Background Peroxisomes are organelles that proliferate continuously and play an indispensable role in human metabolism. Consequently, peroxisomal gene defects can cause multiple, often severe disorders, including the peroxisome biogenesis disorders. Currently, 13 different PEX proteins have been implicated in various stages of peroxisome assembly and protein import. Defects in any of these proteins result in a peroxisome biogenesis disorder. The authors present here a novel genetic defect specifically affecting the division of peroxisomes.
Methods The authors have studied biochemical and microscopical peroxisomal parameters in cultured patient fibroblasts, sequenced candidate PEX genes and determined the consequence of the identified PEX11β gene defect on peroxisome biogenesis in patient fibroblasts at different temperatures.
Results The patient presented with congenital cataracts, mild intellectual disability, progressive hearing loss, sensory nerve involvement, gastrointestinal problems and recurrent migraine-like episodes. Although microscopical investigations of patient fibroblasts indicated a clear defect in peroxisome division, all biochemical parameters commonly used for diagnosing peroxisomal disorders were normal. After excluding mutations in all PEX genes previously implicated in peroxisome biogenesis disorders, it was found that the defect was caused by a homozygous non-sense mutation in the PEX11β gene. The peroxisome division defect was exacerbated when the patient's fibroblasts were cultured at 40°C, which correlated with a marked decrease in the expression of PEX11γ.
Conclusions This novel isolated defect in peroxisome division expands the clinical and genetic spectrum of peroxisomal disorders and indicates that peroxisomal defects exist, which cannot be diagnosed by standard laboratory investigations.
- Peroxisome biogenesis
- organelle division
- Zellweger syndrome spectrum
- metabolic disorders
- clinical genetics
- academic medicine
- molecular genetics
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- Peroxisome biogenesis
- organelle division
- Zellweger syndrome spectrum
- metabolic disorders
- clinical genetics
- academic medicine
- molecular genetics
Peroxisomes are single-membrane-bound organelles found in virtually all eukaryotic cells, which play an important role in numerous essential metabolic pathways such as, the α- and β-oxidation of fatty acids and the biosynthesis of ether phospholipids and bile acids in humans.1 Defects in human genes encoding peroxisomal proteins can cause a number of different peroxisomal disorders that are grouped in two main classes, that is, the single peroxisomal enzyme deficiencies, in which usually only one metabolic pathway is affected, and the peroxisome biogenesis disorders, including the Zellweger spectrum disorders, in which multiple metabolic pathways are defective due to impaired assembly of the organelle.1–3
Peroxisome biogenesis disorders can be caused by mutations in any of 13 different PEX genes encoding proteins involved in peroxisome assembly and/or peroxisomal protein import.2 4 In addition to these PEX genes, a number of proteins have been identified in different species that are involved in different peroxisomal processes including proliferation of the organelles, but for which no human disorders have been identified.5 8 It is believed that peroxisome proliferation occurs through the growth and division of pre-existing peroxisomes9 10 and by de novo synthesis from the endoplasmic reticulum.6 11 The division of peroxisomes involves elongation of existing peroxisomes followed by membrane constriction and, finally, fission of peroxisomal tubules.5–7 Dynamin-related protein 1 (DLP1/DRP1) and Fission 1 (hFis1) have been shown to be involved in the fission of peroxisomal tubules, while PEX11 proteins have been implicated in peroxisome elongation and constriction in different organisms.5 10 12 In humans, three different PEX11 isoforms are known, which share considerable sequence similarity and are encoded by the PEX11α, PEX11β and PEX11γ genes.13 14 In this study, we report the identification of the first patient with a defect of peroxisome division due to a homozygous non-sense mutation in the PEX11β gene.
Subjects and methods
The patient is a 26-year-old Dutch man with mild intellectual disability and a normal karyotype. He is the fourth child of non-consanguineous parents and has two healthy brothers and one healthy sister. The mother had three miscarriages. He was born at term by normal vaginal delivery after an uncomplicated pregnancy. Birth weight was 3.5 kg. At 6 weeks of age, bilateral congenital cataracts were noted followed by cataract extraction at 4 and 5 months. Since then no other ophthalmological problems occurred, but his vision remained <10%. He showed normal early development and walked at the age of 1.5 years. At that age, he underwent surgery for a hydrocele testis. After the surgery, a remarkable regression occurred: he lost his speech and ability to walk, and it took half a year for him to reach his former level. In general, when ill, his condition becomes rather poor and it takes relatively long for him to recover. He has a remarkably dry skin with scaling of hands and feet. At the age of 7 years, progressive bilateral perceptive hearing loss was noted, requiring hearing devices. At the age of 12 years, he was referred for extensive clinical and laboratory evaluation. Main features on neurological examination were nystagmus with a rotatory component, normal muscular strength, normal sensibility and low symmetrical reflexes in the upper extremities, and weak muscular strength, sensory abnormalities and areflexia in the lower extremities. There was no evidence for cardiomyopathy. Cerebral MRI revealed a Chiari malformation type I. An EEG was normal. EMG showed low normal motor conduction velocity and absent sensory responses. A combined muscle/nerve biopsy showed a predominance of type I muscle fibres and a reduced number of myelinated fibres. In the subsequent years, he became wheelchair-bound for longer distances. He often suffered from gastrointestinal problems and urinary incontinence. Since the age of 15 years, he has had recurrent severe migraine-like episodes with photophobia, headaches and vomiting, often following mental stress or physical exertion. Since the age of 16.5 years, he has been treated with valproic acid, co-enzyme Q10 and carnitine. Although his overall condition improved with this medication, and the frequencies of the migraine-like episodes decreased, they still occurred 2–5 times per year, on average.
The patient and his parents provided written informed consent for this study and publication of the results.
Metabolic, biochemical and microscopical analyses
Concentrations of very-long-chain fatty acids, phytanic and pristanic acid and C27-bile acid intermediates in plasma,15 and of plasmalogens in erythrocytes,16 were measured as described previously. Concentrations of very-long-chain fatty acids,17 β-oxidation of cerotic acid (C26:0), palmitic acid (C16:0) and pristanic acid,18 α-oxidation of phytanic acid19 and dihydroxyacetonephosphate acyltransferase activities20 were measured in cultured fibroblasts as described previously. To determine the ratio between intra-peroxisomal β-oxidation and extra-peroxisomal elongation of very-long-chain fatty acids, fibroblasts were incubated with 30 μmol/l deuterium-labelled docosanoic acid (D3-C22:0) for 72 h in triplicate.21 As a measure for intra-peroxisomal β-oxidation, the concentration of intracellular palmitic acid (D3-C16:0; in nmol/mg of protein) was determined. As a measure for extra-peroxisomal elongation, the concentration of intracellular cerotic acid (D3-C26:0; in nmol/mg of protein) was determined. Fatty acid concentrations were measured as described previously.22
Primary skin fibroblasts from the patient and from healthy volunteers were cultured in Dulbecco's modified Eagle's medium plus 25 mm HEPES buffer (Gibco, Invitrogen, Carlsbad, California, USA) containing 10% fetal bovine serum (FBS, Bio-Whittaker/Lonza, Basel, Switzerland), 100 U/ml penicillin, and 100 μg/ml streptomycin, in a humidified atmosphere of 5% CO2 and at 37°C or 40°C. Peroxisomes were examined by means of immunofluorescence microscopy using antisera against the peroxisomal matrix protein catalase and the peroxisomal membrane protein PMP70.23
Immunoblot analysis was performed with homogenates of cultured fibroblasts, separated by SDS-PAGE and transferred onto nitrocellulose by semidry blotting. Antiserum against PEX11β (Abcam, Cambridge, UK) and antiserum against PEX11γ (Proteintech Group, Chicago, Illinois, USA) were both used at a 1:500 dilution. As a control for equal protein loading, we simultaneously probed immunoblots with a monoclonal antibody against tubulin (Sigma, St Louis, Missouri, USA) using a 1:2000 dilution or a monoclonal antibody against β-actin (Sigma) using a 1:10 000 dilution. Antigen-antibody complexes were visualised with IRDye 800CW goat anti-rabbit secondary antibody for PEX11β and PEX11γ and IRD 680CW donkey anti-mouse secondary antibody for tubulin and β-actin using the Odyssey Infrared Imaging System (LI-COR Biosciences, Nebraska, USA), which allows sensitive and reliable determination and quantification of protein amounts in a non-saturated manner.
All exons, plus flanking intronic sequences of the PEX1, PEX2, PEX3, PEX5L, PEX6, PEX10, PEX11α, PEX11β, PEX11γ, PEX12, PEX13, PEX14, PEX16, PEX19, PEX26, DLP1 and FIS genes, were sequenced after amplification by PCR from genomic DNA.4 PCR fragments were sequenced using BigDye Terminator cycle sequencing kits (Applied Biosystems, Foster City, California, USA). PEX11β sequence data were compared with the reference PEX11β sequence (GenBank accession No. NM_003846) with nucleotide numbering starting at the first adenine of the translation initiation codon ATG.
The coding regions of wild-type PEX11α, PEX11β and PEX11γ were amplified by PCR from reverse-transcribed RNA isolated from control fibroblasts and sub-cloned into a eukaryotic expression vector (pcDNA3; Invitrogen). The patient's fibroblasts cultured at 37°C were co-transfected with the pEGFP-SKL vector and pcDNA3 containing either the PEX11α, PEX11β or PEX11γ cDNA using the nucleofector technology (Lonza, Basel, Switzerland). pEGFP-SKL codes for an enhanced green fluorescent protein containing a carboxy-terminal peroxisomal targeting sequence.8 After transfection, the patient's fibroblasts were cultured on cover slips at 37°C. After 16 h, the medium was refreshed and the temperature of the culture was raised to 40°C. Cells were examined by means of fluorescence microscopy 6 days after transfection to determine the sub-cellular localisation of the peroxisomal reporter protein EGFP-SKL.
Expression of PEX11γ at different temperatures
Patient and control fibroblasts were cultured at 37°C and 40°C for 7 days; RNA was isolated from the cells and total cDNA fractions were prepared which were used for real-time RT-PCR using the LightCycler 480 SYBR Green I Master kit (Roche, Mannheim, Germany) to determine the messenger RNA levels of PEX11γ. The PPIB gene encoding cyclophilin B was used as house-keeping gene to adjust for variations in the amount of input RNA. All samples were analysed in triplicate. The data were analysed using linear regression calculations as described previously.24 In addition, protein homogenates from the cells were prepared and these were used for immunoblotting experiments to determine the protein levels of PEX11γ.
Metabolic, biochemical and microscopical analyses
Based on the occurrence of congenital cataract, the early sensory neuronal hearing loss, the sensory nerve involvement and the recurring migraine-like attacks, the patient was suspected to be suffering from a mitochondrial or peroxisomal disorder. Extensive metabolic investigations were performed, but initially no indication for this was found. Mitochondrial oxidation rates were slightly reduced in a muscle biopsy of the patient, but ATP and creatine phosphate production from pyruvate were within normal range as were the activities of the mitochondrial complexes (not shown). Moreover, all standard biochemical peroxisomal parameters were normal, including plasma concentrations of very-long-chain fatty acids, phytanic and pristanic acid, and the C27-bile acid intermediates, as were the concentrations of plasmalogens in erythrocytes (table 1). Only on one single occasion, 1 week after having suffered two severe migraine-like episodes in 4 days, a slightly elevated ratio of cerotic acid (C26:0) to docosanoic acid (C22:0) (0.028; normal range 0.0–0.02) was measured in plasma, suggesting peroxisomal involvement. A few hours before onset of the first episode, this ratio was within the normal range (0.013). However, subsequent biochemical investigations of primary skin fibroblasts from the patient revealed no abnormalities in the peroxisomal β-oxidation rates of cerotic acid and pristanic acid, α-oxidation rate of phytanic acid, and dihydroxyacetonephosphate acyltransferase activity (table 1). In contrast, immunofluorescence microscopical analyses of the patient's fibroblasts using antisera against the peroxisomal matrix protein catalase and the peroxisomal membrane protein PMP70 revealed an aberrant peroxisomal phenotype. Peroxisomes varied markedly in size and number with few fibroblasts having normal numbers of normal sized peroxisomes, whereas most fibroblasts had lower numbers of enlarged and elongated peroxisomes (figure 1). In ∼10% of the patient's fibroblasts, catalase was not located in peroxisomes but in the cytosol, although all fibroblasts of the patient contained peroxisomal membranes (figure 1). Culturing of the patient's fibroblasts at 40°C exacerbated the aberrant peroxisomal phenotype.25 Already after 3 days of culturing the cells at 40°C, a complete absence of catalase-containing peroxisomes was noted in ∼90% of the fibroblasts, although all cells still contained enlarged, elongated peroxisomal membrane structures (figure 1). When cells cultured at 40°C were shifted to 37°C, it took 8 days for catalase-containing peroxisomes to reappear in ∼90% of the cells. After 3 days, only ∼30% of cells showed catalase-containing peroxisomes. Culturing of control fibroblasts at 40°C had no effect on the peroxisomal phenotype (see online supplementary figure 1).
To determine the extent of peroxisomal dysfunction in the patient's fibroblasts at the different temperatures, cells were incubated for 72 h with D3-C22:0 and the ratio was measured between the concentration of D3-C16:0, produced from D3-C22:0 by intra-peroxisomal β-oxidation, to the concentration of D3-C26:0, produced from D3-C22:0 by extra-peroxisomal elongation. When cultured at 37°C, the ratio of D3-C16:0 to D3-C26:0 concentrations in the patient's fibroblasts (87.4±14.2) was decreased when compared with the ratio in control fibroblasts (148.1±3.3), but higher than the ratio observed in peroxisome-deficient fibroblasts from patients with a peroxisome biogenesis disorder (0.0±0.1). Culturing of the fibroblasts at 40°C caused a significant decrease in the ratio of D3-C16:0 to D3-C26:0 concentrations in the patient's fibroblasts (4.6±0.8) to a similar ratio as that observed in peroxisome-deficient fibroblasts from patients with a peroxisome biogenesis disorder (0.1+0.02), while the ratio in control fibroblasts increased (254.1±13.2).
Mutation analysis revealed a p.Q22X mutation in PEX11
To exclude that the observed defect was due to mutations in any of the 13 PEX genes previously implicated in peroxisome biogenesis disorders, the coding regions of these genes were sequenced, which did not reveal mutations. Because the peroxisomal phenotype in the patient's cells suggested a defect in peroxisome division, a defect in one of the genes previously implicated in this process was considered. All exons, plus flanking intronic sequences of the DLP1, FIS1, PEX11α and PEX11γ genes from the patient were sequenced, but did not contain mutations. However, sequence analysis of the PEX11β gene of the patient identified a homozygous c.64C>T mutation (figure 2). Both parents were heterozygous for this mutation, confirming the homozygosity in the patient. The c.64C>T mutation changes the glutamine at amino acid position 22 into a non-sense mutation (p.Q22X), as a consequence of which no PEX11β protein is produced. This was confirmed by immunoblot analysis of protein homogenates of the patient's fibroblasts, using antiserum against PEX11β (figure 2).
Functional complementation studies with PEX11 isoforms
Over-expression of wild-type PEX11β in the patient's fibroblasts cultured at 40°C changed the mutant peroxisome-deficient phenotype to the normal wild-type phenotype, confirming the defect in PEX11β as the underlying cause. Over-expression of wild-type PEX11α in the patient's fibroblasts cultured at 40°C did not change the peroxisome-deficient mutant phenotype. However, over-expression of wild-type PEX11γ in the patient's fibroblasts cultured at 40°C showed partial complementation, and resulted in elongated and enlarged peroxisomes in ∼50% of the transfected cells (figure 3). The peroxisomal phenotype observed in the patient's cells over-expressing PEX11γ appeared similar to the one observed in the patient's fibroblasts cultured at 37°C, which suggested that the occurrence of the enlarged, elongated catalase-containing peroxisomes is related to the levels of PEX11γ. This was supported by the surprising observation that both the mRNA and protein levels of PEX11γ are markedly decreased in patient and control fibroblasts cultured at 40°C when compared with fibroblasts cultured at 37°C (figure 3). The corresponding mRNA levels of PEX11α and PEX11β are shown in the online supplementary figure 2. These findings indicate that the peroxisomal phenotype observed at 37°C is the consequence of an isolated PEX11β deficiency, whereas at 40°C a combined PEX11β and PEX11γ deficiency occurs, which results in the absence of functional, catalase-containing peroxisomes. To further study the role of PEX11γ, attempts were made to knock down PEX11γ in patient and control cells cultured at 37°C using different short hairpin RNAs separately and in different combinations. Unfortunately, this only resulted in 50% knock down at the protein level of PEX11γ. This level of knock down did not result in the absence of catalase-containing peroxisomes.
We describe the first patient with a novel isolated genetic defect of peroxisome division due to a defective PEX11β. The patient showed mild intellectual disability, congenital cataracts, progressive hearing loss and polyneuropathy, all of which are clinical features that are also observed in patients with a mild peroxisome biogenesis disorder.26–29 In addition, he had recurrent migraine-like episodes following mental stress or physical exertion, which is not common for peroxisomal disorders. Unexpectedly, although the peroxisomal appearance in the patient's fibroblasts was clearly aberrant, all peroxisomal biochemical parameters commonly used to diagnose peroxisomal defects were within normal range. A similar observation was made previously in a patient with a dominant negative mutation in DLP1, who, in addition to a severe lethal defect in the fission of mitochondria, also displayed a defect in the fission of peroxisomes.8 This makes it very likely that patients with a defect in peroxisome division have so far been missed at diagnosis, because the diagnosis requires microscopical investigations of cells, which is not a standard procedure. Moreover, because the clinical presentation of our index patient is neither very specific nor very severe, our findings imply that a peroxisomal disorder should always be considered in patients with mild intellectual disability, congenital cataracts, progressive hearing loss and/or sensory nerve involvement even when standard laboratory investigations do not provide an indication for this.
Remarkably, a previously reported PEX11β knockout mouse showed neonatal lethality and exhibited many of the severe pathological features typically observed in lethal peroxisome-deficient Zellweger syndrome mice, including neuronal migration defects, hypotonia, enhanced neuronal apoptosis and developmental delay, although biochemically the PEX11β knockout mouse also appeared only mildly affected in peroxisome metabolism.30 31 Apart from a mild defect in the peroxisomal β-oxidation and ether-phospholipid synthesis and a twofold reduction of peroxisome abundance, all peroxisomal biochemical parameters were normal and no effect on peroxisomal protein import was observed in this PEX11β knockout mouse.30 31 It is unclear why the PEX11β knockout mouse shows such a severe clinical phenotype when compared with our patient.
Despite a complete deficiency of PEX11β in the patient's fibroblasts, peroxisome division/biogenesis was only partly disturbed at 37°C, which appeared to be related to the levels of PEX11γ. This follows from our observation that the defect in peroxisome biogenesis in the patient's fibroblasts was exacerbated when these were cultured at 40°C, during which the mRNA and protein levels of PEX11γ are markedly lower, and deficient, respectively, when compared with growth at 37°C. The combined PEX11β and PEX11γ deficiency at 40°C resulted in the complete disappearance of catalase-containing peroxisomes and a significant decrease in the fatty acid β-oxidation to elongation ratio, while enlarged peroxisomal membrane structures were still present. In addition, over-expression of PEX11γ in the patient's fibroblasts at 40°C partially restored peroxisome biogenesis resulting in a similar peroxisomal phenotype as observed at 37°C. Our observation that the patient's fibroblasts incubated at 40°C only recovered slowly after shifting the temperature to 37°C, may explain the clinical impression that our patient recuperates rather slowly after a period of illness. Moreover, this seems to corroborate well with the finding of slightly elevated ratios of C26:0 to C22:0 concentrations after two severe episodes of migraines.
Peroxisomes are believed to arise through growth and division of pre-existing peroxisomes (major or default pathway), or de novo from pre-peroxisomal vesicles originating from the endoplasmic reticulum (minor or salvage pathway) (figure 4). It was previously reported that over-expression of PEX11α and PEX11β in mammalian cells results in an increase in peroxisome numbers, but that PEX11γ over-expression does not have an effect on peroxisome abundance.30 In line with previous reports on its function,10 our findings indicate that PEX11β functions in the growth and division pathway and is involved in peroxisome elongation and constriction. In addition, our findings suggest that PEX11γ may function in the de novo pathway and somehow mediates or promotes the import of peroxisomal matrix proteins into the pre-peroxisomal vesicles that are derived from the endoplasmic reticulum. Figure 4 shows a hypothetical model for peroxisome proliferation based on these findings.
To conclude, our discovery of a novel isolated genetic defect in peroxisome division, which cannot be diagnosed by standard laboratory analysis, further expands the clinical and genetic spectrum of peroxisomal disorders and indicates that additional peroxisomal defects exist that can only be detected by specific diagnostic workups including microscopical analysis in fibroblasts. Consequently, we expect that additional patients with mutations in PEX11β or other genes involved in peroxisome division will be identified by the application of next-generation sequencing technologies to identify the genetic causes in patients with unresolved defects.
Professor Smit is holder of the Metakids chair. We thank Petra Mooijer, Connie Dekker and Inge Dijkstra for their valuable contributions, and the patient and his parents for their permission to conduct these studies and publication of the results.
Funding This work was supported by the “Prinses Beatrix Fonds” Grant Number MAR 03_0216 and by the 6th Framework Program of the European Union Grant Number LSHG-CT-2004-512018.
Competing interests None.
Patient consent The patient and his parents provided (in Dutch) written informed consent for this study and for publication of the results.
Provenance and peer review Not commissioned; externally peer reviewed.
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