Myoclonus epilepsy with ragged-red fibers
Jun. 10, 2021
At vero eos et accusamus et iusto odio dignissimos ducimus qui blanditiis praesentium voluptatum deleniti atque corrupti quos dolores et quas.
This article includes discussion of Refsum disease, heredopathia atactica polyneuritiformis, and phytanic acid storage disease. The foregoing terms may include synonyms, similar disorders, variations in usage, and abbreviations.
Refsum disease is a rare, autosomal recessive disorder characterized clinically by retinitis pigmentosa, peripheral polyneuropathy, and cerebellar ataxia and biochemically by accumulation in tissues of the dietary branched-chain fatty acid, phytanic acid. Neurologic deficits are often irreversible by the time a diagnosis is established. In this article, the author notes that aggressive therapy can result in neurologic improvement in some cases.
• Refsum disease is a rare, autosomal recessive disorder characterized by retinitis pigmentosa, peripheral polyneuropathy, cerebellar ataxia, and a high cerebrospinal fluid protein concentration without pleocytosis.
• Symptoms typically present in early adulthood and are caused by toxic levels of the branched-chain fatty acid, phytanic acid, which accumulates in tissue lipids secondary to deficient activity of the peroxisomal fatty acid alpha-oxidation pathway.
• Most Refsum disease patients have a mutation in Phyh, the gene encoding the peroxisomal alpha-oxidation enzyme, phytanoyl-CoA alpha-hydroxylase.
• Phytanic acid in humans comes solely from dietary sources, including ruminant meats and fats, dairy products, and certain fish, but not from consumption of chlorophyll-containing vegetables.
• Refsum disease is managed mainly by life-long dietary avoidance of foods containing phytanic acid and is supplemented by lipid apheresis when acute lowering of phytanic acid levels is indicated.
Refsum disease, originally termed "heredopathia atactica polyneuritiformis," is a familial neurologic syndrome first described by Sigvald Refsum in 1946 (73). The 4 Norwegian cases originally reported by Refsum had what is now considered to be a diagnostic tetrad of clinical findings that include retinitis pigmentosa, peripheral polyneuropathy, cerebellar ataxia, and a high cerebrospinal fluid protein concentration without pleocytosis.
Until 1963 the pathogenesis and pathophysiology of Refsum disease was not known. Postmortem studies of liver and kidney tissue from a patient diagnosed with this disorder revealed fatty infiltrates composed mainly of neutral lipids, providing the first evidence that Refsum disease was a lipidosis. More than half of the total fatty acids isolated from liver lipids were a single, unusual species not previously reported in human tissues and subsequently identified as phytanic acid (3,7,11,15-tetramethylhexadecanoic acid).
Refsum disease must not be confused with the peroxisomal disorder "infantile Refsum disease." Scotto and colleagues found elevated levels of phytanic acid in 3 infants and suggested that this "infantile phytanic acid storage disease" was a variant of Refsum disease (85). Other biochemical defects not found in typical Refsum disease patients were present in these cases, including elevated plasma levels of long-chain fatty acids and pipecolic acid, decreased plasmalogen synthesis, and abnormal subcellular catalase distribution (51). As a result of these additional findings, it is now recognized that phytanic acid accumulation in these children is not the primary defect but is secondary to a disorder of peroxisome biogenesis. This group of patients also includes the Zellweger syndrome and neonatal adrenoleukodystrophy phenotypes (51). The term "infantile Refsum disease" is now generally used to describe the longest surviving subset of this group.
As originally described by Refsum, all patients with the disorder bearing his name have a diagnostic tetrad of clinical findings: retinitis pigmentosa, peripheral polyneuropathy, cerebellar ataxia, and a high protein concentration in the cerebrospinal fluid without an increased number of cells. Absent from the original description of the disease, but now required for diagnosis, is elevated plasma concentrations of phytanic acid. In most cases, cardiac involvement and nerve deafness are also present. Several findings found less commonly are anosmia, pupillary abnormalities, cataracts, ichthyosis, and skeletal abnormalities (Table 1). Obligate heterozygotes for Refsum disease do not manifest clinical signs or symptoms of neurologic disease and generally have normal plasma phytanic acid levels.
• Retinitis pigmentosa+
• Cerebellar ataxia+
• Peripheral polyneuropathy (motor and sensory)+
• Cardiac involvement (nonspecific electrocardiogram abnormalities)++
• Symptoms of cranial nerve involvement:
- Neurogenic hearing loss++
- Abnormal pupillary reflex+++
• Skeletal malformations+++
• Skin changes (dry skin, ichthyosis)+++
• Increased CSF protein without pleocytosis+
• Elevated plasma phytanic acid concentration+
Key: +Always present, ++Usually present, +++Sometimes present
Clinical course. Night blindness is typically the first symptom noticed by patients. Other early manifestations include ataxia and other cerebellar signs, frequently overlooked as simply "clumsiness" in the initial stages of the disease. Subsequently, patients develop a peripheral neuropathy that eventually leads to wasting and distal paralysis. The first appearance of symptoms can range from early childhood to the sixth decade, but they usually begin during the second or third decade of life (87). The disease is progressive with gradual deterioration if untreated. Even without treatment, acute exacerbation of symptoms followed by nearly complete remission is not uncommon. These exacerbations are frequently associated with stress, such as pregnancy or a viral illness. Respiratory failure is the cause of death in some patients, and sudden death has been observed in several cases (74). With proper treatment, life expectancy of Refsum disease patients is normal.
Primary clinical features. Patients with Refsum disease are always found to have retinitis pigmentosa of the "salt-and-pepper" type and hemeralopia. The degree of retinal involvement and the extent of the visual field defect may depend on the stage of the disease. Retinal changes, once developed, are generally unresponsive to dietary treatment.
The peripheral neuropathy of Refsum disease is of the mixed motor and sensory type and is chronic and progressive if untreated. Symptoms are generally first noted in the distal lower extremity and then in the small muscles of the hand; involvement is usually symmetrical. Deep sensation is disturbed, and deep tendon reflexes are diminished. Electrophysiologic studies reveal slowed motor nerve conduction velocities. Although the possibility has been raised that the ataxia present in all cases is secondary to this polyneuropathy, it is generally thought that the severity of the symptoms cannot be explained on this basis alone and that patients have true cerebellar ataxia. Although cerebellar ataxia is a component of Refsum’s original diagnostic tetrad, 2 siblings with clinical and biochemical features of Refsum disease, but without cerebellar involvement, have been reported (22).
Other clinical findings. Most patients have evidence of cardiac involvement. Common findings include tachycardia, gallop rhythm, systolic murmur, and enlargement of the heart. Evidence of conduction disturbances, sinus tachycardia, nonspecific ST-T changes, or myocardial damage is often present on electrocardiograms. For this reason, it has been proposed that fatal cardiac arrhythmias may be responsible for some cases of sudden death reported in untreated Refsum disease. Symptoms, such as neurogenic hearing loss, anosmia, disturbed pupillary reflex, and miosis, indicate cranial nerve involvement in Refsum disease. The hearing loss, like the retinal degeneration, is usually unresponsive to treatment. More than half of patients exhibit skeletal malformations, including nonspecific (pes cavus or hammer toes) or specific (symmetrical hyperplasia and hypoplasia of fingers, toes, metacarpals, and metatarsals) deformities. Many patients also have skin changes, which range in severity from dry skin to ichthyosis. Hyperkeratosis and accumulation of fat droplets within basal keratinocytes are noted histologically.
Late-onset Refsum disease presenting as a leukoencephalopathy. A single case of a female patient with no neurologic symptoms prior to 69 years of age was reported (05). She presented with balance difficulties and memory problems. She had gait apraxia but no clear cerebellar ataxia. Brain MRI showed a leukoencephalopathy involving the periventricular white matter, subcortical area, and the brainstem with relative sparing of juxtacortical U fibers. Electromyography showed no polyneuropathy, and her electroretinogram was normal. Cerebrospinal fluid (CSF) showed 2 white cells per cubic mm and slightly increased protein content of 0.46 g/L. Plasma lipid analysis showed significantly elevated phytanic acid levels but normal pristanic and very long-chain fatty acid levels. Genetic analysis revealed that she was homozygous for a frameshift mutation in the Refsum disease-causing PHYH gene that resulted in a premature stop codon.
Exacerbation and remission of symptoms with no apparent cause have been noted in Refsum disease; however, exacerbations have also been associated with stress, concurrent illness, or pregnancy. In addition, a paradoxical rise in the plasma phytanic acid concentration and clinical relapse has been observed when a patient on dietary therapy experiences a sudden weight loss. In these situations, plasmapheresis to lower circulating phytanic acid may be useful. If neurologic deficits have not progressed to the point of irreversibility, aggressive therapy (eg, weekly plasma exchange) may yield some improvement.
A small study suggested an association between elevated serum phytanic acid levels and increased risk of prostate cancer (112). This study was undertaken because several previous studies showed increased expression of alpha-methylacyl-CoA racemase, an enzyme required for the complete degradation of dietary phytanic acid, in prostate tumors (56). However, the incidence of prostate cancer in Refsum disease patients has not been investigated.
GK, a 65-year-old white male, developed night blindness in childhood that worsened in his late teens. A diagnosis of retinitis pigmentosa was made at 33 years of age. He developed a mild hearing impairment in his 40s. He had had high foot arches since childhood and had noted dry skin for years. There was no documented consanguinity, although both parents came from the same town in Russia. Physical examination at 42 years of age showed bilateral pes cavus. He had feet slapping when he walked, and he could not stand on his heels. He was not ataxic. His Romberg was negative. Both retinas showed dark pigmentation. His electroretinogram was undetectable. There was severe constriction of his visual fields bilaterally. Slit lamp examination showed bilateral subcapsular lens opacity in both eyes. He had anosmia. There was bilateral calf atrophy, and his leg muscles were weak. His knee and tricep reflexes were trace, and reflexes were absent at the ankles. He had a decreased response to pinprick below the elbows and knees bilaterally, and his vibratory sense was absent below the knees. There was decreased position sense in the toes. He had palpable ulnar and superficial peroneal nerves. A left sural nerve biopsy showed chronic neuropathy with loss of myelinated fibers. Nerve conduction velocities were slow, and there was decreased amplitude of sensory potentials for right median and ulnar nerve. Serum phytanic acid level was determined to 416 µg/ml (normal is less than 3 µg/ml). CSF examination showed an elevation of protein to 127. The patient was started on a low phytanic acid diet. He also underwent a long course of plasmapheresis with improvement in his phytanic acid levels and in his neuropathy. When seen at 52 years of age, his vision had worsened, but his hearing had remained stable.
The cause of Refsum disease is presumed to be elevated tissue concentrations of phytanic acid, most frequently secondary to a defect in phytanoyl-CoA hydroxylase (PHYH; also called phytanoyl-CoA alpha-hydroxylase, PAHX), a peroxisomal enzyme in the phytanic acid alpha-oxidation pathway. Genetic defects affecting PEX7, a protein required for import of a specific group of peroxisomal enzymes including phytanoyl-CoA hydroxylase, are also a recognized cause of Refsum disease (94).
Accumulation of phytanic acid in plasma and tissues. Although the concentration of phytanic acid in plasma of normal individuals is nearly undetectable, this fatty acid can account for up to 30% of the total plasma fatty acids in Refsum disease patients. The hepatic and renal fatty infiltrates found in this disease are neutral lipids containing significant quantities of phytanic acid. Plasma levels presumably reflect both the dietary intake and the tissue stores.
Origin of phytanic acid. The observation that phytanic acid structurally resembles phytol, the side chain of chlorophyll, gave rise to the hypothesis that the diet is the main source of this fatty acid. Studies in both rats and man have shown that both dietary phytol and dietary phytanic acid are efficiently absorbed and that phytol is efficiently converted to phytanic acid in vivo. However, chlorophyll-bound phytol is not well absorbed in either rats or humans, suggesting that green vegetables are not a primary dietary source of phytanic acid. In contrast, ruminant fats and dairy products are considered to be the main source of phytanic acid in the human diet (58; 59). Rumen bacteria efficiently release the phytol side chain of chlorophyll; phytol is then either absorbed or converted to phytanic acid. It was observed that erythrocyte phytanic acid levels in great apes were higher than in humans despite a phytanate-poor diet (105). Colon volume relative to small bowel volume is significantly higher in great apes (approximately 2:1) than in man (approximately 0.33:1). Although colonic absorption of nutrients is relatively minor in humans, it is of major importance in the apes. Thus, it was hypothesized that “hindgut fermentation” of plant material in the colons of great apes is likely responsible for the higher phytanic acid levels in these nonhuman primates.
Phytanic acid degradation pathway. Cells from Refsum disease patients have impaired ability to degrade phytanic acid. As a result of the methyl branch on the 3-carbon of phytanic acid, catabolism by the classic beta-oxidation pathway is not possible. Instead, a single carbon is initially removed in an alpha-oxidation process, shifting the position of the methyl groups and allowing further degradation by beta-oxidation. Successive rounds of beta-oxidation yield alternating 3- and 2-carbon fragments. However, both the enzymatic steps and the subcellular localization of the alpha-oxidation pathway have been controversial. A series of studies conducted in the 1960s by Steinberg and colleagues revealed that in both rats and humans phytanic acid is first converted to alpha-hydroxyphytanic acid and is then decarboxylated, forming pristanic acid (2,6,10,14-tetramethylpentadecanoic acid) and CO2 (87). Pristanic acid is then degraded by beta-oxidation, first via the peroxisomal pathway and, ultimately, in mitochondria (97). Evidence suggests that mitochondrial oxidation of peroxisomally chain-shortened pristanic acid requires long-chain acyl-CoA dehydrogenase (100). Both the 3R, 7R, 11R- and 3S, 7R, 11R- stereoisomers of phytanic acid are found in nature, and both can be converted to the corresponding 2R, 6R, 10R- and 2S, 6R, 10R- stereoisomers of pristanic acid by the alpha-oxidation pathway (20). However, beta-oxidation of pristanate requires the S-configuration about carbon 2. The enzyme alpha-methylacyl-CoA racemase, which is found in peroxisomes and mitochondria and catalyzes the interconversion of 2R and 2S, is, thus, required for the complete disposition of dietary phytanic acid.
Patients with defects in peroxisome assembly have impaired capacity to degrade phytanic acid and, therefore, it was hypothesized that the alpha-oxidation pathway is localized in peroxisomes. Studies published since 1994 have clarified both the biochemical steps and the subcellular location of this pathway. The first step is activation of phytanic acid to its coenzyme A thioester (phytanoyl-CoA) in peroxisomes, a reaction catalyzed by both long- and long-chain acyl-CoA synthetases (104; 103; 88). Phytanoyl-CoA is converted to alpha-hydroxyphytanoyl-CoA by phytanoyl-CoA hydroxylase (PHYH), a 2-oxoglutarate- and Fe++-requiring dioxygenase (62). PHYH is a peroxisomal enzyme that contains a functional peroxisome targeting signal 2 and requires Pex7p, the peroxisome targeting signal 2 receptor, for proper targeting to the organelle (61). Intraperoxisomal sterol carrier protein-2 (SCP-2) may increase the specificity of PHYH for phytanic acid versus other fatty acid substrates (65). Subsequent decarboxylation of alpha-hydroxyphytanoyl-CoA yields an aldehyde, pristanal, and formyl-CoA (14; 98). This reaction proceeds by a unique mechanism that is similar to that catalyzed by 3-hydroxy-3-methylgluatryl-CoA lyase and requires thiamine pyrophosphate as a cofactor. Because this cofactor is not known to be required for any other peroxisomal enzyme, a study was conducted, and it was found that although peroxisomes do contain thiamine pyrophosphate, they cannot phosphorylate thiamine and, therefore, must take up the cofactor in phosphorylated form (26). Formyl-CoA was found to be unstable at pH greater than 5.5, spontaneously breaking down to formate and CoA under physiologic conditions (14). Formate is ultimately translocated to mitochondria and converted to CO2. Pristanal must be oxidized to pristanic acid and reactivated to its CoA thioester for subsequent degradation by peroxisomal beta-oxidation. Pristanic acid reactivation may require long-chain acyl-CoA synthetase (88). Under experimental conditions, pristanal formation from alpha-hydroxyphytanoyl-CoA was detectable only when nicotinamide adenine dinucleotide was excluded from reaction mixtures, suggesting the involvement of a dehydrogenase (98). Most investigators now believe that the complete degradation of phytanic acid (conversion of phytanic acid to formate and pristanic acid and the subsequent beta-oxidation of pristanic acid) occurs in peroxisomes (96). However, some evidence exists advancing the theory that pristanal dehydrogenase may be microsomal. Fatty aldehyde dehydrogenase, the microsomal enzyme defective in the Sjögren-Larsson syndrome, was found to oxidize pristanal; furthermore, phytanic acid oxidation was reported to be defective in fibroblasts from Sjogren-Larsson patients (97). In contrast, Jansen and colleagues reported normal phytanic acid oxidation and residual pristanal dehydrogenase activity in fibroblasts from Sjogren-Larsson patients who completely lacked FALDH (38). This issue was subsequently resolved when it was reported that alternative splicing of the mouse Aldh3A2 gene yields 4 variant forms of Faldh, 1 of which is targeted exclusively to peroxisomes (02). The organization of mouse and human genes are similar, and these authors found that the peroxisomal variant, FALDH-V, plays an essential role in the efficient degradation of branched chain fatty acids and that it protects cells from the damage induced by lipid peroxidation. The more common variant, FALDH-N, was localized to the endoplasmic reticulum (microsomes) and did not catalyze the oxidation of pristanal. Furthermore, these authors detected pristanal dehydrogenase activity in peroxisomal fractions isolated from rat liver. Interestingly, FALDH has been found to be required for the conversion of dietary phytol to phytanic acid (95).
An important role for liver-type fatty acid-binding protein in phytanic acid metabolism has been established (03; 04). Overexpression of Fabp1 (L-FABP) in mice enhanced cellular phytanic acid uptake and stimulated phytanic acid alpha-oxidation but had little effect on esterification of the branched-chain fatty acid. Conversely, in mice in which the Fabp1 gene was disrupted, cells exhibited decreased phytanic acid uptake and decreased alpha-oxidation rates. These findings suggest that other tissue-specific FABP isoforms may be important for the metabolism of phytanic acid in tissues pathologically affected in Refsum disease, eg, the nervous system and heart.
Omega-oxidation of phytanic acid may contribute to overall phytanic acid degradation. In this pathway, the terminal (omega) methyl group is oxidized to a carboxylic acid, yielding a dicarboxylic acid that can be partially degraded from the omega end by beta-oxidation. Evidence from rats and humans suggests that the overall contribution of omega-oxidation to phytanic acid degradation is minor. Humans typically consume 50 to 100 mg of phytanic acid daily. Wierzbicki and colleagues have estimated the omega-oxidation capacity in Refsum patients by measuring urinary excretion of the metabolite 3-methyladipic acid and found it to be around 7 mg/day (108). In vitro studies using liver microsomes confirmed that the omega-oxidation pathway is functional in humans (47; 111); the reactions are NADPH-dependent and involve specific P450 enzymes. In addition, phytanic acid is a good substrate for human UDP-glucuronosyltransferases, suggesting that some phytanic acid might be eliminated by this mechanism (54).
Regulation of peroxisomal phytanic acid and pristanic acid metabolism may in part be controlled by the amount of free fatty acid substrate versus substrate thioesterified to CoA. Peroxisomes contain a novel isozyme of acyl-CoA thioesterase (ACOT6), which is specific for these branched-chain fatty acids (106). As noted above, phytanoyl-CoA and pristanoyl-CoA and not the free fatty acids are the substrates for peroxisomal alpha- and beta-oxidation, respectively.
Enzyme defect in Refsum disease. For nearly 2 decades, it was suspected that the metabolic defect in Refsum disease was at the level of PHYH. Evidence for defective PHYH was obtained in 1997, when a liver biopsy from a Refsum disease patient was found to have no detectable enzyme activity as compared to controls (39). The cDNA encoding this enzyme was independently cloned by 2 laboratories (37; 61), and mutations in the cDNA obtained from at least 22 different Refsum disease patients have been identified (37; 35; 36; 61; 11). About two thirds were missense mutations, and the rest included insertions, deletions, and splice-site mutations that resulted in a large deletion through the skipping of exon 3.
Mukherji and colleagues (64) conducted structure-function analyses of clinically observed PHYH mutations and found that although most mutations caused impaired phytanoyl-CoA hydroxylation, 1 did not. PHYH containing this mutation (P29S) was fully active, but its location near the amino terminus suggests that impaired targeting to peroxisomes produced the clinical disease. Subsequent to this report, the crystal structure of PHYH was solved to 2.5 Å resolution, and many disease-causing mutations were found to cluster around the binding pockets for either Fe++ or 2-oxoglutarate (60).
Wierzbicki and colleagues used linkage analysis to study 8 genetically informative families that included 17 patients with a clinical diagnosis of Refsum disease. Four families (with 8 affected members) exhibited linkage to 10p13, the Refsum disease locus; however, in 3 other families (including 9 affected individuals), linkage was excluded (109). These investigators found linkage to 6q22-24 in 2 of the latter families and subsequently identified their defective gene as PEX7 (94). Horn and colleagues published a detailed article of a Refsum disease patient with PEX7 deficiency and found the phenotype to be indistinguishable from that caused by PHYH deficiency and, therefore, proposed that Refsum disease be subdivided into type 1 (PHYH deficiency) and type 2 (PEX7 deficiency) (31).
PEX7 deficiency is the recognized cause of rhizomelic chondrodysplasia punctata, a disorder that presents at birth with a considerably more severe clinical phenotype. Pex7p, the product of the PEX7 gene, is a cytoplasmic receptor for proteins with peroxisome targeting signal 2 (PTS2), an amino acid sequence residing near the amino-terminus. In the absence of Pex7p, PTS2-containing enzymes are not targeted to peroxisomes, resulting in specific biochemical abnormalities. Known PTS2 proteins include phytanoyl-CoA hydroxylase, alkyl-dihydroxyacetone phosphate synthase, and peroxisomal 3-oxoacyl-CoA thiolase 1. Deficiencies in ADHAPS and thiolase were also found in the 2 families with PEX7 mutations. These findings suggest that a broad phenotypic spectrum exists for PEX7 deficiency and that other tests of peroxisome biochemistry may be indicated in the workup of newly identified Refsum disease patients. It is possible that the remaining patients whose defects have not yet been identified have mutations in the gene encoding alpha-hydroxyphytanoyl-CoA lyase, the enzyme following PHYH in the alpha-oxidation pathway. This enzyme was purified from rat liver and cDNA encoding its human ortholog was cloned (24). At present, no mutations in the lyase gene have been reported.
Pathogenesis. All evidence from biochemical studies suggests that elevated plasma and tissue levels of phytanic acid are directly or indirectly responsible for the clinical manifestations of Refsum disease. Furthermore, reducing the body burden of phytanic acid with dietary therapy leads to clinical improvement. Several potential pathogenic mechanisms have been suggested. Young and colleagues (113) investigated the effects of phytanic acid on cultured retinal cells and found that this fatty acid readily incorporated into cellular phospholipids, resulting in increased membrane fluidity but no increase in susceptibility to lipid peroxidation. They further reported that phytanic acid did not competitively inhibit alpha-tocopherol uptake, weakening the hypothesis that phytanic acid in membranes interferes with vitamin E function. Phytanoyl-CoA, not free phytanic acid, is the substrate for incorporation into phospholipids, triacylglycerol, and other lipids. Phytanoyl-CoA (and pristanoyl-CoA) was found to exhibit high affinity (Kd near 11 nM) for the nuclear receptor, peroxisome proliferator-activated receptor alpha (PPARalpha), whereas the respective free fatty acids showed only weak binding (32). PPARalpha activation upregulates expression of genes encoding enzymes involved in fatty acid beta-oxidation and other lipid metabolic pathways. However, the possibility that PPARalpha activation contributes to the pathogenesis of Refsum disease remains to be explored. Tang and colleagues incubated prostate PC-3 cells with phytanic acid and found decreased rates of cell proliferation that were associated with increased levels of the phytanate ester of retinol, suggesting that this compound could contribute to Refsum disease pathogenesis (92).
Mice deficient in sterol carrier protein-x (which encodes both the putative phytanic acid-binding protein, Scp2, and the 3-ketoacyl-CoA thiolase required for peroxisomal beta-oxidation of pristanic acid) accumulated phytanic acid in phospholipids of myocardial membranes when maintained on a phytol-enriched diet (63). This was associated with bradycardia, impaired AV nodal and intraventricular impulse conduction, and a high incidence of sudden death, strengthening the hypothesis that arrhythmias may explain sudden death in untreated Refsum disease patients. Foulon and colleagues investigated the substrate specificity of phytanoyl-CoA hydroxylase and found that a variety of 3-methyl-branched fatty acyl-CoAs were hydroxylated by this enzyme (and presumably were further metabolized by alpha-oxidation) (25). Thus, accumulation of 3-methyl fatty acids other than phytanic acid could contribute to the pathogenesis of Refsum disease.
Elevated phytanic acid levels may alter the behavior of the plasma membrane ion transporters. Wiersbicki and colleagues initially reported that the maximal velocity of the erythrocyte sodium-lithium countertransporter was affected by increasing plasma phytanic acid concentrations (107). Studies done using purified synaptic vesicles from young rat brain cortex revealed that membrane synaptic Na/K ATPase activity was decreased when either phytanic acid (100 to 200 µm) or its metabolite, pristanic acid (50-200 µm), were present in assays (09; 10). These observations led Busanello and colleagues to conclude that phytanic and pristanic acids may impair synaptic neurotransmission.
Increasing evidence suggests that phytanic acid affects mitochondrial function. Schonfeld and Wojtczak reported that exposure of isolated brain mitochondria to phytanic acid resulted in release of cytochrome c, suggesting that this branched-chain fatty acid activates the mitochondrial route of apoptosis (75; 83). Schonfeld and coworkers also found that very low concentrations of phytanic acid de-energized brain mitochondria, reduced state 3 respiration, and sensitized the mitochondria to rapid permeability transition (82). Subsequent studies by this group indicated that phytanic acid activates intracellular calcium stores, producing mitochondrial depolarization and generating reactive oxygen species (41). They concluded that these factors contribute to the short-term toxicity of phytanic acid and may induce the onset of neurodegeneration. Further studies demonstrated that the phytanic acid-induced increase in reactive oxygen species was associated with partial inhibition of the mitochondrial electron transport chain, most likely by changing membrane fluidity (84). In agreement with this finding, Komen and colleagues reported that phytanic acid decreased ATP synthesis and mitochondrial membrane potential in human skin fibroblasts (46). Nagai reported that in addition to its effect on mitochondrial dysfunction in Neuro2a cells, phytanic acid activated histone deacetylase activity, reducing histone acetylation and promoting cell death (66). The cytotoxic effect of phytanic acid was abolished by sodium butyrate, by a caspase-9 inhibitor, and by apicidin, a Hdac2- and 3-specific inhibitor. Nagai concluded that the toxicity of phytanic acid depends on activation of the Hdac2 and 3 subtypes.
Busanello and colleagues used homogenates of young rat brain cortex to investigate the in vitro effects of phytanic acid and pristanic acid on mitochondrial function (09; 10). Increasing concentrations of phytanic acid decreased respiratory chain activity at complexes I, II, 1-III, II-III, and IV, while increasing concentrations of pristanic acid decreased activity at complexes I, II, and II-III. Pristanic acid treatment also decreased mitochondrial CO2 production from acetate, whereas phytanic acid was without effect. Subsequent work by these investigators showed that both phytanic and pristanic acids behave as uncouplers of oxidative phosphorylation (07; 08). Reiser and coworkers incubated mixed cultures of hippocampal neurons, astrocytes, and oligodendrocytes with phytanic acid or pristanic acid to assess effects on reactive oxygen species production and cellular calcium signaling (78; 49). They found that although reactive oxygen species production was increased by both branched-chain fatty acids, pristanic acid caused a more robust increase. In addition to effects on reactive oxygen species production, Reiser and colleagues showed that both phytanic acid and pristanic acid caused dramatic increases in cellular Ca++ concentrations. The increases were found to be mediated by the inositol triphosphate signaling cascade. Subsequently studies revealed the involvement of a G-protein coupled receptor, GPR40, in this process. HEK 293 cells overexpressing GPR40 had increased Ca++ levels when incubated with phytanic acid or pristanic acid, but not when incubated with the unbranched fatty acid palmitate. Leipnitz and colleagues also investigated oxidative damage caused by incubation of homogenates of rat brain cerebellum and cortex with phytanic acid (53). Phytanic acid increased thiobarbituric acid-reacting substances in both cortical and cerebellar homogenates, an effect that was prevented by the antioxidants alpha-tocopherol and melatonin. Increased carbonyl content and sulfhydryl oxidation was also observed. Glutathione levels were decreased by phytanic acid, and this effect was reversed by antioxidants. These investigators subsequently showed similar phenomena occurred in heart mitochondria, and they proposed that phytanic acid- induced disturbances of cellular energy and redox homeostasis may contribute to the cardiomyopathy seen in many Refsum disease patients (29).
Incubation of vascular smooth muscle cells with phytanic acid-induced TNFalpha activation and secretion markedly increased inducible nitric-oxide synthase mRNA and protein and promoted apoptosis (34). Dhaunsi and coworkers reported that treatment of smooth muscle cells with phytanic acid increased NADPH oxidase activity (16). Phytanic acid also increased total and phosphorylated EGFR, and treatment of cells with EGFR inhibitor blocked the phytanate-induced enhancement of NADPH oxidase activity. Phytanic acid has been found to be a physiological ligand for peroxisome proliferator-activated receptor alpha (PPARalpha), which leads to transcriptional upregulation of many proteins including liver fatty acid binding protein (17; 110; 116). Subsequently, it was reported that PHYH is upregulated in cells incubated with phytanic acid (115; 115b) or dehydroepiandrosterone (15). This physiologic response, which may be necessary to maintain a low body burden of phytanic acid, would not be operative in Refsum patients with PHYH mutations. Further studies suggesting a phytanic acid/PPAR interaction were subsequently reported. Heim and colleagues (30) showed that physiologic concentrations of this fatty acid enhanced 2-deoxy-D-glucose uptake in rat hepatocytes by increasing mRNA expression of glucose transporter-1 and -2, and glucokinase. Lampen and colleagues (50) found that phytanic acid in combination with retinoic acid receptor ligands induced intestinal retinoic acid hydroxylase and retinoic acid metabolism. Schluter and colleagues (80; 79; 81) reported that phytanic acid induced differentiation of both white and brown adipose and that it also induced expression of the uncoupling protein-1 (UCP1) mRNA in brown adipose tissue. Enhancement of UCP1 expression was enhanced by cotransfection of brown adipocytes with a retinoid X receptor expression vector, a finding supported by the observation of Radominska-Pandya and Chen (71), who found that phytanic acid is a natural ligand for retinoid X receptor beta. It is not clear whether these observations have implications for Refsum disease.
PHYH was found to bind to the immunophilin FKBP52, suggesting that it might have a role in cellular signaling pathways (12). Lee and colleagues identified a previously undescribed brain-specific protein, PHYH-AP1, that also binds to PHYH in a yeast 2-hybrid system (52). Subsequently, these investigators reported that PHYH-AP1 (now called BAP4) also interacts with brain-specific angiogenesis inhibitor 1 (BAI1) (44). They postulate that PHYH interacts with BAI1 through BAP4 and that this interaction may be involved in the development of the central neurologic symptoms of Refsum disease such as retinitis pigmentosa, nerve deafness, and cerebellar ataxia. Selective overexpression of PHYH-AP1 in the heart (atrium) of a transgenic mouse model produced tachycardia and increased susceptibility to arrhythmia; this may be related to arrhythmias associated with Refsum disease (43). These investigators found evidence that visual stimulation is essential for expression of this protein and that PHYH-AP1 may be involved in developmental regulation of photoreceptor function (01). This same group also identified a second protein that interacts with murine PHYH as a novel, long-chain fatty acyl-CoA synthetase that is expressed primarily in brain and testis (42). Inhibition of this acyl-CoA synthetase blocked proliferation of cultured neuronal cells, suggesting possible relevance to the development of neurologic symptoms in Refsum disease. A yeast 2-hybrid screen conducted by Chen and colleagues (13) identified PHYH as a protein interacting with human coagulation factor VIII, and further reported that overexpression of PHYH decreased factor VIII production significantly in factor VIII-producing cells. The significance of these observations for Refsum disease is not clear.
Mouse model of Refsum disease. A mouse with disruption of the Phyh gene was produced in 2008 (21). The mouse displayed no phenotypic abnormalities when reared under normal laboratory conditions and fed normal rodent chow, which contains very low levels of phytanic acid and its precursor, phytol. When knockout mice were fed a diet supplemented with 0.25% phytol, the animals lost weight due to lipoatrophy with loss of white adipose tissue. Plasma, liver, kidney, testis, and cerebellum showed significantly elevated levels of phytanic acid. Plasma levels of other lipids, including cholesterol, triacylglycerol, free fatty acids, and total fatty acids, were decreased in knockout mice on the phytol diet. Histologic analysis of livers from knockout mice revealed steatosis, hepatocyte degeneration, and inflammatory infiltrates; the steatosis was microvesicular on a 0.1% phytol diet and macrovesicular on a 0.25% phytol diet. Histologic analysis of testes revealed that knockout mice on either 0.1% or 0.25% phytol diet lacked the full complement of spermatogenic cells. Brains of knockout mice fed the 0.25% phytol diet showed prominent reactive astrocytosis and a striking loss of cerebellar Purkinje cells.
Phyh knockout mice fed phytol exhibited neuromuscular function abnormalities, with an increased number of paw slips while moving on a grid and absent trunk curl (21). These mice had an unsteady gait with reduced paw print areas (both forepaws and hind paws) and reduced base of support for the hind paws. Peripheral motor nerve conduction velocity was decreased in phytol-fed knockout mice, due to increased latency in action potentials. However, no gross evidence for demyelination was observed in sciatic nerve by histology and staining for myelin basic protein. Further studies of this mouse model should yield additional insight into the pathophysiology of Refsum disease.
Although Ferdinandusse and colleagues found essentially no effects of either 0.1% or 0.25% phytol diet on wild-type mice (21), other investigators reported that normal mice fed a diet supplemented with 0.5% or 1.0% phytol exhibited midzonal hepatocellular necrosis and periportal hepatocellular fatty vacuolation (57). In that study, high phytanic and pristanic acid levels correlated with increased PPARalpha-mediated responses, including reduced body weight, hepatomegaly, and peroxisome proliferation.
Genetics. Richterich and colleagues conducted formal genetic studies on a total of 37 patients and concluded that this disorder was inherited in an autosomal recessive manner based on the high parental consanguinity rate, absence of disease in parents, and observed number of affected sibs (76). A similar pattern of inheritance has been observed in patients subsequently diagnosed with Refsum disease. Cultured fibroblasts from obligate heterozygotes show an intermediate reduction in their ability to oxidize phytanic acid, consistent with an autosomal recessive mode of inheritance. The PHYH gene defective in Refsum disease has been localized to human chromosome 10p between D10S249 and D10S466 (61; 11). The PEX7 gene is located at 6q21-q22.2.
Refsum disease is a rare disorder; Refsum reviewed the existing literature in 1975 and found a total of 73 reported cases (74). In 1995 Steinberg estimated the total number of confirmed cases to be about 150 (87).
Prenatal diagnosis of Refsum disease can be established by measuring phytanic acid alpha-oxidation in cultured amniocytes or chorionic villus cells. However, considering the rarity of the disorder and the rather late age of onset of symptoms, identification of at-risk pregnancies is difficult.
Refsum disease must be distinguished primarily from (a) other neurologic syndromes that resemble it but that do not have elevated phytanic acid and (b) other peroxisomal disorders with increased phytanic acid levels. Neurologic disorders that might be considered in the differential diagnosis include Friedreich ataxia, retinitis pigmentosa, multiple sclerosis, Dejerine-Sottas hypertrophic peripheral neuropathy, Charcot-Marie-Tooth syndrome, nonspecific heredoataxia syndromes, abetalipoproteinemia, Tangier disease, amyotrophic lateral sclerosis, Sjogren-Larsson syndrome, Spielmeyer-Vogt disease, and Tay-Sachs disease. PHARC (polyneuropathy, hearing loss, ataxia, retinitis pigmentosa, and cataract) first described in 2008, is a novel Refsum-like disorder caused by mutations in the ABHD12 gene, located on chromosome 20; this disorder should also be considered in the differential diagnosis (23). A finding of elevated plasma phytanic acid would rule out all of these disorders and, particularly in an adolescent or an adult, would indicate a diagnosis of Refsum disease. Two sisters who presented with an acute demyelinating polyneuropathy suggestive of familial Guillain Barré syndrome were subsequently found to have Refsum disease based on elevated phytanic acid levels (99). The differential diagnosis of hereditary neuropathies in adult patients has been reviewed (69).
Phytanic acid accumulation is not unique to Refsum disease. Impaired ability to degrade phytanic acid leading to elevated plasma levels is also observed in peroxisome biogenesis disorders, which include the Zellweger syndrome or neonatal adrenoleukodystrophy or "infantile Refsum disease" phenotypic continuum and rhizomelic chondrodysplasia punctata (51). The former group of patients also has elevated plasma concentrations of long-chain fatty acids and pipecolic acid. Fibroblasts from all of these patients also exhibit decreased plasmalogen synthesis. Furthermore, signs and symptoms of peroxisome biogenesis disorders are typically present at birth. Deficiency of peroxisomal alpha-methylacyl-CoA racemase can also present as an adult-onset sensory motor neuropathy; however, pristanic acid, and phytanic acid levels are elevated in this disorder (18). Mildly elevated plasma phytanic acid was also found in 1 reported case of sterol carrier protein x deficiency (19); this patient presented with leukoencephalopathy, dystonia, and motor neuropathy and had markedly elevated plasma pristanic acid levels. Thus, a diagnosis of Refsum disease can be made if patients exhibit the tetrad of clinical features originally described by Refsum, have elevated plasma phytanic acid or inability to degrade phytanic acid, and have no other biochemical abnormalities suggesting more generalized peroxisome dysfunction.
Because the enzyme deficient in most Refsum patients, PHYH, requires thiamine pyrophosphate, it has been suggested that patients with untreated thiamine deficiency may have elevated phytanic acid levels (86). Classical thiamine deficiency (beriberi) is associated with polyneuropathy but is infrequently seen except among alcoholic patients; thiamine deficiency is also found in inborn errors such as thiamine-responsive megaloblastic anemia.
Patients presenting with symptoms of night blindness, gait disturbance, or peripheral neuropathy should be evaluated for Refsum disease. A complete neurologic examination is indicated to evaluate signs of peripheral motor or sensory neuropathy, ataxia, and cranial nerve dysfunction. Ophthalmologic examination should be performed to ascertain whether the patient has the salt-and-pepper type of retinitis pigmentosa typical of Refsum disease, any visual field defects, miosis, or abnormal pupillary reflexes. Olfactory functional assessment tools such as the quantitative University of Pennsylvania Smell Identification Test may be useful; in a study of 16 Refsum disease patients conducted in 2004, all were found to have complete anosmia or grossly impaired smell function despite a median 15 years of dietary treatment (27). Skin and skeletal systems should be evaluated for the changes sometimes seen in Refsum disease; Jayaram and Downes noted that the presence of hand or foot abnormalities in a patient with autosomal recessive or simplex retinitis pigmentosa is suggestive of Refsum disease (40). All symptoms of this disease do not develop simultaneously. There may be several years between the onsets of different symptoms. Thus, diagnosis is often difficult, and early misdiagnosis is not uncommon.
The primary laboratory test is measurement of the plasma phytanic acid concentration by gas chromatography or gas chromatography coupled to mass spectrometry. A novel method using proton magnetic resonance spectroscopy (MRS) to identify and quantitate plasma phytanic acid levels was reported (68). Analysis of plasma from a Refsum patient yielded results similar to those obtained by gas chromatography, suggesting the possibility that noninvasive MRS diagnostic methods could be developed. If other peroxisomal diseases must be ruled out, plasma long-chain fatty acids, plasma pristanic acid, and fibroblast plasmalogen synthesis should be determined. Fibroblast phytanic acid degradation is useful to confirm diagnosis. Mutation analysis of the phytanoyl-CoA hydroxylase gene should not be required but could be useful in atypical cases. An electrocardiogram should be obtained and evaluated for the nonspecific changes sometimes seen in Refsum disease. Evaluation of CSF protein and cellular content may be helpful, but is probably not necessary if the plasma phytanic acid level is elevated.
Current standard of care. Phytanic acid is entirely of exogenous origin, and, therefore, control of dietary intake of this fatty acid decreases its accumulation in Refsum disease patients. Ingestion of both phytanic acid and free phytol, which can be converted to phytanic acid in man, should be minimized. Patients have a small residual capacity to degrade phytanic acid, and, as a result, these dietary measures will allow the depletion of body stores. Normal daily consumption of phytanic acid is 50 to 100 mg, and estimated residual degradation capacity in patients is 7 to 30 mg/day (28; 108). Thus, dietary intake must be significantly less than 30 mg daily to facilitate mobilization and elimination of stored phytanate.
The main dietary sources of phytanic acid are dairy products, ruminant meats and fats, and fatty fish. For effective management, these must be eliminated from the diet. Green vegetables, due to their high chlorophyll content, were not permitted in the early dietary trials; however, due to the poor bioavailability of chlorophyll-bound phytol, this is probably not necessary. Although phytanic acid is present in many foods, good information on food phytanate content is limited. More detailed information on dietary management of Refsum disease and the amount of phytanic acid in various foodstuffs can be found elsewhere (55; 58; 59; 33; 06; 77). Although nonleafy vegetables typically have a very low phytanate content, phytyl fatty acid esters (trans-phytol esterified with a fatty acid) that could contribute to phytanic acid body burden have been reported in some vegetables (48).
Response to treatment is often difficult to evaluate because reduction in plasma phytanate levels and improvement in clinical symptoms are generally not rapid. The overall plasma concentration is determined by both the dietary intake and the release of fatty acid from endogenous stores. The amount of phytanate stored as triglyceride in adipose can be high, and, as a result, the process of mobilization and clearance can be slow. Sudden weight loss in patients on a low phytanic acid diet can lead to a paradoxical rise in plasma concentration and clinical relapse, indicating that the contribution of adipose mobilization can be significant.
Plasmapheresis has been used in some cases, generally with success, as an adjunct to dietary therapy (55; 33). This type of therapy can be particularly useful in the early management of the disease or in cases of clinical relapses, but it is not a practical substitute for lifelong dietary treatment. Therapeutic apheresis using membrane differential filtration has been shown to be safe and effective in long-term management of Refsum disease (90). Zolotov and colleagues reported that long-term lipid apheresis was beneficial in 4 patients with severe Refsum disease whose symptoms progressed despite their compliance with a low phytanic acid diet (114). Kohlschutter and colleagues used lipid apheresis as an adjunct to dietary management of a 14-year-old girl with night blindness (45). After 30 months of combined therapy, the patient was maintained only on diet. Their work suggests that aggressive measures to keep phytanic acid levels low may prevent more serious sequelae of Refsum disease.
Dietary treatment should begin immediately on diagnosis. Although many symptoms of the disease respond favorably to treatment, the progression of retinal changes and hearing loss generally slow down or stabilize but do not improve. Several reports, however, indicate that patients may benefit from cochlear implantation (72; 67; 89). Aggressive therapy – dietary changes and weekly plasma exchange for 9 months – in a newly-diagnosed 42-year-old female patient with classical symptoms led to near normalization of both cervical vestibular evoked myogenic potential and ocular vestibular evoked myogenic potential latencies (93). In addition to improvement in her vestibular neuropathy, peripheral nerve function and mobility improved as well. Refsum disease is a rare autosomal recessive disorder in which accurate diagnosis is difficult, whereas dietary changes generally are not initiated until some irreversible vision or hearing deficit is already present. Thus, the earlier the intervention, the more favorable the outcome.
Future therapeutic prospects. Perera and colleagues treated 2 brothers (ages 48 and 50 years) with Refsum disease with the intestinal lipase inhibitor orlistat (70). Despite appropriate dietary management and plasmapheresis, their plasma phytanic acid levels remained 10-fold greater than the upper limit of normal. In addition, both siblings had progressive neurologic and dermatologic symptoms. The men then began orlistat treatment (120 mg prior to meals) along with dietary treatment and plasmapheresis. After following this regimen for several years, plasma phytanic acid levels were reduced by more than 50%, and both men reported stabilization or improvement of some neurologic symptoms.
Wanders and coworkers treated Phyh knockout mice fed a high phytanic acid diet with fenofibrate (101). Untreated mice have high levels of phytanic acid in plasma, kidney, muscle, brain, and liver. Fenofibrate treatment decreased phytanic acid levels in plasma, kidney, and muscle, but not brain or liver. Phytanic acid levels were actually higher in livers of fenofibrate-treated mice, and it was suggested that increased peripheral lipolysis along with increased hepatic uptake were responsible for this phenomenon. Lack of effect on brain was attributed to the inability of fenofibrate to cross the blood-brain barrier.
Kemp and coworkers have been investigating the fatty acid omega-oxidation pathway (102). Although no drugs are currently available, they speculated that compounds causing upregulation of specific P450 isozymes could in theory increase the rate of omega-oxidation of phytanic acid. Further investigation of orlistat, fenofibrate, and specific P450 activators in the treatment of Refsum disease seems warranted.
High levels of phytanic acid have been associated with life-threatening cardiac arrhythmia and peripheral neuropathy, requiring emergency plasmapheresis (55). Without therapy, half of untreated patients died before 30 years of age. Steinberg noted that since the institution of dietary therapy for Refsum disease 30 years ago, there have been no deaths directly attributable to the disease (87). Dietary therapy (with or without supplemental plasmapheresis) is effective in decreasing the severity of peripheral nerve dysfunction and ataxia, decreasing the plasma phytanic acid and CSF protein concentrations, and improving the electrocardiogram. Although therapy produces little or no improvement in existing retinal or hearing deficits, evidence is available that long-term dietary treatment may prevent progression the of nervous system damage.
Stress, including pregnancy, has been reported to exacerbate symptoms of Refsum disease (87), but no specific details regarding the effect of Refsum disease on pregnancy or outcome of pregnancy was reported until 2017 (91). The mother’s presentation was unusual in that she was symptomatic by age 3. She had salt and pepper retinitis pigmentosa, sensorineural hearing loss, ichthyosis, mild developmental delay, and bony abnormalities of the hands and feet. A diagnosis of Refsum disease was confirmed at age 10 by biochemical and genetic analyses. Her plasma phytanic acid level were not well controlled by diet, and plasmapheresis was occasionally necessary. She became pregnant at age 27. Her prepregnancy weight was 75.6 kg. During the first trimester, she experienced epigastric pain and lower abdominal pain. Fetal ultrasound indicated normal fetal development. Following advice from a metabolic physician and a metabolic dietitian, she was able to keep her phytanic acid levels at a higher than desirable, but a consistent, level. At 5 months, she had gained 7 kg. In the third trimester she presented with sinus tachycardia (mean, 120/min) and shortness of breath. Through the remainder of her pregnancy she had breathlessness, fluctuating hypertension, dry skin, and pruritus, all of which improved postpartum. Labor was induced at term, and during labor she was given Polycal® and Calogen® supplements to reduce the risk of acute metabolic decompensation. She gave birth to a healthy daughter, whose phytanic acid level was normal at 6 weeks of age. However, she suffered from postpartum depression and had poor appetite. This was of concern as release of phytanic acid from adipose tissue during starvation typically elevates plasma levels. Other postpartum issues included episodes of fatigue and general weakness, dyspareunia that required a refashioning of episiotomy, elevated blood pressure, headaches, dizziness, persistent episodes of nausea, vomiting, and epigastric abdominal pain. The latter symptoms were eventually attributed to irritable bowel syndrome. The child was reportedly healthy and developing normally.
Exacerbation of symptoms has been associated with stress, and, thus, caution should be exercised if a Refsum disease patient must undergo anesthesia.
Paul A Watkins MD PhD
Dr. Watkins of Johns Hopkins University has no relevant financial relationships to disclose.See Profile
Raphael Schiffmann MD
Dr. Schiffmann of Baylor Scott & White Research Institute received research grants from Amicus Therapeutics, Takeda Pharmaceutical Company, Protalix Biotherapeutics, and Sanofi Genzyme.See Profile
Nearly 3,000 illustrations, including video clips of neurologic disorders.
Every article is reviewed by our esteemed Editorial Board for accuracy and currency.
Full spectrum of neurology in 1,200 comprehensive articles.
Childhood Degenerative & Metabolic Disorders
Jun. 10, 2021
Childhood Degenerative & Metabolic Disorders
Wilson disease is caused by mutations in ATP7B that result in abnormal copper metabolism, and subsequent excess free copper is toxic. The main clinical categories of neurologic Wilson disease have been traditionally divided variably into tremor, dysarthria, pseudosclerosis (tremor +/- dysarthria), parkinsonism, dystonia, or chorea. The aim of treatment is to reduce the amount of toxic free copper.
Jun. 09, 2021
Childhood Degenerative & Metabolic Disorders
Apr. 12, 2021
Childhood Degenerative & Metabolic Disorders
Smith-Lemli-Opitz syndrome is an autosomal recessive multiple congenital malformation that is associated with intellectual disability. The primary defect
Apr. 09, 2021
Childhood Degenerative & Metabolic Disorders
Mar. 10, 2021
Childhood Degenerative & Metabolic Disorders
Mar. 04, 2021
Childhood Degenerative & Metabolic Disorders
Feb. 21, 2021
Childhood Degenerative & Metabolic Disorders
Feb. 18, 2021