Hemoglobin (Hb) is a protein responsible for oxygen transportation from lungs to the entire body. It is composed by four globular subunits - the globins - each with a central core containing a heme molecule. Globins are encoded by the α- and β-globin gene clusters located at 16p13.3 and 11p15.5, respectively. The pattern of globin genes expression during development is precisely controlled by the interaction of cis-regulatory genomic regions (located in close proximity to and far from genes) with trans-activating/silencing factors within permissive chromatin domains. In fact, approximately 25-65 kb upstream of the α-globin genes there are four multispecies conserved sequences (MCS-R1 to R4) which are critical for the expression regulation of the downstream globin genes. The main objectives of this work were to characterize the molecular lesions underlying eight unusual cases of α-thalassemia or Hb H disease, and to understand their origin and functional consequences.
Deletions were detected by Multiplex Ligation-dependent Probe Amplification (MLPA) using the SALSA MLPA P140B HBA kit (MCR-Holland). Additionally, specifically designed synthetic MLPA probes, as well as Gap-PCR and Sanger sequencing were performed for fine deletion breakpoint mapping.
We have found seven different deletions (ranging from 3.3 to ≈323 kb), four of them not previously described. The four largest deletions removed all the α-globin genes, whereas the other three deletions removed one or more of the distal regulatory elements keeping the globin genes structurally intact. In one case, only the MCS-R2 (also known as HS-40) was removed and replaced by a 39 nt DNA fragment possibly resulting from a complex rearrangement that introduces new pieces of DNA (probably from Chrs. 3 and 7) bridging the two deletion breakpoints. In the remaining case, no deletion was found and the patient revealed to be a very unusual case of acquired alpha-thalassemia-myelodysplastic syndrome.
It is important to detect individuals with this type of uncommon deletions as there is a 25% risk of having a child with Hb Bart’s hydrops fetalis or Hb H disease if their partner is a carrier of an α0-thal or α+-thal allele, respectively. Moreover, further investigation is currently being developed on one of these natural mutants which is bringing new insights into the long-range regulation mechanism of the globin gene expression and to the pathophysiology of the α-thalassemia.N/
