Journal: bioRxiv

Article Title: Nonsense, but not frameshift, truncating mutations result in Cyclin D2 stabilisation in an induced pluripotent stem cell model of MPPH

doi: 10.1101/2025.08.29.673076

Figure Lengend Snippet: A - Electropherogram showing both alleles (top and bottom) of CCND2 biallelic frameshift line CCND2 FS-T1 compared to wildtype (middle) causing truncation at positions 266 and 272 on allele 1 and allele 2, respectively. B - illustrates the same but for the second CCND2 biallelic frameshift line CCND2 FS-T2 showing an 11 bp deletion on allele 1 (top) and a different 11 bp deletion on allele 2 (bottom) compared to wildtype (middle), causing CCND2 truncation at position 272 and 274 respectively. C - Sanger sequencing electropherograms indicating with arrows the single base pair change c.814G>T in heterozygous (top) and homozygous (bottom) CCND2 E272*Het and CCND2 E272*Hom CRISPR-Cas9 edited lines compared to wildtype (middle).

Article Snippet: A single-guide RNA targeting the final exon of CCND2 (5’-ACGTGACGGATCCAAGTCGG-3’) was ligated in to either the GFP-tagged CRISPR-Cas9 px458 plasmid (Addgene #48138) or the enhanced specificity Cas9 (eSpCas9, Addgene #71814) following digestion with BbsI restriction enzyme (Thermo Fisher Scientific).

Techniques: Sequencing, CRISPR

Journal: bioRxiv

Article Title: Nonsense, but not frameshift, truncating mutations result in Cyclin D2 stabilisation in an induced pluripotent stem cell model of MPPH

doi: 10.1101/2025.08.29.673076

Figure Lengend Snippet: A - 2D genomic and proteomic structure of CCND2, showing location of MPPH disease causing variants at the C-terminus (bottom) ( Mirzaa et al ., 2014 , Zhao et al ., 2024 ) and likely pathogenic variants from ClinVar in italics (top). Green indicates phosphorylation sites Ser269, Ser271 and Thr280, pink indicates ubiquitination site Lys270. B – Illustration of the CCND2 C- terminus showing the site of truncations, at the gene and protein level, in each CRISPR-Cas9 engineered line in comparison to wildtype (WT). Red hatched regions indicate regions of amino acid sequence changes due to a frameshift mutation. Green and pink highlighted regions signify locations of phosphorylation sites (Ser269, Ser271 and Thr280) and ubiquitination site Lys270, respectively.

Article Snippet: A single-guide RNA targeting the final exon of CCND2 (5’-ACGTGACGGATCCAAGTCGG-3’) was ligated in to either the GFP-tagged CRISPR-Cas9 px458 plasmid (Addgene #48138) or the enhanced specificity Cas9 (eSpCas9, Addgene #71814) following digestion with BbsI restriction enzyme (Thermo Fisher Scientific).

Techniques: Phospho-proteomics, Ubiquitin Proteomics, CRISPR, Comparison, Sequencing, Mutagenesis

Journal: bioRxiv

Article Title: Nonsense, but not frameshift, truncating mutations result in Cyclin D2 stabilisation in an induced pluripotent stem cell model of MPPH

doi: 10.1101/2025.08.29.673076

Figure Lengend Snippet: Sections were stained for PAX6, β-Tubulin, TBR1, NeuN and Hoechst on all CCND2 CRISPR-Cas9 edited lines and compared to wildtype. For each, similar results were obtained from 2 independent batches grown. Scale bar 20um at x10 magnification.

Article Snippet: A single-guide RNA targeting the final exon of CCND2 (5’-ACGTGACGGATCCAAGTCGG-3’) was ligated in to either the GFP-tagged CRISPR-Cas9 px458 plasmid (Addgene #48138) or the enhanced specificity Cas9 (eSpCas9, Addgene #71814) following digestion with BbsI restriction enzyme (Thermo Fisher Scientific).

Techniques: Staining, CRISPR