Duchenne Muscular Distrophy

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

Introduction

• Duchenne Muscular Dystrophy(DMD) Factso Incidence/prevalence

• Genotype of DMDo Molecular Makeupo Dystrophin Function

• Phenotype of DMD• Allelic Variants • Diagnosis

o Signs and testso Treatment

• Ongoing research

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 years; wheelchair dependency had a median age of 10 years;

• In this cohort, death occurred at a median age of 17 years.

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.

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 identified the gene that, when flawed — a problem known as a mutation — causes DMD.

• In 1987, the protein associated with this gene was identified and named dystrophin.

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

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.

• The C-terminal: contains the syntrophin binding sight (for binding internal cellular components)

Dystrophin Function• The DMD gene encodes for the

protein dystrophin, found in muscle 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.

o In DMD the damage to muscle cells is so extreme that the supply of stem cells are exhausted and repair can no longer occur.

Genotype of DMD (continued)• Mutations lead to an absence of or defect in the protein

dystrophin, which results in progressive muscle degeneration

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% caseso 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.

Genotype of DMD (continued)

A deletion of part of the DMD geneThis 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.

Genotype of DMD (continued)PCR deletion screen in

Duchenne muscular dystrophyNine 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 duplications to cause loss of function of the gene.

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 in 95% of cases by the last years of life.• Nervous System: 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 et al. (1993) and Pillers et al. (1993).

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 problemso 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.

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.

Allelic Variants

Allelic Variants (Continued)

DMD DiagnosisPCR deletion screen in Duchenne muscular dystrophyNine 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 duplications to cause loss of function of the gene.

DMD Diagnosis - Muscle biopsy

Dystrophin antibody staining of muscle cells

Normal Control 4 year old boy with DMD – No detectable dystrophin

DMD Diagnosis – Prenatal Tests

Flow chart depicting the evaluation necessary for possible prenatal testing for Duchenne's and Becker's muscular dystrophy.

Treatments for DMD

• To improve breathing:o O2 therapyo Ventilatoro Scoliosis surgeryo Tracheotomy

• To improve mobility:o Physical therapyo Surgery on tight jointso Prednisoneo Non-steroidal medicationso Wheelchair

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

impenetrableo Viruses might provoke the immune system

and cause the destruction of muscle fibers with the new genes

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.

Ongoing Research – PTC124 • PTC124 is a

novel, orally administered small-molecule compound that targets a particular genetic alteration known as a nonsense mutation.

Ongoing Research – Antisense Drug

• An antisense drug is a man-made form of 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 blocked, allowing for the dystrophin protein to be produced.

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-Healt

h-Beauty-Therapy/96775-Successful-Treatment-Of-DMD-Duchenne-Muscular-Dystrophy.html

• http://staff.um.edu.mt/acus1/5Mutations.htm

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-i

n-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

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