disorders of peroxisome biogenesis (pbd)

© 2009 Society for Inherited Metabolic Disorders www.simd.org

Disorders of Peroxisome

Biogenesis (PBD)Nancy Braverman, M.S., M.D.

McGill University-Montreal Children's Hospital Research InstituteMontreal, QC


Properties of peroxisomes

Spherical, single membrane bound, Diameter = 0.2 - 1 µm, several hundred/cell All eukaryotes


>2000: Study of protein functions, pathophysiology,applications to management and therapy

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

Timeline of discovery


ER

PxMt

Organelles

Benefits

• concentrate enzymes and substrates

• sequester toxic substances

Requirements

• targeting systems

• transporters, receptors

Consequences

• genetic diseases ofthe components

Nucleus


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


Peroxisomes originate from ER membranes and by fission of existing peroxisomes

adapted from Annu Rev Genet. 2000;34:623-652. Sacksteder KA, Gould SJ.

NEXT >>Click to view animation >>


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.


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)


Properties of peroxisomal matrix proteins

Contain Peroxisome Targeting Sequences (PTS)

Imported as oligomers/fully assembled proteins

Can have dual localizations in mitochondria, cytosol

C - terminal (-SKL)Most matrix proteinsReceptor is PEX5

-SKL

PTS1 PTS2

N - terminal (-R/KLX5 Q/HL-)

Presequence cleaved internally3 enzymes only: Thiolase, PhyH, AGPSReceptor is PEX7

R/KLX5Q/HL-SKL


Peroxisomal β-Oxidation



β-Oxidation pathways depend on the substrate



α- oxidation and auxillary enzymes in β-oxidation


J. Biol Chem. 2001; 276:38115-20. Su HM, Moser AB, Moser HW, Watkins PA


Synthesis of docohexanoic acid (DHA) requires peroxisomal β-oxidation


Plasmalogen

CH2-O-CH=CH-R

CH-O-C-CH2-R2

O

CH2-OPO3-ethanolamine or -choline

Vinyl ether-linkedalkyl chain at C-1

Plasmalogens are abundant in nervous tissue and erythrocyte membranes as• phosphatidyl-choline • phosphatidyl-ethanolamine

Plasmalogens are glycerol-based phospholipids with a vinyl ether-linked alkyl group in the C-1 position

Functions:• antioxidant • DHA storage • lipid messengers (PAF) • vesicle formation

Plasmalogen lipids


Plasmalogen biosynthesis is initiated in peroxisomes



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 racemase deficiency

• Adult Refsum disease

• Hyperoxaluria Type I


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


Zellweger Syndrome Infantile Refsum Disease

Zellweger spectrum disorder (ZSD), a clinical continuum


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


Neuronal migration defects (ZS): Polymicrogyria, pachygyria, heterotopias


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


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


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


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


Rhizomelic Chondrodysplasia Punctata (RCDP)

Dysmorphic facies: frontal bossing, short saddle nose with anteverted nares, congenital cataractsprofound impairment of growth and mental retardation, variable survival


Skeletal changes in RCDP

Rhizomelia, metaphyseal flaring Epiphyseal stippling, small thorax Vertebral coronal clefts Mineralization of intervertebral discs,

contractures


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


PBD phenotypes correlate with biochemical severity


Confirmation of metabolite testing

Establish a fibroblast culture for

• VLCFA content

• Plasmalogen synthesis

• Phytanic acid oxidation

• Catalase solubility

• Immunocytochemistry

• DNA testing


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’)


Immuno-gold staining for alanine-glyoxylate aminotransferase is granular when the enzyme is inside the peroxisome

Credit to Frank Roels

Mosaic pattern of peroxisome matrix proteins in liver biopsy specimen from an IRD patient


Mosaic pattern of peroxisome matrix proteins in cultured fibroblasts from PBD patients

Credit to N. Braverman


Control ZS

PX #and size

Matrix protein import

IRD

In ZSD, phenotype correlates with severity of protein import defect, peroxisome number and size


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


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


In ZSD, phenotype does not correlate to specific PEX gene defects


Role of peroxins in matrix protein import


Gould, Raymond, Valle.In: Metab & Molec Basis of Inh Dis. Ch 129 p. 3190.


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


Pathogenesis of PBD

Tools: mouse models, mammalian cell culture, pathology investigations, other model organisms

Neuron migration and integrityAccumulation of reactive oxygen speciesRole of mitochondria in peroxisome disordersAccumulation of VLCFA and BCFADeficiency of ether phospholipidsPeroxisomes functions in development and differences between tissues/organs


Peroxisome single enzyme defects:

X-linked Adrenoleukodystrophy


X-linked adrenoleukodystrophy (X-ALD)

Medical history

• 8-year old previously healthy, typically developed male

• Attention deficit/hyperactivity apparent within the past year

• Performing poorly in 2nd grade

• Recently began to run clumsily and to walk stiffly

• No recent illnesses

• No medications


Deterioration in writingover a 4 month period

Brain MRI – white matter disease

X-ALD

Dec 29, 1989

Mar 5, 1990

May 3, 1990


X-ALD

Defect in peroxisomal very long chain fatty acid oxidation

Adrenoleukodystrophy protein (ALDP) gene (ABCD1)

• Mapped to Xq28

• Over 200 mutations known, most crm negative

Incidence ~ 1/20,000

All ethnic groups


X-ALD: defective peroxisomal β-oxidation



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


Childhood cerebral form ~35%

• Onset - ~6-12 yrs (survival: several years)

• 90% with adrenal insufficiency

Adrenomyeloneuropathy (AMN) ~50%

• Spastic paraparesis and sphincter dysfunction

• Onset - ~2nd-5th decade (survival: decades)

• 2/3 with adrenal insufficiency

Other phenotypes ~15%

• Addison disease only

• Adult-onset cerebral involvement - dementia

Female heterozygotes- 50% with mild AMN-like Sx

Multiple phenotypes of X-ALD


X-ALD pedigree


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

X-ALD laboratory evaluation


Newborn screening for X-ALD: pilot project stage (Hubbard et al, 2007)

Resolution of lyso-PC’s during LC-MS/MS analysis

LC–MS/MS data from NB blood spots for 26:lyso-PC

Collision assisted decomposition of lyso-PCs results in fragmentation


X-ALD treatment

Dietary therapy

• Restriction of dietary VLCFA intake

• Lorenzo’s oil- 4:1 mix

– Glycerol trioleate (C18:1)

– Glycerol trierucate (C22:1)

• Lowers plasma C26:0 and C24:0 levels


Clinical effect

• Does not stop cerebral degeneration in boys with neurologic symptoms

• May delay onset of cerebral form in asymptomatic boys

• No effect on adrenal function

Ongoing trial in AMN

X-ALD treatment


Develop therapies targeted to metabolic or molecular defects

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

A--------->B


Next 2 lectures:

Molecular biology of peroxisomes: evaluation of the proteinimport system, membrane assembly, modifier genes

Biochemistry and pathophysiology of peroxisome disorders: Model systems of disease, evaluation of therapies

disorders of peroxisome biogenesis (pbd)

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