Gabriel Tamagnini

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.

Dominant β-thalassaemia trait in a Portuguese family is caused by a deletion of (G) TGGCTGGTGT (G) and an insertion of (G) GCAG (G) in codons 134, 135, 136 and 137 of the β-globin gene

Summary We have studied a Portuguese family with a dominant β‐thalassaemia trait that was present in one member of each of three generations. It was characterized by a moderate anaemia, microcytosis and hypochromia, anisopoikilocytosis, Heinz body formation in peripheral red cells, splenomegaly, and a blood transfusion requirement during pregnancy. Sequence analyses of amplified DNA detected a deletion of (G) TGGCTGGTGT(G) at codons 134‐137 (ValAlaGlyVal) and the insertion of (G)GCÀ(G) (GlyArg) at the same location. Thus, the resulting β chain has an abnormal structure only at codons 134‐137 and is two residues shorter than the normal 146 residues. This chain could not be detected in circulating red cells and must be degraded rapidly by proteolysis because the Heinz bodies consisted mainly of a chains.

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.

β-Thalassehia Mutations in the Portuguese; High Frequencies of Two Alleles in Restricted Populations

We report the characterization of seven different beta-thalassemia mutations in 131 newly diagnosed Portuguese beta-thalassemia heterozygotes. Methodology included the detection of abnormal fragments by agar gel electrophoresis of PCR-amplified DNA fragments after digestion with specific restriction endonucleases, as well as hybridization with synthetic nucleotide probes and sequencing of amplified DNA. Four mutations, including the newly discovered TGG-->TGA change at codon 15, occurred in excess of 10% and accounted for some 90% of the beta-thalassemia alleles in this population. The geographical distribution is uneven; the TGG-->TGA mutation at codon 15 was primarily observed in the coastal region north of Lisbon, while the IVS-I-6 (T-->C) mutation was confined to the central part of the country.

HB Coimbra or α2β299(Gl)ASP→GLU, A Newly Discovered High Oxygen Affinity Variant

We have identified a new high oxygen affinity hemoglobin variant in members of a Portuguese family; it is characterized by an Asp→Glu replacement at codon 99 of the β chain which is in the α1β2 interface. The altered functional properties of Hb Coimbra likely result from the inability to form a hydrogen bond between β99Glu and α42Tyr; such a bond is formed in deoxy Hb A between the normally occurring β99Asp and α42Tyr. The two affected members of the family have a distinct erythrocytosis with hemoglobin levels of 18 to 20 g/dl. The mutation in the β-globin gene (GAT→GAA at codon 99) resulting in the Asp→Glu replacement is the seventh type at this specific location. A review of the many variants of the α and β chains identifies primarily aspartic acid and glutatnic acid residues as being most frequently replaced; it is speculated that codons GAC and GAT (for Asp), and GAGand GAA (for Glu) are most susceptible to mutational events.

Hb Vila Real [β36(C2)Pro→His]: A Newly Discovered High Oxygen Affinity Variant

A 15-year-old girl was referred to the hematology out patient clinic for diagnosis of erythrocytosis. She was asymptomatic and submitted to extensive investigations to exclude pulmonary, renal or cardiac disease. Her mother has undergone regular phlebotomies over the past 20 years for polycythemia, with an obstetric history of two miscarriages, a stillborn baby, and two normal children born by elective Caesarean section. The proband and her mother presented a high hemoglobin (Hb) level with normal erythrocyte indices and normal leukocyte and platelet counts. Hb isoelectrofocusing (IEF) did not show any abnormal band. Quantification of Hb A, and Hb F (1 ) was normal (Table I). On cation exchange high performance liquid chromatography (HPLC) a Hb variant eluted with normal Hb A,. Reversed phase HPLC showed a p chain variant with 36% of total p chains. DNA was isolated from peripheral blood leukocytes (2) and amplified by polymerase chain reaction (PCR) using previously described conditions (3). Localization ofthe mutation was done by single strand conformation analysis (SSCA) (4) of DNA fragments encompass-

IN VITRO PROTEOLYSIS OF RED CELL MEMBRANE IN HEREDITARY SPHEROCYTOSIS

zole, or oral neomycin/colistin/nystatin. In these patients the incidence of pseudomembranous colitis has been less than 1%. Because of its cost effectiveness and increased patient acceptability, we recently changed this regimen to oral ciprofloxacin and fluconazole. So far, we have used this in only 20 patients but already we have encountered two with confirmed Cl. diflcile associated colitis. As the use of ciprofloxacin may become more widespread, our own experience should alert others to the possibility of this potentially serious complication. REFERENCES