Disorders of Peroxisome
Biogenesis (PBD)
Nancy Braverman, M.S., M.D.
McGill University-Montreal Children's Hospital Research Institute
Montreal, QC
© 2009 Society for Inherited Metabolic Disorders www.simd.org
Properties of peroxisomes
 Spherical, single membrane bound,
 Diameter = 0.2 - 1 µm, several hundred/cell
 All eukaryotes
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Timeline of discovery
1958: Peroxisomes 1st described
1964: Zellweger cerebro-hepato-renal syndrome
1973: Abnormal peroxisomes in ZS
1983: PBD, a paradigm for metabolic
malformation syndromes
1987: Complementation groups
reflect genetic heterogeneity in PBD
1990: Peroxisome biogenesis (PEX) genes identified in yeast
1994: Human genes identified by yeast homology
>2000: Study of protein functions, pathophysiology,
applications to management and therapy
© 2009 Society for Inherited Metabolic Disorders www.simd.org
Organelles
 Benefits
• concentrate
• sequester
enzymes and substrates
Nucleus
toxic substances
 Requirements
• targeting
systems
• transporters,
Px
receptors
 Consequences
• genetic
diseases of
the components
ER
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Peroxisome assembly (PEX) genes
 Encode proteins (peroxins) required for matrix protein
import, peroxisome division and membrane formation
 14 human PEX genes; 13 thus far responsible for PBD
 26 different PEX genes among eucaryotes
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Peroxisomes originate from ER
membranes and by fission of existing
peroxisomes
Click to view animation >>
NEXT >>
adapted from Annu Rev Genet. 2000;34:623-652.
Sacksteder KA, Gould SJ.
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Role of peroxins in matrix protein
import
Click to view animation >>
Gould, Raymond, Valle.In: Metab & Molec Basis of Inh Dis. Ch
129 p. 3190.
© 2009 Society for Inherited Metabolic Disorders www.simd.org
Enzymatic pathways in
peroxisomes
 Fatty acid oxidation (VLCFA, PA)
 H2O2 detoxification (catalase)
 Docohexanoic acid (DHA) synthesis
 Bile acid synthesis
 Plasmalogen (ether phospholipid) synthesis
 Cholesterol synthesis
 Glyoxylate detoxification
 Lysine catabolism (pipecolic acid)
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Properties of peroxisomal matrix
proteins
 Contain Peroxisome Targeting Sequences (PTS)
PTS1
PTS2
-SKL -SKL
C - terminal (-SKL)
Most matrix proteins
Receptor is PEX5
R/KLX5Q/HL
N - terminal (-R/KLX5 Q/HL-)
Presequence cleaved internally
3 enzymes only: Thiolase, PhyH, AGPS
Receptor is PEX7
 Imported as oligomers/fully assembled proteins
 Can have dual localizations in mitochondria, cytosol
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Peroxisomal β-Oxidation
Click to view animation >>
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β-Oxidation pathways depend
on the substrate
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α- oxidation and
auxillary enzymes in β-oxidation
Click to view animation >>
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Synthesis of docohexanoic acid (DHA)
requires peroxisomal β-oxidation
Click to view animation >>
J. Biol Chem. 2001; 276:38115-20.
Su HM, Moser AB, Moser HW, Watkins PA
© 2009 Society for Inherited Metabolic Disorders www.simd.org
Plasmalogen lipids
Vinyl ether-linked
alkyl chain at C-1
CH2-O-CH=CH-R
O
CH-O-C-CH2-R2
CH2-OPO3-ethanolamine
or -choline
Plasmalogen
Plasmalogens are glycerolbased phospholipids with a
vinyl ether-linked alkyl group
in the C-1 position
Plasmalogens are abundant in
nervous tissue and erythrocyte
membranes as
• phosphatidyl-choline
• phosphatidyl-ethanolamine
Functions:
• antioxidant
• DHA storage
• lipid messengers (PAF)
• vesicle formation
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Plasmalogen biosynthesis is
initiated in peroxisomes
Click to view animation >>
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Genetic disorders of peroxisomes
 Multiple enzyme deficiencies: Peroxisomal
Biogenesis Disorders (PBD)
• Zellweger
spectrum disorder (ZSD) (~1/60,000)
• Rhizomelic
chondrodysplasia punctata spectrum
(RCDP)(~1/100,000)
 Single enzyme deficiencies
• X-linked
adrenoleukodystrophy (X-ALD) (~1/20,000)
• 3-methyl-CoA
• Adult
racemase deficiency
Refsum disease
• Hyperoxaluria
Type I
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Some single enzyme deficiencies
can mimic PBDs
 VLCFA oxidation → Zellweger spectrum disorder
• Acyl-CoA
oxidase
• D-Bifunctional
protein (hydratase/dehydrogenase)
 Plasmalogen biosynthesis → RCDP spectrum
• DHAPAT
(RCDP2)
• ADHAPS
(RCDP3)
 Some PBDs mimic SEDs →
• Adult
Refsum disease causes PEX7 deficiency
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Zellweger spectrum disorder
(ZSD), a clinical continuum
Zellweger Syndrome
Infantile Refsum Disease
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Craniofacial dysmorphism (ZS)
 Widely patent fontanels and sutures
 Prominent high forehead
 Shallow orbital ridges
 Low broad nasal bridge
 Anteverted nares
 Hypertelorism
 Epicanthal folds
 High arched palate
 Micrognathia
 Redundant skin folds of neck
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Neuronal migration defects (ZS):
Polymicrogyria, pachygyria,
heterotopias
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Neonatal adrenoleukodystrophy
 15 mo old with FTT
 Weight: 50th% for 6-mo old
 Height: 10th%
 Frontal bossing
 Wide anterior fontanel
 Depressed nasal bridge
 Epicanthal folds
 Diffuse hypotonia
 White matter changes on MRI
 Developmental delays and
seizure disorder
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Infantile Refsum disease?
 42-yr old woman
 Hearing loss at 2-3 yrs
 Progressive retinal disease
 Legally blind at 11 yrs
 Intermittent behavioral/psychiatric
problems
 Lives in a group home
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Disease course
 About 1/2 of PBD patients have NALD-IRD phenotypes
 Patients show progressive deterioration over time and
become blind, deaf and loose cognitive abilities
 Deterioration may coincide with onset or progression
of leukodystrophy
 If effective treatment was available, it might halt the
disease progression
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Infantile Refsum disease
 Diagnosed ~18 months
 RP and hearing loss
 Walked at ~30 months
 Developed seizures at 4 yrs
 Deterioration in vision
 Moderate to severe MR
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Rhizomelic Chondrodysplasia
Punctata (RCDP)
Dysmorphic facies: frontal bossing, short saddle
nose with anteverted nares, congenital cataracts
profound impairment of growth and mental retardation,
variable survival
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Skeletal changes in RCDP
 Rhizomelia, metaphyseal flaring
 Epiphyseal stippling, small thorax
 Vertebral coronal clefts
 Mineralization of intervertebral discs,
contractures
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Clinical spectrum of RCDP
 6-yr old: moderate MR,
cataracts and CDP, but no
rhizomelia or growth failure
 20-yr old, congenital cataracts,
mild learning disability, normal
stature, no rhizomelia, no CDP
 66-yr old initially diagnosed at
7-yrs with adult RD
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PBD phenotypes correlate with
biochemical severity
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Confirmation of metabolite testing
 Establish a fibroblast culture for
• VLCFA
content
• Plasmalogen
synthesis
• Phytanic
acid oxidation
• Catalase
solubility
• Immunocytochemistry
• DNA
testing
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ZSD: Challenges to Diagnosis
1. Patients with mild or atypical clinical presentation
2. Patients with mild or atypical biochemical profile
3. Patients with biochemical abnormalities in blood,
but normal studies in fibroblasts (peroxisomal
‘mosaicism’)
4. Patients with abnormal peroxisome morphology and
soluble catalase in hepatocytes, but normal studies in
fibroblasts (peroxisomal ‘mosaicism’)
5. Patients with abnormal peroxisome morphology and
soluble catalase in some cells, adjacent to other cells
that are normal in liver and fibroblasts (peroxisomal
‘mosaicism’)
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Mosaic pattern of peroxisome matrix
proteins in liver biopsy specimen
from an IRD patient
Immuno-gold staining for alanine-glyoxylate aminotransferase
is granular when the enzyme is inside the peroxisome
Credit to Frank Roels
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Mosaic pattern of peroxisome matrix
proteins in cultured fibroblasts from
PBD patients
Credit to N. Braverman
© 2009 Society for Inherited Metabolic Disorders www.simd.org
In ZSD, phenotype correlates with severity of
protein import defect, peroxisome number and size
PX #
and
size
Matrix
protein
import
Control
ZS
IRD
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ZSD: Approaches to Mutation
Identification
 1. Complementation by somatic cell hybridization
 2. Complementation by transfection of PEX cDNAs
 3. Targeted sequence analysis of specific PEX genes
Hierarchal algorithm based on common mutations
and frequency of each PEX gene defect
Molecular analysis is used for carrier detection,
prenatal diagnosis, preimplantation genetic diagnosis,
prognostic value, difficult cases
 4. Next generation sequencing platforms
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Mutation analysis of PEX genes
 2 mutations in PEX1 account for 56% of ZSD
 PEX6, 26, 10, 12 account for 26% of ZSD
 2 mutations in PEX7 account for 65% RCDP
 Association of severe mutations with severe disease
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In ZSD, phenotype does not correlate
to specific PEX gene defects
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Role of peroxins in matrix protein
import
Click to view animation >>
Gould, Raymond, Valle.In: Metab & Molec Basis of Inh Dis. Ch
129 p. 3190.
© 2009 Society for Inherited Metabolic Disorders www.simd.org
Contrast ZS and RCDP
 ZS
• both
PTS1 and PTS2 defects
• reduced
number and size of peroxisomes
 RCDP
• PTS2
defect only (PTS1 normal)
• peroxisomal
morphology normal
 RCDP should represent a segment of ZS, but there are
more severe abnormalities of bone, lens, different CNS
defects and skin
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Pathogenesis of PBD
Tools: mouse models, mammalian cell
culture, pathology investigations, other
model organisms
Neuron migration and integrity
Accumulation of reactive oxygen species
Role of mitochondria in peroxisome disorders
Accumulation of VLCFA and BCFA
Deficiency of ether phospholipids
Peroxisomes functions in development and
differences between tissues/organs
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Peroxisome single
enzyme defects:
X-linked Adrenoleukodystrophy
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X-linked adrenoleukodystrophy
(X-ALD)
 Medical history
• 8-year
old previously healthy, typically
developed male
• Attention
deficit/hyperactivity apparent within
the past year
• Performing
• Recently
poorly in 2nd grade
began to run clumsily and to walk stiffly
• No
recent illnesses
• No
medications
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X-ALD
Deterioration in writing
over a 4 month period
Dec 29, 1989
Brain MRI –
white matter disease
Mar 5, 1990
May 3, 1990
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X-ALD
 Defect in peroxisomal very long chain
fatty acid oxidation
 Adrenoleukodystrophy protein (ALDP) gene (ABCD1)
• Mapped
• Over
to Xq28
200 mutations known, most crm negative
 Incidence ~ 1/20,000
 All ethnic groups
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X-ALD: defective peroxisomal
β-oxidation
Click to view animation >>
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ALD protein
 Homology with ABC half-transporter family
 No homology with fatty acyl-CoA synthetases
 Yet, the biochemical defect is in the activation
of very long chain fatty acids to acyl-CoA esters
in peroxisomes
VLCFA + CoA + ATP
VLCFA-CoA
 Exact mechanistic link between ALDP deficiency
and VLCFA activation is yet undefined
• May
be defective transport of VLCFA across
peroxisomal membrane
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Multiple phenotypes of X-ALD
 Childhood cerebral form
• Onset
• 90%
~35%
- ~6-12 yrs (survival: several years)
with adrenal insufficiency
 Adrenomyeloneuropathy (AMN)
• Spastic
• Onset
• 2/3
~50%
paraparesis and sphincter dysfunction
- ~2nd-5th decade (survival: decades)
with adrenal insufficiency
 Other phenotypes
• Addison
~15%
disease only
• Adult-onset
cerebral involvement - dementia
 Female heterozygotes- 50% with mild AMN-like Sx
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X-ALD pedigree
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X-ALD laboratory evaluation
 Plasma VLCFA analysis
• Elevated
C26:0 and C24:0
• Elevated
C26:0/C22:0 and C24:0/C22:0 ratios
 Mutation analysis (ABCD1 gene) useful for
heterozygote detection
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Newborn screening for X-ALD: pilot
project stage (Hubbard et al, 2007)
Collision assisted
decomposition of lyso-PCs
results in fragmentation
 Resolution of lyso-PC’s during LC-MS/MS analysis
LC–MS/MS data from
NB blood spots for 26:lyso-PC
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X-ALD treatment
 Dietary therapy
• Restriction
• Lorenzo’s
of dietary VLCFA intake
oil- 4:1 mix
– Glycerol trioleate (C18:1)
– Glycerol trierucate (C22:1)
• Lowers
plasma C26:0 and C24:0 levels
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X-ALD treatment
 Clinical effect
• Does
not stop cerebral degeneration in boys with
neurologic symptoms
• May
• No
delay onset of cerebral form in asymptomatic boys
effect on adrenal function
 Ongoing trial in AMN
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Develop therapies targeted to metabolic or
molecular defects
A--------->B

Phytanic acid restriction

Reduction in VLCFA

Enhance omega oxidation of VLCFA

Supplementation with DHA, bile acids, plasmalogens

Induce peroxisome proliferation

Enhance activity of a defective PEX protein

Bypass defect in PEX protein
© 2009 Society for Inherited Metabolic Disorders www.simd.org
Next 2 lectures:
Molecular biology of peroxisomes: evaluation of the protein
import system, membrane assembly, modifier genes
Biochemistry and pathophysiology of peroxisome disorders:
Model systems of disease, evaluation of therapies
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