Congenital muscular dystrophy with primary partial laminin alpha2 chain deficiency: molecular study

The authors report a case of congenital muscular dystrophy with mild nonprogressive muscle weakness, white matter hypodensity, and absence of the laminin alpha2 chain in muscle fibers with two antibodies, but not with four others. They identified mutations in LAMA2, which explain the partial laminin alpha2 deficiency. Analysis of this case and two others allows us to refine the epitopes of two of the commercial antibodies, and illustrate the importance of using antibodies directed against different domains of the protein.     

Quantitative proton MRS of cerebral metabolites in laminin alpha2 chain deficiency

Congenital muscular dystrophy (CMD) due to merosin (laminin alpha2 chain) deficiency is an autosomal recessively inherited disorder characterized by severe muscular weakness and hypotonia from birth on. Brain involvement is the rule and characterized by variable T2 hyperintensities of white matter which appears swollen on cranial MRI. The pathophysiology of these white matter changes is not clear. In five patients with laminin alpha2 deficient CMD we performed short-echo time localized proton MRS with determination of absolute metabolite concentrations in grey and white matter. In affected white matter, a consistent pattern of metabolites was detected comprising reduced concentrations of N-acetylaspartate and N-acetylaspartylglutamate, creatine, and phosphocreatine, and to a milder degree of choline-containing compounds. In contrast, concentrations of myo-inositol were in the normal range. Spectra of cortical and subcortical grey matter were normal. The observed metabolite profile is consistent with white matter edema, that is reduced cellular density, and relative astrocytosis. This interpretation is in line with the hypothesis that laminin alpha2 deficiency results in leakage of fluids across the blood-brain barrier and a histopathological report of astrocytic proliferation in CMD.     

Partial laminin alpha2 chain deficiency in a patient with myopathy resembling inclusion body myositis

It is becoming evident that clinical phenotypes associated with partial laminin alpha2 chain deficiency are variable. We recently observed a 29-year-old man with leukoencephalopathy and vacuolar myopathy resembling inclusion body myositis. Laminin alpha2 immunohistochemical analysis showed reduction of the protein on muscle fiber surfaces. Molecular analysis revealed two novel compound heterozygous mutations in the LAMA2 gene. This is the first report linking a mutation in the LaMA2 gene with leukoencephalopathy and inclusion body-like myositis.     

Failure to detect antibodies to myelin basic protein or peptic fragments of myelin basic protein in CSF of patients with MS

Using the sodium sulfate precipitation radioimmunoassay and solid-phase radioimmunoassay, we measured antibody to intact human myelin basic protein and myelin basic protein peptic fragments, residues 1-44, 45-89, and 90-170, in CSF. Comparable levels of binding were obtained for MS and normal CSF by both tests. The increased amount of CSF IgG in MS patients cannot be attributed to specific antibody against myelin basic protein or its peptic fragments.     

Domain-specific antibodies against the B2 chain of laminin inhibit neuronal migration in the neonatal rat cerebellum

Although the spatial and temporal patterns of neuronal migration have been analyzed in great detail, little direct evidence is available as to what extracellular matrix molecules are involved. Because there is indirect evidence implicating the extracellular matrix protein laminin in neuronal migration, we investigated the effects of antibodies against a synthetic peptide derived from a neurite outgrowth domain of the B2 chain of laminin on neuronal migration in living cerebellar slices. We show by using infrared video microscopy that divalent Fab₂ fragments of these antibodies inhibit granule neuronal movement in living slices of (P8) rat cerebellum. This inhibition of neuronal movement manifests itself by cessation of both radial and horizontal translocations of nuclei inside the granule neuronal processes. Fab₂ fragments of antibodies against the intact (native) laminin molecule or Fab₂ fragments from the preimmune serum do not affect nuclear translocation. Immunocytochemistry shows binding of the divalent Fab₂ fragments of the B2 chain-specific antibodies to the Purkinje and Bergmann glial cell areas, and as punctate deposits in between the cells of the external granule cell layer. Native laminin antibodies bind to the basement membranes, and binding of the Fab₂ fragments from the preimmune sera cannot be demonstrated. These results indicate that neuronal migration in the postnatal rat cerebellum in vivo involves nuclear translocation that can be inhibited by antibodies against a neurite outgrowth domain of the B2 chain of laminin. Thus, migration of cerebellar granule neurons may depend on the interaction between a neurite outgrowth domain of the B2 chain of laminin and neuronal cytoskeleton involved in nuclear movement. © 1995 Wiley-Liss, Inc.     

Clinical relevance of antibodies against serotonin and gangliosides in patients with primary fibromyalgia syndrome

The fibromyalgia syndrome (FMS) is a non-articular rheumatic disorder associated with disturbances in serotonin metabolism. In order to evaluate whether patients with FMS suffer from an autommune disorder, we tested sera from 50 clinically well-defined FMS patients for non-organ-specific and organ-specific antibodies by enzyme-linked immunosorbent assay and immunofluorescence test. Common antibodies against nuclei, mitochondria, and microsomes were not increased in these patients compared to healthy controls. However, 74% had antibodies against serotonin and gangliosides. The clinical and diagnostic relevance of these antibodies is supported by the absence of anti-serotonin antibodies in other rheumatic disorders such as rheumatoid arthritis, polymyalgia rheumatica, and collagen diseases. These antibodies may belong to the group of antireceptor antibodies, considering the fact that gangliosides are an important component of the serotonin receptor. It remains to be determined whether these antibodies are of pathogenetic relevance, interfering with serotonin binding and thereby inducing symptoms associated with FMS.     

Detection of cerebrospinal fluid antibodies against myelin basic protein in patients with AIDS dementia complex

We evaluated cerebrospinal fluid (CSF) antibody levels against a lipid-free, denatured form of myelin basic protein (LF-MBP) in 11 patients with AIDS dementia complex (ADC) by using an enzymelinked immunosorbent assay (ELISA). In 9 out of 11 patients, anti-LF-MBP antibody levels were significantly higher than those observed both in 15 human immunodeficiency virus (HIV)-infected patients without neurological disorders and in 9 anti-HIV-negative subjects affected by other neurological diseases. Furthermore, we followed up anti-MBP levels in 5 out of the 11 ADC patients and detected a strict relationship with the encephalopathy progression. At the same time, with the aim to detect early demyelinating events we investigated CSF antibody levels against a lipid-bound, native-like form of MBP (LB-MBP). Results did not show any significant difference between LF-MBP and LB-MBP in terms of antibody reactivity. The detection of anti-MBP antibodies in CSF may provide the opportunity to assess a diagnostic tool for discovering demyelinating lesions in ADC patients.     

Immunocytochemical localization of the mammalian voltage-dependent sodium channel using polyclonal antibodies against the purified protein

Antibodies were raised in rabbits against the purified voltage-dependent sodium channel from rat skeletal muscle sarcolemma. The resultant antiserum reacted with the purified channel in a solid-phase radioimmunoassay and precipitated the sodium channel from a crude mixture of solubilized membrane proteins. Crude membrane proteins separated according to size under nondenaturing conditions by chromatography on Sepharose CL-6B contained a single peak of immunoreactivity that coincided with the native channel. On immunoblots of sarcolemmal membrane proteins, the antiserum reacted predominantly with a diffuse high molecular weight band that was comparable in migratory characteristics to the large glycoprotein subunit of the purified channel. Using immunocytochemical techniques, binding of this polyclonal antiserum was localized to the surface membrane of rat skeletal muscle. This staining was specifically blocked by pre-incubation of the antiserum with the purified channel protein. The antiserum also stained the surface membrane of rat cardiac muscle and the nodes of Ranvier in rat peripheral nerve. Species cross-reactivity was seen with mouse, human, and guinea pig skeletal muscle while chicken, rabbit, and frog muscle was not stained. The antiserum also reacted with the surface membranes of fetal rat muscle in tissue culture. These results indicate that sodium channels in adult mammalian skeletal muscle, cardiac muscle, and peripheral nerve and in fetal muscle in culture all share common antigenic determinants. The antiserum should prove useful for topographical studies of sodium channel distribution in these tissues.     

Detection of elevated antibodies against SR protein kinase 1 in the serum of Alzheimer's disease patients

Autoantibodies targeting specific cellular antigens are often present in sera and cerebrospinal fluids (CSFs) of patients with Alzheimer's disease (AD) and could play a role in the onset and/or progression of the disease. In this study we identified SR Protein Kinase 1 (SRPK1) as a new autoantigen elevated in AD. SRPK1, the prototype of the serine/arginine family of kinases, has been implicated in the regulation of multiple cellular processes such as pre-mRNA splicing, cell proliferation, chromatin structure, nuclear import and germ cell development. Using an ELISA assay, anti-SRPK1 antibodies, targeting mainly the first catalytic domain of the kinase, were detected in sera of patients with AD, at significantly elevated levels as compared to control subjects. The findings of this study document for the first time the existence of antibodies targeting SRPK1 in human sera and are indicative of a correlation between the levels of a-SRPK1 antibodies and the incidence of AD.     

Screening of protein S deficiency using a functional assay in patients with venous and arterial thrombosis

Protein S is the vitamin K-dependent cofactor of activated protein C which functions as a potent anticoagulant by degrading activated factors V and VIII in a Ca2+ and phospholipid-dependent reaction. Protein S circulates under two forms, free (approximately 40%) or bound to C4b-binding protein (C4b-bp); only the free form supports the cofactor activity for activated protein C. Total protein S antigen is usually measured by rocket immunoelectrophoresis. Free protein S antigen is measured by the same technique but after precipitation of the protein S-C4b-bp complex by PEG 8000. However, these immunological assays do not detect functional alterations of protein S which can be responsible for thrombosis. This paper describes a functional assay for free protein S based on its ability to promote the prolongation of clotting time following factor Va inactivation by activated protein C when coagulation is triggered by factor Xa. Using this assay a prolongation of about 100 s between 0 and 1 U/ml protein S is measured, allowing a reliable and rapid determination of functional protein S. The correlation coefficient between functional protein S and free antigenic protein S is 0.921. This functional protein S assay has allowed the detection of 34 cases of protein S deficiency, confirmed by immunological assays, and their classification. The striking observation is the high frequency (approximately 25%) of arterial thrombosis in these patients. The rapid determination of functional protein S in patients with venous or arterial thrombosis is of diagnostic interest and should allow the detection of mutant protein S in combination with an immunological assay.     

Synaptic plasticity in the dy2J mouse model of laminin alpha2-deficient congenital muscular dystrophy

Laminin alpha2-deficient congenital muscular dystrophy is a debilitating disease affecting both muscle and neural tissue as a result of mutations in the LAMA2 gene. It presents at or soon after birth with muscle weakness and is further characterised by clinical central nervous system involvement. Laminin alpha2 is part of the extracellular matrix, linked to the cellular cystoskeleton via dystroglycan which is an integral part of the dystrophin-glycoprotein complex (DGC). We examined both short- and long-term synaptic plasticity in the C57BL6J/dy(2J) mouse, an animal model of laminin alpha2 deficient congenital muscular dystrophy. Using a cerebellar slice preparation, we show that the pre-synaptically mediated paired-pulse facilitation (PPF) was no different between dy(2J) and littermate controls. Approximately half (7/12) the dy(2J) Purkinje cells displayed a blunted LTD compared to littermate controls, and one third (4/12) of dy(2J) Purkinje cells displayed LTP. This study demonstrates that a defective laminin alpha2 causes a disruption in long-term synaptic plasticity at the Purkinje cell-parallel fibre synapse.     

Dopamine D2 receptor imaging with SPECT using IBZM in 16 patients with Parkinson disease

The striatal D2 receptor was investigated for variability of behavior in 16 patients with Parkinson disease by means of SPECT using 123I-IBZM, a recently synthesized radioligand with high affinity and specificity for this type of receptor. All the patients underwent routine laboratory tests, EEG and cranial CT scanning as well as SPECT. To ensure accurate clinical assessment we used the Hoehn and Yahr, and Webster scales, NUDS and Mini Mental State examination. Our preliminary data indicate:

- increased uptake by the striatal dopamine receptor in untreated patients with early PD compared with controls (striatum/cerebellum ratio of 1.77±0.12 vs 1.59±0.13);

- a slight but significant lowering of D2 receptor binding when L-Dopa is started (1.49±0.09);

- low D2 uptake values in the more severely affected patients;

- absence of asymmetries in hemiparkinsonism.

     

Poor relationship between phenotypes of protein S deficiency and mutations in the protein S alpha gene

By single strand conformational polymorphism, nucleotide sequencing and enzyme restriction, we analyzed the protein S alpha gene in 17 protein S-deficient probands and in their available family members. The relationship between genotype and phenotype was also evaluated. Twelve different sequence variations were identified in 17 probands. Ten were putative causal mutations distributed in 16 probands: 4 were nonsense, 5 missense and one a splice site mutation. In most families in which a mutation was identified, more than one phenotype of PS deficiency was present. The same splice site mutation (intron j G-A, exon 10+5) was associated with type I deficiency in one family and with type I/III in another unrelated family. A phenotypic discrepancy was also observed for the Arg474Pro, Gly597Asp and Arg410stop mutations. Glu26Ala, previously reported in kindreds with type I deficiencies, was found in association with I, II and III phenotypes in four unrelated kindreds. Phenotypic analysis of protein S deficiency is poorly related to the underlying genetic defect.     

Myasthenia gravis patients with a thymoma have antibodies against a high molecular weight protein in sarcoplasmic reticulum.

Our purpose was to investigate whether components of the sarcoplasmic reticulum (SR) are relevant antigens in myasthenia gravis (MG). Using enzyme-linked immunosorbent assay (ELISA), 75 MG sera and 120 control sera were examined for IgG antibodies against SR prepared from rabbit skeletal muscle. 16/30 thymoma MG patients had IgG antibodies that reacted with SR. 1/30 MG patients with thymic hyperplasia and 3/15 MG patients with thymic atrophy had SR antibodies in low concentrations. Control sera were negative. Using immunoblot, SR antibodies were detected in the thymoma group only. 14/30 sera from thymoma patients reacted with a protein of 320 kDa relative molecular weight. The only reported SR protein with similar electrophoretic mobility is the subunit of the spanning protein which links junctional SR to sarcolemma and functions as a calcium-release channel.     

Duration of oral anticoagulant treatment in patients with venous thromboembolism and a deficiency of antithrombin, protein C or protein S--a decision analysis

Patients with a first venous thromboembolic event and a deficiency of the coagulation inhibitors antithrombin, protein C or protein S have an increased risk of recurrent venous thromboembolism compared to patients without such a deficiency. A decision analysis was performed to assess the effect of continuing treatment with vitamin K antagonists on mortality by a reduction in fatal recurrent pulmonary embolism and an induction of fatal haemorrhages associated with their use. The treatment decision involves continuation or discontinuation of vitamin K antagonists in patients with a first spontaneous or secondary venous thromboembolism and an antithrombin, protein C or S deficiency. Although the efficiency of oral anticoagulation is high in all age groups early after the first thromboembolic event, it decreases over time. Our analysis indicates that the optimal treatment duration will vary, depending on the type of the initial event (spontaneous or secondary; deep venous thrombosis or pulmonary embolism), age, and time passed since the initial thromboembolic episode. Moreover, life-long duration of the prophylaxis seems not warranted in all patients.     

Clinical significance of anti-protein Z antibodies in patients with lupus anticoagulant

INTRODUCTION: Protein Z serves as cofactor for the inactivation of factor Xa by the plasma protein Z-dependent protease inhibitor. Deficiency of protein Z was reported to exhibit a clinical manifestation like lupus anticoagulant characterised by thrombosis and fetal loss. As anti-protein Z antibodies may be associated with low protein Z levels, we hypothesised that anti-protein Z antibodies might play a role in lupus anticoagulant (LA). MATERIALS AND METHODS: Anti-protein Z antibodies were measured by commercially available ELISA in 102 LA-patients (69 with and 33 without thrombosis) and 33 healthy volunteers. RESULTS: Elevated anti-protein Z IgG and/or IgM, IgG and IgM antibody levels were more prevalent among LA-patients (62%, 35%, 45%) than among controls (50%, 25%, 25%), but the difference was only statistically significant for the IgM subtype (p=0.037). Anti-protein Z IgG (odds ratio [OR] 0.77, 95% confidence interval [CI] 0.33-1.82) and IgM (OR 0.82, CI 0.35-1.88) antibody levels in the highest quartile of controls did not indicate an increased risk for thrombosis among LA-patients. Anti-protein Z IgG (OR 2.0, CI 0.5-7.6) and IgM (OR 1.8, CI 0.5-6.6) antibody levels in the highest quartile of controls were more prevalent in women with pregnancy loss than in those with normal pregnancy, but the difference was not statistically significant. CONCLUSION: Our data indicate that anti-protein Z antibodies are not associated with thrombosis in LA. However, women with LA and pregnancy loss show a tendency towards elevated anti-protein Z antibody levels.