The molecular basis of the muscular dystrophies

M2M 2015

Muscular Dystrophies Theme Lecture 2

COMMONWEALTH OF AUSTRALIA

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Laing and Nowak Bioessays 2005

Dalakas et al NEJM 2000

Source: Prof K North

Genes and proteins

Genes are transcribed into mRNA, which is then translated to make a protein

The order of amino acids in the protein is determined by the nucleoKde sequence of the gene

The order of amino acids determines shape of the protein Shape of the protein determines its funcKon

Source: biowiki.ucdavis.edu

The average gene

Consists of 8-9 exons

Is spread across 3,000 bases

Produces a processed gene message ~ 1000 leWers (amino acids) long

Dystrophin gene

Localized to chromosome Xp21

Second largest gene known, occupying 1% of the X-chromosome and 0.1% of the enKre genome 79 exons spanning 2.4 megabases. mRNA is over 14kb. Exons account for only 0.6% of the gene- rest large intronic regions Large size makes it suscepKble to mutaKons: 1/3 of all mutaKons de novo

Diering transcripts occur in dierent Kssues

MutaKons in dystrophin cause DMD and BMD DMD: Duchenne muscular dystrophy BMD: Becker muscular dystrophy (milder phenocopy of DMD) Also: X-linked cardiomyopathy, X-linked cramps-myalgia syndrome, isolated quadriceps myopathy

Also

Duchenne muscular dystrophy

The most common human MD The dystrophin gene product is also known as dystrophin The large DMD gene causes producKon of several isoforms

Isoform = variant forms of the same protein Formed by alternaKve promoter usage and splicing of pre-mRNA

4 long isoforms (l, m, c, p): skeletal, cardiac, smooth muscle, brain

Smaller isoforms: central nervous system, reKna, kidney The predominant isoform found in skeletal and cardiac muscle is a 427 kDa protein predicted to contain 3685 amino acids.

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Dystrophin gene architecture (HUGE) 79 exons spanning 2.4Mbp, 14kb mRNA

Dystrophin has mulKple isoforms

Chakkalakal et al FASEB 2005 Chakkalakal et al FASEB 2005

Dystrophin has mulKple domains

Full-length dystrophin has four structural domains

1) A (vital) N-terminal acKn binding domain

2) a middle rod domain of spectrin-like repeats

- Shorter forms with fewer repeats remain variably funcKonal

3) a cysteine-rich domain

4) a carboxyl-terminal domain allowing assembly of the DAPC

Chakkalakal et al FASEB 2005

Dystrophin 2 500 000 base pairs, 79 exons, 427 kd protein

Most important bits

Most important bits

(Can be variably dispensed with)

X DMD

BMD

Structure of the dystrophin gene transcript indicaKng the reading frame and major funcKonal domains. Boxes represent in-frame exons, whereas interlocking forward and reverse arrows and notches indicate codons

spanning the exon:exon juncKons. JuncKon codon sequences are shown above the exons.

The geneKcs of Duchenne muscular dystrophy

In coding regions of genes there are three types of base subsLLons: Silent: i.e. no change in protein product Missense: Amino acid change in protein product Nonsense: Causes a premature stop in protein producKon

Other sorts of mutaKons in DMD: DeleKons/ inserKons

Frameshih: base added or lost from amino acid sequence Code out of frame (shihed) downstream

DuplicaKons

Nonsense mutaKons Premature stop signals

Normal : IiiiiiiTHEiiiiiBADANDiiiiiOLDDiiiiiOGATETiiiiiiiHEFATiiiiCATENDiiii

MutaKon changing A to E iiiiiTHEiiiiBADENDiiiiiiiOLDDiiiiOGATETiiiiiiiiiiHEFATiiiiiiiiCATENDiiii

THE BAD END OLD DOG ATE THE FAT CAT END CorrupKon of message, loss of gene product

15% of all gene mutaKons in DMD Usually not picked up on MLPA

Frameshih mutaKons

iiiiiiiiiTHEiiiiiiiiiiiBADANDiii------------iiiiiiiiiOGATETiiiiiiiiiiiiiiHEFATiiiiiiiiiiCATENDiiiiiiiii

Example: DELETION OF EXON 3 (OLD D)

THE BAD AND OGA TET HEF ATC ATE NDi iii

CorrupKon of geneKc message, loss of product - occurs in ~60% of DMD cases and is out of frame - Most common mutaKon picked up on MLPA

In-frame deleKons

iiiiiiiiiTHEiii----------------iiiiiiiiiiiiiiOLDDiiiiiiiiiOGATETiiiiiiiiiiiiHEFATiiiiiiiiiiiiiiiiCATENDiiiiiiiii

DELETION OF EXON 2 (BAD AND)

THE OLD DOG ATE THE FAT CAT END

DeleKon doesnt change overall gist of message

Dystrophin sKll produced but shorter than usual (= truncated protein)

Typical of BMD mutaKons

DuplicaKons

Responsible for about 5% of all DMD cases

DuplicaKons shihing the reading frame cause DMD

DuplicaKons preserving the reading frame cause BMD.

2nd most common DMD mutaKon idenKed on MLPA

http://www.wikidoc.org/index.php/File:Gene-duplication.png

Dystrophin acts as a cellular anchor

Dystrophin links the internal cytoskeleton to the extracellular matrix

The N (amino)-terminus of dystrophin binds to F-acKn and the carboxyl (C) terminus to the dystrophin-associated protein complex (DAPC) at the sarcolemma

The DAPC links the acKn cytoskeleton to the extracellular matrix

This stabilizes the sarcolemma during cycles of contracKon and relaxaKon

This also transmits force generated in the muscle sarcomeres to the extracellular matrix

The DAPC is also involved in cell signalling

Loss of dystrophin causes loss of the DAPC at the sarcolemma

Loss of this physical link renders the sarcolemma fragile Muscle bres become suscepKble to injury and degeneraKon during repeated cycles of muscle contracKon and relaxaKon

Nowak and Davies. EMBO reports 5:872-876, 2004

Dystrophin and calcium homeostasis

Dystrophin has also been proposed to play a role in calcium homeostasis

mdx mice are the animal model for DMD In mdx mice and DMD paKents resKng intracellular calcium levels are elevated in muscle

mdx muscles show enhanced calcium inux through calcium/stretch-acKvated channels, causing acKvaKon of the inammatory response

Elevated expression of inammatory mediators and chemoaWractants is seen in dystrophin-decient muscles prior to the onset of weakness

Dystrophic changes in muscle may relate to inammaKon due to aberrant calcium homeostasis

The consequence of dystrophin mutaKons

Absence of structural proteins in muscle Membrane instability

Calcium inux Apoptosis, necrosis InammaKon Fibrosis

Disrupted muscle architecture Signaling defects Secondary loss of other proteins Weakness

Dystrophin mutaKons cause DMD and BMD

MutaKons disrupKng the reading frame (= frame-shih) of dystrophin cause loss of dystrophin and cause DMD

In BMD mutaKons maintain the reading frame (= in-frame mutaKons) abnormal but partly funcKonal dystrophin

65% of DMD is caused by large parKal deleKons 5% is caused by duplicaKons of one or more exons DeleKons and duplicaKons: detected by mulKplex ligaKon-dependent probe amplicaKon (MLPA) analysis

Other paKents have small duplicaKons/ deleKons or point mutaKons

Not detected by MLPA, may be found by other methods i.e targeted sequencing

MulKplex PCR: DeleKon analysis in DMD

A B C D

B

A

C

D

Authors own

MulKplex PCR of the dystrophin gene Primer sets of selected exons Gel electrophoresis

Authors own

MulKplex ligaKon-dependent probe amplicaKon in DMD

/wiki/File:MLPA_in_GeneMarker.jpg Determines the relaKve copy number of all exons within gene simultaneously

/wiki/File:MLPA_in_GeneMarker.jpg

Normal muscle biopsy

Authors own

Dystrophic muscle biopsy

Authors own

Muscle pathology in DMD CharacterisKc ndings in dystrophies

Variable bre size Hypercontracted (opaque) muscle bres Muscle bre degeneraKon and regeneraKon Muscle bre internal architecture: Normal or immature Absent dystrophin in DMD Other membrane proteins

Sarcoglycans: Reduced Aquaporin 4: Reduced

Increased brosis within muscle

Immunohistochemistry = Fluorescent anKbody staining

1

2

3

Normal muscle

Absent protein

Decreased/ incomplete staining

Authors own

Early pathologic changes in DMD

Phagocytosis: Invasion of bres by macrophages

NecroKc muscle bres are pale on NADH stain

Images: hWp://neuromuscular.wustl.edu/

Images: hWp://neuromuscular.wustl.edu/

Phagocytosis: Invasion of bres by macrophages

Necro>c muscle bres are pale on NADH stain

Cellular inltrate Many small regeneraKng bres

Images: hWp://neuromuscular.wustl.edu/

H & E stain Acid phosphatase

Late dystrophic changes

Staining proper>es of immature muscle bres include: 1. H & E (leI): Basophilic bres 2. Alkaline phosphatase posi>ve (centre) 3. 2C bres: Intermediate staining on ATPase pH 4.3 (right)

Images: hRp://neuromuscular.wustl.edu/

Late-stage muscle pathology in DMD

Increased endomysial connec>ve >ssue Variable bre size Hypercontracted muscle bres Images: hRp://neuromuscular.wustl.edu/

Muscular dystrophy Immunostaining (dystrophin)

Images: hWp://neuromuscular.wustl.edu

Normal dystrophin staining around the rim of muscle fibers

Absent (DMD)

Images: hWp://neuromuscular.wustl.edu

Reduced (BMD)

Western blosng Dys 6-10 Dys 2

Coomassie

250 250

Myosin QuanKes the amount of a protein in specic Kssue Determines size of protein

Dystrophin

Authors own

Summary: Duchenne muscular dystrophy

Most common human muscular dystrophy Caused by out-of-frame mutaKons in dystrophin gene on Xp21 Most commonly large deleKons Less commonly duplicaKons or point mutaKons In-frame mutaKons cause Becker MD (milder phenocopy)

Second largest gene and protein product known to man Dystrophin is vital for structural integrity of skeletal muscle bres

Dystrophin loss causes a progressive dystrophic process in muscle bres