TY - JOUR
T1 - CRISPR single base editing, neuronal disease modelling and functional genomics for genetic variant analysis
T2 - pipeline validation using Kleefstra syndrome EHMT1 haploinsufficiency
AU - Fear, Vanessa S.
AU - Forbes, Catherine A.
AU - Anderson, Denise
AU - Rauschert, Sebastian
AU - Syn, Genevieve
AU - Shaw, Nicole
AU - Jamieson, Sarra
AU - Ward, Michelle
AU - Baynam, Gareth
AU - Lassmann, Timo
PY - 2022/12
Y1 - 2022/12
N2 - Background: Over 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. Methods: In 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. Results: As 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. Conclusion: The 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.
AB - Background: Over 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. Methods: In 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. Results: As 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. Conclusion: The 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.
KW - CRISPR SNV editing
KW - Functional genomics
KW - Inducible pluripotent stem cells
KW - Kleefstra syndrome
KW - Rare genetic diseases
KW - Translational genetics
KW - Variant of uncertain significance
UR - http://www.scopus.com/inward/record.url?scp=85124282324&partnerID=8YFLogxK
U2 - 10.1186/s13287-022-02740-3
DO - 10.1186/s13287-022-02740-3
M3 - Article
C2 - 35139903
AN - SCOPUS:85124282324
SN - 1757-6512
VL - 13
JO - Stem Cell Research and Therapy
JF - Stem Cell Research and Therapy
IS - 1
M1 - 69
ER -