Autori: Risso Sebastiano Tiranti Giorgio
Duchenne muscular dystrophy is a severe X-linked recessive disease caused by
mutations in the dystrophin gene . The gene is
the largest in the human genome, encompassing 2.6 million base
pairs of DNA and containing 79 exons. Approximately 60% of dystrophin
mutations are large insertions or deletions that lead to
frameshift errors downstream, whereas approximately 40% are
point mutations or small frameshift rearrangements. The vast majority of DMD patients lack the dystrophin protein resulting in progressive muscular atrophy, wasting, and death by age 20, often related to respiratory system–due to compromised diaphragm activity, or cardiac failure; calf and deltoid muscles display the typical finding of pseudohypertrophy.
Duchenne muscular dystrophy (DMD) affects 1 in
every 3500 live male births.
Symptoms usually appear in boys aged 1 to 6. Typically, the first noticeable symptom is delay of motor milestones, including sitting and standing independently. The mean age for walking in boys with Duchenne muscular dystrophy is 18 months. There is progressive muscle weakness of the legs and pelvic muscles, which is associated with a loss of muscle mass (wasting). This muscle weakness causes a waddling gait and difficulty climbing stairs. Muscle weakness also occurs in the arms, neck, and other areas, but not as severely or as early as in the lower half of the body. There is a steady decline in muscle strength between the ages of 6 and 11 years. By age 10, braces may be required for walking, and by age 12, most boys are confined to a wheelchair. Bones develop abnormally, causing skeletal deformities of the spine and other areas.Calf muscles initially enlarge and the enlarged muscle tissue is eventually replaced with fat and connective tissue. Early signs may include pseudohypertrophy (enlargement of calf muscles). Muscle contractures occur in the legs, making the muscles unusable because the muscle fibers shorten and fibrosis occurs in connective tissue. Occasionally, there can be pain in the calves.
Muscular weakness and skeletal deformities frequently contribute to breathing disorders. Cardiomyopathy (enlarged heart) occurs in almost all cases, beginning in the early teens in some, and in all after the age of 18 years. Intellectual impairment may occur, but it is not inevitable and does not worsen as the disorder progresses.
Intellectual disabilities only affect a minority of boys with Duchenne type muscular dystrophy, but are more common than in other children.
In some centers, muscle biopsy is no longer routine in the diagnostic workup of DMD if genetic testing is positive and the clinical phenotype is consistent. Muscle biopsy is then only performed where genetic testing is negative, or the clinical phenotype is atypical. Others, however, advocate for muscle biopsy to remain a routine investigation in DMD, as it remains the gold standard for diagnosis.
On light microscopy early changes include degenerating necrotic muscle fibers with invasion by macrophages, as well as clusters of small- to intermediate-sized regenerating muscle fibers which have basophilic cytoplasm. Increased variability of muscle fiber size is also seen, initially with larger than normal, then smaller than normal fibers as the disease progresses.Type 1 fiber predominance is observed, as are hypercontracted muscle fibers. Eventually there is significant replacement of muscle fibers by fat and endomysial connective tissue.
Absent or markedly reduced dystrophin in muscle biopsies of boys with DMD can be demonstrated on immunostaining and/or Western blot analysis, using antibodies directed against different epitopes of dystrophin. Generally, antibodies recognizing the amino-terminus, carboxy-terminus and rod domains are used. Immunostaining using the amino-terminus or rod domain antibodies shows faint sarcolemmal staining in up to 60% of DMD patients. Immunoreactivity to carboxy-terminal antibodies however is absent in DMD and is therefore useful in differentiating DMD from BMD. Western blot analysis allows quantification of the amount of dystrophin protein as well assessment of size of the protein present. In DMD less than 5% of the normal quantity of dystrophin is present when carboxy-terminal antibodies are used, whilst up to 25% of normal dystrophin levels is seen with the use of rod domain antibodies.
Electromyography and nerve conduction studies are rarely required in the diagnosis of DMD. Needle electromyography findings are myopathic, with short duration, low amplitude polyphasic motor unit potentials, particularly in proximal muscles. Abnormal spontaneous activity in the form of fibrillation potentials, positive sharp waves and complex repetitive discharges may be detected due to denervation and some reinnervation in necrotic muscles. This may also result in the presence of satellite motor unit potentials. Over time the motor units become very small and some areas become electrically silent.
Nerve conduction studies are normal in early DMD. As the disease progresses, compound muscle action potentials decrease in amplitude.
MUSCLE MAGNETIC RISONANCE
Muscle MRI is usually not performed in DMD for diagnosis, but may be a useful noninvasive tool to evaluate progression of muscle involvement over time. Abnormalities in signal are seen on T1 and T2 images, with initial selective involvement of the gluteus maximus, adductor magnus, quadriceps, biceps femoris, rectus femoris and gastrocnemii muscles.
SERUM MUSCLE ENZYME
The characteristic finding in DMD is a markedly raised serum CK level, at least 10 to 20 times (and often 50 to 200 times) the upper limit of normal before the age of five years. Serum CK concentrations are high even in newborns and prior to any symptoms. The high CK levels at birth can form the basis of neonatal screening for DMD. Levels peak at two to three years of age and then decline with increasing age, due to progressive loss of dystrophic muscle fibers. A serum CK less than 10 times normal in a child with suspected DMD in the first three years of life should raise the question of an alternate diagnosis.
Serum alanine transaminase and aspartate transaminase levels are raised in DMD and tend to correlate with CK levels. Other enzymes raised in DMD include aldolase and lactate dehydrogenase. Most of these are not specific for muscle and are generally not useful in the diagnosis of DMD.
MOLECULAR GENETIC TESTING
Molecular genetic testing is now the mainstay of diagnosis in most centers. A multiplex polymerase chain reaction (PCR), covering 18 exons at the deletion hotspots developed by Chamberlain and Beggs detected 90-98% of all deletions, although duplications were not identified by this method. More recently, the development of multiplex ligation-dependent probe amplification (MLPA) has provided a more sensitive technique for detecting deletions. All 79 exons are covered by two sets of probes, with individual exons depicted as a single peak. This allows gene dosage abnormalities to be detected, allowing detection of duplications and testing of carrier individuals as well as for deletions. Occasionally, point mutations will also be detected as single exon deletions, with further analysis allowing more specific delineation of the point mutation.
If MLPA testing is negative, the DMD gene can be tested for point mutations. Direct sequence analysis of the DMD gene is generally available on a research basis only, due to its labor-intensive and costly nature. Several groups have developed strategies to target exonic regions for direct sequencing after the use of initial screening methods.
A targeted high-density oligonucleotide comparative genomic hybridization microarray that allows high-resolution analysis of the DMD gene has also been developed recently, allowing identification of deletions and duplications but also previously unidentified deep intronic mutations.
Dystrophin has a major structural role in muscle as it links the internal cytoskeleton to the extracellular matrix. The amino-terminus of dystrophin binds to F-actin and the carboxyl terminus to the dystrophin-associated protein complex (DAPC) at the sarcolemma. The DAPC includes the dystroglycans, sarcoglycans, integrins and caveolin, and mutations in any of these components cause autosomally inherited muscular dystrophies. The DAPC is destabilized when dystrophin is absent, which results in diminished levels of the member proteins. This in turn leads to progressive fibre damage and membrane leakage. The DAPC has a signalling role, the loss of which also contributes to pathogenesis. Dystrophin is postulated to be essential for force transduction by providing an indirect link between the contractile apparatus in the muscle fibre with the extracellular matrix. The mutations in the dystrophin gene which result in DMD cause disruption of the reading frame, resulting in a severe reduction or complete absence of dystrophin in the skeletal and cardiac muscle, which in turn leads to mechanically induced sarcolemmal damage, loss of intracytoplasmic calcium homeostasis, and muscle fibre degeneration. Several dystrophin isoforms are also expressed in brain and their deficiency in this tissue is responsible for the mental retardation which complicates the course of DMD in approximately one third of cases. Approximately 65% of patients with DMD have intragenic out-of-frame (gross rearrangements) deletions and approximately another 10% have duplications of one or more exons of the dystrophin gene. The remaining patients have point mutations or other smaller gene rearrangements (pure intronic deletions, insertions of repetitive sequences, splice site mutations). As a general rule out-of-frame dystrophin gene mutations lead to a severe reduction or absence of dystrophin in the muscle resulting in the DMD phenotype, whereas in-frame mutations lead to the expression of abnormal but partly functional truncated dystrophin protein, resulting in the milder Becker muscular dystrophy (BMD). The frame shift hypothesis holds true for over 90% of cases and is commonly used both for diagnosis and for differentiating between DMD and BMD. However, there are important exceptions to the frame shift rule: in-frame mutations in the gene coding for the crucial actin-binding domain of dystrophin protein may cause the Duchenne severity phenotype, whereas some out-of-frame mutations are associated with BMD. The X linked recessive inheritance of DMD is well recognised, but there is a high incidence of new mutations and two thirds of patients do not have a positive family history at presentation. Whether dystrophin and its associated proteins have a direct role in the regulation of calcium ions, calcium channels or intracellular calcium stores, or indirectly alters calcium regulation through enhancement of membrane tearing, remains unclear .
Duchenne muscular dystrophy is inherited in what is known as an X-linked recessive pattern. The defective gene is found on the X chromosome. Because women have two X chromosomes, if one contains a normal copy of the gene, that gene will make enough of the protein to prevent symptoms. But boys have an X chromosome from their mother and a Y from father, so if the X chromosome is defective, there is no second X to make up for it and they will develop the disease. The sons of carrier females (women with one defective chromosome but no symptoms themselves) each have a 50% chance of having the disease, and the daughters each have a 50% chance of being carriers. Because this is an inherited disorder, risks include a family history of Duchenne muscular dystrophy.
1 - Joint contractures are usual.
2 - Respiratory: Chronic respiratory insufficiency due to restrictive lung disease is inevitable in all patients. Vital capacity increases as predicted until around age 10 years; after this time it starts to decrease at a rate of 8-12% per year. When vital capacity reaches less than 1 liter the risk of death within the next one to two years is relatively high.
Obstructive sleep apnea is the predominant cause of sleep disordered breathing in the first decade, occurring in up to one-third of patients, with occurring in the second decade. Four stages of hypercapnic chronic respiratory failure are typically described: Stage 1, sleep disordered breathing without hypercapnia; Stage 2, sleep disordered breathing with hypercapnia during rapid eye movement (REM) sleep; Stage 3, with hypercapnia during REM and Non-REM sleep; and Stage 4, diurnal hypercapnia. At Stage 4, mean survival is less than 12 months without respiratory support.
3 - Cardiac : Cardiac disease consists of dilated cardiomyopathy due to cardiac fibrosis as well as disturbances of rhythm and conduction.
Clinically apparent cardiomyopathy is first evident after 10 years of age, affects one-third of patients by age 14 years, and is present in all patients over 18 years of age. Preclinical cardiac involvement is seen in 25% of patients under six years of age, with a persistent tachycardia commonly noted. Atrial and ventricular arrhythmias occur, including premature ventricular beats and more complex or sustained ventricular ectopy, which increase with age and ventricular dysfunction. Despite the high frequency of cardiac involvement, most patients are relatively asymptomatic due to physical inactivity.
4 - Smooth muscle can also be affected, causing gastrointestinal symptoms such as gastric dilation or pseudo-obstruction.
5 - Nutritional problems and weight loss can occur in the late stage of DMD.
6 - Intellectual disability: Intellectual disability is seen in 30% of boys with DMD, with the average intelligence quotient (IQ) being 85, normally distributed one standard deviation below the population norms. Verbal IQ is more impaired than performance IQ. Intellectual disability is not correlated with the severity of weakness. Boys with DMD also have a higher incidence of attention deficit hyperactivity disorder
7 - Ophthalmic: there may be an increased incidence of colour blindness.
8 - Ortophedic: Scoliosis develops in almost all children with DMD, and impacts on respiratory vital capacity. It progresses significantly after boys lose ambulation, and maintenance of ambulation slows the rate of progression. Long bone fractures are common and usually due to falls, affecting 21-44% of boys. Half of the fractures occur in independently ambulatory boys, with 20-40% losing ambulation as a result. Osteoporosis is present in most children with DMD. Loss of bone mineral density begins even when boys are still ambulant, and continues to diminish with age.