Diagnosis Of Congenital Dyserythropoietic Anemia Research Articles

CoDysAn: A Telemedicine Tool to Improve Awareness and Diagnosis for Patients With Congenital Dyserythropoietic Anemia.

Congenital Dyserythropoietic Anemia (CDA) is a heterogeneous group of hematological disorders characterized by chronic hyporegenerative anemia and distinct morphological abnormalities of erythroid precursors in the bone marrow. In many cases, a final diagnosis is not achieved due to different levels of awareness for the diagnosis of CDAs and lack of use of advanced diagnostic procedures. Researchers have identified five major types of CDA: types I, II, III, IV, and X-linked dyserythropoietic anemia and thrombocytopenia (XLDAT). Proper management in CDA is still unsatisfactory, as the different subtypes of CDA have different genetic causes and different but overlapping patterns of signs and symptoms. For this reason, we developed a new telemedicine tool that will help doctors to achieve a faster diagnostic for this disease. Using open access code, we have created a responsive webpage named CoDysAn (Congenital Dyserythropoietic Anemia) that includes practical information for CDA awareness and a step-by-step diagnostic tool based on a CDA algorithm. The site is currently available in four languages (Catalan, Spanish, Italian, and English). This telemedicine webpage is available at http://www.codysan.eu.

Whole Exome Sequencing Identifies a Novel Variant in the Pklr Gene Which Worsen the Anemia Status in a Congenital Dyserthropoietic Anemia Patient

Congenital dyserythropoietic anemia (CDAs) is a group of hereditary disorders characterized by ineffective erythropoiesis and distinct morphological abnormalities of erythroblasts in the bone marrow. Diagnosis of CDA is based primarily on the morphology of bone marrow erythroblasts but genetic tests might have more and more important roles recently. Here, we describe a 31-year old female with atypical CDA under regular blood transfusions. The results of blood tests were as followed: RBC: 3080000/µL, Hb: 9.9 g/dL, Hct: 26.8%, MCV: 87.0 fl, MCH:32.2 pg, and MCHC: 36.9 g/L. In this study, analysis of CDA-related genes, including SEC23B, CDAN1, KLF1, and C15orf41, were analyzed by exome sequencing but no pathogenic variant was found. In additional, we analyzed RBC-related genes and a novel variant in the PKLR p.A468G (NP_000289) was found which was also confirmed by Sanger sequence. Variant of PKLR gene has been reported as the cause of pyruvate kinase (PK) deficiency anemia. PK deficiency is the most cause of congenital nonspherocytic hemolytic anemia with GMAF (global minor allele frequency) of 0.0001. The clinical features of PK-deficient patients are highly variable degree of chronic hemolysis with severe neonatal jaundice and fetal anemia at birth. In this case, PKLR p.A468G heterozygous variant was detected in a patient with PK deficiency, as an example of precision diagnosis by exome sequencing. DisclosuresNo relevant conflicts of interest to declare.

Evaluation of Phenotype-Genotype Correlation in Two Common PIEZO1 Mutations p.R2456H and p.L2495_E2495dup

Hereditary xerocytosis (HX) is a rare autosomal dominant hemolytic anemia caused by mutations in the mechanosensitive cation channel PIEZO1 or, less commonly, in the Ca2+-gated K+ channel KCNN4 (Gardos channel). It is a clinically heterogeneous condition, characterized by erythrocyte dehydration. As erythrocytes traverse narrow capillaries and sinusoids, PIEZO1 is thought to be activated by mechanical stimuli, leading to increased intracellular Ca2+ which then may activate KCNN4, causing K+ efflux and water loss. The p.R2456H and p.L2495_E2496dup PIEZO1 mutations are likely the most common of the PIEZO1 disease-causing mutations and both result in delayed channel inactivation (Glogowska et al., 2017, Blood 130:1845). The prolonged channel activity in response to mechanical stimulation is likely to contribute to erythrocyte dehydration.Ten patients with PIEZO1-associated HX (5 with p.R2456H and 5 with p.L2495_E2496dup PIEZO1 heterozygous mutations) had their diagnosis established utilizing a Next Generation sequencing panel for hereditary hemolytic and congenital dyserythropoietic anemias (HHA/CDA panel). These patients, along with family members who had similar clinical characteristics (a total of 8 patients with p.R2456H and 6 patients with p.L2495_E2496dup) ranging from 2 - 63 years of age, were enrolled in an IRB-approved study to explore the phenotype-genotype correlation of the disease. We collected patient and family histories and laboratory data and performed phenotypic testing including ektacytometry and erythrocyte cation determinations by atomic spectroscopy.Most of the patients in our cohort had compensated hemolysis without anemia at the time of evaluation and had not required any transfusions. All of the patients had elevated reticulocyte counts ranging from 5.7 to 15.7 %. Reticulocyte counts of individuals with the p.R2456H mutation were significantly higher than those with the p.L2495_E2496dup mutation (Student's t test, p=0.01). Some of the patients had splenomegaly, but it was not a universal finding. Although elevated MCHC is often considered a hallmark of HX, most of the individuals in this cohort had increased MCV and MCH but only high-normal MCHC. Varying percentages of hyper dense cells were detected in blood samples analyzed using an Advia Hematology System and stomatocytes were notable in most peripheral blood smears.Several complications have been associated with HX including hydrops fetalis, hemolytic episodes, thrombosis (especially after splenectomy), gallstones, and iron overload. One patient in our cohort had experienced ascites and a pleural effusion in the perinatal period and one was diagnosed with gallstones at age 11. Nearly all the patients had an increase in total bilirubin. Ferritin levels were increased in the majority of the patients tested after 15 years of age. The three patients older than 40 years of age had iron overload verified by T2* MRI imaging of the liver.HX presents several diagnostic challenges due to its heterogeneity. Two of the subjects in our study had been previously diagnosed with hereditary spherocytosis (HS) and one had the diagnosis of congenital dyserythropoietic anemia (CDA) prior to genetic evaluation. Misdiagnosis of individuals with HX as having HS is especially problematic since splenectomy is contraindicated in HX due to a greatly increased incidence of thromboembolic complications. Another diagnostic consideration concerns the use of osmotic fragility tests. Although osmotic fragility tests are frequently used to diagnose HX, ektacytometry provides greater accuracy. In the present study two patients who had ektacytometry results characteristic of HX had reportedly normal osmotic fragility tests in previous work-up. All individuals in our cohort with confirmed disease-causing mutations in PIEZO1 demonstrated the classic left shifted ektacytometry osmoscan indicative of erythrocyte dehydration. In addition, all individuals with these mutations demonstrated the expected decrease in erythrocyte potassium content not associated with a proportional increase in sodium. This illustrates the importance of specialized testing (ektacytometry and/or intracellular cation determinations) and the use of appropriately designed genetic panels to establish an accurate diagnosis in patients with hemolytic anemia of ambiguous phenotype, especially when splenectomy is being contemplated. DisclosuresQuinn:Amgen: Research Funding; Global Blood Therapeutics: Research Funding; Silver Lake Research Corporation: Research Funding.

Whole-exome analysis to detect congenital hemolytic anemia mimicking congenital dyserythropoietic anemia

Congenital dyserythropoietic anemia (CDA) is a heterogeneous group of rare congenital disorders characterized by ineffective erythropoiesis and dysplastic changes in erythroblasts. Diagnosis of CDA is based primarily on the morphology of bone marrow erythroblasts; however, genetic tests have recently become more important. Here, we performed genetic analysis of 10 Japanese patients who had been diagnosed with CDA based on laboratory findings and morphological characteristics. We examined 10 CDA patients via central review of bone marrow morphology and genetic analysis for congenital bone marrow failure syndromes. Sanger sequencing for CDAN1, SEC23B, and KLF1 was performed for all patients. We performed whole-exome sequencing in patients without mutation in these genes. Three patients carried pathogenic CDAN1 mutations, whereas no SEC23B mutations were identified in our cohort. WES unexpectedly identified gene mutations known to cause congenital hemolytic anemia in two patients: canonical G6PD p.Val394Leu mutation and SPTA1 p.Arg28His mutation. Comprehensive genetic analysis is warranted for more effective diagnosis of patients with suspected CDA.

An Unusual Hydrops Fetalis Associated with Compound Heterozygosity for Krüppel-like Factor 1 mutations

Hydrops fetalis is commonly due to Hb Bart’s (γ4) disease in South East Asia. Here, we report an unusual case of hydrops fetalis due to congenital dyserythropoietic anemia (CDA) associated with compound heterozygosity for Krüppel-like factor 1 (KLF1) gene mutations. Fetal cardiomegaly was first detected on routine mid-trimester scan in a pregnant woman with normal mean corpuscular volume (MCV) and Rhesus positive status. The fetus subsequently developed hydrops fetalis, and cordocentesis showed severe fetal anemia with a hemoglobin (Hb) level of 3.4 g/dL. Common causes of fetal anemia including Hb Bart’s disease, parvovirus infection, and red cell antibodies were excluded. In view of the marked increase in erythroblasts at various stages of erythropoiesis, the diagnosis of CDA was suspected. We screened the couple for previously reported KLF1 gene mutations, showing that the mother was heterozygous for the c.525_526insCGGCGCC, p.Gly176Argfs*179 mutation, and her husband heterozygous for c.1012C>A, p.Pro338Thr mutation. The fetus was a compound heterozygote for these two KLF1 mutations. After counseling, repeated intrauterine transfusions were given at 27, 29, and 34 weeks’ gestation; the hydrops fetalis was resolved. The baby was delivered at 34 weeks’ gestation and required monthly blood transfusions but was otherwise thriving. Bone marrow aspiration at 10 months of age showed the features of ineffective erythropoiesis, compatible with CDA. In conclusion, hydrops fetalis can rarely be due to CDA associated with a compound heterozygous mutation for KLF1 gene mutations, and be managed by repeated intrauterine transfusions. Our present report adds to the wide clinical spectrum of KLF1 mutations.

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. DisclosuresNo relevant conflicts of interest to declare.

The morphological diagnosis of congenital dyserythropoietic anemia: results of a quantitative analysis of peripheral blood and bone marrow cells

The congenital dyserythropoietic anemias comprise a group of very rare hereditary disorders characterized by ineffective erythropoiesis and by distinct morphological abnormalities of the erythroblasts in the bone marrow. The classification proposed in 1968[1][1] is still used today.[2][2], [3][3]

INTERFERON THERAPY IN CONGENITAL DYSERYTHROPOIETIC ANEMIA TYPE I/II

Congenital dyserythropoietic anemia (CDA) is a rare disorder, characterized by the association of ineffective erythropoiesis, variable degree of anemia, and erythroblastic morphological abnormalities. α-Interferon has been reported to be effective in type I CDA, but efficacy in other types of CDA is uncertain. Encouraged by the reports, we evaluated the efficacy of α-interferon in 6 children with CDA. Diagnosis of CDA was established on the basis of clinical profile, distinct morphological findings on light microscopy, and the Ham's test, following the exclusion of the more common causes of hemolysis. Erythrocyte agglutinability and lysis to anti-i and anti-l sera, electron microscopy, and SDS–polyacrylamide gel electrophoresis were not performed, due to nonavailability. There were 3 cases, each, with type I and type II CDA. The mean age was 5.5 years (range: 6 months to 11.5 years). Five of the 6 patients were transfusion dependent. α-Interferon was administered subcutaneously for a mean duration of 19 weeks (range: 12–30). The dose ranged from 2.6 to 6.5 million IU/m2/dose. The frequency of injections varied from thrice weekly to alternate days. No favorable effect on hemoglobin, reticulocyte count, or transfusion frequency was observed. α-Interferon therapy was found to be ineffective in all the patients. These observations question the use of interferon in CDA until further studies in a larger number of patients establish its efficacy.