Accelerating the Genetic Diagnosis of Hereditary Spastic Paraplegia Through Combining Short- and Long-Read Whole Genome Sequencing with Functional Characterization of Identified Variants using CRISPRi in iPSC-derived neurons

• Applicant: Dr. Luca Magdalena Schierbaum
• Subject Area: Pediatric and Adolescent Medicine
• Term: since 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 536105452

Introduction: Hereditary spastic paraplegia (HSP) comprises a group of more than 80 genetic disorders characterized by progressive weakness and spasticity in the lower limbs. The complexity of childhood-onset HSP, with over 80 known causative genes, poses challenges for accurate diagnosis and understanding the underlying genetic mechanisms. Current diagnostic rates for HSP are only around 40-50%, partly due to the difficulty in determining the pathogenicity of identified gene variants. This research project aims to enhance the genetic diagnosis of HSP by employing a combination of short-read and long-read whole genome sequencing (srWGS, lrWGS), followed by functional characterization of the identified variants using CRISPR interference (CRISPRi) in induced pluripotent stem cell (iPSC)-derived neurons. Methods and expected results: The project will involve conducting srWGS on HSP patients who have previously undergone negative clinical exome testing. Data obtained from srWGS will be filtered using established pipelines for single nucleotide variants (SNVs), structural variants (SVs), and repeat expansions (REs) in known genomic regions. Additionally, new filtering algorithms will be developed to analyse variants in non-coding regions, novel repeat expansions, and mitochondrial variants. Variants will be assessed for their deleterious potential based on various criteria, such as frequency in population databases, in-silico prediction tools, constraint scores, protein and gene function, regulatory elements, and associated diseases and phenotypes. Variants of uncertain significance that could potentially impact splicing will undergo further assessment through lrWGS and RNA sequencing. To functionally characterize the identified loss-of-function variants, a pipeline will be established using CRISPRi in iPSC-derived neurons. CRISPRi clones will be generated by integrating pC13N-dCas9-BFP-KRAB at the CLYB locus in iPSCs carrying a doxycycline-inducible mouse neurogenin 2 at the AAVS1 locus. Second, single guide RNAs (sgRNA) will be delivered using lentiviral vectors. Automated, non-invasive imaging and measurement of neurite outgrowth after re-plating will serve as a standardized and quantifiable readout. Conclusion: This comprehensive approach aims to identify potential disease-causing variants in HSP and provide insights into the genetic mechanisms underlying the condition. The outcomes of this study will contribute to the development of more accurate and scalable diagnostic methods, as well as personalized treatment strategies for individuals with HSP.

• DFG Programme: WBP Fellowship
• International Connection: USA
• Host: Professor Darius Ebrahimi-Fakhari, Ph.D.