Biochemical Studies in Fibroblasts to Interpret Variants of Unknown Significance in the ABCD1 Gene.

Due to newborn screening for X-linked adrenoleukodystrophy (ALD), and the use of exome sequencing in clinical practice, the detection of variants of unknown significance (VUS) in the ABCD1 gene is increasing. In these cases, functional tests in fibroblasts may help to classify a variant as (likely) benign or pathogenic. We sought to establish reference ranges for these tests in ALD patients and control subjects with the aim of helping to determine the pathogenicity of VUS in ABCD1. Fibroblasts from 36 male patients with confirmed ALD, 26 healthy control subjects and 17 individuals without a family history of ALD, all with an uncertain clinical diagnosis and a VUS identified in ABCD1, were included. We performed a combination of tests: (i) a test for very-long-chain fatty acids (VLCFA) levels, (ii) a D3-C22:0 loading test to study the VLCFA metabolism and (iii) immunoblotting for ALD protein. All ALD patient fibroblasts had elevated VLCFA levels and a reduced peroxisomal ß-oxidation capacity (as measured by the D3-C16:0/D3-C22:0 ratio in the D3-C22:0 loading test) compared to the control subjects. Of the VUS cases, the VLCFA metabolism was not significantly impaired (most test results were within the reference range) in 6/17, the VLCFA metabolism was significantly impaired (most test results were within/near the ALD range) in 9/17 and a definite conclusion could not be drawn in 2/17 of the cases. Biochemical studies in fibroblasts provided clearly defined reference and disease ranges for the VLCFA metabolism. In 15/17 (88%) VUS we were able to classify the variant as being likely benign or pathogenic. This is of great clinical importance as new variants will be detected.

Identification of an unusual variant peroxisome biogenesis disorder caused by mutations in the PEX16 gene.

BACKGROUND: Zellweger syndrome spectrum disorders are caused by mutations in any of at least 12 different PEX genes. This includes PEX16, which encodes an integral peroxisomal membrane protein involved in peroxisomal membrane assembly. PEX16-defective patients have been reported to have a severe clinical presentation. Fibroblasts from these patients displayed a defect in the import of peroxisomal matrix and membrane proteins, resulting in a total absence of peroxisomal remnants. OBJECTIVE: To report on six patients with an unexpected mild variant peroxisome biogenesis disorder due to mutations in the PEX16 gene. Patients presented in the preschool years with progressive spastic paraparesis and ataxia (with a characteristic pattern of progressive leucodystrophy and brain atrophy on MRI scan) and later developed cataracts and peripheral neuropathy. Surprisingly, their fibroblasts showed enlarged, import-competent peroxisomes. RESULTS: Plasma analysis revealed biochemical abnormalities suggesting a peroxisomal disorder. Biochemical variables in fibroblasts were only mildly abnormal or within the normal range. Immunofluorescence microscopy revealed the presence of import-competent peroxisomes, which were increased in size but reduced in number. Subsequent sequencing of all known PEX genes revealed five novel apparent homozygous mutations in the PEX16 gene. CONCLUSIONS: An unusual variant peroxisome biogenesis disorder caused by mutations in the PEX16 gene, with a relatively mild clinical phenotype and an unexpected phenotype in fibroblasts, was identified. Although PEX16 is involved in peroxisomal membrane assembly, PEX16 defects can present with enlarged import-competent peroxisomes in fibroblasts. This is important for future diagnostics of patients with a peroxisomal disorder.

Enzymatic diagnosis of Sjögren-Larsson syndrome using electrospray ionization mass spectrometry.

BACKGROUND: Sjögren-Larsson syndrome is a metabolic disorder characterized by accumulation of long-chain fatty alcohols in plasma of patients due to mutations in the ALDH3A2 gene, that codes for a microsomal fatty aldehyde dehydrogenase (FALDH). Recent studies have demonstrated that FALDH is involved in the last step of the conversion of 22-hydroxy-C22:0 into the dicarboxylic acid of C22:0 (C22:0-DCA). METHODS: FALDH activity was determined by incubating fibroblast homogenates with omega-hydroxy-C22:0 in the presence of NAD(+). Electrospray ionization mass spectrometry (ESI-MS) was used to quantify the amounts of C22:0-DCA produced. RESULTS: All SLS patients were deficient in C22:0-DCA productions with activities ranging from 3.2-26.3% of mean control. CONCLUSIONS: The new assay described in this paper has substantial advantages over previous assays, and allows for the easy, reliable and rapid diagnosis of SLS.

Genotype-phenotype correlation in PEX5-deficient peroxisome biogenesis defective cell lines.

Proteins destined for the peroxisomal matrix are targeted by virtue of a peroxisomal targeting sequence type 1 (PTS1) or type 2 (PTS2). In humans, targeting of either class of proteins relies on a cytosolic receptor protein encoded by the PEX5 gene. Alternative splicing of PEX5 results in two protein variants, PEX5S and PEX5L. PEX5S is exclusively involved in PTS1 protein import, whereas PEX5L mediates the import of both PTS1 and PTS2 proteins. Genetic complementation testing with over 500 different fibroblast cell lines from patients diagnosed with a peroxisome biogenesis disorder (PBD) identified 11 cell lines with a defect in PEX5. The aim of this study was to characterize these cell lines at a biochemical and genetic level. To this end, the cultured fibroblasts were analyzed for very long chain fatty acid (VLCFA) concentrations, peroxisomal beta-and alpha-oxidation, dihydroxyacetone-phosphate acyltransferase (DHAPAT) activity, peroxisomal thiolase, and catalase immunofluorescence. Mutation analysis of the PEX5 gene revealed 11 different mutations, eight of which are novel. PTS1- and PTS2-protein import capacity was assessed by transfection of the cells with green fluorescent protein (GFP) tagged with either PTS1 or PTS2. Six cell lines showed a defect in both PTS1 and PTS2 protein import, whereas four cell lines only showed a defect in PTS1 protein import. The location of the different mutations within the PEX5 amino acid sequence correlates rather well with the peroxisomal protein import defect observed in the cell lines.

Clinical and biochemical spectrum of D-bifunctional protein deficiency.

OBJECTIVE: D-bifunctional protein deficiency is an autosomal recessive inborn error of peroxisomal fatty acid oxidation. Although case reports and small series of patients have been published, these do not give a complete and balanced picture of the clinical and biochemical spectrum associated with this disorder. METHODS: To improve early recognition, diagnosis, prognosis, and management of this disorder and to provide markers for life expectancy, we performed extensive biochemical studies in a large cohort of D-bifunctional protein-deficient patients and sent out questionnaires about clinical signs and symptoms to the responsible physicians. RESULTS: Virtually all children presented with neonatal hypotonia and seizures and died within the first 2 years of life without achieving any developmental milestones. However, within our cohort, 12 patients survived beyond the age of 2 years, and detailed information on 5 patients with prolonged survival (> or =7.5 years) is provided. INTERPRETATION: Biochemical analyses showed that there is a clear correlation between several biochemical parameters and survival of the patient, with C26:0 beta-oxidation activity in cultured skin fibroblasts being the best predictive marker for life expectancy. Remarkably, three patients were identified without biochemical abnormalities in plasma, stressing that D-bifunctional protein deficiency cannot be excluded when all peroxisomal parameters in plasma are normal.

Identification of the molecular defect in patients with peroxisomal mosaicism using a novel method involving culturing of cells at 40 degrees C: implications for other inborn errors of metabolism.

The peroxisome biogenesis disorders (PBDs), which comprise Zellweger syndrome (ZS), neonatal adrenoleukodystrophy, and infantile Refsum disease (IRD), represent a spectrum of disease severity, with ZS being the most severe, and IRD the least severe disorder. The PBDs are caused by mutations in one of the at least 12 different PEX genes encoding proteins involved in the biogenesis of peroxisomes. We report the biochemical characteristics and molecular basis of a subset of atypical PBD patients. These patients were characterized by abnormal peroxisomal plasma metabolites, but otherwise normal to very mildly abnormal peroxisomal parameters in cultured skin fibroblasts, including a mosaic catalase immunofluorescence pattern in fibroblasts. Since this latter feature made standard complementation analysis impossible, we developed a novel complementation technique in which fibroblasts were cultured at 40 degrees C, which exacerbates the defect in peroxisome biogenesis. Using this method, we were able to assign eight patients to complementation group 3 (CG3), followed by the identification of a single homozygous c.959C>T (p.S320F) mutation in their PEX12 gene. We also investigated various peroxisomal biochemical parameters in fibroblasts at 30 degrees C, 37 degrees C, and 40 degrees C, and found that all parameters showed a temperature-dependent behavior. The principle of culturing cells at elevated temperatures to exacerbate the defect in peroxisome biogenesis, and thereby preventing certain mutations from being missed, may well have a much wider applicability for a range of different inborn errors of metabolism.