Atlas of Genetic Diagnosis and Counseling || Dystrophinopathies

Dystrophinopathies

The dystrophinopathies include a spectrum of muscle

disease caused by mutations in the DMD gene that

encodes the protein dystrophin. They are characterized

by a spectrum of muscle disease that ranges from mild

to severe. The mild end of the spectrum includes the

phenotypes of asymptomatic increase in serum con-

centration of creatine phosphokinase (CK) and muscle

cramps with myoglobinuria and isolated quadriceps

myopathy. The severe end of the spectrum includes

progressive muscle diseases that are classified as

Duchenne muscular dystrophy (DMD) or Becker

muscular dystrophy (BMD) when skeletal muscle is

primarily affected and as X-linked dilated cardiomy-

opathy (XLDCM) when the heart is primarily affected.

In this chapter, I will limit my discussion on DMD

and BMD.

DMD is one of the most common types of muscular

dystrophy in childhood, primarily affecting skeletal

and cardiac muscle. It is one of the most common of

all clinical genetic disorders. Its incidence is estimated

to be approximately 1 in 3,500 live male births. BMD

is a milder allelic form of dystrophin deficiency, affect-

ing 1 in 30,000 male births.

Synonyms and Related Disorders

Becker and Duchenne muscular dystrophy

Genetics/Basic Defects

1. DMD

a. Inheritance

i. X-linked recessive

ii. Exceptionally high mutation rate of 10�4 in

both sperm and eggs

iii. Approximately 1/3 of cases are due to new

genetic mutations

iv. Approximately, 2/3 of cases occurring by

inheritance of the disease-causing gene

from the carrier mother

v. Only males affected (as a rule)

b. DMD gene

i. Observation of a series of young females

affected clinically as Duchenne muscular

dystrophy with an X-autosome translocation

a) Breakpoint in the X chromosome in the

same place (Xp21.1)

b) Different autosomes involved for each

affected female

c) Mapping of dystrophin gene to chromo-

some Xp21

ii. The largest human gene, covering 2.5

megabases and including 79 exons

iii. The enormity of the DMD gene along with the

spontaneous mutation rate of each base pair

allows a high frequency of novel mutations.

c. Dystrophin, the product (protein) of the human

dystrophin gene (dys)i. Loss of dystrophin at the muscle membrane

clearly related to mutations in the gene

encoding dystrophin at band Xp21

ii. Cloning of the dystrophin gene by positional

cloning in the late 1980s constituted the ini-

tial proof that deletions in the Xp21 region

were associated with the disease

iii. The dystrophin gene (Muntoni et al. 2003)

a) The largest gene of the 30,000 genes that

encode proteins in the human genome

H. Chen, Atlas of Genetic Diagnosis and Counseling, DOI 10.1007/978-1-4614-1037-9_76,# Springer Science+Business Media, LLC 2012

687

b) Consisting of 79 exons spanning

more than 2.6 million bp of genomic

sequence

c) Correspond to about 0.1% of the total

human genome

d) Correspond to about 1.5% of the entire

X chromosome

iv. Southern blot analysis of affected boys with

a complete set of cDNA probes

a) Over 60% with detectable deletions

b) Six percent with duplications

c) A number of point mutations described

recently

v. Consequences of absent dystrophin in

skeletal and cardiac muscles in affected

patients

a) Muscle contraction leading to membrane

damage and activation of the inflamma-

tory cascade

b) Progressing to muscle necrosis, fibrosis,

and loss of function

d. The DMD phenotype most frequently due to

mutations that cause a disruption in the reading

frame (Wagner 2002)

2. BMD

a. Inheritance: X-linked recessive (same as DMD).

b. Also caused by mutations in the gene for dystro-

phin at Xp21.1.

c. The BMD phenotype most often due to muta-

tions that preserve the open reading frame but in

which portions of the protein are deleted. Frame

deletions of the long rod segment of the gene are

particularly forgiving and produce a mild pheno-

type (Wagner 2002).

3. Mechanism for female DMD and BMD

a. Turner syndrome with a dystrophin mutation

on the remaining X chromosome (Chelly et al.

1986)

b. Skewed X inactivation either in the female DMD

mutation carriers (Yoshioka et al. 1998)

c. Balanced X-autosome translocation patients

(Verellen-Dumoulin et al. 1984)

d. Uniparental disomy of the entire X chromosome

with mutations (Quan et al. 1997)

e. Co-occurrence of mutations in both dystrophin

and androgen receptor genes (Katayama et al.

2006)

f. Double dystrophin mutations on both

X chromosomes (Fujii et al. 2009)

Clinical Features

1. DMD (Roland 2000)

a. During early infancy

i. Asymptomatic

ii. Normal motor milestones

iii. Rare global developmental delay or

delayed achievement of early motor

milestones

b. Four to five years of age: onset of symptoms

i. A waddling gait

ii. Difficulty in climbing stairs due to pelvic

weakness

iii. Difficulty in running

iv. Toe walking resulting from tight Achilles

tendon

v. Inability to jump

vi. Frequent falling

vii. Neck flexor weakness with marked head

lag when pulling to sit from the supine

position

c. Progressive difficulty in rising from the floor

secondary to weakness of proximal pelvic gir-

dle and proximal leg muscle, resulting in the

Gower’s maneuver requiring the use of the

hands to “climb up the legs”

d. Pseudohypertrophy of muscles, especially calf

muscles: unusually firm and rubbery consis-

tency on palpation

e. Compensatory lumbar lordosis to maintain an

upright posture secondary to weakness of hip

extensors

f. Weakness of the arms (proximal more severely

affected than distal) apparent as the disease

progresses

g. Marked laxity of the shoulder girdle muscula-

ture with prominent spontaneous winging of the

scapulae

h. Inability to walk usually before 13 years of age

i. Rapid development of fixed skeletal deformities

following loss of ambulation

i. Equinovarus deformities of the feet

ii. Scoliosis

iii. Wheelchair confinement by adolescence

j. Unexplained cardiac arrest and/or

myoglobinuria: features of malignant hyper-

thermia during general anesthesia in rare

instances

688 Dystrophinopathies

k. Progressive and restrictive respiratory deficit

with nocturnal hypoventilation in the latter

teens to early 20s secondary to weak intercostal

muscles

l. Eventual respiratory failure requiring assisted

ventilation

m. Some degree of mental impairment is usually

present. Approximately 25% of patients have

IQ below 75, presumably due to the lack of

dystrophin in the brain.

n. Natural history

i. Progressive and predictable deterioration of

muscle function

ii. Cause of death: cardiopulmonary insuffi-

ciency in the late second or third decade

o. Female carriers

i. Usually asymptomatic

ii. Manifesting female carriers (rare): Occa-

sional, slow progressive myopathy of moder-

ate severity with elevated CPK (>1,000) and

associated symptoms in about 8% of carriers.

Following conditions induce expression of

the disease phenotype:

a) Random X-inactivation: The extent of

clinical involvement is dependent in

part upon the degree of skewed

X-chromosome inactivation in somatic

cells.

b) Turner syndrome having a single

X chromosome

c) X-autosome translocation that disrupts

the dystrophin gene and causes

nonrandom inactivation of the normal

allele on the other X chromosome

2. BMD

a. A more benign and variable presentation with

later onset and slower progression

b. Onset of symptoms after age 6–12

c. Progressive symmetrical muscle weakness and

atrophy

i. Proximal greater than distal

ii. Often with calf hypertrophy

iii. Weakness of quadriceps femoris may be the

only sign.

d. Activity induced cramping in some patients

e. Flexion contractures of the elbows may occur

late in the course

f. Wheelchair dependency, if present, after

16 years of age

g. Preservation of the neck flexor muscle strength

in BMD, differentiating it from DMD

h. Rare BMD patients not diagnosed until adult-

hood and never lose ambulation

i. A proportion of cases have some degree of men-

tal impairment

j. Heart failure from dilated cardiomyopathy,

a common cause of morbidity and the most

common cause of death, despite the milder skel-

etal muscle involvement

k. Death usually in the fourth or fifth decade

l. Female carriers

i. Clinical features similar to DMD female

carriers.

ii. About 5–10% of female carriers show some

degree of muscle weakness.

iii. Often with calf hypertrophy.

iv. May develop dilated cardiomyopathy.

Diagnostic Investigations

1. Determination of serum creatine kinase (CK-MM)

a. Fifty-fold elevation (BMD) to 100-fold eleva-

tion (DMD) in affected boys, resulting from

leakage of the muscle isoform

b. Serum creatine kinase levels: range of values

overlapping with the normal range in carrier

females, making the test less than definitive

2. Determination of other enzymes: grossly elevated

aldolase, SGOT, lactic dehydrogenase, and pyru-

vate kinase

3. Electrocardiography and echocardiography to

detect cardiac involvement

a. Electrocardiography

i. Abnormalities in the early stage of the

disease

a) Tall R-wave

b) Deep Q-wave

ii. Arrhythmias

iii. Progressive cardiomyopathy in the mid-

teens

a) Myocardial dilatation

b) Myocardial thickening

b. Echocardiography

i. Left ventricular dilatation and dysfunction

ii. Mitral regurgitation secondary to dilated

cardiomyopathy or associated mitral valve

prolapse

Dystrophinopathies 689

4. Radiography for scoliosis

5. Pulmonary function testing with negative inspira-

tory force and forced vital capacity as disease

progresses

6. Cytogenetic analysis

a. Males affected with DMD and other X-linked

disorders such as retinitis pigmentosa, chronic

granulomatous disease, McLeod red cell phe-

notype, glycerol kinase deficiency, and adrenal

hypoplasia as part of contiguous gene deletion

syndrome

i. High resolution chromosome studies

a) To detect visible deletions

b) To detect chromosome rearrangements

involving Xp21.1

ii. FISH analysis with probes covering the GK

andNRDB1 genes in addition to exons in the

DMD gene

b. Females with classic DMD

i. May have X chromosome rearrangement

ii. May have deletion involving Xp21.1

iii. May have complete absence of an

X chromosome (45,X)

iv. May have complete uniparental disomy of

the X chromosome

v. Warrant high-resolution chromosome studies

7. Electromyography (EMG) to distinguish a myo-

pathic process from a neurogenic disorder

a. Not diagnostic

b. Demonstrating characteristic myopathy

c. Reduction in the duration and amplitude of

motor unit action potentials

d. An enhanced frequency of polyphasic potentials

8. DNA analyses for diagnostic confirmation

a. Deletion of the dystrophin gene in 60% of

patients: 98% of all deletions which occur in

hotspots within the dystrophin gene can be rec-

ognized by multiplex PCR which amplifies

18–25 of the gene’s 79 exons from genomic

DNA obtained from blood samples

b. Duplications (6% of cases): the next most com-

mon mutation of the dystrophin gene, also

detectable by multiplex PCR amplification

c. Point mutations (1/3 of patients) difficult to

identify due to the large size of the gene

d. Enhanced single-strand conformation polymor-

phism (SSCP) and heteroduplex analysis:

highly sensitive and possible to detect approx-

imately 90% of patients with DMD

e. RNA-based methods, such as reverse-

transcriptase PCR (RT-PCR)

f. Protein truncation test: to rapidly screen the

DMD gene for translation terminating mutations

9. Muscle biopsy (needle biopsy vs. open biopsy under

general anesthesia), frequently performed when a

clinically suspected DMD patient does not have a

largedeletionorduplicationbygenomicDNAtesting

a. Histology

i. Rounding of muscle fibers

ii. Marked variability in muscle fiber size

iii. Increased central nucleation

iv. Fiber splitting

v. Presence of necrotic and regenerating fibers

along with large, round hyaline fibers

vi. Virtual replacement of muscle fibers by

fatty and fibrous tissue in late stage

b. Dystrophin determination by Western blot

analysis or immunostaining

i. Usually little or no detectable dystrophin in

patients with DMD

ii. Dystrophin reduced in amount or abnormal

in size in patients with BMD

10. Carrier testing (Mathews 2003)

a. Carrier testing of young girls or genetic testing

of siblings of patients with DMD should be

delayed until they are old enough to participate

in the decision-making process.

b. Appropriate to test the mother who has an

affected boy with deletion or duplication iden-

tified by standard DNA screening, especially if

there are other female relatives of childbearing

age who are at risk for being carriers.

c. Possibility of gonadal mosaicism (up to 15%)

exists when the mother does not carry the boy’s

mutation

i. Her sisters could not have inherited the

mutation.

ii. Her daughter may have inherited the

mutations.

d. Offer linkage analysis to modify risk of carrier

status if no mutation is known or if tissue for

DNA analysis is not available

i. Knowledge of a childhood CPK level in the

at risk girl helpful

ii. Elevated CPK in an at-risk female is pre-

sumptive evidence of her being a carrier

e. Muscle biopsy: not a helpful test in determining

carrier status.


Genetic Counseling

1. Recurrence risk

a. Patient’s sib given that the mother is a carrier

(has a disease-causing mutation) (the carrier

mother with one defective gene and one normal

gene is usually not affected)

i. Fifty percent risk to her son to receive the

disease gene and express the disease.

ii. Fifty percent risk to her daughter to receive

the disease gene and become a carrier.

iii. Mother, regardless of proven carrier status or

does not have a DMDmutation detectable in

her DNA, has an empiric risk of 15–20% of

having an affected male fetus due to pres-

ence of maternal germinal mosaicism.

iv. Mother with concomitant somatic and

germline mosaicism: The risk to sibs of

inheriting a DMD mutation may be higher

than if the mother has germline mosaicism

only (van Essen et al. 2003).

b. Patient’s offspring

i. Males with DMD: Patient usually succumb

or too debilitated to reproduce

ii. Males with BMD

a) May reproduce.

b) None of the sons will inherit the

mutation.

c) All the daughters are carriers.

c. Germline mosaicism (den Helderman-van

Enden et al. 2009)

i. Causes the presence of multiple affected

offspring from apparently noncarrier

parents.

ii. In X-linked DMD/BMD, the recurrence risk

for noncarrier females due to germ line

mosaicism has been estimated to be between

14% and 20% (95% confidence interval)

(Darras and Francke 1987; Bakker et al.

1987, 1989 van Essen et al. 1992) if the

risk haplotype is transmitted.

iii. A recurrence risk of 8.6% (4.8–12.2) if the

risk haplotype is transmitted with

a remarkable difference between proximal

(15.6%) (4.1–27.0) and distal (6.4%)

(2.1–10.6) deletions (den Helderman-van

Enden et al. 2009). Overall, most mutations

originated in the female. Deletions occur

more often on the X chromosome of the

maternal grandmother, whereas point muta-

tions occur on the X chromosome of the

maternal grandfather. In unhaplotyped de

novo DMD/BMD families, the risk of recur-

rence of the mutation is 4.3%.

2. Prenatal diagnosis

a. In case of a known and readily detectable gene

defect

i. Amniocentesis and mutation analysis of

amniocytes, usually by multiplex PCR

ii. Preimplantation diagnosis by single-cell

PCR of blastomere or polar body

iii. Fetal nucleated erythrocytes from maternal

blood analyzed by multiplex PCR

b. In case of unknown gene defect

i. Fetal sexing, allowing females to proceed

to term

ii. Linkage analysis

iii. Fetal muscle biopsy for quantitative dystro-

phin analysis may serve as a diagnostic

option (Nevo et al. 1999)

c. Comparative genomic hybridization (CGH)

(Bovolenta et al. 2010)

i. Molecular diagnosis of DMD: requires

a great deal of effort due to enormous size

of the gene and to allele heterogeneity.

ii. While multiplex ligation-dependent probe

amplification (MLPA) represents the stan-

dard molecular technique for detecting

exonic DMD gene rearrangements (Kesari

et al 2008), several comparative genomic

hybridization (CGH) platforms have recently

been reported to rapidly screen the entire

DMD gene, as well as neighboring

sequences, for deletions and duplications

(Hegde et al 2008; del Gaudio et al 2008;

Bovolenta et al 2008).

3. Management (Kapsa et al. 2003; Mathews 2003)

a. Largely supportive

i. Physical therapy to eliminate the need for

surgical release of contractures

a) Night splints with ankle foot orthoses

(AFOs)

b) Daily stretching

c) Crucial to maintain ambulation as long as

possible because its loss is associated with

contractures and scoliosis, and, in turn,

associated with restrictive lung disease


ii. Regular use of an incentive spirometer at

home to prolong pulmonary function

iii. Continuous positive airway pressure

(CPAP)

iv. Bilevel positive airway pressure BiPAP:

more physiologic

v. Psychological support

b. American College of Chest Physician Statement

on the respiratory and management of patients

with Duchenne dystrophy undergoing anesthesia

(Birnkrant 2009)

c. Treatment of dystrophic cardiomyopathy

i. An angiotensin-converting enzyme inhibitor

ii. With or without a b-blockeriii. A diuretic

d. Pharmaceutical agents (steroids) shown great

promise in delaying the progression of DMD

i. Prednisone

ii. Deflazacort (not FDA approved but used

widely in Europe and Canada)

e. Orthopedic surgery

i. Release of joint contractures

ii. Spine fusion to minimize painful spinal

deformity and secondary pulmonary

complications

f. Prescribe wheelchair when dependency

becomes inevitable

g. Careful monitoring of pulmonary function

h. Ventilation for respiratory failure

i. Genetic therapies in DMD: Use of viral and

plasmid vectors to deliver dystrophin to

dystrophin–deficient muscle in vivo (Kapsa

et al. 2003)

i. Truncated dystrophin genes (minidystrophin

and microdystrophin transgenes): improve

force output and other features of the dystro-

phic mdx phenotype

ii. Tissue-specific promoters: Targeted trans-

gene expression via muscle-specific

promoters, a good platform for vector-

mediated therapeutic delivery of dys to

dystrophic muscle

iii. Plasmid vectors

iv. Viral vectors

a) Adenoviral vectors

b) Adenoassociated vectors

c) Retroviral vectors

d) Lentiviral vectors

e) Other viral vectors including herpes

simplex virus, Epstein-Barr virus, and

chimeric adeno-retrovirus

j. Corrective gene therapy (Kapsa et al. 2003;

Nelson et al. 2009)

i. Targeted corrective gene conversion

therapies

a) Introduction of construct of homologous

DNA containing a nonhomologous

sequence into mammalian cells in vitro

induces specific genetic transformations

in the host chromosomal DNA.

b) An attractive therapeutic strategy for

DMD if the DNA can be delivered to

the muscles efficiently.

ii. Small fragment homologous replacement

(SFHR): involves the application of PCR

amplicons to correct mutant loci in vitro or

in vivo

iii. Chimeraplasty with hybrid RNA-DNA

molecules (chimeraplasts) that promote

gene conversion via intranuclear DNA

mismatch repair mechanisms

iv. Gentamicin

a) An aminoglycoside antibiotics targeting

functional complexes (typically

ribosomes)

b) Causes a relaxation in codon

recognition

c) Enables stop-codon read-through of

the mdx nonsense mutation (a mutation

that produces a stop codon in the

transcribed mRNA) in exon 23 of

the dystrophin gene in the mdx mouse

v. Cell-mediated delivery of dys

a) Use cell transplantation to deliver nor-

mal (non-dystrophic) dys to dystrophic

muscle

b) Use donor myogenic precursor cells to

remodel the dystrophic muscle of the

recipient

c) Systemic applications of stem-cell

subpopulations

d) Use autologous cells (as an alternative

to donor cells) which can be isolated,

genetically engineered, and used to

deliver functional dystrophin to the dys-

trophic muscle


vi. Muscle derived precursor cells

a) Delivery of normal dystrophin by

the transplantation of non-dystrophic

muscle derived precursor cells

b) Resulting in some recovery of normal

function in dystrophic muscle

c) Greatly compromised by host immune

response

vii. Non-muscle stem cells

a) Induction of a few systemically

injected bone-marrow cells to enter

muscle after regeneration induced by

injury.

b) These cells with neuronal, osteogenic,

myogenic, and haemopoietic expression

profiles may provide alternatives for

cell-based delivery of non-dystrophic

loci to dystrophic muscle.

viii. Utrophin

a) A 395 kDa ubiquitous protein homo-

logue of dystrophin.

b) Utrophin expression has a widespread

sarcolemmal distribution in human dys-

trophic muscle.

ix. Other alternative approaches that may be

useful in the support of functional improve-

ment in dystrophic muscle include:

a) a7b1 integrin

b) Myoprotective or myoproliferative

cytokine factors such as leukemia inhib-

itory factor and insulin-like growth fac-

tor-1

c) Inhibition of myostatin

x. Emerging genetic therapies to treat DMD

a) A drug, PTC124, was identified that

suppresses nonsense codon translation

termination. PTC124 can lead to resto-

ration of some dystrophin expression in

human Duchenne muscular dystrophy

muscles with mutations resulting in pre-

mature stops.

b) Two drugs developed for exon skipping,

PRO051 and AVI-4658, result in the

exclusion of exon 51 from mature

mRNA. They can restore the transla-

tional reading frame to dystrophin tran-

scripts from patients with a particular

subset of dystrophin gene deletions and

lead to some restoration of dystrophin

expression in affected boys’ muscle

in vivo.

c) Both approaches have concluded

phase I trials with no serious adverse

events.

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a b c

Fig. 1 (a–c) A 10-year-old boy with DMD showing Gower

sign maneuver. He walked before 16 months of age and had

trouble getting up. Radiography showed mild cardiomegaly.

Muscle biopsy showed absence of dystrophin. Molecular

genetic analysis revealed exons 45–50 deletion of the dystrophin

gene

Fig. 2 A 12-year-old boy with DMD showing moderate calf

hypertrophy. He began to fall frequently at school, could not get

up from sitting position and had waddling gait, proximal muscle

weakness, prominent lordosis, decreased deep tendon reflexes,

and mild mental retardation. Muscle biopsy revealed absence of

dystrophin

Fig. 3 A 10-year-old boy with DMD starting to use wheelchair

for ambulation. He had tip toe walking at age of 6 and markedly

elevated CPK at 24,000–26,000. Muscle biopsy of quadriceps

femoris muscle showed marked variation in fiber size, moderate

number of necrotic fibers, occasional regeneration fibers and

hyalinized fibers, mild increase in internal nuclei, a few split

fibers, and moderate increase in perimysial and endomysial

connective tissue. Molecular genetic analysis revealed deletion

of exon 45 of the dystrophin gene


a b c

Fig. 4 (a–c) A male with DMD at 4-, 10-, and 29-year-old showing the progression of the disease. He has deletion of exons 49–54

of the dystrophin gene

Fig. 5 Biopsy of left calf

muscle of another patient at

age 4 showed widening of

perimysium (large arrows)and endomysium with fibrosis;

variation of fiber size with

presence of large rounded

fibers (small arrows).Degenerative fibers were often

seen (H & E, x400)


Fig. 6 Enzyme histochemical

stain showed both type I

(dark stained) and type II

(light stained) fibers arerandomly affected and there is

a remarkable variation of fiber

size even at the early stage

(myosin ATPase at 4.6, x400)


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Atlas of Genetic Diagnosis and Counseling || Dystrophinopathies