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deletion is present in approximately 85% of patients with HNPR HNPP patients have rarely been shown to have point mutations in PMP22, with seven novel mutations reported thus far. Linkage to 17p11.2 has been excluded in one Dutch family with HNPR This study aimed to ascertain how commonly PMP22 point mutations were found in patients with a phenotypic diagnosis of HNPP but were negative for the common 17p 11.2 deletion. Methods: We studied 68 unrelated patients with a clinical diagnosis of HNPP referred for genetic testing. All cases were shown to be negative for the 17pl 1.2 deletion using the polymerase chain reaction and microsatellite markers. The coding regions of PMP22 were sequenced in all patients using techniques reported previously. Results: One patient had a 1-nucleotide insertion at codon 94 (c281_282insG). This frameshift mutation has been previously reported in 2 European pedigrees with HNPP. Conclusions: Point mutations in PMP22 are rare causes of HNPP. Routine sequencing of PMP22 in 17p11.2 deletion-negative patients is not recommended.
of congenital hypomyelination neuropathy with separation of terminal myelin loops from the axon affecting the axon-Schwann cell unit as the initial change. In CMT4A, axonal degeneration and regeneration with minor evidence of demyelination and remyelination was considered as the main structural change. In HMSN L, we observed more demyelinating than axonal changes. Conclusions: The variability of structural changes in the small number of sural nerves studied thus far in molecular genetically defined, recessively inherited peripheral neuropathies correlates with the remarkably large spectrum of clinical features.
59 15:40 A Homozygous T l l 8 M Mutation in the Peripheral Myelin Protein (PMP22) Causes an Axonal Neuropathy
P-601 Characterization of Tubular Aggregates in Human and Mouse Skeletal Muscle
M. Scavina* 1, A. Clark 1, K.M. Kxajewski 2, M.E. Shy 2. 1Wilmington,
12. Chevessier*, L Marty, M. Fardeau, D. Hanta, M. Verdi re-Sahuqu .
United States; 2Detroit, United States
Paris, Grenoble, France
Objective: To describe an individual with a homozygous T 118M mutation
Tubular aggregates are observed in various muscle disorders and are generally considered as a secondary molphological alteration of the sarcoplasmic reticulum. In some patients, the primitive origin of this pathology is still unknown especially those suffering from "tubular aggregates myopathy". These alterations are also present in fast twitch skeletal muscle of aged inbred male mice. The aim of this study was to better characterize the tubular aggregates in skeletal muscle of three patients characterized as "tubular aggregates myopathy" and of C57B16 inbred male mice. Different muscles were stained with Gomori trichrome method to reveal the general molphology of muscle and immunolabeling of cryostat sections was performed using different sarcoplasmic reticulum markers. A set of specific antibodies against proteins involved in calcium homeostasis were used: anti-SERCA1 and 2 ATPases, anti-calsequestrin, antiryanodine receptor (RyR1) and anti-triadin antibodies. An immunocytochemical labeling was observed with all these antibodies in type I and II muscle fibers in human but restricted to type IIB and fast skeletal muscle in mouse. Identification of type fiber was done using slow and fast myosin heavy chain antibodies. In conclusion, all specific components of excitation-contraction coupling or intracellular calcium flux regulation in muscle fibers are present in tubular aggregates.
in the PMP22 gene. Methods: An 11 year old female with no family history of neuropathy was born with vertical tall and underwent numerous surgical procedures on her lower extremities. She remained stable until age 10 when she became progressively weaker. Exam reveals full strength in the upper extremities, atrophy and weakness in the distal lower extremities, pes cavus and hammertoes. Reflexes are absent. Vibration and proprioception are intact while temperature and pinprick sensation are diminished. Results: Electromyogram and nerve conduction velocities (EMG and NCVs) at age 5 were normal. NCVs at age 10 were normal while EMG showed chronic partial denervation in all muscles tested. Motor units showed high amplitude, long duration and rapid firing rates in the 30 to 40 Hertz range with maximal effort. Sequencing of PMP22 revealed a homozygous alteration at codon position 118, resulting in an amino acid change from threonine to methionine. Conclusions: The T118M mutation in PMP22 is not a benign polymorphism but causes significant disease in a homozygous individual. Despite PMP22 expression in myelinating Schwann cells, the neuropathy is clinically axonal, suggesting PMP22 mutations can disrupt the interaction between Schwann cells and axons without disrupting myelination.
THURSDAY, JULY 11, 2002
Poster Presentations Muscle Histochemistry
Molecular-Pathologic Correlations in Recessively Inherited Peripheral Neuropathy Type CMT4A (GDAP1), CMT4F (Periaxin), and HMSN L (NDRG1)
P-602 The Relationship of Clinical Severity to Dystrophin Immunohistochemistry and Western Blotting in Duchenne and Becker MD
J.M. Schroder*. Aachen, Germany
Ahmet Hoke*, Sheila Kumar, Thomas Crawford. Baltimore, USA
Background: Meanwhile 16 genes and more than 30 chromosomal loci
Rationale: The correlation between clinical severity in Duchenne and
have been detected as being involved in causing hereditary motor and sensory peripheral neuropathies (HMSN). Objectives: Genes detected in 2001 as being affected in recessively inherited neuropathies comprise periaxin (PRX) causing Charcot-MarieTooth neuropathy type 4F (CMT4F), and the ganglioside-induced differentiation-associated protein 1 gene (GDAP1) causing CMT1A. The gene involved in causing HMSN Lore, i.e., N-myc downstream regulated 1 (NDGR1), has already been identified one year before. Methods: From a large series of more than 6,000 sural nerve biopsies, we used DNA extracted from paraffin-embedded material of selected cases to identify mutations in dominantly inherited neuropathies (Thiex and Schr der 1998) and more recently in severe recessive ones. Results: In periaxin neuropathy (CMT4F), we detected a severe type
Becker's muscular dystrophy and dystrophin abnormalities on immunohistochemistry and Western blotting is not clearly defined. Methods: We stratified 19 patients with an ambiguous dystrophin genotype-phenotype relationship into 5 groups by clinical severity during late childhood to early teen years. Dystrophin Western blotting was classified as either absent or reduced. The quality of dystrophin immunohistochemical staining for rod domain, N-terminus and C-terminus antibodies was sorted into those with normal to moderately reduced dystrophin staining and those with severely reduced or absent dystrophin staining. Clinical and dystrophin classifications were blinded. Results: In this cohort Western blotting has a positive predictive value (ppv) of 67% for BMD and 62% for DMD phenotype. Dystrophin immunohistochemistry has a ppv of 70% and 78% for BMD and
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DMD, respectively. The combination of Western and immunohistochemical data has substantially better ppv, with congruent Westernimmunohistochemical classification having a 100% ppv for both BMD and DMD. Conclusion: In patients with an ambiguous genotype-phenotype relationship the combination of Western and immunohistochemical dystrophin testing has higher predictive value for prognosis than does either test in isolation.
P-605 The Histochemical Characteristics of Muscle in X-Linked Bulbo-Spinal Muscular Atrophy (X-BSMA) and ALS: Multiple Small Groups of Atrophic Fibers with Mixed Fiber Types
P-603 Changes in DMPK Levels during Development and in Congenital Myotonic Dystrophy using Monoclonal Antibodies
Methods: We compared X-BSMA muscle biopsies to those with dener-
W.P. Rakowicz* 1, T. Miller 2, A. Pestronk 2. 1Cambridge, UK; est Louis,
MO, USA Objective: To analyze the histochemical characteristics of muscle from six individuals with genetically-confirmed X-BSMA.
G.E. Morris* 1, Nguyen thi Man 1, Le Thanh Lain 1, D. Furling2, G.S.Butler-Browne 2. 1Wrexham, United Kingdom; 2Paris, France
Objectives: We have previously used a panel of monoclonal antibodies (mAbs) against the myotonic dystrophy protein kinase, DMPK, to show that this is an 85kD protein restricted to skeletal and cardiac muscles. We have now used the mAbs to measure DMPK in fetal human muscle and congenital myotonic dystrophy (DM1). Methods: The levels of expression of 85kD DMPK protein were determined by Western blotting at different fetal stages in normal and DM1 skeletal muscle. Results: DMPK became detectable in skeletal muscle between 9 and 16 weeks of foetal development and remained at high levels into the neonatal period. A 15-fold increase in DMPK was observed during human myoblast differentiation in cell culture. DMPK levels were compared between normal foetuses and age-matched foetuses with severe congenital DM. DMPK in quadriceps of DM 1 patients with large CTG (> 1500) was reduced to 57% of control levels. Conclusions: The results are consistent with greatly reduced expression from the mutant allele and normal expression from the unaffected allele. We suggest that very large CTG repeats cause almost complete retention in the nucleus of DMPK transcripts from the affected allele. Supported by Association Fran aise contre les Myopathies (AFM) and the Muscular Dystrophy Campaign (UK).
ration from other causes by evaluating patterns of muscle fiber atrophy and fiber type distribution. Results: X-BSMA muscles had type 1 fiber predominance. Groups of atrophic fibers were found in 5/6 patients (1.24-1.1 groups per high power field (gps/HPF)). 814-23% of the atrophic groups contained both type 1 and type 2 fibers. Chronic sensorimotor neuropathies had a similar density of grouped atrophic fibers (1.14-1.0 gps/HPF) but only 404-23% of the groups contained mixed fiber types (p<0.005). Groupsof atrophic muscle fibers were especially common in probable or definite ALS (3.34-0.7 gps/HPF; p<0.001 ALS vs other denervation) and 684-20% of these contained both fiber types. Thus the proportion of atrophic groups with mixed fiber types in ALS was similar to X-BSMA but greater than in chronic neuropathies (p<0.05). Conclusion: Small, atrophic groups of muscle fibers are more frequently of mixed type in motor neuron disorders, X-BSMA and ALS, than in other types of denervation. Groups of atrophic fibers with mixed types, when present at high densities, are especially characteristic of ALS and distinguish ALS from other forms of denervation.
P-606 Evaluation of Muscle Biopsies in Myopathic Disorders and its Correlation with Magnet Resonance Spectroscopy M.C. Sharma .1, S.K. Atri I , N.R. Jaganathan 2 , C. Sarkar I . Departments
of 1Pathology and 2NMR, All India Institute of Medical Sciences, New Delhi, India Objectives: Neuromuscular disorders constitute a significant proportion
P-604 The Expression of Tenascin in Biopsied Muscles of Neuromuscular Diseases N. Ohkoshi*, S. Hoshino, A. Ishii, S. Shoji. Tsukuba, Japan
Objective: Tenascin-C, an extracellular matrix protein, is prominent associated with tissue regeneration in embryonic tissue, tumor, and wound healing, suggesting that it has some roles in tissue growth and restructuring. We investigated the expression of tenascin-C in biopsied muscles of neuromuscular diseases. Methods: The expression of tenascin-C was immunohistochemically examined using ABC method in biopsied muscles of dermatomyositis (11 patients), polymyositis (11), muscular dystrophy (11), motor neuron disease (1), and other diseases (12). Results: In dermatomyositis, tenascin was immunostained widespread expression in the interstitial space. The expression showed a network pattern surrounding numerous fibers, predominantly in a perifascicular area and severe inflammatory lesions. Especially, the expression was strong in the lesion of perifascicular atrophy. In polymyositis, tenascin was expressed a certain number of fibers surrounding necrotic or regenerating fibers or severe inflammatory lesions. In progressive muscular dystrophy, tenascin was sporadically expressed surrounding a few necrotic fibers. In neurogenic muscular changes, type 2 atrophy, or mitochondrial myopathy, tenascin was not expressed. Conclusions: Tenascin was expressed in inflammatory and ischemic lesions in dermatomyositis. The expression of tanascin was useful for diagnosis of dermatomyositis and active lesions of polymyositis and progressive muscular dystrophy.
of neurological disorders and is an important cause of morbidity and mortality. There is tremendous development in the molecular genetics of muscle diseases but studies on muscle metabolism are limited especially on in vitro MRS. Hence, this study was undertaken (i) to evaluate muscle biopsies of cases clinically and pathologically diagnosed as myopathic disorders (ii) to study muscle metabolism in these cases using in vitro proton MRS and (iii) to correlate MRS findings with clinical and histological diagnosis. Methods: Of 447 muscle biopsies of 2 years (2000-2001 ), 249 cases were diagnosed as myopathic disorders. In 64 muscle biopsies of myopathic disorders, where material was sufficient, in vitro proton MRS was done. For control 9 cases of normal quadriceps from orthopedic OT and 7 biopsies of cases diagnosed as within normal histological limits were taken. In 32 cases, in vivo proton MRS was also done. Results: (i) There was statistically significant difference in lactate levels in mitochondrial myopathy (mean 17.4), and control (7.84) but not between muscle dystrophy (mean 9.66) and control. (ii) There was no statistically significant difference in glucose levels between control (mean 0.62), dystrophy (0.52) and mitochondrial (mean 1.44). (iii) There was no significant difference in alanin levels of controls (mean 1.58), dystrophy (mean 1.10) and mitochondrial (mean 2.22). Although levels were on higher side in mitochondrial myopathy. (iv) The difference in the levels of GLX was significant between control (2.16), mitochondrial myopathy (4.19) and dystrophy (2.21). (v) The difference in the creatine levels of different groups - control (0.74), dystrophy (0.58) and mitochondrial (1.95) was significant but for acetate it was non-significant. Conclusion: In vitro protein MRS was found to be good technique to be supplemented by morphology. All metabolites were decreased in muscular dystrophies and high levels of lactate, creatine and GLX were higher in mitochondrial myopathy. Levels were also high in neurogenic atrophy.
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P-607 Plasma Cell Predominant Polymyositis Associated with Sjogren's Disease: Immunohistochemical Analysis of Cellular Infiltrate W.C. Yee*, K.L. Boon, L.S. Chua, H.H. Chng, K, Ponnudurai.
Singapore, Republic of Singapore Objective: To characterize the cellular infiltrate in plasma cell predominant polymyositis associated with Sjogren's disease Method: Immunohistochemical localization of CD3, CD4, CD8, CD20, CD25, CD38 and macrophage markers was conducted on muscle sections of a patient with Sjogren's disease, proximal myopathy and histological evidence of polymyositis with numerous plasma cells. The relative proportions of T cells, B cells, plasma cells and macrophages were determined at perivascular, perimysial and endomysial sites of accumulation. Immunohistochemical localisation of kappa and lambda light chains and MHC class I antigen was also performed. Result: At all sites, CD38+ cells (plasma cell associated) were predominant, followed by CD8+ T cells. B cells (CD20+ and CD25+) were the least common. There was a positive gradient for CD8+ cells, but a negative gradient for CD20+ and CD25+ cells between perimysial and endomysial sites. At endomysial sites, CD8+ cells outnumbered CD4+ cells. Plasma cells showed polyclonality. Conclusion: Although a humoral pathophysiology was proposed for similar rare cases previously reported, analysis of the cellular infiltrate in our patient suggests the presence of a T cell mediated immune response against muscle associated antigen(s). The role of plasma cells in the primary process remains uncertain. P-608 Immunohistchemical Analysis of Skeletal Muscles from Canine X-Linked Muscular Dystrophy M. Yoshimura*, Y. Itoh, K. Yuasa, M. Sakamoto, K. Sugie, I. Nonaka, S. Takeda. Tokyo, Japan Objective: Canine X-linked muscular dystrophy (CXMD) exhibits the phenotype of Duchenne muscular dystrophy (DMD) caused by a splice mutation of the DMD gene. We have established a breeding colony of beagle-based CXMD in Japan (CXMDJ) and examined histology of the CXMDJ. Method: The semen from Golden Retriever muscular dystrophy (GRMD) was inseminated into a normal female Beagle. First-generation CXMDJ and control littermates were necropsied at 11-month-old. Serial cryosections of anterior tibial (TA), diaphragm, and extraocular muscles (EOM) were stained with H & E and a battery of monoclonal antibodies against dystrophin, utrophin and dystrophin-associated proteins (DAPs; at- and ~-dystroglycans and at-, [3-, y-, and ~-sarcoglycans). Results: In H & E staining, severe chronic myogenic changes were observed in diaphragm. TA exhibited acute on-going degenerations and EOM exhibited almost normal findings. In immunohistochemical stainings, expressions of all DAPs were decreased from the sarcolemma with absence of dystrophin. Utrophin expression was noted at the sarcolemma from all skeletal muscles examined. Conclusion: We had investigated Beagle-based CXMD in Japan (CXMDJ), and found that CXMDJ revealed a typical pattern of skeletal muscle involvement as shown in GRMD.
Muscle Pain P-609 Efficacy of Amitriptyline in Low Back Pain Induced by Myofascial Pain Syndrome M.M. Mohammadi*, K. Gharagozli, M. Moslemizadeh, N. Abdoli.
Tehran, Iran Objective: Myofascial pain syndromes are one of the most prevalent disorders in general population. Low back pain is a type of this disorder, some
etiologies such as muscular derangement are responsible for this illness. some other causes like depression may have major role in the etiology. This study has tried to investigate role of amitriptyline in control of low back pain form of myofascial pain syndrome in patients who suffered low back pain without any symptoms of depression. Methods: Patients that were refered to low back pain clinic of Loghman Hakim Mdical Center and had normal neurologic examination, Magnetic Resonance Imaging and electromyographic survey, were under the research. For each patients we prescribed 50 milligram amitriptyline at bedtime for about 2 weeks. Before and After two weeks of treatment, patients must to fill the questionnaires including present pain intensity. For evaluation the efficacy of amitriptyline, the results were ststistically compared by t-test. Results: this study showed that Low back pain with etiology of myofascial pain syndrome are higher in women. Amitriptyline was very effective for this patients (p<001). Somnolence and dry mouth was the most adverse effects of therapy. Conclusion: In cases of low back pain, induced by myofascial pain syndromes even if there was not any symptom of depression or psychiatric problem, physicians could try with amitriptyline for improvement the pain. This efficacy may be due to analgesic effects of amitriptyline. P-610 Pain and inherited neuromuscular diseases: results of a postal inquiry among 281 adults V. Tiffreau*, G. Viet, A. Thevenon. Lille, France We present the results of a postal inquiry about pain in adult patients with inherited neuromuscular disease (NMD). Questionnaires were sent to 281 outpatients at the Physical Medicine unit, University Hospital of Lille. Patients were asked about their motor deficiency (Brooke and Vignos scales), pain intensity (VAS, VNS)and localization, the effect of pain on daily activity, about anxiety and depression (HAD score), causes of pain, medical and physical treatment. Results: response rate was 45%. 70% of the subjects were able to walk. Pain frequency was 70% and 60% of subjects suffered from chronic pain. Mean pain intensity was 6,1/10(VNS), 46% of patients were identified as anxious, and 16% as depressed. Major causes of pain were walking, standing and moving limbs. Pain related to walking was identified as chronic. There was no relationship between motor deficiency and pain severity or frequency, but pain effect on daily activity was more important in walking patients. 60% of patients had received at least one analgesic drug. Massage was the more frequent and effective physical treatment. Conclusion: pain is a frequent symptom of NMD adult patients. Pain related to walking seems to he rather chronic and has important effect on daily actity.
Myasthenic Disorders P-611 Acetylocholine Receptor Antibodies as a Useful Parameter in Monitoring the Dynamics of Myasthenia Gravis Malgorzata Bilinska*. Gdansk, Poland Myasthenia gravis (MG) is an acquired autoimmune disease, in which acetylcholine receptors in postsynaptic membrane are blocked by autoantibodies (AChRAB). AChRAB are present in approximately 55-95% of myasthenia patients. Their presence depends on type of M G and correlates with the clinical state of patients. We investigated the presence of AChRAB in 52 MG patients aged 20-77 and 30 healthy volunteers as a control group. In all we performed the neurological examination and studied the disease history to qualify patients to different types of MG according to the Ossermann classification. In order to analyse present clinical condition and further, to perform the compared analysis we used the Oosterhuis M G advanced clinical scale. In all patients and volunteers we repeated the the above mentioned investigations