CLINICAL FEATURES GENETICS/BASIC DEFECTS 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 dis- ease that ranges from mild to severe. The mild end of the spec- trum includes the phenotypes of asymptomatic increase in serum concentration of creatine phosphokinase (CK) and mus- cle cramps with myoglobinuria and isolated quadriceps myopa- thy. 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 3500 live male births. BMD is a milder allelic form of dystrophin deficiency, affecting 1 in 30,000 male births.

GENETICS/BASIC DEFECTS

1. DMD

a. Inheritance

i. X-linked recessive

ii. Exceptionally high mutation rate of 10

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 inheri- tance 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 chromosome 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 dys-

trophin 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 initial proof that deletions in the Xp21 region were associated with the disease

iii. The dystrophin gene

a) The largest gene of the 30,000 genes that encode proteins in the human genome 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) 6% 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 dam- age and activation of the inflammatory cascade b) Progressing to muscle necrosis, fibrosis, and

loss of function

d. The DMD phenotype most frequently due to muta- tions that cause a disruption in the reading frame 2. BMD

a. Inheritance: X-linked recessive (same as DMD) b. Also caused by mutations in the gene for dystrophin

at Xp21.1

c. The BMD phenotype most often due to mutations 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 phenotype.

CLINICAL FEATURES

1. DMD

a. During early infancy i. Asymptomatic

ii. Normal motor milestones

iii. Rare global developmental delay or delayed achievement of early motor milestones

b. 4–5 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

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c. Progressive difficulty in rising from the floor second- ary to weakness of proximal pelvic girdle and proxi- mal 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 consistency 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 musculature 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 hyperthermia during general anesthesia in rare instances

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 insufficiency in the late 2nd or 3rd decade

o. Female carriers

i. Usually asymptomatic

ii. Manifesting female carriers (rare): occasional, slow progressive myopathy of moderate severity with elevated CPK (>1000) and associated symp- toms in about 8% of carriers. Following condi- tions induce expression of the disease phenotype:

a) Random X-inactivation: the extent of clini- cal involvement is dependent in part upon the degree of skewed X-chromosome inacti- vation in somatic cells

b) Turner syndrome having a single X chromo- some

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 adulthood and never lose ambulation

i. A proportion of cases have some degree of mental impairment

j. Heart failure from dilated cardiomyopathy, a common cause of morbidity and the most common cause of death, despite the milder skeletal muscle involvement k. Death usually in the 4th or 5th 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. 50-fold elevation (BMD) to 100-fold elevation

(DMD) in affected boys, resulting from leakage of the muscle isoform

b. Serum creatine kinase levels: range of values overlap- ping 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 pyruvate 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 car-

diomyopathy or associated mitral valve prolapse 4. Radiography for scoliosis

5. Pulmonary function testing with negative inspiratory 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 phenotype, 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 and

NRDB1 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 chromo- some (45,X)

iv. May have complete uniparental disomy of the X chromosome

v. Warrant high-resolution chromosome studies 7. Electromyography (EMG) to distinguish a myopathic

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 recognized by multiplex PCR which amplifies 18 to 25 of the gene’s 79 exons from genomic DNA obtained from blood samples b. Duplications (6% of cases): the next most common

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 polymorphism (SSCP) and heteroduplex analysis: highly sensitive and possible to detect approximately 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 gen- eral anesthesia), frequently performed when a clinically suspected DMD patient does not have a large deletion or duplication by genomic DNA testing

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

a. Carrier testing of young girls or genetic testing of sib- lings 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 identified by stan-

dard 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 presumptive 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 (the carrier mother with one defective gene and one normal gene is usually not affected)

i. 50% risk to her son to receive the disease gene and express the disease

ii. 50% risk to her daughter to receive the disease gene and become a carrier

iii. Mother of a DMD patient, regardless of proven carrier status, has an empiric risk of 10–30% of having an affected male fetus due to presence of maternal germinal mosaicism

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

2. Prenatal diagnosis

a. In case of a known and readily detectable gene defect i. Amniocentesis and mutation analysis of amnio-

cytes, 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 dystrophin analysis may serve as a diagnostic option 3. Management

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, asso- ciated 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. Treatment of dystrophic cardiomyopathy i. An angiotensin-converting enzyme inhibitor ii. With or without a β-blocker

iii. A diuretic

c. 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)

d. Orthopedic surgery

i. Release of joint contractures

ii. Spine fusion to minimize painful spinal deformity and secondary pulmonary complications e. Prescribe wheelchair when dependency becomes

inevitable

f. Careful monitoring of pulmonary function g. Ventilation for respiratory failure

h. Genetic therapies in DMD: use of viral and plasmid vectors to deliver dystrophin to dystrophin–deficient muscle in vivo

i. Truncated dystrophin genes (minidystrophin and microdystrophin transgenes): improve force output and other features of the dystrophic mdx phenotype ii. Tissue-specific promoters: targeted transgene 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

i. Corrective gene therapy

i. Targeted corrective gene conversion therapies a) Introduction of construct of homologous

DNA containing a nonhomologous sequence into mammalian cells in vitro induces spe- cific 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 ampli- cons 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 normal (nondystrophic) dys to dystrophic muscle b) Use donor myogenic precursor cells to

remodel the dystrophic muscle of the recipient c) Systemic applications of stem-cell subpopu-

lations

d) Use autologous cells (as an alternative to donor cells) which can be isolated, geneti- cally engineered, and used to deliver func- tional dystrophin to the dystrophic muscle vi. Muscle derived precursor cells

a) Delivery of normal dystrophin by the trans- plantation of nondystrophic muscle derived precursor cells

b) Resulting in some recovery of normal func- tion in dystrophic muscle

c) Greatly compromised by host immune response

vii. Nonmuscle 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, myo- genic, and haemopoietic expression profiles may provide alternatives for cell-based delivery of nondystrophic loci to dystrophic muscle.

viii. Utrophin

a) A 395 kDa ubiquitous protein homologue of dystrophin

b) Utrophin expression has a widespread sar- colemmal distribution in human dystrophic muscle.

ix. Other alternative approaches that may be useful in the support of functional improvement in dystrophic muscle include:

a) α7β1 integrin

b) Myoprotective or myoproliferative cytokine factors such as leukemia inhibitory factor and insulin-like growth factor-1

c) Inhibition of myostatin

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Fig 1. 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 hypertro- phy. He began to fall frequently at school, could not get up from sit- ting 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.

Fig. 4. A male with DMD at 4-, 10-, and 29-year-old showing the pro- gression of the disease. He has deletion of exons 49–54 of the dys- trophin gene.

Fig. 5. Biopsy of left calf muscle of another patient at age 4 showed widening of perimysium (large arrows) and endomysium with fibro- sis; variation of fiber size with presence of large rounded fibers (small arrows). Degenerative fibers were often seen. (H & E, × 400)

Fig. 6. Enzyme histochemical stain showed both type I (dark stained)

and type II (light stained) fibers are randomly affected and there is a

remarkable variation of fiber size even at the early stage (myosin

ATPase at 4.6, × 400)