genotype-phenotype correlations and carrier detection, as well as prenatal diagnosis. Sanger sequencing of the coding region and adjacent intronic sequences of F9 still remains a valid and effective tool for the molecular study of hemophila B, providing information for appropriate genetic counseling and new insights regarding the molecular basis of the pathology. info:eu-repo/semantics/publishedVersion

Este artigo constitui, no essencial, uma tradução abreviada da publicação: David S, Aguiar P, Antunes L, Dias A, Morais A, Sakuntabhai A, Lavinha J. Variants in the non-coding region of the TLR2 gene associated with infectious subphenotypes in pediatric sickle cell anemia. Immunogenetics. 2018 Jan;70(1):37-51. Epub 2017 Jun 30. https://doi.org/10.1007/s00251-017-1013-7 A anemia falciforme é caracterizada por hemólise crónica, crises vaso- -oclusivas (CVO) e infeções recorrentes frequentemente graves. Uma coorte de 95 doentes pediátricos com anemia falciforme foi estudada quanto à associação genótipo-fenótipo para o subfenótipo “infeção bacteriana grave pelo menos uma vez durante o período de acompanhamento do doente” e três regiões polimórficas não codificantes do gene TLR2, a saber, a indel -196 a -174, o SNP rs4696480 e uma repetição em tandem (GT) n. A ausência do haplótipo [Del] -T- [n≥17] (Hap7) em homozigotia parece proteger contra a infeção bacteriana grave, numa associação estatisticamente significativa, resistindo à correção para testes múltiplos. Além disso, uma redução na taxa de incidência da infeção bacteriana grave foi associada a um aumento do componente hemolítico, aos níveis de hemoglobina fetal (antes do tratamento pela hidroxiureia) e à prevalência da alfa-talassemia de 3,7 kb. Estes resultados poderão vir a ter implicações práticas nas estratégias de cuidados de saúde para reduzir a morbilidade e mortalidade dos doentes com anemia falciforme. Sickle cell anemia (SCA) is characterized by chronic hemolysis, severe vasoocclusive crises (VOC) and recurrent often-severe infections. A cohort of 95 SCA pediatric patients was the background for genotype- -to-phenotype association of the patient’s infectious disease phenotype “severe bacterial infection at least once during the patient’s follow-up” and three noncoding polymorphic regions of the TLR2 gene, the -196 to -174 indel, SNP rs4696480 and a (GT)n short tandem repeat. The absence of the haplotype [Del]-T-[n≥17] (Hap7) in homozygocity protected against severe bacterial infection, in a statistically significant association, resisting correction for multiple testing. Moreover, a reduction in the incidence rate of severe bacterial infection was associated to a rise in the hemolytic score, fetal hemoglobin levels (prior to hydroxyureia treatment) and 3.7-kb alpha-thalassemia. These results could have practical implications in health care strategies to lower the morbidity and mortality of SCA patients. Este estudo foi realizado com o apoio da FCT/MEC através de fundos nacionais e cofinanciado pelo FEDER, no âmbito do Acordo de parceria PT2020 (UIDMULTI/00211/2013). Foi ainda parcialmente financiado por subvenções da FCT (PIC/IC/83084/2007) e do Centro de Investigação em Genética Molecular Humana info:eu-repo/semantics/publishedVersion

A drepanocitose é uma doença genética causada pela mutação c.20A>T, em homozigotia, no gene da beta-globina, que leva à síntese de uma hemoglobina anómala – hemoglobina S. Para além da anemia hemolítica crónica, as manifestações clínicas são diversas e, em crianças, a mais grave é a vasculopatia cerebral que inclui os acidentes vasculares cerebrais (AVC) e os enfartes cerebrais silenciosos. Apesar de monogénica, variantes noutros genes poderão atuar como modificadores do seu curso e gravidade clínica. Neste trabalho, estudámos 70 doentes pediátricos, de origem subsaariana, com drepanocitose e bem caracterizados em termos de vasculopatia cerebral. Procedemos à genotipagem de variantes nos genes VCAM1 e NOS3 envolvidos na ativação do endotélio dos vasos sanguíneos e no tónus vascular. A análise estatística revelou uma associação positiva entre a presença da variante rs1409419_T, bem como do haplotipo 7 de VCAM1, e a ocorrência de AVC. Por outro lado, para o gene NOS3, observámos uma associação negativa entre o VNTR_alelo 4b e o haplotipo V, e a ocorrência de enfarte cerebral silencioso, bem como entre o haplotipo VII e a ocorrência de vasculopatia cerebral. Os resultados obtidos sublinham a importância de VCAM1 e NOS3 como moduladores genéticos, bem como o seu potencial como biomarcadores para a prevenção e prognóstico da vasculopatia cerebral em crianças com drepanocitose. Sickle cell anemia is a genetic disease caused by homozygosity for the mutation c.20A>T in the beta-globin gene which leads to the synthesis of an abnormal hemoglobin – hemoglobin S. In addition to chronic hemolytic anemia, the disease includes various clinical manifestations of which cerebral vasculopathy (that comprises overt stroke and silent cerebral infarction) is the most severe in children. Despite being a monogenic disease, its progression and severity may be modified due to the effect of variants in other genes. In this work, we studied 70 sickle cell anemia pediatric patients of sub-Saharan ancestry well characterized in terms of cerebral vasculopathy. We genotyped variants in the VCAM1 and NOS3 genes, due to the involvement of these genes in blood vessel endothelium activation and vascular tone balance. Statistical analyses showed a positive associative between the presence of the rs1409419_T variant, as well as of the haplotype 7 of VCAM1, and stroke. On the other hand, for the NOS3 gene, we observed a negative association between the presence of the VNTR_4b allele and haplotype V and silent cerebral infarcts. A similar association was observed between haplotype VII and cerebral vasculopathy as a whole. Our results underline the importance of VCAM1 and NOS3 genes as genetic modulators, as well as their role as potential biomarkers for cerebral vasculopathy prevention and prognosis in children with sickle cell anemia. Este trabalho foi parcialmente financiado pela Fundação para a Ciência e a Tecnologia (projeto PIC/IC/83084/2007), pelo Instituto de Saúde Ambiental/Faculdade de Medicina da Universidade de Lisboa e pelo Instituto Nacional de Saúde Doutor Ricardo Jorge (projeto 2012DGH720). info:eu-repo/semantics/publishedVersion

As doenças raras são caracterizadas por uma grande diversidade de sinais e sintomas que variam não só de doença para doença, mas também entre doentes que sofrem de uma mesma patologia. As doenças raras afetam não apenas os indivíduos doentes, mas também têm grande impacto nas famílias, amigos, cuidadores e na sociedade em geral. O diagnóstico precoce e o início de tratamento com base na evidência são fatores importantes para reduzir o impacto de uma doença rara na vida adulta. As doenças raras são um problema de saúde pública reconhecido pela Comissão Europeia e pela Organização Mundial da Saúde ( 1) ( 2 ) . O atraso no diagnóstico pode significar que se desperdiçou a oportunidade de uma intervenção atempada enquanto um diagnóstico correto poderá revelar a existência de uma doença rara subjacente não suspeitada. Neste trabalho pretende-se dar a conhecer as contribuições efetivas a nível nacional do Instituto Nacional de Saúde Doutor Ricardo Jorge (INSA, I.P.), para o Serviço Nacional de Saúde (SNS) na investigação, diagnóstico, prevenção e controlo das doenças raras, realizadas no Departamento de Genética Humana (DGH) desde 1975. Rare diseases are characterized by a wide range of signs and symptoms that var y not only from disease to disease but also among patients suffering from the same clinical condition. Rare diseases not only af fect sick individuals, but also have a major impact on families, friends, caretakers and societ y. Early diagnosis and evidence-based initiation of treatment are impor tant factors in reducing the impact of a rare disease on adulthood. Rare diseases are a public health issue recognized by the European Commission and by the World Health Organization ( 1) ( 2 ) . A delay in diagnosis may represent that an oppor tunit y was missed for a timely inter vention whereas a correct diagnosis may reveal the existence of a rare unsuspected underlying disease. The aim of this work is to present the ef fective contributions of the National Institute of Health Doutor Ricardo Jorge to the National Health System in the contex t of research, diagnosis, prevention and control of rare diseases, which have been per formed since 1975 in the Human Genetics Depar tment. info:eu-repo/semantics/publishedVersion

Ferroportin (FPN1), encoded by the SLC40A1 gene, is the unique cellular iron exporter identified in mammals. FPN1 transfers iron from the intestine and macrophages into the bloodstream. This function is negatively regulated by hepcidin. Mutations in SLC40A1 may affect FPN1 function, originating distinct autosomal dominant diseases: (i) the Ferroportin Disease (FD), due to loss-of-function mutations, is characterized by decreased iron export from enterocytes and severely affected iron transfer in macrophages, giving rise to a marked iron accumulation in spleen and liver; and (ii) the Type 4 Hereditary Hemochromatosis (HH), resulting from gain-of-function mutations conferring resistance to hepcidin-mediated FPN1 degradation and consequently high cellular iron export. In this study, 335 individuals suspected of having non-classic HH were enrolled. Six genes related with iron metabolism were analysed by SSCP, dHPLC or NGS. The latter used TruSeq or Nextera XT libraries and a MiSeq platform (Illumina). Genetic variants found were validated by Sanger sequencing. Predictive consequences at protein level were evaluated using Polyphen-2 and SIFT softwares. From all patients analysed, three SLC40A1 pathogenic variants were detected in heterozygosity in three women: two missense, c.238G>A, p.Gly80Ser and c.610G>A, p.Gly204Ser; and one deletion, c.485_487delTTG; p.Val162del. These variants had been reported in public databases, but they were not known to be present in the Portuguese population. The p.Gly80Ser and the p.Val162del are FPN1 loss-of-function mutations and were found associated with hyperferritinemia and low transferrin saturation (FD). In contrast, the p.Gly204Ser induced a gain of FPN1 function with a full iron export capacity giving the patient a type 4-HH phenotype, which includes iron overload, hyperferritinemia and high transferrin saturation. Detailed clinical evaluation of the suspected patients are useful to unravel the effect of different mutations in FPN1 function, expression and regulation. This work was partially supported by INSA_2013DGH910 and GenomePT (POCI-01-0145-FEDER-022184). info:eu-repo/semantics/publishedVersion

Microcytic anemia is a common condition frequently caused by iron deficiency anemia (IDA) or β-thalassemia trait (BTT). Some mathematical indices have been described as fast and inexpensive tools for distinguishing these two conditions. This approach is very useful in mass screening programs especially in countries with limited resources. This study aimed to evaluate the diagnostic performance of 13 distinct indices: RBC, England&Fraser, Mentzer, Srivastava, Shine&Lal, RDW, Ricerca, Jayabose (RDWI), Green&King (G&K), MDHL, MCHD, Sirdah and Ensani. We investigated 102 adult Portuguese female, presenting anemia (HbA; c.92+6T>C; c.92+110G>A or c.1188C>T) and 51 IDA, being assured that no individual had simultaneously the two conditions. To determine the performance of the indices, sensitivity, specificity, Youden index (YI) and receiver operating characteristic (ROC) curves were calculated. Due to the high values of AUC (Area Under the Curve) from ROC analysis, a cutoff of 0.70 for the YI was established in order to determine the best formulas. We find that the 3 best performing indices to differentiate the 2 groups were RBC (YI=0.71; AUC=0.902), RDWI (YI=0.84; AUC=0.973) and G&K (YI=0.82; AUC=0.972). Our results suggest a similarity with other Mediterranean countries such as Spain and Greece, where G&K and RDWI also performed above our set cutoff. The same is observed in Brazil probably due to its Portuguese ancestry. We conclude that aiming to diagnosis the condition underlying a microcytic anemia in a female population, there is value in using this method to recognize the individuals suspected of BTT and forward them for HbA2 measurement or HBB molecular test. In the future, a robust group of male patients should be added to the analysis in order to extrapolate which of these indices would best apply to the whole adult Portuguese population. This work was partially funded by INSA_2012DGH720 and ISAMB info:eu-repo/semantics/publishedVersion

Iron-refractory iron deficiency anemia (IRIDA) is a rare autosomal recessive anemia usually unresponsive to oral iron intake and partially responsive to parenteral iron treatment. This disease is the result of mutations in TMPRSS6 gene, encoding Matriptase-2, a transmembrane serine protease that plays a key role in down-regulating hepcidin, allowing iron bioavailability for erythropoiesis. Once TMPRSS6 is mutated, the corresponding protein is absent or inactive at the hepatocyte membrane leading to uncontrolled high levels of hepcidin and impaired iron absorption. This study investigates the case of a 4-year-old boy, of sub-Saharan ancestry (Mozambique/Angola), presenting with microcytic hypochromic anemia, low transferrin saturation, normal ferritin, and having a partial response to intravenous iron treatment. The subject is a -α3.7-thalassemia carrier. TMPRSS6 was screened for variants by Next-Generation Sequencing using Nextera XT libraries in a MiSeq platform (Illumina). Genetic variants found were validated by Sanger sequencing. In silico analyses were performed in HSF, SIFT, Poly-Phen2 and Missense3D software. In order to run a Missense3D analysis, we predicted a 3D Structure for matriptase-2 in the software Phyre2. A novel missense mutation (c.871G>A) was found in heterozygosity, in TMPRSS6 exon 8. In silico analysis indicates the conserved amino acid change (G291S) may be damaging to the protein stability. Due to its location in the CUB1 domain, it may also affect the enzyme activation and substrate recognition. Additionally, 3 SNPs previously associated with a greater risk of developing iron deficiency anemia (K253E, V736A and Y739Y) were also identified in TMPRSS6. Although IRIDA is noted as an autosomal recessive disease, we infer that, in this case, the result of a digenic inheritance of the novel damaging mutation (c.871G>A; G291S) and 3 common modulating SNPs in the same gene in addition to the co-inheritance of the α-thalassemia allele, may add up to an IRIDA-like phenotype. Further functional studies of the mutated protein as well as family studies should be conducted. INSA_2013DGH910 e GenomePT (POCI-01-0145-FEDER-022184) info:eu-repo/semantics/publishedVersion

Sickle cell anemia (SCA) is a highly heterogeneous and multifactorial-like monogenic disease that arises from homozygosity for the c.20A>T mutation in the HBB gene. Vascular disease is systemic in SCA, with profound effects in organs like the brain, where stroke is the most severe end of the cerebral vasculopathy (CVA) spectrum. Endothelial dysfunction plays a major role in vasculopathy with several adhesion molecules, such as VCAM1, being produced by the endothelium altered as a response to inflammatory cytokine (e.g., TNF-α) signalling. In previous association studies, we found positive associations between the presence of four specific VCAM1 gene promoter haplotypes and i) high blood flow velocities in the median cerebral artery, and ii) a chronic hemolysis biochemical marker. In this study, we aimed to assess the functional role of those VCAM1 promoter haplotypes in endothelial cell response following endothelial activation through TNF-α stimulation. After molecular cloning of 3 haplotype constructs, using a pGL4 promoterless vector, haplotype sequence was confirmed, by Sanger sequencing, prior to transfection. We used EAhy926, HUVEC and HBEC as different endothelial cell models, and performed transfection experiments for each construct, with and without TNF-α stimulation. Differences in promoter activity were assessed by luciferase reporter assay. All haplotypes showed differences in promoter activity, after TNF-α stimulation, in all cell models. Haplotype 1 showed decreased promoter activity, while haplotypes 7 and 8 showed increased activity after TNF-α stimulation, in all cell models. These results are consistent with lower VCAM1 expression due to haplotype 1, and therefore a protective effect. Conversely, a higher expression due to haplotypes 7 and 8, suggests an increased vasculopathy risk, in a pro-inflammatory milieu. The association between specific haplotypes and endothelial cell response further enhances the modifier effect of VCAM1 on endothelial dysfunction and its impact in SCA pathophysiology, as well as its potential role as a biomarker of SCA vasculopathy risk, severity and prognosis. This work was partially supported by INSA_202DGH720 project. info:eu-repo/semantics/publishedVersion

Iron-refractory iron deficiency anemia (IRIDA) is a rare autosomal recessive anemia often unresponsive to oral iron intake and partially responsive to parenteral iron treatment. The disease originates from mutations in TMPRSS6 gene, encoding Matriptase 2, a transmembrane serine protease that plays an essential role in down-regulating hepcidin. Once TMPRSS6 is mutated, the corresponding protein is absent or inactive at the hepatocyte membrane leading to uncontrolled high levels of hepcidin and impaired iron absorption. This study aimed to investigate a 4-year-old boy of sub-Saharan ancestry (Mozambique/Angola), presenting with microcytic hypochromic anemia, low transferrin saturation, normal ferritin, and having a partial response to intravenous iron treatment. He is a -α3.7-thalassemia carrier. TMPRSS6 was screened for variants by Next-Generation Sequencing using Nextera XT libraries in a MiSeq platform (Illumina). Genetic variants found were validated by Sanger sequencing. In silico analyses were performed in HSF, SIFT, Poly-Phen2 and Missense3D softwares. A novel missense mutation (c.871G>A) was found in heterozygosity, in TMPRSS6 exon 8. In silico analysis indicates the conserved amino acid change (G291S) may be damaging to the protein stability. Due to its location in the CUB1 domain, it may also affect the enzyme activation and substrate recognition. Additionally, 3 SNPs previously associated with a greater risk of developing iron deficiency anemia (K253E, V736A and Y739Y) were also identified in TMPRSS6. Although IRIDA is known as an autosomal recessive disease, we conclude that, in this case, the result of a digenic inheritance of the novel damaging mutation (c.871G>A; G291S) and the 3 common modulating SNPs in the same gene and a co-inheritance of the α-thalassemia HBA deletion may lead to an IRIDA-like phenotype. Further functional studies of the mutated protein as well as family studies should be conducted. This work was partially supported by INSA_2013DGH910 and GenomePT (POCI-01-0145-FEDER-022184). info:eu-repo/semantics/publishedVersion

Sickle-cell disease (SCD) is a clinically heterogeneous autosomal recessive monogenic disorder characterized by recurrent episodes of severe haemolysis, vaso-occlusion and infection. Proteomics promises to offer novel unbiased molecular insights into the pathophysiology of SCD. The objective of this project is to analyze by bioinformatic tools mass spectrometry (MS) proteomics raw data, which has been generated by INSA’s Proteomic Laboratory in order to investigate proteome changes that might be related with SCD vaso-occlusion exacerbation. The MS raw data included in this study was generated by shotgun proteomic analysis on red blood cell (RBC) samples from six child SCD patients at steady-state and vaso-occlusion exacerbation episode and five child control subjects. The RBC samples, the haemoglobin depleted-cytoplasmic fraction and membrane fraction, were analysed by proteomic discovery-based approach using the ESI-LQT Orbitrap XL (Thermo) mass spectrometer. The generated MS raw data files were analysed by the PatternLab for Proteomic 4.0 bioinformatic platform for protein identification and extracted-ion chromatograms (XICs)-based label-free relative quantification. The following “golden parameters” were applied to obtain reliable and trustworthy data: one unique peptide at least should be considered to infer a protein identification, and the identified proteins should be present at least in 80% of the studied groups/conditions. 250 proteins were identified, and the respective normalized ion abundance factor was compared by using the Wilcoxon-T non-parametric statistical test. The differentially expressed proteins in crisis as compared to steady-state (p-value ≤ 5%) were investigated in the light of the Gene Ontology (GO) knowledge base (Database for Annotation, Visualization and Integrated Discovery – DAVID) and Reactome database for protein integration into signaling pathways with biological meaning. The most relevant results indicated that 3 cytoplasmic proteins (Dematin, Moesin and Protein S100-A4) and 9 membrane proteins (Eosinophil cationic protein, Bone marrow proteoglycan, Voltage-dependent anion-selective channel protein 1, Voltage-dependent anion-selective channel protein 2, Reticulon-3, Carbonic anhydrase 2, Haemoglobin subunit alpha, Haemoglobin subunit delta and Eosinophil peroxidase) showed significantly differential expression in crisis as compared to steady-state. Haemoglobin subunit alpha and Carbonic anhydrase 2 (CA2) have been reported as involved in important pathways related with O2/CO2 exchange in erythrocytes. Sickle cell crisis is frequently related to infection, involving Reticulon-3 and Haemoglobin subunit alpha. These proteins were identified to be modulated in the RBC membrane fraction from SCD patients at crisis. The most relevant proteins identified by these computational approaches will be selected for further biochemical verification by using SWATH-MS and/or Western blot methods. These proteins may be promising candidate early biomarkers to identify SCD patients at risk for vaso-occlusion crisis. Fundação para a Ciência e a Tecnologia partially financing this project (Grant PIC/IC/83084/2007 and Doctoral Fellowship - SFRH/BPD/31209/2006). N/A

O presente número do Boletim Epidemiológico Observações veicula, entre outras contribuições, resultados do trabalho recentemente realizado no Instituto Nacional de Saúde Doutor Ricardo Jorge (INSA) sobre perigos ambientais reais ou potenciais (sal na alimentação, desconforto térmico, infeções fúngicas profundas e nanomateriais), doenças raras (hipolipidemias primárias e cistinose) ou literacia em saúde ambiental. (...) info:eu-repo/semantics/publishedVersion

Sickle cell anaemia (SCA) is an autosomal recessive genetic disease that leads to the synthesis of haemoglobin S (HbS). The pathophysiology of the disease is centred on HbS polymerization inside the red blood cells, which become sickle-shaped (SSRBCs), rigid, viscous and adherent-prone to the vascular endothelium, favouring the occurrence of chronic haemolysis and vaso-occlusion. The main vascular problems of SCA arise from several pathways including endothelial dysfunction and nitric oxide (NO) metabolism. Children with SCA have a much higher risk (11% by age 20 years) of developing stroke or silent cerebral infarcts (up to 37%) than the general paediatric population. Abnormal interactions between SSRBCs and the cerebral arterial endothelium lead to endothelial injury, vaso-occlusion and tissue ischemia and result in cerebral vasculopathy (CVA) through a yet unknown pathophysiological mechanism. Current risk screening strategies rely mainly on imaging techniques (transcranial Doppler ultrasonography and magnetic resonance imaging) and children with altered results undergo regular blood transfusion and/or hydroxyurea therapy to reduce stroke risk/recurrence. However, we need more specific/sensitive biomarkers for stroke prediction/prognosis. Genetic modulators may be paramount in SCA pathophysiology and in CVA severity. They include variants in VCAM1 (endothelial dysfunction), ITGA4 (cell-cell adhesion), and NOS3 (nitric oxide metabolism. The main goals of this work are: a) improve the knowledge on the genetic architecture of paediatric cerebral vasculopathy in SCA; b) assessing the consequences of those genetic variants on gene expression/protein function; c) identify genotypic/phenotypic markers of SCA sub-phenotypes; and d) analyse their potential as genetic modulators of disease severity. This would be crucial in assessing potential pharmacological targets specifically aimed to the vascular system and instrumental for the design of novel preventive, prophylactic or therapeutic strategies. INSA_202DGH72 and ISAMB N/A

Sickle cell anaemia (SCA) arises from homozygosity for the mutation c.20A>T in the HBB gene. However, it shows a multifactorial-like behaviour with high heterogeneity of clinical features. Cerebral vasculopathy (CVA), namely paediatric ischemic stroke, is one of its most devastating consequences. The risk of CVA development, specifically stroke or silent cerebral infarction, may be modulated by underlying genetic modifiers, for example those affecting vascular homeostasis. In this study, we aimed to investigate the impact of variants in genes related with endothelial adhesion (VCAM1 and ITGA4) and nitric oxide metabolism (NOS3) on CVA in a group of 70 SCA children well characterized according to their CVA degree. In addition, the effect of the same genetic variants on biochemical/haematological biomarkers of chronic haemolysis was also analysed. Moreover, we also evaluated the putative additional modulating role of variants previously identified as stroke risk factors by genome-wide associated studies: GOLGB1 Y1212C, ENPP1 K173Q and PON1 Q192R. Molecular analysis was performed using PCR, PCR-RFLP, next-generation sequencing and Sanger sequencing. SPSS software was used for statistical analyses and association studies. One of the seven VCAM1 promoter haplotypes found and the VCAM1 promoter rs1409419_T showed association with moderate to high time-averaged mean of maximum velocity in the middle cerebral artery. The same association was observed for the variant ENPP1 K173Q. On the other hand, we observed that ITGA4 variants rs113276800_A and rs3770138_T were associated with stroke events. As for NOS3, one of the six haplotypes and the intron 4 VNTR_4b allele (5 repeats) were associated with lower risk of silent cerebral infarction. Chronic haemolysis biomarker levels also seemed to be influenced by genetic variants. LDH levels was higher in the presence of VCAM1 promoter rs1409419_T and one VCAM1 haplotype but lower in patients with one of the two ITGA4 haplotypes found. Genetic modulation also occurred in total bilirubin levels, which were higher in association with VCAM1 rs3783613_C allele. Our results, namely the association between specific variants with certain cerebral vasculopathy predictors further enhances their putative modulating effect on SCA paediatric stroke risk, severity and prognosis. These findings provide additional clues on the SCA pathophysiology and uncover features in these genes that may prove to be crucial as potential therapeutic targets. INSA_202DGH720 and ISAMB info:eu-repo/semantics/publishedVersion

As epidemias de gripe representam um sério problema de saúde pública com elevados custos económicos. Foi sugerido que um dos alelos do gene IFITM3 (rs12252-C) constitui um impor tante fator de risco de base populacional para as formas graves da gripe por infeção pelo vírus A(H1N1)pdm09. Analisámos variantes do IFITM3 associadas à gravidade da doença em doentes por tugueses (n=41). Foram identificados sete SNPs no amplicão de 352bp do IFITM3 em torno do rs12252. De acordo com HapMap, o SNP rs34481144 per tence ao mesmo bloco de desequilíbrio de ligação (LD) que rs12252, e está em LD com rs6421983. A associação negativa com formas sintomáticas graves em relação a ligeiras obser vada para o alelo rs34481144-A é sugestiva de um efeito protetor no modelo de hereditariedade dominante. Para além disso, o haplótipo Hap4 com rs34481144-A, e sem o rs12252-C, mostrou estar significativamente associado a gripe sintomaticamente ligeira. Por outro lado, apesar de a um nível de significância limiar, o haplótipo Hap1 com rs34481144-G, sem rs12252-C, mostrou-se associado à gripe com sintomatologia grave. Em comparação com a população por tuguesa em geral, foram obser vadas diferenças significativas nas frequências do alelo possivelmente protetor rs34481144-A no grupo com sintomatologia grave, do Hap1 deletério no grupo com sintomatologia ligeira e do Hap4 protetor no grupo com doença grave. A proporção de casos com sintomas graves que poderiam ser evitados se todos os indivíduos da população apresentassem o alelo protetor rs34481144-A foi estimada em 56% e 64%, respetivamente na população em geral e no grupo de indivíduos com doença ligeira. A implicação destas variantes nos fenótipos da doença necessita de estudos de validação, nomeadamente de natureza funcional. Influenza epidemics are a serious global public health and economic problem. The IFITM3 allele (rs12252-C) was suggested as a strong population-based genetic risk factor for severe influenza virus infection by A(H1N1)pdm09. We analyzed IFITM3 variants for association to influenza severity in Por tuguese patients (n=41). Seven SNPs, within the 352bp IFITM3 amplicon around rs12252, were identified. According to HapMap SNP rs34481144 belongs to the same linkage disequilibrium (LD) block as rs12252, and is in strong LD with rs6421983. A negative association with severe relative to mild disease was obser ved for allele rs34481144-A, indicating a protective ef fect under the dominant model. Moreover, haplotype Hap4 with rs34481144-A, not including rs12252-C, was significantly associated to mild influenza. Conversely, although with borderline significance, haplotype Hap1 with rs34481144-G, not including rs12252-C, was associated to severe disease. Moreover, in comparison to the general Por tuguese population, statistical significant dif ferences in the frequencies of the protective allele rs34481144-A in the severe disease group, the deleterious Hap1 in the mild disease group and the protective Hap4 in the severe disease group, were obser ved. The population attributable risk (PAR) for the targeted rs34481144 allele or genotype was of 55.91% and 64.44% in the general population and the mildly infected individuals, respectively. Implication of these variants in disease phenotype needs fur ther validation, namely through functional analysis. Este trabalho foi financiado pela Fundação Luso-Americana para o Desenvolvimento (LACR Award program - 2007) e pela Fundação para a Ciência e Tecnologia (FCT), Portugal, (POCTI/ESP/44826/2002), com a comparticipação dos fundos da Comunidade Europeia (FEDER). info:eu-repo/semantics/publishedVersion

A análise do genoma do patógeno e da genética do hospedeiro tem possibilitado o entendimento da coevolução do Mycobacterium tuberculosis com o seu hospedeiro humano, conduzindo a relações simpátricas mais estáveis entre o hospedeiro e o patógeno. A influência de fatores clínicos (coinfeção pelo VIH e tuberculose multirresistente (MDRTB]) e demográficos (sexo e idade) na relação hospedeiro-patógeno simpátrica foi investigada utilizando o spacer oligonucleotide typing (spoligotyping) enquanto método de genotipagem de isolados do M. tuberculosis pertencendo à linhagem Euro-Americana. Foram analisados 547 casos de tuberculose (TB) e os isolados correspondentes, de uma amostragem consecutiva de seis anos num contexto de elevada prevalência para a coinfecção TB-VIH (32,0%). Entre estes, 62,0% resultaram de infeção causada pelos principais genótipos de M. tuberculosis em circulação. A relação simpátrica foi definida de acordo com o spoligotype em comparação com a base de dados internacional de spoligotype SpolDB4. Foi evidenciada uma associação estatisticamente significativa da MDRTB com os isolados simpátricos, independente da coinfeção pelo VIH. Para além disso, o resultado do estudo da prevalência da distribuição da infeção com a idade foi sugestivo de que os determinantes da predisposição genética do hospedeiro à TB simpátrica eram impulsionados por variantes comuns sob efeito de um “gene principal” (“major gene effect ”). Em termos de saúde pública, estes resultados poderão dar um importante contributo para a elaboração de modelos que preveem a duração dos ciclos de transmissão ativa, nomeadamente associada a casos graves de MDRTB. Major contributions from pathogen genome analysis and host genetics have equated the possibility of Mycobacterium tuberculosis co-evolution with its human host leading to more stable sympatric host–pathogen relationships. We explored the influence on the sympatric host-pathogen relationship of clinical (HIV infection and multidrug-resistant tuberculosis (MDRTB]) and demographic (gender and age) factors using spacer oligonucleotide typing (spoligotyping) for the classification of M. tuberculosis strains within the Euro-American lineage. We analyzed a total of 547 tuberculosis (TB) cases, from six year consecutive sampling in a setting with high TB-HIV coinfection (32.0%). Of these, 62.0% were caused by major circulating M. tuberculosis genotypes. The sympatric relationship was defined according to spoligotype in comparison to the international spoligotype database SpolDB4. A significant association of MDRTB with sympatric strains, regardless of the HIV status, was evidenced. Furthermore, distribution curves of the prevalence of sympatric TB in relation to patients’ age suggested that the host genetic determinisms in the host-pathogen relationship were driven by more common variants under a “major gene” effect. Este trabalho foi financiado pela Fundação Luso-Americana para o Desenvolvimento (LACR Award program - 2007) e pela Fundação para a Ciência e Tecnologia (FCT), Portugal, (POCTI/ESP/44826/2002), com a comparticipação dos fundos da Comunidade Europeia (FEDER). info:eu-repo/semantics/publishedVersion

It is well demonstrated that the frequency of sickle cell allele, HBB*S, reaches the highest values in regions of endemic malaria, due to resistance to the malaria infection conferred by HBB*S. Its presence in Portugal has been explained as the consequence of past population migrations, which in part also explain the uneven distribution of HBB*S across the country, where it tends to increase in frequency from the north towards the south-central regions. In order to better understand how HBB*S was introduced in Portugal, the screening of HBB was performed in 266 individuals from Alentejo (Coruche, Serpa (Pias) and Alcácer do Sal). Then, to further infer the origin of the detected HBB*S chromosomes, their haplotypic background, encompassing the entire cluster of β-globin genes, was examined through a SNaPshot® Multiplex system that was purposely implemented as an alternative to the conventional RFLP-based method, and that allowed to interrogate the most informative positions defining the HBB*S haplotypes. 19 individuals harboured the c.20A>T mutation that underlies HBB*S, whose frequency was 2.2, 2.9 and 7.7% in Coruche, Alcácer do Sal and Serpa, respectively. The last value is the highest up to now reported in the country, where foci of high prevalence of HBB*S carriers have been previously identified in south regions. Among the chromosomes bearing HBB*S, 29% harboured the Benin haplotype, 13.1% the Senegal and 10.5% the Bantu. Remarkably, whereas in Alcáçer do Sal and Coruche only Senegal and Bantu haplotypes were found, in Serpa all haplotypes were Benin. Taking into account the global distribution of HBB*S haplotypes, the findings obtained reinforce a scenario before proposed positing that the introduction of HBB*S in south Portugal was mediated by gene influx events with distinct sources: one from the region encompassing the Mediterranean basin (captured by the Benin haplotype), and other from sub-Saharan Africa, likely afforded by the transatlantic slave trade (captured by the Senegal and Bantu haplotypes). info:eu-repo/semantics/publishedVersion

The widespread application of carbon nanotubes (CNT) on industrial, biomedical, and consumer products can represent an emerging respiratory occupational hazard. Particularly, their similarity with the fiber-like shape of asbestos have raised a strong concern about their carcinogenic potential. Several in vitro and in vivo studies have been supporting this view by pointing to immunotoxic, cytotoxic and genotoxic effects of some CNT that may conduct to pulmonary inflammation, fibrosis, and bronchioloalveolar hyperplasia in rodents. Recently, high throughput molecular methodologies have been applied to obtain more insightful information on CNT toxicity, through the identification of the affected biological and molecular pathways. Toxicogenomic approaches are expected to identify unique gene expression profiles that, besides providing mechanistic information and guiding new research, have also the potentialto be used as biomarkers for biomonitoring purposes. In this review, the potential of genomic data analysis is illustrated by gene network and gene ontology enrichment analysis of a set of 41 differentially expressed genes selected from a literature search focused on studies of C57BL/6 mice exposed to the multiwalled CNT Mitsui-7. The majority of the biological processes annotated in the network are regulatory processes and the molecular functions are related to receptor-binding signalling. Accordingly, the network-annotated pathways are cell receptor-induced pathways. A single enriched molecular function and one biological process were identified. The relevance of specific epigenomic effects triggered by CNT exposure, for example, alteration of the miRNA expression profile is also discussed in light of its use as biomarkers in occupational health studies. Centre for Toxicogenomics and Human Health (ToxOmics); Foundation for Science and Technology (UID/BIM/00009/2013) info:eu-repo/semantics/publishedVersion

Realizou-se no passado dia 11 de maio de 2018 o 1st HBM-PT, tendo reunido no Instituto Nacional de Saúde Doutor Ricardo Jorge (INSA), em Lisboa, mais de oitenta participantes da Academia, Indústria, Saúde Ocupacional e Regulamentação, entre outros. O Encontro partiu da iniciativa do conjunto de parceiros que constituem o denominado National Hub (NH-PT) para o projeto “Human Biomonitoring Initiative - HBM4EU” que inclui a Fundação para a Ciência e a Tecnologia, I.P. (FCT), Instituto Nacional de Saúde Dr. Ricardo Jorge, I.P. (INSA), Direção Geral da Saúde (DGS) e Agência Portuguesa do Ambiente, I.P. (APA), em colaboração com a Faculdade de Medicina, Universidade de Lisboa (FMUL) e Escola Superior de Tecnologia da Saúde Lisboa (ESTeSL), Instituto Politécnico de Lisboa. Este primeiro Workshop visou juntar investigadores reguladores, público em geral e outros atores-chave para discutir a contribuição da biomonitorização humana para as políticas de saúde e ambiente e para a avaliação de risco para a saúde humana. Para além disso, pretendeu informar as partes interessadas acerca do projeto HBM4EU, incluindo aspetos relevantes, tais como a sua estrutura e as atividades já desenvolvidas durante o primeiro ano do projeto ou a desenvolver futuramente. Nesta síntese os autores pretenderam oferecer uma visão geral do evento, através de um breve resumo das apresentações dos oradores convidados e dos temas abordados pelo painel de discussão tecendo ainda algumas considerações finais sobre o evento. The 1st HBM-PT was held on the 11th of May and more than eighty participants from the Academy, Industry, Occupational Health and Regulation, among others, met at the National Institute of Health Dr. Ricardo Jorge, I.P. (INSA) in Lisbon. The meeting was organized by the partners that constitute the National Hub (NH-PT) for the Human Biomonitoring Initiative - HBM4EU project, which includes the Foundation for Science and Technology, I.P. (FCT), the Directorate-General of Health (DGS), the Portuguese Environment Agency, I.P. (APA), and INSA, in collaboration with the Faculty of Medicine, University of Lisbon (FMUL) and the School of Health Technology Lisbon (ESTeSL), Lisbon Polytechnic Institute. This first Workshop aimed to bring together researchers, regulators, the general public and other stakeholders to discuss the contribution of human biomonitoring to health and environment policies and to the risk assessment of chemicals to human health. In addition, it intended to inform interested parties about the HBM4EU project, including relevant aspects of its structure and activities already developed during the first year, or to be developed in the future. In this summary document, the authors intended to give an overview of the event, through a brief summary of the invited speakers presentations and the main topics approached by the discussion panel, ending up with some final remarks of the event. This Workshop was organized by the National Hub under the scope of the H2020 European Joint Program Human Biomonitoring Initiative - HBM4EU (GA 733032). N/A

Sickle cell anemia (SCA) arises from homozygosity for the mutation c.20A>T in the HBB gene which originates hemoglobin S (HbS). In hypoxic conditions, HbS polymerizes inside erythrocytes deforming them and ultimately leading to hemolysis and vaso-occlusion. SCA shows a multifactorial-like behaviour with a high heterogeneity of clinical features, with stroke being the most severe of them. This heterogeneity may arise from underlying genetic modifiers, namely those affecting vascular adhesion/endothelial dysfunction. These include genes encoding the VCAM-1 molecule and its ligand VLA-4 (ITGA4 or integrin α4), increasingly studied due to their expression in activated human endothelium and leucocytes/stress reticulocytes, respectively. The aim of this study was to identify putative genetic modulators of stroke risk by analyzing 70 pediatric SCA patients, grouped according to their degree of cerebral vasculopathy. Molecular analysis was performed using Next-Generation Sequencing (NGS) and Sanger Sequencing. R software was used for statistical analyses and association studies. In silico studies were performed using PHASE, TFbind, PROMO and Human Splicing Finder software tools. We identified six different VCAM1 promoter variants and seven haplotypes. The VCAM1 promoter rs1409419_T allele was associated with stroke events (p=0.008; O.R.= 4.33; C.I.95% =1.391-14.257), while one VCAM1 promoter haplotype was found to be protective of stroke (p=0.011; O.R.=0.22; C.I.95% =0.048-0.784). On the ITGA4 gene, forty variants were found, six of them novel. All patients presented with at least one variant in this gene. We observed co-inheritance of specific sets of ITGA4 variants indicating the presence of haplotypes not previously described. Additionally the presence of specific variants seems to result in a predisposition for either high reticulocyte count, elevated lactate dehydrogenase, raised bilirubin levels or increased transcranial Doppler velocity values. Our results reinforce the role of endothelial molecules and blood cell interaction in SCA severity. The association between specific VCAM1, as well as ITGA4, variants with certain cerebral vasculopathy predictors, further enhances their putative modulating effect on pediatric stroke severity and prognosis. These findings provide additional clues on the SCA pathophysiology and uncover features of both genes that may prove to be crucial as potential therapeutic targets. info:eu-repo/semantics/publishedVersion

21ª Reunião da Sociedade Portuguesa de Genética Humana, 16-18 nov 2017 Sickle Cell Anemia (SCA) is a hereditary anemia caused by a missense mutation in HBB and it is characterized by chronic hemolysis, recurrent episodes of vaso-occlusion and infection. Cerebral vasculopathy is one of the most devastating complications of the disease and even young children with SCA have a high risk of stroke. It is known that both environmental and genetic determinants are able to modulate the onset, course and outcome of the disease. Among those, the level of fetal hemoglobin (HbF) has been proposed as the most significant disease modulator. Thus, in this work, we aimed to investigate if the level of HbF in SCA children is related with the risk of stroke and if it is modulated by variants in genes, such as HBG2, BCL11A, HBS1L-MYB, and KLF1. Sixty-seven children (3 years of age) with SCA were enrolled in this study. Hematological and imaging data were retrospectively obtained from patients’ medical records at Greater Lisbon area hospitals. Patients were grouped according to their degree of cerebral vasculopathy evaluated by transcranial Doppler velocities and magnetic resonance imaging. Molecular analyses were performed using Next-Generation Sequencing, Sanger sequencing and PCR-RFLP. In silico studies and statistical analyses were done using the PolyPhen-2 and SPSS softwares, respectively. The association studies revealed that low HbF levels were associated with stroke events in SCA children (p=0.005). At the molecular level, it was observed that patients with the rarest genotypes in HBG2 (rs7482144_TT+TC) presented higher levels of HbF (p=0.031). Additionally, the rs11886868_C and the rs4671393_A alleles in BCL11A also seemed to predispose to higher HbF levels. Moreover, eleven distinct variants in KLF1 were detected (one of them novel, the p.Q342H) with 83% of the patients having at least one variant in this gene. The group of patients who have co-inherited the above mentioned variants in HBG2 and BCL11A together with at least one KLF1 variant presented the highest HbF levels (p=0.021). Our results corroborate previous studies suggesting that a low level of HbF in SCA patients is a risk factor for stroke. Furthermore, we report for the first time the importance of KLF1 variants in combination with other genetic modifiers to the final phenotypic expression of HbF in SCA children with different degrees of cerebral vasculopathy. Consequently, this study allowed the delineation of a genetic pattern with prognostic value for SCA. info:eu-repo/semantics/publishedVersion

Being subjected to diverse anthropogenic pressures, from industrial to agricultural activities, estuaries have long been regarded as ecosystems particularly sensitive to contamination. A recent study addressed the potential adverse effects of the Portuguese Sado river sediment contaminants on human and ecosystem health. Several complementary approaches were used to assess the effects on human health, including: (i) an epidemiological one to characterize the exposure pathway to the estuary environment; (ii) an in vitro characterization of the genotoxic potential of sediments’ contaminants in a human cell line; (iii) an in vivo study of gene and chromosome alterations induction in LacZ plasmid-based transgenic mice. The epidemiological survey confirmed the occurrence of direct and indirect (through food chain) exposure of the local population to estuarine contaminants. On the other hand, the complex mixture of contaminants extracted from sediments, which contain metals and polycyclic aromatic hydrocarbons, was toxic to human liver cells exposed in vitro, causing cell death, oxidative stress and genetic damage [3, 4]. Furthermore, the results of an in vivo study showed a time-dependent increase in chromosome damage in blood immature erythrocytes but did not show induction of mutations in liver cells or DNA damage in blood, spleen, kidney or liver cells of exposed comparatively to control mice. Although the histopathological analysis of liver tissues did not reveal specific alterations associated with exposure, changes observed in hepatocytes structures indicated that liver function related to carbohydrate metabolism and storage was compromised, therefore revealing an important chronic effect. Overall, the complementary approaches used in this study suggested that the exposure of local populations to the Sado river estuary contaminants may have a negative impact on their health. Nevertheless, there is still a need to perform a biomonitoring study, i.e., obtain and integrate data from exposure and from cellular and molecular biomarkers of early biological effects and susceptibility in the exposed comparatively to a control population, in order to try to derive an indicator of risk of estuary-associated chronic diseases. N/A

Hemoglobin (Hb) is a protein responsible for oxygen transportation from lungs to the entire body. It is composed by four globular subunits - the globins - each with a central core containing a heme molecule. Globins are encoded by the α- and β-globin gene clusters located at 16p13.3 and 11p15.5, respectively. The pattern of globin gene expression during development is precisely controlled by the interaction of cis-regulatory genomic regions (located in close proximity to and far from genes) with trans-activating/silencing factors within permissive chromatin domains. Distally upstream of the α-globin genes there are four multispecies conserved sequences (MCS-R1 to R4) which are critical for the expression of the downstream globin genes. Deletions removing the α-globin genes and/or their distal MCSs give rise to α-thalassemia, one of the most common genetic recessive disorders worldwide, due to a reduced rate of α-globin chain synthesis. The severity of the pathology is variable ranging from a very mild microcytic hypochromic anemia to a moderately severe anemia associated with the formation of β4 tetramers resulting in HbH disease or an even higher reduction or complete absence of α-chains resulting in hemoglobin Bart’s hydrops fetalis, a condition generally incompatible with life. The main objectives of this work were to characterize the molecular lesions underlying ten Portuguese cases of unusual α-thalassemia/HbH disease and to understand their origin and functional consequences. After exclusion the most frequent molecular lesions associated with α-thalassemia, Multiplex Ligation-dependent Probe Amplification (MLPA) using the SALSA MLPA P140B HBA kit (MCR-Holland) was used to search for DNA deletions in the subtelomeric region of chromosome 16p. Additionally, specifically designed synthetic MLPA probes, as well as gap-PCR and Sanger sequencing were performed for more accurate deletion breakpoint mapping. We have found five distinct deletions and one indel, all in heterozygosity. The deletions range from approximately 3.3 to 323 kb and two of them are novel. The three larger deletions remove the entire α-globin cluster whereas the others remove totally or partially the distal regulatory elements keeping the α-globin genes structurally intact. The indel comprises the deletion of the MCS-R2 regulatory element and the insertion of a singular 39 bp DNA fragment possibly originating from a complex rearrangement involving chromosome 3. Finally, no α-globin gene cluster deletion or point mutation were found in a patient who revealed to be a very unusual case of acquired alpha-thalassemia associated with a myelodysplastic syndrome. Our study widens the spectrum of molecular lesions and unusual molecular mechanisms by which α-thalassemia/HbH may occur and emphasizes the importance of diagnosing large α0-deletions to provide patients with appropriate genetic counseling. info:eu-repo/semantics/publishedVersion 22nd Congress of the European Hematology Association, 22-25 June 2017

European Society of Human Genetics, 27-30 May 2017 Introduction: Inherited deletions removing the α-globin genes and/or their upstream regulatory elements (MCSs) give rise to alpha-thalassemia, one of the most common genetic recessive disorders worldwide. The pathology is characterized by microcytic hypochromic anemia due to reduction of the α-globin chain synthesis, which are essential for hemoglobin tetramerization. Material and Methods: In order to clarify the suggestive α-thalassemia phenotype in eleven patients, we performed Multiplex Ligation-dependent Probe Amplification with commercial and synthetic probes, gap-PCR, and Sanger sequencing to search for deletions in the subtelomeric region of chromosome 16p. Results: We have identified six distinct large deletions, three of them novel, and one indel. The deletions range from approximately 3.3 to 323 kb, and i) remove the whole α-globin cluster; or ii) remove exclusively the upstream regulatory elements leaving the α-globin genes structurally intact. The indel consists in the loss of MCS-R2 (HS-40), which is the most important distal regulatory element for the α-globin gene expression, and the insertion of 39 nt, seemingly resulting from a complex rearrangement involving two DNA segments (probably from chromosome 3q), bridging the deletion breakpoints with a CC-bp orphan sequence in between. Finally, in one patient no α-globin deletion or point mutation were found. This patient revealed to have acquired alpha-thalassemia associated with a myelodysplastic syndrome. Conclusions: Our study widens the spectrum of molecular lesions by which α-thalassemia may occur and emphasizes the importance of diagnosing large α0-deletions to provide patients with appropriate genetic counseling. info:eu-repo/semantics/publishedVersion

Editorial sobre a segunda edição do Dia do Jovem Investigador promovido pelo Instituto Nacional de Saúde Doutor Ricardo Jorge a 8 de maio de 2017. A iniciativa teve como objetivo proporcionar o contacto direto com a produção científica e tecnológica dos jovens investigadores, dando a conhecer o seu importante contributo a este Instituto, enquanto Laboratório do Estado no setor da Saúde, laboratório nacional de referência e observatório nacional de saúde. info:eu-repo/semantics/publishedVersion

Hemoglobin (Hb) is a protein responsible for oxygen transportation from lungs to the entire body. It is composed by four globular subunits - the globins - each with a central core containing a heme molecule. Globins are encoded by the α- and β-globin gene clusters located at 16p13.3 and 11p15.5, respectively. The pattern of globin genes expression during development is precisely controlled by the interaction of cis-regulatory genomic regions (located in close proximity to and far from genes) with trans-activating/silencing factors within permissive chromatin domains. In fact, approximately 25-65 kb upstream of the α-globin genes there are four multispecies conserved sequences (MCS-R1 to R4) which are critical for the expression regulation of the downstream globin genes. The main objectives of this work were to characterize the molecular lesions underlying eight unusual cases of α-thalassemia or Hb H disease, and to understand their origin and functional consequences. Deletions were detected by Multiplex Ligation-dependent Probe Amplification (MLPA) using the SALSA MLPA P140B HBA kit (MCR-Holland). Additionally, specifically designed synthetic MLPA probes, as well as Gap-PCR and Sanger sequencing were performed for fine deletion breakpoint mapping. We have found seven different deletions (ranging from 3.3 to ≈323 kb), four of them not previously described. The four largest deletions removed all the α-globin genes, whereas the other three deletions removed one or more of the distal regulatory elements keeping the globin genes structurally intact. In one case, only the MCS-R2 (also known as HS-40) was removed and replaced by a 39 nt DNA fragment possibly resulting from a complex rearrangement that introduces new pieces of DNA (probably from Chrs. 3 and 7) bridging the two deletion breakpoints. In the remaining case, no deletion was found and the patient revealed to be a very unusual case of acquired alpha-thalassemia-myelodysplastic syndrome. It is important to detect individuals with this type of uncommon deletions as there is a 25% risk of having a child with Hb Bart’s hydrops fetalis or Hb H disease if their partner is a carrier of an α0-thal or α+-thal allele, respectively. Moreover, further investigation is currently being developed on one of these natural mutants which is bringing new insights into the long-range regulation mechanism of the globin gene expression and to the pathophysiology of the α-thalassemia. N/A