RETT Syndrome diagnostic testing by DHPLC and sequence analysis of the MECP2 gene. I.M Buyse1,2, P. Fang1,2, K. Hoon1,2, R. Amir2, H. Zoghbi2,3,4, B.B. Roa1,2. 1) Baylor DNA Diagnostic Laboratory; 2) Dept. of Molecular & Human Genetics; 3) Dept. of Pediatrics; 4) Howard Hughes Medical Institute, Baylor college of Medicine,Houston, TX.
Rett syndrome is an X-linked dominant neurodevelopmental disorder that affects females and is usually lethal in males. The disease locus was mapped to the MECP2 gene on chromosome Xq28. To date, MECP2 sequence analysis identified mutations in ~80% of diagnosed Rett patients. Different missense, nonsense and frameshift mutations were identified, with the majority in the methyl CpG-binding or transcriptional repression functional domains in both familial and de novo cases. A diagnostic test was developed to analyze the entire MECP2 coding region by sequencing both forward and reverse directions. To date, a total of 189 unrelated cases (185 females, 4 males) with a possible diagnosis of Rett syndrome were tested in our diagnostic laboratory. A MECP2 coding region mutation was identified in 86/185 (46.5%) female patients of this heterogeneous group and in none of the males tested. Unclassified amino acid substitutions were found in 5/185 patients and analysis of both parents was recommended to evaluate these variants as de novo mutations or polymorphisms. A total of 29 different mutations (6 missense, 8 nonsense, 1 splice-site and 14 frameshifts) were identified. Of these, 14 were novel and 7 were recurrent mutations. A total of 9 polymorphisms were detected. Prenatal diagnosis for a subsequent pregnancy was performed in 2 cases wherein a familial mutation was identified. Parental analysis suggested a de novo mutation and prenatal diagnosis was negative in both cases. Evaluation of the Denaturing High-Performance Liquid Chromatography (DHPLC) test sensitivity showed 100% concordance with sequence analysis for a total of 57 sequence variants analyzed. Thus, we devised a strategy whereby we use DHPLC for initial sequence variation screening. DHPLC variants will be sequenced for mutation identification. For samples that are negative by DHPLC or are found to encode a polymorphism, the entire MECP2 coding region will be sequenced. This provides a robust and efficient strategy for Rett diagnostic testing.