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Autosomal dominant form of SCID due to gain-of-function RAC2 mutation
addition to the hematopoietic defect, patients with RD have bilateral sensorineural deafness.4 We and others have previously demonstrated that bi-allelic mutations in the gene coding for adenylate kinase 2 (AK2) are involved in the RD phenotype by disrupting the ATP production required to sustain the survival and differentiation of hematopoietic stem/progenitor cells (HSPC). The high AK2 expression levels in the inner ear may also account for the sensorineural hearing loss observed in patients with RD.5-7
We have identified an autosomal dominant (AD) mis- sense mutation in the RAC2 gene (coding for Ras-related C3 botulinum toxin substrate 2 [RAC2]) in three SCID patients whose clinical presentation overlaps with the RD SCID but who lack AK2 mutations and deafness. RAC2 belongs to the Rac subfamily of RHO small GTPases. In the inactive GDP-bound state, RAC2 is located in the cytosol. Upon stimulation and activation by guanine nucleotide exchange factors, the active RAC2-GTP-bound form translocates to the plasma membrane. There, active RAC2 protein triggers various signaling pathways until the GTP is hydrolyzed following binding to GTPase-acti- vating proteins (GAP).8-10 Unlike the other members of the Rac subfamily (RAC1 and RAC3), RAC2 is mostly expressed on hematopoietic cells.11,12 Using biochemical and in vitro differentiation assays, we demonstrated that the RAC2 mutation was closely related to an impairment in cell differentiation capacity and defects in cellular and mitochondrial networks. Taken as a whole, our data demonstrate that a dominant gain-of-function (GOF) mutation in the GDP/GTP binding site of the RAC2 pro- tein inhibits HSPC differentiation and leads to a severe AD form of SCID with a clinical presentation of RD.
Methods
Patients and human cord blood samples
The study was conducted in accordance with French legislation and the principles of the Declaration of Helsinki. Informed con- sent was obtained from the patients’ parents or legal guardians, and the study protocol was approved by the regional independent ethics committee and the French Ministry of Research (DC 2014- 2272/2015/ DC-2008-329). Primary fibroblasts were obtained from skin biopsies.
Human cord blood (CB) samples eligible for research purposes were obtained from the Cord Blood Bank at St Louis Hospital (Paris, France; authorization 2014/09/23). Mononuclear cells were isolated by density separation on Lymphoprep (Abcys). CD34+ HSPC were sorted magnetically using the autoMACSpro separa- tor, and cell purity was checked with a MACSQuant analyzer (Miltenyi Biotec) before transduction and culture (Online Supplementary Methods).
Gene sequencing and gene expression in the patient's fibroblasts
Genomic DNA was isolated by phenol/chloroform extraction from primary fibroblasts (from P1, P2 and P3) or peripheral blood mononuclear cells (PBMC, from P3’s father). Whole-exome sequencing was performed with an Illumina TruSeq exome enrichment kit (Illumina), using 100 bp paired-end reads. Eighty- five percent of the target regions were observed with a coverage >20×. Variant calling was not based on a particular genetic model. The mutation was confirmed by Sanger sequencing. For western blot analyses, cells were lysed in Tris buffer (20 mM Tris, pH 7.9;
300 mM NaCl; 1% Nonidet P-40) supplemented with protease and phosphatase inhibitors. Cell extracts were separated by SDS- PAGE, blotted, and stained with anti-RAC2 (ab154711, Abcam) or anti-GAPDH (SC-32233, Santa Cruz, CA, USA) antibodies. After staining with an HRP-conjugated secondary antibody, the immunoblot was developed and quantified in an Odyssey system (LiCor).
RAC2 activation assays and immunoblotting with the HEK293T cell line
The HEK293T cells were cultured overnight prior to transduc- tion with the appropriate lentiviral supernatant (Online Supplementary Methods). After two days of culture, the cell pellets were frozen in liquid nitrogen. Levels of activated RAC2 were determined using the G-LISA® RAC Activation Assay Biochem KitTM (#BK125, Cytoskeleton Inc.) according to the manufactur- er’s instructions, except that RAC2 monoclonal specific antibody (AT2G11, sc-517424 Santa Cruz Biotechnology, Inc.) and HRP- conjugated anti-mouse antibody (#1721011, Bio-Rad) were used to detect the amount of captured active RAC2 (details in the Online Supplementary Methods).
Homology modeling
Three-dimensional homology models were built for the G12R mutant of human RAC2 (Uniprot P15153) using MODELLER soft- ware (version 20, Webb and Sali, 2016, 2017). The crystal structure of wild-type (WT) human RAC2 (PDB code: 1DS6) was used as a template.
Statistical analysis
For all analyses, three or more independent experiments were performed. Data are reported as the mean±standard error of the mean (SEM). Two-tailed, unpaired t-tests were performed using Prism 4 software (GraphPad). P<0.05 was considered statistically significant.
Results
A missense mutation (G12R) in the RAC2 GTPase protein leads to a severe combined immunodeficiencies phenotype
A few members of our cohort of patients with a SCID phenotype do not yet have a molecular diagnosis. Three patients (Figure 1A) from two unrelated kindreds (P1 from one kindred, and mother and daughter P2 and P3 from another kindred) presented with all the immunological and clinical features of RD other than deafness. As observed in patients with RD, the complete absence of neutrophils was responsible for the occurrence of severe infections (Table 1) earlier than is usually observed in other forms of SCID.3 The analysis of other blood cell lin- eages highlighted the absence of T and B lymphocytes and circulating monocytes. The T-cell receptor excision circle (TREC) level was very low (<5 copies/mL in P3, the only patient tested, vs. >34 copies for a control), reflecting defective T-cell maturation. Conversely, the platelet count was normal and (in P3 only) the hemoglobin level was slightly below normal (Table 1). Overall, the peripheral cell count profile was in line with the severe hypoplastic content of the patients’ bone marrow (BM) (Table 1 and Online Supplementary Table S1). HSCT performed soon after birth was successful in P1 (who was previously described as P4 by André-Schmutz et al.13) and P2, with full donor immune reconstitution, demonstrating that the
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