Congenital Anemia Due to Erythropoietin Gene Mutation Presenting With Diamond–Blackfan Anemia-like features

Abstract

Diamond–Blackfan anemia (DBA) is an inherited hypoplastic anemia, caused by mutations in ribosomal protein genes. Other mutations such as mutations in the erythropoietin (EPO) gene can lead to DBA-like (DBAL) through impairment of erythropoiesis. We present a 9-year-old patient with normocytic, normochromic, transfusion-dependent anemia since birth and reticulocytopenia. Bone marrow biopsies showed erythroid hypoplasia thereby excluding myelodysplasia and iron deficiency. Whole-exome sequencing revealed a homozygous mutation (c.530G>A; p.R177Q) in the EPO gene, which encodes a protein involved in erythroid progenitor-cell proliferation, confirmed the diagnosis of the DBAL. Diagnosis of congenital anemias has been complicated by their similar characteristics with DBA, but genetic testing is important in detecting rare causes of these disorders. This diagnosis enabled us to use recombinant human EPO therapy which reduced the need for blood transfusion.

Introduction

Diamond–Blackfan anemia (DBA) is a rare and severe congenital hypoplastic anemia caused by dominant mutations in ribosomal protein genes. DBA-like (DBAL) is a rare disease caused by recessively inherited mutations in the erythropoietin (EPO) gene. DBA and DBAL predominantly occur in infancy or early childhood. ¹

EPO, produced and released in the fetal liver early in gestation and after birth in the kidneys, is responsible for stimulating erythroid progenitor cells in the bone marrow. Mutations or deficiencies of EPO can lead to inadequate erythropoiesis ² and phenotypes similar to those of DBA.

Several theories have been proposed regarding the etiology of DBA. DBA was historically considered a ribosomopathy due to genetic mutations in 1 of 19 ribosomal proteins affecting ribosome synthesis. However, subsequent investigations have reported 3 other noncoding gene mutations, GATA1 (an erythroid transcription factor), TSR2 (which encodes a direct binding partner of RPS26), and EPO. The characteristic hematological findings of DBA include severe normochromic macrocytic anemia and reticulocytopenia, isolated erythroid aplasia in the marrow, and increased erythrocyte adenosine deaminase levels. ³

Case Presentation

We report a case of a 9-year-old boy with a longstanding history of anemia, which began as early as 7 months of age. His mother took him to seek medical care after observing that he appeared pale and was increasingly tired. Blood tests at that time showed extreme anemia, necessitating a transfusion. He has required many transfusions and iron supplementation since. Despite several investigations, no one was able to successfully identify the exact cause of his anemia.

His past surgical history is significant for an orchiopexy for an undescended testis and a tonsillectomy at the age of 6, and he has no other past medical history aside from his anemia. He had no history of serious infections, developmental delays, or other major health problems. His parents say that, between transfusions, he was active and participated in everyday life but tired more quickly than his peers, and they also reported that his condition affected his school performance.

When first evaluated in 2015, he appeared generally well but pale. His liver or spleen were not enlarged, there were no signs of bleeding and the rest of the examination was unremarkable. Blood work showed anemia with an inappropriately low reticulocyte count (1%) for anemia—which meant his bone marrow wasn’t producing enough red blood cells (RBCs). Bone marrow biopsy in 2016 demonstrated a slightly hypocellular bone marrow (70%) for his age, trilineage hematopoiesis with normal maturation, no evidence of myelodysplasia or marrow infiltration, decreased stainable iron, and no ring sideroblasts.

Follow-up on the patient was lost from 2016 to 2023. He was reexamined when he returned in early 2024. His anemia persisted. His bone marrow biopsy showed the same previous 2016 bone marrow biopsy findings: mild bone marrow suppression without evidence of significant disease. The occult blood test was positive and a genetic test for EPO gene confirmed homozygous mutation. The patient, who was born to consanguineous parents, was diagnosed with DBAL, which was caused by loss-of-function mutation in the EPO gene.

Whole-exome sequencing identified a homozygous mutation at chromosome 7:100723081 in the EPO gene. This was a c.530G-to-A transition in exon 5, leading to amino acid substitution from arginine to glutamine (R177Q), which was subsequently confirmed by using Sanger sequencing.

With this new diagnosis, a way forward seemed clearer. Testing for his EPO levels confirmed a normal EPO level which is 40.6 mIU/ml. He also required an extensive gastrointestinal workup to determine the source of blood loss. Family genetic testing might help determine if his parents carried the mutation. Because EPO is produced in the kidneys, his renal function also had to be evaluated.

When considering his options, a trial of recombinant human EPO (rhEPO) was discussed to potentially stimulate RBC production and reduce his dependence on transfusions. If this was unsuccessful, a trial of corticosteroids may still be considered although less likely to be beneficial if the EPO pathway is disrupted and Iron levels also need to be managed, especially if there is ongoing, low-grade gastrointestinal blood loss.

The patient was started on darbepoetin alfa and at 0.45 µg/kg once weekly. Treatment was well tolerated and the regimen was modified to a dose of 0.75 µg/kg every 2 weeks. Hemoglobin levels increased from 9.7 to 11.4 g/dL over 9 weeks, indicating a response to therapy, and suggesting preserved erythropoietic capacity despite the EPO gene mutation.

Discussion

Congenital hypoplastic anemia due to EPO gene mutation is rare, but it can have considerable clinical effects. It can be caused by a mutation in the EPO gene or its relatives, such as EPAS1. The anemia associated with this mutation is characterized by normocytic normochromic anemia, reticulocytopenia, and relative EPO deficiency. ²

The risk factors for developing anemia due to EPO gene mutation are geographical location and ethnicity, in which certain mutations are linked to a certain population, such as novel mutations in the EPO receptor (EPO-R), VHL, and EPAS1 genes that have been identified in cohort studies in India. ⁴ The other risk factors are genetic predisposition, family history, and consanguinity. ²

Many cases of rare congenital hypoplastic anemia are due to non-RP mutated such as GATA1, EPO, ADA2. ⁵ As in our case, we found a patient with loss-of-function EPO mutation which causes DBAL, and this mutation is inherited in an autosomal recessive manner. ⁶

The EPO is an important hormone working as a growth factor for erythroid progenitor cells. ⁷ Human (EPO) is a glycoprotein with an acidic nature and a molecular weight of 30.4 kDa. It consists of a peptide core structure, which contains single chain of 165 amino acids, and has a specific binding property to the EPO-R, ⁸ which is important for RBC production.

Expression of the EPO gene is related to oxygen concentration, ⁷ which is regulated by inhibitory and stimulatory transcription factors. Inhibitory factors like GATA-2 and NF-κB help control the production of EPO, and stimulatory factors like hypoxia-inducible transcription factor-2 and hepatocyte nuclear factor-4 alpha which play a dominant role in boosting EPO expression. ⁸

Two different mutations may occur in EPO gene expression, the first gain-of-function mutations, which cause erythrocytosis. ² This form of erythrocytosis is inherited in an autosomal dominant pattern. ⁹ The second is loss-of-function mutations, which cause congenital hypoplastic anemia. ²

In a study by Kim et al, the researchers found a homozygous mutation in the EPO gene (located on chromosome 7), specifically a c.530G-to-A transition in exon 5. This mutation leads to an amino acid change, from arginine to glutamine (R150Q). This is similar to our case, the researchers ruled out other mutations in genes known to cause DBA, and they performed functional studies of the EPO protein in the lab, by using recombinant wild type and mutant EPO. They found the R150Q mutation mildly affected its binding affinity to the EPO-R, but severely affect the kinetics of binding to the receptor, with faster dissociation compared to wild type. Further testing with cultured erythroid cells and hematopoietic stem cells showed that even though there was a higher concentration of the mutant EPO protein, it couldn’t correct the defect in RBC production. Additionally, by using flow cytometry and Western blotting, experiments were done and showed that the R150Q mutation caused decreased phosphorylation of important proteins (JAK2, STAT1, and STAT3) that are part of the signaling pathway activated by the EPO-R. Also, the mutation caused impairment of the dimerization of the EPO-R, which is important for activating JAK2 and triggering the downstream signaling needed for proper RBC production. ¹⁰

In the past 20 years, the recombinant EPO therapy had a good impact on people who suffer from anemia caused by chronic kidney disease, and this therapy decreased the need for blood transfusion and improved heart function and cognitive ability. ⁸ Also, there is good response and improvement in congenital hypoplastic anemia in some patients who were treated with recombinant EPO therapy. ¹⁰ On the other hand, some patients are treated with corticosteroids, which stimulate early erythroid progenitors independently of EPO, leading to improvement in anemia. ¹¹

As a summary, the management of this type of congenital anemia is by regular monitoring of the hemoglobin and RBC levels, blood transfusion as needed, and, in some cases, rhEPO therapy can be used. Although its effect is variable based on the response of the EPO-R and the genetic mutation, that is, present. ¹²

Conclusion

This case illustrates a rare and under-recognized genetic cause of anemia that does not cause bone marrow failure but instead it results in an abnormality in EPO signal transduction. This clinical recognition of congenital EPO mutation is particularly relevant as it identifies a target for treatment with EPO, with the aim to improve the patient’s quality of life and reduce his transfusion burden. This case demonstrates the value of genetic testing in the evaluation of unexplained anemia in childhood, as it can diagnose a rare disease that could go undiagnosed for years.