Diagnostic testing for Rett syndrome by DHPLC and direct sequencing analysis of the MECP2 gene: Identification of several novel mutations and polymorphisms

Rett syndrome (RTT) is an X-linked dominant neurodevelopmental disorder aff ecting 1/10,000-15,000 girls. The disease-causing gene was identified as ME CP2 on chromosome Xq28, and mutations have been found in similar to 80% of patients diagnosed with RTT. Numerous mutations have been identified in de novo and rare familial cases, and they occur primarily in the methyl-CpG-bi nding and transcriptional-repression domains of MeCP2. Our first diagnostic strategy used bidirectional sequencing of the entire MECP2 coding region. Subsequently, we implemented a two-tiered strategy that used denaturing hig h-performance liquid chromatography (DHPLC) for initial screening of nucleo tide variants, followed by confirmatory sequencing analysis. If a definite mutation was not identified, then the entire MECP2 coding region was sequen ced, to reduce the risk of false negatives. Collectively, we tested 228 unr elated female patients with a diagnosis of possible (209) or classic (19) R TT and found MECP2 mutations in 83 (40%) of 209 and 16 (84%) of 19 of the p atients, respectively. Thirty-two different mutations were identified (8 mi ssense, 9 nonsense, 1 splice site, and 14 frameshifts), of which 12 are nov el and 9 recurrent in unrelated patients. Seven unclassified variants and e ight polymorphisms were detected in 228 probands. Interestingly, we found t hat T203M, previously reported as a missense mutation in an autistic patien t, is actually a benign polymorphism, according to parental analysis perfor med in a second case identified in this study. These findings highlight the complexities of missense variant interpretation and emphasize the importan ce of parental DNA analysis for establishing an etiologic relation between a possible mutation and disease. Overall, we found a 98.8% concordance rate between DHPLC and sequence analyses. One mutation initially missed by the DHPLC screening was identified by sequencing. Modified conditions subsequen tly enabled its detection, underscoring the need for multiple optimized con ditions for DHPLC analysis. We conclude that this two-tiered approach provi des a sensitive, robust, and efficient strategy for RTT molecular diagnosis .