A Deletion Of About 11kb Starting From 0.5 Kb 5’ To The β-Globin Gene Reactivates Foetal Haemoglobin and Leads To β Thalassemia Intermedia When Associated To a β-Zero Thalassemic Mutation

Thalassemia intermedia is characterized by severe but not transfusion dependent anemia secondary to seriously decreased production of hemoglobin (Hb). In the majority of cases, thalassemia intermedia concerns β-globin gene pathology. The molecular basis of thalassemia intermedia is heterogeneous. Here we describe a case of an adopted child native of Myanmar suffering from β-thalassemia intermedia which was proved to be secondary to a β-zero thalassemia associated with a not yet described deletional form of HPFH.

Male child born in 1994 with Hb varying between 50 and 60 g/l, with Hb A2 of 2.1% and Hb F of 97.9%. No α-thalassemia or α-gene triplication was found. Sequencing of β-globin gene put in evidence the IVS-I-1 (G>T) or c.92+1G>T mutation in a “homozygous” state. This mutation is known to produce a β-zero thalassemia.

The patient was treated with hydroxyurea as well as with erythropoietin and the Hb value was improved up to 86 g/l with normal leucocytes and platelets count. No transfusion was given during this period of treatment. Because the clinical phenotype was not typical for β-thalassemia major homozygous for the above mentioned mutation, we analyzed β-globin cluster looking for the presence of a possible deletion responsible for Hb F activation.

Patient’s DNA was extracted with commercial columns from peripheral blood cells. Analysis of deletion in the beta cluster was performed by MLPA (Multiplex Ligation Probe Analysis) MRC-Holland P-102 probe mix. The data obtained were analyzed with the Coffyanalyzer software. The exact size of the deletion was determined by PCR with the primers: DelHBB_F: 5’-AGGCTTGGCTCCTGTTTAGT-3’, DelHBB_R: 5’-TGAGAG CTGCTGAGTTGTGT-3’

A heterozygous deletion in the beta-globin cluster has been detected by MLPA. This deletion was located between the coordinated 5,237,089 and 5,251,133 on chromosome 11 - (GRCh37/hg19 Assembly). The deletion starts about 0.5 kb 5’ upstream the HBB gene, between HBB and HBD genes, and ends about 9 kb downstream the 3’ end of HBB gene. The density of the MLPA probes is not sufficient to determinate the exact size of the deletion (between 14.3kb and 9.6 kb). A PCR using the primers DelHBB_F and DelHBB_R determined the size of this deletion to around 11kb.

Our molecular biology studies confirmed our clinical suspicion of association of HPFH with β-zero thalassemia. In fact, we put in evidence a not yet described (to our knowledge) 11kb deletion, which is very similar to the 12.6kb deletion of the Dutch β-zero thalassemia (Br J Haematol 67:369;1987) and to the Asian Indian 10.3kb deletion described by Craig et al (Br J Haematol 82:735;1992). Our deletion starts between δ and β-globin gene, almost 0.5 kb upstream of the β-gene, and goes about 9 kb downstream of 3’ end of the β-gene. The exact borders of the deletion are currently under investigation by PCR and appropriate primers.

The pathophysiology of reactivation of γ-globin genes in our case is not yet known. We raise the following hypothesis: does this deletion bring an enhancer located 3’ to β-globin gene, close enough to the γ-genes, so that transcription of these genes continues after birth? In vitro studies in expression systems (constructs) are currently performed to elucidate the exact mechanism of γ-globin activation.

No relevant conflicts of interest to declare.