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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.

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.

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.