The molecular basis of variable phenotypic severity among common missense mutations causing Rett syndrome

Human Molecular Genetics, 2016, Vol. 25, No. 3 558–570 doi: 10.1093/hmg/ddv496 Advance Access Publication Date: 8 December 2015 Original Article ORIGINAL ARTICLE The molecular basis of variable phenotypic severity among common missense mutations causing Rett syndrome Wellcome Trust Centre for Cell Biology, University of Edinburgh, Michael Swann Building, Max Born Crescent, Edinburgh EH9 3BF, UK *To whom correspondence should be addressed. Tel: +44 1316505670; Fax: +44 1316505379; Email: Abstract Rett syndrome is caused by mutations in the X-linked MECP2 gene, which encodes a chromosomal protein that binds to methylated DNA. Mouse models mirror the human disorder and therefore allow investigation of phenotypes at a molecular level. We describe an Mecp2 allelic series representing the three most common missense Rett syndrome (RTT) mutations, including first reports of Mecp2[R133C] and Mecp2[T158M] knock-in mice, in addition to Mecp2[R306C] mutant mice. Together these three alleles comprise ∼25% of all RTT mutations in humans, but they vary significantly in average severity. This spectrum is mimicked in the mouse models; R133C being least severe, T158M most severe and R306C of intermediate severity. Both R133C and T158M mutations cause compound phenotypes at the molecular level, combining compromised DNA binding with reduced stability, the destabilizing effect of T158M being more severe. Our findings contradict the hypothesis that the R133C mutation exclusively abolishes binding to hydroxymethylated DNA, as interactions with DNA containing methyl-CG, methyl-CA and hydroxymethyl-CA are all reduced in vivo. We find that MeCP2[T158M] is significantly less stable than MeCP2[R133C], which may account for the divergent clinical impact of the mutations. Overall, this allelic series recapitulates human RTT severity, reveals compound molecular aetiologies and provides a valuable resource in the search for personalized therapeutic interventions. Introduction Mutations in the X-linked MECP2 gene are implicated in several human disorders characterized by developmental delay and intellectual disability, including Rett syndrome (RTT) (1) and MECP2 duplication syndrome (2). RTT is a condition with postnatal onset that predominantly affects girls, as males fail to survive beyond infancy. Animal models have proved useful for improving our understanding of MeCP2 function and for explaining in molecular terms the origin of the RTT phenotype. The first mouse models were simple loss-of-function alleles caused by gross deletion of most of the coding sequence (3,4), but knock-in mutations corresponding to specific RTT-causing mutations (5,6) offer the opportunity for deeper understanding. Of particular interest are missense RTT mutations leading to the substitution of a single amino acid, as these pinpoint critical regions of the protein that cannot be deduced from frameshift and nonsense mutations, or † These authors contributed equally to this work. Received: August 24, 2015. Revised: November 9, 2015. Accepted: November 30, 2015 © The Author 2015. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. 558 Kyla Brown†, Jim Selfridge†, Sabine Lagger, John Connelly, Dina De Sousa, Alastair Kerr, Shaun Webb, Jacky Guy, Cara Merusi, Martha V. Koerner and Adrian Bird* Human Molecular Genetics, 2016, Vol. 25, No. 3 Results WT MeCP2-GFP mice are essentially phenotypically WT We generated an allelic series in which endogenous Mecp2 or mutant Mecp2 genes were fused in frame with EGFP at their C-termini (Fig. 1A; Supplementary Material, Fig. S1). Mice expressing knock-in WT MeCP2-EGFP fusion genes have been reported (13,17), but without extensive characterization. We initially looked for phenotypic defects due to the fusion of WT MeCP2 with EGFP by monitoring male mice at the molecular and whole organism levels. Analysis of hemizygous males has the advantage that phenotypic severity is not influenced by the pattern of X chromosome inactivation, and so severity of individual mutations can be assessed in an unbiased fashion. Quantitative polymerase chain reaction (PCR) and western blots indicated that both mRNA and protein products of the Mecp2-GFP gene (WT-GFP) were expressed in brain, though at somewhat higher levels than the endogenous Mecp2 gene (WT) (Fig. 1B and C). Quantitative western blots suggested that the level of WT-GFP is ∼1.6-fold higher than in untagged WT littermates. At the whole organism level, we analysed cohorts of WT-GFP mice back-crossed for four generations to give a genetic background that is ∼94% C57BL/6J. WT-GFP knock-in mice were fertile and showed normal survival but tended to be smaller than WT littermates (Fig. 1D and E). Cohorts were monitored using a phenotypic scoring methodology that records breathing, tremor, gait, hindlimb clasping, mobility and general condition (18). This series of observational tests has the advantage that it is not affected by learning and can therefore be performed weekly over long periods, giving reproducible results. Using this method, WT-GFP mice showed no significant phenotypic deterioration compared with WT littermates (Fig. 1F), reinforcing the view that, despite the presence of the EGFP tag, they are essentially WT. To search for neurological phenotypes in more detail, we subjected WT-GFP mice to a series of motor coordination and behavioural tests (Fig. 1G–I). Performance on the elevated plus maze was indistinguishable from WT and on the accelerating rotarod was also not significantly different from WT littermates. The hanging-wire test showed a weak but reproducible reduction in the ability to engage hindlimbs with the wire. We noted in addition that there was a trend towards a mild reduction in weight and a trend towards defective rotarod performance, but neither achieved significance. These very weak phenotypic effects may be attributable to the over-expression of MeCP2-GFP relative to untagged protein. Taking the findings together, however, we conclude that the addition of the C-terminal EGFP epitope and the moderate over-expression of the protein have minimal phenotypic consequences by these assays. Allelic series of RTT missense mutations recapitulates severity in humans Using the same knock-in technology, we generated the following mouse lines: Mecp2[T158M]EGFP, Mecp2[R306C]EGFP and Mecp2 [R133C]EGFP, referred to as T158M-GFP, R306C-GFP and R133CGFP, respectively. Both MeCP2 isoforms, which differ only at their extreme N-termini, are affected by the knock-in. Each line was back-crossed to obtain a predominantly C57BL/6J genetic background equivalent to that of the WT-GFP mice (94%). Each of the mutants gave rise to males that exhibited overt phenotypic defects from ∼6 (...truncated)