wk4-Duchenne Muscular Dystrophy

Summary

-DMD= a severe and progressive X-linked genetic disorder caused by mutations in the dystrophin gene resulting in protein deficiency -wheel chair dependent in early teens -central portion of the protein acts as a linker between important functional domains at the N- and C-terminal ends -dystrophin gene=prone to internal deletion because of its very lrg size and long introns with repetitive sequences -genetic testing can determine underlying disease causing mutation >can make big databases and records of the many diseases that might occur

Multiple ligation-dependent probe amplification(MLPA)

-DNA sequencing unable to identify mutations -paired blue=normal -red peaks=sample(125-475bp for individual exons) -large exons can require partly overlapping probes >can use overlapping probes -deletion of seven exons=arrows -blue peak reduced by ~50% =heterozygous deletion -peak order is sometimes not the same as for gene exons -always include internal control→make sure that experiment has worked

The effect of in-frame deletions of central exons in the dystrophin gene

-Large deletions in dystrophin gene: >can remove may of the central exons >are associated with Becker muscular dystrophy(DMD) if reading frame is not disrupted(in-frame deletion) >lose the spectrum and not the C and N terminal regions >reading frame not disrupted >no premature termination codon(TER)

the effect of out-of frame deletions of central exons in the dystrophin gene

-Large out of frame deletions of central exons in the dystrophin gene: >single central exon with a total # of nucleotides not exactly divisible by 3 is deleted→frameshift >premature termination codon(pTER)→occurs within next exon→trigger nonsense-mediated decay of dystrophin mRNA >failure to make protein→DMD

Potential Complications of DMD

-Scoliosis: curvature due to contractures + muscle weakness -Cardiac: •dilated cardiomyopathy •arrhythmia-shortness of breath and fatigue -Respiratory: •progressive weakening of the diaphragm •pneumonia/other respiratory infections •respiratory failure -cognitive impairment(non-progressive) -permanent, progressive disability •decreased mobility •decreased ability to take care of self -death: •DMD affected individuals are killed by failure of the heart muscles by age 25

Example of DMD pedigree 2

-X linked muscular dystrophy: • the women marked with dots are obligate carriers of the disease gene •carriers→have offspring who are affected/carriers(female)

Diagnosis of DMD

-aim=to provide an accurate and prompt diagnosis→initiation of appropriate interventions, counting support and education and minimising length and impact of potentially protracted diagnostic process -should be done by a neuromuscular specialist→assess child clinically and rapidly access and interpret appropriate investigations in context of clinical presentation -family follow up and support after diagnosis is often augmented by supported from geneticists and genetic counsellors

Functional role of dystrophin protein

-anchors the cytoskeleton of muscle cells to the ECM via the dystrophin glycoprotein complex -complex includes sarcoglycans(mutations cause limb-girdle muscular dystrophies) and dystroglycans. -muscle cells that lack dystrophin are mechanically fragile, and fail after a few years causing progressive weakness -contractions and relaxations will completely disrupt structure

Confirmation of diagnosis

-blood samples: >testing with blood samples is always necessary even if DMD is first confirmed by the absence of dystrophin protein expression on muscle biopsy -genetic test: >results of genetic test provide the clinical info required for genetic counselling, prenatal diagnosis, and consideration for future mutation specific therapies -muscle biopsy: >could be done depending on clinical situation, availability of genetic testing and the facilities in the centre where patient is seen >needle biopsy might be appropriate if testing is for DMD only >conchotome technique provides a larger sample than single-core needle biopsy and does not require an open surgical procedure

Cause of Duchenne Muscular Dystrophy

-by mutation in the gene that produces an important muscle protein called dystrophin >mainly deletions in the dystrophin gene (DMD; locus Xp21.2) -dystrophin is a common regulator of beta-catenin and APC: •downregulation of dystrophin downregulates APC •genetic modifier of APC mutation

Summary of inheritance of muscular dystrophy

-carrier mother + X unaffected father •mother: 1 mutated X + 1 normal X •Father:1 normal X + 1 normal Y Possible offsrping: 1-unaffected female: •normal X from mother + normal X from father 2-Carrier female: •normal X from father + mutated X from mother •normal X→enough functional dystrophin produced→not affected a lot but may show symptoms 3-unaffected male: •normal X from mother+ normal Y from father 4-affected male: •mutated X from mother + normal Y from father •X not normal→no dystrophin production

Gene identification facilitated by disease associated chromosomal abnormality

-chromosome: an affected boy with a cytogenetically visible deletion at Xp21.3 and a woman with a balanced Xp21;21p12 translocation -comments: positional cloning strategies identified genes within the deletion/translocation breakpoint→implicate that giant dystrophin gene -Balanced translocations and inversions are very useful→no net loss of DNA -underlying gene is expected to be close to or located at breakpoint -size of gene and fact that there are many exons to ligate to make gene functional →deep sequencing and exome sequencing

Symptoms:Early

-delayed onset walking -difficulty in performing a standing jump -waddling when walking up -enlarged calves

Multiplex ligation-dependent probe amplification(MPLA)

-detects copy # changes over broad ranges of DNA lengths -can scan for intragenic deletions and duplications by assessing copy # of exons >used to show that u can have exon duplication -useful 4 disorders with high frequency intragenic deletions e.g. DMD -can complement ability of DNA sequencing to scan for point mutations

Post diagnosis for patients of DMD

-diagnosed by muscle biopsy and genetic testing of dystrophin if necessary

Symptoms: Later

-difficulty getting up from a chair -loss of ability to climb stairs -wide gaied w/ balance problems

Locus of DMD on the X chromosome and other diseases associated with the X chromosome

-dystrophin gene (DMD; locus Xp21.2) -other disease associated with X chromosome: •Hemophilia: caused by mutation in the gene encoding coagulation factor VIII(F8, Xq28). •Fragile X: caused by mutation in FMR1 gene(FMR1,Xq27.3) •Rett syndrome(RTT): caused by mutation in the gene encoding methyl-CpG-binding protein-2 (MECP2, Xq28)

More symtoms

-fatigue -mental retardation -muscle weakness: begins in legs and pelvis, less severe in arms, neck -poor motor skills(running, hopping, jumping) -frequent falls -rapidly worsening weakness -progressive difficulty walking by age 10→might need braces→soon after most confined to wheelchair

Dystrophin protein structure

-functionally important parts are domains in the N-terminal and C-terminal regions •actin binding domain •lots of spectrin-like repeats •domains that bind DAP components to form link with ECM -dystrophin provides a structural link between actin filaments of cytoskeleton and through membrane bound dystrophin-associated protein (DAP) complex the ECM

What is muscular dystrophy?

-genetic disease that affects skeletal muscles -characterized by: •progressive muscle weakness •wasting and loss of motor skills -X-linked recessive most common •Duchenne Muscular Dystrophy(DMD) •Becker's Muscular dystrophy(BMD) -incidence: 1/3500 males born -onset age from infancy to adulthood -most end up wheelchair-bound

Gower Maneuver

-kind of test for DMD -patients work out a process to stand up -affected boys stand up by bracing their arms against their legs because of weakness in proximal muscles -go up stairs on all 4 years

Summary of genetic causes of muscular dystrophy

-main cause: •X linked disease •mutation in X chromosome inherited from parents •recessive mutations -female carriers/affected •females affected only if both X-chromosomes mutated •single mutation(carrier): normal as enough functional dystrophin produced >usually unaffected but carrier of disease> affects later generation •double mutation(affected): very unlikely and rare -males-single X chromosome(Affected): •mutation in X-chromosome→no dystrophin produced

Duchenne Muscular Dystrophy

-most common of several childhood muscular dystrophies -inherited X-linked recessive disorder -progressive degeneration of muscle -onset generally b4 6yrs -muscle loss and low muslce tone usually not noticed until observation of unusual walking and/or talking ~ age 3

Clinical Features

-most common type of dystrophy:1/3500 -early onset:signs appear b4 6yrs of age -delayed development of motor skills -difficulty in keeping balance -progressive muscle weakness/fatigue -respiratory muscles eventually involved -pseudohypertrophy:muscles become swollen with deposits of fat and fibrous tissue -contractures:shortening and hardening of muscles, tendons, or other tissue→leading to deformity and rigidity of joints -Wheelchair dependent by early teens -death in late teens, early 20s

Muscle histology of DMD patients

-muscle biopsies: •take muscle biopsy sample and embed in paraffin wax •then slice it making 5ul sections •place sections on a slide and do immunohistochemistry •immunohistochemistry: use antibody against dystrophin→hybridizes to dystrophin →brown stain -if positive control works then we are right=normal one -carrier is in between -Normal: regular cellular architecture with dystrophin(brown) on all outer membranes -DMD: Irregular architecture and dystrophin absent from surface of muscle fibers -Carrier: Biopsy from a female carrier of DMD showing patchy stains around outer cell membrane of cells

Effect of DMD on the muscle fiber membrane

-muscles are made up of fibre bundles (cells) -interdependent proteins along the membrane surrounding each fiber serves to keep muscle cells working properly -one of the proteins=dystrophin >if dystrophin is missing→DMD -consequences of DMD=constant muscle contraction/relaxation→weaken and destroys muscle

Pedigree analysis: Jason's family tree

-referral by GP to paediatrician -reported observations: •jason aged 3 yrs •delayed walking compared to 2 older female siblings •difficulty running and riding tricycle •climbs up stairs on all 4s -pedigree shows: •Jason has picked up a de novo mutation→parents are not carriers

Example of DMD pedigree 1

-shaded squared=affected males -dots in circles=carrier females -the X chromosome carrying the disease-causing mutation can be tracked through the family

Screening for exon deletions in DMD using PCR

-using exon specific primers nine selected exons of the dystrophin gene were amplified from the DNA of 20 affected boys -PCR product run on electrophoretic gel >each exon is characterized to give a band of specific size -boys have single X chromosome→deletion=missing bands >exon deletions=arrows -Lane 3: large deletion/technical failure >could be because they are continuous -Boys with no deletions may have deletions in other exons/point mutations/duplications causing loss of function of dystrophin

2 Types of Muscular Dystrophy-(Bushby et al., 2010)

-variable phenotyic expression relates to types of mutations and its effect on production of dystrophin -disorder in affected girls is much milder than in boys 1-BMD(Becker muscular dystrophy): inability to produce functional dystrophin •dystrophin: not 100% functional •limited function→less severe than DMD •milder allelic form of disease •loss of ambulation over 16yrs 2-DMD(Duchenne muscular dystrophy): inability to produce dystrophin •no dystrophin production/production of only non-functional dystrophin •no function→more severe disease •loss of independent ambulation by age of 13yrs

Incidence rates

-~1/3,500-5000 males is born with DMD -much rarer in females: •some symptoms:weaker muscles in the back, legs, and arms that fatigue easily -even carriers are affected in a way: •carriers may have heart problems, shortness of breath/failure to moderate exercise •10% of female carriers show some disease manifestations that might include or even exclusively affect cognitive and/or cardiac function •untreated heart problems: can be serious/life-threatening -spontaneous/inherited: •30% due to de novo(spontaneous) mutations •mostly inherited

Diagnosis of DMD

1-Blood creatine phosphokinase(CPK) test: •damaged muscles can release creatine kinase into blood •elevated levels of creatine kinase=muscle injury: trauma/muscular dystrophy •used by athletes 2-Electromyography(EMG): •measuring electric signaling to and from the muscle can rule out neurodegenerative diseases and confirm a muscle disease •painful 3-Muscle biopsy: •the microscopic analysis of a sample of muscle tissue can identify absence of dystrophin and characteristics associated with muscular dystrophies 4-Genetic testing: •test for mutations in muscular dystrophy related genes •can determine exact form of muscular dystrophy

Principle of MLPA

1-each target sequence(exon) has a pair of primers that hybridise to adjacent sequences within the target and carry unique end sequences 2-stuffer fragment provides a DNA spacer of variable length→different sizes of PCR products are generated 3-DNA ligase seals left and right probes→continuous sequence flanked by unique end sequences 4-probe pairs for multiple target sequences(exons) are simultaneously hybridised to their target sequences >they are then ligated→continuous sequences→simultaneously amplified in a multiplex rxn 5-PCR products are separated by capillary gel electrophoresis