STICKLER SYNDROME Corrine Fillman, M.S., C.G.C. Connective Tissue Gene Tests (CTGT) 6580 Snowdrift Road, Suite 300 Allentown, PA 18106 1

OUTLINE Stickler Syndrome Clinical Findings Genes Connective Tissue & Collagen Autosomal Dominant Stickler syndrome Marshall syndrome Autosomal Recessive Stickler syndrome How is Stickler & Marshall Syndrome Diagnosed? Clinical Diagnosis Molecular Diagnosis-CTGT 2 2

STICKLER SYNDROME AFFECTS MANY PARTS OF THE BODY: Eyes: Nearsightedness Cataracts Retinal detachment Oral/Facial: Cleft palate Bifid uvula Small chin Pierre-Robin sequence Flat cheeks Flat nasal bridge Ears: Hearing Loss Joints: Early-onset arthritis Bones: Chondrodysplasia 3 3

STICKLER SYNDROME A GENETIC DISORDER A condition that can be passed from one generation to the next within a family A condition caused by a defect (mutation) in a gene 4 4

Stickler syndrome Genes COL11A1 COL9A2 COL2A1 COL11A2 COL9A1 COL9A3 5

STICKLER SYNDROME A CONNECTIVE TISSUE DISORDER The genes associated with Stickler syndrome are responsible for making proteins called collagens. Specifically collagen type II, XI & IX. There are many types of collagens and collagens are found throughout the body in our connective tissue. Connective tissue is a material that provides strength and support for organs and tissues. The collagen types associated with Stickler syndrome are found primarily in cartilage, part of the clear gel that fills the eyeball (the vitreous) and the inner ear. 6 6

STICKLER SYNDROME COLLAGEN PROTEIN The collagen protein is made of 3 protein chains wrapped around one another like a rope. Collagen II protein is made of 3 identical protein chains made from the COL2A1 gene. Collagen XI protein is made of 3 different protein chains. 1 chain is made from the COL11A1 gene 1 chain is made from the COL11A2 gene 1 chain is made from the COL11A3 gene Collagen IX protein is made of 3 different protein chains. 1 chain is made from the COL9A1 gene 1 chain is made from the COL9A2 gene 1 chain is made from the COL9A3 gene 7

STICKLER SYNDROME AUTOSOMAL DOMINANT The majority of individuals with Stickler syndrome have autosomal dominant Stickler syndrome. The genes associated with autosomal dominant Stickler syndrome are COL2A1, COL11A1 and COL11A2. First-degree relatives (children, siblings and parents) have a 50% chance of also having Stickler syndrome. 8 8

Stickler syndrome types There are differences in an individual’s clinical findings depending upon the affected gene and mutation Stickler syndrome, type I 80-90% of individuals with Stickler syndrome have mutations in the COL2A1 gene. Autosomal dominant inheritance. Individuals may have a “membranous” type 1 vitreous anomaly. Mutations in the COL2A1 gene cause Stickler syndrome, type I. Individuals with mutations in exon 2 of the COL2A1 gene have an ocular variant of Stickler syndrome. Individuals may present with typical eye findings including an empty vitreous and/or early-onset retinal detachment but there are minimal or absent findings in the ears, bones, joints and oral/facial structures. 9 9

THERE ARE DIFFERENCES IN AN INDIVIDUAL’S CLINICAL FINDINGS DEPENDING UPON THE AFFECTED GENE AND MUTATION

Stickler syndrome, type I In the case of COL2A1, the type of mutation and the location within the gene will result in different clinical findings: COL2A1 mutations are associated with more than one disorders including: Stickler syndrome, type I Stickler syndrome, type I, nonsyndromic ocular Other Conditions: Achondrogenesis, type II / Hypochondrogenesis Spondyloepiphyseal dysplasia congenita Spondyloepimetaphyseal dysplasia, Strudwick type Kniest dysplasia Spondyloepiphyseal dysplasia late onset Avascular necrosis of femoral head Early-onset osteoarthritis 11

STICKLER SYNDROME TYPES There are differences in an individual’s clinical findings depending upon the affected gene and mutation Stickler syndrome, type II Mutations in the COL11A1 gene cause Stickler syndrome, type II. 10-20% of individuals with Stickler syndrome have mutations in the COL11A1 gene. Autosomal dominant inheritance. Individuals may have a “beaded” type 2 vitreous anomaly. 12 12

THERE ARE DIFFERENCES IN AN INDIVIDUAL’S CLINICAL FINDINGS DEPENDING UPON THE AFFECTED GENE AND MUTATION In the case of COL11A1, the type of mutation and the location within the gene will result in different clinical findings: COL11A1 mutations are associated with more than one disorder including: Stickler syndrome, type II Other Conditions: Marshall syndrome Fibrochondrogenesis 13

Mutations in the COL11A1 gene cause Marshall syndrome. Shares many features in common with Stickler syndrome including: nearsightedness, retinal detachment, hearing loss, cleft palate and Pierre-Robin sequence. In addition, patients may have early-onset hearing loss, short stature, abnormalities in cranial ossification, and more pronounced facial features including markedly flat cheeks with a flat nasal bridge and a short upturned nose. Mutations in the COL11A1 gene cause Marshall syndrome. Mutations in this gene also cause Stickler syndrome, type II. Autosomal dominant inheritance and first-degree relatives (children, siblings & parents) have a 50% chance of also having Marshall syndrome. A -B-Stickler syndrome C-F- Marshall syndrome 14 Annunen et al., 1999. Am J Hum Genet 65:974-983. 14

STICKLER SYNDROME TYPES There are differences in an individual’s clinical findings depending upon the affected gene and mutation Stickler syndrome, type III Mutations in the COL11A2 gene cause a non-ocular form of Stickler syndrome. Autosomal dominant inheritance. Individuals have cleft palate and/or Pierre-Robin sequence, hearing loss and early-onset osteoarthritis. Individuals do not have eye findings typically associated with Stickler syndrome. 15 15

THERE ARE DIFFERENCES IN AN INDIVIDUAL’S CLINICAL FINDINGS DEPENDING UPON THE AFFECTED GENE AND MUTATION In the case of COL11A2, the type of mutation and the location within the gene will result in different clinical findings: COL11A2 mutations are associated with more than one disorder including: Stickler syndrome, type III Other Conditions: Otospondylomegaepiphyseal dysplasia Weissenbacher-Zweymuller syndrome 16

STICKLER SYNDROME AUTOSOMAL RECESSIVE It is not known how many individuals with Stickler syndrome have autosomal recessive inheritance. Mutations in the COL9A1, COL9A2 and COL9A3 genes cause Stickler syndrome, autosomal recessive. Siblings of an affected individual have a 25% chance of also having Stickler syndrome and a 50% chance of being an unaffected carrier. Individuals present with clinical symptoms similar to Stickler syndrome type I/II. 17 17

STICKLER SYNDROME AUTOSOMAL RECESSIVE 2006-COL9A1 Van Camp et al., 2006. Am J Hum Genet 79:449-457. Severe myopia, vitreoretinal degeneration,hearing loss, chondrodysplasia 2010- COL9A2 & COL9A3-Canine studies Goldstein et al., 2010. Mamm Genome 21:398-408. Oculoskeletal dysplasia similar to STL1 & STL2 segregates as an autosomal recessive trait in Labrador Retriever and Samoyed dog breeds Vitreous dysplasia, retinal detachment, cataracts, short-limb dwarfism 2011- COL9A2 Baker et al., 2011. Am J Med Genet Part A 9999: 1-5. Severe myopia,vitreoretinal degeneration, retinal detachment, hearing loss 18 18

STICKLER SYNDROME AUTOSOMAL RECESSIVE 19 Baker et al., 2011. Am J Med Genet Part A 9999: 1-5 19

HOW IS STICKLER SYNDROME AND MARSHALL SYNDROME DIAGNOSED?

Clinical Diagnosis: made by evaluating a patient’s medical and family history and having a thorough physical exam by a physician and/or specialists. A physician can use published literature and references that describe the clinical findings to help make a diagnosis. There is a wide range of clinical findings, and individuals in the same family may have different findings and varying degrees of severity. Molecular Diagnosis (genetic testing): made by identifying a mutation in one of the associated genes. Molecular testing is available in CLIA approved laboratories and requires a blood sample from the individual suspected to have a diagnosis. Can confirm a suspected clinical diagnosis. Results from a molecular diagnosis can be used for surveillance and management. Results can be used to confirm or rule out the diagnosis in at risk family members. 21 21

CONNECTIVE TISSUE GENE TESTS Director of Research, Development and Technology Leena Ala-Kokko, M.D., Ph.D. CEO and Medical Director James Hyland, M.D., Ph.D. CTGT was established in June, 2004 and is located in Allentown, PA. About 20 employees. CLIA & CAP certified. Currently test for over 100 different genetic disorders. Offer many different technologies for testing. CTGT in continually adding new tests and developing new technologies. CTGT offers family testing and prenatal testing for all our genes. CTGT has a rapid turnaround time averaging about 2 weeks. 22 22

CONNECTIVE TISSUE GENE TESTS MOLECULAR DIAGNOSIS 2 Test Requests: Sequencing Deletion/Duplication 23

CONNECTIVE TISSUE GENE TESTS SEQUENCING Probe: COL11A1 Gene 24

CONNECTIVE TISSUE GENE TESTS SEQUENCING COL11A1 Gene The cat ran home. The mat ran home. 25

CONNECTIVE TISSUE GENE TESTS SEQUENCING Result: COL11A1 IVS50+1G>A Test request: Marshall syndrome COL11A1 Clinical Findings: 3 year old boy Robin Sequence (cleft palate & small chin) Flat face Bilateral conductive hearing loss Severe myopia (legally blind) Result: COL11A1 IVS50+1G>A 26

CONNECTIVE TISSUE GENE TESTS DELETION/DUPLICATION Test Sample Control Sample 27

CONNECTIVE TISSUE GENE TESTS DELETION/DUPLICATION Clinical Findings: 2 year old boy Flat face Myopia at 15 months old Short upturned nose Cleft palate Sequencing Results: COL2A1 sequencing negative COL11A1 sequencing negative COL11A2 sequencing negative Cleft palate Flat face Deletion/Duplication Results: COL2A1 deletion/duplication negative COL11A1 deletion exons 47-62 Deletion/Duplication Results: COL11A1 deletion exons 47-62 28

CONNECTIVE TISSUE GENE TESTS DELETION/DUPLICATION COL11A1 gene no deletion 29 COL11A1 gene deletion of exons 47-62

CONNECTIVE TISSUE GENE TESTS SEQUENCING VS. DELETION/DUPLICATION Sequencing-looks for small spelling errors in the gene’s set of instructions. Deletion/Duplication-looks for large sections of the instructions that are missing. 30

CONNECTIVE TISSUE GENE TESTS STICKLER SYNDROME TESTING Gene Sequencing Deletion/Duplication COL2A1 X X COL11A1 X X COL11A2 X X COL9A1 X X COL9A2 X X COL9A3 X X CTGT offers both sequencing and deletion/duplication testing for all the genes currently associated with Stickler syndrome. Having a genetic test is a choice not a requirement. Genetic testing is a personal decision and the decision is different for each family and each person within a family. 31

Connective Tissue Gene Tests (CTGT) THANK YOU Corrine Fillman, M.S., C.G.C. Connective Tissue Gene Tests (CTGT) 6580 Snowdrift Road, Suite 300 Allentown, PA 18106