Delhi course 2020
SSIEM Academy
Peroxisomal investigation
Delhi, 6-9 January 2020
Christine Vianey-Saban, CHU Lyon [email protected] Metabolic functions of peroxisomes
Biosynthesis Bile acids Plasmalogens (etherphospholipids) Docosahexaenoic acid (C22:6w3 or DHA) : essential FA Catabolism a-oxidation of phytanic acid b-oxidation of very-long-chain fatty acids, and pristanic acid Detoxification of glyoxylate (alanine glyoxylate-aminotransferase : AGT) Oxidation of pipecolic acid (pipecolate oxidase in cerebral tissue)
H2O2 (catalase, peroxidase, …) D-amino acids, polyamines, some leukotrienes and prostaglandins, ….
3 C Vianey-Saban SSIEM Academy Delhi, 6-9 January 2020 Biosynthesis of bile acids
Mitochondria Peroxisome
AMACR b-ox Conj. (R-DHCA) (S-DHCA)
Cholesterol (25R)-dihydroxycholestanoyl-CoA (R-DHCA) AMACR b-ox Conj. (R-THCA) (S-THCA)
(25R)-trihydroxycholestanoyl-CoA (R-THCA) C27 C24 AMACR = a-methylacyl-CoA racemase b-ox = peroxisomal b-oxidation (4 steps) Conj. = conjugation to glycine and taurine
4 C Vianey-Saban SSIEM Academy Delhi, 6-9 January 2020 Biosynthesis of plasmalogens Plasmalogens (special type of etherphospholipid) = 18% of phospholipids Mainly in cerebral tissue : constituent of myelin Also constituents of cell membranes of heart, lungs, kidneys and muscles
DHAP : dihydroxyacetone phosphate; DHAP-AT : dihydroxyacetone phosphate acyltransferase
5 C Vianey-Saban SSIEM Academy Delhi, 6-9 January 2020 a-oxidation of phytanic acid
phytanic acid LCS phytanoyl-CoA
? ?
phytanic acid VLCS phytanoyl-CoA Phytanoyl-CoA 2-hydroxylase 2-OH-phytanoyl-CoA 2-hydroxy phytanoyl-CoA lyase Pristanal Pristanal dehydrogenase Pristanic acid VLCS AMACR (2S)-pristanoyl-CoA (2R)-pristanoyl-CoA Peroxisome
Phytanic acid = 3,7,11,15-tetramethylhexadecanoic acid (derives from phytol) Pristanic acid = 2,6,10,14-tetramethylpentadecanoic acid LCS : long-chain acyl-CoA synthetase VLCS : very-long-chain acyl-CoA synthetase AMACR : a-methylacyl-CoA racemase
6 C Vianey-Saban SSIEM Academy Delhi, 6-9 January 2020 b-oxidation of VLCFA, pristanic and bile acids VLCF-CoA ACBD5? VLCF-CoA (25S)-DHC-CoA (25S)-THC-CoA (2S)-pristanoyl-CoA
ACOX1 ACOX2 ACOX2 ACOX3
DBP DBP DBP
ACAA1 SCPx SCPx SCPx
Chenodeoxy Choloyl-CoA choloyl-CoA 4,8,12-trimethyl Propionyl-CoA Propionyl-CoA Acetyl-CoA (n-2)VLCF-CoA tridecanoyl-CoA
ACBD5: Acyl-CoA Binding Domain contain protein 5 ; ACOX1 : straight-chain acyl-CoA oxidase ; ACOX2/3: branched- chain acyl-CoA oxidase ; DBP: D-bifunctional protein ; ACAA1 : 3-ketoacyl-CoA thiolase 1 ; SCPx : sterol carrier protein X 7 C Vianey-Saban SSIEM Academy Delhi, 6-9 January 2020 Adapted from Wanders et al, Biochim Biophys Acta 2010;1801:272 Inborn errors of peroxisomal metabolism
Subdivided into 2 groups The peroxisome biogenesis disorders (PBD) The single peroxisomal enzyme deficiencies
8 C Vianey-Saban SSIEM Academy Delhi, 6-9 January 2020 Peroxisome biogenesis disorders (PBD)
Inheritance : autosomal recessive Subdivided into 2 subgroups The Zellweger spectrum disorders (ZSDs) Zellweger syndrome Neonatal adrenoleukodystrophy (NALD) Infantile Refsum disease Peroxisome biogenesis is fully impaired, although to variable extents Same PEX genes are involved : PEX1, PEX2, PEX3, PEX5, PEX6, PEX10, PEX12, PEX13, PEX14, PEX16, PEX19, PEX26 Rhizomelic chondrodysplasia punctata (RCDP) type 1 and 5 Peroxisome biogenesis is partially impaired Only plasmalogen biosynthesis and phytanic a-oxidation are impaired Type I: PEX7 mutations Type V: PEX5L mutations Sometimes : clinical spectrum less severe # adult Refsum disease
9 C Vianey-Saban SSIEM Academy Delhi, 6-9 January 2020 Single peroxisome enzyme deficiencies
Inheritance : autosomal recessive except for X-ALD : X-linked Peroxisomal b-oxidation deficiencies X-linked adrenoleukodystrophy (X-ALD) = ALDP protein deficiency Acyl-CoA Binding Domain Containing 5 (ACBD5) deficiency Acyl-CoA oxidase 1 (ACOX1) deficiency D-bifunctional protein (DBP) deficiency Sterol carrier protein (SCPx) deficiency 2-methyl acyl-CoA racemase (AMACR) deficiency Plasmalogen biosynthesis deficiencies Rhizomelic chondrodysplasia punctata (RCDP) type 2 = DHAP-AT deficiency Rhizomelic chondrodysplasia punctata (RCDP) type 3 = Alkyl-DHAP synthetase deficiency Rhizomelic chondrodysplasia punctata (RCDP) type 4 = FAR1 (fatty acyl- CoA reductase) deficiency Phytanic a-oxidation deficiencies Refsum disease = phytanoyl-CoA-2-hydroxylase deficiency 10 C Vianey-Saban SSIEM Academy Delhi, 6-9 January 2020 Single peroxisome enzyme deficiencies
Bile acid biosynthesis deficiencies Acyl-CoA oxidase 2 (ACOX2) deficiency Peroxisomal membrane protein 70 (PMP70/ABCD3) defect Bile acid CoA:amino acid N-acyltransferase (BAAT) deficiency Glyoxylate detoxification deficiencies Hyperoxaluria type I = alanine glyoxylate-aminotransferase deficiency
11 C Vianey-Saban SSIEM Academy Delhi, 6-9 January 2020 Peroxisomal disorders (biogenesis)
Disorder Protein, enzyme, transporter Gene
ZSDs Peroxins 13 PEX genes RCDP type 1 Peroxin 7 (PTS2-receptor) PEX7 RCDP type 5 Peroxin 5L (PTS1-receptor long) PEX5
12 C Vianey-Saban SSIEM Academy Delhi, 6-9 January 2020 Peroxisomal disorders (single enzyme)
Disorder Protein, enzyme, transporter Gene
X-ALD Protein ALDP ABCD1 ACOX1 deficiency Straight chain acyl-CoA oxidase ACOX1 Acyl-CoA Binding Domain ACBD5 ACBD5 Containing 5 DBP deficiency D-bifunctional protein HSD17B4 SCPx deficiency Sterol carrier protein SCP2 AMACR deficiency 2-methylacyl-CoA racemase AMACR RCDP type 2 DHAP-AT GNPAT RCDP type 3 Alkyl-DHAP synthase AGPS RCDP type 4 FattyAcyl-CoAReductase1 FAR1 Refsum disease Phytanoyl-CoA-2-hydroxylase PAHX
13 C Vianey-Saban SSIEM Academy Delhi, 6-9 January 2020 Peroxisomal disorders (single enzyme)
Disorder Protein, enzyme, transporter Gene
ACOX2 deficiency Acyl-CoA oxidase 2 ACOX2 PMP70 defect Peroxisomal membrane protein 70 ABCD3 Bile acid CoA:amino acid N- BAAT deficiency BAAT acyltransferase Hyperoxaluria type I AGT AGXT
14 C Vianey-Saban SSIEM Academy Delhi, 6-9 January 2020 Biochemical investigation in blood (1)
Peroxisomal b-oxidation Very-long-chain fatty acids C26:0 hexacosanoic or cerotic acid C24:0 tetracosanoic or lignoceric acid C22:0 docosanoic or behenic acid Ratio C26:0 / C22:0 and C24:0 / C22:0 GC, GC/MS (stable isotope dilution) or LC-MS/MS Pristanic acid GC, GC/MS or LC-MS/MS Bile acids : dihydroxy- and trihydroxycholestanoic acids GC, GC/MS (after hydrolysis of Gly and Tau conjugates) or LC-MS/MS Peroxisomal a-oxidation Phytanic acid GC, GC/MS or LC-MS/MS
15 C Vianey-Saban SSIEM Academy Delhi, 6-9 January 2020 Biochemical investigation in blood (2)
Plasmalogen and DHA biosynthesis (erythrocytes) C16:0- and C18:0-dimethylacetal (after transmethylation) Ratio to the corresponding FA C22:6w3 (DHA) after transmethylation Pipecolic acid GC/MS stable isotope dilution Amino acid analysis : lack of sensitivity if ninhydrine without methylcellosolve LC-MS/MS Acylcarnitines ZSD C16:0 and C18:0 dicarboxylacylcarnitines C24:0 and C26:0 acylcarnitines LC-MS/MS (multi reaction monitoring : MRM)
16 C Vianey-Saban SSIEM Academy Delhi, 6-9 January 2020 Plasma acylcarnitines
C2
C0 Peroxisomal biogenesis disorder Zellweger syndrome * *
* * * C16DC C18DC * * * C16 C18:1 C26
17 C Vianey-Saban SSIEM Academy Delhi, 6-9 January 2020 Biochemical investigation in blood (3)
C26:0-lysophosphatidylcholine (C26:0-lysoPC): bloodspot PBD, X-ALD, etc (all defects with VLCFA accumulation) LC-MS/MS (multi reaction monitoring : MRM) X-ALD PBD / ZSD
Huffnagel 2017 Mol Genet Klouwer 2017 Metab 122 JIMD 40 875-881 209-215
18 Courtesy Dr Frederic Vaz Biochemical investigation in urine (1)
Organic acids PBD 3,6-epoxydicarboxylic acids : C10, C12, C13, C14 saturated and unsaturated 2-hydroxysebacic Odd dicarboxylic acids : pimelic (C7) and azelaic (C9) acids Increase of sebacic / adipic acid ratio 4-hydroxyphenyllactic +/- 4-hydroxyphenylpyruvic Refsum disease 2,6-dimethylsuberic acid and 3-methyladipic acid
19 C Vianey-Saban SSIEM Academy Delhi, 6-9 January 2020 Biochemical investigation in urine (2)
Bile acids LC-MS/MS Acylcarnitines PBD and D-bifunctional protein deficiency C14:0, C16:0 and C18:0 dicarboxylacylcarnitines (DC-carnitines) C22:0, C24:0 and C26:0 acylcarnitines LC-MS/MS Detoxification of glyoxylate Oxalic and glycolic acid (GC/MS stable isotope dilution)
20 C Vianey-Saban SSIEM Academy Delhi, 6-9 January 2020 ERNDIM quality controls
Special assays in plasma VLCFA Pristanic acid Phytanic acid Pipecolic acid Special assays in urine Pipecolic acid
21 C Vianey-Saban SSIEM Academy Delhi, 6-9 January 2020 VLCFA, pristanic, and phytanic acids in plasma: Technical aspects
Fasting or fed state Anti-coagulant EDTA or heparine Rapid centrifugation Influence of haemolysis Sample storage Stable compounds : plasma can be sent at room temperature Longer storage (> 48 hours) : stored frozen Circulating VLCFA and branched-chain fatty acids Mainly as esterified forms: sphingomyelin, phospholipids, triglycerides and carnitine If free : bound to albumin Measurement after acidic and alkaline hydrolysis
22 C Vianey-Saban SSIEM Academy Delhi, 6-9 January 2020 VLCFA, pristanic, and phytanic acids in plasma: interpretation of results
VLCFA Reference range is not age-dependent False positive results: increase of C26 if hypertriglyceridemia but normal ratios Phytanic and pristanic acids Derived from diet (phytol) Reference range is age-dependent their level can be normal in young patients with PBD (or DBP, SCPx and AMACR deficiency)
23 C Vianey-Saban SSIEM Academy Delhi, 6-9 January 2020 Results of biochemical investigation (1)
Peroxisome biogenesis disorders (PBD)
Pristanic Phytanic THCA Plasmal Pipec Disorder VLCFA acid acid DHCA ogens olic ZSDs N - N - RCDP type 1 N - N N - N N RCDP type 5 N - N N - N N
• Zellweger spectrum disorders all peroxisomal functions are affected • RCDP type I and 5: primary (I) and secondary (5) PEX7 deficiency all PTS2-related protein functions affected
C Vianey-Saban SSIEM Academy Delhi, 6-9 January 2020 24 Results of biochemical investigation (2) Single peroxisomal enzyme deficiency
Pristanic Phytanic THCA Plasmal Pipec Disorder VLCFA acid acid DHCA ogens olic X-ALD N N NNN ACOX1 N N NNN ACBD5 N N N N N DBP N - * N - N - N N - SCPx N N - * N - N - N N AMACR N N - * N - N N RCDP type 2 N N N N N RCDP type 3 N N N N N RCDP type 4 N N N N N Refsum disease N N N N N * Pristanic > phytanic C Vianey-Saban SSIEM Academy Delhi, 6-9 January 2020 25 Results of biochemical investigation (3)
Pristanic Phytanic THCA Plasmal Pipec Disorder VLCFA acid acid DHCA ogens olic ACOX2 N N N N N PMP70 N N N N N BAAT N N N N NN Hyperoxaluria type I N N N N N N
• In BAAT deficiency: bile acid spectrum shows only unconjugated bile acids • In Hyperoxaluria type I: oxalic and glycolic acid urinary excretion are elevated
C Vianey-Saban SSIEM Academy Delhi, 6-9 January 2020 26 Confirmation of diagnosis (1)
ZSDs Fibroblasts Beta-oxidation of C24:0 or C26:0 or d3-C22:0 loading test
Measurement of VLCFA and/or C26:0-lysoPC Control (catalase IF) Immunofluorescence microscopy analysis (catalase/ABCD1) Beta-oxidation of pristanic acid and a-oxidation of phytanic acid DHAP-AT activity Immunoblotting for thiolase
Complementation analyses Patient Courtesy Dr Frederic Vaz DNA « PEX screen » : investigation of the more frequently mutated genes by Sanger sequencing Sequencing of the gene, if complementation analyses has been performed NGS : panel or genes or exome sequencing
27 C Vianey-Saban SSIEM Academy Delhi, 6-9 January 2020 Confirmation of diagnosis (2)
RDCP Biochemical studies in fibroblasts are possible Alkyl-DHAP synthetase, DHAP-AT and phytanic acid oxidation activity and immunoblot of thiolase in fibroblasts DNA analysis (NGS or targeted) is most commonly used
28 C Vianey-Saban SSIEM Academy Delhi, 6-9 January 2020 Confirmation of diagnosis (3)
Single peroxisome enzyme deficiency Fibroblasts Beta-oxidation with different substrates (C26:0, pristanic acid) Measurement of the suspected deficient enzyme Western blot of the suspected deficient enzyme DNA Sequencing of the corresponding gene X-ALD VLCFA can be normal in carriers (in approximately 15% of women, according to literature) : ALDP protein / ABCD1 gene C26:0-lysoPC When a symptomatic patient is identified mutation analysis of ABCD1 gene has to be performed the identified mutation has to be searched in all family members for an adequate genetic counselling
29 C Vianey-Saban SSIEM Academy Delhi, 6-9 January 2020 Diagnostic flow chart
• SED = Single enzyme deficiency with phenotypical ZSD similarities like ACOX1 deficiency and DBP deficiency • Next generation sequencing (NGS) of all PEX genes is advised when complementation analysis is not practicable
Klouwer 2015 Orphanet J Rare Dis 10 151 30 C Vianey-Saban SSIEM Academy Delhi, 6-9 January 2020 Conclusion (1) Peroxisomal investigation in blood
At minimum VLCFA Phytanic and pristanic acid If possible Plasmalogens in erythrocytes C26-lysoPC in bloodspot C27 bile acids: DHCA and THCA (+plasma and urine spectrum) Pipecolic acid
⇒ Analysis of VLCFA, phytanic acid, pristanic acid and plasmalogens allows to detect most peroxisomal disorders
31 C Vianey-Saban SSIEM Academy Delhi, 6-9 January 2020 Conclusion (2)
Further investigation in fibroblasts: oxidation studies, enzyme activities, western blot, … Mutation analyses Specialized labs
Essential to provide an accurate genetic counselling and eventually prenatal diagnosis
32 C Vianey-Saban SSIEM Academy Delhi, 6-9 January 2020 Conclusion (3)
Although peroxisomal disorders are individually rare diseases (except for X-ALD), they are not so rare as a group Peroxisomal investigation has to be performed In neonates With classical signs of Zellweger syndrome or RCDP But also when unexplained hypotonia, dysmorphic features, seizures In infants and children Retinitis pigmentosa, sensory neural deafness, hepatopathy, dysmorphic features, psychomotor retardation In adults Ataxia, cerebellar syndrome, neuropathy, retinitis pigmentosa
33 C Vianey-Saban SSIEM Academy Delhi, 6-9 January 2020