Congenital dyserythropoietic anemia type III (CDA III) is one of the rarest types of CDA. The autosomal dominant form of CDA III, is due to mono-allelic mutations in the KIF23 gene (MIM:105600); two such mutations described so far in a total of three families.1,2 KIF23 encodes mitotic kinesin-like protein (MKLP1), which dimerizes and combines with a homodimer of the RACGAP1 protein (Rac GTPase-activating protein 1), to form the centralspindlin complex regulating Rho GTPase activity and required for cytokinesis.3,4 Sporadic cases with CDA III pathology have been reported, suggesting a different genetic alteration.5 All reported CDA III cases present with a core phenotype consisting of variable degree of macrocytic anemia, signs of intravascular hemolysis, and giant multinucleated erythroblasts in the bone marrow. Additional symptoms such as multiple myeloma, monoclonal gammopathy, angioid streaks, hemosiderinuria, hepatosplenomegaly, iron overload or cirrhosis were described in some CDA III patients.6-9 Using next generation sequencing (NGS) in combination with ex vivo erythroid differentiation we identified two pathogenic missense mutations in the RACGAP1 gene in three unrelated families affected with the recessive form of CDA III.
Written informed consent was obtained for all participants in this study that was conducted in accordance with the Declaration of Helsinki’s ethical principles, and the protocol was approved by the Ethics Committee associated with the UIC on 04/08/2021.
The first patient is an 18-year-old male (Family A, II.2, Figure 1A) diagnosed at the age of 4 months suffering from severe macrocytic anemia and hepatosplenomegaly. The parents come from a small village in the South of Spain; with a possible common ancestor 11 generations prior to the proband. Peripheral blood and bone marrow showed multiple erythroid abnormalities, including multinucleated erythroblasts (Online Supplementary Table S1; Figure 1B; Online Supplementary Figure S1A to H). At 18 months of age, radiography of the skull showed a hair-on-end appearance and a computed tomography scan revealed widening of the diploic space (data not shown), both characteristic features of chronic hemolytic disease with ineffective erythropoiesis. Hemolysis was confirmed by increased plasma bilirubin, lactate dehydrogenase (LDH) and undetectable haptoglobin. Serum ferritin levels are normal at 18 years of age and iron overload is not a clinical problem as there is loss of iron via hemosiderinuria (Online Supplementary Figure S1J; Online Supplementary Table S1). Currently, the patient presents with splenomegaly, but not obvious ophthalmological problems or biliary lithiasis. Overall, the hematological and cytomorphological studies support the diagnosis of CDA type III. The second patient is a 35-year-old female (Family B, II.1, Figure 1A), with unknown parental consanguinity and a history of antiphospholipid syndrome and papillary thyroid cancer. At birth, she presented with aregenerative macrocytic anemia (Online Supplementary Table S1) and significant hepatosplenomegaly. At the age of 5 months, she was diagnosed with CDA III based on bone marrow morphology (Figure 1B). Splenectomy, done at 9 years of age, reduced her transfusion requirements but moderate macrocytic anemia has persisted (hemoglobin [Hb] 96-101 g/L, mean corpuscular volume [MCV] 123 fL). She has short stature and skull defects secondary to increased extramedullary hematopoiesis. Electron microscopy of bone marrow images show irregular heterochromatin and iron-filled mitochondria.10,11
A 40-year-old male of Sephardic Jewish descent with the diagnosis of CDA III since childhood (Family C, II.3, Figure 1A), based on bone marrow evaluation, was enrolled in the CDA Registry of North America (CDAR).12 Family history was negative for anemia; reportedly his parents were second cousins. He was born at full-term and was diagnosed with significant anemia at 4 months of age, requiring transfusion. He continued to receive three to four transfusions per year for a Hb trough <60 g/L up to 24 years of age (Online Supplementary Table S1). He had hepatosplenomegaly and skull changes due to bone marrow expansion. Despite deferoxamine infusion subcutaneously 5 nights/week at 12-18 years of age, magnetic resonance imaging (MRI) at 24 years of age showed significant iron overload. Therefore, he was started on a chronic transfusion regimen every 2-4 weeks, with the goal to maintain Hb >100 g/L to suppress ineffective erythropoiesis and on deferoxamine continuous intravenous infusion for 9 months, followed by deferasirox up to 34 years of age when he received hematopoietic stem cell transplant.13 The patient reports azoospermia discovered at 24 years of age.
Common clinical hematological features in our three reported patients are: macrocytic anemia, aberrant giant multinucleated erythroblasts in the bone marrow and skull defects secondary to severe anemia with ineffective erythropoiesis. Aside from these core features, a clinical variability exists in both KIF23 and RACGAP1 patients and a deeper phenotypic characterization will require further cases.
Initially, NGS panels failed to detect pathogenic mutations in known genes associated with congenital anemia (e.g., CDAN1, CDIN1, SEC23B, KIF23, KLF1 and GATA1). In Family A, whole exome sequencing (WES) was performed and predicted pathogenic variants with a minor allele frequency (MAF) <0.03, and proper segregation were selected. RAC-GAP1 gene was prioritized as a candidate gene due to its role in cytokinesis and its partnering with MKLP1. A very rare homozygous missense mutation (c.1294C>T;p.Pro432Ser) in RACGAP1 was found in proband A.II.2 (Figure 1A; Online Supplementary Figure S1K). In patient B.II.1, another rare homozygous missense mutation, (c.658A>G;p.Thr220Ala) was found (Figure 1A; Online Supplementary Figure S1K). By means of WES in patient C.II.3, the same homozygous p.Thr220Ala mutation was found (Online Supplementary Figure S1K). Carrier status for relatives in Family C could not be evaluated as no DNA was available.
p.Pro432Ser and p.Thr220Ala are rare genetic variants (rs760038605 and rs1264268274) with a very low allele frequency (MAF 0.000008 and 0.000004; GnomAD_exomes). Both classified as pathogenic according to ACMG rules (PM2, PP3 and PS3) with CADD scores of 27.3 and 23.4, respectively. Mutations were submitted to ClinVar database (accession number: SCV002540790 and SCV002540791). Pro432 and Thr220 are 100% conserved among vertebrates and are located in the GTPase-activating domain and in the basic region of the RACGAP1 protein, respectively (Figure 1C). The RACGAP1 basic region is required for the interaction with the RHOA–GEF ECT2,14 among other proteins. Computational modeling of RAC-GAP1 in complex with RHOA, CDC42, and RAC1 GTPases suggests that the mutation p.Pro432Ser induces local structural changes affecting two RACGAP1 loops involved in complex formation with each of these GTPases (Online Supplementary Figure S2A). Modeling of the Thr220Ala mutation is not possible as the crystal structure containing this region of RACGAP1 (or homologous protein) is not available.
Given the erythroid-restricted phenotype associated with CDA III, we investigated by ex vivo CD34+ erythroid differentiation whether the identified RACGAP1 mutations cause alterations like those observed in our patients using flow cytometry and cytospin methods (Figure 2). Both mutations impaired cell growth (data not shown), especially the p.Thr220Ala mutation, therefore, only the erythroid differentiation of A.II.2 patient was fully assessed. At terminal differentiation (day 14), A.II.2-derived-erythrocytes (CD71-;CD235a+ cells) were reduced compared with control cells (Figure 2A, lane 13 and 14). Concomitantly, the number of enucleated cells in cytospins was also reduced (Figure 2B). Lentiviral transduction with wild-type (WT) RACGAP1 gene restored the erythrocyte population and reduced multinucleated cells enucleating back to control levels at day 14 (Figure 2A, lanes 14 to 16; Figure 2B). This data is supported by cytospin evaluation showing significant multinucleation in ex vivo erythropoiesis, which improved after lentiviral transduction of the WT RACGAP1 gene (Figure 2C to D). Multinucleation defects were also observed in siRNA RACGAP1-silenced HeLa cells. Cytokinesis failure was rescued by WT RACGAP1 construct but not by p.Pro432Ser or p.Thr220Ala mutated RACGAP1 constructs (Online Supplementary Figure S2B to D). Importantly, we observed that ex vivo erythropoiesis from A.II.2 CD34+ cells phenocopied patient macrocytosis. Lentiviral transduction of WT RACGAP1 gene also rescued the macrocytic phenotype (Figure 2E and F). Collectively, this data indicates that RACGAP1 mutations are responsible for the macrocytosis, multinucleation and erythroid defects seen in our patients.
RACGAP1 is a known GTPase regulator required for RHOA activation and for RAC1 inactivation to promote cytokinesis. In patient-derived lymphoblastoid cell lines (LCL) we observed decreased levels of active RHOA and CDC42 GTPases, positive regulators of cytokinesis, and increased levels of active RAC1, an inhibitor of cytokinesis (Figure 3A to C). Our data suggests that RACGAP1 mutations results in a GTPase imbalance leading to cytokinesis blockage, which could explain the multinucleation defects observed in patient’s bone marrow erythroblasts in HeLa cells and in the ex vivo CD34+-differentiation.
While preparing this submission, it came to our attention a recent report of a single sporadic case of CDA III with p.Leu396Gln and p.Pro432Ser RACGAP1 mutations, published as a letter to the editor in the Blood journal.15 The coincidence of the p.Pro432Ser mutation found in both works (Spanish and South American families) may represent a “hot spot” or a founder mutation in the RACGAP1 gene. The work by Wontakal et al.,15 done independently and in parallel to ours, reinforces our findings and gives some functional data based on HeLa and control erythroid cells. Our work contributes with extensive clinical data in three unrelated patients, detailed characterization of the erythropoiesis defects using patient-derived CD34+ cells, and provides a deeper insight into the molecular mechanisms of these alterations by showing in patient-derived cells a GTPase imbalance that could result in defective cytokinesis.
Overall, our work demonstrates that mutations in the RAC-GAP1 gene cause a recessively inherited form of CDA III, characterized by moderate to severe anemia due to cytokinesis failures producing multinucleated erythroblast and subsequently ineffective erythropoiesis.
- Received April 29, 2022
- Accepted September 23, 2022
No conflicts of interest to disclose.
Study concept and research design by MS. Patients’ clinical data, blood and bone marrow images by IP-S, R-MM-C, MM, F-AG-F, AV and TAK. Sequencing experiments and mutation validation by XF-C, MS, AH, TAK and CT. WES data analysis by GH, CT, AH, and TAK. Molecular experimental work, data plot and statistical analyses by GH, MD, OQ-B and LR-C. Computational modelling GH and MO. Writing of the manuscript by GH, MS, VV, LR-C and TAK. Funding recruitment by MS, SP-M, J-CS, CT and TAK. All authors participated in data discussion, read, and approved the manuscript.
Data will be shared upon reasonable request.
We are very grateful to all families and patients who kindly contributed to this study. We thank Dr Vladimir Benes from European Molecular Biology Laboratory (EMBL), Genomics Core Facility, Heidelberg, Germany for his excellent service in whole exome sequencing. We thank Dr Katie G. Seu and Dr Mary Risinger from Cincinnati Children’s Hospital Medical Center for their assistance in proof reading of this manuscript.
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