Human Molecular Genetics, 2000, Vol. 9, No. 8 1195-1200
© 2000 Oxford University Press
Human phytanoyl-CoA hydroxylase: resolution of the gene structure and the molecular basis of Refsum’s disease
University of Amsterdam, Academic Medical Centre, Departments of 1Pediatrics (Emma Children’s Hospital) and 2Clinical Chemistry, Laboratory for Genetic Metabolic Diseases (Room F0-224), PO Box 22700, 1100 DE Amsterdam, The Netherlands, 3Free University Hospital, Department of Clinical Chemistry, Metabolic Unit, Amsterdam, The Netherlands and 4Department of Pediatrics, Institute of Clinical Biochemistry, Rikshospitalet, Oslo, Norway
Received 10 January 2000; Revised and Accepted 13 March 2000.
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ABSTRACT |
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Refsum’s disease (RD) is an inherited neurological syndrome biochemically characterized by the accumulation of phytanic acid in plasma and tissues. Patients with RD are unable to degrade phytanic acid due to a deficient activity of phytanoyl-CoA hydroxylase (PhyH), a peroxisomal enzyme catalysing the first step of phytanic acid
-oxidation. To enable mutation analysis of RD at the genome level, we have elucidated the genomic organization of the PHYH gene. The gene is ~21 kb and contains nine exons and eight introns. Mutation analysis of PHYH cDNA from 22 patients with RD revealed 14 different missense mutations, a 3 bp insertion, and a 1 bp deletion, which were all confirmed at the genome level. A 111 bp deletion identified in the PHYH cDNA of several patients with RD was due to either one of two different mutations in the same splice acceptor site, which result in skipping of exon 3. Six mutations, including a large in-frame deletion and five missense mutations, were expressed in the yeast Saccharomyces cerevisiae to study their effect on PhyH activity. The results showed that all these mutations lead to an enzymatically inactive PhyH protein. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Refsum’s disease (RD) is a rare inherited neurological disorder, characterized by a tetrad of clinical abnormalities including retinitis pigmentosa, peripheral neuropathy, cerebellar ataxia and an elevated protein concentration in the cerebrospinal fluid [for review see (1)]. The only biochemical abnormality found in RD is the accumulation of phytanic acid (3,7,11,15-tetramethylhexadecanoic acid) in tissues and body fluids.
Phytanic acid is a branched-chain fatty acid, which is a normal constituent of the human diet. Because of the methyl branch at the third carbon atom of this fatty acid, its breakdown can not proceed via the ß-oxidation pathway, which is the common degradation route for most fatty acids. Instead, phytanic acid undergoes one round of -oxidation to produce its n–1 analogue, pristanic acid (2,6,10,14-tetramethylpentadecanoic acid) (see refs 1–3 for reviews). Prior to -oxidation, phytanic acid is activated to its coenzyme A-ester, phytanoyl-CoA (4). The subsequent -oxidation pathway consists of three steps. In the first step, phytanoyl-CoA is converted to 2-hydroxyphytanoyl-CoA, a reaction catalyzed by phytanoyl-CoA hydroxylase (PhyH) (5,6). The second step involves the conversion of 2-hydroxyphytanoyl-CoA to pristanal plus formyl-CoA, and is catalyzed by the enzyme 2-hydroxyphytanoyl-CoA lyase (7–11). In the third and last step, pristanal is dehydrogenated to form pristanic acid by an NAD(P)+-dependent aldehyde dehydrogenase (9,12).
Previously, we and others have demonstrated that phytanic acid accumulation in patients with RD is caused by a deficient PhyH activity (13) resulting from mutations in the PHYH gene (14–17). Due to the lack of the genomic structure of the PHYH gene, however, all these mutations were identified in PHYH cDNA from patients. To be able to confirm the zygosity of those mutations that appeared homozygous at the cDNA level and to resolve the molecular basis for aberrant cDNA species observed in some patients, we elucidated the genomic organization of the PHYH gene. Furthermore, we have extended the mutation analysis to a total of 22 patients with RD which led to the identification of numerous novel mutations including splice site mutations. The effect of several of these mutations on the enzyme activity of PhyH was finally studied by heterologous expression in the yeast Saccharomyces cerevisiae.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Genomic organization of the PHYH gene
To resolve the PHYH genome structure, two PHYH gene-containing PAC clones were identified by PCR screening, and exon–intron junctions were subsequently determined as described in Materials and Methods. The PHYH gene spans ~21 kb and comprises nine exons and eight introns (Table 1; Fig. 1). All exon–intron junctions conform to the GT and AG consensus sequence for 5'-donor and 3'-acceptor splice sites, respectively.
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PHYH mutation analysis in patients with RD
In our previous studies on eight patients with RD, mutation analysis of PHYH cDNA revealed five different mutations including a single nucleotide deletion resulting in a premature stop codon, an in-frame deletion of 111 nucleotides that results in a putative protein lacking 37 internal amino acids, and three different missense mutations all causing an amino acid substitution (14,16). We have now extended our studies to a total of 22 patients with RD including two pairs of sibs. PhyH activity measurements in homogenates of cultured skin fibroblasts from these patients showed that all patients are deficient in PhyH activity (results not shown). Mutation analysis of the PHYH cDNAs from these patients revealed 14 different missense mutations, a 1 bp deletion and a 3 bp insertion, of which the homozygous mutations were confirmed at the genome level (Table 2). Interestingly, we observed co-segregation of the c.530A
G and the c.734GA mutations in four unrelated patients suggesting a common ancestor. A frequent mutation observed at the cDNA level was a 111 bp deletion, c.135–245del, which we identified in six patients (three homozygotes and three compound heterozygotes). Comparison with the genome structure of the PHYH gene revealed that this deletion comprises the entire exon 3, suggesting a mutation that causes exon skipping. Indeed, when we sequenced the splice site junctions of exon 3 of these patients, we detected two different mutations in the splice acceptor site of intron 2, IVS2-2ag and IVS2-1gc (Table 2), each resulting in skipping of exon 3. From the 16 different mutations in the PHYH open reading frame (ORF) nine were located in exon 6, five in exon 7, one in exon 2 and one in exon 3 (Fig. 1). One patient (no. 1) was a homozygote for a c.636ag mutation. This mutation was also observed in heterozygous form in three additional unrelated patients with different genotypes, who have not been included in this paper. The fact that this mutation does not result in an amino acid substitution indicates that it represents a polymorphism. This was confirmed by the analysis of the PHYH cDNAs from 64 control individuals, which revealed an incidence of ~10% for this mutation.
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Heterologous expression of the PHYH cDNA in S.cerevisiae
In order to confirm that the missense mutations and the in-frame 111 nucleotide deletion in the PHYH cDNA are the underlying cause of the deficient PhyH activity, a heterologous expression system was developed. To this end, the human PHYH ORF was cloned in a yeast expression vector and expressed under the transcriptional control of the oleate-inducible catalase A (CTA1) promoter in S.cerevisiae. When cell lysates were prepared from either untransformed yeast cells or from yeast cells transformed with the expression vector without the PHYH ORF, no PhyH activity could be detected. When the PHYH ORF was expressed, however, a PhyH activity of 3.4 ± 0.3 nmol/h/mg protein could be measured in the yeast lysates (Fig. 2A). The PhyH protein was readily detectable by immunoblot analysis using a polyclonal antiserum raised against a PhyH polypeptide of 18 aa and has an apparent Mw of 38.5 kDa (Fig. 2B).
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The same expression system was subsequently used to study the effect on the PhyH protein of several of the mutations found in the patients with RD (Fig. 2A). When the cDNA with the in-frame 111 bp deletion was expressed, no PhyH activity could be detected. Upon immunoblot analysis, a single immunoreactive band was detected with an Mw of 36.5 kDa, which is ~2 kDa smaller than the wild-type human PhyH protein (Fig. 2B). This finding is in agreement with the calculated size after the deletion of 37 internal aa due to the 111 bp deletion. Although the encoded mutant protein was clearly detectable, its expression level was much lower than that of the wild-type protein, suggesting that the mutant protein is less stable and degraded more rapidly. Expression of cDNAs with three different missense mutations, including c.823C
T, c.824GA and c.610GA, resulted in mutant PhyH proteins without any detectable PhyH activity (Fig. 2A). In all cases normal amounts of the mutant PhyH protein were found when compared with the wild-type PhyH protein, indicating that these mutant PhyH species were not unstable, at least, when expressed in S.cerevisiae. Slight differences in electrophoretic mobility during SDS–PAGE were observed after immunoblot analysis, reflecting different mutations (Fig. 2B). It has been shown previously that with SDS–PAGE visible changes in apparent molecular weight can already occur when a single amino acid is changed (18).
In four patients, co-segregation of two missense mutations, c.530AG and c.734GA, was found. Two of these patients were compound heterozygotes (patients 5 and 6) and two patients were homozygotes (patients 4 and 7) for these mutations (Table 2), confirming that both mutations are present on one allele. In order to determine their specific effects on PhyH activity, cDNAs with either both or only one of these mutations were expressed in S.cerevisiae. In all three cases, PhyH protein was present in normal amounts (Fig. 2B). PhyH activity measurements showed that when the two mutations were expressed together, no PhyH activity could be detected. When expressed as a single mutation, however, only the c.530AG transition resulted in a complete inactivation of PhyH. The c.734GA transition caused a partial deficiency (~75% PhyH activity when compared with the wild-type PhyH). These results indicate that the c.530AG transition is responsible for the inactivation of PhyH activity in the patients.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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In this paper we report the genomic organization of the PHYH gene and its use to confirm mutations identified at the cDNA level of a group of 22 patients with RD, most of whom have not been analyzed before. In total, we identified 18 mutations, including 14 missense mutations, a 1 bp deletion, a 3 bp insertion and two different splice acceptor site mutations, both giving rise to skipping of exon 3, which comprises 111 bp. Mihalik and coworkers (15) also reported a 111 nucleotide deletion, but at a different position of the PHYH cDNA (c.158–169del). Inspection of the surrounding cDNA sequence and the adjacent intervening sequence as described in their mutation report, however, revealed that these authors have assigned the wrong position numbers to this fragment, and in fact also found the deletion of exon 3, which in their case is caused by the 5'-splice site mutation g.IVS2-2A
G (see Table 2). Chahal and coworkers reported a deletion of 88 aa in two patients with RD (patients 1 and 3 in ref. 17). Analysis of the PHYH cDNA sequence and comparison with the genome structure revealed that this is due to the deletion of exons 5 plus 6 (82 + 182 = 264 nucleotides; see Table 1) resulting in c.415–678del. As a consequence, a polypeptide lacking aa I139 to E226 will be produced (and not aa 138–225, as erroneously stated in ref. 17). Most likely, this case of exon skipping is caused by mutations in one of the splice sites, which can now be studied at the genome level.
PhyH activity measurements in fibroblast homogenates from patients with missense mutations showed that in all cases the enzyme activity is completely deficient. Heterologous expression of five missense mutations in the yeast S.cerevisiae showed that mutant PhyH is produced in all cases, and that all but one of these mutations result in an enzymatically inactive PhyH protein, at least in this expression system. The c.734GA mutation, which produced a protein with 75% residual activity in S.cerevisiae, was found in four patients. In all these patients, however, this mutation co-segregated with the c.530AG,which was shown to cause the complete inactivation of PhyH. Neither in the patients with RD nor in control alleles was the c.734GA transition found as an isolated mutation.
Although we did not express all newly identified missense mutations, the observation that all patients with RD carried mutations in the PHYH gene plus the fact that the corresponding patients’ fibroblasts were found to lack PhyH activity strongly indicate that the mutations found indeed cause the enzymic PhyH deficiency. As most missense mutations were found in exons 6 and 7, it is likely that the part of the PhyH protein encoded by these exons is important for enzyme activity. Future studies on the three dimensional structure of PhyH could benefit from these mutations and eventually will reveal the basis for the inactivation caused by these mutations.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Patients
All patients studied in this paper were diagnosed with RD based on clinical findings and elevated phytanic acid levels in plasma. Some patients were previously described in detail: patients 19 and 20 as patients 1 and 2 by Jansen and coworkers (14), and patients 16, 17 and 12 as cases 2, 4 and 11 by Skjeldal and coworkers (19). Mutation analysis performed on PHYH cDNA derived from fibroblasts from the five patients mentioned above has been described previously (14). More detailed information on both clinical and biochemical data of the other 17 patients included in this study can be obtained from the authors upon request.
Sequence analysis and organization of the PHYH gene
In order to obtain PHYH-containing clones from a genomic PAC library, a PCR protocol was developed allowing amplification of a sequence containing part of an exonic sequence plus part of the adjacent intervening sequence. PCR screening of the human PAC library was performed by a commercial vendor (GenomeSystems, St Louis, MO) and two positive clones were obtained (16924, 16925). PCR analysis using primers complementary to the 5'-end and the 3'-end cDNA sequences plus several cDNA sequences in between, was done to verify the presence of the complete PHYH gene. Exon–intron junctions were subsequently determined using one of the following methods: (i) Direct sequencing with the entire PAC clone as template using BigDyeTerminator chemistry (Perkin Elmer Benelux, Gouda, The Netherlands) and primers designed on the PHYH cDNA sequence; (ii) PCR amplification of a PAC fragment using primers designed on the PHYH cDNA sequence, followed by sequence analysis using BigDye Primer chemistry (Perkin Elmer); and (iii) PCR amplification of a PAC fragment using primers designed on the PHYH cDNA sequence, followed by subcloning of this fragment in the vector pGEM-T (Promega) and subsequent sequence analysis using BigDyeTerminator chemistry. For some sequence reactions, ‘GC-Melt’ buffer (Advantage-GC cDNA PCR kit, Clontech) was used. All exon–intron junctions were verified by PCR with primer combinations complementary to both an exon and an intron sequence, using both the PAC clone and human DNA isolated from cultured skin fibroblasts as template, followed by sequence analysis. The DNA sequences of the various exons and the flanking intron sequences have been submitted to the GenBank and are available under accession nos AF242379–AF242386.
Enzyme activity measurements
PhyH activity measurement in cell lysates from yeast cultures was performed according to a radiochemical HPLC based assay procedure used for PhyH measurement in human liver homogenates (20).
PHYH cDNA and genomic DNA mutation analysis
Sequence analysis of PHYH cDNA derived from cultured skin fibroblasts from control individuals and patients with RD was carried out as previously described (14). To identify mutations causing missplicing and to confirm apparent homozygous mutations at the genome DNA level, (parts of) the various exons containing the mutations were amplified by PCR from genome DNA isolated from the patients and sequenced using Big Dye Primer chemistry using either –21M13 or M13-Rev primers. The PCR program used for exon amplification started with 2 min of denaturation at 96°C followed by 5 cycles of 30 s at 96°C, 30 s at 55°C and 1–3 min at 72°C and 25 cycles of 30 s at 94°C, 30 s at 55°C and 1–3 min at 72°C with a final extension step at 72°C for 15 min.
Exon 3 sequences were amplified with primers IVS2-FW (5'-TGTAAAACGACGGCCAGTCAGGGTTTCATCATGTTGCC-3') and c.185-Rev (5'-CATAAAATTTTCTCTGTTCCAGG-3'). Exon 6 sequences were amplified with primers IVS5-FW (5'-TGTAAAACGACGGCCAGTGCAGCAGTACAATCATAGC-3') and IVS6-Rev (5'-CAGGAAAC- AGCTATGACCACAGATCACCTGAGGTCAGG-3') Exon 7 sequences were amplified with primers IVS6-FW (5'-TGTAAAACGACGGCCAGTTGTGATCATGGCTCACTGG-3') and c.944-Rev (5'-CAGGAAACAGC- TATGACCAGCTCCCAAAGAATTTATGT-3').
Heterologous expression of the PHYH cDNA in S.cerevisiae
The complete coding sequence of human PHYH was amplified from cDNA obtained from cultured skin fibroblasts using XbaI and HindIII tagged primers (5'-AAA TCT AGA AAA ATG GAG CAG CTT CGC GCC GC-3' and 5'-AAA AAG CTT TCA AAG ATT GGT TCT TTC TCC-3', respectively) and cloned into the plasmid pGEM-T (Promega, Leiden, The Netherlands). The PHYH sequence was subsequently verified by sequence analysis using T7 and SP6 primers. The PHYH coding sequence was then released from pGEM-T as an XbaI–HindIII fragment and subcloned into the XbaI and HindIII sites of the yeast expression plasmid pEL30 (low copy plasmid containing CEN-sequence) (21) under the transcriptional control of the oleate-inducible promoter pCTA1. The expression constructs and, as a control, the vector pEL30 were transformed into S.cerevisiae strain BJ1991 (MAT, leu2, trp1, ura3-52, prb1-1122, pep4-3) (22). Transformants were grown in minimal essential medium containing 3 g/l glucose and 6.7 g/l yeast nitrogen base without amino acids (Difco Laboratories, Detroit, MI) supplemented with appropriate amino acids at 30°C. In order to induce expression, cells were harvested by centrifugation and transferred into a medium containing 1.2 g/l oleic acid, 1.2 g/l Tween-40, 5 g/l potassium phosphate (pH 6.0), 3 g/l yeast extract, and 5 g/l peptone. Cells were grown at 30°C and harvested by centrifugation after the culture had reached a spectrophotometric absorbance of ~0.5 at 600 nm. The cells were resuspended in 250 µl buffer containing 20 mM Tris–HCl pH 7.5, 5 mM dithiothreitol, 1 mg/ml leupeptin, 2 mg/ml Pefabloc (Merck NL, Amsterdam, The Netherlands) and 10% (v/v) glycerol. After addition of 200 ml glass beads, the suspension was vortexed for 30 min at 4°C, centrifuged for 2 min at 12 000 g at 4°C, and the supernatant containing human PhyH was taken for PhyH activity measurements and further experiments.
In order to clone mutant alleles from patients with RD, the protocol as described above was followed but then using cDNA derived from fibroblasts of the patients.
Generation of anti-PhyH antiserum
The deduced PHYH amino acid sequence was analyzed for putative antigenic sites using MacVector sequence analysis software (Oxford Molecular Group, Oxford, UK). According to the antigenic index, aa 160 to 177 (NKPPDSGKKTSRHPLHQD) of PhyH were predicted to comprise a potential antigenic polypeptide. This 18 aa polypeptide was synthesized chemically and coupled to ovalbumin (OVA).
Female New Zealand white rabbits were primed with 180 mg of the OVA-PhyH polypeptide in 0.8 ml PBS mixed with an equal volume of Freund’s complete adjuvant. After 2 weeks, the immunization was followed by a boost injection of 180 mg of the OVA-PhyH polypeptide mixed with Freund’s incomplete adjuvant. Two more boost injections were given at 4 week intervals. Eight days after each boost injection a 10 ml blood sample was taken and serum was prepared.
Immunoblot analysis
Samples were subjected to SDS–PAGE on a 10% polyacrylamide gel and blotted onto a nitrocellulose filter. Non-specific binding sites were blocked for 1 h using a PBS solution containing 1 g/l Tween-20 (PBST), supplemented with 30 g/l non-fat dried milk (NFDM). Primary (polyclonal anti-PhyH) and secondary (goat anti-rabbit IgG coupled to alkaline phosphatase) antibody incubations were performed in PBST. After each incubation, the blots were washed extensively in 3 g/l NFDM/PBST. Antigen–antibody complexes were visualized using alkaline phosphatase staining in a buffer containing 0.1 M Tris–HCl pH 9.5, 0.1 M NaCl, 5 mM MgCl2, 0.33 g/l 4-nitro blue tetrazolium chloride, 0.17 g/l 5-bromo-4-chloro-3-indolyl-phosphate (disodium salt).
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ACKNOWLEDGEMENT |
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This study was financially supported by a grant from the Netherlands Organization for Scientific Research (NWO, project no. 901-03-133).
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FOOTNOTES |
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+ To whom correspondence should be addressed. Tel: +31 20 5664197; Fax: +31 20 6962596; Email: wanders@amc.uva.nl
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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