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Metabolic and molecular basis of

peroxisomal disorders: A review
Ronald Wanders
American Journal of Medical Genetics

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 American Journal of Medical Genetics 126A:355 – 375 (2004)
Research Review
Metabolic and Molecular Basis of Peroxisomal Disorders:
A Review*
Ronald J.A. Wanders**
University of Amsterdam, Academic Medical Centre, Departments of Clinical Chemistry and Pediatrics, Emma Children’s Hospital,
Laboratory of Genetic Metabolic Diseases, Amsterdam, The Netherlands
The group of peroxisomal disorders now includes
17 different disorders with Zellweger syndrome as
prototype. Thanks to the explosion of new infor-
mation about the functions and biogenesis of
peroxisomes, the metabolic and molecular basis
of most of the peroxisomal disorders has been
resolved. A review of peroxisomal disorders is
provided in this paper. ß 2004 Wiley-Liss, Inc.
KEY WORDS: peroxisomes; genetic diseases;
phospholipids; fatty acids; Zell-
weger syndrome
INTRODUCTION
A characteristic feature of eukaryotic cells is the presence of
specialized subcompartments which all play their own distinct
role in cell physiology. In order to fulfil their separate
functions, the different subcellular compartments (mitochon-
drion, lysosome, nucleus, cytosol, and endoplasmatic reticu-
lum) are all equipped with a unique set of enzymes to carry out
their different tasks. After long being underrated as relic
organelles of little physiological relevance, the true function of
peroxisomes in cell physiology has become more clear in recent
years. Most of the functions of peroxisomes involve lipid
metabolism, and defects in one or more peroxisomal functions
are associated usually with severe clinical manifestations. The
prototype of the group of peroxisomal disorders is the cerebro-
hepato-renal syndrome or Zellweger syndrome (ZS), first
described in 1964 as a familial syndrome of multiple congenital
defects in two pairs of sibs [Bowen et al., 1964].
The group of peroxisomal disorders now includes 17 different
diseases listed in Table I. Although different classifications
have been proposed through the years, there is growing
Grant sponsor: The Princess Beatrix Fund; Grant sponsor: The
Dutch Foundation for Scientific Research (NWO); Grant numbers:
901-03-159, 901-03-133; Grant sponsor: The European Union;
Grant numbers: QLG1-CT-2001-1277, QLG3-CT-2002-00696.
*After this paper was submitted, the gene defective in the last
complementation group (Table II) was reported [Matsumoto et al.
(2003) Nat Cell Biol 5:454–460; Matsumoto et al. (2003) Am J
Hum Genet 73:233–246]. The gene involved, PEX26, codes for a
peroxisomal membrane protein which recruits the Pex1p–Pex6p
AAA ATPase complex to the peroxisomal membrane.
**Correspondence to: Prof., Dr. Ronald J.A. Wanders, Lab
Genetic Metabolic Diseases, Academic Medical Centre, Meiberg-
dreef 9, 1105 AZ Amsterdam, The Netherlands.
E-mail: r.j.wanders@amc.uva.nl
Received 30 June 2003; Accepted 9 September 2003
DOI 10.1002/ajmg.a.20661
ß 2004 Wiley-Liss, Inc.
consensus to use a classification in which two groups of
disorders are distinguished: (1) the disorders of peroxisome
biogenesis and (2) the single peroxisomal enzyme deficiencies.
It is the purpose of this review to update the reader on the
clinical, metabolic, and molecular aspects of peroxisomal
disorders. The major basic aspects of peroxisome biogenesis
and function will be discussed first.
Peroxisome Biogenesis
Identification of genes and proteins involved in peroxisomes
biogenesis has been helped by the conservation of peroxisome
biogenesis among eukaryotic species, from yeasts and plants to
mammals. In fact, studies in yeasts have set the stage followed
by the subsequent identification of corresponding human
genes and proteins. To avoid confusion, a unified nomenclature
has been agreed in which genes involved in peroxisome
biogenesis are called PEX-genes and the encoded proteins
peroxins.
The first principle of peroxisome biogenesis is that perox-
isomal proteins are encoded by nuclear genes, synthesized on
free cytosolic polyribosomes, and imported posttranslationally.
This applies to both peroxisomal matrix and peroxisomal
membrane proteins. The implication is that newly synthesized
peroxisomal proteins must have specific targeting signals
that direct them to and into peroxisomes. It turns out that
peroxisomal matrix proteins contain one of two different
signals called PTS1 and PTS2. The PTS1 signal is a C-terminal
tripeptide with the sequence Ser-Lys-Leu (SKL), or a con-
served variant thereof, used by most (>90%) of the human
peroxisomal proteins. A few matrix proteins are targeted by a
distinct signal, PTS2, located near the N-terminus which
conforms to the motif Arg/Lys-Leu/Val/Ile-X5-Gln/His-Leu in
which X denotes any amino acid (Fig. 1). The second principle of
peroxisome biogenesis is that peroxisomal proteins are taken
up into pre-existing peroxisomes thereby expanding the
peroxisomal compartment until a critical size is reached which
results either in fission of peroxisomes into two daughter
peroxisomes or in budding from the peroxisome reticulum
followed by subsequent growth. Conceptually, the complicated
process of peroxisome biogenesis can be subdivided into
different steps including: (1) recognition of the PTS1 and
PTS2 signals by soluble receptors; (2) docking of the receptors
loaded with the PTS1 or PTS2 protein to the peroxisomal
membrane; (3) dissociation of the receptor–ligand complexes
followed by transport of the ligand across the peroxisomal
membrane; and (4) recycling of the receptors. Two different
strategies have been productive in identification of human
genes involved in peroxisome biogenesis. The first strategy
involved homology probing making use of the information
available from the different genetic studies in yeasts. Com-
puter-based searches of mammalian sequence databases
with yeasts peroxin sequences have identified several human
homologs [Gould and Valle, 2000]. The second strategy
involved peroxisome-deficient Chinese Hamster Ovary
(CHO) cells and mammalian cDNA expression libraries to
complement the defect. These two strategies have led to
356 Wanders

TABLE I. The Peroxisomal Disorders

Disorder Abbreviation Protein involved Gene Chromosome MIM

Disorders of peroxisome
biogenesis
Zellweger syndrome ZS Peroxins PEX-genes Multiple loci 214100
Neonatal adrenoleukodystrophy NALD Peroxins PEX-genes Multiple loci 214110
Infantile Refsum disease IRD Peroxins PEX-genes Multiple loci 202370
Hyperpipecolic acidaemiaa HPA Peroxins PEX-genes Multiple loci 266510
Rhizomelic chondrodysplasia RCDP type 1 Pex7p PEX7 6q21-q22.2 215100
punctata type 1
Single peroxisomal enzyme/
protein deficiencies
X-linked adrenoleukodystrophy X-ALD ALDP ABCD1 Xq28 300100
Acyl-CoA oxidase deficiency ACOX1-deficiency Straight-chain acyl-CoA ACOX1 17q25.1 264470
(pseudo-neonatal ALD) oxidase (SCOX/
ACOX1)
D-bifunctional protein D-BP deficiency D-BP/MF2/MFEII/ HSD17B4 5q2 261515
deficiency D-PBE
2-methylacyl-CoA racemase Racemase AMACR AMACR 5p13.3-p12 604489
deficiency deficiency
Rhizomelic chondrodysplasia RCDP type 2 DHAPAT GNPAT 1q42.1-42.3 222765
punctata type 2 (DHAPAT
deficiency)
Rhizomelic chondrodysplasia RCDP type 3 ADHAPS AGPS 2q33 600121
punctata type 3 (alkylDHAP
synthase deficiency)
Refsum disease (phytanoyl-CoA ARD Phytanoyl-CoA PHYH/PAHX 10p15-p14 266500
hydroxylase deficiency) hydroxylase (PhyH)
Hyperoxaluria type 1 PH1 Alanine glyoxylate AGXT 2q37.3 259900
aminotransferase
(AGT)
Glutaric acidemia type 3 GA3 ? ? ? 231690
Mevalonate kinase deficiency MK-deficiency Mevalonate kinase MVK 12q24.1 251170, 260920
Acatalasaemia — Catalase CAT 11p13 115500
Mulibrey nanism MUL Trim37p TRIM 17q22-23 253250
a
At least the four original HPA-patients described by Gatfield et al. [1968], Burton et al. [1981], and Thomas et al. [1975] are now known to be affected by a
disorder of peroxisome biogenesis. As described in the text, additional HPA-patients have been described in literature in whom peroxisome biogenesis is
normal. The primary defect in these patients remains to be identified.

the identification of many human PEX-genes and allowed the
virtually complete resolution of the molecular basis of the
peroxisome biogenesis disorders (PBDs). Furthermore, our
understandings about the functions of peroxins have rapidly
grown. Figure 1 is a schematic representation of peroxisome
biogenesis in humans and the proposed function of the
different peroxins [Gould and Valle, 2000].
FUNCTIONS OF PEROXISOMES
Peroxisomes play an indispensable role in human metabo-
lism as stressed by the devastating consequences of the
absence of peroxisomes in Zellweger patients. The main
functions of peroxisomes in humans are briefly discussed
below.
Fatty Acid Beta-Oxidation
Peroxisomes contain a fatty acid b-oxidation machinery just
as mitochondria do. The mechanism of b-oxidation in the two
organelles is identical and involves four consecutive reactions:
dehydrogenation, hydration (of the double bond), dehydro-
genation again, and thiolytic cleavage. Through this four-step
mechanism a two-carbon unit is split from the fatty acid in the
form of an acetyl-CoA unit, which can then be degraded in the
citric acid (Krebs) cycle to produce CO2 and H2O plus energy
(ATP).
The peroxisomal and mitochondrial b-oxidation systems
have different functions as they catalyze the b-oxidation of
different fatty acids and fatty acid derivatives. Indeed mito-
chondria catalyze the b-oxidation of the bulk of fatty acids
derived from the diet including palmitate, oleate, linoleate, and
linolenate. Peroxisomes play an important role by oxidizing a
different set of fatty acids and fatty acid derivatives: (1) very-
long-chain fatty acids (VLCFA), notably hexacosanoic acid
(C26:0); (2) pristanic acid (2, 6, 10, 14-tetramethylpentadeca-
noic acid) as derived from dietary sources either directly or
indirectly (from phytanic acid) and; (3) di- and trihydroxycho-
lestanoic acid (DHCA and THCA). The latter two compounds
are intermediates in the formation of the primary bile acids,
cholic acid and chenodeoxycholic acid from cholesterol in the
liver (Fig. 2).
Another major function of the peroxisomal b-oxidation
system concerns ‘‘biosynthesis’’ of polyunsaturated fatty acids
including docosahexaenoic acid (C22:6o3). Recent studies have
established that the formation of C22:6o3 from linolenic acid
(C18:3o3) involves the active participation of the peroxisomal
fatty acid oxidation (FAO) system (Fig. 2). It should be noted
that the peroxisomal FAO system is only able to chain-shorten
fatty acids and not able to degrade fatty acids to completion.
This is most convincingly demonstrated for pristanic acid
which undergoes three cycles of b-oxidation in peroxisomes to
produce 4,8-dimethylnonanoyl-CoA which is then shuttled to
the mitochondria in the form of its carnitine-ester for full
oxidation to CO2 and H2O. The same is true for VLCFA which
are probably chain shortened to a medium-chain acyl-CoA-
ester followed by transport to the mitochondria as carnitine-
ester to allow complete oxidation to CO2 and H2O (Fig. 2).

Fig. 1. Current model of peroxisome biogenesis. PTS1-proteins synthe-
sized on free polyribosomes are recognized and bound by the PTS1-receptor
(either Pex5pS or Pex5pL). Pex5pS is capable of targeting PTS1-proteins to
the peroxisomal docking machinery with Pex14p as the first binding
partner, whereas Pex5pL is needed for PTS2-import with Pex7p as the
PTS2-receptor. In the absence of Pex5pL, the Pex7p/PTS2-protein complex
is unable to dock at the peroxisomal membrane. After binding of the Pex5pS/
PTS1-protein or Pex5pL/PTS1-protein/Pex7p/PTS2-protein complex to
Peroxisomal Disorders 357
Pex14p and subsequently to Pex13p, the complexes dissociate and the PTS1-
and PTS2-proteins (ligands) translocate across the peroxisomal membrane,
a process dependent on Pex2p, Pex10p, and Pex12p. The receptors Pex5pS,
Pex5pL, and Pex7p recycle back to the cytosol. Pex1p and Pex6p are
supposed to be involved in the latter process. Not shown are Pex3p, Pex16p,
and Pex19p which are involved in the correct targeting of peroxisomal
membrane proteins (PMP).
358 Wanders

Fig. 2. Schematic representation of the role of the peroxisomal b-oxidation system in the oxidation of VLCFAs, pristanic acid, tetracosahexaenoic acid
(C24:6o3), and di- and trihydroxycholestanoic acid (DHCA and THCA) and the interaction between peroxisomes and mitochondria, the ultimate site of
oxidation of the end products of peroxisomal b-oxidation to CO2 and H2O.

Fig. 3. Enzymology of the peroxisomal fatty acid b-oxidation system.
Human peroxisomes contain two acyl-CoA oxidases, one specific for straight-
chain fatty acyl-CoAs and called straight-chain acyl-CoA oxidase (SCOX),
and a second, specifically reactive with 2-methyl branched-chain fatty acyl-
CoAs (pristanoyl-CoA, di- and trihydroxycholestanoyl-CoA). The products
Figure 2 also shows chenodeoxycholoyl-CoA and choloyl-CoA
to be the products of b-oxidation of the bile acid intermediates
dihydroxycholestanoyl-CoA and trihydroxycholestanoyl-CoA.
In peroxisomes, chenodeoxycholoyl-CoA and choloyl-CoA are
Peroxisomal Disorders 359
of the two acyl-CoA oxidases, i.e., the enoyl-CoA esters of C26:0, pristanic
acid and di- and trihydroxycholestanoic acid, are handled by a single en-
zyme, i.e. D-bifunctional protein (D-BP) whereas two thiolases are involved
in the final cleavage step.
converted into taurine or glycine conjugates which are then
exported from peroxisomes into bile.
Figure 3 depicts the enzymology of the peroxisomal fatty acid
b-oxidation system. Human peroxisomes contain 2 acyl-CoA
360 Wanders
oxidases, one specific for straight-chain fatty acids like C26:0,
therefore called straight-chain acyl-CoA oxidase (SCOX or
ACOX1) and a second one, catalyzing the dehydrogenation of
2-methyl branched-chain fatty acids like pristanoyl-CoA and
di- and trihydroxycholestanoyl-CoA. The enoyl-CoA-esters of
C26:0, pristanic acid, DHCA, and THCA are all handled by a
single bifunctional enzyme harboring both enoyl-CoA hydra-
tase and 3-hydroxyacyl-CoA dehydrogenase activity. This
bifunctional protein named DBP is the single enzyme involved
in both oxidation of C26:0, pristanic acid, DHCA, and THCA.
The role of the originally identified L-bifunctional enzyme
(LBP) remains to be resolved.
Ether-Phospholipid Biosynthesis
Ether-phospholipids are a special class of phospholipids
which differ from the regular diacylphospholipids in an ether-
linkage rather than an ester-linkage at the sn-1 position of
the glycerol backbone. Two groups of ether phospholipids
can be distinguished with either an 1-O-alkyl or an 1-O-alk-10 -
enyl-linkage. The latter phospholipids (1-O-alk-10 -enyl-2-acyl-
phosphoglycerides) with an a, b-unsaturated ether-bond are
also known as plasmalogens. Platelet-activating factor (PAF;
1-O-alkyl-2-acetylglycerophosphocholine) is the best known
ether-phospholipid. Figure 4 depicts the enzymology of the
etherphospholipid biosynthetic pathway with part of the
enzymes localized in peroxisomes and another part in
the endoplasmatic reticulum. Biosynthesis of ether-phospho-
lipids starts in the peroxisome with production of dihydrox-
yacetonephosphate (DHAP) to acyl-DHAP as catalyzed by the
peroxisomal enzyme dihydroxyacetonephosphate-acyltrans-
ferase (DHAPAT), followed by the introduction of the typical
ether-bond by the enzyme alkyl-DHAP synthase. Both
enzymes are strictly peroxisomal. The third step is catalyzed
by the enzyme alkyl/acyl-DHAP:NAD(P)H oxidoreductase
which has a bimodel distribution in both peroxisomes and
endoplasmatic reticulum. The product alkylglycerol-3-phos-
phate (alkyl-G-3P) undergoes subsequent conversion into
plasmalogens.
Fatty Acid a-Oxidation
3-methyl-branched fatty acids cannot be b-oxidized directly
due to the presence of a methyl group at the 3-position. Instead,
3-methyl fatty acids first undergo a-oxidation thereby remov-
ing the terminal carboxyl group as CO2. One of the most
important 3-methyl-branched fatty acids is phytanic acid, long
known to accumulate in Refsum disease as well as in other
peroxisomal disorders (see below). The mechanism involved in
the oxidative decarboxylation of 3-methyl fatty acids has been
resolved in recent years. Figure 5 depicts the structure and
enzymology of the peroxisomal fatty acid a-oxidation system.
Phytanic acid first undergoes activation to phytanoyl-CoA, via
the long-chain acyl-CoA synthetase (LACS) present at the
outer aspect of the peroxisomal membrane and then enters
the peroxisomal matrix probably via a carrier protein. Once
inside peroxisomes, phytanoyl-CoA undergoes hydroxylation
followed by cleavage to pristanal and formyl-CoA. Subse-
quently, pristanal is converted into pristanic acid which is then
activated to pristanoyl-CoA. Pristanoyl-CoA undergoes three
cycles of b-oxidation in the peroxisome to produce 4,8-
dimethylnonanoyl-CoA which leaves the peroxisome as carni-
tine-ester to undergo full oxidation to CO2 and H2O in
mitochondria. Evidence holds that the complete pathway
from phytanoyl-CoA to 4,8-dimethylnonanoyl-CoA is intra-
peroxisomal (Fig. 5). Most enzymes involved in peroxisomal
fatty acid a-oxidation and their encoding genes have been
identified with the exception of pristanal dehydrogenase
[Wanders et al., 2001b,c].
Biosynthesis of Cholesterol
and Other Isoprenoids
Peroxisomes may play a major role in isoprenoid biosynth-
esis as enzymes like mevalonate kinase, phosphomevalonate
kinase, and mevalonate pyrophosphate decarboxylase have
been claimed to be localised in peroxisome. If the first part of
the de novo isoprenoid biosynthesis pathway from acetyl-CoA
to farnesylpyrophosphate is indeed localised in peroxisomes,
mevalonate kinase is a peroxisomal disorder (see footnote1
however).
Detoxification of Glyoxylate
Glyoxylate is a toxic metabolite as it is a substrate for the
ubiquitous enzyme lactate dehydrogenase which interconverts
pyruvate and lactate but also converts glyoxylate into oxalate.
Oxalate rapidly precipitates as calcium oxalate with severe
consequences for the tissues involved. Under normal condi-
tions, glyoxylate is rapidly converted into glycine via the
peroxisomal enzyme alanine glyoxylate aminotransferase
(AGT). In hyperoxaluria type 1, this enzyme is deficient,
glyoxylate accumulates, and is either oxidized to oxalate or
reduced to glycolate (Fig. 6).
L-Pipecolic Acid Oxidation
L-lysine is normally degraded via the saccharopine pathway
involving the sequential action of: (1) L-lysine: 2-oxoglutarate
reductase and (2) saccharopine dehydrogenase. However, L-
lysine may also be degraded through the L-pipecolic acid
pathway which may especially be important in the brain since
the activity of the saccharopine pathway is low in the brain. In
the L-pipecolic acid pathway, L-pipecolic acid is produced from
L-lysine in 2 steps, and then oxidized by L-pipecolate oxidase, a
peroxisomal enzyme in man [Mihalik et al., 1989; Wanders
et al., 1989]. The structure and function of the L-pipecolic acid
pathway remains incompletely understood.
THE PEROXISOMAL BIOGENESIS
DISORDERS (PBDs)
Zellweger Syndrome (MIM 214100)
ZS was first described by Bowen et al. [1964] in two pairs of
siblings. Subsequently, many additional patients have been
described in the literature (for reviews see [Heymans, 1984;
Wilson et al., 1986; Wanders et al., 1988a]). The clinical
presentation of ZS is dominated by the typical craniofacial
features and neurological aberrations. Facial features include
a large anterior fontanel with widely spaced sutures (96%), a
broad, full forehead (97%), micrognathia (100%), external ear
deformity (97%), low and broad nasal bridge (100%), shallow
orbital ridges (100%), and redundant skin folds in the neck
(100%). The neurological picture is dominated by profound
hypotonia (99%) resulting in poor sucking (97%) requiring
gavage feeding, depressed neonatal and deep tendon reflexes,
and a flat occiput. Other neurological abnormalities include an
abnormal Moro response (100%), hypo/areflexia (98%), and
seizures (92%). Ocular abnormalities including corneal cloud-
ing, congenital cataracts, and glaucoma, whereas Brushfield
spots and abnormal retinal degeneration are also frequently
encountered. The electroretinogram was extinguished in vir-
tually all patients.
1
A recent report by Hogenboom et al. [2004] provides convincing
evidence in favour of a cytosolic localisation of mevalonate kinase,
which implies that mevalonate kinase deficiency can no longer be
considered a peroxisomal disorder.
 Peroxisomal Disorders 361

Fig. 4. Pathway for the synthesis of etherphospholipids. Biosynthesis starts in the peroxisome by acyldihydroxyacetonephosphate (acylDHAP)
formation using dihydroxyacetonephosphate acyltransferase (DHAPAT), and then the ether-bond is introduced by alkyldihydroxyacetonephosphate
synthase (ADHAPS) generating alkylDHAP. Both DHAPAT and ADHAPS are strictly peroxisomal in contrast to the other enzymes.

The liver is enlarged (78%) and fibrotic (78%) with severe
micronodular cirrhosis. Renal cysts were observed in 78 of
80 patients who were studied pathologically [Heymans, 1984;
Wanders et al., 1988a]. Calcific stippling of the patellae and
other bones occurs in 50–60% of patients.
The most conspicuous pathological abnormalities are found
in the brain [Liu et al., 1976; Evrard et al., 1978; Aubourg et al.,
1985; Powers et al., 1989]. Macroscopic abnormalities include
deviant sulci and gyri such as deep, almost vertical parietal
clefts and pachymicrogyria. Microscopic abnormalities show
362 Wanders
Fig. 5. Structure and enzymology of the peroxisomal phytanic acid a-
oxidation system. Phytanic acid first undergoes activation to phytanoyl-
CoA, a reaction probably carried out by the long-chain acyl-CoA synthase
(LACS) present at the outer aspect of the peroxisomal membrane.
Phytanoyl-CoA then enters the peroxisome via an unknown mechanism
after which phytanoyl-CoA is hydroxylated to 2-hydroxyphytanoyl-CoA via
the enzyme phytanoyl-CoA hydroxylase. Subsequently, 2-hydroxyphyta-
noyl-CoA is cleaved by the enzyme 2-hydroxyphytanoyl-CoA lyase (2HPCL)
into formyl-CoA and pristanal, which is converted into pristanic acid via
an ill-defined aldehyde dehydrogenase. Pristanic acid is converted into its
CoA-ester, probably by the enzyme very-long-chain acyl-CoA synthetase
(VLACS) followed by b-oxidation.

Fig. 6. Metabolism of glyoxylate into glycine, glycolate, and oxalate. Under normal conditions, glyoxylate is converted into glycine within the peroxisome
via the enzyme alanine glyoxylate-aminotransferase (AGT). If AGT is functionally inactive, either through loss of catalytic activity or through
mislocalization to mitochondria, glyoxylate accumulates followed by oxidation to oxalate or reduction to glycolate.
two types of abnormalities: dysontogenetic and degenerative.
The former type affects neuromigration at different levels and
in different areas. The neocortex migration defect consists of
medium and large sized neurons immediately beneath the
molecular layer, marginal glial heterotopia, simple microgyri
with apparently fused first layers, microgyria of the cloverleaf
type, and subcortical and deep nodular neuronal heterotopia.
Germinolytic cysts in the subependymal areas of the cerebral
hemispheres relate to early loss of cells inhabiting the germinal
matrix. Purkinje cell heterotopia and dysplastic inferior
olivary nuclei are found. The degenerative features are those
of storage and cell degeneration and cell death: storage as
neutral fat in astrocytes, glycogen in cortical neurons, and
lamellar-lipid and lamellar membrane inclusions in lateral
cuneate and Clarke’s column neurons, cell degeneration in
cortical neuronal loss, fibrous gliosis, globoid cells, lipid
phagocytes, and glial nodules.
Neonatal Adrenoleukodystrophy (MIM 202370)
Another PBD is neonatal adrenoleukodystrophy (NALD),
first described by Ulrich et al. [1978] as connatal ALD in a boy
with hypotonia, convulsions, absent grasp reflexes, and little
spontaneous movements at birth. The patient had all the signs
characteristic for adrenoleukodystrophy including demyelina-
tion of the central nervous system (CNS) white matter, atrophy
of the adrenal cortex, ballooned adrenocortical cells, and
splinter-like lamellar elements composed of electron-dense
leaflets separated by a clear space. However, there were also a
number of CNS abnormalities not described in X-linked
adrenoleukodystrophy (XALD) such as cerebral heterotopias,
micropachygyria, and other gray matter changes reflecting
generalized cerebral maldevelopment. We know now that
XALD and NALD are different disorders, NALD belonging to
the PBDs and XALD to the single peroxisomal dysfunction
disorders [Kelley et al., 1986].
Infantile Refsum Disease (MIM 266510)
Infantile Refsum disease (IRD) was first described in 1982 by
two groups of investigators [Boltshauser et al., 1982; Scotto
et al., 1982]. Scotto et al. [1982] described three patients
including a 5-year-old boy with an enlarged liver, mental
retardation, sensorineural deafness, pigmented retina, anos-
Peroxisomal Disorders 363
mia, and dysmorphic features. Ultrastructural analysis of a
liver biopsy specimen revealed lamellar structures that
resembled those normally found in plant chloroplasts, which
are known to contain bound phytol. This prompted the authors
to measure plasma phytanic acid, which turned out to be highly
elevated (50–100 mg/ml: normal:
3 mg/ml). This finding led to
the name ‘‘infantile phytanic acid storage disease,’’ or IRD,
which name is confusing because of the implied relationship to
adult Refsum disease. The patients described by Scotto et al.
[1982] were later re-described in more detail by Poll-Thé et al.
[1987].
Further studies demonstrated that the accumulation of
phytanic acid is a phenomenon secondary to a defect in
peroxisome biogenesis. Indeed, hepatic peroxisomes are
severely diminished in number [Roels et al., 1986], plasmalo-
gen synthesis is impaired, very long chain fatty acids, bile acid
intermediates, and pipecolic acid are elevated and phytanic
acid oxidation is deficient. These data underline the conclusion
that ZS, NALD, and IRD are phenotypic variants resulting
from a defect in peroxisome biogenesis with partly overlapping,
partly divergent features [Aubourg et al., 1986; Kelley et al.,
1986]. This is exemplified by the autopsy findings in a patient
with IRD who had, at 11 years of age, severe growth re-
tardation, normocephaly, sunken eyes, tapetoretinal degen-
eration, high prominent nasal bridge, numerous ectatic veins,
redundant skin folds in the neck, and a small mouth [Torvik
et al., 1988]. The patient did not speak or walk, gave no reaction
to visual or sound stimuli, and was spastic with myoclonic fits.
There were no malformations of the cerebral cortex or
heterotopias, no cortical renal cysts, no skeletal changes, and
no liver damage. So this case [Torvik et al., 1988] also differed
from NALD in many respects.
Distinction between Zellweger syndrome, neonatal
adrenoleukodystrophy, and infantile Refsum disease.
Because of the phenotypic overlap among the PBDs it is often
difficult to classify patients, especially since phenotypic
variants have been described which do not belong to either
known PBD [Barth et al., 2001]. ZS, NALD, and IRD all have
liver disease, variable neurodevelopmental delay, retinopathy,
and perceptive deafness with onset in the first months of life.
In addition, patients with ZS are severely hypotonic and
weak from birth and have distinct facial features, periartic-
ular calcifications, severe brain dysfunction associated with
364 Wanders
neuronal migration disorder and die before 1 year of age.
Patients with NALD have hypotonia, seizures, may have
polymicrogyria, have progressive white matter disease, and
usually die in late infancy. Patients with IRD may have
external features reminiscent of ZS, no neuronal migration
disorder, and no progressive white matter disease. The
cognitive and motor development varies between severe global
handicaps and moderate learning disabilities with deafness
and visual impairment due to retinopathy. Major points of
difference [Raymond and Moser, 1997] are: (1) survival. In
general, the mean age of death of NALD patients is much later
compared to classical ZS, while in IRD patients survival to the
second decade is not uncommon. Several patients who are now
over 20 years of age are known to us; (2) NALD and IRD
patients have hypotonia and some degree of psychomotor
development. Most achieve some head control, are able to sit
up, and may achieve the ability to stand and walk indepen-
dently, and to engage in some form of alternative communica-
tion, although only a few develop the capacity to speak; (3)
NALD and IRD patients do not have the profound and
characteristic defect of neuronal migration as in ZS [Evrard
et al., 1978], although some milder degrees of migration defects
may be present; (4) facial dysmorphism is less severe in NALD
and IRD than in classical ZS; (5) neonatal seizures are present
in both ZS and NALD, but not in IRD; (6) renal cysts and
chondrodysplasia punctata are present in most patients with
ZS, but not in the milder variants. Children with NALD/IRD do
have evidence of a chondrodysplasia in short stature, flat face,
and delayed eruption of primary teeth; (7) severe hearing
impairment and pigmentary degeneration of the retina appear
to be present in all patients with the milder variants. Retinal
abnormalities are probably present in all patients with
classical ZS, but may not be recognized before the patients’
early death; (8) leukodystrophy may develop in the children
with the more mild phenotypes: variable age of onset, loss of
already acquired milestones, development of spasticity, and
progressive course often leading to death.
Hyperpipecolic Acidemia (pMIM 239400)
Several reports have appeared on what was first presumed
to be isolated hyperpipecolic acidaemia (HPA), the first one
[Gatfield et al., 1968] on a male infant with hepatomegaly,
retinopathy, and a progressive neurological disorder. Three
additional patients [Thomas et al., 1975; Burton et al., 1981]
had hepatomegaly, retinopathy, developmental delay, and
dysmorphic features. When L-pipecolic acid was later found to
be elevated in PBD patients too, these cases were re-evaluated
and in each a deficiency of peroxisomes was found [Wanders
et al., 1988b, 2001a]. Hepatic peroxisomes were stated to be
normal in the two Burton et al. cases [Challa et al., 1983], but
these results are hard to reconcile with data from several
groups showing a generalized loss of peroxisomal functions in
fibroblasts from the two Burton et al. cases and the absence of
punctate immunofluorescence indicating a true disorder of
peroxisome biogenesis. Others [Rao and Chang, 1992] reported
also that both L-pipecolate oxidase and acyl-CoA oxidase were
deficient in fibroblasts from the first Burton patient. Recently,
we have performed more extensive studies in fibroblasts from
the Thomas et al. case by complementation studies: this
allowed identification of the defective gene (PEX1) (Wanders
et al., unpublished). Based on these results, we conclude that
probably all above patients were affected by a peroxisome
biogenesis defect. HPA does not represent a distinct pheno-
type among the disorders of peroxisome biogenesis and has
become obsolete in the classification of such disorders, espe-
cially since true, isolated HPA may well exist. Indeed, in the
literature, several patients have been described with isolated
pipecolic acidaemia.
Increased pipecolic acid levels have been reported [Roesel
et al., 1991] in plasma and urine of a patient with Dyggve–
Melchior–Clausen syndrome, characterized by mental retar-
dation, and spondylometaepiphyseal dysostosis. We have since
tested more than 10 patients with this syndrome and have not
found elevated pipecolic acid levels suggesting that the earlier
finding may have been fortuitous.
Vallat et al. [1996] reported the serendipitous discovery of
major HPA in a 44-year-old man, his major health problem
being recurrent embolism possibly related to a moderate
fasting hyperhomocysteinemia (20 mmol/L; normal: <14).
Plasma pipecolic acid levels were 220–250 mmol/L (normal:
<2.5) in the presence of normal lysine levels. The chirality of
the pipecolic acid accumulating in the patient was determined
to be L-. The authors concluded that L-HPA may be a benign
trait. However, others [Kerckaert et al., 2000] identified three
patients with diverse neurological abnormalities all showing
isolated HPA. In two, the pipecolic acid in CSF was also
strongly elevated. Hepatic peroxisomes were normally pre-
sent. The exact consequences of isolated HPA remain to be
established.
Biochemical and molecular basis of ZS, NALD, and
IRD. Peroxisomes are strongly reduced in number and size,
or absent in ZS, NALD, and IRD. This causes a generalized loss
of peroxisomal functions in peroxisomal disorders, which is
consequently reflected in a series of abnormalities:
* Elevated plasma very long chain fatty acids: peroxisomes
are the site of oxidation of very long chain fatty acids and
oxidation of these fatty acids is deficient in fibroblasts of ZS,
NALD, and IRD-patients, although the extent of the defi-
ciency differs in the order ZS > NALD > IRD [Wanders et al.,
1995], which explains that on average VLCFAs are highest
in plasma from ZS-patients [Lazarow and Moser, 1995].
* Elevated plasma di- and trihydroxycholestanoic acid
(DHCA/THCA): in peroxisomes these acids normally under-
go chain-shortening to chenodeoxycholic acid and cholic
acid, respectively (Figs. 2 and 3). In peroxisomal disorders,
oxidation of DHCA and THCA is deficient, which leads to the
accumulation of these cholestanoic acids in plasma.
* Elevated plasma pristanic acid and phytanic acid: pristanic
acid b-oxidation and phytanic acid a-oxidation is restricted
to peroxisomes, which explains why pristanic acid and
phytanic acid accumulate in ZS, NALD, and IRD-patients.
However, levels may vary markedly and may even be
normal, which is explained by the dietary origin of phytanic
acid and pristanic acid (either as pristanic acid itself or as
phytanic acid).
* Elevated L-pipecolic acid: the deficient activity of L-pipecolate
oxidase in peroxisome-deficient cells leads to elevated
plasma L-pipecolic acid levels. The levels in plasma from ZS,
NALD, and IRD patients varies widely for unknown reasons.
* Deficient docosahexaenoic acid (DHA) levels in plasma and
erythrocytes: peroxisomes are essential in DHA-formation,
which explains the deficiency of DHA in Zellweger patients
although in milder affected patients levels may be normal.
The deficiency is usually more pronounced in erythrocytes
as compared to plasma.
* Deficient erythrocyte plasmalogens: fibroblasts studies
have shown that biosynthesis of etherphospholipids includ-
ing plasmalogens, is severely impaired in ZS-patients but
less so in NALD and IRD patients. Erythrocyte plasmalogen
levels are clearly deficient in ZS-patients but may only be
partially reduced or even normal in milder affected patients
[Wanders et al., 1986].
The increased knowledge on peroxisome biogenesis and the
proteins involved has also led to more insight in the genetics of

TABLE II. Complementation Groups in Peroxisome Biogenesis Disorders (PBD)
Complementation groups PTS import
Gifu KKI Adam PTS1 PTS2
A 8 VI  
B 7 (¼5) VII  
C 4 (¼6) III  
D 9 VIII  
E 1 II  
F 10 V   þ
G 12 IX  
H 13 X   þ
J 14 XI  
2 IV  **
3 XII  
R 11 I þ  þ
*See Addendum.
**A single patient has been described in literature in which only the PTS1 import route was blocked [see Gould and
Valle, 2000].
PBDs [Gould and Valle, 2000]. A major step was the application
of complementation analysis to the study of the genetic basis of
PBDs. A first study [Brul et al., 1988] performed with
fibroblasts from seven patients, revealed five different genetic
complementation groups (CGs) indicative of marked genetic
heterogeneity. A similar conclusion was reached in subsequent
studies [Roscher et al., 1989; Yajima et al., 1992]. A col-
laborative study showed that the CGs identified in the three
centres could be assigned to nine unique CGs [Shimozawa
et al., 1993], to which in subsequent years three additional
groups have been added. In 11 of the 12 GCs, the gene in-
volved has been identified [Gould and Valle, 2000], and the
PTS1- and PTS2 import routes as well as the phenotypes
associated with each CG are listed in Table II. One group,
number 12, is different from the 11 other groups in terms of its
associated cell biology (only the PTS2 pathway is defective).
Furthermore, this group is associated with a single peroxiso-
mal disorder, i.e., rhizomelic chondro-dysplasia punctata
(RCDP). We have performed complementation studies now in
301 PBD-patients of which all 80 RCDP Type 1 patients
belonged to group 12 and had a mutation in PEX7. The
remaining 221 patients, with phenotypes ranging from ZS to
IRD, were found to belong to the 11 remaining CGs: major
groups were 139 (63%) in the PEX1 group, 22 (10%) in the
PEX6-group and 12 (5.4%) in the PEX12-group (Gootjes and
Wanders, unpublished).
Mutation analysis has shown two common mutations in the
PEX1 gene in patients of the PEX1 group, including a missense
mutation (G843D) [Portsteffen et al., 1997; Reuber et al., 1997]
which causes a partial dysfunction of Pex1p, and a 1 bp in-
sertion (C.2097insT) [Maxwell et al., 1999] which causes a
complete loss of function of Pex1p. Most other mutations were
private. Interestingly, patients homozygous for the G843D
mutation invariably show a mild phenotype, usually of the
IRD-type, whereas patients homozygous for C.2097insT show
the severe Zellweger-type.
Rhizomelic Chondrodysplasia Punctata (RCDP)
Type 1 (MIM 215100)
Chondrodysplasia punctata (CDP) refers to a heterogeneous
group of bone dysplasias of which stippling of the epiphyses in
infancy is the common feature. Two major types are the
Conradi-Hünermann type (McKusick 118650) with autosomal
dominant inheritance and the autosomal recessive rhizomelic
type (McKusick 215100). In addition, X-linked recessive
(McKusick 302950) and X-linked dominant (lethal in males)
forms of chondrodysplasia punctata have been identified.
Peroxisomal Disorders 365
Gene
Ghosts involved Phenotypes
þ PEX 26* ZS/NALD/IRD
þ PEX 10 ZS/NALD/IRD
þ PEX 6 ZS/NALD/IRD
 PEX 16 ZS
þ PEX 1 ZS/NALD/IRD
PEX 2 ZS
 PEX 3 NALD
PEX 13 ZS/NALD
 PEX 19 ZS
þ PEX 5 ZS/NALD/IRD
þ PEX 12 ZS/NALD/IRD
PEX 7 RCDP
Peroxisomal abnormalities are only found in the rhizomelic
type. This type is clinically characterized by a disproportion-
ally short stature primarily affecting the proximal parts of the
extremities, facial features such as a broad nasal bridge, highly
arched palate, dysplastic ears, and micrognathia, congenital
contractures, and severe mental retardation with spasticity.
Roentgenological studies have shown symmetrical shortening
of femur and humerus with irregular and broad metaphysics,
calcific stippling mainly of the epiphyses, absent femur head
nucleus, coronal clefts of vertebrae, increased intravertebral
disc spaces, cupping of dorsal ribs, and a barrel-formed thorax.
Review of unpublished cases from our own center and patients
described in literature shows that calcific stippling of the
epiphyses and periarticular areas is present in all patients
below 18 months of age and disappears in older patients. The
femoral heads remain severely deformed and all patients also
lack a clear nucleus of the femur head. No coronal cleft was
noted in the majority of patients beyond 3 years of age. In most
cases the spinal column showed irregular and/or small discs
with increased distances between the vertebrae. In patients
beyond 10 years of age, radiographs of the chest show signs of
chronic infections and a barrel-shaped thorax with severe
torsion scoliosis.
Biochemical and molecular basis of RCDP type 1. The
identification of RCDP as a peroxisomal disorder dates back
to when Heymans et al. [1985] reported that erythrocyte
plasmalogen levels were markedly deficient in RCDP patients.
Phytanic acid was also strongly elevated suggesting the
involvement of two different peroxisomal pathways. Later
studies led to the identification of distinct peroxisomal
abnormalities including deficiencies in: (1) alkyl-DHAP
synthase; (2) phytanoyl-CoA hydroxylase; (3) DHAPAT (par-
tial deficiency); and (4) mature 41 kDa thiolase (only the
precursor 44 kDa form is present). This perplexing combina-
tion of deficiencies was resolved in 1997 when independently
three groups found PEX7 as the defective gene. A loss of Pex7p
function blocks the PTS2 import pathway, and explains the
deficiency of the enzymes. The reduced activity of DHAPAT
is the secondary consequence of the absence of alkyl-DHAP
synthase [de Vet et al., 1999]. RCDP is genetically hetero-
geneous (see below). However, in all RCDP patients identified
by us until now, erythrocyte plasmalogen levels have been
found deficient making plasmalogen analysis a highly reliable
start for diagnosis.
Molecular studies have identified many different mutations
[Braverman et al., 2002; Motley et al., 2002], although a
single mutation, L292ter, accounts for about half of the
mutant PEX7 genes in RCDP probands. Haplotype analysis
366 Wanders
has shown in every case that this mutation is on the same
background, strongly suggesting a founder effect in Northern
Europeans.
SINGLE PEROXISOMAL ENZYME DEFICIENCIES
The Disorders of Peroxisomal Beta Oxidation
X-linked adrenoleucodystrophy (MIM 300100). X-
linked adrenoleucodystrophy (X-ALD) is the most common
single peroxisomal disorder with a minimum incidence of
1:21,000 males in the USA [Bezman et al., 2001] to 1:15,000
males in France (Aubourg, cited in Kemp et al., 2001). The
phenotype varies widely. At present, at least six phenotypic
variants can be distinguished [Moser et al., 2000]. This
classification is somewhat arbitrary and based on age of onset
and the organs principally involved. The two most frequent
phenotypes are childhood cerebral ALD (CCALD) and adreno-
myeloneuropathy (AMN). Onset of CCALD is between 3 and
10 years of age with progressive behavioral, cognitive,
and neurologic deterioration, often leading to total disability
within 3 years. The cerebral phenotype is not only observed in
childhood but may also present later in life in adolescence
(adolescent cerebral ALD) or adulthood (adult cerebral ALD).
There is a marked difference between the cerebral phenotypes
and AMN since the cerebral phenotypes show an inflammatory
reaction in the cerebral white matter which resembles what
is observed in multiple sclerosis. In contrast to CCALD, the
inflammatory response is absent or mild in AMN which has a
much later age of onset (28  9 years) and a much slower rate of
progression. Nevertheless approximately 40% of AMN
patients do develop some cerebral involvement associated
with a more rapid downhill progression. It is important to
emphasize that approximately 40% of women heterozygous for
XALD develop AMN-like symptoms in middle age or later.
Cerebral involvement and adrenocortical insufficiency are
rare, however.
The biochemical hallmark of X-ALD is the accumulation of
VLCFAs in plasma and different cell types. Analysis of plasma
VLCFAs has proved to be a powerful diagnostic method with
very few if any false negatives [Moser et al., 1999]. In 15–20%
of obligate heterozygotes, however, test results are falsely
negative which makes mutation analysis the only reliable test
for heterozygote identification.
The accumulation of VLCFAs is due to the defective
oxidation of VLCFAs in peroxisomes. Indeed, when C26:0
beta-oxidation is studied in fibroblasts, the rate of oxidation in
X-ALD fibroblasts is 20–30% of control compared to a virtually
complete deficiency in ZS fibroblasts. The X-ALD-gene was
identified in 1993 using a positional cloning strategy [Mosser
et al., 1993]. The protein proved to belong to the superfamily of
ABC transmembrane transporter proteins. Functionally,
eukaryotic ABC transporters contain two homologous halves,
each with a transmembrane domain (TMD), and an ATP
binding domain (ATP). Most functional ABC transporters have
the following topology: TMD–ATP–TMD–ATP. In some, the
entire transporter is encoded by a single gene, in others two
ABC half-transporters homodimerize to generate a functional
ABC transporter. ALDP with one membrane domain and one
ATP binding domain has the structure of an ABC half-
transporter.
Three additional mammalian peroxisomal ABC half-trans-
porters have been identified, closely related by nucleic acid
and protein sequence: ALDRP; PMP70; and PMP69 or
PMP70R. These four transporters may form different hetero-
dimeric combinations in the peroxisomal membrane and are
probably involved in the transport of different metabolites.
Although definitive evidence is lacking, ALDP, as homodimer
or as heterodimer, seems to transport VLCFA across the
peroxisomal membrane either as free acid or as CoA-ester.
Mutation analysis of the ABCD1-gene has shown many
different, often private mutations [Kemp et al., 2001], which
can be studied in the mutation database for X-ALD (http://
www.XALD.nl).
Acyl-CoA Oxidase Deficiency
(Pseudo NALD) (MIM 264470)
Acyl-CoA oxidase deficiency was first described [Poll-Thé
et al., 1988] in two siblings with neonatal hypotonia, severe
delayed motor development, sensory deafness, and retinopa-
thy, without dysmorphisms. Both patients showed some initial
progress in the first 2 years of life followed by a progressive
neurological regression, including progressive hypodensity of
cerebral white matter in neuroimaging. This led to the presum-
ptive diagnosis neonatal adrenoleucodystrophy (NALD), and
elevated plasma VLCFA levels were found. However, in
contrast to NALD [Aubourg et al., 1986] levels of the bile acid
intermediates, phytanic acid, pristanic acid, and pipecolic acid
were normal. Furthermore, peroxisomes were found to be
present in liver biopsy specimens although they were enlarged
in size and decreased in number. These patients were found to
be deficient for acyl-CoA oxidase 1 (ACOX1), the first enzyme
involved in the b-oxidation of VLCFA. This explains the nor-
mal presence of all peroxisomal metabolites except VLCFAs
(Fig. 3). In a second family [Wanders et al., 1990], the index
patient showed generalized hypotonia, failure to thrive, deaf-
ness, hepatomegaly, psychomotor retardation, absent reflexes,
frequent convulsions, and demise at 3 11/12 year. Two ad-
ditional cases [Suzuki et al., 1994] had profound hypotonia,
dysmorphic features (hypertelorism, low nasal bridge, low-set
ears, and polydactyly), and initial moderate developmental
delay with later motor regression. Subsequently, three similar
cases were identified [Watkins et al., 1995].
Plasma abnormalities in acyl-CoA deficiency are restricted
to the accumulation of VLCFAs (Fig. 3). There is only a single
report in literature on the molecular basis of acyl-CoA oxidase
deficiency [Fournier et al., 1994] showing a large deletion in the
acyl-CoA oxidase 1 gene in the original patients described by
Poll-Thé et al. [1988].
D-Bifunctional Protein Deficiency
Bifunctional protein deficiency was first described in a
male with severe hypotonia and therapy refractory seizures
[Watkins et al., 1989]. No developmental progress was ob-
served. There was no hepatomegaly, fontanelles were large
with open metopic sutures, visual evoked responses and
brainstem auditory evoked responses were grossly abnormal,
and brain biopsy at 6 weeks showed polymicrogyria and
heterotopias. The patient died at 5 months of age. Autopsy
showed minute glomerular cysts in the kidneys, adrenal
atrophy with ballooned, lipid laden cells, and some portal
fibrosis in the liver. Based on the clinical features, neonatal
ALD was considered. In plasma, elevated levels of VLCFA
and bile acid intermediates levels were found, and there were
abundant peroxisomes in a liver biopsy. Immunoblot studies
subsequently revealed the absence of L-bifunctional protein
(LBP) in postmortem liver material. Many additional patients
have been described in the literature: data on 44 patients
[Wanders et al., 2001a] indicated that children show severe
central nervous system involvement with profound hypotonia
(98%), uncontrolled seizures (95%), and failure to acquire any
significant development (100%). Children are usually full term
and show no evidence of intrauterine growth retardation.
Dysmorphic features include macrocephaly, high forehead,
flat nasal bridge, low-set ears, large open fontanelles, and
micrognathia. Abnormal neuronal migration (88%) as poly-
microgyria, heterotopia, and other symptoms were found in
cerebrum and cerebellum [Kaufmann et al., 1996].

Two studies [Suzuki et al., 1997; van Grunsven et al., 1998]
independently identified the deficiency of D-bifunctional pro-
tein (D-BP). D-BP is involved in the peroxisomal b-oxidation of
all known peroxisomal substrates including VLCFAs, DHCA,
and THCA, and pristanic acid (Fig. 2) which explains the
accumulation of all metabolites in most patients. The group
of bifunctional protein deficiency is heterogeneous consisting
of three subgroups, explained by the two catalytic activities
(a hydratase and 3-hydroxyacyl-CoA dehydrogenase activity)
of the protein [Watkins et al., 1989]. Accordingly, in subgroup
A D-BP is completely absent causing loss of both activities
[van Grunsven et al., 1999a,b]. Subgroup B represents D-BP
enoyl-CoA hydratase deficiency and in subgroup C D-3-
hydroxyacyl-CoA dehydrogenase is functionally inactive
[van Grunsven et al., 1998]. Molecular studies have identified
a variety of mutations including a rather frequent mutation
found in patients belonging to subgroup C [Wanders et al.,
2001a].
Peroxisomal Thiolase Deficiency
(Pseudo Zellweger Syndrome) (MIM 261510)
In 1986, a girl with ‘pseudo-ZS’ was reported [Goldfischer
et al., 1986] showing marked facial dysmorphism, muscle
weakness, hypotonia, no psychomotor development, and
demise at 11 months. At autopsy, the patient had renal cysts,
atrophic adrenals with striated cells, minor or little fibrosis,
hypomyelination in the cerebral white matter, foci of neuronal
heterotopia, and a sudanophilic leucodystrophy. Plasma
analysis showed elevated VLCFAs, but peroxisomes were
found to be abundantly present in the liver. Subsequent
immunoblot analysis indicated selective deficiency of perox-
isomal thiolase suggesting peroxisomal thiolase deficiency.
However, re-investigation of this patient [Ferdinandusse et al.,
2002] disclosed that the true defect in this patient is not at the
level of peroxisomal thiolase but in the D-BP. As a consequence,
peroxisomal thiolase deficiency can no longer be considered a
distinct peroxisomal disorder.
Peroxisomal 2-Methylacyl-CoA Racemase
(AMACR) Deficiency
A new defect in the peroxisomal fatty acid beta-oxidation
pathway in patients suffering from an adult-onset sensory
motor neuropathy was recently identified [Ferdinandusse
et al., 2000]. Sensory motor neuropathy may occur in X-linked
adrenoleucodystrophy and Refsum disease, resulting from
accumulation of VLCFA and phytanic acid, respectively.
Analysis of two patients with adult onset sensory motor
neuropathy and additional signs suggesting Refsum dis-
ease and X-linked adrenoleucodystrophy showed normal
VLCFAs, marginally elevated phytanic acid, and definitely
increased levels of the 2-methyl branched chain fatty acids,
pristanic acid and di- and trihydroxycholestanoic acid
[Ferdinandusse et al., 2000]. This suggested a specific defect
in the peroxisomal beta-oxidation of branched-chain fatty
acids. Fibroblast studies showed complete deficiency of
racemase activity (AMACR). This enzyme is not directly
involved in beta-oxidation itself but is important in beta-
oxidation of both pristanic acid and di- and trihydroxychol-
estanoic acid. Subsequent molecular studies led to the
identification of mutations in the gene coding for AMACR
[Ferdinandusse et al., 2000].
DISORDERS OF FATTY ACID a-OXIDATION
Refsum Disease (MIM 266500)
Refsum disease was first delineated as a distinct entity
clinically by Sigvald Refsum in the 1940s under the name
Peroxisomal Disorders 367
heredopathia-atactica-polyneuritiformis. Cardinal manifes-
tations include retinitis pigmentosa, cerebellar ataxia, chronic
polyneuropathy, and an elevated CSF protein level. Less
constant features include sensory neural hearing loss, anos-
mia, ichtyosis, skeletal malformations, and cardiac abnormal-
ities. The clinical picture of Refsum disease is often that of
a slowly developing, progressive peripheral neuropathy man-
ifested by severe motor weakness and muscular wasting,
especially of the lower extremities. Onset has occasionally
been detected in early childhood, is observed most before
20 years, and can be in the 5th decade in others [Wanders et al.,
2001b].
The accumulation of phytanic acid in Refsum patients was
discovered in the early 1960s [Wanders et al., 2001b], but the
enzyme deficiency of phytanoyl-CoA hydroxylase [Jansen et al.,
1997], and mutation analysis [Jansen et al., 2000] were more
recent. Studies have shown that in a subset of patients the
defect is not in phytanoyl-CoA hydroxylase but involves PEX7
[Braverman et al., 2002; van den Brink et al., 2003].
DISORDERS OF ETHERPHOSPHOLIPID
BIOSYNTHESIS
Rhizomelic Chondrodysplasia Punctata (RCDP)
Type 2 DHAPAT-Deficiency) (MIM 222765)
RCDP was first found to be genetically heterogeneous in
1992 when isolated DHAPAT-deficiency was described in a
patient showing symptoms of RCDP [Wanders et al., 1992]. All
craniofacial abnormalities were present, he was extremely
hypotonic, had cataracts, and pronounced rhizomelic short-
ening especially of upper limbs. Radiologically all abnormal-
ities consistent with RCDP were seen except for stippled
calcifications of patellae and acetabulum. Deficient erythro-
cyte plasmalogens but normal phytanic acid level were found.
Fibroblast studies showed isolated DHAPAT-deficiency with
normal values for alkyl DHAP synthase, phytanic acid a-
oxidation, and peroxisomal thiolase 1 [Wanders et al., 1992]. A
second patient [Barr et al., 1993] also had the severe
phenotype, but two sibs [Clayton et al., 1994] who have another
affected sib at follow-up [Sztriha et al., 1997] were less severely
affected, and a mild case has been reported [Moser et al., 1995;
Elias et al., 1998] who lacked rhizomelia and craniofacial
dysmorphism.
In all patients with RCDP type 2, DHAPAT activity is
severely deficient, which is associated with the inability to
synthesize ether-phospholipids including plasmalogens.
This is reflected in deficient plasmalogen levels in all tissues
including erythrocytes. In all patients studied so far, erythro-
cyte plasmalogen levels have been clearly deficient. The
identification of the human DHAPAT cDNA [Thai et al.,
1997] allowed molecular studies showing a variety of muta-
tions [Ofman et al., 1998]. In severely affected cases [Wanders
et al., 1992; Barr et al., 1993], we have found mutations (848 TT
insertion and 567del204bp deletion, respectively) that cause
complete absence of DHAPAT activity, in mildly affected pa-
tients [Clayton et al., 1994; Sztriha et al., 1997] a homozygous
632G!A mutation was found, which gave some residual
activity in DHAPAT-activity. Therefore, there may be some
phenotype/genotype correlation.
Rhizomelic Chondrodysplasia Punctata
(RCDP) Type 3 (Alkyl DHAP Synthase
Deficiency) (MIM 600121)
A first case of isolated alkyl DHAP synthase deficiency was
found in a patient with classical RCDP, completely deficient
erythrocyte plasmalogen levels, and normal plasma phytanic
acid levels [Wanders et al., 1994]. Fibroblast studies indicated
368 Wanders
absent alkyl DHAP synthase and normal DHAPAT, phytanoyl-
CoA hydroxylase, and peroxisomal thiolase. Three unpub-
lished patients have been identified by Dr. H.W. Moser
(Baltimore), who also show classical RCDP with early demise.
Another patient [de Vet et al., 1999] is less affected and is still
alive at 7 years, showing mild rhizomelia, generalized con-
tractures, inability to sit or crawl, cataract, seizures, and
profound developmental delay.
In the original patient [Wanders et al., 1994], alkyl DHAP
synthase was fully deficient, but in a subsequent patient [de
Vet et al., 1999] not only alkyl DHAP synthase was deficient
but also DHAPAT activity was low (15%). Complementation
studies indicated that the patient did belong to RCDP group 3.
Antibodies against the alkyl DHAP synthase [de Vet et al.,
1997] showed that alkyl DHAP synthase and DHAPAT form a
complex within the peroxisome [Biermann et al., 1999a,b].
Detailed further studies [de Vet et al., 1997, 1998, 1999;
Biermann et al., 1999a,b] have shown that the deficient
activity of DHAPAT in the De Vet case is secondary to the
complete absence of the alkyl DHAP synthase protein, and in
the Wanders patient the protein is normally expressed and
localized in peroxisomes but functionally inactive (due to a
G1256A mutation). As DHAPAT is stable only in a complex
with alkyl-DHAP synthase within the peroxisome (Fig. 4),
DHAPAT activity is normal in the Wanders patient but
deficient in the De Vet patient [Ofman et al., 1998, 1999].
Hyperoxaluria Type 1 (Alanine Glyoxylate
Aminotransferase Deficiency) (MIM 259900)
Primary hyperoxaluria describes two rare disorders char-
acterized by recurrent calcium oxalate nephrolithiasis and
nephrocalcinosis, frequently leading to progressive renal in-
sufficiency and death before the age of 20 years. In both forms,
there is excessive synthesis and excretion of oxalate. In type 1
(glycolic aciduria), excessive amounts of glyoxylic and glycolic
acids are also found in the urine, in type II (L-glyceric aciduria),
large amounts of L-glyceric acid but normal amounts of
glyoxylic and glycolic acid are excreted. The enzymatic basis
in the two types involves the peroxisomal enzyme alanine:
glyoxylate aminotransferase in PH1 and the cytosolic enzyme
D-glycerate dehydrogenase/glyoxylate reductase in PH2,
respectively.
PH1 is clinically variable both in symptoms and timing. In
most patients, first symptoms start before 5 years of age. There
is an enormous spread, however, in the ages at which the
disease becomes apparent, which can be as early as the first
year of life or may happen much later in life even beyond the
fifth decade. Deposition of calcium oxalate within the kidney
parenchymatous tissue (nephrocalcinosis) or the renal pelvis/
urinary tract (urolithiasis) continues until eventually renal
function is impaired causing subsequent sequelae like uremia
and systemic oxalosis. The failure to clear oxalate from the
body leads to its further deposition in almost all areas of the
body affecting multiple tissues in addition to the kidneys and
urinary tract including the myocardium with heart block,
myocarditis, and cardioembolic stroke, the nerves (peripheral
neuropathy), bone (bone pain, multiple fractures, osteosclero-
sis), the eyes (retinopathy, optic atrophy), bone marrow, and
other organs [Danpure and Purdue, 1995].
The primary defect of PH1 is a mutation in the alanine
glyoxylate aminotransferase gene. As a consequence, perox-
isomal glyoxylate accumulates and glyoxylate is either oxidiz-
ed to oxalate, which precipitates as calcium oxalate or is
reduced to glycolate (Fig. 6). Definitive diagnosis requires a
liver biopsy since the enzyme is only expressed in liver. In most
patients (60%), there is virtually no residual AGT activity but
in the remainder, activities are between 2 and 48% of controls
[Danpure and Purdue, 1995]. In most of the patients belonging
to this last group, AGT is mistargeted to the mitochondria
where the enzyme may well be active but incapable to detoxify
the glyoxylate produced in the peroxisome, and, thus, patients
with relatively high residual activities still present with a
severe form of hyperoxaluria Type 1 [Danpure et al., 1993].
The identification of the cDNA [Takada et al., 1990] and
the resolution of the exon/intron structure of the AGT gene
[Purdue et al., 1991] has allowed molecular studies which
have shown a series of different mutations [Danpure and
Purdue, 1995].
Glutaric Aciduria Type 3 (MIM 231690)
A single patient was described at 11 months of age because of
failure to thrive and postprandial vomiting [Bennett et al.,
1991]. She was found to be homozygous for b-thalassemia but
also had significant glutaric aciduria. Fibroblasts showed
normal glutaryl-CoA dehydrogenase activity and glutaryl-CoA
oxidase activity was not detectable. This study has not been
followed up at the protein or DNA level. No additional cases
have been identified so far.
Mevalonate Kinase Deficiency
(MIM 251170 and MIM 260920)
Mevalonate kinase deficiency is the only disorder involv-
ing the peroxisomal part of isoprenoid biosynthesis. There
is the classical form of mevalonate kinase deficiency (MIM
251170) associated with profound developmental delay, facial
dysmorphism, cataract, hepatosplenomegaly, lymphade-
nopathy, and early death [Hoffmann et al., 1993], and
mevalonate kinase deficiency has been observed in hyperim-
munoglobulinaemia D and periodic fever syndrome (HIDS;
MIM 260920) [Drenth et al., 1999; Houten et al., 1999a,b]
showing recurrent episodes of fever associated with lympha-
denopathy, arthralgia, gastrointestinal dismay, and skin
rash. In HIDS patients, mevalonate kinase was found to
be strongly reduced (1.2–3.4% of normal) but not fully
deficient as in classical mevalonate kinase deficiency [Drenth
et al., 1999; Houten et al., 1999a,b]. Laboratory diagnosis
involves measurement of mevalonic acid in urine, mevalonate
kinase activity measurements in leukocytes, platelets, or
fibroblasts, and eventually molecular analysis [Waterham,
2002].
Acatalasemia (MIM 115500)
Acatalasemia is a rare disease which has mainly been
described in Japan and Switzerland. In Japan, acatalasaemia
is associated with ulcerating, often gangrenous, oral lesions
whereas these abnormalities were not seen in Swiss patients
[Eaton and Mouchou, 1995].
Mulibrey Nanism (MIM 253250)
Muscle-liver-brain-eye (mulibrey) nanism (MUL) is an
autosomal recessive disorder with severe growth failure of
perinatal onset, characteristic dysmorphic features, pericar-
dial constriction, and hepatomegaly. Other common features
include J-shaped sella turcica, yellowish dots in the ocular
fundi, slight muscular weakness, cutaneous naevi flammei,
and enlargement of cerebral ventricles. Psychomotor develop-
ment of patients with Mulibrey nanism is within normal limits.
Mulibrey nanism occurs world-wide but is most common in the
Finnish population. Mutations in the TRIM37 gene (previously
designated MUL), a RING-B-box-coiled-coil subfamily of zinc-
finger proteins, on chromosome 17q22-23, underlie Mulibrey
nanism [Avela et al., 2000]. Recent studies [Kallijarvi et al.,
 Peroxisomal Disorders 369

2002] have shown that the TRIM37 protein is localized in
peroxisomes. The possibility that the disorder may be a
peroxisomal disorder was considered earlier [Schutgens et al.,
1994], although normal plasma VLCFA and normal peroxiso-
mal functions in fibroblasts were found. It seems that TRIM37
is not involved in one of the known peroxisomal functions
and does not produce any abnormalities similar to other
peroxisomal disorders and its true function remains to be
established.
III. Rhizomelic chondrodysplasia punctata. This includes all
types.
III. X-linked adrenoleucodystrophy complex. This includes all
types.
IV. The remaining peroxisomal disorders. This includes
Refsum disease, 2-methylacyl-CoA racemase (AMACR)
deficiency, hyperoxaluria type 1, glutaryl-CoA oxidase
deficiency, mevalonate kinase deficiency, acatalasaemia,
and Mulibrey nanism.

LABORATORY DIAGNOSIS OF Laboratory Diagnosis of the Disorders

PEROXISOMAL DISORDERS
No single laboratory test is capable of identifying all
peroxisomal disorders. The selection of laboratory analyses
should be based on the clinical presentation of the patient
involved. We have introduced the concept of diagnostic groups
[Wanders et al., 1996] in order to develop logical guidelines
for the laboratory diagnosis of the various peroxisomal
disorders. Although not further specified here prenatal diag-
nostic laboratory tests can be done in virtually all peroxisomal
disorders.
These diagnostic groups are:
III. the disorders of peroxisome biogenesis and of the per-
oxisomal beta-oxidation. This group include ZS, NALD,
IRD, acyl-CoA oxidase deficiency, and D-bifunction pro-
tein deficiency, but not X-ALD and AMACR-deficiency.
of Diagnostic Group I
The similarity in clinical presentation of these two groups
of disorders dictate the grouping (Table III). VLCFA are
abnormal in all disorders listed: we have now studied over
>500 patients with either a defect in peroxisome biogenesis or
a peroxisomal beta-oxidation disorder, and VLCFA-levels have
always been abnormal. Normal VLCFA-levels rule out a PBD
or peroxisomal beta-oxidation defect. If VLCFAs are abnormal,
additional tests have to be performed for further discrimina-
tion. This includes analysis in erythrocytes (plasmalogens) and
plasma (bile acid intermediates, pristanic acid, and phytanic
acid). If erythrocytes are available and plasmalogen levels are
deficient, the patient definitely suffers from a ‘‘disorder of
peroxisome biogenesis’’. This should be followed by detailed
studies in fibroblasts, to establish whether the defect is ex-
pressed in fibroblasts, for complementation studies, and to

TABLE III. Biochemical Characteristics of Different Peroxisomal Disorders

Diagnostic group

1 2 3

RCDP RCDP RCDP

ZS NALD/IRD AOXD DBPD type 1 type 2 type 3 X-ALD

Plasma
Very-long chain fatty acids " " " " N N N "
Di- and trihydroxy " " N "a N N N N
cholestanoic acid
Phytanic acid N-"b N-"b N N-"b,d N-"b N N N
Pristanic acid N-"c N-"c N N-"c N N N N
Erythrocytes
Plasmalogen level # N N N # # # N
Liver
Peroxisomes Deficient Deficient Present but Present but Present Present Present Present
abnormal abnormal
Fibroblasts
Plasmalogen synthesis # # N N # # # N
DHAPAT # # N N # # #e N
Alkyl DHAP synthase # # N N # N # N
C26:0 b-oxidation # # # # N N N #
Pristanic acid b-oxidation # # N # N N N N
Acyl-CoA oxidase 1 # # # N N N N N
D-bifunctional protein # # N # N N N N
Phytanic acid a-oxidation # # N N # N N N
Phytanoyl CoA hydroxylase # # N N # N N N
Peroxisomes Deficient Deficient Present but Present but Present Present Present Present
abnormal abnormal
Abbreviations: ZS, Zellweger syndrome; NALD, neonatal adrenoleukodystrophy; IRD, infantile Refsum disease; AOXO, acyl-CoA oxidase 1 deficiency;
DBPD, D-bifunctional protein deficiency; RCDP, rhizomelic chondrodysplasia punctata.
a
Di- and trihydroxycholestanoic acid are not elevated in all DBPD-patients (see [van Grunsven et al., 1999a,b]).
b
Phytanic acid is derived from dietary sources only and may, therefore, vary from normal to elevated in patients in whom phytanic acid a-oxidation is
deficient.
c
Pristanic acid is derived from dietary sources only either directly or indirectly from phytanic acid via a-oxidation and may, therefore, vary from normal to
elevated if pristanic acid b-oxidation is deficient.
d
Phytanic acid is often elevated if pristanic acid b-oxidation is impaired even if phytanic acid a-oxidation per se is normal.
e
In all patients with RCDP type 3, except one [Wanders et al., 1994], DHAPAT is deficient as a consequence of its instability in the absence of alkyl DHAP
synthase (see text).
370 Wanders

Fig. 7. Flow chart for the differential diagnosis of patients suffering from a peroxisome biogenesis disorder (PBD) or a peroxisomal b-oxidation disorder
(POD).
 Peroxisomal Disorders 371

Fig. 8. Flow chart for the differential diagnosis of rhizomelic chondrodysplasia punctata (RCDP) type 1, 2, and 3.

establish the gene defective in this patient. If plasmalogen the milder disorders of peroxisomal biogenesis (mainly IRD)
levels are normal, a disorder of peroxisomal beta-oxidation can [Wanders et al., 1986]. In such cases, the question whether one
be expected (Fig. 7). However, the erythrocyte plasmalogen is dealing with a disorder of peroxisomal b-oxidation or a
levels may be completely normal in patients affected by one of disorder of peroxisome biogenesis, can only be resolved via
372 Wanders
detailed studies in fibroblasts (Fig. 7). Furthermore, identifica-
tion of the precise defect in the peroxisomal b-oxidation
pathway can also be resolved through studies in fibroblasts
only (Fig. 7).
Laboratory Diagnosis of the Disorders
of Diagnostic Group II
The clinical similarities of the three RCDP types warrants
inclusion into a single diagnostic group. The biochemical
characteristics of these disorders are listed in Table III.
Erythrocyte plasmalogens are deficient in each type, so this
is a reliable initial laboratory test. Our experience in >100
RCDP-patients shows that erythrocyte plasmalogen levels
were always deficient even in milder affected cases as in the
patient described by Smeitink et al. [1992]. As shown in the
flowchart of Figure 8, detailed fibroblast studies are required
for further discrimination. Only in Type 1 also other perox-
isomal functions including phytanic acid alpha-oxidation are
impaired. Furthermore, peroxisomal thiolase is in its 44 kDa
precursor form in RCDP type 1 but in its mature 41 kDa form in
types 2 and 3. Molecular analysis of the PEX7 gene has to follow
[Braverman et al., 1997, 2002; Motley et al., 1997, 2002;
Purdue et al., 1997]. Discrimination between types 2 and 3
requires measurement of both DHAPAT and alkylDHAP
synthase in fibroblasts homogenates, followed by molecular
analysis [Wanders et al., 2001a].
Laboratory Diagnosis Of Diagnostic Group
III (X-Linked Adrenoleucodystrophy)
Studies in hundreds of patients have shown that analysis of
plasma VLCFA is a reliable initial test to verify whether a
patient is affected by X-ALD. If abnormal, one may proceed
with fibroblasts studies and molecular analysis. Fibroblasts
studies, however, are not obligatory and direct molecular
studies in blood cells can be performed too.
Heterozygote detection is not as straightforward. Plasma
VLCFA-levels have been found to be normal in about 15%
of obligate heterozygotes. We advocate to perform direct
molecular studies and omit VLCFA-analysis in families in
which a molecular defect has been established. In case the
family history is negative, first plasma VLCFA-analysis can
be performed, if needed followed by fibroblast studies includ-
ing immunofluorescence using antibodies against the ALD-
protein. This method may especially be rewarding since the
product of the mutant X-ALD allele often produces an unstable
protein so that in heterozygotes a mosaic pattern is observed
[Kemp et al., 2001].
Laboratory Diagnosis of Disorders of Group IV
Disorders of group IV are clinically heterogeneous and all
require separate laboratory test for diagnosis described below.
Refsum disease. In most, but not all Refsum disease
patients phytanoyl-CoA hydroxylase is deficient leading to the
impaired degradation of phytanic acid. As phytanic acid is
derived from dietary sources, plasma phytanic acid levels may
vary widely. However, normal phytanic acid levels in proven
Refsum patients are not reliably reported, and plasma phy-
tanic acid analysis appears to be a reliable test for Refsum
disease. Definite diagnosis requires measurement of phyta-
noyl-CoA hydroxylase in fibroblasts and molecular analyses
[Jansen et al., 2000].
Primary hyperoxaluria type 1 (PH1). In patients with
hyperoxaluria type 1, alanine glyoxylate-aminotransferase is
deficient which leads to a block in glyoxylate detoxification,
and, hence, oxidation to oxalate or reduction to glycolate. In
most, but not all patients, there is increased urinary excretion
of all three acids. Definitive diagnosis of PH1 requires a liver
biopsy for assessment of AGT-activity.
Glutaric aciduria type 3 (glutaryl-CoA oxidase defi-
ciency). In the single patient with this defect there was
increased urinary excretion of glutaric acid not due to glutaric
aciduria type 1 or type 2. Glutaryl-CoA oxidase was subse-
quently found to be deficient.
Mevalonate kinase deficiency (MK-deficiency). In the
classic form of MK-deficiency, urinary mevalonic acid is
extremely high in all patients. If abnormal, mevalonate kinase
activity should be measured in blood cells (leukocytes, lym-
phocytes, thrombocytes) or in fibroblasts, followed by molecu-
lar studies [Houten et al., 1999a,b]. In hyper IgD-periodic fever
syndrome (HITS), mevalonate kinase is also deficient, but
urinary mevalonic acid may be completely normal, which
implies that diagnosis should be based on direct enzyme
analysis in white blood cells, platelets, and/or fibroblasts.
Peroxisomal 2-methyl-CoA racemase (AMACR) defi-
ciency. Patients with a deficiency of AMACR are unable to
degrade pristanic acid and the bile acid intermediates. Plasma
level studies should, therefore, include analysis of pristanic
acid by GC-MS or bile acid intermediates preferably by
tandem-MS. If abnormal, fibroblast studies, including mea-
surements of racemase activity, and molecular studies should
be performed [Ferdinandusse et al., 2000].
ACKNOWLEDGMENTS
The author gratefully acknowledges Mrs. Maddy Festen, Iet
van der Gracht, and Susan Gersen-van Zadel for expert pre-
paration of the manuscript and Mr. Jos Ruiter for preparation
of the figures.
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