Congenital Dyserythropoietic Anemias: Molecular Diagnosis and Diagnostic Approach in a Cohort of Italian Patients

Introduction. Congenital dyserythropoietic anemias (CDAs) are  hereditary rare  erythropoietic disorder characterized  by distinct morphological abnormalities of the bone marrow cells, ineffective erythropoiesis and systemic iron overload. This conditions is characterized by a maturation arrest during erythropoiesis with a reduced reticulocyte production in contrast with erythroid hyperplasia in bone marrow.  Three types of CDA are known: types 1, 2 and 3.The identification of their causative genes provided evidence that these conditions have different molecular mechanisms that induce abnormal cell maturation and division.  We describe the clinical and laboratory manifestations, the diagnosis procedure, the therapeutic approaches and the clinical phenotype in some cases of congenital dyserythropoietic anemia (CDAs) in Italian patients. The molecular analysis allow us to identify several genetic variants some of which have never been described previously. This report highlights the importance of recognizing CDAI even in countries where thalassemia is common. Among the different tools genetic study of CDA-related genes remains the main approach for pursuing the right diagnosis.

Methods. Peripheral blood samples from 100 normal adults and  20 patients with  different types of anemia were collected. Blood samples were analyzed by  EMA binding test and by polyacrylamide gel electrophoresis in presence of sodium dodecyl sulphate ( SDS-PAGE). SDS PAGE  was carried out in a 3.5–17% exponential gradient gel in Fairbanks buffer Genomic DNA was extracted from mononuclear cells of peripheral blood samples, by using a  phenol-chloroform method. Polymerase chain reaction (PCR) products were sequenced directly using Big-Dye terminator 3.1 cycle sequencing kit and run on ABI PRISM 3130 DNA analyzer. The bone marrow of all patients was analysed  with light or electron microscopy. The diagnosis of CDAs was based on the presence of mild to moderate anemia ineffective erythropoiesis and bone marrow erythroblasts morphological abnormalities. SEC23B , CDAN1, KLF1, BCL11A gene sequencing analysis was performed to highlight the presence of nucleotide variations and their relationship with the clinical presentation.  

Results and Discussion. We collected blood samples from 20 Italian  patients with suspect of CDAs and 100 samples  belonging to the healthy  control subjects. None  mutation in the genes analyzed was found in the samples controls. We identified SEC 23 B mutations in 4 out of  20  patients analysed. In two patients with suspect of CDAII we found two nucleotide variations; SDS PAGE in these patients  identified  the presence of abnormal band 3   and the EMA binding test resulted pathologic. Bone marrow (BM) light microscopy revealed more than 10% mature binucleated erythroblasts. In the patients suspected of CDA1, several gene variants  were found  in the CDAN1 gene, some described in the literature  as mutations or polymorphisms, others of uncertain significance. In a patient diagnosed with CDA1  two novel mutations was found.  EMA binding test resulted normal in  all patients with CDA1. Light microscopy of the BM of CDA1 patients showed erythroid hyperplasia, presence of internuclear bridges between intermediate erythroblasts and characteristic pattern of spongy “swiss cheese” hetrochromatin  in electron microscopy. None KLF1 and BCL11A mutations correlated with atypical CDAs was identified. In this last patients the EMA binding test resulted normal. CDAs are  rare clinical hereditary disorders whose correct diagnosis is often delayed to adolescence or  adulthood, when significant iron overload and end organ damage may have been occurred. Patients are often  misdiagnosed with other congenital haemolytic anaemias. Inaccurate diagnosis can lead to inappropriate therapies, such as iron supplements, aggressive transfusion or splenectomy. However even if the gold standard  for the CDAs diagnosis is the electronic microscopy, the identification of the mutated genes involved in the majority of CDAs patients made in recent years has improved the possibility of detecting these diseases.