Tom Cullup Guy’s Hospital DNA Laboratory

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

5. Collagen VI • Heterotrimeric • Extracellular matrix protein • Genes: • COL6A1/COL6A2/COL6A3 • Similar structure • COL6A1 and COL6A2: 21q22.3 (head to tail) • COL6A3: 2q37

6. TH Cys vWF A α1 α2 α3 Kunitz Protease inhibitor motif Fibronectin type III motif

7. Macromolecular Structure • Assembly of Collagen VI multi-step process: • Assembly of triple-helical monomer (1 x α1, α2, α3) • 2 x monomers assemble into antiparallel dimers • 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. AAA Col6a1 AAA Col6a2 AAA Col6a3 Practical Overview Reverse Transcription Extraction cDNA Fibroblast sample mRNA 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 FIII KPI vWFA TH Het In-frame del/splice Het missense (TH Glycine residues) Hom In-frame del/splice Hom Out-of-frame del/splice Hom missense N C

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