Smith-Magenis SyndromePresented by: Sara Mickelson

What is SMS?Syndrome was first described in 1982 by Ann Smith(genetic counselor) and Dr. Ruth MagenisOccurs in 1 in 25,000 peopleArises from a spontaneous heterozygous deletion of part of chromosome 17p11.2 through non-allelic homologous recombinationNo correlation between the size of the deletion and the severity of the phenotypePeople with chromosome 17p11.2 duplicated have a mild phenotype

PhenotypesWide range of phenotypesMental retardation (IQ 20-78)Behavioral abnormalitiesAggressive and Self-inflicted injuriesSelf-hugging, polyembolokoilamaniaSleep disturbancesMelatonin imbalance resembles jet lagDelayed speech and motor developmentDistinct physical characteristics

Journal of Medical Genetics 36

Cloning the SMS regionCommon deletion region is ~4Mb as detected by pulsed-field gel electrophoresis and FISHSomatic rodent:human hybrid cell lines with the chromosome 17 deletion determined that the critical region is ~1.1Mb16 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

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 ESTSequence data analysis also identified three low copy repeat sequencesSomatic cell hybrid analysis revealed that the SMS breakpoints occurred within the distal and proximal SMS-REPs

Transcription MapFigure 1 Lucas et al EJHG 9 (2001)

Repeated SequencesHighly homologous (98%) and chromosome 17 contains several Low Copy Repeats which act as substrates for NAHRThree low copy repeat sequences: proximal (256kb), middle(241kb), and distal(176kb)Middle SMS-REP is an inverted copyEach SMS-REP contains roughly 14 genesCritical deletion region occurs between the proximal and middle SMS-REPsDivergence from a progenitor 40-65mya

Location of SMS-REPsFigure 7 Bi et al. 2002

SMS-REPs cont.hotspots for NAHR within SMS-REPs12kb region within the ~34kb KER gene clusterContains >300bps with perfect identity along with polymorphic nucleotides 2.1kb AT rich inverted repeats flank proximal & middle but not the distalHairpin formation initiates NAHR event

Figure 2 Bi et al. 2002

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

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

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

Candidate GenesUsed 6 markers from chromosome 17p11-17p12 regionsPlasmid clones of 14 ESTs in critical region were sequenced and obtained commerciallySequence analysis and tissue expression (Northern blot) was used to identify 6 possible candidate genes within the SMS critical region

What are the Candidate Genes?FLII: actin binding & severing in fly; cell adhesion and protein-protein interactionLLGLI: associated with cytoskeleton and serine kinaseDRG2: GTP binding proteinRASD1: ras related protein in GTPasesNT5M: dephosphorylation of T & U as a mitochondrial deoxyribonucleotidaseTheir roles in SMS are still unknown

The Candidate Gene: RAI1Retinoic-acid induced 1 gene is expressed in all adult tissuesHomologous to mouse Rai1 gene that influences neuronal differentiation Haploinsufficiency accounts for facial, otolaryngological, neurological, and behavioral abnormalitiesHeart & renal defects due to other genes within chromosome 17p11.2 since >90% of people with SMS have part of the critical region deleted

Lucas et al EJHG 9 (2001)

RAI1 cont.8kb transcript that is caused by alternative splicing to produce an 1863 AA proteinContains CAG repeats & nuclear localization signalsSequence similar to transcriptional coactivator TCF20RAI1 may interact with other DNA-binding proteins to exert effects on transcription

Mutation causes SMS?Mutated RAI1 in three individuals with SMS, but no deletion of critical regionDeletion in exon 3 of RAI1 on one alleleCauses the protein to be truncated due to dominant frameshift mutationNone of the parents carried any of these mutations

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

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

Screening for SMSScreening for SMS among patients with mental retardation of unknown causes SMS is often under diagnosed because of subtle and variable expressionInitially used Southern blotting and dosage comparison between markers, SMS deletion specific and chromosome X control probesConfirmatory testing used FISH and/or PCR microsatellitle genotyping1 in 569 were detected to have SMS

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

ReferencesAllanson, 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.

NAHR is also known as unequal crossing overShow pictures of people with the diseaseCertain gene or EST in EcoRI-digested BAC or PAC DNAThis is just the map from the middle REP to the distal REPFigure 7 Hypothetical model for evolution of SMS-REPs. Our data indicate that in the first step, a progenitor SMS-REP must have arisen in an ancient chromosome. Its structure was almost the same as the proximal SMS-REP at the present time, but included the B region flanking sequences on both sides similar to the distal SMS-REP. The distal SMS-REP resulted from the deletions of two large areas between the A and B, and C and D regions. Secondly, deletion of both flanking sequences of the B region in the progenitor resulted in the proximal SMS-REP. Finally, two terminal deletions and one interstial deletion involving the UPF3A gene accompanied by interchromosomal insertional duplication together with an inversion generated the middle SMS-REP. Figure 2 Bacterial artificial chromosome/P1 artificial chromosome (BAC/PAC) contig map spanning three SMS-REPs. Thick bold lines represent minimal-tiling-path, large-insert clones utilized for genomic finished sequence of SMS-REPs. Clones are designated with their clone name and their GenBank accession number. Markers in boxes represent SMS-REP-flanking sequences used to determine SMS-REP orientations. Final orientation was determined by the construction of a complete BAC/PAC contig spanning the common deletion (Bi et al. 2002 ).

Figure 5 Repeats flanking the KER gene cluster. A, The 2.1-kb AT-rich (38% GC) inverted repeats (black arrows) with 98% homology flank the sequence blocks B and C, including the KER gene cluster, in both the proximal and the middle SMS-REPs, but not in the distal SMSREP. Note that the sizes of the homology blocks and the flanking inverted repeats are not drawn to scale. Within the inverted repeat, there are 646-bp internal inverted repeats with 93% homology (blue arrows). One repeat centromeric to the block C is separated from block C with a 91-bp segment (brown arrows) that shares 98% homology with a segment within the inverted repeat and 17 additional nucleotides, including 4 adenines (indicated as A4N13). The 4 adenines are within a tract of 19 continuous adenines, in which 15 are located within block C, the homologous region between the distal and proximal SMS-REPs. B, Each of the inverted repeats can theoretically form hairpin structures as indicated. The 12-kb hotspot region is adjacent to the inverted repeat centromeric to the block C. The long adenine tract and the hairpin structures may be more sensitive to DSBs, which might serve as the initiation event for NAHR.

Dosage sensitive because of the haploinsufficiency of the syndromeFigure 2 genetic organisation EJHGFig 3:northern blotPic of protein?Cool fact: RAI1 is also involved with schizophreniaShow pictures!!!!FIG. 3. Craniofacial abnormalities. (A and B) Df(11)17/+ and wild-type (wt) animals are pictured together. (A) Note the positions of the snouts and the broad distance between the eyes (hypertelorism) of the Df(11)17/+ mouse compared with that between the eyes of the wild-type littermate. (B) The short distance between the eyes and the nose of the Df(11)17/+ mouse compared with the distance between those of the wild-type littermate can also be visualized. Skeletal preparations of wild-type (C, D, and I), Df(11)17/+ (E, F, and J), and Df(11)17/Dp(11)17 (G and H) skulls of 3-month-old male animals are shown for comparison. The different positions of the noses in the Df(11)17/+ mice can be explained by the shape of the nasal bone (F, arrow) compared with that of the wild type (D). (H) This phenotype is completely rescued with the addition of an extra copy of the genes that are deleted [Df(11)17/Dp(11)17 animals]. (K) The different landmarks pictured in panels C and I were used to objectively measure the distances between the landmarks. Cranial landmarks are as follows: a and h, nasal; b, c, and i, anterior notch on frontal process situated laterally in relation to the intraorbital fissure; d, intersection of parietal and intraparietal bones; e, intersection of interparietal and occipital bones at the midline; j, bregma; and k, intersection of maxilla and sphenoid on inferior alveolar ridge. The relative distances (in centimeters; see Materials and Methods) were used for the statistical analysis, and the averages of the distances are shown in the table. The asterisks denote that significant differences (P of