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

Stabilisation of Cyclin D2 is the underlying cause of a range of neurodevelopmental disorders, characterised by megalencephaly, cortical migration defects and overgrowth. Intracellular CCND2 is vital for mTOR pathway signalling, with inhibition of mTOR resulting in CCND2 phosphorylation and degradation by the ubiquitin proteasome system. Mutations at the regulatory c-terminus of CCND2, and in proteins that regulate mTOR such as PTEN, PIK3CA, AKT3 and TSC1/2, result in CCND2-stabilisation and overgrowth. To determine the molecular and cellular mechanisms underpinning the neurodevelopmental defects observed in Megalencephaly-Polymicrogyria-Polydactyly-Hydrocephalus (MPPH) syndrome, we generated human induced pluripotent stem cell (iPSC) derived models of CCND2-associated disease. Using CRISPR-Cas9 we generated lines containing either a pathogenic CCND2 variant (c.814G>T, p.Glu272Ter) or frameshift variants in the final exon of CCND2, all of which truncate CCND2 before the critical Thr-280 residue, required for its phosphorylation and degradation. We observed truncating frameshift variants do not result in CCND2 stabilization, whereas the single nucleotide c.814G>T, p.Glu272Ter substitution does, mimicking the effect seen in MPPH patients. Differentiation into human cortical spheroids (hCS) revealed all CCND2-truncating lines continued to express PAX6 beyond the neural progenitor (NP) expansion phase. Furthermore, both the homozygous and heterozygous p.Glu272Ter hCS failed to produce mature Tbr-1 expressing neurons, while some expression was observed in the frameshift hCS, highlighting differences in neurogenesis between frameshift and nonsense lines. Despite all lines truncating CCND2 and removing Thr-280, our data implies that frameshift truncations do not stabilise CCND2. In comparison, truncation of CCND2 through introduction of a single nucleotide nonsense variant results in CCND2 stabilisation, mimicking MPPH.