s r e d r o s i Survey of diagnosis of lysosomal D c storage disorders i l o b a t e M d Milan Elleder e t Institute of Inherited Metabolic Disorders i r e Charles University, 1st Faculty of Medicine and University Hospital h Prague n I f o e t u t i t s n I October 5, 2006
s r e d prehistory – empirical part of the story r o s clinical reports by Tay (1881), Gaucher (1882) and Sachs (1896) i D and by others c i l o b modern history of the lysosomes a their discovery: C. de Duve et al. t e (Biochem. J. 60, 604, 1955) M Nobel Prize 1974 d e t i r modern history of the lysosomal storage e h • H.G. Hers et al. (1963) Acid glucosidase deficiency in GSD II n I • Austin et al. (1963) Arylsulphatase deficiency in MLD f o e t u t present state of the art (2006) – 48 defined entities i t s of different molecular basis (groups Ia,b and II) n I
Iduronate-2-sulphate sulphatase neuronal enzymopathies s ceroid tripeptidylpeptidase I r lipofuscinoses lysosomal storage e due to mutant P d a Cathepin D disorders Ia α -L-iduronidase l m enzyme r i t o o heparan N-sulfatase y l protein - p NAc- α -D-glucosaminidase s MPS r o t i e GSD II (n=30) i n=10 D n t h a i c o i d e CoA: α -glucosaminide NAc-transferase c s α t - 1 e , 4 r i - a g s l l u e c o o s i d a s b e a GlcNAc- 6-sulphate sulphatase c i d l i a p a s e β -glucosylceramidase t e lysosome M GalNAc-6-sulphate sulphatase s e a d m i expanded by a e r c G d a l N A c - * 4 storage - β -galactosylceramidase s u l p h a t e e s β -glucuronidase u l p h a t a s e e s t a * n i i h l y r e a l y u arylsulfatase A r m * o e n i d o a g s N e n h ( - h i * a y h a c l p u e aspartylglucosaminidase r s t o B n y n i l c - a α c α -Mannosidase e i - d g I ) s NAc- β -glucosaminidase A a lipidoses a l β -Mannosidase a A c d f t o n=9 i e e s n o a e s m e s i s a i m s a n a i d a d d a e a d d s i s i e n i o s i s t c o i s o m u u o t t l c c c g a a t u - a r β l i l F u a - a t c - g g e A s α n - - N α α β β 29 hydrolases - * n α I 2006 glycoproteinoses 1 transferase n=7
I.A lysosomal enzymopathies caused by mutation of the s enzyme protein r e d 20. cathepsin D mutated enzymes degrading lipids, r o GPs, proteins, and glycogen mutated enzymes degrading GAGs s i 21. α -L-iduronidase D 1. ceramidase (N-Acyl-sfingoid-Cer) (DS, HS) 2. β -glucosylceramidase (GlcCer) 22.Iduronate-2-sulphate sulphatase( DS,HS) c 3. β -galactosylceramidase (GalCer) i 23. heparan N-sulphatase (HS) l o 4. arylsulphatase A (SGalCer) 24. NAc- α -D-glucosaminidase (HS) b 5. NAc- β -glucosaminidase A (GM2) a 25. CoA: α -glucosaminid NAc-transferase (HS) t 6. NAc- β -glucosaminidase B (GM2,GP) e 26. GlcNAc- 6-sulphate sulphatase (HS) M 7. β -galactosidase (GM1, OLS, GP, KS) 27. GalNAc-6-sulphate sulphatase (KS,C6S) 8. α -galactosidase A ( Gb3Cer) d 28. GalNAc-4-sulphate sulphatase (DS) e 9. sphingomyelinase (P-cholin-cer) 29. β -glucuronidase (DS,HS,C4S,C6S) t i 10. acid lipase (CE, TAG) r 30. hyaluronidase (hyaluronic acid) e 11. α -neuraminidase (GP, gangliosides?) h 12. N-acetyl- α -galactosaminidase (GP, n I blood gr. A: α GalNAc- [Fuc α ]- β gal-;) 30 lysosomal enzymes f o 13. acid α -1,4-glucosidase (glycogen) 29 hydrolases +1 transferase e 14. α -Mannosidase (GP) t 30 proteins 30 genes 30 entities 15. β -Mannosidase u (GP) t 16. α -Fucosidase (GP) i t memento – there is more than 40 s 17. aspartylglucosaminidase (GP) n lysosomal enzymes known !! 18 tripeptidylpeptidase I – TPP (prot.) I 2006 19. palmitoyl-protein thioesterase-PPT
s r e I.B deficient enzyme associated functions (n=8) d r o deficient posttranslational processing (n= 2) s i D • abnormal targeting of lys. enzymes extracellularly (deficient c synthesis of Mannose-6P label – mucolipidosis II/III i l o • deficient synthesis of active site in a group of sulphatases (special b a enzyme in ER: FGE formylglycine generating enzyme or SUMF1 t e factor 1): cystein → formylglycine M sulphatase modifying d (PSD = MPS + sulphatidosis, ect) e t i r deficient protection=increased degradation (galactosialidosis) e h (protection by cathepsine A) (n=1) n I f o deficient lysosomal enzyme activators (n=5) e • SAPs A-D (pSap deficiency): A (GALC ase ); B (ASA, α Gal ase ); t u t C (GC ase ); D (ceramidase) i t s • hexosaminidase activator (alternat. Tay-Sachs) n I
s r e d r o total 38 enzymopathic entities to be diagnosed s i D prime importance: enzyme activity assay c i l o b a t e mechanism of the deficient M activity should be specified d e t i r e h DNA analysis is of n I f secondary importance o e t u t i t s n I 2006
s II. lysosomal disorders due to deficiency of r e noncatalytic membrane components d r (n = 10) o s i D A. transporters across the lysosomal membrane(n=2) c i • cystinosis l o • sialic acid storage disease b a t e B. mutant lysosomal membrane proteins operating in the M membrane lipid trafficking and by so far unknown (albeit d e essential) mechanisms (n<8) t i r NCL3, NCL5, NCL6, NCL 8 (neuronal ceroid lipofuscinoses) e h proteins, ML IV, NPC1, NPC2 (Niemann-Pick disease type C), n I LAMP 2 (Danon disease) f o e total 10 entities to be diagnosed at the protein/metabolite levels t u final diagnosis the DNA level t i t (gene coding the dysfunctional protein) s n !! activities of all the lysosomal enzymes are in the normal range !! I
s r e II. (n=10) I. A, B (n=38) d r deficiencies of noncatalytic deficiencies o s lysosomal membrane in breakdown i D functions catalysis c i 48 l o entities b a t e M hall mark: „storage“- overfilling d and gradual lysosomal expansion by: • undegraded e t i enzyme substrates r e h • unremovable n enzyme products I f o • misshandeled e N heterogenous t u N t compounds i t s n normal cell I 2006 storage cell
Lysosomal s enzymopathy r A B e cytology cytology - EM d A B A B r o A A B A B s i A B A B A B D A B B A B c i l A B A B o A B b A B A B ultrastructure a A B t of the stored material e M d e t i r e h n OLS I GM2 Gb 3 Cer f o e t u t i t s CE n I low power EM: cell in advanced SU GlcCer stage of lysosomal storage
s r e d r o s i D c ISSD NPC1 NCL6 ML IV i l loss of mucolipin function loss of NPC1 protein function o loss of NCL 6 protein loss of SA transporter retention of sialic acid (function unknown) (altered lipid strafficking) (function unknown) b mixture of lipids stored mixture of lipids stored SCMAS storage a t e EM patterns in group II LSDs M d e basic cytological patterns t of lysosomal storage i r e h n striated pattern-GC (2D distension) EM patterns in uncleaved foamy pattern (3D distension) I substrate accumulation f o e t u t i t s n I NCL6 Gaucher (GlcCer) GSD II (glycogen) MPS (GAGs) CESD (CE storage) Tay-Sachs (GM2)
s r e d r o s i D c i each of the molecular defects is a l o b a specific biological entity informing t e M indirectly about the level of a critical d e lysosomal function (e.g. degradative, t i r e trafficking) in normal human h n I (eukaryotic) tissues f o e t u t i t s n I
s r e d r o s i D lysosomal storage disorders c the diagnostic approach i l o b a decisions at the decisions at the t decisions at the tissue level e clinical level biochemical/molecular M • storage cell analysis (biopsy) (phenotype analysis) levels (48 entities !!) • results of urine analysis d e t Intermediate auxiliary i r enzyme e direction indicator h deficiency „at a crossroad“ n (and its I mechanism!) f o deficient lysosomal e t noncatalytic u membrane t protein i t s n I
s r specific storage pattern at the cell level: e d uniform storage of SM liquid crystals r o in a b.m. histiocyte s i D recommendation: c i l ASM activity evaluation o b a t e Niemann-Pick type A M hepatosplenomegly d e t i r e h n I f o e t u t i t s n I uniform staining foamy storage pattern birefringence of SM liquid crystals for phospholipids
s r e d r o s i D c i l o autofluorescence (ceroid) phase contrast birefringence (absent) b a variable storage t e of phospholipids M bone marrow storage In isotropic state d pattern in NPC (admixture of ceroid) e t cytology and i r e staining h Filipin test – NPC1,2 gene n Giemsa I f o e t u t i t s n I irregular accumulation of phospholipids (ferric hematoxylin)
Recommend
More recommend
