Molecular Biology & Genomics (SEC. 061)Presentation by Imran * Jimmy
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
Duchenne MuscularDystrophy 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.• Its 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
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
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, its 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 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.
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% 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.
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 all100-200 bp long, and so if drawn to scale each exon would be represented bya line occupying less than 0.05% of the width of the figure. Random deletionbreakpoints therefore almost always fall in introns. Their effect is to removeone or more complete exons from the mature mRNA. The deletion shownremoves exons 45-47 from the mature mRNA, while leaving all the other exonsintact.
Genotype of DMD (continued)PCR deletion screen inDuchenne musculardystrophyNine selected exons of the DMDgene have been amplified fromthe DNA of a panel of 20 affectedboys. When the product is run onan electrophoretic gel each exongives a band of a characteristicsize. Because a boy has only asingle X chromosome, anydeletion shows up as missingbands. Different exon deletionscan be seen in lanes 1, 5, 11, 12,19 and 20. Lane 3 may be a largedeletion or a technical failure. Theboys with no deletion on this gelmay have others of the 79dystrophin exons deleted, or mayhave point mutations or
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
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 75points.o Memory problemso Carrying out daily functionsIncrease in bone fractures due to the decrease in bonedensity.Increase in serum CK (creatine phosphokinase) levelsup 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.
DMD DiagnosisPCR deletion screen inDuchenne musculardystrophyNine selected exons of the DMDgene have been amplified fromthe DNA of a panel of 20 affectedboys. When the product is run onan electrophoretic gel each exongives a band of a characteristicsize. Because a boy has only asingle X chromosome, anydeletion shows up as missingbands. Different exon deletionscan be seen in lanes 1, 5, 11, 12,19 and 20. Lane 3 may be a largedeletion or a technical failure. Theboys with no deletion on this gelmay have others of the 79dystrophin exons deleted, or mayhave point mutations or
DMD Diagnosis - Muscle biopsyDystrophinantibodystaining ofmuscle cells Normal Control 4 year old boy with DMD – No detectable dystrophin
DMD Diagnosis – Prenatal TestsFlow chart depicting theevaluation necessaryfor possible prenataltesting for Duchennesand Beckers musculardystrophy.
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
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
Ongoing Research – Exon skippingThe exon skipping technique tries to change a Duchenne mutationinto a Becker mutation. If a variation disturbs the reading frame andthus causes Duchenne dystrophy, the reading frame can be restoredby artificially removing from the messenger RNA one or more exonsdirectly in front or after the deletion, the duplication, or the exonwhich 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
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