Muscular dystrophy

1. Muscular dystrophy Dr. Derakhshandeh

2. Muscular dystrophy (MD) • a group of rare inherited muscle diseases • muscle fibers are unusually susceptible to damage • Muscles, primarily voluntary muscles, become progressively weaker • In some types of muscular dystrophy, heart muscles, other involuntary muscles and other organs are affected

3. voluntary & in voluntary muscles

4. Duchenne's muscular dystrophy (Xp21.2) • The types of muscular dystrophy: • a genetic deficiency of the protein dystrophin : • dystrophinopathies • Duchenne's muscular dystrophy : • the most severe form of dystrophinopathy. • It occurs mostly: • in young boys

5. Dystrophin • a large (427 kD) cytoskeletal protein • structure with an actin-binding domain at the amino terminus (N) • The carboxy-terminal domains associate with a large transmembrane complex of glycoproteins • directly bind with elements of the extracellular • Dystrophin: likely plays a critical role in establishing connections between the internal, actin-based cytoskeleton and the external basement membrane • Its absence may lead to increased membrane fragility

6. Dystrophin

7. Duchenne's muscular dystrophy • Difficulty getting up from a lying or sitting position • Weakness in lower leg muscles, resulting in difficulty running and jumping • Waddling gait • Mild mental retardation, in some cases

8. Waddling gait

9. In the late stages of muscular dystrophy, fat and connective tissue often replace muscle fibers.

11. DMD

12. Orthopaedic management of patients with Duchenne's muscular dystrophy

13. Duchenne's muscular dystrophy • X-linked inheritance Prevalence 0.003-0.05/1,000 total • Signs and symptoms of Duchenne's usually appear between the ages of 2 and 5 • It first affects the muscles of the pelvis, upper arms and upper legs. • By late childhood, most children with this form of muscular dystrophy are unable to walk.

14. Most die by their late teens or early 20s, often from pneumonia, respiratory muscle weakness or cardiac complications. • Some people with Duchenne's MD may exhibit curvature of their spine (scoliosis).

15. Becker's muscular dystrophy • This type of muscular dystrophy is a milder form of dystrophinopathy. • It generally affects older boys and young men, and progresses more slowly, usually over several decades. • Signs and symptoms of Becker's MD are similar to those of Duchenne's. • The onset of the signs and symptoms is generally later, from age 2 to 16.

16. Multiplex PCR imagesIranian J Publ Health, Vol. 32, No. 3, pp.47-53, 2003S Kheradmand kia , DD Farhud , S Zeinali , AR Mowjoodi, H Najmabadi ,F Pourfarzad, P Derakhshandeh-Peykar ,

17. -/- +/+ -/+ +/y -/+ +/y -/- +/+ -/+ -/y +/+ +/y Iranian J Publ Health, Vol. 32, No. 3, pp.47-53, 2003

18. Iranian J Publ Health, Vol. 32, No. 3, pp.47-53, 2003

20. MLPA Multiplex Ligation-dependent Probe Amplification

21. MLPA

22. MLPA analysis of the human DMD-gene in a normal male

23. Agarose-gel analysis of DMD deletion patient

24. The MLPA reaction & five major steps 1) DNA denaturation and hybridisation of MLPA probes 2) ligation reaction 3) PCR reaction 4) separation of amplification products by electrophoresis 5) data analysis

25. The MLPA reaction I • first step: the DNA is denatured and incubated overnight with a mixture of MLPA probes • MLPA probes consist of two separate oligonucleotides, each containing one of the PCR primer sequences • The two probe oligonucleotides hybridize to immediately adjacent target sequences • Only when the two probe oligonucleotides are both hybridised to their adjacent targets can they be ligated during the ligation reaction • only ligated probes will be exponentially amplified during the subsequent PCR reaction

26. The MLPA reaction II • the number of probe ligation products is a measure for the number of target sequences in the sample • The amplification products are separated using capillary electrophoresis • Probe oligonucleotides that are not ligated only contain one primer sequence. As a consequence, they cannot be amplified exponentially and will not generate a signal. • The removal of unbound probes is therefore unnecessary in MLPA and makes the MLPA method easy to perform.

27. Advantages of MLPA • methods which were primarily developed for detecting point mutations, such as sequencing and DHPLC (denaturing high-performance liquid chromatography), generally fail to detect copy numbers changes • Southern blot analysis, will not always detect small deletions and is not ideal as a routine technique • comparing MLPA to FISH, MLPA not only has the advantage of being a multiplex technique, but also one in which very small (50-70 nt) sequences are targeted • Moreover, MLPA can be used on purified DNA • The over 300 probe sets now available are dedicated to applications ranging from the relatively common (Duchenne, DiGeorge syndrome, SMA)

28. MAPH Multiplex Amplifiable Probe Hybridisation

29. MAPH • Detection of deletions/duplication mutations in Duchenne Muscular Dystrophy using: MAPH

30. MAPH • Although ~95% of deletions can be detected in males using multiplex PCR • other methods must be used to determine duplications, as well as the carrier status of females • The most commonly applied methods are quantitative multiplex PCR and quantitative Southern blotting

31. MAPH • Using high-quality Southern blots it is possible to perform a quantitative analysis and detect duplications • this technique is time consuming • it is difficult to exactly determine the duplication • it can be difficult to detect duplications in females and triplications will be missed Armour et al (Nucl.Acids Res. 2000)

32. system for analyzing all 79 exons of the DMD gene for deletions and duplications • MAPH is based on a quantitative PCR of short DNA probes recovered after hybridization to immobilized genomic DNA

34. 1 ug of denatured genomic DNA is spotted on a small nylon filter • hybridized overnight in a solution containing one of the probe mixes • Following stringent washing the next day the filter is placed in a PCR tube • and a short PCR reaction is performed • This releases the specifically-bound probes into the solution • An aliquot of this is transferred to a second, quantitative PCR reaction

35. alterations can be examined by using a set of short probes (140-600 bp) • After washing and PCR the differently sized products resolved and quantified measured • The amount of probe amplified depends on the number of hybridising targets and therefore on the copy number of the corresponding locus in the test DNA

37. MAPH dystrophin probe sets A/B: The two probes sets encompassingall exons in normal individuals

38. A relative comparison is made between the band intensities or peak heights

40. Outline of the MAPH technique

41. A: a female deleted for exons 49 and 51 B: a control female C: a female duplicated for the exons 49 and 51 D: a male deleted for exons 49 and 51

42. Analysis of exon products on a micro-arrayPCR-fragments containing DMD exons are spotted in triplicate on each array top left exons 1-24 top right exons 25-48 bottom left exons 49-72 bottom right exons 73-79

43. Applications • areas such as cancer risk (BRCA1 and HNPCC) • learning disability (US: "mental retardation") • muscular dystrophy (DMD/BMD) neuromuscular disorders (SMA)