The

molecular basis of the

muscular dystrophies

M2M 2015
Muscular Dystrophies Theme
Lecture 2

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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

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.

Dystrophin gene architecture (HUGE)

79 exons spanning 2.4Mbp, 14kb mRNA

1........................................................................................2.....................................................................................3........................................................................
..........................4.................................................................................................................................................................5............................................................
.....................................................................................................6...............................................................................................................7...................................
...........................................................................................................................................................................................................................................................
...........................................................................................................................................................................................................................................................
...........................................................................................................................................................................................................................................................
..........................................8................................9.....................................................................................................................................10....................................
.......................11.......................................................................12................................................................................................................................................13.
...........................................................14.15.............................................................16................................................................................17..................................
..........................18............................................................................................................19...........................................................20..............................................
..............21.....................................22........23....................................24..............25..............................................26....................................27................................
.....28.......................................29..........................30................................31.........32.................................................................33...................................................
................34..................................................................35...................................................................36..........................................................................................
........37..............................................................................................................................................................................................................................................
38.................................................................................39..................................................................40............................................................................................
41..........................................................................................................................42..........................................................................................43...........................
.....................................................................................................................................................................44..................................................................................
...........................................................................................................................................................................................................................................................
...........................................................................................................................................................................................................................................................
...........................................................................................................................................................................................................................................................
...........................................................................................................................................................................................................................................................
...........................................................................................................................................................................................................................................................
.............................................45.............................................................................................................................................................................................46.........
............................................................................................................................47...........................................................................................................................
..............................48.............................................................................................................................49........................................................................................
............................................................................................................................50...........................................................................................................................
51....................................................................52..................................................................................53.............................................54............55........................
.............56..........................................57.....................................................58..................59.........................................60..............................................61..............
..............62...........................................................................................................................63..........................................................................................................
..................64................................................................65.......................................................................................................66......................................................
..........................................................67.............................................................................................................................................................................................
............................68..................................................................69..................................................................70...............................................................................
.............................................71...................................................................72....................................................................73...........................................................
...........................................74..............................................................................75.........................................................................................................76.............
.....................................................................................................................................77...................................................................78...........................................
...........................................................................79

Dystrophin has mulKple isoforms

Chakkalakal
l FASEB
Chakkalakal
et ael t Fa
ASEB
2005 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

X DMD
BMD

(Can be variably dispensed with)

Most
important bits

Most
important bits

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

Example: DELETION OF EXON 3 (OLD D)
iiiiiiiiiTHEiiiiiiiiiiiBADANDiii------------iiiiiiiiiOGATETiiiiiiiiiiiiiiHEFATiiiiiiiiiiCATENDiiiiiiiii

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
DELETION OF EXON 2 (BAD AND)
iiiiiiiiiTHEiii----------------iiiiiiiiiiiiiiOLDDiiiiiiiiiOGATETiiiiiiiiiiiiHEFATiiiiiiiiiiiiiiiiCATENDiiiiiiiii

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
A
C
D

MulKplex PCR of the dystrophin gene

Primer sets of selected exons

Gel electrophoresis

Authors own

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

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/
Phagocytosis: Invasion of
Necro>c muscle bres are
bres by macrophages
pale on NADH stain
Images: hWp://neuromuscular.wustl.edu/

H & E stain

Cellular inltrate
Many small regeneraKng bres
Images: hWp://neuromuscular.wustl.edu/

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)

Normal

dystrophin staining
around the rim of
muscle fibers

Reduced
(BMD)

Images: hWp://neuromuscular.wustl.edu

Absent
(DMD)

Western blosng
Dys 6-10

Dys 2
Dystrophin

250

QuanKes the amount of a

protein in specic Kssue
Determines size of protein

250

Myosin
Coomassie
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