ESR8: ABCA4 transcript analysis of retinal cells derived from mono-allelic STGD1 cases
Partner
NewCells Biotech Ltd, Newcastle, United Kingdom (newcellsbiotech.co.uk) Supervisor Prof. Dr. M. Lako |
Avril Watson
I’m originally from Dublin, Ireland. I graduated from Trinity College Dublin with a BA (Mod.) in Genetics and an MSc. in Immunology, both with a research focus on retinal degeneration under the supervision Prof. Jane Farrar and Prof. Sarah Doyle, respectively. As part of the StarT ITN, I have moved to Newcastle Upon Tyne to carry out my studies on Stargardt Disease under the supervision of Prof. Majlinda Lako and Newcells Biotech. My PhD research project involves identifying accompanying DNA mutations in unresolved monoallelic STGD1 or CRD cases using long-read sequencing (LRS) technology. These mutations are predicted to be deep intronic variants that affect the splicing of ABCA4. Ten patient-specific iPSC cell lines have been already generated by P2-RUMC. I aim to expand these iPSCs and differentiate them to photoreceptor precursor cells (PPCs) using the 3D retinal organoid protocol. We have data that shows sufficient ABCA4 expression after 2 months that will enable me to conduct transcript analysis on these samples. RNA from these PPCs will be isolated and sequenced using PacBio LRS to identify any splicing defects, which should in theory allow me to identify the accompanying DNA variant in these mono-allelic cases. I feel both very excited and fortunate to begin my work on Stargardt Disease with Prof. Lako as part of this ITN and I also look forward to hearing of the progress made by other ESRs to the overall goal of this consortia. Best wishes, Avril. |
Abstract
ABCA4 locus sequencing in genetically unexplained STGD1 cases on average yields three rare sequence variants per patient. RNA splice site prediction programs can select variants that potentially affect RNA splicing. Currently, two functional assays are being used to assess the effect of putative splice variants. First, employing splice midigenes containing wild-type or mutant sequences that are transfected into HEK293T cells, splice defects are visualised using RT-PCR and gel analysis. Second, photoreceptor precursor cells (PPCs) can be derived from induced pluripotent stem cells (iPSCs) that were obtained through reprogramming of patient skin fibroblasts or blood cells. Currently, the ABCA4 transcript is analysed using PCR-amplification of tiled cDNA segments. Here, ESR8 will augment ABCA4 transcript analysis by generating PPCs and mature photoreceptor cells from ten patient-derived iPSCs of mono-allelic STGD1 cases, available from P2-RUMC. Using PacBio single molecule (long-read) sequencing technology, ESR8 will assess the entire spectrum of aberrant transcripts in PPCs of unsolved STGD1 cases. ABCA4 locus sequencing on genomic DNA will be performed in collaboration with ESR5.
ABCA4 locus sequencing in genetically unexplained STGD1 cases on average yields three rare sequence variants per patient. RNA splice site prediction programs can select variants that potentially affect RNA splicing. Currently, two functional assays are being used to assess the effect of putative splice variants. First, employing splice midigenes containing wild-type or mutant sequences that are transfected into HEK293T cells, splice defects are visualised using RT-PCR and gel analysis. Second, photoreceptor precursor cells (PPCs) can be derived from induced pluripotent stem cells (iPSCs) that were obtained through reprogramming of patient skin fibroblasts or blood cells. Currently, the ABCA4 transcript is analysed using PCR-amplification of tiled cDNA segments. Here, ESR8 will augment ABCA4 transcript analysis by generating PPCs and mature photoreceptor cells from ten patient-derived iPSCs of mono-allelic STGD1 cases, available from P2-RUMC. Using PacBio single molecule (long-read) sequencing technology, ESR8 will assess the entire spectrum of aberrant transcripts in PPCs of unsolved STGD1 cases. ABCA4 locus sequencing on genomic DNA will be performed in collaboration with ESR5.
