Abstract
BackgroundOver 400 million people worldwide are living with a rare disease. Next Generation Sequencing (NGS) identifies potential disease causative genetic variants. However, many are identified as variants of uncertain significance (VUS) and require functional laboratory validation to determine pathogenicity, and this creates major diagnostic delays.MethodsIn this study we test a rapid genetic variant assessment pipeline using CRISPR homology directed repair to introduce single nucleotide variants into inducible pluripotent stem cells (iPSCs), followed by neuronal disease modelling, and functional genomics on amplicon and RNA sequencing, to determine cellular changes to support patient diagnosis and identify disease mechanism.ResultsAs proof-of-principle, we investigated an EHMT1 (Euchromatin histone methyltransferase 1; EHMT1 c.3430C > T; p.Gln1144*) genetic variant pathogenic for Kleefstra syndrome and determined changes in gene expression during neuronal progenitor cell differentiation. This pipeline rapidly identified Kleefstra syndrome in genetic variant cells compared to healthy cells, and revealed novel findings potentially implicating the key transcription factors REST and SP1 in disease pathogenesis.ConclusionThe study pipeline is a rapid, robust method for genetic variant assessment that will support rare diseases patient diagnosis. The results also provide valuable information on genome wide perturbations key to disease mechanism that can be targeted for drug treatments.
Highlights
Over 400 million people worldwide are living with a rare disease
We propose a new and rapid pipeline for variants of uncertain significance (VUS) assessment using a pipeline of Clustered regularly interspaced short palindromic repeats (CRISPR) single base editing, inducible pluripotent stem cell disease modelling, and functional genomics
To demonstrate this pipeline we assess a known patient genetic variant EHMT1_c.3430C > T (p.Gln1144*) CRISPR edited into human inducible pluripotent stem cells, and determine genome wide changes in neural progenitor cell differentiation by functional genomics
Summary
Over 400 million people worldwide are living with a rare disease. Generation Sequencing (NGS) identifies potential disease causative genetic variants. Fear et al Stem Cell Research & Therapy (2022) 13:69 and classified as variants of uncertain significance (VUS) meaning the patient cannot receive a diagnosis [1]. These identified VUS require functional validation to determine disease pathogenicity, a process currently performed using functional assays in highly specialised laboratories, that is undertaken in ad hoc fashion. This requirement for functional validation of VUS leads to a significant delay in rare diseases patient diagnosis of > 5 years, if not decades [2]. To demonstrate this pipeline we assess a known patient genetic variant EHMT1_c.3430C > T (p.Gln1144*) CRISPR edited into human inducible pluripotent stem cells (iPSCs), and determine genome wide changes in neural progenitor cell differentiation by functional genomics
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have