A new test method for biochemical analysis of plasmalogens in dried blood spots and erythrocytes from patients with peroxisomal disorders.

Measurement of plasmalogens is useful for the biochemical diagnosis of rhizomelic chondrodysplasia punctata (RCDP) and is also informative for Zellweger spectrum disorders (ZSD). We have developed a test method for the simultaneous quantitation of C16:0, C18:0, and C018:1 plasmalogen (PG) species and their corresponding fatty acids (FAs) in dried blood spots (DBS) and erythrocytes (RBC) by using capillary gas chromatography-mass spectrometry. Normal reference ranges for measured markers and 10 calculated ratios were established by the analysis of 720 and 473 unaffected DBS and RBC samples, respectively. Determination of preliminary disease ranges was made by using 45 samples from 43 unique patients: RCDP type 1 (DBS: 1 mild, 17 severe; RBC: 1 mild, 6 severe), RCDP type 2 (DBS: 2 mild, 1 severe; RBC: 2 severe), RCDP type 3 (DBS: 1 severe), RCDP type 4 (RBC: 2 severe), and ZSD (DBS: 3 severe; RBC: 2 mild, 7 severe). Postanalytical interpretive tools in Collaborative Laboratory Integrated Reports (CLIR) were used to generate an integrated score and a likelihood of disease. In conjunction with a review of clinical phenotype, phytanic acid, and very long-chain FA test results, the CLIR analysis allowed for differentiation between RCDP and ZSD. Data will continue to be gathered to improve CLIR analysis as more samples from affected patients with variable disease severity are analyzed. The addition of DBS analysis of PGs may allow for at-home specimen collection and second-tier testing for newborn screening programs.

Dicarboxylic acylcarnitine biomarkers in peroxisome biogenesis disorders.

The peroxisome is an essential eukaryotic organelle with diverse metabolic functions. Inherited peroxisomal disorders are associated with a wide spectrum of clinical outcomes and are broadly divided into two classes, those impacting peroxisome biogenesis (PBD) and those impacting specific peroxisomal factors. Prior studies have indicated a role for acylcarnitine testing in the diagnosis of some peroxisomal diseases through the detection of long chain dicarboxylic acylcarnitine abnormalities (C16-DC and C18-DC). However, there remains limited independent corroboration of these initial findings and acylcarnitine testing for peroxisomal diseases has not been widely adopted in clinical laboratories. To explore the utility of acylcarnitine testing in the diagnosis of peroxisomal disorders we applied a LC-MS/MS acylcarnitine method to study a heterogenous clinical sample set (n = 598) that included residual plasma specimens from nineteen patients with PBD caused by PEX1 or PEX6 deficiency, ranging in severity from lethal neonatal onset to mild late onset forms. Multiple dicarboxylic acylcarnitines were significantly elevated in PBD patients including medium to long chain (C8-DC to C18-DC) species as well as previously undescribed elevations of malonylcarnitine (C3-DC) and very long chain dicarboxylic acylcarnitines (C20-DC and C22-DC). The best performing plasma acylcarnitine biomarkers, C20-DC and C22-DC, were detected at elevated levels in 100% and 68% of PBD patients but were rarely elevated in patients that did not have a PBD. We extended our analysis to residual newborn screening blood spot cards and were able to detect dicarboxylic acylcarnitine abnormalities in a newborn with a PBD caused by PEX6 deficiency. Similar to prior studies, we failed to detect substantial dicarboxylic acylcarnitine abnormalities in blood spot cards from patients with x-linked adrenoleukodystrophy (x-ald) indicating that these biomarkers may have utility in quickly narrowing the differential diagnosis in patients with a positive newborn screen for x-ald. Overall, our study identifies widespread dicarboxylic acylcarnitine abnormalities in patients with PBD and highlights key acylcarnitine biomarkers for the detection of this class of inherited metabolic disease.

Multivariate analysis and model building for classifying patients in the peroxisomal disorders X-linked adrenoleukodystrophy and Zellweger syndrome in Chinese pediatric patients.

BACKGROUND: The peroxisome is a ubiquitous single membrane-enclosed organelle with an important metabolic role. Peroxisomal disorders represent a class of medical conditions caused by deficiencies in peroxisome function and are segmented into enzyme-and-transporter defects (defects in single peroxisomal proteins) and peroxisome biogenesis disorders (defects in the peroxin proteins, critical for normal peroxisome assembly and biogenesis). In this study, we employed multivariate supervised and non-supervised statistical methods and utilized mass spectrometry data of neurological patients, peroxisomal disorder patients (X-linked adrenoleukodystrophy and Zellweger syndrome), and healthy controls to analyze the role of common metabolites in peroxisomal disorders, to develop and refine a classification models of X-linked adrenoleukodystrophy and Zellweger syndrome, and to explore analytes with utility in rapid screening and diagnostics. RESULTS: T-SNE, PCA, and (sparse) PLS-DA, operated on mass spectrometry data of patients and healthy controls were utilized in this study. The performance of exploratory PLS-DA models was assessed to determine a suitable number of latent components and variables to retain for sparse PLS-DA models. Reduced-features (sparse) PLS-DA models achieved excellent classification performance of X-linked adrenoleukodystrophy and Zellweger syndrome patients. CONCLUSIONS: Our study demonstrated metabolic differences between healthy controls, neurological patients, and peroxisomal disorder (X-linked adrenoleukodystrophy and Zellweger syndrome) patients, refined classification models and showed the potential utility of hexacosanoylcarnitine (C26:0-carnitine) as a screening analyte for Chinese patients in the context of a multivariate discriminant model predictive of peroxisomal disorders.

Adrenal Insufficiency in Peroxisomal Disorders: A Single Institution Case Series.

INTRODUCTION: There are two major categories of peroxisomal disorders (PDs): peroxisomal biogenesis disorders (PBDs) due to defects in peroxisomal (PEX) genes and deficiency of other peroxisomal enzymes (such as D-bifunctional enzyme deficiency due to HSD17B4). PDs are characterized by abnormal elevations of very-long-chain fatty acids (VLCFA). We aimed to evaluate the clinical phenotype of adrenal insufficiency in patients with PD and to assess any genotype-phenotype correlations with adrenal insufficiency. CASE PRESENTATION: We performed a retrospective electronic medical record review at a single university medical center, of data over 12 years and identified 7 patients with PD. Of the 7 patients identified, 6 patients had a diagnosis of PBD and one had a single peroxisomal enzyme deficiency, HSD17B4. The average age of the patients at diagnosis were 0.61 ± 0.66 years. Four patients (66.7%) had primary adrenal insufficiency: 3, out of the 4, patients had elevated baseline ACTH. Three patients failed to have increased response after the Cortrosyn™ stimulation test. Three patients were on daily hydrocortisone replacement, and 1 patient was on stress-dose hydrocortisone only as needed. Specific genetic variant analysis revealed that all the 3 patients with PBD and adrenal insufficiency who were on steroid supplementation had the compound heterozygous pathogenic variant in exon 13 of PEX1 c.2097dupT (p.Ile700Tyrfs*42) and c.2528G>A (p.Gly843Asp), while the 1 patient with peroxisomal enzyme deficiency and adrenal insufficiency had compound heterozygous pathogenic variants in HSD17B4 c.1369A>T (p.Asn457Tyr) and c.1210 - 1G>A (splice acceptor). Two of these patients with PEX1 variants also required mineralocorticoid supplementation. The 3 PBD patients without adrenal insufficiency did not have a PEX1 variant. DISCUSSION/CONCLUSION: Primary adrenal insufficiency is common in patients with PD. Based on our data, patients with the compound heterozygous PEX1 pathogenic variants of exon 13 (c.2097dupT and c.2528G>A) tend to have adrenal insufficiency. Aldosterone deficiency, though rare, can occur in PD.

Development of a system adapted for the diagnosis and evaluation of peroxisomal disorders by measuring bile acid intermediates.

OBJECTIVE: Bile acid intermediates, 3α,7α,12α-trihydroxycholestanoic acid (THCA) and 3α,7α-dihydroxycholestanoic acid (DHCA), are metabolized in peroxisomes. Some peroxisomal disorders (PDs), such as Zellweger spectrum disorder (ZSD), show an accumulation of bile acid intermediates. In particular, ABCD3 deficiency and acyl-CoA-oxidase 2 deficiency are characterized by these metabolite abnormalities. In patients with ZSD, levels of bile acid intermediates can be lowered by a primary bile acid supplementation treatment; therefore, measuring their levels could help evaluate treatment effectiveness. Here, we established a method for the quantitative determination of bile acid intermediates (THCA/DHCA) for differentiating PDs and assessing bile acid treatment. METHODS: Serum samples, obtained from patients with several forms of ZSD as well as peroxisomal ß-oxidation enzyme deficiencies, were deproteinized and analyzed using liquid chromatography-mass spectrometry. RESULTS: Levels of the bile acid intermediates increased significantly in patients with Zellweger syndrome (ZS) and slightly in patients with neonatal adrenoleukodystrophy and infantile Refsum disease (IRD), reflecting the severity of these diseases. One patient with ZS treated with primary bile acids for 6 months showed slightly decreased serum DHCA levels but significantly increased serum THCA levels. One patient with IRD who underwent living-donor liver transplantation showed a rapid decrease in serum THCA and DHCA levels, which remained undetected for 6 years. In all controls, THCA and DHCA levels were below the detection limit. CONCLUSION: The analytical method developed in this study is useful for diagnosing various PD and validating bile acid treatment. Additionally, it can help predict the prognosis of patients with PD and support treatment strategies.

Quantification of Very-Long-Chain and Branched-Chain Fatty Acids in Plasma by Liquid Chromatography-Tandem Mass Spectrometry.

Peroxisomal disorders are a heterogeneous group of genetic disorders caused by impaired peroxisomal biogenesis or by defects in single peroxisomal proteins. The most common peroxisomal disorders are Zellweger spectrum disorders (ZSDs), due to pathogenic variants in one of the 13 PEX genes, and X-linked adrenoleukodystrophy/adrenomyeloneuropathy (X-ALD/AMN), due to pathogenic variants in ATP-binding cassette transporter type D1 (ABCD1) gene. Peroxisomes perform multiple essential cellular functions, including ß-oxidation of very-long-chain fatty acids (VLCFAs), pristanic acid and some bile acid intermediates, and α-oxidation of phytanic acid. In most patients, abnormal levels of VLCFAs and/or branched-chain fatty acids (BCFAs, e.g., phytanic and pristanic acids) are present; hence, measuring these analytes is critical when suspecting a peroxisomal disorder. This chapter describes a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to quantify VLCFAs and BCFAs in plasma or serum for the diagnosis of peroxisomal disorders. The method consists of an acid hydrolysis step to release the fatty acids from their coenzyme A esters followed by derivatization using oxalyl chloride, dimethylaminoethanol, and then methyl iodide. The trimethyl-amino-ethyl (TMAE) iodide ester derivatives are analyzed using UPLC-MS/MS in positive electrospray ionization and multiple reaction-monitoring (MRM) mode. Quantitation is performed using a five-point calibration curve after normalizing with deuterated internal standards.

Beyond rare disorders: A new era for peroxisomal pathophysiology.

Metabolism is emerging as a central influencer of multiple disease states in humans. Peroxisomes are central metabolic organelles whose decreased function gives rise to severe peroxisomal diseases. Recently, it is becoming clear that, beyond such rare inborn errors, the deterioration of peroxisomal functions contributes to multiple and prevalent diseases such as cancer, viral infection, diabetes, and neurodegeneration. Despite the clear importance of peroxisomes in common pathophysiological processes, research on the mechanisms underlying their contributions is still sparse. Here, we highlight the timeliness of focusing on peroxisomes in current research on central, abundant, and society-impacting human pathologies. As peroxisomes are now coming into the spotlight, it is clear that intensive research into these important organelles will enable a better understanding of their contribution to human health, serving as the basis to develop new diagnostic and therapeutic approaches to prevent and treat human diseases.

Late onset AMACR deficiency with metabolic stroke-like episodes and seizures.

Alpha-methylacyl-CoA racemase (AMACR) deficiency is a rare peroxisomal disorder causing pristanic acid accumulation. Only 16 cases have been described so far. A female in her seventh decade presented with episodes of dysphasia, headache and sensory disturbance inconsistent with migraine, epilepsy or transient ischaemic attack. An MRI demonstrated unusual changes in the pons, red nuclei, thalami and white matter. Mitochondrial disease was suspected but detailed testing was negative. After eight years of symptoms, she developed a febrile encephalopathy with hemispheric dysfunction, focal convulsive seizures and coma. Her condition stabilised after one month. Lacosamide was continued for seizure prevention. The diagnosis remained elusive until whole genome sequencing revealed AMACR deficiency. Pristanic acid levels were highly elevated and dietary modification was recommended. Genetic peroxisomal disorders can present in older age; our patient is the oldest in the AMACR deficiency literature. Novel features in our case include central apnoea, dystonia and rapid eye movement behaviour disorder.

Newly defined peroxisomal disease with novel ACBD5 mutation.

Peroxisomal disorders are a heterogeneous group of diseases caused by mutations in a large number of genes. One of the genetic disorders known to cause this situation is ACBD5 (Acyl-CoA binding-domain-containing-5) gene mutations that have been described in recent years. Here, we report two siblings with a novel homozygous nonsense variation (c.1297C>T, p.Arg433*) in ACBD5 (NM_145698.4) gene using Clinical Exome Sequencing (Sophia Genetics).

Prevalence of patients with lysosomal storage disorders and peroxisomal disorders: A nationwide survey in Japan.

INTRODUCTION: Lysosomal storage disorders and peroxisomal disorders are rare diseases caused by the accumulation of substrates of the metabolic pathway within lysosomes and peroxisomes, respectively. Owing to the rarity of these diseases, the prevalence of lysosomal storage disorders and peroxisomal disorders in Japan is unknown. Therefore, we conducted a nationwide survey to estimate the number of patients with lysosomal storage disorders and peroxisomal disorders in Japan. METHODS: A nationwide survey was conducted following the "Manual of nationwide epidemiological survey for understanding patient number and clinical epidemiology of rare diseases (3rd version)". A questionnaire asking for detailed information, such as disease phenotypes and medical history, was created and sent to 504 institutions with doctors who have experience in treating patients with lysosomal storage disorders and peroxisomal disorders. Result A total of 303 completed questionnaires were collected from 504 institutions (response rate: 60.1%). The number of patients was estimated by calculating the rate/frequency of overlap. The estimated number of patients was 1658 (±264.8) for Fabry disease, 72 (±11.3) for mucopolysaccharidosis I, 275 (±49.9) for mucopolysaccharidosis II, 211 (±31.3) for Gaucher disease, 124 (±25.8) for Pompe disease, 83 (±44.3) for metachromatic leukodystrophy, 57 (±9.4) for Niemann-Pick type C, and 262 (±42.3) for adrenoleukodystrophy. In addition the birth prevalence was calculated using the estimated number of patients and birth year data for each disease, and was 1.25 for Fabry disease, 0.09 for mucopolysaccharidosis I, 0.38 for mucopolysaccharidosis II, 0.19 for Gaucher disease, 0.14 for Pompe disease, 0.16 for metachromatic leukodystrophy, 0.16 for Niemann-Pick type C, and 0.20 for adrenoleukodystrophy. DISCUSSION: Among the diseases analyzed, the disease with the highest prevalence was Fabry disease, followed by mucopolysaccharidosis II, adrenoleukodystrophy, Gaucher disease and metachromatic leukodystrophy. In particular, the high prevalence of mucopolysaccharidosis II and Gaucher disease type II was a feature characteristic of Japan. CONCLUSION: We estimated the number of patients with lysosomal storage disorders and peroxisomal disorders in Japan. The details of the age at diagnosis and treatment methods for each disease were clarified, and will be useful for the early diagnosis of these patients and to provide appropriate treatments. Furthermore, our results suggest that supportive care and the development of an environment that can provide optimal medical care is important in the future.

Twins with PEX7 related intellectual disability and cataract: Highlighting phenotypes of peroxisome biogenesis disorder 9B.

Peroxisome biogenesis disorders (PBDs) are a group of autosomal recessive disorders caused due to impaired peroxisome assembly affecting the formation of functional peroxisomes. PBDs are caused by a mutation in PEX gene family resulting in disease manifestation with extreme variability ranging from the onset of profound neurologic symptoms in newborns to progressive degenerative disease in adults. Disease causing variations in PEX7 is known to cause severe rhizomelic chondrodysplasia punctata type 1 and PBD 9B, an allelic disorder resulting in a milder phenotype, often indistinguishable from that of classic Refsum disease. This case report highlights the variability of PEX7 related phenotypes and suggests that other than RCDP1 and late onset phenotype similar to Refsum disease, some cases present with cataract and neurodevelopmetal abnormalities during childhood without chondrodysplasia or rhizomelia. This report also underlines the importance of considering PBD 9B in children presenting with neurodevelopmental abnormalities especially if they have congenital cataract.

The peroxisomal disorder spectrum and Heimler syndrome: Deep phenotyping and review of the literature.

The spectrum of peroxisomal disorders is wide and comprises individuals that die in the first year of life, as well as people with sensorineural hearing loss, retinal dystrophy and amelogenesis imperfecta. In this article, we describe three patients; two diagnosed with Heimler syndrome and a third one with a mild-intermediate phenotype. We arrived at these diagnoses by conducting complete ophthalmic (National Eye Institute), auditory (National Institute of Deafness and Other Communication Disorders), and dental (National Institute of Dental and Craniofacial Research) evaluations, as well as laboratory and genetic testing. Retinal degeneration with macular cystic changes, amelogenesis imperfecta, and sensorineural hearing loss were features shared by the three patients. Patients A and C had pathogenic variants in PEX1 and Patient B, in PEX6. Besides analyzing these cases, we review the literature regarding mild peroxisomal disorders, their pathophysiology, genetics, differential diagnosis, diagnostic methods, and management. We suggest that peroxisomal disorders are considered in every child with sensorineural hearing loss and retinal degeneration. These patients should have a dental evaluation to rule out amelogenesis imperfecta as well as audiologic examination and laboratory testing including peroxisomal biomarkers and genetic testing. Appropriate diagnosis can lead to better genetic counseling and management of the associated comorbidities.

Serum very long-chain fatty acids (VLCFA) levels as predictive biomarkers of diseases severity and probability of survival in peroxisomal disorders.

CONCLUSION: VLCFA levels correlate with the severity of the clinical course of ZS, DBP and mild ZSD. The best predictive value for estimating the projected disease severity and survival time is a concentration of C26:0.

Laboratory diagnosis of disorders of peroxisomal biogenesis and function: a technical standard of the American College of Medical Genetics and Genomics (ACMG).

Peroxisomal disorders are a clinically and genetically heterogeneous group of diseases caused by defects in peroxisomal biogenesis or function, usually impairing several metabolic pathways. Peroxisomal disorders are rare; however, the incidence may be underestimated due to the broad spectrum of clinical presentations. The inclusion of X-linked adrenoleukodystrophy to the Recommended Uniform Screening Panel for newborn screening programs in the United States may increase detection of this and other peroxisomal disorders. The current diagnostic approach relies heavily on biochemical genetic tests measuring peroxisomal metabolites, including very long-chain and branched-chain fatty acids in plasma and plasmalogens in red blood cells. Molecular testing can confirm biochemical findings and identify the specific genetic defect, usually utilizing a multiple-gene panel or exome/genome approach. When next-generation sequencing is used as a first-tier test, evaluation of peroxisome metabolism is often necessary to assess the significance of unknown variants and establish the extent of peroxisome dysfunction. This document provides a resource for laboratories developing and implementing clinical biochemical genetic testing for peroxisomal disorders, emphasizing technical considerations for sample collection, test performance, and result interpretation. Additionally, considerations on confirmatory molecular testing are discussed.

Variant analysis of PEX11B gene from a family with peroxisome biogenesis disorder 14B by whole exome sequencing.

BACKGROUND: Peroxisome biogenesis disorder 14B (PBD14B) is an autosomal recessive peroxisome biogenesis disorder characterized clinically by mild intellectual disability, congenital cataracts, progressive hearing loss, and polyneuropathy peroxisome biogenesis disorders are genetically heterogeneous group of disorders caused by biallelic mutations in peroxin (PEX) genes. METHODOLOGY/LABORATORY EXAMINATION: DNA of the family was extracted and sequenced by whole exome sequencing. The results were validated with Sanger sequencing analyzed with Bioinformatics software. RESULTS: Sequencing result showed that the patient has carried a homozygous variant of c.277C>T of the PEX11B gene. The patient's brother has carried a homozygous variant of c.277C>T of the PEX11B gene and their variants of c.277C>T of the PEX11B gene were inherited, respectively, from his mother and father. DISCUSSION AND CONCLUSION: The homozygous variant of c.277C>T of the PEX11B gene probably underlie the disease in this child and her brother.

Adrenoleukodystrophy in the era of newborn screening.

PURPOSE OF REVIEW: Adrenoleukodystrophy (ALD) is a peroxisomal disorder with varying clinical presentations, including adrenal insufficiency, neurologic disease, and testicular dysfunction. The present review is intended to describe the current knowledge of the pathophysiology of ALD and provide an update regarding newborn screening, diagnosis, monitoring, and treatment. RECENT FINDINGS: New York State initiated newborn screening for ALD on December 30, 2013. Successful ALD newborn screening has led to its addition on other state newborn screens and recommendations for universal screening. Initial incidence reports, based on newborn screening, suggest ALD may be more common than previously described. The Pediatric Endocrine Society has published guidance for monitoring newborn males with ALD and case reports suggest biochemical adrenal insufficiency can be present during early infancy. Allogeneic hematopoietic stem cell transplant and gene therapy have been effective at halting the progression of cerebral ALD. SUMMARY: Early diagnosis and monitoring for progression of ALD can prevent adrenal crisis and treat the cerebral form of the disease. Initial guidelines for surveillance are likely to evolve as newborn screening not only aids in early detection and therapeutic interventions for ALD, but also expands our knowledge of the natural history of ALD.

Visual loss as first clinical manifestation of X-linked adrenoleukodystrophy / Perda visual como primeira manifestação clínica de adrenoleucodistrofia ligada ao X

Abstract X-linked adrenoleukodystrophy (X-ALD) represents a group of diseases characterized by the accumulation of very long chain fattyacids (VLCFAs) in the tissues. Its clinical manifestations are usually manifold. Visual changes may be present, but they often appear later in the disease. We describe here the case of a 9-year-old boy with X-ALD, whose first symptom was visual loss, which began at 8 years of age. His ophthalmologic evaluation revealed no alterations. Shortly thereafter, he suffered a head injury. The magnetic resonance imaging of brain revealed findings that led to the suspicion of X-ALD. The plasma VLCFA dosage confirmed this diagnosis. This report aims toshow that in cases of visual loss with a normal ophthalmic examination, a high index of suspicion should be given for conditions suchas X-ALD, since it affects the cortical routes related to vision. Fundoscopy findings appear late in X-ALD.

Resumo A adrenoleucodistrofia ligada ao X (X-ALD) representa um grupo de doenças caracterizadas pelo acúmulo de ácidos graxos de cadeia muito longa (VLCFAs) nos tecidos. Suas manifestações clínicas costumam ser múltiplas. Alterações visuais podem estar presentes, contudo costumam surgir mais tardiamente na doença. Descrevemos aqui o caso de um menino de 9 anos com X-ALD, cujo primeiro sintoma foi perda visual, iniciada aos 8 anos de idade. A sua avaliação oftalmológica não revelou alterações. Pouco tempo depois, ele sofreu um traumatismo craniano. A imagem de ressonância magnética de encéfalo revelou achados que levaram a suspeita de X-ALD. A dosagem dos VLCFAs no plasma confirmou este diagnóstico. Este relato tem como objetivo mostrar que em casos de perda visual com um exame oftalmológico normal, deve-se ter um alto índice de suspeita para condições como a X-ALD, pois a mesma afeta as vias corticais relacionadas à visão. Nessa doença, os achados da fundoscopia aparecem mais tardiamente.

Ataxia with novel compound heterozygous PEX10 mutations and a literature review of PEX10-related peroxisome biogenesis disorders.

OBJECTIVES: To describe the clinical and genetic features of a Chinese peroxisome biogenesis disorder 6B patient with PEX10 mutations and review PEX10-related peroxisomal disorders. PATIENTS AND METHODS: The proband is a 7-year-old boy with mild mental retardation and gait instability, intention tremor and nystagmus. An extensive clinical and laboratory evaluation including molecular genetic studies was performed. Genomic DNA was extracted from peripheral blood using the standardized phenol/chloroform extraction method, and the coding region of the PEX10 gene was sequenced in three family members. RESULTS: Cerebral MRI showed cerebellar atrophy. Magnetic resonance spectroscopy revealed a decreased N-acetyl aspartate peak in the cerebellum. Nerve conduction velocity examination found prolonged motor and sensory nerve potential latencies (proximal obvious), decreased potential amplitude, and slow nerve conduction velocity. Routine blood tests and biochemistries were abnormal. The PEX10 gene test showed compound heterozygous mutations (c.209 G > A, p. G70E and c.830 T > C, p. L277 P). The mutation c.830 T > C, p. L277 P has been previously reported, whereas c.209 G > A, p. G70E is novel. CONCLUSION: We identified an ataxia case of peroxisome biogenesis disorder 6B caused by novel compound heterozygous mutations of the PEX10 gene. Peroxisome biogenesis disorders should be considered in the differential diagnosis of autosomal recessive ataxia, especially cases with early onset.

Atypical PEX16 peroxisome biogenesis disorder with mild biochemical disruptions and long survival.

BACKGROUND: Mutations in PEX16 cause peroxisome biogenesis disorder (PBD). Zellweger syndrome characterized by neurological dysfunction, dysmorphic features, liver disease and early death represents the severe end of this clinical spectrum. Here we discuss the diagnostic challenge of atypical PEX16 related PBD in 3 patients from highly inbred kindred and describe the role of specific metabolites analyses, fibroblasts studies, whole-exome sequencing (WES) and metabolomics profiling to establish the diagnosis. METHODS AND PATIENTS: The proband is a 12-year-old male born to consanguineous parents. Despite normal development in the first year, regression and progressive spastic diplegia, poor coordination and dysarthria occurred thereafter. Patient 2 (3-year old female) and Patient 3 (19-month old female) shared similar clinical course with the proband. Biochemical studies on plasma and fibroblasts, WES and global metabolomics analyses were performed. RESULTS: Very-long-chain fatty acids analysis showed subtle elevations in C26 and C26/C22. Global Metabolomics-Assisted Pathway profiling was not remarkable. Immunocytochemical investigations on fibroblasts revealed fewer catalase and PMP70-containing particles indicating aberrant peroxisomal assembly. Complementation studies were inconclusive. WES revealed a novel homozygous variant in PEX16 (c.859C>T). The biochemical profiles of Patient 2 and Patient 3 were similar to the proband and the same genotype was confirmed. CONCLUSION: This paper highlights the diagnostic challenge of PEX16 patients due to the widely variable clinical and biochemical phenotypes. It also emphasizes the important roles of combined biochemical assays with next generation sequencing techniques in reaching diagnosis in the context of atypical clinical presentations, subtle biomarker abnormalities and consanguinity.

Application of machine learning algorithms for the differential diagnosis of peroxisomal disorders.

We have established diagnostic thresholds of very long-chain fatty acids (VLCFA) for the differential diagnosis of peroxisomal disorders using the machine learning tools. The plasma samples of 131 controls and 90 cases were tested for VLCFA using gas chromatography-mass spectrometry following stable isotope dilution. These data were used to construct association rules and for recursive partitioning. The C26/22 in healthy controls ranged between 0.008 and 0.01. The C26 levels between 1.61 and 3.34 µmol/l and C26/C22 between 0.05 and 0.10 are diagnostic of X-linked adrenoleukodystrophy (X-ALD). Very high levels of C26 (>3.34 µmol/l) and C26/C22 ratio (>0.10) are diagnostic of Zellweger syndrome (ZS). Significant elevation of phytanic acid was observed in Refsum (t = 6.14, P < 0.0001) and Rhizomelic chondrodysplasia punctata (RCDP) (t = 16.72, P < 0.0001). The C26/C22 ratio is slightly elevated in RCDP (t = 2.58, P = 0.01) while no such elevation was observed in Refsum disease (t = 0.86, P = 0.39). The developed algorithm exhibited greater clinical utility (AUC: 0.99-1.00) in differentiating X-ALD, ZS and healthy controls. The algorithm has greater clinical utility in the differential diagnosis of peroxisomal disorders based on VLCFA pattern. Plasmalogens will add additional value in differentiating RCDP and Refsum disease.