Peroxisomal disorders disorders Peroxisomal Guy Besley Guy Besley - - PowerPoint PPT Presentation

Peroxisomal Peroxisomal disorders disorders

Guy Besley Guy Besley Willink Biochemical Genetics Unit, Willink Biochemical Genetics Unit, Manchester Children’s Hospital, Manchester Children’s Hospital, Manchester M27 4HA Manchester M27 4HA

BIMDG-York 3 July 2003

Peroxisomes Peroxisomes

• Found in all nucleated mammalian cells

Found in all nucleated mammalian cells

• Initially identified

Initially identified histologically histologically as as microbodies microbodies in 1950s in 1950s

• First evidence of biological function in 1960s

First evidence of biological function in 1960s

• Catalase

Catalase ‘marker’ enzyme to remove H ‘marker’ enzyme to remove H2

2O

O2

2

generated by several generated by several oxidases

• xidases
• Many other important functions now known

Many other important functions now known

Peroxisome Peroxisome morphology morphology

• Generally appear as spherical single

Generally appear as spherical single membrane membrane-

• bound, bodies of 0.1

bound, bodies of 0.1 – – 1.0 1.0µ µm in m in diameter diameter

• The number per cell varies depending on

The number per cell varies depending on

• demand. Most numerous in liver and kidney,
• demand. Most numerous in liver and kidney,

few in fibroblasts few in fibroblasts

• Localised by

Localised by histochemical histochemical staining for staining for catalase catalase (DAB) or using (DAB) or using immunocytochemical immunocytochemical methods methods

• Peroxisomes

Peroxisomes are formed from pre are formed from pre-

• existing

existing

• rganelles
• rganelles
• In rodents but not man induction can be

In rodents but not man induction can be stimulated by stimulated by clofibrate clofibrate, , plasticisors plasticisors or

• r

hypolipidaemic hypolipidaemic drugs drugs

Liver EM Liver EM – – DAB stain DAB stain

Peroxisomal Peroxisomal functions functions

• The importance of these functions is

The importance of these functions is illustrated by the severe consequences of illustrated by the severe consequences of peroxisomal peroxisomal dysfunction, especially in dysfunction, especially in peroxisome peroxisome biogenesis defects biogenesis defects

• Although these defects were first reported

Although these defects were first reported in 1973 by in 1973 by Goldfischer Goldfischer, it was not until a , it was not until a decade later that the biochemical decade later that the biochemical abnormalities were really recognised abnormalities were really recognised

Peroxisomal Peroxisomal functions in man functions in man

• Distinct fatty acid

Distinct fatty acid β β-

• oxidation system,
• xidation system,

especially of very long straight especially of very long straight-

• chain

chain fatty acids (C12 fatty acids (C12-

• 27)

27)

• Biosynthesis of ether phospholipids

Biosynthesis of ether phospholipids ( (plasmalogens plasmalogens) )

• The

The α α-

• oxidation of 3
• xidation of 3-
• methyl branched

methyl branched chain fatty acids ( chain fatty acids (phytanate phytanate) )

• Biosynthesis of

Biosynthesis of isoprenoids isoprenoids

Other Other peroxisomal peroxisomal functions in functions in man man

• Removal of

Removal of glyoxylate glyoxylate via via alanine alanine: :glyoxylate glyoxylate amino transferase amino transferase

• Oxidation of

Oxidation of pipecolic pipecolic acid derived from L acid derived from L-

• lysine

lysine

• Oxidation of

Oxidation of glutaryl glutaryl-

• CoA

CoA

• Elongation of fatty acids

Elongation of fatty acids

• Unlike mitochondria, the energy generated by

Unlike mitochondria, the energy generated by various oxidation steps generates heat and various oxidation steps generates heat and H H2

2O

O2

2, not ATP.

, not ATP.

Very long chain fatty acid Very long chain fatty acid metabolism metabolism

• Very long chain fatty acids (C22

Very long chain fatty acids (C22-

• 26) are

26) are preferentially oxidised by the preferentially oxidised by the peroxisome peroxisome. .

• They are taken up into

They are taken up into peroxisomes peroxisomes following following activation to their activation to their CoA CoA ester ester

• Uptake is via a specific ABC

Uptake is via a specific ABC-

• transporter in

transporter in the peroxisomal membrane, which is ATP the peroxisomal membrane, which is ATP-

• dependent

dependent

• Referred to as ALDP which exists as a homo

Referred to as ALDP which exists as a homo-

• r
• r heterodimer

heterodimer

Pristanic acid branched chain acyl-CoA oxidase Straight chain acyl-CoA oxidase bifunctional enzyme (hydratase + dehydrogenase) Thiolase 2 (Sterol carrier protein X) Thiolase 1

Peroxisome β-oxidation

Phytanic acid THCA VLCFA (C26) VLCFA (C24)

Trimethyl tridecanoyl-CoA choloyl-CoA

Phytanic acid α-oxidation

Phytanic acid Phytanoyl-CoA phytanoyl-CoA hydroxylase 2-hydroxy-phytanoyl-CoA Pristanic acid lyase and dehydrogenase

Plasmalogens Plasmalogens – – ether ether-

• linked

linked phospholipids phospholipids

H2COCH=CHR R’COOCH O H2C-O-P-CH2CH2NH3 O Plasmalogens represent 5 – 20% membrane phospholipids (30% in myelin) and protect against photosensitivity

Plasmalogen Plasmalogen biosynthesis biosynthesis

Acyl Acyl-

• CoA

CoA Gly Gly-

• 3

3-

• P

P DHAP DHAP DHAP DHAP-

• AT

Acyl Acyl-

• DHAP

DHAP AT CoASH CoASH Alkyl Alkyl-

• DHAP

DHAP Long Long-

• chain alcohol

Alkyl Alkyl-

• Gly

Gly-

• 3

3-

• P

P Alkyl Alkyl-

• DHAP

DHAP synthase synthase chain alcohol Fatty acid Fatty acid Alkyl/ Alkyl/acyl acyl-

• DHAP:

DHAP: NAD(P)H NAD(P)H oxidoreductase plasmalogens plasmalogens

• xidoreductase

Peroxisome Peroxisome biogenesis biogenesis

• Peroxisomal

Peroxisomal proteins are synthesised on free proteins are synthesised on free polyribosomes polyribosomes

• They must carry a specific peptide signal to

They must carry a specific peptide signal to direct them to pre direct them to pre-

• existing

existing peroxisomes peroxisomes

• Most matrix proteins carry the

Most matrix proteins carry the Peroxisome Peroxisome Targetting Targetting Signal (PTS1) serine Signal (PTS1) serine-

• lysine

lysine-

• leucine

leucine (SKL) (SKL)

• A second signal (PTS2) is required for some

A second signal (PTS2) is required for some enzyme proteins, including enzyme proteins, including thiolase thiolase, , phytanoyl phytanoyl-

• CoA

CoA hydroxylase hydroxylase and DHAP and DHAP-

• AT, alkyl

AT, alkyl-

• DHAP

DHAP synthase synthase

• Peroxisome

Peroxisome membrane proteins do not use PTS1 membrane proteins do not use PTS1

• r PTS2 for
• r PTS2 for targetting

targetting

PEX genes PEX genes

• Through studies on

Through studies on peroxisome peroxisome function and function and defects in biogenesis a number of genes have defects in biogenesis a number of genes have been identified been identified

• These genes encode mostly

These genes encode mostly peroxisome peroxisome integral membrane proteins essential for the integral membrane proteins essential for the assembly and biosynthesis of assembly and biosynthesis of peroxisomes peroxisomes

• The proteins are referred to as

The proteins are referred to as peroxins peroxins and and the genes PEX genes the genes PEX genes

• Mutations in PEX genes are responsible for

Mutations in PEX genes are responsible for peroxisome peroxisome biogenesis disorders ( biogenesis disorders (Zellweger Zellweger) ) and RCDP and RCDP

Peroxisomal Peroxisomal disorders and PEX disorders and PEX gene disorders: two major gene disorders: two major categories categories (www.

(www.peroxisome peroxisome.org) .org)

• PEX gene defects

PEX gene defects

– – Peroxisome Peroxisome biogenesis disorders including: biogenesis disorders including: Zellweger Zellweger syndrome, neonatal syndrome, neonatal adrenoleukodytrophy adrenoleukodytrophy and infantile and infantile Refsum Refsum disease and disease and Rhizomelic Rhizomelic chondrodysplasia chondrodysplasia punctata punctata (PTS2 defect (PTS2 defect -

• PEX7 mutations)

PEX7 mutations)

• Single enzyme defects (X

Single enzyme defects (X-

• ALD,

ALD, β β-

• xidation defects,
• xidation defects, Refsum

Refsum etc) etc)

Peroxisome Peroxisome biogenesis disorders biogenesis disorders

• Zellweger

Zellweger syndrome syndrome – – most severe phenotype most severe phenotype

– – Typical Typical dysmorphology dysmorphology, profound , profound hypotonia hypotonia, , hepatic dysfunction, neonatal presentation hepatic dysfunction, neonatal presentation

• Neonatal

Neonatal adrenoleukodystrophy adrenoleukodystrophy

– – Intermediate phenotype Intermediate phenotype

• Infantile

Infantile Refsum Refsum disease disease – – milder phenotype milder phenotype

– – Retinopathy, deafness, mild Retinopathy, deafness, mild dysmorphism dysmorphism

• 11 complementation groups, group 1 (PEX1

11 complementation groups, group 1 (PEX1 mutations account for 65% patients) includes mutations account for 65% patients) includes all phenotypes all phenotypes

Zellweger Zellweger syndrome syndrome

• Typical facial

Typical facial dysmorphism dysmorphism: high forehead, : high forehead, large anterior large anterior fontanelle fontanelle, , hypoplastic hypoplastic supraorbital supraorbital ridges, epicanthic folds ridges, epicanthic folds

• Profound

Profound hypotonia hypotonia and seizures and seizures

• Retinopathy, cataracts, hearing loss

Retinopathy, cataracts, hearing loss

• Enlarged liver

Enlarged liver

• Renal cysts

Renal cysts

• Punctate

Punctate stippling esp. patella and epiphyses stippling esp. patella and epiphyses

Zellweger Zellweger -

• punctate

punctate stippling stippling

Normal and Normal and Zellweger Zellweger liver liver

83% 83% 80% 80% 100% 100% Liver Liver 20% 20% 82% 82% 80% 80% Seizures Seizures 52% 52% 82% 82% 99% 99% Hypotonia Hypotonia + + ++ ++ +++ +++ Psychomotor delay Psychomotor delay 93% 93% 100% 100% 100% 100% Deafness Deafness 95% 95% 80% 80% 70% 70% Retinopathy Retinopathy 7% 7% 45% 45% 80% 80% Cataract Cataract + + + + ++ ++ Dysmorphism Dysmorphism 6.5y 6.5y 2y 2y <1y <1y Age of death Age of death IRD IRD NALD NALD ZS ZS Feature Feature

Trilamellar Trilamellar body in IRD body in IRD

Biochemical abnormalities Biochemical abnormalities

• All PBD patients have the same basic

All PBD patients have the same basic biochemical abnormalities, however biochemical abnormalities, however some abnormalities maybe less marked some abnormalities maybe less marked in milder patients in milder patients

• They all lack functional

They all lack functional peroxisomes peroxisomes

• Many of the 50 matrix proteins are lost

Many of the 50 matrix proteins are lost

• However

However peroxisome peroxisome membrane proteins membrane proteins ( (PMPs PMPs) may remain as ghosts ) may remain as ghosts

Diagnosis of PBD patients Diagnosis of PBD patients

• Plasma very long chain fatty acid

Plasma very long chain fatty acid analysis analysis

• Plasma

Plasma phytanic phytanic acid acid

• Bile acid analysis

Bile acid analysis

• Erythrocyte

Erythrocyte plasmalogens plasmalogens

• DHAP

DHAP-

• AT activity

AT activity

Rhizomelic Rhizomelic chondrodysplasia chondrodysplasia punctata punctata

• Type 1

Type 1

– – PEX7 gene defect with PTS2 receptor defect PEX7 gene defect with PTS2 receptor defect

• Plasmalogen

Plasmalogen biosynthesis impaired, low DHAP biosynthesis impaired, low DHAP-

• AT activity

AT activity

• Phytanate

Phytanate raised but raised but pristanate pristanate low and low and VLCFAs VLCFAs normal normal

• Immature

Immature thiolase thiolase

– – Note some patients present with Note some patients present with Refsum Refsum phenotype phenotype

• Type 2

Type 2

– – DHAP DHAP-

• AT isolated deficiency, low

AT isolated deficiency, low plasmalogens plasmalogens

• Type 3

Type 3

– – Alkyl DHAP Alkyl DHAP synthase synthase deficiency, low deficiency, low plasmalogens plasmalogens

Rhizomelic Rhizomelic chondrodysplasia chondrodysplasia punctata punctata

• Proximal limb shortening, joint

Proximal limb shortening, joint contractures contractures

• Punctate

Punctate calcific calcific stippling esp. of stippling esp. of epiphyses, skeletal epiphyses, skeletal dysplasia dysplasia

• Microcephaly

Microcephaly, psychomotor retardation , psychomotor retardation and FTT and FTT

• Cataracts, deafness

Cataracts, deafness

• Ichthyosis

Ichthyosis

Single Single peroxisomal peroxisomal defects defects

• X

X-

• linked

linked adrenoleukodystropy adrenoleukodystropy

• Peroxisome

Peroxisome β β-

• oxidation defects
• xidation defects

– – Acyl Acyl-

• CoA

CoA oxidase

• xidase deficiency

deficiency – – Bifunctional Bifunctional protein deficiency protein deficiency – – Thiolase Thiolase deficiency deficiency

• Refsum

Refsum disease disease

• Racemase

Racemase deficiency deficiency

X X-

• linked

linked adrenoleukodystrophy adrenoleukodystrophy

(www.x (www.x-

• ald

ald. .nl nl) )

• Mutations in the ALDP gene

Mutations in the ALDP gene

• Phenotype: classical juvenile to milder

Phenotype: classical juvenile to milder adrenomyeloleukodystrophy

• adrenomyeloleukodystrophy. Female carriers (15%

. Female carriers (15% may manifest symptoms) may manifest symptoms)

• Diagnosis by plasma VLCFA analysis

Diagnosis by plasma VLCFA analysis

• ALDP expression in fibroblasts (70% cases

ALDP expression in fibroblasts (70% cases -

• ve

ve) )

• Mutation analysis, but mostly private (over 500)

Mutation analysis, but mostly private (over 500)

• Prenatal diagnosis on cultured cell VLCFA or by

Prenatal diagnosis on cultured cell VLCFA or by mutation analysis mutation analysis

Acyl Acyl-

• CoA

CoA oxidase

• xidase deficiency

deficiency ( (pseudo

pseudo-

• neonatal

neonatal adrenoleukodystrophy adrenoleukodystrophy) )

• Peroxisomes

Peroxisomes present present

• Diagnosis by increased

Diagnosis by increased VLCFAs VLCFAs, but , but normal normal pristanate pristanate, , phytanate phytanate, THCA and , THCA and normal DHAP normal DHAP-

• AT activity/

AT activity/plasmalogen plasmalogen levels levels

Acyl-CoA oxidase deficiency

Phytanic acid THCA VLCFA (C26) Pristanic acid branched chain acyl-CoA oxidase bifunctional enzyme (hydratase + dehydrogenase) Thiolase 2 Thiolase 1 Straight chain acyl-CoA oxidase VLCFA (C24)

Trimethyl tridecanoyl-CoA choloyl-CoA

Bifunctional Bifunctional protein deficiency protein deficiency

• Severe disorder, presenting like

Severe disorder, presenting like Zellweger Zellweger. .

• Dysmorphia

Dysmorphia, seizures, , seizures, hypotonia hypotonia, death in , death in first year, disordered neuronal migration. first year, disordered neuronal migration.

• Most common of

Most common of β β-

• oxidation defects
• xidation defects
• Peroxisomes

Peroxisomes present present

• Diagnosis by increased

Diagnosis by increased VLCFAs VLCFAs, increased , increased pristanate pristanate and THCA, but normal DHAP and THCA, but normal DHAP-

• AT or

AT or plasmalogens plasmalogens

Bifunctional enzyme deficiency

Phytanic acid THCA VLCFA (C26) Pristanic acid branched chain acyl-CoA oxidase bifunctional enzyme (hydratase + dehydrogenase) Thiolase 2 Thiolase 1 Straight chain acyl-CoA oxidase VLCFA (C24)

Trimethyl tridecanoyl-CoA choloyl-CoA

Thiolase Thiolase deficiency deficiency

• Rare, 1 patient originally reported

Rare, 1 patient originally reported

• Peroxisomes

Peroxisomes present present

• Diagnosis similar to

Diagnosis similar to bifunctional bifunctional protein protein defect (increased defect (increased VLCFAs VLCFAs, THCA and , THCA and ? ?pristanate pristanate) )

• Identification of defect by fibroblast

Identification of defect by fibroblast complementation studies. complementation studies.

Refsum Refsum disease disease

• Retinitis

Retinitis pigmentosum pigmentosum, ataxia, chronic , ataxia, chronic polyneuropathy polyneuropathy, deafness, , deafness, anosmia anosmia, , ichthyosis ichthyosis, , cardiac abnormalities, skeletal abnormalities cardiac abnormalities, skeletal abnormalities (metatarsal shortening) (metatarsal shortening)

• Onset usually in late childhood to adults

Onset usually in late childhood to adults

• Diagnosis: raised

Diagnosis: raised phytanate phytanate, but normal , but normal VLCFAs VLCFAs but low but low pristanate pristanate

• Note some patients have PEX7 mutation

Note some patients have PEX7 mutation (RCDP variant) (RCDP variant)

Phytanic acid α-oxidation

Phytanic acid Phytanoyl-CoA phytanoyl-CoA hydroxylase 2-hydroxy-phytanoyl-CoA Pristanic acid lyase and dehydrogenase

2 2-

• methylacyl

methylacyl-

• CoA

CoA racemase racemase deficiency deficiency

• Presentation similar to

Presentation similar to adrenomyeloneuropathy adrenomyeloneuropathy and and Refsum Refsum disease disease

• Increased

Increased pristanate pristanate and bile acids (THCA) but and bile acids (THCA) but normal normal VLCFAs VLCFAs

• Suggests a defect in branched

Suggests a defect in branched-

• chain

chain oxidase

• xidase, but this

, but this was normal was normal

• However the 2

However the 2-

• methyl esters exist as 2R and 2S

methyl esters exist as 2R and 2S stereoisomers stereoisomers, but , but β β-

• oxidation requires conversion to
• xidation requires conversion to

2S form by 2S form by racemase racemase enzyme enzyme

• Defect in the

Defect in the methylacyl methylacyl-

• CoA

CoA racemase racemase was recently was recently identified identified

Diagnostic flow chart Diagnostic flow chart

Clinical suspicion Clinical suspicion

normal normal

VLCFAs VLCFAs

increased increased

Check for Check for

• ther disorders
• ther disorders
• Phytanate

Phytanate/ /Pristanate Pristanate

• Bile acids

Bile acids

• Pipecholic

Pipecholic acid acid Peroxisome Peroxisome biogenesis biogenesis disorder disorder Bile acids Bile acids Pristanate Pristanate Oxidase Oxidase def def Bifunctional Bifunctional Enz

• Enz. def.

. def.

normal normal

Peroxisome Peroxisome β β-

• oxidation
• xidation

defect defect

normal normal

Plasmalogens Plasmalogens DHAP DHAP-

• AT

AT

deficient deficient high high

Clinical suspicion Clinical suspicion

Flow chart for RCDP Flow chart for RCDP

Atypical bone Atypical bone dysplasia dysplasia, , cataracts, mental retardation cataracts, mental retardation Classical RCDP Classical RCDP plasmalogens plasmalogens DHAP DHAP-

• AT

AT phytanate phytanate Excludes RCDP Excludes RCDP Types 1, 2 and 3 Types 1, 2 and 3 Check for other Check for other types of types of chondrodysplasia chondrodysplasia eg eg sterol analyses sterol analyses Type 2 RCDP Type 2 RCDP Type 3 RCDP Type 3 RCDP Classical type 1 RCDP Classical type 1 RCDP

deficient deficient normal normal normal normal deficient deficient normal normal raised raised

That’s all, folks That’s all, folks