1. Stickler Syndrome Study at the National Institutes of Health Nazli McDonnell M.D., Ph.D. Laboratory of Clinical Investigation National Institute on Aging National Institutes of Health Baltimore, Maryland

2. Introduction Stickler Syndrome is an autosomal dominant hereditary disorder that effects the connective tissue 1 in 10,000 individuals in North America (Hermann et al, 1975) are thought to have Stickler Syndrome Stickler Syndrome may affect your eyes, your hearing, your bones and joints

3. Intro cont. Known mutations that lead to Sticklers are found on the Col2A1, Col11A1 and the Col11A2 genes They are genes code for the collagens that are found in your eyes, joints and bones Mutations on these genes account for around 50% of the cases of Stickler Syndrome. Other causes for Sticklers syndrome are unknown at this time.

4. Manifestations Stickler Syndrome effects many parts of the body. In the eyes, Stickler syndrome may cause near-sightedness (myopia), vitreous degeneration, retinal detachments and retinal tearing, premature cataracts and glaucoma People with Stickler Syndrome may be born with cleft palates and/or bifid uvulas

5. Manifestations cont. The most noticeable manifestations of Stickler are the facial manifestations. People with Sticklers Syndrome often have a flattened facial profile, flattened and broadened nasal bridge and small chins. Patients also have problems with high frequency hearing loss and hypermobile tympanic membranes

6. Diagnosis is often missed Stickler syndrome is among the most common autosomal dominant connective tissue disorders but is often unrecognized and therefore not diagnosed by clinicians. Ten percent of patients with isolated cleft palate and 12% with the Pierre-Robin sequence were found to have undiagnosed Stickler syndrome in one series [Kronwith et al., 1990; Sheffield et al., 1987] The actual incidence is higher.

7. Background Collagens are the most common proteins in the extracellular matrix Collagen Type II is abundant in the vitreous of the eye, the spinal column, cartridge, and inner ear Type II is also present in many tissues during embryological development Collagen XI is found in association with Collagen II.

8. The Collagens Collagens consists of three polypeptide chains which are folded into a rod-like triple helical molecule Each of the constituent chains of the triple helix are called alpha chains and are coiled in a left handed helix with three amino acids per turn. The constituent amino acids are regularly arranged in the order Gly-X-Y such that glycine, which is the smallest of all amino acids, occupies the restricted space in which the three helical chains come together. This arrangement is crucial for the stability of the macromolecule.

9. Collagen structure Side view shows: Primary structure = (X-Y-gly) n X,Y are often lysine or proline Top end view shows: Secondary structure is a collagen helix with pitch of 3.0 residues per turn

10. Collagen Fibrils

11. Collagens and Stickler Syndrome Type 2 collagen is a homotrimer of three COL2A1 gene products, whereas type 11 collagen is a heterotrimer containing one each of the COL2A1, COL11A1, and COL11A2 gene products. Both type 2 and 11 collagens are members of the fibrillar collagens, which are primarily found in cartilage, vitreous, and nucleus pulposus (soft, gelatinous central portion of an intervertebral disk).

12. The diagnostic criteria Orofacial Abnormalities (2 points maximum) (2 points)Cleft palate (open cleft, submucous cleft, or bifid uvula) (Major) (1 point)Characteristic facies (malar hypoplasia, broad nasal bridge, Micro/retrognathia) Ocular Abnormalities (2 points maximum) (2 points)Characteristic vitreous changes or retinal abnormalities (lattice degeneration, retinal holes, or retinal tear, retinal detachment) (Major) Auditory Abnormalities (2 points maximum) (2 points)High frequency sensorineural hearing loss (Major) Age < 20: threshold  20 dB at 4-8 kHz Age 20-40:threshold  30 dB at 4-8 kHz Age > 40:threshold  40 dB at 4-8 kHz (1 point)Hypermobile tympanic membranes Skeletal Abnormalities (2 points maximum) (1 point)History of femoral head failure (slipped epiphysis or Legg-Perthes like disease) (1 point)Radiographically demonstrated osteoarthritis before age 40 (1 point)scoliosis, spondylolisthesis, or Scheuermann-like kyphotic deformity Family History / Molecular Data (1 point)Independently affected 1 st degree relative in a pattern consistent with autosomal dominant inheritance or presence of COL2A1, COL11A1, or COL11A2 mutation associated with Stickler syndrome Diagnosis requires: 5 or more points total up to 9 points At least one 2-point major manifestation Absence of features suggestive of a skeletal dysplasia (e.g. stature <5% centile)

13. Genotype/Phenotype Correlations Study involved 48 families suspected of having Stickler Syndrome Consent was received from each individual to allow the NIA to collect data and genetic material from each patient Patients underwent a detailed clinical genetics examination, dilated ophthalmology exam, audiology and otolaryngology evaluations

14. Materials and methods contd. In most cases, radiographs of the spine and hip were available The enrollment and tests took place before the development of the diagnostic criteria for Stickler syndrome. Forty eight probands were selected from each family for molecular genetic analysis Col2A1 and Col11A1 genes were amplified by polymerase chain reaction (PCR) and were screened for mutations

15. Results 23 mutations in Col2A1 and 4 mutations in Col11A1 was found in our cohort of 48 probands. All persons included in the study had orofacial features, while clefting of palate/uvula showed intra and inter family variability All persons also had ocular involvement in the form of vitreous or retinal changes.

16. Types of Mutations STOP = 16 Splice Site = 8 Insertion = 1 Arg/Cys = 1 Other missense =1

17. Results contd. Hearing loss, premature osteoarthritis, and skeletal involvement was common. Femoral head failure occurred in 3 families. Hip or knee pain needs to be taken very seriously in children with Stickler syndrome as it can be a manifestation of femoral head failure.

18. Summary of Findings Facial features29/29 Open Cleft11/29 Submucus Cleft7/29 Bifid Uvula5/29 Vitreous Change24/29 Retinal Change24/29 Vitreous OR Retinal Change29/29 High frequency sensory neural hearing loss17/29 Hypermobile Tympanic Membranes2/29 Femoral Head Failure3/29 Premature Osteoarthritis16/29 Skeletal Abnormalities14/29

19. Discussion The mutation detection rate in this Stickler Syndrome cohort was 56% All subjects with a mutation in Col2A1 or Col11A1 met the recently published Stickler Diagnostic Criteria The majority of the mutations detected resulted in a splice site aberration (often implicated in exon skipping) or a premature termination codon.

20. Discussion Contd. The phenotype analysis revealed that ocular involvement and craniofacial dysmorphisms are core features of Stickler syndrome. Comparison with a large pedigree without a Col2A1/Col11A1 mutation in the proband reveals that this family has a phenotype indistinguishable from the subjects with such mutations

21. Mutations in Stickler Syndrome All of the mutations found resulted in haploinsufficiency (inadequate amount of collagen, as opposed to abnormal collagen) Dominant negative mutations in Collagen II result in the dwarfisms This opens the door to the possibility that symptoms of Stickler syndrome may be treated by a mechanism that increases collagen production from the “good copy” of the gene.

22. Future directions The next project will be to study the genes of the individuals who lack known mutations. We are studying a candidate gene, CSPG2, which has been implicated in a family with Wagner syndrome which is a disease that affects the eye very similarly to Stickler syndrome.

23. Future Directions Tissues from patients with the known mutations will be analyzed to learn more about how the mutations effect the workings of the collagen Phenotype analysis will continue to better understand the syndrome

24. Enrollment of new patients We will start to enroll new patients soon Eye exam is now available for the study in its new location in Baltimore, however we are working on the details of transportation of participants Hope to enroll 20 patients in 2008

25. Acknowledgements Ben Griswold, Minna Männikko, Jeremy Wells, Marja Majava-Elo, Joseph Tran, Katherine Mandel, Peter S. Rose, Howard P. Levy,Joie Davis, Yvonne Szymko, Benjamin Rubin, Ekaterini Tsilou, Muriel Kaiser, Andrew J. Griffith, Ruth Altshuler Liberfarb, Leena Ala-Kokko, Clair A. Francomano 1 Laboratory of Clinical Investigation, National Institute on Aging, National Institutes of Health 2 Department of Medical Biochemistry and Molecular Biology, University of Oulu 3 Department of Genetics, Massachusetts General Hospital 4 Clinical Research Branch, National Institute on Aging, National Institutes of Health 5 Department of Orthopedic Surgery, Mayo Clinic, Rochester, Minnesota 6 Department of Internal Medicine, Johns Hopkins University School of Medicine 7 National Human Genome Research Institute, National Institutes of Health 8 National Eye Institute, National Institutes of Health 9 National Institute on Deafness and Other Communication Disorders, National Institutes of Health 10Laboratory of Genetics, National Institute on Aging, National Institutes of Health 11Ursinus College, Collegeville, Pennsylvania