Transcript MUSCULAR DYSTROPHY, DUCHENNE TYPE

Duchenne
muscular dystrophy
(DMD)
Richard C. Arceo, M. D.
DESCRIPTION
Duchenne muscular dystrophy
(DMD) is a severe recessive X-linked
form of muscular dystrophy
characterized by rapid progression of
muscle degeneration, eventually
leading to loss of ambulation and
death.
Incidence/Prevalence
• Although reliable prevalence data
are lacking, the prevalence of DMD
is generally estimated at 1:3,500 live
male births (Emery 1991).
• The birth prevalence of DMD in
northern England is one in 5,618 live
male births.
Pathogenesis
• DMD is caused by mutations in the dystrophin
gene which is the largest human gene, spanning
2,200 kb on the X chromosome and occupying
roughly 0.1% of the genome.
• The gene is composed of 79 exons and 8 tissuespecific promoters [Koenig et al., 1987].
• The primary transcript measures about 2,400
kilobases and takes 16 hours to transcribe, the
mature mRNA measures 14.0 kilobases.
• The 79 exons code for a protein of over 3500 amino
acid residues.
Where is the DMD gene located?
Cytogenetic Location: Xp21.2
• Dystrophin is a rod-shaped cytoplasmic
protein, and a vital part of a protein
complex that connects the cytoskeleton
of a muscle fiber to the surrounding
extracellular matrix through the cell
membrane.
• Dystrophin provides structural stability
to the Dystroglycan complex (DGC),
located on the cell membrane
Abnormal gene product
• Mutations will lead to lack of
dystrophin expression causing DMD,
whereas those that lead to abnormal
quality or quantity of dystrophin lead
to BMD.
Much investigative work determined that
dystrophin is involve in the release of
calcium from the sarcoplasmic
reticulum in muscle fibers.
The lack of dystrophin causes calcium to
leak into the cell, which promotes the
action of an enzyme that dissolves muscle
fibers.
When the body attempts to repair the tissue,
fibrous tissue forms, and this cuts off the
blood supply so that more and more cells die.
CLINICAL FEATURES
SKELETAL MUSCLE
• The most distinctive feature of
Duchenne muscular dystrophy is a
progressive proximal muscular
dystrophy with characteristic
pseudohypertrophy of the calves
The first symptoms of DMD appear during
preschool years.
The disorder affects the legs first.
A boy has trouble walking and
maintaining balance.
In most cases, he begins walking
three to six months later than average.
As his muscles begin to weaken, he may
change the way he walks.
He places his legs farther apart in order to
maintain balance.
Walking this way produces a waddling
effect that is characteristic of DMD.
Contractures usually begin at
about the age of five or six.
This forces a boy to walk on his tiptoes.
Balance becomes more of a problem.
As a result, falls and broken bones
become common at this age.
By the age of nine or ten, a boy with DMD
might not be able to climb stairs
Or stand by himself.
By age 10, braces may be required to
aid in walking but most patients are
wheelchair dependent by age 12.
Early common sign of
muscular dystrophy
To get up from the ground, the child
‘walks up' his thighs with his hands.
This is mainly because of weak thigh muscles.
The Baskaran family
South East of England
Jamie
Oliver
• May develop a severe curve
of the spine.
• Heart and breathing muscles
also get weak.
• Child usually dies before age 20 from
heart failure or pneumonia.
NERVOUS SYSTEM
• Mental retardation of mild degree is a
pleiotropic effect of the Duchenne gene
(Zellweger and Niedermeyer, 1965)
• As indicated later, the finding of dystrophin
mRNA in brain may bear a relationship to
the mental retardation in DMD patients.
• In 50 DMD patients with a mean age of 11.1
years (range 3.5 to 20.3), Bresolin et al. (1994)
found that 31% had a Wechsler full
intelligence quotient (FIQ) lower than 75 and
that only 24% had appropriate IQ levels by
this index
Bushby et al. (1995) studied 74 boys with
DMD, 18% of which had a full scale IQ of
below 70.
The authors found no significant IQ
difference between the patients with
promoter deletions and those without, nor
did they find a relationship between the
length of the deletion and full scale IQ.
They found, however, that boys with distal
deletions were more likely to be mentally
retarded than were those with proximal
deletions
CARDIAC MUSCLE
• Myocardial involvement appeared in a high percentage of
DMD patients by about 6 years of age; it was present in
95% of cases by the last years of life. (Nigro et al., 1983).
• Mirabella et al. (1993) noted that electrocardiographic
abnormalities had been reported in 6.6 to 16.4% of DMD
heterozygous females and that in one carrier female
severe cardiomyopathy had been described in
association with muscle weakness.
• They reported 2 carriers with dilated cardiomyopathy
and increased serum CK but no symptoms of muscle
weakness. Heart biopsies in both patients showed absence
of dystrophin in many muscle fibers
SMOOTH MUSCLE
• Noting that in DMD functional
impairment of smooth muscle in the
gastrointestinal tract can cause acute gastric
dilatation and intestinal
pseudoobstruction that may be fatal,
Barohn et al. (1988) studied gastric emptying
in 11 patients with DMD.
• Strikingly delayed gastric emptying times
were observed.
Boland et al. (1996) studied a retrospective cohort
of 33 male patients born between 1953 and 1983.
The mean age at DMD diagnosis was 4.6 years;
wheelchair dependency had a median age of 10
years;
cardiac muscle failure developed in 15% of patients
with a median age of 21.5 years;
smooth muscle dysfunction in the digestive or
urinary tract occurred in 21% and 6% of the patients,
respectively, at a median age of 15 years.
In this cohort, death occurred at a median age of 17
years.
Diagnosis
1. Serum creatine phosphokinase (CK)
concentration
2. Electromyography (EMG)
is useful in distinguishing a myopatic
process from a neurogenic disorder.
This is done by demonstrating shortduration, low-amplitude, polyphasic,
rapidly recruited motor unit potentials.
3. Skeletal muscle biopsy
• Muscle histology early in the disease shows
nonspecific dystrophic changes,
including variation in fiber size, foci of
necrosis and regeneration, hyalinization,
and, later in the disease, deposition of fat
and connective tissue.
Findings in the Dystrophin Protein from
Skeletal Muscle Biopsy
Molecular Genetic Testing
• Gene:
DMD is the only gene known to be associated with DMD
• Clinical testing: Deletion/duplication Analysis
1. Multiplex PCR [Multicenter Study Group 1992],
2. Southern blotting [Darras et al 1988], and
3. FISH (with probes covering DMD exons 3-6, 8, 12, 13, 17, 19, 3234, 43-48, 50, 51, and 60)
can be used to detect deletions, which account for approximately
65% of mutations in individuals with DMD.
Approximately 98% of deletions are detectable by these
methodologies.
Southern blotting and quantitative PCR
analysis can be used to detect duplications.
Duplications may lead to in-frame or out-offrame transcripts and account for the
disease-causing mutations in approximately
6%-10% of males with DMD or BMD.
In one study [Galvagni et al 1994], duplications were
detected in 8.18% of individuals with DMD.
In a series of individuals already screened for
deletions and point mutations, duplications
were detected in 87% of cases [White et al 2006].
• New testing methods including single-condition
amplification internal primer sequencing
(SCAIP) [Flanigan et al 2003] and denaturing
gradient gel electrophoresis (DGGE)-based wholegene mutation scanning [Hofstra et al 2004] aim
at detecting the remaining 30%-35% of the DMD
mutations in a semiautomatic, rapid, accurate,
and economical fashion.
• A muscle biopsy-based diagnostic approach
was developed and optimized to increase the
mutation detection frequency to nearly 100%
[Deburgrave et al 2007].
• To date, 501 deletions, 8 duplications,
and 989 point mutations have been
documented in the dystrophin gene
(Leiden muscular dystrophy database;
www.dmd.nl).
• 5 exons commonly deleted in deletion-
type Duchenne muscular dystrophy
(DMD).
• The five DMD gene exons (17, 19, 44, 45
and 48) can be analysed in separate
duplex PCR reactions
Molecular Genetic Testing
• The current methodologies used for detecting
mutations in the dystrophin gene include
multiplex PCR, Southern blotting [Stockley et al.,
2006], multiplex ligation-dependent probe
amplification (MLPA) [Gatta et al., 2005; Janssen et
al., 2005; Schwartz and Duno, 2004], detection of
virtually all mutations-SSCP (DOVAM- S) [Buzin et
al., 2000, 2005; Liu et al., 1999], denaturing highperformance liquid chromatography (DHPLC)
[Bennett et al., 2001], single condition
amplification/internal primer sequencing (SCAIP)
[Flanigan et al., 2003], and Sanger sequencing
[Hamed and Hoffman, 2006; Stockley et al., 2006].
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HUMAN MUTATION 0,1^9,2008
Signs and Symptoms in Carriers of Duchenne
and Becker Muscular Dystrophy
DMD Carriers
• None
76%
• Muscle weakness
19%
• Myalgia/cramps
5%
• Left ventricle dilation 19%
•
Dilated cardiomyopathy
8%
From Hoogerwaard et al [1999b)
BMD Carriers
81%
14%
5%
16%
0
Carrier Testing
• A reliable and simple method based on
quantitative real-time PCR detects
deletions/duplications in 100% of
DMD/BMD carriers [Joncourt et al 2004].
• Carrier testing for deletions may also be
performed by FISH [Voskova-Goldman et
al 1997].
• Carrier testing for point mutations may be
performed by sequence analysis.
Genotype-Phenotype Correlations
• In males with DMD, phenotypes are best correlated with the
degree of expression of dystrophin, which is largely
determined by the reading frame of the spliced message obtained
from the deleted allele [Monaco et al 1988, Koenig et al 1989].
• Very large deletions may lead to absence of dystrophin
expression.
• Mutations that disrupt the reading frame include stop
mutations, some splicing mutations, and deletions or
duplications.
• They produce a severely truncated dystrophin
protein molecule that is degraded, leading to the
more severe DMD phenotype.
• Data suggest that dystrophin deletions
involving the brain distal isoform
Dp140 are associated with intellectual
impairment [Felisari et al 2000
Testing Strategy
Establishing the diagnosis of DMD:
• For individuals with clinical findings suggesting a
dystrophinopathy and an elevated serum CK
concentration, the first step in diagnosis is molecular
genetic testing of the DMD gene:
• If a disease-causing mutation is identified, the
diagnosis is established;
• If no DMD disease-causing mutation is identified,
skeletal muscle biopsy of individuals with
suspected DMD is warranted for western blot and
immunohistochemistry studies of dystrophin.
Management
Evaluations Following Initial Diagnosis
To establish the extent of disease in an individual diagnosed
with a dystrophinopathy, the following evaluations
are recommended:
• Physical therapy assessment
• Developmental evaluation before entering elementary
school for the purpose of designing an individualized
educational plan, as necessary
• If the individual is older than age ten years at diagnosis,
evaluation for cardiomyopathy by electrocardiography,
chest radiography, cardiac echocardiography, pulmonary
function studies, and/or MRI [Towbin 2003]
• Medications
• Prednisone. Studies have shown that
prednisone improves the strength and
function of individuals with DMD.
It is hypothesized that prednisone has a
stabilizing effect on membranes and
perhaps an anti-inflammatory effect.
Whether the improvement is the result of an
immunosuppressive effect remains unclear,
as individuals treated with azathioprine did
not have a beneficial effect.
• In a randomized double-blind six-month trial, prednisone
administered at a dose of either 0.75 mg/kg/day or 1.5
mg/kg/day increased strength and reduced the rate
of decline in males with DMD [Mendell et al 1989].
• The improvement begins within ten days of starting the
treatment, requires a single dose of 0.75 mg/kg/day of
prednisone for maximal improvement, reaches a plateau
after three months, and can be sustained for as long as
three years in those children maintained on doses of 0.5
and 0.6 mg/kg/day [Fenichel et al 1991].
• One open-label study suggested that therapy with
prednisone could prolong ambulation by two years.
• Side effects include weight gain (>20% of baseline) (40%),
hypertension, behavioral changes, growth retardation,
cushingoid appearance (50%), and cataracts [Mendell et al
1989, Griggs et al 1993].
• Pulmonary:
• Baseline pulmonary function testing
before confinement to a wheelchair
(usually age ~9-10 years)
• Evaluation by a pediatric pulmonologist
twice yearly after any one of the
following: confinement to a wheelchair,
reduction in vital capacity below 80%
predicted, and/or age 12 years [Finder et
al 2004]
• Deflazacort:
• Deflazacort, a synthetic derivative of prednisolone used in Europe
but not currently available in the US, is thought to have fewer side
effects than prednisone, particularly with regard to weight gain
[Angelini 2007].
• A larger study comparing deflazacort to prednisone, carried out in
Europe, showed that the two medications were similarly or equally
effective in slowing the decline of muscle strength in DMD.
• Another European multicenter, double-blind, randomized trial of
deflazacort versus prednisone in DMD showed equal efficacy in
improving motor function and functional performance [Bonifati et
al 2000].
• A more recent study of deflazacort treatment showed efficacy in
preserving pulmonary function as well as gross motor function
[Biggar et al 2006].
• In a comparison of two different protocols of
deflazacort treatment in DMD, a 0.9-mg/kg/day
dose was more effective than a dose of 0.6
mg/kg/day for the first 20 days of the month and
no deflazacort for the remainder of the month
[Biggar et al 2004]; 30% of children on the
highest dose developed asymptomatic cataracts
that required no treatment.
• A systematic review and meta-analysis of 15
studies showed that deflazacort improves
strength and motor function more than
placebo; whether it has a benefit over prednisone
on similar outcomes remains unclear [Campbell &
Jacob 2003].
Therapies Under Investigation
• Aminoglycosides. Up to 15% of individuals with DMD
exhibit the gene mutation known as a premature stop codon.
• Suppression of stop codons has been demonstrated with
aminoglycoside treatment of cultured cells; the treatment creates
misreading of RNA and thereby allows alternative amino
acids to be inserted at the site of the mutated stop codon.
• In the mdx mouse, in vivo gentamicin therapy resulted in
dystrophin expression at 10%-20% of that detected in normal
muscle [Barton-Davis et al 1999], a level that provided some
degree of functional protection against contraction-induced
damage.
• Aminoglycoside therapy has been suggested as an
alternative to gene therapy but could be aimed only at
individuals with premature stop codons.
In a preliminary study in which gentamicin (7.5
mg/kg/day) was administered to four individuals for
two weeks, full-length dystrophin did not appear in the
muscles of the treated individuals [Wagner et al 2001].
Some authors, unable to reproduce the results previously
published for the mouse model of DMD, have called for
more preclinical investigation of this potential therapy
[Dunant et al 2003].
In an in vitro study [Kimura et al 2005], dystrophin
expression was detected in myotubes of males with
DMD using gentamicin; however, the treatment was
more effective in persons with the nonsense mutation
TGA than TAA or TAG.
• PTC124 is a new, orally administered
non-antibiotic drug that appears to promote
ribosomal read-through of nonsense (stop)
mutations.
Preclinical efficacy studies in mdx mice have yielded
encouraging results [Barton et al 2005, Welch et
al 2007]. A Phase I multiple-dose safety trial is
ongoing [Hirawat et al 2005].
• Morpholino antisense oligonucleotides
mediate exon skipping [Aartsma-Rus et al 2006a]
and have improved the mdx mouse model of DMD
[Wilton & Fletcher 2005, Alter et al 2006].
• Oxandrolone, an anabolic (androgenic) steroid with a
powerful anabolic effect on skeletal muscle myosin
synthesis [Balagopal et al 2006], was shown in a pilot study
to have effects similar to prednisone, with fewer side effects
[Fenichel et al 1997].
• A randomized, prospective, controlled trial showed that
oxandrolone did not produce a significant change in the
average manual muscle strength score of males with DMD, as
compared with placebo; however, the mean change in
quantitative muscle strength was significant [Fenichel et al
2001].
• The investigators conducting this study felt that oxandrolone
may be useful before initiating therapy with corticosteroid
because it is safe in the short term, accelerates linear growth,
and may be beneficial in slowing the progression of weakness.
• However, the long-term effects of oxandrolone in the treatment
of DMD have not been studied.
• Gene Therapy: Experimental gene therapies
are currently under investigation [Gregorevic
& Chamberlain 2003, Tidball & Spencer 2003, van
Deutekom & van Ommen 2003, Nowak & Davies
2004].
A mouse model for DMD exists and is
proving useful for furthering our
understanding on both the normal function
of dystrophin and the pathology of the
disease.
In particular, initial experiments that
increase the production of utrophin, a
dystrophin relative, in order to
compensate for the loss of dystrophin in the
mouse are promising and may lead to the
development of effective therapies for this
devastating disease.
• Stem cell therapy: is under
investigation but remains experimental
[Gussoni et al 1997, Gussoni et al 1999,
Gussoni et al 2002, Skuk et al 2004].
• PM R. 2009 Jun;1(6):547-59.
•
Mesenchymal stem cells: emerging
therapy for duchenne muscular dystrophy.
Markert CD, Atala A, Cann JK, Christ G,
Furth M, Ambrosio F, Childers MK.
Department of Neurology, School of Medicine,
and Wake Forest Institute for Regenerative
Medicine, Wake Forest University Health
Sciences, Winston-Salem, NC(dagger).
• Other:
• Immunosuppression with azathioprine is not beneficial.
• Myoblast transfer has been inefficient.
• Creatine monohydrate has been studied as potential
treatment in muscular dystrophies and neuromuscular
disorders [Tarnopolsky & Martin 1999, Walter et al 2000,
Louis et al 2003].
In a recent randomized, controlled, cross-over trial, 30 boys
with DMD were given creatine (~0.1 g/kg/day) for four
months and placebo for four months [Tarnopolsky et al
2004].
Treatment with creatine resulted in improved grip
strength of the dominant hand and increased fat-free
mass when compared to placebo; however, no functional
improvement was noted. Given the limited data and
modest benefit, treatment with creatine monohydrate
cannot be recommended for treatment of DMD.
• Cyclosporin was reported to improve
clinical function in children with DMD who
received the medication for eight weeks.
Nevertheless, because of the rare reports of
cyclosporin-induced myopathy in
individuals receiving the medication for
other reasons, the use of cyclosporin in
treating DMD remains controversial.
• Histone deacetylase inhibitors have
improved the mdx mouse by inducing the
expression of the myostatin inhibitor
follistatin [Minetti et al 2006].
Genetic Counseling
• Mode of Inheritance:
The dystrophinopathies are inherited in an
X-linked manner.
Genetic counseling is advised for people with a
family history of the disorder.
Duchenne muscular dystrophy can be detected
with about 95% accuracy by genetic studies
performed during pregnancy.
Carrier females have a 50% chance of
transmitting the DMD mutation in each
pregnancy.
Sons who inherit the mutation will be affected;
daughters who inherit the mutation are
carriers and may or may not develop
cardiomyopathy.
Prenatal Testing
• Prenatal testing is possible for pregnancies of
women who are carriers if the DMD mutation has
been identified in a family member or if linkage
has been established.
• The usual procedure is to determine fetal sex by
karyotype or specialized studies to identify the sex
chromosomes from cells obtained by chorionic
villus sampling (CVS) at approximately ten to
12 weeks' gestation or by amniocentesis
usually performed at approximately 15-18 weeks'
gestation.
• If the karyotype is 46,XY, DNA extracted
from fetal cells can be analyzed for the
known disease-causing mutation or using
the linkage previously established.
Preimplantation genetic diagnosis
(PGD)
Preimplantation genetic diagnosis may be
available for families in which the diseasecausing mutation has been identified.
Preimplantation genetic diagnosis (PGD)
is a new alternative to conventional prenatal
diagnosis particularly for those couples for
whom termination of pregnancy is not
acceptable.
PGD is currently available for a wide range of
single gene disorders including many Xlinked disorders, cystic fibrosis, and ßthalassaemia (Handyside et al., 1989 , 1992 ;
Cui et al., 1995 ; Coonen et al., 1996 ; Ray et
al., 1996a ; Kuliev et al., 1998 ).
• Sexing of embryos for PGD (Handyside et al.,
1989 ) has allowed the transfer of healthy
female embryos where embryos are at risk of
X-linked diseases such as DMD.
• However, with a gender-only selection
strategy, all male embryos will be discarded
even though half of these are not affected and
female embryos are transferred regardless of
carrier status.
Summary
• What is Duchenne muscular dystrophy?
• Duchenne muscular dystrophy (DMD) is a rapidly
progressive form of muscular dystrophy that
occurs primarily in boys.
• It is caused by a mutation in a gene, called the
DMD gene that can be inherited in families in an
X-linked recessive fashion, but it often occurs in
people from families without a known family
history of the condition.
Molecular Genetic Testing
• There is no known cure for
Duchenne muscular dystrophy,
although recent
stem-cell research is showing
promising vectors that may
replace damaged muscle tissue.
• Gene Therapy: Experimental gene
therapies are currently under
investigation [Gregorevic & Chamberlain
2003, Tidball & Spencer 2003, van Deutekom
& van Ommen 2003, Nowak & Davies 2004].
Treatment
is generally aimed at
controlling the onset of
symptoms to
maximize the quality of
life.
The Baskaran family
South East of England
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Parents, Ben and Debby,
have four sons:
Jason, 19, Jamie, 13,
Oliver, 12
and Konnor, aged 10
Both Oliver and Jamie
have Duchenne muscular
dystrophy.
Thank You
• Normal allelic variants. The DMD gene spans 2.4 Mb of DNA
and comprises 79 exons. It has at least four promoters. It is the
largest known human gene. Innumerable intragenic variants
have been described, many of which are useful as markers for
genetic linkage analysis.
• Pathologic allelic variants. Disease-causing alleles are
highly variable, including deletion of the entire gene, deletion or
duplication of one or more exons, and small deletions, insertions,
or single-base changes. In both DMD and BMD, partial deletions
and duplications cluster in two recombination hot spots, one
proximal at the 5' end of the gene, comprising exons 2-20 (30%),
and one more distal, comprising exons 44-53 (70%) [Den Dunnen
et al 1989]. Duplications cluster near the 5' end of the gene, with
duplication of exon 2 being the single most common duplication
identified [White et al 2006]. More than 4,700 mutations have
been identified [Aartsma-Rus et al 2006b].
CYTOGENETICS
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Greenstein et al. (1977) found DMD in a 16-year-old girl with a reciprocal X;11
translocation. The mother was thought not to be a carrier. Possibly the break at
Xp21 caused a null mutation; the normal X chromosome was inactivated. Verellen
et al. (1978) reported the same situation with X;21 translocation and break at Xp21.
Canki et al. (1979) described similar findings in a girl with X;3 translocation with
break at Xp21. The mother was thought to be heterozygous.
Zneimer et al. (1993) used a combination of conventional and molecular
cytogenetic techniques to investigate the twins first reported by Richards et al.
(1990). The twins carried a deletion of approximately 300 kb within the dystrophin
gene on one X chromosome. A unique DNA fragment generated from an exon
within the deletion was hybridized in situ to metaphase chromosomes of both twins,
a probe that would presumably hybridize only to the normal X chromosome and not
to the X chromosome carrying the deletion. The chromosomes were identified by
reverse-banding (R-banding) and by the addition of 5-bromodeoxyuridine in culture
to distinguish early and late replicating X chromosomes, corresponding to active
and inactive X chromosomes, respectively. The experiment showed predominant
inactivation of the normal X chromosome in the twin with DMD. With an improved
method of high resolution R-banding, Werner and Spiegler (1988) showed deletion
of Xp21.13 in an 8-year-old boy with normal intelligence and no disorder other than
DMD. His healthy mother was heterozygous for the deletion, which was subject to
random X inactivation in lymphocytes.
• Zneimer et al. (1993) used a combination of conventional and molecular
cytogenetic techniques to investigate the twins first reported by Richards et
al. (1990). The twins carried a deletion of approximately 300 kb within the
dystrophin gene on one X chromosome.
• A unique DNA fragment generated from an exon within the deletion was
hybridized in situ to metaphase chromosomes of both twins, a probe that
would presumably hybridize only to the normal X chromosome and not to
the X chromosome carrying the deletion.
• The chromosomes were identified by reverse-banding (R-banding) and by
the addition of 5-bromodeoxyuridine in culture to distinguish early and late
replicating X chromosomes, corresponding to active and inactive X
chromosomes, respectively.
• The experiment showed predominant inactivation of the normal X
chromosome in the twin with DMD. With an improved method of high
resolution R-banding, Werner and Spiegler (1988) showed deletion of
Xp21.13 in an 8-year-old boy with normal intelligence and no disorder other
than DMD. His healthy mother was heterozygous for the deletion, which was
subject to random X inactivation in lymphocytes.
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MAPPING
Duchenne muscular dystrophy is not linked to colorblindness or G6PD (Emery et
al., 1969; Zatz et al., 1974). No linkage with Xg has been found; total lod scores
were -14.6 and -2.4 for theta of 0.10 and 0.30, respectively (Race and Sanger,
1975).
Lindenbaum et al. (1979) found DMD with X-1 translocation and suggested that
the DMD locus is at Xp1106 or Xp2107. A number of females with X-autosome
translocations with the breakpoint in the Xp21 band have shown Duchenne
muscular dystrophy. One interpretation is that the gene locus is in that region and
that the locus on the normal X is inactivated. Murray et al. (1982) found linkage of
DMD with a restriction enzyme polymorphism at a distance of about 10 cM. The
cloned DNA sequence bearing the polymorphism (lambda RC8) was assigned to
Xp22.3-p21 by study of somatic cell hybrids. Spowart et al. (1982) outlined
reasons for doubting the location of the DMD gene at Xp21.
Wieacker et al. (1983) studied the linkage between the restriction fragment length
polymorphism defined by the cloned DNA sequence RC8 and X-linked ichthyosis.
At least 2 crossovers were found among 9 meioses in an informative family,
suggesting that RC8 and STS may be about 25 cM apart. Since STS is 15 cM
proximal to the Xg locus and since the RC8 and Duchenne muscular dystrophy are
closely linked, DMD may be 50 cM or more from Xg. Worton et al. (1984) studied
a female with DMD and an X;21 translocation which split the block of genes
encoding ribosomal RNA on 21p. Thus, ribosomal RNA gene probes can be used to
identify a junction fragment from the translocation site and to clone segments of
the X at or near the DMD locus.
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Kingston et al. (1983, 1984) found linkage of BMD with the cloned sequence L1.28 (designated
DXS7 by the seventh Human Gene Mapping Workshop in Los Angeles; D = DNA, X = X
chromosome, S = segment, 7 = sequence of delineation). The interval was estimated to be about
16 cM, which is also the approximate interval between DXS7 and DMD. DXS7 is located
between Xp11.0 and Xp11.3. Thus, these 2 forms of X-linked muscular dystrophy appeared to be
allelic, a possibility also supported by the finding of both severe and mild disease (Duchenne
and Becker, if you will) in females with X-autosome translocations. Contrary to reports of
others, Kingston et al. (1984) found no evidence of linkage of BMD to colorblindness; Xg also
showed no linkage.
Francke et al. (1985) studied a male patient with 3 X-linked disorders: chronic granulomatous
disease with cytochrome b(-245) deficiency and McLeod red cell phenotype, Duchenne muscular
dystrophy, and retinitis pigmentosa. A very subtle interstitial deletion of part of Xp21 was
demonstrated as the presumed basis of this 'contiguous gene syndrome.' That this was a
deletion and not a translocation was demonstrated by the absence of 1 DNA probe from the
genome of the patient. Since this probe (called 754) was clearly very close to DMD and
recognized a RFLP of high frequency, it proved highly useful for linkage studies of DMD. The
close clustering of CGD, DMD, and RP suggested by these findings was inconsistent with
separate linkage data, which indicated that McLeod and CGD were close to Xg and that DMD
and RP are far away (perhaps at least 55 cM) and as much as 15 cM from each other. At least 4
possible explanations of the discrepancy were proposed by Francke et al. (1985). One suggestion
was that the deletion contained a single defect affecting perhaps a cell membrane component
with the several disorders following thereon.
Mulley et al. (1988) reported the recombination frequencies between DMD and intragenic
markers from 8 informative families containing 30 informative meioses. No recombinants were
observed. The authors commented that the average theta between intragenic markers and DMD
may be 1 to 2%. Grimm et al.(1989) reported a recombination rate of 4% between 2 subclones of
the DNA segment DXS164 within the dystrophin locus, indicating a hotspot for recombination.
Analysis of five Duchenne muscular dystrophy exons and gender
determination using conventional duplex polymerase chain
reaction on single cells
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Nicole D. Hussey1,6, Hu Donggui1,3, David A.H. Froiland1, Damian J. Hussey2, Eric A.
Haan4, Colin D. Matthews1 and Jamie E. Craig5
1 Department of Obstetrics and Gynaecology and 2 Department of Medicine, University of Adelaide,
The Queen Elizabeth Hospital, Woodville 5011, South Australia, Australia, 3 Institute of Obstetrics and
Gynaecology, The 2nd People's Hospital, Guangzhou, 510150, People's Republic of China and 4 South
Australian Clinical Genetics Service, The Women's and Children's Hospital, North Adelaide, 5006,
South Australia
Abstract
We have developed five conventional duplex polymerase chain reaction (PCR) protocols on single
lymphocytes and blastomeres from embryos, in order to analyse five exons commonly deleted in
deletion-type Duchenne muscular dystrophy (DMD). The five DMD gene exons (17, 19, 44, 45 and 48)
can be analysed in separate duplex PCR reactions together with the sex-determining region Y (SRY)
gene which enables simultaneous gender assignment. We present here PCR amplification results from
single lymphocytes isolated from a normal male (220 cells), a normal female (24 cells) and a male
DMD patient (40 cells) carrying a deletion of exons 46–49 within the DMD gene. The method failed to
produce a PCR signal for the SRY gene in 8/220 normal male cells (3.6%) and for a DMD exon in 0–
4.5% of normal male cells. One negative control out of 112 was positive. When this method was used to
analyse two blastomeres from each of five embryos, concordant results were obtained for each pair of
blastomeres. All embryos produced signals for the DMD exon tested with four of the embryos found to
be male and one female. This method is therefore suitable for preimplantation genetic diagnosis and
will allow the transfer of healthy embryos (both male and female) in families carrying DMD gene
deletions involving at least one of the five exons 17, 19, 44, 45 and 48.
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Abstract
We have developed five conventional duplex polymerase chain reaction (PCR)
protocols on single lymphocytes and blastomeres from embryos, in order to
analyse five exons commonly deleted in deletion-type Duchenne muscular
dystrophy (DMD).
The five DMD gene exons (17, 19, 44, 45 and 48) can be analysed in separate duplex
PCR reactions together with the sex-determining region Y (SRY) gene which
enables simultaneous gender assignment. We present here PCR amplification
results from single lymphocytes isolated from a normal male (220 cells), a normal
female (24 cells) and a male DMD patient (40 cells) carrying a deletion of exons
46–49 within the DMD gene. The method failed to produce a PCR signal for the
SRY gene in 8/220 normal male cells (3.6%) and for a DMD exon in 0–4.5% of
normal male cells.
One negative control out of 112 was positive. When this method was used to
analyse two blastomeres from each of five embryos, concordant results were
obtained for each pair of blastomeres.
All embryos produced signals for the DMD exon tested with four of the embryos
found to be male and one female. This method is therefore suitable for
preimplantation genetic diagnosis and will allow the transfer of healthy embryos
(both male and female) in families carrying DMD gene deletions involving at least
one of the five exons 17, 19, 44, 45 and 48.
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Introduction
Duchenne or Becker muscular dystrophy (DMD/BMD) is one of the most
common X-linked lethal genetic diseases with a worldwide frequency of one
in 3500 live male births (Harper, 1989 ). Since no effective therapy exists thus
far, most patients die at ~20 years of age. Mutations in the DMD gene can be
divided into three different catagories of deletions, duplications and point
mutations. Deletions within the 79 exon DMD gene account for ~60% of all
DMD cases, 98% of which can be detected by two sets of multiplex
polymerase chain reaction (PCR) reactions (Beggs et al., 1990 ; Chamberlain
et al., 1990 ). Prenatal diagnosis using these two multiplex PCR protocols can
determine whether a male pregnancy is affected when the deletion mutation
for the family is known (Abbs, 1996 ).
Preimplantation genetic diagnosis (PGD) is a new alternative to conventional
prenatal diagnosis particularly for those couples for whom termination of
pregnancy is not acceptable. PGD is currently available for a wide range of
single gene disorders including many X-linked disorders, cystic fibrosis, and
ß-thalassaemia (Handyside et al., 1989 , 1992 ; Cui et al., 1995 ; Coonen et al.,
1996 ; Ray et al., 1996a ; Kuliev et al., 1998 ).
Sexing of embryos for PGD (Handyside et al., 1989 ) has allowed the transfer
of healthy female embryos where embryos are at risk of X-linked diseases
such as DMD. However, with a gender-only selection strategy, all male
embryos will be discarded even though half of these are not affected and
female embryos are transferred regardless of carrier status.
Pleiotropy: Gene that affects more than
one characteristic of an individual
Example:
1. Sickle cell disease
2. Cystic fibrosis
Dystroglycan Complex: In muscles, a complex of
transmembrane glycoproteins links a network of dystrophin
and actin filaments on the inside of the plasma membrane to two
proteins of the extracellular basal lamina, alpha2 laminin and
agrin.
These protein associations stabilize the muscle plasma
membrane from inside and outside.
This muscle membrane skeleton resembles in concept and function
the actin-spectrin network or red blood cells.
Genetic defects or deficiencies in dystrophin, transmembrane linker
proteins of the dystroglycan/sarcoglycan complex, or alpha
laminin cause muscular dystrophy in humans, most likely due to
the mechanical instability of the membrane leading to
cellular damage and eventual atrophy of the muscle.
Cytogenetics is a branch of genetics that is
concerned with the study of the structure
and function of the cell, especially the
chromosomes[1].
It includes routine analysis of G-Banded
chromosomes, other cytogenetic
banding techniques, as well as
molecular cytogenetics such as fluorescent
in situ hybridization (FISH) and
comparative genomic hybridization (CGH).
• Advent of banding techniques
• In the late 1960s Caspersson developed banding techniques which
differentially stain chromosomes. This allows chromosomes of
otherwise equal size to be differentiated as well as to elucidate the
breakpoints and constituent chromosomes involved in
chromosome translocations.
• Deletions within one chromosome could also now be more
specifically named and understood. Deletion syndromes such as
DiGeorge syndrome, Prader-Willi syndrome and others were
discovered to be caused by deletions in chromosome material.
• Diagrams identifying the chromosomes based on the banding
patterns are known as cytogenetic maps. These maps became
the basis for both prenatal and oncological fields to quickly move
cytogenetics into the clinical lab where karyotyping allowed
scientists to look for chromosomal alterations.
• Techniques were expanded to allow for culture of free
amniocytes recovered from amniotic fluid, and elongation
techniques for all culture types that allow for higher resolution
banding.
Human Male Karyotype
• Beginnings of molecular cytogenetics
• In the 1980s advances were made in molecular
cytogenetics. While radioisotope-labeled probes had
been hybridized with DNA since 1969, movement was
now made in using fluorescently labeled probes.
Hybridizing them to chromosomes preparations made
using existing techniques came to be known as
fluorescent in situ hybridization (FISH).
• This change significantly increased the usage of
probing techniques as fluorescently labeled
probes are safer and can be used almost indefinitely.
Further advances in micromanipulation and
examination of chromosomes led to the technique of
chromosome microdissection whereby aberrations in
chromosomal structure could be isolated, cloned and
studied in ever greater detail.
• Mosaicism
• Presence of 2 kinds of chromosome
constitution in the same individual
(zygote)
• Routine analysis
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Routine chromosome analysis refers to analysis of metaphase chromosomes
which have been banded using trypsin followed by Giemsa, Leishmanns, or a
mixture of the two. This creates unique banding patterns on the chromosomes.
The molecular mechanism and reason for these patterns is unknown, although it
likely related to replication timing and chromatin packing.
Several chromosome-banding techniques are used in cytogenetics laboratories.
Quinacrine banding (Q-banding) was the first staining method used to produce
specific banding patterns. This method requires a fluorescence microscope and is no
longer as widely used as Giemsa banding (G-banding).
Reverse banding (R-banding) requires heat treatment and reverses the usual
white and black pattern that is seen in G-bands and Q-bands. This method is
particularly helpful for staining the distal ends of chromosomes.
Other staining techniques include C-banding and nucleolar organizing region
stains (NOR stains). These latter methods specifically stain certain portions of the
chromosome.
C-banding stains the constitutive heterochromatin, which usually lies near the
centromere, and NOR staining highlights the satellites and stalks of acrocentric
chromosomes.
High-resolution banding involves the staining of chromosomes during prophase
or early metaphase (prometaphase), before they reach maximal condensation.
Because prophase and prometaphase chromosomes are more extended than
metaphase chromosomes, the number of bands observable for all chromosomes
increases from about 300 to 450 to as many as 800. This allows the detection of less
obvious abnormalities usually not seen with conventional banding.
• Karyotype
• Is the use of nomenclature to describe the normal
or abnormal, constitutional or acquired,
chromosome complement of an individual, tissue or
cell line
• 46,XX or 46,XY
• 47,XX,+21
• Karyogram
• A systematized array of the chromosomes prepared
either by drawing, digitized imaging or by
photography
• Ideogram
• Diagrammatic representation of a karyotype/
chromosome
Chromosome Designation
9q34.2
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Chrom number
Chrom arm
Chrom region
Chrom band
Sub-band
Linkage Map/Chromosome Map
Gene linkage: the existence of several genes on the same chromosome.
The genes on the same chromosome form a linkage group because these
genes tend to be inherited together.
Linkage Map/Chromosome Map: Tells the order of gene loci
on chromosomes. To construct a chromosome map, investigators can
sometimes rely on crossing over.
Crossing-over occurs between nonsister chromatids when
homologous pair of chromosomes pair prior to separation during
meiosis. During crossing-over, the nonsister chromatids exchange
genetic materials and therefore genes.
Following crossing-over, recombinant chromosomes occur. Recombinant
chromosomes contribute to recombinant gametes. Recombinant means
a new combination of alleles.
All the genes on one chromosome form a linkage group that tends to stay
together, except when crossing-over occurs.
Blotting: Transfer step method to detect
molecules separated by gel
electrophoresis. Specific proteins are often
detected with antibodies.
Typically proteins are transferred
electrophoretically from the
polyacyrlamide gel to a sheet of
nitrocellulose or nylon before reaction
with antibodies.
A deletion is a mutation in which a part of a chromosome
or a sequence of DNA is missing. Deletion is the loss of genetic material.
Any number of nucleotides can be deleted, from a single base to an entire piece of
chromosome. Deletions can be caused by errors in chromosomal crossover during
meiosis. This causes several serious genetic diseases.
The three major single chromosome mutations; deletion (1),
duplication (2) and inversion (3).
The two major two chromosome mutations;
insertion (1) and translocation (2).
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Numerical abnormalities
When an individual is missing either a chromosome from a pair
(monosomy) or has more than two chromosomes of a pair (trisomy,
tetrasomy, etc). An example of a condition caused by a numerical
anomaly is Down Syndrome, also known as Trisomy 21 (an individual
with Down Syndrome has three copies of chromosome 21, rather than
two). Turner Syndrome is an example of a monosomy where the
individual is born with only one sex chromosome, an X.
Structural abnormalities
When the chromosome's structure is altered. This can take several
forms:
Deletions: A portion of the chromosome is missing or deleted. Known
disorders include Wolf-Hirschhorn syndrome, which is caused by
partial deletion of the short arm of chromosome 4; and Jacobsen
syndrome, also called the terminal 11q deletion disorder.
Duplications: A portion of the chromosome is duplicated, resulting in
extra genetic material. Known disorders include Charcot-Marie-Tooth
disease type 1A which may be caused by duplication of the gene
encoding peripheral myelin protein 22 (PMP22) on chromosome 17.
• Gene duplication (or chromosomal duplication or gene
amplification) is any duplication of a region of DNA that
contains a gene; it may occur as an error in homologous
recombination, a retrotransposition event, or duplication of an
entire chromosome.
• The second copy of the gene is often free from selective pressure
— that is, mutations of it has no deleterious effects to its host
organism. Thus it mutates faster than a functional single-copy
gene, over generations of organisms.
• A duplication is the opposite of a deletion. Duplications arise
from an event termed unequal crossing-over that occurs during
meiosis between misaligned homologous chromosomes.
• The chance of this happening is a function of the degree of
sharing of repetitive elements between two chromosomes. The
product of this recombination are a duplication at the site of the
exchange and a reciprocal deletion.[2]
Gene duplication as amplification
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Gene duplication doesn't necessarily constitute a lasting change in a species'
genome. In fact, such changes often don't last past the initial host organism. From
the perspective of molecular genetics, amplification is one of many ways in which a
gene can be overexpressed. Genetic amplification can occur artificially, as with the
use of the polymerase chain reaction technique to amplify short strands of DNA in
vitro using enzymes, or it can occur naturally, as described above. If it's a natural
duplication, it can still take place in a somatic cell, rather than a germline cell
(which would be necessary for a lasting evolutionary change).
Also, in either event, duplications can be and often are marginally or severely
detrimental. For instance, duplications of oncogenes are a common cause of many
types of cancer, as is the case with P70-S6 Kinase 1 amplification and breast
cancer.[8] In such cases the genetic duplication occurs in a somatic cell and affects
only the genome of the cancer cells themselves, not the entire organism, much less
any subsequent offspring.
Genomic microarrays detect Duplications
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Technologies such as genomic microarrays, also called array comparative genomic
hybridization (array CGH), are used to detect chromosomal abnormalities, such as
microduplications, in a high throughput fashion from genomic DNA samples. In
particular, DNA microarray technology can simultaneously monitor the expression
levels of thousands of genes across many treatments or experimental conditions,
greatly facilitating the evolutionary studies of gene regulation after gene
duplication or speciation [9][10].
• Translocations: When a portion of one chromosome is transferred
to another chromosome. There are two main types of
translocations. In a reciprocal translocation, segments from
two different chromosomes have been exchanged. In a
Robertsonian translocation, an entire chromosome has attached
to another at the centromere; these only occur with chromosomes
13, 14, 15, 21 and 22.
• Inversions: A portion of the chromosome has broken off, turned
upside down and reattached, therefore the genetic material is
inverted.
• Rings: A portion of a chromosome has broken off and formed a
circle or ring. This can happen with or without loss of genetic
material.
• Isochromosome: Formed by the mirror image copy of a
chromosome segment including the centromere.
• Chromosome instability syndromes are a group of disorders
characterized by chromosomal instability and breakage.
They often lead to an increased tendency to develop certain types
of malignancies.
Inheritance
• Most chromosome anomalies occur as an
accident in the egg or sperm, and are therefore
not inherited.
• Therefore, the anomaly is present in every cell
of the body. Some anomalies, however, can
happen after conception, resulting in
mosaicism (where some cells have the anomaly
and some do not).
• Chromosome anomalies can be inherited from
a parent or be "de novo". This is why
chromosome studies are often performed on
parents when a child is found to have an
anomaly.
• G-banding is technique used in cytogenetics to produce a
visible karyotype by staining condensed chromosomes.
The metaphase chromosomes are treated with trypsin (to partially
digest the protein) and stained with Giemsa. Dark bands that
take up the stain are strongly A,T rich (gene poor). The reverse
of G-bands is obtained in R-banding.
Banding can be used to identify chromosomal abnormalities,
such as translocations, because there is a unique pattern of light
and dark bands for each chromosome.
• It is difficult to identify and group chromosomes based on
simple staining because the uniform color of the structures
makes it difficult to differentiate between the different
chromosomes.
• Therefore, techniques like G-banding were developed that made
'bands' appear on the chromosomes. These bands were same in
appearance on the homologous chromosomes, thus,
identification became easier and more accurate. The
acid/saline/giemsa protocol reveals G-bands.
When the proband's DMD mutation is not known. Linkage
analysis can be offered to at-risk females to determine carrier status in
families with more than one affected male with the unequivocal
diagnosis of DMD/BMD/DMD-associated DCM. Linkage studies are
based on accurate clinical diagnosis of DMD/BMD/DMD-associated
DCM in the affected family members and accurate understanding of the
genetic relationships in the family. Linkage analysis relies on the
availability and willingness of family members to be tested. The
markers used for linkage in DMD/BMD/DMD-associated DCM are
highly polymorphic and informative, and lie both within and flanking
the DMD locus; thus, they can be used in most families with
DMD/BMD/DMD-associated DCM [Kim et al 2002].
Note: The large size of the DMD gene leads to an appreciable risk of
recombination. It has been estimated that the gene itself spans a genetic
distance of 12 centimorgans [Abbs et al 1990]; thus, multiple
recombination events among different members of a family may
complicate the interpretation of a linkage study. Linkage testing is not
available to families in which there is a single affected male.
• Immunosuppressive therapy:
The following recommendations for immunosuppressive
therapy are in accordance with the national practice
parameters regarding corticosteroid therapy
developed by the American Academy of Neurology and
the Child Neurology Society [Moxley et al 2005].
• Boys with DMD who are older than age five years
should be offered treatment with prednisone
(0.75/mg/kg/day).
• Prior to the initiation of therapy, the potential benefits
and risks of corticosteroid treatment should be
carefully discussed with each individual.