Rett syndrome (RTT) is an X-linked dominant neurodevelopmental disorder 
affecting 1/10,000-15,000 girls. The disease-causing gene was identified as 
MECP2 on chromosome Xq28, and mutations have been found in approximately 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-binding 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 high-performance liquid 
chromatography (DHPLC) for initial screening of nucleotide variants, followed by 
confirmatory sequencing analysis. If a definite mutation was not identified, 
then the entire MECP2 coding region was sequenced, to reduce the risk of false 
negatives. Collectively, we tested 228 unrelated female patients with a 
diagnosis of possible (209) or classic (19) RTT and found MECP2 mutations in 83 
(40%) of 209 and 16 (84%) of 19 of the patients, respectively. Thirty-two 
different mutations were identified (8 missense, 9 nonsense, 1 splice site, and 
14 frameshifts), of which 12 are novel and 9 recurrent in unrelated patients. 
Seven unclassified variants and eight polymorphisms were detected in 228 
probands. Interestingly, we found that T203M, previously reported as a missense 
mutation in an autistic patient, is actually a benign polymorphism, according to 
parental analysis performed in a second case identified in this study. These 
findings highlight the complexities of missense variant interpretation and 
emphasize the importance 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 
subsequently enabled its detection, underscoring the need for multiple optimized 
conditions for DHPLC analysis. We conclude that this two-tiered approach 
provides a sensitive, robust, and efficient strategy for RTT molecular 
diagnosis.