BY Mr P G GEEGI ASST PROF DEPT

BY Mr. P. G. GEEGI, ASST. PROF, DEPT OF BIOCHEMISTRY St, JOSEPH’S COLLEGE, TRICHY. Course title: Molecular diagnostics( UNIT i) CLASS: I M. Sc BIOCHEMISTRY.

� DMD affects mostly males at a rate of 1 in 3, 500 births. � There are over 200 types of mutations that can cause any one of the forms of muscular dystrophy. There also mutations that occur within the same gene that cause other disease types. � DMD is the most severe and common type of muscular dystrophy. � DMD is characterized by the wasting away of muscles. � DMD is the most aggressive form of muscular dystrophy. � Diagnosis in boys usually occurs between 16 months and 8 years. Parents are usually the first to notice problem. � Death from DMD usually occurs by age of 30.

Females carry the DMD gene on the X chromosome. Females are carriers and have a 50% chance of transmitting the disease in each pregnancy. Sons who inherit the mutation will have the disease. Daughters that inherit the mutation will be carriers. The DMD gene is located on the Xp 21 band of the X chromosome. Mutations which affect the DMD gene. 96% are frameshift mutations 30% are new mutations 10 -20% of new mutations occur in the gametocyte (sex cell, will be pass on to the next generation). The most common mutation are repeats of the CAG nucleotides.

� During �It the translocation process, a mutation occurs. leads to the absence of dystrophin gene. Very Large Deletions (lead to absence of dystrophin) �Mutations causing reading errors (causes a degraded, low functioning DMD protein molecule) are Stop mutation Splicing mutation Duplication Deletion Point Mutations

� � � � � Delays in early childhood stages involving muscle use, in 42% of patients. Delays in standing alone Delays in sitting without aid Delays in walking (12 to 24 months) � Toe walking or flat footednees. Child has a hard time climbing. 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. Memory problems 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. Reparatory problems and cardiomyopathy leading to congestive heart failure are the usual cause of death.

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. It is thought to give the rod its flexibility. 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)

DMD GENE AND 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. Under normal wear and tear stem cells within the muscle regenerate new muscle cells and repair the damage. In DMD the damage to muscle cells is so extreme that the supply of stem cells are exhausted and repair can no longer occur.

ALLELIC VARIANTS Disease Mutation Effect of Mutation Phenotype Duchenne Muscular Dystrophy Very Large Deletions caused by: Stop mutations Splicing mutations Deletions Duplications Severely Functionally Impaired Dystrophin Protein As Discussed In Prior Slides Becker Muscular Dystrophy Deletion or Duplication That Change In-Frame Exons Creates A Protein That Is Partially Functional Same As But Less Sever Then DMD But Onset At Greater Then 7 Years Old DMD Related Dilated Cardiomyopathy Effects The Cardiac Muscle Promoter and The First Exon No Dystrophin Transcriptions Being Carried Out In Cardiac Muscle Tachycardia (Fat Heart Beat) Leads To Congestive Hear Failure Limb-Girdle Muscular Dystrophy In Gene That Encodes Scarcoglycans and Other Proteins of Muscle Cells Decrease In Scarcoglycans Proteins Pelvic and Shoulder Girdle Can Look Like DMD or BMD

Disease Mutation Effect of Mutation Phenotype Proximal Myotonic Myopathy Repeats In The Gene That Encodes For Zinc Finger Protein 9 Lack of Zinc Finger Protein 9 Causes Weakness In Muscle Cells Stiffness or Pain In Limb Girdle Distribution Myotonic Dystrophy Increase In CTG Nucleotide Repeats of CTG Cause Neurological Disorders Frontal Balding, Cataracts, Diabetes, Distal Limb Weakness Emery-Dreifuss Muscular Dystrophy (EDMD) EMD That Codes For Emerin and LMNA Which Codes For Lamins A Lack of Specificity of The Dystrophic Changes Observed. Joint Contractures Leading To Muscle Weakness and Wasting Usually Some Cardiac Involvement Spinal Muscular Atrophy Mutation In The SMN Gene Degeneration of Motor Neurons Which Are Nerve Cells In The Spinal Cord. Poor Muscle Tone, Absence of Deep Tendon Reflexes

� � OMIM MUSCULAR DYSTROPHY, DUCHENNE TYPE; DMD #310200 http: //www. ncbi. nlm. nih. gov/entrez/dispomim. cgi? id=310200 DYSTROPHIN; DMD #300377 http: //www. ncbi. nlm. nih. gov/entrez/dispomim. cgi? cmd=entry&id=300377. Pub. Med Houben F, Ramaekers FC, Snoeckx LH, Broers JL. Role of nuclear lamina-cytoskeleton interactions in the maintenance of cellular strength. Biochim Biophys Acta. 2006 Sep 19; Maeda M, Nakao S, Miyazato H, Setoguchi M, Arima S, Higuchi I, Osame M, Taira A, Nomoto K, Toda H. Cardiac dystrophin abnormalities in Becker muscular dystrophy assessed by endomyocardial biopsy. Am Heart J. 1995 Apr; Kanagawa M, Toda T. The genetic and molecular basis of muscular dystrophy: roles of cellmatrix linkage in the pathogenesis.

J Hum Genet. 2006 Sep 13; Beroud C, Tuffery-Giraud S, Matsuo M, Hamroun D, Humbertclaude V, Monnier N, Moizard MP, Voelckel MA, Calemard LM, Boisseau P, Blayau M, Philippe C, Cossee M, Pages M, Rivier F, Danos O, Garcia L, Claustres M Multiexon skipping leading to an artificial DMD protein lacking amino acids from exon 45 through 55 could rescue up to 63% of patients with Duchenne muscular dystrophy. Hum Mutat. 2006 Oct 13; Ervasti JM. Dystrophin, its interactions with other proteins, and implications for muscular dystrophy. Biochim Biophys Acta. 2006 Jun 7;