M. Letícia Ribeiro

βs haplotypes in various world populations

SummaryWe have determined the βs haplotypes in 709 patients with sickle cell anemia, 30 with SC disease, 91 with S-β-thalassemia, and in 322 Hb S heterozygotes from different countries. The methodology concerned the detection of mutations in the promoter sequences of the Gγ- and Aγ-globin genes through dot blot analysis of amplified DNA with 32P-labeled probes, and an analysis of isolated Hb F by reversed phase high performance liquid chromatography to detect the presence of the AγT chain [Aγ75 (E19) Ile→Thr] that is characteristic for haplotype 17 (Cameroon). The results support previously published data obtained with conventional methodology that indicates that the βs gene arose separately in different locations. The present methodology has the advantage of being relatively inexpensive and fast, allowing the collection of a vast body of data in a short period of time. It also offers the opportunity of identifying unusual βs haplotypes that may be associated with a milder expression of the disease. The numerous blood samples obtained from many SS patients living in different countries made it possible to compare their hematological data. Such information is included (as average values) for 395 SS patients with haplotype 19/19, for 2 with haplotype 17/17, for 50 with haplotype 20/20, for 2 with haplotype 3/3, and for 37 with haplotype 31/31. Some information on haplotype characteristics of normal βA chromosomes is also presented.

A new PKLR gene mutation in the R-type promoter region affects the gene transcription causing pyruvate kinase deficiency.

Mutations in the PKLR gene responsible for pyruvate kinase (PK)‐deficient anaemia are mainly located in the coding regions: 11 are in the splicing sites and, recently, three mutations have been described in the promoter region. We now report a novel point mutation A→G on nucleotide 72, upstream from the initiation codon of the PKLR gene, in four Portuguese PK‐deficient patients. This new regulatory mutation occurs within the most proximal of the four GATA motifs (GATA‐A element) in the R‐type promoter region. In two patients who were homozygous for this mutation, a semiquantitative reverse transcription polymerase chain reaction (PCR) procedure was used to evaluate the amount of R‐PK mRNA transcript in the reticulocytes. The mRNA level was about five times lower than in normal controls, demonstrating that the PKLR gene transcription is severely affected, most probably because the −72A→G point mutation disables the binding of the erythroid transcription factor GATA‐1 to the GATA‐A element. Supporting these data, the two patients homozygous for the −72A→G mutation had severe haemolytic anaemia and were transfusion dependent until splenectomy. Two other patients who were compound heterozygous for this mutation and the previously described missense mutation 1456C→T had a mild condition.

Genetic basis of Congenital Erythrocytosis mutation update and online databases

Congenital erythrocytosis (CE), or congenital polycythemia, represents a rare and heterogeneous clinical entity. It is caused by deregulated red blood cell production where erythrocyte overproduction results in elevated hemoglobin and hematocrit levels. Primary congenital familial erythrocytosis is associated with low erythropoietin (Epo) levels and results from mutations in the Epo receptor gene (EPOR). Secondary CE arises from conditions causing tissue hypoxia and results in increased Epo production. These include hemoglobin variants with increased affinity for oxygen (HBB, HBA mutations), decreased production of 2,3‐bisphosphoglycerate due to BPGM mutations, or mutations in the genes involved in the hypoxia sensing pathway (VHL, EPAS1, and EGLN1). Depending on the affected gene, CE can be inherited either in an autosomal dominant or recessive mode, with sporadic cases arising de novo. Despite recent important discoveries in the molecular pathogenesis of CE, the molecular causes remain to be identified in about 70% of the patients. With the objective of collecting all the published and unpublished cases of CE the COST action MPN&MPNr‐Euronet developed a comprehensive Internet‐based database focusing on the registration of clinical history, hematological, biochemical, and molecular data (http://www.erythrocytosis.org/). In addition, unreported mutations are also curated in the corresponding Leiden Open Variation Database.

PK-LR gene mutations in pyruvate kinase deficient Portuguese patients.

In nine unrelated Portuguese patients with pyruvate kinase (PK) deficient anaemia, whose symptoms ranged from a mild chronic haemolytic anaemia to a severe anaemia presenting at birth and requiring multiple transfusions, the PK‐LR gene mutations were identified and correlated with their phenotypes. Five different mutations were identified, three of them for the first time: a missense mutation 1670G → C on exon 12 and two 5′ splice donor site (GT) mutations on intron 8 [IVS8(+2)T → G] and intron 10 [IVS10(+1)G → C]. Two previously described missense mutations, 1456C → T and 993C → A, were also found. The genotype/phenotype correlation showed that patients with two missense mutations or with a missense mutation and a splicing mutation had a mild haemolytic anaemia. The three patients with severe anaemia, who were transfusion dependent until splenectomy, were homozygous for the splicing site mutations IVS10(+1)G → C or IVS8(+2)T → G.

The cytoskeletal binding domain of band 3 is required for multiprotein complex formation and retention during erythropoiesis

Band 3 is the most abundant protein in the erythrocyte membrane and forms the core of a major multiprotein complex. The absence of band 3 in human erythrocytes has only been reported once, in the homozygous band 3 Coimbra patient. We used in vitro culture of erythroblasts derived from this patient, and separately short hairpin RNA-mediated depletion of band 3, to investigate the development of a band 3-deficient erythrocyte membrane and to specifically assess the stability and retention of band 3 dependent proteins in the absence of this core protein during terminal erythroid differentiation. Further, using lentiviral transduction of N-terminally green fluorescent protein-tagged band 3, we demonstrated the ability to restore expression of band 3 to normal levels and to rescue secondary deficiencies of key proteins including glycophorin A, protein 4.2, CD47 and Rh proteins arising from the absence of band 3 in this patient. By transducing band 3-deficient erythroblasts from this patient with band 3 mutants with absent or impaired ability to associate with the cytoskeleton we also demonstrated the importance of cytoskeletal connectivity for retention both of band 3 and of its associated dependent proteins within the reticulocyte membrane during the process of erythroblast enucleation.

Polymorphic variations influencing fetal hemoglobin levels: association study in beta-thalassemia carriers and in normal individuals of Portuguese origin.

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<1.7%; n=45) model, showed nominal significant associations for BCL11A SNPs rs11886868 (OR=4; P=0.001), rs766432 (OR=3.7; P=0.002) and rs7606173 (OR=0.36; P=0.032). KLF1 rs3817621 was not found associated with HbF levels. Our results suggest that in Portuguese β-thal carriers the HBG2 XmnI polymorphism is strongly associated with HbF levels. In normal individuals, BCL11A polymorphisms, but not HMIP or HBG2 (XmnI) loci, are nominally associated with HbF expression.

Chronic hemolytic anemia is associated with a new glucose-6-phosphate dehydrogenase in-frame deletion in an older woman

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 patients 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.

SLC40A1 Q248H allele frequencies and associated SLC40A1 haplotypes in three West African population samples

Background: 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. Aim: To increase information about Q248H mutation frequency in native sub-Saharan populations examining three West African populations. Subjects and methods: 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. Results: 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. Conclusions: 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.

Molecular diagnosis of haemophilia A at Centro Hospitalar de Coimbra in Portugal: study of 103 families – 15 new mutations

Summary.  Haemophilia A (HA), the most commonly inherited bleeding disorder, has well known phenotype heterogeneity, influenced by the type of mutation, modulating factors and development of inhibitors. Nowadays, new technologies in association with bioinformatics tools allow a better genotype/phenotype correlation. With the main objective of identifying familial carrier women and to offer prenatal diagnosis, 141 HA patients belonging to 103 families, followed or referred to the Haemophilia Centre of CHC, E.P.E., were studied. Molecular diagnosis strategy was based on HA severity: IVS22 and IVS1 inversions, direct sequencing and MLPA technique. New missense and splicing mutations were further analyzed using molecular modelling. Genotype/phenotype correlation was assessed taking into account the known modulating factors. During this study, mutations were detected in 102/103 families, carrier status was determined in 83 women and 14 prenatal diagnoses were performed. In a total of 46 different mutations identified, 15 have not been reported previously by the HAMSTeRS and HGMD. Genotype/phenotype correlation revealed two cases with a clinical picture less severe than expected by the type of mutation identified. Six patients developed inhibitors: five severe (IVS22, IVS1, large deletion) and one mild (p. Gln2265Lys). The adopted strategy allowed the identification of 99% of the molecular alterations underlying the HA phenotype (98% detection rate for severe and 100% for moderate and mild). Evaluation of genotype–phenotype correlation was complemented with structural protein modelling of newly identified missense mutations, contributing to better understanding of the disease‐causing mechanisms and to deepening knowledge on protein structure‐function.

Mutations and haplotype diversity in 70 Portuguese G6PD-deficient individuals: an overview on the origin and evolution of mutated alleles

G6PD deficiency mutational profile and haplotype diversity using 6 RFLPs (FokI/PvuII/BspHI/PstI/BclI/NlaIII) and a (CTT)n microsatellite, were investigated in 70 G6PD-deficient Portuguese individuals. All but one G6PD A-376G/202A variants (44/45) have a single haplotype (+/+/–/+/–/+/195). G6PD Betica376G/968C alleles (n=10) have a single RFLP haplotype (+/–/–/+/–/+) and 4 different (CTT)n repeats. Age estimates based on microsatellite variation suggest that Betica mutation arose 900 generations ago. G6PD SantaMaria376G/542T allele was found on haplotype (+/–/–/+/–/+/201) and 10 G6PD variants on RFLP haplotypes (–/–/+/+/–/–), (–/–/+/+/–/+) and (–/–/+/+/+/+).