From PGT-M Discovery to Mechanism: Functional Validation of novel compound heterozygous RAG1 Mutations in Severe Combined Immunodeficiency

From PGT-M Discovery to Mechanism: Functional Validation of novel compound heterozygous RAG1 Mutations in Severe Combined Immunodeficiency | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article From PGT-M Discovery to Mechanism: Functional Validation of novel compound heterozygous RAG1 Mutations in Severe Combined Immunodeficiency Yongxiang Liu, Xiongwei Shan, Guilan Huang, Yuwei Duan, Yunshi Li, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9021280/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 9 You are reading this latest preprint version Abstract Purpose Severe combined immunodeficiency(SCID) is a life-threatening primary immunodeficiency disorder. This study aimed to identify novel recombination activating gene 1 ( RAG1 ) variants in a Chinese pedigree and characterize their impact on protein structure and function, providing a genetic basis for preimplantation genetic testing for monogenic (PGT-M) cycle. Methods Potential RAG1 mutations of the probands were screened by whole-exome sequencing (WES) and confirmed by Sanger sequencing. Configuration predictions of the variants were achieved using SWISS-MODEL. PROVEAN, PolyPhen-2 and MutationTaster were used to predict their pathogenicity. Isogenic pre-B cell lines carrying the mutations were established via CRISPR-Cas9 RNP editing. Functional impacts were assessed through Western blotting, proliferation ability, and apoptosis analysis. Results We identified novel compound heterozygous RAG1 variants c.946T>G (p.C316G) and c.1194_1196del (p.L399del) in two affected siblings with typical SCID. Familial genotyping confirmed autosomal recessive inheritance, with each parent as an asymptomatic carrier of one variant. Both mutations were highly conserved and predicted to be pathogenic. Structural modeling revealed disruption of RAG1 secondary and tertiary structure, affecting zinc-binding (p.C316G) and hydrogen-bonding (p.L399del) interactions. Functional studies demonstrated markedly reduced RAG1 protein expression, synergistic impairment of RAG2 expression, and significantly elevated apoptosis in double-mutant pre‑B cells. Further investigation indicated dysregulation of the PI3K/AKT/FOXO1 pathway, evidenced by increased phosphorylation of AKT and FOXO1. Conclusions Our study provides genetic and functional evidence that biallelic RAG1 p.C316G and p.L399del mutations act synergistically to cause SCID through protein destabilization, disruption of RAG1/RAG2 complex integrity, and induction of pre‑B cell apoptosis likely mediated by PI3K/AKT/FOXO1 signaling dysregulation. These findings expand the mutational spectrum of RAG1 and support the clinical application of PGT-M for affected families. Severe combined immunodeficiency RAG1 Compound heterozygous mutations Functional analysis Apoptosis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Severe combined immunodeficiency (SCID) is an immune deficiency disorder that cause severe dysfunction of the immune system, resulting in the loss or abnormal function of T cells and B cells in the thymus and bone marrow, presenting in early infancy with opportunistic infections and failure to thrive [1, 2]. Approximately 50% of patients with SCID have been reported absence of B/T lymphocytes as a result of RAG mutations [3]. The recombinase complex, composed of RAG1 and RAG2 proteins, initiates V(D)J recombination by binding and cleaving recombination signal sequences (RSSs) flanking the V(D)J gene segments. This process is essential for the development of a diverse repertoire of immunoglobulin (Ig) and T lymphocyte receptor (TCR) fragment rearrangement [4, 5]. In humans, different RAG1 variants cause variable clinical penetrance and immunophenotypes depending on the impacts of complete or partial loss of recombinant enzyme activity. Currently, numerous RAG1 variants are documented in the ClinVar database, yet only a limited number have been experimentally validated in patients, underscoring the need for further clinical and experimental assessment of their pathogenicity. The core domains of RAG1 include a RING finger with E3 ubiquitin ligase activity, two zinc fingers (ZFA and ZFB), and DNA-binding regions (NBR and HBR) [5, 6]. The RING finger (amino acids 293-331) and ZFA mediate multimerization [7]. Critically, the NBR (amino acids 387-457), which exhibits a relatively high mutation rate, confers sequence-specific RSS binding and is essential for DNA cleavage during V(D)J recombination [8, 7]. The residues 316 and 399del, which we investigated in this study, are located in the RING finger and the NBD domain, respectively. At present, many different RAG1 mutations and their clinical phenotypes have been found [9-12], however, the pathogenic mechanisms and functional studies with compound heterozygous variants of C316G and L399del in RAG1 in our study have not yet been reported. In this study, we aim to bridge this knowledge gap not only by detailed clinical assessments and pathogenic prediction guided by the American College of Medical Genetics and Genomics (ACMG) guidelines [13] , but also by conducting functional experiments in establishment of Pre-B cell lines harboring these variants via Cas9 RNP strategy. Our research involves meticulous related protein expression, proliferation ability and apoptosis rate, intending to gain a deeper understanding of the effect of these mutations on the clinical phenotypes of these patients. Overall, our study strengthens the genetic and functional evidence for the C316G and L399del variants in RAG1 that may synergistically cause SCID, and help inform the development of innovative therapeutic strategies for these patients. Materials and Methods Study participants and sample collection The probands’ parents gave informed consent and agreed to participate in this study. To further explore the etiology of the disease, peripheral blood samples were taken from the couple, and their clinical manifestations and biochemical parameters were collected for analysis. Sanger sequencing was performed on the obtained samples. Identified variants were assessed using public databases (ClinVar, gnomAD, 1000 genomes project, and HGMD database), and all procedures were conducted under the principles of the Declaration of Helsinki. Bioinformatics analysis To assess the RAG1 variants, we obtained the reference sequence (NM_000448.3) from NCBI GenBank. Conservation analysis was performed by aligning orthologs from six species (human, chimpanzee, pig, cattle, rat, mouse) using DNAMAN. We generated wild-type and mutant 3D structures via SWISS-MODEL, visualized in PyMOL Viewer, and employed PROVEAN, PolyPhen-2, and MutationTaster to predict functional consequences. Variant pathogenicity and annotation were evaluated according to the classification criteria and guidelines of ACMG. Cell culture and nucleofection The human RAG1 + Pre-B cell line (Nalm6) was cultured in RPMI 1640 medium (Cellgro, USA) supplemented with 10% fetal bovine serum (Hyclone, USA), 100 U/mL penicillin and 100 μg/mL streptomycin (Gibco, US) at 37°C in a humidified atmosphere of 5% CO2. Upon reaching a density of 5×10⁵ ~ 1×10⁶ cells/mL, cells were nucleofected using the Lonza 4D-Nucleofector™ X Unit system (Lonza, Germany) with the manufacturer's recommended program for Nalm6 cells. Immediately post-nucleofection, pre-warmed complete medium was added, and the cells were gently transferred to a 60 mm dish. After 24 hours, the cells were collected by centrifugation (800 rpm, 5 min) and resuspended in fresh complete medium without HDR Enhancer V2 for continued culture. CRISPR-Cas9 gene editing Single-guide RNAs (gRNAs) and single-stranded oligodeoxynucleotides (ssODNs) were designed using the IDT Alt-R CRISPR HDR Design Tool. Chemically modified gRNAs and high-fidelity Ultramer™ ssODN HDR templates (Supplemental Table 1) were co-delivered via nucleofection of the RNP complex. We assessed initial editing efficiency with the T7 Endonuclease I ( T7E I) assay and then isolated single-cell clones. The target locus was PCR-amplified from genomic DNA and verified by Sanger sequencing. We first established a homozygous C316G positive clone, then used the same strategy to introduce the L399del mutation into this clone to create the double mutant. RNA extraction and quantitative real-time PCR Total RNA was extracted using Trizol reagent (TaKaRa, Japan) and reverse-transcribed into cDNA using a commercial synthesis kit (Accurate Biology, China). Quantitative polymerase chain reaction (qPCR) analysis was carried out in a 7500 real-time PCR system (Applied Biosystems, USA) using SYBR Green PCR Core Reagents (Accurate Biology, China). All samples were tested in triplicate, and average CT values were calculated and normalized to the housekeeping gene β-actin according to the 2-ΔΔCt method. Primer sequences are listed in Supplemental Table 1. Western blotting Total protein was extracted from cells using RIPA buffer supplemented with 1% PMSF (Beyotime, China). Protein concentration was determined by a BCA assay (Sangon Biotech, China). Denatured proteins were separated by 8% SDS-PAGE and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, USA). After blocking with 5% skim milk in TBST for 1 hour at room temperature, the membranes were incubated overnight at 4°C with the following primary antibodies: anti-RAG1 (Boster, 1:1500), anti-RAG2 (Proteintech, 1:800), anti-FOXO1 (Proteintech, 1:4000), or anti-GAPDH (Proteintech, 1:10000). Subsequently, the membranes were probed with an HRP-conjugated secondary antibody (Proteintech, 1:4000) for 1 hour at room temperature. Protein signals were visualized using an enhanced chemiluminescence (ECL) detection system. Cell viability and apoptosis assays Cell proliferation was assessed using the CCK-8 assay. Log-phase cells were seeded in 96-well plates at a density of 5×10⁵ cells per well. Cell viability was quantified at 24, 48, and 72 h by measuring the absorbance at 450 nm according to the manufacturer's instructions. Apoptosis was detected using an Annexin V-FITC/PI Apoptosis Detection Kit. Cells were stained and analyzed by flow cytometry with appropriate FITC and PI channels. The apoptosis rate was calculated as the sum of early apoptosis (lower right quadrant) and late apoptosis (upper right quadrant) percentages. As a positive control for apoptosis, cells were treated with 30 μM cisplatin (DNA cross-linking apoptosis inducer) for 12 hours prior to flow cytometric analysis. Statistical analysis Quantitative data were analyzed using GraphPad Prism 9.5 (GraphPad Software, USA). Differences among multiple groups were analyzed by one-way ANOVA followed by Tukey’s post hoc test. P -Values less than 0.05 was considered statistically significant. All experiments were repeated at least three times. Results Clinical features The couple, a 33-year-old male and a 29-year-old female with normal karyotypes, presented with a history of primary infertility. The female had irregular menstruation, and the male was diagnosed with oligoasthenoteratospermia. Their first pregnancy resulted in a full-term male infant who was diagnosed with "congenital immunodeficiency syndrome" at three months of age and died at six months. No genetic testing was performed. A subsequent intrauterine insemination (IUI) pregnancy resulted in a female infant diagnosed with T-B severe combined immunodeficiency (SCID) at two months of age, who also died at six months. Compound heterozygous mutations in the RAG1 gene were identified in the second infant. Consequently, the couple was referred to the Prenatal and Genetic Clinic of our hospital and was recommended to pursue preimplantation genetic testing for monogenic disorders (PGT-M). Prior to entering the PGT-M cycle, to investigate the etiology of the congenital immunodeficiency syndrome in the proband, whole-exome sequencing report from an external institution revealed compound heterozygous mutations in the RAG1 gene in the proband: c.1194_1196del (p.L399del) and c.946T>G (p.C316G), which were confirmed by Sanger sequencing. The couple showed no related symptoms. Following informed consent, their peripheral blood samples were subjected to karyotyping and Sanger sequencing. Variant pathogenicity was classified according to ACMG guidelines. All procedures were conducted in accordance with the Declaration of Helsinki. Identification of RAG1 mutations and pathogenicity analysis To establish a genetic diagnosis for the probands (II-1 and II-2) in this pedigree (Fig. 1A), whole-exome sequencing revealed novel compound heterozygous variants in exon 2 of the RAG1 gene: a heterozygous c.946T>G (p.C316G) and a heterozygous c.1194_1196del (p.L399del). Sanger sequencing of parental peripheral blood confirmed the female carrier (I-1) harbored the heterozygous c.946T>G variant, while the male (I-2) carried the heterozygous c.1194_1196del variant (Fig. 1A, B). Cross-referencing these two mutations with the dbSNP, ClinVar or the Human Gene Mutation Database (HGMD) confirmed that neither variant had been previously reported or documented in the literature, establishing them as novel pathogenic variants. Multiple sequence alignment of RAG1 across six vertebrate species demonstrated that Cys316 and Leu399 reside in highly conserved regions (Fig. 1C). Bioinformatic tools including PROVEAN, PolyPhen-2, and MutationTaster consistently predicted both variants to be damaging (Fig. 1D). According to ACMG/AMP guidelines, the c.946T>G (p.C316G) variant was classified as a variant of uncertain significance (VUS), with the following supporting evidence: PP1_Moderate (co-segregation with disease in multiple affected family members), PM2_Supporting (absent from ESP, 1000 genomes and ExAC databases), PP3 (multiple computational predictions support a deleterious effect), and PP4 (the phenotype of the patients was highly specific for a monogenic disorder). The c.1194_1196del (p.L399del) variant was classified as likely pathogenic based on PM4 (protein length change due to an in-frame deletion in a non-repeat region), PP1_Moderate, PM2_Supporting, and PP4. Subsequent screening of our institutional whole-exome database of infertility patients did not identify these RAG1 variants. Impacts of the mutations on the secondary and 3D structure of RAG1 protein To elucidate the structural impact of the identified RAG1 mutations, we performed computational analyses. Secondary structure prediction using PSIPRED v4.0 indicated that both p.C316G (hereinafter referred to as mut1) and p.L399del (hereinafter referred to as mut3) reside within alpha-helical regions (Fig. 2A). Domain analysis via Pfam and SMART databases localized the p.C316G mutation within the RING-type zinc finger domain (amino acids 293-331) and the p.L399del mutation within a DNA-binding domain (amino acids 392-467). We next modeled the tertiary structural changes using SWISS-MODEL. Due to the inability of a single template to cover the full length of the RAG1 amino acid sequence, the homology modeling method was employed using templates with database IDs 1rmd.1.A and 6oeo.1.C as homologous templates to predict the tertiary structure of the mutant human RAG1 protein. The 1rmd.1.A template covers amino acids 268 to 383 of RAG1 (the region containing mut1). Results indicate that the mut1 mutation leads to the substitution of the hydrophobic residue cysteine with the hydrophilic yet non-polar glycine. This alteration induces a change in the tetrahedral coordination geometry of the zinc finger structure, resulting in a loss of zinc ion binding capacity (Fig. 2B). The Zn²⁺ ion within the zinc finger domain plays a critical role: it not only governs protein folding and provides the structural scaffold but also stabilizes the alpha-helical conformation. This stabilization enables the alpha-helix to dock into the major groove of DNA, allowing the zinc finger protein to achieve specific binding to DNA or RNA. Consequently, the mut1 mutation ultimately disrupts the zinc finger structure, inhibits its specific DNA-binding function, and compromises the regulation of gene expression. The 6oeo.1.C template covers amino acids 396 to 1043 of RAG1 (the region containing mut3). Within this structure, two hydrogen bonds are formed between Histidine 398 and Serine 401. The mut3 mutation, which causes a deletion of Leucine 399, leads to the loss of one of these hydrogen bonds (Fig. 2C). The formation and stability of the protein's tertiary structure rely heavily on non-covalent interactions, including hydrogen bonding. Disruption of these hydrogen bonds can cause the protein's spatial configuration to become less compact and more disordered, reducing its structural stability and potentially impairing its biological function. Establishment of Monoclonal Pre-B Cell Lines with the RAG1 Targeted Mutations To further investigate the impact of the mutation on protein function, the optimized Cas9 ribonucleoprotein (RNP) strategy was used to generate intended mutations into pre-B Cells. Following confirmation of efficient genome editing by T7E1 cleavage assay, mutant cells were subjected to limiting dilution to isolate single clones. Individual monoclonal colonies were expanded to sufficient density, and genomic DNA was extracted, amplified via PCR, and sequenced to validate the mutations. As shown in Figure 3B, T7E1 cleavage analysis revealed high editing activity at both gRNA target sites. Quantification of band intensity using ImageJ indicated editing efficiencies of 77.63% for the c.946T>G (p.C316G) target and 80.12% for the c.1194_1196del (p.L399del) target. Sequencing results (Figure 3A) confirmed the successful establishment of four monoclonal pre-B cell lines harboring the intended mutations: (1) mut1: a homozygous c.946T>G (p.C316G) mutant line; (2) mut2: a homozygous c.946T>G (p.C316G) line carrying an additional synonymous nucleotide change (C>A); (3) mut3: a homozygous c.1194_1196del (p.L399del) mutant line; and (4) mut4: a homozygous double-mutant line containing both c.946T>G (p.C316G) and c.1194_1196del (p.L399del) mutations. The mutations affect RAG1 and RAG2 expression Quantitative PCR analysis revealed distinct expression patterns of RAG1 and RAG2 across the mutant cell lines (Figure 3C, 3D). While RAG1 mRNA levels in mut1, mut2, and mut3 cells showed no significant difference (ns, P >0.05) compared to wild-type controls, a statistically significant reduction ( P <0.05) was observed in the mut4 double mutant. In contrast, RAG2 mRNA expression was significantly decreased ( P <0.05) in mut1, mut2, and mut4 cells relative to wild-type controls. Western blot analysis corroborated these findings at the protein level (Figure 3E, 3F). RAG1 protein (located at ~120 kDa) expression remained unchanged in mut1, mut2, and mut3 cells but was significantly reduced ( P <0.05) in the mut4 double mutant. Notably, RAG2 protein (located at ~ 59 kDa) expression was significantly diminished ( P G (p.C316G) and c.1194_1196del (p.L399del) mutations in RAG1 significantly impair RAG2 expression in pre-B lymphocytes. The impaired expression of RAG2 is likely a direct consequence of the functional impairment of mutant RAG1 protein, consequently affecting the stability or expression of its essential molecular partner, RAG2. Flow cytometric analysis demonstrated a significant increase in apoptosis in RAG1 -mutant pre-B cell lines As illustrated in Figure 4F and 4G, cisplatin-treated positive control cells exhibited markedly elevated levels of both early and late apoptosis, with a combined apoptotic rate of 19.86%. In contrast, wild-type pre-B cells showed a baseline apoptosis rate of 0.63%. Introduction of RAG1 mutations resulted in progressively increased apoptotic rates across all mutant lines: mut1 (1.79%), mut2 (1.26%), mut3 (2.62%), and the double mutant (3.48%). These findings indicate that both the c.946T>G (p.C316G) and c.1194_1196del (p.L399del) mutations in RAG1 significantly enhance susceptibility to apoptosis in pre-B cells. The observed mutation-dependent increase in apoptosis suggests a potential mechanism through which RAG1 deficiencies may impair B cell development by promoting cell death at the pre-B cell stage, thereby contributing to the immunodeficiency phenotype. RAG1 variants do not impair pre-B cell proliferation capacity Assessment of cellular proliferation by CCK-8 assay revealed no significant differences between RAG1-mutant pre-B cells and wild-type controls at 24, 48, or 72 hours post-seeding ( P >0.05; Figure 4H). These results indicate that the c.946T>G (p.C316G) and c.1194_1196del (p.L399del) mutations in RAG1 do not substantially affect the proliferative capacity of pre-B cells. Together with our previous observation of enhanced apoptosis in these mutants, these findings suggest that the reduction in mature B lymphocytes associated with RAG1 deficiency is likely mediated through increased apoptotic susceptibility rather than impaired proliferation, providing a mechanistic insight into the development of immunodeficiency in affected patients. RAG1 mutations potentially modulate the PI3K/AKT1/FOXO1 signaling pathway and promote apoptosis The PI3K/AKT/FOXO1 signaling pathway plays a crucial role in regulating cell survival and apoptosis [14]. To examine whether RAG1 mutations influence this pathway, we analyzed the expression of key pathway components. Q-PCR analysis revealed significant upregulation of AKT1 and FOXO1 mRNA levels in both mut3 and double mutant cells ( P <0.05; Figure 3C, 3D). However, at the protein level, Western blot analysis presented a more complex picture: while total FOXO1 protein was decreased in the double mutant group ( P <0.05; Figure 3E, 3F), the levels of phosphorylated FOXO1 (p-FOXO1) were significantly elevated (Figure 5A, 5B). This critical finding indicates that the reduction in total FOXO1 is likely attributable to post-translational regulation. Consistent with enhanced FOXO1 inactivation, we also observed a marked increase in the expression of both total AKT and phosphorylated AKT (p-AKT) in the double mutant (Figure 5A, 5B). In contrast, acetylated FOXO1 (Ac-FOXO1) levels remained unchanged across all groups. Collectively, these results demonstrate that the double mutation in RAG1 leads to constitutive activation of AKT, which subsequently promotes FOXO1 phosphorylation and degradation, thereby disrupting normal PI3K/AKT/FOXO1 signaling and contributing to apoptosis in pre-B lymphocytes. Discussion While the clinical spectrum of Severe Combined Immunodeficiency (SCID) associated with RAG1 mutations has been well-documented, functional validation of specific variants remains limited [15-17]. In the present study, we identified two novel compound heterozygous mutations of RAG1 gene, c.946T>G/p.C316G and c.1194_1196del/p.L399del, in a Chinese family presenting with a typical SCID phenotype. These mutations are not reported in public Genome Aggregation Database (gnomAD) or on the Exome Variants Server (EVS), and their functional impact has not been experimentally characterized. Notably, it has been proven that the RAG1/2 mutations can lead to multiple forms of immunological and clinical phenotypes [9, 11, 18]. According to the clinical descriptions of the patients and their children, neither heterozygous parent exhibited immunodeficiency, suggesting that neither variant alone is sufficient to cause disease. However, the compound heterozygous state in the probands led to full SCID manifestation, consistent with an autosomal recessive mode of inheritance. This pattern, in which the disease phenotype manifests only in the compound heterozygous state, is consistent with previous reports by several authors [19, 17, 20] . Bioinformatic analysis indicated that both mutations detected in our study are evolutionarily conserved across vertebrates and are located in functionally critical domains: p.Cys316Gly mutation within the RING-type zinc finger domain, which is essential for zinc ion coordination and RAG1-RAG2 complex assembly [21], while p.L399del mutation localizes to the nonamer DNA-binding region (NBR) of the core RAG1, responsible for specific recognition of recombination signal sequences (RSS) and proper targeting of V(D)J recombination adhere to the 12/23 rule [22]. Consistently, configuration and pathogenicity prediction also revealed the damaging impacts of both RAG1 variants on the protein’s 3D structure and stability. The p.C316G mutation probably contributes to the substitution of a hydrophobic cysteine residue with a hydrophilic yet non-polar glycine is predicted to abolish the domain’s ability to bind zinc ions. The p.L399del mutation reduces hydrogen bonding with adjacent residues such as His398 and Ser401, potentially impairing interaction efficiency with DNA substrates/RAG2 and complex stability, and ultimately impair the overall efficacy of V(D)J recombination. To functionally validate these predictions, we successfully established these homozygous mutations into human pre-B cell lines using a CRISPR-Cas9 RNP approach. While single mutants did not significantly alter RAG1 expression, the double mutant exhibited markedly reduced levels of both RAG1 and RAG2 proteins, accompanied by increased apoptosis. In conjunction with the previously constructed secondary and tertiary structure models, it suggested that the mutations potentially induce apoptosis in pre-B lymphocytes through aberrant protein structure and destabilization of RAG complex, thereby impairing B lymphocyte survival. Given the notable decrease in RAG2 expression, we propose that the RAG1 c.946T>G (p.C316G) and c.1194_1196del mutations may coordinately affect the expression of RAG2, and that their synergistic effect disrupts normal B lymphocyte differentiation and development. Interestingly, we observed a significant decrease in FOXO1 protein expression in double-mutant cells. Previous studies have established FOXO1 as a crucial transcription factor in B cell development and a key regulator of RAG1/2 expression at the pre-B cell stage [23-25]. Consistent with this, Foxo1 –/– pro‑B cells exhibit increased apoptosis and show impaired V H to D H J H gene rearrangement [23], underscoring the essential role of FOXO1 in both gene recombination and cell survival. Based on these findings and our data, we hypothesize that the double mutation in RAG1 limits RAG1/2 expression, and that a subsequent negative feedback mechanism suppresses FOXO1 expression. This downregulation of FOXO1 may then inhibit gene recombination and ultimately contribute to pre-B cell apoptosis. This model is firmly supported by established signaling pathways and our experimental data. Signals emanating from the interleukin-7 receptor (IL7R) and the pre-B-cell receptor (pre-BCR) regulate the dynamic pattern of RAG1 and RAG2 expression, which involves phosphoinositide-3 kinase (PI3K) and protein kinase B (PKB, also known as AKT) impinging on FOXO transcription factors, themselves critical for RAG expression and gene recombination [26, 24, 27]. In line with this, we found that double-mutant cells exhibited increased activation of AKT, as evidenced by elevated levels of phosphorylated AKT (p-AKT). Most importantly, and in direct support of our hypothesis, we detected a significant increase in phosphorylated FOXO1 (p-FOXO1) in these cells. This finding provides direct molecular evidence that the functional impairment caused by the RAG1 double mutation leads to aberrant activation of the PI3K/AKT signaling pathway. The consequent AKT-mediated phosphorylation of FOXO1 triggers its nuclear export and proteasomal degradation [28-30], which perfectly explains the concurrent decrease in total FOXO1 protein we observed. This persistent inactivation and degradation of FOXO1, a known regulator of genes controlling cell cycle arrest [31] and apoptosis [32], provides a compelling mechanistic link between the RAG1 mutations and the increased apoptosis in pre-B lymphocytes. This study has certain limitations. The initial WES data for the probands were obtained from an external clinical report, and original DNA samples were unavailable for further validation. Furthermore, all functional assays were performed in an in vitro pre-B cell model, future studies should employ animal models to better recapitulate the systemic immune pathology. In conclusion, our study identifies two novel RAG1 mutations, p.C316G and p.L399del, that act synergistically to disrupt protein stability, impair RAG2 expression, and promote pre-B cell apoptosis. We provide direct evidence that this process is mechanistically driven by the constitutive activation of the PI3K/AKT pathway, leading to AKT-mediated FOXO1 phosphorylation and degradation. These findings expand the mutational spectrum of RAG1 and elucidate a key pathological pathway in RAG-deficient SCID. Declarations Funding This work was funded by the National Traditional Chinese Medicine Inheritance and Innovation Center, the First Affiliated Hospital of XXX University (grant number: 2023QN02), the Administration of Traditional Chinese Medicine of Guangdong Province (grant numbers: 20241103, 20242036), and the Medical Scientific Research Foundation of Guangdong Province (grant number: A2023401). Ethical approval and consent to participate All experiments were approved by the Ethics Committee of the First Affiliated Hospital of XXX University (No. ZYYEC-ERK-2022-093) and all procedures were in accordance with the ethical guidelines of the Helsinki Declaration. The parents were informed and consented to participate. Consent for publication Written informed consent was obtained from the parents for publication of this study, including any personal or clinical details. Data availability Data will be made available on request. 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Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genet Med. 2015;17(5):405-24. https://doi:10.1038/gim.2015.30. Wang W, Jiang X, Xia F, Chen X, Li G, Liu L, et al. HYOU1 promotes cell proliferation, migration, and invasion via the PI3K/AKT/FOXO1 feedback loop in bladder cancer. Mol Biol Rep. 2023;50(1):453-64. https://doi:10.1007/s11033-022-07978-x. Zhang X, Kang X, Yang M, Cai Z, Song Y, Zhou X, et al. A variant of RAG1 gene identified in severe combined immunodeficiency: a case report. BMC Pediatr. 2023;23(1):56. https://doi:10.1186/s12887-022-03822-0. Meng X, Melsen JE, van der Holst R, de Mooij B, Vloemans S, van Eggermond M, et al. RAG1 lentiviral gene therapy restores T cell development of RAG1-SCID patient cells in artificial thymic organoids. Blood Adv. 2025. https://doi:10.1182/bloodadvances.2025016970. Akarsu A, Sonmez G, Schiefer AI, Ozcan HN, Üner M, Esenboga S, et al. Rubella-associated granuloma in a patient with a compound heterozygous RAG1 defect and review of the literature. Immunol Res. 2025;73(1):150. https://doi:10.1007/s12026-025-09711-9. Lee YN, Frugoni F, Dobbs K, Walter JE, Giliani S, Gennery AR, et al. A systematic analysis of recombination activity and genotype-phenotype correlation in human recombination-activating gene 1 deficiency. J Allergy Clin Immunol. 2014;133(4):1099-108. https://doi:10.1016/j.jaci.2013.10.007. Mou W, Yang Z, Wang X, Hei M, Wang Y, Gui J. Immunological assessment of a patient with Omenn syndrome resulting from compound heterozygous mutations in the RAG1 gene. Immunogenetics. 2023;75(4):385-93. https://doi:10.1007/s00251-023-01309-5. Shen J, Jiang L, Gao Y, Ou R, Yu S, Yang B, et al. A Novel RAG1 Mutation in a Compound Heterozygous Status in a Child With Omenn Syndrome. Front Genet. 2019;10:913. https://doi:10.3389/fgene.2019.00913. Fayyaz H, Zaman A, Shabbir S, Khan ZK, Haider N, Saleem AF, et al. Mutational analysis in different genes underlying severe combined immunodeficiency in seven consanguineous Pakistani families. Mol Biol Rep. 2024;51(1):302. https://doi:10.1007/s11033-024-09222-0. Eastman QM, Leu TM, Schatz DG. Initiation of V(D)J recombination in vitro obeying the 12/23 rule. Nature. 1996;380(6569):85-8. https://doi:10.1038/380085a0. Dengler HS, Baracho GV, Omori SA, Bruckner S, Arden KC, Castrillon DH, et al. Distinct functions for the transcription factor Foxo1 at various stages of B cell differentiation. Nat Immunol. 2008;9(12):1388-98. https://doi:10.1038/ni.1667. Amin RH, Schlissel MS. Foxo1 directly regulates the transcription of recombination-activating genes during B cell development. Nat Immunol. 2008;9(6):613-22. https://doi:10.1038/ni.1612. Feng M, Yang K, Wang J, Li G, Zhang H. First Report of FARSA in the Regulation of Cell Cycle and Survival in Mantle Cell Lymphoma Cells via PI3K-AKT and FOXO1-RAG1 Axes. Int J Mol Sci. 2023;24(2). https://doi:10.3390/ijms24021608. Herzog S, Hug E, Meixlsperger S, Paik JH, DePinho RA, Reth M, et al. SLP-65 regulates immunoglobulin light chain gene recombination through the PI(3)K-PKB-Foxo pathway. Nat Immunol. 2008;9(6):623-31. https://doi:10.1038/ni.1616. Verkoczy L, Duong B, Skog P, Aït-Azzouzene D, Puri K, Vela JL, et al. Basal B cell receptor-directed phosphatidylinositol 3-kinase signaling turns off RAGs and promotes B cell-positive selection. J Immunol. 2007;178(10):6332-41. https://doi:10.4049/jimmunol.178.10.6332. Ochodnicka-Mackovicova K, Bahjat M, Bloedjes TA, Maas C, de Bruin AM, Bende RJ, et al. NF-κB and AKT signaling prevent DNA damage in transformed pre-B cells by suppressing RAG1/2 expression and activity. Blood. 2015;126(11):1324-35. https://doi:10.1182/blood-2015-01-621623. Herzog S, Reth M, Jumaa H. Regulation of B-cell proliferation and differentiation by pre-B-cell receptor signalling. Nat Rev Immunol. 2009;9(3):195-205. https://doi:10.1038/nri2491. Coffer PJ, Burgering BM. Forkhead-box transcription factors and their role in the immune system. Nat Rev Immunol. 2004;4(11):889-99. https://doi:10.1038/nri1488. Kops GJ, Medema RH, Glassford J, Essers MA, Dijkers PF, Coffer PJ, et al. Control of cell cycle exit and entry by protein kinase B-regulated forkhead transcription factors. Mol Cell Biol. 2002;22(7):2025-36. https://doi:10.1128/mcb.22.7.2025-2036.2002. Dong P, Zhang X, Zhao J, Li D, Li L, Yang B. Anti-microRNA-132 causes sevoflurane‑induced neuronal apoptosis via the PI3K/AKT/FOXO3a pathway. Int J Mol Med. 2018;42(6):3238-46. https://doi:10.3892/ijmm.2018.3895. Additional Declarations No competing interests reported. Supplementary Files Supplementaltable1.docx Supplemental table 1 Primers and ssODNs used in this study. Westernblottingfigures.rar ExperimentofT7EIenzymedigestion.rar Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 31 Mar, 2026 Reviews received at journal 25 Mar, 2026 Reviews received at journal 24 Mar, 2026 Reviewers agreed at journal 18 Mar, 2026 Reviewers agreed at journal 17 Mar, 2026 Reviewers invited by journal 17 Mar, 2026 Editor assigned by journal 09 Mar, 2026 Submission checks completed at journal 09 Mar, 2026 First submitted to journal 03 Mar, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9021280","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":608327475,"identity":"859e713d-ac11-4c7f-ba3d-cd77778f8bf3","order_by":0,"name":"Yongxiang Liu","email":"","orcid":"","institution":"The First Affiliated Hospital of Guangzhou University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Yongxiang","middleName":"","lastName":"Liu","suffix":""},{"id":608327478,"identity":"e0120dd2-c4c7-4006-9d05-eb175dadb530","order_by":1,"name":"Xiongwei Shan","email":"","orcid":"","institution":"The Second Affiliated Hospital of Guangzhou University of Chinese Medicine/Guangdong Province Hospital of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Xiongwei","middleName":"","lastName":"Shan","suffix":""},{"id":608327481,"identity":"a3967e50-d0e6-4a2d-bd06-cd6520b5cf7c","order_by":2,"name":"Guilan Huang","email":"","orcid":"","institution":"The First Affiliated Hospital, Sun Yat-sen University","correspondingAuthor":false,"prefix":"","firstName":"Guilan","middleName":"","lastName":"Huang","suffix":""},{"id":608327485,"identity":"c9d7d4a4-46ed-4124-9881-128326a7af0c","order_by":3,"name":"Yuwei Duan","email":"","orcid":"","institution":"The First Affiliated Hospital, Sun Yat-sen University","correspondingAuthor":false,"prefix":"","firstName":"Yuwei","middleName":"","lastName":"Duan","suffix":""},{"id":608327486,"identity":"cac8f8cf-afe5-4b17-b7e3-84626b2e7b85","order_by":4,"name":"Yunshi Li","email":"","orcid":"","institution":"The First Affiliated Hospital of Guangzhou University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Yunshi","middleName":"","lastName":"Li","suffix":""},{"id":608327487,"identity":"79df3a5c-76f2-4a44-b4f5-e16343cb91f0","order_by":5,"name":"Wenhui Hou","email":"","orcid":"","institution":"The First Affiliated Hospital of Zhengzhou University","correspondingAuthor":false,"prefix":"","firstName":"Wenhui","middleName":"","lastName":"Hou","suffix":""},{"id":608327488,"identity":"93af7eb7-0f39-4f6e-80c6-73bb7dfa9a8c","order_by":6,"name":"Zishuo Li","email":"","orcid":"","institution":"The First Affiliated Hospital of Guangzhou University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Zishuo","middleName":"","lastName":"Li","suffix":""},{"id":608327490,"identity":"d7085c6f-0293-4665-a415-da909fcb2e04","order_by":7,"name":"Shuting Zhang","email":"","orcid":"","institution":"The First Affiliated Hospital of Guangzhou University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Shuting","middleName":"","lastName":"Zhang","suffix":""},{"id":608327492,"identity":"aaeb340c-f99e-4ce2-92f3-173030249917","order_by":8,"name":"Zhiwei Weng","email":"","orcid":"","institution":"The First Affiliated Hospital of Guangzhou University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Zhiwei","middleName":"","lastName":"Weng","suffix":""},{"id":608327493,"identity":"ae4019ac-fd5b-4c4a-a214-8c5a6bcfe74e","order_by":9,"name":"Shaohu Zhou","email":"","orcid":"","institution":"The First Affiliated Hospital of Guangzhou University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Shaohu","middleName":"","lastName":"Zhou","suffix":""},{"id":608327494,"identity":"091d2286-282b-422f-ad9c-1b83adf31578","order_by":10,"name":"Xuekun Huang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAsklEQVRIiWNgGAWjYBACAwY2IFlhI8fG3n6AFC1n0oz5eM4kkKCFse1w4jwJBwPitJhLpKVJF5xJS2+TYEhg+FGxjbAWy55jx6RnVNjktkk3HmDsOXObCIcdb2+T5jmTltsmcyCBmbGNGC2H2dukedsOp7NJJBgQqeV42zGQlgQStJw5lmwNdJhhGzCQDxLnlxtphrd5Kmzk5dvbDz74UUGEFhRwgET1o2AUjIJRMApwAQD4VDsZCYAuwAAAAABJRU5ErkJggg==","orcid":"","institution":"The First Affiliated Hospital of Guangzhou University of Chinese Medicine","correspondingAuthor":true,"prefix":"","firstName":"Xuekun","middleName":"","lastName":"Huang","suffix":""}],"badges":[],"createdAt":"2026-03-03 14:25:26","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9021280/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9021280/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":105047108,"identity":"4959b95d-8a74-45c0-8beb-e6c9b387d9fe","added_by":"auto","created_at":"2026-03-20 09:14:27","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2193974,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIdentification of RAG1 mutation and bioinformatics-based pathogenicity prediction. \u0026nbsp;\u003c/strong\u003e(A) Pedigree map of the family. The female infant and male infant were probands (II-1 and II-1). Squares represent males and circles indicate females. A single diagonal line indicates deceased individuals. (B) Sanger sequencing results of RAG1 variants, and arrows indicate the site of mutations. Their father (I-2) and mother (I-1) harboring the heterozygous c.1194_1196del/p.L399del and c.946T\u0026gt;G/p.C316G mutations, respectively. (C) Schematic structure of RAG1 protein and cross-species conservation analysis of RAG1 around C316G and L399del. The amino acids of mutations were marked in red boxes and both located within a highly conserved region. RING: RING finger; ZFA: zinc finger A;NBR: a nonamer DNA-binding region; HBR: a hexamer DNA-binding region; ZFB: zinc finger B; (D) Prediction of the pathogenicity of the p.C316G and p.L399del mutations found in the RAG1 gene.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-9021280/v1/ef6095920deb6b377f064ff4.png"},{"id":105047109,"identity":"1ed84673-7efc-4d5b-9035-e2bbf7f7eb01","added_by":"auto","created_at":"2026-03-20 09:14:27","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":3902163,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of RAG1 variants on the protein structure. \u003c/strong\u003e(A) The secondary structure and solvent accessibility of RAG1 by PSIPRED v4.0 online database analysis. (B, C) The alterations of the 3D-structure of c.946T\u0026gt;G (p.C316G) and c.1194_1196del (p.L399del) mutation proteins by SWISS-MODEL and homologous modeling analysis.The red squares confirm RAG1 mutation sites.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-9021280/v1/3f5643b3ebb38808d2d10edc.png"},{"id":105047113,"identity":"c0057ea0-80e1-4f69-bc34-3faf22228452","added_by":"auto","created_at":"2026-03-20 09:14:27","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2348579,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eImpaired gene expression by the two mutations in pre-B lymphocyte cells. \u003c/strong\u003e(A)Sanger sequencing analysis confirmed the successful construction of the RAG1 mutants (mut1: c.946T\u0026gt;G/p.C316G, mut2: c.946T\u0026gt;G (p.C316G) mutant containing a synonymous (C\u0026gt;A) nucleotide change, mut3: c.1194_1196del/p.L399del, and double mut: c.946T\u0026gt;G/p.C316G and c.1194_1196del/p.L399del mutation). (B) Experiment of \u003cem\u003eT7E\u003c/em\u003e I enzyme digestion. M: 1.0 Kb marker; 1: PCR product (non-\u003cem\u003eT7E\u003c/em\u003e I digestion); 2: \u003cem\u003eT7E\u003c/em\u003e I enzyme digestion of wild type group; 3 and 4: \u003cem\u003eT7E\u003c/em\u003e I enzyme digestion of c.946T\u0026gt;G (p.C316G) and c.1194_1196del/p.L399del targets, respectively. (C, D) The qRT-PCR results of the gene expression of \u003cem\u003eRAG1\u003c/em\u003e, \u003cem\u003eRAG2\u003c/em\u003e, \u003cem\u003eFOXO1\u003c/em\u003eand \u003cem\u003eAKT\u003c/em\u003e between wt and mutant groups. (E, F) The western blot results of the protein expression of \u003cem\u003eRAG1\u003c/em\u003e, \u003cem\u003eRAG2\u003c/em\u003e, and \u003cem\u003eFOXO1\u003c/em\u003ebetween wt and mutant groups. A significant difference was represented by *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, while ns indicates no significant difference.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-9021280/v1/788273b86593fa25dcdbe424.png"},{"id":105562609,"identity":"a4f46e67-99d2-45a2-a32b-3a2c37863b27","added_by":"auto","created_at":"2026-03-27 12:43:37","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":971555,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe apoptosisand proliferation between wild-type and mutants in Pre-B lymphocyte cells were detected by flow cytometry and CCK-8 assay, respectively. \u003c/strong\u003e(A-G) The apoptosis rates among wild-type, mut1, mut2, mut3, double mutant, and cisplatin-induced apoptosis groups were detected by flow cytometry analysis. The apoptosis rate was calculated by early and late apoptosis rate.（H）CCK-8 assay was used to detect the proliferation of wild-type and mutants at 24h, 48h and 96h after plating. A significant difference was represented by *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, while ns indicates no significant difference.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-9021280/v1/c202edd484dceb84e8115601.png"},{"id":105047111,"identity":"b15d54d1-bda7-4e19-8821-6fcfb5017fbe","added_by":"auto","created_at":"2026-03-20 09:14:27","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":163378,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRAG1 mutations induce hyperactivation of the PI3K/AKT pathway and promote FOXO1 inactivation. \u003c/strong\u003e(A, B) The protein levels of p-FOXO1, AKT1, and p-AKT in WT and mutated groups were performed by western blotting. A significant difference was represented by *\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, while ns indicates no significant difference.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-9021280/v1/a15b89a393f871e8b6087dee.png"},{"id":106723477,"identity":"332b9873-3806-4bac-813e-054720f876f2","added_by":"auto","created_at":"2026-04-12 17:51:11","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":10155595,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9021280/v1/93b63c0b-ba5d-4533-973a-4119f7d91d1d.pdf"},{"id":105903847,"identity":"09c5276e-b26a-4fc7-a2dc-bdfc67eb4711","added_by":"auto","created_at":"2026-04-01 09:55:18","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":18722,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplemental table 1\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePrimers and ssODNs used in this study.\u003c/p\u003e","description":"","filename":"Supplementaltable1.docx","url":"https://assets-eu.researchsquare.com/files/rs-9021280/v1/ef4a6db64d4f27daf5848034.docx"},{"id":105047115,"identity":"9a2c175e-a708-42e1-92bc-f901df08b18c","added_by":"auto","created_at":"2026-03-20 09:14:28","extension":"rar","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":37559652,"visible":true,"origin":"","legend":"","description":"","filename":"Westernblottingfigures.rar","url":"https://assets-eu.researchsquare.com/files/rs-9021280/v1/b3bb3746e1cf7ad4b643f0b8.rar"},{"id":105047114,"identity":"892887fb-a54e-4939-b8e8-a9b9d5bc8a84","added_by":"auto","created_at":"2026-03-20 09:14:27","extension":"rar","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":685049,"visible":true,"origin":"","legend":"","description":"","filename":"ExperimentofT7EIenzymedigestion.rar","url":"https://assets-eu.researchsquare.com/files/rs-9021280/v1/650443a19b2a2ad24c538978.rar"}],"financialInterests":"No competing interests reported.","formattedTitle":"From PGT-M Discovery to Mechanism: Functional Validation of novel compound heterozygous RAG1 Mutations in Severe Combined Immunodeficiency","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSevere combined immunodeficiency (SCID) is an immune deficiency disorder that cause severe dysfunction of the immune system, resulting in the loss or abnormal function of T cells and B cells in the thymus and bone marrow, presenting in early infancy with opportunistic infections and failure to thrive [1, 2]. Approximately 50% of patients with SCID have been reported absence of B/T lymphocytes as a result of \u003cem\u003eRAG\u0026nbsp;\u003c/em\u003emutations [3]. The recombinase complex, composed of RAG1 and RAG2 proteins, initiates V(D)J recombination by binding and cleaving recombination signal sequences (RSSs) flanking the V(D)J gene segments. This process is essential for the development of a diverse repertoire of immunoglobulin (Ig) and T lymphocyte receptor (TCR) fragment rearrangement [4, 5]. In humans, different \u003cem\u003eRAG1\u0026nbsp;\u003c/em\u003evariants cause variable clinical penetrance and immunophenotypes depending on the impacts of complete or partial loss of recombinant enzyme activity. Currently, numerous \u003cem\u003eRAG1\u003c/em\u003e variants are documented in the ClinVar database, yet only a limited number have been experimentally validated in patients, underscoring the need for further clinical and experimental assessment of their pathogenicity.\u003c/p\u003e\n\u003cp\u003eThe core domains of RAG1 include a RING finger with E3 ubiquitin ligase activity, two zinc fingers (ZFA and ZFB), and DNA-binding regions (NBR and HBR)\u0026nbsp;[5, 6]. The RING finger (amino acids 293-331) and ZFA mediate multimerization\u0026nbsp;[7]. Critically, the NBR (amino acids 387-457), which exhibits a relatively high mutation rate, confers sequence-specific RSS binding and is essential for DNA cleavage during V(D)J recombination [8, 7]. The residues 316 and 399del, which we investigated in this study, are located in the RING finger and the NBD domain,\u0026nbsp;respectively.\u003c/p\u003e\n\u003cp\u003eAt present,\u0026nbsp;many different RAG1 mutations\u0026nbsp;and their clinical phenotypes\u0026nbsp;have been found [9-12], however, the pathogenic mechanisms\u0026nbsp;and functional studies with\u0026nbsp;compound heterozygous\u0026nbsp;variants of C316G and L399del in RAG1 in our study have not yet been\u0026nbsp;reported. In this study, we aim to bridge this knowledge gap not only by detailed clinical assessments\u0026nbsp;and\u0026nbsp;pathogenic prediction\u0026nbsp;guided by the American College of Medical Genetics and Genomics (ACMG) guidelines\u0026nbsp;[13]\u0026nbsp;, but also by conducting\u0026nbsp;functional experiments\u0026nbsp;in establishment of Pre-B cell lines\u0026nbsp;harboring these variants via Cas9 RNP\u0026nbsp;strategy.\u0026nbsp;Our research involves meticulous related protein expression,\u0026nbsp;proliferation ability and apoptosis rate, intending to gain a deeper understanding of the effect of these mutations on the clinical phenotypes of these patients. Overall, our study strengthens the genetic and functional evidence for the C316G and L399del variants in \u003cem\u003eRAG1\u0026nbsp;\u003c/em\u003ethat may synergistically cause SCID, and help inform the development of innovative therapeutic strategies for these patients.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003eStudy participants and sample collection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe probands\u0026rsquo; parents gave informed consent and agreed to participate in this study. To further explore the etiology of the disease, peripheral blood samples were taken from the couple, and their clinical manifestations and biochemical parameters were collected for analysis. Sanger sequencing was performed on the obtained samples. Identified variants were assessed using public databases (ClinVar, gnomAD, 1000 genomes project, and HGMD database), and all procedures were conducted under the principles of the Declaration of Helsinki.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBioinformatics analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo assess the \u003cem\u003eRAG1\u003c/em\u003e variants, we obtained the reference sequence (NM_000448.3) from NCBI GenBank. Conservation analysis was performed by aligning orthologs from six species (human, chimpanzee, pig, cattle, rat, mouse) using DNAMAN. We generated wild-type and mutant 3D structures via SWISS-MODEL, visualized in PyMOL\u0026nbsp;Viewer, and employed PROVEAN, PolyPhen-2, and MutationTaster to predict functional consequences.\u0026nbsp;Variant pathogenicity and annotation were evaluated according to the classification criteria and guidelines of ACMG.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell culture and nucleofection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe human RAG1\u003csup\u003e+\u003c/sup\u003e Pre-B cell line (Nalm6) was cultured in RPMI 1640\u0026nbsp;medium (Cellgro, USA)\u0026nbsp;supplemented with 10%\u0026nbsp;fetal bovine serum (Hyclone, USA), 100 U/mL penicillin and 100 \u0026mu;g/mL streptomycin (Gibco, US) at 37\u0026deg;C\u0026nbsp;in a humidified atmosphere of 5% CO2. Upon reaching a density of 5\u0026times;10⁵ ~ 1\u0026times;10⁶ cells/mL, cells were nucleofected using the Lonza 4D-Nucleofector\u0026trade; X Unit system (Lonza, Germany) with the manufacturer\u0026apos;s recommended program for Nalm6 cells. \u0026nbsp;Immediately post-nucleofection, pre-warmed complete medium was added, and the cells were gently transferred to a 60 mm dish. After 24 hours, the cells were collected by centrifugation (800 rpm, 5 min) and resuspended in fresh complete medium\u0026nbsp;without HDR Enhancer V2\u0026nbsp;for continued culture.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCRISPR-Cas9 gene editing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSingle-guide RNAs (gRNAs) and single-stranded oligodeoxynucleotides (ssODNs) were designed using the IDT Alt-R CRISPR HDR Design Tool.\u0026nbsp;Chemically modified gRNAs and high-fidelity Ultramer\u0026trade; ssODN HDR templates (Supplemental Table 1) were co-delivered via nucleofection of the RNP complex. We assessed initial editing efficiency with the \u003cem\u003eT7\u0026nbsp;\u003c/em\u003eEndonuclease I (\u003cem\u003eT7E\u003c/em\u003e I) assay and then isolated single-cell clones. The target locus was PCR-amplified from genomic DNA and verified by Sanger sequencing. We first established a homozygous C316G positive clone, then used the same strategy to introduce the L399del mutation into this clone to create the double mutant.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRNA extraction and quantitative real-time PCR\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal RNA was extracted using Trizol reagent (TaKaRa, Japan) and reverse-transcribed into cDNA using a commercial synthesis kit (Accurate Biology, China). Quantitative polymerase chain reaction (qPCR) analysis was carried out in a 7500 real-time PCR system (Applied Biosystems,\u0026nbsp;USA) using SYBR Green PCR Core Reagents (Accurate Biology, China). All samples were tested in triplicate, and average CT values were calculated and normalized to the housekeeping gene\u0026nbsp;\u0026beta;-actin according to the 2-\u0026Delta;\u0026Delta;Ct method. Primer sequences are listed in Supplemental Table 1.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWestern blotting\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal protein was extracted from cells using RIPA buffer supplemented with 1% PMSF (Beyotime, China). Protein concentration was determined by a BCA assay (Sangon Biotech, China). Denatured proteins were separated by 8% SDS-PAGE and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, USA). After blocking with 5% skim milk in TBST for 1 hour at room temperature, the membranes were incubated overnight at 4\u0026deg;C with the following primary antibodies: anti-RAG1 (Boster, 1:1500), anti-RAG2 (Proteintech, 1:800), anti-FOXO1 (Proteintech, 1:4000), or anti-GAPDH (Proteintech, 1:10000). Subsequently, the membranes were probed with an HRP-conjugated secondary antibody (Proteintech, 1:4000) for 1 hour at room temperature. Protein signals were visualized using an enhanced chemiluminescence (ECL) detection system.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell viability and apoptosis assays\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCell proliferation was assessed using the CCK-8 assay. Log-phase cells were seeded in 96-well plates at a density of 5\u0026times;10⁵ cells per well. Cell viability was quantified at 24, 48, and 72 h by measuring the absorbance at 450 nm according to the manufacturer\u0026apos;s instructions. Apoptosis was detected using an Annexin V-FITC/PI Apoptosis Detection Kit. Cells were stained and analyzed by flow cytometry\u0026nbsp;with appropriate FITC and PI channels. The apoptosis rate was calculated as the sum of early apoptosis (lower right quadrant) and late apoptosis (upper right quadrant) percentages. As a positive control for apoptosis, cells were treated with 30 \u0026mu;M cisplatin (DNA cross-linking apoptosis inducer) for 12 hours prior to flow cytometric analysis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eQuantitative\u0026nbsp;data were analyzed using GraphPad Prism 9.5 (GraphPad Software, USA). Differences among multiple groups were analyzed by one-way ANOVA followed by Tukey\u0026rsquo;s post hoc test.\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003cem\u003eP\u003c/em\u003e-Values less than 0.05 was considered statistically significant. All experiments were repeated at least three times.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eClinical features\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe couple, a 33-year-old male and a 29-year-old female with normal karyotypes, presented with a history of primary infertility. The female had irregular menstruation, and the male was diagnosed with oligoasthenoteratospermia. Their first pregnancy resulted in a full-term male infant who was diagnosed with \"congenital immunodeficiency syndrome\" at three months of age and died at six months. No genetic testing was performed. A subsequent intrauterine insemination (IUI) pregnancy resulted in a female infant diagnosed with T-B severe combined immunodeficiency (SCID) at two months of age, who also died at six months. Compound heterozygous mutations in the\u003cem\u003e\u0026nbsp;RAG1\u003c/em\u003e gene were identified in the second infant. Consequently, the couple was referred to the Prenatal and Genetic Clinic of our hospital and was recommended to pursue preimplantation genetic testing for monogenic disorders (PGT-M).\u003c/p\u003e\n\u003cp\u003ePrior to entering the PGT-M cycle, to investigate the etiology of the congenital immunodeficiency syndrome in the proband, whole-exome sequencing report from an external institution revealed compound heterozygous mutations in the \u003cem\u003eRAG1\u003c/em\u003e gene in the proband: c.1194_1196del (p.L399del) and c.946T\u0026gt;G (p.C316G), which were confirmed by Sanger sequencing. The couple showed no related symptoms. Following informed consent, their peripheral blood samples were subjected to karyotyping and Sanger sequencing. Variant pathogenicity was classified according to ACMG guidelines. All procedures were conducted in accordance with the Declaration of Helsinki.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIdentification of \u003cem\u003eRAG1\u0026nbsp;\u003c/em\u003emutations and pathogenicity analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo establish a genetic diagnosis for the probands (II-1 and II-2) in this pedigree (Fig. 1A), whole-exome sequencing revealed novel compound heterozygous variants in exon 2 of the \u003cem\u003eRAG1\u003c/em\u003e gene: a heterozygous c.946T\u0026gt;G (p.C316G) and a heterozygous c.1194_1196del (p.L399del). Sanger sequencing of parental peripheral blood confirmed the female carrier (I-1) harbored the heterozygous c.946T\u0026gt;G variant, while the male (I-2) carried the heterozygous c.1194_1196del variant (Fig. 1A, B). Cross-referencing these two mutations with the dbSNP, ClinVar or the Human Gene Mutation Database (HGMD) confirmed that neither variant had been previously reported or documented in the literature, establishing them as novel pathogenic variants.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMultiple sequence alignment of RAG1 across six vertebrate species demonstrated that Cys316 and Leu399 reside in highly conserved regions (Fig. 1C). Bioinformatic tools including PROVEAN, PolyPhen-2, and MutationTaster consistently predicted both variants to be damaging (Fig. 1D). According to ACMG/AMP guidelines, the c.946T\u0026gt;G (p.C316G) variant was classified as a variant of uncertain significance (VUS), with the following supporting evidence: PP1_Moderate (co-segregation with disease in multiple affected family members), PM2_Supporting (absent from ESP, 1000 genomes and ExAC databases), PP3 (multiple computational predictions support a deleterious effect), and PP4 (the phenotype of the patients was highly specific for a monogenic disorder). The c.1194_1196del (p.L399del) variant was classified as likely pathogenic based on PM4 (protein length change due to an in-frame deletion in a non-repeat region), PP1_Moderate, PM2_Supporting, and PP4. Subsequent screening of our institutional whole-exome database of infertility patients did not identify these \u003cem\u003eRAG1\u003c/em\u003e variants.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImpacts of the\u003c/strong\u003e\u003cstrong\u003emutations on the secondary and 3D structure of RAG1 protein\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo elucidate the structural impact of the identified RAG1 mutations, we performed computational analyses. Secondary structure prediction using PSIPRED v4.0 indicated that both p.C316G (hereinafter referred to as mut1) and p.L399del (hereinafter referred to as mut3) reside within alpha-helical regions (Fig. 2A). Domain analysis via Pfam and SMART databases localized the p.C316G mutation within the RING-type zinc finger domain (amino acids 293-331) and the p.L399del mutation within a DNA-binding domain (amino acids 392-467).\u003c/p\u003e\n\u003cp\u003eWe next modeled the tertiary structural changes using SWISS-MODEL. Due to the inability of a single template to cover the full length of the RAG1 amino acid sequence, the homology modeling method was employed using templates with database IDs 1rmd.1.A and 6oeo.1.C as homologous templates to predict the tertiary structure of the mutant human RAG1 protein. The 1rmd.1.A template covers amino acids 268 to 383 of RAG1 (the region containing mut1). Results indicate that the mut1 mutation leads to the substitution of the hydrophobic residue cysteine with the hydrophilic yet non-polar glycine. This alteration induces a change in the tetrahedral coordination geometry of the zinc finger structure, resulting in a loss of zinc ion binding capacity (Fig. 2B). The Zn²⁺ ion within the zinc\u0026nbsp;finger domain plays a critical role: it not only governs protein folding and provides the structural scaffold but also stabilizes the alpha-helical conformation. This stabilization enables the alpha-helix to dock into the major groove of DNA, allowing the zinc finger protein to achieve specific binding to DNA or RNA. Consequently, the mut1 mutation ultimately disrupts the zinc finger structure, inhibits its specific DNA-binding function, and compromises the regulation of gene expression.\u0026nbsp;The 6oeo.1.C template covers amino acids 396 to 1043 of RAG1 (the region containing mut3). Within this structure, two hydrogen bonds are formed between Histidine 398 and Serine 401. The mut3 mutation, which causes a deletion of Leucine 399, leads to the loss of one of these hydrogen bonds (Fig. 2C). The formation and stability of the protein's tertiary structure rely heavily on non-covalent interactions, including hydrogen bonding. Disruption of these hydrogen bonds can cause the protein's spatial configuration to become less compact and more disordered, reducing its structural stability and potentially impairing its biological function.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEstablishment of Monoclonal Pre-B Cell Lines with the RAG1 Targeted Mutations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo further investigate the impact of the mutation on protein function, the optimized Cas9 ribonucleoprotein (RNP) strategy was used to generate intended mutations into pre-B Cells.\u0026nbsp; Following confirmation of efficient genome editing by T7E1 cleavage assay, mutant cells were subjected to limiting dilution to isolate single clones. Individual monoclonal colonies were expanded to sufficient density, and genomic DNA was extracted, amplified via PCR, and sequenced to validate the mutations. As shown in Figure 3B, T7E1 cleavage analysis revealed high editing activity at both gRNA target sites. Quantification of band intensity using ImageJ indicated editing efficiencies of 77.63% for the c.946T\u0026gt;G (p.C316G) target and 80.12% for the c.1194_1196del (p.L399del) target. Sequencing results (Figure 3A) confirmed the successful establishment of four monoclonal pre-B cell lines harboring the intended mutations: (1) mut1: a homozygous c.946T\u0026gt;G (p.C316G) mutant line; (2) mut2: a homozygous c.946T\u0026gt;G (p.C316G) line carrying an additional synonymous nucleotide change (C\u0026gt;A); (3) mut3: a homozygous c.1194_1196del (p.L399del) mutant line; and (4) mut4: a homozygous double-mutant line containing both c.946T\u0026gt;G (p.C316G) and c.1194_1196del (p.L399del) mutations.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe mutations affect \u003cem\u003eRAG1\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003eand\u0026nbsp;\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eRAG2\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e\u0026nbsp;expression\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eQuantitative PCR analysis revealed distinct expression patterns of \u003cem\u003eRAG1\u0026nbsp;\u003c/em\u003eand \u003cem\u003eRAG2\u0026nbsp;\u003c/em\u003eacross the mutant cell lines (Figure 3C, 3D). While \u003cem\u003eRAG1\u0026nbsp;\u003c/em\u003emRNA levels in mut1, mut2, and mut3 cells showed no significant difference (ns, \u003cem\u003eP\u003c/em\u003e\u0026gt;0.05) compared to wild-type controls, a statistically significant reduction (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05) was observed in the mut4 double mutant. In contrast, \u003cem\u003eRAG2\u003c/em\u003e mRNA expression was significantly decreased (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05) in mut1, mut2, and mut4 cells relative to wild-type controls. Western blot analysis corroborated these findings at the protein level (Figure 3E, 3F). RAG1 protein (located at ~120 kDa) expression remained unchanged in mut1, mut2, and mut3 cells but was significantly reduced (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05) in the mut4 double mutant. Notably, RAG2 protein (located at ~ 59 kDa) expression was significantly diminished (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05) across all mutant cell lines, including mut1, mut2, mut3, and mut4.\u003c/p\u003e\n\u003cp\u003eThese results indicate that both the c.946T\u0026gt;G (p.C316G) and c.1194_1196del (p.L399del) mutations in RAG1 significantly impair RAG2 expression in pre-B lymphocytes. The impaired expression of RAG2 is likely a direct consequence of the functional impairment of mutant RAG1 protein, consequently affecting the stability or expression of its essential molecular partner, RAG2.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFlow cytometric analysis demonstrated a significant increase in apoptosis in \u003cem\u003eRAG1\u003c/em\u003e-mutant pre-B cell lines\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs illustrated in Figure 4F and 4G, cisplatin-treated positive control cells exhibited markedly elevated levels of both early and late apoptosis, with a combined apoptotic rate of 19.86%. In contrast, wild-type pre-B cells showed a baseline apoptosis rate of 0.63%. Introduction of RAG1 mutations resulted in progressively increased apoptotic rates across all mutant lines: mut1 (1.79%), mut2 (1.26%), mut3 (2.62%), and the double mutant (3.48%). These findings indicate that both the c.946T\u0026gt;G (p.C316G) and c.1194_1196del (p.L399del) mutations in \u003cem\u003eRAG1\u0026nbsp;\u003c/em\u003esignificantly enhance susceptibility to apoptosis in pre-B cells. The observed mutation-dependent increase in apoptosis suggests a potential mechanism through which RAG1 deficiencies may impair B cell development by promoting cell death at the pre-B cell stage, thereby contributing to the immunodeficiency phenotype.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eRAG1\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;variants do not impair pre-B cell proliferation capacity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAssessment of cellular proliferation by CCK-8 assay revealed no significant differences between RAG1-mutant pre-B cells and wild-type controls at 24, 48, or 72 hours post-seeding (\u003cem\u003eP\u003c/em\u003e\u0026gt;0.05; Figure 4H). These results indicate that the c.946T\u0026gt;G (p.C316G) and c.1194_1196del (p.L399del) mutations in RAG1 do not substantially affect the proliferative capacity of pre-B cells. Together with our previous observation of enhanced apoptosis in these mutants, these findings suggest that the reduction in mature B lymphocytes associated with RAG1 deficiency is likely mediated through increased apoptotic susceptibility rather than impaired proliferation, providing a mechanistic insight into the development of immunodeficiency in affected patients.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eRAG1\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;mutations potentially modulate the PI3K/AKT1/FOXO1 signaling pathway and promote apoptosis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe PI3K/AKT/FOXO1 signaling pathway plays a crucial role in regulating cell survival and apoptosis [14]. To examine whether RAG1 mutations influence this pathway, we analyzed the expression of key pathway components. Q-PCR analysis revealed significant upregulation of AKT1 and FOXO1 mRNA levels in both mut3 and double mutant cells (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05; Figure 3C, 3D). However, at the protein level, Western blot analysis presented a more complex picture: while total FOXO1 protein was decreased in the double mutant group (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05; Figure 3E, 3F), the levels of phosphorylated FOXO1 (p-FOXO1) were significantly elevated (Figure 5A, 5B). This critical finding indicates that the reduction in total FOXO1 is likely attributable to post-translational regulation. Consistent with enhanced FOXO1 inactivation, we also observed a marked increase in the expression of both total AKT and phosphorylated AKT (p-AKT) in the double mutant (Figure 5A, 5B). In contrast, acetylated FOXO1 (Ac-FOXO1) levels remained unchanged across all groups. Collectively, these results demonstrate that the double mutation in RAG1 leads to constitutive activation of AKT, which subsequently promotes FOXO1 phosphorylation and degradation, thereby disrupting normal PI3K/AKT/FOXO1 signaling and contributing to apoptosis in pre-B lymphocytes.\u0026nbsp;\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eWhile the clinical spectrum of Severe Combined Immunodeficiency (SCID) associated with \u003cem\u003eRAG1\u003c/em\u003e mutations has been well-documented, functional validation of specific variants remains limited [15-17]. \u0026nbsp;In the present study, we identified two novel compound heterozygous mutations\u0026nbsp;of\u0026nbsp;\u003cem\u003eRAG1\u0026nbsp;\u003c/em\u003egene, c.946T\u0026gt;G/p.C316G\u0026nbsp;and\u0026nbsp;c.1194_1196del/p.L399del, in a Chinese family presenting with a typical SCID phenotype. These mutations are not reported in public Genome Aggregation Database (gnomAD) or on the Exome Variants Server (EVS), and their functional impact has not been experimentally characterized.\u003c/p\u003e\n\u003cp\u003eNotably, it has been proven that the RAG1/2 mutations can lead to multiple forms of immunological and clinical phenotypes [9, 11, 18]. According to the\u0026nbsp;clinical descriptions of the patients and their children,\u0026nbsp;neither heterozygous parent exhibited immunodeficiency, suggesting that neither variant alone is sufficient to cause disease. However, the compound heterozygous state in the probands led to full SCID manifestation, consistent with an autosomal recessive mode of inheritance. This pattern, in which the disease phenotype manifests only in the compound heterozygous state, is consistent with previous reports by several authors\u0026nbsp;[19, 17, 20]\u0026nbsp;.\u003c/p\u003e\n\u003cp\u003eBioinformatic analysis indicated that both mutations detected in our study are evolutionarily conserved across vertebrates and are located in functionally critical domains: p.Cys316Gly mutation within the RING-type zinc finger domain, which is essential for zinc ion coordination and RAG1-RAG2 complex assembly [21], while\u0026nbsp;p.L399del\u0026nbsp;mutation localizes to the nonamer DNA-binding region (NBR) of the core RAG1, responsible for specific recognition of recombination signal sequences (RSS) and proper targeting of V(D)J recombination adhere to the 12/23 rule\u0026nbsp;[22]. Consistently, configuration and pathogenicity prediction also revealed the damaging impacts of both \u003cem\u003eRAG1\u003c/em\u003e variants on the protein\u0026rsquo;s 3D structure and stability.\u0026nbsp;The p.C316G\u0026nbsp;mutation probably contributes to the substitution of a hydrophobic cysteine residue with a hydrophilic yet non-polar glycine is predicted to abolish the domain\u0026rsquo;s ability to bind zinc ions. The p.L399del mutation reduces hydrogen bonding with adjacent residues such as His398 and Ser401, potentially impairing interaction efficiency with DNA substrates/RAG2 and complex stability, and ultimately impair the overall efficacy of V(D)J recombination.\u003c/p\u003e\n\u003cp\u003eTo functionally validate these predictions, we successfully established these homozygous mutations into human pre-B cell lines using a CRISPR-Cas9 RNP approach. While single mutants did not significantly alter RAG1 expression, the double mutant exhibited markedly reduced levels of both RAG1 and RAG2 proteins, accompanied by increased apoptosis. In conjunction with the previously constructed secondary and tertiary structure models, it suggested that the mutations potentially induce apoptosis in pre-B lymphocytes\u0026nbsp;through aberrant protein structure and destabilization of RAG complex, thereby impairing B lymphocyte survival. Given the notable decrease in RAG2 expression, we propose that the RAG1 c.946T\u0026gt;G (p.C316G) and c.1194_1196del\u0026nbsp;mutations may coordinately affect the expression of RAG2, and that their synergistic effect disrupts normal B lymphocyte differentiation and development.\u003c/p\u003e\n\u003cp\u003eInterestingly, we observed a significant decrease in FOXO1 protein expression in double-mutant cells. Previous studies have established FOXO1 as\u0026nbsp;a crucial transcription factor in B cell development and a key regulator of RAG1/2 expression at the pre-B cell stage\u0026nbsp;[23-25]. Consistent with this, Foxo1\u003csup\u003e\u0026ndash;/\u0026ndash;\u003c/sup\u003e pro‑B cells exhibit increased apoptosis and show impaired V\u003csub\u003eH\u003c/sub\u003e to D\u003csub\u003eH\u003c/sub\u003eJ\u003csub\u003eH\u003c/sub\u003e gene rearrangement [23], underscoring the essential role of FOXO1 in both gene recombination and cell survival. \u0026nbsp;Based on these findings and our data, we hypothesize that the double mutation in \u003cem\u003eRAG1\u003c/em\u003e limits RAG1/2 expression, and that a subsequent negative feedback mechanism suppresses FOXO1 expression. This downregulation of FOXO1 may then inhibit gene recombination and ultimately contribute to pre-B cell apoptosis.\u003c/p\u003e\n\u003cp\u003eThis model is firmly supported by established signaling pathways and our experimental data.\u0026nbsp;Signals emanating from the interleukin-7 receptor (IL7R) and the pre-B-cell receptor (pre-BCR) regulate the dynamic pattern of RAG1 and RAG2 expression, which involves phosphoinositide-3 kinase (PI3K) and protein kinase B (PKB, also known as AKT) impinging on FOXO transcription factors, themselves critical for \u003cem\u003eRAG\u003c/em\u003e expression and gene recombination [26, 24, 27]. In line with this, we found that double-mutant cells exhibited increased activation of AKT, as evidenced by elevated levels of phosphorylated AKT (p-AKT). Most importantly, and in direct support of our hypothesis, we detected a significant increase in phosphorylated FOXO1 (p-FOXO1) in these cells. This finding provides direct molecular evidence that the functional impairment caused by the RAG1 double mutation leads to aberrant activation of the PI3K/AKT signaling pathway. The consequent AKT-mediated phosphorylation of FOXO1 triggers its nuclear export and proteasomal degradation\u0026nbsp;[28-30], which perfectly explains the concurrent decrease in total FOXO1 protein we observed. This persistent inactivation and degradation of FOXO1, a known regulator of genes controlling cell cycle arrest [31] and apoptosis [32], provides a compelling mechanistic link between the \u003cem\u003eRAG1\u003c/em\u003e mutations and the increased apoptosis in pre-B lymphocytes.\u003c/p\u003e\n\u003cp\u003eThis study has certain limitations. The initial WES data for the probands were obtained from an external clinical report, and original DNA samples were unavailable for further validation.\u0026nbsp;Furthermore, all functional assays were performed in an \u003cem\u003ein vitro\u0026nbsp;\u003c/em\u003epre-B cell model, future studies should employ animal models to better recapitulate the systemic immune pathology.\u003c/p\u003e\n\u003cp\u003eIn conclusion, our study identifies two novel \u003cem\u003eRAG1\u003c/em\u003e mutations, p.C316G and p.L399del, that act synergistically to disrupt protein stability, impair RAG2 expression, and promote pre-B cell apoptosis. We provide direct evidence that this process is mechanistically driven by the constitutive activation of the PI3K/AKT pathway, leading to AKT-mediated FOXO1 phosphorylation and degradation. These findings expand the mutational spectrum of \u003cem\u003eRAG1\u0026nbsp;\u003c/em\u003eand elucidate a key pathological pathway in RAG-deficient SCID.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eThis work was funded by the National Traditional Chinese Medicine Inheritance and Innovation Center, the First Affiliated Hospital of XXX University (grant number: 2023QN02), the Administration of Traditional Chinese Medicine of Guangdong Province (grant numbers: 20241103, 20242036), and the Medical Scientific Research Foundation of Guangdong Province (grant number: A2023401).\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval and consent to participate\u003c/strong\u003e All experiments were approved by the Ethics Committee of\u0026nbsp;the First Affiliated Hospital of XXX University\u0026nbsp;(No. ZYYEC-ERK-2022-093) and\u0026nbsp;all procedures were in accordance with the ethical guidelines of the Helsinki Declaration.\u0026nbsp;The parents were informed and consented to participate.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u0026nbsp;\u003c/strong\u003eWritten informed consent was obtained from the parents for publication of this study, including any personal or clinical details.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e Data will be made available on request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAranda CS, Gouveia-Pereira MP, da Silva CJM, Rizzo M, Ishizuka E, de Oliveira EB, et al. 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Int J Mol Med. 2018;42(6):3238-46. https://doi:10.3892/ijmm.2018.3895.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":false,"email":"","identity":"journal-of-assisted-reproduction-and-genetics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Journal of Assisted Reproduction and Genetics","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"VoR Journals","inReviewEnabled":false,"inReviewRevisionsEnabled":false},"keywords":"Severe combined immunodeficiency, RAG1, Compound heterozygous mutations, Functional analysis, Apoptosis","lastPublishedDoi":"10.21203/rs.3.rs-9021280/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9021280/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003ePurpose\u003c/strong\u003e Severe combined immunodeficiency(SCID) is a life-threatening primary immunodeficiency disorder. This study aimed to identify novel recombination activating gene 1 (\u003cem\u003eRAG1\u003c/em\u003e) variants in a Chinese pedigree and characterize their impact on protein structure and function, providing a genetic basis for preimplantation genetic testing for monogenic (PGT-M) cycle.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e Potential \u003cem\u003eRAG1\u003c/em\u003e mutations of\u003cstrong\u003e \u003c/strong\u003ethe probands were screened by whole-exome sequencing (WES) and confirmed by Sanger sequencing. Configuration predictions of the variants were achieved using SWISS-MODEL. PROVEAN, PolyPhen-2 and MutationTaster were used to predict their pathogenicity. Isogenic pre-B cell lines carrying the mutations were established via CRISPR-Cas9 RNP editing. Functional impacts were assessed through Western blotting, proliferation ability, and apoptosis analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/em\u003eWe identified novel compound heterozygous \u003cem\u003eRAG1\u003c/em\u003e variants c.946T\u0026gt;G (p.C316G) and c.1194_1196del (p.L399del) in two affected siblings with typical SCID. Familial genotyping confirmed autosomal recessive inheritance, with each parent as an asymptomatic carrier of one variant. Both mutations were highly conserved and predicted to be pathogenic. Structural modeling revealed disruption of RAG1 secondary and tertiary structure, affecting zinc-binding (p.C316G) and hydrogen-bonding (p.L399del) interactions. Functional studies demonstrated markedly reduced RAG1 protein expression, synergistic impairment of RAG2 expression, and significantly elevated apoptosis in double-mutant pre‑B cells. Further investigation indicated dysregulation of the PI3K/AKT/FOXO1 pathway, evidenced by increased phosphorylation of AKT and FOXO1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions\u003c/strong\u003e Our study provides genetic and functional evidence that biallelic \u003cem\u003eRAG1\u003c/em\u003e p.C316G and p.L399del mutations act synergistically to cause SCID through protein destabilization, disruption of RAG1/RAG2 complex integrity, and induction of pre‑B cell apoptosis likely mediated by PI3K/AKT/FOXO1 signaling dysregulation. These findings expand the mutational spectrum of RAG1 and support the clinical application of PGT-M for affected families.\u003c/p\u003e","manuscriptTitle":"From PGT-M Discovery to Mechanism: Functional Validation of novel compound heterozygous RAG1 Mutations in Severe Combined Immunodeficiency","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-20 09:14:21","doi":"10.21203/rs.3.rs-9021280/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-04-01T01:11:01+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-25T06:47:58+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-24T10:42:37+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"215966982041596915195602720697851905018","date":"2026-03-18T14:15:16+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"205578133213627328780773736886343838620","date":"2026-03-18T02:46:53+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-03-18T00:43:27+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-03-10T01:23:24+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-03-10T01:22:28+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Assisted Reproduction and Genetics","date":"2026-03-03T14:17:26+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":false,"email":"","identity":"journal-of-assisted-reproduction-and-genetics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Journal of Assisted Reproduction and Genetics","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"VoR Journals","inReviewEnabled":false,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"97c872e9-c94e-4ec6-9490-26a4e84c9365","owner":[],"postedDate":"March 20th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-04-22T13:25:21+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-20 09:14:21","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9021280","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9021280","identity":"rs-9021280","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}