Peroxisome Biogenesis Defects

18.1 Clinical Features

and Laboratory Investigations Peroxisome biogenesis disorders are genetically het- erogeneous diseases with an autosomal recessive mode of inheritance. They include Zellweger syn- drome (ZS, also called cerebrohepatorenal syn- drome), neonatal adrenoleukodystrophy (NALD), and infantile Refsum disease (IRD). The clinical pic- tures of these disorders show similarities, but an im- portant difference is a difference in severity, the clin- ical course being most severe in ZS and mildest in IRD. Exceptional patients present with a still milder phenotype.

After birth children with ZS show profound mus- cular hypotonia. Most patients lie motionless with weak or absent Moro reflex, tendon reflexes, and sucking and swallowing reflexes. Tube feeding is nec- essary. Typically, the children have craniofacial dys- morphism with a high and bulging forehead, flat occiput, upslanting palpebral fissures, puffy eyelids, hypoplastic supraorbital ridges, and a low and broad nasal bridge with hypertelorism and epicanthus folds, giving them a mongoloid appearance. In addi- tion, Brushfield spots, peripheral pigmentary retinopathy, optic nerve dysplasia or hypoplasia, glaucoma, corneal clouding, cataracts, low-set mal- formed ears, high arched palate, micrognathia, and widely patent sutures and fontanels are present.

Macrocephaly may be present. Some children have a cleft soft palate. The children have a severe visual and hearing deficit. Nystagmus is often present. He- patomegaly, prolonged neonatal or later-onset icterus, and hemorrhages due to hypoprothrombine- mia are common. Limb anomalies include cubitus valgus, camptodactyly, single transverse palmar creases, and talipes equinovarus. Failure to thrive and severe psychomotor retardation are conspicuous.

Seizures are frequent. Cardiac defects are not fre- quent, but ventricular septum defect, patent ductus arteriosus, and patent foramen ovale may occur.

Cryptorchidism is frequently observed in boys, cli- toromegaly and labial hypoplasia in girls. About 90%

of the patients die within the first year of life, death occurring in the majority within the first few months.

Most children with NALD have neurological ab- normalities at birth, but some are initially near-nor- mal. Hypotonia is moderate to severe, reflexes are hy- poactive. Craniofacial dysmorphism is milder than in

ZS. Widely patent fontanels are uncommon. The af- fected children show feeding problems, failure to thrive, hepatomegaly, and sometimes jaundice. Fur- thermore, the disease is characterized by seizures, sensorineural hearing loss, decreased vision with nystagmus, optic atrophy or dysplasia, and pigmen- tary retinal degeneration. The posterior eye segment abnormalities are identical to those of ZS, but anteri- or segment abnormalities are lacking. Macrocephaly may be present. Within the first year of life severe de- velopmental retardation becomes apparent, although most infants reach some milestones before neurolo- gical deterioration occurs. The age at which regres- sion begins varies from 12 months to more than 7 years. Progressive neurological dysfunction is char- acterized by cerebellar ataxia, spasticity of the arms and legs with truncal hypotonia, increased deep ten- don reflexes, extensor plantar reflexes, and sensory defects. If not present from birth onwards, seizures usually occur in this period. Visual dysfunction pro- gresses to blindness. Adrenal insufficiency is rarely clinically manifest. Exceptional patients are initially entirely normal or close to normal with onset of rapid neurological deterioration in the second year of life.

The course of disease is more protracted in NALD than in ZS, death occurring between the ages of 1.5 and more than 10 years.

IRD is the mildest variant of the three mentioned disorders. The disease is usually not manifest at birth but presents itself within the first 6 months of life with psychomotor retardation, minor facial dysmor- phism, mild hypotonia, sensorineural deafness, and visual impairment with retinal pigmentary de- generation, optic dysplasia, and nystagmus. Hepato- megaly and failure to thrive with growth retardation are common. Seizures occur but epilepsy is not as severe as in ZS. Many patients are able to sit and walk independently after several years, whereas others never acquire these abilities. The gait is usually ataxic and broad-based, and cognitive function in the se- verely retarded range. Life expectancy is considerably longer than in ZS and NALD, up to more than 2 decades.

Milder variants of peroxisome biogenesis defects with later onset and more protracted disease course have been described. One of the mildest variants reported concerns a family in whom adults with a normal or borderline intelligence only demonstrate sensorineural hearing loss and retinitis pigmentosa,

Peroxisome Biogenesis Defects

leading to a diagnosis of Usher syndrome (Raas- Rothschild et al. 2002).

In ZS, laboratory investigations may reveal many different, in themselves nonspecific biochemical ab- normalities, such as hyperbilirubinemia, elevated liv- er enzymes, hypoprothrombinemia, reduced albumin level, hypocarnitinemia, hypocholesterolemia, gener- alized amino aciduria, and elevated CSF protein.

These abnormalities are not necessarily all present.

Elevated serum iron, iron saturation, and transferrin may be found, but these findings are inconsistent and transient. As a rule, an abnormally low cortisol re- sponse to ACTH stimulation is found despite normal basal cortisol level. More specific abnormalities are directly related to generalized deficiency of peroxiso- mal function. Plasma levels of very long-chain fatty acids are increased with an elevation of the C

:C

and C

:C

fatty acid ratio. Saturated as well as mo- nounsaturated and polyunsaturated very-long-chain fatty acids are increased. Plasma pipecolic acid and phytanic acid may be normal initially but increase with age. Plasma dicarboxylic acids are raised.Abnor- mal bile acids such as dihydroxycholestanoic acid and trihydroxycholestanoic acid are elevated. In urine elevated levels of dicarboxylic acids, dihydroxyc- holestanoic acid, and trihydroxycholestanoic acid are present. In platelets and red blood cells a deficiency of the peroxisomal enzyme dihydroxyacetone phos- phate acyltransferase can be shown. The plasmalogen content of red blood cells is decreased in the first few months of life. The plasmalogen level of the red blood cells increases with age and may be normal in pa- tients who are 4 months or older. The synthesis of platelet activating factor by leukocytes is deficient.

The activity of dihydroxyacetone phosphate acyl- transferase can be shown to be deficient in leukocytes and thrombocytes. In cultured fibroblasts a de- creased content of plasmalogen and increased levels of very-long-chain fatty acids can be demonstrated.

The b-oxidation of very-long-chain fatty acids, the de novo biosynthesis of plasmalogens, and the activity of dihydroxyacetone phosphate acyltransferase, alkyl dihydroxyacetone phosphate synthase, and phytanic acid oxidation can be shown to be deficient in fibro- blasts. Finally, in fibroblasts catalase activity is not found in organelles, but in the cellular cytoplasm.

Laboratory abnormalities are essentially the same in NALD and IRD, but milder than in ZS. In ZS the accumulation of very-long-chain fatty acids includes the saturated and monounsaturated C

fatty acid and is associated with a decrease in the C

saturated fatty acid concentration. In NALD the rise in very-long- chain fatty acids is not associated with an increase in monounsaturated C

fatty acid and the C

fatty acid is on average higher than normal. In NALD, mild adrenal insufficiency and low cortisol response in ACTH stimulation is usually found, although appar-

ently normal adrenal function is not incompatible with the diagnosis. In IRD adrenal function is nor- mal.

X-ray examinations in ZS often reveal calcific stip- pling of bony epiphyses. Stippled, irregular calcifica- tion of particularly the patellae, greater trochanters, triradiate cartilages, acetabulum, scapula, and ster- num is seen in 50–70% of the ZS patients. Ultrasound may detect multiple small renal cortical cysts, but they are often difficult to find. In NALD and IRD no calcific stippling of bony epiphyses is present. No renal cysts are found. ERG is extinguished at a very early age in ZS and the EEG is highly abnormal with epileptic discharges. BAEP shows reduced responses.

In NALD and IRD the ERG becomes extinguished and BAEP is abnormal. Nerve conduction velocity may be decreased in NALD; it is normal in IRD.

Prenatal diagnosis can be performed with the help of various biochemical investigations in cultured chorionic villus cells and amniocytes. DNA tech- niques can be applied in families with known muta- tions.

18.2 Pathology

Brain weight in ZS is normal or may exceed normal.

External examination reveals abnormalities in the cerebral convolutional pattern with areas of pachy- gyria and polymicrogyria. Polymicrogyria is typical- ly present in the opercular regions of the frontal, pari- etal, and temporal lobes and within the insular re- gion, with an increased number of gyri and decreased amplitude of the gyri. In the superior region, over the frontoparietal convexities, the polymicrogyric cortex merges with a pachygyric cortex, where convolutions are abnormally broad and reduced in number. There is a failure of full opercularization of the insula and the sylvian fissure is abnormally vertical in orienta- tion. Otherwise the gyral pattern of the cerebral hemispheres is normal. Often the cerebellum is hy- poplastic and the cerebellar cortex has areas of polymicrogyria. External appearance of the brain stem is normal.

On sectioning the gyral abnormalities are con- firmed with moderate thickening of the cortical plate in the areas of abnormal gyration. The lateral ventri- cles are mildly enlarged and have a mildly colpo- cephalic configuration. There is a moderate symmet- rical decrease in volume of the white matter. Olfacto- ry bulbs and tracts and optic nerves are thin, as is the corpus callosum. Periventricular subependymal cysts, so-called germinolytic cysts, are often present over the heads of the caudate nucleus. In the brain stem, hypoplasia and dysplasia of the inferior olives is apparent.

18.2 Pathology 155

Microscopic examination reveals that the cerebral cortex has a normal cytoarchitectonic pattern in the normally convoluted areas. The cytoarchitecture of polymicrogyric and pachygyric cortex is abnormal and there are heterotopias in the subcortical white matter under the regions of cortical abnormality. The migrational abnormality has principally affected neurons destined for the outer cortical layers. Many cells normally found in the outer cortical layers (lay- ers II and III) are distributed in heterotopic positions within the deep cortical layers (layers V and VI) and in the white matter below the cortex. However, some of the neurons destined for the deep cortical layers are in their normal laminar positions. The difference between the pachygyric and polymicrogyric cortices is related to differences in intracortical cell patterns.

The outer cortical layers are relatively more cellular in polymicrogyric cortex than in pachygyric cortex, whereas the reverse is true with regard to the deeper cortical layers. Within the cortex a marked astrocytic gliosis is present. Within the proliferated astrocytes, an excess of sudanophilic lipid material is seen. PAS- positive deposits are seen in the cortex of some of the patients related to glycogen deposition in the cyto- plasm and nucleoplasm of neurons and astrocytes.

Clusters of unusual histiocytes and multinucleated giant cells have been described with cytoplasm that is in part homogeneously PAS-positive and in part foamy and sudanophilic.

The white matter is characterized by a profound deficiency of myelin and presence of gliosis. Myelin deficiency is caused by delayed and disturbed myeli- nation; active loss of myelin cannot be demonstrated.

Throughout the white matter many astrocytes are present which are hypertrophic and contain sudano- philic lipid material in their cytoplasm. Lipid deposits are also seen in histiocytes and macrophages. No perivascular infiltration with inflammatory cells is present. The white matter abnormality is usually dif- fuse and generalized. In some cases the periventricu- lar white matter is most severely involved with no myelin left in that area. Oligodendrocytes are reduced in number. There is a decrease of axons in the white matter corresponding to an area of pachygyria or polymicrogyria. The ventricles are partially denuded of ependyma and regionally the ependyma is a pseu- dostratified columnar epithelium resembling the epi- thelium seen in midfetal life. In many patients germi- nolytic cysts are present in subependymal areas.

The basal ganglia have a normal configuration, but at microscopic examination changes similar in nature to those in the cortex are seen, with an increase in astrocytes and the presence of lipid-laden cells. The optic nerves, chiasm, and optic tracts may show dif- fuse deficiency of myelin and presence of gliosis. The brain stem is normal in architecture, except for hy- poplasia and dysplasia of the inferior olives, which

lack the usual delicately convoluted pattern. The brain stem is poorly myelinated. A diffuse increase in the number of astrocytes is found in gray and white matter and lipid-laden foamy cells are present. The cerebellum is often hypoplastic and its convolutional pattern abnormal. Areas of polymicrogyria are com- mon and microscopic examination shows that corti- cal lamination is markedly abnormal at these points.

Many heterotopic Purkinje cells are present in the subcortical white matter. A marked increase in astro- cytes is present in the cerebellar cortex, and many glial cells and macrophages have vacuolated foamy cytoplasm filled with lipid droplets. The changes in the cerebellar white matter are similar to those in the white matter of the cerebral hemispheres. The dentate nuclei may be hypoplastic and dysplastic, showing the same lack of convolutions as observed in the infe- rior olivary nuclei.

Electron microscopy reveals that the lipid-laden cells contain typical trilamellar inclusions along with heterogeneous material. These inclusions are in- tralysosomal. The trilamellar structures are com- posed of two parallel electron-dense lines separated by an electron-lucent zone. They are identical to those encountered in other peroxisomopathies and may contain very-long-chain fatty acids. Structurally ab- normal mitochondria have been reported in some cases.

Following birth hepatic cirrhosis may develop

rapidly, although not invariably. The liver is enlarged

in most ZS patients. Histological findings vary from

near-normal to diffusely abnormal, dependent on the

age of the patient. In the first 2 months of life, micro-

scopic abnormalities are absent or mild and include

fibrosis, cholestasis, and intrahepatic bile duct hy-

poplasia. In older patients fibrosis and distortion of

liver architecture is more severe, ending in micro-

nodular cirrhosis. Inconstant excess of hemosiderin

in hepatocytes and in particular in Kupffer cells and

macrophages may be related to age, with the greatest

prominence between 5 and 18 weeks of age. In excep-

tional cases, accumulation of glycogen is found. On

ultrastructural examination, no peroxisomes are seen

in hepatocytes, where they are normally found in

abundance. Invariably, abnormal mitochondrial mor-

phology is seen. Intralysosomal trilamellar inclusions

are present in Kupffer cells and macrophages. In the

kidney multiple small cysts are present in the cortex,

especially in the subcapsular region. The average di-

ameter is 3 mm, with some reaching 8 mm. They are

predominantly of tubular, occasionally of glomerular

origin. Ultrastructural examination reveals no perox-

isomes in renal tubular epithelium, where they are

normally found in large numbers. Adrenal glands are

either normal in size or small. The medulla is unre-

markable. In the zona reticularis and inner fascicula-

ta large striated cells are seen with dense inclusions.

On ultrastructural examination, these cells contain trilaminar inclusions. Hyperplasia of pancreatic islets and pancreatic fibrosis have been observed in isolat- ed patients. In muscle tissue myopathic changes and presence of abnormal mitochondria are occasionally observed.

In NALD brains, the gyrational abnormalities are much milder than in ZS. Areas of polymicrogyria and pachygyria, in particular in the area of the sylvian fissure, and a few islands of heterotopic neurons are seen within the white matter. The cerebral cortex is otherwise normal. Few cerebellar heterotopias are seen. Inferior olives may be dysplastic.

White matter degenerative changes are, however, much more severe in NALD than in ZS. Diffuse de- myelination involves cerebellar white matter, brain stem, and cerebral hemispheres. Demyelination oc- curs in zoned lesions in X-linked adrenoleukodystro- phy, but is more diffuse in NALD. The process is most severe in the cerebellar white matter, the pyramidal tracts in the internal capsule, brain stem and spinal cord, and in the parieto-occipital region. The cerebral arcuate fibers are relatively spared. In the areas of demyelination, the axons are relatively intact, but if demyelination is severe, axons may also be destroyed and cavitation may occur. In the affected areas gliosis is present and there is an accumulation of lipids, predominantly in histiocytes and macrophages, but little, if any, in astrocytes. Perivascular infiltration with mononuclear inflammatory cells may be pre- sent, but is then less severe than in X-linked adreno- leukodystrophy. On ultrastructural examination tri- lamellar inclusions have been found in vacuoles with- in histiocytes and macrophages in areas of demyeli- nation. A polyneuropathy with thin myelin sheaths and trilamellar inclusions in Schwann cells and fibro- blasts has been found in some of the patients.

Liver disease in NALD is less severe than in ZS. He- patic fibrosis and micronodular cirrhosis may occur, but are not obligatory. Evidence of glycogen deposits may be present. Mitochondria have been described as either normal or abnormal in morphology. In most cases, absence or a marked reduction in number and size of peroxisomes has been reported, but enlarged hepatic peroxisomes have also been found. Histio- cytes and macrophages that contain lipid storage ma- terial are present and may be abundant. On ultra- structural examination these cells are shown to con- tain the typical trilamellar inclusions. No renal corti- cal cysts are present. The adrenal cortex is atrophic and contains ballooned, lipid-laden cells. Electron microscopy reveals trilamellar inclusions in adreno- cortical cells and in macrophages. Myopathic changes and mitochondrial abnormalities have been found in muscle tissue.

NALD is characterized by a generalized, systemic infiltration of many tissues by lipid-laden macro-

phages not seen in ZS. The storage cells harbor tri- lamellar inclusions and heterogeneous material. They are seen in multiple sites of the reticuloendothelial system and are not confined to sites of active degen- eration such as the CNS. They occur in liver, spleen, lungs, lymph nodes, and gastrointestinal mucosa.

In IRD no malformations of the cerebral cortex are present and no neuronal heterotopias within the white matter. The white matter may be hypoplastic.

White matter changes are mild. Myelin content is diminished, but there are no signs of active demyeli- nation. In the areas of myelin deficiency and gliosis, one finds macrophages surrounding vessels and con- taining trilaminar lamellae. There may be cerebellar atrophy with reduced numbers of granular cells.

In the liver in IRD fibrotic changes may be present.

Trilamellar lipid inclusions are seen in macrophages, Kupffer cells, and hepatocytes. Peroxisomes are ab- sent or reduced in number and size. Mitochondria have abnormal morphology. No cortical renal cysts are seen. No adrenal degeneration is present. Foamy histiocytes may be seen in multiple organs.

18.3 Pathogenetic Considerations

The assembly of peroxisomes requires the interaction of a set of biogenesis proteins, peroxins, which are en- coded by PEX genes. Peroxisomal matrix proteins are synthesized on free polyribosomes and are directed to the peroxisome by specific targeting signals, PTS1 and PTS2. Most peroxisomal proteins use PTS1;

phytanoyl-CoA hydroxylase, alkyldihydroxyacetone phosphate synthase, and peroxisomal thiolase are the only three peroxisomal enzymes that use a PTS2 targeting sequence. PTS1 consists of a C-terminal tripeptide, SKL, that is recognized by the PTS1 recep- tor. There is a third mechanism for importation of peroxisomal membrane proteins (PMPs).

In peroxisome biogenesis disorders there is a defect in peroxisomal membrane synthesis or the matrix protein import. Complementation studies by somatic cell fusion studies have been extremely important in the elucidation of the basic defects in peroxisome biogenesis defects. In complementation studies cultured skin fibroblasts from different pa- tients are fused and the resulting multinucleated cells are collected. Complementation is said to have oc- curred when the multinucleated cells show a restora- tion of function or structural features that were defi- cient in the unfused cell lines. Eleven different com- plementation groups have so far been identified for ZS, NALD, and IRD, and the underlying genes for the complementation groups are: PEX1 (complementa- tion group 1 or E), PEX2 (group 10 or F), PEX3 (group 12 or G), PEX5 (group 2), PEX6 (group 4 or C), PEX10 (group 7 or B), PEX12 (group 3), PEX13 (group 13 or

18.3 Pathogenetic Considerations 157

H), PEX16 (group 9 or D), PEX19 (group 14 or J), and PEX26 (group 8 or G). Almost all complementation groups are associated with more than one clinical phenotype and often with all three. Rhizomelic chon- drodysplasia punctata patients belong to a separate complementation group (complementation group 11), related to the gene PEX7.

PEX1, located on chromosome 7q21–22, encodes Pex1p, a protein of the AAA ATPase family involved in peroxisome matrix protein import (AAA stands for ATPases associated with diverse cellular activities).

PEX2, located on chromosome 8q21.1, encodes Pex2p, an integral peroxisomal membrane protein involved in matrix protein import. PEX3, located on chromo- some 6q23–24, encodes Pex3p, a peroxisomal mem- brane protein factor for the proper localization of peroxisomal membrane proteins and involved in peroxisomal membrane biogenesis. PEX5, located on chromosome 12p13, encodes the PTS1 receptor, in- volved in peroxisome matrix protein import. PEX6, located on chromosome 6p21.1, encodes Pex6p, a pro- tein of the AAA ATPase family involved in peroxi- some matrix protein import. PEX10, located on chro- mosome 1p36, encodes Pex10p, an integral peroxiso- mal membrane protein involved in peroxisome ma- trix protein import. PEX12, located on chromosome 17q11–12, encodes Pex12p, an integral peroxisomal membrane protein involved in matrix protein import.

PEX13, located on chromosome 2p14–16, encodes Pex13p, an integral peroxisomal membrane protein involved in matrix protein import. PEX16, located on chromosome 11p12–2, encodes Pex16p, an integral peroxisomal membrane protein involved in peroxiso- mal membrane biogenesis. PEX19, located on chro- mosome 1q22, encodes a peroxisomal membrane protein receptor involved in membrane biogenesis.

PEX26 encodes Pex26p, a peroxisomal membrane protein involved in peroxisome matrix protein im- port. It recruits Pex1p and Pex6p AAA ATPase com- plexes to peroxisomes.

Complementation group 1 is the largest and con- tains about 65% of the patients. The related clinical phenotype is highly variable and covers the entire clinical spectrum from ZS to NALD to IRD. There is some genotype–phenotype correlation. Patients with two null mutations have a more severe phenotype than patients with a residual function of Pex1p.

With defects in PEX3, PEX16, and PEX19 no perox- isomal membrane structures are present at all. In the other complementation groups, the defect involves peroxisomal protein import but not the synthesis of peroxisomal membranes, and in cells belonging to these complementation groups remnant peroxisomal membrane structures are present, also called peroxi- somal ghosts. These structures can be demonstrated in cultured fibroblasts by using antibodies to peroxi- somal membrane proteins. These structures are

largely empty. They lack most of the peroxisomal ma- trix proteins; they contain the unprocessed precursor form of thiolase, unprocessed acyl-CoA oxidase, and residual dihydroxyacetone phosphate acyltransferase activity. Some catalase has also been found in the interior of peroxisomal remnant structures.

The absence of normal peroxisomes is associated with defective function of multiple peroxisomal en- zymes. Several of the enzyme proteins, which are nor- mally located in the peroxisomal matrix, are free in the cytosol and are stable and biologically active. This is the case with catalase,

-amino acid oxidase, ala- nine glyoxylate aminotransferase, polyamine oxidase and

-a-hydroxy acid oxidase. In contrast, other per- oxisomal enzymes are synthesized normally, but are unstable in the cytosol, are rapidly degraded, and their enzyme activities are decreased. This is the case with the peroxisomal b-oxidation enzyme proteins, alkyldihydroxyacetone phosphate synthase and dihy- droxyacetone phosphate acyltransferase, and the plasmalogen synthesizing enzymes.

In the peroxisome biogenesis disorders, mitochon- drial abnormalities are also found. Mitochondria are often morphologically abnormal. It is generally as- sumed that the mitochondrial abnormalities are sec- ondary. There is a metabolic interdependence of mitochondria and peroxisomes. Participation in fatty acid metabolism is a property of both organelles. Per- oxisomes shorten very-long-chain fatty acids prior to their oxidation by mitochondria. Defects in peroxiso- mal b-oxidation lead to accumulation of long-chain fatty acyl-CoAs, which have regulatory effects on a number of mitochondrial enzymes. The mitochon- drial abnormality may contribute to the clinical dis- ease. In some patients muscle pathology with abnor- mal mitochondria is observed, suggesting a mito- chondrial myopathy. However, no changes in lactate, pyruvate, 3-hydroxy butyrate, and acetoacetate have ever been reported in patients, indicating that in vivo the proposed mitochondrial dysfunction is usually of no or only minor importance.

The peroxisome biogenesis defects are histopatho- logically characterized by a combination of malfor- mative and degenerative abnormalities. The dysonto- genetic or malformative changes include facial dys- morphia, renal cortical cysts, gray matter migrational disturbances, and abnormalities in myelin deposi- tion. The degenerative, regressive changes include the pigmentary retinal degeneration, the liver fibrosis and cirrhosis, the adrenal cortical atrophy, and storage, demyelination, and neuronal degeneration.

In ZS the dysontogenetic abnormalities predominate

in the CNS. In NALD there are some dysontogenetic

changes, but CNS pathology is dominated by degen-

eration and storage phenomena. In IRD both are mild

or absent in the nervous system.

Mechanisms which interfere with migration in ZS and NALD do so to a partial degree only, as some neu- rons are in their normal position and only some of the neurons of a given class fail to complete their migrations. Neuronal migration is not disturbed in rhizomelic chondrodysplasia punctata. This observa- tion makes a disturbance of plasmalogen synthesis or phytanic acid oxidation improbable as the cause of the migrational defect. The disturbance of neuronal migration in patients with D-bifunctional protein de- ficiency directs attention to the very-long-chain fatty acid and bile acid abnormalities. Neuronal migra- tional abnormalities may reflect the effect of accumu- lated very-long-chain fatty acids, since elevations consistently accompany the peroxisomal disorders with disturbed migration. The exception is X-linked adrenoleukodystrophy, in which very-long-chain fat- ty acids are elevated and no migrational disturbance is present. However, in X-linked adrenoleukodystro- phy the increase in very-long-chain fatty acids is not as severe as in ZS or NALD, and is more restricted. In ZS, saturated, monounsaturated and polyunsaturated fatty acids are increased. In NALD and IRD, saturated and monounsaturated fatty acids are elevated, where- as in X-linked adrenoleukodystrophy, only saturated fatty acids are elevated. A possible role of accumula- tion of bile acid intermediates must also be consid- ered. It is hypothesized that an accumulating sub- stance interferes with the cell adhesion molecule in- teractions and linkages, which are necessary for nor- mal migration of neurons along radial glial fibers. In ZS, ependymal abnormalities are found which are qualitatively similar but quantitatively less extensive than those found in classical lissencephaly. The ab- normal ependyma may be a primary factor in the pathogenesis of migrational disturbances.

The white matter abnormalities vary among the disorders of peroxisomal biogenesis from predomi- nantly deficient and disturbed myelination in ZS to predominant demyelination in NALD and variable white matter gliosis in IRD. In ZS myelin is severely deficient, but no signs of active demyelination are seen; in NALD the process of myelination is initially relatively normal, but demyelination follows. The dif- ference between the two may be related to the degree of abnormality of membrane composition. Abnormal membrane composition is related to decreased avail- ability of plasmalogens and accumulation of very- long-chain fatty acids and phytanic acid in the vari- ous membrane lipids. The more severe abnormality in ZS results in disturbed myelin formation, whereas in NALD myelin is laid down but subsequently bro- ken down as a consequence of increasing instability.

In IRD the biochemical abnormalities are mildest and white matter pathology only mild or minor. The in- flammatory response in NALD may be related to the liberation of lipids containing very-long-chain fatty

acids in the process of myelin breakdown. These may be immunogenic and elicit an inflammatory re- sponse, as seen in X-linked adrenoleukodystrophy.

ZS, NALD, and IRD are all characterized by the presence of trilamellar inclusions in lysosomes, the amount of which increases with age. They consist of cholesterol-bound very-long-chain fatty acids. The storage is, as a rule, more abundant in macrophages than in parenchymal cells.

Hepatic fibrosis and cirrhosis are probably related to abnormal bile acid oxidation. Bile acid intermedi- ates such as trihydroxycholestanoic acid with known hepatic toxicity may be important pathogenetically for both the development of bile duct paucity and hepatocellular injury.

The adrenal cortex is affected in all peroxisomal disorders with an elevation of very-long-chain fatty acids. The increase in these fatty acids causes an in- crease in membrane viscosity in adrenocortical cells, which in turn results in a decreased number of hormone receptor sites, subsequently leading to a decreased ability to respond to ACTH. Adrenal insuf- ficiency followed by atrophy is due to the lack of response to ACTH.

18.4 Therapy

In treating patients with a disorder of peroxisome biogenesis, first of all supportive care is essential. In addition, it would seem rational to try to compensate as far as possible for the biochemical abnormalities that have been brought about by the peroxisomal dys- function. Treatment would include oral supplementa- tion of ether lipids and bile salts and dietary restric- tion of very-long-chain fatty acids and phytanic acid.

The treatment has so far not resulted in definite clin- ical improvement or prolongation of life. Treatment with docosahexaenoic acid ethyl ester has been advo- cated on the basis of improvement of biochemical parameters and some neurological improvement in treated children. The rationale of the treatment is the observation of severe docosahexaenoic acid deficien- cy in tissues of children with disorders of peroxiso- mal biogenesis, while this substance is known to be an important constituent of brain membrane phospho- lipid and of photoreceptor cells. So far, the results of a controlled trial are lacking.

Administration of clofibrate fails to induce liver peroxisomes in ZS patients. Administration of 4- phenylbutyrate, a human peroxisome proliferator, increased the number of peroxisomes in fibroblasts of patients with a peroxisome biogenesis disorder (Wei et al. 2000). In NALD and IRD fibroblasts, but not in ZS fibroblasts, there was an increase in very-long- chain fatty acid b-oxidation and plasmalogen con- centrations, and a decrease in very-long-chain fatty

18.4 Therapy 159

acid concentrations. These data suggest that pharma- cological agents that induce peroxisome proliferation may have a therapeutic potential in the treatment of patients with milder variants of peroxisome biogene- sis defects.

The problem of any therapeutic trial in the disor- ders of peroxisomal biogenesis is that the dysontoge- netic abnormalities cannot be changed by treatment and that only the degenerative changes acquired post- natally can, hopefully, be prevented.

18.5 Magnetic Resonance Imaging

In ZS the migrational derangement is well depicted by MRI. A very characteristic abnormality is the peri- sylvian polymicrogyria, which appears as a thickened cortical mantle consisting of many little dots (Figs. 18.1–18.3). The dots are cross-sections of the microgyri. The polymicrogyric cortex merges with

pachygyric cortex in the frontoparietal region (Figs. 18.1 and 18.2). The pachygyric cortex is visual- ized as broad convolutions of mildly thickened cor- tex. The cortex bordering the interhemispheric fis- sure and the occipital cortex are relatively normal.

The gyral abnormalities are very extensive and seri- ous in exceptional cases (Fig. 18.1). Small dots of ec- topic gray matter may also be seen under the cortex and in the subependymal region (Figs. 18.2 and 18.3).

The ventricular system is mildly enlarged and tends to have a primitive form with mildly enlarged occipi- tal horns, which have a squared-off configuration.

The ventricles are rarely markedly enlarged. Germi- nolytic cysts are often seen in the caudatothalamic groove in the early stages (Figs. 18.1 and 18.3). The corpus callosum is thin. The width of the white mat- ter is reduced. Myelination is delayed and may be patchy, consistent with a disturbed myelination.

In NALD, CT has been shown to reveal progressive white matter hypodensities, particularly in the peri-

Fig. 18.1. Sagittal (first row) and axial (second row) T2-weighted images in a neonate with ZS. The lateral aspects of the brain are diffusely pachygyric and polymicrogyric. The sylvian fissure is wide open. Note the huge subependymal cysts on both sides.

(Courtesy of Dr. M.A. Breukels, Department of Pediatrics, and Drs. J.P. Westerhof and F. Kok, Department of Radiology, Elkerliek Hospital, Helmond, The Netherlands)

ventricular area, centrum semiovale, and cerebellum.

The presence of extensive contrast enhancement in centrum semiovale, internal capsules, and cerebral peduncles has been reported. MRI may either show evidence of polymicrogyria in the area of the sylvian fissure (Fig. 18.4) or fail to show evidence of gyral abnormalities (Fig. 18.5). When demyelination starts, the earliest changes are seen in the cerebellum, in- volving both the hilus of the dentate nucleus and the peridentate white matter (Figs. 18.4 and 18.5). Other structures that become involved are brain stem tracts, in particular the pyramidal tracts, the posterior limb of the internal capsule, and the posterior cerebral white matter more than the anterior white matter (Figs. 18.4 and 18.5). On sequential MRI, the abnor- malities are rapidly progressive.

In IRD MRI is normal in some patients, but is ab- normal in others. MRI does not show migrational ab- normalities. Symmetrical abnormalities in signal in- tensity may be seen in the hilus of the dentate nuclei and peridentate cerebellar white matter (Fig. 18.6).

These seem to be the first and sometimes the only ab- normalities present. Patchy, ill-defined abnormalities may be seen in the periventricular cerebral white matter (Fig. 18.6). The abnormal white matter merges into the normal white matter without sharp demarca- tion. The corpus callosum and posterior limb of the internal capsule may also be involved. There is no contrast enhancement. The white matter abnormali- ties may be slowly progressive over time, not neces- sarily associated with clinical decline. Profound cere- bral and cerebellar atrophy may occur (Fig. 18.7).

18.5 Magnetic Resonance Imaging 161

Fig. 18.2. Axial T2-weighted images in a 3-month-old boy with a PEX1 defect and the typical clinical course of ZS.The im- ages show some ventricular enlargement. Myelination is sparse. In the frontal area the gyri are too coarse (pachygyria),

whereas polymicrogyria is present in the perisylvian region.

There are several minor neuronal heterotopias in the periven- tricular region. The cerebellar vermis is hypoplastic

Fig. 18.3. A 1-week-old baby boy with ZS, related to a PEX1 mutation. The upper row (T1-weighted sagittal images) shows a thin corpus callosum, a hypoplastic vermis, and a sub- ependymal cyst in the thalamocaudate notch. The sylvian fis- sure has a more vertical orientation than normal and is bor- dered by polymicrogyric cortex. The axial T2-weighted images

show mildly enlarged lateral ventricles and absence of a large part of the vermis with an abnormally shaped fourth ventricle.

The gyral deformity is most marked in the region of the sylvian fissure, with a combination of pachygyria and polymicrogyria.

The left image of the third row shows a germinolytic cyst over the caudate nucleus and some blood in the occipital horns

18.5 Magnetic Resonance Imaging 163

Fig. 18.4. A 2-year-old boy with a PEX5 defect and progressive neurological deterioration with death at the age of 2.8 years.

The images show white matter abnormalities in the hilus of the dentate nucleus, the cerebellar hemispheric white matter,

and the posterior cerebral white matter.The cortex in the area of the sylvian fissure appears mildly dysplastic with evidence of some polymicrogyria. Courtesy of Dr. P.G. Barth [Barth et al.

2001 (patient 3), 2004 (patient 4), with permission]

Fig. 18.5. A 2.5-year old girl with a PEX5 defect and progres- sive neurological deterioration who died at the age of 3 years.

The images show signal abnormalities in the hilus of the den- tate nucleus, the cerebellar hemispheric white matter, pyrami-

dal tracts of the brain stem, and the posterior and central cere- bral white matter. There is no evidence of cortical dysplasia.

Courtesy of Dr. P.G. Barth [Barth et al. 1990, 2004 (patient 1), with permission]

18.5 Magnetic Resonance Imaging 165

Fig. 18.6. A 10.5-year-old boy with a PEX1 defect and a stable clinical course. Note the signal abnormalities in the hilus of the dentate nucleus, the corticospinal tracts at the level of the midbrain, the posterior limb of the internal capsule, the poste-

rior cerebral white matter, and splenium of the corpus callo- sum. Apart from the hilus of the dentate nucleus, the signal changes are mild and poorly demarcated. Courtesy of Dr. P.G.

Barth [Barth et al. 2004 (patient 13), with permission]

Fig. 18.7. T2-weighted images of a 14-year-old boy with a PEX1 defect and a very slow downhill course.The images show dilated ventricles and cerebral and cerebellar atrophy and widespread signal abnormalities in the atrophic cerebral

white matter, extending into the arcuate fibers.The hilus of the dentate nucleus is abnormal in signal. Courtesy of Dr. P.G. Barth [Barth et al. 2004 (patient 11), with permission]