Diagnostic challenges and disease management in patients with a mild Zellweger spectrum disorder phenotype.

Peroxisome Biogenesis Disorders-Zellweger spectrum disorder (PBD-ZSD) is a rare, autosomal recessive peroxisome biogenesis disorder that presents with variable symptoms. In patients with PBD-ZSD, pathogenic variants in the PEX family of genes disrupt normal peroxisomal function, impairing α- and ß-oxidation of very-long-chain fatty acids and synthesis of bile acids, resulting in increased levels of toxic bile acid intermediates and multisystem organ damage. The spectrum of severity in PBD-ZSD is variable, with some patients dying in the first year of life, while others live into adulthood. Symptoms of mild PBD-ZSD include various combinations of developmental delay, craniofacial dysmorphic features, visual impairment, sensorineural hearing loss, liver disease, and adrenal insufficiency. Disease progression in mild PBD-ZSD is generally slow, and may include extended periods of stability in some cases. The presence and extent to which symptoms occur in mild PBD-ZSD represents a diagnostic challenge that can cause delays in diagnosis with potential significant implications related to disease monitoring and treatment. There is some support for the pharmacologic therapies of Lorenzo's oil, docosohexanoic acid, and batyl alcohol in altering symptoms; however, systematic long-term studies are lacking. Cholic acid (CA) therapy has demonstrated treatment efficacy in patients with PBD-ZSD, including decreased toxic bile acid intermediates, transaminase levels, and liver inflammation, with improvement in growth parameters. However, these responses are most apparent in patients diagnosed and treated at a young age. Advanced liver disease may limit the efficacy of CA, underscoring the need to diagnose and treat these patients before significant liver damage and other related complications occur. Here we discuss the signs and symptoms of PBD-ZSD in patients with mild disease, standard diagnostic tools, factors affecting disease management, and available pharmacological interventions.

The cholic acid extension study in Zellweger spectrum disorders: Results and implications for therapy.

INTRODUCTION: Currently, no therapies are available for Zellweger spectrum disorders (ZSDs), a group of genetic metabolic disorders characterised by a deficiency of functional peroxisomes. In a previous study, we showed that oral cholic acid (CA) treatment can suppress bile acid synthesis in ZSD patients and, thereby, decrease plasma levels of toxic C27 -bile acid intermediates, one of the biochemical abnormalities in these patients. However, no effect on clinically relevant outcome measures could be observed after 9 months of CA treatment. It was noted that, in patients with advanced liver disease, caution is needed because of possible hepatotoxicity. METHODS: An extension study of the previously conducted pretest-posttest design study was conducted including 17 patients with a ZSD. All patients received oral CA for an additional period of 12 months, encompassing a total of 21 months of treatment. Multiple clinically relevant parameters and markers for bile acid synthesis were assessed after 15 and 21 months of treatment. RESULTS: Bile acid synthesis was still suppressed after 21 months of CA treatment, accompanied with reduced levels of C27 -bile acid intermediates in plasma. These levels significantly increased again after discontinuation of CA. No significant changes were found in liver tests, liver elasticity, coagulation parameters, fat-soluble vitamin levels or body weight. CONCLUSIONS: Although CA treatment did lead to reduced levels of toxic C27 -bile acid intermediates in ZSD patients without severe liver fibrosis or cirrhosis, no improvement of clinically relevant parameters was observed after 21 months of treatment. We discuss the implications for CA therapy in ZSD based on these results.

Zellweger spectrum disorder patient-derived fibroblasts with the PEX1-Gly843Asp allele recover peroxisome functions in response to flavonoids.

Zellweger spectrum disorder (ZSD) results from biallelic mutations in PEX genes required for peroxisome biogenesis. PEX1-G843D is a common hypomorphic allele in the patient population that is associated with milder disease. In prior work using a PEX1-G843D/null patient fibroblast line expressing a green fluorescent protein (GFP) reporter with a peroxisome-targeting signal (GFP-PTS1), we demonstrated that treatments with the chemical chaperone betaine and flavonoid acacetin diacetate recovered peroxisome functions. To identify more effective compounds for preclinical investigation, we evaluated 54 flavonoids using this cell-based phenotype assay. Diosmetin showed the most promising combination of potency and efficacy (EC50 2.5 µM). All active 5',7'-dihydroxyflavones showed greater average efficacy than their corresponding flavonols, whereas the corresponding flavanones, isoflavones, and chalcones tested were inactive. Additional treatment with the proteostasis regulator bortezomib increased the percentage of import-rescued cells over treatment with flavonoids alone. Cotreatments of diosmetin and betaine showed the most robust additive effects, as confirmed by three independent functional assays in primary PEX1-G843D patient cells, but neither agent was active alone or in combination in patient cells homozygous for the PEX1 c.2097_2098insT null allele. Moreover, diosmetin treatment increased PEX1, PEX6, and PEX5 protein levels in PEX1-G843D patient cells, but none of these proteins increased in PEX1 null cells. We propose that diosmetin acts as a pharmacological chaperone that improves the stability, conformation, and functions of PEX1/PEX6 exportomer complexes required for peroxisome assembly. We suggest that diosmetin, in clinical use for chronic venous disease, and related flavonoids warrant further preclinical investigation for the treatment of PEX1-G843D-associated ZSD.

Coagulopathy in Zellweger spectrum disorders: a role for vitamin K.

INTRODUCTION: Zellweger spectrum disorders (ZSDs) are caused by an impairment of peroxisome biogenesis, resulting in multiple metabolic abnormalities. This leads to a range of symptoms, including hepatic dysfunction and coagulopathy. This study evaluated the incidence and severity of coagulopathy and the effect of vitamin K supplementation orally and IV in ZSD. METHODS: Data were retrospectively retrieved from the medical records of 30 ZSD patients to study coagulopathy and the effect of vitamin K orally on proteins induced by vitamin K absence (PIVKA-II) levels. Five patients from the cohort with a prolonged prothrombin time, low factor VII, and elevated PIVKA-II levels received 10 mg of vitamin K IV. Laboratory results, including thrombin generation, at baseline and 72 h after vitamin K administration were examined. RESULTS: In the retrospective cohort, four patients (13.3%) experienced intracranial bleedings and 14 (46.7%) reported minor bleeding. No thrombotic events occurred. PIVKA-II levels decreased 38% after start of vitamin K therapy orally. In the five patients with a coagulopathy, despite treatment with oral administration of vitamin K, vitamin K IV caused an additional decrease (23%) of PIVKA-II levels and increased thrombin generation. CONCLUSION: Bleeding complications frequently occur in ZSD patients due to liver disease and vitamin K deficiency. Vitamin K deficiency is partly corrected by vitamin K supplementation orally, and vitamin K administered IV additionally improves vitamin K status, as shown by further decrease of PIVKA-II and improved thrombin generation.

Oral Cholic Acid Is Efficacious and Well Tolerated in Patients With Bile Acid Synthesis and Zellweger Spectrum Disorders.

OBJECTIVES: Patients with bile acid synthesis disorders (BASDs) due to single enzyme defects (SEDs) or Zellweger spectrum disorders (ZSDs) accumulate hepatotoxic atypical bile acids resulting in potentially fatal progressive liver disease. We evaluated the efficacy and safety of oral cholic acid in patients with BASD. METHODS: In this phase 3, open-label, single-arm, nonrandomized, noncomparative study conducted over 18 years, patients were administered cholic acid orally 10 to 15 mg ·â€Škg ·â€Šday. The primary efficacy variables were changes from pre- to post-treatment in atypical urinary bile acids, liver chemistries (serum aspartate aminotransferase, alanine aminotransferase), and height and weight. Additional efficacy variables included changes in serum bilirubin and liver histology. RESULTS: Of the 85 enrolled patients (63 with SED and 22 with ZSD), 79 received at least 1 dose of study medication; 70 patients (50 with SED and 20 with ZSD) were included in the modified intent-to-treat dataset. Cholic acid significantly improved urine bile acid metabolite scores (P < 0.0001) and serum aspartate aminotransferase and alanine aminotransferase (P < 0.0001) in patients with SED and ZSD. Cholic acid also improved height and weight percentiles in both groups, but only the change in weight was significant (P < 0.05). Serum direct bilirubin decreased significantly post-treatment (P < 0.001) in the intent-to-treat population, and liver biopsies showed either stable findings or histologic improvement in all parameters except bridging fibrosis. The overall safety profile of cholic acid was favorable, with no study drug-related serious adverse events or drug-related deaths reported. CONCLUSIONS: Oral cholic acid is a safe, efficacious, and well-tolerated treatment for BASD due to SED and ZSD.

Cholic acid therapy in Zellweger spectrum disorders.

INTRODUCTION: Zellweger spectrum disorders (ZSDs) are characterized by a failure in peroxisome formation, caused by autosomal recessive mutations in different PEX genes. At least some of the progressive and irreversible clinical abnormalities in patients with a ZSD, particularly liver dysfunction, are likely caused by the accumulation of toxic bile acid intermediates. We investigated whether cholic acid supplementation can suppress bile acid synthesis, reduce accumulation of toxic bile acid intermediates and improve liver function in these patients. METHODS: An open label, pretest-posttest design study was conducted including 19 patients with a ZSD. Participants were followed longitudinally during a period of 2.5 years prior to the start of the intervention. Subsequently, all patients received oral cholic acid and were followed during 9 months of treatment. Bile acids, peroxisomal metabolites, liver function and liver stiffness were measured at baseline and 4, 12 and 36 weeks after start of cholic acid treatment. RESULTS: During cholic acid treatment, bile acid synthesis decreased in the majority of patients. Reduced levels of bile acid intermediates were found in plasma and excretion of bile acid intermediates in urine was diminished. In patients with advanced liver disease (n = 4), cholic acid treatment resulted in increased levels of plasma transaminases, bilirubin and cholic acid with only a minor reduction in bile acid intermediates. CONCLUSIONS: Oral cholic acid therapy can be used in the majority of patients with a ZSD, leading to at least partial suppression of bile acid synthesis. However, caution is needed in patients with advanced liver disease due to possible hepatotoxic effects.

Docosahexaenoic acid therapy in peroxisomal diseases: results of a double-blind, randomized trial.

OBJECTIVES: Peroxisome assembly disorders are genetic disorders characterized by biochemical abnormalities, including low docosahexaenoic acid (DHA). The objective was to assess whether treatment with DHA supplementation would improve biochemical abnormalities, visual function, and growth in affected individuals. METHODS: This was a randomized, double-blind, placebo-controlled trial conducted at a single center. Treatment groups received supplements of DHA (100 mg/kg per day). The primary outcome measures were the change from baseline in the visual function and physical growth during the 1 year follow-up period. RESULTS: Fifty individuals were enrolled and randomized. Two were subsequently excluded from study analysis when it was determined that they had a single enzyme disorder of peroxisomal beta oxidation. Thirty-four returned for follow-up. Nine patients died during the trial of their disorder, and 5 others were lost to follow-up. DHA supplementation was well tolerated. There was no difference in the outcomes between the treated and untreated groups in biochemical function, electroretinogram, or growth. Improvements were seen in both groups in certain individuals. CONCLUSIONS: DHA supplementation did not improve the visual function or growth of treated individuals with peroxisome assembly disorders. CLASSIFICATION OF EVIDENCE: This interventional study provides Class II evidence that DHA supplementation did not improve the visual function or growth of treated individuals with peroxisome assembly disorders during an average of 1 year of follow-up in patients aged 1 to 144 months.

Recovery of PEX1-Gly843Asp peroxisome dysfunction by small-molecule compounds.

Zellweger spectrum disorder (ZSD) is a heterogeneous group of diseases with high morbidity and mortality caused by failure to assemble normal peroxisomes. There is no therapy for ZSD, but management is supportive. Nevertheless, one-half of the patients have a phenotype milder than classic Zellweger syndrome and exhibit a progressive disease course. Thus, patients would benefit if therapies became available and were instituted early. Recent reports indicate several interventions that result in partial peroxisome recovery in ZSD fibroblasts. To identify drugs that recover peroxisome functions, we expressed a GFP-peroxisome targeting signal 1 reporter in fibroblasts containing the common disease allele, PEX1-p.Gly843Asp. The GFP reporter remained cytosolic at baseline, and improvement in peroxisome functions was detected by the redistribution of the GFP reporter from the cytosol to the peroxisome. We established a high-content screening assay based on this phenotype assay and evaluated 2,080 small molecules. The cells were cultured in chemical for 2 days and then, were fixed and imaged by epifluorescent microscopy on a high-content imaging platform. We identified four compounds that partially recover matrix protein import, and we confirmed three using independent assays. Our results suggest that PEX1-p.G843D is a misfolded protein amenable to chaperone therapy.

Drug delivery to peroxisomes: employing unique trafficking mechanisms to target protein therapeutics.

Peroxisomes are multifunctional organelles of all human cells, responsible for a variety of essential biochemical and metabolic processes including alpha- and beta-oxidation of specific fatty acids, plasmalogen biosynthesis and glyoxylate detoxification. Inborn errors of biogenesis or in the ability to synthesize or properly traffic specific enzymes to peroxisomes result in devastating human disease. The organelle has also emerged as a contributor to cellular oxidative stress through its ability to generate hydrogen peroxide. Unlike most other organelles, the peroxisome's import apparatus will accommodate fully folded, oligomeric and co-factor-bound substrates. The strategies outlined here are designed to take advantage of this unique mechanism to target protein therapeutics. Emphasis is also placed on how to deliver these bioactive molecules into cells to engage the peroxisomal protein import machine. The critical antioxidant enzyme catalase has been successfully delivered and targeted by many of the approaches detailed herein; these examples will be discussed.

Bile acid treatment alters hepatic disease and bile acid transport in peroxisome-deficient PEX2 Zellweger mice.

UNLABELLED: The marked deficiency of peroxisomal organelle assembly in the PEX2(-/-) mouse model for Zellweger syndrome provides a unique opportunity to developmentally and biochemically characterize hepatic disease progression and bile acid products. The postnatal survival of homozygous mutants enabled us to evaluate the response to bile acid replenishment in this disease state. PEX2 mutant liver has severe but transient intrahepatic cholestasis that abates in the early postnatal period and progresses to steatohepatitis by postnatal day 36. We confirmed the expected reduction of mature C24 bile acids, accumulation of C27-bile acid intermediates, and low total bile acid level in liver and bile from these mutant mice. Treating the PEX2(-/-) mice with bile acids prolonged postnatal survival, alleviated intrahepatic cholestasis and intestinal malabsorption, reduced C27-bile acid intermediate production, and prevented older mutants from developing severe steatohepatitis. However, this therapy exacerbated the degree of hepatic steatosis and worsened the already severe mitochondrial and cellular damage in peroxisome-deficient liver. Both untreated and bile acid-fed PEX2(-/-) mice accumulated high levels of predominantly unconjugated bile acids in plasma because of altered expression of hepatocyte bile acid transporters. Significant amounts of unconjugated bile acids were also found in the liver and bile of PEX2 mutants, indicating a generalized defect in bile acid conjugation. CONCLUSION: Peroxisome deficiency widely disturbs bile acid homeostasis and hepatic functioning in mice, and the high sensitivity of the peroxisome-deficient liver to bile acid toxicity limits the effectiveness of bile acid therapy for preventing hepatic disease.

Inhibition of peroxisomal functions due to oxidative imbalance induced by mistargeting of catalase to cytoplasm is restored by vitamin E treatment in skin fibroblasts from Zellweger syndrome-like patients.

Many of the peroxisomal diseases exhibit excessive oxidative stress leading to neurological alterations and dysfunction. The role of peroxisomal oxidative stress in cellular function was highlighted by the loss of metabolic functions in peroxisomes of mutant cell lines, where catalase is mistargeted to the cytoplasm, but restored to peroxisomes by genetic manipulation (Sheikh et al. [Proc. Natl. Acad. Sci. USA 95 (1998) 2961)]. We report here that two human skin fibroblast cell lines from Zellweger syndrome-like patients are defective in the import of catalase into peroxisomes, causing impairment of metabolic function of this organelle. However, by lowering the cell culturing temperature (30 degrees C) the targeting of catalase to peroxisomes was restored, and with it the metabolic functions. Furthermore, mislocalization of catalase induces an oxidative imbalance in the cells which on treatment with a natural antioxidant, alpha-tocopherol (vitamin E), resulted in reduction of the oxidative levels and restoration of metabolic function (peroxisomal beta-oxidation and levels of very long chain fatty acids and plasmalogen as well as alpha-oxidation of branched-chain fatty acids). However, restoration of peroxisomal functions was not associated with the targeting of catalase to peroxisomes. Therefore, our finding suggests that correction of mistargeted catalase to peroxisomes is a temperature sensitive event and supports the hypotheses that its location outside peroxisomes induces an oxidative imbalance that results in metabolic dysfunction. The imbalance can be reversed by treatment with vitamin E, leading to normalization of peroxisomal functions. These findings open a novel approach for therapeutic treatment of certain peroxisomal disorders where gene or hypothermic therapies are not an option.

Restoring the DHA levels in the brains of Zellweger patients.

Patients with the Zellweger syndrome and its variants have very low levels of docosahexaenoic acid (DHA) in the brain, retina, and other tissues. Such a marked DHA deficiency could be related to the pathogenesis of peroxisomal disorders. Therefore, restoring the DHA levels in these patients can probably improve the clinical course of the disease. With this rationale, 20 patients with generalized peroxisomal disorders have been treated to date with DHA ethyl ester, at daily doses of 100-500 mg, for variable periods of time. Treatment has been always accompanied by a nutritious diet, normal for the age, in order to provide all the necessary nutrients and avoid a polyunsaturated fatty acid (PUFA) imbalance. The most constant improvement has been normalization of the DHA levels and liver function. Vision has improved in about half the patients and muscle tone has generally increased. Magnetic resonance imaging (MRI) examination revealed improvement of myelination in 9 patients. Significantly, the clinical improvement has been most marked in those patients who started the treatment before 6 mo of age. Biochemically, the plasma very long-chain fatty acids (VLCFA) 26:0 and 26:1n-9 decreased markedly despite the complete diet provided. In erythrocytes, the plasmalogen ratio 18: ODMA/18:0 increased in most cases, and sometimes even normalized. All these beneficial effects suggest that DHA deficiency plays a fundamental role in the pathogenesis of peroxisomal disease. Because DHA accretion is maximal during early brain development, it is essential to initiate the treatment as soon as possible. Otherwise, restoration of brain DHA levels and prevention of further damage will not be possible.

Therapeutic developments in peroxisome biogenesis disorders.

Clinically, peroxisome biogenesis disorders (PBDs) are a group of lethal diseases with a continuum of severity of clinical symptoms ranging from the most severe form, Zellweger syndrome, to the milder forms, infantile Refsum disease and rhizomelic chondrodysplasia punctata. PBDs are characterised by a number of biochemical abnormalities including impaired degradation of peroxide, very long chain fatty acids, pipecolic acid, phytanic acid and xenobiotics and impaired synthesis of plasmalogens, bile acids, cholesterol and docosahexaenoic acid. Treatment of PBD patients as a group is problematic since a number of patients, especially those with Zellweger syndrome, have significant neocortical alterations in the brain at birth so that full recovery would be impossible even with postnatal therapy. To date, treatment of PBD patients has generally involved only supportive care and symptomatic therapy. However, the fact that some of the milder PBD patients live into the second decade has prompted research into possible treatments for these patients. A number of experimental therapies have been evaluated to determine whether or not correction of biochemical abnormalities through dietary supplementation and/or modification is of clinical benefit to PBD patients. Another approach has been pharmacological induction of peroxisomes in PBD patients to improve overall peroxisomal biochemical function. Well known rodent peroxisomal proliferators were found not to induce human peroxisomes. Recently, our laboratory demonstrated that sodium 4-phenylbutyrate induces peroxisome proliferation and improves biochemical function (very long chain fatty acid beta-oxidation rates and very long chain fatty acid and plasmalogens levels) in fibroblast cell lines from patients with milder PBD phenotypes. Dietary supplementation and/or modification and pharmacological induction of peroxisomes as treatment strategies for PBD patients will be the subject of this review.

Docosahexaenoic acid therapy in docosahexaenoic acid-deficient patients with disorders of peroxisomal biogenesis.

A patient with classic Zellweger syndrome was treated with docosahexaenoic acid ethyl ester (DHA-EE) for three months. Five other patients with Zellweger variants (four of them less than one-year-old and a five-year-old) were treated with DHA-EE until normalization of the DHA levels in erythrocytes. When arachidonic acid (AA) concentration decreased, AA was added to the diet. Thereafter, a combined treatment with DHA plus AA followed, in a variable proportion that allowed the high levels of DHA in erythrocytes to be maintained. In the patient with Zellweger syndrome, DHA therapy produced an increase in plasmalogen and a decrease in 26:0 and 26:1. No clear clinical improvement could be detected in this patient during the short period of treatment with DHA-EE. The most consistent clinical effect produced by DHA therapy in the other patients with disorders of peroxisomal biogenesis was visual improvement, even in those patients that were virtually blind before the treatment. In general, the developmental curve began to accelerate. The infants became more alert, acquired better visual and social contact and muscular tone improved, with the beginning of good head control. The liver tests tended to normalize and some patients showed a reduction of hepatomegaly. All these favorable changes occurred when the patients were taking the DHA-EE alone. In some of the patients, muscular tone seemed to improve further after introducing AA supplements. From the biochemical point of view, the plasmalogen levels increased in most cases in erythrocytes, and the two ratios 26:0/22:0 and 26:1/22:0 decreased in plasma. In some patients there was a tendency for 26:1 to increase in plasma and for 18:0 plasmalogen to decrease in erythrocytes when AA was introduced in the diet. The significance of these findings remains to be elucidated, but they stress the importance of strict monitoring and control of the polyunsaturated fatty acids status during DHA therapy.

[Congenital deficiencies in bile acid synthesis: from diagnosis to treatment]. / Les déficits congénitaux de synthèse des acides biliaires: du diagnostic au traitement.

Bile acids represent the principal excretion pathway of cholesterol. Their synthesis involves hydroxylations of the sterol nucleus and shortening of the lateral chain transforming an highly insoluble and immiscible molecule in an hydrophilic and detergent one. Congenital defect of one of the enzymes of bile acid synthesis (26-alpha-hydroxylase) constitutes the etiology of cerebrotendinous xanthomatosis, associating degenerative disease of the central nervous system, cutaneous xanthomas and cataract; the degenerative process may be stopped by a substitutive therapy with chenodeoxycholic acid. In another syndrome characterised by a lack of peroxisomes (Zellweger disease and associated syndromes), some bile acid metabolites accumulate due to the enzymatic block. Some cases of malabsorption due to a congenital defect of bile sterol synthesis are reported; such a case was jointly investigated in Brussels and Groningen.

Oral bile acid treatment and the patient with Zellweger syndrome.

The cerebrohepatorenal syndrome of Zellweger is a congenital syndrome of multiple manifestations, including hepatomegaly and liver dysfunction. Treatment is generally of a supportive nature, aimed at improving nutrition and growth, controlling the central nervous system symptoms and limiting progression of liver disease. Because the liver disease in Zellweger syndrome may be attributed to an overproduction and accumulation of cholestanoic acids, exacerbated by diminished primary bile acid synthesis, we hypothesized that primary bile acid administration would be beneficial in improving liver function by a mechanism involving down-regulation in the synthesis of these atypical bile acids. We report here the clinical and biochemical responses to primary bile acid administration in a 2-mo-old boy who was seen with the typical signs of Zellweger syndrome. Liver disease was evident from hepatomegaly and elevated serum liver enzymes and bilirubin. The diagnosis was supported by markedly elevated serum very long chain fatty acids and the bile acids dihydroxycholestanoic acid and trihydroxycholestanoic acid. Confirmation of the lack of peroxisomes was established by electron microscopy. When the patient was 6 mo old, the primary bile acids cholic acid and chenodeoxycholic acid, (100 mg each/day) were administered orally. A significant improvement in biochemical indices of liver function occurred with a normalization of the serum bilirubin and liver enzymes and a histological improvement in the extent of inflammation and bile duct proliferation and disappearance of cannalicular plugs. Serum and urinary cholestanoic acids showed a significant decrease within a few days. A striking and sustained increase in growth was observed after therapy, and an improvement in neurological symptoms was noted. In conclusion, this study indicates that primary bile acid therapy improves liver function and growth in the patient with peroxisomal dysfunction and should be considered in the supportive therapies for this condition.