establishment of a screening service for bethlem myopathy and ullrich congenital muscular dystrophy...
TRANSCRIPT
Establishment of a screening service for Bethlem Myopathy and Ullrich Congenital Muscular Dystrophy
Tom Cullup
Guy’s Hospital DNA Laboratory
Introduction
• UCMD and BM– Phenotypes – The Collagen VI genes
• Testing Strategy
• Initial Results
• Discussion on cDNA sequencing
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
Collagen VI
• Heterotrimeric• Extracellular matrix protein• Genes:
– COL6A1/COL6A2/COL6A3– Similar structure– COL6A1 and COL6A2: 21q22.3 (head to tail)– COL6A3: 2q37
Gene Exons Amino Acids
COL6A1 35 1028
COL6A2 28 1019
COL6A3 44 (43) 3177
Total 107 5224
vWF A
TH
Fibronectin type III motifKunitz Protease inhibitor motif
Cys
α1
α2
α3
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
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
Practical Overview
AAA
AAA
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
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
Deletion of Ex10 (COL6A2) at the RNA level
No definitive change at DNA level - ?mosaic splicing mutation
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
vWFA vWFATH FIII KPI
Het In-frame del/splice
Hom In-frame del/splice
Hom Out-of-frame del/splice
Het missense (TH Glycine residues)
Hom missense
N C
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
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
Acknowledgments
• Guy’s DNA lab:– Michael Yau– Steve Abbs
• Hammersmith Neuromuscular unit:– Prof. Francesco Muntoni– Cecilia Jimenez-Mallebrera– Lucy Feng