Alpha-thalassemia due to novel deletions and complex rearrangements in the subtelomeric region of chromosome 16p

2º Dia do Jovem Investigador do Instituto Nacional de Saúde Doutor Ricardo Jorge, INSA, 8 maio 2017Introduction: Inherited deletions removing the α-globin genes and/or their upstream regulatory elements (MCSs) give rise to alpha-thalassemia, one of the most common genetic recessive disorders worldwide. The pathology is characterized by microcytic hypochromic anemia due to reduction of the α-globin chain synthesis, which are essential for hemoglobin tetramerization. Material and Methods: In order to clarify the suggestive α-thalassemia phenotype in eleven patients, we performed Multiplex Ligation-dependent Probe Amplification with commercial and synthetic engineered probes, gap-PCR, and Sanger sequencing to search for deletions in the subtelomeric region of chromosome 16p. Results: We have identified five distinct large deletions, two of them novel, and one indel. The deletions range from approximately 3.3 to 323 kb, and i) remove the whole α-globin cluster; or ii) remove exclusively the upstream regulatory elements leaving the α-globin genes structurally intact. The indel consists in the loss of MCS-R2 (HS-40), which is the most important distal regulatory element for the α-globin gene expression, and the insertion of 39 bp, seemingly resulting from a complex rearrangement involving two DNA segments (probably from chromosome 3q) bridging the deletion breakpoints with a CC-bp orphan sequence in between. Finally, in one patient no α-globin deletion or point mutation were found. This patient revealed to be a very unusual case of acquired alpha-thalassemia associated with a myelodysplastic syndrome. Conclusions: Our study widens the spectrum of molecular lesions by which α-thalassemia may occur and emphasizes the importance of diagnosing large α-zero-deletions to provide patients with appropriate genetic counseling.info:eu-repo/semantics/publishedVersio

Diagnóstico genético da diabetes tipo MODY (Maturity-onset diabetes of the young) em Portugal

Departamento de Promoção da Saúde e Prevenção de Doenças Não Transmissíveis, Instituto Nacional de Saúde Doutor Ricardo Jorge, IP

Unusual molecular mechanisms in the origin of alpha-thalassemia

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 gene 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. Distally upstream of the α-globin genes there are four multispecies conserved sequences (MCS-R1 to R4) which are critical for the expression of the downstream globin genes. Deletions removing the α-globin genes and/or their distal MCSs give rise to α-thalassemia, one of the most common genetic recessive disorders worldwide, due to a reduced rate of α-globin chain synthesis. The severity of the pathology is variable ranging from a very mild microcytic hypochromic anemia to a moderately severe anemia associated with the formation of β4 tetramers resulting in HbH disease or an even higher reduction or complete absence of α-chains resulting in hemoglobin Bart’s hydrops fetalis, a condition generally incompatible with life. The main objectives of this work were to characterize the molecular lesions underlying ten Portuguese cases of unusual α-thalassemia/HbH disease and to understand their origin and functional consequences. After exclusion the most frequent molecular lesions associated with α-thalassemia, Multiplex Ligation-dependent Probe Amplification (MLPA) using the SALSA MLPA P140B HBA kit (MCR-Holland) was used to search for DNA deletions in the subtelomeric region of chromosome 16p. Additionally, specifically designed synthetic MLPA probes, as well as gap-PCR and Sanger sequencing were performed for more accurate deletion breakpoint mapping. We have found five distinct deletions and one indel, all in heterozygosity. The deletions range from approximately 3.3 to 323 kb and two of them are novel. The three larger deletions remove the entire α-globin cluster whereas the others remove totally or partially the distal regulatory elements keeping the α-globin genes structurally intact. The indel comprises the deletion of the MCS-R2 regulatory element and the insertion of a singular 39 bp DNA fragment possibly originating from a complex rearrangement involving chromosome 3. Finally, no α-globin gene cluster deletion or point mutation were found in a patient who revealed to be a very unusual case of acquired alpha-thalassemia associated with a myelodysplastic syndrome. Our study widens the spectrum of molecular lesions and unusual molecular mechanisms by which α-thalassemia/HbH may occur and emphasizes the importance of diagnosing large α0-deletions to provide patients with appropriate genetic counseling.info:eu-repo/semantics/publishedVersion22nd Congress of the European Hematology Association, 22-25 June 201

Widening the spectrum of deletions and molecular mechanisms underlying alpha-thalassemia

Inherited deletions of α-globin genes and/or their upstream regulatory elements (MCSs) give rise to α-thalassemia, an autosomal recessive microcytic hypochromic anemia. In this study, Multiplex Ligation-dependent Probe Amplification performed with commercial and synthetic engineered probes, Gap-PCR, and DNA sequencing were used to characterize lesions in the subtelomeric region of the short arm of chromosome 16, possibly explaining the α-thalassemia/HbH disease phenotype in ten patients. We have found six different deletions, in heterozygosity, ranging from approximately 3.3 to 323 kb, two of them not previously described. The deletions fall into two categories: one includes deletions which totally remove the α-globin gene cluster, whereas the other includes deletions removing only the distal regulatory elements and keeping the α-globin genes structurally intact. An indel was observed in one patient involving the loss of the MCS-R2 and the insertion of 39 bp originated from a complex rearrangement spanning the deletion breakpoints. Finally, in another case no α-globin gene cluster deletion was found and the patient revealed to be a very unusual case of acquired alpha-thalassemia-myelodysplastic syndrome. This study further illustrates the diversity of genomic lesions and underlying molecular mechanisms leading to α-thalassemia.info:eu-repo/semantics/publishedVersio