Mutations in the gene encoding peroxisomal sterol carrier protein X (SCPx) cause leukencephalopathy with dystonia and motor neuropathy.

In this report, we describe the first known patient with a deficiency of sterol carrier protein X (SCPx), a peroxisomal enzyme with thiolase activity, which is required for the breakdown of branched-chain fatty acids. The patient presented with torticollis and dystonic head tremor as well as slight cerebellar signs with intention tremor, nystagmus, hyposmia, and azoospermia. Magnetic resonance imaging showed leukencephalopathy and involvement of the thalamus and pons. Metabolite analyses of plasma revealed an accumulation of the branched-chain fatty acid pristanic acid, and abnormal bile alcohol glucuronides were excreted in urine. In cultured skin fibroblasts, the thiolytic activity of SCPx was deficient, and no SCPx protein could be detected by western blotting. Mutation analysis revealed a homozygous 1-nucleotide insertion, 545_546insA, leading to a frameshift and premature stop codon (I184fsX7).

Linoleic acid supplementation of Barth syndrome fibroblasts restores cardiolipin levels: implications for treatment.

The object of this study was to investigate whether the levels of cardiolipin in cultured skin fibroblasts of patients with Barth syndrome (BTHS) can be restored by addition of linoleic acid to growth media. To this end, fibroblasts from controls and BTHS patients were grown in the presence or absence of linoleic acid. High-performance liquid chromatography-electrospray ionization tandem mass spectrometry was used for quantitative and compositional analysis of cardiolipin. Incubation of cells from both BTHS and controls with different concentrations of linoleic acid led to a dose- and time-dependent increase of cardiolipin levels. The increased levels of cardiolipin in fibroblasts of BTHS patients after treatment with linoleic acid indicate that an increased amount of linoleic acid in the diet might be beneficial to BTHS patients.

An efficient cDNA-AFLP-based strategy for the identification of putative pathogenicity factors from the potato cyst nematode Globodera rostochiensis.

A new strategy has been designed to identify putative pathogenicity factors from the dorsal or subventral esophageal glands of the potato cyst nematode Globodera rostochiensis. Three independent criteria were used for selection. First, genes of interest should predominantly be expressed in infective second-stage juveniles, and not, or to a far lesser extent, in younger developmental stages. For this, gene expression profiles from five different developmental stages were generated with cDNA-AFLP (amplified fragment length polymorphism). Secondly, the mRNA corresponding to such a putative pathogenicity factor should predominantly be present in the esophageal glands of pre-parasitic juveniles. This was checked by in situ hybridization. As a third criterion, these proteinaceous factors should be preceded by a signal peptide for secretion. Expression profiles of more than 4,000 genes were generated and three up-regulated, dorsal gland-specific proteins preceded by signal peptide for secretion were identified. No dorsal gland genes have been cloned before from plant-parasitic nematodes. The partial sequence of these three factors, A4, A18, and A41, showed no significant homology to any known gene. Their presence in the dorsal glands of infective juveniles suggests that these proteins could be involved in feeding cell initiation, and not in migration in the plant root or in protection against plant defense responses. Finally, the applicability of this new strategy in other plant-microbe interactions is discussed.

Linkage analysis by genotyping of sibling populations: a genetic map for the potato cyst nematode constructed using a "pseudo-F2" mapping strategy.

A mapping strategy is described for the construction of a linkage map of a non-inbred species in which individual offspring genotypes are not amenable to marker analysis. After one extra generation of random mating, the segregating progeny was propagated, and bulked populations of offspring were analyzed. Although the resulting population structure is different from that of commonly used mapping populations, we show that the maximum likelihood formula for a normal F2 is applicable for the estimation of recombination. This "pseudo-F2" mapping strategy, in combination with the development of an AFLP assay for single cysts, facilitated the construction of a linkage map for the potato cyst nematode Globodera rostochiensis. Using 12 pre-selected AFLP primer combinations, a total of 66 segregating markers were identified, 62 of which were mapped to nine linkage groups. These 62 AFLP markers are randomly distributed and cover about 65% of the genome. An estimate of the physical size of the Globodera genome was obtained from comparisons of the number of AFLP fragments obtained with the values for Caenorhabditis elegans. The methodology presented here resulted in the first genomic map for a cyst nematode. The low value of the kilobase/centimorgan (kb/cM) ratio for the Globodera genome will facilitate map-based cloning of genes that mediate the interaction between the nematode and its host plant.

Secretory granule proteins from the subventral esophageal glands of the potato cyst nematode identified by monoclonal antibodies to a protein fraction from second-stage juveniles.

Sodium dodecyl sulfate-extracted proteins from second-stage juveniles (J2) of the potato cyst nematode Globodera rostochiensis were fractionated by preparative continuous flow electrophoresis, and monoclonal antibodies (MAbs) were raised against the 38- to 40.5-kDa protein fraction. Screening of the hybridoma culture fluids by immunofluorescence microscopy of J2 resulted in the identification of 12 MAbs that bound specifically to the subventral esophageal glands. On Western blots of J2 these MAbs identified four protein bands with apparent molecular masses of 30, 31, 39, and 49 kDa. Immunoelectron microscopy with one of these MAbs showed an intense labeling of the electron dense core of the secretory granules in the subventral gland cells of J2. It is concluded that one or more of these proteins are localized within these secretory granules. Immunofluorescence microscopy of J2 from other plant parasitic nematode species showed that most of these MAbs also bind to the subventral glands of G. pallida and G. tabacum but not of Heterodera schachtii, H. glycines, Meloidogyne incognita, or M. hapla.