1. Clinical Trials 2008: Genotype/Phenotype Jeffrey L. Neul M.D., Ph.D. Assistant Professor Division of Neurology Department of Pediatrics Assistant Medical Director Blue Bird Circle Rett Center Baylor College of Medicine

2. 8 mutations account for 55-65% cases in typical RTT MECP2 C terminal truncations account for 8-10% R106W R133C T158M R168X R255X R270X R294X R306C missense truncating Large deletions account for 7-10% Genotype/phenotype correlation in typical RTT

3. Genotype/Phenotype comparison Goal: Determine if there is any clinical difference between specific mutations Goal: Determine if there is any clinical difference between specific mutations Large cohort (285 cases) allows analysis between common specific mutations Large cohort (285 cases) allows analysis between common specific mutations Participants: Girls and Women with Rett Syndrome seen at UAB or TCH/BCM Participants: Girls and Women with Rett Syndrome seen at UAB or TCH/BCM Clinical Severity Score assessed Clinical Severity Score assessed Complete mutation testing Complete mutation testing Exon 1 Exon 1 Large DNA rearrangements (deletions) Large DNA rearrangements (deletions) Neul et al., Neurology 2008; 70: 1313-1321. Neul et al., Neurology 2008; 70: 1313-1321.

4. Typical Rett Syndrome: Clinical Criteria Normal pre/perinatal period Normal pre/perinatal period Postnatal deceleration of head growth Postnatal deceleration of head growth Loss of purposeful hand skills Loss of purposeful hand skills Stereotypic hand movements Stereotypic hand movements Communication dysfunction Communication dysfunction Social withdrawal Social withdrawal Absence of other disease processes Absence of other disease processes

5. Clinical Severity Score-Percy Clinical scoring system Clinical scoring system Rated from 0 (better) to 5 (worse) Rated from 0 (better) to 5 (worse) Thirteen categories Thirteen categories Regression OnsetSomatic Growth Head GrowthSitting AmbulationHand Use ScoliosisLanguage Nonverbal Respiratory AutonomicStereotypies Epilepsy  Overall score is a sum of all categories

6. Clinical Severity Score-example Regression Onset Regression Onset 0 – No regression 1 - > 30months old 2 – between 18 and 30 months 3 – between 12 and 18 months 4 – between 6 and 12 months 5 – less than 6 months old

7. Genotype/Phenotype comparison RTT Type Mutation No mutation Total Typical 237 (97%) 8 (3%) 245 Atypical 30 (75%) 10 (25%) 40

9. Random XCI cases: R168X (n=12), R133C (n=7), R306C (n=8), Large Deletions (n=8)

10. Genotype/Phenotype What categories of the clinical severity score are different? What categories of the clinical severity score are different? Regression OnsetSomatic Growth Head GrowthSitting AmbulationHand Use ScoliosisLanguage Nonverbal Respiratory AutonomicStereotypies Epilepsy

11. Genotype/Phenotype What categories of the clinical severity score are different? What categories of the clinical severity score are different? Regression OnsetSomatic Growth Head GrowthSitting AmbulationHand Use ScoliosisLanguage Nonverbal Respiratory AutonomicStereotypies Epilepsy p<0.0038 Kruskal Wallis Bonferroni corrected

13. AmbulationHand UseLanguage 0Acquired<18moAcquired and conserved preserved 118mo<walks alone<30mo Acquired on time, partially conserved Short phrases 2>30mo walks alone Acquired late, partially conserved Single words 3>50mo or walks with help Acquired and lostVocalization/ babbling 4lostNever acquiredScreaming/none 5Never acquired

14. AmbulationHand UseLanguage 0Acquired<18moAcquired and conserved preserved 118mo<walks alone<30mo Acquired on time, partially conserved Short phrases 2>30mo walks alone Acquired late, partially conserved Single words 3>50mo or walks with help Acquired and lostVocalization/ babbling 4lostNever acquiredScreaming/none 5Never acquired Absent/lost Full/partially preserved

15. Clinical categoryRetained function or mildly affected RegressionRegression after 18 mo of life Somatic growthweight decrease less than 2SD Head growthOFC greater than the 10 th % > 24 mo of life Motor/sittingsitting maintained Ambulationwalks independently Hand Useat least partially conserved Scoliosisless than 20% Language skillsat least single words Nonverbal communicationmaintains eye contact > 5 seconds Respiratory dysfunctionminimal respiratory dysfunction (<10%) Autonomic symptomsno autonomic abnormality Stereotypystereotypy onset after 36 mo of life Seizuresabsent or less than monthly

19. Genotype/Phenotype Molecular Conclusions R106W R133C R168X R255X R270X R294X R306C C terminal truncations MILD-hypomorphic SEVERE-null Large Deletions

20. Genotype/Phenotype Importance for clinical trials Specific common mutations have different clinical severity. Specific common mutations have different clinical severity. Balanced study design Balanced study design Intra-subject paired design Intra-subject paired design Compare pre- and post-treatment Compare pre- and post-treatment Each participant functions as own control. Each participant functions as own control.

21. Acknowledgements Blue Bird Circle Rett Center Daniel Glaze Judy Barrish Gay Horelica O’Brian Smith UAB Rett Center Alan Percy Jane Lane

22. Walks alone (%) Uses words (%) At least partially conserved hand use (%) R133C7550*92* R294X86*50*86* C-term78*67*#83 Del4112 #53 R168X28*3*38* *, # pair-wise p<0.05

24. Mutation groupn% Age of Exam (mo) Mean (SD) R106W93.7 173.5 (97.9) R133C124.9 124.8 (71.0) T158M2912.2 104.9 (66.2) R168X2911.8 132.5 (101.5) R255X3213.1 99.5 (76.7) R270X187.4 146.3 (167.2) R294X145.7 183.8 (79.3) R306C218.6 141.6 (122.1) Large rearrangements186.9 101.1 (61.0) C-term truncations187.3 136.7 (101.3) Other mutations3815.1 105.1 (70.0) no mutation83.3 158.3 (97.9) Total245100 125.5 (95.9)

25. Severity of Mutations p=0.0007 */# p<0.05 Differences for R133C, R168X, and large deletions hold up when only random XCI cases considered.

26. * * *

27. * * * * * * * * Preserved Short phrases None Single words Vocalizations

28. * * * *

29. genotypen walks alone (%) uses hands (%) uses words (%) R106W9335633 R133C1275 92* 50 * T158M30605027 R168X29 28 * 38* 3 * R255X32385928 R270X18446722 R294X14 86 *86 50 * R306C216752 10 # c-term. trunc.17 82 * 88* 71 *# large del.174153 12 # other3743 78* 41 * no mutation9337833