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Nephropathic cystinosis: 10 th anniversary of the CTNS gene Elena Levtchenko, MD, PhD University Hospital Leuven, Belgium.

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Presentation on theme: "Nephropathic cystinosis: 10 th anniversary of the CTNS gene Elena Levtchenko, MD, PhD University Hospital Leuven, Belgium."— Presentation transcript:

1 Nephropathic cystinosis: 10 th anniversary of the CTNS gene Elena Levtchenko, MD, PhD University Hospital Leuven, Belgium

2 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 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 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 5 Treatment with cysteamine cystine cytoplasm cystinosin lysosome NH 2 COOH CH CH 2 S CH 2 CH NH 2 COOH + HS CH 2 NH 2 SH CH 2 CH NH 2 COOH CH CH 2 S CH 2 NH 2 + cystinecysteaminecysteine cysteine – cysteamine cysteine transporter CAT- transporter cytoplasm lysosome

6 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 g/m2) - gastro-intestinal complaints (Dohil et al. 2006) - bad breath and sweat odor due to formation of dimethylsulfide (Besouw et al. 2007)

7 cystinosis heterozygotes controls Cystine dysposal from granular fraction of normal, heterozygous and cystinotic cells loaded with [ 35 S] cystine dimethyl ester


9 9 CTNS gene structure (17p13, 23 kb) 123c3c ATG TAG Cystinosin: predicted structure GYDQL YFPQA Chergui et al Most common mutation in European population: 57 kb deletion: CTNS gene, CARKL gene (Town et al. 1998, Wamelink et al. 2008)

10 Functional assay of cystinosin C N C N [ 35 S] - C-S-S-C lysosome cytoplasm Kalatzis et al. 2001, H +


12 Expression of Cystinosin-LKG is not restricted to lysosomes Taranta et al. 2008

13 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

14 14 lysosome mitochonrion nucleus cystine cysteine cystine reductase cystinosin GCS -glut-cys GS GSH oxoglutarate carrier I IIIIIIV V ATP O 2 -. O2-.O2-. H2O2H2O2 SOD GSSG GSH peroxidase GSH GSH reductase dicarboxilate carrier GSSG oxidative redox state GSSG/GSH influence on gene expression Disturbed ATP metabolism O 2 -. glucose pyruvate glycolytic pathway GSH citric acid cycle NADH FADH 2 Stimulation of apoptosis H2O2H2O2 SOD lipid peroxides H+H+ e-e- e-e- H+H+ ADP + P O2-.O2-. pyruvate carrier H+H+ H2O2H2O2 lumen interstitium cytosole cystine Cysteinylation of PKC- - LKG cystine

15 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

16 Molecular therapy, vol.16, No.8, , 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

17 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.

18 Thanks to Cystinosis research group Nijmegen/Leuven L.MonnensM. WilmerM. BesouwB. Van den Heuvel Bambino Gesù Children hospital, Rome F. Emma A. Taranta VUMC, Amsterdam H. Blom M. Wamelink


20 Lysosomal membrane transport lysosome H+H+ H+H+ ATP ADP Acid hydrolases: Proteases Glycosidases Nucleases Phosphatases Sulphatases H+H+ Substrates: Amino acids Monosaccharides Nucleosides Di/tripeptides Inorganic ions Vitamins ?

21 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)

22 22 corneal cystine crystals cystinotic kerathopathy cystinotic retinopathy

23 23 nucleus H+H+ H+H+ cystine cysteine lysosome cytoplasma cystinosin protein degradation cystine cysteine

24 24 Markello et al n= 67 n = 32 n = 17


26 Cystine accumulation in cystinosis Kidney: x normal Liver: x normal Muscle: x normal Brain: x normal Stokes et al Wilmer et al. 2008

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