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Introdução: A deficiência de glicose-6-fosfato desidrogenase (G6PD) é o defeito enzimático mais comum no mundo, afetando mais de 500 milhões de pessoas. Em Portugal, a frequência populacional da deficiência de G6PD no sexo masculino foi estimada em cerca de 0,5%, e desde o ano 2000 têm vindo a ser descritas diversas variantes G6PD causadoras da deficiência. O principal objetivo deste estudo foi melhorar o conhecimento sobre a heterogeneidade molecular da deficiência de G6PD na população portuguesa.Material e Métodos: Análise retrospetiva do perfil mutacional de 138 indivíduos não-aparentados de naturalidade portuguesa (101 homens e 37 mulheres), sem ascendência subsaariana conhecida, diagnosticados com deficiência de G6PD entre 1994 e 2020 na Unidade de Hematologia Molecular do Centro Hospitalar e Universitário de Coimbra (CHUC). O estudo molecular foi feito por sequenciação direta de Sanger ou análise por PCR-RFLP.Resultados: Identificaram-se 21 mutações patogénicas diferentes. Destas, 20 são mutações missense, que levam à troca de aminoácido, e uma é uma deleção in-frame de 18 nucleótidos no exão 10. As três mutações mais frequentes pertencem ao haplótipo subsaariano G6PD c.376A>G, nomeadamente as variantes G6PD: A- (c.202G>A; p.68Val>Met) (58,6%), Betica (c.968T>C; p.323Leu>Pro) (12,1%) e Santamaria (c.542A>T; p.181Asp>Val) (4,3%).Conclusão: Existe uma elevada heterogeneidade molecular da deficiência de G6PD em Portugal.

Hb F production is under the influence of major quantitative trait loci (QTL). The present study aims: i) to replicate the association with Hb F for representative genetic variants in the three major Hb F QTLs in a Portuguese sample of β-thalassemia (β-thal) carriers; and ii) to test different genetic multi-locus models to account for the genetic component of Hb F variation. A population sample of 79 Portuguese β-thal carriers (39 males, 40 females), aged between 2 to 70 years old, were genotyped for polymorphisms in the locus control region (LCR)-5' hypersensitive site 4 (5'HS4) rs16912979

In the already identified quantitative trait loci (QTL), modulating Hb F levels are cis-acting haplotypes of the β-globin gene cluster itself, although the single nucleotide polymorphisms (SNPs) accounting more for the association, remain uncertain. In this study, the role in Hb F production of previously reported candidate SNPs within the β-globin gene cluster was reexamined, along with a yet poorly studied variation in the BGLT3 gene. In a sample of β-thalassemia (β-thal) carriers, we succeeded in replicating the significant association between increased Hb F levels and rs7482144 (C>T) (HBG2 XmnI), which is the most well-established variation in the cluster influencing the trait. This SNP was found to be in strong linkage disequilibrium (LD) with a variation in the HBBP1 gene [rs10128556 (G>A)], which consistently revealed a similar association signal. Remarkably, much stronger than the latter associations were those involving both rs968857 (T allele) (3' HBBP1) and rs7924684 (G allele) (BGLT3), two SNPs that were also in strong LD. As the pattern of LD detected in the β-globin gene cluster does not correlate with a tight linkage between markers, complex interactions between SNPs at the cluster seem to modulate Hb F. Seeing that no such associations were detected in normal subjects, the question can be raised on whether, under erythropoiesis stress, epigenetic mechanisms contribute to change the regulation of the entire β-globin gene cluster. In conclusion, we provide statistical evidence for a new player within the β-globin gene cluster, BGLT3, that in cooperation with other regions influences Hb F levels in β-thal carriers.

Three major loci have been associated with HbF levels, including − 158C/T (XmnI) at HBG2 promoter region, and several polymorphisms at BCL11A intron-2 and HBS1L-MYB (HMIP) intergenic region. Mutations in the KLF1 gene were recently associated with increased HbF levels. This study aims to evaluate whether genetic variability at these loci influences HbF levels in β-thalassemia carriers and in normal individuals of Portuguese origin. Sixty five β-thalassemia carriers, HbF levels ranging from 0.2% to 9.5%, and 60 individuals with normal hematological parameters, HbF levels ranging from 0.2% to 7.4%, were selected for this study. In β-thal carriers linear regression models revealed a strong statistical significant association for HBG2 (XmnI) rs7482144 (β = 0.455; P = 5.858 × 10− 7), and nominal significance for BCL11A rs766432 (β = 0.215; P = 0.029) and HMIP rs9399137 (β = 0.209; P = 0.011). In normal individuals, a case (HbF > 2%; n = 15) vs. control (HbF

We herein present a case of congenital erythrocytosis caused by haemoglobin (Hb) Bethesda in a Japanese family. A 55-year-old asymptomatic man was referred to our hospital for the investigation of erythrocytosis, which was present in other members of his family. The patient's serum erythropoietin level was normal, and the JAK2 V617F mutation was not detected. His P50 value was mildly decreased, thus we suspected the presence of an Hb variant with a high oxygen affinity. The high-performance liquid chromatography analysis showed an abnormal Hb, and by direct sequencing we identified the Hb Bethesda variant in this patient. For the differential diagnosis, we recommend the estimation of the P50 value as a practical and useful test.

article Glucose-6-phosphate dehydrogenase (G6PD) deficiency, an X-linked disorder, is usually observed in hemizygote males and very rarely in females. The G6PD class 1 variants, very uncommon, are associated with chronic hemolytic anemia. Here we report a Portuguese woman who suffered in her sixties from a chronic hemolytic anemia due to G6PD deficiency. Molecular studies revealed heterozygosity for an in-frame 18-bp deletion, mapping to exon 10 leading to a deletion of 6 residues, 362-367 (LNERKA), which is a novel G6PD class 1 variant, G6PD Tondela. Two of her three daughters, asymptomatic, with G6PD activity within the normal range, are heterozygous for the same deletion. The patient's leukocyte and reticulocyte mRNA studies revealed an almost exclusive expression of the mutant allele, explaining the chronic hemolytic anemia. Patient whole blood genomic DNA HUMARA assay showed a balanced pattern of X chromosome inactivation (XCI), but granulocyte DNA showed extensive skewing, harboring the mutated allele, implying that in whole blood, lymphocyte DNA, with a very long lifetime, may cover up the current high XCI skewing. This observation indicates that HUMARA assay in women should be assessed in granulocytes and not in total leukocytes.

Ferroportin is a transmembrane protein responsible for iron export from enterocytes and macrophages. Mutation c.744G → T (Q248H), located in exon 6 of the ferroportin gene SLC40A1, is found as a polymorphism in populations of African origin. This mutation has been extensively analysed in African-Americans, but poorly studied in native African populations.To increase information about Q248H mutation frequency in native sub-Saharan populations examining three West African populations.Samples from S. Tomé e Príncipe (n = 115), Angola (n = 156) and Republic of Guinea (n = 170) were analysed for Q248H mutation and for two polymorphisms, IVS1( - 24)G → C and microsatellite (CGG)(n), using standard molecular methodology.The estimated frequencies of Q248H allele were 2.2% in S. Tomé e Príncipe, 3.5% in Angola and 4.1% in Republic of Guinea. Analysis of polymorphisms IVS1( - 24)G → C and (CGG)(n) showed mutation allele c.744T to be strongly associated with haplotype IVS1( - 24)G/(CGG)(7).This study confirmed the presence of Q248H mutation at polymorphic frequencies in three native sub-Saharan populations. Analysis of two additional markers in the same gene support a single origin of the mutant allele c.744T in the haplotype background IVS1( - 24)G/(CGG)(7).

Introduction Congenital erythrocytosis can be classified as primary, when the defect is intrinsic to the RBC progenitors and independent of the serum erythropoietin (Epo) concentration, or secondary, when the erythrocytosis is the result of an upregulation of Epo production. Primary erythrocytosis is associated with mutations in the EPOR gene, secondary CE can de due to mutations that stabilize the hemoglobin in the oxygenated form or to mutations in the genes that control the transcriptional activation of the EPO gene – VHL, EGLN1, EPAS1. Chuvash polycythemia, caused by mutations in VHL gene, shares features of both primary and secondary erythrocytosis, with increased Epo production but also hypersensitivity of progenitors to Epo. Material and Methods With the main objective of describing the etiology and molecular basis of CE, we have studied 70 consecutive unrelated patients presenting with idiopathic erythrocytosis from our hematology clinic or referred from other centers. According to a study algorithm, we have sequenced all the genes described as associated with CE. Results and Discussion Erythrocytosis molecular etiology was identify in 25 (36%) of the 70 subjects. High-affinity Hb variants were the most common cause, present in 20% of the cases. New mutations were identified in the JAK2, EPOR, VHL, and EGLN1 genes. Conclusions High-affinity hemoglobin variants are a very rare cause of secondary CE, but it seems likely that their incidence may be underestimated. Our experience shows that in erythrocytosis with a dominant inheritance and normal or inappropriate high Epo levels, the HBB and HBA genes should be the first to be studied. In spite of the seven genes known to be involved in CE, the majority of the cases have unknown etiology.

Neonatal cyanosis in healthy newborns can be associated either with methemoglobin due to cytochrome b5 reductase deficiency or to M-hemoglobin, a group of hemoglobin variants resulting from mutations in the globin chain genes. We report the clinical case of a neonate with cyanosis and normal cardiac and respiratory function. At birth the hematological parameters were normal; however, the methemoglobinemia was 16%. Spontaneously, the cyanosis gradually decreased and by the fifth month of age the methemoglobin level was normal. A heterozygous G gamma-globin gene (HBG2) missense mutation 87 C-A (Leu28Met) was identified. His father, with a history of transfusion in the neonatal period, is heterozygous for the same mutation. This hemoglobin variant, not previously described, was called Hb F Viseu and is the sixth G gamma-chain variant reported in association with neonatal cyanosis.

Reliable and accurate epidemiological data is a prerequisite for a cost effective screening program for inherited disorders, which however, is lacking in a number of developing countries. Here we report the first detailed population study in the Republic of Guinea, a sub-Saharan West African country, designed to assess the frequency of glucose-6-phosphate dehydrogenase (G6PD) deficiency and hemoglobinopathies, including screening for thalassemia. Peripheral blood samples from 187 Guinean adults were screened for hemoglobin (Hb) variants by standard hematological methods. One hundred and ten samples from males were screened for G6PD deficiency by the fluorescent spot test. Molecular analysis was performed for the most common α-thalassemia (α-thal) deletions, β-globin gene mutations, G6PD variants B (376A), A (376G), A- (376G/202A) and Betica (376G/968C), using polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP) or sequencing. Of the 187 subjects screened, 36 were heterozygous for Hb S [β6(A3)Glu→Val, GAGGTG] (allele frequency 9.62%). Sixty-four subjects were heterozygous and seven were homozygous for the -α(3.7) kb deletion (allele frequency 20.85%). β-Thalassemia alleles were detected in five subjects, four with the -29 (AG) mutation (allele frequency 1.07%) and one with codon 15 (TGGTAG) (allele frequency 0.96%). The G6PD A- and G6PD Betica deficient variants were highly prevalent with a frequency of 5.7 and 3.3%, respectively. While we did not test for ferritin levels or α(0)-thal, four females (5.2%) had red cell indices strongly suggestive of iron deficient anemia: Hb9.7 g/dL; MCH19.3 pg; MCV68.2; MCHC31.6 g/dl; RDW19.8%. Our results are consistent with high frequency of alleles such as Hb S, α-thal and G6PD deficient alleles associated with malaria resistance. Finding a 9.6% Hb S allele frequency supports the notion for a proficient neonatal screening to identify the sickle cell patients, who might benefit from early prophylactic treatment for infections. The incidence of significant iron deficient anemia in women is lower than expected in an under developed country.

Hemoglobinopathies are priority genetic diseases for prevention programs in at-risk populations. We implemented an accurate and simple methodology to identify hemoglobin (Hb) variants and to quantify HbA2 and HbF in capillary blood samples stored at room temperature for up to 7 days after collection. This methodology is particularly indicated for screening for carriers in primary care medical centers in which facilities for collecting venous blood are not available.

Four different gene mutations were identified in five unrelated Portuguese patients with pyrimidine 5'-nucleotidase type I (P5'N-I) deficient chronic hemolytic anemia. Mutations 502G--C (168Gly--Arg), 773T--C (258Ile--Thr) and the insertion of an Alu element in exon 9, leading to skipping of this exon in the mRNA transcript, are newly described mutations whereas mutation 425T--C (142Leu--Pro) has been previously reported.

The prevalence of β-thalassemia carriers among the Portuguese was around 2–3%, but nowadays, due to the immigrant populations coming from Africa, Brazil, East Europe and Asia, with different forms of thalassemias and Hb variants, hemoglobinopathies are much more common and with different genetic characteristics. Concerned with the risk of having an increasing number of patients with the severe forms of hemoglobinopathies, we decided to screen carriers, after personal informed consent, among pregnant women until 18-weeks of gestation and young adults attending primary care services. The goal of the project is to screen approximately 50000 blood samples in order to: know the hemoglobinopathies frequency and heterogeneity in the population living in Central Portugal (~2500000 inhabitants), and, based on these data, to develop a future cost/effective strategy for carrier identification; identify carriers to provide genetic information and counseling. In primary care medical centers there are no facilities to collect venous blood samples and we needed to establish a methodology to identify Hb variants and to correctly quantify HbA2 and Hb F in capillary blood samples, which has to be send by ordinary mail to our Lab Center. Working with the “HbA1c Capillary Collection System” from BioRad, we settled an accurate procedure to perform HPLC analysis on capillary blood stored at room temperature until 7 days after collection. To validate the technique we tested, in diverse conditions, more than 200 random and known controls samples: for the same individuals, HPLC results in capillary blood, collected with this system, and in EDTA peripheral blood samples, were identical. This methodology will detect β- and δβ-thalassemias and Hb variants; molecular characterization can be done through blood spots in filter paper (Guthrie spots) collected at the same time. Within 24–48 hours after sample reception in the Lab, results are reported to the respective physician, who is going to establish the correlation with the hematological parameters. Carriers identified through the screening will be urged to have their partner tested. If they are both carriers, they will be sent to our out patient clinic in order to evaluate the risk of having a child with a clinically significant hemoglobinopathy. If indicated, underling mutation will be identified, genetic counseling provided and prenatal diagnosis offered. The project, supported by the national program Saúde XXI/FEDER/FSE, started last April, was preceded by training sessions for doctors and nurses working in primary care centers and maternity hospitals. Brochures and posters are distributed for public information. As reported in some European surveys, we are willing to be effective in preventing the birth of affected infants with sickle cell disease and severe forms of thalassemia in Portugal.