A novel mutation of −50 (G→A) in the direct repeat element of the β-globin gene identified in a patient with severe β-thalassemia

Abstract

Dear Editor, β-Thalassemia is a heterogeneous genetic disease associated with defective expression of the β-chain of human hemoglobin. So far, more than 200 mutations have been defined and affect almost every known stage of β-globin gene expression resulting in a reduction (β) or complete absence (β) of β-chain synthesis from the affected allele [1, 2]. In southern China, the carrier rate of β-thalassemia in Guangdong province was 2.54% [3], with five common mutations accounting for 90% of all β-thalassemia individuals. Identification of new mutations and update of the mutation spectrum of thalassemia in one specific ethnic population is always needed for improving genetic counseling and prenatal diagnosis. We here describe a novel promoter mutation of −50 (G→A) within the conserved direct repeat element (DRE) leading to β-thalassemia major when associated with a β-thalassemia mutation. A 3-year-old Chinese boy with his parents was seen at our genetics clinic. The boy suffered from β-thalassemia major and had been transfused every 2 months since the age of 6 months. Molecular analyses performed by reverse dot blots (RDB) at other clinics revealed that he was a heterozygote for the common mutation of codons (CDs) 41–42 (−TTCT) of the β-globin gene, but the other mutational allele was not determined. At this referral, the family was planning for another pregnancy and counseled about prenatal diagnosis. Blood samples were obtained from all three family members. Hematological investigations showed that both of the parents had a classical β-thalassemia trait and the son manifested with severe β-thalassemia (Table 1). Determination of the β-thalassemia mutations was performed using the polymerase chain reaction (PCR)–RDB method which can simultaneously detect 17 types of β-globin gene mutations found in Chinese population. This confirmed the heterozygous presence of the CDs 41–42 (−TTCT) mutation in the son and the father, but the mutation was not found in the mother. Gap PCR was also undertaken in the three family members to exclude the coinheritance of αthalassemia, and this did not reveal any of the three common Chinese α-globin gene deletion mutations (−α, −α, –). To further investigate the underlying mutation in this family, the β-globin gene was amplified by PCR from DNA of the mother, and the product was analyzed by direct nucleotide sequencing using the BigDyeTM Terminator Cycle Sequencing Kit and the ABI PRISMTM 310 genetic analyzer (Applied BioSystems). Direct sequencing of the β-globin gene showed the normal sequence except for the heterozygous G→A mutation at position −50 relative to the Cap site (Fig. 1). This so far undescribed mutation was also revealed in the son by DNA sequencing. Thus the patient was confirmed to be a compound heterozygote with a genotype of −50 (G→A)/CDs 41–42 (−TTCT). In order to evaluate the functional effect of this novel mutation, two other family members were investigated. A semiquantitative reverse transcription PCR assay was used to measure β globin mRNA in the reticulocytes from three heterozygous subjects (the mother, grandmother, and an aunt) and eight normal individuals. These analyses showed a mildly reduced β-globin mRNA level (23.4 %) in heterozygotes compared with that in normal subjects. Ann Hematol (2009) 88:1149–1150 DOI 10.1007/s00277-009-0732-8