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

      Abstract

      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 bloodcells. 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.

      Keywords

      Background

      Peroxisome structure and function

      Peroxisomes are ubiquitous single membrane-bound organelles responsible for several anabolic and catabolic reactions.
      • Waterham H.R.
      • Ferdinandusse S.
      • Wanders R.J.
      Human disorders of peroxisome metabolism and biogenesis.
      The most well-characterized peroxisomal pathways are those used in clinical evaluation, including β-oxidation of very long–chain fatty acids (VLCFAs), α-oxidation of methyl-branched phytanic acid, and the synthesis of mature bile acids (BAs) and plasmalogens
      • Waterham H.R.
      • Ferdinandusse S.
      • Wanders R.J.
      Human disorders of peroxisome metabolism and biogenesis.
      (Fig. 1 Supplemental).
      VLCFAs, defined as having a carbon chain with 22 carbons or more, are “tethered” to the peroxisome membrane by ACBD5
      • Ferdinandusse S.
      • Falkenberg K.D.
      • Koster J.
      • et al.
      ACBD5 deficiency causes a defect in peroxisomal verylong-chain fatty acid metabolism.
      and transported into the peroxisome by the integral peroxisome membrane protein ABCD1.
      • Mosser J.
      • Douar A.M.
      • Sarde C.O.
      • et al.
      Putative X-linked adrenoleukodystrophy gene shares unexpected homology with ABC transporters.
      Once in the peroxisome, fatty acid shortening occurs by a mechanism similar to mitochondrial β-oxidation although producing H2O2, which is further catabolized into water and oxygen by peroxisomal catalase.
      • Wen J.K.
      • Osumi T.
      • Hashimoto T.
      • Ogata M.
      Molecular analysis of human acatalasemia. Identification of a splicing mutation.
      Phytanic acid and C27-bile acid intermediates, dihydroxycholestanoic acid (DHCA) and trihydroxycholestanoic acid(THCA), require auxiliary modifications before they can undergo β-oxidation.
      • Waterham H.R.
      • Ferdinandusse S.
      • Wanders R.J.
      Human disorders of peroxisome metabolism and biogenesis.
      ,
      • Vaz F.M.
      • Ferdinandusse S.
      Bile acid analysis in human disorders of bile acid biosynthesis.
      These substrates enter the peroxisome by ABCD3.
      • Ferdinandusse S.
      • Jimenez-Sanchez G.
      • Koster J.
      • et al.
      A novel bile acid biosynthesis defect due to adeficiency of peroxisomal ABCD3.
      Phytanoyl-CoA requires α-oxidation byphytanoyl-CoA hydroxylase (PhyH);
      • Mihalik S.J.
      • Morrell J.C.
      • Kim D.
      • Sacksteder K.A.
      • Watkins P.A.
      • Gould S.J.
      Identification of PAHX, a Refsum diseasegene.
      the resulting pristanoyl-CoA, as well as DHCA and THCA-CoA derivatives, require racemization from R to S forms by α-methylacyl-CoA racemase (AMACR).
      • Waterham H.R.
      • Ferdinandusse S.
      • Wanders R.J.
      Human disorders of peroxisome metabolism and biogenesis.
      ,
      • Vaz F.M.
      • Ferdinandusse S.
      Bile acid analysis in human disorders of bile acid biosynthesis.
      ,
      • Ferdinandusse S.
      • Denis S.
      • Clayton P.T.
      • et al.
      Mutations in the gene encoding peroxisomal alpha-methylacyl-CoA racemase cause adult-onset sensory motor neuropathy.
      The first step in peroxisomal β-oxidation utilizes an oxidase: acyl-CoA oxidase (ACOX) 1
      • Fournier B.
      • Saudubray J.M.
      • Benichou B.
      • et al.
      Large deletion of the peroxisomal acyl-CoA oxidase gene in pseudoneonatal adrenoleukodystrophy.
      for straight-chain fatty acids, ACOX2
      • Vaz F.M.
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      Bile acid analysis in human disorders of bile acid biosynthesis.
      ,
      • Ferdinandusse S.
      • Denis S.
      • van Roermund C.W.T.
      • et al.
      A novel case of ACOX2 deficiency leads to recognition of a third human peroxisomal acyl-CoA oxidase.
      for bile acids, and ACOX2 and ACOX3 for pristanic acid. The next steps, hydration and dehydrogenation, are accomplished by the bifunctional enzyme (DBP),
      • Ferdinandusse S.
      • van Grunsven E.G.
      • Oostheim W.
      • et al.
      Reinvestigation of peroxisomal 3-ketoacyl-CoA thiolase deficiency: identification of the true defect at the level of d-bifunctional protein.
      followed by thiolysis (Sterol Carrier Protein X [SCPx]
      • Ferdinandusse S.
      • Kostopoulos P.
      • Denis S.
      • et al.
      Mutations in the gene encoding peroxisomal sterol carrier protein X (SCPx) cause leukencephalopathy with dystonia and motor neuropathy.
      or ACAA1.
      • Vaz F.M.
      • Ferdinandusse S.
      Bile acid analysis in human disorders of bile acid biosynthesis.
      ) After several β-oxidation cycles, fatty acids shortened to approximately 16 carbons exit the peroxisome as carnitine derivatives. The C27-bile acid intermediates, DHCA and THCA, and the docosahexaenoic acid (DHA) precursor (C24:6n-3) undergo only one cycle of β-oxidation, which results in the production of the C24 mature bile acids, 3α,7α,12α-trihydroxy-5β-cholanoic acid (CA) and 3α,7α-dihydroxy-5β-cholanoic acid (CDCA)
      • Vaz F.M.
      • Ferdinandusse S.
      Bile acid analysis in human disorders of bile acid biosynthesis.
      and DHA (C22:6n-3).
      • Su H.M.
      • Moser A.B.
      • Moser H.W.
      • Watkins P.A.
      Peroxisomal straight-chain Acyl-CoA oxidase and D-bifunctional protein are essential for the retroconversion step in docosahexaenoic acid synthesis.
      Subsequently, CA and CDCA are conjugated to glycine or taurine by the bile acid-CoA:amino acid N-acyltransferase (BAAT).
      • Carlton V.E.
      • Harris B.Z.
      • Puffenberger E.G.
      • et al.
      Complex inheritance of familial hypercholanemia with associated mutations in TJP2 and BAAT.
      An additional oxidative pathway occurring in peroxisomes is the oxidation of L-pipecolic acid, an intermediate of lysine catabolism, to Δ1-piperidine-6-carboxylate (P6C).
      • Fujita T.
      • Hada T.
      • Higashino K.
      Origin of D- and L-pipecolic acid in human physiological fluids: a study of the catabolic mechanism to pipecolic acid using the lysine loading test.
      The main pathway for lysine degradation occurs in liver mitochondria with conversion to saccharopine, and then to α-aminoadipic semialdehyde (AASA).
      • Sacksteder K.A.
      • Biery B.J.
      • Morrell J.C.
      • et al.
      Identification of the alpha-aminoadipic semialdehyde synthase gene, which is defective in familial hyperlysinemia.
      The two pathways converge on AASA, since the P6C ring spontaneously opens forming AASA. AASA is a substrate for the enzyme AASA dehydrogenase, encoded by ALDH7A1and found both in the cytosol and within mitochondria. Pathogenic variants inALDH7A1 cause pyridoxine-dependentepilepsy (PDE; OMIM 266100),
      • Plecko B.
      • Paul K.
      • Paschke E.
      • et al.
      Biochemical and molecular characterization of 18 patients with pyridoxine-dependent epilepsy and mutations of the antiquitin (ALDH7A1) gene.
      ,
      • Mills P.B.
      • Struys E.
      • Jakobs C.
      • et al.
      Mutations in antiquitin in individuals with pyridoxine-dependent seizures.
      an infantile epilepsy unresponsive to standard anticonvulsants.
      Peroxisomes are also responsible for the initial steps of plasmalogen biosynthesis. Plasmalogens are a subset of glycerophospholipids distinguished by the presence of a vinyl-ether bond at the sn-1 position and enriched polyunsaturated fattyacids at the sn-2 position. They are abundantin cellular and subcellular membranes of many tissues. Synthesis of plasmalogens begins with two enzymes associated with the peroxisomal luminal membrane, dihydroxyacetonephosphate acyltransferase (DHAPAT)
      • Ofman R.
      • Hettema E.H.
      • Hogenhout E.M.
      • Caruso U.
      • Muijsers A.O.
      • Wanders R.J.
      Acyl-CoA:dihydroxyacetonephosphate acyltransferase: cloning of the human cDNA and resolution of the molecular basis in rhizomelic chondrodysplasia punctata type 2.
      and alkyl-dihydroxyacetonephosphate synthase (AGPS).
      • de Vet E.C.
      • Ijlst L.
      • Oostheim W.
      • Wanders R.J.
      • van den Bosch H.
      Alkyl-dihydroxyacetonephosphate synthase. Fate in peroxisome biogenesis disorders and identification of the point mutation underlying a single enzyme deficiency.
      In forming avinyl-ether bond, AGPS utilizes fatty alcohols made by fatty acyl-CoA reductase (FAR1)
      • Buchert R.
      • Tawamie H.
      • Smith C.
      • et al.
      A peroxisomal disorder of severe intellectual disability, epilepsy, and cataracts due to fatty acyl-CoA reductase 1 deficiency.
      from fatty acyl-CoAs specifically of chain-lengths C16:0, C18:0, and C18:1. FAR1 is a membrane protein located on the cytoplasmic surface of the peroxisome membrane; it displays feedback inhibitionto cellular plasmalogen levels and thus regulates this pathway. The 1-alkyl-DHAP product, generated from the reaction catalyzed by AGPS, is reduced toalkyl-glycerophosphate by an acyl/alkyl-DHP reductase. Further modifications occur in the endoplasmic reticulum to generate a mature vinyl-etherglycerophospholipid (plasmalogen).
      Lastly, glyoxylate detoxification to glycine by the enzymealanine-glyoxylate aminotransferase (AGT) takes place predominantly in liverperoxisomes. Deficiencies in AGT cause the kidney condition primaryhyperoxaluria type 1 (OMIM 259900).
      • Nishiyama K.
      • Funai T.
      • Katafuchi R.
      • Hattori F.
      • Onoyama K.
      • Ichiyama A.
      Primary hyperoxaluria type I due to a point mutation of T to C in the coding region of the serine:pyruvate aminotransferase gene.

      Peroxisome biogenesis

      Peroxisomes are versatile compartments that can increase their capacity in response to the metabolic needs of the cell. Peroxisome biogenesis refers to the formation of new peroxisome membranes, division of existing peroxisomes, and enzyme import into the peroxisomematrix.
      • Waterham H.R.
      • Ferdinandusse S.
      • Wanders R.J.
      Human disorders of peroxisome metabolism and biogenesis.
      ,
      • Steinberg S.J.
      • Dodt G.
      • Raymond G.V.
      • Braverman N.E.
      • Moser A.B.
      • Moser H.W.
      Peroxisome biogenesis disorders.
      These processes require the coordinated action of 16 PEX proteins, or peroxins, encoded by their cognate PEX genes (Table 1 Supplemental). Peroxisomal enzymes are synthesized on cytosolic ribosomes and contain one of two peroxisome-targeting sequences (PTS). The majority of enzymes contain the targeting sequence PTS1, recognized by PEX5 receptor.
      • Dodt G.
      • Braverman N.
      • Wong C.
      • et al.
      Mutations in the PTS1 receptor gene, PXR1, define complementation group 2 of the peroxisome biogenesis disorders.
      ,
      • Baroy T.
      • Koster J.
      • Stromme P.
      • et al.
      A novel type of rhizomelic chondrodysplasia punctata, RCDP5, is caused by loss of the PEX5 long isoform.
      A few enzymes, notably ACAA1, PhyH,
      • Mihalik S.J.
      • Morrell J.C.
      • Kim D.
      • Sacksteder K.A.
      • Watkins P.A.
      • Gould S.J.
      Identification of PAHX, a Refsum diseasegene.
      and AGPS,
      • de Vet E.C.
      • Ijlst L.
      • Oostheim W.
      • Wanders R.J.
      • van den Bosch H.
      Alkyl-dihydroxyacetonephosphate synthase. Fate in peroxisome biogenesis disorders and identification of the point mutation underlying a single enzyme deficiency.
      contain a PTS2 sequence recognized by PEX7 receptor.
      • Braverman N.
      • Steel G.
      • Obie C.
      • et al.
      Human PEX7 encodes the peroxisomal PTS2 receptor and is responsible for rhizomelic chondrodysplasia punctata.
      PEX5 and PEX7 receptors bind to their respective PTS1 or PTS2 targeted enzymes in the cytosol; in addition, PEX7 binds the PEX5 long isoform (PEX5L),
      • Baroy T.
      • Koster J.
      • Stromme P.
      • et al.
      A novel type of rhizomelic chondrodysplasia punctata, RCDP5, is caused by loss of the PEX5 long isoform.
      which is critical for PEX7-mediated import of PTS2-tagged proteins.
      The receptor–enzyme complexes dock at the peroxisome membranes using PEX13
      • Shimozawa N.
      • Suzuki Y.
      • Zhang Z.
      • et al.
      Nonsense and temperature-sensitive mutations in PEX13 are the cause of complementation group H of peroxisome biogenesis disorders.
      ,
      • Liu Y.
      • Bjorkman J.
      • Urquhart A.
      • Wanders R.J.
      • Crane D.I.
      • Gould S.J.
      PEX13 is mutated in complementation group 13 of theperoxisome-biogenesis disorders.
      and PEX14
      • Shimozawa N.
      • Tsukamoto T.
      • Nagase T.
      • et al.
      Identification of a new complementation group of the peroxisome biogenesis disorders and PEX14 as the mutated gene.
      (importomer complex)and release their cargo enzymes into the matrix. Three integral membrane proteins PEX2,
      • Shimozawa N.
      • Tsukamoto T.
      • Suzuki Y.
      • et al.
      A human gene responsible for Zellweger syndrome that affects peroxisome assembly.
      PEX10,
      • Warren D.S.
      • Morrell J.C.
      • Moser H.W.
      • Valle D.
      • Gould S.J.
      Identification of PEX10, the gene defective in complementation group 7 of the peroxisome-biogenesis disorders.
      and PEX12
      • Chang C.C.
      • Lee W.H.
      • Moser H.
      • Valle D.
      • Gould S.J.
      Isolation of the human PEX12 gene, mutated in group 3 of the peroxisome biogenesis disorders.
      participate in monoubiquitination of PEX5, which allows PEX5 (presumably with PEX7) to be recycled into the cytosol by PEX1–PEX6–PEX26
      • Reuber B.E.
      • Germain-Lee E.
      • Collins C.S.
      • et al.
      Mutations in PEX1 are the most common cause of peroxisome biogenesis disorders.
      • Fukuda S.
      • Shimozawa N.
      • Suzuki Y.
      • et al.
      Human peroxisome assembly factor-2 (PAF-2): a gene responsible for group C peroxisome biogenesis disorder in humans.
      • Yahraus T.
      • Braverman N.
      • Dodt G.
      • et al.
      The peroxisome biogenesis disorder group 4 gene, PXAAA1, encodes a cytoplasmic ATPase required for stability of the PTS1 receptor.
      • Matsumoto N.
      • Tamura S.
      • Furuki S.
      • et al.
      Mutations in novel peroxin gene PEX26 that cause peroxisome-biogenesis disorders of complementation group 8 provide agenotype-phenotype correlation.
      (exportomer complex) for another round of import. PEX3, PEX16,
      • Honsho M.
      • Tamura S.
      • Shimozawa N.
      • Suzuki Y.
      • Kondo N.
      • Fujiki Y.
      Mutation in PEX16 is causal in the peroxisome-deficient Zellweger syndrome of complementation group D.
      and PEX19
      • Matsuzono Y.
      • Kinoshita N.
      • Tamura S.
      • et al.
      Human PEX19: cDNA cloning by functional complementation, mutation analysis in a patient with Zellweger syndrome, and potential role in peroxisomal membrane assembly.
      are required to form new peroxisome membranes from the endoplasmic reticulum and from mitochondrial membranes.
      • Sugiura A.
      • Mattie S.
      • Prudent J.
      • McBride H.M.
      Newly born peroxisomes are a hybrid of mitochondrial and ER-derived pre-peroxisomes.
      PEX11 (there are 3 isoforms, PEX11 alpha, beta,
      • Ebberink M.S.
      • Koster J.
      • Visser G.
      • et al.
      A novel defect of peroxisome division due to ahomozygous non-sense mutation in the PEX11beta gene.
      and gamma) is required for fission of existing peroxisomes, a process requiring mitochondrial fission proteins, MFF, FIS1, and DLP1
      • Waterham H.R.
      • Koster J.
      • van Roermund C.W.
      • Mooyer P.A.
      • Wanders R.J.
      • Leonard J.V.
      A lethal defect of mitochondrial and peroxisomal fission.
      (DNM1L).

      Clinical description of peroxisomal disorders

      Peroxisomal disorders are a heterogeneous group of inherited diseases due to defects in peroxisomal biogenesis (PBD, Table 1 Supplemental) or single peroxisomal proteins (SPPD; Table 2 Supplemental). PBDs are divided into two groups: Zellweger spectrum disorders (ZSD) and rhizomelic chondrodysplasia punctata (RCDP). ZSDs are a highly heterogeneous group of disorders due to pathogenic variants in one of several PEX genes (Table 1 Supplemental). Core features of ZSDs include liver dysfunction, neurological abnormalities including developmental delays, adrenocortical dysfunction, and vision and hearing impairment.

      Steinberg SJ, Raymond GV. Braverman NE, Moser AB. Zellweger spectrum disorder. In: Adam MP, Ardinger HH, Pagon RA et al., editors. GeneReviews. Seattle, WA: University of Washington; 1993–2017.

      ,
      • Braverman N.E.
      • Raymond G.V.
      • Rizzo W.B.
      • et al.
      Peroxisome biogenesis disorders in the Zellweger spectrum: an overview of current diagnosis, clinical manifestations, and treatment guidelines.
      RCDP is characterized by proximal shortening of the long bones, a distinctive facial appearance, cataracts, congenital contractures, and intellectual disability. Punctate calcification within the epiphyses of long bones (chondrodysplasia punctata) is apparent on radiographs, particularly in infancy.
      • Wanders R.J.
      • Waterham H.R.
      Peroxisomal disorders I: biochemistry and genetics of peroxisome biogenesis disorders.
      X-linked adrenoleukodystrophy (X-ALD), caused by pathogenic variants in ATP-binding cassette transporter type D1 (ABCD1),
      • Mosser J.
      • Douar A.M.
      • Sarde C.O.
      • et al.
      Putative X-linked adrenoleukodystrophy gene shares unexpected homology with ABC transporters.
      typically presents with one of threephenotypes:
      • Moser H.W.
      • Mahmood A.
      • Raymond G.V.
      X-linked adrenoleukodystrophy.
      (1) childhood cerebral form with progressive neurological impairment (cognition, behavior, vision, hearing, motor function) and adrenal insufficiency; (2) late-adolescent or early-adulthood adrenomyeloneuropathy (AMN) with progressive paraparesis and adrenocortical dysfunction; (3) isolated adrenal insufficiency at any age. Approximately 20–50% of adult females with an ABCD1 variant may develop symptoms of AMN. Frequently, X-ALD is misdiagnosed as attention deficit–hyperactivity disorder (ADHD), multiple sclerosis, or idiopathic adrenal insufficiency. Other SPPDs are less common than X-ALD (Table 2 Supplemental). While some features are common to patients with RCDP, ZSDs, or X-ALD, others are not. For example, ulcerating oral gangrene is typically observed in patients with catalase deficiency. Accumulation of calcium oxalate in the kidney progressing to renal failure is common in patients with hyperoxalurias, but also described in ZSD patients. Retinitis pigmentosa, peripheral neuropathy, cerebellar ataxia, and elevated protein levels in the cerebrospinal fluid are seen in Refsum disease (neurological and visual abnormalities are also seen in ZSD patients). Adult-onset neurodegeneration is present in AMACR deficiency. Elevated serum BA concentrations, itching, and fat malabsorption are seen in familial hypercholanemia. Encephalopathy, developmental delay, and hypotonia are present in patients with encephalopathy due to defective mitochondrial peroxisomalfission 1 (EMPF1).

      Incidence

      Collectively, peroxisomal disorders have an estimated combined incidence of 1:5,000 individuals.
      • Waterham H.R.
      • Ferdinandusse S.
      • Wanders R.J.
      Human disorders of peroxisome metabolism and biogenesis.
      The most common SPPD is X-ALD
      • Mosser J.
      • Douar A.M.
      • Sarde C.O.
      • et al.
      Putative X-linked adrenoleukodystrophy gene shares unexpected homology with ABC transporters.
      ,
      • Moser H.W.
      • Mahmood A.
      • Raymond G.V.
      X-linked adrenoleukodystrophy.
      with an estimated incidence of 1:17,000.
      • Waterham H.R.
      • Ferdinandusse S.
      • Wanders R.J.
      Human disorders of peroxisome metabolism and biogenesis.
      The most common PBDs are ZSDs,
      • Braverman N.E.
      • Raymond G.V.
      • Rizzo W.B.
      • et al.
      Peroxisome biogenesis disorders in the Zellweger spectrum: an overview of current diagnosis, clinical manifestations, and treatment guidelines.
      with an estimated frequency of 1:50,000 in North America.
      • Steinberg S.J.
      • Dodt G.
      • Raymond G.V.
      • Braverman N.E.
      • Moser A.B.
      • Moser H.W.
      Peroxisome biogenesis disorders.
      Although ZSDs are pan-ethnic, the incidence varies by region, ranging from 1:12,000 in the French Canadian region of Quebec
      • Levesque S.
      • Morin C.
      • Guay S.P.
      • et al.
      A founder mutation in the PEX6 gene is responsible for increased incidence of Zellweger syndrome in a French Canadian population.
      to 1:500,000 in Japan.
      • Klouwer F.C.
      • Berendse K.
      • Ferdinandusse S.
      • Wanders R.J.
      • Engelen M.
      • Poll-The B.T.
      Zellweger spectrum disorders: clinical overview and management approach.

      Mode of inheritance

      Most PBDs and SPPDs are autosomal recessive (AR) conditions. The most notable exception to this is X-ALD, which is X-linked; hemizygous males will develop a clinical phenotype and heterozygous females may develop symptomsover time. Other exceptions include autosomal dominant (AD) de novo variants inDLP1 in patients with EMPF1
      • Waterham H.R.
      • Koster J.
      • van Roermund C.W.
      • Mooyer P.A.
      • Wanders R.J.
      • Leonard J.V.
      A lethal defect of mitochondrial and peroxisomal fission.
      and a recently described autosomal dominant form of ZSD due to allelic imbalance for the PEX6-p.Arg860Trp allele(NM_000287.4:c.2578C>T).
      • Falkenberg K.D.
      • Braverman N.E.
      • Moser A.B.
      • et al.
      Allelic expression imbalance promoting a mutant PEX6 allele causes Zellweger spectrum disorder.

      Peroxisomal disorders treatment

      Several symptomatic or supportive interventions are available to treat peroxisomal disorders, such as feeding tubes to ensure adequate nutrition, eye glasses and hearing aids, antiepileptic medications, and treatment for nephrolithiasis.

      Steinberg SJ, Raymond GV. Braverman NE, Moser AB. Zellweger spectrum disorder. In: Adam MP, Ardinger HH, Pagon RA et al., editors. GeneReviews. Seattle, WA: University of Washington; 1993–2017.

      ,
      • Braverman N.E.
      • Raymond G.V.
      • Rizzo W.B.
      • et al.
      Peroxisome biogenesis disorders in the Zellweger spectrum: an overview of current diagnosis, clinical manifestations, and treatment guidelines.
      ,
      • Wanders R.J.A.
      Peroxisomal disorders: improved laboratory diagnosis, new defects and the complicated route to treatment.
      Adrenal hormone replacement for those with adrenal insufficiency is lifesaving. Specific interventions are available for selected diagnoses. Prevention of oxalate calculi with urine alkalinization and hydration preserves renal function in patients with hyperoxaluria type I;
      • Wanders R.J.A.
      Peroxisomal disorders: improved laboratory diagnosis, new defects and the complicated route to treatment.
      clinical trials using RNA-mediated drugs are ongoing (Alnylam Pharmaceuticals 2016, NCT02706886). In patients with Refsum disease, dietary intake of phytanic acid is restricted to reduce accumulation, and fasting is avoided to prevent its mobilization from stored fat.

      Wanders RJA, Waterham HR. Leroy BP. Refsum disease. In: Adam MP, Ardinger HH, Pagon RA et al., editors. GeneReviews. Seattle, WA: University of Washington; 1993.

      Cholic acid therapy has been used in specific disorders of bile acid synthesis, such as AMACR deficiency, to reduce bile precursor accumulation and normalize liver function.
      • Wanders R.J.A.
      Peroxisomal disorders: improved laboratory diagnosis, new defects and the complicated route to treatment.
      Cholic acid therapy reduces the production of bile acid intermediates in patients with ZSD; however, its use may be limited due to possible hepatotoxicity.
      • Berendse K.
      • Klouwer F.C.
      • Koot B.G.
      • et al.
      Cholic acid therapy in Zellweger spectrum disorders.
      ,

      Klouwer FCC, Koot BGP, Berendse K, et al. The cholic acid extension study in Zellweger spectrum disorders: results and implications for therapy.2019;42:303–312.

      Patients with X-ALD and cerebral involvement can benefit from hematopoietic stem cell transplantation (HSCT) if performed prior to significant involvement.
      • Miller W.P.
      • Rothman S.M.
      • Nascene D.
      • et al.
      Outcomes after allogeneic hematopoietic cell transplantation for childhood cerebral adrenoleukodystrophy: the largest single-institution cohort report.
      ,
      • Kuhl J.S.
      • Suarez F.
      • Gillett G.T.
      • et al.
      Long-term outcomes of allogeneic haematopoietic stem cell transplantation for adult cerebral X-linked adrenoleukodystrophy.
      HSCT is associated with up to a 20% mortality rate due to transplant-related complications.
      • Miller W.P.
      • Rothman S.M.
      • Nascene D.
      • et al.
      Outcomes after allogeneic hematopoietic cell transplantation for childhood cerebral adrenoleukodystrophy: the largest single-institution cohort report.
      ,
      • Kuhl J.S.
      • Suarez F.
      • Gillett G.T.
      • et al.
      Long-term outcomes of allogeneic haematopoietic stem cell transplantation for adult cerebral X-linked adrenoleukodystrophy.
      Early results of the STARBEAM study suggest that transplantation with autologous hematopoietic stem cells transfected with elivaldogene tavalentivec (Lenti-D) lentiviral vector is as effective as conventional allogeneic transplantation.
      • Eichler F.
      • Duncan C.
      • Musolino P.L.
      • et al.
      Hematopoietic stem-cell gene therapy for cerebral adrenoleukodystrophy.
      Currently, there is notargeted therapy for AMN.

      Newborn screening for peroxisomal disorders

      In February 2016, the US Secretary of Health and Human Services approved the inclusion of X-ALD in the Recommended Uniform Screening Panel (RUSP) for state newborn screening (NBS) programs.
      • Kemper A.R.
      • Brosco J.
      • Comeau A.M.
      • et al.
      Newborn screening for X-linked adrenoleukodystrophy: evidence summary and advisory committee recommendation.
      Attempts to screen for this condition by analyzing VLCFAs in dried blood spots (DBS) were carried out in the late 1980s and early 1990s; however, cumbersome extraction procedures, long run times, high false positive rates, and the discovery of endogenous VLCFAs in filter paper used for NBS made analysis of VLCFAs in DBS unreliable.
      • Jakobs C.
      • van den Heuvel C.M.
      • Stellaard F.
      • Largilliere C.
      • Skovby F.
      • Christensen E.
      Diagnosis of Zellweger syndrome by analysis of very long-chain fatty acids in stored blood spots collected at neonatal screening.
      For these reasons, other lipid fractions were evaluated and the lysophosphatidylcholine (LPC) species were found not only to be elevated in DBS of patients with X-ALD, but could be measured accurately and efficiently using standard NBS technology.
      • Hubbard W.C.
      • Moser A.B.
      • Tortorelli S.
      • Liu A.
      • Jones D.
      • Moser H.
      Combined liquid chromatography-tandem mass spectrometry as an analytical method for high throughput screening for X-linked adrenoleukodystrophy and other peroxisomal disorders: preliminary findings.
      Current NBS protocols utilize measurement of hexacosanoyl-lysophatidylcholine (C26:0 LPC); other markers, such as the ratio of C26:0 LPC to C20:0 LPC, and hexacosanoylcarnitine (C26-carnitine) may also be useful. New York State began screening for X-ALD at the end of 2013; its experience, together with data on the efficacy of early treatment, was instrumental in obtaining RUSP approval.
      • Kemper A.R.
      • Brosco J.
      • Comeau A.M.
      • et al.
      Newborn screening for X-linked adrenoleukodystrophy: evidence summary and advisory committee recommendation.
      To date, several methods are available to measure C20:0-C26:0 LPCs, either alone or incombination with other analytes,
      • Hubbard W.C.
      • Moser A.B.
      • Liu A.C.
      • et al.
      Newborn screening for X-linked adrenoleukodystrophy (X-ALD): validation of a combined liquid chromatography-tandem massspectrometric (LC-MS/MS) method.
      • Haynes C.A.
      • De Jesus V.R.
      Improved analysis of C26:0-lysophosphatidylcholine in dried-blood spots via negative ion mode HPLC-ESI-MS/MS for X-linked adrenoleukodystrophy newborn screening.
      • Sandlers Y.
      • Moser A.B.
      • Hubbard W.C.
      • Kratz L.E.
      • Jones R.O.
      • Raymond G.V.
      Combined extraction of acyl carnitines and 26:0 lysophosphatidylcholine from dried blood spots: prospective newborn screening for X-linked adrenoleukodystrophy.
      • Turgeon C.T.
      • Moser A.B.
      • Morkrid L.
      • et al.
      Streamlined determination of lysophosphatidylcholines in dried blood spots for newborn screening of X-linked adrenoleukodystrophy.
      • Tortorelli S.
      • Turgeon C.T.
      • Gavrilov D.K.
      • et al.
      Simultaneous testing for 6 lysosomal storagedisorders and X-adrenoleukodystrophy in dried blood spots by tandem mass spectrometry.
      • Haynes C.A.
      • De Jesus V.R.
      Simultaneous quantitation of hexacosanoyl lysophosphatidylcholine, amino acids, acylcarnitines, and succinylacetone during FIA-ESI-MS/MS analysis of dried blood spot extracts for newborn screening.
      • Hong X.
      • Kumar A.B.
      • Ronald Scott C.
      • Gelb M.H.
      Multiplex tandem mass spectrometry assay for newborn screening of X-linked adrenoleukodystrophy, biotinidase deficiency, andgalactosemia with flexibility to assay other enzyme assays and biomarkers.
      and additional states have enacted X-ALDscreening (for a list of states, refer to www.babysfirsttest.org). Guidelines on follow-up evaluation of a positive screen are available.
      • Vogel B.H.
      • Bradley S.E.
      • Adams D.J.
      • et al.
      Newborn screening for X-linked adrenoleukodystrophyin New York State: diagnostic protocol, surveillance protocol and treatment guidelines.
      ,
      • Regelmann M.O.
      • Kamboj M.K.
      • Miller B.S.
      • et al.
      Adrenoleukodystrophy: guidance for adrenal surveillance in males identified by newborn screen.
      Although NBS was designed to identify newborns with X-ALD, LPCs may detect other conditions, such as ZSDs, and ACOX1 and DBP deficiencies. In addition, some neonates with Aicardi Goutières syndromehave elevated levels of C26:0 LPC.
      • Armangue T.
      • Orsini J.J.
      • Takanohashi A.
      • et al.
      Neonatal detection of Aicardi Goutieres syndrome by increased C26:0 lysophosphatidylcholine and interferon signature on newborn screening blood spots.

      Methods

      The laboratory technical standard was informed by a review of the literature, including current guidelines, and expert opinion. Resources consulted included PubMed (search terms: peroxisomes; peroxisome biogenesis; peroxisomal disorders [AND newborn screening]; very long–chain fatty acids [AND liquid chromatography OR gas chromatography]; plasmalogen [AND liquid chromatography OR gaschromatography]; pipecolic acid [AND liquid chromatography OR gas chromatography]; bile acid synthesis [AND peroxisomal disorders]; X-linked adrenoleukodystrophy; Zellweger spectrum disorder; rhizomelic chondrodysplasia), the American College of Medical Genetics and Genomics (ACMG) Standards and Guidelines for Clinical Genetics Laboratories, Clinical and Laboratory Standards Institute (CLSI) guidelines, CLIA regulations, and the Centers for Disease Control and Prevention Morbidity and Mortality Weekly Report (MMWR) on Good Laboratory Practices for Biochemical Genetics Testing and Newborn Screening for Inherited Metabolic Disorders. When the literature provided conflicting evidence about a topic or when there was insufficient evidence,the authors used expert opinion to inform the recommendations. Expert opinion included the coauthors of the document, members of the ACMG Laboratory Quality Assurance Committee, as well as the experts consulted outside of the Committee but acknowledged in this document. The standard Human Genome Variation Society (HGVS) nomenclature
      • den Dunnen J.T.
      • Dalgleish R.
      • Maglott D.R.
      • et al.
      HGVS recommendations for the description of sequence variants: 2016 update.
      was used to describe the sequence variants cited in this document.
      Any conflicts of interest for workgroup members or consultants are listed. A draft was delivered to the ACMG Board of Directors for review and member comment. The draft document was posted on the ACMG website and an email link was sent inviting ACMG members to provide comment. The authors assessed all comments. When appropriate, additional evidence was included to address member comments and the draft was amended. Both member comments and author responses were reviewed by a representative of the ACMG Laboratory Quality Assurance Committee and by the ACMG Board of Directors. The ACMG Board of Directors approved the final document before submission for publication.

      Preanalytical requirements

      Specimen requirements

      Laboratory testing for peroxisomal disorders includes different classes of metabolites that accumulate in different metabolic pathways. Hence, avariety of assays are utilized, each being routinely performed in a certain specimen type depending on analyte abundance, ease of collection, or other analytical and clinical considerations. In general, the laboratory should evaluate sample requirements and factors influencing its collection, handling, shipping, and storage at the time of method validation. Sample requirements mustbe made available to ordering physicians.
      Quantitative analysis of very long–chain fatty acids (VLCFAs) and branched-chain fatty acids (BCFAs; e.g., phytanic and pristanic acids) can be performed in plasma or serum
      • Takemoto Y.
      • Suzuki Y.
      • Horibe R.
      • Shimozawa N.
      • Wanders R.J.
      • Kondo N.
      Gas chromatography/mass spectrometry analysis of very long chain fatty acids, docosahexaenoic acid, phytanic acid and plasmalogen for the screening of peroxisomal disorders.
      separated from venous blood (2–5 mL) ideally within 1 hour from collection to avoid hemolysis; sodium or lithiumheparin (green top) or EDTA (lavender top) tubes can also beused.
      • Hall N.A.
      • Lynes G.W.
      • Hjelm N.M.
      Ratios for very-long-chain fatty acids in plasma of subjects with peroxisomal disorders, as determined by HPLC and validated bygas chromatography-mass spectrometry.
      Metabolite analysis, immunochemical or enzymatic assays can be performed on primary fibroblasts derived from a 2–3 mm skin biopsy under sterile conditions and cultured according to standard protocols. Free and conjugated bile acids (BA) are usually quantified in serumsamples; EDTA plasma and serum (red top) contain similar BA concentrations.
      • Scherer M.
      • Gnewuch C.
      • Schmitz G.
      • Liebisch G.
      Rapid quantification of bile acids and their conjugates in serum by liquid chromatography-tandem mass spectrometry.
      Although plasmalogens can be detected in plasma and serum, these specimens are less sensitive for distinguishing peroxisomal disease states from controls, due to their lower plasmalogen concentrations compared with erythrocytes and possibly due to the influence of diet on plasma/serum levels.
      • Takemoto Y.
      • Suzuki Y.
      • Horibe R.
      • Shimozawa N.
      • Wanders R.J.
      • Kondo N.
      Gas chromatography/mass spectrometry analysis of very long chain fatty acids, docosahexaenoic acid, phytanic acid and plasmalogen for the screening of peroxisomal disorders.
      ,
      • Bjorkhem I.
      • Sisfontes L.
      • Bostrom B.
      • Kase B.F.
      • Blomstrand R.
      Simple diagnosis of the Zellweger syndrome by gas-liquid chromatography of dimethylacetals.
      In general, erythrocytes are separated by centrifugation from 1–5 mL of EDTA anticoagulated blood, washed twice in phosphate-buffered saline to remove residual plasma and buffy coat, flushed with nitrogen, and stored frozen until processing.
      • Steinberg S.
      • Jones R.
      • Tiffany C.
      • Moser A.
      Investigational methods for peroxisomaldisorders.
      Pipecolic acid accumulates in several body fluids. For ease of collection, clinical testing is usually carried out in plasma (sodium or lithium heparin, EDTA, or ACD),
      • Yuzyuk T.
      • Liu A.
      • Thomas A.
      • et al.
      A novel method for simultaneous quantification of alpha-aminoadipic semialdehyde/piperideine-6-carboxylate and pipecolic acidin plasma and urine.
      serum,
      • van den Berg G.A.
      • Breukelman H.
      • Elzinga H.
      • Trijbels J.M.
      • Monnens L.A.
      • Muskiet F.A.
      Determination of pipecolic acid in urine and plasma by isotope dilution mass fragmentography.
      or urine. While pipecolic acid can be measured in cerebral spinal fluid (CSF), normal levels in this specimen type are very low, approximately tenfold less than plasma; thus, detection requires very sensitive methods.
      • Kok R.M.
      • Kaster L.
      • de Jong A.P.
      • Poll-The B.
      • Saudubray J.M.
      • Jakobs C.
      Stable isotope dilution analysis of pipecolic acid in cerebrospinal fluid, plasma, urine and amniotic fluid using electron capture negative ion mass fragmentography.
      Pipecolic acid is not consistently elevated in the urine of patients with PBDs,
      • van den Berg G.A.
      • Breukelman H.
      • Elzinga H.
      • Trijbels J.M.
      • Monnens L.A.
      • Muskiet F.A.
      Determination of pipecolic acid in urine and plasma by isotope dilution mass fragmentography.
      so plasma is preferred with one exception: in neonates, plasma pipecolic acid in affected individuals may be within normal limits, thus urine is recommended (unpublished data from the Peroxisomal Diseases Laboratory, Kennedy Krieger Institute). Pipecolic acid urinary excretion is measured using random urine, and is normalized to creatinine. Urine should be collected in a clean container without preservatives.
      • Yuzyuk T.
      • Liu A.
      • Thomas A.
      • et al.
      A novel method for simultaneous quantification of alpha-aminoadipic semialdehyde/piperideine-6-carboxylate and pipecolic acidin plasma and urine.
      ,
      • van den Berg G.A.
      • Breukelman H.
      • Elzinga H.
      • Trijbels J.M.
      • Monnens L.A.
      • Muskiet F.A.
      Determination of pipecolic acid in urine and plasma by isotope dilution mass fragmentography.
      Molecular genetic studies are typically performed using DNA isolated from leukocytes, usually using 1–2 mL EDTA whole blood. Some laboratories offer testing of RNA by complementary DNA (cDNA) sequencing. Other tissue sources, including cultured chorionic villus sample (CVS) or amniocytes for preimplantation/prenatal diagnosis, can be used if necessary. When nopathogenic variant(s) have been previously identified, biochemical testing in CVS or amniocytes can be used in at-risk pregnancies. When testing CVS oramniocytes, maternal cell contamination must beexcluded.
      • Steinberg S.
      • Katsanis S.
      • Moser A.
      • Cutting G.
      Biochemical analysis of cultured chorionic villi for the prenatal diagnosis of peroxisomal disorders: biochemical thresholds and molecular sensitivity for maternal cell contamination detection.

      Sample handling, shipping, and storage

      Plasma, urine, and CSF should be immediately frozen upon collection, and shipped on dry ice. Stability under different conditions varies among metabolites, and laboratories should establish proper handling, shipping, and storage conditions. VLCFAs and BCFAs are stable up to 15 days at room temperature (18–25 °C) or refrigerated (2–8 °C), and at least 92 days at ≤−20 °C(unpublished data from the Biochemical Genetics Laboratory at Mayo Clinic). Plasmalogens are stable in whole blood at room temperature (18–25 °C) or refrigerated (2–8 °C) for up to 72 hours. However, once erythrocytes have been isolated and washed, they should be stored under nitrogen at ≤−65 °C, where plasmalogens are stable for up to 12 months (unpublished data from the Peroxisomal Diseases Laboratory, Kennedy Krieger Institute). Pipecolic acid and the metabolites P6C and AASA are stable in plasma or urine up to 5 hours at room temperature (18–25 °C) and up to 24 hours refrigerated (2–8 °C); once samples are frozen, they are stable at least 40 days at ≤−20 °C and up to 1 year at ≤−65 °C.
      • Yuzyuk T.
      • Liu A.
      • Thomas A.
      • et al.
      A novel method for simultaneous quantification of alpha-aminoadipic semialdehyde/piperideine-6-carboxylate and pipecolic acidin plasma and urine.
      ,
      • Struys E.A.
      • Bok L.A.
      • Emal D.
      • Houterman S.
      • Willemsen M.A.
      • Jakobs C.
      The measurement of urinary Delta(1)-piperideine-6-carboxylate, the alter ego of alpha-aminoadipic semialdehyde, in Antiquitin deficiency.
      BAs are stable in EDTA plasma up to 24 hours at room temperature (18–25 °C), at least for several weeks once samples are frozen (≤−20 °C).
      • Scherer M.
      • Gnewuch C.
      • Schmitz G.
      • Liebisch G.
      Rapid quantification of bile acids and their conjugates in serum by liquid chromatography-tandem mass spectrometry.
      Cultured primary fibroblasts are shipped at room temperature (18–25 °C) in T-25 flasks filled with culture media. Whole blood for DNA extraction should be shipped at room temperature if delivery is within 24 hours or refrigerated if it takes longer (2–8 °C).

      Preanalytical variables

      In general, fasting or preprandial blood specimens are recommended for metabolite testing; the effect on metabolite concentrations following feeding has not been evaluated for all analytes and may differ among analytes. VLCFAs and BCFAs levels have been shown to increase slightly after a meal.
      • Moser H.W.
      • Moser A.B.
      • Frayer K.K.
      • et al.
      Adrenoleukodystrophy: increased plasma content of saturated very long chain fatty acids.
      BA concentrations also increase after a meal; the increase is modest in healthy individuals, but significant in patients with various liver conditions.
      • Heikkinen J.
      Effect of a standard test meal on serum bile acid levels in healthy nonpregnant and pregnant women and in patients with intrahepatic cholestasis of pregnancy.
      Other factors affect VLCFA and/or BCFA concentrations leading to an abnormal result: a ketogenic diet,
      • Stradomska T.J.
      • Bachanski M.
      • Pawlowska J.
      • Syczewska M.
      • Stolarczyk A.
      • Tylki-Szymanska A.
      The impact of a ketogenic diet and liver dysfunction on serum very long-chain fatty acids levels.
      high peanut butter consumption,
      • Lam C.
      • Wong D.
      • Cederbaum S.
      • Lim B.
      • Qu Y.
      Peanut consumption increases levels of plasma very long chain fatty acids in humans.
      severe liver disease, and hyperlipidemia. Hemolysis increases VLCFAs through release from erythrocyte membranes. Hence, plasma/serum should be separated within 24 hours from collection,
      • Steinberg S.
      • Jones R.
      • Tiffany C.
      • Moser A.
      Investigational methods for peroxisomaldisorders.
      ideally within the first hour, and conditions compromising blood integrity should be avoided. On the other hand, pipecolic acid levels in plasma/serum are not affected by hemolysis or hyperlipidemia,
      • Yuzyuk T.
      • Liu A.
      • Thomas A.
      • et al.
      A novel method for simultaneous quantification of alpha-aminoadipic semialdehyde/piperideine-6-carboxylate and pipecolic acidin plasma and urine.
      and there is no evidence of significant changes with postprandial collection.
      • Fujita T.
      • Hada T.
      • Higashino K.
      Origin of D- and L-pipecolic acid in human physiological fluids: a study of the catabolic mechanism to pipecolic acid using the lysine loading test.
      Hemolyzed blood samples are not optimal for plasmalogen analysis, as loss of erythrocytes will artificially lower absolute levels of plasmalogens measured as C16:0 and C18:0 dimethyl acetals (DMAs). Specimens demonstrating moderate to severe hemolysis should be rejected; erythrocytes collected froms lightly hemolyzed blood may be used if plasmalogen content is evaluated as a ratio to C16 and C18 fatty acids. Testing performed in blood collected following a blood transfusion could result in erroneously normal or partially reduced plasmalogen levels, as transfused erythrocytes can survive for up to 120 days.
      • Steinberg S.
      • Jones R.
      • Tiffany C.
      • Moser A.
      Investigational methods for peroxisomaldisorders.
      Results obtained in this scenario requires clinical correlation and reanalysis at least 4 weeks post-transfusion should be considered.

      Method validation

      Protocols should be established by each laboratory to determine and periodically verify performance characteristics of methods used in peroxisomal disorder testing, in accordance with CLIA and the College of American Pathologists (CAP) regulations (see CLSI document C24

      CLSI. Statistical quality control for quantitative measurement procedures: principles and definitions. 4th Ed. CLSI guideline C24. Wayne, PA: Clinical and Laboratory Standards Institute; 2016.

      ). The laboratory should implement procedures to address values outside of their established criteria for an assay’s performance.
      Gas chromatography–mass spectrometry (GC-MS) and tandem mass spectrometry (MS/MS), with or without liquid chromatography (LC), are routinely used to quantify VLCFAs/BCFAs, plasmalogens, pipecolic acid, and BAs. Quantitation withthese methods requires a calibration curve with purified synthetic standards, andisotope-labeled internal standards. With few exceptions (e.g., the metabolites P6Cand AASA), high-grade standards and internal standards are commercially available from several sources; however, standards may not be available for all components of a multianalyte panel. Reagent performance should be verified by the laboratory prior to clinical use.
      As for any analytical assay, quality control (QC) samples should be included with each batch of patient samples processed. Ideally, QC samples should be of the appropriate sample matrix and include two levels of quality control, typically normal and abnormal. Target ranges for QC samples should be established by analyzing replicates (at least 15–20) over several runs, possibly by multiple operators. Plasma or serum specimens from several normal individuals can be pooled and used as normal QC for metabolites assayed in plasma, serum, or CSF. Similarly, urine from a normal individual is used for metabolites assayed in urine. Often, normal plasma/serum or urine samples spiked with standards are used as abnormal QC, also referred to as “high control,” while the normal control can be also referred to as “low control.” Ideally, the abnormal QC is around 75% of the highest calibrator used, which usually corresponds to the upper limit of quantification, and within the range of values observed in patients. A readily available source of erythrocytes with normal plasmalogen levels can be obtained from expired blood purchased from the American Red Cross. Canine blood, which has low erythrocyte plasmalogen levels relative to human blood, is a potential source for an abnormal control and may be obtained from veterinary clinics.

      Proficiency testing

      Laboratories should participate in an ongoing proficiency testing(PT) program, at least semiannually, as required by regulating agencies. PT outcome should be carefully reviewed to monitor performance and identify results requiring investigation; any remedial action should be carefully documented. The European Research Network for Evaluation and Improvement of Screening, Diagnosis and Treatment of Inherited Disorders of Metabolism (ERNDIM) provides a commercially available, external proficiency testing service for the quantitative analysis of VLCFAs (C:22, C24:0, C26:0), phytanic and pristanicacids in serum, and L-pipecolic acid in serum and urine. Scheduled interlaboratory comparison (alternative PT) can be used when external proficiency testing is unavailable.

      Reference intervals

      Laboratory-specific reference intervals should be established for all analytes and periodically verified following guidelines (e.g., CLSI document EP28-A3c

      CLSI. Defining, establishing, and verifying reference intervals inthe clinical laboratory; approved guideline. 3rd Ed. CLSI document EP28-A3c. Wayne, PA: Clinical and Laboratory Standards Institute; 2008.

      ). Literature-based ranges should be verified using specific methods/platforms employed by the laboratory to validate the test. Anonymized samples from the general population are acceptable oncerelevant conditions affecting test results have been excluded; for some analytes, reference intervals are age-specific. If possible, the range of values observed in affected individuals should also be determined. Since there are often significant differences between sample types, reference intervals should be established or verified for each specimen type used for clinical testing.

      Testing for peroxisomal disorders

      Analysis of very long–chain fatty acids and branched-chain fatty acids in plasma

      Traditionally, the measurement of very long–chain fatty acids(VLCFAs) and branched-chain fatty acids (BCFAs) in plasma/serum was carried out by GC-MS.
      • Steinberg S.
      • Jones R.
      • Tiffany C.
      • Moser A.
      Investigational methods for peroxisomaldisorders.
      This method quantifies fatty acids containing hydrocarbon chains with 8 to 30 carbons, but only a subset is used for diagnosing peroxisomal disorders. The first step of sample preparation consists of hydrolysis of fatty acids bound to complex molecules, using an acid hydrolysis followed by basic hydrolysis and reacidification. After extractionwith hexane, the fatty acids are derivatized using pentafluorobenzyl bromide(PFBBr).
      • Steinberg S.
      • Jones R.
      • Tiffany C.
      • Moser A.
      Investigational methods for peroxisomaldisorders.
      Capillary GC-MS using negative chemicalionization with ammonia as the reagent gas and selected negative-ion monitoring allows separation and detection of the corresponding PFB-esters. Quantitation is achieved using stable isotope-labeled internal standards.
      • Steinberg S.
      • Jones R.
      • Tiffany C.
      • Moser A.
      Investigational methods for peroxisomaldisorders.
      VLCFA/BCFA quantitation by GC-MS requires a lengthy and laborious sample preparation (approximately 8 hours), and run time of approximately 30 minutes per sample. Several methods using positive-ion electrospray ionization tandem mass spectrometry (ESI-MS/MS)
      • Johnson D.W.
      A rapid screening procedure for the diagnosis of peroxisomal disorders: quantification of very long-chain fatty acids, asdimethylaminoethyl esters, in plasma and blood spots, by electrospray tandem mass spectrometry.
      • Johnson D.W.
      • Trinh M.U.
      • Oe T.
      Measurement of plasma pristanic, phytanic and verylong chain fatty acids by liquid chromatography-electrospray tandem mass spectrometry for the diagnosis of peroxisomal disorders.
      • Al-Dirbashi O.Y.
      • Santa T.
      • Rashed M.S.
      • et al.
      Rapid UPLC-MS/MS method for routine analysis of plasma pristanic, phytanic, and very long chain fatty acid markers of peroxisomal disorders.
      or negative-ion electrospray ionization mass spectrometry (ESI-MS)
      • Valianpour F.
      • Selhorst J.J.
      • van Lint L.E.
      • van Gennip A.H.
      • Wanders R.J.
      • Kemp S.
      Analysis of very long-chain fatty acids using electrospray ionization mass spectrometry.
      have been developed to simplify sample preparation and reduce run time. The ESI-MS/MS method originally developed by Johnson,
      • Johnson D.W.
      A rapid screening procedure for the diagnosis of peroxisomal disorders: quantification of very long-chain fatty acids, asdimethylaminoethyl esters, in plasma and blood spots, by electrospray tandem mass spectrometry.
      detecting fatty acids as dimethylaminoethyl ester derivatives, offered a rapid sample preparation (approximately 2 hours) and a short run time (3.5 minutes/sample). However, BCFAs cannot be distinguished from their straight-chain isomers with this method. Method limitations were resolved by the inclusion of a second derivatization step to trimethylaminoethyl ester derivative, and the use of liquid chromatography;
      • Johnson D.W.
      • Trinh M.U.
      • Oe T.
      Measurement of plasma pristanic, phytanic and verylong chain fatty acids by liquid chromatography-electrospray tandem mass spectrometry for the diagnosis of peroxisomal disorders.
      however, these changes resulted in sample preparation and run time comparable with GC-MS methods. The method developed by Al-Dirbashi et al. utilizes ultraperformance liquid chromatography(UPLC) and derivatization with 4-[2-(N,N-dimethylamino)ethylaminosulfonyl]-7-(2-aminoethylamino)-2,1,3-benzoxadiazole (DAABD-AE) to rapidly detect (5 minutes/sample) plasma BCFAs/VLCFAs after a relatively simple sample preparation (approximately3 hours).
      • Al-Dirbashi O.Y.
      • Santa T.
      • Rashed M.S.
      • et al.
      Rapid UPLC-MS/MS method for routine analysis of plasma pristanic, phytanic, and very long chain fatty acid markers of peroxisomal disorders.
      Overall, VLCFA/ BCFA concentrations are comparable among methods.
      • Al-Dirbashi O.Y.
      • Santa T.
      • Rashed M.S.
      • et al.
      Rapid UPLC-MS/MS method for routine analysis of plasma pristanic, phytanic, and very long chain fatty acid markers of peroxisomal disorders.
      VLCFAs/BCFAs analysis is a particularly useful biochemical test for the diagnosis of peroxisomal disorders, since abnormalities in these metabolitesare noted in the majority of patients. Elevations in hexacosanoic acid (C26:0)and the C26:0 to docosanoic acid (C22:0) ratio are typical of X-ALD patients. Some cases, especially adults, might also show an elevation in tetracosanoic acid (C24:0). The test is very sensitive in males, with almost all X-ALD or AMN patients presenting an abnormal profile. However, 15–20% of female heterozygotes have a normal profile, whether or not they are symptomatic.
      • Moser A.B.
      • Kreiter N.
      • Bezman L.
      • et al.
      Plasma very long chain fatty acids in 3,000 peroxisome disease patients and 29,000 controls.
      ,
      • Engelen M.
      • Barbier M.
      • Dijkstra I.M.
      • et al.
      X-linked adrenoleukodystrophy in women: a cross-sectional cohort study.
      Recently, C26:0 LPC in DBS has been shown tobe a more sensitive marker to identify X-ALD heterozygotes compared with C26:0-carnitine in plasma, being abnormal in all X-ALD heterozygotes, including those with normal plasma results.
      • Huffnagel I.C.
      • van de Beek M.C.
      • Showers A.L.
      • et al.
      Comparison of C26:0-carnitine andC26:0-lysophosphatidylcholine as diagnostic markers in dried blood spots from newborns and patients with adrenoleukodystrophy.
      The degree of the VLCFA abnormality does not correlate with age of onset or severity of phenotype,
      • Tran C.
      • Patel J.
      • Stacy H.
      • et al.
      Long-term outcome of patients with X-linkedadrenoleukodystrophy: a retrospective cohort study.
      and cannot be used as a predictor of clinical outcome. Abnormal results for this test are not specific to X-ALD/AMN; peroxisomal biogenesis disorders, ACOX1 deficiency, and DBP deficiency have a similar VLCFA profile but different clinical features and other biochemical abnormalities. Elevated phytanic acid accompanies the abnormal VLCA profile in ZSD and some DBP deficiencies. As a consequence of impaired α-oxidation, isolated elevations in phytanic acid are seen in Refsum disease, RCDP types 1and 5; however, the VLCFA profile is normal.
      • Mihalik S.J.
      • Morrell J.C.
      • Kim D.
      • Sacksteder K.A.
      • Watkins P.A.
      • Gould S.J.
      Identification of PAHX, a Refsum diseasegene.
      ,

      Wanders RJA, Waterham HR. Leroy BP. Refsum disease. In: Adam MP, Ardinger HH, Pagon RA et al., editors. GeneReviews. Seattle, WA: University of Washington; 1993.

      ,
      • Hoefler G.
      • Hoefler S.
      • Watkins P.A.
      • et al.
      Biochemical abnormalities in rhizomelic chondrodysplasia punctata.
      AMACR deficiency is characterized by elevation of BCFAs, in particular pristanic acid, and by an increased pristanicto phytanic acid ratio;
      • Ferdinandusse S.
      • Denis S.
      • Clayton P.T.
      • et al.
      Mutations in the gene encoding peroxisomal alpha-methylacyl-CoA racemase cause adult-onset sensory motor neuropathy.
      similar abnormalities can be seen in SCPx deficiency.
      • Ferdinandusse S.
      • Kostopoulos P.
      • Denis S.
      • et al.
      Mutations in the gene encoding peroxisomal sterol carrier protein X (SCPx) cause leukencephalopathy with dystonia and motor neuropathy.
      Since VLCFA levels do not change over time, age is not a significant factor in result interpretation.
      • Moser A.B.
      • Kreiter N.
      • Bezman L.
      • et al.
      Plasma very long chain fatty acids in 3,000 peroxisome disease patients and 29,000 controls.
      In contrast, BCFAs are low at birth, and increase with dietary intake of phytanic acid; hence, affected newborns may not show BCFA abnormalities. As noted previously, nonfasting and hemolyzed specimens and samples from individuals on a ketogenic diet can produce abnormal results; the pattern of abnormalities in different VLCFA and BCFA species can assist in differentiation from peroxisomal disorders; in particular, the C24/C22 ratio is less influenced by diet. A normal result in plasma does not exclude aperoxisomal disorder, as false negative results have been reported.
      • Rosewich H.
      • Waterham H.R.
      • Wanders R.J.
      • et al.
      Pitfall in metabolic screening in a patient with fatal peroxisomal beta-oxidation defect.
      ,
      • Lusebrink N.
      • Porto L.
      • Waterham H.R.
      • et al.
      Absence of biochemical evidence at an early age delays diagnosis in a patient with a clinically severe peroxisomal biogenesis disorder.

      Analysis of plasmalogens in red blood cells

      Historically, plasmalogen deficiency was identified by measuring plasmalogens as DMAs in erythrocytes.
      • Bjorkhem I.
      • Sisfontes L.
      • Bostrom B.
      • Kase B.F.
      • Blomstrand R.
      Simple diagnosis of the Zellweger syndrome by gas-liquid chromatography of dimethylacetals.
      Several analytical methods for measuring plasmalogen levels have been published and include gas–liquid chromatography (GC), GC-MS, and liquid chromatography/mass spectrometry (LC-MS) techniques.
      • Takemoto Y.
      • Suzuki Y.
      • Horibe R.
      • Shimozawa N.
      • Wanders R.J.
      • Kondo N.
      Gas chromatography/mass spectrometry analysis of very long chain fatty acids, docosahexaenoic acid, phytanic acid and plasmalogen for the screening of peroxisomal disorders.
      ,
      • Bjorkhem I.
      • Sisfontes L.
      • Bostrom B.
      • Kase B.F.
      • Blomstrand R.
      Simple diagnosis of the Zellweger syndrome by gas-liquid chromatography of dimethylacetals.
      ,
      • Moser A.B.
      • Jones D.S.
      • Raymond G.V.
      • Moser H.W.
      Plasma and red blood cell fatty acids in peroxisomal disorders.
      • Caruso U.
      Simple analysis of plasmalogens in erythrocytes using gas chromatography/mass spectrometry with selected-ion monitoring acquisition.
      • Zemski Berry K.A.
      • Murphy R.C.
      Electrospray ionization tandem mass spectrometry of glycerophosphoethanolamine plasmalogen phospholipids.
      • Hui S.P.
      • Chiba H.
      • Kurosawa T.
      Liquid chromatography-mass spectrometricdetermination of plasmalogens in human plasma.
      GC and GC-MS methodologies require solvent extraction of lipids and conversion of fatty acids to fatty acid methyl esters (FAME) and plasmalogens to DMA derivatives by acid methanolysis.
      • Steinberg S.
      • Jones R.
      • Tiffany C.
      • Moser A.
      Investigational methods for peroxisomaldisorders.
      DMA derivatives are separated by GC using a nonpolar capillary column and plasmalogen levels may be expressed as a ratio of C16:0 DMA and C18:0 DMA compared with their corresponding FAME or quantified using a nonendogenous free fatty acid of similar chain length (e.g.,C19:0 free fatty acid). Ratio analysis may have greater sensitivity, as samples from individuals with RCDP have a higher level of long-chain alcohols, which are subsequently converted to C16 fatty acid.
      • Rizzo W.B.
      • Craft D.A.
      • Judd L.L.
      • Moser H.W.
      • Moser A.B.
      Fatty alcohol accumulation in the autosomal recessive form of rhizomelic chondrodysplasia punctata.
      When GC alone is used, appropriate standards must be analyzed concurrently to confirm relative retention times for DMAs and FAMEs. GC-MS methods use selected-ion monitoring (SIM) mass spectrometry to increase assay specificity by utilizing mass assignments, m/z 255 for C16:0 DMA andm/z 283 for C18:0 DMA, in addition to retention time for accurate identification. More recently, LC-MS methods measuring plasmalogens have been published.
      • Zemski Berry K.A.
      • Murphy R.C.
      Electrospray ionization tandem mass spectrometry of glycerophosphoethanolamine plasmalogen phospholipids.
      ,
      • Hui S.P.
      • Chiba H.
      • Kurosawa T.
      Liquid chromatography-mass spectrometricdetermination of plasmalogens in human plasma.
      The advantages of these techniques include reduced sample processing time and more accurate quantification using synthetic plasmalogens as internal standards, although these internal standards also could be used in SIM-GC-MS analysis. The disadvantage is that not all plasmalogens are measured, only the major species of the ethanolamine and choline plasmalogens. One source of variability in analyte measurement is the GC injection port. Recovery may be reduced as a result of inadequate passage of DMAs and FAMEs through the injection port due to interaction with septal particles produced during the injection process. Thus, for GC and GC-MSanalyses, a specialized septum such as the Merlin microseal septum, which is designed to eliminate septal coring, may be required. Likewise, for LC-MS assays, recovery may be influenced by carryover, as plasmalogens may adhere to LC polyetheretherketone (PEEK) tubing.
      Conditions affecting plasmalogen synthesis, including PBDs and isolated deficiencies of DHAPAT, AGPS, or FAR1, can lead to reduced plasmalogen levels and ultimately result in an abnormal phenotype. Plasmalogen levels are typically low at birth, gradually increasing to plateau by 6 months of age.
      • Labadaridis I.
      • Moraitou M.
      • Theodoraki M.
      • Triantafyllidis G.
      • Sarafidou J.
      • Michelakakis H.
      Plasmalogen levels in full-termneonates.
      Because unaffected neonates can have levels that overlap those in older children confirmed to have a PBD, each laboratory, based on their analytical method of choice, must establish age-specific reference intervals. Still, plasmalogen levels in PBD patients may normalize with age,
      • Wanders R.J.
      • Purvis Y.R.
      • Heymans H.S.
      • et al.
      Age-related differences in plasmalogen content of erythrocytes from patients with the cerebro-hepato-renal (Zellweger) syndrome: implications for postnatal detection of the disease.
      decreasing the utility of this assay for the investigation PBDs in older children. In RCDP, the extent of plasmalogen deficiency correlates with phenotypic severity.
      • Braverman N.
      • Chen L.
      • Lin P.
      • et al.
      Mutation analysis of PEX7 in 60 probands with rhizomelic chondrodysplasia punctata and functional correlations of genotype with phenotype.
      While severely affected individuals have a significant reduction in red blood cell (RBC) plasmalogen content, those with less severe phenotypes show a more modest reduction. In very mild cases, the RBC plasmalogen level may be near normal. While RCDP is often the primary clinical indication for measuring plasmalogen levels, this assay also is performed as part of an overall investigation for inborn errors of metabolism affecting peroxisomal function or assembly. Secondary plasmalogen deficiency has been observed in a number of common conditions such as respiratory disease, inflammatory conditions, Parkinson disease, Down syndrome, and Alzheimer disease.
      • Braverman N.E.
      • Moser A.B.
      Functions of plasmalogen lipids in health and disease.

      Analysis of pipecolic acid in urine and plasma

      Amino acid analysis performed with traditional methods, like ion exchange chromatography, can detect L-pipecolic acid when present in large amounts;
      • Hutzler J.
      • Dancis J.
      The determination of pipecolic acid: method and results of hospital survey.
      but several more sensitive methods have been developed using mass spectrometry combined with gas or liquid chromatography.
      • Steinberg S.
      • Jones R.
      • Tiffany C.
      • Moser A.
      Investigational methods for peroxisomaldisorders.
      ,
      • van den Berg G.A.
      • Breukelman H.
      • Elzinga H.
      • Trijbels J.M.
      • Monnens L.A.
      • Muskiet F.A.
      Determination of pipecolic acid in urine and plasma by isotope dilution mass fragmentography.
      ,
      • Kok R.M.
      • Kaster L.
      • de Jong A.P.
      • Poll-The B.
      • Saudubray J.M.
      • Jakobs C.
      Stable isotope dilution analysis of pipecolic acid in cerebrospinal fluid, plasma, urine and amniotic fluid using electron capture negative ion mass fragmentography.
      ,
      • Zee T.
      • Stellaard F.
      • Jakobs C.
      Analysis of pipecolic acid in biological fluids using capillary gas chromatography with electron-capture detection and [2H11] pipecolic acid as internal standard.
      • Rashed M.S.
      • Al-Ahaidib L.Y.
      • Aboul-Enein H.Y.
      • Al-Amoudi M.
      • Jacob M.
      Determination of L-pipecolic acid in plasma using chiral liquid chromatography-electrospray tandem mass spectrometry.
      • Semeraro M.
      • Muraca M.
      • Catesini G.
      • et al.
      Determination of plasma pipecolic acid by an easy and rapid liquid chromatography-tandem mass spectrometry method.
      Pipecolic acid can be measured by GC-MS in negative
      • Steinberg S.
      • Jones R.
      • Tiffany C.
      • Moser A.
      Investigational methods for peroxisomaldisorders.
      ,
      • Kok R.M.
      • Kaster L.
      • de Jong A.P.
      • Poll-The B.
      • Saudubray J.M.
      • Jakobs C.
      Stable isotope dilution analysis of pipecolic acid in cerebrospinal fluid, plasma, urine and amniotic fluid using electron capture negative ion mass fragmentography.
      or positive
      • Zee T.
      • Stellaard F.
      • Jakobs C.
      Analysis of pipecolic acid in biological fluids using capillary gas chromatography with electron-capture detection and [2H11] pipecolic acid as internal standard.
      mode following derivatization of the amine group with methyl chloroformate, acidic ethylacetate extraction, and further derivatization with pentafluorobenzyl bromide. Some LC-MS/MS methods do not require sample derivatization, thus requiring smaller sample volumes and less processing time.
      • Rashed M.S.
      • Al-Ahaidib L.Y.
      • Aboul-Enein H.Y.
      • Al-Amoudi M.
      • Jacob M.
      Determination of L-pipecolic acid in plasma using chiral liquid chromatography-electrospray tandem mass spectrometry.
      ,
      • Semeraro M.
      • Muraca M.
      • Catesini G.
      • et al.
      Determination of plasma pipecolic acid by an easy and rapid liquid chromatography-tandem mass spectrometry method.
      A deuterated stable isotope, like2PA-d11 or2PA-d9, is utilized as internal standard. Quantitation can be performed by stable isotope dilution orusing a calibration curve.2H5-phenylalanine has also been used as internal standard.
      • Rashed M.S.
      • Al-Ahaidib L.Y.
      • Aboul-Enein H.Y.
      • Al-Amoudi M.
      • Jacob M.
      Determination of L-pipecolic acid in plasma using chiral liquid chromatography-electrospray tandem mass spectrometry.
      ,
      • Semeraro M.
      • Muraca M.
      • Catesini G.
      • et al.
      Determination of plasma pipecolic acid by an easy and rapid liquid chromatography-tandem mass spectrometry method.
      A chiral column can specifically discriminate L-pipecolic acid from the D-enantiomer.
      • Rashed M.S.
      • Al-Ahaidib L.Y.
      • Aboul-Enein H.Y.
      • Al-Amoudi M.
      • Jacob M.
      Determination of L-pipecolic acid in plasma using chiral liquid chromatography-electrospray tandem mass spectrometry.
      However, in both affected and normal individuals, the D-enantiomer represents only 2% of pipecolic acid.
      • Armstrong D.W.
      • Zukowski J.
      • Ercal N.
      • Gasper M.
      Stereochemistry of pipecolic acid found in the urine and plasma of subjects with peroxisomal deficiencies.
      Interestingly, elevated pipecolic acid was the first biochemical abnormality described in ZSD patients.
      • Danks D.M.
      • Tippett P.
      • Adams C.
      • Campbell P.
      Cerebro-hepato-renal syndrome of Zellweger. A report of eight cases with comments upon the incidence, the liver lesion, and a fault in pipecolic acid metabolism.
      Patients with single peroxisomal protein defects, like ACOX1 deficiency or DBP deficiency, display pipecolic acid within the normal range.
      • Steinberg S.J.
      • Dodt G.
      • Raymond G.V.
      • Braverman N.E.
      • Moser A.B.
      • Moser H.W.
      Peroxisome biogenesis disorders.
      ,
      • Hoefler G.
      • Hoefler S.
      • Watkins P.A.
      • et al.
      Biochemical abnormalities in rhizomelic chondrodysplasia punctata.
      ,
      • Peduto A.
      • Baumgartner M.R.
      • Verhoeven N.M.
      • et al.
      Hyperpipecolic acidaemia: a diagnostic tool for peroxisomal disorders.
      Pipecolic acid accumulation is not exclusive to peroxisomal biogenesis disorders. In addition to lysine, patients with hyperlysinuria type I accumulate pipecolic acid in plasma and urine.
      • Tondo M.
      • Calpena E.
      • Arriola G.
      • et al.
      Clinical, biochemical, molecular and therapeutic aspects of 2 new cases of 2-aminoadipic semialdehyde synthase deficiency.
      Pipecolic acid accumulates and is considered a secondary biomarker in patients with PDE,
      • Willemsen M.A.
      • Mavinkurve-Groothuis A.M.
      • Wevers R.A.
      • Rotteveel J.J.
      • Jakobs C.
      Pipecolic acid: a diagnostic marker in pyridoxine-dependent epilepsy.
      although normallevels have been described.
      • Mercimek-Mahmutoglu S.
      • Donner E.J.
      • Siriwardena K.
      Normal plasma pipecolic acid level in pyridoxine dependent epilepsy due to ALDH7A1 mutations.
      In PDE, the specific metabolites P6C and AASA are markedly increased,
      • Plecko B.
      • Paul K.
      • Paschke E.
      • et al.
      Biochemical and molecular characterization of 18 patients with pyridoxine-dependent epilepsy and mutations of the antiquitin (ALDH7A1) gene.
      ,
      • Yuzyuk T.
      • Thomas A.
      • Viau K.
      • et al.
      Effect of dietary lysine restriction and arginine supplementation in two patients with pyridoxine-dependent epilepsy.
      and pipecolic acid is routinely assessed as part of multianalyte panels that also include AASA and P6C.
      • Yuzyuk T.
      • Liu A.
      • Thomas A.
      • et al.
      A novel method for simultaneous quantification of alpha-aminoadipic semialdehyde/piperideine-6-carboxylate and pipecolic acidin plasma and urine.
      ,
      • Sadilkova K.
      • Gospe Jr., S.M.
      • Hahn S.H.
      Simultaneous determination of alpha-aminoadipic semialdehyde, piperideine-6-carboxylate and pipecolic acid by LC-MS/MS for pyridoxine-dependent seizures and folinic acid-responsive seizures.
      Elevated pipecolic acid has also been described in a patient with pathogenic variants in NADK2,
      • Tort F.
      • Ugarteburu O.
      • Torres M.A.
      • et al.
      Lysine restriction and pyridoxal phosphate administration in a NADK2 patient.
      a gene encoding a mitochondrial enzyme implicated in several pathways, including lysine degradation.
      • Tort F.
      • Ugarteburu O.
      • Torres M.A.
      • et al.
      Lysine restriction and pyridoxal phosphate administration in a NADK2 patient.
      Elevated pipecolic acid is also seen in chronic liver dysfunction
      • Matsuda Y.
      • Fujita T.
      • Hada T.
      • Higashino K.
      Comparative study on the correlation of plasma gamma-aminobutyric acid and pipecolic acid with liver function in patients with liver cirrhosis.
      and has been reported in an individual without any clinical features.
      • Vallat C.
      • Denis S.
      • Bellet H.
      • Jakobs C.
      • Wanders R.J.
      • Mion H.
      Major hyperpipecolataemia in a normal adult.
      ,
      • Baas J.C.
      • van de Laar R.
      • Dorland L.
      • et al.
      Plasma pipecolic acid is frequently elevated in non-peroxisomal disease.

      Other biochemical tests

      Bile acids (BAs) are hydroxylated steroids synthetized from cholesterol in the liver and conjugated to the amino acids taurine or glycine. BAs facilitate the absorption of fat and fat-soluble vitamins in the small intestine; after being metabolized to unconjugated and dehydroxylated species by the gut bacteria, they are reabsorbed and recycled by the liver. Serum concentrations are directly affected by liver function and represent a sensitive marker for common liver conditions, including intrahepatic cholestasis of pregnancy. The final step of BA synthesis takes place in the peroxisomes, where the C27-bile acid intermediates DHCA and THCA are converted to the primary C24-bile acids. In PBD patients, the C27-bile acid intermediates accumulate and cause progressive liver disease.
      • Wanders R.J.
      • Ferdinandusse S.
      Peroxisomes, peroxisomal diseases, and the hepatotoxicity induced by peroxisomal metabolites.
      Testing for these intermediates specifically aids in the diagnosis of peroxisomal disorders.
      • Courillon F.
      • Gerhardt M.F.
      • Myara A.
      • Rocchiccioli F.
      • Trivin F.
      The optimized use of gas chromatography-mass spectrometry and high performance liquid chromatography to analyse the serumbile acids of patients with metabolic cholestasis and peroxisomal disorders.
      • Bootsma A.H.
      • Overmars H.
      • van Rooij A.
      • et al.
      Rapid analysis of conjugated bile acids in plasmausing electrospray tandem mass spectrometry: application for selective screening of peroxisomal disorders.
      • Johnson D.W.
      • ten Brink H.J.
      • Schuit R.C.
      • Jakobs C.
      Rapid and quantitative analysis of unconjugated C(27) bile acids in plasma and blood samples by tandem mass spectrometry.
      The quantitative analysis of serum BAs by GC-MS is sensitive, accurate, and allows for good discrimination between species.
      • Courillon F.
      • Gerhardt M.F.
      • Myara A.
      • Rocchiccioli F.
      • Trivin F.
      The optimized use of gas chromatography-mass spectrometry and high performance liquid chromatography to analyse the serumbile acids of patients with metabolic cholestasis and peroxisomal disorders.
      However, since GC-MS methods require alarge sample volume and cleavage of conjugated bile acids followed by derivatization, they have been largely replaced by LC-MS/MS methods in the clinical laboratory.
      • Scherer M.
      • Gnewuch C.
      • Schmitz G.
      • Liebisch G.
      Rapid quantification of bile acids and their conjugates in serum by liquid chromatography-tandem mass spectrometry.
      ,
      • Bootsma A.H.
      • Overmars H.
      • van Rooij A.
      • et al.
      Rapid analysis of conjugated bile acids in plasmausing electrospray tandem mass spectrometry: application for selective screening of peroxisomal disorders.
      ,
      • Johnson D.W.
      • ten Brink H.J.
      • Schuit R.C.
      • Jakobs C.
      Rapid and quantitative analysis of unconjugated C(27) bile acids in plasma and blood samples by tandem mass spectrometry.
      BA abnormalities can be severe in ZSDs, and disease severity correlates with the impairment in BA synthesis; mildly affected individuals can have normal BA levels.
      • Berendse K.
      • Engelen M.
      • Ferdinandusse S.
      • et al.
      Zellweger spectrum disorders: clinical manifestations in patients surviving into adulthood.
      Metabolite levels can also be assessed in cultured skin fibroblasts, although these tests are seldom performed using this sample type, since it requires an invasive skin biopsy and time to establish primary fibroblast cultures. In a minority of individuals (<5%) suspected of having a peroxisomal disorder, measurement of peroxisomal metabolites in fibroblasts could be useful (Steven Steinberg, personal communication); several individuals with abnormalities in fibroblast VLCFAs and BCFAs suggestive of a peroxisomal disorder, with normal plasma values, have been reported.
      • Rosewich H.
      • Waterham H.R.
      • Wanders R.J.
      • et al.
      Pitfall in metabolic screening in a patient with fatal peroxisomal beta-oxidation defect.
      ,
      • Lusebrink N.
      • Porto L.
      • Waterham H.R.
      • et al.
      Absence of biochemical evidence at an early age delays diagnosis in a patient with a clinically severe peroxisomal biogenesis disorder.
      Assays have been described that measure peroxisomal enzyme activities in patient fibroblasts, usually using radioisotope-labeled substrates.
      • Ferdinandusse S.
      • Ebberink M.S.
      • Vaz F.M.
      • Waterham H.R.
      • Wanders R.J.
      The important role of biochemical and functional studies in the diagnostics of peroxisomal disorders.
      Only phytanic acid oxidase and pristanic acid oxidase are routinely offered clinically. Fibroblasts can be used to assess the cellular distribution of the enzyme catalase (catalase solubility), which is normally localized to peroxisomes; catalase localization to the cytosol is a sensitive marker of peroxisomal dysfunction.
      • Ferdinandusse S.
      • Ebberink M.S.
      • Vaz F.M.
      • Waterham H.R.
      • Wanders R.J.
      The important role of biochemical and functional studies in the diagnostics of peroxisomal disorders.

      Molecular testing

      Molecular testing can be used to confirm (e.g., X-ALD) or identify (e.g., ZSD) the specific gene defect in individuals with highly suggestive clinical and biochemical findings. In some individuals, molecular testing may be used to clarify equivocal laboratory findings in the presence of a nonspecific clinical presentation. Carrier testing for at-risk relatives by molecular testing is the best approach, since carrier testing by biochemical methods is not possible for most peroxisomal disorders, and has only about 80% sensitivity for X-ALD heterozygotes. The classification of variants can be challenging and may require further research-based studies for clarification, but should be made using the ACMG variant classification guidelines.
      • Richards S.
      • Aziz N.
      • Bale S.
      • et al.
      Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology.
      In families where aperoxisomal defect has been confirmed biochemically (e.g., abnormal VLCFA results in cultured skin fibroblasts from the index case), but molecular testing has identified variants of uncertain clinical significance, predictive prenatal testing using biochemical methods is best, if available. When two definitive pathogenic variants are identified in transin a proband, predictive testing, including preimplantation genetic diagnosis, can be reliably offered to families. For confirmatory testing, test options range from a single-gene test to a multigene panel to exome or genome sequencing. The best approach varies depending on the clinical presentation and certainty of the biochemical profile. Testing ABCD1 in males with a biochemical and clinical diagnosis of X-ALD should be adequate, but males with a mild or dominant presentation of a ZSD may overlap
      • Tran C.
      • Hewson S.
      • Steinberg S.J.
      • Mercimek-Mahmutoglu S.
      Late-onset Zellweger spectrum disorder caused by PEX6 mutations mimicking X-linked adrenoleukodystrophy.
      (PEX6-p.Arg860Trp;NM_000287.4:c.2578C>T) and thus require follow-up of a negative ABCD1 test with a ZSD gene panel. Likewise, individuals with a suspected diagnosis of acatalasemia or primary hyperoxaluria type 1 would be candidates for single-gene testing. For individuals with a suspected diagnosis of ZSD, but who have undergone plasma VLCFA testing only, a broader panel that includes PEX genes and genes associated with SPPD should be considered. Similarly, a patient with a suspected diagnosis of RCDP who has deficient erythrocyte plasmalogens and phytanic acid oxidation (elevated plasma phytanic acid) could have molecular testing limited to PEX7, but if there is uncertainty then a broader RCDP multigene panel is recommended. For individuals with a mild or equivocal biochemical profile, a comprehensive multigene panel that includes most PEX genes and genes associated with SPPDs would be ideal. Exome or genome sequencing may be considered for patients with negative single-gene and multigene panel tests or when the most appropriate single or multigene test cannot be determined. The majority of pathogenic variants associated with peroxisomal disorders involve single-nucleotide substitutions or small insertion/deletions that can be detected by sequencing (either Sanger or next-generation sequencing platforms); however, in some instances, exon-level deletions or duplications are present, and require alternative techniques for detection. Although some laboratories may be able to detect multiexon deletions or duplications using next-generation sequencing platforms, an orthogonal method such as multiplex ligation-dependent probe amplification (MLPA) will be usually needed to confirm a single exoncopy-number alteration. About 5–6% of ABCD1 pathogenic variants are large deletions that can be detected by Southern blot or MLPA; in these cases, ABCD1 sequencing results are negative in a female heterozygote, but would fail to amplify in a male hemizygote. In both cases, further testing to detect large deletions is required. Moreover, when sequencing ABCD1 it is important to be aware of pseudogene regions, homologous to exons 7–10, duplicated on chromosomes 2, 10, 16, and 22.
      • Eichler E.E.
      • Budarf M.L.
      • Rocchi M.
      • et al.
      Interchromosomal duplications of the adrenoleukodystrophy locus: a phenomenon of pericentromeric plasticity.
      Both Sanger and next-generation sequencing platforms necessitate careful validation to be certain that these pseudogene regions are avoided.
      For autosomal recessive disorders, parental testing is useful to be able to offer carrier testing to family members in each line age. If a pathogenic variant is detected in apparent homozygosity, parental targeted testing will confirm that one pathogenic variant was inherited from each parent. If both parents are not carriers, the second variant may be a large deletion; alternatively, the variant could be present in two copies, but of the same parental chromosome (uniparental isodisomy has been reported in association withZSD
      • Turner C.L.
      • Bunyan D.J.
      • Thomas N.S.
      • et al.
      Zellweger syndrome resulting from maternal isodisomy of chromosome 1.
      and RCDP
      • Nimmo G.
      • Monsonego S.
      • Descartes M.
      • Franklin J.
      • Steinberg S.
      • Braverman N.
      Rhizomelic chrondrodysplasia punctata type 2 resulting from paternal isodisomy of chromosome 1.
      ); apparent homozygosity could also be caused by allele dropout due to technical reasons.

      Ethics declarations

      Disclosure

      The following authors direct laboratories that perform laboratory testing for the diagnosis and management of peroxisomal disorders as a fee for service: I.D.B., S.T., S.S., L.K., and K.C.-O. N.B. is a member of the medical and scientific advisory board for the Global Foundation for Peroxisome Disorders.

      Acknowledgements

      The authors gratefully acknowledge Ann Moser for her significant contribution to the section “Analysis of plasmalogens in red blood cells” and for carefully reviewing the final manuscript, and the members of the ACMG Biochemical Genetics Subcommittee (Laboratory Quality Assurance Committee) for their valuable input and editorial assistance.

      Additional information

      The Board of Directors of the American College of Medical Genetics and Genomics approved this technical standard on 15 October 2019.
      Disclaimer:
      This technical standard is designed primarily as an educational resource for clinical laboratory geneticists to help them provide quality clinical laboratory genetic services. Adherence to this standard is voluntary and does not necessarily assure a successful medical outcome. This standard should not be considered inclusive of all proper procedures and tests or exclusive of other procedures and tests that are reasonably directed to obtaining the same results. In determining the propriety of any specific procedure or test, the clinical laboratory geneticist should apply his or her own professional judgment to the specific circumstances presented by the individual patient or specimen.
      Clinical laboratory geneticists are encouraged to document in the patient’s record the rationale for the use of a particular procedure or test, whether or not it is in conformance with this standard. They also are advised to take notice of the date any particular standard was adopted, and to consider other relevant medical and scientific information that becomes available after that date. It also would be prudent to consider whether intellectual property interests may restrict the performance of certain tests and other procedures.
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      Supplementary information

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