Fatty Acid Oxidation DefectsAcid Oxidation Defects And Its .

Fatty Acid Oxidation Defects andIts Clinical SignificanceSeiji YAMAGUCHI,YAMAGUCHI MDProfessorDepartment of Pediatrics,Shimane University, JapanThe 8th International & 13th National Congress on Quality Improvementin Clinical Laboratories、April 22 to 25, 2015, Tehran1 1

Department of PediatricsShimane hima OsakaTandem MSGC/MSSapporo6th ICNS 2006(Awaji)10th ICIEM 2006(Makuhari)

Izumo-Taisha Shrine, Shimane, Japan

1 OutlineFattyy Acid Oxidation Disorder(FAOD)4

Energygy pproduction in mitochondriaGlGlycolysisl leATPRespiratoryRi tchain

Mitochondrial β-oxidationCarnitineLong chain FAMedium chain FAOCTN2plasma m.１CoAcarnitineCPT1outer m.acyl-CoA２acylcarnitineCACTCPT2VLCADinner m.TFPLong chainacylcarnitineacyl-CoAKATcarnitine３CADMedium chainacetyl-CoAlC AHADEHTCARespiratorychainFADH2ATPRespiratorychain (CoQ)ETFETFDH４6

Fatty acid β-oxidationβ oxidation defects (FAODs)β-OxidationMetabolic StepDisorders1) Carnitine cycleCarnitine uptake defect (CUD)CPT1 deficiencyCPT2 deficiencyCACT deficiency2) Long-chain β-oxidationVLCAD deficiencyTFP deficiency3) Medium to short chainMCAD deficiency4) Electron transferGlutaric acidemia type II (GA2)

CLASSIFICATION of FATTY ACID DISORDERSCLINICAL FORMFINDING1) Severe formDeath in early infancyHHypoglycemialiLiver failurey p yCardiomyopathy2) Intermediate formEpisodic attacksAcute encephalopathyS dd iinfantSuddenf tddeathth3) Late onset form(myopathic)Intermittent Episodes of:LethargyMyopathyMyalgiaMMyoglobinurial bi iLiver dysfunction

2 Diagnostic Approaches forMitochondrial fattyy acid oxidationdefects (FAOD)

DiDiagnosticti MethodsM th d1) Blood acylcarnitine analysis (MS/MS)2) Organic acid analysis (GCMS)3) In vitro probe assay (enzyme determination)4) Molecular analysis (immunoblot or gene analysis)5) Muscle biopsy：lipid myopathy

Tandem MS (MS/MS)(GC/MS)GC/MSBlood filter paperacylcarnitine・amino acids（mass screening）Urineorganic acids（confimative）

1) Acylcarnitine (MS/MS)AMCAD deficiency**Blood filter paperC8**B***CPT2 deficiencyC16** * *CC0*＊***C18:1controlC2＊* Internal standardC4C3＊＊C5＊C8＊C16C14 ＊＊

Abnormal metabolites in impaired on] glycineDicarboxylic acid(n-1)-hydroxy-acidacylglycine rated dicarboxylic acidXenoyl-CoAacylcarnitineω-oxidation oxylic acidacetyl-CoA3h d3-hydroxy-acylcarnitineliti

Urinary organic acid profile of MCAD deficiency (GC/MS)7 OH t i7-OH-octanoicoctenedioict di i3-OH-sebacic IS-2MCAD 2.545.047.550.052.5controlIS-2citrici iIS-1C24

In vitro probe acylcarnitine (IVP) assay

In vitro Probe Assay (enzyme determination)Palmitate (C16)loadingGlucose lowFatty acid ed S/MS)

Results ofIn vitro probe assaynmol/mg protA5040C2Palmitate((load))30normal20100BMCAD def（M di ）（Medium）C2 C4 C6C8C10 C12 C14C16C85040C430C620100C2 C4 C6 C8 C10 C12 C14C1650C 4030VLCAD def（long-chain）20C12 C14100C2 C4C6D4030C16C1050Glutaric acidemia type II（short lomg）C8 C10 C12 C14C16C8C4C6C12C1620100 C2 C4C6C8C10 C12 C14C1617

3 TreatmentFatty acid Oxidation Disorders

Treatment for Fatty Acid Disorder1) Avoid “long fasting”(ex.) 8 hrs: before 1 yr of age; 10 hrs: before 2 yrs2) Early infusion of “glucose”In particular, during stress (infection, over-exercise)3) “Carnitine therapy” for some casesExceptp for CPT1 deficiencyy4) Some drugsRiboflavin, CoQ105) Dietary therapy((ex.)) hihighh carbohydrateb h d t / llow lilipidid dietdi t(ex.) MCT milk (oil)19

CASEPresentation

CASE 11-year-old boy with MCAD deficiencySudden death following common cold

1MCAD deficiency: Sudden infant deathCase:1 yr 8 m, boy. Pyrexia due to infection, followed byunconsciousness and convulsion, and suddenly died.Family history: His sister (5 yr old) is severely handicapped,handicapped since acute encephalopathyat 1 year of age.Acute (1y8m)Acylcarnitine analysisC8*(blood filter paper, tandem MS)Elevation of C6,C6 C8,C8 C10,C10 C10:1*C6C10:1** ****Newborn ppaperpC8Urinary organic acid (acute stage)H poketotic dicarboHypoketoticdicarboxyliclic acidaciduriariaElevation of hexanoylglycine etc.**C6* * *C10:1***22

Japanese MCAD deficiency Clinical onset, outcome and genotype (Shimane Univ. 2012)GenotypeAge atonsetAge C TDevelop. delay21y1y( )IVS4 1G Ac.422 A TSudden death1yy8yy 10m(( ))c.449-452delc.449-452delDevelop.p delayy41y 1m1y 1m( )del. ex 11-12del. ex 11-12Develop. delay51y 3m1y 3m(n.a)del. ex 11-12del. ex 11-12Develop. delay1y 4m1y 4m( )c.449-452delc.449-452delDevelop. delay1y 7m1y 7m( )c.275C Tc.157C TDevelop. delay1y 8m1y 8m( )c.449-452delc.449-452delSudden death92y 2m2y 2m( )c.449-452delc.449-452delnormal10—5d((-))c.1085G Ac.843A Tnormal11—5d(-)c.449-452delc.154A Gnormal12—5d(-)IVS3 2T Cc.843 A Tnormal13—5d(-)c.449-452delc.212 G Anormal14—5d()(-)c.449-452del449 452d lc.134134 A Gnormall15—5d(-)c.1085G Ac.1184A Gnormal16—5d(-)c.449-452delIVS3 5G Anormal17—5d(-)c.449-452delc.820 A Cnormal5y c36*a*b78NON‐SympNptomatic18*a:*bAllele 1Allele 2Outcome—a-a, b-b: sibling cases(n.a) data, not available

MCAD deficiencyMedium-chain acyl-CoA dehydrogenase deficiencyIncidence1: 10,000 (Caucasian, a common mutation, 985A G)1: 110,000,((Japanese,p, a common mutation,, 449delCTGA))Acute symptomstriggered by longfastingVomiting, lethargy, acute encephalopathySudden deathLaboratory test(acute)Hypoglycemia, hyperammonemiaS/ SMS/MS(blood AC)BiochemicalMarkers(mass spectrometry) GC/MS(urinary OA)PrognosisC8, C6,CC C10,CC8/C10Hexanoylglycineygy((HG))Suberylglycine (SG)Dicarboxylic acidsAs many as 35% have no episodes lifelongThe 1st attack occurs before 3 to 4 years25% of children suddenly die during the 1st attack

Common mutation in MCAD deficiencyCaucasian985A G(90%)? ?Japan449delCTGA(45%)

CASE 219-year-old woma with VLCAD deficiencyRepeated episodes of myopathy-likemyopathy like illnessafter adolescence

2VLCAD deficiency:y adult onset, myopathic form【Case】 18y female【Family history】 nothing special【Clinical history】Since around 10 yrs of age,Exercise-induced myalgia at legs14 yrs Episodic general fatigue17 yrs〜 Frequent episodes of the fatigue.Sometimes red-colored urine (myoglobinuria)yrs Entered a university,y, and a yearyoff.18 yAdministered for thorough medical check

Routine laboratory testsBiochemistryAST 104ALT 55LDH 726CK2,520,ALD 1.9 （2.7 5.9）BUN 13Total ketone634 μmol/L（ 150）Acetoacetate 195 μmol/L3-OH-butyrate 439 μmol/LBlood carnitine：total62.6 μmol/Lfree44.5 μmol/LUrine myiglobin 11 ng/mL（ ４）Urinary organic acid：WNLECG, Cardiac echo, X-ray:no abnormalities

MS/MSによる 3-C2d3-C2C2C2 (I.S.)C14:2C14 C2C2D9-C8D9-C8((I.S.)((I.S.)))d3-C4d3-C4(I(I.S.)( SS ))(I.S.)C3C3C4C4C8:1C8:1対照

First discoverer of VLCAD and TFP(JBC, 1992)and many of peroxisomal enzymesReportedVLCAD deficiencyBertland C, et al ((BBA, 1993))Aoyama, et al (BBRC, 1993)Yamaguchi S et al (Ped Res, 1993)(1990)Professor Takashi Hashimoto,Department of Biochemistry, Shinshu University, Japan30

Identification of VLCAD deficiencyy(Yamaguchi S, Hahismoto T, Tanaka K, et al. Ped Res, 1993)C 123 4 5 6VLCAD31

CASE 32-year-old boy with glutaric acidemia type IIBezafibrate ((BEZ)) showed dramatic effects

Case: 2y9m boy (intermediate form) Past history– Detected by NBS using MS/MS– 1yy : Repeatedpepisodespof unconsciousness– 1y7m : Lethargy due to hypoglycemia– 2y2my: Respiratorypy failure followingg RSV infection( ICU for 3 weeks) Clinical findings– Muscle weakness : moderate– Unstable gait– Developmental delay : DQ 59 (Enjouji system)

PATHOPHYSIOLOGYGl t i aciduriaGlutaricid i typetII (GA2)FAO pathwaySCAD, MCAD, LCAD, VLCADBCAA pathwayIsovaleryl-CoAIsovalerylCoA DHisobutyryl-CoA DHmethylbutyryl-CoA DHLysine & tryptophanglutaryl-CoAl tl C A DHOtherssarcosine DH2-hydoxyglutarate DHFADETFαETFβFADH2ETFDHCoQRespiratorychainATPDH: dehydrogenaseETF: electron transfer flavoproteinETFDH: ETF dehydrogenase

Topics in treatmentBezafibrate- HypolipidemicHli id i drugd- PPAR agonist(peroxisome proliferation activated receptor)

Changes of Acylcarnitines Before and After Bez additionIn vitro probeassayBefore additionof Bezafibrate(BEZ)VLCAD deficiency(long chain defect)Glutaric acidemia type 2(broad range defect)30302020C12010C16C140C8C410C6C12C160C2In the presenceof BEZC10C4 C6C8 C10 C12 C14C16Bez30C220101000C4 C6C8 C10 C12 C14C8 C10 C12 C14C16C16Bez3020C2C4 C6C2C4 C6C8 C10 C12 C14※ Bez may up-regulate mitochondrial β-oxidation enzymes.C1636

A Clinical trial of Bezafibratefor a 2-year-old boy withBezGlutaric acidemia type 2mg/day1002003002002506 006.00C4Changes ofBlood acylcarnitine3.001.40C40.002 002.00C10, C81.00C100.40C80.00Before 1w2m6m9m12m37

Clinical Trial of Bezafibratein a 2y9m old boy with glutaric acidemia type 2Bezafibrate treatmentBefore4 months afterDevelopmental test(E j ji system))(EnjojiDQ 59(2 9 )(2y9m)79(3 3 )(3y3m)10days after Bez start,M k dl iimprove gait,Markedlyitbecome to climb stairsG itGaitunstablet blhypoglycemia (frequent)―(none for 24 months) ―GGenerall ffatigueti

CASE 4Neonatal osent form of TFP deficiencyPPrenatalt l diagnosisdii off theth nextt 2 pregnancy

Prenatal diagnosis of mitochondrial trifunctionalprotein (TFP)p() deficiencyyOuter membraneACSCPT2carnitinelong-chainfatty acid（長鎖脂肪酸）acyl arnitineVLCADETFenoyl-CoAacyl-CoAInner membraneTFP3-OH-acyl-CoA3-ketoacyl-CoAC16-OHC18 1 OHC18:1-OHLong-chain β-oxidation（C18 C12)acetyl-CoAMedium-chain β-oxidation（C12 C4)

Clinical course of the probandNormal delivery:att 38wks38 k off gestation,t ti BirthBi th weighti ht 2588g25882 days after birth:hypotonia, hypoglycemia, cardiomyopathy6 days of ageDied, despite no response of any treatmentsPostmortem investigation:diagnosis of TFP deficiency

ImmunoblottinggC: controlT: TFP deficiencyV: VLCAD deficiencyE: rat enzymeEnzymes and antibodies aregifted by Professor T. Hashimoto,Biochemistry Shinshu UniversityBiochemistry,42

Prenatal diagnosis for OA and FAOD in ShimaneUniversityAmniocenthesisAminiotic fluid Collected at around 16 wks of gestationplacentaOrganic acid (GC/MS)acylcarnitine (MS/MS）CentrifugesupernatantuterusGene analysispelletamniotic fluidCulturedamnioticcellsEnzyme assayImmunoblottingyGene analysis

Prenatal Diagnosis of TFP deficiency28才28才① IVS16 2 T G②IVS13 1 G ACase 16才Case 2生後6日死亡①IVS16 2 T G② IVS13 1 G A①IVS16 2 T G② IVS13 1 G A① wild② wild

Acylcarnitine analysis of amniotic ase 10.140.12case 10.11case 10 080.080.080.040.0070Normaln 6case 200.0045Normaln 6case200.017Normaln 6case 20

Results of inherited metabolic disease of organic andfattyy acids((1998 to 2014, Shimane University)y)DiseaseNo. of caseaffected（112）（43）Methylmalonic acidemia8032Propionic acidemia168Glutaric acidemia type 1112Multiple carboxylase51（10）（2）TFP deficiencyd fi i41VLCAD deficiency30CACT deficiency21Glutaric acidemia type 210（21）（5）Citrulinemia type 110Infant death of unknown origin (from Asia)205（total）（143）（50）（Organic acidemai）（Fatty acid oxidation defect）（Others）GC/MSMS/MSGene analysisCourtesy byDr.Hasegara, Shimane

Thank youyIwami Kagura Dancing, Shimane

Fatty acidFatty acid β-oxidationoxidation defects (FAODs)defects (FAODs) β-Oxidation Metabolic Step Disorders Metabolic Step Carnitine uptake defect (CUD) CPT1 deficiency 1) Carnitine cycle CPT2 deficiency CACT deficiency 2) Long-chain β-oxidation VLCAD deficiency TFP deficiencyTFP deficiency 3) Medium to short chain MCAD deficiency