Peter B. Kang and Basil T. Darras
Disorders of the peripheral nervous system can generally be divided into 3 major categories, based on anatomic localization:
1. Neurogenic disorders, including motor neuron diseases
2. Disorders of the neuromuscular junction
3. Myopathies, including muscular dystrophies
History and physical examination remain critical in focusing the differential diagnosis of neuromuscular diseases (see also Chapter 569). Polyneuropathies typically present with distal weakness and atrophy, loss of deep tendon reflexes, and sensory deficits. Children with chronic polyneuropathies, especially Charcot-Marie-Tooth disease, may be unaware of sensory loss. Footdrop may develop in advanced cases. Motor neuron disease such as spinal muscular atrophy will cause proximal or generalized weakness. Disorders of the neuromuscular junction are difficult to diagnose in children. Infant botulism typically begins with constipation, followed by a descending pattern of weakness. Juvenile myasthenia gravis may present with a variety of symptoms, including fatigue, but this disorder is almost always accompanied by ocular or bulbar deficits. Myopathies tend to cause proximal or generalized weakness. Neither neuromuscular junction disorders nor myopathies are accompanied by sensory loss.
During the last 20 years, the field of neuro-muscular disease has changed drastically, with advancements in the molecular pathogenesis of myopathies and other neuromuscular disorders.1 These breakthroughs have allowed improved and new diagnostic tests that can confirm clinical diagnoses very accurately and less invasively. In this chapter, we will describe the diagnostic tests currently used in clinical practice.
Measurements of serum muscle enzyme levels may be quite useful in the initial evaluation of children with suspected neuromuscular disease. The most well-known serum marker of neuro-muscular disease is creatine kinase (CK), also known as creatine phosphokinase (CPK). This muscle enzyme is normally found in small amounts in serum (up to 150 or 175 U/L in many diagnostic laboratories) but may be released in large quantities when muscle membrane breakdown occurs in muscular dystrophy and other myopathies.2,3 Mild elevations in CK levels up to 300 U/L are nonspecific and may be a normal finding, but values up to 500 U/L are sometimes found in neuromuscular diseases such as spinal muscular atrophy or congenital myopathies. Moderate (501 to 1000 U/L) to severe (above 1000 U/L) elevations in CK levels are usually associated with primary muscle disease, which can be inherited, as in muscular dystrophy, or acquired, as in acute rhabdomyolysis. CK levels are generally not useful in the evaluation of possible neuropathies and disorders of the neuromuscular junction.
Other enzymes are also released by injured muscle tissue and can be measured in serum. Among these, aldolase may also be elevated in muscle disease.4 Normal aldolase levels typically range up to 12 U/L. Three liver enzymes are also found in muscle tissue and may be mildly increased in muscle disease: alanine aminotransferase, aspartate aminotransferase, and lactate dehydrogenase. Patients who have unexplained mild elevations of liver enzymes should have CK and aldolase levels checked before undergoing invasive testing such as liver biopsy.
Analysis of muscle and nerve histology on tissue obtained at biopsy is an old and somewhat invasive diagnostic test, but it remains a useful and powerful tool in many situations. Approaches to the analysis of muscle tissue include traditional histochemical stains, immunohistochemistry, biochemical assays, and electron microscopy. Testing of cultured fibroblasts obtained from skin biopsy may be useful if metabolic muscle disorders are suspected.
The merits of open versus needle muscle biopsy have been debated for some time. A needle biopsy is less invasive and tends to leave a smaller scar, but the quality of the sample obtained may be variable, especially in cases of advanced disease where the muscle sampled is fibrotic, atrophic, or both. Most pediatric centers continue to rely on open biopsy performed under general anesthesia. The most common muscle sampled is the vastus lateralis; the gastrocnemius is preferred in some centers.
There are several traditional histochemical stains for muscle. Hematoxylin and eosin (H&E) staining highlights general muscle fiber size and morphology, as well as the location of the nuclei. Inflammation and fibrosis will also be evident on H&E. Gomori trichrome highlights fibrous tissue very well. ATPase stains at pH 4.3, 4.6, and 9.4 delineate fiber types. Specific structures within the muscle fiber are highlighted by chemical reactions. Mitochondria are prominent on NADH stains. Abnormal accumulations of glycogen are visible on periodic acid-Schiff (PAS) staining, and lipid droplets appear on oil red O stains.5
The generation of antibodies that bind specific proteins provides a means for estimating the quantity and localization of these proteins in muscle tissue, using immunohistochemical or, less commonly, immunoblot methods. Commonly assessed proteins include dystrophin, merosin, and α-, β-, γ-, and δ-sarcoglycans. Other proteins may be detected, depending on the availability of high-quality antibody stocks and the technical skills of the pathology laboratory. These results may suggest specific genetic defects and can focus subsequent genetic testing.6-8
Biochemical assays may also be performed on muscle tissue and cultured fibroblasts to test for the presence of enzyme deficiencies, and thus may be useful in detecting mitochondrial disorders, glycogen storage disorders, and fatty acid oxidation disorders. These assays are technically difficult and are commercially available in only a few selected centers. The results of such testing should be interpreted with caution. Whenever possible, an enzyme deficiency detected on biochemical testing should be confirmed with genetic analysis.
Electron microscopy of muscle tissue may be used to detect subtle structural abnormalities. Abnormalities in morphology and localization of mitochondria may be illustrated. Abnormal accumulations of material such as glycogen may also be more evident on electron microscopy.
Open incisions are required for nerve biopsies. The most common nerve sampled is the sural nerve, as it is an accessible pure sensory nerve that innervates only a small patch of skin on the lateral side of the foot. In recent years, nerve biopsies have been performed only rarely in children. However, they may still be useful for unexplained neuropathy. Light and electron microscopic examination can reveal demyelinating and axonal patterns of damage, as well as inflammation and abnormal accumulations.
Nerve conduction studies and electromyography (collectively referred to as EMG) remain an essential tool in the diagnosis of peripheral nerve disorders, disorders of the neuromuscular junction, and in some cases, myopathies.9,10Two critical parameters measured during this testing are nerve conduction velocity and action potential amplitude. EMG can help determine whether a neuropathy is present, and if so, provide further information including physiology (demyelinating versus axonal), anatomy (motor versus sensory, generalized versus focal), and chronicity. Inherited and acquired neuropathies usually, but not always, display different characteristics on EMG studies.11-13Repetitive stimulation, which is a specialized form of nerve conduction study, can detect the presence of a neuromuscular junction defect.
In some centers, genetic testing is obtained immediately instead of EMG in cases of suspected inherited nerve disorders such as Char-cot-Marie-Tooth (CMT) disease, and spinal muscular atrophy (SMA), depending on the availability of EMG and its perceived discomfort. However, this is often not a cost-effective approach, as some suspected patients may not have a neuropathy, and a full panel of testing for multiple genes can cost many thousands of dollars. Negative genetic testing does not completely exclude a polyneuropathy. In skilled hands, EMG is a useful and minimally painful diagnostic procedure and can direct focused genetic testing.
Advances in knowledge about the genetic origins of inherited disease have transformed the diagnostic approach to disease over the past 2 decades, primarily in a positive manner, but also with a major drawback. Genetic testing is unique in that it can, in the correct clinical context, provide definitive proof of the diagnosis of an inherited disease. It is also, in most cases, noninvasive, requiring only DNA extracted from blood leukocytes. Except in cases of mitochondrial disease and mosaicism, DNA from other tissue samples does not increase the yield of genetic testing.
Several techniques are commonly used in genetic diagnostic laboratories. Fluorescence in situ hybridization (FISH) and Southern blot analyses may be used to evaluate for whole gene deletions and duplications (eg, PMP22duplications and deletions in inherited neuropathies).14-16 Multiplex polymerase chain reaction (PCR) analysis is helpful in detecting exon deletions and duplications that commonly affect the dystrophin (DMD) gene in cases of Duchenne and Becker muscular dystrophy.17-20 Smaller lesions, including point mutations, may be detected only by direct sequence analysis, which is costly and labor intensive, especially for larger genes.21
The major drawback of genetic testing is the cost. A vast array of genetic tests are available, and a nonfocused approach can lead to high costs without yielding a specific diagnosis. It is critical to continue using basic tools such as the patient history, physical examination, serum markers, electrophysiology, and tissue histology to narrow the possible diagnoses as much as possible before genetic testing.