Mutations in MECP2 exon 1 in classical rett patients disrupt MECP2_e1 transcription, but not transcription of MECP2_e2

Abstract

The overwhelming majority of Rett syndrome cases are caused by mutations in the gene MECP2. MECP2 has two isoforms, termed MECP2_e1 and MECP2_e2, which differ in their N-terminal amino acid sequences. A growing body of evidence has indicated that MECP2_e1 may be the etiologically relevant isoform in Rett Syndrome based on its expression profile in the brain and because, strikingly, no mutations have been discovered that affect MECP2_e2 exclusively. In this study we sought to characterize four classical Rett patients with mutations that putatively affect only the MECP2_e1 isoform. Our hypothesis was that the classical Rett phenotype seen here is the result of disrupted MECP2_e1 expression, but with MECP2_e2 expression unaltered. We used quantitative reverse transcriptase PCR to assay mRNA expression for each isoform independently, and used cytospinning methods to assay total MECP2 in peripheral blood lymphocytes (PBL). In the two Rett patients with identical 11 bp deletions within the coding portion of exon 1, MECP2_e2 levels were unaffected, whilst a significant reduction of MECP2_e1 levels was detected. In two Rett patients harboring mutations in the exon 1 start codon, MECP2_e1 and MECP2_e2 mRNA amounts were unaffected. In summary, we have shown that patients with exon 1 mutations transcribe normal levels of MECP2_e2 mRNA, and most PBL are positive for MeCP2 protein, despite them theoretically being unable to produce the MECP2_e1 isoform, and yet still exhibit the classical RTT phenotype. Altogether, our work further supports our hypothesis that MECP2_e1 is the predominant isoform involved in the neuropathology of Rett syndrome.

Over-Expression of Either MECP2_e1 or MECP2_e2 in Neuronally Differentiated Cells Results in Different Patterns of Gene Expression

“…This contrasts with several previous studies which used quantification of end-point RT-PCR products and showed higher levels of e1 than e2 [2], [3]. Our previous study looking at e1 and e2 mRNA levels in lymphocytes using the ΔΔCt method also showed very similar levels of e1 and e2 [17], also differing from the previous reports [2], [3]. This may not, however, correspond directly to relative amount of the protein isoforms, as e1 is translated with much higher efficiency than e2 [3], and has been shown to be a more stable protein than e2 [10], and as predicted by in silico analysis of the different N-terminal sequences for likelihood of N-methionine excision and N-terminal acetylation and effect on protein stability and longevity [10].…”

Optogenetic approach for functional assays of the cardiovascular system by light activation of the vascular smooth muscle

“…5B). The contractile force generated by blue light (~24 mW/mm 2 ) was comparable to that produced by 1 μM phenylephrine (16) a selective α-adrenergic receptor agonist, and even larger than that produced by 60 mM KCl (Fig. 5C, E).…”

Rett Syndrome and MeCP2

“…Since the discovery of MeCP2E1 isoform, scientists have started to reevaluate the mutation analysis of exon 1. To date, several RTT-causing mutations are reported within exon 1 encoding for MeCP2E1 (Gianakopoulos et al 2012;Saunders et al 2009;Chunshu et al 2006;Quenard et al 2006;Bartholdi et al 2006;Saxena et al 2006;Ravn et al 2005). Studies conducted in [2005][2006] suggested that mutations in exon 1 are rare in RTT, because they could only detect exon 1 mutations in 0.03-1 % of the tested samples from RTT patients (Amir et al 2005;Quenard et al 2006;Evans et al 2005b).…”