CRISPR single base editing, neuronal disease modelling and functional genomics for genetic variant analysis: pipeline validation using Kleefstra syndrome EHMT1 haploinsufficiency.
CRISPR SNV editing
Functional genomics
Inducible pluripotent stem cells
Kleefstra syndrome
Rare genetic diseases
Translational genetics
Variant of uncertain significance
Journal
Stem cell research & therapy
ISSN: 1757-6512
Titre abrégé: Stem Cell Res Ther
Pays: England
ID NLM: 101527581
Informations de publication
Date de publication:
09 02 2022
09 02 2022
Historique:
received:
06
08
2021
accepted:
28
10
2021
entrez:
10
2
2022
pubmed:
11
2
2022
medline:
25
3
2022
Statut:
epublish
Résumé
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. 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. 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. 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.
Sections du résumé
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.
Identifiants
pubmed: 35139903
doi: 10.1186/s13287-022-02740-3
pii: 10.1186/s13287-022-02740-3
pmc: PMC8827184
doi:
Substances chimiques
EHMT1 protein, human
EC 2.1.1.-
Histone-Lysine N-Methyltransferase
EC 2.1.1.43
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
69Subventions
Organisme : Wellcome Trust
Pays : United Kingdom
Informations de copyright
© 2022. The Author(s).
Références
Elife. 2017 May 02;6:
pubmed: 28462777
BMC Bioinformatics. 2013 Apr 15;14:128
pubmed: 23586463
Cell. 2006 Aug 25;126(4):663-76
pubmed: 16904174
BMC Bioinformatics. 2010 Apr 01;11:165
pubmed: 20356413
Am J Hum Genet. 2006 Aug;79(2):370-7
pubmed: 16826528
Curr Opin Cell Biol. 2004 Jun;16(3):293-9
pubmed: 15145354
ChemMedChem. 2009 Oct;4(10):1568-82
pubmed: 19739196
Curr Protoc Bioinformatics. 2014 Sep 08;47:11.12.1-34
pubmed: 25199790
Elife. 2021 Feb 17;10:
pubmed: 33594975
Genes Dev. 2005 Apr 1;19(7):815-26
pubmed: 15774718
Mol Cell. 2008 Dec 5;32(5):718-26
pubmed: 19061646
Dis Model Mech. 2020 Aug 27;13(8):
pubmed: 32651197
PLoS One. 2010 Dec 14;5(12):e14311
pubmed: 21179468
FEBS J. 2019 Sep;286(17):3389-3400
pubmed: 31034753
Genome Biol. 2014;15(12):550
pubmed: 25516281
Nat Biotechnol. 2016 May;34(5):525-7
pubmed: 27043002
Nucleic Acids Res. 2018 Jan 4;46(D1):D1284
pubmed: 29161433
Am J Hum Genet. 2012 Jul 13;91(1):73-82
pubmed: 22726846
Neuron. 2016 Sep 21;91(6):1260-1275
pubmed: 27657450
Stem Cells. 2011 Apr;29(4):660-9
pubmed: 21360626
eNeuro. 2015 Jul 10;2(4):
pubmed: 26465007
Nucleic Acids Res. 2015 Apr 20;43(7):e47
pubmed: 25605792
Genes Dev. 2002 Nov 15;16(22):2893-905
pubmed: 12435631
Bioinformatics. 2011 Jun 15;27(12):1653-9
pubmed: 21543442
Trends Biochem Sci. 2013 Dec;38(12):621-39
pubmed: 24148750
Oncogene. 2020 Apr;39(15):3163-3178
pubmed: 32051553
Proc Int Conf Intell Syst Mol Biol. 1994;2:28-36
pubmed: 7584402
Mol Syndromol. 2012 Apr;2(3-5):202-212
pubmed: 22670141
Nat Commun. 2019 Oct 30;10(1):4928
pubmed: 31666522
J Biol Chem. 2013 Feb 1;288(5):3227-39
pubmed: 23250796
Am J Hum Genet. 2014 Nov 6;95(5):490-508
pubmed: 25307298
Genes Dev. 2011 Apr 15;25(8):781-8
pubmed: 21498567
F1000Res. 2015 Dec 30;4:1521
pubmed: 26925227
Cell. 2002 Oct 4;111(1):91-103
pubmed: 12372303
Cereb Cortex. 2019 Mar 1;29(3):1280-1290
pubmed: 29462287
Cell Syst. 2015 Dec 23;1(6):417-425
pubmed: 26771021
Genome Biol. 2010;11(10):R106
pubmed: 20979621
Biochem Biophys Res Commun. 2011 Aug 19;412(1):86-91
pubmed: 21798247
Nat Genet. 2003 Jul;34(3):267-73
pubmed: 12808457
Genes Dev. 2016 Feb 15;30(4):421-33
pubmed: 26883361
Nat Struct Mol Biol. 2008 Mar;15(3):245-50
pubmed: 18264113
Genome Med. 2019 Dec 31;12(1):3
pubmed: 31892348
Gene. 2016 Dec 31;595(2):131-141
pubmed: 27651234
Proc Natl Acad Sci U S A. 2005 Oct 25;102(43):15545-50
pubmed: 16199517
J Cell Physiol. 2020 Apr;235(4):4011-4021
pubmed: 31625158
Clin Epigenetics. 2019 Nov 27;11(1):165
pubmed: 31775874
Stem Cell Reports. 2018 Aug 14;11(2):380-394
pubmed: 29983389
Bioinformatics. 2013 Jan 1;29(1):15-21
pubmed: 23104886
Nat Biotechnol. 2019 Mar;37(3):224-226
pubmed: 30809026
Nat Commun. 2016 Aug 22;7:12513
pubmed: 27545325
F1000Res. 2016 Jun 13;5:1356
pubmed: 28105305
Development. 2019 Aug 13;146(15):
pubmed: 31371378
Mol Genet Genomic Med. 2016 Dec 26;5(2):130-140
pubmed: 28361099
Nucleic Acids Res. 2016 Jul 8;44(W1):W90-7
pubmed: 27141961
Curr Protoc Mol Biol. 2018 Jan 16;121:31.14.1-31.14.11
pubmed: 29337372
Dev Biol. 2010 Apr 15;340(2):293-305
pubmed: 20122919
Front Mol Neurosci. 2011 May 17;4:6
pubmed: 21629823
J Med Genet. 2009 Sep;46(9):598-606
pubmed: 19264732
Front Cell Dev Biol. 2020 Feb 18;8:79
pubmed: 32133359
Development. 2020 Feb 21;147(4):
pubmed: 32001437
Genome Biol. 2007;8(2):R24
pubmed: 17324271
Orphanet J Rare Dis. 2017 May 3;12(1):83
pubmed: 28468665