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
Cys
vWF A
TH
α1
α2
α3
Fibronectin type III motif
Kunitz Protease
inhibitor motif
Macromolecular Structure
•
Assembly of Collagen VI multi-step process:
1.
2.
3.
•
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
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
Extraction
AAA
Col6a1
AAA
Col6a2
AAA
Fibroblast
sample
Reverse
Transcription
cDNA
Col6a3
mRNA
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
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
vWFA
TH
vWFA
FIII KPI
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