Isabel Picanço

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 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.

The β‐Thalassemia Mutation/Haplotype Distribution in the Moroccan Population

The present study compiles the results of our own research and of a prior study on β‐thalassemia (thal) in Morocco, comprising a total of 187 β‐thalassemic chromosomes. Six major mutations: (β0) codon 39 (C→T), (β+) IVS‐I‐6 (T→C), (β0) frameshift codon (FSC) 6 (− A), (β0) FSC 8 (− AA), (β0) IVS‐I‐1 (G→A) and (β+) − 29 (A→G) account for 75.7% of the independent chromosomes studied. A regional predominance was observed (Gharb and West regions) for the (β+) IVS‐I‐6 (T→C) mutation. Despite an observed heterogeneity of molecular anomalies, a direct method of diagnosis of the prevalent mutations is feasible in this population. The distributions of mutations and haplotypes are in conformity with the geographical location of Morocco and the historical links with both the Mediterranean communities that have successively interspersed with the Berbers, the Phoenicians, the Carthaginians, the Romans, the Arabs, the population of the Iberian Peninsula and, to a lesser degree, the Vandals and the Byzantines and permanently, with the Sub‐Saharan Africans. In the adult population, the levels of fetal hemoglobin (Hb) in heterozygotes vary from trace quantities to 2.38 g/dL of total Hb. With the exception of the (β0) codon 39 (C→T) nonsense mutation, no statistically significant correlation was found, neither between mutation and Hb F levels, nor gender and Hb F levels in heterozygotes. The genetic markers for Hb F increase, located within cis active sites such as the XmnI site at − 158 bp of the Gγ‐globin gene and the ATXTY repeat region at − 540 bp of the β‐globin gene, were assessed. The polymorphism XmnI shows linkage disequilibrium with haplotypes III, IV and IX, as previously observed in the Algerian, Sicilian and Portuguese β‐thal populations. Contrary to what has previously been reported for a population of β‐thal carriers of European descent, this sample does not show a statistically significant correlation between Hb F levels and the presence of the genetic markers XmnI restriction site at − 158 bp of the Gγ‐globin gene and ATXTY alleles at 5′ of the β‐globin gene.

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.

Molecular Basis of A-Thalassa in Portugal

We have estimated the incidence and molecular basis of α-thal-assemia in a Portuguese population, mostly from the Greater Lisbon area. In a group of 100 consecutive cord blood samples, the gene frequency of the rightward deletion (-α3.7) was 0.035, and the leftward deletion (-α4.2) was 0.015. In this group, we have also found four heterozygotes for the triple α-globin gene rearrangement (αααanti3.7 gene frequency 0.020). We have characterized the subtypes of -α3.7 and αααanti3.7 rearrangements. On the whole, these results give an incidence of 10% for deletional a-thalassemia carriers in the studied Portuguese population In a group of 342 subjects presenting β-thalassemia, or Hb S trait, β-thalassemia major, sickle cell disease or low red blood cell indices, the -α3.7, -α4.2, –SEA –MED (αα)MM and αααanti3.7 haplotypes were found in different combinations. Only one nondeletional α-thalassemia determinant (a 5 nucleotide deletion in the α2-globin gene in the second intervening sequence donor site) was detect...

Human α2-globin nonsense-mediated mRNA decay induced by a novel α-thalassaemia frameshift mutation at codon 22

We describe a novel α‐thalassaemia determinant in a 3‐year‐old girl presenting a mild microcytic and hypochromic anaemia, and normal haemoglobin A2 level. Molecular studies revealed heterozygosity for a novel microdeletion (−C) at codon 22 of the α2‐globin gene. As the frameshift mutation generates a premature translation termination codon at position 48/49, we investigated the effect of the nonsense codon on the α2‐globin gene expression. Although it does not affect RNA splicing, the premature nonsense codon induces accelerated mRNA degradation. To our knowledge, this is the first time the nonsense‐mediated mRNA decay has been reported to occur in human α‐globin mRNA.

Combined effect of two different polymorphic sequences within the β globin gene cluster on the level of HbF

β thalassemia and Hb Lepore heterozygotes included in this study exhibit fetal hemoglobin levels varying from trace quantities to 14% (1.74 g/dl) of total hemoglobin in the adult. In this work, we have examined the correlation of DNA sequence polymorphisms with the observed HbF level. The analysis of polymorphic markers within the β globin cluster in 39 individuals heterozygous for β thalassemia or Hb Lepore confirms the previous findings for homozygous β thalassemia: the presence of both an (AT)9 T5 sequence configuration at position −540 of the β globin gene and a (C → T) variation at −158 of the Gγ globin gene is associated with elevated expression of HbF. However, at least one defective β globin gene is required to reveal this association. The best evidence is from the study of individuals heterozygous for Hb Lepore with various levels of HbF. In these individuals it was possible to explore the effect of a single (AT)x Ty motif (the other being absent from the rearranged Lepore chromosome) on HbF expression. The presence of the (AT)9 T5 configuration increases HbF level from a median of 0.515 g/dl observed in (AT)7 T7 subjects, to 1.39 g/dl.

Genetic variation in CD36, HBA, NOS3 and VCAM1 is associated with chronic haemolysis level in sickle cell anaemia: a longitudinal study†

Chronic haemolysis stands out as one of the hallmarks of sickle cell anaemia, a clinically heterogeneous autosomal recessive monogenic anaemia. However, the genetic architecture of this sub‐phenotype is still poorly understood. Here, we report the results of an association study between haemolysis biomarkers (serum LDH, total bilirubin and reticulocyte count) and the inheritance of 41 genetic variants of ten candidate genes in a series of 99 paediatric SS patients (median current age of 9.9 yr) followed up in two general hospitals in Greater Lisboa area (median follow‐up per patient of 5.0 yr). Although in a large number of tests a seemingly significant (i.e. P < 0.05) association was observed, the following ones were confirmed upon correction for multiple comparisons: (i) an increased serum LDH level was associated with haplotype 7 within VCAM1 gene; (ii) a lower total bilirubin was associated with the 3.7‐kb deletion at HBA gene, rs2070744_T allele at NOS3 gene, and haplotype 9 within VCAM1 promoter; and (iii) a diminished reticulocyte count was associated with the 3.7‐kb deletion at HBA, whereas an increased count was associated with rs1984112_G allele at CD36 gene. On the whole, our findings suggest a complex genetic architecture for the sickle cell anaemia haemolysis process involving multiple pathways, namely control of vascular cell adhesion, NO synthesis and erythrocyte volume and haemoglobinisation.

Clinical and laboratory effects of hydroxyurea in children and adolescents with sickle cell anemia: a Portuguese hospital study.

Our aim was to assess the efficacy and safety of hydroxyurea (HU) in children with severe forms of sickle cell anemia followed in a Portuguese hospital. We carried out an open-label, uncontrolled prospective study, which included children with severe forms of sickle cell anemia. Hydroxyurea was started at 15 mg/kg/day and increased to a maximum dose of 25 mg/kg/day. Patients were monitored to assess compliance, clinical and hematological response and toxicity. Nine children and adolescents, five girls and four boys, with a median age of 13 years (range 8 to 16) were enrolled in the study during a period of 24 months. All patients completed at least 15 months of therapy. Hb F was significantly increased, from a mean of 7.0 ± 3.9% to 13.7 ± 5.3% (p = 0.028). Clinically, all patients responded significantly with a reduction of 80% in the number of vaso-occlusive crises (VOC), 69% in hospital admissions, 76% in hospitalization days and 67% in transfusion requirements, without significant toxicity. We concluded that, in our population, HU proved to be effective in increasing Hb F levels, and in decreasing hospitalizations for VOC and transfusion requirements with no major side effects. Long-term clinical follow-up is important to certify benefit maintenance.

Hb Yaoundé [β134(H12)Val→Ala] in Association with Hb C [β6(A3)Glu→Lys] in a Caucasian Portuguese Family

Hb Yaoundé [β134(H12)Val→Ala] is a rare, silent and asymptomatic hemoglobin (Hb) variant. It was previously reported in a Black man from Cameroon, in association with Hb Kenitra [β69(E13)Gly→Arg], and was subsequently found and described as Hb Mataro in a sub‐Saharan child. To date, Hb Yaoundé has not been described in Caucasian people and molecular studies have never been performed. Here we describe a three‐generation Caucasian Portuguese family in whom Hb Yaoundé was found in association with Hb C [β6(A3)Glu→Lys] (in the proband) and in a heterozygous state (in the probands mother). The Hb studies of the probands hemolysate, performed by isoelectric focusing (IEF) and low‐pressure cation exchange chromatography, only revealed an Hb variant identified as Hb C by comparison with a control. However, analysis performed by reversed‐phase high‐performance liquid chromatography (HPLC) showed two different β chain variants and a complete absence of the normal β chain. This uncommon Hb variant was identified as Hb Yaoundé by DNA sequencing analysis of the β‐globin gene (codon 134, GTG→GCG). The β‐globin gene haplotypes were determined in all family members using polymerase chain reaction (PCR)‐based methodology; Hb Yaoundé was found to be associated with the Mediterranean haplotype II. This study is the first description of Hb Yaoundé in Caucasian individuals, and its association with a Mediterranean haplotype supports the hypothesis of an independent genetic origin other than African.