A founder mutation in the PEX6 gene is responsible for increased incidence of Zellweger syndrome in a French Canadian population.

BACKGROUND: Zellweger syndrome (ZS) is a peroxisome biogenesis disorder due to mutations in any one of 13 PEX genes. Increased incidence of ZS has been suspected in French-Canadians of the Saguenay-Lac-St-Jean region (SLSJ) of Quebec, but this remains unsolved. METHODS: We identified 5 ZS patients from SLSJ diagnosed by peroxisome dysfunction between 1990-2010 and sequenced all coding exons of known PEX genes in one patient using Next Generation Sequencing (NGS) for diagnostic confirmation. RESULTS: A homozygous mutation (c.802_815del, p.[Val207_Gln294del, Val76_Gln294del]) in PEX6 was identified and then shown in 4 other patients. Parental heterozygosity was confirmed in all. Incidence of ZS was estimated to 1 in 12,191 live births, with a carrier frequency of 1 in 55. In addition, we present data suggesting that this mutation abolishes a SF2/ASF splice enhancer binding site, resulting in the use of two alternative cryptic donor splice sites and predicted to encode an internally deleted in-frame protein. CONCLUSION: We report increased incidence of ZS in French-Canadians of SLSJ caused by a PEX6 founder mutation. To our knowledge, this is the highest reported incidence of ZS worldwide. These findings have implications for carrier screening and support the utility of NGS for molecular confirmation of peroxisomal disorders.

Bile acid analysis in human disorders of bile acid biosynthesis.

Bile acids facilitate the absorption of lipids in the gut, but are also needed to maintain cholesterol homeostasis, induce bile flow, excrete toxic substances and regulate energy metabolism by acting as signaling molecules. Bile acid biosynthesis is a complex process distributed across many cellular organelles and requires at least 17 enzymes in addition to different metabolite transport proteins to synthesize the two primary bile acids, cholic acid and chenodeoxycholic acid. Disorders of bile acid synthesis can present from the neonatal period to adulthood and have very diverse clinical symptoms ranging from cholestatic liver disease to neuropsychiatric symptoms and spastic paraplegias. This review describes the different bile acid synthesis pathways followed by a summary of the current knowledge on hereditary disorders of human bile acid biosynthesis with a special focus on diagnostic bile acid profiling using mass spectrometry.

2-Hydroxyphytanic acid oxidase activity in rat and human liver and its deficiency in the Zellweger syndrome.

Phytanic acid is a saturated, branched-chain fatty acid which as a consequence of the presence of a methyl group at the 3-position cannot be degraded by beta-oxidation. Instead, phytanic acid first undergoes alpha-oxidation to yield pristanic acid which can be degraded by beta-oxidation. The structure of the alpha-oxidation pathway and its subcellular localization has remained an enigma although there is convincing evidence that 2-hydroxyphytanic acid is an obligatory intermediate. We have now studied the degradation of 2-hydroxyphytanic acid in both rat and human liver. The results show that 2-hydroxyphytanic acid is converted to 2-ketophytanic acid in homogenates of rat as well as human liver. Detailed studies in rat liver showed that the enzyme involved is localized in peroxisomes accepting molecular oxygen as second substrate and producing H2O2. 2-Ketophytanic acid formation from 2-hydroxyphytanic acid was found to be strongly deficient in liver samples from Zellweger patients which lack morphologically distinguishable peroxisomes. The latter results not only provide an explanation for the elevated levels of 2-hydroxyphytanic acid in Zellweger patients but also suggest that the subcellular localization of 2-hydroxyphytanic acid dehydrogenation is identical in rat and man, i.e., in peroxisomes.

Topology of catalase assembly in human skin fibroblasts.

The biogenesis, assembly and import of the peroxisomal enzyme catalase was studied in human skin fibroblasts from control persons and from patients with the Zellweger syndrome. For this purpose, two monoclonal antibodies were generated which are able to discriminate between the monomeric or dimeric form and the tetrameric, enzymically active conformation of the enzyme. Metabolic labelling studies showed that catalase is assembled to the tetrameric conformation within one hour after its synthesis, while it is still in the cytosol of the cell. Subsequently, the enzyme becomes particle-bound in the control cells, a process that is retarded by addition of the catalase inhibitor 3-amino-1,2,4-triazole. However, the tetramer remains in the cytosol in cells from Zellweger patients. It is concluded that newly synthesized catalase can be assembled to a tetramer in the cytosol in human skin fibroblasts. Unfolding of this tetramer prior to import into peroxisomes is indicated.

D-aspartate oxidase, a peroxisomal enzyme in liver of rat and man.

By means of subcellular fractionation D-aspartate oxidase was shown to be localized in peroxisomes in rat and human liver. The oxidase from both sources was most active on D-aspartate and N-methyl-D-aspartate. In different rat tissues, the highest enzyme activity was found in kidney, followed by liver and brain. In these tissues, oxidase activities became detectable 1-4 days after birth, reaching adult values after 4 weeks. Analysis of liver samples from patients with Zellweger syndrome, a generalized peroxisomal dysfunction, demonstrated no significant deficiency of this particular oxidase.

The Zellweger syndrome: deficient conversion of docosahexaenoic acid (22:6(n-3)) to eicosapentaenoic acid (20:5(n-3)) and normal delta 4-desaturase activity in cultured skin fibroblasts.

The metabolism of docosahexaenoic acid (22:6(n-3)) and adrenic acid (22:4(n-6)) was studied in cultured fibroblasts from patients with the Zellweger syndrome, X-linked adrenoleukodystrophy (X-ALD) and normal controls. It was shown that [4,5- 3H]22:6(n-3) is retroconverted to labelled eicosapentaenoic acid (20:5(n-3)) in normal and X-ALD fibroblasts, while this conversion is deficient in Zellweger fibroblasts. [U- 14C]Eicosapentaenoic acid (20:5(n-3)) is elongated to docosapentaenoic acid (22:5(n-3)) in all three cell lines. With [U- 14C]20:5(n-3) as the substrate, shorter fatty acids were not detected. With [4,5- 3H]22:6(n-3) as the substrate, labelled fatty acids were esterified in the phospholipid- and triacylglycerol-fraction to approximately the same extent in all three cell lines. [2- 14C]Adrenic acid (22:4(n-6)) was desaturated to 22:5(n-6) and elongated to 24:4(n-6) in all three cell lines and to the largest extent in the Zellweger fibroblasts. This agrees with the view that the delta 4-desaturase is not a peroxisomal enzyme. The observation that the retroconversion of 22:6(n-3) to 20:5(n-3) is deficient in Zellweger fibroblasts strongly suggest that the beta-oxidation step in the retroconversion is a peroxisomal function. Peroxisomal very-long-chain (lignoceroyl) CoA ligase is probably not required for the activation of 22:6(n-3), since the retroconversion to 20:5(n-3) is normal in X-ALD fibroblasts.

Purification of peroxisomes and subcellular distribution of enzyme activities for activation and oxidation of very-long-chain fatty acids in rat brain.

Brain contains high amounts of very-long-chain (VLC) fatty acids (> C22). Since mitochondria from liver and skin fibroblasts lack lignoceroyl-CoA ligase, in liver and skin fibroblasts fatty acids are exclusively oxidized in peroxisomes. Findings by Poulos and associates [9] suggested that contrary to liver and cultured skin fibroblasts brain mitochondria contain lignoceroyl-CoA ligase and can oxidize lignoceric acid. The present study was undertaken to develop a procedure for the isolation of subcellular organelles of higher purity from brain and to get a better understanding of the subcellular localization of the oxidation of VLC fatty acids in brain. The enzyme activities for activation and oxidation of palmitic and lignoceric acids were determined in peroxisomes, mitochondria, microsomes and a myelin fraction from rat brain and peroxisomes, mitochondria and microsomes purified from rat liver. Like in liver, brain lignoceroyl-CoA ligase activity in microsomes and peroxisomes was approx. 9 times higher than in mitochondria. In addition to palmitoyl-CoA ligase the antibodies against palmitoyl-CoA ligase inhibited the residual mitochondrial lignoceroyl-CoA ligase activity, meaning that lignoceroyl-CoA ligase activity in mitochondria was derived from palmitoyl-CoA ligase. Accordingly, in peroxisomes lignoceric acid was oxidized at 7 times higher rate than in mitochondria. Mitochondria were able to oxidize lignoceric acid efficiently when supplemented with lignoceroyl-CoA ligase activity from microsomes or myelin. These results show that in brain lignoceric acid is oxidized in peroxisomes and that lignoceroyl-CoA ligase activity is localized in peroxisomes and microsomes, but not in mitochondria. Peroxisomes and microsomes contain both lignoceroyl-CoA and palmitoyl-CoA ligases. Similar to peroxisomes and microsomes, the antibodies against palmitoyl-CoA ligase inhibited only the palmitoyl-CoA ligase activity in myelin but not the lignoceroyl-CoA ligase activity. These results suggest that in addition to palmitoyl-CoA ligase, myelin also contains lignoceroyl-CoA ligase.

Phenotypic heterogeneity in cultured skin fibroblasts from patients with disorders of peroxisome biogenesis belonging to the same complementation group.

We have studied fibroblast cell lines derived from a control subject (cell line 85AD5035F) and three patients clinically described as having the Zellweger syndrome (cell line W78/515), the infantile form of Refsum disease (cell line BOV84AD) and hyperpipecolic acidaemia (cell line GM3605), respectively. The mutant cell lines belonged to the same complementation group. The fibroblasts were cultured under identical conditions and were harvested at different time intervals after reaching confluence. Several peroxisomal parameters were determined. In agreement with previous reports, a lowered enzymic activity of acyl-CoA: dihydroxyacetonephosphate acyltransferase and a decrease in latent catalase clearly distinguished the patient cell lines from the control cell line. However, the cell lines exhibited a phenotypic heterogeneity. This was most strikingly encountered when cells were processed for indirect immunofluorescence microscopy and stained with anti-(catalase). The control cells exhibited a punctate fluorescence, which is indicative of the presence of catalase in peroxisomes. In the mutant cell line W78/515 a diffuse fluorescence was observed, indicative of the presence of catalase in the cytosol. In the other two mutant cell lines a punctate fluorescence was observed in some of the cells. Moreover, clear differences in the extent of proteolytic processing of acyl-CoA oxidase were detected. The mutant cell line BOV84AD displayed a control-like pattern with all molecular forms of acyl-CoA oxidase (72, 52 and 20 kDa) present, whereas in the W78/515 cell line only the 72 kDa component could be visualised. The GM3605 cell line was intermediate in this respect.

The neuronal migration defect in mice with Zellweger syndrome (Pex5 knockout) is not caused by the inactivity of peroxisomal beta-oxidation.

The purpose of this study was to investigate whether deficient peroxisomal beta-oxidation is causally involved in the neuronal migration defect observed in Pex5 knockout mice. These mice are models for Zellweger syndrome, a peroxisome biogenesis disorder. Neocortical development was evaluated in mice carrying a partial or complete defect of peroxisomal beta-oxidation at the level of the second enzyme of the pathway, namely, the hydratase-dehydrogenase multifunctional/bifunctional enzymes MFP1/L-PBE and MFP2/D-PBE. In contrast to patients with multifunctional protein 2 deficiency who present with neocortical dysgenesis, impairment of neuronal migration was not observed in the single MFP2 or in the double MFP1/MFP2 knockout mice. At birth, the double knockout pups displayed variable growth retardation and about one half of them were severely hypotonic, whereas the single MFP2 knockout animals were all normal in the perinatal period. These results indicate that in the mouse, defective peroxisomal beta-oxidation does not cause neuronal migration defects by itself. This does not exclude that the inactivity of this metabolic pathway contributes to the brain pathology in mice and patients with complete absence of functional peroxisomes.

Unique multifunctional HSD17B4 gene product: 17beta-hydroxysteroid dehydrogenase 4 and D-3-hydroxyacyl-coenzyme A dehydrogenase/hydratase involved in Zellweger syndrome.

Six types of human 17beta-hydroxysteroid dehydrogenases catalyzing the conversion of estrogens and androgens at position C17 have been identified so far. The peroxisomal 17beta-hydroxysteroid dehydrogenase type 4 (17beta-HSD 4, gene name HSD17B4) catalyzes the oxidation of estradiol with high preference over the reduction of estrone. The highest levels of 17beta-HSD 4 mRNA transcription and specific activity are found in liver and kidney followed by ovary and testes. A 3 kb mRNA codes for an 80 kDa (737 amino acids) protein featuring domains which are not present in the other 17beta-HSDs. The N-terminal domain of 17beta-HSD 4 reveals only 25% amino acid similarity with the other types of 17beta-HSDs. The 80 kDa protein is N-terminally cleaved to a 32 kDa enzymatically active fragment. Both the 80 kDa and the N-terminal 32 kDa (amino acids 1-323) protein are able to perform the dehydrogenase reaction not only with steroids at the C17 position but also with D-3-hydroxyacyl-coenzyme A (CoA). The enzyme is not active with L-stereoisomers. The central part of the 80 kDa protein (amino acids 324-596) catalyzes the 2-enoyl-acyl-CoA hydratase reaction with high efficiency. The C-terminal part of the 80 kDa protein (amino acids 597-737) facilitates the transfer of 7-dehydrocholesterol and phosphatidylcholine between membranes in vitro. The HSD17B4 gene is stimulated by progesterone, and ligands of PPARalpha (peroxisomal proliferator activated receptor alpha) such as clofibrate, and is down-regulated by phorbol esters. Mutations in the HSD17B4 lead to a fatal form of Zellweger syndrome.

Developmental and pathological expression of peroxisomal enzymes: their relationship of D-bifunctional protein deficiency and Zellweger syndrome.

We present the developmental changes of peroxisomal enzymes, catalase, L-bifunctional protein (L-BF) and D-bifunctional protein (D-BF), in the normal brains, and patients with D-BF deficiency, a new peroxisomal disease. D-BF immunoreactivity was observed in controls as early as 13 gestational weeks (GW) and increased with maturation. The adult pattern with fine granule staining of somata and dendrites became apparent in adolescence. L-BF appeared at 20 GW in the cerebral cortex and Purkinje cells and positive glia appeared early in the white matter at 17 GW, and then increased with age. Catalase-positive neurons were identified in the same manner as L-BF, D-BF deficiency in both fetus and infant showed markedly diminished enzyme immunoreactivity. Patients demonstrate reduced D-BF expression. Zellweger syndrome shows decreased expression for the three proteins. This study shows that the peroxisomal enzymes may be closely related to neuronal maturation and gliogenesis in human brain and to disturbance of neuronal migration as seen in Zellweger syndrome significant. D-BF deficiency may exhibit a range of symptoms during the neonatal and early infantile periods some of which may be similar to Zellweger syndrome.

Cyclooxygenase pathway in dermal fibroblasts from patients with metabolic disorders of peroxisomal origin.

Cyclooxygenase metabolism was studied in fibroblasts from patients with metabolic disorders of peroxisomal origin (adrenomyeloneuropathy, X-linked adrenoleukodystrophy, cerebrohepatorenal syndrome of Zellweger and rhizomelic chondrodysplasia punctata). In response to arachidonic acid (6.25-100 microM) or calcium ionophore A23187 (2.5-20 microM) prostaglandin E2 and 6-keto-prostaglandin F1 alpha are the main cyclooxygenase metabolites formed. No formation of thromboxane B2 or 2,3-dinor-thromboxane B2 was found. Apparently due to the heterogeneous nature of peroxisomal disorders no uniform pattern of cyclooxygenase metabolism and eicosanoid concentrations in cell lines from patients with peroxisomal defects was found.

A case of pseudo-Zellweger syndrome with a possible bifunctional enzyme deficiency but detectable enzyme protein. Comparison of two cases of Zellweger syndrome.

Three infants with peroxisomal disorders were investigated clinicobiochemically and neuroradiologically. Two had classical Zellweger syndrome, and cranial CT scans showed typical disproportionate enlargement of the occipital horns of the lateral ventricles (colpocephaly) with marked hypodensity of the white matter. In one female infant, although the clinical findings were similar to those in Zellweger syndrome, some findings, such as elevated transaminase levels, liver fibrosis, the absence of renal cortical cysts and colpocephaly, were negative or milder. Biochemical analyses revealed increased very long-chain fatty acids, dicarboxylic aciduria and impaired beta-oxidation of lignoceric acid. However, peroxisomes were abundantly present in hepatocytes and cultured fibroblasts, and all peroxisomal beta-oxidation enzyme proteins were detected on immunoblot analysis. A cell fusion study suggested that the enzyme responsible for this case of 'pseudo-Zellweger syndrome' is bifunctional.

Polyunsaturated fatty acid changes suggesting a new enzymatic defect in Zellweger syndrome.

The fatty acid composition of red blood cells, fibroblasts, forebrain, liver and kidney were studied in a 3-month-old infant who died from Zellweger Syndrome, and the results were compared with those of age-matched controls. Besides a typical increase in the very long chain fatty acids 26:0 and 26:1 and a great reduction in the plasmalogen levels, confirming the diagnosis of Zellweger Syndrome, some striking changes in the polyunsaturated fatty acid patterns were discovered. The most important was a very drastic decrease in the values of 22:6 omega 3 and 22:5 omega 6, the two products of delta 4-desaturation. In the kidney, the level of 22:6 omega 3 fell below that of 26:0. Consequently, the ratio 26:0/22:6 omega 3 (and 26:1/22:6 omega 3) was most useful in emphasizing the fatty acid anomalies, especially in renal tissue, where the 26:0/22:6 omega 3 ratio increased to almost 200 times the normal values. Other significant, although less consistent fatty acid alterations were increases in 18:2 omega 6, 18:3 omega 6, 20:3 omega 6, 18:4 omega 3 and 20:4 omega 3, and a decrease in 20:4 omega 6 in some tissues. The existence is proposed of a new enzyme defect in peroxisomal disorders, involving the desaturase system of long chain polyunsaturated fatty acids.

[Zellweger syndrome, neonatal adrenoleukodystrophy or infantile Refsum's disease in a case with generalized peroxisome defect?]. / Zellweger-Syndrom, neonatale Adrenoleukodystrophie oder infantile Refsumsche Erkrankung bei einem Fall mit generalisiertem Peroxisomendefekt?

An eleven month-old boy presented clinically with craniofacial dysmorphia, severe psychomotor retardation, neurological deterioration, no response to visual and acoustic stimuli, failure to thrive, hepatomegaly and adrenal insufficiency. Specific biochemical markers for a peroxisomal deficiency disorder (Zellweger's syndrome, neonatal adrenoleukodystrophy, infantile Refsum's disease) revealed pathological results for very long chain fatty acids, phytanic acid, pristanic acid, plasmalogen biosynthesis and catalase, thus confirming the clinical diagnosis. Comparison of clinical and biochemical findings in the patient with the characteristics of the three peroxisomal deficiency disorders showed overlapping with each of these disorders, which corresponds to the current view that these three peroxisomal disorders differ only with respect to onset and severity of the clinical manifestations, but not with regard to the biochemical defects.

Peroxisomal beta-oxidation of polyunsaturated long chain fatty acids in human fibroblasts. The polyunsaturated and the saturated long chain fatty acids are retroconverted by the same acyl-CoA oxidase.

The metabolism of the C22 unsaturated fatty acids erucic acid (22:1(n-9)), adrenic acid (22:4(n-6)), docosapentaenoic acid (22:5(n-3)) and docosahexaenoic acid (22:6(n-3)) was studied in cultured fibroblasts from patients with acyl-CoA oxidase deficiency, the Zellweger syndrome, X-linked adrenoleukodystrophy (X-ALD) and normal controls. [3-14C] 22:4 (n-6) and [3-14C] 22:5 (n-3) were shortened (retroconverted) to [1-14C] 20:4 (n-6) and [1-14C] 20:5 (n-3), respectively, in normal and X-ALD fibroblasts. In Zellweger and acyl-CoA oxidase deficient fibroblasts these reactions were deficient. Since the retroconversion is normal in X-ALD fibroblasts peroxisomal very long chain (lignoceryl) CoA ligase is probably not required for the activation of C22 unsaturated fatty acids. The present work with fibroblasts from patients with a specific acyl-CoA oxidase deficiency, previously shown to have a deficient peroxisomal clofibrate-inducible acyl-CoA oxidase, and which accumulate 24:0 and 26:0 fatty acids, supports the view that this enzyme is responsible for the chain-shortening of docosahexaenoic acid (22:6(n-3)), erucic acid (22:1(n-9)), docosapentaenoic acid (22:5(n-3)), and adrenic acid (22:4(n-6)) as well.

Distinction between peroxisomal bifunctional enzyme and acyl-CoA oxidase deficiencies.

The clinical distinction between patients with a disorder of peroxisome assembly (e.g., Zellweger syndrome) and those with a defect in a peroxisomal fatty acid beta-oxidation enzyme can be difficult. We studied 29 patients suspected of belonging to the latter group. Using complementation analysis, 24 were found to be deficient in enoylcoenzyme A hydratase/3-hydroxyacylcoenzyme A dehydrogenase bifunctional enzyme and 5 were deficient in acyl-CoA oxidase. Elevated plasma very long-chain fatty acids (VLCFA), impaired fibroblast VLCFA beta-oxidation, decreased fibroblast phytanic acid oxidation, normal plasmalogen synthesis, normal plasma L-pipecolic acid level, and normal subcellular catalase distribution were characteristic findings in both disorders. The elevation in plasma VLCFA levels and impairment in fibroblast VLCFA beta-oxidation were more severe in bifunctional-deficient than in oxidase-deficient patients. The clinical course in bifunctional deficiency (profound hypotonia, neonatal seizures, dysmorphic features, age at death approximately 9 months) was more severe than in oxidase deficiency (moderate hypotonia without dysmorphic features, development of a leukodystrophy, age at death approximately 4 yr). Based on these findings, accurate early diagnosis of these deficiencies of peroxisomal beta-oxidation enzymes is possible.

Phytanoyl-CoA hydroxylase is present in human liver, located in peroxisomes, and deficient in Zellweger syndrome: direct, unequivocal evidence for the new, revised pathway of phytanic acid alpha-oxidation in humans.

Phytanic acid (3,7,11,15-tetramethylhexadecanoic acid) is a branched-chain fatty acid which accumulates in a number of inherited diseases in human. Because beta-oxidation is blocked by the methyl group at C-3, phytanic acid first undergoes decarboxylation via an alpha-oxidation mechanism. The structure and subcellular localization of the phytanic acid alpha-oxidation pathway have remained enigmatic through the years, although they have generally been assumed to involve phytanic acid and not its CoA-ester. This view has recently been challenged by the findings that in rat liver phytanic acid first has to be activated to its CoA-ester before alpha-oxidation and by the discovery of a new enzyme, phytanoyl-CoA hydroxylase, which converts phytanoyl-CoA to 2-hydroxyphytanoyl-CoA. We now show that this newly discovered enzyme is also present in human liver. Furthermore, we show that this enzyme is located in peroxisomes and deficient in liver from Zellweger patients who lack morphologically distinguishable peroxisomes, which provides an explanation for the long-known deficient oxidation of phytanic acid in these patients. These results suggest that phytanic acid alpha-oxidation is peroxisomal and that it utilizes the coenzyme A derivative as substrate, thus giving further support in favour of the new, revised pathway of phytanic acid alpha-oxidation.

Molecular basis of Zellweger syndrome, beta-ketothiolase deficiency and mucopolysaccharidoses.

1. A human peroxisome assembly factor-1 (PAF-1) complementary DNA has been cloned that restores the morphological and biochemical abnormalities (including defective peroxisome assembly) in fibroblasts from a patient with group F Zellweger syndrome. The cause of the syndrome in this patient was a point mutation that resulted in the premature termination of PAF-1. The homozygous patient apparently inherited the mutation from her parents, each of whom was heterozygous for that mutation. Furthermore, we cloned and characterized the rat and human cDNAs for peroxisome-assembly factor-2 (PAF-2), which restores peroxisomes of the complementary group C Zellweger cells, by functional complementation, and identified two pathogenic mutations in the PAF-2 gene in two patients. 2. Seventeen mutations have been identified in 13 mitochondrial acetoacetyl-CoA thiolase-deficient patients. 3. We purified N-acetylgalactosamine-6-sulfate (GalNAc6S) sulfatase and cloned the full-length cDNA of human N-acetylgalactosamine-6-sulfate sulfatase (GALNS). The gene encoding GalNAc6S sulfatase has been localized by fluorescence in situ hybridization to chromosome 16q24, and the entire genomic gene structure has been characterized. About 40 different GALNS gene mutations have been identified in the patients with mucopolysaccharidosis IV A.