Janet Pereira

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.

RhD variant caused by an in-frame triplet duplication in the RHD gene.

BACKGROUND: The RHD gene is highly polymorphic and a large number of D variants have already been detected. Several mechanisms are involved in the origin of D variants. In‐frame deletions, resulting in a single‐amino‐acid deletion, have been described associated with RhD and RhCE variants. No in‐frame duplications and/or insertions have been reported in the RH genes to date.

Hereditary nonspherocytic hemolytic anemia caused by red cell glucose-6-phosphate isomerase (GPI) deficiency in two Portuguese patients: Clinical features and molecular study

Glucose-6-phosphate isomerase (GPI) deficiency cause hereditary nonspherocytic hemolytic anemia (HNSHA) of variable severity in individuals homozygous or compound heterozygous for mutations in GPI gene. This work presents clinical features and genotypic results of two patients of Portuguese origin with GPI deficiency. The patients suffer from a mild hemolytic anemia (Hb levels ranging from 10 to 12.7g/mL) associated with macrocytosis, reticulocytosis, hyperbilirubinemia, hyperferritinemia and slight splenomegaly. Genomic DNA sequencing revealed in one patient homozygosity for a new missense mutation in exon 3, c.260G>C (p.Gly87Ala), and in the second patient compound heterozygosity for the same missense mutation (p.Gly87Ala), along with a frameshift mutation resulting from a single nucleotide deletion in exon 14, c.1238delA (p.Gln413Arg fs*24). Mutation p.Gln413Arg fs*24 is the first frameshift null mutation to be described in GPI deficiency. Molecular modeling suggests that the structural change induced by the p.Gly87Ala pathogenic variant has direct impact in the structural arrangement of the region close to the active site of the enzyme.

Gene symbol: NT5C3. Disease: haemolytic anemia.

Department of Neurological and Behavioural Sciences, University of Siena, Policlinico Le Scotte, Viale Bracci 2, 53100 Siena, Italy, email: bianchi22@unisi.it, Tel.: +39-057-7585760, Fax: +39-057-740327 Gross insertions Accession Number Description Hgi0501 In two unrelated Portuguese patients with pyrimidine 5¢-nucleotidase (P5¢N)deficiency we have found the insertion of an Alu element within the exon9 of the structural gene for type-I isoform of P5’N, between nucleotides 743-744. The new Alu element share 98.2 identity with AluYa5 and AluYa1 subfamilies. RT-PCR analysis showed that the Alu insertion leads to the skipping of the exon9 in the mRNA transcript

Congenital dyserythropoietic anemia associated to a GATA1 mutation aggravated by pyruvate kinase deficiency.

Dear Editor, A 12-year-old Spanish male was referred to our department to elucidate the etiology of a congenital macrocytic anemia (MCV 104 fL; Hb 9.0 g/dL) with dyserythropoiesis, needing blood transfusions during the first 4 months of life, and mild thrombocytopenia (73 × 10/L) with large platelets. At the age of 3 months, he presented: Hb 5.9 g/dL, reticulocytes 105 × 10/L, peripheral blood smear with anisocytosis, poikilocytosis, and basophilic stippling. He has a low PK activity (45 % of normal). The bone marrow was suggestive of congenital dyserythropoietic anemia (CDA), with erythroid hyperplasia and dyserythropoiesis and orthochromatic erythroblasts showing an irregular nuclear contour and bior multinucleated erythroblasts; electronic microscopy identified vacuoles in the cytoplasm of erythroblasts. CDAs are rare forms of bone marrow failure syndromes characterized by ineffective erythropoiesis [1, 2]. There are three classical forms of CDA presentation due to mutations in CDANI, C15orf41, SEC23B, and KIF23 [3–5] and rarer forms associated with pathological variants in KLF1 and GATA1. GATA1 variants are associated with deregulation of erythroid and megakaryocytic lineages differentiation [6–8]. We studied the patient and available family members (Fig. 1), results are summarized in Table 1. Sequence analysis of CDANI, SEC23B, C15ORF41, KLF1, HBB, HBA was normal, in PKLR, we found a new pathological variant c.1284delA (Glu429Argfs*19) in the heterozygous state. His mother and sister were also heterozygous for PKLR c.1284delA. The heterogeneity of family’s data affecting both erythroid and megakaryocytic lines prompted us to perform sequencing analysis of GATA1. An A to G transition (c.866 A > G) was identified in the propositus in hemizygous state. Interestingly, PK activity was also decreased in the GATA1 hemizygous relative studied (Table 1—III-2). This pedigree illustrates the difference of the hematological manifestations among the propositus, carrier of mutations at the GATA1 and PKLR, and the other family males, presenting only the GATA1 mutation. This novel GATA1 mutation results in the substitution of histidine for arginine (p.His289Arg) in the C-terminal zinc finger. This residue is conserved across GATA1 orthologs of various species, and bioinformatics analysis classifies the variant as pathological. The mutation was not detected in 100 normal chromosomes and was published by us in the GenBank (Accession Number KC733811). This is the first report of a CDA associated with a mutation in the GATA1 in a region coding for the Cterminal zinc finger domain of GATA1 protein, necessary for DNA binding [9]. GATA1 is an important transcription factor for hematopoiesis regulation, in particular for the erythroid and megakaryocyte lineages. Mutations affecting the C-terminal zinc finger domain are probably not as damaging as those affecting the N-terminal, which would account for the milder phenotypes of hemizygous males, with mild macrocytic anemia. To explain the more severe phenotype of the propositus comparatively to his * Celeste Bento celeste.bento@chuc.min-saude.pt