David Valle, Gerald V. Raymond, and Stephen J. Gould
PEROXISOME BIOLOGY AND METABOLISM
Peroxisomes are single membrane-bound or-ganelles present in all cells except for erythro-cytes. In human cells, peroxisomes are spherical in shape and range in number from a few hundred to a few thousand per cell.1,2 They contain a dense proteinaceous matrix composed of 50 or more enzymes that participate in a variety of metabolic processes.3 Prominent among these is a set of enzymes catalyzing β-oxidation that are analogous to but distinct from those catalyzing mitochondrial β-oxidation and are encoded by different genes. The β-oxidation systems of peroxisomes and mitochondria have distinct but overlapping substrate specificities, with the peroxisomal system oxidizing very-long-chain (C20-C26) and long-chain (C12–C18) fatty acids and the mitochondrial system oxidizing long-chain (C12–C18), medium–chain (C12–C6), and short-chain (C6–C4) fatty acids. An additional difference is that the FAD-linked acyl-CoA oxidase, which catalyzes the first step in the peroxisomal β-oxidation spiral, is reoxidized by molecular oxygen to produce H2O2 while the analogous enzymes in mitochondria transfer their electrons to the respiratory chain via electron transport flavoprotein (ETF) and ETF-dehydrogenase. The H2O2produced in the first step of the peroxisome β-oxidation spiral and by other peroxisomal oxidases is efficiently eliminated by catalase, another peroxisomal matrix enzyme. Additional metabolic processes that involve peroxisomal matrix enzymes include glyoxy-late transamination; β-oxidation of phytanic acid and other β-methyl-branched fatty acids; and synthesis of cholesterol, bile acids, and ether lipids such as plasmalogen and lysine degradation.
GENETIC DISORDERS OF PEROXISOMES: OVERVIEW
Peroxisomal disorders can be divided into two classes: (1) peroxisomal biogenesis disorders (PBD), which are characterized by deficiency of multiple peroxisomal functions, and (2) single-function disorders in which only one peroxisomal function is deficient.1 The PBD are a genetically heterogeneous set of disorders comprising at least 13 complementation groups as determined by somatic cell hybridization studies.1,2,4 All are inherited as autosomal recessive traits and have an aggregate frequency of about 1 in 50,000. Zellweger syndrome (ZS) and rhizomelic chondrodysplasia punctata (RCDP) are examples of the PBD clinical phenotypes. At the cellular level, most PBD complementation groups demonstrate aberrant cytosolic localization of matrix proteins with relatively empty peroxisomes and normal import of peroxisomal membrane proteins and synthesis of peroxisomal membranes. By contrast, a few PBD complementation groups have no detectable peroxisomes or peroxisomal membrane vesicles.
The single-function peroxisome disorders include at least 11 different disorders inherited either as autosomal or X-linked recessive traits, nearly all of which are uncommon, with frequencies of less than 1 in 50,000.3,5 The clinical example for this class of peroxisomal disorders is X-linked adrenoleukodystrophy (X-ALD), a neurological disorder with abnormal accumulation of very-long-chain fatty acids (VLCFA) and with an incidence in males of about 1 in 20,000.6,7 At the cellular level, peroxisomes in the single-function disorders appear normal and have normal import of matrix proteins.
PEROXISOME BIOGENESIS DISORDERS (PBD)
The clinical consequences of the PBD can be organized into two broad phenotypic spectra. The largest of these, the Zellweger spectrum, accounts for about 80% of PBD patients and includes at least three phenotypes originally thought to represent discrete disorders but which are now recognized as segments of a continuous spectrum.1,4,8 Listed from the most to the least severe, these three disorders are Zellweger syndrome (ZS), neonatal adrenoleukodystrophy (NALD), and infantile Refsum disease (IRD). The second PBD phenotypic spectrum, accounting for about 20% of PBD patients, is rhizomelic chondrodysplasia punctata (RCDP). The phenotype of most RCDP patients is severe and relatively uniform, but milder variants have been described.
Zellweger syndrome, a metabolic disorder with dysmorphic features (see Table 176-1), represents the severe end of the Zellweger spectrum. These infants have a characteristic facial appearance, with a high forehead, epicanthal folds, a small nose with a broad nasal bridge, anteverted nares, and micrognathia (Fig. 162-1A and B). The anterior fontanelle is large. Cataracts and a pigmentary retinopathy are common. There is profound hypotonia, feeding problems, and growth failure. Most have neonatal seizures. Liver function is abnormal, with conjugated hyperbilirubinemia. Radiological examination reveals punctate calcifications (“calcific stippling”) in the patella and epiphyses of the long bones. Multiple small renal cysts are common but may not be detected by ultrasound examination. Abnormalities in neuronal migration (neocortical dysplasia and cerebral/cerebellar white matter disease) are common. Infants with ZS rarely live to be 1 year of age.
Neonatal adrenoleukodystrophy (NALD) is similar to, but less severe than, ZS. Dysmorphic facial features are less severe or may even be absent (Fig. 162-1C). Hypotonia and seizures are common. Because of their flat facial features and hypotonia, NALD patients are sometimes thought to have Down syndrome. Survival ranges from several months to several years. The older patients have profound mental retardation often accompanied by sensorineural hearing loss and retinopathy.
Infantile Refsum disease (IRD) patients have mild dysmorphic features and hypotonia (Fig. 162-1D). The predominant manifestations may be hepatomegaly, cholestasis, osteoporosis, and failure to thrive in early infancy. As they get older, virtually all IRD patients develop sensorineural hearing loss and pigmentary retinopathy. They usually learn to walk but have severe mental retardation. Patients with IRD may live into adulthood.
Milder variants of the Zellweger spectrum with normal development and appearance may present in adult life with sensorineural hearing loss and pigmentary retinopathy.
FIGURE 162-1. Facial appearances of PBD patients in the Zellweger spectrum. (A) Four-month old infant with Zellweger syndrome; (B) 3-month-old infant with Zellweger syndrome; (C) 2-year-old child with neonatal adrenoleukodystrophy (NALD); (D) 4-year-old child with infantile Refsum disease (IRD).
RHIZOMELIC CHONDRODYSPLASIA PUNCTATA SPECTRUM (RCPD)
Patients with classic RCDP (see Table 176-1) have severe skeletal involvement at birth that distinguishes them from those in the Zellweger spectrum (Fig. 162-2). There is rhizomelia (shortening of the proximal limbs) and limited range of movement of the large joints of the extremities. Radiological examination shows extensive calcific stippling that involves the epiphyses of long bones; it is most prominent in the knees, elbows, hips, and shoulders. Coronal clefts of the vertebral bodies are apparent on lateral spine films. RCDP patients also have a flat face with frontal bossing. Cataracts are common, and an ichthyotic skin rash may develop after birth. Severe psy-chomotor retardation is present, and most die before 2 years of age. In addition to this classic RCDP phenotype, mildly affected patients with little or no rhizomelia have been described; some have mild intellectual defects as their only manifestation. Classic RCDP is a PBD caused by mutations in PEX7, the gene encoding the receptor for PTS2 proteins. A few patients with the classic RCDP phenotype (< 10% of the total number of patients) have single-function defect in PTS2-targeted peroxisomal matrix enzymes necessary for normal synthesis of plasmalogen.
Over the last two decades, research in model organisms and PBD patients has identified a set of genes and their protein products, termed PEX genes and peroxins, respectively, that are necessary for peroxisome assembly.1,4 Most of these are involved in the targeting and uptake of matrix proteins into the organelle. All of the PBD complementation groups so far defined are explained by mutations in these PEX genes, one particular gene for each complementation group. The number of PEX genes is greater than the number of complementation groups, suggesting there are new groups to recognize. See Weller et al for a review of this topic.4
Peroxisome matrix proteins are synthesized on free cytosolic ribosomes and directed to the organelle by targeting sequences of two types (Fig. 162-3). Peroxisome-targeting signal 1 (PTS1), a C-terminal tripeptide (-SKL or conservative variants thereof), is utilized by more than 90% of the matrix proteins. PTS2, located 5 to 10 residues from the N-terminus, is utilized by a few peroxi-somal matrix proteins, including one involved in β-oxidation (peroxisomal thiolase 1), one involved in α-oxidation, and one involved in plasmalogen synthesis. The PTS1 or PTS2 motifs are bound by specific cytosolic receptors encoded by PEX5 and PEX7, respectively. Docking of the receptor and its bound cargo on the peroxisome membrane is mediated by binding to specific peroxisomal membrane proteins (PEX13, PEX14, PEX17). With the action of additional peroxisomal membrane proteins (PEX2, PEX8, PEX10, PEX12), the newly synthesized matrix proteins are translocated into the organelle, and the receptors are recycled to the cytosol. Recycling of PEX5 requires an unusual monoubiquitination on a conserved cysteine near the N-terminus of the protein.9 The recycling process is facilitated by the action of PEX1, PEX6, PEX22, PEX4, and PEX26. Genetic defects in this subset of PEX genes are responsible for nine PBD complementation groups that are characterized at the cellular level by mislocalization of matrix proteins to the cytosol.
The mechanism and signals involved in targeting peroxisomal membrane proteins to the organelle are less well understood, although PEX genes encoding proteins that function in this process have been identified (PEX3, PEX16, PEX19).4 Not surprisingly, genetic defects in this subset of peroxins are responsible for the three PBD complementation groups characterized by lack of detectable peroxisomes.
Recently, an infant with metabolic features consistent with abnormal peroxisome and mi-tochondrial function has been described and represents a different type of PBD.10 In this patient’s cells, there was a defect of organellar fission of both peroxisomes and mitochondria caused by a dominant negative mutation in the dynamin-like protein 1 gene DLP1. This observation, together with studies in model systems, indicates that normal DLP1 function is required for the division of two different subcellular organelles.
FIGURE 162-2. Radiographs of an infant with rhizomelia chondrodysplasia punctata (RCDP). (A) Forearm showing extreme rhizomelia and punctate calcifications; (B) lateral spine showing coronal clefts of the vertebral bodies; (C) lower extremity.
LABORATORY DIAGNOSIS OF PBD
The most frequently utilized diagnostic laboratory tests for PBD detect abnormalities of peroxisomal metabolic processes, including very-long-chain fatty acid (VLCFA) β-oxidation, phytanic acid β-oxidation, and plasmalogen synthesis.1,2,4 Plasma VLCFA (C26:0 and C26:1) are abnormally increased in Zellweger spectrum patients to an extent roughly correlating with clinical severity. Compared to control levels (C26:0 = 0.22 ± 0.08; C26:1 = 0.12 ± 0.05 μg/mL), VLCFA are elevated about tenfold in ZS, about fivefold in neonatal adrenoleukodystrophy (NALD), and about threefold in IRD. Similarly, plasma phytanic acid (normal = 0.8 ± 0.4 μg/mL) is increased ten- to a hundredfold in Zellweger spectrum patients who are old enough to ingest dietary precursors of this compound. Red cell plasmalogens (expressed as a ratio of dim-ethylacetyl derivatized plasmalogens to fatty acids of the same chain length; normal range of C16:0 DMA/C16:0 fatty acids = 0.051 − 0.90; C18:0 DMA/C18:0 fatty acids = 0.137 − 0.255) are reduced by tenfold or more. Other laboratory abnormalities in Zellweger spectrum patients include increased urinary excretion of medium- and long-chain dicarboxylic acids and pipecolic acid and reduced levels of plasma bile acids.
In RCDP, plasma VLCFA levels are normal, possibly because peroxisomal thiolase 2 (sterol carrier protein X) substitutes for the lack of the PTS2-targeted peroxisomal thiolase 1. RBC plasmalogens are reduced in RCDP to an extent similar to that in Zellweger spectrum patients. In RCDP patients ingesting foods containing phytanic acid precursors, the levels of phytanic acid in plasma are usually higher than in Zellweger spectrum patients.
Confirmation of diagnoses made on the basis of clinical phenotype and the above metabolite assays should be confirmed by studies of peroxisomal β-oxidation and plasmalogen synthesis in cultured skin fibroblasts. These are available at reference labs (see www.genetests.org or www.peroxisome.org) and can be supplemented by immunohistochemical studies localizing PTS1-and PTS2-targeted matrix proteins and peroxisomal membrane proteins. Complementation analysis is a useful preliminary test for molecular studies to identify the responsible PEX gene; however, this has little prognostic value, since patients representing different segments of the Zellweger spectrum have been identified in most complementation groups. Progress in identifying the PEX genes and the relative frequency of disease-causing mutations has led to an efficient hierarchical plan for molecular diagnosis by gene sequencing.8
FIGURE 162-3. Model of import of peroxisomal matrix proteins and of peroxisome membrane biogenesis. Matrix proteins targeted by either a PTS1 or PTS2 signal bind their respective receptors (PEX5, PEX7) in the cytosol. These complexes interact with a set of integral peroxisomal membrane proteins (PEX14, PEX13, PEX2, PEX10, PEX12) to achieve docking to the organelle. The PEX1, PEX6 heterodimer is located at the organelle surface by PEX26 and plays a role in the translocation of the matrix proteins and/or the release of the receptors. The peroxisome membrane protein receptor, PEX19, and two peroxins, PEX3 and PEX1 6, necessary for de novo peroxisomal membrane biogenesis are also shown.
TREATMENT OF PBD
The pathophysiology of the PBDs is complex and often begins in utero. For these reasons, treatment of patients with the severe forms of these disorders is largely supportive. Feeding difficulties are frequent, and placement of a gastros-tomy tube is often indicated to improve nutrition and to facilitate care. Anticonvulsant medications are indicated to control seizures. In older individuals, plasma phytanic acid concentrations should be measured repeatedly and dietary phytanic acid and its precursors (phytol) limited. The consequences of the severe skeletal involvement should be carefully assessed in RCDP patients. For example, moderate cervical spine stenosis has been described in RCDP.
Age at demise is often listed as one of the criteria used to categorize phenotypic severity in the Zellweger spectrum (ZS). At the time of diagnosis, however, the phenotypic features of ZS, NALD, and IRD overlap; thus, in most cases, it is unwise to make precise survival predictions.
All known PBD are inherited as autosomal recessive traits, with a 25% recurrence risk for each subsequent pregnancy of couples who have had one PBD infant. Prenatal diagnosis is possible by biochemical methods and is provided by several reference labs (see Web sites above). If the molecular basis of the index case is known, similar studies can be used for prenatal diagnosis in subsequent pregnancies.
In contrast to the PBD, peroxisomes in this class of disorders have a normal structure and function except for an isolated deficiency or abnormality of a particular peroxisomal protein.3 At least 11 well-defined disorders meet this criterion, and they can be grouped according to the disrupted peroxisomal function (Table 162-1).
This highly variable X-linked neurodegenerative disorder is caused by mutations in adrenoleuko-dystrophy (ALD), the gene encoding ALDP, an ATP-binding cassette (ABC) transporter located in the peroxisomal membrane.6,7
There are multiple phenotypic presentations for males with X-ALD. The most severe is the childhood cerebral form, which is a rapidly progressive, inflammatory, central demyelinat-ing disease that begins between ages 3 and 10. About 35% of X-ALD males manifest this phe-notype with progressive behavioral, cognitive, and neurological abnormalities that lead to total disability within 3 years and eventually to death. Nearly all of these patients have adrenal insufficiency. The T2 weighted MRI shows symmetric areas of increased signal in the parieto-occipital region.
A second, distinct phenotype, known as adre-nomyeloneuropathy (AMN), begins in the third to fourth decade and is characterized by a distil axonopathy, mainly involving the spinal cord. AMN patients manifest a slowly progressive gait disturbance and progressive urinary sphincter dysfunction. About two thirds of AMN patients develop adrenal insufficiency, and about 40% eventually manifest cerebral involvement.
Other phenotypic presentations include adults with only adrenal involvement, but many of these men eventually develop AMN symptoms (see Chapter 576 for detailed description of neurological manifestations).
Table 162-1. Summary of Disorders with Abnormal Function of a Single Peroxisomal Protein
There is no correlation between the nature of the ALD mutation and phenotypic severity. In fact, multiple affected members in a single family, all with the same mutant ALD allele, may manifest the extremes of phenotypic expression; individuals with the childhood cerebral form occur in the same family as individuals with only AMN or adrenal insufficiency.
ALDP, the protein product of ALD, is a peroxisomal membrane transporter.3,6 The functional transporter is either a homodimer of two ALDP subunits or a heterodimer of ALDP, with one of three other related ABC transporters found in the peroxisome membrane. The ligands for these transporters are not known with certainty but are likely to be long-chain fatty acids, fatty acyl-CoAs, or other hydro-phobic substances. Interestingly, recent reports have implicated loss of peroxisomal function in oligodendrocytes as a cause of de-myelination and neuroinflammation.
LABORATORY DIAGNOSIS OF X-ALD
Regardless of their phenotype, X-ALD patients have a marked elevation of plasma VLCFA, particularly C26:0. In cultured skin fibroblasts, β-oxidation of VLCFA is impaired. These observations suggest that ALDP function is required for these fats to enter peroxisomes, where they are degraded by peroxisomal β-oxidation.
Prenatal diagnosis is possible by measuring VLCFA in amniotic fluid and by molecular analysis of ALD.
The highly variable phenotypic manifestations of X-ALD make evaluating any therapy difficult. At this point, it is clear that reducing plasma VLCFA by dietary measures does not arrest cerebral disease once established. Reducing VLCFA as a preventative therapy is presently under study. Current evidence indicates that performing bone marrow transplantation in boys in the early stages of the childhood cerebral phenotype blocks progression of the disease. A successful stem cell gene therapy using a lentiviral vector has been recently reported in two patients.11
X-ALD is inherited as an X-linked recessive trait. The combination of plasma VLCFA determination and molecular analysis can identify all X-ALD hemizygotes and heterozygotes. Reliable prenatal diagnosis of affected individuals can be utilized to prevent transmission of the disease. The inability to predict phenotypic severity complicates this process.
DISORDERS OF PEROXISOMAL FATTY ACID β-OXIDATION
In addition to X-ALD, at least five inherited disorders of peroxisomal fatty acid β-oxidation have been identified. After X-ALD, D-bifunctional protein deficiency is the most common. Interestingly, the phenotype of inherited deficiency of enzymes in the β-oxidation spiral resembles that of PBD patients at the severe end of the Zellweger spectrum (ZS and NALD). This observation suggests that disruption of peroxisomal β-oxidation plays a major role in the pathophysiology of the PBD. Some patients with isolated peroxisomal fatty acid oxidation defects may present in late childhood as isolated peripheral neuropathy mimicking Charcot Marie Tooth disorder.
Diagnosing these patients depends on evidence of peroxisomal β-oxidation disruption (increased VLCFA, pristanic acid, and bile acid precursors) without evidence of lack of other peroxisomal functions (plasmalogen synthesis) or of problems in peroxisomal assembly. All of these disorders are inherited as autosomal recessive traits, and prenatal diagnosis is available.
This abnormality in the peroxisomal β-oxidation of phytanic acid typically presents in the second decade, although about a third of patients experience their first symptoms before 10 years of age.3,5 Virtually all patients develop retinitis pigmentosa with night blindness as an initial symptom. The electroretinogram is extinguished, and the visual field gradually constricts. Progressive combined sensory and motor neuropathy affecting mainly the lower extremities is a later symptom, as is cerebellar dysfunction. Cerebrospinal fluid protein concentration is elevated without a pleocytosis. Other less uniform symptoms include early onset anosmia, cardiomyopathy, mild epi-physeal dysplasia, and ichthyotic skin rash.
The primary defect is deficiency of phytanoyl-CoA hydroxylase caused by mutations in the PAHX gene. Plasma phytanic acid levels are markedly elevated (a hundred- to a thousandfold). The enzymatic defect can be demonstrated in cultured skin fibroblasts.
Refsum disease is an autosomal recessive disorder, and prenatal diagnosis by biochemical and/or molecular methods is possible.
The sole sources of phytanic acid are dietary phytanic acid and its precursor, phytol. Meat, ruminant fat, dairy products, and fish are rich sources of phytanic acid. Diets restricted in these substances produce a gradual but dramatic decline in plasma and tissue phytanic acid. This can be hastened by plasmapheresis. Patients who maintain their phytanic acid at near normal levels often improve clinically. For these reasons, dietary treatment should be instituted as soon as possible and maintained for life.