ESR5: Identification and splice assays of deep-intronic ABCA4 variants in mono-allelic STGD1
Partner
Radboud University Medical Center Nijmegen, The Netherlands (www.ru.nl/donders/research/theme-2-perception-action-control/research-groups-theme-2/blindness-genetics/) Supervisor Prof. Dr. F. Cremers |
Zelia Corradi
I obtained my MSc in Medical Biotechnology at the University of Bologna and completed my internship in the laboratory of Transcriptional Neurobiology of CiBio (Trento). There, I worked on a project aimed at finding and developing a gene therapy approach to treat patients affected by Spinal and Bulbar Muscular Atrophy (SBMA). Since March 2020, I joined prof. dr. Cremers group (Human Genetics Department, RadboudUMC) as a PhD student in the StarT project. My doctoral project focused on the missing heritability of Stargardt disease with the aim of identifying novel variants and investigate their role through in vitro splice assays. I’m very passionate about the help novel techniques can lend to the better understanding of inherited diseases, and ultimately to treatment. Being involved in this project has cemented my passion for human genetics and the unraveling of the molecular causes of inherited diseases. The ITN program itself has been an great opportunity to meet experts in the field and create the foundations of a strong scientific network. Currently, I am excited to continue working in the group of dr. Susanne Roosing and prof. dr. Frans Cremers, now as a postdoctoral researcher, to implement a novel smMIPs platform for the sequencing of genes associated with inherited retinal diseases and additional candidate genes for a total of 358 genes in unsolved IRD cases. I will be defending my PhD these by the end of 2024. |
Abstract
Approximately 25% of STGD1 cases show one or no coding ABCA4 variant. Using ABCA4 locus sequencing, we and others identified deep-intronic variants. We focused on the identification of RNA splice defects and generated a complete set of Gateway-based splice vectors, denoted midigenes, that contain wild-type (WT) ABCA4 multi-exon segments of 4.7 to 11.7 kb. Using a mutagenesis protocol, we rapidly introduced new variants into these vectors and performed in vitro splice assays in HEK293T cells. We assessed the effect of all reported 47 non-canonical ABCA4 splice variants and tested 10 deep-intronic variants identified in 40 mono-allelic Dutch STGD1 cases. Splice defects were visualised by RT-PCR using primers annealing to flanking ABCA4 exons. For selected variants, we also confirmed their effect on patient-derived photoreceptor progenitor cells (PPCs). ESR5 will develop a cost-effective sequencing method for the ABCA4 locus using single molecule Molecular Inversion Probes (smMIPs), and sequence 400 mono-allelic STGD1 cases that have been recruited by P2-RUMC. Sequence data (variants) of ESR5, ESR6 and ESR7 will be compiled. Hundred variants predicted to affect splicing will be introduced into WT midigenes. The effect of selected variants will be analysed in patient-derived PPCs and retinal pigment epithelium (RPE) cells.
Approximately 25% of STGD1 cases show one or no coding ABCA4 variant. Using ABCA4 locus sequencing, we and others identified deep-intronic variants. We focused on the identification of RNA splice defects and generated a complete set of Gateway-based splice vectors, denoted midigenes, that contain wild-type (WT) ABCA4 multi-exon segments of 4.7 to 11.7 kb. Using a mutagenesis protocol, we rapidly introduced new variants into these vectors and performed in vitro splice assays in HEK293T cells. We assessed the effect of all reported 47 non-canonical ABCA4 splice variants and tested 10 deep-intronic variants identified in 40 mono-allelic Dutch STGD1 cases. Splice defects were visualised by RT-PCR using primers annealing to flanking ABCA4 exons. For selected variants, we also confirmed their effect on patient-derived photoreceptor progenitor cells (PPCs). ESR5 will develop a cost-effective sequencing method for the ABCA4 locus using single molecule Molecular Inversion Probes (smMIPs), and sequence 400 mono-allelic STGD1 cases that have been recruited by P2-RUMC. Sequence data (variants) of ESR5, ESR6 and ESR7 will be compiled. Hundred variants predicted to affect splicing will be introduced into WT midigenes. The effect of selected variants will be analysed in patient-derived PPCs and retinal pigment epithelium (RPE) cells.
