Peroxisomal Fatty Acid Oxidation

Peroxisomal β-oxidation enzymes are distinct from their mitochondrial counterparts. The biochemical steps are similar, involving several distinct enzymes including an oxidase, multifunctional enzyme (displaying enoyl-CoA hydratase and 3-hydroxy acyl-CoA dehydrogenase activity), and 3-oxo acyl-CoA thiolase. It recently has become clear that several distinct oxidases, multifunctional proteins, and enzymes catalyzing the thiolytic cleavage exist. The two pathways, l– and d-specific, are depicted in Figure 38-1.

Oxidation of Unsaturated Fatty Acids

Degradation of monounsaturated and polyunsaturated fatty acids has been demonstrated to occur in peroxisomes, in addition to chain shortening of long-chain dicarboxylic acids [Ferdinandusse et al., 2001; Hiltunen et al., 1986; Kolvraa and Gregersen, 1983; Schulz and Kunau, 1987]. An example of a polyunsaturated fatty acid that requires peroxisomal oxidation is docosahexaenoic acid, which has an important role in retina and brain development, and is known to be deficient in certain peroxisomal disorders [Moser et al., 1999; Watkins et al., 2001].

Branched-Chain Fatty Acid Oxidation and Phytanic Acid α-Oxidation

Branched-chain fatty acids are channeled into the d-specific pathway. The relevant enzymes include branched-chain acyl-CoA oxidase, MFE2, and sterol carrier protein, which functions as thiolase (see Figure 38-1). Phytanic acid has a β-methyl group that blocks β-oxidation and therefore must be α-oxidized first. After activation to its CoA derivative, phytanic acid is α-hydroxylated by phytanoyl CoA α-hydroxylase, the enzyme defective in classic Refsum’s disease [Jansen et al., 1997; Mihalik et al., 1997]. The α-hydroxy phytanoyl CoA is then degraded by a lyase to pristanic acid and formate [Croes et al., 1997]. The pristanic acid then can enter β-oxidation through the branched-chain pathway.

Bile Acid Synthesis

Cholesterol is converted in the liver to cholic acid and chenodeoxycholic acid by a series of enzymatic steps localized to several subcellular compartments. Pedersen and Gustafsson [1980] first demonstrated that the peroxisomal fraction of rat liver catalyzes the conversion of an intermediate product of this pathway, tri- or tetrahydroxy-5-β-cholestanoic acid (trihydroxycholestanoic acid), to cholic acid. Subsequently, Kase et al. [1986] reported that the conversion of trihydroxycholestanoic acid to cholic acid, and of dihydrocholestanoic acid to chenodeoxycholic acid also took place in peroxisomes and was deficient in patients with Zellweger’s syndrome. Shortening of the cholesterol side chain by β-oxidation resulting in the formation of bile acids is analogous to fatty acid oxidation and uses the enzymes of the d-specific peroxisomal β-oxidation pathway. The oxidase for trihydroxycholestanoic acid–CoA is separate from that of fatty acids and in humans also is believed to function as a branched-chain fatty acid oxidase (see Figure 38-1). A cytochrome P-450 catalyzing the hydroxylation of the C26/27 carbon of cholesterol in bile acid synthesis and formation of taurine conjugates of bile acid also has been localized to peroxisomes [Gutierrez et al., 1988; Kase and Bjorkheim, 1989]. In peroxisomal disorders, an increase in plasma and urine bile acid intermediates can be used as a diagnostic criterion.

Plasmalogen Synthesis

Plasmalogens are ether phospholipids and constitute 5–20 percent of phospholipids in mammalian cell membranes [Snyder, 1972]. They are abundant in myelin, in which they constitute one-third of the myelin phospholipids [Norton and Autilio, 1966]. Although the role of ether phospholipids is not fully elucidated, they appear to have important roles as antioxidants in membrane dynamics, storage of polyunsaturated fatty acids, and signal transduction [Brites et al., 2004].

Plasmalogen synthesis is initiated by the acylation of dihydroxyacetone phosphate by dihydroxyacetone phosphate acyltransferase. Experimental studies in cultured skin fibroblasts demonstrate that ATP is required to overcome the latency of dihydroxyacetone phosphate acyltransferase, a finding that is compatible with an ATP-linked translocation of the substrate across the peroxisomal membrane [Wolvetang et al., 1990]. The second enzyme, alkyl-dihydroxyacetone phosphate synthase, replaces the acyl group with a long-chain alcohol [Hajra and Bishop, 1982]. Both enzymes involved in these initial steps of introducing the ether bond into ether phospholipids are located on the inner surface of peroxisome membranes. The third step is the reduction of the alkyl-dihydroxyacetone phosphate to 1-alkyl glycerol-3-phosphate by the enzyme acyl/alkyl-dihydroxyacetone phosphate reductase, which is present in both peroxisomes and microsomes [Datta et al., 1990]. All subsequent steps of synthesis occur in the microsomes. A method developed by Roscher et al. [1985] determines the ratio of the activities in peroxisomal and microsomal components, and has proved to be of value in the study of peroxisomal disorders, many of which feature a marked reduction in plasmalogen synthesis.

Prostaglandin Degradation

Peroxisomes are involved in the chain shortening of prostaglandins F_2a [Diczfalusy and Alexson, 1988] and E₂ [Schepers et al., 1986]. Tiffany et al. [1991] demonstrated that basal and interleukin-1-stimulated synthesis of prostaglandin E₂ increased in a group of patients with X-linked adrenoleukodystrophy.

Amino Acid Metabolism

Molecular Etiology of Disorders of Peroxisome Assembly

Cell complementation studies were instrumental in the early understanding of the genetics of peroxisomal assembly disorders. Identification of complementation groups provided cell lines that are homogeneous with respect to their gene defect. In conjunction with the use of other molecular biology tools, including the study of yeast and other cell types and the emergence of computerized genomic databases, a new understanding of the molecular underpinnings of the peroxisome assembly disorders has been attained [Lazarow, 1995]. These disorders result from defects in the PEX genes. A complex interaction of peroxins is necessary for the biogenesis of peroxisomes, and a defect in any of these proteins impairs the process. The final common pathway is peroxisomal dysfunction, with the respective clinical syndromes.

From the study of complementation groups, it was determined that peroxisomal disorders comprise up to 16 complementation groups [Moser et al., 1995]. A majority of the identified groups included persons with Zellweger’s syndrome and milder variations of that condition. One complementation group, however, contained all of the patients with rhizomelic chondrodysplasia punctata, and no overlap between this clinical group and the others was identified. It has subsequently been determined that Zellweger spectrum disorders are secondary to PTS1-mediated pathways including PEX5, and that rhizomelic chondrodysplasia punctata is secondary to mutations in PEX7, the receptor for PTS2 proteins.

The most common causes of Zellweger spectrum disorders are mutations in either PEX1 or PEX6, although to date, 13 PEX genes have been identified as potential causes of these disorders (Table 38-1). PEX1 and PEX6 encode AAA ATPases. Their exact role in matrix protein importation is not certain, but it is evident that they interact. Overexpression of PEX6 or PEX1 can correct mild deficiencies of the other [Germain-Lee et al., 1997; Reuber et al., 1997]. PEX1 defects account for more than 65 percent of cases of such defects [Steinberg et al., 2003; Weller et al., 2003]. A degree of phenotype correlation with the causative mutation has been noted. A single base-pair deletion results in a severe phenotype, and G843D allows a milder phenotype [Collins and Gould, 1999]. Because it is known that PEX1 and PEX6 interact, the mutation G843D affects this interaction [Weller et al., 2003].

Clinical and Pathologic Features

Zellweger’s syndrome

Zellweger’s syndrome is a multiple congenital anomaly syndrome characterized by craniofacial abnormalities, eye abnormalities, neuronal migration defects, hepatomegaly, chondrodysplasia punctata, and near-complete absence of peroxisomes. The craniofacial features include a high forehead, hypoplastic supraorbital ridges, epicanthal folds, midface hypoplasia, and a large fontanel (Figure 38-3). The head circumference usually is normal. Reported ocular abnormalities include cataracts, glaucoma, corneal clouding, Brushfield spots, optic nerve hypoplasia, and pigmentary retinal abnormalities. Severe weakness and hypotonia manifest in the newborn period, often accompanied by seizures and apnea [Heymans, 1984; Wilson et al., 1986]. Most affected infants have oromotor dysfunction and require tube feeding. Little psychomotor development ensues, and the average life span is limited, with most affected children surviving for 12–24 months. The facial appearance, Brushfield spots, and profound hypotonia may lead to a consideration of Down syndrome, although the chromosomal determination will eliminate that as a consideration.

Fig. 38-3 Cerebrohepatorenal syndrome of Zellweger.

A, Note the prominent forehead, large fontanel, hypertelorism, epicanthal folds, hypotonia, and hepatomegaly. B, Milder form of generalized peroxisomal disorder: 10-year-old boy with ataxia, significant mental retardation, visual loss secondary to retinitis pigmentosa, absence of speech, and sensorineural hearing loss. Liver biopsy showed micronodular cirrhosis and very few, abnormally small peroxisomes.

Striking abnormalities of neuronal migration unique to Zellweger’s syndrome are evident in the cerebral hemispheres as areas of pachygyria or polymicrogyria localized to the opercular region. In the cerebellum, the Purkinje cells form scattered heterotopias throughout the cortex and in the granule cell layer. Laminar discontinuities involving the olivary nucleus are noted, which also are unique to Zellweger’s syndrome [Evrard et al., 1978; Volpe and Adams, 1972]. Studies of PEX gene knockout mice have demonstrated similar neuropathologic changes [Baes et al., 1997; Faust, 2003; Faust et al., 2001; Gressens et al., 2000; Janssen et al., 2000, 2003]. N-methyl-d-aspartate (NMDA)-mediated abnormalities may contribute to the clinical findings seen in the most severely affected persons [Gressens et al., 2000].

Multiple other abnormalities have been reported. The eyes demonstrate loss of retinal ganglion cells and gliosis of the optic nerve [Cohen et al., 1983]. Retinal pigmentary degenerative changes are associated with absent electroretinograms [Hittner et al., 1981]. Hepatomegaly (present in 78 percent of patients) with periportal fibrosis may result in significant cholestasis (in 59 percent) and jaundice, micronodular cirrhosis (in 37 percent), and hypoprothrombinemia [Heymans, 1984]. Excessive iron deposition has been noted, which diminishes with age [Gilchrist et al., 1976]. Renal cortical cysts of varied sizes are present in 97 percent of patients studied pathologically but may be missed by ultrasound analyses [Bernstein et al., 1974; Heymans, 1984]. The adrenal gland demonstrates changes similar to those in X-linked adrenoleukodystrophy, with cytoplasmic lamellar inclusions consisting of cholesterol esterified with very long chain fatty acids [Goldfischer et al., 1983]. Skeletal abnormalities include clubfoot, thumb rotation, and stippled chondral calcification of the patella and acetabulum in 50 percent of patients [Heymans, 1984].

Laboratory Diagnosis

The diagnostic abnormalities present in patients with Zellweger spectrum disorders are listed in Table 38-2. A major diagnostic biochemical abnormality is the increased amount of very long chain fatty acids, which are fatty acids with carbon chains of more than 22. They also are elevated in single-enzyme defects of peroxisomal β-oxidation, so their presence is nonspecific, and other studies are required for this diagnosis. Very long chain fatty acid accumulation here is due to the reduction of peroxisomal β-oxidation, which is greater in Zellweger’s syndrome than in milder forms. A virtual absence of peroxisomal acyl-CoA oxidase and bifunctional enzyme has been demonstrated in the liver of patients with Zellweger’s syndrome [Suzuki et al., 1986; Tager et al., 1985]. It should be noted, however, that very long chain fatty acids are not elevated in rhizomelic chondrodysplasia punctata and other peroxisomal disorders (e.g., acatalasemia) that do not involve lipid oxidation, and normal levels do not exclude all peroxisomal disorders.

Because the first two steps of plasmalogen synthesis are peroxisomal functions, a reduction of plasmalogen levels is noted in patients with peroxisome biogenesis defects. Deficient levels of dihydroxyacetone phosphate acyltransferase have been used for diagnostic purposes in skin fibroblasts, amniocytes, chorionic villus samples, red blood cells, leukocytes, and platelets [Besley and Broadhead, 1987; Datta et al., 1984; Schutgens et al., 1985; Wanders et al., 1985, 1986a]. Webber et al. [1987] have demonstrated that the K_m values and other properties of the residual enzyme are normal, suggesting that the enzyme is not defective but is abnormally labile in the cytosol because of failure of transport into the peroxisome. Plasmalogens are reduced to 5 percent of control values in Zellweger’s syndrome [Heymans et al., 1984; Wanders et al., 1986a].

An age-related increase in the levels of phytanic acid occurs in all of the peroxisome biogenesis disorders, including rhizomelic chondrodysplasia punctata, although not to the extent seen in classic adult Refsum’s disease. The accumulation of phytanic acid was demonstrated first in infantile Refsum’s disease [Scotto et al., 1982] and later in Zellweger’s syndrome [Poulos et al., 1984]. Poulos et al. [1988] demonstrated that pristanic acid is elevated in disorders of peroxisome biogenesis. This elevation was later confirmed to be due to an impaired capacity to oxidize pristanic acid [Singh et al., 1990]. These findings are unlike those in classic Refsum’s disease, in which the defect involves the conversion of phytanic acid to pristanic acid. Thus, oxidation of pristanic acid is a peroxisomal function that is disrupted in disorders of peroxisome biogenesis [Watkins et al., 1990].

Normally, bile acid intermediates, such as trihydroxycholestanoic acid and dihydroxycholestanoic acid, are absent or present in low concentrations. In Zellweger spectrum disorders, however, they constitute 30–50 percent of total plasma bile acids [Clayton et al., 1987; Eyssen et al., 1985; Gustafsson et al., 1983; Hanson et al., 1979; Mathis et al., 1980; Monnens et al., 1980; Poulos and Whiting, 1985]. Bile acid intermediates also may be abnormal in single-enzyme defects, such as peroxisomal bifunctional enzyme deficiency.

The impaired activity of l-pipecolic acid oxidase results in increased accumulation of pipecolic acid in plasma and increased excretion of pipecolic acid in urine of patients with Zellweger spectrum disorders [Kelley and Moser, 1984; Mihalik et al., 1989; Wanders et al., 1988a]. Pipecolic acid levels in blood and urine are age-dependent, but an excess can be demonstrated in all patients, irrespective of age [Dancis and Hutzler, 1986]. Medium- and long-chain dicarboxylic acids accumulate and are excreted in urine, suggestive of a partial block in the degradation of long-chain dicarboxylic acids [Bjorkheim et al., 1984; Rocchiccioli et al., 1986].

In Zellweger’s syndrome, the number of catalase-containing particles in liver or kidney biopsy specimens is reduced [Goldfischer et al., 1973; Small et al., 1988], although today biopsy rarely is needed for diagnosis. A reduction in the number of peroxisomes also occurs in neonatal adrenoleukodystrophy and infantile Refsum’s disease but, as expected, is less severe than in Zellweger’s syndrome. The catalase is cytosolic rather than particulate in catalase-containing particles. Although, in the past, peroxisomes were considered absent, Santos et al. [1988] demonstrated membranous structures called peroxisomal ghosts that contain peroxisomal membrane proteins. This finding has been substantiated by other investigators, who demonstrated the presence of major membrane proteins in skin fibroblasts from patients with Zellweger’s syndrome, some of which were quite abundant [Gaertner et al., 1991; Suzuki et al., 1989]. These ghosts lack the matrix enzyme catalase and some or all of the matrix proteins. Schram et al. [1986] have reported that peroxisomal β-oxidation enzymes, which normally are located in the peroxisome matrix, are formed at a normal rate but are rapidly degraded in the cytosol. The mitochondrial abnormalities observed are considered secondary [Trijbels et al., 1983].

Prenatal Diagnosis

Cultured amniocytes or chorionic villus cells can be used to diagnose all of the disorders of peroxisome biogenesis. A variety of biochemical strategies have been used and focus on the type and degree of biochemical abnormality. Two independent techniques, the measurement of very long chain fatty acid β-oxidation [Moser et al., 1984a; Solish et al., 1985] and plasmalogen synthesis [Roscher et al., 1985; Schutgens et al., 1985], have demonstrated accurate diagnosis when performed by laboratories experienced in these methodologies. Molecular genetic techniques may be used in families in which the mutation has been identified and pre-implantation genetic diagnosis has been performed [Al-Sayed et al., 2007].

Therapy

Because many of the abnormalities are present in the affected fetus, potential for therapy is limited at present and likely to remain so. Treatment is primarily supportive, targeting, as appropriate, liver dysfunction with vitamin K for prothrombin deficiency, tube feeding, and antiepileptic medication. In the milder phenotypes, rehabilitative approaches, including communication training and physical and occupational therapy, are helpful.

Attempts to normalize some of the biochemical abnormalities include oral ether lipid therapy [Holmes et al., 1987; Poulos et al., 1990; Wilson et al., 1986] and dietary restriction of very long chain fatty acids [Van Duyn et al., 1984] and phytanic acid [Greenberg et al., 1987; Steinberg, 1989]. These approaches may normalize plasma levels of very long chain fatty acids, phytanic acid, and red blood cell plasmalogens. The clinical effectiveness of these interventions, however, has yet to be demonstrated because of the phenotypic variability of these disorders. Ursodeoxycholic acid can reduce levels of bile acid intermediates and has the potential to prevent liver damage [Colombo et al., 1990]. Dietary therapy with docosahexaenoic acid replaces low levels in plasma and red blood cells of patients with Zellweger’s syndrome, neonatal adrenoleukodystrophy, and infantile Refsum’s disease [Martinez et al., 1993, 1994]; however, it is ineffective in improving clinical function [Mahmood et al., 2010]. Peroxisome proliferators, such as clofibrate, do not induce the formation of catalase-containing peroxisomes or improve clinical status [Lazarow et al., 1985].

Adrenoleukodystrophy

The first clinical cases of what is known as X-linked adrenoleukodystrophy were described in 1923 by Siemerling and Creutzfeldt. The X-linked mode of inheritance was suggested by Fanconi et al. [1963]. A key observation of characteristic lamellar lipid-soluble cytoplasmic inclusions in the adrenal cortical cells and brain macrophages of patients with childhood adrenoleukodystrophy was made by Powers et al. [1980]. Similar inclusions were observed in men with Addison’s disease and spastic paraparesis by Budka et al. [1976], and by Griffin et al. [1977], and the related condition was named adrenomyeloneuropathy. Similar histopathologic changes observed in a symptomatic child and an adult within a kindred strengthened the assertion that, despite the phenotypic variation, the same disease process was present. Igarashi et al. [1976] defined the biochemical defect by demonstrating that the inclusions in the adrenal cortex and cerebral white matter consisted of cholesterol esters with a striking excess of saturated unbranched fatty acids of 24- to 30-carbon chain length, of which C26:0 and C25:0 were the most abundant. This excess of very long chain fatty acids in cultured skin fibroblasts, plasma, or red blood cells is useful for diagnostic purposes [Kawamura et al., 1978; Moser et al., 1981; Tanaka et al., 1986, 1990; Tsuji et al., 1981].

Clinical and pathologic features of X-linked adrenoleukodystrophy and adrenomyeloneuropathy

Table 38-3 summarizes the different phenotypes of patients with X-linked adrenoleukodystrophy. These patients all demonstrate accumulation of very long chain fatty acid, and molecular studies have confirmed ABCD1 gene mutations. Various phenotypes have been recognized as occurring within the same pedigree, so neither the genetic mutation nor the biochemical abnormality predicts the clinical presentation.

Table 38-3 Phenotypes among X-Linked Adrenoleukodystrophy Hemizygotes

Type	Frequency (%)
Childhood cerebral	33–39
Adolescent cerebral	4–7
Adult cerebral	2–3
AMN	26–32
Addison’s only	13–17
Asymptomatic	7–18

AMN, adrenomyeloneuropathy.

(Adapted from the North American data of the Kennedy Krieger Institute [Bezman and Moser, 1998].)

Childhood Cerebral Form of Adrenoleukodystrophy

The childhood cerebral variant is the most common and fulminant form of X-linked adrenoleukodystrophy. Affected boys are normal until 4–8 years of age, when they manifest behavior problems and failure in school as a result of rapid regression of auditory discrimination, spatial orientation, speech, and writing. Seizures occur in 30 percent of patients and in rare instances may be the initial sign. The magnetic resonance imaging (MRI) scan reveals parieto-occipital white matter lesions (in 85 percent of patients) or frontal lesions (in 15 percent) at this stage, with contrast accumulation at the leading edge of the lesion [Duda and Huttenlocher, 1976; Eiben and DiChiro, 1977; Kumar et al., 1987] (Figure 38-5). Rapid clinical deterioration leads to spastic quadriparesis, swallowing difficulty, and visual loss, culminating in a vegetative state usually within 2 years of the initial signs and symptoms. Although males come to medical attention because of the neurologic deficits, impaired cortisol response can be identified by adrenocorticotropic hormone stimulation in 85 percent of this group [Moser et al., 1991b].

Fig. 38-5 Magnetic resonance imaging (MRI) studies in adrenoleukodystrophy.

A, Typical alteration of the blood–brain barrier at the edge of the demyelinated lesion in childhood adrenoleukodystrophy demonstrated on gadolinium-enhanced MRI. B, The discrete but continuous auditory pathway involvement from the lateral lemniscus in the pons to the medial geniculate bodies at the midbrain level to the temporal cortex can be seen. MRI permits precise correlation between the location of the lesion and cognitive deficits; the findings may explain the early auditory comprehension deficiency seen in childhood adrenoleukodystrophy. C, MRI study of the spine in X-linked adrenoleukodystrophy. Views in axial (left) and lateral (right) planes demonstrate cord atrophy, particularly in the thoracic regions.

Adolescent Cerebral Form of Adrenoleukodystrophy

Patients with the adolescent cerebral form of adrenoleukodystrophy manifest signs and symptoms of cerebral involvement, as described previously, between 10 and 21 years of age.

Adult Cerebral Form of Adrenoleukodystrophy

In the adult cerebral form of adrenoleukodystrophy, dementia, psychiatric disturbances, seizures, and spastic paraparesis develop after age 21 [Bresnan and Richardson, 1979; Esiri et al., 1984; James et al., 1984; Kitchin et al., 1987; Sereni et al., 1987; Turpin et al., 1985]. Patients may be misdiagnosed as having multiple sclerosis, brain tumor, or schizophrenia, and demonstrate rapid deterioration similar to that in the childhood cerebral form. Therefore, patients with these clinical presentations and adrenal insufficiency or leukodystrophy should be evaluated for a positive family history; in addition, measurement of plasma very long chain fatty acids is warranted.

Adrenomyeloneuropathy

The neurologic manifestations of adrenomyeloneuropathy, an adult form of adrenoleukodystrophy, consist of an insidious onset and slow progression of spastic paraparesis, impaired vibratory sense in the lower extremity, and bladder or bowel dysfunction. Onset is typically in the third decade of life. The primary pathology involves the spinal cord with loss of myelinated axons in the corticospinal tracts, nucleus gracilis, and dorsal spinocerebellar tracts [Budka et al., 1983; Probst et al., 1980; Schaumburg et al., 1977]. Sural and peroneal nerves reveal a loss of large and small myelinated fibers.

The development of cerebral demyelination has been seen in approximately 15–20 percent of men with adrenomyeloneuropathy [van Geel et al., 2001]. This pathologic change needs to be differentiated from long tract findings on MRI [Loes et al., 2003]. Cerebral disease is similar in time course to the childhood form of the disease and leads to dementia, spasticity, blindness, and death. Approximately half of patients with adrenomyeloneuropathy appear to have some degree of cerebral involvement, with mild to moderate abnormalities noted on MRI in 46 percent [Kumar et al., 1995] (see Figure 38-5). These abnormalities consist most frequently of parietal-occipital white matter and optic radiation involvement.

Adrenal insufficiency, or Addison’s disease, precedes the onset of neurologic symptoms in 42 percent of patients; in some, this may occur 3–35 years earlier. Adrenal insufficiency was evident in 67 percent of patients in our series. Serum testosterone levels were abnormally low in 22 percent of our patients, and early-onset sexual dysfunction occurred in one-third.

Adrenoleukodystrophy with Addison’s Disease Only

The diagnosis of adrenoleukodystrophy with Addison’s disease only includes the patients who have isolated Addison’s disease in the absence of neurologic signs and symptoms, and is more common than was previously recognized. A study conducted in an endocrine clinic found that 5 of 8 male Addisonian patients had the biochemical defect of adrenoleukodystrophy [Sadeghi-Nejad and Senior, 1990]. Recognition of these patients is vital for genetic counseling and to monitor for the development of adrenomyeloneuropathy or cerebral symptoms later on. Onset of adrenal dysfunction does not correlate with neurologic involvement.

Asymptomatic Patients with the Biochemical Defect of Adrenoleukodystrophy

In asymptomatic patients with the biochemical defect of adrenoleukodystrophy, diagnosis may be accomplished by measurement of plasma very long chain fatty acids during screening tests of relatives of symptomatic patients. These persons may be of any age. The elevation of very long chain fatty acid levels in these males is comparable with that in severely affected family members. At present, it is uncertain why the abnormal increase of very long chain fatty acids is not associated with disease in some patients. A 62-year-old man in this category whose brother died at the age of 48 years with the adult cerebral form of adrenoleukodystrophy has been identified. The unaffected brother had no evidence of adrenal insufficiency but, when re-examined by the same neurologist 4 years later, demonstrated signs of corticospinal tract involvement compatible with mild adrenomyeloneuropathy. Several asymptomatic men demonstrate an impaired cortisol response to adrenocorticotropic hormone stimulation, suggesting that they have adrenal involvement and are presumed to be at risk for developing adrenomyeloneuropathy.

Symptomatic Heterozygotes

Between 10 and 20 percent of the women who are carriers for the adrenoleukodystrophy gene manifest myelopathy resembling that in adrenomyeloneuropathy between the third and fifth decades of life [Dooley and Wright, 1985; Heffungs et al., 1980; Morariu et al., 1982; Moser et al., 1980; Naidu et al., 1986; Noetzel et al., 1987; O’Neill et al., 1982, 1984; Penman, 1960; Pilz and Schiener, 1973]. Long tract signs and diminished vibration sense in the legs are present in two-thirds of the patients, although only 25 percent complain of symptoms. Approximately 14 percent have severe spinal involvement requiring assistance with ambulation, and 5 percent have dementia [Naidu and Moser, 1990]. In contrast with males, adrenal insufficiency rarely occurs in these women [El-Deiry et al., 1997].

Elevation of very long chain fatty acids in plasma or skin fibroblasts allowed identification of 85 percent of obligate heterozygotes in one series [Moser et al., 1983]. The remaining cases can be identified by molecular analysis for the gene defect. Mutation analysis helps identify heterozygotes once the mutation in the index case is known [Boehm et al., 1999]. Migeon et al. [1981] studied fibroblast clones from heterozygotes and identified two types of cells; one had a normal fatty acid pattern, and the pattern in the other cell type was similar to that in affected males, reflecting X-inactivation status. To determine if differential X-inactivation accounts for the clinical variability in heterozygotes, an indirect immunofluorescence analysis of ALDP, the protein defective in X-linked adrenoleukodystrophy, has been applied to skin fibroblasts. Previous studies have reported that 70 percent of males with X-linked adrenoleukodystrophy lack immunoreactivity for ALD protein (ALDP). As a result of differential X-inactivation, heterozygotes from these informative families have a mixed population of normal and ALDP-deficient cells. Despite considerable variation, a positive correlation is noted between immunonegative cells and clinical severity. No correlation is seen between immunoreactivity and either very long chain fatty acid levels or age alone. When age is considered, however, the correlation between severity and ALDP immunoreactivity is strengthened. Thus, differences in patterns of X-inactivation appear, in part, to contribute to the variable severity of the disease seen in the X-linked adrenoleukodystrophy heterozygotes [Naidu et al., 1997].

Contiguous Deletion of the XALD Gene

Corzo and co-workers identified three males in the newborn period who demonstrated abnormalities suggestive of a defect of l-bifunctional enzyme [Corzo et al., 2002]. Very long chain fatty acids were elevated; other findings included hypotonia and liver disease. All three patients died in the first year of life. Further investigation demonstrated large deletions of ABCD1, including the promoter region, with extension of these 5′ deletions into contiguous genes, including the DNA marker DXS1356E. The investigators proposed the name “contiguous ABCD1 DXS1357E deletion syndrome” (CADDS) for this phenotype. The specific role of the additional genetic areas is uncertain at present. Two of the mothers were determined to be carriers for these deletions, indicating the importance of correct diagnosis of single-enzyme defects of peroxisomal β-oxidation.

Acyl-CoA Oxidase Deficiency

Two siblings and an unrelated female patient have been reported to have acyl-CoA oxidase deficiency [Kyllerman et al., 1990; Poll-The et al., 1988a]. A large deletion of the gene encoding this enzyme was reported in the siblings [Fournier et al., 1994]. Dysmorphism, hypotonia, feeding difficulties, and intractable neonatal seizures were the presenting features. Little motor or cognitive development occurred in the first 2 years of life. Ocular involvement with extinguished electroretinography and white matter involvement with abnormal contrast enhancement were present. Adrenal insufficiency was not evident, but elevated serum adrenocorticotropic hormone and low cortisol levels were present in one patient. No organomegaly was present. Although the clinical characteristics resembled those in the milder forms of peroxisome biogenesis disorders, the following two features distinguished it from the Zellweger spectrum disorders:

Immunoblot studies revealed absence of acyl-CoA oxidase in the liver, and other peroxisomal β-oxidation enzymes were normal [Poll-The et al., 1988a]. Cultured skin fibroblasts had a reduced capacity to oxidize lignoceric acid and peroxisomal palmitoyl-CoA oxidase. The first prenatal diagnosis of isolated acyl-CoA oxidase deficiency was made by Wanders et al. [1990b]. In amniotic fluid, measurements of very long chain fatty acids were significantly elevated, but levels of bile acid intermediates were normal. In amniocytes, very long chain fatty acids were markedly increased, but plasmalogen biosynthesis was normal. Immunoblot analysis of amniocytes and the aborted fetal liver tissues demonstrated that all components of acyl-CoA oxidase were absent, whereas the bifunctional protein and mature form of thiolase were normal.

Bifunctional Enzyme Deficiency

Watkins et al. [1988] described a patient without dysmorphism or organomegaly who had significant hypotonia and neonatal seizures. Mental retardation was severe, and death occurred at 5½ months. Neuropathologic abnormalities included polymicrogyria and focal cortical heterotopias, similar to those in peroxisome biogenesis disorders [Kaufmann et al., 1996; Naidu et al., 1988b]. The liver had normal-appearing peroxisomes. Biochemical studies revealed elevation of very long chain fatty acids and bile acid intermediates; all other peroxisomal functions were normal. Postmortem immunoblot studies of liver demonstrated the absence of the bifunctional protein; other peroxisomal β-oxidation enzymes were present. The mRNA for the bifunctional protein was normal, indicating defective translation of the mRNA or abnormal post-translational processing, such as defective peroxisomal import mechanisms.

Although bifunctional enzyme deficiency bears some clinical resemblance to the acyl-CoA deficiency with elevation of very long chain fatty acids, a significant difference is the additional accumulation of bile acid intermediates (trihydroxycholestanoic acid) as well [Watkins et al., 1995]. The reason for trihydroxycholestanoic acid accumulation is evident in Figure 38-1, which demonstrates that the initial oxidation step for bile acid intermediates and very long chain fatty acids occurs by separate enzymes, whereas the subsequent step at the level of the bifunctional enzyme is common to both systems. Another patient with similar clinical features was found to have a functionally inactive bifunctional protein, although immunologic studies revealed normal amounts of the enzyme [Wanders et al., 1990c]. This patient is presumed to have a different mutation from the one described previously.

3-Oxoacyl-CoA Thiolase Deficiency

The entity 3-oxoacyl-CoA thiolase deficiency was first described by Goldfischer and co-workers in 1986 and called pseudo-Zellweger syndrome because of the presence of normal peroxisomes in the liver despite a clinical resemblance to Zellweger’s syndrome. Biochemically, the levels of very long chain fatty acids and bile acid intermediates were increased, but phytanic acid level was normal. A re-examination of material from one of these cases demonstrated that the infant, in fact, had a defect in bifunctional enzyme. The clinical existence of a thiolase deficiency is therefore not proved [Ferdinandusse et al., 2002].

Trihydroxycholestanoic Acid-CoA Oxidase Deficiency

Christensen et al. [1990] described an unusual female patient who demonstrated normal development until 18 months of age, when she deteriorated, with clinical manifestations including ataxia, dysarthria, and dry skin. By 5 years of age, she was severely incapacitated, with hearing loss and mental retardation. At this stage she had normal findings on computed tomography (CT) scan, absence of retinal degeneration, and normal liver function and nerve conduction velocities. Because the phytanic acid level was elevated, she was placed on a diet low in phytanate, after which she demonstrated significant improvement in gait, hearing, and speech. The most prominent biochemical abnormality, however, was an elevation in bile acid intermediates with normal very long chain fatty acid concentration, presumed to be secondary to a defect in trihydroxycholestanoic acid-CoA oxidase. Because phytanic acid oxidase levels were normal in cultured fibroblasts, the elevation in phytanic acid is believed to result from inhibition of phytanate oxidation by the accumulating bile acid intermediates.

Peroxisomal α-Methylacyl-CoA Racemase Defect

Several patients with adult-onset neurologic findings, including spasticity, sensory motor neuropathy, seizures, and retinitis pigmentosa, have been described. Very long chain fatty acid levels were normal, but elevations in C27 bile acid intermediates and pristanic acid were found. Phytanic acid was mildly elevated. Because pristanic acid β-oxidation was reduced, the activity of α-methylacyl-CoA racemase (AMACR) was examined and found to be deficient. AMACR is involved in the interconversion of (R)- and (S)-stereoisomers of α-methyl branched-chain fatty acyl-CoA esters, including pristanoyl-CoA, and the interconversion of the CoA esters of dihydroxycholestanoic acid and trihydroxycholestanoic acid. Its physiologic function is to produce the (S)-stereoisomers because only these serve as substrate for branched-chain acyl-CoA oxidase [Ferdinandusse et al., 2000].

Acatalasemia

Acatalasemia demonstrates clinical heterogeneity, with presentations ranging from absence of symptoms (in the Swiss variant) to oral ulcers, presumed to be secondary to peroxide-generating bacteria, in a severe Japanese variant. The Japanese patients have greatly reduced catalase activity; however, the electrophoretic mobility of the residual enzyme is normal. By contrast, the symptom-free patients with the Swiss variant have a large amount of catalase, but it demonstrates greater than normal heat lability. Studies by Crawford et al. [1988] report that the Japanese variant involves a regulatory mutation in which the gene is not transcribed, whereas in the Swiss variant, a structural mutation renders the enzyme unstable. The human catalase gene has been localized to 11p13 and cloned [Junien et al., 1980; Quan et al., 1986].

Hyperoxaluria Type I

Primary hyperoxaluria type I does not involve the nervous system, but does highlight an important pathogenic mechanism in peroxisomal disorders. It is inherited as an autosomal- recessive trait. Clinical onset is in childhood or adolescence. Patients present with urolithiasis and renal failure. Increased urinary excretion of oxalate (more than 50 mg/1.73 m² of body surface per day), glycolate (more than 70 mg/1.73 ² per day), and occasionally glyoxylate is diagnostic. In 1986, Danpure and Jennings demonstrated that the enzyme alanine-glyoxalate aminotransferase, which converts glyoxylate to glycine, is deficient in patients with this disorder [Danpure and Jennings, 1986]. Latta and Brodhel [1990] noted that this disorder accounted for 2–2.7 percent of children with end-stage renal disease. In human liver, this enzyme is peroxisomal [Danpure and Jennings, 1986], and the activity varies in affected patients, correlating with the severity of the disease [Danpure and Jennings, 1988]. A milder pyridoxine-responsive form of the disease has a less severe reduction of the enzyme [Wanders et al., 1988b]. Liver biopsy establishes the enzyme defect, but peroxisomes are present and intact in the liver [Iancu and Danpure, 1969]. The functions of other peroxisomal enzymes are normal. Danpure et al. [1989a] and Cooper et al. [1988] noted that in one-third of the patients with diminished but not absent alanine-glyoxalate aminotransferase, the enzyme is misdirected to the mitochondria, instead of its normal localization in humans to the peroxisome. The alanine-glyoxalate aminotransferase cDNA has been sequenced from eight patients with hyperoxaluria type I in whom the alanine-glyoxalate aminotransferase was localized to the mitochondria, and three new mutations were identified. One of these encoded a glycine to arginine substitution at residue 170 and was unique to these patients, but the two other mutations also were present in 5–10 percent of the control population [Purdue et al., 1990]. Nishiyama et al. [1991] have reported a point mutation encoding a serine to proline mutation at residue 205 in a patient in whom alanine-glyoxalate aminotransferase activity was nearly absent and who thus differed from the other group.

Diagnosis is established by the demonstration of deficient alanine-glyoxalate aminotransferase activity in a liver biopsy specimen [Danpure and Jennings, 1986; Danpure et al., 1987]. Prenatal diagnosis has been achieved by second-trimester fetal liver biopsy [Danpure et al., 1989b] and, in the future, could be achieved by DNA analysis of amniocytes or chorionic villus sampling. The estimated incidence of this disorder is 1 in 5 million to 15 million children; family studies demonstrate an increased incidence of parental consanguinity [Latta and Brodhel, 1990].

An exciting therapeutic advance has been a combination of liver and kidney transplantation with extremely favorable results [McDonald et al., 1989; Morgan and Watts, 1989; Ruder et al., 1990; Watts et al., 1987]. Some of the mildly affected patients respond to pyridoxine [Wanders et al., 1988b; Watts et al., 1979]. MacCollin et al. [1991] have described a patient with normal intelligence, ataxia, peripheral neuropathy, and calcium oxalate stones. Peroxisomes were absent. A marked increase in very long chain fatty acid and pipecolic acid was documented, with catalase present in the cytosolic fraction. This patient’s case is the only recorded case of hyperoxaluria in association with abnormal peroxisome biogenesis and multiple peroxisomal enzyme involvement.

Peroxisomal Glutaryl-CoA Oxidase Deficiency

The first patient with peroxisomal glutaryl-CoA oxidase deficiency was identified at 11 months, when the child was presented for failure to thrive and vomiting (in particular, postprandial vomiting), by Bennett et al. [1991]. Urinary glutaric acid level was 500 mmol per mole of creatinine, whereas the normal level is less than 2 mmol. The glutaricaciduria was responsive to riboflavin 200 mg given twice daily. At the age of 5 years 8 months, the patient was judged to be developmentally normal, although this outcome may have been attributable to the vitamin therapy. Detailed metabolic investigations ruled out glutaricaciduria types I and II. The activity of peroxisomal glutaryl-CoA oxidase was reduced to less than 10 percent of control values. Of interest, loading studies demonstrated that the glutaric acid was derived from lysine, presumably through the same pathway in which pipecolic acid is an intermediate. This case represents a peroxisomal cause of glutaricaciduria distinct from the well-known mitochondrial disorders resulting in glutaricaciduria types I and II.

Classic Adult Refsum’s Disease

Classic adult Refsum’s disease is a rare disorder of autosomal-recessive inheritance; originally described by Refsum [1946], it is characterized by retinitis pigmentosa, peripheral polyneuropathy, cerebellar ataxia, and increased cerebrospinal fluid protein. Biochemical identification is by the increased plasma levels of phytanic acid with normal pristanic acid. In this condition, the capacity for hydroxylation of phytanoyl-CoA has been lost. Low phytanic acid oxidation also is seen in human cells lacking PEX7, the receptor for PTS2, suggesting that the enzyme defective in Refsum’s disease is targeted to peroxisomes by a PTS2.

The enzyme deficiency of phytanoyl-CoA hydroxylase A and the gene defect in this disease have been identified conclusively [Jansen et al., 1997; Mihalik et al., 1997] and are the primary cause of adult Refsum’s disease. The gene PAHX is localized to chromosome 10, and patients with Refsum’s disease are homozygous for inactivating mutations in PAHX. It must be emphasized, however, that a substantial number of adult patients with the same phenotypic features have now been identified as having mutations in PEX7 [Jansen et al., 1997], and mutational analysis is required for definitive diagnosis. Therapy consists of restriction of dietary phytanic acid and symptomatic treatment.

Mulibrey Nanism

Mulibrey nanism is a rare disorder of autosomal-recessive inheritance that manifests with prenatal and postnatal growth failure, hepatomegaly, pericardial constriction, and characteristic facial features. Mulibrey stands for “muscle-liver-brain-eye,” and features of this disorder include yellow dots on the fundi, muscular weakness, enlarged cerebral ventricles, increased incidence of Wilms’ tumor, and cutaneous nevus flammeus [Kallijarvi et al., 2002; Lapunzina et al., 1995]. Patients have normal intelligence. It is more common in the Finnish population. It is due to defects in TRIM37 located on 17q22–q23. This gene encodes RING-B-box-coiled-coil protein, which is localized to the peroxisome. Patients with TRIM37 mutations do have normal peroxisome morphology, and other PTS1-containing matrix proteins are not affected. No abnormality is seen in very long chain fatty acid concentration or other biochemical measures. TRIM37 apparently lacks a PTS1 signal, but uptake of this protein was lacking in cell lines defective for PEX1 and PEX5. The exact role in peroxisomal function is not known at present [Avela et al., 2000; Kallijarvi et al., 2002]. The demonstration of this disorder as involving the peroxisome highlights an important point in going forward in the identification of peroxisomal disorders. Current screening strategies have focused on known biochemical features, and expansion of our understanding will require newer diagnostic modalities.