1. Smith-Magenis Syndrome Presented by: Sara Mickelson

2. What is SMS? Syndrome was first described in 1982 by Ann Smith(genetic counselor) and Dr. Ruth Magenis Occurs in 1 in 25,000 people Arises from a spontaneous heterozygous deletion of part of chromosome 17p11.2 through non- allelic homologous recombination No correlation between the size of the deletion and the severity of the phenotype People with chromosome 17p11.2 duplicated have a mild phenotype

3. Phenotypes Wide range of phenotypes –Mental retardation (IQ 20-78) –Behavioral abnormalities Aggressive and Self-inflicted injuries –Self-hugging, polyembolokoilamania –Sleep disturbances Melatonin imbalance resembles jet lag –Delayed speech and motor development –Distinct physical characteristics

4. Journal of Medical Genetics 36

5. Cloning the SMS region Common deletion region is ~4Mb as detected by pulsed-field gel electrophoresis and FISH Somatic rodent:human hybrid cell lines with the chromosome 17 deletion determined that the critical region is ~1.1Mb 16 BACs and 2 PACs were used to assemble the contig transcription map Known genes and ESTs were hybridized to EcoRI-digested BACs to map the initial contig

6. Cloning cont. Gene order was determined by human genome project sequence analysis and Southern hybridization for the presence or absence of a certain gene or EST Sequence data analysis also identified three low copy repeat sequences Somatic cell hybrid analysis revealed that the SMS breakpoints occurred within the distal and proximal SMS-REPs

7. Transcription Map Figure 1 Lucas et al EJHG 9 (2001)

8. Repeated Sequences Highly homologous (98%) and chromosome 17 contains several Low Copy Repeats which act as substrates for NAHR Three low copy repeat sequences: proximal (256kb), middle(241kb), and distal(176kb) Middle SMS-REP is an inverted copy Each SMS-REP contains roughly 14 genes Critical deletion region occurs between the proximal and middle SMS-REPs Divergence from a progenitor 40-65mya

9. Location of SMS-REPs Figure 7 Bi et al. 2002

10. SMS-REPs cont. “hotspots” for NAHR within SMS-REPs 12kb region within the ~34kb KER gene cluster Contains >300bps with perfect identity along with polymorphic nucleotides 2.1kb AT rich inverted repeats flank proximal & middle but not the distal Hairpin formation initiates NAHR event

11. Figure 2 Bi et al. 2002

12. Figure 5 Bi et al Am.J.Hum.Genet. 73(2003)

13. Cloning cont. Sequence analysis also determined that the deletion region contains ~25 genes and 14 ESTs Critical region of chromosome 17 contains a high average of gene composition compared to the whole human genome

14. Identifying Candidate Genes Looked at genes that are developmentally regulated (mental & behavioral) and expressed in the neural crest (craniofacial & heart development) Dosage sensitive Transcription factors? –Effect development

15. Candidate Genes Used 6 markers from chromosome 17p11- 17p12 regions Plasmid clones of 14 ESTs in critical region were sequenced and obtained commercially Sequence analysis and tissue expression (Northern blot) was used to identify 6 possible candidate genes within the SMS critical region

16. What are the Candidate Genes? FLII: actin binding & severing in fly; cell adhesion and protein-protein interaction LLGLI: associated with cytoskeleton and serine kinase DRG2: GTP binding protein RASD1: ras related protein in GTPases NT5M: dephosphorylation of T & U as a mitochondrial deoxyribonucleotidase Their roles in SMS are still unknown

17. The Candidate Gene: RAI1 Retinoic-acid induced 1 gene is expressed in all adult tissues Homologous to mouse Rai1 gene that influences neuronal differentiation Haploinsufficiency accounts for facial, otolaryngological, neurological, and behavioral abnormalities Heart & renal defects due to other genes within chromosome 17p11.2 since >90% of people with SMS have part of the critical region deleted

18. Lucas et al EJHG 9 (2001)

19. RAI1 cont. 8kb transcript that is caused by alternative splicing to produce an 1863 AA protein Contains CAG repeats & nuclear localization signals Sequence similar to transcriptional coactivator TCF20 RAI1 may interact with other DNA-binding proteins to exert effects on transcription

20. Mutation causes SMS? Mutated RAI1 in three individuals with SMS, but no deletion of critical region Deletion in exon 3 of RAI1 on one allele Causes the protein to be truncated due to dominant frameshift mutation None of the parents carried any of these mutations

21. Mouse knockout Human chromosome 17p11.2 is syntenic to the 32-34 cM region of mouse chromosome 11 and the genetic order is highly conserved Heterozygous knockout of the syntenic deletion region in mouse using the Cre-loxP site-specific system Several SMS phenotypes were observed in mice: –Craniofacial abnormalities, seizures and abnormal EEGs, weight differences, and reduced male fertility

22. Figure 3 Walz et al. Molecular & Cell Biology 23 (2003)

23. Screening for SMS Screening for SMS among patients with mental retardation of unknown causes SMS is often under diagnosed because of subtle and variable expression Initially used Southern blotting and dosage comparison between markers, SMS deletion specific and chromosome X control probes Confirmatory testing used FISH and/or PCR microsatellitle genotyping 1 in 569 were detected to have SMS

24. Figure 5 Struthers et al. J Med Genet 39(2002)

25. References Allanson, Judith, Greenberg, Frank, and Smith, Ann. “The face of Smith-Magenis syndrome: a subjective and objective study.” Journal of Medical Genetics 36:394-397, 1999. Lucas, R., Vlangos, C., Das, P., Patel, P., and Elsea, S. “Genomic organization of the ~1.5 Mb Smith- Magenis syndrome critical interval: transcription map, genomic contig, and candidate gene analysis.” European Journal of Human Genetics 9:892-902, 2001. McBride, Gail. “Melatonin disrupts sleep in Smith-Magenis syndrome.” Lancet 354, 1999. Park, S., et al. “Structure and Evolution of the Smtith-Magenis Syndrome repeat Gene clusters, SMS-REPs.” Genome Research 12(5):729-738, 2002. Shaw, C., Weimin, B., and Lupski, J. “Genetic proof of unequal meiotic crossovers in reciprocal deletion and duplication of 17p11.2.” American Journal of Human Genetics 71:1072-1081, 2002. Slager, Rebecca E., Newton, Tiffany L., Vlangos, Christopher N., Finucane, Brenda, and Elsea, Sarah H. “Mutations in RAI1 associated with Smith-Magenis syndrome.” Nature Genetics 33(4): 466-468, 2003. Struthers, J. L., Carson, N., McGill, M., Khalifa, M. M. “Molecular screening for Smith-Magenis syndrome among patients with mental retardation of unknown cause.” Journal of Medical Genetics 39(59). Walz, K., et al. “Modeling del(17)(p11.2p11.2) and dup (17)(p11.2p11.2) contiguous gene syndromes by chromosome engineering in mice: Pheontypic consequences of gene dosage imbalance.” Molecular and Cell Biology 23(10):3646-3655, 2003. Weimin, B., Park, S., Shaw, C., Withers, M., Patel, P., and Lupski, J. “Reciprocal Crossovers and a Positional Preference for Strand Exchange in recombination events resulting in deletion or duplication of chromosome 17p11.2.” American Journal of Human Genetics 73:1302-1315, 2003. Weimin B., Yan, J., Stankiewicz, P., et al. “Genes in a Refined Smith-Magenis Syndrome Critial Deletion Interval of Chromosome 17p11.2 and the syntenic region of the mouse.” Genome Research 12(5):71-28, 2002.