Paula Faustino

Epidemiology of haemoglobin disorders in Europe: an overview

Objective. As a result of global population movements, haemoglobin disorders (thalassaemias and sickle cell disorders) are increasingly common in the formerly non‐indigenous countries of Northern and Western Europe and in the indigenous countries of Southern Europe. This article presents an overview of the changing picture and a method for assessing service needs. Method. Data on country of birth or ethnic origin of residents are adjusted to obtain the estimated proportions of residents and births in non‐indigenous groups at risk for haemoglobin disorders in European countries. The results are combined with prevalence data in each country of origin to obtain country prevalence estimates. Service indicators (annual tests or other interventions required to ensure equitable delivery of treatment and prevention) are then derived by country. Results. Haemoglobin disorders now occur at comparable frequency throughout Northern, Western and Southern Europe. Annually, there are more affected conceptions in Northern and Western than in Southern Europe, and sickle cell disorders are more common than thalassaemias. There is growing need for health policy‐makers to support motivated professionals working to develop optimal patient care, carrier diagnosis, genetic counselling and access to prenatal diagnosis throughout the Region. Conclusion. There is a strong case for pan‐European collaboration on haemoglobin disorders to share policies, standards and the instruments required to support them. These include methods for needs assessment, service standards, education and information strategies and materials, and methods for evaluating service delivery.

An overview of molecular basis of iron metabolism regulation and the associated pathologies

Iron is essential for several vital biological processes. Its deficiency or overload drives to the development of several pathologies. To maintain iron homeostasis, the organism controls the dietary iron absorption by enterocytes, its recycling by macrophages and storage in hepatocytes. These processes are mainly controlled by hepcidin, a liver-derived hormone which synthesis is regulated by iron levels, inflammation, infection, anemia and erythropoiesis. Besides the systemic regulation of iron metabolism mediated by hepcidin, cellular regulatory processes also occur. Cells are able to regulate themselves the expression of the iron metabolism-related genes through different post-transcriptional mechanisms, such as the alternative splicing, microRNAs, the IRP/IRE system and the proteolytic cleavage. Whenever those mechanisms are disturbed, due to genetic or environmental factors, iron homeostasis is disrupted and iron related pathologies may arise.

Genetic and biochemical markers in patients with Alzheimer's disease support a concerted systemic iron homeostasis dysregulation

Alzheimers disease (AD) is the most common form of dementia in the elderly individuals, resulting from a complex interaction between environmental and genetic factors. Impaired brain iron homeostasis has been recognized as an important mechanism underlying the pathogenesis of this disease. Nevertheless, the knowledge gathered so far at the systemic level is clearly insufficient. Herein, we used an integrative approach to study iron metabolism in the periphery, at both genotypic and phenotypic levels, in a sample of 116 patients with AD and 89 healthy control subjects. To assess the potential impact of iron metabolism on the risk of developing AD, genetic analyses were performed along with the evaluation of the iron status profile in peripheral blood by biochemical and gene expression studies. The results obtained showed a significant decrease of serum iron, ferritin, and transferrin concentrations in patients compared with the control subjects. Also, a significant decrease of ferroportin (SLC40A1) and both transferrin receptors TFRC and TFR2 transcripts was found in peripheral blood mononuclear cells from patients. At the genetic level, significant associations with AD were found for single nucleotide polymorphisms in TF, TFR2, ACO1, and SLC40A1 genes. Apolipoprotein E gene, a well-known risk factor for AD, was also found significantly associated with the disease in this study. Taken together, we hypothesize that the alterations on systemic iron status observed in patients could reflect an iron homeostasis dysregulation, particularly in cellular iron efflux. The intracellular iron accumulation would lead to a rise in oxidative damage, contributing to AD pathophysiology.

Novel promoter and splice junction defects add to the genetic, clinical or geographic heterogeneity of β-thalassaemia in the Portuguese population

SummaryIn order to delineate the spectrum and the relative abundance of β-globin gene defects causing thalassaemia in the Portuguese population, a representative sample was analysed including 51 β-thalassaemia carriers along with 26 patients representing different clinical phenotypes. Seven mutations were identified, four of which [codon 39 (C→T), 39%; intervening sequence (IVS)1 nucleotide (nt) 1 (G→A), 26%; IVS1 nt 110 (G→A), 17%; IVS1 nt6 (T→C), 15%] account for 97% of 93 β-thalassaemia chromosomes. Two previously undescribed mutations, namely a C→T substitution at position — 90 in the proximal CACCC box, and the deletion of nucleotides 4 and 5 (AG) in IVS 2 were identified. The uncommon, though ubiquitous, G→T transversion at codon 121 was found once upon haplotype V. Direct prenatal diagnosis can be offered to 95% of couples at risk of bearing a thalassaemic child.

Non-classical hereditary hemochromatosis in Portugal: novel mutations identified in iron metabolism-related genes

The most frequent genotype associated with Hereditary hemochromatosis is the homozygosity for C282Y, a common HFE mutation. However, other mutations in HFE, transferrin receptor 2 (TFR2), hemojuvelin (HJV) and hepcidin (HAMP) genes, have also been reported in association with this pathology. A mutational analysis of these genes was carried out in 215 Portuguese iron-overloaded individuals previously characterized as non-C282Y or non-H63D homozygous and non-compound heterozygous. The aim was to determine the influence of these genes in the development of iron overload phenotypes in our population. Regarding HFE, some known mutations were found, as S65C and E277K. In addition, three novel missense mutations (L46W, D129N and Y230F) and one nonsense mutation (Y138X) were identified. In TFR2, besides the I238M polymorphism and the rare IVS5 −9T→A mutation, a novel missense mutation was detected (F280L). Concerning HAMP, the deleterious mutation 5’UTR −25G→A was found once, associated with Juvenile Hemochromatosis. In HJV, the A310G polymorphism, the novel E275E silent alteration and the novel putative splicing mutation (IVS2 +395C→G) were identified. In conclusion, only a few number of mutations which can be linked to iron overload was found, revealing their modest contribution for the development of this phenotype in our population, and suggesting that their screening in routine diagnosis is not cost-effective.

The Peopling of São Tomé (Gulf of Guinea): Origins of Slave Settlers and Admixture with the Portuguese

The geographic origins of African slave settlers and the Portuguese genetic contribution to the population of São Tomé (Gulf of Guinea) were assessed through the analysis of b-globin haplotypes in 44 chromosomes bearing the bS allele and through the study of the genetic variation in eight autosomal markers (APOA1, AT3, FY, LPL, OCA2, RB1, Sb19.3, and GC) informative for admixture in a sample of 224 individuals. The observed bS haplotype distribution (36.4% Bantu, 52.3% Benin, 4.5% Cameroon, 4.5% Senegal, and 2.3% atypical) is in accordance with the historical information on the major geographic sources of slave settlers of São Tomé, although it captures a more important contribution of Central-West Africa regions than previously anticipated. European admixture, estimated to be 10.7 ± 0.9%, has created a considerable level of genetic structure, as indicated by the finding of significant linkage disequilibrium between 33% of unlinked marker loci pairs. Recent admixture was found to have an important contribution to these values, since removal of individuals with Portuguese or Cape Verdian parents or grandparents from the sample dropped the miscegenation level to 6.5 ± 0.8% and reduced significant linkage disequilibrium to 11% of unlinked marker pairs. Taken together, these results indicate that the peopling of São Tomé might have provided one of the first examples of the combination of diverse African contributions and European admixture that emerged from the overseas population relocations promoted by the Atlantic slave trade.

The role of HFE mutations on iron metabolism in beta-thalassemia carriers

AbstractHereditary hemochromatosis (HH) is an autosomal recessive disorder of iron metabolism characterized by increased iron absorption and progressive storage resulting in organ damage. HFE gene mutations C282Y and H63D are responsible for the majority of HH cases. A third HFE mutation, S65C, has been associated with the development of a mild form of hemochromatosis. The beta-thalassemia trait is characterized by mild, ineffective erythropoiesis that can induce excess iron absorption and ultimately lead to iron overload. The aim of this study was to evaluate the effect of genetic markers (HFE mutations C282Y, H63D, and S65C) on the iron status of beta-thalassemia carriers. A total of 101 individuals heterozygous for beta-thalassemia and 101 normal control individuals were studied. The allelic frequencies of C282Y (1.5 versus 3.5%), H63D (15.3 versus 18.3%), and S65C (1.0 versus 1.5%) did not differ significantly between beta-thalassemia carriers and normal controls. Serum iron (P=0.029) and transferrin saturation (P=0.009) were increased in beta-thalassemia carriers heterozygous for H63D mutation. The number of subjects carrying C282Y or S65C mutations was too low to conclude their effect on the iron status. These results suggest that the beta-thalassemia trait tends to be aggravated with the coinheritance of H63D mutation, even when present in heterozygosity.

Novel large deletions in the human α-globin gene cluster: Clarifying the HS-40 long- range regulatory role in the native chromosome environment

Globin genes, which encode the protein subunits of hemoglobin (Hb), are organized in two different gene clusters and present a coordinated and differential pattern of expression during development. Concerning the human alpha-globin gene cluster (located at chromosome region 16p13.3), four upstream highly conserved elements known as multispecies conserved sequences (MCS-R1-4) or DNase I hypersensitive sites (HSs) are implicated in the long-range regulation of downstream gene expression. However, only the absence of the MCS-R2 site (HS-40) has proven to drastically downregulate the expression of those genes, and consequently, it has been regarded as the major and crucial distal regulatory element. In this study, Multiplex Ligation-dependent Probe Amplification was used to screen for deletions in the telomeric region of the short arm of chromosome 16, in an attempt to explain the alpha-thalassemia or the HbH disease present in a group of Portuguese patients. We report four novel and five uncommon deletions that remove the alpha-globin distal regulatory elements and/or the complete alpha-globin gene cluster. Interestingly, one of them occurred de novo and removes all HSs except HS-10, while other eliminates only the HS-40 site, the latter being replaced by the insertion of a 39 nucleotide orphan sequence. Our results demonstrate that HS-10 alone does not significantly enhance the alpha-globin gene expression. The absence of HS-40 in homozygosity, found in a patient with Hb H disease, strongly downregulates the expression of alpha-globin genes but it is not associated with a complete absence of alpha-globin chain production. The study of naturally occurring deletions in this region is of great interest to understand the role of each upstream regulatory element in the native human erythroid environment.

Mutational spectrum of delta‐globin gene in the Portuguese population

The phenotype of increased Hb A2 typical of β‐thalassaemia (β‐thal) carriers can be reduced to normal or borderline values because of the co‐inheritance of a δ‐globin gene (HBD, MIM #142000) mutation, which may lead to misinterpretation of diagnostic results. To know the spectrum of δ‐globin mutations in the Portuguese population we performed a mutational analysis of the δ‐globin gene in a group of 51 Portuguese β‐thal carriers presenting microcytosis, hypochromia and a normal/borderline Hb A2 level and in another group of 15 individuals suspected to have δ‐globin structural abnormalities. The heterozygosity for the β+IVS‐I‐6T→C (HBB:c. 92+6T>C) mutation was the main cause for the mentioned atypical β‐thal carrier phenotype. Furthermore, eight individuals were double heterozygous for one common β‐thal mutation and the δ+Cd27G→T mutation (Hb A2–Yialousa; HBD:c.82G>T). One of them also presented a novel δ‐globin gene promoter mutation,−80G→A (HBD:c.−130G>A), responsible for about 25% decrease of the promoter activity in transient expression assays. One the other hand, in the other group of 15 individuals suspected to have δ‐globin structural abnormalities observed by biochemical methods, some known Hb A2 variants were identified – Hb A2′ (HBD:c.49G>C), Hb A2‐Babinga (HBD:c.410G>A), and Hb A2‐Wrens (HBD:c.295G>A), and the novel Hb A2‐Fogo [δ64(E8)(Gly→Ser); (HBD:c.193G>A)]. This novel Hb A2 variant was observed segregating in linkage with Hb E (HBB:c.79G>A) in a three generation family. In conclusion, six different δ‐globin mutations were found, being two of them new molecular defects. All δ‐alleles identified were found linked to the expected β‐globin cluster haplotype. All mutations caused a low Hb A2 level and through this could lead to misdiagnosis when inherited together with a β‐thal allele.

β‐Thalassaemia unlinked to the β‐globin gene interacts with sickle‐cell trait in a Portuguese family

Summary An autosomally transmitted hypochromic microcytic mild anaemia with elevated haemoglobin (Hb) A2 and globin chain imbalance has been observed in a three‐generation family of Portuguese origin. Extensive DNA analysis of the β‐globin gene cluster, including the complete sequencing of the,β‐globin gene and flanking regions, failed to reveal any genetic alteration. The co‐segregation of sickle‐cell trait in this family enabled us to postulate a defective erythroid trans‐acting factor was playing a role in the down‐regulation of both βA‐ and βs‐globin genes. Among the transcription factors that could possibly have caused the reported phenotype, NF‐E2 is unlikely to be implicated, whereas Nrf1 and Nrf2 cannot be ruled out. Thus, this family carries a novel β‐thalassaemia autosomal determinant unlinked to the β‐globin gene. This observation reinforces the notion of the haemoglobinopathies as single gene disorders under polygenic regulation.