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Autosomal dominant form of SCID due to gain-of-function RAC2 mutation
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Figure 3. The G12R mutation inhibits hematopoietic stem/progenitor cell (HSPC) proliferation and differentiation. (A) Proliferation of CD34+ cells in an 8-day culture. Flow cytometry was used to analyze the change over time in the percentage (%) of GFP-express- ing cells (GFP+) from day 2 (when the transduc- tion efficiency was measured) until day 8 (the end of the culture). (B) The proportions of “ROS low”, “DILC1(5) low” and annexin-V-positive GFP+ live cells were determined on day 4. All the analyses were performed in the live cell (7-AAD- negative) gate. The results are quoted as the mean±standard error of mean(SEM) of four independent experiments. (C) Neutrophil differ- entiation in a 7-day culture. Flow cytometry was used to analyze the change over time in the per- centage (%) of GFP+ cells from day 2 (when the transduction efficiency is measured) until day 8 (end of the culture) and the number of granulo- cytes (CD11b+CD15+) among the GFP+ live cells. (D) The proportions of “ROS low”, “DILC1(5) low” and annexin-V-positive cells among the GFP+ live cells were analyzed on day 4. Results are quoted as the mean±SEM of three inde- pendent experiments. (E) T-cell differentiation in a 7-day culture (n=3). Flow cytometry was used to analyze the change over time in the percent- age (%) of GFP+ from day 2 (when the transduc- tion efficiency is measured) until day 7 (end of the culture). The number of T-cell progenitors (CD7+) was evaluated by flow cytometry on day 7 in the GFP+ live cell gate (7-AAD-negative). The results are quoted as the mean±SEM of three independent experiments. *P<0.05; **P<0.01; ***P<0.001. WT: wild-type.
Discussion
In three SCID patients with innate and adaptive immune system defects, we identified a heterozygous, dominant missense mutation (p.G12R) in the highly con- served GDP/GTP binding domain of the RAC2 protein. Given the severity of the clinical presentation, the patients had to undergo HSCT in the first few weeks of life. The observation of AD inheritance broadens the clinical spec- trum of RD, as we can now distinguish between two forms: a recessive syndromic form associated with deaf- ness and AK2 mutations, and a non-syndromic disease associated with a hypoplastic BM and a specific AD G12R mutation in RAC2. The absence of sensorineural hearing loss in the latter form might be correlated with the pre- dominant expression of RAC2 in hematopoietic lineages. These data fit with the observation whereby in murine models, RAC1 and RAC3 (but not RAC2) are involved in the development of the inner ear;25 however, this has yet to be confirmed in human studies. The HSCT performed as soon as possible after birth in two patients with the G12R mutation restored hematopoiesis and highlights the non-redundant regulatory role of RAC2 in this process. In line with this observation, we demonstrated the drastic effect of the G12R RAC2 mutation on cell proliferation and survival, especially in cord blood HSPC. These results are in agreement with previous reports on the role of RAC2 in the regulation of HSPC10,26 and during T-cell dif-
ferentiation via the activation of Wnt/β-catenin pathways.9 Furthermore, the constitutive active form of RAC2 G12V has already been described as preventing thymocyte differentiation and inducing apoptosis in the mouse.27 It should be noted that, despite the observation of fibroblast defects in vitro, skin alterations have not been noted for P1 and P2 more than 10 years after HSCT, sug- gesting that, in vivo, other RHO family members (or per- haps other molecules present in the microenvironment) provide compensatory survival signals to non-hematopoi- etic cells. The same dichotomy has been reported in RD: fibroblasts from RD patients displayed impairments in apoptosis and mitochondrial function in vitro,5 whereas the patients having undergone HSCT have not reported any skin or tissue lesions (24 years after HSCT, for the case with the longest follow-up). Taken as a whole, these data demonstrate that RAC2 is mandatory at different hematopoietic checkpoints; hence, the unregulated activi- ty of GTP-bound RAC2 driven by the G12R mutation might explain the severe BM hypoplasia and the absence of circulating leukocytes in P1, P2, and P3.
In HSPC, RHO GTPases also regulate cell trafficking and ROS production.9,26 In particular, the literature data show that RAC2 mutant cells are associated with unbalanced actin cycling and hyper-segmented nuclei,15,16,18,19,28 two fea- tures associated with defective cytokinesis, a process involving RHO GTPases.29-31 The highly segmented nuclear membranes found in P3’s fibroblasts are consistent
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