defining the peroxisome - uza cema disease… · brown etal (1982) johns hopkins med. j. 151,...

PEROXISOMAL DISEASES

Ronald JA Wanders

Laboratory for Genetic Metabolic Disorders

Department of Pediatrics & Clinical ChemistryDepartment of Pediatrics & Clinical Chemistry

Academic Medical Center

University Hospital Amsterdam,

Defining the peroxisome

10

20

30

40

50

enzy

me a

ctiv

ity (%

of to

tal)

katalase

glutDH

PGI

B-Hex

esterase

peroxisomes mitochondria

1954 Ultrastructural identification of peroxisomes as small cellular bodies (“microbodies”) by Rhodin

1960s Biochemical characterisation of peroxisomes byDeDuve and co-workers

1976 Identification of a complete fatty acid ß-oxidation system in rat liver peroxisomes

00 2 4 6 8 10 12 14 16 18 20

CEREBRO-HEPATO-RENAL SYNDROME OF ZELLWEGER = ZELLWEGER SYNDROME (ZS)

First described in 1964

More than 200 patients described in literature

Zellweger patients show a great number of abnormalities including craniofacial, neurological, ocular, hepatological and skeletal aberrations

Patients usually die early in life within the first year

Peroxisomes are absent in all body cells of Zellweger patients as first shown by Goldfischer etal. (1973) Science 182, 62-64 in hepatocytes and kidney tubule cells.

KEY FINDINGS INDICATING THAT ZELLWEGER SYNDROME IS TRUELY A DISEASE OF THE PEROXISOME.

Brown etal (1982) Johns Hopkins Med. J. 151, 344-351

1. ACCUMULATION OF HEXACOSANOIC ACID (C26:0)IN PLASMA FROM ZELLWEGER PATIENTS

CONTROL

ZELLWEGER

C26:0 (µmol/L) in plasma

0 6 12

KEY FINDINGS INDICATING THAT ZELLWEGER SYNDROME IS TRUELY A DISEASE OF THE PEROXISOME.

2.PLASMALOGEN DEFICIENCY IN ZELLWEGER SYNDROMEHeijmans etal (1983) Nature 306, 69-70.

E +

pP

E)

60

Per

cen

tag

e p

PE

of

(P

0

10

20

30

40

50

brain kidney liver muscle heart

Zellweger Controls

ABNORMAL METABOLITES IN ZELLWEGER SYNDROME

Zellweger

1. Accumulation of very-long-chain fatty acids

2. Deficiency of plasmalogens

Fatty acid ß-oxidationin peroxisomes

Ether-phospholipid biosynthesis

syndrome


Zellweger





syndrome

3. Accumulation phytanic acidFatty acid

alpha-oxidation


Zellweger



3. Accumulation phytanic acid



Fatty acid l h id tisyndrome

p y

4. Increased urinary excretion of oxalate and glycolate

alpha-oxidation

Glyoxylatedetoxification

5. Increased L-pipecolic acidL-Pipecolic acid

oxidation

METABOLIC FUNCTIONS OF PEROXISOMES IN HUMANS

Fatty acid ß-oxidation

Fatty acidalpha-oxidation

Etherphospho-lipid

biosynthesis

METABOLIC


Otherfunctions

L-Pipecolic acid oxidation

METABOLIC FUNCTIONS

OF PEROXISOMES IN HUMANS

Metabolic abnormalities in ZS

Erythrocyte plasmalogens

VLCFA

Bile acid intermediates

Pristanic acid *

METABOLIC


Etherphospho-lipid

biosynthesis

Phytanic acid *

* Maybe normal, dependant on diet

Urinary oxalate and glycolate #

#Moderately elevated, AGT normal

L-Pipecolic acid

FUNCTIONS OF PEROXISOMES

IN HUMANS



L-pipecolic acid oxidation

Metabolic abnormalities in ZS

Erythrocyte plasmalogens

VLCFA

Bile acid intermediates

Pristanic acid *

METABOLIC


Etherphospho-lipid

biosynthesis

UNIQUE SETOF

Phytanic acid *

* Maybe normal, dependant on diet

Urinary oxalate and glycolate #

#Moderately elevated, AGT normal

L-Pipecolic acid

FUNCTIONS OF PEROXISOMES

IN HUMANS



L-pipecolic acid oxidation

OFPEROXISOMALBIOMARKERS

LIST OF PEROXISOMAL DISORDERSPEROXISOME BIOGENESIS DISORDERS

- Zellweger syndrome (ZS)- Neonatal adrenoleukodystrophy (NALD)- Infantile Refsum disease (IRD)- Rhizomelic chondrodysplasia punctata (RCDP)

SINGLE PEROXISOMAL ENZYME DEFICIENCIES

- X-linked adrenoleukodystrophy- D-bifunctional protein deficiency- Acyl-CoA oxidase deficiency- 2-Methylacyl-CoA racemase deficiency- Sterol carrier protein x deficiency- DHAPAT-deficiency (RCDP Type 2)- Alkyl DHAP-synthase deficiency (RCDP Type 3)- Refsum disease- Hyperoxaluria Type 1- Acatalasaemia

NEWLY IDENTIFIED PEROXISOMAL DISORDERS

DISORDERS OF PEROXISOMAL DYNAMICS(FISSION, ELONGATION ETC)

- DLP1 deficiency (Waterham etal 2007 New Engl.J.Med. 356, 1736-1738- PEX11ß deficiency (Ebberink etal 2012 J Med Genet 2012 May;49(5):307-13 )PEX11ß deficiency (Ebberink etal 2012 J Med Genet. 2012 May;49(5):307 13 )

PEROXISOMAL DYNAMICS

ELONGATION &

CONSTRICTION

FISSION

PEX11B FIS1

DLP1

MITOCHONDRIAL DYNAMICS

FIS1DLP1

Maintenance of a functional mitochondrial tubular network is the balanced result of the opposing actions of proteins promoting mitochondrial fusion and those

promoting mitochondrial fission

MFN1MFN2OPA1


LIST OF PEROXISOMAL DISORDERS

PEROXISOME BIOGENESIS DISORDERS


- X-linked adrenoleukodystrophy- D-bifunctional protein deficiency

Pathway affected

Zellweger spectrum disorders

D bifunctional protein deficiency- Acyl-CoA oxidase deficiency- 2-Methylacyl-CoA racemase deficiency-Sterol carrier protein x deficiency

- DHAPAT-deficiency (RCDP Type 2)-Alkyl DHAP-synthase deficiency (RCDP Type 3)

- Refsum disease

-Hyperoxaluria Type 1

- Acatalasaemia

Beta-oxidation

Plasmalogen biosynthesis

Alpha-oxidation

H2O2 metabolism

Glyoxylate metabolism





- X-linked adrenoleukodystrophy- D-bifunctional protein deficiency

Pathway affected


D bifunctional protein deficiency- Acyl-CoA oxidase deficiency- 2-Methylacyl-CoA racemase deficiency-Sterol carrier protein x deficiency

- DHAPAT-deficiency (RCDP Type 2)-Alkyl DHAP-synthase deficiency (RCDP Type 3)

- Refsum disease

-Hyperoxaluria Type 1

- Acatalasaemia

Beta-oxidation


Alpha-oxidation

Glyoxylate metabolism

H2O2 metabolism

ZELLWEGER SPECTRUM DISORDERS :

ZELLWEGER SYNDROME, NEONATAL ADRENOLEUKODYSTROPHY AND INFANTILE REFSUM DISEASE

:

PHENOTYPIC VARIANTS WITH OVERLAPPING CLINICAL SIGNS AND SYMPTOMS RANGING IN SEVERITY FROM ZS TO NALD AND IRD

Common features include neurodevelopmental delay, retinopathy, perceptive deafness, and variable liver disease

IDENTIFICATION OF HUMAN PEROXISOME BIOGENESIS

GENES BY SEQUENCE SIMILARITY

CG Gifu Baltimore A’dam Gene involved

1. A 8 IX PEX26/15

2. B 7/5 XI PEX10

3. C 4/6 III PEX6

4. D 9 X PEX16

5 E 1 II PEX15. E 1 II PEX1

6. F 10 V PEX2

7. G 12 VIII PEX3

8. H 13 VII PEX13

9. J 14 XIII PEX19

10. - 2 IV PEX5

11. - 3 VI PEX12

12. - - XII PEX14

RCDP NA NA NA PEX7

ZSDs

5s

5s

19

5s

Cargobinding

Recycling

HUMAN PEROXISOME BIOGENESIS

PTS1 proteinMembrane protein

5s

14 13 102 12

6

26

1

316

Docking Import

PTS1 protein

5L

7

5L7

19

Cargobinding

Recycling



5L7

14 13 102 12

6

26

1

5L

316

Docking Import

PTS2 proteinPTS2 protein

5s

5L5s

7

5L7 19

5s

Cargobinding

Recycling


PTS1 protein PTS2 proteinMembrane protein

14 13 102 12

6

26

1

316

Docking Import

PTS1 protein PTS2 protein

COMPLEMENTATION ANALYSIS OF PBD CELLS

Co-transfection of PEXx and GFP-SKL

Re-appearance of peroxisomes:

Defective PEXxGFP-SKL

pcDNA3 PEXx

no peroxisomes:

No defective PEXx

repeat with other PEXX cDNAs

Patient cell CG ?

GFP-SKL

+pcDNA3-PEXx

+pEGFP-SKL

COMPLEMENTATION ANALYSIS OF PBD CELLS

Co-transfection of PEXX and GFP-SKL

Patient cell + pEGFP-SKL+ pPEX1(2 days after transfection)

PEX1 not defective

Patient cell + pEGFP-SKL + pPEX1(2 days after transfection)

PEX1 defective

PBD GENETIC COMPLEMENTATION ANALYSIS

Results after analysis of 600 PBD patients (PEG + GFP-SKL)

PEX gene

1 PEX1

2 PEX2

3 PEX3

4 PEX5

Phenotype

ZS, NALD, IRD

ZS, NALD, IRD

ZS

ZS NALD IRD

Number of cell lines

355

23

2

13

Frequency (%)

59

4

0.5

24 PEX5

5 PEX6

6 PEX10

7 PEX12

8 PEX13

9 PEX14

10 PEX16

11 PEX19

12 PEX26/15

ZS, NALD, IRD

ZS, NALD, IRD

ZS, NALD

ZS, NALD, IRD

ZS, NALD

ZS

ZS, NALD

ZS, IRD

ZS, IRD

13

97

18

51

8

2

3

4

20

2

16

3

9

1

0.5

1

1

3

Data from Ebberink etal Hum Mutat. 2011 Jan;32(1):59-69

PBD GENETIC COMPLEMENTATION ANALYSIS

Results after analysis of 600 PBD patients (PEG + GFP-SKL)

PEX gene

1 PEX1

2 PEX2

3 PEX3

4 PEX5

Phenotype

ZS, NALD, IRD

ZS, NALD, IRD

ZS

ZS NALD IRD

Number of cell lines

355

23

2

13

Frequency (%)

59

4

0.5

24 PEX5

5 PEX6

6 PEX10

7 PEX12

8 PEX13

9 PEX14

10 PEX16

11 PEX19

12 PEX26/15

ZS, NALD, IRD

ZS, NALD, IRD

ZS, NALD

ZS, NALD, IRD

ZS, NALD

ZS

ZS, NALD

ZS, IRD

ZS, IRD

13

97

18

51

8

2

3

4

20

2

16

3

9

1

0.5

1

1

3

Data from Ebberink etal Hum Mutat. 2011 Jan;32(1):59-69

PEROXISOME BIOGENESIS DISORDERS AND MUTATION ANALYSIS OF THE PEX1 GENE

MUTATED IN 59% OF PBD-PATIENTSMany private mutations

Two frequently occurring mutations identified

c.2528 G>A (p.G843D)*

ll l f i ti t l ti 36%

1

- allele frequency in our patient population : 36%

- G843D substitution causes instability of Pex1p with partial reduction in function

- patients homozygous for c.2528 G A all show MILD phenotype (IRD)

c.2097_2098insT (p.I700fsX41)**

- allele frequency in our patient population : 17%

- truncated protein with full loss of Pex1p function

- patients homozygous for c.2097 insT all show SEVERE phenotype (ZS)

* Reuber etal. (1997) Nat. Genet. 17, 445-448 ; Portsteffen etal (1997) Nat. Genet. 17, 449-452

** Maxwell etal (1999) Hum. Genet 105, 38-44; Collins etal. (1999) Hum. Mutat. 14, 45-53

2



:


c.2528G>A / c.2528G>Ac.2097_2098insT / c.2097_2098insT

CATALASE IMMUNOFLUORESCENCE IN FIBROBLASTS FROM PATIENTS WITH DIFFERENT MUTATIONS IN PEX1

PEX1 p.I700fsX41 (c.2097_2098insT) PEX1 p.G843D (c.2528G>A)

0 20 40 60 80 100 120

Control

Pristanic acid ß-oxidation (%)

PRISTANIC ACID ß-OXIDATION IN FIBROBLASTS FROM PATIENTS WITH DIFFERENT MUTATIONS IN PEX1

PEX1 p.I700fsX41 (c.2097_2098insT)

PEX1 p.G843D (c.2528G>A)

37°C

EFFECT OF A SHIFT IN TEMPERATURE (37°C 30°C) ON THE NUMBER OF PEROXISOMES IN FIBROBLASTS FROM

A PATIENT HOMOZYGOUS FOR THE MILD c.2528G>A (G843D) MUTATION

37°C 30°C

Gootjes et al. HUMAN MUTATION 24:130-139 (2004)

0 20 40 60 80 100 120

Control

PEX1 p.I700fsX41 (c.2097_2098insT)

PEX1 p.G843D (c.2528G>A)

Pristanic acid ß-oxidation (%)

PRISTANIC ACID ß-OXIDATION IN FIBROBLASTS FROM PATIENTS WITH DIFFERENT MUTATIONS IN PEX1

37°C

Control

Control

40°C

37 C

30°CPEX1 p.I700fsX41 (c.2097_2098insT)

PEX1 p.G843D (c.2528G>A)

PEX1 p.I700fsX41 (c.2097_2098insT)

PEX1 p.G843D (c.2528G>A)

CONCLUSION

A shift in temperature (37°C 30°C) not only induces an increase in the number of peroxisomes but also restores the functional capacity of peroxisomes

QUESTIONIs it possible to restore peroxisome biogenesis in fibroblasts from patients homozygous for PEX1p.G843D at 37°C?

Zhang etal Proc Natl Acad Sci U S A. 2010 Mar 23;107(12):5569-74.

Screened a large library of small compounds (2080) for their capacity to restore peroxisome formation in cells homozygous for c.2528 G>A (p.G843D)

2 new compounds identified : Ro31-8220, GF109203X

PEROXISOMES IN FIBROBLASTS FROM PATIENTS WITH DIFFERENT MUTATIONS IN PEX1 AND THE EFFECT OF

GLYCEROL AND ARGININE

Untreated

Arginine 40 mM

Glycerol 5% (v/v)

PEROXISOMES IN FIBROBLASTS FROM PATIENTS WITH DIFFERENT MUTATIONS IN PEX1 AND THE EFFECT OF INCREASING CONCENTRATIONS OF ARGININE (37°C)

Glycerol and arginine but also other compounds are able to (partially) restore peroxisome formation and function (!) in cells from patients homozygous for p.G843D

CONCLUSION

CURRENT RESEARCH

Practical approach :

1. Continue on the arginine line….

2. Test arginine in vivo (mouse model!)

Fundamental approach

Resolve the folding pathway for PEX1 and identify which factors are involved



:


Common features include neurodevelopmental delay, retinopathy, perceptive deafness, and variable liver disease

Third child of non-consanguineous parents

Normal neurological examination at 14 years

At age 18, cerebellar signs with impaired gait, dysmetria, ataxia of the trunc, dysarthria, and slow saccades without oculomotor apraxia and hyporeflexia


MARKED HETEROGENEITY IN CLINICAL SIGNS AND SYMPTOMS

EXAMPLE : PBD DEFECT IN A PATIENT WITH ISOLATED CEREBELLAR ATAXIA

a d s o saccades t out ocu o oto ap a a a d ypo e e a

ICARS score 17/100

No other neurological symptoms

WISC-IV analysis : full, verbal, and performance IQ were 80, 74 and 75 (normal range)

Brain MRI: isolated cerebellar atrophy at 18 years

Normal electroretinogram, peripheral nerve conduction and visual, brainstem auditory, and somatosensory evoked potentials

Molecular studies of cerebellar ataxia genes including frataxin and aprataxin : normal

Peroxisomal parameters in plasma : abnormal !


A MARKEDLY HETEROGENEOUS GROUP OF DISEASES


Sevin C, Ferdinandusse S, Waterham HR, Wanders RJ, Aubourg P. Orphanet J Rare Dis. 2011 Mar 10;6:8.


A MARKEDLY HETEROGENEOUS GROUP OF DISEASES


Detailed studies in fibroblasts : very mild abnormalities with the virtual normal presence of peroxisomes

Sequencing of candidate PEX genes revealed a homozygous c.865_866insA mutation in the PEX2 gene leading to a frameshift 17 codons upstream of the stop codon. PEX gene mutations usually result in a severe neurological phenotype (Zellweger spectrum disorders).

PS Similar patient with isolated cerebellar ataxia identified by Regal etal Ann Neurol. 2010 Aug;68(2):259-63

PSEUDO ZELLWEGER SYNDROME(Goldfischer etal (1986) J.Pediatr.108, 25-32)

Infant girl with the full spectrum of clinical and pathological abnormalities described for Zellweger syndrome including craniofacial dysmorphia, neuronal migration defect, etc.

Abnormal VLCFA’s and bile acid intermediates

However, abundant peroxisomes in liver parenchymal cells !

PSEUDO ZELLWEGER SYNDROME(Goldfischer etal (1986) J.Pediatr.108, 25-32)

Infant girl with the full spectrum of clinical and pathological abnormalities described for Zellweger syndrome including craniofacial dysmorphia, neuronal migration defect, etc.

Ab l VLCFA’ d bil id i t di tAbnormal VLCFA’s and bile acid intermediates

However, abundant peroxisomes in liver parenchymal cells !

2002 : Identification of true defect in this patient at the level of D-bifunctional protein

(Ferdinandusse etal (2002), Am.J.Hum.Genet. 70, 1589-1593)

PEROXISOMAL FATTY ACID ß-OXIDATION IN HUMAN PEROXISOMES AND ITS DEFICIENCIES

C26:0 pristanic acid THCA

cholesterol

CoASH

CoASH

ATP

AOX1 AOX2

DBP

pTH1 pTH2

acylCoA oxidase deficiency D-bifunctional protein deficiency

Racemase deficiency

X-ALD

ALDPracemase

KAPLAN-MEIER SURVIVAL ANALYSIS IN D-BIFUNCTIONAL PROTEIN DEFICIENCY

D-BIFUNCTIONAL PROTEIN DEFICIENCY(Ferdinandusse etal (2006), Ann.Neurol. 59, 92-104)

126 patients with D-BP deficiency identified in our laboratory

Questionnaires sent out.

Outcome :

Neonatal hypotonia

Seizures in first month of life

83/85 (98%)

79/85 (93%)

External dysmorphia

Visual system failure

Neocortical dysplasia

Liver disease

Demyelination cerebral hemispheres

Cerebellar atrophy

Early death (< 2year)

53/79 (67%)

40/73 (55%)

12/45 (27%)

18/69 (26%)

9/47 (19%)

8/49 (16%)

104/116 (90%)

PEROXISOMAL FATTY ACID ß-OXIDATION IN HUMAN PEROXISOMES AND ITS DEFICIENCIES

C26:0 pristanic acid THCA

cholesterol

CoASH

CoASH

ATP

AOX1 AOX2

DBP

pTH1 pTH2

acylCoA oxidase deficiency D-bifunctional protein deficiency

Racemase deficiency

X-ALD

ALDPracemase

Initial test PLASMA VLCFA-analysis

Abnormal

Definitely ZSD, DBP deficiency,

LABORATORY DIAGNOSIS OF THE ZELLWEGER SPECTRUM DISORDERS (ZS,NALD,IRD), D-BIFUNCTIONAL PROTEIN

DEFICIENCY AND ACYL-CoA OXIDASE DEFICIENCY

Definitely ZSD, DBP deficiency,or ACOX deficiency

Full study in fibroblasts

Biogenesis defect

Complementation analysis

DNA analysis (PEX genes)

Single enzyme defect

DBP and ACOX activity

DNA analysis

Initial test PLASMA VLCFA-analysis

Abnormal

Definitely ZSD, DBP deficiency, ZSD, DBP deficiency, or

Normal

LABORATORY DIAGNOSIS OF THE ZELLWEGER SPECTRUM DISORDERS (ZS,NALD,IRD), D-BIFUNCTIONAL PROTEIN


Definitely ZSD, DBP deficiency,or ACOX deficiency


Biogenesis defect

Complementation analysis

DNA analysis (PEX genes)

Single enzyme defect

DBP and ACOX activity

DNA analysis

ACOX deficiency not excluded


Normal

ZSD, DBP deficiency,or ACOX deficiency

virtually excluded

Abnormal

PLASMA VLCFA MAY BE ENTIRELY NORMAL IN PATIENTS AFFECTED BY THE ZELLWEGER SPECTRUM DISORDERS (ZS,NALD,IRD), D-BIFUNCTIONAL PROTEIN


Example : Rosewich etal (2006) Neuropediatrics 37, 95-98

2 Month old boy

Dysmorphic features including high forehead broad nasal bridge high archedDysmorphic features including high forehead, broad nasal bridge, high-arched palate, syndactyly (second and third toes on both feet)

Psychomotor retardation, muscular hypotonia, clonic seizures

MRI : typical pattern showing pachygyria, perisylvian polymicrogyria, cerebral and cerebellar white matter abnormalities

Retinopathy

Repetitive measurement of VLCFA’s and other peroxisomal metabolites :

NO ABNORMALITIES

Detailed studies in fibroblasts : acyl-CoA oxidase deficiency, splice-site mutation

Other examples : Soorani-Lunsing etal (2005) J.Inher.Metab.Dis. 28, 1172-1174, etc.






X-LINKED ADRENOLEUKODYSTROPHY

(XALD)

Most frequent peroxisomal disorder

Marked phenotypic heterogeneity

Two phenotypes most frequent including :p yp q g

1. Childhood cerebral ALD (CCALD)

2. Adrenomyeloneuropathy (AMN)

In all XALD phenotypes : accumulation of very-long-chain fatty acids due to their deficient oxidation in peroxisomes

Caused by mutations in the ABCD1 gene wich codes for the peroxisomal half-ABC-transporter ALDP (Mosser etal 1993, Nature 361, 726-730)

X-LINKED ADRENOLEUKODYSTROPHY

Accumulation of very-long-chain fatty acids in plasma

CONTROL

X-ALD

C26:0 (µmol/L) in plasma0 1.5 3

CONTROL

X-ALD

Activity (%)

Deficient oxidation of C26:0 in intact fibroblasts

C26:0

Acetyl-CoA

Peroxisomal ß-oxidation (? cycles)

FATTY ACID ß-OXIDATION OF C26:0 INCLUDING THE ROLE OF ALDP AND THE CROSSTALK BETWEEN MITOCHONDRIA AND PEROXISOMES

C26:0-CoA

TE

C26:0-CoA

ALDP ALDP

AcetateMedium chain

fatty acidsTE Medium chain

Acyl-CoA

Deficient in X-ALD

y

Acetyl-carnitine

Acetyl-carnitine

Krebscycle

CO2

CAT

Mitochondrion

Acetate

CAT

Peroxisome

CoASH

Medium chain Acyl-carnitine

COT

fatty acids

Medium chain Acyl-carnitine

CPT2

ß-oxidation

CoASH

Acyl CoA

Medium chain fatty acids

CACT

Medium chain Acyl-CoA

CO2

Acetyl-CoA

1 2 3 4 5 6

Matrix

Membrane

PUTATIVE SECONDARY STRUCTURE OF ALDP IN THE PEROXISOMAL MEMBRANE

Walker A

Walker B

NH2

COOH

Cytosol

C sequence

MOLECULAR ANALYSIS OF ABCD1 IN X-ALD

587 different mutations identified (http://www.x-ald.nl)

300 (51%) missense mutations

166 (28%) frame shift mutations

68 (12%) nonsense mutations

35 (6%) small insertions/deletions

18 (3%) larger deletions of one or more exons

1 2 3 4 5 6

Matrix

Membrane

PUTATIVE SECONDARY STRUCTURE OF ALDP IN THE PEROXISOMAL MEMBRANE

Arg113fs

Walker A

Walker B

NH2

COOH

Cytosol

C sequence

Asp194His

Arg660Trp

Arg554His

Glu609GlyAla616Thr

EFFECT OF TEMPERATURE ON THE AMOUNT OF ALDP PROTEIN IN FIBROBLASTS FROM PATIENTS CARRYING

DIFFERENT MISSENSE MUTATIONS IN ABCD1

EFFECT OF TEMPERATURE ON C26:0 BETA-OXIDATION IN FIBROBLASTS FROM PATIENTS CARRYING DIFFERENT

MISSENSE MUTATIONS IN ABCD1

37°C 30°C

2% → 17%0% → 0%

60% → 75%

EFFECT OF TEMPERATURE ON THE C26:0/C22:0 RATIO IN FIBROBLASTS FROM PATIENTS CARRYING DIFFERENT

MISSENSE MUTATIONS IN ABCD1

2% → 19%

1% → 12%5% → 34%

CONCLUSIONS

A shift in temperature (37°C 30°C) leads to increased amounts of ALDP in fibroblasts from some but not all patients carrying different missense mutations in ABCD1

Increased amounts of ALDP do not necessarily imply improved catalytic functioning of ALDP

CURRENT RESEARCH

Practical approach : search for compounds able to counteract misfolding of ALDP at 37°C

Fundamental approach : identification of the factors involved in folding of ALDP






Rhizomelic chondrodysplasia

punctata (RCDP)

- Growth retardation, dwarfism

- Facial dysmorphia

- Rhizomelic shortening of

upper extremities

- Severe mental retardation

MUTANT GENE : PEX7

CODES FOR THE PTS2 RECEPTOR

REQUIRED FOR THE CORRECT TARGETING OF 3 PEROXISOMALPROTEINS

PLASMALOGENS

1=peroxisomal thiolase

2=alkyl DHAP synthase

3=phytanoyl-CoA hydroxylase

PHYTANIC ACID

5L

7

5L7

19

Cargobinding

Recycling



5L7

1.Fytanoyl-CoA hydroxylase

2.Alkyl-DHAP synthase

3.Peroxisomal thiolase

14 13 102 12

6

26

1

5L

316

Docking Import

ACYLCoA

ACYLCoA

ACYLDHAP

DHAPAT

?

DHAP

CoASH

LONG-CHAIN ALCOHOL

NADPH

NADP

ACYLCoA REDUCTASE

?

PEROXISOMES AND THEIR ROLE IN PLASMALOGEN BIOSYNTHESIS

TRANSPORTTO ER

ALKYLDHAPSYNTHASE

ACYLDHAP

ALKYLDHAP

PLASMALOGENS

ERLONG-CHAINALCOHOL

FATTY ACID

PEROXISOME

ACYLCoA

ACYLCoA

ACYLDHAP

DHAPAT

?

DHAP

CoASH

LONG-CHAIN ALCOHOL

NADPH

NADP

ACYLCoA REDUCTASE

?

PEROXISOMES AND THEIR ROLE IN PLASMALOGEN BIOSYNTHESIS

RCDP type 2

TRANSPORTTO ER

ALKYLDHAPSYNTHASE

ACYLDHAP

ALKYLDHAP

PLASMALOGENS

ERLONG-CHAINALCOHOL

FATTY ACID

PEROXISOMERCDP type 3

LABORATORY DIAGNOSIS OF PEROXISOMAL

CHONDRODYSPLASIA PUNCTATA

Initial test PLASMALOGEN analysis in erythrocytes

Abnormal

Definitely RCDP type 1,2 or 3 RCDP type 1,2 or 3

Normal

Definitely RCDP type 1,2 or 3


RCDP type 1

PEX7 ADHAPS

not excluded


Normal

RCDP type 1,2 or 3virtually excluded

Abnormal

RCDP type 2 RCDP type 3

GNPAT




SINGLE PEROXISOMAL ENZYME DEFICIENCIESPathway affected


Pathway affected

Beta-oxidation


Alpha-oxidationGlyoxylate metabolismH2O2 metabolism

ACKNOWLEDGEMENTS

Biogenesis

Merel EbberinkHans Waterham

DNA

Hans Waterham

Clinic

Bwee-Tien Poll-ThePeter Barth

Mouse models

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