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Investigation of Muscle Disease

Investigation of Muscle Disease

25626ournal of , Neurosurgery, and Psychiatry 1996;60:256-274 J Neurol Neurosurg Psychiatry: first published as 10.1136/jnnp.60.3.256 on 1 March 1996. Downloaded from NEUROLOGICAL INVESTIGATIONS

Investigation of muscle

F L Mastaglia, N G Laing

Various pathological processes, some geneti- such as the dystrophinopathies, encompassing cally determined and others acquired, may the Duchenne and Becker forms of muscular Australian affect the function of the skeletal muscles and dystrophy, the sarcoglycanopathies, which Neuromuscular may manifest in different ways. Some, such as include many cases of limb-girdle muscular Research Institute, the University congenital , produce dystrophy, and the comprising Departments of and at birth whereas others do not the periodic paralyses and myotonic syn- Medicine and cause functional abnormalities until - dromes. Department of hood, , or adult life. With the This review focuses on the modern Neurology and Clinical application of modem molecular biological approach to the clinical and laboratory investi- Neurophysiology, techniques major advances have taken place in gation of patients with muscle with Queen Elizabeth II Medical Centre, Perth, the identificaton of the genetic particular emphasis on the application of mole- Western Australia responsible for many of the hereditary muscle cular techniques in diagnosis. F L Mastaglia diseases and new mutations in nuclear or N G Laing mitochondrial DNA are being reported on a to Correspondence regular basis. 2 These discoveries have had a Clinical evaluation Department of Medicine, major impact on the diagnostic approach to The investigation of a patient with muscle dis- Centre, GBlock, Nedlands, patients with these disorders and have led to ease should always commence with a detailed Western Australia 6009. the definition of new categories of history which, in the case of known hereditary disorders, may provide an immediate indica- tion of the nature of the patient's condition. Table 1 Drug induced muscle disorders Moreover, a history of heavy alcohol con- Disorder Inducing drug sumption or administration of drugs with known myotoxic actions (table 1) may point to Suxamethonium, danazol, clofibrate, salbutamol, lithium, captopril, a toxic and therefore potentially reversible colchicine, procainamide, metolazone, cytotoxics, zidovudine, http://jnnp.bmj.com/ isoetherine, zimeldine, labetalol, pindolol, cimetidine, penicillamine, aetiology for the patient's symptoms.3 A his- gold, enalapril, rifampicin, i.-tryptophan, nifedipine Diazacholesterol, ,¢blockers,* «-agonists (fenoterol, ritodrine),* tory of a thyroidectomy or parathyroidectomy, clofibrate,t (frusemide, ethacrynic acid, mersalyl, or symptoms of or hyperthy- )t Necrotising Alcohol, gemfibrozil, lovastatin, simvastatin, clofibrate, E-aminocaproic roidism, should alert the to the possi- myopathy acid, cyclosporin, zidovudine, cocaine, emetine bility of an endocrine cause whereas a history Zidovudine of chronic diarrhoea, purgative misuse, or Inflammatory excessive consumption of liquorice or other

myopathy D-penicillamine, i.-tryptophan, others rarely on September 23, 2021 by guest. Protected copyright. Autophagic myopathy Chloroquine, vincristine, colchicine, amiodarone, perhexiline preparations containing glycyrrhizinic acid Type 2 Corticosteroids such as snuff, chewing tobacco, and certain Localised myopathy Intramuscular antibiotics, narcotics traditional Chinese medicines, should suggest *May exacerbate myotonia. the possibility of hypokalaemic myopathy. A tMay cause myotonia in animals. history of malignancy, of a systemic connec- tive tissue disease, other autoimmune disease, or state may indicate a pre- Table 2 Disorders in which muscle may be a prominentfeature immunodeficiency disposition to an . Inflammatory Hereditary When muscle is a feature, hypothyroid, Viral Disorders of glycogen pain palmityl transferase deficiency osteomalacic or other metabolic myopathy, Parasitic myositis Myoadenylate deaminase deficiency infestation example, trichinosis), Polymyositis/dermatomyositis Mitochondrial myopathy parasitic (for Granulomatous myositis or a toxic myopathy or neuromyopathy should Interstitial myositis myotonia be considered, (table 2), although in many Localised nodular myositis Vasculitis patients no specific aetiology will be found Eosinophilic fasciitis Others even with complete investigation. Myalgia, Toxic Polymyalgia rheumatica , or developing after Acute alcoholic myopathy Postviral myalgia/fatigue an acute viral also raises the possibil- Acute/subacute drug induced myopathies Muscle overuse syndromes Myopathies due to envenomation Myopathy with tubular aggregates ity of an inflammatory myopathy but in many Endocrine such patients when fatigue and reduced exer- Hypothyroidism cise tolerance are the major symptoms, a diag- Osteomalacia nosis of postviral chronic fatigue syndrome Hyperparathyroidism will usually be reached. Investigation of muscle disease 257 J Neurol Neurosurg Psychiatry: first published as 10.1136/jnnp.60.3.256 on 1 March 1996. Downloaded from In certain instances it may be possible to other types of or spinal reach a definitive diagnosis on the basis of the muscular atrophy, whereas more generalised pattern of muscle involvement found on clinical hypertrophy is common in myotonia con- examination or the finding of other distinctive genita. A hypertrophic myopathy may occa- features such as myotonia, fatiguability, muscle sionally occur in patients with amyloidosis, , or other systemic features. sarcoidosis, or cysticercosis. Muscle contrac- Although the distribution of muscle involve- tures occur, especially in Emery-Dreifuss mus- ment in most of the acquired myopathies is rel- cular dystrophy, and are also a feature of the atively non-selective, in the genetic myopathies fibrosing myositis associated with scleromyx- certain patterns of muscle weakness are distinc- oedema.8 It is always worth looking for muscle tive and may be diagnostically helpful although it tenderness, which, when confined to certain is being increasingly recognised that the pheno- muscles such as those of the calves, may indi- typic manifestations of specific genetic defects cate a focal inflammatory or vasculitic process, may be very variable (for example, the dys- whereas the characteristic pattern of myofas- trophinopathies). Involvement of the extraocu- cial tenderness in patients with fibromyalgia is lar and eyelid muscles is seen characteristically virtually diagnostic of that condition. in oculopharyngeal muscular dystrophy, usually Depressed deep tendon reflexes or sensory associated with dysphagia and often with limb abnormalities in a patient with a myopathy muscle involvement. They are also involved in suggest the presence of an associated periph- the syndrome of chronic progressive external eral neuropathy. This combination can occur ophthalmoplegia, which is usually due to a in patients with drug induced neuromyopathy, mitochondrial myopathy and may occur in iso- connective tissue disease, inclusion body lation, or with a limb myopathy, or other sys- myositis, a paraneoplastic syndrome, or mito- temic features such as pigmentary retinopathy, chondrial myopathy. block, cerebellar , and sensorineural hearing loss as in the Kearns-Sayre syndrome. Involvement of the facial muscles is usually a Biochemical studies prominent feature in facioscapulohumeral mus- cular dystrophy but may also occur in myasthe- The serum concentration of creatine kinase is nia gravis, when it is usually associated with the most reliable biochemical indicator of involvement of the extraocular muscles, and muscle disease. The highest concentrations often of the bulbar and limb muscles; fatigua- occur in patients with acute , bility is a prominent feature. In myotonic dys- inflammatory or drug-induced myopathies, trophy there is often also involvement of the and Duchenne muscular dystrophy in the facial muscles and, characteristically, there is early stages when the patient is still ambulant. atrophy and weakness of the sternomastoids High concentrations may also occur in some and of the distal limb muscles in the later stages metabolic myopathies such as hypokalaemic or of the disease. Other systemic features which hypothyroid myopathy. Moderately raised point to the diagnosis include cataracts and, in concentrations may also be found in patients men, frontal baldness and testicular atrophy. with chronic neuropathic conditions such as

Severe weakness of the neck extensor muscles or motor neuron dis- http://jnnp.bmj.com/ leading to the "dropped head syndrome"6 may ease, although it is rare for the creatine kinase occasionally be the presenting feature in to exceed 10 times the normal maximum con- patients with inflammatory myopathy and may centration in these conditions.9 Raised crea- also occur in motor neuron disease and long- tine kinase concentrations may also be found standing . Weakness confined in some people, without clinical evidence of to or most severe in the distal limb muscles also '0 and may be useful in occurs in the distal myopathies and scapuloper- detecting those with a genetic risk of malig- oneal syndrome. nant hyperthermia, presymptomatic muscular on September 23, 2021 by guest. Protected copyright. The limb-girdle syndrome, in which there is dystrophy, carriers of Duchenne and Becker involvement of the girdle and proximal limb muscular dystrophy, and early inflammatory muscles, is relatively non-specific and may be myopathy (table 3). Serum creatine kinase seen in several genetic and acquired concentrations are normal in most congenital myopathies. Although in most cases of myopathies, myotonic syndromes, and cor- polymyositis and dermatomyositis there is a ticosteroid and . predominantly proximal pattern of muscle Slight increases in serum creatine kinase (up involvement, in the dis- to about three times the normal maximum tribution of muscle weakness is characteristi- concentration) are not necessarily due to mus- cally selective, at least in the earlier stages ofthe cle disease and may occur transiently as a condition, with involvement especially of the result of strenuous exercise, minor muscle quadriceps femoris muscles in the lower limbs trauma including intramuscular injections, and the forearm flexors, particularly the flexor and insertion of EMG needle electrodes, or digitorum profundus, in the upper limbs.7 viral illnesses.9 In a situation where the crea- When present, the characteristic rash of tine kinase concentration is unexpectedly dermatomyositis over the face and the extensor raised it is usual to repeat the test after an aspects of the metacarpophalangeal and inter- interval of a week during which the patient is phalangeal joints is diagnostic of that condition. advised not to engage in strenuous physical , when confined to the exercise, and to consider embarking on further calves, is seen most typically in Duchenne and investigations only if the concentration Becker dystrophy, but occasionally also in remains high or is rising. 258 Mastaglia, Laing J Neurol Neurosurg Psychiatry: first published as 10.1136/jnnp.60.3.256 on 1 March 1996. Downloaded from Table 3 Causes ofsustained increased serum creatine with kinase concentrations in subjects without clinical signs of myophosphorylase deficiency or a gly- muscle disease colytic defect. The rise in venous ammonia concentrations that occurs after exer- Physical exercise normally Muscle trauma cise is absent or reduced in patients with Pressure deaminase Falls myoadenylate deficiency in which Injections ammonia production during exercise is Acute psychosis/delirium 13 Dyskinesiae impaired. Drugs Alcohol NUCLEAR MAGNETIC RESONANCE Others* Hypothyroidism SPECTROSCOPY Hypokalaemia With NMR it is Presymptomatic spectroscopy possible to mon- Malignant hyperthermia itor changes in muscle metabolite concentra- Muscular dystrophies tions exercise Inflammatory myopathies during non-invasively (for McArdle's disease example, inorganic phosphate, phosphocrea- Carrier state tine, ATP, lactate and intra- Duchenne, Becker dystrophy concentrations, Herditary hyperCKaemia cellular pH).'4 ' This may be useful in the evaluation of with *See table 1. patients disorders of gly- colytic or mitochondrial metabolism. The technique is also useful in the evaluation of Serum concentrations of other patients with fatigue and reduced exercise tol- such as aspartate and alanine aminotrans- erance to determine whether there is evidence ferase, aldolase, and lactate dehydrogenase are of an underlying defect of muscle energy of less value than the creatine kinase concen- metabolism. tration but may provide a clue to the presence of muscle disease if they are found to be raised OTHER STUDIES as part of an initial biochemical screen in a Other biochemical studies which may be rele- patient with no other indication of liver dis- vant in the investigation of some patients with ease. muscle symptoms include serum potassium, calcium, phosphate, thyroxine, and cortisol MYOGLOBIN concentrations, as well as urea and creatinine Myoglobinaemia and myoglobinuria may be concentrations and urinary creatine and 3- due to various causes1' and result in urinary methyl histidine excretion as indices of skeletal pigmentation and a positive benzidine dip muscle mass and breakdown.'6 stick test on the urine as does haemoglobinuria and haematuria. Confirmation of myoglobin- uria requires a specific myoglobin radioim- Pharmacological testing munoassay. Serum myoglobin concentrations Although in some families with malignant are raised in patients with inflammatory'2 and hyperthermia mutations have been found in other necrotising myopathies but are not of the and at risk sub- any additional value to the creatine kinase jects can therefore be detected using molecular

concentration in diagnosis or in monitoring genetic techniques, in many other families as http://jnnp.bmj.com/ the response to treatment. well as in those without a known family history who are suspected of being at risk, it is still LACTATE CONCENTRATIONS necessary to perform in vitro muscle contrac- Venous lactate concentrations may be raised ture testing. This involves the exposure of at rest and after low levels of exercise in muscle tissue obtained at to caffeine patients with mitochondrial myopathy and and halothane which, in at risk subjects, defects of the respiratory chain. induce an exaggerated contractile

response. on September 23, 2021 by guest. Protected copyright. Conversely, lactate production is absent or Abnormal responses to caffeine and calcium diminished in metabolic myopathies due to ions have also been described using the defects in glycogenolysis (for example, skinned muscle fibre technique.'7 myophosphorylase or phosphorylase b kinase testing should also be performed in those with deficiency) or of the glycolytic pathway (for unexplained episodes of muscle stiffness or example, phosphofructokinase, phosphoglyc- rhabdomyolysis during or after anaesthesia, erate mutase, phosphoglycerate kinase, or lac- with , or after exercise or heat expo- tate dehydrogenase deficiency) and is the basis sure, and in patients with central core or multi- for the forearm exercise test. This was previ- core disease who are also at risk. ously performed under ischaemic conditions but, because of the occurrence of severe rhab- domyolysis in some patients with glycogen Electrodiagnostic studies metabolic defects, this is no longer recom- mended.9 Venous samples for estima- Conventional EMG is an important investiga- tion of lactate and ammonia are taken at rest tive procedure in patients with suspected mus- and at 1, 2, 4, 6, and 10 minute intervals after cle disease. Firstly, it will often provide a one minute period of repetitive maximum confirmation of a primary myopathic basis for isometric contractions of the forearm flexor the patient's condition and allow differentia- muscles. Whereas there is normally a twofold tion from a neurogenic disorder. Charac- to threefold rise in the lactate concentration teristically, the duration of motor unit action within the first two minutes after exercise, this potentials (MUAPs) is diminished as is response is absent or diminished in patients MUAP amplitude although this is often less Investigation ofmuscle disease 259 J Neurol Neurosurg Psychiatry: first published as 10.1136/jnnp.60.3.256 on 1 March 1996. Downloaded from pronounced and more variable, and there is an which can be performed either with voluntary increased number of polyphasic motor unit muscle activation or with nerve stimulation, potentials. With voluntary contraction there is characteristically shows increased jitter and early recruitment of increased numbers of intermittent blocking indicating failure of trans- short duration MUAPs and an unduly full mission at single motor endplates, and is more interference pattern which is often reduced in sensitive than repetitive nerve stimulation studies amplitude. Reduced motor unit recruitment in the diagnosis of neuromuscular transmission and electrical excitability of the muscle are disorders. found during attacks of weakness in patients In myasthenia gravis SFEMG shows an with periodic . Spontaneous fibrilla- abnormality in up to 95% of patients with limb tion potentials, positive sharp waves, and com- involvement and a normal SFEMG study in a plex repetitive discharges may be found in patient with clearcut limb weakness and fatigue some myopathic disorders and are particularly is strongly against the diagnosis of myasthe- prominent in active inflammatory myopathies nia.'8 However, similar abnormalities may be and certain metabolic and toxic myopathies found in several myopathies and other neuro- (for example, hypothyroid and chloroquine muscular disorders and it is therefore essential myopathy), in some cases of Duchenne mus- that the findings are interpreted in the context cular dystrophy and distal myopathy, and in of the particular clinical situation. myotonic disorders in which they are associ- ated with a pronounced increase in insertional NERVE CONDUCTION STUDIES activity and the diagnostic waxing and waning Motor and sensory nerve conduction studies as ("dive bomber") myotonic discharges result- well as F wave and H reflex studies may be ing from electrical instability of the muscle appropriate to exclude the possibility that the membrane.'8 The occurrence of spontaneous patient's symptoms are due to a peripheral neu- potentials in non-myotonic myopathies has ropathy. Moreover, a been attributed to functional of may coexist in some patients with myotonic muscle fibres which have been disconnected dystrophy, mitochondrial myopathy, drug from their motor innervation as a result of seg- induced myopathy, inclusion body myositis, or mental necrosis. Regenerating muscle fibres inflammatory myopathy associated with con- which have yet to be innervated may also be a nective tissue diseases or malignancy. source of spontaneous potentials. The distribution of EMG changes within individual muscles and in representative proxi- Muscle imaging mal and distal limb and axial muscles provides The techniques of CT and MRI can provide an indication of the extent of the myopathic information on the cross sectional area of limb process and pattern of muscle involvement. or axial muscles and may therefore be useful in This may be useful in selecting a muscle for detecting muscle atrophy or hypertrophy and biopsy, and may also indicate whether the dis- defining selective patterns of muscle involve- ease process is one which is affecting the mus- ment in certain conditions such as the muscular cles in a patchy manner (often the case in the dystrophies and inclusion body myositis.20 21

inflammatory myopathies) or more diffusely These techniques may also show differences in http://jnnp.bmj.com/ (as in the muscular dystrophies or metabolic muscle properties between conditions such as myopathies). The coexistence in the same the muscular dystrophies in which there is muscle of typical myopathic MUAPs and extensive fatty infiltration of muscle, and the longer duration polyphasic potentials is a inflammatory myopathies, but the changes are potentially confusing combination sometimes not sufficiently specific to be of diagnostic encountered in patients with very longstanding value.22 The techniques are, however, of use in denervating conditions such as spinal muscu- detecting and defining the extent of

suppurative on September 23, 2021 by guest. Protected copyright. lar atrophy,'8 and in inclusion body myositis in lesions of muscle (pyomyositis). Radioisotopic which there may also be prominent fibrillation techniques with muscle scanning after adminis- potentials as well as increased fibre density tration oftechnitium pyrophosphate can also be and jitter.'9 used to detect such lesions as well as areas of Although in many patients with well estab- active inflammatory myopathy in patients with lished myopathies the EMG changes are florid polymyositis or dermatomyositis, and areas of and unmistakable, in some patients with muscle infarction or pressure necrosis (crush milder forms of myopathy the motor unit syndrome). Isotopic scanning may also be used changes are less conspicuous. It is in the evalu- to show the extent of subcutaneous calcinosis in ation of such patients that quantitative tech- patients with dermatomyositis and to monitor niques employing automated measurement of changes with treatment. MUAP variables and motor unit recruitment for given levels of effort are useful.'8 NEUROMUSCULAR TRANSMISSION STUDIES A biopsy is still required to provide a definitive Repetitive nerve stimulation studies should be diagnosis in the case of many muscle diseases. performed if muscle fatigue is prominent or if This is especially so in the case of the inflam- there are other features suggestive of myasthe- matory myopathies, in sporadic cases of muscu- nia gravis or of the Lambert-Eaton myasthenic lar dystrophy, suspected metabolic myopathies, syndrome or other and in patients in whom there is still uncer- disorder. tainty as to whether the condition is primarily Single fibre electromyography (SFEMG), neuropathic or myopathic after clinical and 260 Mastaglia, Laing J Neurol Neurosurg Psychiatry: first published as 10.1136/jnnp.60.3.256 on 1 March 1996. Downloaded from EMG evaluation. In neuropathic disorders a and Becker dystrophy clinically, whereas anti- biopsy from an affected muscle will show the bodies can make the distinction very easily. characteristic changes of denervation including Up until recently these cases were among the angulated fibres, grouped fibre atrophy, and, if residual of patients all muscle clinics have who the condition is protracted, fibre type grouping, are difficult to categorise. For example, but will not necessarily differentiate between Bonnemann et al24 in their recent characterisa- anterior horn cell and peripheral nerve disor- tion of mutations in ,8-sarcoglycan screened 62 ders. In the more chronic neurogenic disor- patients with possible ,B-sarcoglycan involve- ders-such as the spinal muscular - ment out of a bank of 2500 muscle to histological changes resembling those found in identify their one ,B-sarcoglycan patient. In our chronic myopathic disorders may also be pre- own clinic, screening nine families undiag- sent (secondary myopathic change). nosed for years, with antibodies to adhalin, The effectiveness of muscle biopsy in estab- showed that three of the families were a-sarco- lishing the diagnosis of myopathy is deter- glycan (adhalin) negative (RD Johnson and mined by the correct choice of muscle, the BA Kakulas, unpublished observations) and correct biopsy technique, and the application thus are at risk from a sarcoglycanopathy. of the appropriate staining and other proce- Immunohistochemistry or immunoblotting dures to the biopsy.23 In selecting a suitable can also predict the severity of disease for biopsy site the ideal muscle is one which is example, in both the dystrophinopathies25 27 only moderately affected (for example, with and the sarcoglycanopathies2' 28 On the other MRC grade 4 muscle power), and which has hand, molecular analysis cannot always pre- not been the site of previous intramuscular dict severity of disease, as people with appar- injections or needle EMG studies. Muscles ently the same can have different which are too severely weakened or atrophic severities of disease29 perhaps due to alternate should be avoided as the histological changes splicing around the mutation.3" in such muscles are often non-specific and dif- ficult to interpret. Selection of a needle or open biopsy is determined by personal choice, Molecular diagnosis experience, and availability. Whereas needle Molecular biology, particularly positional biopsy requires only a minor incision and the cloning, has led to the recent major increase in procedure can be repeated at multiple sites understanding of the causes of inherited dis- and on more than one occasion, the size of the eases, through identifying the mutated tissue sample obtained is limited and histologi- in these diseases and thus the faulty proteins.32 cal interpretation may be difficult, particularly In the muscle clinic, similar molecular tech- in diseases in which the muscle involvement is niques can be used for four major purposes: patchy. In many instances therefore, and espe- * Identifying the precise disease affecting an cially in the case of suspected inflammatory individual patient and family. myopathy or vasculitis, an open biopsy is * Accurate, simple prenatal diagnosis preferable, having the advantage of providing a * Identifying asymptomatic members of fam- larger tissue sample and thereby reducing ilies who are at risk from having more severely

sampling error. affected offspring http://jnnp.bmj.com/ To maximise the information derived from * Presymptomatic diagnosis. the muscle biopsy a battery of histological and All of these lead to more accurate and appro- histochemical stains should be applied rou- priate genetic counselling. tinely to cryostat sections of frozen tissue. In addition, in some cases it will be necessary to IDENTIFYING THE PRECISE DISEASE AFFECTING apply other selected histochemical or AN INDIVIDUAL PATIENT AND FAMILY immunohistochemical techniques using mon- Molecular diagnosis is especially useful for oclonal antibodies for the diagnosis of specific conditions which are difficult to distinguish on September 23, 2021 by guest. Protected copyright. enzyme deficiencies, storage disorders, or clinically or by other laboratory techniques. other hereditary defects such as the dys- The sarcoglycanopathies,33 in which mutations trophinopathies and sarcoglycanopathies. in adhalin, 8B-sarcoglycan, and y-sarcoglycan Electron microscopic examination of muscle all look similar by immunohistochemistry, are a tissue is also necessary especially in cases of good example of diseases only distinguishable suspected mitochondrial myopathy, inclusion by molecular analysis.34 body myositis, and in some congenital myopathies. Tissue should therefore be taken ACCURATE, SIMPLE PRENATAL DIAGNOSIS routinely for this purpose at the time of the Identifying the precise mutation or mutations biopsy and fixed in glutaraldehyde.23 In meta- causing the disease in a family also provides bolic and other inherited myopathies it is also the only really practical route to prenatal diag- prudent to obtain an additional tissue sample nosis where that is appropriate. The only cur- for quantitative biochemical analyses or molec- rent alternative for inherited muscle diseases ular studies should these be required. would be fetal muscle biopsy. 33 In the hereditary myopathies, immunohisto- chemistry of muscle biopsies should be used IDENTIFYING ASYMPTOMATIC MEMBERS OF whenever possible to identify the patient or FAMILIES WHO ARE AT RISK FROM HAVING family muscle disease. For example, the auto- MORE SEVERELY AFFECTED OFFSPRING somal recessive Duchenne-like muscular dys- Molecular diagnosis allows the identification trophies and the limb-girdle dystrophies can of minimally affected or asymptomatic per- be very difficult to distinguish from Duchenne sons. This is important for identifying carriers Investigation ofmuscle disease 261 J Neurol Neurosurg Psychiatry: first published as 10.1136/jnnp.60.3.256 on 1 March 1996. Downloaded from

of X linked diseases such as Duchenne and (4) Hypokalaemic , in which Becker muscular dystrophy, Emery-Dreifuss mutations to two residues in the dihydropyri- dystrophy, and X linked centronuclear myopa- dine receptor (CACNL1A3) thy who are at risk of having affected male off- account for all cases so far described.48 spring. It is also important for tracing at risk (5) Mitochondrial myopathies, in which the subjects-for example, in common point mutations can be tested for pedigrees-through cascade screening. using PCR followed by enzyme digestion41 49 or SSCP analysis.50 PRESYMPTOMATIC DIAGNOSIS Presymptomatic diagnosis using molecular Difficult diseases to diagnose by molecular analysis techniques has been applied, for example, to diagnose the cause of the raised creatine Diseases in which most cases are caused by many kinase and to screen other family members different missense mutations-Finding the muta- after neonatal or screening for tions causing disease in such conditions is the Duchenne muscular dystrophy.3637 Presymp- most difficult task for molecular analysis. This tomatic diagnosis is also possible by molecular is because the entire gene sequence has to be techniques in any of the later onset diseases, screened, and the difficulty is therefore such as hypokalaemic periodic paralysis.38 increased if the gene message is large, which is The polymerase chain reaction (PCR),39 the case for many of the structural proteins of which amplifies specific regions of DNA, has muscle. The difficulty of identifying missense played a major part in the molecular revolu- mutations was reinforced again in a recent tion and is being manipulated for ever more summary of current methods of screening purposes-such as reverse transcriptase PCR genes for unknown mutations,5' which states (RT-PCR) which allows PCR to amplify spe- that, "Detection of mutations, particularly at cific cDNA messages40 or the creation of the 100% level is time consuming and expen- restriction enzyme sites to discriminate sive" and "The 'one best method' still remains between normal and mutant alleles.4' elusive". Indeed, the plethora of techniques currently on the market for identifying DEGREES OF EASE OF MOLECULAR DIAGNOSIS unknown mutations in genes indicates that There are perhaps three degrees of difficulty none is ideal. A previous review52 summarised for molecular diagnosis: easy, difficult, and the techniques then current such as the simple average. Easy diseases to diagnose are those in single strand conformation analysis (SSCA), which most cases are caused by a single muta- denaturing gradient gel electrophoresis tion, or a few mutations, or mutations which (DGGE), heteroduplex analysis (HA), are simple to detect. Difficult diseases to diag- RNAase A cleavage, chemical mismatch cleav- nose are: (a) those in which most cases are age (CMC), and direct sequencing. Tech- caused by many different missense mutations niques which should perhaps be added to the which alter only a single and thus list include restriction endonuclease finger- do not truncate the protein; (b) diseases in printing (REF),53 dideoxyfingerprinting,54 and which linkage to a particular locus has been enzymatic mismatch cleavage (EMC).5556

identified but the disease gene has not been The heroic way to look for mutations is to http://jnnp.bmj.com/ identified; or (c) diseases with significant search genomic DNA. This will often involve genetic heterogeneity-that is, where the same synthesising PCR primers for regions flanking clinical may be caused by muta- each of the exons of the gene followed by, for tions in a number of different genes. Average example, SSCP analysis of each exon. This diseases to diagnose are those caused in most would necessitate synthesising 80 primers for a cases by nonsense mutations which produce a gene like the cardiac ,B-myosin heavy chain premature stop codon and therefore truncated gene (MYH7) involved in familial hyper- proteins. trophic -as MYH7 has 40 on September 23, 2021 by guest. Protected copyright. exons-and then performing 40 separate Easy diseases to diagnose by molecular analysis SSCPs. The short cut alternative is to examine Diseases which currently fall into this category the cDNA for mutations.52 This reduces the are: number of base pairs that have to be screened (1) Myotonic dystrophy, in which the triplet compared with examining the genomic repeat expansion can be detected and sized by sequence of the gene. Analysis of cDNA will a PCR reaction, and/or Southern blotting also instantly disclose any deletions or duplica- using EcoRI, BglI, or BamHI digestion.42 tions of the message and splice site mutations (2) Duchenne and Becker muscular dystro- which also significantly alter the size of the phy (for most patients) as most disease caus- transcript, leading to rapid identification of ing mutations are large deletions of the one type of single base mutation.5758 To exam- gene. These deletions can be ine the cDNA however, a source of RNA is detected by multiplex PCR analysis of DNA required from the patient in whom the candi- from affected boys to identify the deleted date gene is expressed. This should be rela- exons.25 43-45 tively simple, as nearly all patients with a (3) Spinal muscular atrophy (autosomal muscle disease should have had a muscle recessive), in which the common can biopsy, and RNA can easily be extracted from be detected by two single strand conformation frozen muscle tissue and cDNA synthesised polymorphism (SSCP) analyses, one for exon for examination. Analysis of cDNA made from 7 of the survival motor neuron gene and one illegitimate transcription messages59 should for exon 8.46 47 always be considered as a possible means of 262 Mastaglia, Laing J Neurol Neurosurg Psychiatry: first published as 10.1136/jnnp.60.3.256 on 1 March 1996. Downloaded from obtaining cDNA for muscle disease genes. cular dystrophies may also be included in this However, in some cases it may be more appro- category. Some of the different types can now priate to analyse cDNA from muscle, (for be identified as sarcoglycanopathies or dystro- example, the mitochondrial myopathies), as phinopathies by immunohistochemistry, but it the mutations in may be differ- is still a large task to precisely identify the gene ent and more relevant than those in circulating involved by molecular diagnosis.

lvmphocvtes.60-J -. Suggesting that a in any Average diseases to diagnose by molecular analysis message is responsible for disease-especially Average diseases are those in which most muta- in a giant message like the dystrophin mes- tions causing disease are nonsense mutations, sag;e61_iis fraught with difficulties.62 The criteria as these can be detected using the protein trun- noi used to decide that an2ridentified cation test (PTT).7576 This test uses an in vitro rmally altceration in a DNA sequence is a disease translation system to synthesise protein from cauising mutation include a major change of an cDNA. If there is a mutation in the protein amino acid, change of an amino acid con- coding region leading to a premature stop served in many species, the change not being codon, the protein synthesised in vitro will be see:n in a large number of controls, and the shorter than normal and can be identified by chsange being in a candidate gene. However, electrophoresis. The PTT was first applied evem when all these criteria are fulfilled, the widely to the identification of nonsense, prema- chsange may still not be disease causing.62 A ture stop codon producing, mutations in familial missense mutation found only in a single adenomatous polyposis coli75 and Duchenne paltient, or a small family, probably requires a muscular dystrophy,76 (fig 1), but it has also fur.ictional assay (not necessarily a transgenic been applied to .7778 The mc)use) or at least characterisation in an PTTl should perhaps be used for all recessive exjpression system to be certain that the mis- disorders, especially severe ones, as these are sernse mutation causes the disease.63 Unless often caused by nonsense mutations. For exam- the,re is this degree of certainty about the sta- ple, the PTT would have detected many of the tuss of the identified alteration in DNA, it mutations so far described in the sarcogly- shlould not be used for prenatal diagnosis. canopathies, especially the severe cases,24283379 Diseases in which there is only linkage but the in which prenatal diagnosis would be most pre?cise disease gene has not been identified- appropriate and there would be most likelihood Diistal myopathy,64 65 dominant limb-girdle of pressure to rapidly find the family mutation. mlascular dystrophy,6667 X linked centronu- Obviously some diseases fit into both the cle ar (myotubular) myopathy,68 oculopharyn- easy and difficult categories or both the easy geral muscular dystrophy,69 and recessive and average categories. Duchenne muscular nemaline myopathy7' are all diseases in which dystrophy is both easy for most mutations and there is at present linkage but no identified average for the minority. Mitochondrial dissease gene. When there is only linkage for a myopathies are easy for most cases and diffi- diEsease, the use of linkage for diagnosis should cult for the others. The common mtDNA perhaps be restricted to individual families in point mutations can be tested relatively easily, mutations are diffi- wIhich the significant LOD score of 3 has been but all the other possible http://jnnp.bmj.com/ obtained, especially if there is evidence of cult to screen for and to separate from poly- genetic heterogeneity for the disease. morphisms not causing disease.80 This is also Diseases with significant genetic heterogeneity- true for other diseases. However, testing only Pe!rhaps the best example of this at present is for the common known mutations becomes a m,alignant hyperthermia, in which although self fulfilling prophecy and unless the rest of many families show linkage to the ryanodine the transcript is screened for mutations, many receptor gene on chromosome 1971 many oth- mutations causing disease may be missed. ers apparently do not72 73 and virtually only sin- As emphasised by Forrest et all' it is essential on September 23, 2021 by guest. Protected copyright. glie families have shown linkage to other that comprehensive databases of gene muta- regions ofthe .74 The limb-girdle mus- tions and perhaps, even more importantly, databases of normal polymorphisms should be created and widely disseminated, so that indi- Figure 1 Protein can cross check truncation for the deletion ~3 1 vidual diagnostic laboratories prone part of the whether a base change identified in a patient is dystrophin gene carried out a known mutation causing disease, a known on mRNA from muscle or new. 1 and 2 are 4I' polymorphism, entirely biopsies. Lanes _ftMWd'I'Momoma; from control muscle immmmww :.... biopsies. Lanes 3, 4, 5, .... :....--.-.--.- and 6 show variously truncated proteins in four Muscular dystrophies patients with Duchenne The classification of the muscular dystrophies muscular dystrophy. has undergone considerable change with the recognition of the disease genes and molecular basis for some of these conditions-in particu- lar, the X linked and the limb-girdle dystro- phies (table 4).' The availability of genetic or molecular markers for many of these condi- tions has meant that the diagnosis can now be established with greater precision and that dis- eases manifesting with similar can Investigation ofmuscle disease 263 J Neurol Neurosurg Psychiatry: first published as 10.1136/jnnp.60.3.256 on 1 March 1996. Downloaded from Table 4 Muscular dystrophies normal concentrations may be found. The high Dystrophy type Locus Gene serum creatine kinase in affected boys with Duchenne dystrophy at birth has been the X linked: Dystrophinopathies Xp21-2 Dystrophin basis for the development of neonatal screen- Duchenne ing programmes for newborn males in some Becker Quadriceps myopathy countries.82 The detection of isolated cases by Myalgia and neonatal screening makes it possible to prevent HyperCKaemia Emery-Dreifuss Xq28 Emerin the birth of secondary cases in families and it Xq28 ? has been estimated that it is possible to prevent Autosomal dominant: Facioscapulohumeral 15 to 20% of new cases of Duchenne dystro- FSHD 4q35 ? phy with this type of screening. FSHD2 ? ? Limb-girdle* In most instances definitive diagnosis of a LGMD1A 5q22-q34 ? muscular dystrophy and recognition of the par- LGMD1B ? ? Distal* ticular type still requires a muscle biopsy for MPD1 14q ? routine histology and immunohistochemical Oculopharyngeal* OPMD 14ql 1-2-q13 ? staining for dystrophin and for the other pro- Autosomal recessive teins of the dystrophin-glycoprotein complex Limb-girdle LGMD2A 15qI5-1-q21-1 Calpain such as the sarcoglycans. The immunohisto- LGMD2B 2q13-pI6 ? chemical demonstration of complete or partial LGMD2C (SCARMD) 13ql2 rsarcoglycan LGMD2D 17ql2-q21-33 Adhalin (a- dystrophin deficiency is the gold standard for sarcoglycan) establishing the diagnosis of Duchenne and LGMD2E 4q12 ,B-sarcoglycan Congenital Becker muscular dystrophy. Complete or virtu- LAMA2 6q2 Merosin ally complete absence of dystrophin is charac- Fukuyama 9q31-q33 ? Distal (Myoshi)* 2p ? teristic of Duchenne dystrophy whereas in Oculopharyngeal* Becker dystrophy there is usually patchy *Autosomal dominant or recessive. preservation of the protein on the sarcolemma of muscle fibres. Dystrophin immunohisto- chemistry may therefore be of prognostic as be readily distinguished. For example, in some well as diagnostic value in differentiating patients with Becker dystrophy who have a Duchenne and Becker dystrophy in young dystrophin mutation the clinical phenotype boys. The importance of using a panel of anti- may closely resemble that of limb-girdle mus- bodies to different regions of the dystrophin cular dystrophy, which is now known to have molecule from the N to the C terminus has at least seven different genetic varieties (table been emphasised.83 4). Other examples include the differentiation Immunoblotting for dystrophin in muscle of merosin deficiency from a dystrophinopathy tissue is a more sensitive technique, which may in with congenital muscular dystrophy show more subtle changes in the molecular size and the differentiation of adhalin deficiency or amount of dystrophin. Hoffman et al26 from dystrophinopathy in the severe childhood showed that patients with Duchenne muscular autosomal recessive form of muscular dystro- dystrophy had less than 3% of the normal classified as LGMD2C). The molec- phy (now quantity of dystrophin, intermediate patients http://jnnp.bmj.com/ ular definition of these conditions has also had had up to 60% of normal concentrations, important implications for the identification of whereas patients with Becker dystrophy most heterozygote carriers and for prenatal diagno- often had abnormal sized dystrophin rather sis, especially in the X linked dystrophies. It than abnormal quantities. has also led to the recognition that there is a wide range of phenotypic expression and clini- MOLECULAR DIAGNOSIS cal severity, particularly in the dystro- Dystrophinopathies phinopathies and sarcoglycanopathies.798' The main applications of molecular diagnosis on September 23, 2021 by guest. Protected copyright. are accurate diagnosis if other methods have DIAGNOSTIC APPROACH failed or are inappropriate, accurate diagnosis The diagnostic approach to the patient with a of asymptomatic subjects (in the case of these suspected muscular dystrophy initially involves X linked diseases this largely means carriers), a detailed clinical assessment of the affected and prenatal diagnosis. patient and family history which, when posi- The dystrophin gene was the first major dis- tive, may point to an X linked disorder or to ease gene to be identified by positional cloning. one of the distinctive forms of dominantly The dystrophin message and the distribution inherited myopathy such as facioscapulo- of deletion mutations which cause about 70% humeral dystrophy or distal myopathy. of cases of Duchenne and Becker muscular Electromyography is helpful in confirming the dystrophy were first described in 1987.84 myopathic nature of the condition and exclud- However, pulsed field gel electrophoresis had ing myotonic or chronic neurogenic con- already indicated that most mutations causing ditions. A raised serum creatine kinase Duchenne dystrophy were large deletions.85 concentration, although not specific, may also The identification of the dystrophin gene be a diagnostic pointer particularly if the con- immediately allowed much more accurate dif- centration is very high as is usually the case in ferential diagnosis of Duchenne and Becker the early stages of Duchenne or Becker muscu- muscular dystrophy using the available cDNA lar dystrophy when concentrations of up to probes.86-88 Molecular diagnosis of boys 300 times normal may occur. However, in the affected with dystrophinopathies is now carried other dystrophies less pronounced increases or out by multiplex PCR of genomic DNA (fig 2), 264 Mastaglia, Laing J Neurol Neurosurg Psychiatry: first published as 10.1136/jnnp.60.3.256 on 1 March 1996. Downloaded from Figure 2 Duchenne muscular dystrophy multiplex PCR. Lane 1: size standard; lanes 2, 4, 6, 8: patients with Duchenne muscular dystrophy; lanes 3, 5, 7, 9: control: lanes 2 and 3 (from top down) promotor, exons 3, 8, 50, 53: lanes 4 and 5: exons 13, 16, 74; lanes 6 and 7: exons 48, 43, 47, 60; lanes 8 and 9: exons 19, 42, 4. Patient is deletedfor at least exons 48 and 50 (arrows).

which gives a diagnosis in hours rather than the detected in patients with LGMD2A.1'0 107 previous days or weeks.23 Prenatal diagno- There are four other localised or identified sis is also achieved with multiplex PCR when a recessive limb-girdle muscular dystrophy family deletion has been identified, and by genes: LGMD2C, LGMD2D, and LGMD2E linkage using microsatellites89 for the dys- are the sarcoglycanopathies whereas trophin gene in the large minority of families in LGMD2B, localised to the short arm of 2p,108 which deletions have not been identified. is an as yet unidentified gene. Splice site mutations in the gene can be identi- There are at least two genes for autosomal fied by RT-PCR.5758 dominant limb-girdle muscular dystrophy, one Identification of carriers of dystro- (LGMD1A) localised on chromosome 5.6667 phinopathies has been achieved with dosage The other (LGMD1B) is not yet localised.'9 Southern blotting,90 92 presence of junction fragments,85 and non-inheritance of alleles.93 Sarcoglycanopathies http://jnnp.bmj.com/ However, as with all aspects of molecular diag- Adhalinopathy (a-sarcoglycanopathy) was the nosis, carrier detection has moved to PCR; first sarcoglycanopathy to be identified, ini- PCR dosage has been used with great accuracy tially by deficiency in immunohistochemical to identify carriers9495 as has RT-PCR from staining"0 and later by mutations in the illegitimate transcription of the dystrophin adhalin gene.28111112 Adhalinopathy is also message in circulating lymphocytes.40 known as recessive limb-girdle muscular dys- The increasing diversity of dystrophinopathy trophy type D (LGMD2D). Mutations in the phenotypes has been reviewed by Beggs et al96 other two sarcoglycans have now been demon- on September 23, 2021 by guest. Protected copyright. and includes quadriceps myopathy,97 cramps strated. Mutations in the /B-sarcoglycan gene and myalgia,98 99 hyperCKaemia,'00 and X on chromosome 4 are associated with linked cardiomyopathy.'10 102 LGMD2E, the second limb-girdle muscular dystrophy found among the Amish34 and also Limb-girdle muscular dystrophies other communities.24 Mutations in the ysarco- The first recessive limb-girdle muscular dystro- glycan gene on chromosome 13 are associated phy gene to be localised (LGMD2A) was on with severe congenital autosomal recessive chromosome 15103104 through linkage in fami- muscular dystrophy (SCARMD, LGMD2C).33 lies from the island of Reunion. Mutations in In each of these diseases there is a reduction of the calcium activated protease gene calpain immunohistochemical staining for all three were subsequently identified both in the fami- sarcoglycans. Thus reduction of immunostain- lies from Reunion and in the Amish families ing for any of the sarcoglycans in the absence which showed linkage to the chromosome 15 of reduction in dystrophin staining indicates region. 305 (Two different autosomal recessive mutation in one of, at present, three genes. As limb-girdle muscular dystrophies segregate in Lim et al 34 state in the discussion of their the Amish community-the other is paper, only molecular investigation of all the LGMD2E.34) The identification of calpain as candidate genes can provide a definitive diag- the mutant gene in LGMD2A was the first nosis. implication of a non-structural protein in a muscular dystrophy.105 No abnormalities in the Congenital muscular dystrophy dystrophin associated glycoproteins were Congenital muscular dystrophies are charac- Investigation ofmuscle disease 265 J Neurol Neurosurg Psychiatry: first published as 10.1136/jnnp.60.3.256 on 1 March 1996. Downloaded from

terised by severe dystrophic changes of muscle Myotonic syndromes from birth."3 Tome et al'07 were the first to Table 5 gives a classification of these condi- show merosin (IAMA2) deficiency in cases of tions, based on the underlying molecular congenital muscular dystrophy of the occidental defects. Differentiation of the various types is type. However, not all of the clinically indistin- usually possible on the basis of clinical features guishable cases showed merosin deficiency, such as the age of onset, pattern of inheri- and this heterogeneity is yet to be explained on tance, provocative factors for the myotonia, a molecular basis. It is estimated that 40% of and the presence or absence of dystrophic occidental congenital muscular dystrophy muscle weakness or other systemic features. cases show LAMA2 deficiency."13 Sub- sequently mutations of the LAMA2 gene were MYOTONIC DYSTROPHY identified in patients with congenital muscular This is the most common of the genetic forms dystrophy. 114 of myotonia and is transmitted as an autoso- The LAMA2 gene maps to the long arm of mal dominant trait with considerable variability chromosome 6, the Japanese form of congeni- in the degree of phenotypic expression and age tal muscular dystrophy, Fukuyama muscular of onset of symptoms. The congenital form, in dystrophy to the long arm of chromosome 9,115 which there is generalised hypotonia and and thus a different as yet unidentified gene weakness, often with facial diplegia and a dis- must be involved. Fukuyama muscular dystro- tinctive tent shaped mouth, may occur in the phy and Walker-Wallberg syndrome may be offspring of either affected males or females genetically identical."6 but the more severe cases are usually the off- spring of female heterozygotes who may them- Facioscapulohumeral muscular dystrophy selves be only very mildly affected.'24 125 A gene for facioscapulohumeral muscular dys- Mental retardation and delay in motor and trophy (4q35) was first mapped in 1990 to speech development are common in early chromosome 4 near the telomere." 7 Sub- onset cases. Myotonia is not present in sequently, altered sized fragments involving a affected infants but is usually readily demon- repeated sequence with homeodomain homol- strable in the tongue, hand, and forearm mus- ogy were identified in familial and sporadic cles in patients presenting during adolescence cases of facioscapulohumeral muscular dystro- or early adult life, and tends to become pro- phy."18 119 The precise gene or genes affected by gressively less severe with the passage of time. the chromosomal rearrangements have not, Progressive weakness and atrophy of the fore- however, been identified and thus the mecha- arm, calf, and sternomastoid muscles occur as nism of pathogenesis remains a mystery. In the condition progresses and in some cases addition, recombination with the altered sized weakness of the facial, bulbar, and respiratory fragments has been documented'20 and not all muscles also develops and may be associated facioscapulohumeral muscular dystrophy fami- with irregular breathing patterns, sleep lies show linkage to 4q35. 121 Facioscapulo- apnoea, and .'26 Ventilatory humeral muscular dystrophy is thus still a function studies as well as monitoring of problematic disease for molecular diagnosis. breathing and arterial blood gases during sleep

should be performed if these problems are sus- http://jnnp.bmj.com/ Oculopharyngeal muscular dystrophy pected. A gene for oculopharyngeal muscular dystro- Other distinctive features in the adult include phy among French Canadians has been linked frontal baldness and testicular atrophy in men to the proximal long arm of chromosome 14 and distinctive subcapsular cataracts, which are close to the genes for cardiac a-myosin and ,B best seen with slit lamp examination.'25 Cardiac myosin69 and in the same region as a gene for involvement also occurs and electrocardio- dominant distal myopathy.65 graphic abnormalities which include atrioven- tricular and intraventricular conduction defects on September 23, 2021 by guest. Protected copyright. Emery-Dreifuss muscular dystrophy and atrial or ventricular arrhythmias, are com- The Emery-Dreifuss muscular dystrophy gene mon and may cause Stokes-Adams attacks or has been identified and its protein product even sudden death.'25 127 The incidence of named emerin.'22 Emerin seems to be a trans- mitral valve prolapse is also increased in some membrane protein but its function is uncer- families.'28 Investigation of cardiac function, tain. including ECG and echocardiography, should therefore be performed routinely and, when Distal myopathy indicated, ECG monitoring and radionuclide Two genes for distal muscular dystrophy have been localised. One (MPD1) with a phenotype similar to that originally described by Gowers65 and the other for the recessive Miyoshi myopa- Table 5 Classification ofhereditary myotonias thy.64 It is interesting that the MPD 1 gene Myotonia Gene to a a maps similar region of chromosome 14 as myotonias: gene for oculopharyngeal muscular dystrophy69 Autosomal dominant (Thomsen) CLCN1 Autosomal recessive (Becker) CLCN1 and that the Miyoshi myopathy gene is in a Sodium channel myotonias: similar region of chromosome 2 to a gene for SCN4A Myotonia fluctuans SCN4A autosomal recessive limb-girdle muscular dys- Myotonia permanens SCN4A trophy (LGMD2B) 108 raising the possibility Dystrophic myotonias: that the two Myotonic dystrophy Myotonin protein kinase conditions may be caused by Proximal myotonic myopathy ? mutations in the same gene.'23 266 Mastaglia, Laing J Neurol Neurosurg Psychiatry: first published as 10.1136/jnnp.60.3.256 on 1 March 1996. Downloaded from angiocardiography may also be indicated. to diagnose, with molecular techniques requir- Other systemic manifestations which may ing screening of the entire coding region of the require investigation in their own right include gene. * mellitus, disorders of the or immune function, and gastrointestinal and gen- SODIUM CHANNEL MYOTONIA itourinary abnormalities.'25 These varieties of myotonia have all been Myotonic dystrophy is one of the easy mus- localised to the same locus and have been cle diseases to diagnose by molecular tech- associated with different mutations in the a niques, as virtually all cases have variations of subunit of the sodium muscle channel gene the same mutation: the expansion of the CTG (SCN4A) on chromosome 17.48 137 In paramy- triplet repeat in the myotonin protein kinase otonia congenita myotonia is characteristically gene on .129-131 The large triplet induced by cold exposure, particularly in the repeat expansions causing severe disease can facial and hand muscles, and may be associ- be identified by Southern blotting after diges- ated with attacks of weakness (cold paresis) tion of genomic DNA with EcoRI, and smaller which improve with warming. In most cases expansions can be identified by digestion with the myotonia is increased by repeated muscle BglI or BamHl.41 The PCR can identify nor- contraction (paradoxical myotonia) rather mal subjects by the presence of two alleles than improving as in most other myotonic dis- within the normal size range and the very orders. In potassium sensitive paramyotonia small expansions in minimally affected or (paralysis periodica paramyotonica) paradoxi- asymptomatic subjects (fig 3).131 132 Often, this cal myotonia and episodes of weakness are identifies where the disease came from in a precipitated by potassium administration. family with someone other than the person the Such cases account for the overlap between family has always suspected. Thus by cascade paramyotonia and hyperkalaemic periodic screening it is possible to detect and warn paralysis in some families. those family members at risk from possible A third variety of sodium channel myotonia cardiac complications and sudden death. has been designated myotonia fluctuans. In A dominantly inherited myotonic myopathy this condition, which is dominantly inherited, with proximal muscle weakness and cataracts myotonia is of variable severity at different without the characteristic trinucleotide repeat times, may be associated with painful muscle expansion has recently been described'33 134 , with a tendency to increase in severity and has been shown not to be allelic with the after exercise, ingestion of potassium, and genes for myotonic dystrophy or for the chlo- administration of depolarizing agents, and is ride or sodium channelopathies.'33 responsive to treatment with acetazolamide'37 or, in some families, mexilitine.'38 A fourth CHLORIDE CHANNEL MYOTONIA variety of sodium channel myotonia with con-

Wa. _It is now known that both the autosomal dom- tinuous severe myotonia, muscle stiffness, and inant (Thomsen) and recessive (Becker) forms hypertrophy has also been described and des- of are caused by muta- ignated myotonia permanens. 48 tions in the muscle chloride channel (CLCN1) The mutations in these conditions are a of the coding 5 gene on chromosome 7.48 Symptoms may be spread through large part http://jnnp.bmj.com/ 1 2 3 4 present from birth in the dominant form but region of the SCN4A gene making molecular Figure 3 Analysis of the often do not develop until early childhood or diagnosis difficult, although two mutations, myotonic dystrophy triplet adolescence. The myotonia is widespread, Thri 31 3Met and Glyl 306Val, predominate repeat expansion by PCR. usually painless, and more severe in the reces- in paramyotonia congenita and should be ini- Lane 1: grandmother tially screened for. showing two normal sized sive form beig accentuated by rest, cold, and alleles; lane 2: emotion and improving with repeated muscle asymtomatic grandfather contraction. It is commonly associated with showing one normal sized diffuse muscular hypertrophy, particularly in Periodic paralysis on September 23, 2021 by guest. Protected copyright. allele and a small expansion; lane 3: uncle of affected males. Muscle weakness may develop Some conditions may cause episodic weakness proband (son) oflanes 1 after exercise, particularly in cases of the reces- or paralysis of the limb muscles (table 6). It is and 2 showing non- sive form of the condition, and is associated difficult to evaluate the importance of a com- inheritance of grandfather's allele with a reduction in the amplitude of the plaint of episodic weakness unless the patient (expansion too bigfor evoked CMAP and twitch tension.'35 In addi- is watched during an attack. Most patients PCR); lane 4: mother of tion, some degree of fixed weakness and atro- who complain of episodes of weakness are in proband (daughter) of fact to In those who lanes 1 and 2 showing phy with EMG evidence of myopathy referring fatigue. patients non-inheritance of develops, particularly in the forearm and ster- do have episodes of documented weakness it is grandfather's allele nomastoid muscles, in about two thirds of first necessary to exclude disorders of neuro- (expansion too big . . . . for . muscular transmission such as myasthenia PCR); lane 5: proband patients with the recessive condition."6 (daughter oflane 4) To date, a total of 19 separate mutations in gravis, demyelinating disorders of the periph- showing non-inheritance of the CLCN1 gene have been identified, six eral or central , transient mother's allele, expansion being associated with dominant disease and 13 ischaemic episodes, or attacks of hysterical with the recessive disease.48 Most (four out of weakness. If these conditions can be excluded, five) of the truncating mutations cause reces- the possibility of one of the primary periodic sive disease. The mutations are spread paralyses arises, particularly if there are other throughout the coding region of the gene and affected family members (table 6). many have been found in single families, indi- Differentiation between the main varieties cating that other mutations are highly likely to of primary periodic paralysis is usually possible be found in other families. Myotonia con- on the basis of the duration of the attacks of genita is therefore one of the difficult diseases weakness, provocative factors, and serum Investigation ofmuscle disease 267 J Neurol Neurosurg Psychiatry: first published as 10.1136/jnnp.60.3.256 on 1 March 1996. Downloaded from Table 6 Classification ofperiodic paralyses periodic paralysis. Vacuolar change may be Hereditary: found even in some family members without Hypokalaemic (DHP receptor; 1 q3 1-32) definite attacks of weakness.'4' Although the Potassium sensitive (SCN4A; 17q23-25) Hyperkalaemic biopsy changes are distinctive, a biopsy is sel- Paramyotonia congenita dom necessary as the results of provocative Paralysis periodica paramyotonica Andersen's syndrome tests and molecular genetic analysis are usually Acquired: diagnostic. Hypokalaemic Hyperkalaemic As the plasma potassium concentrations Thyrotoxic during an attack of weakness are sometimes misleading, provocative tests remain impor- tant in evaluating people with a negative family potassium concentrations during attacks. In history or those from families in which the hypokalaemic periodic paralysis the attacks are mutation is not known. In hypokalaemic peri- often precipitated by carbohydrate ingestion odic paralysis, attacks of weakness may be and tend to be less frequent and of longer induced by giving an oral glucose load (1-5 duration (up to 24 hours) than in the potas- gm/kg over three minutes; maximum 100 g).9 sium sensitive periodic paralyses (for example, Muscle strength in selected groups, serum hyperkalaemic periodic paralysis) in which the electrolytes, and ECG should be monitored attacks are often precipitated by fasting or by every 30 minutes for three to five hours. If resting after exercise.'3 Although the serum weakness does not develop, an intravenous potassium concentration during an attack of glucose load (3 g/kg in water) is given over a weakness may help to distinguish the period of one hour. If weakness does not hypokalaemic from the hyperkalaemic form, develop after 30 minutes, intravenous insulin potassium concentrations are often normal in (0 1 U/kg) is given and repeated at 60 minutes the second and may occasionally also be nor- if weakness fails to develop. Regular measure- mal in the hypokalaemic form.9 The serum ments of muscle strength and of plasma elec- creatme kinase concentration is often raised in trolyte and glucose concentrations, and ECG severe attacks of both forms and may be some- should be performed throughout the test and what raised even between attacks. Myotonia, the period of induced weakness.9 13 when present clinically or evident on EMG, is In the potassium sensitive forms of periodic more suggestive of hyperkalaemic periodic paralysis attacks of weakness may be induced paralysis or paramyotonia but may occasion- by oral potassium loading.913 A widely used ally occur in the eyelid muscles in protocol involves giving 0 05 g/kg of potas- hypokalaemic periodic paralysis.9 sium chloride in a glucose free solution over a When carried out during an attack of weak- period of three minutes. Muscle strength, ness EMG shows a progressive reduction in serum electrolytes, and ECG are monitored motor unit recruitment and in the amplitude every 15 minutes in the first two hours and of the compound muscle action potential every 30 minutes in the next two hours and evoked by motor nerve stimulation, whereas weakness usually develops after 90 to 180 repetitive nerve stimulation shows a transient minutes. If the initial test is negative, higher

potentiation of the compound muscle action potassium loads of 0 1 to 0 15 g/kg may be http://jnnp.bmj.com/ potential.'39 A myopathic EMG pattern may used. The test is contraindicated in patients be found even during the attack free interval with insulin dependent diabetes mellitus or particularly in patients who develop fixed mus- renal insufficiency. Cold testing, involving cle weakness, as often occurs in those who immersion of the arm in water at 10°C for 30 have had the condition for several years. minutes, may be useful in confirming the diag- Changes in the evoked compound muscle nosis of paramyotonia in patients with a his- action potential also occur with exercise and tory of episodes of cold induced weakness or on September 23, 2021 by guest. Protected copyright. are the basis for the hand exercise test myotonia.9 described by McManis et al.'40 Serial measure- The molecular basis for both the ments of compound muscle action potential hypokalaemic and hyperkalaemic forms of amplitudes show a greater than normal periodic paralysis have now been defined. increase during exercise and decrease in the Hypokalaemic periodic paralysis has been post-exercise period in patients with periodic shown to be caused by three mutations in the paralysis. The test was found to be abnormal dihydropyridine receptor calcium channel in 70% of cases of periodic paralysis but did gene (CACNLIA3) on chromosome 1 q3 1-32 not distinguish between the different forms. affecting two amino acid residues.3848 142 143 The Another form of exercise testing, which is mutations can be detected by PCR followed useful in detecting some cases of hypo- by enzyme digestion as they either create or kalaemic periodic paralysis, including asymp- destroy restriction enzyme sites'42 or by tomatic cases, is that described by Kantola SSCP.38 Mutations in the gene encoding the a and Tarssanen,'4' which involves a 30 minute subunit of the skeletal muscle sodium channel period of bicycle ergometry. Affected persons (SCN4A) causing hyperkalaemic periodic do not show the normal rise in plasma potas- paralysis were first described in 1991.144145 A sium concentrations after the exercise period. recent review by Lehmann-Horn and Riidel48 Muscle biopsy shows a characteristic vacuo- catalogues the five mutations of SCN4A now lar change in muscle fibres, and sometimes known to be associated with hyperkalaemic necrotic muscle fibres, particularly during an periodic paralysis. Two of the mutations, attack of weakness, but sometimes even during Thr7O4Met and Met1 592Val, predominate attack free intervals, in both major forms of and can be screened for first. There is perhaps 268 Mastaglia, Laing J Neurol Neurosurg Psychiatry: first published as 10.1136/jnnp.60.3.256 on 1 March 1996. Downloaded from a degree of genetic heterogeneity in hyper- to chromosome 1149 and a mutation in the a- kalaemic periodic paralysis as one family with tropomyosin gene TPM3 has been shown to a convincing clinical diagnosis of the disease segregate with the disease.'50 A gene for reces- did not map to the SCN4A gene.62 Mutations sive has been localised to in the same gene have also been found in some chromosome 2.70 Neither of the loci map to families with paramyotonia congenita48 and the known locations for the a-actinin genes.'51 in some cases of hyperkalaemic periodic This and the mutation in tropomyosin may paralysis associated with cardiac arrhythmias indicate that nemaline myopathy is a disease (Andersen's syndrome) 146 147 of the thin filament rather than of the Z disc.'23 was first linked to the same region of chromosome 19 as malignant Congenital myopathies hyperthermia'52 153 and then mutations associ- The congenital myopathies are a heteroge- ated with central core disease were identified neous group of disorders which may result in in the RYRi gene.'54-'56 Thus both malignant hypotonia in infancy or may present later in hyperthermia and central core disease are childhood with weakness and delayed motor caused by mutations in the RYRI gene and milestones (table 7). Some, such as central whether central cores manifest or not may core disease, are benign and relatively non- depend on the degree of calcium loading of progressive whereas others such as X linked the individual muscle fibres.'55 centronuclear myopathy are usually fatal in infancy. Skeletal abnormalities such as talipes, , and as well as dys- Mitochondrial myopathies morphic features are not uncommon. Since the discovery of the first mitochondrial and ophthalmoplegia are often a feature in DNA mutations in 1988"157 58 it has become centronuclear myopathy and may also occur in apparent that the clinical spectrum of mito- minicore disease. Respiratory insufficiency chondrial diseases is extremely diverse, rang- may occur, particularly in nemaline myopathy, ing from relatively mild and slowly progressive centronuclear myopathy, and cytoplasmic myopathies confined to the extraocular mus- body myopathy. The congenital myopathies cles (chronic progressive external ophthalmo- should be considered in the differential diag- plegia) to severe fatal infantile myopathies (for nosis of the floppy infant and need to be dis- example, deficiency) tinguished from other neuromuscular and and multisystem encephalomyopathies (such CNS disorders which may produce infantile as the Kearns-Sayre, MEIAS, and MERFF hypotonia. syndromes9 159--162) (table 8). In addition, defi- In all of these conditions a definitive diag- ciencies of mitochondrial enzymes encoded by nosis relies on a muscle biopsy with full nuclear DNA have been identified (for exam- histochemical and electron microscopic exam- ple, succinate dehydrogenase deficiency). ination of muscle tissue. These will identify specific morphological changes such as central CLINICAL PHENOTYPES AND GENOTYPES cores, nemaline rods, central nucleation, and Table 9 shows that mitochondrial defects may

abnormalities of myofibre differentiation and be associated not only with myopathic disor- http://jnnp.bmj.com/ fibre type proportions or distinctive inclusion ders but with a wide variety of other neurolog- bodies (for example, cytoplasmic bodies, fin- ical and non-neurological manifestations. ger print bodies, spheroid bodies, hyaline bod- Although some classic syndromes have been ies, reducing bodies, zebra bodies, and tubular defined (table 8), there is a certain amount of aggregates). This will also exclude other con- overlap between these and new phenotypic ditions such as congenital muscular dystrophy, combinations are still being identified. metabolic myopathies, and neurogenic disor- Moreover, there is considerable variability in ders such as spinal muscular atrophy and con- the age of onset, rate of progression, and on September 23, 2021 by guest. Protected copyright. genital peripheral neuropathies.9 148 extent of phenotypic expression in different The genetic basis for some of the congenital tissues even within the major syndromes. This myopathies such as central core disease, nemaline myopathy, and the neonatal form of centronuclear myopathy have now been Table 8 Mitochondrial myopathies and defined. To date, one gene for autosomal encephalomyopathies dominant nemaline myopathy has been linked Progressive external ophthalmoplegia Sporadic Familial Kearns-Sayre syndrome Ophthalmoplegia Table 7 Congenital myopathies Pigmentary retinopathy Cardiac conduction defects Central core disease* Nemaline myopathy Sensorineural deafness Centronuclear myopathy Limb-girdle myopathy Minicore disease* Infantile myopathy Congenital fibre type disproportion Benign reversible Myopathies with cytoplasmic inclusions Severe fatal Fingerprint body myopathy MERFF syndrome Reducing body myopathy Cytoplasmic body myopathy Hyaline body myopathy Myopathy Myopathy with tubular aggregates MELAS syndrome Zebra body myopathy Myopathy Sarcotubular myopathy -like episodes *Predisposition to malignant hyperthermia. Investigation ofmuscle disease 269 J Neurol Neurosurg Psychiatry: first published as 10.1136/jnnp.60.3.256 on 1 March 1996. Downloaded from Table 9 Clinical manifestations of are often raised in the MELAS syndrome and Neurological Non-neurological may also be raised in Kearns-Sayre syndrome and in patients with a proximal myopathy or External ophthalmoplegia Short stature Limb myopathy Cardiac conduction encephalomyopathy.I59 A muscle biopsy is the Fatiguability and poor exercise defects most important diagnostic investigation, the tolerance Cardiomyopathy Cerebellar ataxia Pigmentary retinopathy characteristic findings being of ragged red Epilepsy Cataracts fibres with the modified Gomori Myoclonus Lactic acidosis trichrome Sensorineural deafiess Diabetes mellitus stain, with abnormally prominent subsar- Peripheral neuropathy colemmal mitochondrial Renal tubular defects aggregates with the Stroke Episodic nausea and oxidative enzyme stains (NADH dehydroge- Vascular vomiting nase, succinate dehydrogenase) and abnormal Basal ganglion calcification Intestinal pseudo- mitochondrial morphology with mitochondrial obstruction inclusions on electron microscopy. In addi- Multiple tion, histochemical staining for cytochrome oxidase activity shows a characteristic mosaic pattern of fibres lacking enzyme activity in most cases. Immunohistochemical techniques heterogeneity is characteristic of this group of may be applied to determine whether nuclear disorders'63 and is attributable to the random or mtDNA encoded subunits of cytochrome distribution of mitochondria containing the oxidase are absent. In situ hybridisation with mutated DNA within different tissues and to probes to different parts of the mtDNA mole- the admixture of normal and abnormal popula- cule may also be used to study the distribution tions of mitochondria within the cells of these of mutant mitochondria in muscle fibres. tissues () as a result of which dif- The identified common mutations in the ferent tissues in the same patient and different mitochondrial genome associated with members of the same family may be involved MERRF or MELAS can be tested using PCR to different degrees. followed by enzyme digestion41 49 or SSCP The classic syndromes have been associated analysis. 168 The major deletions of the mtDNA with different types of genomic defect. For associated with Kearns-Sayre syndrome or example, patients with the pure form of CPEO may be identified either using Southern chronic progressive external ophthalmoplegia blotting80 or PCR'69 including long range (CPEO) usually have single major PCR, which can amplify the entire 16 569 deletions'59 160 but point mutations have been base pairs of the mtDNA in one reaction.'70 found in some patients.2 Similarly, those The severity of the disease relates to the per- patients with the Kearns-Sayre syndrome have centage of mutant mitochondria,60 which can single large mtDNA deletions which occur be estimated either after Southern blotting, or sporadically.'57 164 In patients with familial enzyme digestion after PCR, or from SSCP.167 CPEO two distinct syndromes have been iden- Whether all the base changes shown to be tified. In the first, which is maternally inher- associated with mitochondrial related disease ited, deletions are not found, whereas in the actually cause disease may be debatable; some

second, which is dominantly inherited, large may merely be linked to other disease causing http://jnnp.bmj.com/ scale multiple mtDNA deletions are usually mutations.'7' On the other hand, the polymor- present and are thought to be secondary to a phisms of mtDNA may contribute to dis- mutation in a nuclear gene encoding a factor ease. 172 The mitochondrial genome is thus which controls mtDNA replication.'65 Such a problematic for molecular diagnosis, the rele- gene has recently been linked to chromosome vance of some mutations to disease being 1Oq23 3-24-3 in a Finnish family with autoso- unclear. mal dominant CPEO.50 Cases presenting purely with a proximal myopathy may also be on September 23, 2021 by guest. Protected copyright. dominantly inherited'66 but are uncommon Acute rhabdomyolysis and myoglobinuria and the genotypic basis for these cases has not Various hereditary and acquired disorders may been defined. In the encephalomyopathy syn- lead to episodes of severe widespread muscle dromes different point mutations have been fibre destruction and myoglobinuria." found in tRNA-lys (nucleotides 8344 or 8356) Clinically, there is widespread muscle weak- in the MERRF syndrome'67 and tRNA-leu ness which may be profound and in severe (nucleotides 3243, 3251, 3252, 3271 or 3291) cases there is often severe muscle pain, tender- in the MELAS syndrome.2160 ness, and depression of the deep tendon reflexes. When myoglobinuria is severe it may DIAGNOSTIC APPROACH lead to the development of acute renal failure. In patients with CPEO, Kearns-Sayre syn- In patients with malignant hyperthermia mus- drome, or one of the other classic syndromes, cle rigidity, hyperpyrexia, and rhabdomyolysis the clinical features are often sufficiently dis- usually develop during anaesthesia when sus- tinctive to strongly suggest the diagnosis of a ceptible subjects are exposed to halothane and mitochondrial disorder, and in familial cases, a some other inhalational anaesthetic agents, maternal pattern of transmission would also but episodes of rhabdomyolysis may also be raise the possibility of such a diagnosis. A precipitated by stress, strenuous physical raised fasting venous lactate concentration is activity, or systemic infective illnesses. an additional diagnostic pointer. Lactate con- Patients presenting in this way for the first centrations are usually normal in cases of time should undergo a detailed clinical assess- CPEO, but venous and CSF concentrations ment for a drug induced, toxic, or infective 1270 Mastaglia, Laing J Neurol Neurosurg Psychiatry: first published as 10.1136/jnnp.60.3.256 on 1 March 1996. Downloaded from cause and hypokalaemia, hypothyroidism, and forearm exercise test (see above) and assays of other metabolic disorders should be excluded. carnitine and carnitine palmityl transferase Patients in whom no obvious cause can be activity as well as a muscle biopsy for histo- identified-especially those in whom there is a chemical and biochemical studies to confirm history of previous episodes-should have a the diagnosis of these metabolic disorders. muscle biopsy and appropriate biochemical With molecular studies mutations causing dis- investigations for an underlying genetic disor- ease have been identified in some of these con- der such as malignant hyperpyrexia or a disor- ditions but such studies are not usually der of glycolysis or fatty acid metabolism." 173 necessary for the diagnosis. A muscle biopsy is Susceptibility to malignant hyperthermia in also necessary for the diagnosis of mitochon- humans was first linked to the region contain- drial myopathy, malignant hyperthermia, dys- ing the ryanodine receptor calcium release trophin deficiency, and tubular aggregates, channel on chromosome 1971 174 after the which may sometimes present with unex- mutation for swine malignant hyperthermia plained myalgia, cramps, or exercise intoler- was linked to the glucose phosphate isomerase ance. gene which maps in humans to chromosome The vast majority of patients with com- 19. A few mutations have now been identified plaints of muscle pain, stiffness, or cramping in the relatively large (15 kb) RYRI gene mes- do not have any significant muscular weakness sage'75 in a minority of families with malignant or other evidence of muscle disease. It is hyperthermia.74 The aim ofmolecular diagnosis important in such patients to look for evidence in this condition must be to reduce the of muscular and myofascial tenderness. The reliance on the extremely invasive current gold finding of point tenderness in the typical sites standard of the muscle contracture test, with is diagnostic of the condition of fibromyalgia which the molecular diagnosis is not always in and invasive investigations such as EMG and agreement.7274 However, until a clearer and muscle biopsy can usually be avoided. more comprehensive list of mutations in the Similarly, a pattern ofproximal or axial muscle RYR1 gene causing malignant hyperthermia is pain and stiffness with in elderly available, including resolution of the apparent patients with preserved muscle strength and a genetic heterogeneity, and the discrepancies raised erythrocyte sedimentation rate is diag- between molecular diagnosis and the contrac- nostic of polymyalgia rheumatica and warrants ture test are resolved, molecular diagnosis for a therapeutic trial ofprednisone without muscle susceptibility to malignant hyperthermia will biopsy, although the possibility of an underly- remain problematical. ing connective tissue or or malignancy should always be considered and investigated appropriately. In a third group of Painful muscle conditions patients, with the postviral myalgia/fatigue Muscle pain may be a feature of some syndrome, there are no diagnostic clinical or myopathies and other conditions (table 2). laboratory abnormalities. Muscle biopsy and The pain may be focal or diffuse and may EMG may, however, be indicated in such occur at rest, during, or after exercise. When patients to exclude an inflammatory or meta-

myalgia develops after an infective illness, an bolic myopathy, particularly when symptoms http://jnnp.bmj.com/ alcoholic binge, exposure to a myotoxic drug, are disabling and if the serum creatine kinase or after an episode of envenomation, the cause concentration is raised. is usually apparent. A wide variety of drugs may cause myalgia and muscle cramps without significant muscular weakness and the symp- Inflammatory myopathies toms usually resolve promptly after withdrawal The diagnosis of an inflammatory myopathy of the offending agent (table 1). The finding of may be readily made on clinical grounds in significant muscular weakness, tenderness, patients with the characteristic skin changes of on September 23, 2021 by guest. Protected copyright. and increase in serum creatine kinase suggests a dermatomyositis or with the typical pattern of necrotising myopathy and a muscle biopsy is muscle weakness seen in inclusion body usually warranted to confirm the diagnosis. A myositis and may also be suspected in patients biopsy is also indicated when an inflammatory with a connective tissue disorder such as sys- myopathy is suspected and should include the temic lupus erythematosus, progressive sys- overlying fascia, if there is a possibility of fasci- temic sclerosis, mixed connective tissue itis. Muscle pain and stiffness are common disease, Sj6gren's syndrome, or other autoim- symptoms in hypothyroidism and in patients mune disease who develop a proximal myopa- with metabolic disease. The diagnosis in thy and would be supported by the finding of such patients can usually be confirmed by raised serum creatine kinase and an abnormal appropriate biochemical and other studies EMG (see above). However, it is important without resorting to muscle biopsy. that the diagnosis should be firmly established Muscle pain and cramping which develop on histological grounds and a muscle biopsy during exercise suggest a disorder of muscle should always be performed before commenc- energy metabolism, such as deficiency of ing treatment. An open biopsy, usually from myophosphorylase, phosphofructokinase, or the deltoid or vastus lateralis muscle, is prefer- other glycolytic enzyme defect, or of carnitine able for the diagnosis of inflammatory myopa- palmityl transferase, especially if there is a his- thy. In addition to routine histological tory of discolouration of the urine after exer- preparations, mononuclear cell populations, cise, suggesting myoglobinuria. Such patients immune complex deposition, MHC, and warrant further investigation including the intercellular adhesion molecule expression can Investigation of muscle disease 271 J Neurol Neurosurg Psychiatry: first published as 10.1136/jnnp.60.3.256 on 1 March 1996. Downloaded from be assessed with immunohistochemical tech- Ms L Kelloway for fig1 and to Mr M Davis, Ms J Dench, and niques.176 177 Ms N Harber for figs 2 and 3.. Other investigations are directed towards 1 Neuromuscular disorders: gene location. Neuromuscul Disord 1995;6:I-III. identifying any underlying immune or infec- 2 Mitochondrial encephalomyopathies: gene mutation. tive disorder or malignancy. These should Neuromuscul Disord 1995;6:V-VI. 3 Argov Z, Mastaglia FL. Drug-induced neuromuscular dis- include an autoantibody screen for antinuclear orders in man. In: WaltonJN, Karpati G, Hilton-Jones factor, rheumatoid factor, anti-RNP, and, D, eds. Disorders of voluntary muscle. 6th ed. 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Aust NZ J Med HTLV-1 infection may develop an inflamma- 1986;16:329-35. tory myopathy similar to polymyositis,'79 sero- 9 Griggs RC, MendellJR, Miller RG. Evaluation and treat- ment of myopathies. Contemporary neurology series. logical testing for HIV and HTLV-1 infection Philadelphia: FA Davis, 1995. as well as studies of lymphocyte subsets are 10 Sunohara N, Takagi A, Nonaka I, Sugita H, Satoyoshi E. Idiopathic hyperCKemia. Neurology 1984;34:544-7. also indicated, especially in areas where infec- 11 Tien I, DiMauro S, Rowland LP. Myoglobinuria. In: tion with these agents is prevalent. The possi- Rowland LP, DiMauroS, eds. Handbook of clinical neu- rology. Amsterdam: Elsevier, 1992:553-93. bility of an underlying malignancy should be 12 Kagen U. Myoglobinaemia in inflammatory myopathies. considered, particularly in adults presenting J7AMA 1977;237:1448-52. 13 Moxley RT. Metabolic and endocrine myopathies. 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