CFTDE-Necker X.03

Differences phenotypiques

enfants adultes

Jean-Marie Saudubray

SFEIM Grenoble Juin 2016

CFTDE-Necker X.03

De la chimie cosmique au DACU

(d’après Christian de Duve)

Chimie cosmique déterministe et

reproductible, non biosélective :C,H,O,N,

mais aussi Sucres,Acides aminés,Acides

gras ,Bases azotées

ARN (1ere molécule porteuse

d’informations réplicable)

Chimie +Sélection

Formation du Dernier Ancétre Commun

Universel (DACU)

CFTDE-Necker X.03

Les étapes de l’histoire de la vie:

des unicellulaires au mystére de la conscience

( Christian de Duve)

CFTDE-Necker X.03

Le minimum vital du DACU

Une membrane

Une source d’énergie

Une molécule porteuse d’information

pouvant étre répliquée (ARN) et un

archivage des informations (ADN)

Des substances nutritives

CFTDE-Necker X.03

La leçon d’anatomie Rembrandt 1632

CFTDE-Necker X.03

La cellule humaine 2016

From Dr. Joe Clarke

Lysosome Localization of hydrolytic enzymes involved in

the degradation of large, complex substrates,

such as proteins, glycoconjugates, nucleic

acids, complex lipids

Mitochondria 1. Oxidative phosphorylation (respiratory chain)

2. Citric acid cycle

3. β-Oxidation of fatty acids, with production of acetyl-

CoA

4. Oxidative degradation of some amino acids

5. Proximal steps of urea biosynthesis

6. Biosynthesis of some amino acids

7. Proximal and distal steps of porphyrin biosynthesis

Plasma membrane 1. Limiting loss of intracellular components of the

cell,

ensuring concentration and preservation of

optimum local milieu

2, Regulating uptake of amino acids

3. Neurotransmitter binding and uptake (nervous

tissue)

4. Regulating uptake of metabolites, drugs,

chemicals, hormones, etc.

5. Uptake (endocytosis) and intracellular

trafficking of material suspended in extracellular

milieu

Peroxisomes 1. Synthesis of bile acids

2. Synthesis of some steroid hormones

3. Synthesis of plasmalogens

4. Transmission of glyoxylate to glycine

5. Catalase

6. Oxidation of D-amino acids

7. α-Oxidation of branched chain fatty acids, e.g.,

phytanic acid

8. β-Oxidation of long-chain and very long-chain fatty

acids

9. Oxidation of pipecolic acid

10. Spermine and spermidine oxidation

Golgi complex Post-translational modification of N-linked

oligosaccharides of nascent glycoproteins,

including glycosaminoglycan biosynthesis

Rough endoplasmic

reticulum (ER) Translation (mRNA-directed synthesis

of polypeptides)

Smooth endoplasmic

reticulum (ER)

1. Post-translational modification of nascent

polypeptides, including N- and O-glycosylation

2. Biosynthesis of cholesterol, phospholipids,

triglycerides, glycosphingolipids

3. Steroid hormone biosynthesis (endocrine tissues)

4. Detoxifications (P450, liver)

5. Glucose-6-phosphate (liver)

6. Calcium sequestration (sarcoplasmic reticulum,

muscle)

7. Porphyrin degradation

CFTDE-Necker X.03

Auscultation

Thoracique

par Laennec

en 1819

R A Thom’s

tableau

1954

CFTDE-Necker X.03

New Technologies 2016

La Metabolomique:

Mayo clinic’s biochemical genetics laboratory (Courtesy J Vockley)

CFTDE-Necker X.03

Le métabolisme intermédiaire

C ’ est l'ensemble des transformations

chimiques de dégradation (catabolisme) et de

fabrication (anabolisme) qui permettent un

cycle continu d'échanges entre les cellules et

les substances apportées par l'alimentation:

glucides, lipides et protides.

Immense réseau interconnecté

CFTDE-Necker X.03

Oxaloacetate

citrate

Pyruvate

Acetyl-CoA

NADH

NAD+

Isocitrate

PDH

IDH ATP

CTP

citrate

+

Glucose 1-P

Glucose 6-P

F 1,6-BP

PFK

GA3-P

6-P-

gluconolactone

Ribulose 5-P

NADPH

NADP+

G6PD -

Citrate lyase

Acetyl-CoA

Acetyl -CoA

carboxylase

Malonyl-CoA

Ceramide

Sphingolipids LCFA

VLCFA

Elongases

ELOV 1-6

NADP+

NADPH

Acyl-DHAP

Alkyl-DHAP

Plasmalogens

DHAPAT

ADHAPS

Fatty acyl-CoA

FAS NADP+

NADPH

Aceto-

acetyl-CoA

HMG-CoA

Mevalonate

Cholesterol

Ubiquinone

Dolichol

HMG-CoA

synthase

HMG-CoA

reductase

Co A

DHAP G3P DHase

Glycerol 3-P

TPI

phosphopantetheine

Vitamine B5

PANK2

CoA synthase

Serine

1,3-DPG

3-PG

Glyceraldehyde

3-P-DHase

P-Glycerate

kinase

Serine palmitoyl

transferase

Phospholipids

Diacylglycerol

Phosphatidate

SERAC-1

AGK

Cardiolipin

G3-P Acyltransferase

Triglycerides

CFTDE-Necker X.03

De novo PC/serine synthesis

From Lamari in Saudubray 2016

Serine

CFTDE-Necker X.03

Phospholipids from membrane remodeling

From Lamari et al in Saudubray 2016

Phosphatidyl choline and Eicosanoids Phosphatidyl-inositides

CFTDE-Necker X.03

Classification of IEMs >780 diseases

1. Intoxication: IEM of intermediary metabolism

(small molecules) >180 disorders

2. Energetic disorders: IEM of intermediary

metabolism >250 disorders (mostly mitochondrial)

3. Complex molecules: IEM of organelles metabolism >350 disorders (April 2016) (mostly complex lipids and glycosylation disorders)

CFTDE-Necker X.03 From Stephan Kolker

CFTDE-Necker X.03

From F Mochel and F Sedel Chapter 2 in Saudubray et al 2016

,Leigh syndrome

CFTDE-Necker X.03

CFTDE-Necker X.03

Acute crisis

,or nothing

CFTDE-Necker X.03

Phenotypic variations in IEM

1. Within a group of disorders :

Example : lysine, ornithine, arginine metabolism

pathophysiology of symptoms

2. Within a family with the same disorder

- With different residual enzyme activiy

genetic mechanisms

- With same residual enzyme activity

environmental factors

metabolic networks

development (ontogenic changes)

CFTDE-Necker X.03

Urea cycle interactions

12.Carbonic anhydrase VA (CAVA)

12

CO2 CO3H

Phenotypic variations within metabolic pathways

involved in hyperammonemia

After D Rabier

CFTDE-Necker X.03

Potential genetic mechanisms

- family bearing same mutation(s)-

1. Random X inactivation : OTC,PDH

2. Genetic imprinting : Niemann Pick A/B

3. Composite heterozygocity : MSUD

4. Mitochondrial segregation: NARP/LEIGH

5. Unfolding:TPI

6. Individual variations of cell quality control

7. Others …

CFTDE-Necker X.03

OTC deficiency: random X inactivation

v

1

1

1

III

II

I neonatal

death neonatal

death

2 3

I 1 :

I 2-3:

34

II 1:

as

III 1:

Permanent orotic aciduria

Brothers died in neonatal

period

Asymptomatic. No orotic

aciduria after protein loads.

Index case: hyperammonemic

coma at day 7; OTC activity =

12%

Molecular biology: Full deletion of OTC gene

CFTDE-Necker X.03

PDH : a family affair

CFTDE-Necker X.03

PDH activity and symptoms

Courtesy Dr. Brian Robinson

CFTDE-Necker X.03

Genetic imprinting in Sphingomyelinase deficiency

(Simonaro et al. Am J Human Genet 2006; 78:865-70)

The only allele expressed is that of the mother

Severe

Mild

CFTDE-Necker X.03

Composite Heterozygocity in MSUD

(Frezal et al; Hum Genet. 1985;71:89-91

CFTDE-Necker X.03

Composite Heterozygocity in MSUD

(Frezal et al; Hum Genet. 1985;71:89-91

CFTDE-Necker X.03

Mitochondrial segregation

2.5-year-old proband

Weakness

Developmental delay

Hypotonia

Ptosis

Mild lactic acidemia

TCA cycle

intermediates

on urine organic acids

Courtesy Dr. Anita Beck

CFTDE-Necker X.03

Affected 12-month-old brother

Failure to thrive

Developmental

regression

Hypotonia

Seizures

Case 3

12

months 2.5

years

5

years

CFTDE-Necker X.03

Family history

Mother and younger

brother

(at age 12 months)

Courtesy Dr. Anita Beck Case 3

CFTDE-Necker X.03

Head MRI

Bilateral putamen and

globus pallidus

abnormalities:LEIGH

Case 3 Courtesy Dr. Anita Beck

CFTDE-Necker X.03

Clinical spectrum for T8993G

Range of clinical

phenotypes

Based on percentage

of mutant mitochondria

in blood

Carelli V et al. Arch Neurol 2002;59:264–70 Case 3

CFTDE-Necker X.03

Case 3

80%

NARP

94%

Leigh

31%

carrier

?

Percentage blood

heteroplasmy

CFTDE-Necker X.03

Family matters

Phenotypically normal

sister

Case 3

CFTDE-Necker X.03

Tissue variation of 8993T>G

mtDNA

Case 3

CFTDE-Necker X.03

Conformational versus metabolic disease

Triose phosphate isomerase defect

F. Orosz et al. BBA 1792 (2009) 1168–1174

Null mutations:

Severe anemia

No neuro signs

Mild mutations:

No/mild anemia

Severe neuro signs

CFTDE-Necker X.03

Few potential ontogenic and

environmental mechanisms

Metabolic « switch » during development

Intercurrent events (fast,catabolism,diet,drugs)

Residual enzyme activity (+/-limitant step)

Acute versus slow progressive intoxication

Over energy expenditure

CFTDE-Necker X.03

Normal values change with age

CFTDE-Necker X.03

Nitrogen excretion related to age

Relationship between

urinary urea nitrogen

excretion and

body surface area

CFTDE-Necker X.03

Nitrogen production and outcome

100 g muscle contain about 20 g of proteins

Nitrogen protein content is 16%

20 g protein fully catabolized produces 3.2 g

nitrogen

3.2 g nitrogen = 228 mmol N (3200/14)

228 mmol nitrogen produces 114 mmol urea

(228/2) Urea:CO(NH2)2 114 mmol = 6.4 g

(93×60)

24h urea excretion allows to calculate the protein

catabolism

CFTDE-Necker X.03

1 g N = 6.25 g protein = 30 g muscle

Muscle amino acids

0

100

200

300

400

500

600

Leucine Valine Isoleucine Phenylalanine Methionine

mg AA/gN

(%/g N)

(8.5%) Leu MSUD: 500–700 mg/d

(6%) Val MSUD:300–400 mg/d

(4%) Phe PKU: 200–400 mg/d

(3.2%) Meth HSC: 120–250 mg/d

(5.9%) Ileu MSUD: 280–400 mg/d

Amino acid tolerance in IEM strictly reflects the muscle

amino acid composition

CFTDE-Necker X.03

Protein synthesis

Muscle protein content is 20g%

Nitrogen protein content is 16g%

1 g nitrogen = 6.25 g protein = 30 g muscle

Amino acid composition of natural proteins is

genetically determined (doesn’t depend on

the diet)

Amino acids from food are firstly and mostly

directed to protein synthesis

Essential amino acids are limiting factors

CFTDE-Necker X.03

Catabolism in control and PKU

Influence of diffusion space on blood

AA concentration

CONTROL

Muscle 100 g

Protein 20 g

20 AA 3.2 N

CO2+H2O UREA

(100 mmol)

PKU

Muscle 100 g

Protein 20 g

19 AA PHE (4%) 3.2 g N

(LEU, ILU…) 800 mg (186 mmol)

CO2+H2O UREA

(100 mmol)

LEU, ILU, PHE.. 186 mmol

Adult 60 kg 30 liters 800/30= 22 mg/l

Infant 4kg 3 liters 800/3 = 220 mg/l

CFTDE-Necker X.03

Anabolism in PKU is limited by

Phe intake

CLASSICAL PKU

Daily natural protein intake 5–8 g

(Phe 4% = 200–320 mg)

Phe free AA mixture 15g

8 essential AA Phe:300mg 3.2 g N

Maximum potentially

Synthesized protein 5–8 g

Muscle 25–35 g

CONTROL (theorical model)

Daily natural protein intake 20 g

9 essential AA 3.2 g N

No limitant factor

(Phe 4% about 800 mg)

Maximum potentially

synthesized protein 20 g

Muscle 100 g

CFTDE-Necker X.03

Coma

Tanyel Zubarioglu Indian J Pediatr 2016

DOI 10.1007/s12098-016-2077-3

OAT deficiency mimicking neonatal OTC

CFTDE-Necker X.03 From M Baumgartner et al chapter 21 in Saudubray et al 2016

CFTDE-Necker X.03

CFTDE-Necker X.03

20-50 years

• Pyramidal signs (77%)

• Cerebellar ataxia (64%)

• Cognitive decline (61%)

• Polyneuropathy (45%)

• Psychiatric signs (25%)

• Epilepsy (25%) • Learning difficulties (65%)

• Juvenile cataract (84%)

• Chronic diarrhea (40%)

• Tendon xanthomata (45%)

• Neonatal cholestasis

Childhood

Adulthood

Clinical spectrum of

Cerebrotendinous xanthomatosis

Estimated from a series of 135

published cases (Amador)

Courtesy F Sedel

CFTDE-Necker X.03

Mild intellectual

disability

Mild pyramidal signs

Chronic diarrhea

Mild intellectual

disability

Chronic diarrhea

Liver cholestasis

Chronic diarrhea CTX

Courtesy Dr Fanny Mochel

CFTDE-Necker X.03

Cerebrotendinous xanthomatosis

Su

rviv

al P

rob

ab

ility

0.0

0

.2

0.4

0

.6

0.8

1

.0

0 10 20 30 40 50 60

Time to Event (Year)

Psychiatric Symptoms

Walking Difficulties

Cataract

School Difficulties

Diarrhea

Courtesy Fanny Mochel

Cholestan

ol

Natural history of

cerebrotendinous

xanthomatosis:

a paediatric disease

diagnosed in adulthood

Degos et al. Orphanet

Journal of Rare Diseases

(2016) 11:41

CFTDE-Necker X.03

Neutral Lipid Storage Diseases

Steatosis,Myopathy Oxysterol

Spastic paraparesis

Cerebrotendinous

xanthomatosis

Cataract, Spasticity

Ataxia.Psychiatric signs

Niemann Pick C

Pyramidal sign, Ataxia

Cognitive decline

Neuropathy

PBD/IRD

RP. Deafness

Mental retardation

Transient neonatal to early infantile

Cholestasis or Liver failure

followed by neurodegeneration

MEGDEL

Hypotonia

Dystonia

Leigh like

Citrin

Late onset

neuropsychiatric signs

LCHAD

Myoglobinuria

RP.Neuropathy

Arginase

Spastic paraplegia

CFTDE-Necker X.03

Psychiatric signs,

dementia, ataxia,

dystonia

0 1 2 3 6 10 20 30 40 50

Vertical supranuclear gaze palsy

Early infantile

Late infantile

Juvenile

Adult

Systemic signs in NPC

Splenomegaly

Motor

delay Walking

problems,

clumsiness,

speech delay

Learning problems,

ataxia, seizures,

cataplexia

Age (years)

Neonatal

Fetal hydrops/neonatal cholestasis

Courtesy MT Vanier

CFTDE-Necker X.03

Few potential environmental

mechanisms

Metabolic « switch » during development

Intercurrent events (fast,catabolism,diet,drugs)

Residual enzyme activity (+/-limitant step)

Acute versus slow progressive intoxication

Over energy expenditure

CFTDE-Necker X.03

Glucose sources with fasting

4 8 12 16 20 24 28 32 2 8 16 24 0

10

20

30

40

Hours Days

Exogenous

Glucagon Catecholamines

Glucagon Catecholamines

Cortisol

GH

I II III IV

Insulin

Glycogen Gluconeogenesis

MEAL

Glucose used g/h

From JM Saudubray et al J.Inher.Metab.Dis 2000;23:197-214 . J..

Phase I: Post-prandial II: Short to

middle fast

III: Long fast IV: Very long fast

Glucose Source Exogenous Glycogen

Gluconeogenesis

Gluconeogenesis

(hepatic)

Glycogen

Gluconeogenesis

(hepatic and renal)

Consuming

tissues

All All but liver muscle Brain, Blood cell,

Adrenal medulla

Brain, Blood cell,

Adrenal medulla

Greatest brain

nutrient

Glucose Glucose Glucose Ketone bodies

Glucose

CFTDE-Necker X.03

Phase III: Long fast IV: Very long fast

Glucose Source Gluconeogenesis

(hepatic)

Gluconeogenesis

(hepatic and renal)

Consuming

tissues

Brain, Blood cells,

Adrenal medulla

Brain, Blood cells,

Adrenal medulla

Greatest brain

nutrient

Glucose Ketone bodies

Glucose

10

20

30

40

Late energy sources with fasting

4 8 12 16 20 24 28 32 2 8 16 24

Hours Days

Glucose used g/h I II III IV

MEAL

FFAs

Ketones Ketogenesis

Lipolysis

0

1

2

3

4

0

mmol/L

Gluconeogenesis

Courtesy of JM Saudubray, Paris Necker Hospital From JM Saudubray et al J.Inher.Metab.Dis 2000;23:197-214 . J..

CFTDE-Necker X.03

Few potential environmental

mechanisms

Metabolic « switch » during development

Intercurrent events (fast,catabolism,diet,drugs)

Residual enzyme activity (+/-limitant step)

Acute versus slow progressive intoxication

Over energy expenditure

CFTDE-Necker X.03

Late onset OTC

Born in 1951.

Died at 12 years old

from sudden, unexplained

coma. No previous history

JY 49

years

CFTDE-Necker X.03

K Died at 19 yr

Reye Syndrome

OTC deficiency — MALE to FEMALE transmission

CFTDE-Necker X.03

Few potential environmental

mechanisms

Metabolic « switch » during development

Intercurrent events (fast,catabolism,diet,drugs)

Residual enzyme activity (+/-limitant step)

Acute versus slow progressive intoxication

Over energy expenditure

CFTDE-Necker X.03

Sphingolipids synthesis From Vannier,Caillaud Levade Chap 38 in Saudubray et al 2016)

(Sialic acid)

Cer

Globosides

Sulfatides

Sphingomyelin

Glycosphingolipids

Gangliosides

+ALA

orGLY

CFTDE-Necker X.03

Serine palmitoyltransferase (SPT)

deficiency

Causes adult hereditary sensory autonomic

neuropathy type I (autosomal dominant trait

HSAN I)

Progressive intoxication by deoxysphingoid bases

treatable by high doses of serine

Deoxysphingoid bases levels measured by tandem

MS in plasma are elevated (diagnostic test)

Diagnosis confirmed by molecular analysis of the

SPTLC1 and C2 genes

CFTDE-Necker X.03

Phospholipids from membrane remodeling: PLA 2G6

From Lamari et al in Saudubray 2016

Phosphatidyl choline and Eicosanoids Phosphatidyl-inositides

CFTDE-Necker X.03

Biochemistry

PLA2G6 encodes

– iPLA2b enzyme phospholipase group 4 (calcium

dependent)

– Alternative name phospholipase A2B

Releases FFA from phospholipids and

lysophospholipids membranes

Play a role in

– Intracellular signaling:

arachidonate,eicosanoids

– Inflammatory processes

– Apopotosis

CFTDE-Necker X.03

Phospholipase A2B deficiency

Three main clinical phenotypes:

Infantile neuroaxonal dystrophy (INAD)

Neurodegeneration with brain iron

accumulation (NBIA)

INAD and NBIA are caused by loss of

ability of PLA2 to catalyse FA release

which predicts accumulation of PL

(neuroaxonal spheroids):Intoxication ?

Dystonia-Parkinsonism in adults .

Mutations do not directly impair catalytic

function

CFTDE-Necker X.03

PLAN: gen PLA2G6

1- 6 years 4 – 40 years <3 years

PROTOCOL NBIA PLAN: gen PLA2G6

Dystonia))Parkinsonism)

INAD) Atypical)

AREP

Pure

Parkinsonism

L-dopa

responsive

Parkinsonism)

Courtesy Angela Garcia

CFTDE-Necker X.03

Infantile neuroaxonal dystrophy

Courtesy of Isabelle Desguerre, Paris

Cerebellar atrophy with signal hyperintensity in cerebellar cortex

occasional hypointensity in the pallida and substantia nigra ( iron deposits)

CFTDE-Necker X.03

Few potential environmental

mechanisms

Metabolic « switch » during development

Intercurrent events (fast,catabolism,diet,drugs)

Residual enzyme activity (+/-limitant step)

Acute versus slow progressive intoxication

Over energy expenditure

CFTDE-Necker X.03

Changing spectrum of PDH

Male, 25 years old, has normal intelligence

From age 15 months to adulthood:

– Recurrent episodes of ataxia, hypotonia, weakness,

lethargy when febrile (about once a year)

– Between episodes: progressive polyneuropathy,

scoliosis

– Diagnosis: Charcot-Marie-Tooth, type II

At age 19 years, developed left optic

neuropathy

At age 24 years: right optic neuropathy

Case F Sedel M Brivet

CFTDE-Necker X.03

Metabolic studies

Amino acids — elevated alanine (789 µM)

Fasting Postprandial

Lactate

(nl <1.8 mM)

1.6 mM 2.8 mM

Pyruvate

(nl <67 mM)

0.173 mM 0.250 mM

Lac/Pyr (nl<20) 9 11

Case 10

CFTDE-Necker X.03

Treatment

• Treatment with

thiamin (B1) — 500

mg/day

• Visual activity

before treatment:

Right = 4/10

Left = 1/10

• Visual activity

after treatment:

Right = 8/10 (+4)

Left = 6/10 (+5)

Case 10

CFTDE-Necker X.03

Late onset biotinidase deficiency

22-year-old man presented with a subacute

extensive myelopathy with bilateral optic

neuropathy

Mimicking seronegative neuromyelitis optica.

Metabolic evaluation revealed biotidinase

deficiency,

Treatment with high doses of biotin

progressively improved the clinical condition.

Multiple Sclerosis Journal 2015, Vol. 21(12) 1608–1609

CFTDE-Necker X.03

High dose biotin to the rescue of

demyelinating processes?

High doses of biotin (100–300 mg/day) in patients with

SPMS and PPMS have an impact on disease progression

in those with optic neuropathy,homonymous hemianopia,

and spinal cord involvement *

A phase III trial of high-dose biotin in progressive MS

shows improvement in MS-related disability outcomes (13

% of the exposed group vs 0 % of placebo group had

improvement of Expanded Disability Status Scale or timed

25-foot walk at month 9 and12)**.

*Sedel F et al. Mult Scler Relat Disord 2015;4:159–169.

**Tourbah A et al. The 67th Annual Meeting of the American Academy of Neurology, 2015

Tourbah A (2015) Biotin and demyelinating diseases – a new connection? Mult Scler J 21:1608-1609

CFTDE-Necker X.03

L P Saint Paul et al (2016) high-dose biotin in the treatment of progressive multiple sclerosis,

Expert Opinion on Drug Metabolism& Toxicology, 12:3, 327-344

CFTDE-Necker X.03

Clinical circumstances of care

in adult practice

1. IEM diagnosed in the pediatric age and

reaching adulthood :

treated / untreated natural history

2. IEM striking in adulthood :

- unknown presentations

- methods of investigations adapted to adults

3. IEM diagnosed in adulthood but starting at the

pediatric age

CFTDE-Necker X.03

Challenge of adult IEM patient care

1.Long term treatment maintenance is difficult

2. Natural history of treated patients is unknown

3. Pediatricians don’t know late complications

4.Late onset forms are missed by adult physicians

5.Pediatric forms undiagnosed by pediatricians

6.Diagnostic criteria adapted to adult mild forms

(control values)

CFTDE-Necker X.03

Conclusions

Cette différence phenotypique

« …qui est causée par l'âcreté des humeurs

engendrées dans la concavité du

diaphragme, il arrive que ces vapeurs...

Ossabandus, nequeyrs, nequer, potarimum,

potsa milus. Voilà justement pourquoi votre

fille est muette. »

From Sganarelle in Moliére

Le médecin malgré lui

CFTDE-Necker X.03

Conclusions:

Déjà Necker…!

Cette différence phenotypique

« …qui est causée par l'âcreté des humeurs

engendrées dans la concavité du

diaphragme, il arrive que ces vapeurs...

Ossabandus, nequeyrs, nequer, potarimum,

potsa milus. Voilà justement pourquoi votre

fille est muette. » From Sganarelle in Moliére

Le médecin malgré lui

CFTDE-Necker X.03

Chapters 1 and 2

6th edition

CFTDE-Necker X.03

THE END