1. © 2009 NHS National Genetics Education and Development CentreGenetics and Genomics for Healthcare www.geneticseducation.nhs.uk Duchenne Muscular Dystrophy A clinical and molecular overview This PowerPoint file contains a number of slides that may be useful for teaching of genetics concepts. You may use these slides and their contents for non-commercial educational purposes.

2. © 2009 NHS National Genetics Education and Development CentreGenetics and Genomics for Healthcare www.geneticseducation.nhs.uk Duchenne Muscular Dystrophy This presentation includes: Clinical features and Inheritance Pedigrees showing X-linked inheritance pattern Clinical photographs showing Gowers manoeuvre Muscle histology slides showing staining in normal and affected tissue samples Deletion of dystrophin gene, and PCR images Image of the dystrophin molecule Case scenario.

3. © 2009 NHS National Genetics Education and Development CentreGenetics and Genomics for Healthcare www.geneticseducation.nhs.uk Duchenne Muscular Dystrophy (DMD) Clinical Features: Progressive muscle weakness. Mainly in a wheelchair by early teens. Respiratory muscles eventually involved. Death usually in late teens, early twenties. Inheritance: X linked recessive condition, hence males affected and females are carriers (see pedigree on next slide).

4. © 2009 NHS National Genetics Education and Development CentreGenetics and Genomics for Healthcare www.geneticseducation.nhs.uk The X chromosome carrying the disease-causing mutation can be tracked through the family. Note: Shaded squares = affected males: dots in circles = carrier females. DMD Pedigree

5. © 2009 NHS National Genetics Education and Development CentreGenetics and Genomics for Healthcare www.geneticseducation.nhs.uk Fig. 1.10 ©Scion Publishing Ltd Pedigree of Martin Daviess family Assuming this is X-linked muscular dystrophy, the women marked with dots are obligate carriers of the disease gene – that is, they must be carriers because they have offspring who are affected or carriers.

6. © 2009 NHS National Genetics Education and Development CentreGenetics and Genomics for Healthcare www.geneticseducation.nhs.uk Fig. 1.4 ©Scion Publishing Ltd Histology photos courtesy of Dr Richard Charlton. Duchenne muscular dystrophy (a) Affected boys stand up by bracing their arms against their legs (Gowers manoeuvre) because their proximal muscles are weak. (b) and (c) Muscle histology (Gomori trichrome stain). Normal muscle (b) shows a regular architecture of cells with dystrophin (brown stain) on all the outer membranes. (c) Shows muscle from a 10-year-old affected boy. Note the disorganisation, invasion by fibrous tissue and complete absence of dystrophin. Histology photos courtesy of Dr Richard Charlton, Newcastle upon Tyne.

7. © 2009 NHS National Genetics Education and Development CentreGenetics and Genomics for Healthcare www.geneticseducation.nhs.uk Fig. 7.11 ©Scion Publishing Ltd Photo. courtesy of Dr Richard Charlton A muscle biopsy from a female carrier of Duchenne muscular dystrophy stained with an antibody against dystrophin Note the patchy distribution of staining around the outer membranes of cells (compare with the sections from an affected boy and a normal control in next slide).

8. © 2009 NHS National Genetics Education and Development CentreGenetics and Genomics for Healthcare www.geneticseducation.nhs.uk Immunolabeling of Muscle Biopsy Sections Normal Control Dystrophin antibody staining of muscle cells 4 year old boy with DMD – No detectable dystrophin

9. © 2009 NHS National Genetics Education and Development CentreGenetics and Genomics for Healthcare www.geneticseducation.nhs.uk Fig. 3.9 ©Scion Publishing Ltd A deletion of part of the dystrophin gene This figure shows a 500 kb region containing exons 41-50. These exons are all 100-200 bp long, and so if drawn to scale each exon would be represented by a line occupying less than 0.05% of the width of the figure. Random deletion breakpoints therefore almost always fall in introns. Their effect is to remove one or more complete exons from the mature mRNA. The deletion shown removes exons 45-47 from the mature mRNA, while leaving all the other exons intact.

10. © 2009 NHS National Genetics Education and Development CentreGenetics and Genomics for Healthcare www.geneticseducation.nhs.uk Fig. 6.4 ©Scion Publishing Ltd The dystrophin molecule anchors the cytoskeleton of muscle cells to the extracellular matrix, via the dystrophin glycoprotein complex. This includes the sarcoglycans (mutations in which cause limb- girdle muscular dystrophies) and dystroglycans. Muscle cells that lack dystrophin are mechanically fragile, and fail after a few years, hence progressive muscle weakness.

11. © 2009 NHS National Genetics Education and Development CentreGenetics and Genomics for Healthcare www.geneticseducation.nhs.uk Fig. 4.17 ©Scion Publishing Ltd PCR deletion screen in Duchenne muscular dystrophy Nine selected exons of the dystrophin gene have been amplified from the DNA of a panel of 20 affected boys. When the product is run on an electrophoretic gel each exon gives a band of a characteristic size. Because a boy has only a single X chromosome, any deletion shows up as missing bands. Different exon deletions can be seen in lanes 1, 5, 11, 12, 19 and 20. Lane 3 may be a large deletion or a technical failure. The boys with no deletion on this gel may have others of the 79 dystrophin exons deleted, or may have point mutations or duplications to cause loss of function of the gene.

12. © 2009 NHS National Genetics Education and Development CentreGenetics and Genomics for Healthcare www.geneticseducation.nhs.uk 18-exon Multiplex PCR Assay DMD Reaction 1 Reaction 2 Reaction 3 NC 1 2 3 NC 1 2 3 NC 1 2 3 Patient 2 deleted for exons 47, 48 and 50

13. © 2009 NHS National Genetics Education and Development CentreGenetics and Genomics for Healthcare www.geneticseducation.nhs.uk Jasons family tree Jason 15.11.99 Rachel 14.3.74 Tony 26.9.73 Susie 3.6.95 Fay 7.1.98 Jason, aged 3 yrs referred to a paediatrician by his GP Later walking than older siblings Difficulty running and riding tricycle Always climbs up stairs on all fours.