Dystrophinopathies Workup

Updated: Nov 13, 2019
  • Author: Dinesh G Nair, MD, PhD; Chief Editor: Nicholas Lorenzo, MD, CPE, MHCM, FAAPL  more...
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Laboratory Studies

Serum creatine phosphokinase (CPK), as follows:

  • This level is always increased in patients with Duchenne muscular dystrophy or Becker muscular dystrophy, probably from birth. It often is increased to levels that are 50-100 times the reference range (ie, as high as 20,000 mU/mL). In late stage DMD very little muscle mass remains to give rise to an elevated serum CPK level. Recognizing that the natural history of serum CPK in DMD is known to decrease over time, especially for longer-term clinical trials, is important.

  • A child or young adult with a CPK level within the reference range does not likely have a dystrophinopathy.

  • Strongly suspect Duchenne muscular dystrophy in a child with proximal weakness and very elevated levels of CPK. Perform further specific diagnostic testing, including DNA mutation analysis, to confirm the underlying diagnosis (see Other Tests).

There is new enthusiasm to consider newborn screening given the promise of earlier treatment with steroids, molecular therapy, or gene therapy. A 2-tiered system of analysis has been proposed that analyzes newborn CPK from dried blood spots followed up with DNA analysis from that same dried blood spot if the CPK is elevated. [6] Currently, newborn screening is not performed in the United States.


Imaging Studies

Scoliosis frequently ensues in patients with Duchenne muscular dystrophy, particularly after they are wheelchair dependent. Radiographs of the spine are important for screening and evaluating the degree of scoliotic deformity.

As the disease progresses and dyspnea becomes a complaint, chest radiography is also likely to become a part of the evaluation.

Beyond imaging for scoliosis and dyspnea, imaging studies are of little help in making the diagnosis.

Imaging studies of the brain are usually unremarkable.

Dual energy x-ray absorptiometry is a radiographic technique to estimate bone mineral density. Individuals with dystrophinopathies can have accelerated osteopenia/osteoporosis/fracture risk, especially long-bones and vertebral compressions, due to the sedentary condition, fall risk, vitamin D deficiency, calcium intake deficiency, poor sunlight exposure, and chronic corticosteroid treatment.


Other Tests


Electromyography (EMG), even though not diagnostic, narrows the differential diagnosis by effectively excluding primarily neurogenic processes such as spinal muscular atrophy.

In general, the proximal muscles of the lower extremities may exhibit the more prominent EMG findings. A sufficient number of muscles need to be sampled to establish the presence of a diffuse process such as a dystrophy. The more revealing findings will be obtained in muscles of intermediate involvement with respect to weakness.

The motor unit action potentials (MUAPs) in patients with Duchenne or Becker muscular dystrophy are typically of short duration, particularly the simple (ie, nonpolyphasic) MUAPs. MUAP amplitudes are variable (normal to reduced) and they are typically polyphasic from the variability in muscle fiber diameters, resulting in longer MUAP durations. Early recruitment of MUAPs may be seen. If muscle fiber loss is severe, then what appears to be a loss of motor units may be seen with fast firing individual spikes. The latter are distinguished from neurogenic processes by their generally lower-than-normal amplitudes and reduced area of spikes.

Fibrillation potentials and positive sharp waves, which represent spontaneously depolarizing muscle fibers bereft of nervous innervation, are encountered in active disease as necrosis engulfs the motor endplate or separates the endplate from other portions of the muscle fiber. These may be difficult to see in some muscles, requiring higher-than-usual sensitivity settings on the amplifier.

Molecular diagnosis

Individuals with Duchenne or Becker muscular dystrophy can be reliably and accurately detected from peripheral blood samples in nearly all cases. If uninformative deletion/duplication genetic tests have resulted, direct sequencing of the dystrophin gene is a viable option. Other innovative methods have been devised for accurate noninvasive diagnosis.

Currently, most laboratories use multiplex PCR amplification to examine deletion "hotspots," which account for approximately 59% of all mutations. This method has a 98% detection rate for deletions.

Duplications, which account for 5% of mutations, can be detected by several different quantitative techniques, including Southern blot, quantitative PCR, multiplex amplifiable probe hybridization (MAPH), and multiplex ligation-dependent probe (MLPA). These techniques are also highly sensitive for detecting deletions.

The remaining one third of the mutations are composed of subexonic sequences, of which 34% are nonsense mutations, 33% are frameshifts, 29% are splice site mutations, and 4% are missense mutations. These mutations can be screened for by using techniques such as denaturing high-performance liquid chromatography (dHPLC); single- stranded conformational polymorphism analysis with single condition amplification internal primers (SCAIP) or detection of virtually all mutations (DOVAM), a robotically enhanced multiplexed method; or denaturing gradient gel electrophoresis.

Recently, 96% of mutations in patients with Duchenne muscular dystrophy have been shown to be noninvasively identified by using these techniques in a 3-tiered approach. Tier 1 is PCR amplification to detect large deletions, tier 2 would use DOVAM to rapidly scan for point mutations, and tier 3 would use MAPH to define duplications. Other similar techniques can be substituted for any of the tiers. For example, MAPH can be substituted with Southern blot. This same approach can also be applied to the patient with Becker muscular dystrophy. While most of these techniques were originally used for research purposes, many are now available clinically.

In patients without detectable mutations of the dystrophin gene, diagnosis requires muscle biopsy for dystrophin protein quantification (see muscle biopsy in Procedures). For some families of a young boy found to have a dystrophin mutation, the muscle biopsy can provide critically important dystrophin protein information such as molecular weight size and abundance with a western blot. Immunolabeling of frozen muscle sections can enable epitope identification. This information can offer prognostic value if a predicted DNA mutation is in- or out-of-frame as some software modelling predictions and DNA sequencing techniques do indeed have a small error rate.

Electrocardiogram and echocardiogram

Electrocardiogram (ECG) provides a simple means for uncovering sinus arrhythmias and also may demonstrate deep Q waves and elevated right precordial R waves.

Transthoracic echocardiography yields a clearer and more dynamic view of the heart, often revealing small ventricles with prolonged diastolic relaxation.

A Holter monitor is valuable for paroxysmal arrhythmias.

Cardiac MRI and gadolinium enhancement are new noninvasive technologies that can better characterize cardiac tissue changes in dystrophinopathy and may implicate earlier treatments or prophylactic regimens to stabilize the heart.

Carrier detection

Carrier detection is an important aspect of the care and evaluation of patients with Duchenne muscular dystrophy and Becker muscular dystrophy and their family members.

A small minority of female carriers are symptomatic, but even in these symptomatic patients, correct diagnosis requires appropriate testing.

For many years, CPK testing was the best method for carrier detection; however, it is elevated in only two thirds of female carriers and the results can be difficult to interpret in ethnic and racial groups with normally elevated CPK levels. For example, African Americans have a higher reference range than whites; CPK levels of African Americans may exceed the laboratory-stated normal limits without the presence of any pathology.

In families in which an affected male has a known deletion or duplication of the dystrophin gene, testing for carrier status is performed accurately by testing possible carriers for the same deletion or duplication, the absence of which generally excludes them as a carrier. These methods can also be used in prenatal diagnosis but gonadal mosaicism does occur in less than 8% of women and a negative blood DNA tests can be falsely reassuring

If the affected males in the family are unavailable for deletion or duplication testing, the female still can be tested, but the absence of a DNA abnormality does not exclude them as carriers. Obviously, the presence of a deletion or duplication in a female always conveys carrier status.

In families in which the affected male has no detectable deletion or duplication, muscle immunofluorescence for dystrophin can be used in some cases. Carrier females should exhibit a mosaic pattern, with some myofibers being normal and some being abnormal. This is subject to sampling error, and again, normal biopsy findings do not exclude carrier status.

Unfortunately, dystrophin immunoblot quantitation, which is very useful in affected males, is not helpful in carrier detection as even female carriers manifesting the disease may have levels within the reference range.

If all else fails, linkage analysis comparing polymorphic DNA markers on the X chromosome of an affected patient with those of his mother or sister permits detection of asymptomatic carriers. This can be performed using PCR techniques but requires blood from at least one affected male in the family. On occasion, the results are uninformative (eg, if the mother is homozygous for all markers, discerning which X chromosome harbors the defective gene is impossible).



Muscle biopsy

Despite the specificity of molecular genetic diagnosis, up to 10% of boys with a clinical picture of dystrophinopathies may have no detectable deletions on DNA testing. Therefore, muscle biopsy, while supplanted as the criterion standard, remains an important adjunctive tool, both for quantifying the amount of muscle dystrophin as well as for detecting asymptomatic female carriers. Depending on the purpose of the biopsy, proper site selection is crucial.

For detection of female carriers, strong muscles may exhibit no pathology, and very weak muscles may be too devoid of fibers for adequate analysis. For affected males, a very weak muscle may have inadequate tissue for immunoblot and immunofluorescent testing. In addition, the acquisition of muscle tissue from a muscle already severely weak may precipitate further weakness. Therefore, the ideal muscle to biopsy is one that is easily accessible and exhibits moderate weakness (ie, has 80% strength).

Two methods are available for assessing dystrophin in muscle tissue.

Immunostaining of the muscle using antibodies directed against the rod domain, carboxy-terminals, and amino-terminals of dystrophin protein shows absence of the usual sarcolemmal staining in boys with Duchenne muscular dystrophy. Patients with Becker muscular dystrophy show more fragmented and patchy staining of sarcolemmal regions. See the image below.

(A) Normal dystrophin staining.(B) Intermediate dy (A) Normal dystrophin staining.(B) Intermediate dystrophin staining in a patient with Becker muscular dystrophy.(C) Absent dystrophin staining in a patient with Duchenne muscular dystrophy.

Some consider the most accurate lab method for differentiating Duchenne from Becker muscular dystrophy to be the immunoblot of muscle homogenates. Patients with Duchenne muscular dystrophy have greatly decreased or absent amounts of truncated dystrophin, whereas patients with Becker muscular dystrophy protein reveal moderately reduced amounts of dystrophin, which may be smaller (ie, deletion of the dystrophin gene) or larger (ie, duplications of the dystrophin gene) than normal. Clinical correlation is more important as there are exceptions to this notion.


Histologic Findings

Few muscle biopsies are as instantly recognizable as those of patients with Duchenne muscular dystrophy. Features of Duchenne muscular dystrophy are reminiscent of a tissue battlefield after a major conflict, with necrotic muscle fibers littering the landscape. Widespread muscle necrosis leads to angulated fibers, central nuclei, and considerable fiber size variation, with regenerating cells in different stages of atrophy and regrowth.

Fibers that are too damaged to regenerate may become empty skeletal remnants or ghost cells. Actively regenerating fibers often display cytoplasmic basophilia, with large nuclei and prominent nucleoli. Damaged fibers exhibit reduced histochemical staining for oxidative enzymes. Initially, macrophages and cluster of differentiation 8-positive (CD8+) T lymphocytes invade necrosing muscle fibers. In time, this cellular response is supplanted by endomysial and perimysial fibrosis and fatty tissue replacement, which convey the macroscopic appearance of pseudohypertrophy.

Aside from linkage analysis, fluorescent immunostaining for dystrophin protein can be a way to diagnose carrier status in a family with no known gene deletion or duplication. Antibody staining for portions of the dystrophin molecule at the sarcolemmal membrane reveals the conspicuous absence of various portions of the dystrophin complex.

In boys with Duchenne muscular dystrophy, the sarcolemma is virtually devoid of staining (see section C in image below).

(A) Normal dystrophin staining.(B) Intermediate dy (A) Normal dystrophin staining.(B) Intermediate dystrophin staining in a patient with Becker muscular dystrophy.(C) Absent dystrophin staining in a patient with Duchenne muscular dystrophy.

In contrast, carrier females exhibit a more variable mosaic pattern consisting of normal and abnormal fibers.

Immunoblot analysis of muscle tissue, available through commercial laboratories, can determine the size and quantity of the dystrophin molecule. Patients with Duchenne muscular dystrophy exhibit no dystrophin. In patients with Becker muscular dystrophy, variable amounts of dystrophin are present but with an altered molecular size. Carriers of Duchenne muscular dystrophy exhibit mosaicism for dystrophin expression and usually have enough functional dystrophin to be within normal limits on Western blot testing, making this a generally poor method for carrier detection.