Pancreatic neuroendocrine tumors in French VHL mutation carriers: a multicentric retrospective study.

BACKGROUND: Von Hippel-Lindau disease (VHL) is a rare autosomal dominant hereditary cancer-predisposition syndrome caused by germline pathogenic variants (PV) in VHL gene. It is associated with a high penetrance of benign and malignant vascular tumors in multiples organs, including pancreatic neuroendocrine tumors (PanNETs), whose long-term natural history is ill-known. METHODS: Patients with both documented germline PV in VHL gene and PanNETs included in the French PREDIR database between 1995 and 2022 were included. Primary endpoint was the proportion of patients with PanNET-related metastases and secondary endpoint was overall survival (OS). Genotype/phenotype correlations were studied. RESULTS: We included 121 patients with 259 PanNETs. Median age at diagnosis was 38 years. Median follow-up was 89.5 months. PanNET surgical resection was performed in 51 patients. Overall, 29 patients (24%) had metastases (5 synchronous, 10 metachronous), with a higher risk in case of larger PanNET size (p=0.0089; best threshold 28 mm) and grade 2 PanNET (p=0.048), and a pejorative prognostic impact (p=0.043). Patients with PV in VHL exon 1 had larger PanNETs (p=0.018), more often metastatic disease (48% vs 11.5%; p < 0.001) and a trend toward shorter OS (p=0.16). CONCLUSION: The risk of metastases associated to VHL-related PanNETs remains low (24%) but increases with tumor size >28 mm, higher grade and in case of PV located VHL exon 1. These data might help improving the management of these patients, who should be referred to an expert center.

DCLRE1B/Apollo germline mutations associated with renal cell carcinoma impair telomere protection.

Hereditary renal cell carcinoma (RCC) is caused by germline mutations in a subset of genes, including VHL, MET, FLCN, and FH. However, many familial RCC cases do not harbor mutations in the known predisposition genes. Using Whole Exome Sequencing, we identified two germline missense variants in the DCLRE1B/Apollo gene (ApolloN246I and ApolloY273H) in two unrelated families with several RCC cases. Apollo encodes an exonuclease involved in DNA Damage Response and Repair (DDRR) and telomere integrity. We characterized these two functions in the human renal epithelial cell line HKC8. The decrease or inhibition of Apollo expression sensitizes these cells to DNA interstrand crosslink damage (ICLs). HKC8 Apollo-/- cells appear defective in the DDRR and present an accumulation of telomere damage. Wild-type and mutated Apollo forms could interact with TRF2, a shelterin protein involved in telomere protection. However, only ApolloWT can rescue the telomere damage in HKC8 Apollo-/- cells. Our results strongly suggest that ApolloN246I and ApolloY273H are loss-of-function mutants that cause impaired telomere integrity and could lead to genomic instability. Altogether, our results suggest that mutations in Apollo could induce renal oncogenesis.

Update of the UMD-VHL database: classification of 164 challenging variants based on genotype-phenotype correlation among 605 entries.

BACKGROUND: The von Hippel-Lindau (VHL) disease is a hereditary tumour syndrome caused by germline mutations in VHL tumour suppressor gene. The identification of VHL variants requires accurate classification which has an impact on patient management and genetic counselling. METHODS: The TENGEN (French oncogenetics network of neuroendocrine tumors) and PREDIR (French National Cancer Institute network for Inherited predispositions to kidney cancer) networks have collected VHL genetic variants and clinical characteristics of all VHL-suspected patients analysed from 2003 to 2021 by one of the nine laboratories performing VHL genetic testing in France. Identified variants were registered in a locus-specific database, the Universal Mutation Database-VHL database (http://www.umd.be/VHL/). RESULTS: Here we report the expert classification of 164 variants, including all missense variants (n=124), all difficult interpretation variants (n=40) and their associated phenotypes. After initial American College of Medical Genetics classification, first-round classification was performed by the VHL expert group followed by a second round for discordant and ambiguous cases. Overall, the VHL experts modified the classification of 87 variants including 30 variants of uncertain significance that were as (likely)pathogenic variants for 19, and as likely benign for 11. CONCLUSION: Consequently, this work has allowed the diagnosis and influenced the genetic counselling of 45 VHL-suspected families and can benefit to the worldwide VHL community, through this review.

Inhibition of type I PRMTs reforms muscle stem cell identity enhancing their therapeutic capacity.

In skeletal muscle, muscle stem cells (MuSC) are the main cells responsible for regeneration upon injury. In diseased skeletal muscle, it would be therapeutically advantageous to replace defective MuSCs, or rejuvenate them with drugs to enhance their self-renewal and ensure long-term regenerative potential. One limitation of the replacement approach has been the inability to efficiently expand MuSCs ex vivo, while maintaining their stemness and engraftment abilities. Herein, we show that inhibition of type I protein arginine methyltransferases (PRMTs) with MS023 increases the proliferative capacity of ex vivo cultured MuSCs. Single cell RNA sequencing (scRNAseq) of ex vivo cultured MuSCs revealed the emergence of subpopulations in MS023-treated cells which are defined by elevated Pax7 expression and markers of MuSC quiescence, both features of enhanced self-renewal. Furthermore, the scRNAseq identified MS023-specific subpopulations to be metabolically altered with upregulated glycolysis and oxidative phosphorylation (OxPhos). Transplantation of MuSCs treated with MS023 had a better ability to repopulate the MuSC niche and contributed efficiently to muscle regeneration following injury. Interestingly, the preclinical mouse model of Duchenne muscular dystrophy had increased grip strength with MS023 treatment. Our findings show that inhibition of type I PRMTs increased the proliferation capabilities of MuSCs with altered cellular metabolism, while maintaining their stem-like properties such as self-renewal and engraftment potential.

Protein arginine methyltransferase 1 regulates B cell fate after positive selection in the germinal center in mice.

Positively selected germinal center B cells (GCBC) can either resume proliferation and somatic hypermutation or differentiate. The mechanisms dictating these alternative cell fates are incompletely understood. We show that the protein arginine methyltransferase 1 (Prmt1) is upregulated in murine GCBC by Myc and mTORC-dependent signaling after positive selection. Deleting Prmt1 in activated B cells compromises antibody affinity maturation by hampering proliferation and GCBC light zone to dark zone cycling. Prmt1 deficiency also results in enhanced memory B cell generation and plasma cell differentiation, albeit the quality of these cells is compromised by the GCBC defects. We further demonstrate that Prmt1 intrinsically limits plasma cell differentiation, a function co-opted by B cell lymphoma (BCL) cells. Consistently, PRMT1 expression in BCL correlates with poor disease outcome, depends on MYC and mTORC1 activity, is required for cell proliferation, and prevents differentiation. Collectively, these data identify PRMT1 as a determinant of normal and cancerous mature B cell proliferation and differentiation balance.

Preimplantation genetic testing in patients with genetic susceptibility to cancer.

Data on preimplantation genetic testing (PGT-M) in patients with genetic susceptibility to cancer are scarce in the literature, while there is, in our experience, a growing familiarity with assisted reproduction techniques (ART) among pathogenic variant heterozygotes. We performed a retrospective multicenter study of PGT-M outcomes among French patients with genetic susceptibility to cancer. Our objectives were to collect data on this complex issue, and to help cancer geneticists counsel their patients of reproductive age. We also wanted to increase awareness regarding PGT-M among cancer genetics professionals. Patients from three university hospital cancer genetics clinics who had requested PGT-M between 2000 and 2019 were included retrospectively. Data were extracted from medical records. Patients were then contacted directly to collect missing and up-to-date information. Out of 41 eligible patients, 28 agreed explicitly to participate when contacted and were therefore included. They carried PV in VHL (n = 9), APC (n = 8), CDH1 (n = 5), STK11 (n = 2), AXIN2, BRCA1, MEN1, and FH (n = 1). Seven patients were denied PGT-M based on multidisciplinary team meetings or subsequently by the ART hospital teams, two changed their minds, and two were yet to start the process. PGT-M was successful in seven patients (25%), with a mean age at PGT-M request of 27. Most had von Hippel-Lindau. PGT-M failed in the remaining ten, with a mean age at PGT-M request of 32. The main reason for failure was non-implantation of the embryo. Of these, four patients were pursuing PGT-M at the time of last contact. PGT-M outcomes in patients with cancer susceptibility syndromes were satisfactory. These patients should be informed about PGT-M more systematically, which would imply greater awareness among cancer genetics professionals regarding ART. Our series was not representative of cancer susceptibility syndromes in general; the predominance of cases with syndromes characterized by early-onset, highly penetrant disease is explained by the restrictive French guidelines.

Percutaneous Thermal Ablation for Renal Tumors in Patients with Birt-Hogg-Dubé Syndrome.

BHD syndrome is characterized by an increased risk of bilateral and multifocal renal cell carcinoma (RCCs), but is rarely metastatic. Our report aims to analyze the outcome of patients with BHD syndrome who underwent percutaneous thermal ablation (TA). The present report included six BHD syndrome patients (five men) with a mean age of 66 ± 11 (SD) years who had a proven germline FLCN gene mutation and underwent TA for a renal tumor. Nineteen renal tumors (median two tumors per patient; range: 1-3), including seven chromophobe RCCs, five clear-cell RCCs, four papillary RCCs, two clear-cell papillary RCC, and one hybrid oncocytic/chromophobe tumor were treated in 14 ablation sessions. The mean size of the tumors was 21 ± 11 (SD) mm (median: 20 mm; interquartile range (IQR): 14-25 mm) for a mean volume of 7 ± 11 (SD) mL (median: 3; IQR: 1-5 mL). Technical success was achieved in all ablation sessions (primary success rate, 100%). The procedure was well tolerated under conscious sedation with no significant Clavien-Dindo complication (grade 2, 3, 4). All patients were alive with no distant metastasis during a median follow-up period of 74 months (range: 33-83 months). No local tumor progression was observed. The mean decrease in estimated glomerular filtration rate was 8 mL/min/1.73 m2. No patients required dialysis or renal transplantation. In this case series, percutaneous TA appeared as a safe and efficient nephron-sparing treatment for treating RCCs associated with BHD syndrome, even in the case of advanced chronic kidney disease.

Minors at risk of von Hippel-Lindau disease: 10 years' experience of predictive genetic testing and follow-up adherence.

Von Hippel-Lindau (VHL) disease is one of the most common cancer predisposition syndromes. Penetrance is high with around 20% of children presenting detectable and curable manifestations of the disease at 15 years old. VHL predictive genetic testing (PGT) is recommended during childhood from age 5 years in France. Insufficient compliance to surveillance of VHL pathogenic variant (PV) carriers is associated with severe outcome. PGT experienced by children and their parents is probably critical in influencing future acceptance of the result and adherence to surveillance. We conducted a retrospective study on minors tested (aged 5 to 16 years old) from 2010 to 2020, in a multidisciplinary oncogenetics consultation which follows a 3-step protocol based on psychological familial support. The objectives were to assess the adherence to follow-up within the National Expert Center for inherited predispositions to renal tumors (PREDIR) network of VHL PV carriers and its benefit through tumor detection and medical interventions. A VHL PGT was carried out in 34 children. Among the 16 children diagnosed as VHL PV carriers addressed to the PREDIR network, none had discontinued surveillance after a median of 41 months. Follow-up examinations detected 11 tumors in 6 children, 4 have been surgically treated. All had a favorable outcome. Our data suggest that a specific and adapted procedure for PGT in at-risk VHL children as well as a follow-up, organized within a specialized expert network, fosters a complete adherence to the surveillance protocol and thus lead to a favorable clinical outcome.

The Influence of Arginine Methylation in Immunity and Inflammation.

Exploration in the field of epigenetics has revealed that protein arginine methyltransferases (PRMTs) contribute to disease, and this has given way to the development of specific small molecule compounds that inhibit arginine methylation. Protein arginine methylation is known to regulate fundamental cellular processes, such as transcription; pre-mRNA splicing and other RNA processing mechanisms; signal transduction, including the anti-viral response; and cellular metabolism. PRMTs are also implicated in the regulation of physiological processes, including embryonic development, myogenesis, and the immune system. Finally, the dysregulation of PRMTs is apparent in cancer, neurodegeneration, muscular disorders, and during inflammation. Herein, we review the functions of PRMTs in immunity and inflammation. We also discuss recent progress with PRMTs regarding the modulation of gene expression related to T and B lymphocyte differentiation, germinal center dynamics, and anti-viral signaling responses, as well as the clinical relevance of using PRMT inhibitors alone or in combination with other drugs to treat cancer, immune, and inflammatory-related diseases.

Arsenic 3 methyltransferase (AS3MT) automethylates on cysteine residues in vitro.

Arsenic toxicity is a global concern to human health causing increased incidences of cancer, bronchopulmonary, and cardiovascular diseases. In human and mouse, inorganic arsenic (iAs) is metabolized in a series of methylation steps catalyzed by arsenic (3) methyltransferase (AS3MT), forming methylated arsenite (MAsIII), dimethylarsenite (DMAIII) and the volatile trimethylarsine (TMA). The methylation of arsenic is coordinated by four conserved cysteines proposed to participate in catalysis, namely C33, C62, C157, and C207 in mouse AS3MT. The current model consists of AS3MT methylating iAs in the presence of the cofactor S-adenosyl-L-methionine (SAM), and the formation of intramolecular disulfide bonds following the reduction of MAsV to MAsIII. In the presence of endogenous reductants, these disulfide bonds are reduced, the enzyme re-generates, and the second round of methylation ensues. Using in vitro methylation assays, we find that AS3MT undergoes an initial automethylation step in the absence of iAs. This automethylation is enhanced by glutathione (GSH) and dithiothreitol (DTT), suggesting that reduced cysteines accept methyl groups from SAM to form S-methylcysteines. Following the addition of iAs, automethylation of AS3MT is decreased. Furthermore, using a Flag-AS3MT immunoprecipitation coupled to MS/MS, we identify both C33 and C62 as acceptors of the methyl group in vivo. Site-directed mutagenesis (C to A) revealed that three of the previously described cysteines were required for AS3MT automethylation. In vitro experiments show that automethylated AS3MT can methylate iAs in the presence of SAM. Thus, we propose that automethylated may represent an active conformation of AS3MT.

PRMT7 ablation stimulates anti-tumor immunity and sensitizes melanoma to immune checkpoint blockade.

Despite the success of immune checkpoint inhibitor (ICI) therapy for cancer, resistance and relapse are frequent. Combination therapies are expected to enhance response rates and overcome this resistance. Herein, we report that combining PRMT7 inhibition with ICI therapy induces a strong anti-tumor T cell immunity and restrains tumor growth in vivo by increasing immune cell infiltration. PRMT7-deficient B16.F10 melanoma exhibits increased expression of genes in the interferon pathway, antigen presentation, and chemokine signaling. PRMT7 deficiency or inhibition with SGC3027 in B16.F10 melanoma results in reduced DNMT expression, loss of DNA methylation in the regulatory regions of endogenous retroviral elements (ERVs) causing their increased expression. PRMT7-deficient cells increase RIG-I and MDA5 expression with a reduction in the H4R3me2s repressive histone mark at their gene promoters. Our findings identify PRMT7 as a regulatory checkpoint for RIG-I, MDA5, and their ERV-double-stranded RNA (dsRNA) ligands, facilitating immune escape and anti-tumor T cell immunity to restrain tumor growth.

Microexon alternative splicing of small GTPase regulators: Implication in central nervous system diseases.

Microexons are small sized (≤51 bp) exons which undergo extensive alternative splicing in neurons, microglia, embryonic stem cells, and cancer cells, giving rise to cell type specific protein isoforms. Due to their small sizes, microexons provide a unique challenge for the splicing machinery. They frequently lack exon splicer enhancers/repressors and require specialized neighboring trans-regulatory and cis-regulatory elements bound by RNA binding proteins (RBPs) for their inclusion. The functional consequences of including microexons within mRNAs have been extensively documented in the central nervous system (CNS) and aberrations in their inclusion have been observed to lead to abnormal processes. Despite the increasing evidence for microexons impacting cellular physiology within CNS, mechanistic details illustrating their functional importance in diseases of the CNS is still limited. In this review, we discuss the unique characteristics of microexons, and how RBPs participate in regulating their inclusion and exclusion during splicing. We consider recent findings of microexon alternative splicing and their implication for regulating the function of small GTPases in the context of the microglia, and we extrapolate these findings to what is known in neurons. We further discuss the emerging evidence for dysregulation of the Rho GTPase pathway in CNS diseases and the consequences contributed by the mis-splicing of microexons. This article is categorized under: RNA Processing > Splicing Mechanisms RNA Processing > Splicing Regulation/Alternative Splicing RNA in Disease and Development > RNA in Disease.

POGZ promotes homology-directed DNA repair in an HP1-dependent manner.

The heterochromatin protein HP1 plays a central role in the maintenance of genome stability but little is known about how HP1 is controlled. Here, we show that the zinc finger protein POGZ promotes the presence of HP1 at DNA double-strand breaks (DSBs) in human cells. POGZ depletion delays the resolution of DSBs and sensitizes cells to different DNA-damaging agents, including cisplatin and talazoparib. Mechanistically, POGZ promotes homology-directed DNA repair by retaining the BRCA1/BARD1 complex at DSBs in an HP1-dependent manner. In vivo CRISPR inactivation of Pogz is embryonically lethal. Pogz haploinsufficiency (Pogz+ /delta) results in developmental delay, impaired intellectual abilities, hyperactive behaviour and a compromised humoral immune response in mice, recapitulating the main clinical features of the White Sutton syndrome (WHSUS). Pogz+ /delta mice are further radiosensitive and accumulate DSBs in diverse tissues, including the spleen and brain. Altogether, our findings identify POGZ as an important player in homology-directed DNA repair both in vitro and in vivo.

Novel germline MET pathogenic variants in French patients with papillary renal cell carcinomas type I.

Hereditary papillary renal cell carcinoma (HPRC) is a rare inherited renal cancer syndrome characterized by bilateral and multifocal papillary type 1 renal tumors (PRCC1). Activating germline pathogenic variants of the MET gene were identified in HPRC families. We reviewed the medical and molecular records of a large French series of 158 patients screened for MET oncogenic variants. MET pathogenic and likely pathogenic variants rate was 12.4% with 40.6% among patients with familial PRCC1 and 5% among patients with sporadic PRCC1. The phenotype in cases with MET pathogenic and likely pathogenic variants was characteristic: PRCC1 tumors were mainly bilateral (84.3%) and multifocal (87.5%). Histologically, six out of seven patients with MET pathogenic variant harbored biphasic squamoid alveolar PRCC. Genetic screening identified one novel pathogenic variant MET c.3389T>C, p.(Leu1130Ser) and three novel likely pathogenic variants: MET c.3257A>T, p.(His1086Leu); MET c.3305T>C, p.(Ile1102Thr) and MET c.3373T>G, p.(Cys1125Gly). Functional assay confirmed their oncogenic effect as they induced an abnormal focus formation. The genotype-phenotype correlation between MET pathogenic variants and PRCC1 presentation should encourage to widen the screening, especially toward nonfamilial PRCC1. This precise phenotype also constitutes a strong argument for the classification of novel missense variants within the tyrosine kinase domain when functional assays are not accessible.

Clear cell and papillary renal cell carcinomas in hereditary papillary renal cell carcinoma (HPRCC) syndrome: a case report.

BACKGROUND: Hereditary papillary renal cell carcinoma (HPRCC) is a rare autosomal dominant disease characterized by the development of multiple and bilateral papillary type I renal cell carcinomas (RCC) and papillary adenomas caused by activating mutations in the MET proto-oncogene. Classically, distinctive histological features of RCC are described according to the familial renal cell carcinoma syndrome. To date, no clear cell RCC has been reported in HPRCC syndrome. CASE PRESENTATION: We describe the case of a 51-year-old man with a germline MET mutation detected on peripheral blood testing, and no germline VHL mutation, who developed numerous papillary tumors but also unexpectedly clear cell renal cell carcinomas. During the follow-up, an adrenal metastasis was observed 7 years after the initial diagnosis corresponding to a clear cell RCC metastasis. By immunohistochemistry, clear cell tumors showed focal cytokeratin 7, moderate racemase, and diffuse and membranous CAIX expression, while papillary tumors expressed strong diffuse cytokeratin 7 and racemase without CAIX positivity. Using FISH, VHL deletion was observed in one of the clear cell tumors, and the metastatic clear cell tumor presented a trisomy of chromosomes 7 and 17. These last genomic alterations are usually detected in papillary RCC, highlighting the potential link between both histological subtypes of tumors and the HPRCC syndrome. CONCLUSIONS: The pathologist must be aware that the presence of a non-papillary RCC associated with numerous papillary tumors should not exclude the diagnostic suspicion of HPRCC and thus to perform a thorough genomic study.

Involvement of PBRM1 in VHL disease-associated clear cell renal cell carcinoma and its putative relationship with the HIF pathway.

Von Hippel-Lindau (VHL) disease is the main cause of inherited clear-cell renal cell carcinoma (ccRCC) and is caused by germline mutations in the VHL tumor suppressor gene. Bi-allelic VHL alterations lead to inactivation of pVHL, which plays a major role by downstream activation of the hypoxia inducible factor (HIF) pathway. Somatic VHL mutations occur in 80% of sporadic ccRCC cases and the second most frequently mutated gene is polybromo 1 (PBRM1). As there is currently no data regarding PBRM1 involvement in VHL disease-associated ccRCC, the aim of the present study was to assess the PBRM1 mutational status, and PBRM1 and HIF expression in VHL disease-associated ccRCC series compared with a sporadic series. PBRM1 gene was screened by Sanger sequencing for 23 VHL-disease-associated ccRCC and 22 sporadic ccRCC cases. Immunohistochemical studies were performed to detect the expression of PBRM1, HIF1 and HIF2 for all cases. In VHL-associated tumors, 13.0% (n=3/23) had PBRM1 somatic mutations and 17.4% (n=4/23) had a loss of PBRM1 nuclear expression. In sporadic cases, 27.3% (n=6/22) showed PBRM1 somatic mutations and 45.5% (n=10/22) had a loss of PBRM1 nuclear expression. Loss of PBRM1 was associated with an advanced tumor stage. HIF1-positive tumors were observed more frequently in the VHL-associated ccRCC than in the sporadic series. Furthermore, in the VHL cohort, PBRM1 expression appeared to be associated more with HIF1 than with HIF2. Given that hereditary tumors tend to be less aggressive, these results would suggest that co-expression of PBRM1 and HIF1 may have a less oncogenic role in VHL-associated ccRCC.

Cellular pathways influenced by protein arginine methylation: Implications for cancer.

Arginine methylation is an influential post-translational modification occurring on histones, RNA binding proteins, and many other cellular proteins, affecting their function by altering their protein-protein and protein-nucleic acid interactions. Recently, a wealth of information has been gathered, implicating protein arginine methyltransferases (PRMTs), enzymes that deposit arginine methylation, in transcription, pre-mRNA splicing, DNA damage signaling, and immune signaling with major implications for cancer therapy, especially immunotherapy. This review summarizes this recent progress and the current state of PRMT inhibitors, some in clinical trials, as promising drug targets for cancer.

Germline mutation in the NBR1 gene involved in autophagy detected in a family with renal tumors.

Hereditary Renal Cell Carcinomas (RCC) are caused by mutations in predisposing genes, the major ones including VHL, FLCN, FH and MET. However, many families with inherited RCC have no germline mutation in these genes. Using Whole Exome Sequencing on germline DNA from a family presenting three different histological renal tumors (an angiomyolipoma, a clear-cell RCC and an oncocytic papillary RCC), we identified a frameshift mutation in the Neighbor of BRCA1 gene 1 (NBR1), segregating with the tumors. NBR1 encodes a cargo receptor protein involved in autophagy. Genetic and functional analyses suggested a pathogenic impact of the mutation. Indeed, functional study performed in renal cell lines showed that the mutation alters NBR1 interactions with some of its partners (such as p62/SQSTM1), leading to a dominant negative effect. This results in an altered autophagic process and an increased proliferative capacity in renal cell lines. Our study suggests that NBR1 may be a new predisposing gene for RCC, however its characterization needs to be further investigated in order to confirm its role in renal carcinogenesis.

Deletion of RBMX RGG/RG motif in Shashi-XLID syndrome leads to aberrant p53 activation and neuronal differentiation defects.

RNA-binding proteins play important roles in X-linked intellectual disability (XLID). In this study, we investigate the contribution of the XLID-associated RBMX in neuronal differentiation. We show that RBMX-depleted cells exhibit aberrant activation of the p53 pathway. Moreover, we identify that the RBMX RGG/RG motif is methylated by protein arginine methyltransferase 5 (PRMT5), and this regulates assembly with the SRSF1 splicing factor into higher-order complexes. Depletion of RBMX or disruption of the RBMX/SRSF1 complex in PRMT5-depleted cells reduces SRSF1 binding to the MDM4 precursor (pre-)mRNA, leading to exon 6 exclusion and lower MDM4 protein levels. Transcriptomic analysis of isogenic Shashi-XLID human-induced pluripotent stem cells (hiPSCs) generated using CRISPR-Cas9 reveals a dysregulation of MDM4 splicing and aberrant p53 upregulation. Shashi-XLID neural progenitor cells (NPCs) display differentiation and morphological abnormalities accompanied with excessive apoptosis. Our findings identify RBMX as a regulator of SRSF1 and the p53 pathway, suggesting that the loss of function of the RBMX RGG/RG motif is the cause of Shashi-XLID syndrome.