Danon disease: case report and detection of new mutation.

Danon disease is an X-linked disorder resulting from mutations in the lysosome-associated membrane protein-2 (LAMP2) gene. We report a male patient with skeletal myopathy, mental retardation, and massive hypertrophic obstructive cardiomyopathy necessitating heart transplantation. Immunohistochemistry of skeletal muscle and leukocytes, western blot analysis of leukocytes and cardiac muscle, flow cytometry, and DNA sequencing were performed. Muscle biopsy revealed autophagic vacuolar myopathy and lack of immunohistochemically detectable LAMP-2. Diagnosis of Danon disease was confirmed by western blot analysis of myocardial tissue and peripheral blood sample of the patient showing deficiency of LAMP-2 in myocardium and leukocytes. Moreover, absence of LAMP-2 in lymphocytes, monocytes and granulocytes was shown by flow cytometric analysis. Genetic analysis of the LAMP2 gene revealed a novel 1-bp deletion at position 179 (c.179delC) at the 3' end of exon 2, resulting in a frameshift with a premature stop codon.

Late-onset metachromatic leukodystrophy: genotype strongly influences phenotype.

BACKGROUND: P426L and I179S are the two most frequent mutations in juvenile and adult metachromatic leukodystrophy (late-onset MLD), which, in contrast to infantile MLD, show marked phenotypic heterogeneity. OBJECTIVE: To search for genotype-phenotype correlations in late-onset MLD. METHODS: The authors reviewed the clinical course of 22 patients homozygous for mutation P426L vs 20 patients heterozygous for mutation I179S, in which the second arylsulfatase A (ASA) mutation had also been determined. RESULTS: P426L homozygotes principally presented with progressive gait disturbance caused by spastic paraparesis or cerebellar ataxia; mental disturbance was absent or insignificant at the onset of disease but became more apparent as the disease evolved. In contrast, compound heterozygotes for I179S presented with schizophrenia-like behavioral abnormalities, social dysfunction, and mental decline, but motor deficits were scarce. Reduced peripheral nerve conduction velocities and less residual ASA activity were present in P426L homozygotes vs I179S heterozygotes. CONCLUSION: The characteristic clinical differences between homozygous P426L and compound heterozygous I179S patients establish a distinct genotype-phenotype correlation in late-onset metachromatic leukodystrophy.

Analysis of EEG and CSF 14-3-3 proteins as aids to the diagnosis of Creutzfeldt-Jakob disease.

OBJECTIVE: To improve diagnostic criteria for sporadic Creutzfeldt-Jakob disease (CJD). METHODS: Pooled data on initial and final diagnostic classification of suspected CJD patients were accumulated, including results of investigations derived from a coordinated multinational study of CJD. Prospective analysis for a comparison of clinical and neuropathologic diagnoses and evaluation of the sensitivity and specificity of EEG and 14-3-3 CSF immunoassay were conducted. RESULTS: Data on 1,003 patients with suspected CJD were collected using a standard questionnaire. After follow-up was carried out, complete clinical data and neuropathologic diagnoses were available in 805 cases. In these patients, the sensitivity of the detection of periodic sharp wave complexes in the EEG was 66%, with a specificity of 74%. The detection of 14-3-3 proteins in the CSF correlated with the clinical diagnosis in 94% (sensitivity). The specificity (84%) was higher than that of EEG. A combination of both investigations further increased the sensitivity but decreased the specificity. CONCLUSIONS: Incorporation of CSF 14-3-3 analysis in the diagnostic criteria for CJD significantly increases the sensitivity of case definition. Amended diagnostic criteria for CJD are proposed.

Molecular mimicry in infectious encephalitis and neuritis: binding of antibodies against infectious agents on Western blots of human nervous tissue.

OBJECTIVES: To study the occurrence of cross-reactivities of antibodies against infectious agents with human nervous tissue. METHODS: Binding of 25 antibodies against 17 neurotropic pathogens comprising Borrelia burgdorferi, Toxoplasma gondii, and various DNA and RNA viruses to Western blots of human cortex and myelin from central and peripheral nervous system was investigated. RESULTS: Fourteen of the 25 antibodies tested showed binding to Western blots of human nervous tissue, suggesting the presence of shared epitopes. Binding of 11 antibodies against 10 pathogens to cortex and/or myelin correlated with the tissue targeted by neuropathological lesions. Three antibodies did not show such correlation; 11 antibodies did not bind at all. CONCLUSIONS: Our results suggest that shared epitopes between infectious agents and human nervous tissues are more common than previously expected. Thus, molecular mimicry should be considered more frequently as a possible pathogenetic mechanism, among others, inducing tissue damage in encephalitis and neuritis caused by various pathogens.

Ganglioside-induced antiganglioside antibodies from a neuropathy patient cross-react with lipopolysaccharides of Campylobacter jejuni associated with Guillain-Barré syndrome.

Antiganglioside serum antibodies from a patient treated with gangliosides were examined for cross-reactivity with lipopolysaccharides (LPSs) of Campylobacter jejuni strains associated with Guillain-Barré syndrome (GBS). The patient had no preceding infection with C. jejuni and developed chronic progressive motor polyneuropathy following parenteral ganglioside treatment. Serum IgG antibodies recognised GM1 and GD1b gangliosides as well as asialo-GM1, and cross-reactivity was observed with LPSs from C. jejuni O:2, O:4, O:19 and O:41. The results give a clear indication that gangliosides and LPSs from C. jejuni serotypes associated with GBS share common epitopes.

Distinct immunoglobulin class and immunoglobulin G subclass patterns against ganglioside GQ1b in Miller Fisher syndrome following different types of infection.

We studied serum antibodies against gangliosides GQ1b and GM1 in 13 patients with Miller Fisher syndrome (MFS) and in 18 patients with Guillain-Barré syndrome (GBS) with cranial nerve involvement. Anti-GQ1b titers were elevated in all patients with MFS cases (immunoglobulin G [IgG] > IgA, IgM), and in 8 of the 18 with GBS. Lower frequencies of increased anti-GM1 titers were observed in MFS patients (3 of 13), as well as in GBS patients (5 of 18). During the course of MFS, anti-GQ1b titers of all Ig classes decreased within 3 weeks after onset. By contrast, anti-GM1 titers (mainly IgM) transiently increased during the course of MFS in five of six patients, suggesting a nonspecific secondary immune response. In patients with MFS following respiratory infections, IgG was the major anti-GQ1b Ig class (six of six patients) and IgG3 was the major subclass (five of six). In contrast, four of five patients with MFS following gastrointestinal infections showed predominance of anti-GQ1b IgA or IgM over IgG and predominance of the IgG2 subclass; anti-GQ1b IgG (IgG3) prevailed in one patient only. These distinct Ig patterns strongly suggest that different infections may trigger different mechanisms of anti-GQ1b production, such as via T-cell-dependent as opposed to T-cell-independent pathways. Thus, the origin of antibodies against GQ1b in MFS may be determined by the type of infectious agent that precipitates the disease.

Coincidence of two novel arylsulfatase A alleles and mutation 459+1G>A within a family with metachromatic leukodystrophy: molecular basis of phenotypic heterogeneity.

In a family with three siblings, one developed classical late infantile metachromatic leukodystrophy (MLD), fatal at age 5 years, with deficient arylsulfatase A (ARSA) activity and increased galactosylsulfatide (GS) excretion. The two other siblings, apparently healthy at 12(1/2) and 15 years, respectively, and their father, apparently healthy as well, presented ARSA and GS values within the range of MLD patients. Mutation screening and sequence analysis disclosed the involvement of three different ARSA mutations being the molecular basis of intrafamilial phenotypic heterogeneity. The late infantile patient inherited from his mother the frequent 0-type mutation 459+1G>A, and from his father a novel, single basepair microdeletion of guanine at nucleotide 7 in exon 1 (7delG). The two clinically unaffected siblings carried the maternal mutation 459+1G>A and, on their paternal allele, a novel cytosine to thymidine transition at nucleotide 2435 in exon 8, resulting in substitution of alanine 464 by valine (A464V). The fathers genotype thus was 7delG/A464V. Mutation A464V was not found in 18 unrelated MLD patients and 50 controls. A464V, although clearly modifying ARSA and GS levels, apparently bears little significance for clinical manifestation of MLD, mimicking the frequent ARSA pseudodeficiency allele. Our results demonstrate that in certain genetic conditions MLD-like ARSA and GS values need not be paralleled by clinical disease, a finding with serious diagnostic and prognostic implications. Moreover, further ARSA alleles functionally similar to A464V might exist which, together with 0-type mutations, may cause pathological ARSA and GS levels, but not clinical outbreak of the disease.

Antiglycosphingolipid immune responses in neurology. The Vienna experience with isotypes, subclasses, and disease.

IgM, IgG, IgA, and IgG subclass anti-GM1, anti-GQ1b, and anti-asialo-GM1 (anti-GA1) antibodies, respectively, were investigated by ELISA in serum from neurological and other patients. Increased anti-GM1 occurred mostly in approximately 15-35% of the cases without statistical differences; high percentages were found in Guillain-Barré syndrome (GBS) preceded by gastrointestinal infection and multifocal motor neuropathy. Roughly, IgM anti-GM1 was most frequent; however, distinct IgG and IgA reactions were found i.a. in GBS. A particular IgM anti-mono- and disialoganglioside pattern occurred in a patient with sensorimotor neuropathy and paraproteinemia. Anti-GQ1b was elevated in all Miller-Fisher patients, with some prevalence of IgG2 among IgG subclasses. Cross-reactivity of anti-GQ1b was demonstrated with Campylobacter jejuni lipopolysaccharides. Increased anti-GM1 and/or anti-GA1 was more frequent in systemic lupus erythematosus with central nervous system involvement than without. Incidence of anti-GM1 and anti-GA1 in X-adrenoleukodystrophy was relatively high. Although anti-GSL antibodies seem to have limited diagnostic value, studies of isotypes, subclass patterns, and cross-reactivities may lead to further insight into the origin of (auto) immune responses and their immunepathogenetic role in disease.

[Cerebrospinal fluid diagnosis of Creutzfeldt-Jakob disease]. / Liquordiagnostik der Creutzfeldt-Jakob-Krankheit (CJK).

Diagnosis of Creutzfeldt-Jakob disease (CJD) at lifetime according to the international diagnostic criteria may be greatly improved by the additional assay of 14-3-3 protein in cerebrospinal fluid (CSF). Occurrence of 14-3-3 protein in CSF may be observed in etiologically different conditions of brain damage, but confers high diagnostic specificity in cases of suspected CJD based on the diagnostic criteria. We investigated the occurrence of 14-3-3 protein in CSF of 20 patients with an accompanying diagnosis "suspected CJD", of whom 5 cases had to be classified as neither probable nor possible CJD according to the international diagnostic criteria, as well as in 18 control cases with diseases other than CJD. Assay of 14-3-3 comprised SDS-PAGE, western blot, immunostaining with specific antibody, and luminiscence detection. With regard to case histories at the end of our study, 8 definitive and probable CJD cases were 14-3-3 positive and 2 possible CJD cases 14-3-3 negative. Of the 10 cases with final diagnosis other than CJD, 8 cases (in part with manifest or suspected brain damage; one case with hemorrhagic CSF) were 14-3-3 positive. Of the 18 controls with diseases other than CJD, 6 patients, characterized by brain tissue lesions or meningitis, respectively, were found to be 14-3-3 positive. Our observations, the first in Austria and on a limited number of patients yet, are in accordance with previous reports in the literature and further support the use of the 14-3-3 protein assay in CSF as a diagnostic tool for CJD, provided that probands had been pre-evaluated positively by the international diagnostic criteria for CJD.

Serum antibodies against gangliosides and Campylobacter jejuni lipopolysaccharides in Miller Fisher syndrome.

Seven patients with Miller Fisher syndrome (MFS), six in the acute phase and one in the recovery phase, were investigated for serum antibodies against gangliosides and purified lipopolysaccharides (LPS) from different strains of Campylobacter jejuni, including the MFS-associated serotypes O:2 and O:23. Immunoglobulin G antibodies against gangliosides GT1a and GQ1b were found in five of six patients in the acute phase of disease. Three of these patients also displayed antibodies to ganglioside GD2, a finding not previously reported for MFS. All anti-GT1a- and anti-GQ1b-seropositive patients showed antibody binding to C. jejuni LPS, predominantly to O:2 and O:23 LPS. Antibody cross-reactivity between gangliosides GT1a and GQ1b and O:2 and O:23 LPS was demonstrated by adsorption studies. This cross-reactivity between gangliosides and C.jejuni LPS, which is obviously due to oligosaccharide homologies, may be an important pathogenetic factor in the development of MFS after C. jejuni infection.

Occurrence, distribution, and phenotype of arylsulfatase A mutations in patients with metachromatic leukodystrophy.

Occurrence, distribution, and phenotype of arylsulfatase A (ASA) mutations were investigated in 27 patients with metachromatic leukodystrophy (MLD) from Central Europe, mainly from Austria (n = 15) and Poland (n = 9). Genomic DNA from leukocytes, fibroblasts, or paraffin-embedded, formalin-fixed brain or nerve tissue, respectively, was tested by natural or mutated primer-modulated PCR restriction, fragment length polymorphism for the eight most common European mutations: R84Q, S96F, 459+1G > A, I179S, A212V, 1204+1G > A, P426L, and 1401del11bp. The overall identification rate of unrelated MLD alleles was the highest, in adult (90%), medium in juvenile (50%), and lowest in late infantile (36%) MLD patients. The two common alleles, 459+1G > A and P426L, together accounted for 42% of all 50 unrelated MLD alleles investigated; I179S was observed in 6 of 50 MLD alleles (12%). Thus, I179S was far more frequent than hitherto thought and appears to be a third common mutation in Europe. Moreover, a different allelic distribution between Austrian and Polish juvenile patients was disclosed, indicating genetic heterogeneity of MLD even within Central Europe. The genotype-phenotype correlation suggested by Polten et al. [N Engl J Med 324:18-22, 1991] was not followed by all of our MLD patients. Moreover, some MLD patients with identical ASA mutations presented with different phenotypes. This may be due, at least in some cases, to the presence of an additional mutation on individual mutant alleles. Therefore, prediction of the clinical course from single mutation analysis is not possible.

Targeted inactivation of the X-linked adrenoleukodystrophy gene in mice.

In its severe form, X-linked adrenoleukodystrophy (ALD) is a lethal neurologic disease of children, characterized by progressive cerebral demyelination and adrenal insufficiency. Associated with a biochemical defect of peroxisomal beta-oxidation, very long-chain fatty acids (VLCFA) build up in tissues that have a high turnover of lipids, such as central nervous system (CNS) white matter, adrenal cortex, and testis. Whether the abnormal accumulation of VLCFA is the underlying cause of demyelination or merely an associated biochemical marker is unknown. ALD is caused by mutations in the gene for a peroxisomal membrane protein (ALDP) that shares structural features with ATP-binding-cassette (ABC) transporters. To analyze the cellular function of ALDP and to obtain an animal model of this debilitating disease, we have generated transgenic mice with a targeted inactivation of the ald gene. Motor functions in ALDP-deficient mice developed at schedule, and unexpectedly, adult animals appeared unaffected by neurologic symptoms up to at least 6 months of age. Biochemical analyses demonstrated impaired beta-oxidation in mutant fibroblasts and abnormal accumulation of VLCFAs in the CNS and kidney. In 6-month-old mutants, adrenal cortex cells displayed a ballooned morphology and needle-like lipid inclusions, also found in testis and ovaries. However, lipid inclusions and demyelinating lesions in the CNS were not a feature. Thus, complete absence of ALDP expression results in a VLCFA storage disease but does not impair CNS function of young adult mice by pathologic and clinical criteria. This suggests that additional genetic or environmental conditions must be fulfilled to model the early-onset and lethality of cerebral ALD in transgenic mice.

A new polymorphism of arylsulfatase A within the coding region.

A 10-year-old boy with juvenile metachromatic leukodystrophy (MLD) presented with the 459 + 1G-->A arylsulfatase A (ASA) mutation on one allele. To detect his complete genotype, the other ASA allele was sequenced and a T-to-C transition at nucleotide 376 in exon 2 was identified. This missense mutation results in a substitution of leucine 76 by proline. Of 20 MLD unrelated controls, 18 carried the L/P76 mutation either in the homozygous (n = 6) or heterozygous (n = 12) state. The presence or absence of L/P76 did not influence leukocyte ASA activity or urinary sulfatide excretion. Apparently, the substitution of leucine 76 by proline is a common ASA polymorphism, neither being related to MLD nor creating ASA pseudodeficiency. However, because of its frequency and location, L/P76 may be of particular importance in genetic studies requiring the differentiation of the ASA alleles within a kindred. Further studies are directed to the as yet unresolved genotype of the index case with juvenile MLD.

T-cell-mediated ganglionitis associated with acute sensory neuronopathy.

A 67-year-old man presented with acute painful sensory loss, areflexia, ataxia, urinary retention, and severe constipation and became unable to walk within 2 weeks. He died suddenly 5 weeks after the onset of symptoms. Autopsy revealed widespread inflammation of sensory and autonomic ganglia with immunocytochemical evidence of a CD8+ T cell-mediated cytotoxic attack against ganglion neurons. This observation suggests a novel pathogenetic mechanism of immune-mediated human ganglion cell damage comparable to mechanisms operating in polymyositis.

Differentiation of rare leukodystrophies by post-mortem morphological and biochemical studies: female adrenoleukodystrophy-like disease and late-onset Krabbe disease.

Two 6-year-old patients with clinical signs of leukodystrophy had no nosological diagnoses in vivo. Neuropathological studies revealed scavenger cells to be clustered in perivascular regions of the demyelinated brains. Histochemical and ultrastructural details of the non-metachromatic storage macrophages suggested lipid storage and prompted a biochemical analysis of cerebral tissue. The detection of increased amounts of very long chain fatty acids in the cholesterol ester fraction from formalin tissue in one patient was consistent with a diagnosis of an adrenoleukodystrophy-like condition, while the marked reduction in beta-galactocerebrosidase activity in a frozen brain sample of the second patient indicate Krabbe disease. The diagnostic potential of post-mortem studies in rare leukodystrophies is addressed.

Detection of IgA anti-PGL-I specific antigen to Mycobacterium leprae in mangabey monkeys inoculated with M. leprae.

Using sera from 4 pairs of mangabey monkeys inoculated with titrated doses of Mycobacterium leprae we demonstrated that IgA antibodies against M. leprae specific PGL-I antigen were present in 75% of inoculated monkey's sera. High IgA antibody was detected in 50% (3/6) of infected animals and all three developed lepromatous leprosy (LL). Antibody titers correlated with PGL-I antigen in serum. The highest IgA peak appeared late and corresponded to the beginning of treatment, and in two of them appeared shortly after or corresponded with neurological damage. Low IgA response was found in the other 3 monkeys (50%-3/6), two of which developed indeterminate leprosy (I) and the other one LL. Low IgA levels appeared late after IgG and IgM, and shortly after neurologic signs. Both I monkeys were negative for PGL-I in serum. The remaining 2 monkeys (25%-2/8) did not show an IgA response; one of them developed LL but the disease regressed to I. IgM seemed to correspond to the appearance of PGL-I in serum. The other animal did not develop clinical symptoms of leprosy, and PGL-I in serum was negative. Although there was no clear relation between the development of anti-PGL-I IgA and experimental leprosy, the finding of a high IgA response in some animals suggests that further studies are needed to evaluate the role of antigen-specific IgA in the disease process.