Most PBD complementation groups are represented by only a few patients. This is not surprising given their rarity and genetic heterogeneity. Because positional cloning strategies often rely on the availability of large numbers of patients and/or well-studied kindreds with multiple affected individuals, researchers interested in identifying the defective genes in PBD patients have developed alternative approaches to identify the affected genes. Over the past 8 years, these have led to the identification of the genes that are defective in 11 of the 12 known PBD complementation groups and more than 95 percent of PBD patients (Table 129-1).
To date, all of the genes known to be defective in PBD patients have been identified by either or both of two related strategies: homology probing100 and mammalian cell complementation.101 The homology-probing approach relies heavily on studies of peroxisome biogenesis in lower eukaryotes and the sequences of peroxisome biogenesis factors from these other organisms. It also relies heavily on the availability of large sequence databases of human expressed sequence tags, which can be searched for genes capable of encoding proteins with significant sequence similarity to the yeast peroxins. Full-length versions of candidate human PEX genes can then be obtained though routine molecular biologic approaches. Expression of the candidate clones in fibroblasts from PBD patients can be used to determine whether the gene can complement the phenotypes of the cell line; if it can, then analysis of the patient's DNA and RNA analysis can reveal whether the gene is mutated in these patients. This general approach was first used to identify PEX5 as the gene responsible for CG2 of the PBDs104 and was subsequently used to identify the genes defective in CG1 (PEX1155,321), CG3 (PEX12141), CG4 (PEX6154), CG7 (PEX10140,322), CG9 (PEX1656,172), CG11 (PEX7129– 131), CG12 (PEX357), and CG13 (PEX13135,136).
The mammalian cell functional complementation approach uses mutant Chinese hamster ovary (CHO) cell lines rather than yeast as the experimental system. Briefly, mutant CHO cells that are unable to import peroxisomal matrix proteins are identified and placed into complementation groups by cell fusion.94 These CHO pex mutants are then rescued by transfection with mammalian cDNA expression libraries, usually from rat liver.101 The complementing cDNA can be isolated by a variety of techniques, including the screening of progressively smaller subpools of the cDNA library. The identification of the CHO pex mutants and cloning of the rat PEX genes is generally considered more difficult than the analogous procedures in yeast. However, the identification of human orthologs of rat PEX genes is straightforward, due to the high sequence identity between human and rodent genes, whereas identifying human homologs of yeast PEX genes can be difficult. The CHO pex mutant functional complementation strategy was used to clone the very first PBD gene, PEX2, which is mutated in patients from CG10 of the PBDs.102 It has since been used to identify the genes defective in CG1 (PEX1323), CG3 (PEX12324,325), CG4 (PEX6326), and CG14 (PEX1958).
Defects in Peroxisomal Matrix Protein Import
Eleven distinct PEX gene deficiencies have been identified within the PBDs over the past 8 years (Table 129-1). We discuss those that specifically affect peroxisomal matrix protein import first, in their hypothesized order of action.
Human PEX5 cDNA were identified independently by several groups104,327,328 and the PEX5 gene has been mapped to chromosome 12p13 by a variety of techniques.327,329 Two forms of the PEX5 mRNA, PEX5S and PEX5L, differ by the presence or absence of 111 bp within the ORF and are generated by alternative splicing of exon 8.111 PEX5L contains an additional 37 amino acids that are inserted between residues 214 and 215 of PEX5S. PEX5 proteins are predominantly cytoplasmic, partly peroxisomal receptors for newly synthesized peroxisomal matrix proteins. PEX5 binds with high affinity to the type-1 peroxisomal targeting signal (PTS1),72 the targeting signal that is present on most matrix proteins.173 A line diagram of PEX5 shows its various functional domains, together with the sites of known PEX5 mutations in PBD patients (Fig. 129-19). A variety of studies have mapped the site of interaction between PEX5 and the PTS1 to the C-terminal TPR domain103,104,108 and a molecular replacement model of the C-terminal three TPRs predicts that they recognize the PTS1 peptide backbone.109 PEX5 requires PEX14 for docking to peroxisome membranes.118 There appear to be multiple PEX14-binding sites within the N-terminal half of PEX5,137 which may correspond to the repeating tryptophan motif noted by Dodt et al.104 PEX5 also binds to PEX12, an integral PMP that is required for peroxisomal matrix protein import and that is mutated in patients from complementation group 3 of the PBDs.145 This interaction, however, is mediated by the C-terminal TPR domain of PEX5.
Diagram of the human PTS1 receptor, PEX5 protein. The gray boxes indicate the 7 C-terminal tetratricopeptide repeat (TPR) motifs. The triangle indicates the position of the 37-amino-acid segment encoded by the alternatively spliced exon 8.111 The brackets indicate regions of the PEX5 protein that interact with other proteins. The positions of three known missense mutations causing clinical phenotypes are indicated; a homozygote for R390K had ZS; a homozygote for N489K had NALD; and a homozygote for S563W had IRD.104
PEX5 deficiency has been described for just four PBD patients.104,244,330 PEX5-deficient patients span the phenotypic range of the Zellweger spectrum from ZS to IRD. At the cellular level, all PEX5-deficient patients contain numerous PMP-containing peroxisomes.23 However, the four PEX5-deficient patients differ in the extent of their peroxisomal matrix protein import defect.
Complete PEX5 deficiency is displayed by PBD005 cells, which were derived from a severely affected ZS patient and are homozygous for a PEX5 nonsense mutation, R390ter.104 PBD005 cells are unable to import either PTS1- or PTS2-containing peroxisomal matrix proteins but contain numerous PMP-containing peroxisomes.23,104 The translated product of the PEX5 R390ter allele would be expected to encode the N-terminal half of PEX5. However, PEX5 mRNA levels in PBD005 are below the limit of detection by northern blot, presumably due to nonsense-mediated RNA decay.331 Expression of PEX5S is sufficient to rescue PTS1 protein import in these cells but restores PTS2 protein import at only 1 percent of the frequency with which it rescues PTS1 protein import.104,111 In contrast, PEX5L expression rescues both PTS1 and PTS2 protein import in PBD005 cells at equal efficiency.111 Overexpression of a PEX5L transcript containing the R390ter mutation rescued PTS2 protein import in PBD005 cells, indicating that the severe clinical and cellular phenotypes of PBD005 are due to the combined effects of the PEX5/R390X mutation and nonsense mediated RNA decay.111
Two NALD patients, PBD018 and PBD093, are both homozygous for a missense mutation in the PEX5 PTS1-binding domain, N489K.104,244 These patients import PTS2-containing proteins normally but are unable to import any PTS1-containing proteins. The milder clinical phenotypes of these NALD patients are likely due to the more restricted matrix protein import defect in these cells. Asparagine 489 is though to play a critical role in the binding of PTS1 peptides by PEX5.109 This residue, together with arginine R520, is predicted to facilitate PTS1-binding by interacting with the C-terminal carboxylate of the PTS1. Biochemical studies reveal that the N489K mutation does not severely affect PEX5 folding but does reduce the affinity of PEX5 for the PTS1 by several orders of magnitude.332 More recently, an even more mildly affected PEX5-deficient patient was identified, designated 2-03.330 This IRD patient was homozygous for a missense mutation, S563W, which lies downstream of the last TPR domain of PEX5. This residue is not thought to participate directly in PTS1 recognition. The effects of the S563W mutation appear to be more pronounced for inefficiently imported PTS1-containing proteins such as catalase,330 whereas the import of efficiently imported PTS1 proteins, such as acyl-CoA oxidase, are barely affected by this mutation. PTS2 protein import is unaffected in this patient.
The human PEX7 cDNA was identified independently by three groups, in each instance by homology probing approaches.129– 131 The human PEX7 gene contains 10 exons spanning 102 kb at chromosome 6q21-6q22.2.132 A number of alternatively spliced forms of PEX7 mRNA have been identified.132 However, only the cDNAs with the full coding potential are functional in vivo and the shorter transcripts do not interfere with PEX7 function, indicating that they may represent splicing by-products rather than biologically significant transcripts. In yeast, PEX7 encodes the physical receptor for the type-2 peroxisomal targeting signal, PTS2,123,124 and a similar activity is expected for human PEX7. Human PEX7 appears to be predominantly cytoplasmic,129 a distribution that has also been observed for the yeast PTS2 receptor,121,123 but additional studies of PEX7 activity and distribution are necessary. PEX7 appears to interact with PEX5, the PTS1 receptor,333 an interaction that may explain the essential role of PEX5 in PTS2 protein import.104 This interaction has not been shown to be direct and may be mediated by other proteins.
RCDP patients represent approximately 20 percent of all PBD cases (Table 129-1) and PEX7 deficiency has been described for more than 40 RCDP patients.129– 131 Although isolated defects in plasmalogen synthesis enzymes cause clinical phenotypes that are indistinguishable from those of RCDP,274,334 these differ from RCDP in that they have no peroxisome biogenesis defect and are mutated in the two genes that encode peroxisomal enzymes involved in etherphospholipid synthesis.334,335 All known RCDP patients are mutated in PEX7. RCDP patients all contain numerous PMP-containing peroxisomes, import PTS1 proteins normally, and display severe to mild defects in PTS2 protein import.23,127
Complete PEX7 deficiency is displayed by numerous RCDP patients. Braverman et al.129 reported the identification of a common PEX7 mutation, L292X, that is associated with severe forms of this disease. This mutation is present in approximately two-thirds of RCDP patients and is present on approximately 50 percent of the mutated PEX7 alleles in these individuals. Genotyping studies revealed that the L292X mutation is always found on one particular haplotype, indicating that this mutation arose just once.132 The high frequency of the L292X allele in RCDP suggests that its prevalence of this allele in populations of European descent may be somewhat elevated.
Braverman et al.129 also reported the presence of a missense mutation, G217R, in five probands, all of whom were compound heterozygotes for the L292X allele. Three probands contained another missense mutation, A218V, two of whom exhibited relatively mild RCDP phenotypes. Motley et al.131 reported the identification of the L292X and A218V mutations in RCDP patients and Purdue et al.130 reported the L292X mutation, as well as an unexplained mutation that results in the PEX7 mRNAs that are 100 bp shorter than normal. More recently, a severely affected RCDP patient with a new mutation, R232X, was reported.336
During the initial identification and characterization of yeast PEX13, an integral PMP required for peroxisomal matrix protein import, two groups reported human PEX13 cDNAs.113,116 The PEX13 gene has been localized to chromosome 2p14-16 by both FISH and radiation hybrid mapping, and is contained on at least four exons spanning approximately 11 kb.337 Human PEX13 is an approximately 44-kDa integral PMP that appears to span the peroxisome membrane twice and extends both its N- and C-termini into the cytoplasm.113,117 The C-terminal extension of PEX13 contains an SH3 domain, which appears to mediate its interaction with PEX5113 and with PEX14.117 The N-terminal extension of PEX13 appears to interact with PEX7, either directly or indirectly.117 In yeast, loss of PEX13 causes a significant reduction in the levels of peroxisome-associated PEX5 and PEX7, indicating that PEX13 may function as a docking factor for these PTS receptors.
There are only two known cases of PEX13 deficiency, one ZS patient (H-02) and one NALD patient (H-01/PBD222), who together define CG13 of the PBDs.135,136 H-02 cells have numerous PMP-containing peroxisomes but fail to import peroxisomal matrix proteins.135 These cells are homozygous for a nonsense mutation in PEX13, W234ter, which truncates PEX13 upstream of its second transmembrane span and removes its SH3 domain. H-01/PBD222 cells display a much milder matrix protein import defect and in fact import detectable levels of both PTS1- and PTS2-containing matrix proteins.135,136 These cells are homozygous for a missense mutation in the SH3 domain, I326T, and a PEX13/I326T cDNA retains significant PEX13 activity,136 as one might expect from the mild phenotypes of the H-01/PBD222 patient. The I326T mutation only slightly reduces the import of efficiently import peroxisomal matrix proteins but has a more pronounced effect on the import of inefficiently imported matrix proteins such as catalase338 and glycolate oxidase, HAOX1.82
Human PEX10 cDNAs were isolated independently by two different groups,140,322 in both cases by searching for human homologs of yeast pex10 genes. A sequence tagged site (STS) marks the PEX10 gene, sgc30638, located at the top of the chromosome 1 linkage map. This STS is located 10.26 cR from the top of the chromosome 1 linkage group. The PEX10 coding region is contained on seven exons that are distributed across approximately 8 kb of genomic DNA.339 Two PEX10 mRNAs, PEX10 and PEX10L, differ by 57 bp and are generated by use of different splice acceptor sites at the 3′ end of intron 3. The shorter form accounts for approximately 95 percent of the PEX10 mRNA in cells.339 The PEX10L transcript encodes a protein with an additional 19 amino acids inserted following the tyrosine residue at position 200 and appears to be slightly less functional than the shorter, more abundant PEX10 transcript.
PEX10 encodes an integral PMP that spans the membrane twice with its N- and C-termini exposed to the cytosol.140,322 The C-terminal domain of PEX10 contains a zinc RING motif that is essential for PEX10 function.339 PEX10 interacts with PEX12 via its C-terminal RING-containing domain.142 PEX12 is another integral PMP that is required for peroxisomal matrix protein import,141 and its role in the PBDs is discussed below. Loss of PEX10 results in a severe defect in peroxisomal matrix protein import but no defect in peroxisome synthesis, PMP import, or PTS receptor docking.23,140,142 Thus, PEX10 appears to participate in an essential step of peroxisomal matrix protein import that occurs after PTS receptor docking. In the yeast Pichia pastoris, loss of PEX10 results in the most severe matrix protein import defect yet described.138
PEX10 deficiency has been reported in four ZS patients and 1 NALD patient, all of whom were placed in CG7 of the PBDs by cell fusion complementation analysis. The positions of the known PEX10 mutations are shown relative to the structural domains of the PEX10 product (Fig. 129-20). The sole NALD patient, PBD052, imports residual levels of both PTS1- and PTS2-containing peroxisomal matrix proteins.140 PBD052 is a compound heterozygote for a severe nonsense mutation, R125X, and a missense mutation that replaces a zinc-coordinating histidine of the PEX10 zinc RING domain with a glutamine residue, H290Q.140 This latter mutation only partially reduces PEX10 function.339 Although the R125X mutation was also reported to retain PEX10 activity, this has since been shown to be an artifact of the cDNA expression system that was used and the R125X mutation actually appears to eliminate PEX10 function.339
Diagram of the human PEX10 protein. TMD1 and TMD2 indicate transmembrane domains 1 and 2; the large gray rectangle indicates the C-terminal RING domain. The bracket indicates the region of PEX10 that interacts with PEX12. The location of PEX10 mutations in PBD patients is shown above (see Warren et al.140). A patient with the NALD phenotype was a compound heterzygote for R125X and H290Q. A patient homozygous for the exon 1 rearrangement had ZS.
All four PEX10-deficient ZS patients have mutations that completely eliminate PEX10 activity. PBD100, which is often used as a benchmark PEX10 null cell line,23 is homozygous for a splice site mutation at the 5′ boundary of PEX10 intron 3 (GT → AT).140 This mutation induces aberrant splicing of the PEX10 transcript, leading to the production of a PEX10 mRNA that lacks exon 3 and is 407 bp shorter than normal. A PEX10 cDNA with this mutation lacks activity in vivo. PBD117 is a compound heterozygote for a complicated deletion/insertion/frameshift mutation in exon 1 and a frameshift mutation in exon 4, 704insA, that eliminates the C-terminal zinc RING of PEX10.339 PBD116 (also known as PBDB-01322) and PBD121 are each homozygous for a 2 bp deletion in exon 5 that also precludes expression of the C-terminal zinc RING domain.339 In fact, all alleles in PEX10-deficient ZS patients preclude the expression of proteins with a zinc RING domain, the region of PEX10 that interacts with PEX12. Three other patients had been placed in complementation group 7 by cell fusion complementation analysis, but these have since been shown to be defective in PEX12, PEX1, and PEX6.339
Human PEX12 cDNAs were identified both by homology probing141 and by functional complementation of a CHO pex mutant.324 The PEX12 gene contains three exons and spans 2.5 kb.141 There is no evidence for alternative splicing of the PEX12 transcript. The chromosomal location of the PEX12 gene has yet to be determined. PEX12 appears to be an integral PMP that spans the membrane twice and extends its N- and C-termini into the cytoplasm. PEX12 contains an unusual form of the zinc RING motif within its C-terminus. Zinc RING domains typically coordinate two zinc atoms145,340 but three of the four cysteine residues that normally bind the second zinc atom are missing in the PEX12 RING.139,141 Nevertheless, the C-terminal RING-containing domain of PEX12 is essential for biological function139,141,325 and appears to mediate physical interactions between PEX12 and both PEX5, the PTS1 receptor, and PEX10.142
PEX12-deficient patients belong to CG 3 of the PBDs. There are currently 10 known PEX12-deficient patients and they display clinical phenotypes that span the range of the Zellweger spectrum (Table 129-1).18,67,141,142 Peroxisome membrane synthesis and PMP import are unaffected by loss of PEX12, but all patients with mutations in PEX12 display severe-to-mild defects in peroxisomal matrix protein import.23,67,141 Additional studies have shown that cells lacking PEX12 have normal levels of peroxisome-associated PEX5, the PTS1 receptor.142 Taken together, the phenotypes of cells lacking PEX12, the localization of PEX12 to the peroxisome membrane, and the physical interaction of PEX12 with both PEX5 and PEX10 suggest that PEX12 acts in peroxisomal matrix protein import downstream of receptor docking, most likely in matrix protein translocation.145
Of the 10 known PEX12-deficient patients, 6 display severe ZS phenotypes, 2 display NALD phenotypes, and 2 display IRD phenotypes. The deduced products of all mutant PEX12 alleles in these patients have been identified67,141,142,324,325 (Fig. 129-21). The five ZS patients carry an array of splice site, frameshift, and nonsense mutations that share one common feature: the inability to express PEX12 proteins that contain their zinc RING domain.145 In contrast, the NALD and IRD patients all express forms of PEX12 that do include this protein interaction module. The NALD patient PBD095 is homozygous for a mutation that deletes the leucine codon at position 68, L68Δ, and the NALD patient PBD054 is homozygous for a missense mutation in the zinc RING domain.67 The IRD patient PBD099 initially presented a paradox because this individual is a compound heterozygote for a severe splice-site mutation at the 5′ end of intron 1 and a 2-bp deletion in intron 1.67 However, translation initiation at an internal ATG codon appears to generate an active, N-terminally truncated form of PEX12 from the second allele, providing an explanation for the relatively mild phenotypes of this patient. The second IRD patient is a sibling of PBD099 and is presumed to have the same alleles and generate PEX12 activity through a similar mechanism.
Diagram of the consequences of all currently known PEX12 mutations on the PEX12 protein. The positions of transmembrane domain 1 (TM1) and the 2 (TM2) and the C-terminal RING domain are indicated. The horizontal rectangles indicate the wild type or mutant PEX12 protein; the thin horizontal black line extending from the C-terminal indicates peptide encoded by an alternative reading frame extending to the indicated premature stop codon (see references67,141, and 145 for details). The PBD numbers refer to specific PBD probands and their phenotypes are indicated in the parentheses below.
The PEX12-deficient NALD patient PBD054 was originally placed in CG7 by cell fusion complementation analysis.142,339 While this could represent an error of the cell fusion technique, we think not. PBD054 cells are phenotypically rescued by overexpression of PEX10, the gene that is normally defective in CG7, and express a missense mutation, S320F, in the PEX12 zinc RING domain, the region of PEX12 that physically interacts with PEX10.142 Furthermore, this mutation disrupted the ability of PEX12 to bind PEX10, as well as its interaction with PEX5. Thus, we feel that the placement of PBD054 in CG7 might be due to extragenic noncomplementation with the CG7 test cell line PBD052. It is interesting to note that PBD052 cells express a mutant form of PEX10 that has a missense mutation, H290Q, in its zinc RING domain,140,339 the region that interacts with zinc RING of PEX12.145
The analysis of PEX12-deficient patients provided the first detailed study of phenotype-genotype relationships in the PBDs.67 This study concluded that there is a relatively straightforward phenotype-genotype relationship in these diseases, with greater loss of gene function causing more severe defects in peroxisomal matrix protein import, which in turn lead to more pronounced metabolic, physiologic, and developmental deficits. Similar trends are apparent in other complementation groups of the PBDs, and are particularly evident from the analysis of PEX1-deficient patients (see below).68,155
PEX2 was the first PBD gene identified and is defective in patients from CG10 of the PBDs.101,102 The human PEX2 cDNA was cloned via functional complementation of a CHO pex mutant.101,341 The PEX2 gene has been mapped to chromosome 8q21.1 by FISH.342 Although we were unable to delineate the PEX2 gene structure from the literature, the mouse PEX2 gene has been characterized and contains 5 exons with exon 5 containing the entire PEX2 ORF.343 The product of the human PEX2 gene is a 35-kDa integral PMP.344 Like PEX10 and PEX12, PEX2 extends its N- and C-termini to the cytoplasm and carries a zinc RING domain within its cytoplasmically exposed C-terminus. Loss of PEX2 is associated with a severe defect in peroxisomal matrix protein import but no defect in peroxisome membrane synthesis or PMP import.23 Furthermore, cells lacking PEX2 display increased levels of peroxisome-associated PEX5, the PTS1 receptor,114 indicating that PEX2 functions in peroxisomal matrix protein import downstream of PTS receptor docking.
PEX2 deficiency can manifest with severe or mild phenotypes within the Zellweger spectrum and has been reported for seven patients (Fig. 129-22). The initial PEX2-deficient patient, variously referred to as MM,102 F-01,146,147 and PBD062,23,114,244 is homozygous for a nonsense mutation, R199X, that terminates translation less than half-way through the protein.102,147 Two other unrelated patients, PBD094 and PBD410, are also homozygous for the same R199X mutation.345 The ZS patient F-04 is a compound heterozygote for the R119X mutation and a distinct nonsense mutation, R125X.147 The ZS patient F-08 is homozygous for a frameshift mutation, 550delC, that truncates the PEX2 protein following the isoleucine at position 183, effectively deleting the second transmembrane span and the zinc RING domain.146
Diagram of the PEX2 protein. The transmembrane domain 1 (TMD1) and 2 (TMD2) and C-terminal RING domain are indicated. The locations of mutations that cause abnormal peroxisome biogenesis are indicated above. See references102 and 146 for details.
Peroxisomal matrix protein import was severely affected in all five PEX2-deficient ZS patients, but was only mildly affected in the two PEX2-deficient IRD patients, F-05 and F-06. F-05 is a compound heterozygote for the R119X mutation and a PEX2 missense mutation, E55K.147 Glutamate 55 is conserved in virtually all PEX2 proteins including those from protozoans, yeast, plants, insects, and mammals. F-06 is homozygous for a frameshift mutation, 642delG, that terminates translation following glutamine 214 and deletes the PEX2 zinc RING domain.146 Previous mutational studies of PEX2 reported that the zinc RING domain could be removed without affecting PEX2 activity and was therefore dispensable for function.346 The cellular and clinical phenotypes of F-06 demonstrate that loss of the PEX2 zinc RING domain does, after all, have a significant effect on PEX2 function, though such loss does not abrogate PEX2 activity altogether.
The PEX1 gene was first identified in yeast and was in fact the first PEX gene to be identified.159 The human PEX1 cDNA was identified independently by two groups using homology probing strategies155,321 and was also identified via functional complementation of a CHO pex mutant.323 The PEX1 gene resides on chromosome 7 (7q21-q22) and is marked by two sequence tagged sites, WI-16251 and sWSS3932.155,321 The PEX1 gene consists of 24 exons that are distributed over 150 kb of genomic DNA. PEX1 encodes a member of the AAA ATPase protein family and can be found both on the peroxisome and in the cytoplasm.347 PEX1 physically interacts with PEX6, another AAA ATPase that is required for peroxisomal matrix protein import,156,158 and overexpression of PEX1 or PEX6 can suppress defects in the other in an allele-specific manner.156 These two AAA ATPases appear to act late in peroxisomal matrix protein import.120 Loss of human PEX1 has no effect on peroxisome membrane synthesis or PMP import but reduces the efficiency of both PTS1 and PTS2 matrix protein import.23,155 PEX5, the PTS1 receptor, is destabilized in PEX1-deficient cells, indicating that PEX1 plays an important role in PEX5 function and matrix protein import.114
PEX1 deficiency is responsible for disease in approximately two-thirds of all PBD patients.18,68,155,348– 350 The high frequency of PEX1 deficiency appears to be caused by the presence of two mutated PEX1 alleles in the general population, one that has a PEX1 missense mutation, G843D,155 and another that carries a PEX1 frameshift mutation, 2097insT.68 These PEX1 alleles each represent approximately 30 percent of the PEX1 alleles, and one or the other of these alleles are found in roughly 80 percent of PEX1-deficient patients and in 50 percent of all Zellweger spectrum patients. Additional studies have since confirmed the high incidence of the G843D348,349 and 2097insT348 mutations in PEX1-deficient patients. The G843D and 2097insT mutations are associated with distinct haplotypes,68 indicating that they arose once during human evolution and have since expanded in the Caucasian population, the major contributor to our patient population.
The G843D substitution mutation is enriched in IRD and NALD patients.68,155 In fact, PEX1/G843D homozygotes are among the most mildly affected PBD patients known. One patient who appears to be homozygous for this mutation has survived into her forties and was capable of semi-independent living into her thirties.18 Using a functional complementation assay, Reuber et al.155 demonstrated that the G843D mutation does not eliminate PEX1 function and that the PEX1/G843D cDNA retains nearly 15 percent of WT activity. Geisbrecht et al.156 demonstrated that the G843D mutation reduced the ability of PEX1 and PEX6 to bind to one another in vitro and found that overexpression of PEX6 partially suppressed the PEX1/G843D mutation in vivo.
While the frequency of the PEX1/G843D mutation provides an explanation for the large numbers of mildly affected PEX1-deficient patients, the abundance of PEX1-deficient ZS patients is explained by the second common PEX1 mutation, 2097insT. This mutation was first described by Collins and Gould,68 who detected it in approximately two-thirds of the PEX1-deficient ZS patients and found that it comprised 30 percent of the PEX1 alleles in PEX1-deficent patients. The high frequency of the 2097insT mutation was subsequently confirmed in a group of Australian patients.348 This exon 13 frameshift mutation results in low steady state PEX1 mRNA levels,68,348 presumably due to nonsense mediated RNA decay.331,351 However, the 2097insT mutation does not appear to induce aberrant splicing of the PEX1 transcript.68 The 2097insT mutation is enriched in severely affected CG1 patients and all 2097insT homozygotes display ZS phenotypes.68 Using a functional complementation assay, Collins and Gould68 established that a PEX1/2097insT cDNA lacks detectable PEX1 activity.
In addition to these two common PEX1 mutations, numerous other mutations have been identified in the PEX1 gene, some of which are shown here (Fig. 129-23). These include a missense mutation, L664P,323 a 9-bp insertion in exon 12,155,321 a splice donor mutation in intron 18,155,321 a 14-bp insertion in exon 15,155 a nonsense mutation in exon 14,155 a 1-bp insertion in exon 20,155 and a splice donor mutation in intron 20.155
Diagram of the human PEX1 protein. The location of the two nucleotide binding folds (NBF1 and NBF2) are indicated, each comprised of a Walker A and B ATP-binding consensus sequence. The C-terminal AAA motif is indicated. The vertical lines indicate the consequences of the known PEX1 mutation. (See references68 and 155.)
Together, the PEX1 mutation data are consistent with the phenotype-genotype relationship suggested by the recessive nature of the PBDs and prior cell fusion complementation studies.16– 19 Specifically, mutations that are predicted to cause the most severe loss of gene function cause the most severe defects in peroxisomal matrix protein import and are associated with the most severe clinical phenotypes. That one or the other of the two common PEX1 mutations can be found in roughly 80 percent of PEX1-deficient patients and 50 percent of Zellweger spectrum patients indicates that genetic testing technologies may be useful in the PBDs.
Human PEX6 cDNAs were identified both by homology probing154 and by the CHO cell functional complementation assay.326 The PEX6 gene has been mapped to chromosome 6 both by RFLP analysis154 and by FISH, which placed it at 6p21.1.326 This gene contains 17 exons that are dispersed along 14 kb of genomic DNA.352 PEX6 encodes a 104-kDa member of AAA ATPase protein family154,326 that physically interacts with PEX1.156,158 It is therefore not surprising that loss of PEX6 results in phenotypes that are similar to those observed in PEX1 deficiency. PEX6-deficient cells display defects in peroxisomal matrix protein import that vary with allele severity but do not exhibit any detectable defect in peroxisome membrane synthesis or PMP import.23,154 PEX6 is required for proper stability of PEX5154 and acts late in peroxisomal matrix protein import.120
PEX6 deficiency is the second most common cause of the Zellweger spectrum disorders (Table 129-1).18,154,326 More than 20 PBD patients have been identified who are defective in PEX6. The peroxisomal matrix protein import defect in these patients varies from severe to mild, but in all cases, it is possible to detect slight import of peroxisomal matrix proteins. For example, Yahraus et al.154 described a PEX6-deficient ZS patient (PBD106) who lacked detectable levels of PEX6 mRNA and was a compound heterozygote for two frameshift mutations in PEX6, each of which would preclude the synthesis of its essential C-terminal AAA motif. Nevertheless, residual levels of PTS1 protein import could be detected in fibroblasts from this patient.
Additional studies have identified a number of PEX6 mutations in PBD patients, although there is no evidence for any common PEX6 alleles in the CG4 population. Fukuda et al.326 reported mutations in two ZS patients, one of whom was homozygous for a 1-bp insertion at nucleotide 511 of the PEX6 cDNA sequence (511insT) and another in whom a PEX6 splice site mutation was identified (IVS3 + 1G > A). More recently, Zhang et al.352 reported an extensive analysis of PEX6 gene structure in 10 PEX6-deficient patients. The mutations described include a variety of splice site, frameshift, nonsense, and missense mutations that are dispersed throughout the gene. Once again, the phenotype-genotype relationships appear to support a straightforward model in which mutations that are expected to create the most significant loss in protein function are associated with the most severe cellular and clinical phenotypes.
CG 8, the Unknown PEX Gene Deficiency.
The gene defect in CG 8 of the PBDs has yet to be reported. There are at least 11 known CG 8 patients and these individuals span the phenotypic range of Zellweger spectrum (Table 129-1).18 Cellular studies of CG 8 cells have found that they all have numerous PMP-containing peroxisomes but display a variable defect in peroxisomal matrix protein import.23,244 Furthermore, CG 8 cells have reduced levels of PEX5, the PTS receptor, a phenotype that is also observed in PEX1- and PEX6-deficient cells, but not in cells lacking PEX2, PEX7, PEX10, PEX12, or PEX16.114
Other Anticipated PEX Gene Deficiencies.
Although there is only one complementation group of PBD patients for which the gene is not yet known, it is likely that additional complementation groups will be identified in the future. Human homologs of other yeast PEX genes are likely candidates for being defective in these patients. Currently, PEX14,62– 64 PEX11α,28 and PEX11β are the only known human PEX genes that are not defective in at least one patient. In yeast, PEX14 participates in peroxisomal matrix protein import, and we would expect a similar role for its human homolog. Loss of yeast PEX11 does not affect peroxisomal protein import, making it unclear whether loss of human PEX11α or PEX11β would result in a disease phenotype.
There are a number of other PEX genes that are required for peroxisomal matrix protein import in yeast but for which the human PEX genes are not known, including the PEX4,119,151,152 PEX9,165 PEX15,166 PEX17,167 PEX18,133 PEX20,134 PEX21,133 PEX22,153 and PEX23168 genes and it is not a certainty that humans have all such genes. However, because these yeast genes all participate in peroxisomal matrix protein import, their human homologs would represent excellent candidates for a PBD gene.
Defects in Peroxisome Membrane Synthesis
Defects in peroxisome membrane synthesis have been observed in just four PBD patients, all of whom display ZS phenotypes. These patients are defective in any of three genes, PEX3, PEX16, and PEX19.
The human PEX16 cDNA was identified independently by two groups, in each instance by homology probing approaches.56,172 The PEX16 gene has yet to be mapped and its organization remains to be determined. The PEX16 product is an integral PMP 56 that extends its N- and C-termini into the cytoplasm. There is only one known PEX16-deficient patient, referred to as either PBD06156 or PBDD-01.172 This individual displayed severe ZS phenotypes and was homozygous for the nonsense mutation R176X, which eliminated PEX16 function in vivo. Fibroblasts from this patient lacked detectable peroxisomes, as determined by immunofluorescent staining with antibodies specific for the integral peroxisomal membrane protein, PMP70.56,172 By immunoblot, these cells lacked detectable levels of PMP70 and of a different integral PMP, P70R, and failed to import a wide variety of integral PMPs.56
Reexpression of the PEX16 cDNA restored peroxisome membrane synthesis and PMP import, followed by restoration of peroxisomal matrix protein import.56 Phenotypic rescue generally required at least 24 h and was not maximal until 72 h after transfection. In contrast, PEX16 targeting to peroxisomes in normal human fibroblasts could be detected as soon as 2 h after transfection.56 The extreme kinetic difference between PEX16 targeting to peroxisomes and PEX16-mediated peroxisome membrane synthesis suggests that the two processes occur by distinct mechanisms. In addition, the COPI-inhibitor brefeldin A had no effect on either PEX16 targeting to peroxisomes or PEX16-mediated peroxisome membrane synthesis.56
The human PEX19 cDNA was first identified as HK33, a housekeeping gene expressed in many tissues.353 The PEX19 gene localizes to chromosome 1q22,353,354 contains eight exons, and spans approximately 9 kb.354 Three splice variants of PEX19 have been identified, but their functional significance, if any, remains to be determined. PEX19 encodes a predominantly cytoplasmic, partly peroxisomal protein that binds a wide array of PMPs, including PMPs that are involved in peroxisome biogenesis and PMPs that function in metabolite transport.59 PEX19 binds PMPs with high affinity, displaying a Kd of 500 nM for the PMP Pex14.
A single PEX19-deficient ZS patient has been identified, PBDJ-01.58 Cells from this patient are homozygous for a frameshift mutation in PEX19, 764insA, that effectively deletes the C-terminal 45 amino acids from its product.58 PBDJ-01 cells, which have also been referred to as PBD399 cells,59 lack detectable peroxisomes, as determined by immunofluorescent staining with antibodies specific for a variety of integral PMPs.58,59 PMP70, PEX13, and PEX11β simply could not be detected in these PEX19-deficient cells and other PMPs that could be detected, such as PEX14, were mislocalized to their mitochondria. Reexpression of PEX19 restores peroxisome biogenesis in a stepwise fashion with peroxisome membrane synthesis and PMP import preceding peroxisomal matrix protein import.58
The human PEX3 cDNA was identified independently by two groups, each by homology probing using yeast PEX3 sequences as probes.190,355 The PEX3 gene is located at chromosome 6q23-24 and contains 12 exons that span approximately 14 kb.356 PEX3 encodes an integral PMP that physically interacts with PEX19, the putative PMP receptor.59,190 Two PEX3-deficient patients have been identified, PBD400 and PBD401, both of whom display severe ZS phenotypes.57 PBD400 fibroblasts are homozygous for a frameshift mutation, 542insT, that would be expected to delete the C-terminal half of PEX3. PBD401 fibroblasts are homozygous for a nonsense mutation, R53X, which would delete the C-terminal 5/6 of PEX3. Both mutations eliminate PEX3 activity.
Reexpression of PEX3 restores peroxisome biogenesis in both PBD400 and PBD401 cells.57 The earliest time at which rescue could be detected was 24 h after transfection. Complete phenotypic rescue was not maximal until 3 days after transfection. In contrast, PEX3 targeting to peroxisomes could be detected as early as 1 h after transfection, indicating that PEX3 import and PEX3-mediated peroxisome synthesis occur through kinetically distinct mechanisms. PEX3-mediated peroxisome biogenesis occurred in a stepwise fashion, with peroxisome membrane synthesis and PMP import occurring first and peroxisomal matrix protein import detected at later time points. PEX3-medated peroxisome synthesis was unaffected by inhibitors of COPI-mediated and COPII-mediated vesicular transport,57 raising even more doubts about the origin of the nascent peroxisomes during these instances of de novo peroxisome biogenesis.
Although we are relatively early in our understanding of the pathophysiology of PEX gene mutations, some correlations between clinical phenotypic severity, cellular phenotypic severity and genotype are emerging. In CG 1 of the Zellweger spectrum, a frequent PEX1 missense allele, G843D, has been associated with a mild phenotype.68,155 Homozygotes for G843D tend to be in the milder region of the Zellweger spectrum (NALD, IRD, and milder still) and, at the cellular level, their peroxisomes appear to import some amount of residual matrix proteins. By contrast, patients homozygous for the common PEX1 null allele, 2097insT, have typical ZS and their peroxisomes fail to import matrix proteins.68,348 Similarly, in CG 3, a PEX12 allele with an early frameshift is apparently rescued by translational initiation at an internal methionine yielding an N-terminal truncated PEX12 protein with residual activity (≈15 percent).67 A patient who was a genetic compound for this allele and a PEX12 null allele had an IRD phenotype and was the least affected of 7 CG 3 probands.
A similar pattern is suggested by early studies in RCDP. Again there is a common allele, PEX7 L292ter, which produces reduced amounts of a PEX7 protein with no residual function.129,131 This allele has a high frequency due to a founder effect and accounts for about half of all the PEX7 genes causing RCDP.132 Homozygotes for L292ter have a severe cellular defect in the peroxisomal import of PTS2 targeted matrix proteins and have the classical, severe RCDP clinical phenotype.129,132 By contrast, certain PEX7 missense alleles (A218V, S25F, H39P, G41V) encode mutant PTS2 receptors that have residual activity and are found in patients with milder phenotypic variants of RCDP.126,357 The N-terminal location of several of these suggests that this region of the PTS2 receptor may be more tolerant of variation. This prediction is supported by consideration of the structure of proteins homologous to PEX7.
The potential of postnatal treatment is limited by the multiple malformations and defects that originate in fetal life. Nevertheless, it may prove of value for patients with milder phenotypes. Wilson et al.197 and Holmes358 administered ether lipids orally to mildly affected patients with ZS, and achieved a partial normalization of red blood cell plasmalogen levels. Plasma VLCFA can also be normalized at least in part by a dietary regimen which has been tried in X-linked ALD.359 This regimen also minimizes phytanic acid intake and has brought about normalization of phytanic acid levels (H.W. Moser, unpublished observation). This dietary approach was used in some patients with the somewhat milder forms of disordered peroxisomes biogenesis.360 However, given the variability of disease, it has been difficult to evaluate effectiveness.
Two additional approaches were recently proposed, and both have demonstrated positive biochemical effects and anecdotal clinical benefit in studies that have involved single patients. Setchell et al.361 administered oral cholic and deoxycholic acids in a dosage of 100 mg each per day. They reported improved liver function and an unspecified improvement in neurologic status. Martinez et al.362 administered oral docosahexaenoic acid in a dosage of 250 mg/day to two patients, one patient died soon after the institution of therapy, the other, a 6-year-old boy with NALD, was reported to show improved alertness, motor performance, vocabulary, and visual-evoked responses. The same group also reported improvement in MRI.363 In contrast, others have reported that no demonstrable effect. The need for controlled trial of this therapy as well as others has been highlighted.364
Administration of clofibrate has failed to induce liver peroxisomes in Zellweger disease patients.240,365 An interesting observation was that the administration of 4-phenylbutyrate increased the number of peroxisomes in cells of patients with X-ALD.366
Often overlooked, symptomatic therapy has been of benefit in children with ZS, NALD, IRD, and RCDP.