1. Establishment of a screening service for Bethlem Myopathy and Ullrich Congenital Muscular Dystrophy Tom Cullup Guy’s Hospital DNA Laboratory

2. Introduction UCMD and BM –Phenotypes –The Collagen VI genes Testing Strategy Initial Results Discussion on cDNA sequencing

3. UCMD and BM UCMD Ullrich Congenital Muscular Dystrophy BM Bethlem Myopathy Inheritance:AR (AD)Inheritance:AD Symptoms:Muscle weakness Proximal joint contractures Hyperelasticity of distal joints Walking never achieved Death from respiratory failure Symptoms:Hypotonia Delayed motor milestones Weakness Muscle atrophy Contractures (temporary/permanent) Differential diagnosis: Other CMDs/Myopathies SMA Ehlers Danlos Syndrome Marfan Syndrome Differential diagnosis: Sarcoglycanopathies Calpainopathy Dysferlinopathy XL/AD Emery Dreyfus MD

5. Collagen VI Heterotrimeric Extracellular matrix protein Genes: –COL6A1/COL6A2/COL6A3 –Similar structure –COL6A1 and COL6A2: 21q22.3 (head to tail) –COL6A3: 2q37 GeneExonsAmino Acids COL6A1351028 COL6A2281019 COL6A344 (43)3177 Total1075224

6. vWF A TH Fibronectin type III motif Kunitz Protease inhibitor motif Cys α1α1 α2α2 α3α3

7. Macromolecular Structure Assembly of Collagen VI multi-step process: 1.Assembly of triple-helical monomer (1 x α1, α2, α3) 2.2 x monomers assemble into antiparallel dimers 3.2 x dimers align to form tetramers Cysteine residues in all 3 chains thought to be involved in dimer/tetramer formation/stability

8. Testing Strategy Options: –Pre-screen (TGCE) + genomic seq –Genomic seq (+ dosage assay) –cDNA seq cDNA seq: –Should pick up same mutations as genomic seq + demonstrates splice + large del/dup –Potential to reduce sequencing load Genomic: 107 fragments cDNA: 26 fragments

9. Practical Overview AAA Col6a1 Col6a2 Col6a3 Fibroblast sample Extraction mRNA Reverse Transcription cDNA Overlapping 1°PCR primers Tagged, nested 2°PCR primers 2°PCR Fragments Sequenced F+R Using Tag primers

10. Initial Screen Results Initial cohort: 16 patients 14 have definite pathogenic mutations 87.5% pick-up (previous studies: 62%) Why so high? –Patient selection Phenotype screened by Hammersmith Immunohistochemical analysis –Screening strategy 1 patient with het del – no confirmed DNA change 1 patient with -10 change causing splicing defect - ? Classed as mutation if only seen on DNA

11. Deletion of Ex10 (COL6A2) at the RNA level No definitive change at DNA level - ?mosaic splicing mutation

12. Results Interpretation Large proportion of heterozygous mutations for UCMD cases (8 het vs 5 hom) –Previously thought of as AR –UCMD/BM now thought of as continuous phenotypic spectrum –Location of mutations as well as mutation type important

13. vWFA TH FIIIKPI Het In-frame del/splice Hom In-frame del/splice Hom Out-of-frame del/splice Het missense (TH Glycine residues) Hom missense NC

14. Theories on genotype-phenotype correlation “Classical” UCMD: –2 x PTC mutations → No functional protein “Classical” BM: –1 x Missense/in-frame del/splice → Weak dom-neg effect Glycine missense in TH domain: –Evidence that N-term Glycine changes cause ‘kinking’ of tetramers → dominant neg effect –Only 1 example of hom glycine change Het del/splice: –Similar effect to Glycine missense –Preservation of Cys residue allows secretion of abnormal tetramers → dom neg effects on microfibrillar assembly

15. Benefits and drawbacks of cDNA sequencing Benefits: –Smaller number of fragments to sequence –Demonstrates splicing mutations –Shows large rearrangements Drawbacks –RNA unstable –Alternative splicing –Does not fit into lab high-throughput processes –Checking overlapping fragments

16. Acknowledgments Guy’s DNA lab: –Michael Yau –Steve Abbs Hammersmith Neuromuscular unit: –Prof. Francesco Muntoni –Cecilia Jimenez-Mallebrera –Lucy Feng

17. Extracellular Matrix