Workshop on

diagnostic approach
to peroxisomal disorders

Calvin Chong
Chemical Pathology, PMH
No, I wont do molecular genetics

in this presentation,
lest all the fun be spoilt
 The cruel facts
These are the tests available in Hong Kon
g
for peroxisomal disorders:

Plasma very long chain fatty acids

Phytanic acid and pristanic acid
Urine pipecolic acid
Urine organic acid profile
 I suppose it is tempting, if the
only tool you have is a hammer, to
treat everything as if it were a
Maslow A.H. (1962)nail.
Toward a Psychology of being
 What about it in Hong Kong?

Other than X-linked adrenoleukodystroph

y spectrum disease all rare
Biochemical analysis available at two un
iversity hospitals (QMH, PWH)
Some common ground

Some common ground


 Some common ground

Two groups of peroxisome disorders

1. Peroxisome biogenesis disorders

in which everything fails (sort of)
2. Single peroxisome enzyme disorders
in which a particular function fails
 Some common ground

Functions of peroxisomes

Ether phospholipid synthesis

Beta oxidation
Alpha oxidation
Metabolism of pipecolic acid
(and so on)
Some common ground
 #1
Disorders of etherphospholipid b
iosynthesis
Disorders of etherphospholipid biosynth
esis
Rhizomelia chondrodys
plasia punctata

Four types
RCDP 1 PEX7 def
RCDP 2 Glycerone-3-P
acyltransferase def
RCDP 3 Alkylglyceron
e-3-P synthase def
RCDP 4 PEX5L def

From: radiopaedia.org
Etherphospholipids & Plasmalogens

What are plasmalogens?

Phospholipids modified in the sn-1 positi
on to hold an unsaturated alkenyl by ethe
r bond

Usual
Plasmaloge
(diacyl)
ns
phospholipi
ds
Endoplasmic Peroxisom
reticulum Acyl-CoA e

RCDP 2

Acyl-DHA-P

RCDP 3 PTS2
RCDP 1
Alkyl-DHA-P Alkyl-DHA-P
RCDP 4

Alkyl-G-3-P Alkyl-G-3-P

Plasmalogens
 #2
Abnormal peroxisomal
beta oxidation
 Peroxisomal beta oxidation
Very long straight chain fatty acids
Acetyl-CoA + [n-2] VLCFA
Pristanic acid
Propionyl-CoA + Acetyl-CoA +
4,8-dimethyl-nonanoyl-CoA

(Ratios are more helpful at times)

(Concentration used for illustrations here)
 C26:0 Pristanic acid

C26:0-CoA Pristanoyl-CoA
X-linked P Biogenesis Cytoplasm
ALD def Peroxisom
C26:0-CoA Pristanoyl-CoA e

ACOX1 def
2-Pristenoyl-
-C26:1-CoA
2
CoA
D-BP
def
3-keto-C26:0- 3-keto-
CoA pristenyol-CoA
(PTS2)
4,8,12-
C24:0-CoA trimethyltridecanoyl
-CoA
 2.47 C26:0 Pristanic acid 0.21
RI: <1.3 RI: <3.0
mol/L mol/L

C26:0-CoA Pristanoyl-CoA
X-linked P Biogenesis Cytoplasm
ALD def Peroxisom
C26:0-CoA Pristanoyl-CoA e

ACOX1 def
2-Pristenoyl-
-C26:1-CoA
2
CoA
D-BP
def
3-keto-C26:0- 3-keto-
CoA pristenyol-CoA Case 1:

(PTS2)12-year-old boy
4,8,12- Clumsy
C24:0-CoA trimethyltridecanoyl Impaired
-CoA
proprioception
Adrenal insufficiency
 12.9 C26:0 Pristanic acid 2.9
RI: <1.3 RI: <0.6
mol/L mol/L

C26:0-CoA Pristanoyl-CoA
X-linked P Biogenesis Cytoplasm
ALD def Peroxisom
C26:0-CoA Pristanoyl-CoA e

ACOX1 def
2-Pristenoyl-
-C26:1-CoA
2
CoA
D-BP
def
3-keto-C26:0- 3-keto-
CoA pristenyol-CoA Case 2:

(PTS2)7-week old infant

4,8,12- Central hypotonia
C24:0-CoA trimethyltridecanoyl Neonatal seizure
-CoA
MRI: no abnormality
 1.42 C26:0 Pristanic acid 7.4
RI: <1.3 RI: <1.5
mol/L mol/L

C26:0-CoA Pristanoyl-CoA
X-linked P Biogenesis Cytoplasm
ALD def Peroxisom
C26:0-CoA Pristanoyl-CoA e

ACOX1 def
2-Pristenoyl-
-C26:1-CoA
2
CoA
D-BP
def
3-keto-C26:0- 3-keto-
CoA pristenyol-CoA
Case 3:
(PTS2)
4,8,12- 15-month-old girl
C24:0-CoA trimethyltridecanoyl
Global delay,
-CoA
hypotonia
No seizure
 Caveats

VLCFA: Beware of false positives

Pristanic acid: Beware of false negatives

 #3
Abnormal peroxisomal
alpha oxidation
 Peroxisomal alpha oxidation

3-methyl branched chain fatty acids

Alpha-oxidation to release terminal
carboxyl group as CO2

Phytanic acid
 Pristanic acid Phytanic acid

Pristanoyl-CoA Phytanoyl-CoA
Cytoplasm

Peroxisom
Pristanoyl-CoA Phytanoyl-CoA e
Refsum
(PTS2)
ds
2-Pristenoyl- 2-OH-Phytanoyl-
CoA CoA
D-BP
def
3-keto-
Pristanal
pristenyol-CoA

4,8,12-
trimethyltridecanoyl Pristanic acid
-CoA
 #4
Pipecolic acid
 Pipecolic acid

Minor pathway product of lysine

Elevated in peroxisomal disorders
Pipecolic acid oxidase
 -oxo-
Lysine aminohexanoat
e

Saccharopine Pipecolic acid

Cytoplasm
Pipecolic acid
oxidase Peroxisom
-aminoadipic Piperidine-6- e
Semialdehyde carboxylic acid

-aminoadipic
acid

-oxo-adipic
acid

Glutaryl-CoA
 Pipecolic acid

Elevated also in
Antiquitin deficiency (Pyridoxine-R epilepsy)
Hyperlysinemias
Sulfite oxidase deficiency
Hyperprolinemia type 2

(and so on)
 -oxo-
Lysine aminohexanoat
e

Saccharopine Pipecolic acid

Cytoplasm
Pipecolic acid
oxidase Peroxisom
-aminoadipic Piperidine-6- e
Semialdehyde carboxylic acid

-aminoadipic
acid

-oxo-adipic
acid

Glutaryl-CoA
 #5
Putting it altogether
 Putting it together (1)

Four pathways examined in this worksho

p
Plasmalogen synthesis
Fatty acid beta oxidation
Fatty acid alpha oxidation
Pipecolic acid degradation

Many other investigations available

 Putting it together (2)

Peroxisomal biogenesis defects

All four pathways involved

Plasma VLCFA plus pristanic/phytanic acid
Pipecolic acid
Urine organic acid: dicarboxylic acids &
3,6-epoxy-C14-dicarboxylic acid
 Putting it together (3)
RCDP disorders

Type 1 and 4 are related to PTS2 signaling

Type 2 and 3 are individual enzyme defect
s

Type 1 and 4 thus affects also phytanic aci

d (because phytanoyl-CoA hydroxylase P
TS2)
 Putting it together (4)

Isolated increase in phytanic acid

Normal or low pristanic acid

Refsum disease
RCDP type 1 and 4
 12.9 C26:0 Pristanic acid 2.9
RI: <1.3 RI: <0.6
mol/L mol/L

C26:0-CoA Pristanoyl-CoA
X-linked Cytoplasm
ALD Peroxisom
C26:0-CoA Pristanoyl-CoA e

ACOX1 def Phytanic

2-Pristenoyl- acid
-C26:1-CoA
2
1.3 mol/L
CoA
P Biogenesis
D-BP def (<5.3)
def
3-keto-C26:0- 3-keto-
CoA pristenyol-CoA Case 2:

(PTS2) 7-week old infant

4,8,12- Central hypotonia
C24:0-CoA trimethyltridecanoyl Neonatal seizure
-CoA
MRI: no abnormality
 1.42 C26:0 Pristanic acid 7.4
RI: <1.3 RI: <1.5
mol/L mol/L

C26:0-CoA Pristanoyl-CoA
X-linked Cytoplasm
ALD Peroxisom
C26:0-CoA Pristanoyl-CoA e

ACOX1 def
2-Pristenoyl- Pipecolic
-C26:1-CoA
2
CoA acid
P Biogenesis
D-BP def 13.1 mol/L
def
3-keto-C26:0- 3-keto-
CoA pristenyol-CoA
Case 3:
(PTS2)
4,8,12- 15-month-old girl
C24:0-CoA trimethyltridecanoyl
Global delay,
-CoA
hypotonia
No seizure
 #5
Some final words
 Exomes or enzymes?

Both are helpful diagnostic tools

But both are expensive

Local availability exomes

or Panel Testing?
 GC-MS or LC-MS/MS?

Techniques any differences?

 On biochemical testing

Tests are often built differently

Dried blood spot metabolic screening is a
sample type, not a test (per se)

Interesting test results are usually interpr

eted
 Acknowledgements

Dr Angel Chan (QMH)

Dr Eric Law (QMH)
Dr Doris Ching (PMH)
Dr Teresa Tsui (PWH)
Mr CK Lai (PMH)
Thank you very much