Molecular and cellular characterization of congental dyserythropoietic anaemia type 1

Ahmed, Momin (2009). Molecular and cellular characterization of congental dyserythropoietic anaemia type 1. PhD thesis The Open University.

DOI: https://doi.org/10.21954/ou.ro.00010035

Abstract

Congenital dyserythropoietic anaemia type I (CDA I) is an autosomal recessive disorder of erythropoiesis characterized by a clinical picture of anaemia secondary to ineffective erythropoiesis, haemolysis and striking morphology of erythroid precursors (internuclear chromatin bridges, spongy heterochromatin, invaginations of nuclear membrane) and erythrocytes (macrocytosis, anisocytosis, basophilic stippling) in the bone marrow and peripheral blood smears, respectively.

The aim of this work was to 1) collect patients and characterize this rare disease, 2) understand the genetics of CDA I using linkage analysis, 3) identify the CDAN1 gene mutations and finally 4) to study the molecular perturbations which lead to dyserythropoiesis.

Four consanguineous (16 affected and 45 unaffected members) and 6 sporadic families were recruited. Linkage analysis revealed evidence of genetic heterogeneity in two of these families. During the course of this project, a large sequencing project looking at putative candidates in the linked locus (Chr 15q15) cloned the CDAN1 gene and identified mutations in this gene associated with CDA I disease. We confirm the presence of mutations in CDAN1 gene in majority of the patients. We have shown founder effect of the R1041W mutation in the Middle Eastern families whilst the European families show a high degree of allelic heterozygosity. There is a clear lack of well characterized null mutations in CDAN1 gene, which suggests a critical function. CDAN1 is very well conserved through evolution in vertebrates with no clearly recognised functional domain. In silico analysis reveal features of the putative promoter and suggested several potential co-regulated genes providing clues to its function. Analysis of 5’ upstream sequence identified several evolutionary conserved regions likely to harbor cis-regulatory elements.

To understand the dyserythropoiesis of CDA I, I used a liquid culture system to recapitulate terminal differentiation of erythroid precursors and studied gene expression in CDA I cells using oligonucleotide microarrays. Data analysis revealed striking changes in genome wide gene expression profiles from terminally differentiating CDA I erythroid precursors. Changes were noted in the pattern of gene expression affecting numerous systems and transcriptional networks associated with erythroid stress responses, including alterations to the expression of genes involved in regulating the cell cycle. This data furthers the understanding of mechanisms that distinguish normal erythropoiesis and dyserythropoiesis.

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