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 Table of Contents  
Year : 2022  |  Volume : 2  |  Issue : 2  |  Page : 102-106

Congenital dyserythropoietic anemia type IV with kruppel-like factor 1 E325K mutation in a preterm neonate: Case and literature review

1 Department of Neonatology, Bharati Vidyapeeth University Medical College, Pune, Maharashtra, India
2 Department of Pediatrics, Bharati Vidyapeeth University Medical College, Pune, Maharashtra, India

Date of Submission08-Feb-2022
Date of Decision26-Apr-2022
Date of Acceptance04-May-2022
Date of Web Publication30-May-2022

Correspondence Address:
Dr. Suprabha K Patnaik
Department of Neonatology, Bharati Vidyapeeth Deemed University Medical College and Hospital, Dhankawadi, Pune - 411 043, Maharashtra
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/ipcares.ipcares_43_22

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Background: Clinical, pathologic, and genetic heterogeneity is a challenge in identifying and classifying congenital dyserythropoietic anemia (CDA). CDA type IV, the rarest CDA with only 11 reported cases, results from KLF1 gene mutation. Clinical Description: A male preterm neonate presented with jaundice, anemia, pulmonary hypertension and hepatosplenomegaly in the immediate postnatal period, requiring multiple red blood cell transfusions. Management and Outcome: The workup for non-immune haemolytic anemia including red blood cell structural and enzymatic studies and were normal, with peripheral blood smear showing multiple polychromatic cells and numerous nucleated red blood cells including binucleate ones and fetal haemoglobin of 91.2%. Genetic testing revealed KLF1 E325K mutation suggestive of CDA type IV, though parental testing was normal, suggesting de novo mutation. The infant has been receiving packed RBC transfusion every three to four weeks initially and then every two months. The baby is now of twelve months of age, and receives oral vitamin B12 and folic acid supplementation for ineffective erythropoiesis. Though his weight is in the 3rd centile for age and height, he has been developmentally normal. Conclusions: Our report, the first description of a CDA type IV diagnosis in the neonatal period, adds to the limited knowledge of this disorder, which we also comprehensively review. The report highlights the phenotype of the disorder and the importance of neonatal genetic testing in a case of transfusion dependent anemia, having ruled out other causes.

Keywords: Congenital dyserythropoietic anemia, congenital dyserythropoietic anemia type IV, Kruppel-like factor 1 mutation; neonate

How to cite this article:
Garegrat R, Patnaik SK, Malshe N, Bartakke SP, Bafna V, Suryawanshi P. Congenital dyserythropoietic anemia type IV with kruppel-like factor 1 E325K mutation in a preterm neonate: Case and literature review. Indian Pediatr Case Rep 2022;2:102-6

How to cite this URL:
Garegrat R, Patnaik SK, Malshe N, Bartakke SP, Bafna V, Suryawanshi P. Congenital dyserythropoietic anemia type IV with kruppel-like factor 1 E325K mutation in a preterm neonate: Case and literature review. Indian Pediatr Case Rep [serial online] 2022 [cited 2022 Jul 2];2:102-6. Available from: http://www.ipcares.org/text.asp?2022/2/2/102/346257

Congenital dyserythropoietic anemia (CDA) is a heterogeneous group of anemia's characterized by the impaired differentiation and proliferation of the erythroid cell lineage, leading to monolinear cytopenia.[1] The hallmarks of ineffective erythropoiesis include morphologically abnormal erythroblasts (in the bone marrow) and circulating erythrocytes and evidence of hemolysis. However, this particular hematological profile can be seen in several other blood disorders such as hereditary hemolytic anemia, hereditary stomatocytosis, Diamond–Blackfan anemia, Fanconi anemia, other inherited bone marrow failure syndromes, hereditary spherocytosis, thalassemia, and even iron-deficiency anemia. Therefore, arriving at a diagnosis can be quite challenging and requires a rational approach.

CDA is classified into several categories based on clinical, pathologic, and genetic features: Types Ia, Ib, II, III, IV, V, VI, and other variants involving multiple genes such as CDAN1, c15orf41, SEC23B, KIF23, Kruppel-like factor 1 (KLF1), and GATA-1. Genetic confirmation is essential due to the many non-CDA hematological differentials listed earlier and the fact that considerable heterogeneity exists in the various types of CDA.

Type IV CDA is an autosomal disorder that results from mutations in the KLF1 gene, located on chromosome 19p13.2 which encodes for KLF1. This factor is a master regulator of terminal erythroid differentiation, affecting key cellular pathways and structures, such as division, iron metabolism, heme and globin synthesis, plasma membrane, and cytoskeleton. KLF1 directly activates beta-globin gene expression by binding to the gene's promoter, and indirectly silences gamma-globin gene expression by activating BCL11A that encodes a gamma-globin gene repressor, thus bringing about the switch from fetal to adult hemoglobin (Hb) expression.[2] As of December 2019, about 66 variants of the KLF1 gene had been reported and linked with a variety of phenotypes besides CDA Type IV, including embryonic lethality, hereditary persistence of fetal Hb, borderline HbA2, and inhibitor of the Lutheran blood group. An exhaustive literature search found only 11 patients reported in the PubMed database, out of which 9 had genetic confirmation.

We report a preterm neonate presenting with transfusion-dependent anemia who was diagnosed with this disorder. Not only does this add to our limited knowledge of CDA IV, but none have been diagnosed during the neonatal period till date. In addition, we provide a comprehensive review of the nine genetically confirmed cases.

  Clinical Description Top

A baby boy was born at 35-week gestation by emergency cesarean section (indication being meconium-stained liquor, with fetal distress. The pregnancy of the 31-year-old primiparous mother had been uneventful until she had developed preeclampsia in the third trimester of pregnancy. There were no risk factors for sepsis before the birth. The baby weighed 2.8 kg, was nonvigorous at birth and required positive-pressure ventilation for 30 s. The Apgar score was 7 and 9 at 1, and 5 min, respectively. Following this, he developed respiratory distress and was shifted to the neonatal intensive care unit. He was supported with noninvasive ventilation (continuous positive pressure airway) and was hemodynamically stable. In view of the clinical setting, the diagnosis kept was meconium aspiration syndrome. Other supportive measures such as empirical antibiotics, intravenous fluids, and trophic feeds were started, and investigations were planned. The chest X-ray was nonspecific, and the sepsis markers were not suggestive of sepsis.

The baby was noted to have icterus at 7 h of life, with serum bilirubin of 12.2 mg/dL, and managed with intensive phototherapy. Relevant clinical history was probed into, examination performed, and investigations planned to determine the cause of the pathological jaundice. There was no history of consanguinity, unexplained abortions or infantile deaths, significant family history of jaundice within 24 h of birth, requiring double volume exchange transfusion, or severe anemia requiring multiple blood transfusions. Apart from the respiratory parameters, the vitals were stable, and there was no evidence of congestive heart failure. There was no pallor, ecchymosis, cephalhematoma, bulging fontanelle, or edema. There were no major or minor congenital anomalies. However, hepatosplenomegaly (liver and spleen 4 cm and 1 cm below the costal margin, respectively) and a hyperdynamic precordium were noted. The remaining systemic examination was normal.

There was no blood group incompatibility as both mother and baby were A positive. The total serum bilirubin was 12.2 mg/dL with 0.5 mg/dl direct component and without any liver enzyme elevation. The preliminary hemogram showed a Hb of 12.5 g/dL and 15% reticulocytes with normal total leukocyte and platelet counts. Investigations were planned to rule out hemolytic anemia. Abdominal ultrasonography confirmed hepatosplenomegaly with coarse hepatic echotexture. Functional echocardiography revealed pulmonary hypertension, with a pulmonary arterial pressure of 40 mmHg. The baby was on full enteral feeds by day 3 days of life. Till then, he had not displayed any features of hypoxic-ischemic encephalopathy; hence based on the clinical profile and available investigations, a clinical diagnosis of meconium aspiration syndrome with secondary pulmonary hypertension was kept. The cause of pathological jaundice, mild anemia, and hepatosplenomegaly was still uncertain, pending awaited reports.

  Management and outcome Top

Osmotic fragility testing demonstrated poor osmotic resistance in the red blood cells (RBCs) in buffered normal saline. RBC enzyme studies (glucose-6-phosphate dehydrogenase and pyruvate kinase) were normal. Direct agglutination test (direct Coombs test) and indirect coombs test were negative. The report of the high-performance liquid chromatography (HPLC) that had been sent on the 2nd day of life showed 91.2% Hb F, 4.3% HbA0, and undetectable HbA2 [Figure 1]c, which was considered normal for a newborn. A minor fraction of Hb was eluted in the S window, suggesting Hb variant mimicking sickle Hb, and eluting in the HbS window. The significance of this finding was uncertain. Thus, the peripheral smear picture, and normal RBC fragility, RBC enzyme, RBC membrane tests, and the hemoglobin HPLC ruled out immune hemolytic anemias, thalassemia, hereditary spherocytosis, stomatocytosis, and iron-deficiency anemia. Diamond–Blackfan and Fanconi anemia were excluded clinically based on the absence of typical dysmorphisms and absence of other typical features. Secondary dyserythropoiesis due to the perinatal hypoxia and its respiratory sequelae resulting from the meconium aspiration syndrome, though hypothetically plausible, did not explain the persistent anemia and falling Hb levels (that could not be attributed to blood sampling either).
Figure 1: Congenital dyserythropoietic anemia type IV in a preterm neonate. (a) Blood Hb and total bilirubin measurements were obtained during the first 1.5 months of life. Days on which blood transfusion and phototherapy were received are indicated. (b) Microscopy of Leishman-stained peripheral blood smear showing multiple polychromatic cells and numerous nucleated red blood cells including binucleate ones (arrow). (c) Blood hemoglobin profile in high-performance liquid chromatography suggesting an HbF concentration of 92.1%. (d) Aligned blood genomic DNA sequencing reads at the chromosome 19 position where the KLF1 E325K allele variant (C > T sequence change in the minus chromosome strand, in red) is detected in 53% of the reads. Shown is a screenshot of aligned read data in the Integrative Genomics Viewer software. Gray indicates no sequence change compared to the reference GRCh37 genome. Identifiers of GenBank reference RNA and protein sequences are noted. (e) Screenshots of Sanger DNA sequencing chromatograms of a KLF1 amplicon in blood of the case's parents indicating absence of the E325K allele. Hb: Hemoglobin, KLF1: Kruppel-like factor 1

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Packed RBCs were transfused at 15 ml/kg on day 5 of life for increasing anemia (Hb having fallen to10.6 g/dL and other cell lines maintained) in the presence of requirement of respiratory support and 40% FiO2 oxygen. The baby's respiratory distress gradually improved. Respiratory support was weaned off by day 9 of life and feeding transitioned to exclusive breastfeeding. Phototherapy continued as per the bilirubin levels [Figure 1]a and was stopped on day 10 of life. The baby was discharged on day 15 of life with a provisional diagnosis of nonimmune hemolytic anemia, under evaluation. On day 19 of life, the baby came for his first follow-up visit, though asymptomatic appeared pale, was found to have Hb of 7.3 g/dL (indicating ongoing hemolysis) and was given a blood transfusion.

In view of the nonimmune hemolytic picture without any identifiable RBC enzyme or membrane defect, genetic testing was planned. Targeted exon DNA sequencing of a 298-gene-panel (Orion Focus, Neuberg Diagnostics, Chennai, India) on Illumina platform and sequencing data analysis were performed as a commercial service by Neuberg Diagnostics. Of 64 million reads that were obtained, 95% aligned with the GRCh37/UCSC hg19 reference genome with high mapping quality. Sequencing coverage was 100% for 291 genes, including KLF1, and 90%–100% for 7 genes. Variant calling and filtering were as per the company's proprietary ORIONSeek algorithm that incorporates criteria defined by the American College of Medical Genetics. The sequencing revealed a single likely-pathogenic sequence variant. This G > A variant in the coding strand of KLF1 gene at chr19:12995815 (hg19 reference), had an allele frequency of 0.53 at a sequencing depth of 101 [Figure 1]b and [Figure 1]d, and is predicted to result in the substitution of glutamic acid with lysine at amino acid 325 (GenBank sequence NM_006563.4: C.973G>A; p. E325K). Sanger DNA sequencing of the parents' peripheral blood did not show this variant [Figure 1]e, which suggested that the baby's heterozygous KLF1 mutation was de novo. No sequence variation was seen in exons of the HBA1, HBB, or HBD Hb genes (that were also covered by the sequencing panel).

The infant has been on regular follow-up and receiving packed RBC transfusion and oral Vitamin B12 and folic acid supplementation as supportive management for ineffective erythropoiesis. The frequency of transfusion has reduced from the initial 4 weeks' interval to 2 months now. He is now 12 months old, with weight for age and length for age below the 3rd centile, age-appropriate acquisition of developmental milestones and mild splenomegaly. At present, the baby is not on iron chelators. It will be started after the tenth blood transfusion. Hematopoietic stem-cell transplant is being planned.

  Discussion Top

Ineffective erythropoiesis is reduction in the production of mature erythrocytes originating from a pool of immature erythroblasts, whereas dyserythropoeisis refers to abnormal morphology and/or maturation of erythrocytes that is associated with ineffective erythropoiesis. The preliminary diagnosis of CDA is based on both: morphological indicators of ineffective erythropoiesis (nucleated erythroblasts in PBS) and dyserythropoiesis (the presence of multinucleated erythroblasts, atypical cytoplasmic inclusions, and intercellular bridges in bone marrow smear).[2] The PBS in our case showed multiple polychromatic cells and numerous nucleated RBCs with binucleate forms. We did not consider bone marrow aspiration (BMA) as the monocytopenia, early transfusion dependency, and clinical exclusion of differentials that may have warranted a BMA, which prompted us to consider CDA and plan the specific genetic testing early on.

Nine genetically proven CDA Type IV cases have been described in the literature.[1],[3],[4],[5],[6],[7],[8],[9],[10] The clinical details of these and the present case are in [Table 1]. We have not included two cases that were reported as CDA Type IV on the basis of bone marrow study without genetic confirmation.[11],[12] The timing of clinical presentation is variable ranging from in utero (manifesting as hydrops in three cases), the neonatal period, and early childhood. The various manifestations and number of children in which they were seen include organomegaly,[6] anemia on 1st day of life,[5] jaundice,[3] pulmonary hypertension,[2] urogenital anomalies,[2] short stature,[2] and cardiomyopathy.[1] Complications of CDA IV are aplastic crisis, iron overload, hyperbilirubinemia, gallstones, and splenomegaly. This warrants frequent clinical and diagnostic monitoring.
Table 1: Profiles of reported cases of KLF1 mutation-associated congenital dyserythropoietic anemia Type IVb

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Since transfusion dependency is apparent early in life, the management of CDA Type IV is mainly frequent blood transfusions and iron chelation therapy.[2] Two children and one adult underwent splenectomy at 4, 4, and 36 years of age, respectively. Although hematopoietic stem-cell transplantation, interferon-alpha, activin receptor ligand trap, and gene therapy have been tried in other CDAs with variable success, their utility in Type IV remains unevaluated due to the condition's rarity.

Like in our case, all except one of the cases had the heterozygous NM006563: c.973G>A/p. E325K KLF1 mutation; the other two had heterozygous (c. 883delinsTT/p. H295 Lfs*58 and c. 902G>T/p. R301 L) mutations. The E325K variant is believed to arise de novo, and its inheritance has not been reported in any case, including ours [Table 1]. The E325 position is in the second zinc finger domain of KLF1 and is critical for its site-specific DNA binding. It is noteworthy that in the genetic code, only the c.973G>A type of single-nucleotide change can generate the E325K amino acid change and a change of the E325 acidic amino acid to another basic amino acid besides lysine (histidine and asparagine) is impossible with a single-nucleotide change.

Thus, genetic testing to identify such gene mutations is important to arrive at a diagnosis in transfusion-dependent anemia, once CDA is suspected. In our case, prompt genetic testing not only facilitated quick and accurate diagnosis but also let us avoid the highly invasive bone marrow biopsy procedure in a vulnerable neonate.

Declaration of patient consent

The authors certify that they have obtained all appropriate patient consent forms. In the form the patient(s) has/have given his/her/their consent for his/her/their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.

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Conflicts of interest

There are no conflicts of interest.

  References Top

Belgemen-Ozer T, Gorukmez O. A very rare congenital dyserythropoietic anemia variant-type IV in a patient with a novel mutation in the KLF1 gene: A case report and review of the literature. J Pediatr Hematol Oncol 2020;42:e536-40.  Back to cited text no. 1
Iolascon A, Andolfo I, Russo R. Congenital dyserythropoietic anemias. Blood 2020;136:1274-83.  Back to cited text no. 2
Wickramasinghe SN, Illum N, Wimberley PD. Congenital dyserythropoietic anaemia with novel intra-erythroblastic and intra-erythrocytic inclusions. Br J Haematol 1991;79:322-30.  Back to cited text no. 3
Arnaud L, Saison C, Helias V, et al. A dominant mutation in the gene encoding the erythroid transcription factor KLF1 causes a congenital dyserythropoietic anemia. Am J Hum Genet 2010;87:721-7.  Back to cited text no. 4
Jaffray JA, Mitchell WB, Gnanapragasam MN, et al. Erythroid transcription factor EKLF/KLF1 mutation causing congenital dyserythropoietic anemia type IV in a patient of Taiwanese origin: Review of all reported cases and development of a clinical diagnostic paradigm. Blood Cells Mol Dis 2013;51:71-5.  Back to cited text no. 5
de-la-Iglesia-Iñigo S, Moreno-Carralero MI, Lemes-Castellano A, et al. A case of congenital dyserythropoietic anemia type IV. Clin Case Rep 2017;5:248-52.  Back to cited text no. 6
Russo R, Andolfo I, Manna F, et al. Multi-gene panel testing improves diagnosis and management of patients with hereditary anemias. Am J Hematol 2018;93:672-82.  Back to cited text no. 7
Ravindranath Y, Johnson RM, Goyette G, et al. KLF1 E325K-associated congenital dyserythropoietic anemia type IV: Insights into the variable clinical severity. J Pediatr Hematol Oncol 2018;40:e405-9.  Back to cited text no. 8
Ortolano R, Forouhar M, Warwick A, et al. A case of congenital dyserythropoeitic anemia type IV caused by E325K mutation in erythroid transcription factor KLF1. J Pediatr Hematol Oncol 2018;40:e389-91.  Back to cited text no. 9
Jamwal M, Aggarwal A, Sharma P, et al. Congenital dyserythropoietic anemia type IV with high fetal hemoglobin caused by heterozygous KLF1 p.Glu325Lys: First report in an Indian infant. Ann Hematol 2021;100:281-3.  Back to cited text no. 10
Benjamin JT, Rosse WF, Daldorf FG, et al. Congential dyserythropoietic anemia-type IV. J Pediatr 1975;87:210-6.  Back to cited text no. 11
Mc Bride JA. Congenital dyserythropoietic anemia type IV. Blood 1971;38:837.  Back to cited text no. 12


  [Figure 1]

  [Table 1]


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