Transcript The types of muscular dystrophy

Muscular
dystrophy
Dr. Derakhshandeh
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Muscular dystrophy
(MD)
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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)
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The types of muscular dystrophy:
 a genetic deficiency of the protein
dystrophin :
 dystrophinopathies
Duchenne's muscular dystrophy :
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the most severe form of dystrophinopathy.
It occurs mostly:
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in young boys
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Dystrophin
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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
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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 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
,
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-/- +/+
-/+ +/y -/+ +/y
-/- +/+
-/+ -/y +/+ +/y
Iranian J Publ Health, Vol. 32, No. 3, pp.47-53, 2003
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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 DMDgene 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
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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
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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
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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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MAPH
 Detection
of
deletions/duplication mutations
in Duchenne Muscular
Dystrophy using: MAPH
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MAPH
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Although ~95% of deletions can be detected in
males using multiplex PCR
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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
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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
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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)
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After washing and PCR the differently sized
products resolved and quantified measured
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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 encompassing
all 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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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
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Analysis of exon products on a micro-array
PCR-fragments containing DMD exons are spotted in triplicate on each array
top left exons 1-24
bottom left exons 49-72
top right exons 25-48
bottom right exons 73-79
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Applications
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areas such as cancer risk (BRCA1 and
HNPCC)
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learning disability (US: "mental retardation")
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muscular dystrophy (DMD/BMD)
neuromuscular disorders (SMA)
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db-Thalassemia
Disorders of Hemoglobin
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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Thehuman
best characterized
The
globin loci 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
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α-globin gene
γ-globin genes, Gand A
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(B) The α-globin locus is shown with the ζ- and 2 αglobin genes on chromosome 16
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b-Globin gene expression
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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
 and/or
 BM
α-globin accumulation
reactivating γ-globin production
transplantation
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Decreasing excess α-globin
accumulation
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Unequal crossing over in meiosis:
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deletion of the α-globin gene
reduces α-globin synthesis in patients
Homozygous for β-thalassemia (Major) +
decreases the α-globin excess
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 decreased
severity of anemia
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Increasing human γ-globin
expression
 reduce
anemia and cure human βthalassemia
 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)
-globin genes at the same
level in adult life as in fetal life
 express
 Some
HPFH homozygotes have only
HbF (a2g2) and no anemia!
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Doesn't cause any health
problem
 HPFH
/ bThalassemia (no problem)
 HPFH
/ HPFH
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HPFH
as a δβ-globin Disease
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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
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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
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HPFH
 group
of disorders
 characterized
by a decreased or absent:
 b-chain synthesis
 a variable compensatory increase in
g-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. THEIN
Department 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 e,Gg and Ag, d and
b genes, arranged in the order of their developmental expression
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Gap-PCR
db-thalassaemias:
 the
common HPFH
 Hb Lepore
-a 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
918
639 bp
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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
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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%)
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The molecular defects
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the presence of 2 nucleotide
substitutions:
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-196C>T in the promoter of the Ag-globin gene
39C>T nonsense mutation in b-globin gene
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*
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The absence of typical thalassemia
clinical findings
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high Hb F output: which compensated for the
absence of chains
The near absence of Hb A2:
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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
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The absence of typical thalassemia
clinical findings
imbalance in the ratio of a to g
 similar to that in heterozygous
thalassemia
 explains the reduction in MCV
 mean corpuscular Hb
 The
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Patient with nondeletion
homozygous db0 thalassemia
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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
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