Available Assays

The MECP2 gene is composed of four exons,which give rise to two distinct MeCP2 isoforms.23 Given the gene structure and mutation profile for MECP2, diagnostic testing for Rett syndrome is recommended to begin with analysis of the entire MECP2 coding region (exons 1 through 4) by mutation scanning or DNA sequencing. One strategy for mutation scanning is denaturing high performance liquid chromatography (DHPLC), wherein sequence variants that give rise to heteroduplex DNA molecules can be detected with a high degree of sensitivity (95% to 100% under by DHPLC analysis. Elution profiles of MECP2 exon 4PCR products corresponding to the wild-type (red and green) and the mutant (yellow) sequences are shown. (b) Identification of the corresponding MECP2 nonsense mutation by DNA sequencing. A single base change of C to T at nucleotide 880 is identified (N), which predicts an arginine to stop substitution at residue 294 of the MeCP2 protein (880C^T, R294X).

optimized conditions). The specificity of DHPLC is low, however, which requires that positive results be confirmed by DNA sequencing to identify the exact nucleotide change. The use of DHPLC coupled with sequencing has identified multiple recurrent and novel MECP2 mutations of different types (missense, nonsense, splice-site, frameshifting deletions, and insertions).24 Figure 6-4 shows representative data for DHPLC and DNA sequence analyses, demonstrating a heterozygous nonsense mutation in the MECP2 gene (880C^T, R294X). This represents one of the more common truncating mutations seen in Rett syndrome patients. Sequencing is largely considered the gold standard for point mutation detection. Approximately 85% of classic Rett syndrome patients have mutations that are detectable by mutation scanning or sequencing of the MECP2 gene, which is performed by clinical molecular laboratories (http://www.genetests.org/).

To increase overall mutation detection for the MECP2 gene, additional testing is available for large MECP2 gene rearrangements, present in approximately 10% of classic Rett patients. Deletions, insertions, or duplications involving all or part of the MECP2 gene have been identified by dosage-sensitive DNA testing methods. The classic Southern analysis method has been used to detect copy number differences corresponding to deletions or duplications of specific MECP2 exons, with detection of associated junction fragments in some cases. Alternative methods for dosage-sensitive analysis of the MECP2 gene include multiplex ligation-dependent probe amplification (MLPA; MCR Holland, the Netherlands) or quantitative real-time PCR analysis.

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