Unit:
Κατεύθυνση Ιατρική Γενετική: Κλινική και Εργαστηριακή ΚατεύθυνσηLibrary of the School of Health Sciences
Supervisors info:
Ιωάννα-Ραχήλ Traeger-Συνοδινού, Καθηγήτρια, Ιατρική, ΕΚΠΑ
Ελένη Φρυσίρα, Αναπληρώτρια Καθηγήτρια, Ιατρική, ΕΚΠΑ
Μαρία Τζέτη, Αναπληρώτρια Καθηγήτρια, Ιατρική, ΕΚΠΑ
Original Title:
Αξιολόγηση της νέας παραλλαγής c.92+7A>G (IVS-I-7A>G) στο γονίδιο της β-σφαιρίνης (ΗΒΒ) μέσω μελέτης RNA με RT-PCR σε ανώριμα ερυθρά κύτταρα
Translated title:
Evaluation of the new variant c.92+7A>G (IVS-I-7A>G) in the β-globin gene (HBB) by RNA study with RT-PCR on immature red cells
Summary:
Hemoglobinopathies are a heterogenous group of monogenic diseases resulting from mutations in the globin genes. Due to high carrier frequencies worldwide, they consist the commonest monogenic disorders. They are characterized by abnormal synthesis and structure of hemoglobin chains.
β-Thalassemia is inherited in autosomal recessive manner and it is resulted from absent or reduced synthesis of β-chain. More than 200 mutations of β-Thalassemia have been described and most of them are point mutations. A wide variety of mutations interfere with mRNA processing which affected the normal splicing.
The present thesis aimed to investigate the pathogenicity of a novel variant HBB:c.92+7A>G (IVS-I-7A>G). The variant was identified in a child with hematological findings indicative of a possible state of β-Thalassemia Intermediate who had also inherited in trans a common typical beta-thalassemia mutation HBB:c.118C>T (traditionally called codon 39C>T). The evaluation of the pathogenicity of novel DNA variants is challenging and very important in the light of the new DNA technologies such as next-generation sequencing. The evaluation of pathogenicity can be based on many parameters including family segregation studies, population frequency studies, in-silico model studies and finally experimental studies; these approaches are incorporated into various guidelines, such as those of the American College of Medical Genetics and Genomics (ACMG). The specific variant HBB:c.92+7A>G is located very close to the donor splice site of exon 1 of HBB. Thus it could potentially affect splicing of the beta-globin RNA transcript. Based on this assumption, investigating the presence of abnormally spliced mRNA of red-cell precursors which express very high levels of globin RNA. To this end mononuclear cells from peripheral blood from the proband and his father, who was heterozygous for the novel variant HBB:c.92+7A>G were isolated and cultured. The total RNA was isolated from the precursor erythroid cells and reverse transcription PCR (RT-PCR) was performed. To determine the present of aberrantly spliced RNA, polyacrylamide gel electrophoresis (PAGE), sequencing and Fluorecence PCR (F-PCR) were used.
Based on the results of analysis using by PAGE and Sanger sequencing no abnormally spliced beta-globin mRNA were detected. However, the analysis of beta-globin mRNA transcripts using fragment analysis of fluorescently labeled RNA indicated a very small peak which could represent a tiny amount of abnormally spliced RNA. Overall, the results are inconclusive, highlighting the challenges of characterizing the pathogenicity of novel DNA variants.
Main subject category:
Health Sciences
Keywords:
β-Thasassemia, Splicing, mRNA
