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. 2023 Feb 1;108(2):581-587.
doi: 10.3324/haematol.2022.281277.

Mutations in the RACGAP1 gene cause autosomal recessive congenital dyserythropoietic anemia type III

Gonzalo Hernández  1 , Lídia Romero-Cortadellas  2 , Xènia Ferrer-Cortès  1 , Veronica Venturi  2 , Mercedes Dessy-Rodriguez  3 , Mireia Olivella  4 , Ammar Husami  5 , Concepción Pérez De Soto  6 , Rosario M Morales-Camacho  7 , Ana Villegas  8 , Fernando-Ataulfo González-Fernández  8 , Marta Morado  9 , Theodosia A Kalfa  10 , Oscar Quintana-Bustamante  3 , Santiago Pérez-Montero  11 , Cristian Tornador  11 , Jose-Carlos Segovia  3 , Mayka Sánchez  12
Affiliations

Mutations in the RACGAP1 gene cause autosomal recessive congenital dyserythropoietic anemia type III

Gonzalo Hernández et al. Haematologica. .
No abstract available

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Figures

Figure 1.
Figure 1.
RACGAP1 is mutated in autosomal recessive congenital dyserythropoietic anemia III cases. (A) Pedigrees of the 3 families with affected individuals shown in black and healthy carriers shown in half-filled symbols. The family members shown with white symbols were not tested. RACGAP1 genotypes at the protein level are indicated below each individual. Squares denote males, circles denote females, and triangle denotes pregnancy loss. (B) Bone marrow film images showing a multinucleated erythroid cell (top left), an inter-cytoplasmic chromatin bridge between two multinucleated erythroid cells (top right), a multinucleated erythroid form with karyorrhexis (bottom left) and a giant erythrocyte (bottom right) in patient A.II.2 and multinucleated and dysplastic erythroid progenitors in patient B.II.1. Magnification 100x, scale bars represent 5 µm. Additional images are available in the Online Supplementary Figure S1 for patient A.II.2. Bone-marrow images from C.II.3 patient are reported elsewhere. (C) Top: schematic representation of RACGAP1 protein domains indicating the localization of the p.Pro432Ser and p.Thr220Ala mutations (CC = coiled-coil, BR = basic region, C1 = cysteine-rich domain, GAP = GTPase-activating domain). Bottom: phylogenetic protein sequence alignment (CLUSTAL omega) of partial RACGAP1 protein sequences showing conservation for Pro432 and Thr220 amino acids. An asterisk (*) denotes fully conserved residue; a colon (:) indicates conservation between amino-acids with strongly similar properties and a period (.) indicates conservation between amino-acids with weakly similar properties.
Figure 2.
Figure 2.
In vitro erythroid differentiation of patients’ CD34+ cells derived from peripheral blood. (A) Cytometry data of the in vitro erythroid differentiation at different time points showing the proportions of erythroid progenitors, erythroblasts, and erythrocytes in the sample (n=1). Results show 10.3% erythrocytes in controls vs. 4.9% in A.II.2 cells and 1.92% erythroid progenitors in controls vs. 10.3% in A.II.2 cells at day 11 and 41.7% erythrocytes in controls vs. 30.7% in A.II.2 cells and 0.79% erythroid progenitors in controls vs. 10.96% erythroid progenitors in A.II.2 cells at day 14. Chi-square test with 2 degrees of freedom was performed. (B) Quantification of late erythroblast cells at various stages of the enucleation process in cytospins prepared at day 14. Chi-square test with 3 degrees of freedom was performed (n=484-492). (C) Cytospin preparations stained with modified Wright stain from patients A.II.2 and B.II.1 at day 14 showing multinucleation, nuclear deformations, and mitotic alterations. Scale bars represent 5 mm. (D) Quantification of the percentage of multinucleation at day 14 from the cytospins shown in (C). Chi-square test with 2 degrees of freedom was performed. (E) Cytospin preparations at day 14 showing enucleated cells and cells undergoing enucleation. Scale bars represent 5 mm. (F) Quantification of the area of the enucleated cells from panel (E) showing macrocytosis in the cultures from patient A.II.2’s CD34+ cells that is corrected after transduction with the wild-type (WT) RACGAP1 lentiviral construct (n=252-257). Cell area was calculated using Fiji software by selecting cells using a threshold and watershed separation followed by area determination using the Measure particle program. Due to lack of normality of the data (Kolmogorov-Smirnov test), a Krustal-Wallis test with a Dunn’s multiple comparisons test was performed instead of a one-way ANOVA. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.
Figure 3.
Figure 3.
Patients’ RACGAP1 mutations alter normal GTPase balance status and model of GTPase regulation in cytokinesis. (A) Amount of active GTPases (RHOA, RAC1 and CDC42) determined by G-LISA in patient-derived lymphoblastoid cell lines (LCL) from A.II.2 and B.II.1 patients normalized to the mean of 2 different controls. G-LISA experiments were done in quadruplicate (n=4). The Mann-Whitney U test was performed to compare GTPase activation levels in each patient with respect to controls. Dotted lines represent the reference of control levels set to 1.0. Error bars represent the mean +/- standard deviation. *P<0.05, **P<0.01, ***P<0.001. (B) The intermediate step of GTPase activation assessment required for the results shown in (A). Immunoblot of total amounts of RHOA, RAC1 or CDC42 in patients and controls. Three independent protein extracts were generated for each patient/control. The mean amount of each total GTPase was then used to correct the corresponding GTPase activity levels represented in (A). (C) Model for GTPase activation/inactivation in cytokinesis. Correct GTP balance is crucial for cytokinesis. In wild-type (WT) cells (upper panel), RACGAP1 activates RHOA by recruiting the GEF protein ECT2 and directly inactivates RAC1 through its GAP domain, reducing the RAC1-mediated RHOA inhibition. PRC1 inhibits the GAP activity inactivating CDC42. These events generate a strong positive signal to assemble the contractile ring required for cytokinesis. Both mutations (bottom panel) could diminish the RACGAP1-PRC1 interaction, releasing the inhibition to inactivate CDC42. The activation levels of RHOA and CDC42 are diminished while the activation levels of RAC1 are increased, leading to a stronger negative signal towards cytokinesis that leads to a higher degree of multinucleation.
See this image and copyright information in PMC

References

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