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Human Molecular Genetics Pages 2089-2094  

Temperature-sensitive mutation in PEX1 moderates the phenotypes of peroxisome deficiency disorders
Introduction
Results And Discussion
   Temperature-sensitive complementation of CG1 IRD fibroblasts
   Biogenesis of peroxisomal protein in IRD fibroblasts
   CG1 IRD patient analysis
   PEX1G843D renders CG1 CHO cell mutants temperature sensitive
Materials And Methods
   Cell lines
   Morphological analysis
   Mutation analysis
   Transfection of PEX1
   Other methods
Acknowledgements
Abbreviations
References

Temperature-sensitive mutation in PEX1 moderates the phenotypes of peroxisome deficiency disorders

Temperature-sensitive mutation in PEX1 moderates the phenotypes of peroxisome deficiency disorders

Atsushi Imamura1,2, Shigehiko Tamura3, Nobuyuki Shimozawa1, Yasuyuki Suzuki1, Zhongyi Zhang1, Toshiro Tsukamoto2, Tadao Orii4, Naomi Kondo1, Takashi Osumi and Yukio Fujiki3,5,*

1Department of Pediatrics, Gifu University School of Medicine, Gifu 500-8076, Japan, 2Department of Life Science, Himeji Institute of Technology, Kamigori, Hyogo 678-1297, Japan, 3Department of Biology, Faculty of Science, Kyushu University, Fukuoka 812-8581, Japan, 4Faculty of Human Welfare, Chubu Gakuin University, Seki, Gifu 501-3936, Japan and 5CREST, Japan Science and Technology Corporation, Tokyo 170-0013, Japan

Received June 23, 1998; Revised and Accepted September 4, 1998

The peroxisome biogenesis disorders (PBDs), including Zellweger syndrome (ZS), neonatal adrenoleukodystrophy (NALD) and infantile Refsum disease (IRD), are autosomal recessive diseases caused by deficiency of peroxisome assembly as well as malfunction of peroxisomes, where >10 genotypes have been reported. ZS patients manifest the most severe clinical and biochemical abnormalities, while those with NALD and IRD show the least severity and the mildest features, respectively. PEX1 is the causative gene for PBDs of complementation group I (CG1), the highest incidence PBD, and encodes the peroxin, Pex1p, a member of the AAA ATPase family. In the present work, we found that peroxisomes were morphologically and biochemically formed at 30 but not 37°C, in the fibroblasts from all CG1 IRD patients examined, whereas almost no peroxisomes were seen in ZS and NALD cells, even at 30°C. A point missense mutation, G843D, was identified in the PEX1 allele of most CG1 IRD patients. The mutant PEX1, termed HsPEX1G843D, gave rise to the same temperature-sensitive phenotype on CG1 CHO cell mutants upon transfection. Collectively, these results demonstrate temperature-sensitive peroxisome assembly to be responsible for the mildness of the clinical features of PEX1-defective IRD of CG1.

INTRODUCTION

A peroxisome is a single membrane-bounded, ubiquitous intracellular organelle, catalyzing various metabolic pathways such as [beta]-oxidation of very long chain fatty acids (1). The functional importance of human peroxisomes is emphasized by fatal genetic diseases, including Zellweger syndrome (ZS), neonatal adrenoleukodystrophy (NALD) and infantile Refsum disease (IRD), that are linked to failure of peroxisome assembly (2,3). Patients with ZS show severe neurological abnormalities, characteristic dysmorphism and hepatomegaly, and rarely survive, with an average life span of only 6 months. NALD patients have symptoms similar to ZS patients, but they survive a little longer, to early childhood. In contrast, patients with IRD do not manifest significant abnormalities in the central nervous system, and survive with the longest average life, 3-11 years (2). Genetic heterogeneity is seen in subjects with peroxisome biogenesis disorders (PBDs), ZS, NALD and IRD, represented by >10 complementation groups (CGs) (3). Thirteen CGs have been identified in mammals: 11 of these were defined by analysis of patient-derived fibroblasts (4-7) as well as peroxisome-deficient Chinese hamster ovary (CHO) cell mutants (4,8-12) and two distinct CHO mutant cell lines (13). Therefore, >13 genes are likely to be required for mammalian peroxisome biogenesis.

To investigate the molecular mechanisms involved in peroxisome biogenesis and the genetic cause of PBDs, we have to date cloned several peroxisome assembly factor genes, PEX2, PEX6, PEX12 and PEX1, by genetic phenotype-complementation assay of CHO cell mutants, Z65, ZP92, ZP109 and ZP107, respectively (14-18). All CHO mutants resemble fibroblasts from patients with PBD, with regard to defects in biogenesis and functions of peroxisomes. Several human PEX genes, including PEX5 coding for the PTS1 receptor (19), PEX12 (20), PEX7 coding for the PTS2 receptor (21-23), and PEX10 (24), were also cloned recently by a homology search of the human expressed sequence tag database, using yeast PEX genes. Several groups of investigators, including ours, then demonstrated six PEX genes, i.e. PEX1, PEX2, PEX5, PEX6, PEX7 and PEX12, to be responsible for the genetic events in patients with PBD (16-23,25-30). More recently, inactivation of PEX10 was shown to be the primary cause of CG-B PBD (the same group as CG7 in the USA) (24). However, little attention has been paid to determining at the molecular level the basis for the variable severity in clinical features between the severest ZS, NALD, and the mildest, IRD. Very recently, Imamura et al. (31) found restoration of peroxisome biogenesis in a temperature-dependent manner, in fibroblasts from patients with the milder form of PBDs in several CGs, including CG1. In addition, a frequent mutation was found in several CG1 patients with milder forms of PBD, including IRD (25).

Table 1. Temperature sensitivity of peroxisome biogenesis and G843D mutation analysis in fibroblasts from CG1 PBD patients
Patient Phenotype Peroxisome-positive cells (%) tsa Age at death or last follow-upb C24c DHAP-ATased 843G or 843D allele
37°C 30°C 37°C 30°C 37°C 30°C
E-08 ZS 0 0 - 5 m         G/G
E-09 ZS 0 0 -           G/G
E-29 ZS 0 0 - >8 m         G/G
E-14 ZS 0 0e - 4 m 56.0 58.0 0.19 0.27 G/G
E-13 NALD 1 1e - 1 y 8 m         G/G
E-01 NALD 5 5e - 2 y 9 m         G/G
E-10 ZS/NALD 0 30 + >2 y 6 m         D/G
E-26 IRD 0 50e + >6 y 1 m 43.1 312[uarr]     G/G
E-25 IRD 0 60e + >10 y 7 m         G/G
E-24 IRD 0 90e + >1 y 7 m 30.8 305[uarr] 0.32 1.70[uarr] D/G
E-05 IRD 0 90e +   58.8 363[uarr]     D/G
E-27 IRD 0 100 + 3 y 8 m         D/G
E-11 IRD 0 100 + >9 y         D/G
E-06 IRD 5 100 + >10 y 174 420[uarr]     D/D
  Control 1 100 100 -   253 360 1.69 1.82  
  Control 2 100 100 -   395 378 2.01 1.24  
ats, temperature sensitivity.
bAge: y, year; m, month.
cC24, activity of lignoceric acid oxidation (pmol/h/mg protein); [uarr], distinct increase.
dDHAP-ATase, dihydroxyacetonephosphate acyltransferase (nmol/2 h/mg protein).
eFrom ref. 31.

We have identified a missense PEX1 mutant allele, the same as reported (25), in most of the CG1 IRD patients analyzed. The mutation leads to temperature-sensisitive assembly of peroxisomes. The relevance of the temperature-sensitive phenotype to the clinical mildness in CG1 PBD is discussed.

RESULTS AND DISCUSSION

Temperature-sensitive complementation of CG1 IRD fibroblasts

Fibroblasts from a CG1 IRD patient (PBDE-06) were morphologically restored for peroxisome assembly with respect to import of catalase, when cultured for 3 days at 30°C, but not at 37°C (Fig. 1a and b), consistent with our earlier observation (3132) (data not shown). The PBDE-06 fibroblasts were morphologically restored for peroxisome assembly at 37°C, by transfection of human PEX1, HsPEX1 (18) (data not shown), indicating that dysfunction of PEX1 is responsible for peroxisome deficiency, consistent with the result of CG analysis by cell fusion (N. Shimozawa et al., unpublished data). Complementation of peroxisomes was likewise observed by immunostaining of fibroblasts from six unrelated IRD patients (PBDE-26, -25, -24, -05, -27 and -11) of CG1 after 3 days at 30°C, with a temperature sensitivity of 50-100% (Table ). In contrast, no peroxisomes were found in fibroblasts from a ZS patient (PBDE-29), even at 30°C (Fig. 1c and d), while numerous peroxisomes were present in fibroblasts from a normal control at both temperatures (data not shown). Fibroblasts from all other CG1 ZS patients examined (PBDE-08, -09 and -14) showed no apparent distinct changes in phenotype after the temperature shift to 30°C (Table 1). Fibloblasts from two CG1 NALD patients (PBDE-13 and -01) showed no temperature-sensitive type catalase import, although a very low level of punctate staining was noted at 37°C, apparently due to slight leakiness (Table 1). In patient PBDE-10, diagnosed as ZS or NALD, only one-third of fibroblasts were restored for peroxisomes at 30°C (Table 1) (see below). Taken together, these data strongly suggest that the cellular phenotype of PEX1-defective CG1 IRD is temperature sensitive. It is noteworthy that CG1 IRD patients, including patients PBDE-26, -25, -27, -11 and -06, had a longer life span than patients with ZS and NALD (Table 1).


Figure 1. Immunofluorescence staining of fibroblasts from CG1 peroxisome deficiency patients. Cells were cultured for 3 days, at 37 (a and c) or 30°C (b and d), then stained with an antibody to human catalase. Fibroblasts were from an IRD patient (PBDE-06) (a and b) and a ZS patient (PBDE-29) (c and d). Bar, 10 µm.

Biogenesis of peroxisomal protein in IRD fibroblasts

In PBD fibroblasts, peroxisomal proteins are mislocalized to the cytosol, and rapidly degraded or not converted to mature forms, despite normal synthesis (4,17,18). Acyl-CoA oxidase (AOx) is synthesized as a 75 kDa polypeptide (A component) and is proteolytically converted into 53 kDa B and 22 kDa C polypeptides in peroxisomes (4,17,18,33). All three polypeptide components, A, B and C, were evident in fibroblasts from an IRD patient, PBDE-06, after cell culture for 3 days at 30°C, as in normal fibroblasts cultured at both temperatures, whereas only the A component was seen in these IRD cells at 37°C (Fig. 2A, lanes 1-4). In contrast, AOx-A component was not converted to B and C even at 30°C, in fibroblasts from an NALD patient PBDE-13 (lanes 5 and 6). Similar conversion of AOx-A to B and C components was detected at 30°C, in fibroblasts from other CG1 IRD patients such as PBDE-24 and -05 (data not shown).


Figure 2. Biogenesis of peroxisomal protein. Fibroblasts (~105 cells) were cultured for 3 days at 37 (lanes 1, 3 and 5) or 30°C (lanes 2, 4 and 6). Cell lysates were subjected to SDS-PAGE and transferred to a polyvinylidene difluoride membrane. (A) Immunoblot analysis was performed using rabbit anti-rat AOx antibody. Lanes 1 and 2, a normal control; lanes 3 and 4, IRD patient PBDE-06; lanes 5 and 6, NALD patient PBDE-13. A, B and C designate AOx components. An unknown protein with a slightly lower mobility, as compared with AOx-A, is indicated by an asterisk; a non-specific band, but lower in amount, co-migrating with AOx-C polypeptide was discernible in lane 3. (B) Immunoblot using antibody to rat 3-ketoacyl-CoA thiolase. Lanes 1 and 2, normal control; lanes 3 and 4, IRD patient PBDE-06. Open and solid arrowheads in (B) indicate a larger precursor (P) and mature protein (M) of 3-ketoacyl-CoA thiolase, respectively.

Peroxisomal 3-ketoacyl-CoA thiolase is synthesized as a 44 kDa precursor with an N-terminal, cleavable type 2 signal (32,34), and is processed to its final size, 41 kDa, in peroxisomes (4,9,17,18). In normal fibroblasts, only the mature thiolase was detected at both temperatures (Fig. 2B, lanes 1 and 2), thereby demonstrating rapid processing of the precursor form. In fibroblasts from IRD patient PBDE-06, only the larger precursor was found at 37°C (Fig. 2B, lane 3), implying a defect in import and processing activity. When the fibroblasts were cultured for 3 days at 30°C, only the mature form of the thiolase was discerned, reflecting the complementation (lane 4). The data confirm that the temperature-sensitive phenotype is characteristic of IRD cells.

The activity of two peroxisome-specific enzymes, one catalyzing [beta]-oxidation of lignoceric acid, a very-long-chain fatty acid, and dihydroxyacetonephosphate acyltransferase (DHAP-ATase), was determined. In fibroblasts from four CG1 IRD patients (PBDE-26, -24, -05 and -06), lignoceric acid oxidation was markedly enhanced, up to 10-fold, when cultured for 3 days at 30°C, as compared with 37°C (Table 1). The activity was as high as in cells from two normal controls, indicating complementation of lignoceric acid oxidation in a temperature-sensitive manner. DHAP-ATase activity was increased similarly in cells from IRD patient PBDE-24 cultured at 30°C. In contrast, no significant, temperature-sensitive change was observed in either enzyme activity in fibroblasts from ZS patient PBDE-14 and two controls, although the activity in ZS cells was only 10-15% of normal. Thus, these data confirmed temperature-sensitive complementation of peroxisomal enzymes.

Taken together, these results demonstrate that cell culturing at the permissive temperature can complement the abnormality in biogenesis of peroxisomal proteins in CG1 IRD cells.

CG1 IRD patient analysis

To determine the dysfunction of PEX1 in CG1 IRD patient PBDE-06, we examined PEX1 cDNA from fibroblasts, by means of RT-PCR. By subsequent sequencing, we detected a missense mutation in all 10 cDNA clones isolated: G->A at position 2528, in a codon (GGT) for Gly843, resulted in a codon (GAT) for Asp843 (data not shown). We termed this mutated PEX1 HsPEX1G843D. Genomic PCR between nucleotide residues 2453 and 2646 in the PEX1 open reading frame resulted in a single type of PCR product containing A2528, indicating that patient PBDE-06 was a homozygote for the G843D mutation (Table ). The same type of G843D homozygote was also reported for three CG1 IRD and one NALD patient (25). Of six other IRD patients (PBDE-26, -25, -24, -05, -27 and -11), four were heterozygotes, apparently demonstrating that mutation G843D is common (Table ). It is possible that RNA from the other allele in these four IRD patients was not expressed due to a defect at the level of transcription and/or splicing, or could be transcribed normally but degraded rapidly. It would be intriguing to find another mutation(s) in PBDE-26 and -25 manifesting temperature-sensitive complementation of peroxisomes. One patient, PBDE-10, with apparently milder ZS or NALD was found to be a G843D heterozygote showing a partial temperature-sensitive restoration of peroxisomes, presumably due to a lower level of G843D PEX1 mRNA expression (Table 1). Nearly a dozen NALD and ZS patients with a G843D PEX1 allele reported by Reuber et al. (25) may likewise show a partially temperature-sensitive phenotype or possess non-temperature-sensitive type mutations such as a null mutation in the other allele, thereby possibly manifesting more severe NALD or ZS disorders. In contrast, four ZS patients (PBDE-08, -09, -29 and -14) and two NALD patients (PBDE-13 and -01) did not show the G843D mutation. Taken together, a PEX1G843D mutant allele is more likely to be responsible for a distinct temperature-sensitive phenotype of fibroblasts from IRD patients (PBDE-24, -05, -27, -11 and -06). Moreover, the G843D mutation apparently inactivated PEX1 at 37°C, as assessed by transfection to CG1 CHO cell mutants (see below), thereby demonstrating that PEX1G843D is the genetic cause of peroxisome deficiency, at least in one case with a homozygous mutation, in CG1 IRD patients.

PEX1G843D renders CG1 CHO cell mutants temperature sensitive

To assess the temperature-sensitive phenotypic property of Pex1p with mutation G843D, a CG1 CHO cell mutant, ZP101 (10), was stably transfected with HsPEX1G843D. Numerous peroxisomes were detected by immunofluorescent staining of catalase, after 3 day culture only at 30, but not 37°C (Fig. 3Ac and d), while normal HsPEX1-transfected ZP101 cells showed restoration of peroxisomes at both temperature (a and b). These results demonstrated that HsPEX1G843D transfectants had morphologically normal peroxisomes, as numerous as in the wild-type CHO-K1 cells, in a temperature-dependent manner. The peroxisomal [beta]-oxidation activity of lignoceric acid, a very-long-chain fatty acid, as well as DHAP-ATase activity markedly increased in HsPEX1G843D-expressing ZP101 cells cultured at 30°C, as compared with those at 37°C, thereby indicating complementation of peroxisomes (Table 2). The reason for elevated activity of lignoceric acid oxidation at 37°C in PEX1G843D transformants of ZP101 cells is presently unknown, although partial complementation is plausible.


Figure 3. Transformation of CG1 CHO cell mutants with PEX1G843D. (A) PEX1 cDNAs derived from a normal control and IRD patient PBDE-06, PEX1G843D, were transfected into CHO cell mutant ZP101 of CG1; stable transformants were isolated. Cells were cultured for 3 days at 37 (a and c) or 30°C (b and d) and stained with anti-rat catalase antibody. (a and b) PEX1-transfected ZP101; (c and d) PEX1G843D-transfected ZP101. Bar, 10 µm. Several unknown, punctate structures are discernible in (c). (B) A CG1 CHO cell mutant, ZP107, and ZP107 transiently transfected with PEX1G843D were cultured for 3 days at 30°C; cell lysates (~105 cells) were analyzed by immunoblot using anti-3-ketoacyl-CoA thiolase. Lane 1, ZP107; lane 2, PEX1G843D-transfected ZP107. Open and solid arrowheads designate a larger precursor (P) and mature protein (M) of thiolase, respectively.

In ZP107 cells, another CG1 CHO mutant cell line, both a larger precursor and a mature thiolase were detected when transiently transfected with HsPEX1G843D and cultured at 30°C (Fig. 3B, lane 2), thereby reflecting rapid processing of the thiolase precursor in complemented cells, while only the larger precursor was found in ZP107 cells (lane 1). Restoration of biogenesis of peroxisomal proteins is compatible with the morphological findings shown in Figure 3A. Taken together, HsPEX1G843D expression can confer a temperature-sensitive phenotype on CG1 CHO mutants. It is noteworthy that a missense mutation in PEX2 likewise produced a CG10 CHO mutant temperature-sensitive peroxisome assembly (31), compatible with the findings on Pex1p in the present work.

Table 2. Temperature-sensitive enzyme activity of an IRD-derived PEX1 transfectant of CHO cell mutant ZP101
Cell [beta]-oxidation of lignoceric acid
(pmol/h/mg protein)		 DHAP-ATase
(nmol/2 h/mg protein)
37°C 30°C 37°C 30°C
ZP101 5.63 12.0 0.18 0.23
ZP101-PEX1G843Da 108 831 0.10 1.5
aZP101 cells were transfected with PEX1G843D; the transfectants were selected in the presence of hygromycin.

Pex1p may function as a peroxisome biogenesis factor in the peroxisomal protein import process, possibly by interacting with other Pex proteins (3,35,36), including recently cloned Pex10p (24). Very recently, we demonstrated that human Pex1p indeed interacts with another AAA family peroxin Pex6p (37), as was the case for yeast Pex1p (38). It would be intriguing to investigate whether Pex1pG843D alters such an interaction in a temperature-dependent manner. Structural configuration, trafficking kinetics or stability of the peroxins may affect the biological activity in a temperature-sensitive manner, despite possessing a full-length but missense mutation. It is interesting to note that a temperature-sensitive phenotype has been reported in a genetic disease, epidermolysis bullosa simplex, manifesting the worst disturbance of the skin caused by aberrant keratin at a higher temperature (39).

Collectively, the data in the present work demonstrate that the temperature-sensitive phenotype of PEX1 is responsible, partly if not totally, for peroxisome deficiency in the cells of CG1 IRD patients. Such a temperature-sensitive property is likely to be tightly linked to the clinically mildest symptoms of IRD in PBD, possibly reflecting the extended life span noted in IRD patients. It is noteworthy that an allelic mutation, G843D, was found recently in nearly half of CG1 PBD patients (25), suggesting PEX1 mutation G843D to be the most common case in CG1 PBD. Furthermore, given the findings in the present work, the severity of PBD in newborn patients can be prognostic by examining the temperature-sensitive complementation of peroxisomes in patient fibroblasts. Such a prognostic tool may also encourage pediatricians to treat the milder PBD variants with therapies such as an oral administration of docosahexaenoic acid (2).

MATERIALS AND METHODS

Cell lines

Skin fibroblast cell lines from normal controls and patients were cultured in complete medium [Dulbecco's modified Eagle's medium (DMEM) high glucose supplemented with 10% fetal calf serum] as described (17,29). Fibroblasts from five CG1 patients, PBDE-08 (GM8040), PBDE-10 (GM6094), PBDE-05 (GM8770), PBDE-11 (GM8769) and PBDE-06 (GM8772), were purchased from The Human Genetic Mutant Cell Repository. Patients' fibroblasts were classified by complementation analysis by cell fusion as described (4). CHO cells were cultured as described (9-11).

Morphological analysis

Peroxisomes in human fibroblasts and CHO cells were visualized by indirect immunofluorescence light microscopy, as described (4). We used rabbit antibodies to human catalase (4), rat liver 3-ketoacyl-CoA thiolase (9) and rat catalase (9). Antigen-antibody complex was detected by fluorescein isothiocyanate-labeled sheep anti-rabbit immunoglobulin G antibody (Cappel), under a Carl Zeiss Axioskop FL microscope.

Mutation analysis

RT-PCR. Poly(A)+ RNA was obtained from fibroblasts from CG1 IRD patient PBDE-06, using a QuickPrep mRNA purification kit (Pharmacia Biotech). RT-PCR using poly(A)+ RNA was performed with a pair of human PEX1-specific PCR primers: sense F2, 5[prime]-AGCCATGTTCCCATGTGG-3[prime]; and antisense R2, 5[prime]-TGTGAAGCTGTCCTTAAC-3[prime], to amplify the sequence between nucleotide residues 284 and 3545. The nucleotide sequence of the PCR products cloned into pBluescript was determined by the dideoxy chain termination method using a Dye-terminator DNA sequencing kit (Applied Biosystems). Patient-derived PEX1 cDNA, HsPEX1G843D, was cloned into pCMVSPORT-I vector by replacing the BglII-XhoI fragment (at residues 1327-3192) of PEX1 from a normal control with that of the patient PEX1 in pBluescript (18).

G843D mutation analysis of the PEX1 gene. Genomic DNA was prepared from cultured fibroblasts (29) using a SepaGene kit (Sanko Jun-Yaku, Tokyo). To determine whether patients had the PEX1G843D allele, i.e. G->A transition at residue position 2528, PCR amplification of the sequence of residues 2453-2646 was done using a pair of PEX1-specific primers: sense, 5[prime]-TCCGCGGATTTCTTCCTGCGTCTT-3[prime]; and antisense, 5[prime]-CGGACCATACAACAGTATTCCTGT-3[prime]. PCR products were sequenced directly.

Transfection of PEX1

Co-transfection of plasmid pUcD2Hyg and either pCMVSPORTHsPEX1 (18) or pCMVSPORTHsPEX1G843D to CG1 CHO cell mutant ZP101 (10) was performed using a Gene PulserII electroporator (Bio-Rad) at 300 V and 400 µF; stable transformants were isolated by selection in the presence of hygromycin. Transfection of pCMVSPORTHsPEX1G843D to another CG1 CHO mutant ZP107 (11) was performed using Lipofectamine (Gibco BRL), as recommended by the manufacturer.

Other methods

Western blot analysis was performed using rabbit antibodies, including anti-AOx antibody (9) and a second antibody, donkey anti-rabbit IgG antibody conjugated to horseradish peroxidase (Amersham), using ECL western blotting detection reagent (Amersham Pharmacia Biotech). Assays of lignoceric acid [beta]-oxidation (40) and DHAP-ATase (41) were performed as described.

ACKNOWLEDGEMENTS

We thank T. Sakaguchi and N. Matsumoto for technical assistance. This work was supported in part by Grants-in-Aid for Scientific Research from The Ministry of Education, Science, Sports and Culture and a CREST grant (to Y.F.) from the Japan Science and Technology Corp.

ABBREVIATIONS

AOx, acyl-CoA oxidase; CG, complementation group; CHO, Chinese hamster ovary; DHAP-ATase, dihydroxyacetonephospahte acyltransferase; IRD, infantile Refsum disease; NALD, neonatal adrenoleukodystrophy; PBD, peroxisome biogenesis disorders; ZS, Zellweger syndrome.

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*To whom correspondence should be addressed at: Department of Biology, Faculty of Science, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan. Tel: +81 92 642 2635; Fax: +81 92 642 4214, or 2645; Email: yfujiscb@mbox.nc.kyushu-u.ac.jp

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