1 / 78

Muscular dystrophy

2. Muscular dystrophy (MD) . a group of rare inherited muscle diseases muscle fibers are unusually susceptible to damageMuscles, primarily voluntary muscles, become progressively weakerIn some types of muscular dystrophy, heart muscles, other involuntary muscles and other organs are affected. 3

sylvain
Download Presentation

Muscular dystrophy

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

    1. 1 Muscular dystrophy Dr. Derakhshandeh

    2. 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. 3 voluntary & in voluntary muscles

    4. 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. 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. 6 Dystrophin

    7. 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. 8 Waddling gait

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

    10. 10

    11. 11 DMD

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

    13. 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. 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. 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. 16 Multiplex PCR images Iranian J Publ Health, Vol. 32, No. 3, pp.47-53, 2003 S Kheradmand kia , DD Farhud , S Zeinali , AR Mowjoodi, H Najmabadi , F Pourfarzad, P Derakhshandeh-Peykar

    17. 17

    18. 18

    19. 19

    20. 20 MLPA Multiplex Ligation-dependent Probe Amplification

    21. 21 MLPA

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

    23. 23 Agarose-gel analysis of DMD deletion patient

    24. 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. 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. 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. 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. 28 MAPH Multiplex Amplifiable Probe Hybridisation

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

    30. 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. 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. 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

    33. 33

    34. 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. 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

    36. 36

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

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

    39. 39

    40. 40 Outline of the MAPH technique

    41. 41

    42. 42 Analysis of exon products on a micro-array PCR-fragments containing DMD exons are spotted in triplicate on each array

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

    44. 44 db-Thalassemia

    45. 45 Understanding globin regulation in ß-thalassemia: it’s as simple as a, ß, ?, d Arthur Bank The Journal of Clinical Investigation http://www.jci.org Volume 115 Number 6 June 2005

    46. 46 The human globin loci The best characterized in the human genome at the gene and protein levels The ß–locus control region (ß-LCR): A dominant control region located upstream of the globin structural genes a strong enhancer of the expression of the downstream

    47. 47 . The human globin locus and their role in ß-thalassemia (A) The ß-LCR and structural genes (e, G?, A?, d, and ß) in the ß-globin locus on chromosome 11

    48. 48 The major genes expressed throughout fetal life The a-globin gene 2 ?-globin genes, G? and A?

    49. 49

    50. 50 (B) The a-globin locus is shown with the ?- and 2 a-globin genes on chromosome 16

    51. 51 b-Globin gene expression between cis-acting sequences: The ß-LCR trans-acting factors: including transcription factors

    52. 52 C) In early fetal life, the a- and ?-globin chains combine to form HbF (a2?2), the main ß-globin–like globin during the remainder of fetal life and early postnatal life

    53. 53 In fetal life

    54. 54 In Adult life

    55. 55

    56. 56 The current therapy for ß-thalassemia Blood transfusions + iron Chelation Decreasing a-globin accumulation and/or reactivating ?-globin production BM transplantation

    57. 57 Decreasing excess a-globin accumulation Unequal crossing over in meiosis: deletion of the a-globin gene reduces a-globin synthesis in patients Homozygous for ß-thalassemia (Major) + decreases the a-globin excess >> decreased severity of anemia

    58. 58 Increasing human ?-globin expression reduce anemia and cure human ß-thalassemia increase in human ?-globin gene expression >> restoration of HbF Point mutations in the ?-globin gene promoter: increase ?-globin expression, but not by agreat amount

    59. 59 Hereditary persistence of fetal hemoglobin (HPFH) express ?-globin genes at the same level in adult life as in fetal life Some HPFH homozygotes have only HbF (a2g2) and no anemia!

    60. 60 Doesn't cause any health problem HPFH / ?Thalassemia (no problem) HPFH / HPFH

    61. 61 HPFH as a dß-globin Disease Large deletions at the ß-globin locus from the region close to the human A? gene to well downstream of the human ß-globin gene and including deletion of the structural d- and ß-globin genes

    62. 62 HPFH Heterozygotes: a normal level of HbA2 even higher levels of HbF (15 to 30 %) Homozygotes: clinically normal albeit with reduced MCV and MCH Compound heterozygotes with b thalassemia: clinically very mild

    63. 63

    64. 64 HPFH group of disorders characterized by a decreased or absent: b-chain synthesis a variable compensatory increase in g-chain synthesis

    65. 65 Intergenic ?d sequences: ?-globin gene regulation Corfu: homozygous for the Corfu deletion a deletion of 7.2 kb DNA upstream of the d-globin homozygotes were shown to possess 88%-90% HbF only mild anemia Did not require blood transfusion

    66. 66 Corfu deletion

    67. 67 Molecular diagnosis of haemoglobin disorders Clin. Lab. Haem. 2004, 26, 159–176 B. E. CLARK, S. L. THEIN Department of Haematological Medicine, King’s College Hospital and GKT School of Medicine, Denmark Hill, London, UK

    68. 68 The beta locus on chromosome 11 p15.4 with the e,Gg and Ag, d and b genes, arranged in the order of their developmental expression

    69. 69 Gap-PCR db-thalassaemias: the common HPFH Hb Lepore -a Thalassemia, …

    70. 70

    71. 71

    72. 72 Homozygosity for nondeletion db0 thalassemia resulting in a silent clinical phenotype BLOOD, 1 SEPTEMBER 2002 VOLUME 100, NUMBER 5 Renzo Galanello, Susanna Barella, Stefania Satta, Liliana Maccioni, Carlo Pintor, and Antonio Cao

    73. 73 Nondeletion Sardinian db0 thalassemia a homozygous state for nondeletion Sardinian db0 thalassemia a symptomless clinical phenotype with pattern (Hb F: 99.8% and Hb A2: 0.2%)

    74. 74 The molecular defects the presence of 2 nucleotide substitutions: -196C>T in the promoter of the Ag-globin gene 39C>T nonsense mutation in b-globin gene

    75. 75

    76. 76 The absence of typical thalassemia clinical findings high Hb F output: which compensated for the absence of chains The near absence of Hb A2: alterations in the globin gene transcriptional : Activation of g-globin genes and suppression of d-globin genes or preferential survival of red blood cells with the highest Hb F and low Hb A2 level

    77. 77 The absence of typical thalassemia clinical findings The imbalance in the ratio of a to g similar to that in heterozygous thalassemia explains the reduction in MCV mean corpuscular Hb

    78. 78 Patient with nondeletion homozygous db0 thalassemia Had almost no HbA2 (0.3%) the suppressive effect of the in cis Ag -196CT mutation This suppressive in cis effect has already been reported for similar mutations, such as the -202 Gg HPFH

More Related