Inherited Muscular Dystrophies, Lectures 11-12, Dr. B.
Inheritance
Incidence
Onset
Duchenne MD
XLR
1/3000 males
3-5yrs
Becker MD
XLR
1/5 of DMD
Later
Limb-girdle
(LGMD)(ARMD)
AR
Presents Initially
Slow Running, Difficult to rise (Gower’s sign) and climbing stairs
Progression Wheelchair by age 12
IQ
Specific
Symptoms
Death
Investigation of patient with muscular dystrophy
Histological
Primary Cause
20 points below normal

Pseudohypertrophy of calf muscles

Muscle weakness in limb girdles and proximal limbs

Serum Creatine Kinase elevated 50-100 fold due to leak from damaged muscle fibers

Segmental Necrotic loss of muscle fibers
-Necrotic loss of muscle fibers
-Early attempts @ regeneration fail
-Fibers of reduced caliber produced
-Adipose/connective tissue eventually replaces
Mutation in Dystrophin Gene
95% frameshift deletion/duplication a.
OUT-OF-FRAME
Deletion/duplication (premature termination of translation
Similar but milder
Ambulant after 16
Normal

Cardiomyopathy more frequent

Muscle weakness in limb girdles and proximal limbs

Serum Creatine Kinase elevated 50-100 fold due to leak from damaged muscle fibers

Segmental Necrotic loss of muscle fibers
By age 17
1.
Characteristic clinical phenotype and high serum CK a.
Multiplex PCR i.
Deletion of duplication
1.
In frame = Becker
2.
Out of frame = Duchenne ii.
No deletion
- muscle biopsy
2. Muslce biopsy
a-dystrophin deficient
i-western blot
1-Absent dystrophin = Duchenne
2-Truncated dystrophin = Becker
ii-Multiplex PCR
1-Deletion or duplication
a- Inframe = Becker
b-Outframe = Duchene
2-No Deletion
a. Furhter DNA Analysis
If patient has clinical phenotype, high serum CD and Myopathic EMG
1. Muscle biopsy followed by dystrophin immunostaining. If no staining then do combined multiplex PCR and Westernblot (no sure)
Mutation of Dystrophin Gene
85% have in reading frame and milder phenotype
“IN-FRAME” deletion/duplication therefore intact
1-Variable severity
2-Involves shoulder and pelvic girdle muscles
3. eventually difficult to differentiat frm BMD
Characteristic clinical phenotype, high serum CK and myopathic EMG
1.
muscle biopsy
2.
dystrophin immunostain
3.
normal
4.
alpha sarcoglycan immunostain
5.
deficient
6.
immunostan, western blot for alpha,beta,gam ma and sigmal sarcoglycans
7.
sarcoglycanopat hy
Sarcoglycanopathy
(SCARMD also) alpha mutation in sarcoglycan
Emery-Dreifuss (EMD)
XLR
1-Mild
2-Like BMD
3-Elbow,neck, spine
4-scapulohumeroperoneal weakness, joint contracture, cardiac arrhythmias
Characteristic clinical phenotype, high serum CK and Myopathic EMG
1.
Muscle biopsy
2.
dystrophin immunostain
3.
normal
4.
alpha sarcoglycan immunostain
5.
Normal
6.
Both Merosin and Calpain immunostain
7.
Normal
8.
Emerin immunostain
9.
Deficient = EmeryDreifuss
10.
Normal =?
Oculopharyng eal (OPMD)
AD
Greater than 50 yr
1-Eyelid ptosis
2-dysphagia
Fascioscapulohumera
AD
1/20,000
1Fascial weakness
a. This is what separates it from other MD
2-shoulder weakness
a-Scapula Winging
Gene is unknown
Deletion of DRZ4 , a 3.2 kb tandem repeat
Located on 4Q near the telomere

Pathophysiology of Disease
Immunohistoche mical detection of dystrophin in sarcolymmal stain
Diagnostic
Testing:
Confirmation of clinical tests
Pre-natal
Diagnosis
70% of Both DMD and BMD are caused by large disruptions of the dystrophine gene a.
Detected by multiplex PCR or Souther Blot i. both identifiy deleted exons in patients
30% have small gene disruptions (deletions/insertions, splice mutations and point mutations)
a. detected like other mutations (PCR, sequencing)
b. RT-PCR has the added adv of detecting abnormal splicing
-PTT (protein truncation Test)
a. In vitro transcription and translation to detect premature termination of translation (seen in majority of DMD)
2 hotspots:1-20, 45-53
60% Deletion of 1 or more exons
6% Duplication of one or more exon
Caused by total lack of dystrophin
Secondary deficiency of Sarcoglycan complex formation b/c dystrophin req’d for assembly
Associated with shorter dystrophin molecule:
Both ends intact, partially functional
Western Blot will show band for
Dystrophin in BMD patient, lower
(faster moving) b/c smaller
1. Sarcolemmal membrane damage during muscle contraction
2. Calcium influx
3. Actiation of intracellular proteases leads to necrosis
4. Small fibers suffer less mechanical stress and may be spared
Interrupted staining Lack if Dysrophin observed in DMD patients, revertant fibers occasionally observed from 2 nd
mutation that restores reading frame
Patchy staining in symptomatic female carriers

XLR and raised CK and one of:

Immunohistochemical
Same:
Will show shorter Dystrophin in
Western Blotting staining showing deficiency in Dystrophin

Western Blot showing
Defficiency

Deletion/Duplication on multiplex PCR/Southern

Other Delete. Mutations by systematic screening of gene
1.
Known mutation a.
DNA diagnosis at CVS/Amniocentesis
2.
Unknown mutation a. Fetal muscle biopsy
Lack of alpha sarcoglycan
Dystrophin bridge cannot from properly
Dystrophin immunostaining is normal
Muscle staining looks relatively normal at first
Later looks like BMD with interrupted staining
Dystrophin Protien
1.
rod shaped molc
2.
localized in srcolemmal membrane in skeletal muscle fibers
3.
N-terminus binds cytoskeletal actin
4.
Rod-shaped domain contains 24 units of a 109aa seq
5.
H1-4 are for praline rich, hinge regions that add flexibility
6.
Cystein rich domain
7.
C-terminus is highly conserved in evolution (rodents, birds) a.
associates with complex group of sarcolemmal glycoprotiens i.
form two distinct subcomplex:
1.
sarcoglycans
2.
dystroglycans
Dystrophin acts as a mechanical bridge connecting the actin cytoskeleton and the extracellular matrix
MECHANICAL REINFORCEMENT OF THE SARCOLEMMA

Carrier
Gene discovery
Carrier Detection
1.
Difficult to establish a.
a women amy have more than 1 affected child despite not being a carrier i.
G onada mosaic proportion of her germ cells may hae systrophin gene mutation ii.
iii.
Approximately 14% of mothers
Emperical risk of 10-30% is quoted if it is determine that a subsequent child has inheritied the same X-chromosome as an older affected brother iv.
PRENATLA TESTING may be offered to all mothers with a DMD child irrespective of carrier status.
2.
Pedigree analysis may indicate obligate carrier a.
affected son + another affected male relative
3.
pedigree analysis may be strongly suggestive (if 2 or more sons effected)
4.
elevated serum CK (often overlaps with normal level)
5.
Patch immunohistochemical staining (muscle biopsy)
6.
dosage analysis of souther bloot to detech ½ the signal
1/3 of all DMD patients are sporadic cases
a. Their mothers are not carriers
b. De novo germline mutations
2/3 of patients inherit their mutations from a carrier mother afamily history may be positive
FEMALES may have disease
May express phenotype due to skewed x-inactivation
Rarely x autosome translocation
8% of female carriers
Females have increased risk of cardiac involvement in carriers
Clinical Correlation
DMD Management
A.
Weight control to prevent obesity
B.
Physical therapy to promote mobility and prevent contractures
C.
IEP, such as speech therapy
D.
Routine monitoring for cardiomyopathies
E.
Early non-invasive pulmonary care
Postional Cloning
Xq21
Gene is the largest in the human genome
Large size make it a frequent target for mutations
Knowing exactly wh exons are dleted allows one to figure out if the translational reading frame is maintained.
IV. Clinical Features
A.
Neonatal period:
1.
High serum CK (creatine kinase)
2.
Muscle fiber necrosis
B.
0-3 years:
1.
Motor development delay, e.g. the child does not begin walking until 18-19 months old
2.
Abnormal gait and clumsiness
3.
Frequent falling
4.
Difficulty running and/or climbing stairs
C.
3-6 years:
1.
Lordosis- increases with increasing age
2.
Appearance of Gower’s sign
3.
Enlargement of muscles- calves
4.
Tendency to toe-walk, with feet externally-rotated
D.
6-11 years:
1.
Progressive weakness of limbs and torso
2.
Sparing of cranial muscles
3.
Sparing of external anal sphincter
4.
Disappearance of stretch reflexes
5.
Abrupt decrease in functioning
6.
Appearance of joint contractures
E.
Second decade of life:
1.
Quit walking
2.
Arm weakness
3.
Respiratory difficulties
4. Paraspinal muscle weakness leads to kyphoscoliosis