Diagnostic and prognostic value of in vivo proton MR spectroscopy for Zellweger syndrome spectrum patients.

Defects in the biogenesis of peroxisomes cause a clinically and genetically heterogeneous group of neurometabolic disorders, the Zellweger syndrome spectrum (ZSS). Diagnosis predominantly is based on characteristic clinical symptoms, a typical biochemical profile, as well as on identification of the molecular defect in any of the 12 known human PEX genes. The diagnostic workup can be hindered if the typical clinical symptoms are missing and predicting the clinical course of a given patient is almost unfeasible. As a safe and noninvasive method to analyze specific chemical compounds in localized brain regions, in vivo proton magnetic resonance spectroscopy (MRS) can provide an indication in this diagnostic process and may help predict the clinical course. However, to date, there are very few reports on this topic. In this study, we performed localized in vivo proton MRS without confounding contributions from T1- and T2-relaxation effects at 2 Tesla in a comparably large group of seven ZSS patients. Patients' absolute metabolite concentrations in cortical gray matter, white matter, and basal ganglia were assessed and compared with age-matched control values. Our results confirm and extend knowledge about in vivo MRS findings in ZSS patients. Besides affirmation of nonspecific reduction of N-acetylaspartate + N-acetylaspartylglutamate (tNAA) in combination with lipid accumulation as a diagnostic hint for this disease group, the amount of tNAA loss seems to reflect disease burden and may prove to be of prognostic value regarding the clinical course of an already diagnosed patient.

A novel mutation in the XPA gene results in two truncated protein variants and leads to a severe XP/neurological symptoms phenotype.

BACKGROUND: The nucleotide excision repair (NER) pathway repairs UV-induced DNA lesions in an accurate fashion and prevents UV-irradiated areas of the skin from tumour formation. The XPA protein plays a major role in DNA damage demarcation as well as stabilization of other NER factors and was found to be defective in xeroderma pigmentosum (XP) complementation group A patients. OBJECTIVE: Characterization of four new XP-A patients. METHODS: Genomic and cDNA sequencing, post-UV cell survival of living cells, host-cell reactivation of patients' fibroblasts and Western blotting. RESULTS: One of the four investigated patients shows a novel mutation leading to two different truncated protein variants. Three patients contain the already described p.R228X mutation. All patient cell lines exhibit a strong UVC sensitivity and reduced NER capability. In most of the cases stable protein expression was detected. CONCLUSION: We discovered four new XP-A patients and a novel XPA mutation resulting in two diverse patient alleles.

Genetic and clinical aspects of Zellweger spectrum patients with PEX1 mutations.

OBJECTIVE: To analyse the PEX1 gene, the most common cause for peroxisome biogenesis disorders (PBD), in a consecutive series of patients with Zellweger spectrum. METHODS: Mutations were detected by different methods including SSCP analyses as a screening technique on the basis of genomic or cDNA, followed by direct sequencing of PCR fragments with an abnormal electrophoresis pattern. RESULTS: 33 patients were studied. Two common mutations, c.2528G-->A, G843D and c.2098_2098insT, I700YfsX42, accounted for over 80% of all abnormal PEX1 alleles, emphasising their diagnostic relevance. Most PEX1 mutations were distributed over the two AAA cassettes with the two functional protein domains, D1 and D2, and the highly conserved Walker motifs. Phenotypic severity of Zellweger spectrum in CG1 depended on the effect of the mutation on the PEX1 protein, peroxin 1. PEX1 mutations could be divided into two classes of genotype-phenotype correlation: class I mutations led to residual PEX1 protein levels and function and a milder phenotype; class II mutations almost abolished PEX1 protein levels and function, resulting in a severe phenotype. Compound heterozygote patients for a class I and class II mutation had an intermediate phenotype. CONCLUSIONS: Molecular confirmation of the clinical and biochemical diagnosis will allow the prediction of the clinical course of disease in individual PBD cases.

Double-blind study of oral gamma-vinyl GABA in the treatment of dystonia.

Six patients with different forms of dystonia were treated with gamma-vinyl GABA, a specific enzyme-activated inhibitor of GABA-transaminase, in a double-blind, placebo-controlled crossover study. gamma-Vinyl GABA therapy, 2 g daily for 2 weeks, was compared with placebo by weekly assessments. There were no consistent changes in three evaluation scores. Agents that augment CNS GABA are unlikely to benefit patients with generalized dystonia.

Phase I study of methylacetylenic putrescine, an inhibitor of polyamine biosynthesis.

In a phase I clinical trial, nine patients with advanced malignancies not amenable to alternative therapy received alpha-methyl-delta-acetylenic putrescine (MAP), an enzyme-activated, irreversible inhibitor of ornithine decarboxylase (ODC). MAP was given orally in increasing doses to successive groups of three patients as follows: 375 mg, 750 mg and 1500 mg/day, given as three equally divided doses for 4 weeks. Doses of 375 and 750 mg/day were well tolerated, with no detectable toxicity. Of three patients receiving 1500 mg/day, two experienced moderate to severe myelosuppression; one of these also became anuric, requiring the discontinuation of therapy after 9 days. Both effects were reversible after treatment was stopped. No objective responses were observed, with five patients having stable disease and four, progressive disease during the study period. In the seven patients in whom it could be calculated, the plasma elimination half-life t1/2 of MAP measured on the last day of treatment was between 3.9 and 9.2 h in six patients (mean, 5.6 h) and 26.1 h in the seventh. Mean steady-state trough concentrations of MAP were 2.3 mumol after the 375 mg/day dose, 7.1 mumol after 750 mg/day and 16.6 mumol after dosing with 1500 mg/day for 4 weeks, the levels after each treatment schedule being sufficient to inhibit ODC as demonstrated by increases in the urinary excretion of decarboxylated S-adenosylmethionine (dc-SAM). MAP treatment was associated with mean maximal increases in the urinary excretion of dc-SAM of 2.6-, 9.3- and 17.9-fold after 375, 750 and 1500 mg/day for 4 weeks, respectively, but no consistent changes in the urinary excretion of the polyamines, putrescine, spermidine or spermine were observed. Thus, the 24-h urinary excretion of dc-SAM may be used as a conveniently accessible marker of ODC inhibition in cancer patients.

GJA12 mutations are a rare cause of Pelizaeus-Merzbacher-like disease.

BACKGROUND: Pelizaeus-Merzbacher-like disease (PMLD) is a genetically heterogeneous disorder within the group of hypomyelinating leukoencephalopathies. Mutations of the gap junction protein alpha 12 (GJA12) gene are known to cause one autosomal recessive PMLD form. Few patients with GJA12 mutated PMLD have been reported, and to date, the frequency as well as the genotypic and phenotypic spectrum of GJA12 related PMLD is unclear. METHODS: We report mutation analysis of the GJA12 gene in a clinical and radiologic well-characterized multiethnic cohort of 193 patients with PMLD from 182 families. RESULTS AND CONCLUSIONS: Only 16 patients (8.3%) from 14 families (7.7%) carry GJA12 mutations including five families where we detected only one mutated allele. Among those, we identified 11 novel alterations. Thus, GJA12 mutations are a rather rare cause for Pelizaeus-Merzbacher-like disease. The clinical phenotype of patients with a GJA12 mutation was evaluated and is overall comparable to the clinical features seen in mild forms of proteolipid protein 1 (PLP1) related disorder but with better cognition and earlier signs of axonal degeneration.

Rapidly progressive vanishing white matter disease in a child with previously inconspicuous brain MRI.

In this pediatric case of vanishing white matter disease with early onset, rapidly progressive course, and fatal outcome, the white matter vanishing process in patient was for the first time documented morphologically in detail: An initial magnetic resonance imaging documented a normal appearing brain maturation. Rapid progressive brain lesions initiated morphologically DE NOVO in the former well myelinated deep white matter were observed six months later after disease onset, including concentric ongoing signs of restricted proton diffusion cytotoxic edema on diffusion weighted imaging. Cyst-like defects at the lesion center of the deep white matter were detected more clearly on MRI ten months later. A pathomechanism like tumor necrosis factor induced oligodendrocyte apoptosis and primary demyelination was postulated. The case demonstrates that in the presence of clinically progressive symptoms, the development of VWM is possible even if first MRI findings are negative.

Maternal segmental disomy in Leigh syndrome with cytochrome c oxidase deficiency caused by homozygous SURF1 mutation.

Cytochrome c oxidase deficiency (COX) is the most frequent cause of Leigh syndrome (LS), a mitochondrial subacute necrotizing encephalomyelopathy. Most of these LS (COX-) patients show mutations in SURF1 on chromosome 9 (9q34), which encodes a protein essential for the assembly of the COX complex. We describe a family whose first-born boy developed characteristic features of LS. Severe COX deficiency in muscle was caused by a novel homozygous nonsense mutation in SURF1. Segregation analysis of this mutation in the family was incompatible with autosomal recessive inheritance but consistent with a maternal disomy. Haplotype analysis of microsatellite markers confirmed isodisomy involving nearly the complete long arm of chromosome 9 (9q21-9tel). No additional physical abnormalities were present in the boy, suggesting that there are no imprinted genes on the long arm of chromosome 9 which are crucial for developmental processes. This case of segmental isodisomy illustrates that genotyping of parents is crucial for correct genetic counseling.

Autoantibodies to human manganese superoxide dismutase (MnSOD) in children with facial palsy due to neuroborreliosis.

AIM: Acute peripheral facial palsy due to neuroborreliosis is associated with a distal neuritis. In patients with Lyme disease the activity of antioxidant enzymes is decreased. With respect to the pathogenesis of neuroborreliosis, sera of children with acute peripheral facial palsy were investigated for autoantibodies against human manganese superoxide dismutase (MnSOD), which were suspected of raising the oxidative injury of infected tissues. METHODS: Sera of 20 children with acute peripheral palsy with neuroborreliosis, sera of 20 children with facial palsy without reference to Lyme disease and sera of 14 blood donors were tested for antibodies against human MnSOD using an ELISA. RESULTS: The concentrations of IgM autoantibodies to MnSOD of the children with neuroborreliosis were significantly increased, compared with the two control groups. CONCLUSIONS: We propose that the antibodies detected block the protective effects of MnSOD resulting in an increased oxidative inflammation.

Characterization of the mitochondrial genome in childhood multiple sclerosis. II. Multiple sclerosis without optic neuritis and LHON-associated genes.

The occurrence of mitochondrial mutations with primary pathogenic significance for Leber's hereditary optic neuropathy in patients with a multiple sclerosis-like phenotype and the preferential maternal transmission points to an involvement of the mitochondrial genome in conferring increased susceptibility to MS. To evaluate the link between MS and mtDNA variations we investigated a total of thirteen children with MS as well as twenty controls by sequencing eight mitochondrial encoded genes which are known to be the loci for LHON-associated mutations. Numerous synonymous nucleotide substitutions and common polymorphisms were excluded from comparative analyses. No primary LHON mutations were found. Secondary LHON mutations were identified more frequently in control subjects than in the children with MS. The remaining eight discrete missense mutations were chosen for further characterization. Only two of them were found in more than one patient. Our results suggest that nucleotide substitutions within the ND1, ND2, ND4, ND5, ND6, COI, COIII or cytochrome b genes of mtDNA do not contribute to the etiology of typical MS. However, the association of LHON mutations with visual impairment in MS as well as the relationship between phenotypic diversity in certain subgroups of patients with individual mtDNA genotypes merits further investigations.

Characterization of the mitochondrial genome in childhood multiple sclerosis. III. Multiple sclerosis without optic neuritis and the non-LHON-associated genes.

The higher maternal transmission of multiple sclerosis in familial cases and the coincidence of a MS-like phenotype with mitochondrial point mutations in patients with Leber's hereditary optic neuropathy has inspired a detailed assessment of the mitochondrial genome as an etiological factor in the pathogenesis of MS. To further elucidate the contribution of maternally transmitted mutations to MS susceptibility, we sequenced five protein- and all RNA-coding genes of the mtDNA from thirteen children with MS and twenty unaffected individuals. After excluding several synonymous mutations and common polymorphisms, a total of ten ambiguous missense or protein synthesis mutations were selected and analysed. By defining minimal criteria for pathogenity--incidence, location and degree of evolutionary conservation--we conclude that sequence variations in COII, ATPase6 and 8, ND3, or ND4L subunits of oxidative phosphorylation as well as in rRNA and tRNA genes are unlikely to increase susceptibility for the development of MS.

Characterization of the mitochondrial genome in childhood multiple sclerosis. I. Optic neuritis and LHON mutations.

The occurrence of optic neuropathy in patients with MS-like disorders who carry one of the pathogenetically significant LHON mutations as well as the higher incidence of maternal transmission in familial cases of MS support the hypothesis that mitochondrial genes may be implicated in susceptibility to MS. We sequenced the entire mtDNA of six children with MS who developed optic neuritis as early and prominent visual involvement. The analysis revealed a high degree of nucleotide variations relative to the standard mtDNA sequence. After excluding various synonymous nucleotide changes and common neutral polymorphisms, eight discrete novel missense mutations within the protein coding, tRNA or rRNA genes were detected. None of the eight polymorphic sites were found in common between the patients with MS. Of particular interest was the observation that five of six children carried a total of nine secondary LHON mutations at nucleotide positions 4216, 4917 or 13708. We conclude that variation in mtDNA is unlikely to contribute to genetic predisposition for MS. However, secondary LHON mutations may be regarded as additional risk factor for developing prominent optic nerve involvement. The association of individual sets of mtDNA variations with phenotypic presentation in certain subgroups of MS patients remains to be clarified.

Amplifying role of edible dormice in Lyme disease transmission in central Europe.

To determine whether the presence of edible dormice (Glis glis) amplifies the risk of human infection by the Lyme disease spirochete (Borrelia burgdorferi), the capacity of dormice as reservoir hosts was compared with that of other potential reservoirs in a central European site where risk of human infection is intense. Dormice appeared to be more heavily infested by subadult vector wood ticks (Ixodes ricinus) than were other reservoir hosts. Although their spirochete competence was similar to other reservoir hosts, field-derived dormice infected more ticks than did other rodents in the site, because nymphal ticks most readily feed on them. Spirochetes isolated from dormice appeared identical to those implicated as agents of Lyme disease. Subadult wood ticks become replete and detach from dormice during late afternoon when dormice are at rest in their nests. Thus, the presence of edible dormice in Central Europe amplifies transmission of the agent of Lyme disease and intensifies the risk of human infection.

Evaluation of the polymerase chain reaction for the detection of Borrelia burgdorferi in cerebrospinal fluid of children with acute peripheral facial palsy.

UNLABELLED: Neuroborreliosis occasionally represents a diagnostic problem, especially in the early stage of the infection. The polymerase chain reaction (PCR) offers an attractive alternative to antibody testing. The aim of our study was to investigate the diagnostic potential of PCR in comparison to antibody tests in CSF of children with facial palsy. In contrast to other manifestations of neuroborreliosis, facial palsy is a well-defined clinical entity in which CSF findings allow an early distinction according to aetiology. The study included 17 children with neuroborreliosis, defined by the detection of specific IgM antibodies in CSF, and 20 children with facial palsy of unknown cause. Primers used for the nested PCR were generated from conserved sequences of the OspA-gene. Most of the cases in both subgroups have been examined within a few days after the onset of the paresis. Only in 2 out of 17 cases with neuroborreliosis could specific DNA be amplified. The PCR gave negative results in all cases of the control group. CONCLUSION: The IgM capture ELISA is superior to PCR to support the clinical diagnosis of neuroborreliosis.

Transmission of the agent of Lyme disease on a subtropical island.

To determine whether the agent of Lyme disease (Borrelia burgdorferi) perpetuates on a subtropical island, we recorded prevalence of spirochetal infection in Ixodes ricinus ticks collected on Madeira Island and identified local reservoir hosts of both pathogen and vector tick. Spirochetes infect at least 1.3% of the nymphal ticks collected in a particular site. Subadult ticks frequently parasitized Norway as well as black rats on the island, and Lyme disease spirochetes were found in these hosts. Each was competent as reservoir host. Canaries, lizards and domestic ungulates, on the other hand, were incompetent for Madeiran spirochetal isolates. Madeiran spirochetes stimulated rodents to recognize the same antigens as did spirochetes isolated on the European continent. The polymerase chain reaction amplified identical specific sequences of Madeiran spirochetes as of European-derived spirochetes. Rats appear to be the main reservoir hosts of the agent of Lyme disease on Madeira Island, and cattle and sheep serve as definitive hosts of the vector tick. The agent of Lyme disease is enzootic on this subtropical island some 10 degrees of latitude south of the northern Mediterranean coast. Because I. ricinus ticks frequently attack people on Madeira Island, Lyme disease should be considered as a cause of locally acquired human illness.

Nucleotide sequence of the ospAB operon of a Borrelia burgdorferi strain expressing OspA but not OspB.

The nucleotide sequence of a 1.6-kb clone containing the gene for outer surface protein A (OspA) of a German strain (GO2) of Borrelia burgdorferi was determined. The deduced amino acid sequence showed a homology of 82% to the OspA molecules from three other B. burgdorferi strains. The best-conserved region was recognized at the 36-amino-terminal amino acids of OspA. OspB could not be identified in the strain investigated, probably because the nucleotide sequence of the ospAB operon prevented expression of the OspB gene.

Nondifferentiation between Lyme disease spirochetes from vector ticks and human cerebrospinal fluid.

To determine whether Lyme disease neuropathogenesis may result from infection by a particular segment of the locally extant population of spirochetes, genetic markers of spirochetes found in cerebrospinal fluid (CSF) of 12 pediatric patients were compared with those in spirochetes from 40 vector ticks sampled in the vicinity of their homes. The primary structure of the outer surface protein A served as the marker of variation; a fragment of the corresponding gene was amplified by nested polymerase chain reaction and the products sequenced. Tick-derived variants clustered in seven distinct categories, of which four were present in CSF. One of the CSF variants differed from any found in ticks. Coinfection by different spirochete variants was infrequent in ticks and absent in human samples. Spirochetal neuropathology in children in our study site does not correlate with a particular segment of the tickborne pathogens present in nature.

Point mutations and nucleotide insertions in the MDM2 zinc finger structure of human tumours.

This study investigates the human oncoprotein MDM2, which interferes with regulation of cell division and apoptosis. Fifteen mixed-type follicular non-Hodgkin's lymphomas, ten leukaemias, two hepatocellular carcinomas, one osteosarcoma, and ten normal cell lines (fibroblasts, osteoblasts, mesothelium, peripheral lymphocytes) were tested for MDM2 expression and MDM2 gene mutation by reverse transcriptase-polymerase chain reaction (RT-PCR), immunocytochemistry, and nucleotide sequence analysis. Two follicular lymphomas, three leukaemias, both hepatocellular carcinomas, and the osteosarcoma sample showed transcription of the activated MDM2 gene. These samples lacked amplified MDM2 genes and carried mis-sense, non-sense and frame-shift mutations in a zinc finger region of MDM2, altering the amino acid sequence or causing premature termination of transcription. The mis-sense mutations were found in tumour cells that showed significant accumulation of MDM2 and lack of nuclear p53. Non-sense mutations and frame-shift mutations were found in tumours lacking MDM2 proteins. The mutations may affect the biological properties of MDM2 proteins.

Risk of urban Lyme disease enhanced by the presence of rats.

To determine whether Norway rats contribute to the risk of human Lyme disease in a central European city park, densities of endemic rodents were compared as were feeding densities of vector ticks and prevalence of infection by the Lyme disease spirochete. Only Norway rats and yellow-necked mice were abundant, and three times as many mice as rats were present. More larval ticks fed on rats than on mice, and far more nymphs engorged on the rats. All rats but only about half of the mice infected ticks. Each rat was more infectious than each infectious mouse. Infected rats were distributed throughout the city. Spirochetes infected about a quarter of the questing nymphal ticks. The capacity of rats to serve as reservoir hosts for the Lyme disease spirochete, therefore, increases risk of infection among visitors to this and other urban parks.