Main

Peroxisomes are ubiquitous components of eukaryotic cells and are involved in multiple metabolic processes (1). Examples are hydrogen peroxide–based respiration, β-oxidation of VLCFA, and biosynthesis of ether phospholipids, bile acids, and isoprene compounds. Biogenesis of peroxisomes appears to proceed mainly by growth and division of preexisting peroxisomes (1, 2). Newly synthesized peroxisomal proteins contain specific PTS—PTS1 or PTS2 for matrix enzymes and mPTS for membrane proteins—that direct them to and into peroxisomes. The proteins involved in and necessary for peroxisome biogenesis are peroxins, encoded by PEX genes. To date, 15 human PEX genes have been identified.

In humans, defective peroxisome biogenesis results in PBD, a group of autosomal-recessive diseases, including the Zellweger spectrum with four overlapping clinical phenotypes, classical Zellweger syndrome, atypical Zellweger syndrome (“pseudo”-Zellweger syndrome), neonatal adrenoleukodystrophy, and infantile Refsum disease, as well as a distinct phenotype, rhizomelic chondrodysplasia punctata (1). Patients with the classical Zellweger syndrome display characteristic dysmorphic features, severe neurologic dysfunction, eye abnormalities, hepatorenal defects, and patellar calcific stippling. They rarely survive the first year of life. Patients with atypical Zellweger syndrome, neonatal adrenoleukodystrophy, and infantile Refsum disease exhibit similar clinical characteristics to those of classical Zellweger syndrome patients, but can survive up to several decades (1, 3). Zellweger spectrum patients are characterized biochemically by defective import of virtually all peroxisomal matrix enzymes, as well as by the inability of many peroxisomal enzymes to function properly when mislocalized to the cytosol. Patients with rhizomelic chondrodysplasia punctata have unique clinical symptoms, including proximal shortening of the limbs (rhizomelia) and cataracts. At the biochemical level, these patients have a limited set of impaired peroxisomal pathways, namely plasmalogen biosynthesis and branched-chain fatty acid oxidation (1, 3).

Somatic cell fusion studies indicate that the Zellweger spectrum is genetically heterogeneous. Thus far, 11 complementation groups have been established, with complementation group 1 accounting for more than two thirds of Zellweger spectrum patients (1, 4). Mutations in the PEX1 gene are responsible for the defects in this group, and a variety of mutant alleles have been described (5–13). The PEX1 gene encodes a member of the AAA protein family of ATPases, peroxin 1, and interacts with another ATPase of this AAA protein family, peroxin 6 (13–15). Although the exact function of peroxin 1 is unknown, several recent studies favor a role in the process of peroxisomal matrix enzyme import.

The purpose of this study is to examine the relationship between the genotype and the clinical as well as the biochemical phenotype of Zellweger spectrum patients contained within complementation group 1. We have performed mutation and haplotype analyses of the PEX1 gene in 16 thoroughly documented patients who represent the broad range of phenotypic variation within this complementation group. The study has been approved by the local ethical committee.

METHODS

Subjects.

The study includes sixteen patients of German, Swiss, or Turkish origin with clinical and biochemical findings characteristic for the Zellweger spectrum of PBD. Biochemical assays of peroxisomal function were carried out at the time of diagnosis and included at least VLCFA and phytanic acid concentrations, catalase latency, and plasmalogen biosynthesis. All patients were assigned to complementation group 1 by cell fusion analyses.

Mutation analyses.

Genomic DNA and total cellular RNA were isolated from peripheral blood samples or cultured skin fibroblasts according to standard protocols. cDNA was synthesized using the Superscript TM II reagents and protocol (Invitrogen, Carlsbad, CA, U.S.A.). For mutation and polymorphism screening, we amplified PEX1 cDNA fragments covering the entire coding region or the 24 exons of the PEX1 gene. The oligonucleotide primers are listed in Table 1, A and B. Twenty-five microliters of total reaction mixture contained 200 ng of genomic DNA or 50–100 ng of cDNA, specific primers (50 pmol each), reaction buffer (400 μM of each deoxyribonucleoside triphosphate, 10 mM Tris HCl, 1.5 mM MgCl2, 50 mM KCl, 0.1% Triton X-100, 2% formamide) and 1 U Taq DNA polymerase. Thirty cycles were performed (94°C for 60 s, 50–54°C for 60 s, 72°C for 60 s) followed by a final extension of 2 min at 72°C. The annealing temperatures of the oligonucleotide primers and the length of the amplification products are included in Table 1, A and B. The amplified genomic or cDNA fragments were subjected to SSCP analysis (16). Four to 8 μL of PCR product and 4 μL of SSCP buffer (0.25 mg bromphenol blue/xylene cyanol, 74% formamide) were denatured at 94°C for 3–5 min. Electrophoresis for SSCP was performed on 10% polyacrylamide-bisacrylamide gels (29:1) with 3 M urea in 1× TBE buffer (89 mM Tris HCl, 89 mM borate, 2.5 mM Na EDTA) at 4°C. Some of the gels also contained 5% glycerol to enhance migration differences between wild-type and mutant strands (17). DNA bands were detected by silver staining. PCR products showing an aberrant SSCP pattern were sequenced directly by the Sanger dideoxy chain termination method using reagents and the protocol of U.S. Biochemical Corporation (Cleveland, OH, U.S.A.) (18) or by semiautomated sequencing using reagents and the protocol of Applied Biosystems (Foster City, CA, U.S.A.).

Table 1A Oligonucleotides for PEX1 cDNA amplificatio
Full size table
Table 1B Oligonucleotides for exon amplification of the PEX1 gen
Full size table

Allele-specific oligonucleotide hybridization.

14mer or 19mer oligonucleotides were synthesized to the human mutant sites in exons 13, 14, and 15 and the corresponding human wild-type sites. The allele-specific oligonucleotides (ASO) used are 5′-AAGATTGGTGGGTT-3′ (wild-type ASO) and 5′-AAGATTGATGGGTT-3′ (mutant ASO) for G843D, 5′-TAAAAGAGTTTATCTCCAT-3′ (wild-type ASO) and 5′-TAAAAGAGTTTTATCTCCA-3′ (mutant ASO) for c.2097–2098insT, and 5′-AACTGGAGGGTTTG-3′ (wild-type ASO) and 5′-AACTGGCGGGTTTG-3′ (mutant ASO) for G777G. The oligonucleotides were end-labeled as described (19). Samples of PCR-amplified cDNA or genomic fragments for the regions of interest were arrayed on a nitrocellulose filter with a slot blotter. Filters were prehybridized for 2 h and hybridized for 1 h in 1% SDS, 1 M NaCl, and 10% dextran sulfate at 2°C below the calculated Tm of the wild-type or the mutant probe, respectively. The filters were rinsed twice in 2× SSC and 1% SDS at room temperature, and finally washed once for 10 min at the Tm in 2× SSC, 1% SDS. Samples showing the mutant pattern were sequenced for confirmation as described.

RESULTS

Clinical phenotype.

To establish a possible genotype-phenotype correlation, we defined clinical criteria allowing us to score the severity of the clinical phenotype in every Zellweger spectrum patient in complementation group 1 (Table 2, A and B). We made the phenotypic assignment exclusively on the patients' present age or the age at death. A patient was scored as mildly affected if he survived beyond the age of 1 y. A patient was scored as severely affected if he died before that age. From a total of 16 thoroughly documented complementation group 1 patients, 9 could be classified as mildly affected and 7 as severely affected. The nine mildly affected patients had previously been assigned as atypical Zellweger syndrome, neonatal adrenoleukodystrophy, and infantile Refsum disease, and the 7 severely affected patients as classical Zellweger syndrome.

Table 2A Zellweger spectrum patients with mild clinical phenotype
Full size table
Table 2B Zellweger spectrum patients with severe clinical phenotype
Full size table

Although clear differences appear between the ages of survival, the boundaries for the clinical manifestations between mildly and severely affected patients are imprecise. Their clinical signs, including dysmorphic features as well as cerebral, ocular, hepatorenal, and skeletal abnormalities, show considerable overlap. Typical dysmorphic features are present in both patient groups. Cerebral dysfunctions are less profound in mildly affected patients. In the mildly affected group, four of seven patients needed gavage feeding, five of seven patients were hypotonic, and only one of seven patients developed neonatal seizures. In contrast, severe psychomotor retardation is a consistent sign in severely and mildly affected patients. Regarding ocular signs, retinitis pigmentosa and optic atrophy are a common feature of mildly affected patients, whereas cataracts are exclusively present in severely affected patients. Renal cysts and calcific stippling are also typical features of severely affected patients.

Biochemical phenotype.

We also defined biochemical criteria for scoring the severity of disease in Zellweger spectrum patients in complementation group 1. As for the characteristic abnormal clinical features of Zellweger spectrum patients, biochemical abnormalities could also not be taken into account for scoring. There was no clear difference in the extent of impairment of specific peroxisomal metabolic functions. As an example, the measurements of accumulated VLCFA in plasma revealed that four of seven patients with G843D have lower C26:0/C22:0 ratios than all other patients. Nevertheless, this is not a distinguishable marker. The C26:0/C22:0 ratio in plasma in the mildly affected patient group is 0.249 ± 0.163 (mean ± SD) and in the severely affected patient group 0.379 ± 0.078.

Mutations in the PEX1 gene.

We found eight different mutations in the 16 Zellweger spectrum patients in complementation group 1. The mutational spectrum includes one missense mutation, two nonsense mutations, two insertions, and three deletions. The most common mutation in 8 of 16 patients is a G to A transition at cDNA bp 2528 in exon 15 producing G843D. The mutation is located between the two nucleotide binding folds of peroxin 1 (Fig. 1). Six patients are homozygous for G843D. The second common mutation in our patient group is a 1 bp insertion in exon 13, c.2097–2098insT, which results in a frameshift and a premature termination codon 41 amino acids downstream at codon 740. The mutation results in a truncated protein containing only one of the two nucleotide binding folds (Fig. 1). Five patients are heterozygous for c.2097–2098insT, only one patient is homozygous. One of the patients studied (patient PBD-G005) includes both of these common mutations. He is homozygous for G843D and also heterozygous for c.2097–2098insT. His mother is heterozygous for the missense mutation and his father heterozygous for the insertion. The other PEX1 gene mutations we identified include two nonsense mutations, R872X and Y1126X, a 190 bp deletion in exon 14, c.2227–2416del, a 5 bp deletion in exon 18, c.2814–1818delCTTTG, a 1 bp deletion in exon 19, c.2916delA, and a 9 bp insertion c.1960–1961insCAGTGTGGA in exon 12. Three of the eight mutations identified in our patient group, namely G843D, c.2097–2098insT, and c.1960–1961insCAGTGTGGA, have also been described in patients from other groups (5–9, 11–13). Twenty-two of the 30 PEX1 gene mutations described by others and us are located within the two nucleotide binding domains of peroxin 1 (Fig. 1).

Figure 1
figure 1

Distribution of mutations in the PEX1 gene over the functional protein domains. The boxes represent the coding region of 1283 amino acids, including the two nucleotide binding folds (NBF) and the AAA protein family signature (AAA). Asterisks mark the two most common PEX1 mutations, G843D and c.2097–2098insT. Filled symbols represent the mutations from this study and open symbols represent further mutations reported in the literature (5–13).

Full size image

Genotype-phenotype correlation.

To study the genotype-phenotype correlation, we investigated how the clinical and biochemical phenotypes of the 16 Zellweger spectrum patients are distributed among the different PEX1 mutations. We found that the missense mutation G843D gives rise to a milder phenotype than the truncation mutations, insertions, and deletions. When correlated with G843D or the other PEX1 gene mutations, the present age or the age at death is the strongest indicator of mild or severe illness (Fig. 2). Figure 3 underlines that there is no discernible correlation between genotype and biochemical phenotype. Thus, the type of PEX1 mutation can be helpful in predicting the assignment of an individual case to mild or severe disease.

Figure 2
figure 2

Age of death or age of last known survival of Zellweger spectrum patients in relation to the identified PEX1 gene mutations. A correlation between the age of survival and the genotype could be shown. Filled symbols represent the age of patients who were alive at the time of this study and open symbols the age of death for already deceased patients.

Full size image
Figure 3
figure 3

Concentrations of VLCFA in plasma of Zellweger spectrum patients at the time of diagnosis in relation to the PEX1 gene mutations. There was no correlation between the biochemical phenotype and the patients' genotype. Filled symbols represent the VLCFA ratio of patients who were alive and open symbols the VLCFA ratio of those patients who were already deceased at the time of this study.

Full size image

PEX1 haplotypes.

During the course of our mutation analyses work, we also identified two polymorphic synonymous mutations in the PEX1 gene. These polymorphisms have already been reported in part (9, 20). The first polymorphism lies within exon 14. It is an A to C transition at cDNA bp 2331 and has a frequency in population of 0.08. Because GGA and GGC both code for glycine, this alteration produces the synonymous mutation G777G. The second polymorphism lies within intron 11. It is represented by the presence or absence of a 16 bp insertion upstream of the intron 11 donor splice site, IVS11+142insAGAAATTTTAAGTCTT (IVS11ins16bp). The frequency of this polymorphism in population is 0.86.

Among our 16 Zellweger spectrum patients in complementation group 1, the association of the G843D and c.2097–2098insT mutations with the G777G and IVS11ins16bp polymorphisms was striking (Table 3). Five patients who are homozygous for G843D are also homozygous for the GGA codon at G777 and the IVS11ins16bp. An identical haplotype association could be observed in patients PBD-G015 and PBD-G016, who are both compound heterozygous for G843D and c.1960–1961insCAGTGTGGA. Exceptions to this kind of haplotype association are patients PBD-G010 and PBD-G005. Patient PBD-G010 is homozygous for G843D, also homozygous for the GGA codon at G777, but compound heterozygous for the IVS11ins16bp. Patient PBD-G005, with three PEX1 gene mutations, G843D homozygote, and c.2097–2098insT heterozygote, is heterozygous for the two polymorphisms. By contrast to the haplotype association of G843D, patient PBD-G003, who is homozygous for c.2097–2098insT, and patients PBD-G007 and PBD-G002, who are compound heterozygous for c.2097–2098insT, are homozygous for the GGC codon at G777 and the absence of IVS11ins16bp. Patients PBD-G011 and PBD-G013, who are also compound heterozygous for c.2097–2098insT, are compound heterozygous for the two polymorphisms. These comprehensive analyses confirm a distinct haplotype association for the two most common PEX1 mutations, G843D and c.2097–2098insT.

Table 3 PEX1 haplotypes in Zellweger spectrum patients
Full size table

DISCUSSION

So far, 30 different PEX1 mutations have been reported in Zellweger spectrum patients in complementation group 1. The spectrum includes missense and nonsense mutations, deletions, insertions, and splicing mutations. G843D is the most common mutation. The reported allele frequency ranges from 25% to 37% for Caucasian patients in the North American, Australasian, Dutch, and German population (6, 7, 9–12). The second common mutation in this group is c.2097–2098insT, with an allele frequency between 22% and 32%(9, 11, 12). All other PEX1 mutations are private, occurring mostly in single patients.

The presence of common mutations in a population suggests either a high mutation rate at a single nucleotide position or a common evolutionary relationship between mutant alleles. Known mutation hot spots are at CpG dinucleotides or in regions that play an important role in the breakage and rejoining of DNA (21). PEX1 sequence analyses on either side of these two most common patient mutations did not reveal motifs characteristic of hot spots. Two polymorphisms have been identified in the PEX1 gene, G777G and a 16 bp insertion variation in intron 11 (9, 20). Comprehensive haplotype analyses revealed that there is a distinct relationship between the two most common PEX1 mutations, G843D and c.2097–2098insT, and these polymorphisms. G843D alleles are associated with the GGA codon at G777 and the 16 bp insertion in intron 11, whereas c.2097–2098insT alleles are associated with the GGC codon at G777 and the absence of a 16 bp insertion in intron 11. The distinct haplotype associations for G843D and c.2097–2098insT suggest that a founder effect may have contributed to the high frequency of these mutations. The mutations may have arisen once during human evolution and since expanded among the Caucasian population, the origin of our Zellweger spectrum patients. An interesting example is patient PBD-G005, with three PEX1 mutations. The two mutant alleles inherited from his parents are consistent with the founder-effect hypothesis. Furthermore, the paternal allele carries G843D as an additional de novo mutation. Although a founder effect for G843D is most likely, this de novo mutation points to a mutation hot spot in this region with so far unidentified DNA alteration elements.

The PEX1 gene is located on chromosome 7q21–q22 and encodes peroxin 1, a member of the AAA protein family of ATPases. Peroxin 1 is involved in peroxisome biogenesis and appears to act in peroxisomal matrix protein import (22, 23). Given the important function of peroxin 1 in organelle biogenesis, together with the various types of PEX1 mutations and the broad clinical spectrum of abnormalities encountered in cases of mutations, we analyzed the genotype-phenotype correlation in PEX1-associated disease. We studied clinical, biochemical, and genetic characteristics in 16 Zellweger spectrum patients and compared the data with those described in the literature. PEX1 mutations cause predictable clinical phenotypes and we propose to subdivide these mutations in two classes.

Class I mutations include only missense mutations. The presence of missense mutations on both alleles in a given patient causes mild disease. Eleven of 12 Zellweger spectrum patients with PEX1 missense mutations on both alleles including G843D survived the first year of life, and their age of last known survival ranges from 2 to 45 y (the present study, 12). In addition, these patients had a lower frequency of clinical symptoms compared with patients with other forms of PEX1 mutations (Table 2A). Class I mutations result in a peroxin 1 with residual protein levels and function. Most of these alleles carry G843D, and there is only a limited observation for other missense mutations. Fibroblasts of patients homozygous for class I mutations had detectable protein levels for peroxin 1 and the import of catalase, a peroxisomal matrix enzyme, was partially retained (6, 12, 13).

Class II mutations include the truncation mutations, insertions, deletions, and splicing mutations. The presence of a class II mutation on both patient alleles excludes mild disease. All 16 Zellweger spectrum patients homozygous for class II mutations died within the first 12 mo of age (the present study, 12). All these patients had profound cerebral dysfunctions. Cataracts, renal cysts, and calcific stippling are almost exclusive clinical signs in this patient group (Table 2B). Class II mutations lead to a total loss of peroxin function. In fibroblasts of nine patients homozygous for class II mutations, there were no detectable protein levels and peroxin 1 activities (9, 12).

The presence of both class I and class II mutations in a Zellweger spectrum patient gives rise to an intermediate and less well predictable clinical phenotype. The presence of the G843D allele or other class I mutations appears to have a moderating effect on the phenotype, whereas the presence of class II mutations aggravates disease. Patient PBD-G005 and 7 of 11 reported patients who are compound heterozygous for the G843D missense allele and the c.2097–2098insT allele have been diagnosed with an intermediate phenotype (9, 12). The difficulties in predicting the clinical phenotype for patients in this group with a broad variation in disease severity may be due to the fact that the amount of residual protein levels and function depend on the type of interactions of the two mutations of peroxin 1 or on secondary factors involved. To examine these possibilities, more information regarding the physiologic functions and intracellular interactions of peroxin 1 must be acquired.

In conclusion, phenotypic severity of Zellweger spectrum patients in complementation group 1 seems rather correlated with the consequences of the mutation on the function of peroxin 1 than with secondary factors. PEX1 mutations that are expected to create the most significant loss in protein function are associated with the most severe clinical and cellular phenotypes. This information should help clinicians make informed decisions and recommendations to families that carry PEX1 gene mutations. Furthermore, the presence of one or the other of the two most common PEX1 mutations in more than two thirds of Zellweger spectrum patients improves our ability to offer genetic testing and prenatal diagnosis to all of these families.