Synonymous mutations are generally considered as non-pathogenic, and are not expected to change the protein’s function. This paradigm has been challenged in recent years, with evidence that changes in codon usage may have consequences for the efficiency and speed of translation, that, in turn, may then affect protein folding and function [20, 21]. In addition, the possible pathogenic mechanism of single nucleotide substitutions at codon wobble sites creating aberrant splice sites, is frequently overlooked, but while there are a number of reports with evidence for this at missense (non-synonymous) sites, there are fewer reports of such a mechanism in human disease involving synonymous changes . To date, no synonymous mutation in MECP2 has been proven to be pathogenic.
In this report, we have described the functional consequences of a synonymous mutation in MECP2 exon 1, c.48C>T in a girl with a typical, albeit relatively mild form of RTT. This C>T transition changes the codon GGC into GGT, coding for the same amino acid i.e. glycine (p.Gly16Gly) (Figure 2A). At the transcriptional level this silent change clearly affects the gene expression by introducing a premature splice-donor site, resulting in the removal of 16nt of coding sequence from MECP2_E1 transcripts r.[=, 48_63del], This causes a frameshift, and premature truncation is predicted after 16 amino acid residues (KKSQKTSRTNPSSLKR*) (p.Glu17Lysfs*16) (Figure 2D). The effect of the mutation is similar for splice variant MECP2_E2, in that the last 16nt of exon 1 are removed, however, as this lies within the 5′UTR, there is no predicted effect on the protein. However, as the frame in the 5′UTR changes with the mutation and 16nt deletion, this may have an effect on the translation efficiency of MECP2_E2. The number and position of stop codons upstream to the translation start site remains unchanged, but while WT MECP2_E2 has no apparent upstream ORF, the mutant form does have a 27 amino acid upstream ORF, which may compete for translational machinery with the correct ORF beginning in exon 2. Thus, while reduction in MeCP2_E1 protein levels is the most likely etiologically relevant consequence of the mutation in Patient 1, we cannot exclude that a reduction in MeCP2_E2 protein levels caused by translational interference is also contributing to the phenotype.
Reduced MeCP2 expression is likely one of the main pathogenic mechanisms in RTT . Quantitative analysis of MECP2_E1 and E2 mRNA for Patient 1 suggest that this aberrant splice event is specific to the patient, and occurs in roughly half of the transcripts during post-transcriptional processing. A modest increase in MECP2_E2 transcripts were observed in Patient 1 (Figure 3). It is possible that removal of the 16nt at the end of exon 1 may remove inhibitory sequences, permitting increased transcription of the E2 mRNA, which could possibly be partially compensating for the loss of MECP2_E1, resulting in a milder RTT phenotype. Also, skewed X-inactivation may favor the WT allele in Patient 1, however information on X-inactivation was not available for this patient.
In summary, we have found a synonymous substitution in MECP2 exon 1 coding region, which results in a splicing defect, which is predicted to lead to a truncated protein. We cannot, however, exclude the possibility that other mechanisms are also involved, for instance the possible effect on translation timing and efficiency (and thus protein folding and function) of the switch in codon usage at Gly16 from a high to low frequency codon [20, 21], or a contributory effect of translational competition for the MECP2_E2 splice variant from the mutated allele. We recommend the re-evaluation of all de novo synonymous substitutions in MECP2. In particular, through in silico analysis of all silent changes in MECP2 reported on the Rettbase website and in the NHLBI ESP6500 exome sequence database, we found that the change c.627G>A (p.Val209Val) increases the donor splice site prediction score (Splice Site Prediction by Neural Network ) from 0.36 to 0.91 (out of 1.0), c.948C>G (Val316Val) changes the score from 0.09 to 0.42., and c.999G>T (Gly333Gly) changes the score from 0 to 0.59. These three substitutions should clearly be re-evaluated for possible MECP2 mRNA splicing aberrations. Also, we recommend that algorithms used in the analysis of next generation sequence data be updated to predict the effect on splicing machinery at exonic sites away from the known splice donor and acceptor sites.