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Guide to Duchenne Genetics
Jo McCauley
Clinical Scientist
DNA organisation
Chromosomes
DNA structure
https://commons.wikimedia.org/wiki/File:Difference_DNA_RNA-EN.svg
What is a gene?
• Sequence of specific nucleotides
• Vary in size from few hundred to over 2 million bases
• Genes have areas which code for the protein called exons and non-coding
regions called introns
Intron 1
Intron 2
https://commons.wikimedia.org/wiki/File:Genetic_code.svg
DNA to proteins
•
•
•
•
The building blocks of DNA are nucleotides (A, G, C and T)
The building blocks of proteins are amino acids (there are 20)
A single nucleotide cannot code for a single amino acid
Therefore consecutive groups of 3 nucleotides in the linear sequence are decoded for each amino acid
Duchenne and Becker Muscular Dystrophy
0
0.5
2
1
10
20
1.5
30 40
2
50
2.5 Mb
60
70
• Both DMD and BMD are caused by mutations in dystrophin gene located at Xp21.2
• X-linked recessive disorder
• Dystrophin gene spans ~2.5 million bp (0.1% of total human genome)
• 79 exons (+ 8 tissue-specific promoters)
• 99% of gene is introns
• DMD is estimated at 1:3,500 live male births1
• BMD is estimated at 1:12,000 live male births2
1 Moser et al Hum Genet (1984);66:17-40
2 Emery Lancet (2002);359:687-695
Anatomy of dystrophin
•N-terminal actin-binding domain
A classical calponin-homology (CH) actinbinding domain, capable of high-affinity
binding to F-actin in the cytoskeleton.
•Cysteine-rich region
Contains a WW-domain, four
divergent EF-hands, and a ZZ domain.
I tera ts with β-dystroglycan in the
sarcolemma.
CH2
CH1
•Rod domain
Contains 20-24 modified spectrin-like
repeats, forming a rigid helical rod.
•C-terminal region
Contains several leucine heptad
repeats. Interacts with dystrobrevin
and the syntrophins.
Dystrophin protein
Dystrophin-associated protein complex
C-terminus
Mole ular ge eti s a d
eta olis , O’Brie a d Ku kel
Cysteinerich domain
Central rod
domain
N-terminus
What is the function of Dystrophin?
•Mechanical scaffold
Connection between cytoskeleton, plasma membrane,
extracellular matrix.
Loss of dystrophin gives loss of entire complex.
•Signal conduction
Connections with numerous signaling molecules:
Nitric oxide, DAG, Na+, agrin,
X-linked recessive inheritance
Unaffected father
Unaffected son
Unaffected daughter
Carrier mother
Carrier daughter
Affected son
Mutation spectrum
Type and frequency of mutations
Splice site
2.8%
Nonsense
10.2%
Missense
0.4%
Mid-intronic
0.3%
Small insertion
1.8%
Small deletion
5.0%
Large duplication
11.0%
Large deletion
68.5%
• Majority of mutations are deletions
• 2 hotspots:
• 5’ - exons 2-19 (common breakpoint introns 2 + 7)
• 3’ – exons 45-55 (common breakpoint intron 44)
• Most families have a different mutation.
• In 33% of cases the mutation within a family is new.
In-frame or out-of-frame?
If the number of bases that are deleted or duplicated is divisible by 3, this is described as being in-frame (BMD)
300bp
Exon 3
Exon 2
Exon 1
……….gactcg
gatctg…..gta
caactg…..gta
Exon 1
……….gactcg
Exon 3
gatctg…..gta
in-frame deletion - BMD
In-frame or out-of-frame?
299bp
Exon 3
Exon 2
Exon 1
……….gactcg
Exon 1
……….gactcg
gatctgg...gta
caactg……..gt
Exon 3
X
gatctgg...gta
gatctgg...gta
Out-of-frame
Premature termination codon
Truncated protein
DMD
DMD or BMD?
X
Deletion ex74
BMD
X
Deletion ex52
DMD
http://www.musculardystrophyuk.org
Reading Frame Hypothesis
•Proposed by Monaco et al 1988
•Holds true in 92% of cases
•Exceptions include
 in-frame deletions that disrupt critical functional domains
 ex3-7 deletion can use alternative translation start site in ex8
Monaco AP, Bertelson CJ, Liechti-Gallati S, Moser H, Kunkel LM. An explanation for the phenotypic differences between
patients bearing partial deletions of the DMD locus. Genomics. 1988;2(1):90–9
Clinical trials and therapies
Exon skipping - induced by antisense oligonucleotides (ASOs)
• restoring the reading frame by generating shortened but functional protein
• can convert the DMD phenotype into a milder BMD phenotype.
• applicable to up to 83% of all DMD mutations
• first clinically developed ASOs that target exon 51, as considered to be applicable to the
largest subset of patients
Therapies to restore dystrophin expression – exon
skipping
Shimizu-Motohashi et al., Am J Transl Res 2016;8(6):2471-2489
Testing strategy
• MLPA - all 79 exons in two MRC Holland kits
• Detects 65% of DMD mutations; 85% of BMD mutations
• Full screen by Sanger sequencing
• RNA analysis from muscle biopsy or hair roots
• Array testing
• Detects ~30% of DMD mutations (missense, nonsense and
splicing mutations); ~10% of BMD mutations
MLPA
Del ex47-50 (male)
Dup ex16-17 (male)
Het del ex46-50 (female)
Sequencing
Genotype/phenotype correlations
• No relation between size of deletion and severity of disease
• Deletion of a single exon can cause DMD (eg Del ex44)
• Deletion of large parts of the rod-domain (eg Del ex32-44, 48-53) associated with very mild
symptoms
• Phenotype depends on whether mutation disrupts reading frame
• In-frame mutations generally result in abnormal but partly functional dystrophin – BMD
• Out-of-frame mutations result in unstable RNA and almost no protein production – DMD
• Correlation holds for ~92% of cases
X-linked dilated cardiomyopathy
• Some rare mutations produce exclusive cardiac phenotype
• Different mRNA processing in skeletal and cardiac muscle
• Different functions of specific domains in skeletal and cardiac muscle
• Mutations that affect dystrophin splicing/transcription specifically in heart
• Cluster towards 5’ end of gene
• Mutations that preferentially/exclusively disrupt cardiac muscle
• e.g. nonsense mut in ex29, in-frame del ex 49-51, 48-49, 45-48, 45-55, 48, 48-51, 48-53
DMD Registry
• Database for all patients diagnosed with DMD/BMD and manifesting female
carriers
• Register online
• Required to consent to Action Duchenne contacting your clinician and
geneticist for medical and genetic information
• Keep your information up-to-date
https://www.dmdregistry.org/login
Any questions?