Докторская диссертация на немецком языке, посвященная влиянию гена CIC (капикуа) на транспорт фолата (сентябрь 2024 года). Мутации CIC были отмечены у детей с расстройством психического развития, а потом были описаны случаи церебральной фолатной недостаточности у детей с мутациями CIC. Автор диссертации попытался исследовать, как ген CIC влияет на экспрессию генов, отвечающих за перенос фолата.
Der Einfluss genetischer Mutationen im CIC-Gen auf den Folattransport (Leonie Linnea Elisabeth Herholz) (PDF)
Аннотация на английском:
In this dissertation, we studied the influence of the capicua protein on the expression of proteins that are involved in transcellular folate transport. To this end, various expressions of the capicua gene were studied: two missense mutations, one nonsense mutation, and the wildtype of the capicua gene were expressed in different cell lines, and the effect on the expression of further genes was compared. The three mutations were detected in patients with cerebral folate deficiency. This disease is characterized by a low concentration of 5-MTHF in the cerebrospinal fluid, concurrent with a normal hematogenous 5-MTHF level. The disease first manifests clinically in early infancy and may lead to progressive psychomotor retardation, movement disorders like ataxia, and epilepsy when untreated. MRI showed cerebellar and cerebral atrophy and focal demyelination in the white matter. Folate substitution is an effective therapy when started early. The above-mentioned patients did not show mutations in the genes classically associated with folate transport across the blood-brain barrier. However, genome sequencing revealed mutations in the CIC gene. The genes of the folate transport proteins FRα, RFC1, and PCFT all have a binding site for CIC in their regulatory regions. For this reason, a CIC mutation can cause a cerebral folate deficiency. The nonsense mutation p.R353X and the two missense mutations p.G580C and p.T990I were analyzed. Using lentiviral transduction, the mutations were transferred into HeLa cells. To characterize the mutations, immunofluorescences, western blot analyses, q-RT-PCR, and radioactive binding assays were performed. Thereby it was possible to investigate the influence of CIC and the CIC mutations on the localization of CIC, the expression of CIC and the folate transport proteins FRα, RFC1, and PCFT, as well as on the folate binding capacity of the cells. In the wild-type CIC, an upregulation of the expression of FRα was shown. The missense mutations led to a reduced transcription of the FOLR1 gene, but the expression of the FRα protein was equally high as in the wild-type CIC. While this suggests a low-grade functional limitation to the expression of FRα, which could only partially be detected with the present methodology. The nonsense mutation led to the expression of a truncated CIC protein. Due to the loss of the nuclear localization sequence, the truncated CIC protein accumulated ubiquitarily in the cell. In contrast, CIC was detected exclusively in the nucleus in all other cell lines. The expression of FRα was significantly reduced in the cells of the nonsense mutation. The nonsense mutation likely leads to a loss of function of the CIC protein. Further, the truncated protein seems to have a negative effect on the endogenous CIC, as the expression of FRα and the folate binding capacity were even lower than in empty vector-transfected cells. The results of the qRT-PCR and the binding assays were consistent with this finding. Similar results were obtained using a different vector and using a different cell line (Caco-2 cells). We did not detect any effects on the other folate transport proteins, RFC1 and PCFT. Thus, CIC seems to activate the expression of FRα but not of RFC1 or PCFT. Further studies should analyze the molecular mechanisms behind the influence of CIC on folate transport. As CIC has been shown to suppress transcription variously, further proteins could feasibly be involved in the activating function of CIC. A better understanding of the causes of the disease should ultimately enable easier identification of affected patients and allow for the initiation of early and individualized therapy.