Functional Investigation of Variants Identified in Patients with Retinal Dystrophy: using ciliopathies and stem cells as a model system

Amalie Brunbjerg Hey

Publikation: Bog/antologi/afhandling/rapportPh.d.-afhandling


Retinal dystrophy is a leading cause of blindness in the western world. A large number of genes have been found associated with this disease and a specific gene panel has been designed in order to evaluate DNA samples from individuals with suspected retinal dystrophy. Through targeted next generation sequencing DNA is screened for genetic variants in this gene panel. Many patients receive their diagnosis this way. However, for some gene variants it is difficult to assess the pathogenicity. Through donation of a skin biopsy, fibroblast cells from an individual without a diagnosis, can be investigated through different cellular assays to help elucidate the cellular phenotype and ultimately provide a molecular genetic diagnosis. In this dissertation one such case is presented; a missense variant in the RAB28 gene showed altered subcellular localization of the RAB28 protein in fibroblast cells from two individuals homozygous for the variant establishing this specific RAB28 missense variant as likely pathogenic. An example which underlines the
importance of functional assays in this type of work.
However, some genes are only expressed in certain tissues and therefore fibroblast cell may not always be the best system to investigate potential effects in specific differentiated cells and tissues. In these cases, it can be useful to generate induced pluripotent stem cells (iPSC) and differentiate these cells into the cell type of interest and subsequently apply functional assays. To set up the methods for this strategy, fibroblast cells from individuals suffering from Bardet-Biedl syndrome (BBS) were reprogrammed into iPSC using electroporation of the reprogramming factors OCT3/4, SOX2, KLF4, LIN28, L-Myc and a short hairpin RNA targeting P53.Investigation of undifferentiated BBS fibroblast cells showed that these cells had disturbed Hedgehog signaling and that the length of the primary cilium may be disturbed as a consequence of the BBS gene variants.
As both RAB28 and genes with BBS variants have important impact on retinal function and maintenance, iPSC generated from the BBS-fibroblast cells were differentiated into retinal pigment epithelial (RPE) cells and used as model system for the investigations in this dissertation. RPE cells derived from the BBS-iPSC were generated and investigated (data from one experiment). A directed differentiation approach was chosen and mature RPE cells with pigmentation, tight junctions and phagocytic capability from control iPSC. Interestingly, RPE cells derived from the BBS-iPSC were not able to differentiate into mature RPE cells. The morphology of the BBS-RPE differed substantially from the
control-RPE as BBS-RPE were larger and more elongated. The BBS-RPE cells did gain pigment in the first growth phase, but this was later lost. Electron microscopy revealed loose cellular junctions. This finding was confirmed in RNA expression studies where expression of genes associated with adherens unctions were increased compared to genes associated with tight junctions. The BBS-RPE also showed poor phagocytic capabilities and they had no expression of genes associated with mature RPE cells. The BBS-RPE had very long primary cilia compared to control cells, showed dysregulated Hedgehog (Hh) and Wnt ignaling and signs of mitochondrial stress. To improve maturation, prolonged culture of control- and BBS-RPE with agonists and antagonists targeting Wnt and Hh signaling was tested. Control-RPE seemed to benefit from Wnt inhibition, but BBS-RPE showed no sign of improved RPE maturation. Even though these data are preliminary, this indicates that the BBS proteins are important for cilia and RPE cell function, differentiation and maturation.

This work shows that research in rare genetic variants is important for two reasons; first, it helped clarify pathogenicity of one gene variant, and second, basic research will provide more information that in the future may lead to development of treatments for persons with retinal dystrophy.
ForlagRoskilde Universitet
Antal sider122
StatusUdgivet - 2020

Bibliografisk note

Vejledere: Jesper Troelsen & Lisbeth Birk Møller

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