An almost final solution to theEthylmalonic acid syndrome

Valeria TirantiERNDIM Meeting

October 22-23, 2009Basel

CNS•early-onset hypotonia. developmental delay•later spasticity, then global failure

Vascular system•acrocyanosis•petechiae, microematuria, internal bleedings

Gastrointestinal system•chronic diarrhoea

Biochemistry•COX deficiency (only in muscle)•lactic acidosis•ethylmalonic aciduria•elevated C4-C5 acylcarnitine

0123456789

Abundance

urea

ethylmalonate

I.S.

2-ketoglutarate

I.S.isobutyrylglycineisovalerylglycine

Time

EthylmalonateButyryl-CoA CO2 ->CH3-CH2-CH2-CO-SCoA + CH3-CH2-CH-COOH

COOH

CoASH

Ethylmalonic Encephalopathy (EE)

EX1-7del

440del11

11 22 33 44 55 66 77

3G>T

113A>G 131delAG222insA

406A>G

488G>A

487C>T 604insG

559insC375+5G>A

505+1G>A

505+1G>T

554T>G

EX4del

GCCCCdel 376-1G>T187C>T

482G>A230delA34C>T

ETHE1: the gene50 EEpatients28 non-EEEMA patients

HeLa

ETHE1 Mitotrackermerge

Ethe1: the protein

It works as an homodimer

It is a mitochondrial matrix protein

Fe++

It binds iron

Tiranti et al 2004-2006, Mineri et al 2008

days

Surv

ival

pro

babi

lity

(%) +/+

-/-

ETHE1-/- mouse phenotype

Metabolites in body fluids

C4 acylcarnitine

CTR KO CTR EE-patientsmouse human

02468

1012141618

µM

/L

C5 acylcarnitine

CTR KO CTR EE-patients00,5

11,5

22,5

33,5

44,5

µM

/L

mouse human

Ethylmalonic acid

0

50

100

150

200

250

300

350

400

µg/

mg

crea

tinine

CTR KO CTR EE-patientsmouse human

gram

s

weeks02468101214

1 2 3 4

+/+

-/-

Brain

-/- -/-+/+ +/+CI/CS CIV/CS

0

50

100

150

200 Liver

-/- -/-+/+ +/+CI/CS CIV/CS

0

50

100

150

200 Muscle

-/- -/-+/+ +/+CI/CS CIV/CS

Kidney

-/- -/-+/+ +/+CI/CS CIV/CS

-/- -/-+/+ +/+CI/CS CIV/CS

Heart

0

50

100

150

200

** **

**

** p<0.0004

liver

brain

muscle

colon

jejunum-/- +/+

Respiratory chain complex activities in mouse tissues

Western-blot analysis of COX assembly inETHE1 KO mice

+/+ -/- +/+ -/- +/+ -/-

COI

SDH

BRAIN LIVER MUSCLE

+/+

-/-

I dimension

II dimension on Brain

BRAIN

COI

S1 COIHeme AHeme A3 Cox10p

Cu B Sco1pSco2p

Cox17p

COIV

S2 COII Sco1pSco2p

Cox17pCu ACOIII

COVa, Vb, VIc, VIIa, VIII

COVIbCOVIIc

S3 COVIIbCOVIa

S4

Cox11pCox14pCox15p

Pet100pPet117pPet191pSurf1

Oxa1p

LonAfg3Rca1

?

?

?

?

Bioinformatic searchETHE1-like proteins are present in operons containing Rhodanese-likeproteins or as Chimeric proteins ETHE1/Rhodanese

Bradyrhizobium japonicum 27376118 9Legionella pneumophila 54293763 9Legionella pneumophila 52841017 9Danio rerio 47086181 9Caenorhabditis elegans 17538952 9Apis mellifera 48140497 9Drosophila melanogaster 24652740 9Drosophila melanogaster 27819799 9Anopheles gambiae 31210533 9Anopheles gambiae 55240363 9Oryza sativa 56202167 9Oryza sativa 34910930 9Arabidopsis thaliana 12324040 9Arabidopsis thaliana 1644427 9Arabidopsis thaliana 22655048 9Xenopus laevis 27371293 9Pan troglodytes 55649315 9Homo sapiens ETHE1Mus musculus 53236965 9Rattus norvegicus 27676450 9Burkholderia cepacia 46322841 9 FBurkholderia cepacia 46317848 9 FRalstonia eutropha 45515657 9Ralstonia metallidurans 48771425 9Nostoc punctiforme 23128919 9 FSynechococcus elongatus 56750614 9Nostoc sp. 17231396 9Anabaena variabilis 45507470 9Synechocystis sp. 16330445 9Thermosynechococcus elongatus 22299169 9

Polaromonas sp. 54032671 9 F

??

Fusion events Operon??soxY soxA ??soxBsoxZsoxX

??

??

??

Zn-hydrolase domain Rhodanese-related sulfurtransferase domain ‏

Domain of unknown functionPredicted permease‏Gluthation S-transferase ‏

Sulfur oxidase gene cluster

RHODANESE(TST, Thiosulphate Sulfurtrasferase) ‏

Mitochondrial matrix protein

Unknown biological function (Cyanide

detoxyfication, Iron-sulfur cluster

formation, energetic metabolism)

It is involved in sulfur metabolism

Sulfur metabolism

Szabo, Nat Rev Drug Discov 2007

Thiosulphate in urines

EE-Patients

nmol

/mg

crea

tini

ne

0

500

1000

1500

2000

2500

CTR0

2000

4000

6000

8000

10000

12000

14000

16000

18000

20000

nmol

/mg

crea

tini

ne

CTR KO mice

kidneybrain liver serummuscle

0

40

80

120

160

200

+/+ +/+ +/+ +/+ +/+-/- -/- -/- -/- -/-

µM

and tissues

LIVER

0,0

500,0

1000,0

1500,0

2000,0

2500,0

3000,0

3500,0

MUSCLE

0,0

500,0

1000,0

1500,0

2000,0

2500,0

CTR KOCTR KO

p<0.0007 p=0.00007

BRAIN

0,0

10,0

20,0

30,0

40,0

50,0

60,0

70,0

80,0

90,0

CTR KO

p<0.0016

H2S concentration in tissues ofETHE1 knockout mice

H2S

(nM

)

H2S inhibits cytochrome c oxidase(COX)

Szabo, Nat Rev Drug Discov 2007

H2S inhibits enzymatic activities

0 200 400 600 800 1000 12000

20

40

60

80

100

120

NaSH (µM)

residu

al a

ctivity SCAD in liverCOX/CS

NaSH (µM)

residu

al a

ctivity

120

0 1 2 3 4 5 6 7 8 9 10 11 12 130

20

40

60

80

100

NaCN

MUSCLE

BRAIN

LIVER

0 2 4 6 8 10 12

0

20

40

60

80

100

120

NaSH (µM)

residu

al a

ctivity

CI and CS in muscle

CI/CS

CS

Conclusions 1

•The pathogenetic mechanism of Ethylmalonic Encephalopathy(EE) relies on high levels of H2S, that are toxic for both COXand SCAD

•H2S can act as a vasodilator thus explaining the acrocyanosisand can be toxic for the microvessels thus explaining thepetechiae

Which is the role of ETHE1????

H2S (sulfide) is the first inorganic substrate formitochondrial respiratory chain in mammals

H2SSQR RSSH+H2O+O2 H2SO3

SDO

H2S2O3

TST sulfide quinoneoxidoreductase

sulfur dioxygenase

sulfurtranferase

matrixO2 2H2O

2H2S

intermembrane space CIII CIV

SQR

S S

inner mitochondrialmembrane

H2SO3

H2S2O3

-SSHR

e-

e- e-

Cyt cUQH2

UQ

e-

R

H2O

O2

TST/Rhodanese(Thiosulfate Sulfur Transferase)

SDO(SDO: Sulfur Dioxygenase)

(thiosulfate)

(sulfite)

(hydrogen sulfide) (persulfide)-SSHR(persulfide)

(SQR: Sulfide quinone Oxidoreductase)

SDO activity in liver of ETHE1 -/-homogenates

0

1

2

3

4

5

6

7

8

9

10

liver homogenate

SDO a

ctivity

(nmol/m

g/min)

+/+ -/-

Sulfide

ETHE1

SDH

Liver-/- +/+

matrixO2 2H2O

H2S

intermembrane space CIII CIV

SQR

S S

inner mitochondrialmembrane

H2SO3

H2S2O3

-SSHR

e-

e- e-

Cyt cUQH2

UQ

e-

R

Rhodanese(Thiosulfate Sulfur Transferase)

ETHE1(SDO: Sulfur Dioxygenase)

(sulfite)

(persulfide)-SSHR(persulfide)

(SQR: Sulfide quinone Oxidoreductase)

Conclusions 2ETHE1, encodes a mitochondrial sulfur dioxygenase thattakes part in aerobic energetic exploitation of, anddetoxification from, H2S

EE is the first example of a mitochondrial disorder causedby genetically determined poisoning of the respiratory chain

The creation of organ-specific conditional KO animals, canbe useful to identify the main source of H2S generation inEE, e.g. exogenous absorption from the gut flora vs.endogenous production

COX

KOKO

WTWT

1011-1012 cfu/ml

104-107 cfu/ml

101-103 cfu/ml

H&E

H2S-producing gut flora and mucosalCOX

The toxic mechanism underpinning EthylmalonicEncephalopathy makes effective therapy a realisticgoal: for instance antibiotic control of H2S-producingbacterial flora vs. H2S neutralization using GSH as asulfide acceptor

(1) the diffusion of H2S through the colonicmucosa could be a major, but not the onlycause of disease in Ethe1-/- mice andpatients

(2) the restriction of bacterial production ofH2S and its neutralization by NAC, may betherapeutically beneficial in the mouse, andpossibly human, disease condition.

Final considerations and futureperspectives

A specific treatment against the proliferation ofH2S-producing anaerobic bacterial flora in the largeintestine leads to substantial improvement of theclinical conditions in experimental murine EE

CollaborationsTatjana HildebrandtMike LevittMarco RimoldiAlessandro PrelleGigliola FagiolariCecilia Tiveron

THANK YOU!!Unit of Molecular Neurogenetics

Egill BriemRossana Mineri

Carlo Viscomi

Eleonora Lamantea

Ivano DiMeo

Barbara Garavaglia

All the clinicians world-widewho referred the patients toour Centre

Massimo Zeviani