Muscular Dystrophy

Mechanisms of Disease
DG O’Donovan
Consultant Neuropathologist
 Presentation (1)
• A mother brought her 4 year old
son to clinic because he was not
able to play like the other children:
He could not run as well, could not
hop, and he walked on tip toes.
• O/E his calf muscles were big, he
was weak, and could not rise from
sitting on the floor when his arms
were crossed in front of him.
 Presentation (2)
• He was normal at birth.
• But he began to walk at 2 years old – quite
late (normal 97% walking at 18 months)
• Serum Total Creatine Kinase: 16,000 iu/l
(normal 0-200 iu/l) ie 80x normal CK
• Muscle Biopsy:
Muscle Biopsy (a)

F/S H&E obj x10

Muscle Biopsy (a)

F/S H&E obj x40

Muscle Biopsy (a)

F/S H&E obj x40

 Presentation (3)

• His elder brother 5 ½ years was

also examined and found to be
weak
• CK 11,000 iu/l
• Muscle biopsy:
Muscle Biopsy (b)

F/S H&E obj x10

 Muscle Biopsies
• Both muscles were tested with antisera to
Dystrophin, using Dys 1, 2 & 3 antibodies
• All three antibodies gave negative results
in both boy’s muscle samples
 Questions
• Why can they not rise without using
their arms?
• What did Gowers describe?
• What is the diagnosis?
• How would you investigate the
brothers further?
 Answers
• Weakness in knee and hip extensor muscles:
mainly quadriceps femoris
• Gowers’ manoever / sign qv
• Muscular Dystrophy with absence of
Dystrophin = Duchenne Muscular Dystrophy
 Investigations
• Test sample of DNA from peripheral
blood leucocytes or Muscle Tissue DNA
• Multiplex Polymerase Chain Reaction to
look for deletion or duplication in gene
– picks up hostspots 59% mutations…
• If negative do Protein Truncation Test
[PTT] (RNA strategy) or Single
Stranded Conformation Polymorphism
[SSCP] (genomic DNA strategy)
• Muscle Western Blot Analysis
 Investigations
• PTT or SSCP to look for point
mutations, small deletions, small
insertions, splice site mutations, intronic
rearrangements or promoter mutations
• ~90% of cases may be diagnosed by
these genetic methods
• Detection of Virtually All Mutations
(DOVAM)

• Muscle Western Blot Analysis (protein)

 Muscular Dystrophy
• Genetically determined disorder of muscle
(a form of myopathy)
• Generally progressive ->can be eventually
fatal
• Leads to destruction of the contractile
elements of muscle (i.e.muscle fibres)
• Replacement by non contractile elements
(fat and fibrous tissue)
 MD Types & Inheritance
1) Duchenne Muscular Dystrophy XR
2) Becker Muscular Dystrophy XR
3) Emery-Dreifuss XR & AD
4) Limb-Girdle Muscular Dystrophy AD & AR
 Leyden-Möbius (pelvifemoral)
 Erb (Scapulohumeral)
5) Landouzy-Dejerine (Facioscapulohumeral)AD
6) Congenital Muscular Dystrophies AR
7) Oculopharyngeal Muscular Dystrophy AD/AR
8) ? Dystrophia Myotonica AD
 Diseases of Interest for this
Seminar

1) Duchenne Muscular Dystrophy (DMD)

2) Becker Muscular Dystrophy (BMD)
3) X-linked Dilated Cardiomyopathy (XLDCM)
(NB not strictly a muscular dystrophy – it is
a cardiomyopathy)
 DMD 1
• X-linked recessive disorder affects Boys
• Incidence 1/3,500 live male births
• Average age of presentation 3.5 years
• Delayed motor function (late to walk), frequent
falls
• Gower’s Manoeuvre positive (using hands to
push up on thighs legs in order attain an erect
posture) (sign of muscle weakness in pelvis)
 DMD 2
• Typical hypertrophy / pseudohypertrophy of calf
muscles – enlarged due to fibrous and fatty
tissue
• Limb girdle distribution of muscle weakness
• Winging of scapulae
• Inexorable progression of weakness from 7-8
years
• Wheel chair dependent from 10 to 12 years of
age
• Have characteristic contractures due to the
fibrosis
 DMD 3

• Scoliosis
• Cardiomyopathy often develops
• Intellectual impairment
• Recent discovery of night blindness in
patients due to retinopathy and red/green
colour blindness
• Death (in spite of modern medical
management) at 16 to 21 (25) years of age
Duchenne Musclar Dystrophy
 Other Manifestations of DMD
• Smooth muscle: bladder paralysis,
paralytic ileus, gastric dilatation
• Vascular : Trophic changes in skin,
cyanotic mottling, risk of severe blood loss
(blood vessels don’t contract down
properly)
• Severe osteoporosis of long bones (due to
inactivity of limbs)
 DMD Histopathology
• Fibre size variation
• Muscle fibre necrosis
• Hypercontracted hyaline fibres
– Very eosinophilic fibres may be seen
• Muscle fibre regeneration
– Basophilic fibres?
• Immature internal fibre architecture
• Internalised nuclei
• Endomysial fibrosis and fat replacement
• Myopathic grouping
Duchenne Muscular Dystrophy
Dystrophin I (Pos Control) Dystrophin I (Test)
 BMD

• X-linked recessive disorder

– affects Boys
• Incidence 1/17,500 live male births
• Able to walk after 12 years of age
• Typical hypertrophy / pseudohypertrophy
of calf muscles – like Duchenne
• Wheel chair bound 18 to 20’s
• Life expectancy early 40’s
 BMB 2
Phenotype now recognised to be wider than
previously recognised:

• Patients with quadriceps wasting and

weakness
• Patients with muscle hypertrophy alone
• Patients with exercise induced cramps
• Increased longevity (>40 years but
Cardiomyopathy is significant and heart
transplants may be necessary)
 Probable Becker
Muscular Dystrophy
D/BMD
BMD Histopathology

• Similar to DMD but milder

• More chronic features
histologically:
– gross fibre size variation
– fibre splitting
Mild Dystrophinopathy
 Mild Dystrophinopathy

Beta Dystroglycan Dystrophin I Dystrophin III

 Mild Dystrophinopathy

Dystrophin III (Pos control) Dystrophin III (Test)

Objective x40
 XLDCM
Rapidly progressive almost exclusively a
cardiac disorder
Onset in teenage males
Heart failure due to cardiac muscle
weakness & cardiac dilation
Cardiac arrhythmias
Death from congestive cardiac failure in 1
to 2 years of diagnosis
 Initial Cardiac Abnormalities in
DMD & BMD
Sinus tachycardia
Tall R1 in V1 on electrocardiogram (ECG)
Prominent Q in I, aVL, V6 or II, III & aVF
Increased QT dispersion
Echo: Normal or Regional wall motion
abnormalities
57% of patients over 18 years have cardiac
abnormalities
 Histopathology of Myocardium

Left ventricular posterobasal and lateral walls:

Replacement of myocardium by:
Fibrous tissue
Fat
 What are the links between
these three conditions?
• Cardiomyopathy is a feature of all three
• In 1984 Kingston & Harper proved that DMD
and BMD were allelic as had been suspected
by Peter Emil Becker
• XLDCM is also allelic
• In 1987 the gene responsible was cloned and
named “Dystrophin”
• Dystrophin is implicated in all three conditions
• Knowledge of the various mutations and an
idea of their pathogenesis is developing
Genomics & Proteomics

Dystrophin Gene
 Xp21.3

5’ 3’ DNA
79 Exons 3Mb

mRNA
14kb

N C Protein
427kDa
3685AA
 Dystrophin Protein
Cysteine Rich Region
Rod

NH2- -COOH

Actin b dystroglycan Actin

a Actinin
Syntrophin
a Dystrobrevin
 Mutation Rate

• There is a high mutation rate in such a

large gene so that:
• 1/3 of cases have a family history
• 1/3 are new mutations in the mother
• 1/3 are new mutations in the boy
 Dystrophin Isoforms
Dp427l Lymphoblastoid (no protein)
Dp427c Brain Cerebral Cortex
Dp427m Muscle
Dp400 Muscle
Dp427p Brain Purkinje Cells Cerebellar Cortex
Dp260 Retina
Dp140 Peripheral Nerve Axons
Dp116 Peripheral Nerve Schwann Cells
Dp71 Liver, Brain, etc
Dp40 Early Embryonic
 In Frame Deletion
THELAWMAYLIEANDLETMENDIE
Translation
THE LAW MAY LIE AND LET MEN DIE
Deletion
Del (MAYLIEAND) 9 bases

THE LAW LET MEN DIE

 Frame Shifting Deletion

THELAWMAYLIEANDLETMENDIE
Translation
THE LAW MAY LIE AND LET MEN DIE
Deletion
Del (LIEANDL) 7 bases

THE LAW MAY ETM END IE

Stop Codon
 Dystrophin Gene

BMD

D/BMD

DMD

Hot Spots for Mutations

Genetic Mechanisms DMD
Frame shift deletions

Premature stop codon

Arrests protein synthesis

No Dystrophin protein in membrane

 Genetic Mechanisms BMD

In frame deletions

Protein misses an internal section

Abnormal Dystrophin protein in membrane

Abnormal in amount and partial function

 D/BMD
Intermediate Severity
A) In frame deletions

Loss of important functional segment

B) Frame shift deletions

Recovery due to exon skipping

 Manifesting Carriers

Females carrying one abnormal gene

May be symptomatic (adverse Lyonisation)
Manifesting Carrier Status may be familial!
ie Lyonisation under genetic control
17% muscle weakness
8% cardiomyopathy
Mosaic of Pos & Neg fibres by IHC i.e.
multiclonal – some of the cells are
expressing the mutated proteins and
some of them are not.
Manifesting Carriers

• CK range 10-2137 iu/l

• 62.4% of 125 carriers had
[CK] > 95 percentile for adult
women (86 iu/l)
vs
• 5.5% of 200 controls
Interesting phenomenon
 Revertant Fibres
Exon Skipping

DMD: Frame Shift Deletion with loss of

protein (-exon 44)

Exon skipping in a few fibres

(–exons 44-45, 43-44 or 42-44)

Some fibres able to produce Dystrophin

 Muscle Fibre Structure
Satelite cell
DAG’s Dystrophin

Actin Myosin
Myonuclei
Muscle Fibre Necrosis

Ca2+ Delta Lesion

Influx of Calcium Ions due to defective membrane

Exacerbated by Exercise
Muscle Fibre Regeneration

Proliferation and differentiation of Satellite cells

 End Stage Muscle Disease

Replacement by Fat and Fibrous Scar Tissue

 Tissue Specific Isoforms

• Non progressive mental retardation

Dp71 & Dp140 synaptic plasticity
Electroretinogram abnormalities
?? Night blindness
Dp260 (outer plexiform layer of retina)
 Tissue Specific Isoforms
XLDCM

Mutations in the muscle (Dp427m) promoter

Selective cardiac muscle disease

C & P promoters produce Dp427c & Dp427p

Near normal skeletal muscle

 Different Mutations in One Gene may cause
wide phenotypic spectrum
In the case of A/C Lamin several disorders are
produced:
• Autosomal Dominant Emery Dreifuss
Muscular Dystrophy ADEDMD
• Cardiomyopathy
• Dunnigan Type Lipodystrophy
• (familial partial lipodystrophy limbs & trunk, not face)
• etc
 The Future

Heuristic approach to therapy

 References
• Tews DS & Goebel HH Diagnostic
immunohistochemistry in neuromuscular disorders
Histopathology 2005;46:1-23.
• Duchenne Muscular Dystrophy Emery A & Muntoni F 3rd
Edn Oxford University Press, Oxford UK, 2003
• Neuromuscular Disorders: clinical & molecular genetics
Emery AEH, John Wiley & Sons, Chichester UK, 1998
• Handbook of muscle diseases Lane RJM, Marcel Dekker
New York, USA, 1996
 References
• Pathology & Genetics: Karpati G, Structural and
Molecular Basis of Skeletal Muscle Diseases ISN
Neuropath Press, Basel 2002.