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Genetics and Molecular Biology

Print version ISSN 1415-4757On-line version ISSN 1678-4685

Genet. Mol. Biol. vol. 21 n. 4 São Paulo Dec. 1998 

Mild clinical expression of S-b thalassemia in a Brazilian patient with the b+ IVS-I-6 (T®C) mutation


Maria de Fátima Sonati1, Jaspal Kaeda2, Elza Miyuki Kimura1, Fernando Ferreira Costa3 and Lucio Luzzatto2
1Departamento de Patologia Clínica, Faculdade de Ciências Médicas, Universidade Estadual de Campinas, UNICAMP, Caixa Postal 6111, 13083-970 Campinas, SP, Brasil. Send correspondence to M.F.S. Fax: +55-19-239-3114.
2Department of Haematology, Royal Post-Graduate Medical School, Hammersmith Hospital, London, England.
3Departamento de Clínica Médica, Faculdade de Ciências Médicas, Universidade Estadual de Campinas, UNICAMP, Campinas, SP, Brasil.




We report on an eight-year-old Brazilian girl with S-b+ thalassemia. The patient had a steady 10.1 g/dl hemoglobin with 57% HbS. Direct sequence analysis of b-globin gene showed her to be heterozygous for the IVS-I-6 (T®C) mutation. This b+ thalassemia mutation, sometimes referred to as the Portuguese type, was found to be associated with the C®T polymorphism at codon 2. In combination with the bS gene, this mutation results in very mild sickle cell disease symptoms.




HbS-b thalassemia, a sickle cell syndrome, primarily affects people of African and Mediterranean origin. This disorder is very heterogeneous both clinically and genetically. While the clinical course of S-b0 thalassemia can be similar to that of SS disease, patients with S-b+ thalassemia have milder clinical manifestations, and in some patients sickle cell crises are rare (Bunn and Forget, 1986; Stamatoyannopoulos et al., 1994). In Brazil, most patients with S-b thalassemia have a severe clinical course (Zago et al., 1980). Here we report the molecular basis of b-thalassemia in a Brazilian patient with a very mild form of S-b+ thalassemia.



An eight-year-old Black girl, clinically asymptomatic, was referred to the Hematology Clinic because there was a case of sickle cell anemia in her family. Her medical history revealed that she had been found to be anemic at the ages of six months and at two years. She was treated with oral iron therapy. She had never required blood transfusion. Clinical examination revealed normal development, without splenomegaly. The hematological data showed mild microcytic anemia (see Table I), with target cells and microcytic and hypochromic red cells. Hemoglobin analysis (Pembrey et al., 1972; Weatherall and Clegg, 1981) revealed 3.2% HbA2, 57.0% HbS, 1.2% HbF and 38.6% HbA. The reticulocyte count was 4.0% and serum ferritin was normal. Both parents regarded themselves as Black and were clinically normal. No white ancestry was known. The mother was found to be heterozygous for b+-thalassemia, with microcytic and hypochromic red cells and slightly raised HbA2 (3.8%). The father was an HbS heterozygote (see Table I).

21n4at1.GIF (11271 bytes)


DNA analysis

DNA was extracted from peripheral blood leucocytes with phenol-chloroform and amplified by PCR (Saiki et al., 1985), from -158 to IVS-II-115 positions of the b-globin gene, with primers described elsewhere (Tamagnini et al., 1993). Single strand DNA was obtained by using dynabeads-streptavidin (Dynal Inc., Oslo, Norway), according to the manufacturer's instructions, and direct sequencing carried out by the method of Sanger et al. (1977) with the Sequenase kit-version 2.0 (United States Biochemical, Cleveland, OH). Sequence analysis of the b globin gene revealed the b+ IVS-I-6 (T®C) mutation in the patient and in her mother. This was confirmed by digestion with the restriction enzyme Sfa NI, followed by electrophoresis in 3% agarose gel (Atweh and Forget, 1986). As this enzyme also recognizes the C®T polymorphism at codon 2 (CD 2) (which is found in the framework three of the b gene) (Orkin et al., 1982; Atweh and Forget, 1986), its presence was identified in the thalassemic alleles and also in the normal allele of the mother (see Figures 1, 2 and 3). As a-thalassemia can be one of the ameliorating factors of the clinical course of b-thalassemia, its presence was excluded by PCR, as previously described (Baysal and Huisman, 1994).

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Figure 1 - Sfa NI restriction map of the human b-globin gene.


21n4a2f2.GIF (9817 bytes)

Figure 2 - Sfa NI restriction analysis of the patient and her parents. M = DNA size marker; lane 1 = PCR product (undigested); lane 2 = patient's father; lane 3 = patient's mother; lane 4 = patient; lane 5 = normal control.


21n4a2f3.GIF (13228 bytes)

Figure 3 - DNA sequencing of the patient's mother, showing heterozygosis for the b+ IVS-I-6 (T®C) mutation and homozygosis for the C®T polymorphism at codon 2.



The b-thalassemia mutation detected in this family is commonly found in Portuguese and Mediterranean populations, and is frequently referred to as "Portuguese" b-thalassemia (Tamagnini et al., 1983, 1993). This mutation reduces the efficiency of normal splicing, because the base substitution is close to the 5'splice junction, between exon I and intron I and within a conserved consensus sequence. However, a significant fraction of the primary transcript is still processed normally, and therefore the expression is mild (Orkin et al., 1982; Treisman et al., 1983). In a previous study we demonstrated that the b+ IVS-I-6 (T®C) mutation is the 3rd most prevalent b-thalassemia mutation in Southeast Brazil (Martins et al., 1993). It was detected mainly in individuals of Italian descent. In people of African descent, very mild b+-thalassemia has mostly been associated with mutations that either flank the 5' region of the b gene, affecting the rate of transcription, or that are located within the polyadenylation site, altering mRNA processing or stability (Orkin et al., 1984, 1985; Antonarakis et al., 1984). In our patient, instead, very mild S-b+ thalassemia results from the association of the S gene with a splicing mutation. It is not surprising that a genotype combining an "African" gene with a "Portuguese" gene is found in Brazil. Given the history of the Brazilian population, it is also perhaps not surprising that a White mutation is found in a Black person. However, this particular genotypic combination has not been previously reported in Brazil, as far as we know. This is of clinical interest since the resulting clinical phenotype is fortunately quite mild.



Research supported by FAPESP and CNPq. Publication supported by FAPESP.




Nós caracterizamos a base molecular da talassemia b em uma paciente negra brasileira, de 8 anos de idade, com um quadro clínico extremamente benigno de S-b+ talassemia. A paciente tinha uma hemoglobina total de 10,1 g/dl, com 57% de HbS. O sequenciamento direto do DNA, amplificado por PCR, revelou que a paciente é heterozigota da mutação b+ IVS-I-6 (T®C). Esta mutação, algumas vezes referida como o tipo português de talassemia, foi encontrada em associação com o polimorfismo C®T no codon 2 do gene b e, de nosso conhecimento, não havia sido previamente descrita na população negra brasileira.




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(Received April 15, 1997)

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