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Muscular dystroph
y Dr. Derakhshandeh
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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
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voluntary & in voluntary muscles
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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
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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
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Dystrophin
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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
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Waddling gait
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In the late stages of muscular dystrophy, fat and connective tissue often replace muscle fibers.
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DMD
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Orthopaedic management of patients with Duchenne's muscular dystrophy
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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.
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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).
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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.
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Multiplex PCR imagesIranian 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
,
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Iranian J Publ Health, Vol. 32, No. 3, pp.47-53, 2003
/+ +/- +/+ -/-y -/+ +/y -/- +/+ -/+ -/y +/+ +/y
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Iranian J Publ Health, Vol. 32, No. 3, pp.47-53, 2003
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MLPA
Multiplex Ligation-dependent Probe Amplification
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MLPA
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MLPA analysis of the human DMD-gene in a normal male
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Agarose-gel analysis of DMD deletion patient
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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
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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
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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.
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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)
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MAPH
Multiplex Amplifiable Probe Hybridisation
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MAPHDetection of
deletions/duplication mutations in Duchenne Muscular Dystrophy using: MAPH
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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
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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)
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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
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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
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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
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MAPH dystrophin probe sets A/B: The two probes sets encompassingall exons in normal individuals
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A relative comparison is made between the band intensities or peak heights
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Outline of the MAPH technique
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B: a control female
D: a male deleted for exons 49 and 51
A: a female deleted for exons 49 and 51
C: a female duplicated for the exons 49 and 51
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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
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Applications areas such as cancer risk (BRCA1 and
HNPCC)
learning disability (US: "mental retardation")
muscular dystrophy (DMD/BMD) neuromuscular disorders (SMA)
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-Thalassemia-Thalassemia
Disorders of Disorders of HemoglobinHemoglobin
Dr. Pupak Derakhshandeh
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Understanding globin regulation in β-thalassemia:
it’s as simple as α, β, γ, δ
Arthur Bank
The Journal of Clinical Investigation http://www.jci.org Volume 115 Number 6 June 2005
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The human globin loci The best characterized in the human genome at the gene and protein levels
The β–locus control region (β-LCR): A dominant control region located
upstream of the globin structural genes
a strong enhancer of the expression of the downstream
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. The human globin locus and their role in β-thalassemia
(A) The β-LCR and structural genes (ε, Gγ, Aγ, δ, and β) in the β-globin locus on chromosome 11
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The major genes expressed throughout fetal life
The α-globin gene
2 γ-globin genes, Gγ and Aγ
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)B (The α-globin locus is shown with the ζ- and 2 α-globin genes on chromosome 16
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-Globin gene expression
between cis-acting sequences: The β-LCR
trans-acting factors: including transcription factors
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C) In early fetal life, the α- and γ-globin chains combine to form HbF (α2γ2), the main β-globin–like globin during the remainder of
fetal life and early postnatal life
Severe anemia results >>
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In fetal life
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In Adult life
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The current therapy for β-thalassemia
Blood transfusions + iron Chelation
Decreasing α-globin accumulation
and/or reactivating γ-globin production
BM transplantation
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Decreasing excess α-globinaccumulation
Unequal crossing over in meiosis: deletion of the α-globin gene reduces α-globin synthesis in patients
Homozygous for β-thalassemia (Major) + decreases the α-globin excess
>> decreased severity of anemia
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Increasing human γ-globinexpression
reduce anemia and cure human β-thalassemia
increase in human γ-globin gene expression
>> restoration of HbF Point mutations in the γ-globin gene
promoter: increase γ-globin expression, but not
by agreat amount
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Hereditary persistence of fetal hemoglobin (HPFH)
express γ-globin genes at the same level in adult life as in fetal life
Some HPFH homozygotes have only HbF (2γ2) and no anemia!
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Doesn't cause any health Doesn't cause any health problemproblem
HPFH / HPFH / Thalassemia (no problem)Thalassemia (no problem)
HPFH / HPFHHPFH / HPFH
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HPFH as a δβ-globin Disease
Large deletions at the β-globin locus from the region close to the human Aγ gene
to well downstream of the human β-globin gene and including deletion of the structural
δ- and β-globin genes
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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 thalassemia: clinically very mild
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HPFH
group of disorders
characterized by a decreased or absent:-chain synthesis a variable compensatory increase in γ-chain synthesis
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Intergenic γδ sequences: γ-globin gene regulation
Corfu: homozygous for the Corfu deletion a deletion of 7.2 kb DNA upstream of the δ-globin homozygotes were shown to possess
88%-90% HbF only mild anemia Did not require blood transfusion
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Corfu deletion
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Molecular diagnosis of haemoglobin disorders
Clin. Lab. Haem. 2004, 26, 159–176
B. E. CLARK, S. L. THEINDepartment of Haematological Medicine, King’s College Hospital and GKT School of Medicine,
Denmark Hill, London, UK
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The beta locus on chromosome 11 p15.4 with the,Gγ and Aγ, and genes, arranged in the order of their developmental expression
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Gap-PCR
-thalassaemias:
the common HPFH Hb Lepore
Thalassemia, …
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Gap-PCR for the African HPFH-2 deletion
N D N N D N N D D D N D D N N
639 bp
918
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Homozygosity for nondeletion 0 thalassemia resulting in a silentclinical phenotype
BLOOD, 1 SEPTEMBER 2002 VOLUME 100, NUMBER 5
Renzo Galanello, Susanna Barella, Stefania Satta, Liliana Maccioni, Carlo Pintor, and Antonio Cao
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Nondeletion Sardinian 0 thalassemia
a homozygous state for nondeletion Sardinian 0 thalassemia
a symptomless clinical phenotype with
pattern (Hb F: 99.8% and Hb A2: 0.2%)
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The molecular defects the presence of 2 nucleotide
substitutions: -196C>T in the promoter of the Aγ-globin
gene 39C>T nonsense mutation in -globin gene
* *
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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 γ-globin genes and suppression of-globin genes or preferential survival of red blood cells with the
highest Hb F and low Hb A2 level
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The absence of typical thalassemia clinical findings
The imbalance in the ratio of to γ similar to that in heterozygous
thalassemia explains the reduction in MCV mean corpuscular Hb
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Patient with nondeletion homozygous 0 thalassemia
Had almost no HbA2 (0.3%) the suppressive effect of the in cis Aγ196CT
mutation This suppressive in cis effect has already been
reported for similar mutations, such as the -202 Gγ HPFH
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