Technical paper

1. Molecular Biology & Genomics (SEC. 061)
Presentation by Imran * Jimmy

2. Introduction
• Duchenne Muscular Dystrophy(DMD) Facts
o Incidence/prevalence
• Genotype of DMD
o Molecular Makeup
o Dystrophin Function
• Phenotype of DMD
• Allelic Variants
• Diagnosis
o Signs and tests
o Treatment
• Ongoing research

3. Duchenne Muscular
Dystrophy Facts
• Duchenne muscular dystrophy (DMD) is a
severe recessive X-linked form of
muscular dystrophy
• DMD is characterized by rapid progression
of muscle degeneration, eventually leading
to loss of ambulation and death.
• It's caused by mutations in the DMD gene,
the largest gene in the human body.
• DMD affects mostly males at a rate of 1 in
3,500 births.
• The mean age at DMD diagnosis was 4.6

4. DMD Facts (continued)
• Any mother who is a carrier for muscular
dystrophy will have a 50:50 chance giving
birth to a son with muscular dystrophy and a
50:50 chance of giving birth to a daughter
who is a carrier.

5. Incidence/Prevalence
• The first historical account of muscular
dystrophy appeared in 1830, when Sir
Charles Bell wrote an essay about an
illness that caused progressive
weakness in boys.
• DMD is named after the French
neurologist Guillaume Benjamin Amand
Duchenne (1806–1875), who first
described the disease in 1861
• Until the 1980s, little was known about
the cause of any kind of muscular
dystrophy.
• In 1986, MDA-supported researchers

6. Genotype of DMD
• Mapping and molecular genetic
studies indicate that DMD is a
recessive x-linked disorder
caused by a mutation in the
gene at locus Xp21 that codes
for the muscle protein
dystrophin.
• More specifically, it's located at
position at position 21.2 from
base pair 31,047,265 to base
pair 33,267,646 on the X
chromosome

7. Molecular Makeup of
DMD Gene
• There are 79 exons: which makeup
0.6% of the entire gene.
• There are 8 promoters (binding sights).
• Introns: make up 99.4% of the entire
gene.
• Genomic DNA: 2.2 million base pairs.
• N-terminal or actin binding sight: binds
dystrophin to membranes surrounding
striated muscle fiber.
• Rod Domain: contains 24 proteins that
repeat and maintain molecular
structure.
o It is thought to give the rod its flexibility.
o The main rod is interrupted by 4 hinge
regions.
• The cysteine-rich domain: regulates
ADAM protease which are cell
membrane anchors that are important
in maintaining cell shape and structure.

8. • The DMD gene encodes for the
protein dystrophin, found in muscle
Dystrophin Function
cells and some neurons.
• Dystrophin provides strength to
muscle cells by linking the internal
cytoskeleton to the surface
membrane.
• Without this structural support, the
cell membrane becomes
permeable.
• As components from outside the
cell are allowed to enter the
internal pressure of the cell
increases until the cell bursts and
dies.
o Under normal wear and tear stem
cells within the muscle regenerate
new muscle cells and repair the
damage.

9. Genotype of DMD
(continued)
• Mutations lead to an absence of or defect in the protein
dystrophin, which results in progressive muscle
degeneration

10. Genotype of DMD
(continued)
• Mutations which affect the DMD gene.
o The most common type of disease-
causing mutation of the DMD gene is
deletion of 1 or more exons, identified in
approximately 60 to 65% of patients
o Duplications is found in 9% cases
o nonsense or splice site mutations in
16% and 5% cases, respectively
o The most common mutation are repeats
of the CAG nucleotides.
• Although there is no clear correlation
found between the extent of the
deletion and the severity of the
disorder, DMD deletions usually result
in frameshift.

11. Genotype of DMD
(continued)
A deletion of part of the DMD gene
This figure shows a 500 kb region containing exons 41-50. These exons are all
100-200 bp long, and so if drawn to scale each exon would be represented by
a line occupying less than 0.05% of the width of the figure. Random deletion
breakpoints therefore almost always fall in introns. Their effect is to remove
one or more complete exons from the mature mRNA. The deletion shown
removes exons 45-47 from the mature mRNA, while leaving all the other exons
intact.

12. Genotype of DMD
(continued)
PCR deletion screen in
Duchenne muscular
dystrophy
Nine selected exons of the DMD
gene have been amplified from
the DNA of a panel of 20 affected
boys. When the product is run on
an electrophoretic gel each exon
gives a band of a characteristic
size. Because a boy has only a
single X chromosome, any
deletion shows up as missing
bands. Different exon deletions
can be seen in lanes 1, 5, 11, 12,
19 and 20. Lane 3 may be a large
deletion or a technical failure. The
boys with no deletion on this gel
may have others of the 79
dystrophin exons deleted, or may
have point mutations or

13. Phenotype of DMD
• Skeletal Muscle :The most distinctive feature of Duchenne
muscular dystrophy is a progressive proximal muscular dystrophy
with characteristic pseudohypertrophy of the calves.
• Cardiac Muscle : Myocardial involvement appeared in a high
percentage of DMD patients by about 6 years of age; it was
• present inSystem:cases by the last years of life.
Nervous 95% of Mental retardation
of mild degree is a pleiotropic effect of
the DMD gene. The finding of
dystrophin mRNA in brain may bear a
relationship to the mental retardation
in DMD patients.
• Retinal Function: Abnormal retinal
neurotransmission as measured by
electroretinography (ERG) was
observed in boys with DMD by Cibis

14. Phenotype of DMD
(continued)
Delays in early childhood stages involving muscle use,
in 42% of patients.
Learning difficulties in 5% of patients.
Speech problems in 3% of patients.
Leg and calf pain.
Mental development is impaired. IQ’s usually below 75
points.
o Memory problems
o Carrying out daily functions
Increase in bone fractures due to the decrease in bone
density.
Increase in serum CK (creatine phosphokinase) levels
up to 10 times normal amounts.
Wheelchair bound by 12 years of age.
Cardiomyopathy at 14 to 18 years.
Few patients live beyond 30 years of age.
o Reparatory problems and cardiomyopathy leading to
congestive heart failure are the usual cause of death.

15. Phenotype of DMD
(continued)
• (a) Affected boys stand up by bracing their arms against their legs because
their proximal muscles are weak.
• Normal muscle (b) shows a regular architecture of cells with Dystrophin
(brown stain) on all the outer membranes.
• (c) Shows muscle from a 10-year-old affected boy. Note the
disorganisation, invasion by fibrous tissue and complete absence of
Dystrophin.

16. Allelic Variants

17. Allelic Variants
(Continued)

18. DMD Diagnosis
PCR deletion screen in
Duchenne muscular
dystrophy
Nine selected exons of the DMD
gene have been amplified from
the DNA of a panel of 20 affected
boys. When the product is run on
an electrophoretic gel each exon
gives a band of a characteristic
size. Because a boy has only a
single X chromosome, any
deletion shows up as missing
bands. Different exon deletions
can be seen in lanes 1, 5, 11, 12,
19 and 20. Lane 3 may be a large
deletion or a technical failure. The
boys with no deletion on this gel
may have others of the 79
dystrophin exons deleted, or may
have point mutations or

19. DMD Diagnosis - Muscle
biopsy
Dystrophin
antibody
staining of
muscle cells
Normal Control 4 year old boy with DMD – No
detectable dystrophin

20. DMD Diagnosis –
Prenatal Tests
Flow chart depicting the
evaluation necessary
for possible prenatal
testing for Duchenne's
and Becker's muscular
dystrophy.

21. Treatments for DMD
• To improve breathing:
o O2 therapy
o Ventilator
o Scoliosis surgery
o Tracheotomy
• To improve mobility:
o Physical therapy
o Surgery on tight joints
o Prednisone
o Non-steroidal medications
o Wheelchair

22. Ongoing Research -
Gene Therapy
• Researches have developed "minigenes,"
which carry instructions for a slightly
smaller version of dystrophin, that can fit
inside a virus
• Researchers have also created the so-
called gutted virus, a virus that has had
its own genes removed so that it is
carrying only the DMD gene
• Problems with Gene Therapy :
o Muscle tissue is large and relatively
impenetrable
o Viruses might provoke the immune system

23. Ongoing Research –
Exon skipping
The exon skipping technique tries to change a Duchenne mutation
into a Becker mutation. If a variation disturbs the reading frame and
thus causes Duchenne dystrophy, the reading frame can be restored
by artificially removing from the messenger RNA one or more exons
directly in front or after the deletion, the duplication, or the exon
which contains a point mutation.

24. Ongoing Research –
PTC124
• PTC124 is a
novel, orally
administered
small-molecule
compound that
targets a
particular
genetic
alteration
known as a
nonsense

25. Ongoing Research –
• An antisense drug is a
Antisense
man-made form of Drug
RNA that interacts
with the genetic code.
This particular drug
was able to bind to an
area of the defective
gene and prevent it
from being integrated
into the DNA of the
cell. Therefore, the
abnormality in the
genetic code that
prevented the
production of normal
dystrophin was

26. Acknowledgements
• http://www.ncbi.nlm.nih.gov/omim/310200
• http://www.ncbi.nlm.nih.gov/omim/300377
• http://www.biology.iupui.edu/biocourses/n100h/ch11humgenetics.
html
• http://nature.ca/genome/03/d/10/03d_14b_e.cfm
• http://www.genomebc.ca/education/glossary/
• http://www.biologyreference.com/Fo-Gr/Gene.html
• http://www.medicine.uiowa.edu/research/researchweek/mysterydi
agnosis/case1/index.htm
• http://thecodyfoundation.com/dmdoverview.php
• http://www.meramaal.com/193/posts/4-Services-Classes-/59-
Health-Beauty-Therapy/96775-Successful-Treatment-Of-DMD-
Duchenne-Muscular-Dystrophy.html
• http://staff.um.edu.mt/acus1/5Mutations.htm

27. Acknowledgements
(continued)
• http://www.muscular-dystrophy.org/about_muscular_dystrophy
• http://emedicine.medscape.com/article/1178994-diagnosis
• http://www.cbs.dtu.dk/staff/dave/roanoke/genetics980225.html
• http://orthoinfo.aaos.org/topic.cfm?topic=a00384
• http://ladybugkatia.com/StemCellTransplant.html
• http://www.med.upenn.edu/pmi/members/khurana.shtml
• http://www.actionduchenne.org/duchennepedia/article/15/exon
-skipping
• http://chattahbox.com/science/2009/03/17/new-breakthrough-
in-cure-for-muscular-dystrophy/
• http://www.disaboom.com/muscular-dystrophy/experimental-
antisense-drug-targets-duchenne-muscular-dystrophy
• http://www.nature.com/nrg/journal/v4/n10/fig_tab/nrg1180_F2.
html

28. Question and Answers