CRISPR single base editing, neuronal disease modelling and functional genomics for genetic variant analysis: pipeline validation using Kleefstra syndrome EHMT1 haploinsufficiency

Stem Cell Research & Therapy

Sarra Jamieson¹  Sebastian Rauschert¹  Denise Anderson¹  Genevieve Syn¹  Nicole Shaw²  Catherine A. Forbes²  Vanessa S. Fear²  Timo Lassmann³  Michelle Ward⁴  Gareth Baynam⁵ 

[1] Computational Biology, Precision Health, Telethon Kids Institute, Perth Children’s Hospital, 6009, Nedlands, WA, Australia;Translational Genetics, Precision Health, Telethon Kids Institute, Northern Entrance, Perth Children’s Hospital, 15 Hospital Avenue, 6009, Nedlands, WA, Australia;Translational Genetics, Precision Health, Telethon Kids Institute, Northern Entrance, Perth Children’s Hospital, 15 Hospital Avenue, 6009, Nedlands, WA, Australia;Computational Biology, Precision Health, Telethon Kids Institute, Perth Children’s Hospital, 6009, Nedlands, WA, Australia;Undiagnosed Diseases Program, Genetic Services of WA, Subiaco, Australia;Western Australian Register of Developmental Anomalies, King Edward Memorial Hospital, 6008, Subiaco, WA, Australia;Undiagnosed Diseases Program, Genetic Services of WA, Subiaco, Australia;

关键词: Rare genetic diseases;    Translational genetics;    Kleefstra syndrome;    CRISPR SNV editing;    Variant of uncertain significance;    Inducible pluripotent stem cells;    Functional genomics;   

DOI  :  10.1186/s13287-022-02740-3

来源: Springer

【 摘 要 】

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.

【 授权许可】

CC BY