Browsing by Author "Batalha-Reis, Ana"
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- Novel and rare large deletions in the globin gene clusters causing different types of thalassemiaPublication . Coelho, Andreia; Fernandes, Emília; Batalha-Reis, Ana; Sonesson, Annika; Picanço, Isabel; Miranda, Armandina; Faustino, PaulaThe major component of the red blood cells is hemoglobin A which consists of 2α- and 2β-globin chains encoded by α- and β-globin genes located in two different gene clusters (16p13.3 and 11p.15.5, respectively). Molecular defects (usually point mutation or short deletion) that give rise to a quantitative reduction of the corresponding globin chain, result in a hereditary hypochromic and microcytic anemia called thalassemia. However, rarely, the molecular basis of the pathology could be a large deletion affecting several globin genes and/or their distal regulatory sequence. Four patients with hematological phenotypes suggestive of thalassemia, in whom no globinic molecular abnormalities had been found by standard diagnostic procedures, were screened for deletions in the telomeric region of chromosome 16 and 11, by Multiplex Ligation-dependent Probe Amplification (MLPA) assay. To further characterize the breakpoints of the deletions found, we employed synthetic MLPA probemixes designed in our laboratory, as well as PCR and DNA sequencing. We identified two cases of α-thalassemia caused by two distinct large deletions which remove all α-like structural genes and their distal regulatory sites: both are telomeric, one presents at least 271.14 kb of length and the other, at least, 231 kb. Concerning β-globin cluster screening, two deletions were found: one has at least 186 kb, encloses the entire cluster and its locus control region, and gives rise to a εγδβ0-thalassemia. The other presents at least 3 kb, has its 5’ breakpoint located within the second intron of the β-globin gene and its 3’ end within the L1 repetitive region of the cluster. Both α- and β-cluster larger deletions are novel and were named --CMB/αα and PORTUGUESE εγδβ0-Thal, respectively. The other two smaller deletions, given the uncertainty regarding their breakpoints, might be similar to others already published. In all patients, genotypes are well correlated with the different thalassemic phenotypes presented. MLPA proves to be a useful technique to identify known and unknown large deletions affecting globin gene clusters.
- Novel deletions and unusual genetic mechanisms underlying alpha-thalassemiaPublication . Ferrão, José; Silva, Marisa; Gonçalves, Lúcia; Gomes, Susana; Coelho, Andreia; Miranda, Armandina; Seuanes, Filomena; Batalha-Reis, Ana; Valtonen-Andrá, C.; Sonesson, A.; Pina, Francisca; Forjaz-Lacera, João; Maia, Raquel; Kjollerstrom, Paula; Lavinha, João; Gonçalves, João; Faustino, PaulaHemoglobin (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.