1st National Conference on Neurogenetics, SESSION : FLOPPY BABY

Dr. Sheffali Gulati ,
Associate Professor of Pediatrics, All India Institute of Medical Sciences.
Email: sheffaligulati@gmail.com

The role of the clinician in the diagnosis and treatment of a weak child is as important today as it was in the 19 th century. Fundamental tools are a detailed medical history and a carefully performed physical examination. The clinical assessment has three possible immediate outcomes: Reassurance; determine natural history of the process; arrange for further diagnostic procedures.

Neuromuscular disorders can be due to disorder anywhere in the motor unit which is illustrated in Figure 1 below. The anterior horn cell involvement can produce spinal muscular atrophy, Amyotrophic lateral sclerosis; nerve involvement can produce various neuropathies; neuromuscular junction involvement can produce myasthenia gravis, congenital myasthenic syndromes; muscle fiber involvement can produce various dystrophies, myotonic syndromes, and can be due to mitochondrial, congenital, metabolic or autoimmune disorders.

We will be confining ourselves to disorders related to the muscle only. While approaching a child with a muscle weakness , one should consider following questions in one’s mind:

• What is the clinical pattern?
• Any involvement outside skeletal muscle?
• Disease course?
• Mode of inheritance?
• Means for securing a diagnosis?
• Treatment options available?

Initial history should include complaints of the patient, age at onset, symptoms at presentation, course of disease, family history. Mode of inheritance can be variable: It is X-Linked recessive in Duchenne muscular dystrophy, Backer muscular dystrophy; autosomal dominant in Facioscapulohumeral muscular dystrophy, central core disease; autosomal receive in sarcoglycanopathies, congenital muscular dystrophies, to name a few. It is essential to establish a detailed pedigree, which we commonly miss in our clinics.

should be complete and should include: Gower’s sign, waddling gait, scapular wining, facial weakness, tongue/calf hypertrophy, contractures, ophthalmoplegia, ptosis, myotonia, cardiac, respiratory or CNS involvement, Various phenotypes are easily recognizable like Duchenne- Becker muscular dystrophy, Emery Dreifuss Muscle dystrophy, Facio scapulo humeral dystrophy, myotonic dystrophy. Depending upon pattern of muscle involvement, facial or ocular involvement, contractures or wasting, cardiac/respiratory involvement, rate of progression we can form an initial clinical impression.

Traditional diagnostic tests include nerve conduction studies, electromyography (EMG) and open muscle biopsy. Recently there has been lot of advances in molecular neurogenetics.

can be assessed by Electrodiagnostic testing: Electromyography, repetitive nerve stimulation, and nerve conduction testing; imaging; serum creatine kinase (CK) and muscle biopsy. On muscle biopsy diagnosis is made by specific morphologic features, immunohistochemistry (absent or reduced staining for specific protein) and histochemistry (absent or reduced enzyme function). Other diagnostic tests in include antibodies in serum or CSF (may define specific immune neuromuscular disorders) and genetic testing (defines specific hereditary disorders).

Electrophysiology has certain cardinal rules:

• Electromyography and Nerve conduction studies are an extension of the clinical examination.
• Whenever in doubt: think about technical factors; reexamine the patient.
• Always thinks of clinical electrophysiologic correlation.

Goals of electrophysiology include: Localization of the lesion: Nerve/neuromuscular junction/muscle; Assessment of severity; Assessment of temporal course. It helps us to distinguish neuropathic Vs myopathic pattern, neuromuscular junction blocks, myotonic discharges, decremental response, assess nerve conduction velocities (motor & sensory) and sensory potentials.

Various techniques are available: Ultrasound; Computerized tomography; Magnetic resonance imaging. It is potentially useful to select site of muscle biopsy (especially in inflammatory); to demonstrate some selectivity; to follow up disease progression.

It has much inter-individual variability. If elevated suggests muscular dystrophy.

CK: very high

• Hereditary: Duchenne and Becker dystrophy, Limb girdle dystrophies, Miyoshi Distal Myopathy, Acid maltase deficiency.
• Chronic acquired disorders: Inflammatory Myopathies (Except: Inclusion body myositis), Hypothyroid
• Acute muscle damage (Highest CK): Rhabdomyolysis, Trauma, Injection.

Creatine kinase: Unexpectedly High

• Asymptomatic high CK; If persists after rest perform: EMG, Muscle biopsy.
• High CK with few or no symptoms
• Endocrine: Hypothyroid, Hypoparathyroid
• Exercise
• Muscle Trauma: Injections
• Myopathies (asymptomatic): Dystrophy (Dystrophinopathy; Limb-Girdle MD); Metabolic (Glycogen Storage Disorders); Other hereditary myopathy (Central core, Mitochondrial disease, Myopathy with tubular aggregates, Myotonia)
• Acquired disorders: Inflammatory, Drug toxicity
• Denervation: Motor neuron diseases
• Idiopathic

Creatine kinase: Low

• Muscle disease: Reduced mass: End-stage disease, Corticosteroid treatment, Dermatomyositis
• Hyperthyroidism
• Rheumatic diseases: Active inflammation: Rheumatoid arthritis, Systemic lupus erythematosus, Fascitis, Perimyositis.

Selection of muscle to biopsy:

• Chronic disease: Muscle with moderate, but not severe, weakness
• Acute disease: Muscle with severe or moderate weakness
• Best specific muscles: Deltoid; Biceps; Quadriceps
• MRI can be used to select pathological muscle site in difficult cases
• Avoid: Muscles that were site of EMG, injections, or trauma (Biopsy: left side; EMG: right side)

How to read a muscle biopsy?

• Are muscle fibers abnormal?
• Is the pathologic process:
• Neurogenic or Myopathic?
• Acute or Chronic?
• Producing specific diagnostic features?
• What is the distribution of the pathology?
• Are there diagnostic features?
• Is there inflammation or excess cellularity?
• Is there storage material?
• Is there pathology in structures other than muscle fibers?

Muscle biopsy stains

• Morphology : Hematoxylin and eosin; Verhof van Giesson (VvG); Modified Gomori trichrome
• Fiber Typing : Myofibrillar ATPase; ATPase pH 9.4/pH 4.6/pH4.3
• Enzymes: Oxidative : NADH-TR, Succinate dehydrogenase, Cytochrome Oxidase
• Enzymes: Glycolytic : Myophosphorylase, Phosphofructokinase, Amylopectinase
• Enzymes: Hydrolytic : Acid phosphatase, Non-specific esterase, Acetylcholinesterase, Alkaline phosphatase
• Storage material : PAS, Alcian blue, Sudan Black B, Oil red O
• Other : Congo red, Myoadenylate deaminase, Methyl green pyronine, Acridine orange, Von Kossa
• Fixed muscle : H & E.

Immunohistochemistry

• Extracellular molecules
• Laminins: Laminin al-chain; Laminin a2 (Merosin); Laminin-a1
• Collagen
• Perlecan
• Sarcolemma-related proteins
• Dystrophin: 3 Antibodies used, Vs C-terminal, Rod & N-terminal domains of dystrophin; Western blot: Useful for detecting reduced size/reduced amount of dystrophin
• a-Dystronglycan:
• Sarcoglycan
• Dysferlin
• Caveolin-3
• Cytoplasmic proteins: Soluble & Myofibrillar
• Actin
• Z-disk proteins
• Myosin: Fiber typing
• Titin
• Calpain-3 (western blot)
• Desmin
• Nuclear proteins
• Emerin
• Lamin A/C

Western blotting
On muscle specimen, is a semi-quantitative method; detects reduction of protein level or size/weight abnormalities; for a limited number of proteins: Calpain, Dysferlin useful; Dystrophin, Sarcoglycans.

Electron microscopy
Not routinely performed: expensive, time consuming; especially useful ion congenital myopathies, storage myopathies, mitochondrial myopathies, protein aggregating myopathies.

• Myasthenia Gravis: Anti-Acetylcholine Receptor (IgG); Anti-Striational (IgG): With Thymoma
• Lambert-Eaton Myasthenic Syndrome: Anti-P-type Calcium Channel (IgG)
• Myositis associated antibodies
• Antibodies associated with connective tissue disorders
• Disorders of the nerve
• Monoclonal antibodies (M-proteins): Serum or urine
• Cryoglobulins: if detected, test for Monoclonal antibodies; Hepatitis C; Collagen Vascular Screen
• Antibodies vs. Glycolipids & Glycoproteins
• Antibodies vs. Intracellular antigens
• Antibodies vs. Receptors & Channels
• Vasculitis & Autoantibodies

• Chromosomes (FISH)
• DNA (Restriction fragments; PCR; sequencing denaturing techniques; protein truncation test)
• mRNA (Northern blot; microarray expression analysis)
• Protein (Immunohistochemistry; enzyme functional assays; western blot).

Mutational diversity of a gene: Duplication; Deletion; Point mutation; Null mutation; Missense mutation; Triplet expansion; Out of frame or in frame.

• DMD/BMD: DNA;M Biopsy
• LGMD:DNA; M Biopsy; EMG/NCS
• EDMD: DNA; M Biopsy; ENG/NCS
• FSHMD: DNA; EMG/NCS
• MyD: DNA; EMG/NCS
• Periodic paralysis/myotonia: DNA;EMG/NCS
• Metabolic myopathies: M Biopsy; DNA; EMG/NCS
• Congenital myopathies: EMG/NCS; M Biopsy; DNA
• DM/PM:MRI; M Biopsy; EMG/NCS
• Indeterminate proximal weakness: EMG/NCS;RMNS;M Biopsy/DNA
• Neuromuscular transmission disorders: EMG/NCS;RMNS; Antibodies.

1st National Conference on Neurogenetics - Conference Abstracts.Pediatric Oncall [serial online] 2007 [cited 15 February 2007(Supplement 2)];4. Available from:
http://www.pediatriconcall.com/fordoctor/Conference_abstracts/
NEUROGENETICSDEL/muscular.asp