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.

A lethal defect of mitochondrial and peroxisomal fission.

We report on a newborn girl with microcephaly, abnormal brain development, optic atrophy and hypoplasia, persistent lactic acidemia, and a mildly elevated plasma concentration of very-long-chain fatty acids. We found a defect of the fission of both mitochondria and peroxisomes, as well as a heterozygous, dominant-negative mutation in the dynamin-like protein 1 gene (DLP1). The DLP1 protein has previously been implicated, in vitro, in the fission of both these organelles. Overexpression of the mutant DLP1 in control cells reproduced the fission defect. Our findings are representative of a class of disease characterized by defects in both mitochondria and peroxisomes.

Plasmalogens participate in very-long-chain fatty acid-induced pathology.

Peroxisomes are organelles responsible for multiple metabolic pathways including, the biosynthesis of plasmalogens, a class of phospholipids, and the beta-oxidation of very-long-chain fatty acids (VLCFA). Lack of peroxisomes or dysfunction in any of their normal functions is the cellular basis for human peroxisomal disorders. Here we used mouse models to understand and define the biochemical and cellular determinants that mediate the pathophysiological consequences caused by peroxisomal dysfunctions. We investigated the role and effects of cellular plasmalogens and VLCFA accumulation in liver, testis and nervous tissue using Pex7 and Abcd1 knockout (KO) mice. In addition, we also generated a Pex7:Abcd1 double KO mouse to investigate how different peroxisomal dysfunctions modulate cellular function and pathology. We found that plasmalogens function as fundamental structural phospholipids and protect cells from damage caused by VLCFA accumulation. In testis, plasmalogens protect spermatocytes from VLCFA-induced degeneration and apoptosis. In nervous tissue, we found that gliosis, inflammatory demyelination and axonopathy caused by accumulation of VLCFA are modulated by plasmalogens. Our findings demonstrate the importance of normal peroxisomal functioning and allow the understanding of the pathological causality of peroxisomal dysfunctions. Nervous tissue deficient in plasmalogens is more prone to damage, illustrating the importance of plasmalogens in peroxisomal disorders including Zellweger syndrome and X-linked adrenoleukodystrophy.

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.

Specific combination of compound heterozygous mutations in 17ß-hydroxysteroid dehydrogenase type 4 (HSD17B4) defines a new subtype of D-bifunctional protein deficiency.

BACKGROUND: D-bifunctional protein (DBP) deficiency is typically apparent within the first month of life with most infants demonstrating hypotonia, psychomotor delay and seizures. Few children survive beyond two years of age. Among patients with prolonged survival all demonstrate severe gross motor delay, absent language development, and severe hearing and visual impairment. DBP contains three catalytically active domains; an N-terminal dehydrogenase, a central hydratase and a C-terminal sterol carrier protein-2-like domain. Three subtypes of the disease are identified based upon the domain affected; DBP type I results from a combined deficiency of dehydrogenase and hydratase activity; DBP type II from isolated hydratase deficiency and DBP type III from isolated dehydrogenase deficiency. Here we report two brothers (16½ and 14 years old) with DBP deficiency characterized by normal early childhood followed by sensorineural hearing loss, progressive cerebellar and sensory ataxia and subclinical retinitis pigmentosa. METHODS AND RESULTS: Biochemical analysis revealed normal levels of plasma VLCFA, phytanic acid and pristanic acid, and normal bile acids in urine; based on these results no diagnosis was made. Exome analysis was performed using the Agilent SureSelect 50Mb All Exon Kit and the Illumina HiSeq 2000 next-generation-sequencing (NGS) platform. Compound heterozygous mutations were identified by exome sequencing and confirmed by Sanger sequencing within the dehydrogenase domain (c.101C>T; p.Ala34Val) and hydratase domain (c.1547T>C; p.Ile516Thr) of the 17ß-hydroxysteroid dehydrogenase type 4 gene (HSD17B4). These mutations have been previously reported in patients with severe-forms of DBP deficiency, however each mutation was reported in combination with another mutation affecting the same domain. Subsequent studies in fibroblasts revealed normal VLCFA levels, normal C26:0 but reduced pristanic acid beta-oxidation activity. Both DBP hydratase and dehydrogenase activity were markedly decreased but detectable. CONCLUSIONS: We propose that the DBP phenotype seen in this family represents a distinct and novel subtype of DBP deficiency, which we have termed type IV based on the presence of a missense mutation in each of the domains of DBP resulting in markedly reduced but detectable hydratase and dehydrogenase activity of DBP. Given that the biochemical testing in plasma was normal in these patients, this is likely an underdiagnosed form of DBP deficiency.

A key role for the peroxisomal ABCD2 transporter in fatty acid homeostasis.

Peroxisomes are essential organelles exerting key functions in fatty acid metabolism such as the degradation of very long-chain fatty acids (VLCFAs). VLCFAs accumulate in X-adrenoleukodystrophy (X-ALD), a disease caused by deficiency of the Abcd1 peroxisomal transporter. Its closest homologue, Abcd2, exhibits a high degree of functional redundancy on the catabolism of VLCFA, being able to prevent X-ALD-related neurodegeneration in the mouse. In the search for specific roles of Abcd2, we screened fatty acid profiles in organs and primary neurons of mutant knockout mice lacking Abcd2 in basal conditions and under dietary challenges. Our results indicate that ABCD2 plays a role in the degradation of long-chain saturated and omega9-monounsaturated fatty acids and in the synthesis of docosahexanoic acid (DHA). Also, we demonstrated a defective VLCFA beta-oxidation ex vivo in brain slices of Abcd1 and Abcd2 knockouts, using radiolabeled hexacosanoic acid and the precursor of DHA as substrates. As DHA levels are inversely correlated with the incidence of Alzheimer's and several degenerative conditions, we suggest that ABCD2 may act as modulator/modifier gene and therapeutic target in rare and common human disorders.

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.

Impaired neuronal migration and endochondral ossification in Pex7 knockout mice: a model for rhizomelic chondrodysplasia punctata.

Rhizomelic chondrodysplasia punctata is a human autosomal recessive disorder characterized by skeletal, eye and brain abnormalities. The disorder is caused by mutations in the PEX7 gene, which encodes the receptor for a class of peroxisomal matrix enzymes. We describe the generation and characterization of a Pex7 mouse knockout (Pex7(-/-)). Pex7(-/-) mice are born severely hypotonic and have a growth impairment. Mortality in Pex7(-/-) mice is highest in the perinatal period although some Pex7(-/-) mice survived beyond 18 months. Biochemically Pex7(-/-) mice display the abnormalities related to a Pex7 deficiency, i.e. a severe depletion of plasmalogens, impaired alpha-oxidation of phytanic acid and impaired beta-oxidation of very-long-chain fatty acids. In the intermediate zone of the developing cerebral cortex Pex7(-/-) mice have an increase in neuronal density. In vivo neuronal birthdating revealed that Pex7(-/-) mice have a delay in neuronal migration. Analysis of bone ossification in newborn Pex7(-/-) mice revealed a defect in ossification of distal bone elements of the limbs as well as parts of the skull and vertebrae. These findings demonstrate that Pex7 knockout mice provide an important model to study the role of peroxisomal functioning in the pathogenesis of the human disorder.