Clinical manifestations

Presentation and course

All defects of peroxisomal beta-oxidation involve the nervous system. X-linked adrenoleukodystrophy can be distinguished from other single enzyme defects through significant differences in age of onset and clinical manifestations. This disorder is reviewed separately. Two of the other single enzyme defects (ACOX1/acyl-CoA oxidase deficiency and HSD17B4 deficiency) share the following typical clinical features: the hallmark symptoms, muscle hypotonia and seizures, are present in the neonatal period; affected patients show severe delay in psychomotor development and death occurs typically in infancy or early childhood. In many respects, the clinical presentation in these disorders resembles that seen in the peroxisome biogenesis disorder spectrum, which includes the Zellweger syndrome, neonatal adrenoleukodystrophy, and infantile Refsum disease phenotypes. In contrast, AMACR deficiency appears to be a milder disorder with symptoms developing in adulthood.

ACOX1 (Acyl-CoA oxidase) deficiency and HSD17B4 (D-bifunctional protein) deficiency presenting in infancy. These disorders have many similarities in their clinical presentation and will be discussed together. Relevant clinical findings in these 2 disorders are summarized in Table 1.

Table 1. Clinical Presentation in Patients with ACOX1 Deficiency and HSD17B4 Deficiency

(Adapted from 16; Adapted from 15)

Moreover, the clinical presentations of patients with type I, II, or III HSD17B4/D-bifunctional protein deficiency did not significantly differ (16). In nearly all patients, hypotonia was present in the neonatal period; it is usually generalized and severe. On observation abnormal posture is apparent with abduction of the limbs and a "frog-legged" position. Other signs of hypotonia mentioned in these patients are "complete head lag" (25), "nuchal hypotonia" (08), and "weak neck muscles" (04). In most cases neonatal reflexes and Moro response are absent (25; 88; 43; 82); only Barth and associates reported the presence of a Moro response in their patient (04). Assessment of deep tendon reflexes shows variable results covering a spectrum from normal (62), through decreased (54; 88) to absent (25; 43; 82). In milder cases some antigravity movement, spontaneous or with stimulation, may be observed. In patients who survive into infancy and childhood, hypotonia can be replaced by hypertonia with pyramidal signs (62) or, depending on the cerebral progression of the disease, decerebrate posturing (04).

Several authors report poor suck reflex, and feeding difficulties requiring special feeding techniques, gavage feedings, or placement of a gastrostomy tube for long-term management (25; 54; 04; 43; 82).

Dysmorphic features were present in several patients with HSD17B4/D-bifunctional protein deficiency (25; 54; 43; 82; 68; 16). In their large series published in 2006, Ferdinandusse and colleagues reported that 68% of HSD17B4-deficient patients had dysmorphic features that resembled those found in patients with Zellweger syndrome (16). Although some show only mildly abnormal features such as a high forehead (54), one patient had profound craniofacial abnormalities including a high forehead, epicanthal folds, low and broad nasal bridge, anteverted nostrils, micrognathia, and a high arched palate (82). One of the patients described by Mandel and colleagues had overall coarse features, a high forehead, shallow orbital ridges, a long philtrum, and thick lips (43). Abnormal appearance might also be due to involvement of facial muscles (facial diplegia), causing an expressionless face with incomplete eye closure (25). In 2007, Ferdinandusse and colleagues also reported that 50% of ACOX1-deficient patients had external dysmorphia (15).

Both ACOX1 deficiency and HSD17B4 deficiency are associated with seizures manifesting in the neonatal period (25; 08; 54; 62; 88; 04; 81; 16; 15); the age of onset varies between 15 minutes and 7 days. Typical descriptions of these convulsions consist of "clonic movements" of the extremities accompanied by apnea and "upward deviation of the eyes" (62), "lip smacking followed by twitching of the eye and clonic movements of the upper extremities" (88), or "apneic spells, eye blinking and myoclonic seizure activity" (43). Following the classification of Volpe, most of these episodes can be classified as multifocal and focal clonic seizures (78). Convulsions continue beyond the neonatal period, mainly as focal seizures, sometimes with secondary generalization (54; 04). In most patients the seizures remain unresponsive to or are only poorly controlled with anticonvulsants.

Electroencephalograms show "frequent multifocal spikes" (88), "epileptiform discharges" (62), "burst suppression patterns" (43), and in one patient "multifocal spike discharges and an electrographic focal seizure" and later in the course "lack of maturation, multifocal seizure activity, most prominent in slow wave sleep" (25).

Patients with ACOX1 deficiency generally achieved more motor milestones than did those with HSD17B4 deficiency (89). In the latter disorder, overall psychomotor development is delayed, and in the most severely affected patients there is virtually none (25; 43).

The delay in psychomotor development and especially cognitive development might be exacerbated by hearing and visual impairment, which occurs in both disorders (25; 08; 62; 88; 04; 81; 16; 15). More than half of HSD17B4-deficient patients and more than three fourths of the ACOX1-deficient patients had evidence of vision abnormalities, such as nystagmus, strabismus, or failure to fixate objects at 2 months of age, and a progressive loss of vision and hearing was noted in many patients (16; 15). Hearing was impaired in most ACOX1-deficient patients for whom detailed clinical data were available, and in about half of the HSD17B4-deficient patients. Brainstem auditory evoked responses were normal shortly after birth in one patient (62), but this patient, as well as all others, had abnormal tests indicating sensorineural hearing loss later in life. In multiple patients’ visual evoked responses and electroretinograms were also abnormal. Funduscopic exam was unremarkable in some patients early in life (88; 04; 43) but abnormalities may be developed later. Nonspecific retinopathy (54), abnormal retinal pigmentation (04; 82), and retinal degeneration (62) have been described. Optic atrophy was present in one child with ACOX1 deficiency (62). Myopia and hyperopia have been reported (62), and cataracts were present in one patient (82).

Hepatomegaly has been noticed in a number of patients (25; 08; 54; 62; 81; 43; 68; 16; 15), but seems to be mild and without significant impairment of hepatic function in many patients. Evidence of liver disease such as fibrosis or steatosis was found in about one fourth of HSD17B4-deficient patients (16). Liver function tests are typically normal or only mildly elevated. Symptomatic cirrhosis or hepatic failure has never been reported. In some patients the hepatomegaly resolved after the first few months of life (08). Mild splenomegaly has been described in 2 patients (25; 62).

Although postmortem examination showed adrenal atrophy in some cases (25; 08; 54; 88), there are no reports of clinically significant adrenal insufficiency or Addisonian crisis. In some patients plasma corticotropin was elevated (54; 62), and cortisol was decreased (62). Adrenal stimulation tests may be normal (04) or abnormal (54).

Stippled skeletal calcifications, as reported in Zellweger syndrome or other peroxisomal biogenesis disorders, have not been noticed in patients with single enzyme defects. In patients with prolonged survival, skeletal maturation may be delayed (08; 88). Late closure of the large fontanelle, presence of a metopic suture, and macrocephaly have been reported in cases with HSD17B4 deficiency (08; 54; 82).

Other abnormalities associated with peroxisomal single enzyme defects include cardiac malformations. Ventricular septal defects were present in 2 patients (25; 54). One of these patients developed intermittent cardiac failure and required treatment with diuretics and digoxin (25). Only one patient was born prematurely (at 34 weeks); the neonatal course was complicated by episodes of hypoglycemia and a hemorrhagic diathesis (08).

Watkins and colleagues compared the clinical features of 6 ACOX1-deficient and 15 HSD17B4-deficient patients (89). Although HSD17B4-deficient patients had evidence of neuronal migration defects, alpha-CoA oxidase-deficient patients developed a progressive leukodystrophy between 2 and 3 years of age. White matter abnormalities were found in all ACOX1-deficient patients reported by Ferdinandusse and colleagues (15). In contrast, these authors reported delayed maturation of white matter in about one third of HSD17B4-deficient patients, but only found demyelination in less than 20% of cases (16). Khan and colleagues subsequently reported serial MRI findings in a boy with type III HSD17B4 deficiency who survived 8 years 11 months (37). At 2 years 9 months, his MRI showed posterior white matter changes with involvement of the splenium of the corpus callosum, cerebellum, and brainstem, findings that are more typically seen in X-linked adrenoleukodystrophy. MRIs at 4, 5, and 6 years of age showed posterior to anterior progression of demyelination, also more typical of adrenoleukodystrophy than of HSD17B4 deficiency. Excluding this unusual patient, the average age at death was 17.6 months for HSD17B4 deficiency and 5 years for alpha-CoA oxidase deficiency.

In summary, the phenotype of most patients with alpha-CoA oxidase and HSD17B4, deficiency is variable but seems to share some common features. In any patient with hypotonia and seizures, especially when there are combined with dysmorphic features, hearing deficit, visual impairment, and mild hepatomegaly, a peroxisomal dysfunction has to be ruled out.

Adult ACOX1 deficiency. The first reported diagnosis of ACOX1 deficiency in an adult appeared in 2009 (13). A 52-year-old male was evaluated for mildly impaired cognitive function. Although his early developmental milestones were normal, he was “clumsy” with a progressively unsteady gait and became wheelchair-bound at 28 years of age. Neurologic examination was remarkable for slurring dysarthric speech, jerky head tremor, dystonic posturing of the arms, and ataxia on finger-nose testing. Spasticity was present in the lower extremity; reflexes were brisk but symmetrical. There was bilateral ankle clonus, and extensor plantar responses were elicited. His ophthalmologic examination revealed bilateral retinitis pigmentosa, decreased visual acuity, and gaze nystagmus. The patient’s parents were first cousins, and his 55-year-old sister had very similar clinical manifestations. Her cognition was somewhat worse than that of her brother. Bilateral cataracts prevented visualization of her optic fundi. She was unable to move her legs but had normal reflexes. MRI findings in these adult siblings showed profound atrophy of the cerebellum and brainstem (particularly the pons in the male patient) but only modest cerebral atrophy. These findings differed significantly from the white matter changes seen in childhood ACOX1 deficiency.

Adolescent/adult HSD17B4 deficiency and Perrault syndrome. Although most infants with HSD17B4 deficiency die within the first 2 years, adolescents and adults with milder phenotypes have now been identified, primarily via exome sequencing. An adolescent male, aged 16.5 at time of study, had moderate-to-severe sensorineural hearing impairment since 3.5 years of age (47). He was otherwise normal until 11 years of age when he insidiously developed progressive gait ataxia, which now requires him to use a wheelchair other than for short distances. He had bilateral pes cavus and mild hammertoe foot deformity, and diffuse areflexia, and his plantar reflex was flexor. A progressive sensorimotor polyneuropathy with demyelinating features was revealed by nerve conduction studies. Subclinical retinitis pigmentosa was reported. MRI at 12 years of age revealed cerebellar atrophy. There was no evidence of growth retardation, and he had age-appropriate pubertal development. The patient’s brother, aged 14 when reported, also had moderate-to-severe sensorineural hearing Impairment since 2 years of age (47). He had normal physical activity, but examination revealed very mild pes cavus and hyporeflexia, flexor plantar responses, and mild anterior compartment weakness. Coordination was normal, but nerve conduction studies revealed a mild sensorimotor polyneuropathy with demyelinating features. Although visual acuity remained normal, peripheral retinal atrophy was noted. Although growth was normal, there was no evidence of pubarche at the age of 14. Exome sequencing revealed compound heterozygous mutations in HSD17B4 in both siblings. These boys had a missense mutation in the hydratase domain of one allele along with a missense mutation in the dehydrogenase domain of the other allele, prompting McMillan and colleagues to suggest that they represent a new classification of HSD17B4 deficiency (type IV).

Exome sequencing has also been instrumental in identifying adult HSD17B4-deficient patients. An adult male, aged 35 when reported, had progressive ataxia since childhood and required a wheelchair since 29 years of age (40). He had mild developmental delay with cognitive impairment, and his medical record indicated documented azoospermia. Mild sensorineural hearing loss was detected at 34 years of age, and abnormalities were found on occulomotor examination. Progressive cerebellar volume loss was seen on MRIs obtained between ages 14 and 35. He was suspected of having a mitochondrial disorder, but no genetic abnormality was found in a panel of 18 ataxia and mitochondrial disease genes. Exome sequencing revealed the patient carried a rare HSD17B4 variant at a highly conserved locus along with a 12 kb deletion of exons 10 to 13.

Three adult siblings, 2 female and 1 male, were also identified by exome sequencing as having HSD17B4 deficiency (41). Although their ages ranged from 35 to 39 when reported, hearing loss was noted in childhood (age range 5 to 10), and ataxia was noted a few years later (age range 10 to 14). Disease progression was slow and included cerebellar atrophy, intellectual decline, hypogonadism, hyperreflexia, demyelinating sensory neuropathy, and supratentorial white matter changes. Exome sequencing revealed heterozygous mutations in conserved residues of HSD17B4 in all patients.

Perrault syndrome, also known as XX gonadal dysgenesis, is a rare genetic disorder characterized by sensorineural deafness and ovarian dysgenesis in females but only deafness in males. Several genetic causes of Perrault syndrome have been reported, including mutations in HSD17B4, HARS2, CLPP, and LARS2 (06). HSD17B4 deficiency was first reported in 2 sisters with well-documented Perrault syndrome who were 27 and 16 years of age at time of publication (61). Additional findings in both siblings included short stature, mild mental retardation, and progressive peripheral neuropathy. Whole exome sequencing of genomic DNA from one sister found only 1 gene (HSD17B4) with 2 functional variants. Mutations were verified by PCR of lymphocyte cDNA obtained from both sisters and their parents. Similar to the older HSD17B4-deficient patients and the patients with adult ACOX1 deficiency, the older sister had cerebellar manifestations, including ataxia, dysarthria, and intention tremor; her MRI showed moderately severe atrophy of the cerebellar hemispheres and vermis. Ovarian dysgenesis has not been reported in HSD17B4 deficiency, primarily because few if any female patients survived to puberty. As of 2017, 6 Perrault syndrome patients from 4 families have been described, and their clinical findings have been summarized (06). Despite HSD17B4 mutations in either the enoyl-CoA hydratase domain, the hydroxyacyl-CoA dehydrogenase domain, or both, these patients appear to have normal very long-chain fatty acid (VLCFA) levels (06). In a 2016 report, Amor and colleagues described 5 new patients with “juvenile peroxisomal D-bifunctional enzyme deficiency,” and they summarize the clinical findings in 14 patients from 8 families (02). Patients with adolescent and adult HSD14B deficiency, as well as Perrault syndrome, were considered as 1 group in this report.

Sterol carrier protein X deficiency. In 2006, a report of the first known patient with deficiency of sterol carrier protein X (SCP2) was published (21). Unlike ACOX1 and HSD17B4 deficiency, SCP2 deficiency did not present in the neonatal period. The male patient, diagnosed at age 45, had a 28-year history of spasmodic torticollis and dystonic head tremor. At age 44, he was evaluated because of worsening dystonic symptoms. Neurologic findings at that time included hyposmia, pathological saccadic eye movements, brisk deep tendon reflexes of the upper extremities but diminished lower extremity reflexes, plantar sole responses, reduced vibration sense, slight cerebellar ataxia with intention tremor, and balance and gait impairment. No pareses were present, and superficial sensation was normal. Ophthalmological examination was unremarkable. MRI studied revealed bilateral hyperintense signals in the thalamus, butterfly-like lesions in the pons, and lesions in the occipital region. Nerve conduction studies of both the upper and lower extremities were abnormal. The patient was infertile, with hypergonadotrophic hypogonadism and azoospermia. He had 2 male siblings, one (1 year younger) of whom was said to have similar symptoms; the other brother (4 years younger) is reportedly asymptomatic.

AMACR deficiency. To date, only 7 patients (5 adults and 2 children) with documented alpha-methylacyl-CoA racemase deficiency have been reported. However, some similar clinical features among the adult patients are beginning to emerge. The clinical presentation of this disorder in adults is significantly different from that of ACOX1 deficiency and HSD17B4 deficiency, but is somewhat reminiscent of Refsum disease (70). Patients’ ages at diagnosis ranged from about 34 to 57 years; 3 were female and 2 were male. Their initial presentations varied and included spastic paraparesis, progressive visual failure, tremor, and cognitive decline. Four had evidence of peripheral neuropathy, with demyelination present in 1 patient. Three patients presented with seizures, a feature not seen in Refsum disease; for 2 patients, the onset of seizures occurred in their teenage years. Encephalitic or encephalopathic episodes were also a prominent feature in 3 cases, and in 1 patient these were recurrent and associated with the accumulation of neurologic deficits. Pigmentary retinopathy and headaches were present in 3 patients, whereas depression was reported in 3 cases. Wanders and coworkers first described this defect in 3 patients (14).

The first 2 patients presented with adult-onset sensory motor neuropathy. A male patient, diagnosed at 44 years of age, had retinitis pigmentosa, primary hypogonadism, epileptic seizures, and a widespread axonal neuropathy affecting the legs more than the upper extremity. A more complete case report of this patient was subsequently published (46). The second patient, a female who did not develop symptoms until 48 years of age, developed a spastic paraparesis of the lower extremity, with normal MRI of the cervical spine. Nerve conduction studies revealed evidence of a demyelinating polyneuropathy. Another male patient, diagnosed with schizophrenia at the age of 23, subsequently suffered from recurrent bouts of rhabdomyolysis and stroke-like episodes that eventually led to a diagnosis of AMACR deficiency about 11 years later (36). An MRI at the age of 23 showed T2-weighted increased signal in pons, thalamus, and cerebral peduncles; MRIs following stroke-like episodes at the ages of 33 and 34 showed cortical edema. He had decreased visual acuity and an abnormal electroretinogram, but no pigment clumpings were observed. Following his second stroke-like episode, a right frontal cortex biopsy was performed that showed discrete edema, endothelial cell hyperplasia, microglial activation, axonal balloonings, and reactive gliosis, consistent with acute ischemia without inflammation. The biochemical studies that eventually led to a diagnosis of AMACR deficiency were conducted because the first and third patients’ symptoms resembled those of Refsum disease; the second patient’s symptoms resembled those found in adrenomyeloneuropathy, the X-linked adrenoleukodystrophy phenotype that presents in symptomatic heterozygotes during late adolescence or adulthood.

One of the 2 children with AMACR deficiency was discovered serendipitously (14). This patient, born of doubly consanguineous Asian parents, had documented Niemann-Pick C disease. During biochemical workup, a second genetic disorder was suspected, and AMACR deficiency was confirmed. No symptoms directly related to AMACR deficiency could be detected due to the concurrent Niemann-Pick C disease. The second child was an African-American girl who presented at 2 weeks of age (75; 66). Her primary symptoms were consistent with vitamin K deficiency, and serum levels of all fat-soluble vitamins were low. Liver biopsy was consistent with giant-cell neonatal hepatitis and severe cholestasis, prompting the biochemical analyses that led to a diagnosis of AMACR deficiency. An older sibling was said to have died of vitamin K deficiency. The sibling’s liver was used for orthotropic transplantation into a 28-month-old child with end-stage liver disease, and a posttransplantation biopsy was significant for evidence of acute rejection, bile duct proliferation, and fibrosis. Although not proven enzymatically or biochemically, the sibling likely had AMACR deficiency as well.

ACOX2 deficiency. An 8-year-old male patient ultimately diagnosed with ACOX2 deficiency was described in 2016 (77). His parents were second cousins. Growth (height, weight, and head circumference) was normal. An evaluation at 8 months for vomiting, presumably secondary to acute gastroenteritis, revealed elevated transaminase levels. His transaminase levels were intermittently elevated over subsequent years. A liver biopsy at 6 years revealed many thin fibrous septa, swollen hepatocytes, glycogenated nuclei, and focal acinar transformation, but no obvious cholestasis or steatosis. He had mildly delayed language development and mild intellectual disability. Neurologic evaluation revealed slurred speech, vertical gaze palsy, slight dysmetria, and mild gait ataxia. His performance on the Wechsler Intelligence Test for Children was 66 (mild intellectual disability). Fundoscopy and brain MRI were both normal. Exome sequencing revealed a homozygous premature termination at codon 69 of ACOX2. Consistent with a proposed role for ACOX2 in synthesis of bile acids from cholesterol via beta-oxidation of the aliphatic side chain, the patient’s plasma and urine contained elevated levels of C27 bile acid precursors and low to low-normal levels of mature bile acids. However, branched-chain fatty acid (phytanic and pristanic) levels were normal. Vilarinho and colleagues noted that a trial of primary bile acid therapy, which reduces synthesis of potentially toxic intermediates, was planned for this patient, who was at least 6.5 years of age at the time of the report (77). A second patient with ACOX2 deficiency was a boy who presented at age 16 with persistent hypertransaminasemia of unknown origin (48). A liver biopsy showed only mild intracellular cholestasis. The patient was treated with cholestyramine, which improved the transaminasemia. Subsequent analysis of his bile acids revealed decreased C24 species and elevated C27 precursors, in both serum and urine.

ABCD3 deficiency. Ferdinandusse and colleagues described a female child born of consanguineous parents (20). Early growth and development were normal. Mild jaundice was noted during the neonatal period and around 6 months but was not treated. Abdominal distension was observed at 1 year. While being treated at a hospital at 1.5 years for fever and gastroenteritis due to rotavirus infection, hepatosplenomegaly and paleness were noted. She was found to be anemic and mildly jaundiced, and she had elevated serum transaminases. The anemia was thought to be due to iron deficiency. A liver biopsy at 1.5 years revealed evidence of hepatocellular regeneration, but there was minimal intracanalicular and intracytoplasmic cholestasis. Fibrosis was present, but no necrosis or steatosis. At 2 years of age she was started on ursodeoxycholate treatment. Despite normal growth and development, her liver and spleen enlarged further over the next 2 years, leading to pancytopenia. She developed severe portal hypertension around 3 years of age, again with mild jaundice. Bile acids, and particularly bile acid precursors, were markedly increased in plasma. At 4 years of age the patient rapidly deteriorated. Decompensated cirrhosis and hepatopulmonary syndrome necessitated a liver transplant. The patient died 5 days posttransplant from respiratory complications. The diagnosis of ABCD3 deficiency was not made prior to the patient’s demise. Skin fibroblasts were obtained and were found to have a reduced number of enlarged peroxisomes, similar to those seen in ACOX1 and HSD17B4 deficiency. Routine workup for peroxisomal defects included immunostaining with antibody to ABCD3. Surprisingly, no signal was detected; furthermore, the protein was absent on Western blots. Beta-oxidation of VLCFA was normal, whereas pristanic acid beta-oxidation was reduced. Analysis of genomic DNA revealed a homozygous deletion of 1758 bp in the patient’s ABCD3 gene.

ACBD5 deficiency. Exome sequencing of patients with retinal dystrophy identified 6 previously unknown candidate genes, including ACBD5 (01). An ACBD5 mutation predicted to abolish a consensus splice-donor site was detected in 3 affected siblings who presented with cone-rod dystrophy and psychomotor delay associated with significant white matter involvement (18). No further clinical information on these patients was available. The clinical picture of a fourth patient from a different family was reported in 2016. She was the only affected of 3 siblings from healthy, consanguineous parents. She was normal at birth except for a cleft palate (surgically corrected at 6 months). Abnormal eye movements were noted at 7 months, and retinal rod-cone dystrophy was diagnosed. She had delayed motor skill development and an unsteady gait. By 2 years of age, her gait had become progressively abnormal. By 4 years of age, her vocabulary was limited, and she was dysarthric despite normal hearing. She developed progressive microcephaly with facial dysmorphisms, including a tubular nose, hypotelorism, prominent ears, bilateral ptosis, and rotatory nystagmus. Motor dysfunction was marked, with a positive Gowers and proximal weakness; there was increased extrapyramidal and pyramidal tone in her arms and legs. Gait was wide-based with truncal titubation and waddling. By 9 years of age she could walk only with two-handed assistance or short distances with a walker. Brain MRI at 4 years revealed hypomyelination with diffuse T2 signal abnormality in deep white matter, with relative sparing of the subcortical U fibres. Signal abnormality was also seen to involve the long tracts in the brainstem, including the pyramidal tracts, the medial lemniscus, and the inferior cerebellar peduncles (18). Because of mild very long chain fatty acid (VLCFA) levels, peroxisomal dysfunction was suspected. After likely genetic causes of this patient’s biochemical profile, ACOX1 deficiency or heterozygosity for ABCD1 mutation were ruled out, and ACBD5 was investigated. Indeed, a homozygous mutation deleting exons 7 and 8 of the ACBD5 gene, which encodes a peroxisomal membrane protein with a putative acyl-CoA binding domain, were detected. In vitro studies confirmed that ACBD5 mutations affect peroxisomal beta-oxidation of VLCFA (18). Independently, skin fibroblasts from 1 of the 3 original patients were studied and also found to have defective peroxisomal VLCFA beta-oxidation (90).

Prognosis and complications

Overall, the prognosis for patients with peroxisomal single enzyme defects of ACOX1, HSD17B4 is poor. In contrast, AMACR deficiency and SCP2 deficiency appear to be significantly milder, with at least 4 of the 6 known cases still alive in their fifth decade.

Patients with HSD17B4 deficiency have frequent seizures and show only minimal development; the average age at death is 17.6 months (89; 16). A few patients with HSD17B4 deficiency who have survived longer than 2 years are now known; all have type II or III enzyme deficiency. Longer survival best correlated with low VLCFA levels and a high rate of VLCFA beta-oxidation in fibroblasts (16). Adults with HSD17B4 deficiency showed either normal or only mildly-elevated VLCFA levels (61; 47; 41; 40). Patients with ACOX1 deficiency are somewhat less profoundly affected, developing some milestones; the average age at death is about 5 years (15). As there are only a few cases reported, the long-term prognosis for ABCD3, ACOX2, and ACBD5 deficiency remains unknown.

Clinical vignette

ACOX1 deficiency. The patient, first seen at 15 months of age, was the 7 lb product of a full-term gestation to a 22-year-old G2P1 woman. Mother and father were first cousins of Palestinian descent. The patient was reported to have been active in utero. Labor and delivery were normal. He did well in the newborn period, including feeding, and went home with his mother at 2 days of age. At 2 months of age, he had 3 tonic-clonic seizures in one day. He had had a cold but no fever. He was placed on carbamazepine. He was on that medication for 2 months until his mother stopped the medication. He has had no further seizures and has had slow but steady development. At 9 months he was sitting by himself but did not stand. He knew his mother from strangers and was socially interactive. Vision and hearing were normal. On physical examination, his weight was 9.47 kg (10th to 25th percentile), height was 81 cm (75th percentile), and head circumference was 45.4 cm (10th percentile). He was a thin young boy with no unusual features. There was minimal brachycephaly, but the remainder of the general exam was normal.

Neurologic examination revealed an awake, alert, and bright boy who made "raspberries", had a delightful social smile, did have appropriate stranger anxiety, sought out his mother, and played with toys. His cranial nerves were intact. Motor examination revealed that he was markedly hypotonic with normal muscle mass. He slipped through when held in vertical suspension and arched his back when placed in ventral suspension. There was righting of his head in this position. He could transfer objects between hands. When reaching for objects there was a mild tremor and dysmetria. He could sit unsupported. He could stand with support with his feet apart, but was not interested in cruising. Sensation was intact for light touch and pain. Examination of cerebellar function revealed dysmetria, which appeared to be secondary to low tone. Reflexes were 1+ at the knees, biceps, and ankles, and 0 at the triceps and brachioradialis. His toes were downgoing. Reflexes were symmetric, and no pathologic reflexes were present.

Laboratory studies showed normal hematologic, liver functions, and adrenal function. Plasma VLCFA assay revealed that C26:0 was mildly elevated at 0.51 µg/ml (normal mean 0.22). Magnetic resonance imaging demonstrated abnormal white matter signal involving the parietal occipital regions and centrum semiovale bilaterally. He began to manifest progressive loss of skills including motor and visual function, and was last reported to be in a vegetative state.

HSD17B4 deficiency. The patient, first seen at 10 months of age, had been the full-term 7 lb 8 oz product to a 19 year-old G1P0. No problems were reported with the pregnancy, and the fetus was active in utero. At the time of delivery, there was fetal tachycardia, and she was delivered via caesarian section. She required oxygen and had 2 seizures in the first day of life. She then began having frequent seizures and was also noted to be hypotonic. She spent 1.5 months in the hospital and was only home for a few days, when she had to be readmitted for apnea and seizures. She spent the first 4 months in the hospital. A portion of the time in the hospital was complicated by pneumonia and time on the ventilator. She initially took a bottle for 2 months, but then lost this ability with the onset of the above complications and required a gastrostomy tube.

When seen at 10 months she was no longer having apnea, but she was still having daily seizures. She would turn her head to the side in a fencer posture and then had clonic movements. Her liver functions were normal, there were no skeletal problems, and there were no bleeding abnormalities; these features distinguished her from children with peroxisome biogenesis disorders. On physical examination she was a sleeping infant who was difficult to arouse. She had a full forehead with a small anterior fontanelle. She had a fine rash on her face and trunk. On examination of her eyes, her sclera was clear and the conjunctiva pink. The remainder of her general examination was unremarkable.

When she was finally aroused, she had a 2 minute seizure on awakening. There was no visual tracking although the pupils did react. Extraocular movements were present. There were minimal corneal responses and diminished facial movement. There was no apparent response to voice. She had a decreased gag reflex. Her tongue was in the midline and there were no fasciculations. Generally, there was minimal movement and she was markedly hypotonic with decreased muscle mass. She grimaced to blood drawing. There were no reflexes and her toes were nonreactive. Over the next few weeks, she continued to have uncontrolled seizures and died at eleven months of age.

Biological basis

Etiology and pathogenesis

The underlying cause of peroxisomal single enzyme defects is a genetic defect resulting in lack of expression of a protein or enzyme required for beta-oxidation, or expression of a defective protein. These genetic defects seem to be transmitted in an autosomal recessive fashion.

Mutation analysis has revealed the nature of the genetic defects in many patients with ACOX1, HSD17B4, AMACR, and SCP2 deficiency. Ferdinandusse and colleagues described 20 distinct mutations in 22 patients with ACOX1 deficiency (15). No clear-cut correlation of genotype with clinical phenotype was observed in childhood ACOX1 deficiency. Two transcript variants of the human ACOX1 gene result from alternative splicing of 2 different exons 3 (15; 58). One patient with biochemical features typical of ACOX1 deficiency had a homozygous mutation that deleted 6 amino acids encoded by exon 3II; this patient was still able to produce a functional ACOX1 that contained exon 3I (15). This prompted further investigation of the substrate specificity of the 2 enzyme isoforms. Expression in a yeast system indicated that ACOX1 containing exon 3II was capable of utilizing substrates containing long- and very long-chain acyl-CoA substrates, whereas the enzyme containing exon 3I was primarily active with 6-14 carbon acyl-CoAs. The adult siblings with ACOX1 deficiency were homozygous for a missense mutation that causes an amino acid substitution (p.R210H) in a region that does not affect catalytic function or FAD-binding (13). Exome sequencing has now identified several older patients with previously undiagnosed neurologic symptoms as having HSD17B4 deficiency. Several of these more mildly affected patients were found to carry compound heterozygous mutations.

Before HSD17B4 was discovered, it was thought that patients with deficiency of this enzyme were deficient of L-bifunctional protein (EHHADH) (88). This discrepancy was resolved when Wanders and colleagues (73) found that the original “bifunctional enzyme deficiency” patient had mutations in the HSD17B4 gene. These authors looked at DNA from 9 additional patients and found clear-cut mutations in HSD17B4. Subsequently, a large series of patients was investigated in which 61 different mutations were found in 110 patients (24). These investigators examined the available clinical and biochemical data for each patient along with the predicted effect of the mutation on enzyme activity, protein folding, or protein dimerization. Mutations identified in patients with a relatively mild clinical and biochemical presentation were found to be less detrimental to the protein structure than mutations in severely affected patients. They concluded that the amount of residual activity of HSD17B4 correlated with the clinical severity. No case of EHHADH/L-bifunctional protein deficiency has been confirmed genetically.

Wanders and colleagues also cloned the human AMACR gene and found mutations in DNA from their original 3 patients (14). This group later established a diagnosis of SCP2 deficiency in 1 patient and a diagnosis of ABCD3 deficiency in 1 patient by mutation analysis (21; 20). A mutation in ACOX2 was found by exome sequencing (77).

Primary functions of the peroxisomal fatty acid beta-oxidation pathway. The term “beta-oxidation” refers to the process by which an aliphatic carboxylic acid is shortened by cleavage between the second and third carbon atoms in the aliphatic chain. For a straight-chain fatty acid, one cycle of beta-oxidation yields a product shortened by 2 carbons plus acetyl-CoA. For fatty acids with a methyl branch on the second carbon, beta-oxidation yields a product shortened by 3 carbons plus propionyl-CoA.

In general, the shortened fatty acids can undergo additional cycles of beta-oxidation. However, complete degradation of fatty acids as seen in mitochondria does not occur in peroxisomes. The primary functions of the peroxisomal beta-oxidation pathway are: (1) to catabolize certain fatty acids that cannot be degraded by mitochondria and (2) to carry out key reactions in the synthesis of bile acids. Accumulation of substrates and metabolites proximal to a defective enzyme forms the pathogenesis and pathophysiology of the single enzyme defects in this pathway.

Some fatty acids are not oxidized by the traditional mitochondrial beta-oxidation pathway (used to degrade dietary and stored fatty acids for energy production) either because they cannot be transported into mitochondria or they are not recognized as substrates by the mitochondrial enzymes. The physiologically significant fatty acids in this group are the very long-chain fatty acids (VLCFA; containing 22 or more carbons), phytanic acid (3,7,11,15- tetramethylhexadecanoic acid; a 3-methyl branched chain fatty acid), and pristanic acid (2,6,10,14-tetramethylpentadecanoic acid; a 2-methyl branched chain fatty acid).

VLCFA originate from both the diet and endogenous synthesis. Phytanic acid is solely of dietary origin, and the presence of the 3-methyl branch prevents its degradation by beta-oxidation in either peroxisomes or mitochondria. Pristanic acid is produced when phytanic acid is degraded by alpha-oxidation, a peroxisomal process in which the acid is shortened by one carbon. The methyl group in pristanic acid is now in the 2-position, allowing beta-oxidation to proceed. A primary defect in the alpha-oxidation pathway enzyme phytanoyl-CoA alpha-hydroxylase is the cause of Refsum disease, an adult onset polyneuropathy. However, because pristanic acid arises only from phytanic acid, defects in beta-oxidation that prevent pristanic acid degradation can slow down phytanic acid degradation, causing the latter to accumulate as well.

Bile acids are synthesized from cholesterol in a complex pathway involving several subcellular compartments. Modifications to the steroid nucleus and oxidation of one of the methyl groups in the side chain yield the precursors of primary bile acids. The precursor of cholic acid is trihydroxycholestanoic acid (THCA) and the precursor of chenodeoxycholic acid is dihydroxycholestanoic acid (DHCA). The terminal steps in bile acid synthesis involve shortening of the side chains of THCA and DHCA by 3 carbons. As discussed below, chain shortening is analogous to the first cycle of pristanic acid beta-oxidation. Both bile acid precursors and branched chain fatty acids contain asymmetric carbons; thus, these compounds exist in several stereoisomeric configurations.

Peroxisomal fatty acid beta-oxidation pathways and enzymes. The first step in fatty acid oxidation is activation by thio-esterification to CoA, reactions catalyzed by specific acyl-CoA synthetases (87; 80).

Studies suggest thioesterification occurs outside the peroxisome. ACBD3 is a peroxisomal membrane protein with an acyl-CoA binding domain located in the cytosol; this protein is hypothesized to “capture” cytoplasmic VLCFA-CoA for transport into the organelle (Ferdinandusse et al 2016; 90). CoA derivatives of VLCFA, branched-chain fatty acids, and bile acid precursors then enter the organelle via ATP-binding cassette proteins of the ABCD family. Specifically, saturated VLCFA-CoA are thought to require ABCD1 (the gene defective in X-linked adrenoleukodystrophy) for entry (74) and the CoA esters of phytanic acid, pristanic acid, and bile acid precursors enter via ABCD3 (20). Once inside the organelle, a double bond is inserted between carbons 2 and 3 in the aliphatic chain, a reaction catalyzed either by ACOX1 or by branched-chain acyl-CoA oxidase (ACOX2). ACOX1 acts on VLCFA and other straight-chain fatty acids, whereas ACOX2 acts on pristanic acid and the bile acid precursors. The next 2 enzymatic steps, hydration of the double bond (enoyl-CoA hydratase activity) and oxidation of the hydroxyl group to a keto group (hydroxy-acyl-CoA dehydrogenase activity), are both catalyzed by HSD17B4. Although it was initially thought that products of ACOX1 were processed by L-bifunctional protein (EHHADH) and products of branched-chain acyl-CoA oxidase were processed by HSD17B4, evidence from both biochemical and molecular genetic studies clearly indicate that HSD17B4 utilizes substrates generated by both ACOX1 and ACOX2. The precise role of EHHADH/L-bifunctional protein in peroxisomal fatty acid metabolism is uncertain at this time. A study of mice in which both HSD17B4 and EHHADH were knocked out suggested a role for the latter in bile acid synthesis, perhaps via an alternative pathway (17). Hepatocytes from EHHADH-deficient mice were shown to have reduced capacity to degrade dicarboxylic polyunsaturated fatty acids (56). Subsequent studies revealed that the increase in medium-chain (C6-C8) dicarboxylic acids in plasma and urine normally observed after fasting was virtually absent in EHHADH-deficient mice, confirming a role for EHHADH in the peroxisomal beta-oxidation of long-chain (C16-C18) dicarboxylic acids is disturbed (29).

Products of HSD17B4 or EHHADH (3-ketoacyl-CoAs) are then thiolytically cleaved in a CoA-dependent reaction.

Peroxisomal thiolase (ACAA1) catalyzes this reaction for the 3-keto derivatives of VLCFA and other straight-chain fatty acids, but not for the 3-keto derivatives of pristanic acid and bile acid precursors. Thiolase activity was also found in amino terminal portion of a different peroxisomal protein, the 58 kDa sterol carrier protein X (SCP2); the carboxyl portion of this protein is identical to sterol carrier protein 2. SCP2 thiolase can accept 3-ketoacyl-CoA substrates from both straight-chain and branched-chain fatty acid catabolism, although it shows a preference for the latter. The products of ACAA1 and SCPXT are the CoA derivatives of the chain-shortened substrates. In the case of VLCFA and pristanic acid degradation, these products can then go through several additional rounds of beta-oxidation, utilizing the same enzymes in a cyclic process. The other cleavage products of ACAA1 and SCPXT are the 2-carbon compound, acetyl-CoA (from VLCFA catabolism), or the 3-carbon compound, propionyl-CoA (from pristanic acid or THCA degradation).

Biochemical features of patients with deficiencies of peroxisomal beta-oxidation enzymes are summarized in Table 2.

Table 2. Biochemical Abnormalities in Single Enzyme Defects of Peroxisomal Beta-Oxidation

*The C26:0 level in the single patient with SCP2 deficiency was 1.34, which was just outside the control range of 0.46 to 1.31 (21).

Patients with ACOX1 deficiency accumulate VLCFA but not phytanic acid, pristanic acid, or bile acid precursors (THCA and DHCA) (89; 55). Defects in VLCFA degradation can clearly be demonstrated in cultured skin fibroblasts (89). In contrast, patients with HSD17B4 deficiency accumulate VLCFA, phytanic acid, pristanic acid, and bile acid precursors (89; 55; 80; 16). It should be noted that the amount of phytanic acid (and, thus, pristanic acid) that accumulates is dependent on dietary intake. Foods rich in phytanic acid are primarily those containing fats from ruminant animals (eg, beef, cow’s milk); thus, phytanic and pristanic accumulation is typically not observed in infants. Defects in both VLCFA and pristanic acid degradation can be measured in cultured skin fibroblasts from HSD17B4-deficient patients (89; 72). Conversion of the 27-carbon precursor THCA to the 24-carbon primary bile acid, cholic acid, is also defective in HSD17B4 deficiency, resulting in THCA accumulation in plasma (72). Both the side chains of THCA (and DHCA) and pristanic acid are 2-methyl carboxylic acids, and their degradation proceeds by the same pathway. As bile acid synthesis takes place only in liver, fibroblasts cannot be used to assess the bile acid synthesis defect.

Because they contain asymmetric carbon centers, stereoisomers of phytanic acid, pristanic acid, and bile acid precursors are found in nature. Both 2R- and 2S-stereoisomers of pristanic acid are produced from the alpha-oxidation of phytanic acid, but only the 2S-isomer is a substrate for branched acyl-CoA oxidase (22). Peroxisomal alpha-methyl-acyl-CoA (AMACR) interconverts the 2 stereoisomers, thereby functioning as an auxiliary beta-oxidation enzyme. In pristanic acid degradation, the methyl groups on carbons 6 and 10 are also in the R-configuration. As pristanic acid is progressively shortened via several cycles of beta-oxidation, AMACR is again required to permit further degradation. The bile acid precursors THCA and DHCA also exist as 25R and 25S stereoisomers (because the carbon numbering system differs between fatty acids and steroids, the 2-position of pristanic acid and the 25-position of THCA/DHCA are both alpha to the carboxyl carbon). As with pristanic acid, only the S-stereoisomer can be processed by beta-oxidation into the mature bile acids. Thus, AMACR facilitates the utilization of the R-isomer. Elevations in plasma levels of both pristanic acid and bile acid precursors are consistently found in AMACR deficiency. Since pristanic acid is derived solely from dietary phytanic acid, plasma levels may reflect the patient’s consumption of ruminant meats and dairy products.

No patients with defects in any of the acyl-CoA synthetases or EHHADH have been described thus far. As noted above, the 1 report of a patient with thiolase (ACAA1) deficiency was incorrect.

Peroxisome morphology. Interestingly, in most patients with ACOX1 or HSD17B4 deficiency and in the single patient with ABCD3 deficiency, the morphology of peroxisomes in skin fibroblasts from is characteristic. When examined by indirect immunofluorescence using an antibody to a peroxisomal membrane or matrix protein, these cells contain a significantly reduced number of peroxisomes of larger than usual size (62; 05). Although the reason for this change is unknown, the morphologic appearance of peroxisomes can be valuable in prenatal and antenatal diagnosis.

Pathogenesis. It is likely that one or more compounds that accumulate in tissues of patients with single enzyme deficiencies of peroxisomal beta-oxidation contribute to the pathologic features of these disorders. However, correlations between specific metabolites and specific pathologic processes are lacking. For example, patients with deficiency of peroxisomal ACOX1 or HSD17B4 all accumulate VLCFA, as do patients with X-linked adrenoleukodystrophy. However, the development of the fetus and especially the nervous system is not affected in X-linked adrenoleukodystrophy but is severely disturbed in the other peroxisomal single enzyme defects. Symptoms of X-linked adrenoleukodystrophy develop later in life and are likely due to a combination of accumulation of VLCFA and secondary responses such as an immunologically mediated destruction of myelin (52). Ferdinandusse and colleagues (19) found evidence for increased oxidative stress in skin fibroblasts from patients with HSD17B4 deficiency, including increased levels of thiobarbituric acid-reactive substances and 8-hydroxydeoxyguanosine and decreased levels of the antioxidants alpha-tocopherol and coenzyme Q. In ACOX1-deficient mouse oligodendrocytes, increased production of both reactive oxygen species and reactive nitrogen species was found; these increases were further potentiated by increased VLCFA levels (03). Fibroblasts from ACOX1-deficient patients showed activation of the IL-1 inflammatory pathway, with increased secretion of IL-5 and IL-8 cytokines. Although these findings are suggestive, the mechanistic relationship between oxidative stress, inflammation, and pathogenesis remains to be established.

On neuropathological examination several abnormalities are found in patients with ACOX1 and HSD17B4 deficiency. Generalized or partial cerebral atrophy (54; 81), hypomyelination and demyelination (25; 54; 88), and presence of foamy macrophages have been reported (54; 88). Of particular interest is the presence of microgyria, and focal areas of cortical or cerebellar heterotopias indicating defective neuronal migration (25; 54; 88). These abnormalities have only been reported in patients with HSD17B4 deficiency. Developmental defects such as disturbed neuronal migration might explain some of the clinical deficits associated with these disorders. According to Volpe, seizures are often an early neurologic symptom in neuronal migration defects (78). Neuronal heterotopias in other conditions such as neurofibromatosis, myotonic dystrophy, and other muscular dystrophies have been postulated to be one of the factors resulting in intellectual deficits of these patients (78).

Patients with HSD17B4 deficiency who survive beyond early childhood have progressive neurologic deficits, including ataxia and cerebellar atrophy despite normal or only mildly elevated levels VLCFA and other potentially toxic metabolites (61; 47; 41; 40). Thus, additional properties of HSD17B4 may contribute to the pathophysiology. This is supported by studies of knockout mice. Mice with neuron-specific HSD17B4 deletion developed progressive motor disabilities and ataxia that correlated with loss of Purkinje cells and cerebellar atrophy, whereas mice with oligodendrocyte-specific knockout did not (76). Similar to the observation in humans, cerebellar degeneration in mice did not correlate with VLCFA levels or other substrates of peroxisomal beta-oxidation.

Of further interest is the role of accumulating bile acid intermediates in the pathogenic neuronal development, especially the occurrence of neuronal migration defects. Disturbances of neuronal migration have been reported only in patients with HSD17B4 (25; 08; 54; 88; 84). In the same patients abnormal bile acid intermediates were also present. In contrast, patients with ACOX1 deficiency have normal bile acids, and neuronal migration defects have not been reported. This raises the question of a direct relationship between these 2 factors. Vilarinho and colleagues speculated that the mild ataxia and cognitive impairment seen in the 1 patient with ACOX2 deficiency resulted from increased levels of toxic bile acid synthesis intermediates (77). Somewhat surprisingly, there were no neurologic deficits reported in the 1 patient with ABCD3 deficiency, despite similar elevations of plasma bile acid precursors (20).

Adrenal atrophy is an autopsy finding reported in all beta-oxidation defects, including X-linked adrenoleukodystrophy and disorders of peroxisomal biogenesis (25; 08; 54; 88; 83). Atrophy may be present even if clinical symptoms or abnormal function tests were not detected prior to death. Lipid-containing balloon cells have been observed (88) and are thought to contain the accumulating VLCFA, most of them esterified with cholesterol.

Renal cysts are frequently found in disorders of peroxisomal biogenesis such as Zellweger syndrome. Renal cortical cysts have also been reported in 2 patients deficient in HSD17B4 (25; 08). In another patient with an HSD17B4 defect, small glomerular cysts were detected on autopsy (88). Renal changes are subtle and do not result in functional impairment.

As only 1 patient with SCP2 deficiency has been described thus far, the pathogenesis of this disorder is essentially unknown. It has been suggested that the high level of pristanic acid in AMACR deficiency may be responsible for pathogenesis and that the diet recommended for treatment of Refsum disease may be beneficial. However, plasma exchange to acutely lower plasma pristanate in 1 patient with rapidly deteriorating neurologic status produced no clinical response (07). Kruska and Reiser suggest that toxic effects of branched-chain fatty acids seen in AMACR deficiency and Refsum disease may be the result of abnormal cellular signaling; these fatty acids were found to activate the G-protein coupled receptor GPR40, resulting in increased intracellular calcium (38). Pristanic acid (high in AMACR deficiency) was 4.5-fold more potent in increasing calcium than was phytanic acid (higher in Refsum disease). Interestingly, AMACR mRNA or protein levels have been found to be elevated in several human cancers. Initially found to be a biomarker for prostate carcinoma (42), several reports now describe elevated AMACR levels in colon, liver, and kidney neoplasia (34; 35). Several different AMACR transcripts arising from alternative splicing events have been found in prostate tumors that overexpress AMACR, and only 1 transcript retains the carboxy-terminal peroxisomal targeting signal (53). Further investigation of AMACR in cancer may reveal more information about this interesting enzyme and the pathological consequences arising from its deficiency.

Epidemiology

The absolute incidence of peroxisomal beta-oxidation single enzyme defects is unknown. However, some estimates can be made. As noted above, the clinical presentation of ACOX1 deficiency and HSD17B4 deficiency is similar to that seen in the peroxisome biogenesis disorder spectrum. Moser has estimated that in his series of several hundred patients, 18% of those originally diagnosed as having a peroxisome biogenesis disorder are ultimately found to have HSD17B4 deficiency (50). The incidence of peroxisome biogenesis disorders is estimated to be 1:50,000 live births (27). Therefore, a crude estimate puts the incidence of HSD17B4 deficiency at about 1:250,000. In our series examining 26 patients suspected of having ACOX1 deficiency or HSD17B4 deficiency, 3 were found to have ACOX1 deficiency, and the rest were found to have HSD17B4 deficiency (89). Only 7 cases of AMACR deficiency, 1 case of SCP2 deficiency, 2 cases of ACOX2 deficiency, 4 cases of ACBD5 deficiency, and 1 case of ABCD3 deficiency have been reported.

Prevention

Due to the genetic nature of these disorders, the only measure of prevention is prenatal diagnosis.

ACOX1 deficiency and HSD17B4 deficiency. Using chorionic villus sampling as well as amniocentesis, cells can be obtained and the activity of the peroxisomal beta-oxidation pathway can be measured in vitro using a radiolabeled VLCFA as substrate (67; 28). The amount of VLCFA in cultured cells can also be quantitated to establish the diagnosis (49). Molecular analysis of genomic DNA for prenatal diagnosis of HSD17B4 deficiency has been reported (60). In addition, indirect immunofluorescence analysis using an antibody to a peroxisome matrix protein (eg, catalase) or a peroxisomal membrane protein (eg, PMP70) can establish whether the morphologic appearance of peroxisomes is consistent with a diagnosis of ACOX1 or HSD17B4 deficiency. Due to the relatively low general incidence of peroxisomal single enzyme defects, prenatal diagnosis is usually offered only to parents with a previous affected child. If the diagnosis in the sibling was not established biochemically, prenatal testing might also be offered if the clinical presentation of the sibling was consistent with a peroxisomal defect. The tests performed to rule out a peroxisomal single enzyme deficiency will also detect patients with Zellweger syndrome and other disorders of peroxisomal biogenesis. It should also be noted that several of the parents of patients with a peroxisomal single enzyme defect were consanguineous (25; 08; 62; 04; 81; 43).

AMACR deficiency and other rare gene defects. Prenatal diagnosis for these disorders has not yet been described.

Differential diagnosis

ACOX1 or HSD17B4 deficiency. In the neonatal period, especially if dysmorphic features or early onset seizures are absent, hypotonia will be the presenting symptom.

In the absence of an abnormal obstetrical history or any acute perinatal events (such as complicated or prolonged labor, perinatal depression with possible ischemia, acidosis, and hypoxia) a genetic or neurologic cause of the hypotonia appears more likely (10; 11; 09). In known cases of peroxisomal beta-oxidation defects acute conditions such as severe birth depression, intracerebral bleeding, sepsis, or side effects from maternal medications have not been reported; however, these conditions may develop independent of the underlying genetic defect and may complicate the clinical picture. In 2 patients with an uncharacterized defect of peroxisomal beta-oxidation, delivery was complicated by presence of meconium (43). Gestational age of a hypotonic newborn has to be taken into consideration because premature infants are usually somewhat, but not profoundly, hypotonic.

Diseases of the motor unit including Werdnig-Hoffmann disease, transient neonatal or congenital myasthenia gravis, myotonic dystrophy, and other congenital muscular dystrophies and myopathies have to be differentiated from disorders of the central nervous system associated with hypotonia (78; 91). In addition to hypoxic ischemic events, central causes of hypotonia include other metabolic encephalopathies (consider hyperbilirubinemia and hyperammonemia), infectious encephalitis (bacterial or viral, especially herpes simplex infection), endocrinopathy (hypothyroidism), or other genetic disorders, such as disturbed metabolism of amino acids or fatty acids, gangliosidosis, mucopolysaccharidosis, chromosomal abnormalities (especially trisomy 21), and Prader-Willi syndrome. Congenital malformations of the central nervous system such as neural tube defects, holoprosencephaly, Chiari malformation, Dandy-Walker syndrome, and neuronal migration defects including neuronal heterotopias and lissencephaly, have to be excluded by physical exam or imaging studies (78; 86).

If seizures develop early in the neonatal course the involvement of the central nervous system becomes evident. As for hypotonia, seizures can also be the sequelae of a perinatal or postnatal insult to the central nervous system. Other possible causes of convulsions, such as hypoxic-ischemic encephalopathy, intracranial hemorrhage, hypoglycemia, hypocalcemia, intracranial infection, or drug withdrawal have to be excluded (78). Signs that the hypotonia has already been present during fetal life such as contractures or positional malformations of the extremities as well as decreased fetal movements by maternal report or ultrasound examination, decrease the likelihood that the symptoms are due to acute peri- or postnatal events.

The diagnosis of a peroxisomal single enzyme defect can only be established by biochemical studies or mutational analysis. There are no pathognomonic clinical findings. Although there are some differences between ACOX1 and HSD17B4 deficiency patients with respect to clinical manifestations and clinical course, it is not always possible to differentiate between them. The clinical presentation of disorders of peroxisomal biogenesis (27) is also similar. Patients with Zellweger syndrome and neonatal adrenoleukodystrophy typically show more definite dysmorphic features, and hepatic involvement is frequent and more severe. Skeletal survey might show stippled calcifications especially of the patella. Cardiac defects are more common (ventricular septal defect and abnormalities of the left outflow tract). There are also a greater variety of eye abnormalities, ranging from corneal changes, cataracts, and glaucoma to retinal pigmentations and optic atrophy (27). It has become clear, however, that disorders of peroxisomal biogenesis also show a wide range of phenotypic expression, including some patients with atypical and less severe presentations. Van der Knaap and colleagues examined 6 cases in which brain MRI and clinical presentation was suggestive of an infantile-onset peroxisomal disorder, but without clear-cut biochemical abnormalities characteristic of these diseases; mutational analysis revealed that 3 had HSD17B4 deficiency, whereas the other 3 had a peroxisome biogenesis (Zellweger spectrum) disorder (71). These authors concluded that biochemical studies of skin fibroblasts should be performed if MRI is suggestive of a peroxisomal disorder, but blood tests (VLCFA, etc.) are negative or equivocal.

Of potential diagnostic relevance are observations from the HSD17B4 knockout mouse. Neurodevelopmental abnormalities were not present, and if early postnatal death occurred, it was usually related to abnormal bile acid metabolism (32). However, all mice surviving to the post-weanling period developed progressive motor deficits that included loss of mobility due to abnormal cramping reflexes and died at 6 months of age. Severe astrogliosis and reactive microglia were present mainly in gray matter; no overt neuronal lesions were present. It is, thus, conceivable that mild forms of HSD17B4 deficiency in humans might produce similar manifestations.

As more adults with milder forms of ACOX1 and HSD17B4 deficiency are being identified via exome sequencing, a clearer clinical picture is emerging. In particular, HSD17B4 deficiency should be considered in the differential diagnosis of progressive gait ataxia.

AMACR deficiency and SCP2 deficiency. Refsum disease and adrenomyeloneuropathy (an adult-onset phenotype of X-linked adrenoleukodystrophy) should be considered in adults suspected of AMACR deficiency. The latter should be considered in women as well as men, as neurologic symptoms often develop in heterozygous females in later life (14). Because the biochemical abnormalities in the 2 disorders are similar (See Table 2), the patient with SCP2 deficiency was initially thought to have AMACR deficiency (21). However, a finding of normal AMACR enzyme activity in fibroblasts prompted further investigation that established lack of SCP2 enzyme activity, and a mutation in the SCP2 gene.

Diagnostic workup

For patients in whom a peroxisomal single enzyme defect is considered in the differential diagnosis, the initial evaluation should include a detailed history with emphasis on genetic, family, and obstetrical history, as well as a thorough physical and neurologic examination. In some cases (eg, to rule out myotonic dystrophy) a neurologic examination of the parents might be indicated. Laboratory investigation should include glucose and calcium levels as well as other electrolytes, hematologic evaluation to detect signs of infection, cultures of blood and, in some cases, cerebrospinal fluid and drug screening, if indicated. Review of fetal heart rate tracings, prenatally obtained scalp pH, Apgar scores, cord blood pH, and an early blood gas will help to exclude perinatal distress as contributing factors to the infant's symptoms. An EEG, or if possible a video EEG, should be performed if seizures are present or suspected. Imaging studies such as head ultrasound, computed tomography, and magnetic resonance imaging are indicated to assess for the presence of cerebral hemorrhage, structural abnormalities, or hydrocephalus. In most patients with peroxisomal single enzyme defects, imaging studies were normal shortly after birth (62; 88; 43). Some mild abnormalities were observed in other patients including "mild frontal atrophy" (54), "decreased density of the white matter" (25), and "minimal white matter hypodensities" (62). Later in life white matter abnormalities might be more pronounced (62).

It should be noted that most of the biochemical analyses required to establish an accurate diagnosis of peroxisomal diseases are highly specialized and are only available at a few centers worldwide. The initial test to establish the diagnosis of a peroxisomal defect should be quantitation of VLCFA in plasma (51). If this test is requested early in the evaluation, invasive procedures to establish a diagnosis such as muscle or even brain biopsy can be avoided. The level of VLCFA is strikingly elevated in most patients with ACOX1 deficiency and HSD17B4 deficiency (but not AMACR deficiency or SCP2 deficiency) and is not significantly altered by the timing of the blood drawing, the patient's nutritional status, anticonvulsant medications, or age. If VLCFA levels are abnormally elevated, additional studies to exclude other peroxisomal dysfunctions present in peroxisomal biogenesis disorders should be performed (27). These tests include plasma phytanic acid, pristanic acid, and pipecolic acid, and red blood cell plasmalogens. Phytanic and pristanic acid levels can be elevated in AMACR deficiency, SCP2 deficiency, and HSD17B4 deficiency as well as in peroxisomal biogenesis disorders. Because phytanic acid (and, therefore, its metabolite, pristanic acids) only comes from the diet, the degree of elevation is dependent on the age and diet composition of the patient. It should be noted, however, that in a series of 126 HSD17B4 deficiency patients studied at the Academic Medical Center in Amsterdam, 3 had normal plasma VLCFA, phytanic acid, and pristanic acid levels, although skin fibroblast VLCFA levels were elevated (16). The older sibling with Perrault syndrome described above also had normal serum VLCFA levels when measured at 23 years of age (61). A similar phenomenon was also reported for 1 patient with ACOX1 deficiency (64). Pipecolic acid, which accumulates in peroxisomal biogenesis disorders due to lack of peroxisomal L-pipecolate oxidase, can be mildly elevated in HSD17B4 deficiency for reasons that are unclear (25; 54; 88; 04). Typically, the degree of impairment is greater in the peroxisome biogenesis defects than in the single enzyme deficiencies.

The analysis of bile acids from plasma, urine, or bile will usually reveal abnormal bile acid intermediates (THCA, DHCA, or other metabolites) in patients with HSD17B4, SCP2 deficiency, AMACR deficiency, ACOX2 deficiency, and ABCD3 deficiency (25; 54; 88; 84; 82; 22; 16; 21; 20; 77), but not in patients with defects in ACOX1. However, about one fourth of HSD17B4-deficient patients show no accumulation of plasma THCA or DHCA (16). Although both 25R- and 25S-stereoisomers accumulate in HSD17B4 deficiency and peroxisomal biogenesis disorders, only the 25R-isomer accumulates in AMACR deficiency.

Measurement of urinary acyl-carnitines by multiple reaction monitoring (MRM) electrospray ionization tandem mass spectrometry (ESI-MS/MS) has emerged as a promising method for diagnosis of peroxisome biogenesis disorders and HSD17B4 deficiency (12). In a study of 7 patients with peroxisome biogenesis disorders and 2 patients with HSD17B4 deficiency, significantly elevated levels of 14-. 16-, and 18-carbon dicarboxylylcarnitines and 22-, 24-, and 26-carbon monocarboxylylcarnitines were detected in all but one sample. Only the 16-carbon dicarboxylylcarnitine was markedly elevated in urine from one HSD17B4 patient. Nonetheless, the authors conclude that MRM ESI-MS/MS is a rapid, reliable, and sensitive diagnostic method that discriminates patients with these disorders from controls.

If a diagnosis of peroxisomal disorder is suspected, obtaining a skin biopsy to establish cultured fibroblasts is essential. The overall rate of peroxisomal beta-oxidation of a VLCFA (eg, lignoceric acid; C24:0) or pristanic acid can be measured in fibroblasts using [14C]-labeled substrates (67; 28; 14; 75). Wanders and coworkers developed an HPLC assay using varanoyl-CoA (3-alpha,7-alpha,12-alpha,24-tetrahydroxy-beta-cholestan-26-oyl-CoA), a beta-oxidation intermediate between THCA-CoA and choloyl-CoA in bile acid synthesis, as substrate to diagnose HSD17B4 deficiency (79). Metabolites formed when this substrate is incubated with fibroblasts from suspected HSD17B4-deficient patients indicate whether there is a deficiency of the enzymes’ enoyl-CoA hydratase activity, its 3-hydroxyacyl-CoA dehydrogenase activity, or both.

Antibodies to ACOX1, HSD17B4, ACAA1, SCP2, and AMACR have been developed in several laboratories, allowing immunoblot, immunohistochemical, and immunofluorescent detection of these enzyme proteins in liver biopsy samples, cultured fibroblasts, and postmortem tissues (28; 73; 33; 75). Immunoblotting techniques were used to establish the diagnosis in the index cases of single enzyme defects. In these patients ACOX1 and HSD17B4 deficiency, respectively, were not detectable on immunoblot (65; 62; 88). Although immunoblot and immunofluorescence analysis is often useful in establishing a diagnosis, in some patients all 3 enzyme proteins are often present, suggesting that they are present but catalytically inactive. Complementation techniques were initially used to establish the diagnosis in these patients.

McGuinness and associates and others used this method to establish a diagnosis in several patients suspected of having a single enzyme defect in ACOX1 or HSD17B4 (45; 68; 89). When cultured fibroblasts from 2 patients carrying a different genetic defect are fused, beta-oxidation activity will be restored to normal because each cell line will provide the gene product defective in the other. In one series of 26 patients, a diagnosis of ACOX1 deficiency was established in 3 patients, whereas the remaining 23 patients were diagnosed with HSD17B4 deficiency (89). Molecular diagnosis is now used to establish or confirm a suspected single enzyme deficiency of peroxisomal beta-oxidation.

Management

ACOX1 deficiency and HSD17B4 deficiency. There is no curative treatment for deficiencies of these enzymes. After establishment of a diagnosis, supportive measures should be initiated to assist the parents in coping with the nature and prognosis of their child's condition. Providing care to a child with a severe disorder such as a peroxisomal disease requires enormous adjustments in family life. Assistance should be provided for the general care of the infant and specifically for feeding. Appropriate positioning is essential to prevent secondary deformities. Medical management should focus on seizure control and prevention or early detection of possible complications.

Feeding difficulties are reported frequently in patients with peroxisomal single enzyme defects. Although some patients have sufficient suck and swallow coordination to be bottle fed, others require gavage tube feedings (25; 82). Placement of a gastrostomy tube might be indicated (88). Failure to thrive can complicate the course in these patients (25; 81).

The characteristic severe hypotonia will require the caregiver to provide most of the support when holding the infant. Foam contoured seats, Velcro straps, wedges, or rolls might be necessary to maintain good position in a baby seat. With appropriate support of head, trunk and extremities, secondary complications such as torticollis, scoliosis, contractures of the hips in external rotation and abduction, knee contractures, ankle and feet deformities might be avoided; physical therapy is an additional measure of prevention. In one patient (88) a femur fracture occurred at age 3.5 months without a history of trauma. On skeletal survey, generalized osteopenia was present. Delayed bone maturation has been reported in several cases (08; 88).

Despite generalized hypotonia, respiratory drive and chest excursions seem sufficient in these patients to maintain adequate ventilation. No patient required long-term mechanical ventilation after birth. Chest physical therapy and suctioning might be required in some patients to prevent secondary complications.

Seizure control seems to be extremely difficult if not impossible to achieve in patients with peroxisomal single enzyme defects. Only in one reported case (04) were seizures controlled with phenobarbital treatment. In most patients frequent convulsions occurred despite multi-drug therapy including phenobarbital, phenytoin, and in older patients valproic acid and primidone (54). Due to possible impairment of adrenal function, corticotropin treatment may not be effective. One patient received prednisone instead but with only temporary improvement (54). A ketogenic diet has not been used and should only be considered if VLCFA levels are followed, because elevation of these fatty acids has been observed in some patients receiving a ketogenic diet (69).

Although liver and adrenal function should be followed and might be abnormal in patients with peroxisomal single enzyme defects, liver failure, clinical significant cirrhosis, adrenal insufficiency, or Addison crisis have not been reported.

The clinical course of one patient with HSD17B4 deficiency was complicated by a hemodynamically significant ventricular septal defect leading to intermittent cardiac failure and required treatment with diuretics and digoxin. Another with HSD17B4 deficiency had a ventricular septal defect but did not develop signs of cardiac failure. No other cardiac malformations have been reported in patients with peroxisomal single enzyme defects.

The parents of the patient should be informed of the genetic nature of their infant's disease. Although the family might be concentrating on their affected child's diagnosis, care, and immediate needs, they should receive genetic counseling to understand the risks for consequent pregnancies and their options including prenatal diagnosis.

Hematopoietic stem cell transplant was performed in one patient with ACOX1 deficiency at 3 years of age (85). MRI changes were first noted at 23 months and showed only mild progression at the time of transplant. Despite full engraftment, he continued to decline neurologically. He died of pneumonia at 6.75 years of age. Although transplant did not halt progression of his disease, neuroimaging studies showed evidence of slowing, with less brain inflammation, cortical atrophy, and neuronal loss than observed in his affected older brother, who was not transplanted.

Deficiency of SCP2, ACOX2, and ABCD3. Treatment of the 1 patient with SCP2 deficiency was not described in the report (21). The 1 ACOX2 patient was treated unsuccessfully with ursodeoxycholic acid; nonetheless, the authors speculated that this disorder is potentially treatable with bile acid therapy (77). The rationale for this approach is that mature bile acids would feed back and block early steps in synthesis, decreasing the levels of toxic intermediates. Although the single patient with ABCD3 deficiency died before the diagnosis was established (20), the biochemical similarities between this and ACOX2 deficiency suggest that bile acid therapy might be worth considering.

AMACR deficiency. Pristanic acid, which is suspected but not proven to contribute to pathology, is derived solely from the dietary ingestion of phytanic acid. Lowering plasma (and subsequently tissue) pristanate stores by decreasing phytanate ingestion has been suggested (07; 70). The diet recommended for Refsum disease patients, which avoids the primary sources of dietary phytanic acid such as dairy products and ruminant meats and fats, lowers plasma pristanate, but the clinical benefits are not clear (07). Symptomatic treatment of seizures and other neurologic manifestations is indicated.

The child with AMACR deficiency who presented in the neonatal period with coagulopathy and mild cholestasis responded well to treatment with cholic acid and fat-soluble vitamin supplementation and was reportedly in good health at 7.5 years of age. As noted above, the liver from this patient’s older sibling, who died at 5.5 months of age following an acute episode later ascribed to a coagulopathy, was used for transplantation. Because a posttransplant biopsy revealed evidence of acute rejection, bile-duct proliferation, and fibrosis, the recipient was treated with ursodeoxycholic acid. This child was reportedly doing well 8 years posttransplant and was still receiving ursodeoxycholic acid.

Special considerations

Pregnancy

No female patient with a single enzyme defect in peroxisomal ACOX1, HSD17B4 has survived to reproductive age. Interestingly, male mice lacking HSD17B4 were found to be infertile (31), and the 1 male patient with SCP2 deficiency was infertile (21). Carrying a fetus affected by these disorders seems to cause no specific risks to the mother. The pregnancies are typically described to be uncomplicated. Fetal movements may be decreased as an early sign of hypotonia. Three patients were delivered by cesarean section (25; 54; 88). The indication in one case (54) was breech position; it can be hypothesized that the fetal hypotonia might have contributed to the abnormal breech presentation. Labor was tolerated well in the majority of babies, and in only 2 cases did the presence of meconium indicated possible fetal distress (43). Only one patient with single enzyme defects was born prematurely (at 34 weeks' gestational age) (08). It is not known whether any of the 3 adult females diagnosed with AMACR deficiency had any children or complications of pregnancy.

Anesthesia

No specific information is available. There are no reports of airway abnormalities in patients with peroxisomal single enzyme defects. Depending on the medications used, it should be taken into consideration that these patients might have some impairment of liver function.