Fatty Acid Oxidation Defects and Its Clinical Significance

Seiji YAMAGUCHI , MD Professor Department of Pediatrics, Shimane University, Japan

The 8th International & 13th National Congress on Quality Improvement in Clinical Laboratories、April 22 to 25, 2015, Tehran 1 1 Department of Pediatrics Shimane University

6th ICNS 2006 Sapporo (Awaji) Seoul Shimane Sendai Tokyo Fukuoka 10th ICIEM 2006 Kyoto (Makuhari) Hiroshima Osaka

Tandem MS GC/MS Izumo-Taisha Shrine, Shimane, Japan <1> Outline Fatty Acid Oxidation Disorder (FAOD)

4 Energy production in mitochondria

Glyco lys is

Pyruvate acetyl-CoA TCA cycle ATP

RitRespiratory mitochondrial chain β-oxidation Mitochondrial β-oxidation

Carnitine Long chain FA Medium chain FA

OCTN2 plasma m. １ CoA outer m. CPT1 carnitine acyl-CoA acylcarnitine ２

CACT CPT2 VLCAD TFP inner m. Long chain acylcarnitine acyl-CoA carnitine KAT CAD ３ Medium chain acetyl-CACoA HAD EH

TCA FADH2 ETF ４ Respiratory Respiratory ATP ETF chain chain (CoQ) DH 6 Fatty acid β-oxidation defects (FAODs) β-Oxidation Disorders Metabolic Step

Carnitine uptake defect (CUD) CPT1 deficiency 1) Carnitine cycle CPT2 deficiency CACT deficiency

VLCAD deficiency 2) Long-chain β-oxidation TFP deficiency

3) Medium to short chain MCAD deficiency 4) Electron transfer Glutaric acidemia type II (GA2) CLASSIFICATION of FATTY ACID DISORDERS

CLINICAL FORM FINDING Death in early infancy HlHypoglycem ia 1) Severe form Liver failure Cardiomyypopath y Episodic attacks 2) Intermediate form Acute encephalopathy SddSudden ifinfan tdt dea th Intermittent Episodes of: Lethargy 3) Late onset form Myopathy (myopathic) Myalgia MlbiiMyoglobinuria Liver dysfunction <2> Diagnostic Approaches for Mitochondrial fatty acid oxidation defects (FAOD) Diagnos tic M eth od s

1) 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/MS

Blood filter paper Urine

acylcarnitine・amino acids organic acids （mass screening） （confimative） MCAD deficiency 1) Acylcarnitine (MS/MS) A * * C8 Blood filter paper * * * * *

B * CPT2 deficiency C16 *

C18:1 * * * * * *

control C C2 ＊ C0 ＊ C4 C16 C5 C14 C3＊ ＊ C8 ＊ * Internal standard ＊ ＊ ＊ Abnormal metabolites in impaired β-oxidation

ω-oxidation acyl-CoA Dicarboxylic acid [ω-1]-oxidation (nacylglycine-1)-hydroxy-acid AD + glycine X

+ carnitine acylcarnitine enoyl-CoA ω-oxidation Unsaturated dicarboxylic acid

EH X ω-oxidation 3-hydroxyacyl-CoA 3-hydroxy-dicarboxylic acid + carnitine HAD XX 3-hdhydroxy-acylitilcarnitine

3-ketoacyl-CoA KAT

acyl-CoA acetyl-CoA Urinary organic acid profile of MCAD deficiency (GC/MS)

7-OH-octitanoic octdiitenedioic 3-OH-sebacic 3OH-dodecanedioic (x1,000,000) 1.50

1.25 suberic adipic IS-2 sebacic MCAD deficiency

1.00 HG SG 0.75 IS-1 C24

0.50

0.25

7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 32.5 35.0 37.5 40.0 42.5 45.0 47.5 50.0 52.5

IS-2 control IS-1 ciiitric C24 In vitro probe acylcarnitine (IVP) assay In vitro Probe Assay (enzyme determination)

Palmitate (C16) loading

Glucose low Fatty acid free Enriched carniiitine cultured cell

（β-oxidation） block block M L

C10 C16 Acylcarnitines C2 C8 C14 (MS/MS) C6 C12 C4 Results of nmol/mg prot 50 C2 In vitro probe assay A 40 Palmitate 30 (()load) normal 20 10 0 C2 C4 C6 C8 C10 C12 C14 C16

50 C8 B 40 C4 30 MCAD def 20 C6 （MdiMedium） 10 0 C2 C4 C6 C8 C10 C12 C14 C16 50 C 40 30 VLCAD def 20 C14 （long-chain） 10 C12 C16 0 C2 C4 C6 C8 C10 C12 C14 C16 C10 50 D C8 40 C12 C6 Glutaric acidemia type II 30 C4 C16 （short～lomg） 20 10 17 0 C2 C4 C6 C8 C10 C12 C14 C16 <3> Treatment Fatty acid Oxidation Disorders Treatment for Fatty Acid Disorder 1) Avoid “long fasting” (ex.) < 8 hrs: before 1 yr of age; < 10 hrs: before 2 yrs

2) Early infusion of “glucose” In particular, during stress (infection, over-exercise)

3) “Carnitine therapy” for some cases Except for CPT1 deficiency

4) Some drugs Riboflavin, CoQ 10

5) Dietary therapy (ex.) )hih high car bhdbohydra te /l/ low liidditlipid diet

(ex.) MCT milk (oil) 19 CASE Presentation CASE 1

1-year-old boy with MCAD deficiency Sudden death following common cold 1 MCAD deficiency: Sudden infant death

Case: 1 yr 8 m, boy. Pyrexia due to infection, followed by unconsciousness and convulsion, and suddenly died. Family history: His sister ( 5 yr old) is severely handicapped , since acute encephalopathy at 1 year of age. Acute (1y8m)

C8 Acylcarnitine analysis * C6 (blood filter paper, tandem MS) * C10:1 * Elevation of C6, C8, C10, C10:1 * * * * *

Newborn pppaper C8 * Urinary organic acid (acute stage) * C6 C10:1 Hy poketotic dicarboxy lic acidu ria * Elevation of hexanoylglycine etc. * * * * * 22 Japanese MCAD deficiency Clinical onset, outcome and genotype (Shimane Univ. 2012)

Age at Age at Hypo Genotype Case Outcome onset diagnosis glycemia Allele 1 Allele 2

Symp 1 8m 8m (n.a) c.449-452del c.157C>T Develop. delay 2 1y 1y (+) IVS4+1G>A c.422 A>T Sudden death

t 3 *a 1y 8y 10m (()+) c.449-452del c.449-452del Developpy. delay omatic 4 1y 1m 1y 1m (+) del. ex 11-12 del. ex 11-12 Develop. delay 5 1y 3m 1y 3m (n.a) del. ex 11-12 del. ex 11-12 Develop. delay

6 *b 1y 4m 1y 4m (+) c.449-452del c.449-452del Develop. delay

7 1y 7m 1y 7m (+) c.275C>T c.157C>T Develop. delay 8 *a 1y 8m 1y 8m (+) c.449-452del c.449-452del Sudden death 9 2y 2m 2y 2m (+) c.449-452del c.449-452del normal

N 10 — 5d (-) c.1085G>A c.843A>T normal ON 11 — 5d (-) c.449-452del c.154A>G normal ‐

Sym 12 — 5d (-) IVS3+2T>C c.843 A>T normal 13 — 5d (-) c.449-452del c.212 G>A normal p

tomatic 14 — 5d (-) c.449-452dldel c.134 A>G normal 15 — 5d (-) c.1085G>A c.1184A>G normal 16 — 5d (-) c.449-452del IVS3+5G>A normal 17 — 5d (-) c.449-452del c.820 A>C normal

: — 18 *b 5y 5m (-) c.449-452del c.449-452del normal

a-a, b-b: sibling cases (n.a) data, not available MCAD deficiency Medium-chain acyl -CoA dehydrogenase deficiency

1: 10,000 (Caucasian, a common mutation, 985A>G) Incidence 1: 110,000 ((pJapanese ,a common mutation, 449delCTGA) Acute symptoms Vomiting, lethargy, acute encephalopathy triggered by long Sudden death fasting Laboratory test Hypoglycemia, hyperammonemia (acute) MS/MS C8, C6, C10, Biochemical (blood AC) C8/C10 Markers Hexanoyygylglycine (HG) (mass spectrometry) GC/MS Suberylglycine (SG) (urinary OA) Dicarboxylic acids

As many as 35% have no episodes lifelong Prognosis The 1st attack occurs before 3 to 4 years 25% of children suddenly die during the 1st attack Common mutation in MCAD deficiency

Caucasian Japan 985A>G 449delCTGA (90%) ? ? ? (45%) ? CASE 2

19-year-old woma with VLCAD deficiency Repeated episodes of myopathy -like illness after adolescence 2 VLCAD deficiency: adult onset, myopathic form 【Case】 18y female 【Family history 】 nothing special

【Clinical history】 Since around 10 yrs of age, Exercise-induced myalgia at legs 14 yrs～ Episodic general fatigue 17 yrs〜 Frequent episodes of the fatigue. Sometimes red-colored urine (myoglobinuria) 18 yrs Entered a university, and a y ear off. Administered for thorough medical check Routine laboratory tests

Biochemistry Blood carnitine： AST 104 total 62.6 μmol/L ALT 55 free 44.5 μmol/L LDH 726 CK 2,520 ALD 1.9 （2.7～5.9） Urine myiglobin 11 ng/mL BUN 13 （<４）

Total ketone 634 μmol/L Urinary organic acid：WNL （<150） Acetoacetate 195 μmol/L ECG, Cardiac echo, X-ray: 3-OH-butyrate 439 μmol/L no abnormalities MS/MSによる⾎清アシルカルニチン分析

C14:1C14:1 d3-C2d3-C2 C2C2 (I.S.)(I.S.) 患者

D9-C8D9-C8 (I.S.)(I.S.) d3-C4d3-C4 (I.S.)(I.S.) C14:2 C16 C14: 2 C16 C18:1C18:1 C12C12 C16:1C16:1 C3 C18:2C18:2 C3 C4C4 C8:1C8:1 C12:1C12:1

d3d3-C2-C2 (I.S.)(I.S.) C2C2

D9-C8D9-C8 d3-C4 d3-C4 (()(()I.S.I.S.)) (IS)(I(S)(I..SS..)) 対照

C3C3 C4C4 C8:1C8:1 First discoverer of VLCAD and TFP Reported (JBC, 1992) VLCAD deficiency Bertland C, et al (BBA, 1993) and many of peroxisomal enzymes Aoyama, et al (BBRC, 1993) Yamaguchi S et al (Ped Res, 1993)

(1990)

Professor Takashi Hashimoto, 30 Department of Biochemistry, Shinshu University, Japan Identification of VLCAD deficiency (Yamaguchi S, Hahismoto T, Tanaka K, et al. Ped Res, 1993) C 1 2 34563 4 5 6

VLCAD

31 CASE 3

2-year-old boy with glutaric acidemia type II Bezafibrate (()BEZ) showed dramatic effects Case: 2y9m boy (intermediate form) • Past history – Detected by NBS using MS/MS – 1y ～ : Repeated episodes of unconsciousness – 1y7m : Lethargy due to hypoglycemia – 2y2m : Resppyiratory failure followin g RSV infection (→ ICU for 3 weeks) • Clinical findings – Muscle weakness : moderate – Unstable gait – Developmental delay : DQ 59 (Enjouji system) PATHOPHYSIOLOGY Glut ari cacidur ia type II (GA2)

FAO pathway SCAD, MCAD, LCAD, VLCAD BCAA pathway Isovaleryl-CoA DH FAD isobutyryl-CoA DH methylbutyryl-CoA DH ETFα Lysine & tryptophan ETFDH CoQ gltlutary l-CACoA DH ETFβ Respiratory Others chain sarcosine DH FADH2 2-hydoxyglutarate DH ATP

ETF: electron transfer flavoprotein DH: dehydrogenase ETFDH: ETF dehydrogenase rug Bezafibrate d c i em id p li ypo Hliidi H (peroxisome proliferation activated receptor) - - agonist PPAR Topics in treatment Topics Changes of Acylcarnitines Before and After Bez addition

In vitro probe assay VLCAD deficiency Glutaric acidemia type 2 (long chain defect) (broad range defect)

30 30 C10 Before addition of Bezafibrate 20 20 C8 C12 (BEZ) C12 C6 10 C16 10 C4 C16 C14 0 0 C2 C4 C6 C8 C10 C12 C14 C16 C2 C4 C6 C8 C10 C12 C14 C16

30 Bez 30 Bez In the presence of BEZ 20 20

10 10

0 0 C2 C4 C6 C8 C10 C12 C14 C16 C2 C4 C6 C8 C10 C12 C14 C16

※ Bez may up-regulate mitochondrial β-oxidation enzymes.. 36 A Clinical trial of Bezafibrate for a 2-year-old boy with 300 Glutaric acidemia type 2 Bez 200 200 250 mg/day 100

6.00 C4 Changes of 3.00 Blood acylcarnitine 1.40 C4 0.00 2.00 C10, C8 1.00 C10 0.40 C8 0.00 Before 1w 2m 6m 9m 12m 37 Clinical Trial of Bezafibrate in a 2y9m old boy with glutaric acidemia type 2

Bezafibrate treatment Before 4 months after Developmental test DQ 59 79 (EjjiEnjoji system) (2y9m) (3y3m) 10days after Bez start, GitGait unstbltable MkdliMarkedly improve ga it, become to climb stairs +++ ― hypoglycemia (frequent) (none for 24 months)

GlftiGeneral fatigue ＋ ― CASE 4

Neonatal osent form of TFP deficiency PtldiifthtPrenatal diagnosis of the next 2 pregnancy Prenatal diagnosis of mitochondrial trifunctional pp()rotein (TFP) deficiency

Outer membrane CPT2 ACS carnitine acyl-CoA long-chain fatty acid （長鎖脂肪酸） acylcarnitine

CACT CPT2 VLCAD TFP Inner membrane

3-OH-acyl-CoA ETF CoA 3-ketoacyl-CoA acyl-CoA enoyl-CoA acylcarnitine C16-OH C18:1-OH Long-chain β-oxidation carnitine （C18～C12)

acetyl-CoA Medium-chain β-oxidation （C12～C4) Clinical course of the proband Normal delivery: at 38wkfks of ges ttiBithihttation, Birth weight 2588g

2 days after birth: hypotonia, hypoglycemia, cardiomyopathy

6 days of age Died, despite no response of any treatments

Postmortem investigation: diagnosis of TFP deficiency Immunoblotting

C: control T: TFP deficiency V: VLCAD deficiency E: rat enzyme

Enzymes and antibodies are gifted by Professor T. Hashimoto, Biochemistry, Shinshu University

42 Prenatal diagnosis for OA and FAOD in Shimane University

Amniocenthesis Aminiotic fluid Collected at around 16 wks of gestation

placenta Organic acid (GC/MS) acylcarnitine (MS/MS） Centrifuge

supernatant Gene analysis uterus pellet

amniotic fluid Enzyme assay Cultured Immunoblotting amniotic Gene analysis cells Prenatal Diagnosis of TFP deficiency

28才 28才

① IVS16+2 T>G ②IVS13+1 G>A

Case 1 Case 2

6才 生後6日死亡

①IVS16+2 T>G ①IVS16+2 T>G ① wild ② IVS13+1 G>A ② IVS13+1 G>A ② wild Acylcarnitine analysis of amniotic fluid DBS DBS DBS C16-OH 3.09 C18-OH 0.76 1.9 0.16 C18:1-OH case 1 0.14

0.12 case 1 0.11

0.08 case 1 0.08

0.04

0.007 case 2 0.0045 0.017 case2 case 2 0 0 0 0 Normal Normal Normal n=6 n=6 n=6 Results of inherited metabolic disease of organic and fatty acids (1998 to 2014, Shimane University)

Disease No. of case affected （Organic acidemai （112）（43） Methylmalonic acidemia 80 32 GC/MS Propionic acidemia） 16 8 MS/MS Glutaric acidemia type 1 11 2 Multiple carboxylase 5 1 （Fatty acid oxidation defect （10）（2） TFP d efi ci ency 4 1 VLCAD deficiency 3 0 Gene analysis CACT deficiency） 2 1 Glutaric acidemia type 2 1 0 （Others） （21）（5） Citrulinemia type 1 1 0 Infant death of unknown origin (from Asia) 20 5 Courtesy by （total） （143）（50） Dr.Hasegara, Shimane Thank you

Iwami Kagura Dancing, Shimane