1. Baylor/UAB Folate-Betaine Trial Alan Percy, MD JUNE 23, 2008 RETT SYNDROME CLINICAL TRIALS MINI-SYMPOSIUM

2. Principal Goal to test the hypothesis that increasing methyl group pools might promote transcriptional repression by other methyl- binding proteins or by mutant MeCP2 with altered affinity, ameliorating the clinical features of Rett syndrome (RTT). to test the hypothesis that increasing methyl group pools might promote transcriptional repression by other methyl- binding proteins or by mutant MeCP2 with altered affinity, ameliorating the clinical features of Rett syndrome (RTT).

3. Homocysteine Methionine SAM METHYL TRANSFERASES DNA RNA Proteins Lipids BETAINE Dimethylglycine Choline 5MTHF THF FOLIC ACID 5,10-MTHF DNA synthesis ( m-B12) methionine synthase Purine biosynthesis

4. Study Design 12-month, double blind, placebo-controlled 12-month, double blind, placebo-controlled MECP2 mutation positive females meeting consensus criteria; 73 enrolled MECP2 mutation positive females meeting consensus criteria; 73 enrolled randomization: young (  age 5 years) or old (  5 years) randomization: young (  age 5 years) or old (  5 years) structured assessments at baseline, 3, 6, and 12 months structured assessments at baseline, 3, 6, and 12 months primary outcome measures: primary outcome measures: quantitative: waking breathing and hand movements, growth, anthropometry, motor/behavioral function quantitative: waking breathing and hand movements, growth, anthropometry, motor/behavioral function qualitative:EEG and parent questionnaire. qualitative:EEG and parent questionnaire.

5. The Team Baylor College of Medicine Baylor College of Medicine Daniel Glaze Daniel Glaze Kay Motil Kay Motil Jeff Neul Jeff Neul Rebecca Schultz Rebecca Schultz Judy Barrish Judy Barrish William O’Brien William O’Brien E. O’Brian Smith E. O’Brian Smith UAB UAB Alan Percy Jane Lane Janet Isaacs NIH NIH IRSA IRSA Anonymous Donor Anonymous Donor Girls and women with RS and their families Girls and women with RS and their families

6. Results (in a nutshell) sixty-eight participants completed the study sixty-eight participants completed the study no objective evidence of improvement no objective evidence of improvement subjective improvement in alertness, attention, and behavior (calmer) from the parent questionnaire for < 5 years group. subjective improvement in alertness, attention, and behavior (calmer) from the parent questionnaire for < 5 years group.

7. Mutation Distribution(< 5 yr) MutationDrugPlacebo R106W21 R133C21 T158M52 R168X25 R255X21 R270X03 R306C13 del/ins21 C-terminal12 Exon 1 10 Total1819

8. Mutation Distribution (  5 yr) MutationDrugPlacebo R106W12 R133C20 T158M33 R168X30 R255X12 R270X01 R294X21 R306C41 Other point mutation 13 del/ins11 C-terminal31 Total2115

9. Plasma analyses (  5 yr) (all values in µM) DrugDMGBetainMethH-cystCreatGAA Entry7.855.617.98.173.72.1 3 mos 158.6117323.94.477.85.7 6 mos 120.7968.328.66.876.12.9 1 yr 84.9834.422.03.168.92.2 Plceb Entry8.348.320.08.386.91.9 3 mos 12.048.916.57.584.82.0 6 mos 9.649.420.14.782.32.1 1 yr 12.963.921.96.178.92.5

10. Plasma analyses (  5 yr) (all values in µM) DrugDMGBetainMethH-cystCreatGAA Entry7.860.121.79.297.42.3 3 mos 108.4130827.17.189.96.7 6 mos 106.4102925.54.990.73.6 1 yr 65.2899.830.26.270.94.1 Plceb Entry7.059.920.610.693.02.7 3 mos 7.553.820.27.192.22.9 6 mos 6.446.714.13.8106.42.3 1 yr 6.945.721.47.7100.12.8

11. Parental Qualitative Assessment Treatment Group Worse No change Improved Overall group Placebo31116 Drug*21025  5 years Placebo3511 Drug**0116  5 years Placebo065 Drug***299 * p=0.036 ** p=0.012 *** p=0.98

12. Conclusion For future studies care must be given not only to stratification by age, but also to inclusion of appropriate numbers of participants with specific mutations to minimize the introduction of undue sample bias For future studies care must be given not only to stratification by age, but also to inclusion of appropriate numbers of participants with specific mutations to minimize the introduction of undue sample bias Importance of being inclusive Importance of being inclusive

13. Main folate-betaine DNA methylation pathways