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Disruption of the moonlighting function of CTF18 in a patient with T-lymphopenia

ABSTRACT: Newborn screening for immunodeficiency has led to the identification of numerous cases for which the causal etiology is unknown. Here we report the diagnosis of T lymphopenia of unknown etiology in a male proband. Whole exome sequencing (WES) nominated as the top candidate variants, missense mutations in CHTF18 that were inherited in an autosomal recessive manner. CTF18, encoded by the CHTF18 gene, is a component of a secondary clamp loader, which is primarily thought to function by promoting DNA replication. We determined that the patient’s variants in CHTF18 (CTF18 R751W and E851Q) were damaging to function and severely attenuated the capacity of CTF18 to support hematopoiesis and lymphoid development, strongly suggesting that they were responsible for his T lymphopenia; however, the attenuation of CTF18 function appeared to be unrelated to its role as a clamp loader. DNA-damage, expected when replication is impaired, was not evident by expression profiling in murine Chtf18 mutant hematopoietic stem and progenitor cells (HSPC), nor was development of Ctf18-deficient progenitors rescued by p53 loss. Instead, we observed an expression signature suggesting disruption of HSPC positioning and migration. Indeed, the positioning of HSPC in ctf18 morphant zebrafish embryos was perturbed, suggesting that HSPC function was impaired through disrupted positioning in hematopoietic organs. Accordingly, we propose that T lymphopenia in our patient resulted from disturbed cell-cell contacts and migration of HSPC, caused by a non-canonical function of CHTF18 in regulating gene expression.

ORGANISM(S): Mus musculus

PROVIDER: GSE289457 | GEO | 2025/02/12

REPOSITORIES: GEO

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Project description:Introduction. Pulmonary arterial hypertension (PAH) is a severe cardiopulmonary disease that may be triggered by exposure to drugs such as dasatinib or facilitated by genetic predispositions. The incidence of dasatinib-associated PAH is estimated at 0.45%, suggesting individual predispositions. The mechanisms of dasatinib-associated PAH are still incomplete. This study studied the link between dasatinib and KCNK3 and the consequences of dasatinib exposure or KCNK3 knockdown in pulmonary endothelial cells (PECs) and pulmonary arterial smooth muscle cells (PASMC). Methods. We discovered a KCNK3 gene variant in a patient with dasatinib-associated PAH and investigated the impact of this variant on KCNK3 function using patch-clamp analysis. Additionally, we assessed the effects of dasatinib exposure on KCNK3 expression and function in human and rat pulmonary arteries. In control-human PASMCs and PECs (hPASMCs and hPECs), we evaluated the consequence of KCNK3 knockdown on cell migration, mitochondrial membrane potential, ATP production, and in vitro tube formation. Using mass spectrometry, we determined the KCNK3 interactome. Results. Patch-clamp revealed that the identified KCNK3 variant represents a loss-of-function variant. Dasatinib contributed to pulmonary artery constriction by decreasing KCNK3 function and expression. In control-hPASMCs, KCNK3 knockdown promotes mitochondrial membrane depolarization and glycolytic shift. Additionally, dasatinib exposure or KCNK3 knockdown reduced the number of caveolae in PECs. Moreover, KCNK3 knockdown in control-hPECs reduced migration, proliferation, and in vitro tubulogenesis. Lastly, using proximity ligation assay and mass spectrometry, we identified the KCNK3 interactome revealing that KCNK3 interaction with various proteins across different cellular compartments could impact these cellular functions. Conclusion. We identified a novel pathogenic variant in the KCNK3 gene, suggesting that KCNK3 gene variations could also influence the development of dasatinib-associated PAH. Our results support that one of the mechanisms of action of dasatinib-associated PAH results from the downregulation of KCNK3.

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Disruption of the moonlighting function of CTF18 in a patient with T-lymphopenia

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