Nephropathic cystinosis:
10th anniversary of the CTNS gene
Elena Levtchenko, MD, PhD
University Hospital Leuven, Belgium
Definitions
• Cystinosis – an autosomal recessive disease caused by
lysosomal accumulation of cystine due to defective exodus of cystine out of the lysosomes
• Cystinosis – incidence ~1:200,000– clustering in some populations
• Cystinosis– most common cause of inherited
generalized proximal tubular dysfunction
(renal Fanconi syndrome)
3
Clinical forms
• Infantile form:– Fanconi syndrome ~ 3-6 months– end stage renal disease (ESRD) ~ 10 years
• “Late-onset” (juvenile) form:– later onset (often during puberty)– mild tubulopathy, more pronounced proteinuria,
(even in nephrotic range)– later progression to ESRD
• Ocular form• Overlap between ocular and
juvenile forms (Servais et al. 2008)
4
Extra-renal involvement
Eye– photophobia– kerathopathy– retinal blindness
Endocrine organs– hypothyroidism– diabetes mellitus– male hypogonadism
Neuromuscular disease– myopathy
Neurological complaints– epilepsy – mental deterioration– cerebellar and pyramidal signs– stroke-like episodes
Liver disease, exocrine pancreas deficiency Gahl et al. 2002
5
Treatment with cysteamine
cystine
cytoplasm
cystinosin
lysosomeNH2 COOH
CH
CH2
S
S
CH2
CH
NH2 COOH
+
HS
CH2
CH2
NH2
SH
CH2
CH
NH2 COOH
NH2 COOH
CH
CH2
S
S
CH2
CH2
NH2
+
cystine cysteamine cysteinecysteine –cysteamine
cysteine transporter
CAT-
transporter
cytoplasm
lysosome
Efficiency of cysteamine in cystinosis
• Cysteamine postpones the deterioration of the renal function(Markello et al. 1993)
• Cysteamine protects extra-renal organs and should be administered also after renal transplantation (Gahl et al. 2002)
• Topical cysteamine drops (0.5%) dissolve corneal cystine crystals (Tsilou et al. 2002). New viscous cysteamine eye preparations (Bozdag et al. 2008)
BUT:• Cysteamine does not reverse renal Fanconi syndrome • Many patients still develop ESRD and extra-renal complications (at later age)• Major compliance problems due to:
- dose regimen every 6 hours (daily dose 1.3-1.95 g/m2)
- gastro-intestinal complaints (Dohil et al. 2006)
- bad breath and sweat odor due to formation of dimethylsulfide (Besouw et al. 2007)
cystinosis
heterozygotes
controls
Cystine dysposal from granular fraction of normal, heterozygous and cystinotic cells loaded with [35S] cystine dimethyl ester
9
CTNS gene structure (17p13, 23 kb)
1 2 3c
44
5 6 7 8 9 10
11 12
ATG TAG
Cystinosin: predicted structure
GYDQLYFPQA
Chergui et al. 2001
Most common mutation in European population: 57 kb deletion: CTNS gene, CARKL gene (Town et al. 1998, Wamelink et al. 2008)
Functional assay of cystinosin
C
N
C
N
[35S] - C-S-S-C
lysosome
cytoplasm
Kalatzis et al. 2001, 2004
+ H+
Expression of Cystinosin-LKG is not restricted to lysosomes
Taranta et al. 2008
Pathogenesis of cystinosis• How cystinosin defects lead to cell dysfunction in cystinosis is
still unknown
• Existing hypotheses from in vitro studies: – Cystinosin dysfunction leads to defective gluthation synthesis in
cystinosis enhanced oxidative stress leading to cell damage (Levtchenko et al. 2005, Mannucci et al. 2006, Laube et al. 2006)
– Increased sensitivity of cystinotic cells to apoptotic stimuli due to cysteinylation of protein kinase C (Park et al. 2002, 2006)
enhanced cystinotic cell death
– ATP deficiency cell dysfunction and Fanconi syndrome (Coor et al. 1991, Ben-Nun et al. 1993, Foreman et al. 1995, Wilmer et al. 2006)
– No prove in humans or in ctns -/- mice model
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lysosome
mitochonrion
nucleus
cystine
cystine
cysteinecystine reductase
cystinosin
GCS
-glut-cys
GS
GSHoxoglutarate carrier
I II III IV V
ATPO2-. O2-.H2O2
SOD
GSSGGSH peroxidase
GSH
GSH reductase
dicarboxilate carrier
GSSG
oxidative redox state
GSSG/GSH
influence on gene expression
Disturbed ATP metabolismO2-.
glucose
pyruvate
glycolytic pathwayGSH
citric acid cycle
NADH
FADH2
Stimulation of apoptosis
H2O2
SOD
lipid peroxides
H+
e-
e-
H+
ADP
+
P
O2-.
pyruvate carrier
H+
H2O2
lumen interstitium
cytosole
cystine
Cysteinylation of PKC-
- LKG
- LKG
cystine
Possible functions of cystinosin-LKG
• Cystinosin-LKG has probably other functions in the cell additionally to lysosomal cystine transport:
– Cystine-binding in the cytosole: regulate cystine - cysteine pool in the cell and prevent the formation of disulfide compounds
– Alternative plasma membrane cystine transporter
Molecular therapy, vol.16, No.8, 1372-1381, aug.2008
Liver from 3-month old ctns -/- mice
Liver from 6-month old ctns -/- mice
Obstacles of gene therapy in cystinosis:
• Gene should be delivered to all organs
• Use of viral vectors
• Immune response
• Short duration of cystine-lowering effect
• Gene transfer should be administered at early age
Summary
• Discovery of cystinosin in 1998 opened a new chapter of diseases caused by defects of lysosomal transport proteins.
• New data indicate that the expression of cystinosin is not restricted to the lysosomal membrane. Ongoing studies will identify new functions and regulators of cystinosin activity new insights into the pathogenesis of cystinosis.
• Gene therapy in cystinosis is feasible and is a subject of intensive research. Many obstacles should be overcome.
Thanks to
Cystinosis research group Nijmegen/Leuven
L.Monnens M. Wilmer M. Besouw B. Van den Heuvel
Bambino Gesù Children hospital, Rome
F. Emma
A. Taranta
VUMC, Amsterdam
H. Blom
M. Wamelink
QUESTIONS?
Lysosomal membrane transport
lysosome
H+
H+
ATPADP
Acid hydrolases:ProteasesGlycosidasesNucleasesPhosphatasesSulphatases
H+
Substrates:Amino acidsMonosaccharidesNucleosidesDi/tripeptidesInorganic ionsVitamins
?
New insights into the lysosomal membrane transport
• Competition experiments in isolated lysosomes predicted ~20 lysosomal transporters (Pisoni, Thoene 1991)
• Analogous to cystinosin: functional assays of lysosomal membrane proteins (genotype-phenotype correlations in sialic acid storage disease (Morin et al. 2004)
• Proteomics studies of lysosomal membrane (Schroder et al. 2007)
– Identification of new transporters (glucose (GLUT8), myo-inisitol (HMIT), Zn transporter, KCC1…)
• New genes responsible for lysosomal transport disorders (mucolipidosis type IV (MCOLN1), infantile malignant osteopetrosis (ClC7), late infantile neuronal ceroid lipofiscinosis (CLN7)
22corneal cystine crystals cystinotic kerathopathy cystinotic retinopathy
23
nucleus
H+
H+
cystine
cystine
cysteine
lysosome
cytoplasma
cystinosin
protein degradation
cystine
cystine
cysteine
cysteine
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Markello et al. 1993
n= 67
n =
32
n = 17
Cystine accumulation in cystinosis
Kidney: 200 - 400 x normal
Liver: 80 - 1000 x normal
Muscle: 40 - 70 x normal
Brain: 5 - 20 x normal Stokes et al. 2008
Wilmer et al. 2008
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