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