Parent-of-origin effects in retinoblastoma in an in vitro stem cell model

• Applicant: Dr. Deniz Kanber, since 5/2020
• Subject Area: Human Genetics
• Term: from 2017 to 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 325511214

Biallelic inactivation of the RB1 gene results in retinoblastoma, a childhood tumor of the retina. The human RB1 gene is imprinted, resulting in stronger maternal than paternal RB1 expression. Despite the fact that RB1 expression is regulated in a parent-of-origin dependent manner, parent-of-origin effects in retinoblastoma have been reported only for rare specific mutations. All these reports demonstrate a higher risk for tumor development when the first mutation in RB1 is present on the paternal allele. We assume that genomic imprinting on the molecular level is linked to the observed parent-of-origin effect on the phenotypic level of retinoblastoma initiation. Elucidating the mechanistic processes underlying this link will help us to understand the mechanisms why maternal inheritance of some alleles results in a milder phenotype and incomplete penetrance, but paternal inheritance of the same mutation results in a tumor.This proposal aims to determine the molecular function of the paternal-specific RB1-E2B transcript and to explore its role in parent-of-origin effects in retinoblastoma. For this purpose, cells of the neural retina, which represent the cells of origin of retinoblastoma, will be generated by directed differentiation of human embryonic stem cells (hESCs). Studying the function of RB1-E2B in hESCs comes with two drawbacks: first, RB1-E2B is generally expressed on a very low level and second, CpG85 is almost fully methylated in hESCs, abrogating expression of RB1-E2B in these cells. To overcome these limitations, we will replace CpG85 by the strong, constitutively active EEF1A1 promoter. We will examine how strong expression of RB1-E2B influences skewing of regular RB1 expression and if this alternative transcript can be translated into a truncated RB1 protein. In the next step, to examine if RB1-E2B is the link to parent-of-origin effects, the mutation showing the strongest parent-of-origin effect, IVS6+1G>T, will be introduced into the hESCs carrying the EEF1A1 promoter. Both genetic modifications, promoter exchange and mutation, will be engineered employing the CRISPR/Cas9 system. These double-modified hESCs will then be differentiated into NR. The process and outcome of differentiation will be followed and compared quantitatively. Employing the stem cell model, these experiments will provide insight into the function of RB1-E2B and how mutations in RB1 influence early steps in NR formation.

• DFG Programme: Research Grants
• Ehemalige Antragstellerin: Professorin Dr. Laura Steenpaß, until 4/2020