congenital dyserythropoietic anemias ( cdas ) are inherited disorders that result from anomalies during the final stages of erythropoiesis and consequently present defective production of red blood cells 1 .
cdas are characterized by chronic hyporegenerative anemia with inadequate reticulocyte count for the degree of anemia , except for cda type iv , and mild hemolysis .
the most common complications associated with cdas are iron overload , cholelithiasis , and splenomegaly 2 , 3 .
dyserythropoiesis and morphological features of erythroblasts are commonly identified in other related anemias ( hereditary hemolytic anemia , hereditary stomatocytosis , diamondblackfan anemia , fanconi anemia , and other inherited bone marrow failure syndromes ) that should be excluded in clinical practice .
moreover , the overlapping phenotypes shown in cdas , even in patients with the same genetic variation , can make differential diagnosis difficult 1 .
the identification of the causative genes of cdas allowed the classification of these diseases , as well as increasing understanding of pathogenesis and clinical management of cdas 4 , 5 , 6 , 7 , 8 .
three classic types of cdas are distinguished by morphological abnormalities in erythroblasts ; the most recent classification recognizes six different types of cdas : ia , ib , ii , iii , iv , and thrombocytopenia xlinked with or without dyserythropoietic anemia , resulting from variants of cdan1 , c15orf41 , sec23b , kif23 , klf1 , and gata1 genes , respectively 1 .
congenital dyserythropoietic anemias are a rare group of anemias , and their estimated frequency in europe is variable , the lowest 0.08 cases / million in scandinavia to the highest 2.60 cases / million in italy 9 . to date , more than 100 cases of cda type ia have been reported 3 , several members of a family with cda type ib 6 , more than 400 cases of cda type ii 10 , members of two families with cda type iii 7 , and only four patients with cda type iv 8 , 11 .
the different genetic variants causing disease are compiled in the human gene mutation database ( http://www.hgmd.cf.ac.uk/ac/ ) : thirtyfour in the cdan1 gene , eightyeight in the sec23b gene , and sixtythree in klf1 gene but only one variant responsible for dyserythropoietic anemia .
we report the case of a patient diagnosed with unclassified cda ; genetic study allowed the identification of the pathogenic variant c.973g > a , p.glu325lys , in the heterozygous state in the klf1 gene , this being the fifth case reported with cda type iv .
a female patient aged 13 exhibited systolic murmur , hepatosplenomegaly , polyarthralgia , weight loss as result of high temperature , and signs of anemia onset , dyserythropoiesis , and mild hemolysis . in the neonatal period
her twin brother and other siblings have no signs of anemia , her mother has iron deficiency anemia , and her father and grandparents have no known diseases .
the blood count data of this patient at 14 years of age were indicative of normocytic hyperegenerative anemia : decreased red blood cells , hemoglobin and hematocrit , increased red distribution width , and reticulocytes ; other parameters were within the normal range ( table 1 ) .
serological data were distinctive from hemolysis : increased lactate dehydrogenase , hyperbilirubinemia due to indirect fraction , and haptoglobin was undetectable ( table 1 ) .
there were no signs of iron overload , transferrin saturation , and iron and ferritin values were within the normal range ( table 1 ) .
the hemoglobin electrophoresis test was normal except for increased fetal hemoglobin , 12% ( normal range of hbf 0.82 ) .
normal ranges in brackets : red blood cells ( rbc 3.55 ) , hemoglobin ( hb 1215.5 ) , hematocrit ( ht 3646 ) , red distribution width ( rdw 10.514.5 ) , reticulocytes ( retic .
01.5 ) , median corpuscular volume ( mcv 8098 ) , median corpuscular hemoglobin ( mch 2733 ) , median corpuscular hemoglobin concentration ( mchc 3235 ) , platelet ( ptl 120450 ) , total bilirubin ( 01.2 ) , lactate dehydrogenase ( ldh 10250 ) , transferrin saturation ( tf sat .
2045 ) , iron ( 37130 ) , ferritin ( 10160 , females ) , hepcidin ( 226 ) .
data morphology of peripheral blood cells of the patient aged 13 was as follows : presence of poikilocytosis , anisocytosis , basophilic stippling , polychromia , and mild macrocytosis with 10% erythroblasts .
bone marrow morphology aspirated from the patient aged 18 showed intense hyperplasia of red cell series mostly at the last maturation stages , myeloid cells / nucleated erythroid cells ratio of 0.1 ( m / e normal range in adults from 1.2 to 5 ) .
binuclearity appears in 15% of orthochromatic erythroblasts , multinuclearity , and abundant iron in mononuclear phagocyte cells with a small number of sideroblasts ( < 5% ) ( fig .
analysis by electron microscopy revealed ultrastructural abnormalities of erythroblasts , nuclear and chromatin alterations : pyknosis , karyorrhexis , sponge nuclei , euchromatin areas connecting nuclear membrane , pores in the nuclear membrane through which cytoplasm seems to penetrate into nuclei and intercellular bridges .
( a ) may  grnwald stain of bone marrow aspirate ( 40x ) : erithroid hyperplasia , dyserythropoiesis , erythroblastic multinuclearity .
( b ) ultrastructural abnormalities of erythroblasts , nuclear and chromatin alterations : pyknosis , karyorrhexis , sponge nuclei , euchromatin areas connecting nuclear membrane , pores in the nuclear membrane through which cytoplasm seems to penetrate into nuclei , intercellular bridges were also observed but no double nuclear membrane . at 24 ,
the patient had a premature twin birth , one twin died at 72 h and the other at 40 days because of hydrops fetalis , respiratory failure and sepsis .
, this 44yearold patient needs occasional blood transfusions , whenever infectious processes ( urinary infection , at the initial treatment for virus c hepatitis ) but is not transfusion dependent .
she presents hyperferritinemia ( 797 ng / ml ) although iron ( 77 g / dl ) and transferrin saturation ( 30% ) are within the normal ranges .
the patient has been diagnosed with cda type iv after a genetic study by ion torrent next generation sequencing ion ampliseq ( life technologies ) with a customized panel for cdas ( cdan1 , c15orf41 , sec23b , kif23 , and klf1 genes ) .
a pathogenic allelic variant in the heterozygous state was identified in the klf1 gene ( c.973g > a , p.glu325lys ) .
congenital dyserythropoietic anemias are rare inherited disorders characterized by a reduced reticulocyte production and hyperplasia in bone marrow . the anemia and hyperbilirubinemia are usually observed in childhood and young adults .
the clinical presentation in these patients could lead to misdiagnosis such as hemolytic anemia , thalassemia , hereditary spherocytosis , or iron deficiency anemia , and as consequence , to inappropriate therapies .
therapies for cda patients are red cell transfusions , iron chelation to prevent organ damage , splenectomy to abrogate transfusion requirements , or special therapies such as interferon in cda i patients 12 or stem cells transplantation in severe cases of cda 13 , 14 , 15 .
the morphological abnormalities of bone marrow erythroblasts support the differential diagnosis of cdas , mainly represented by cda types i and ii .
cda type i presents megaloblastic binucleated erythroblasts ( 25% ) , chromatin bridges between nuclei , spongy , or  swisscheese  appearance of heterochromatin and invagination of cytoplasm into the nucleus .
cda type ii presents normoblastic binucleated and multinucleated erythroblasts ( 1035% ) and peripheral double plasma membranes .
cda type iii presents giant multinucleated erythroblasts , intranuclear clefts into heterochromatin , autophagic vacuoles , and karyorrhexis .
cda type iv presents dyserythropoietic morphology similar to cda types i and ii : binucleated erythroblasts , rare immature erythroid cells with marked heterochromatin 2 , 3 .
the analysis of erythrocyte membrane proteins facilitates the diagnosis given that the hypoglycosylation of band 3 is the diagnostic hallmark of cda type ii 16 .
the cda type iv ( omim 613673 ) is an autosomaldominant disorder caused by variants in the klf1 gene ( chr 19p13.2 ) which encodes for the krppellike factor 1 ( klf1 ) , an essential erythroidspecific transcriptional factor member of the krppellike family .
klf1 can act as a transcriptional activator and repressor of erythroid gene expression , an activator of genes for heme and globin synthesis , blood group antigens , and other erythroid factors 17 , 18 .
sixtythree variants on klf1 gene have been described so far and result in different phenotypes such as : hereditary persistence of fetal hemoglobin , borderline elevated levels of hemoglobin a2 , microcytosis and/or hypochromia , lutheran blood type , congenital hemolytic anemia , congenital dyserythropoietic anemia type iv 19 , 20 .
recently , homozygosity for null variants has been associated with embryonic lethality 21 . to date , four patients with cda iv have been reported , all of them exhibiting the same autosomaldominant variant c.973g > a , p.glu325lys , in the heterozygous state in the klf1 gene , 4 , 10 , the same genetic variant as the case reported in the present work .
this pathogenic variant is found in the second zinc finger of klf1 , an essential region for binding to dna motif in the regulatory regions of many erythroid genes .
all of the patients reported as cda type iv , and the patient in this work , showed normocytic anemia , normal or slightly increased reticulocyte count , elevated values of hbf , elevated ldh , hyperbilirubinemia , and reduced haptoglobin . in two cases ,
anemia had been found at birth and required repeated blood transfusions . in another case , anemia was associated with hydrops fetalis and treated with intrauterine transfusions ; thalassemic facies and female genitalia with a male karyotype were also presented 8 .
almost all patients exhibit hepatomegaly and splenomegaly , one was splenectomized aged 4 8 , and the present patient was splenectomized aged 36 . in one case ,
the liver showed a moderate iron overload , not caused by blood transfusion therapy 11 .
these patients showed similar features in peripheral blood smear and in bone marrow aspirate : anisopoikilocytosis , polychromasia , erythroid hyperplasia , dyserythropoiesis signs , basophilic stippling of erythroblasts and erythrocyte , bi and multinucleated erythroblasts with internuclear bridges and enlarged nuclear pores .
moreover , erythrocytes of patients reported had low protein expression of cd44 and aqp1 water channel , both expression genes regulated by klf1 8 , 11 .
as klf1 is a transcriptional regulator of the switch from fetal to adult hemoglobin , and of many red blood cell membrane proteins , the clinical characteristics of cda type iv are a combination of hemoglobinopathy , red blood cell defect , and hereditary persistence of fetal hemoglobin features .
these conditions together distinguish cda type iv from other diseases such as thalassemia , hereditary spherocytosis , and hereditary persistence of hbf 11 .
the wide variety of phenotypes observed in cda patients makes differential diagnosis difficult ; the identification of the genetic variants plays a crucial role in the clinical management of patients .
genetic diagnosis would contribute to classify cda patients , and even to identify genetic factors that modify phenotype severity .
the detection of a genetic variant in misdiagnosed or unclassified cda patients would mean these inherited disorders were more frequent than at present .
sdlii : acquired , analyzed , and interpreted the clinical data and morphological images , and revised the manuscript .
mjmj : designed the genetic study , analyzed and interpreted genetic variants , and wrote the article .
 key clinical messagecongenital dyserythropoietic anemias ( cdas ) are displayed by ineffective erythropoiesis . 
 the wide variety of phenotypes observed in cda patients makes differential diagnosis difficult ; identification of the genetic variants is crucial in clinical management . 
 we report the fifth case of a patient with unclassified cdas , after genetic study , with cda type iv . 
Introduction
Case History
Discussion
Authorship
Conflict of Interest