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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?