Survey of diagnosis of lysosomal storage disorders

Milan Elleder Institute of Inherited Metabolic Disorders Charles University, 1st Faculty of Medicine and University Hospital Prague

October 5, 2006 prehistory – empirical part of the story clinical reports by Tay (1881), Gaucher (1882) and Sachs (1896) and by others

modern history of the their discovery: C. de Duve et al. (Biochem. J. 60, 604, 1955) Nobel Prize 1974

modern history of the lysosomal storage •H.G. Hers et al. (1963) Acid glucosidase deficiency in GSD II •Austin et al. (1963) Arylsulphatase deficiency in MLD

present state of the art (2006) – 48 defined entities of different molecular basis (groups Ia,b and II)

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n=9 β ceroid lipidoses neuronal GSD II GSD lipofuscinoses 2006 I.A lysosomal enzymopathies caused by mutation of the enzyme protein mutated degrading lipids, 20. cathepsin D GPs, proteins, and glycogen mutated enzymes degrading GAGs 1. (N-Acyl-sfingoid-Cer) 21. α-L- (DS, HS) 2. β- (GlcCer) 22.Iduronate-2-sulphate sulphatase(DS,HS) 3. β- (GalCer) 23. heparan N-sulphatase (HS) 4. arylsulphatase A (SGalCer) 24. NAc-α-D-glucosaminidase (HS) 5. NAc-β-glucosaminidase A (GM2) 25. CoA:α-glucosaminid NAc-transferase (HS) 6. NAc-β-glucosaminidase B (GM2,GP) 26. GlcNAc- 6-sulphate sulphatase (HS) 7. β-galactosidase (GM1, OLS, GP, KS) 27. GalNAc-6-sulphate sulphatase (KS,C6S) 8. α-galactosidase A ( Gb3Cer) 28. GalNAc-4-sulphate sulphatase (DS) 9. sphingomyelinase (P-cholin-cer) 29. β-glucuronidase (DS,HS,C4S,C6S) 10. acid lipase (CE, TAG) 30. (hyaluronic acid) 11. α- (GP, ?) 12. N-acetyl-α-galactosaminidase (GP, blood gr. A: αGalNAc- [Fucα]- βgal-;) 30 lysosomal enzymes 13. acid α-1,4-glucosidase (glycogen) 29 hydrolases +1 transferase 14. α- (GP) 15. β-Mannosidase (GP) 30 proteins 30 30 entities 16. α- (GP) 17. (GP) memento – there is more than 40 18 tripeptidylpeptidase I – TPP (prot.) lysosomal enzymes known !! 19. palmitoyl-protein thioesterase-PPT 2006 I.B deficient enzyme associated functions (n=8) deficient posttranslational processing (n= 2) •abnormal targeting of lys. enzymes extracellularly (deficient synthesis of Mannose-6P label – II/III •deficient synthesis of in a group of sulphatases (special enzyme in ER: FGE formylglycine generating enzyme or SUMF1 sulphatase modifying factor 1): cystein→ formylglycine (PSD = MPS + sulphatidosis, ect) deficient protection=increased degradation (galactosialidosis) (protection by cathepsine A) (n=1) deficient lysosomal enzyme activators (n=5) •SAPs A-D (pSap deficiency): A (GALCase); B (ASA, αGalase); C (GCase); D (ceramidase) • activator (alternat. Tay-Sachs) total 38 enzymopathic entities to be diagnosed prime importance: enzyme activity assay

mechanism of the deficient activity should be specified

DNA analysis is of secondary importance

2006 II. lysosomal disorders due to deficiency of noncatalytic membrane components (n = 10)

A. transporters across the lysosomal membrane(n=2) • cystinosis • sialic acid storage disease B. mutant lysosomal membrane proteins operating in the membrane lipid trafficking and by so far unknown (albeit essential) mechanisms (n<8) NCL3, NCL5, NCL6, NCL 8 (neuronal ceroid lipofuscinoses) proteins, ML IV, NPC1, NPC2 (Niemann-Pick disease type C), LAMP 2 (Danon disease) total 10 entities to be diagnosed at the protein/metabolite levels final diagnosis the DNA level ( coding the dysfunctional protein) !! activities of all the lysosomal enzymes are in the normal range !! I. A, B (n=38) II. (n=10) deficiencies deficiencies of noncatalytic in breakdown lysosomal membrane catalysis functions 48 entities

hall mark: „storage“- overfilling and gradual lysosomal expansion by: • undegraded enzyme substrates • unremovable enzyme products • misshandeled N heterogenous N compounds normal cell

storage cell 2006 Lysosomal enzymopathy A B cytology cytology - EM A B A B A B A A B A B A B A B A B B A B A B A B A B A B A B ultrastructure A B of the stored material

OLS GM2 Gb3Cer

CE

low power EM: cell in advanced SU GlcCer stage of lysosomal storage ML IV NPC1 NCL6 ISSD loss of mucolipin function loss of NPC1 protein function loss of NCL 6 protein loss of SA transporter (function unknown) (altered lipid strafficking) (function unknown) retention of sialic acid mixture of lipids stored mixture of lipids stored SCMAS storage

EM patterns in group II LSDs

basic cytological patterns of lysosomal storage

striated pattern-GC (2D distension) EM patterns in uncleaved foamy pattern (3D distension) substrate accumulation

GSD II (glycogen) Gaucher (GlcCer) MPS (GAGs) CESD (CE storage) NCL6Tay-Sachs (GM2) each of the molecular defects is a specific biological entity informing indirectly about the level of a critical lysosomal function (e.g. degradative, trafficking) in normal human (eukaryotic) tissues lysosomal storage disorders the diagnostic approach

decisions at the decisions at the tissue level decisions at the clinical level biochemical/molecular (phenotype analysis) •storage cell analysis (biopsy) •results of urine analysis levels (48 entities !!) Intermediate auxiliary direction indicator enzyme deficiency „at a crossroad“ (and its mechanism!)

deficient lysosomal noncatalytic membrane protein specific storage pattern at the cell level: uniform storage of SM liquid crystals in a b.m. histiocyte

recommendation: ASM activity evaluation

Niemann-Pick type A hepatosplenomegly

foamy storage pattern birefringence of SM liquid crystals uniform staining for phospholipids autofluorescence (ceroid) phase contrast birefringence (absent)

variable storage of phospholipids bone marrow storage In isotropic state pattern in NPC (admixture of ceroid) cytology and staining Filipin test – NPC1,2 gene Giemsa

irregular accumulation of phospholipids (ferric hematoxylin) storage pattern in NCL2 (TPP I deficiency)

control activity of AcPhos - cryostat sections NCL

autofluorescent storage material EM intralysosomal curvilinear bodies Grays Anatomy, TB Johnston et al., eds.,1958

urine analysis = „chemical biopsy of the kidney“ positive finding means presence of lysosomal storage in the tubular and glomerular cells; detached storage cells should be the main source of the diagnostic stored compound Lipidoses free of urinary findings Gaucher negative Krabbe negative

Lipidoses with positive findings in the urine (currently not utilized for screening) NPA/B /cholesterol CESD/Wolman cholesteryl esters

Lipidoses with positive findings in the urine (recommended for screening)

Fabry (incl. SapB def.) Gb3Cer MLD (incl. SapB def.) sulphatide Farber GM1 gangliosidosis βgal - OLS GM2 gangliosidosis GlcNAc – OLS kidney (frozen section) in the urinary sediment storage in glomerules

storage in medullary tubules

Fabry disease PAS + results of kidney Gb3Cer Gb Cer and urinary 3 analysis

N N C C C H N normal control; C carriers; H hemizygote

section of the kidney

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desqumated storage cell birefringence of in situ EM – storage in tubular epithelium tubular cell storage Gb3Cer in urines of Fabry (FD) hemizygotes, female carriers and controls.

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300 controls FD carriers FD hemiz. 200

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METHOD: Extraction of total urinary lipids – Ð

Lipid separation – HPTLC orcinol detectionÐ quantification reversed-phase chromatography Ð densitometrically (CAMAG II)

Berná L. et al.,Anal Biochem, 269, 304-311 (1999) kidney tubules and urinary sediment in sulphatidosis

Cresyl violet birefringence

Urinary sediment Urinary sediment

¨storage in kidney tubules (SGalCer) in urines of pacients (P) with sulfatidosis (C1,2=controls)

A. Orcinol detection, B. Azur A detection (specific), Stand.= and GL standard

Berná L. et al.,Anal Biochem, 269, 304-311 (1999) urine in mucopolysaccharidoses (GAGs) KS CS DS2 HS DS1 Hep CONTROL - + - -/± -/±- MPS I - + + + variable + - MPS II - + + + variable + - MPS III - + - + !! - + MPS IVA + ++ -- -- MPS IV B -/± +- - - - MPS VI - + +!! - +!! - MPS VII - ++ +!! + +!! - KS – keratan sulphate; DS – dermatansulphate; CS – chondroitinsulphate; HS – heparan sulphate; Hep - heparin MPS I – Hurler disease (α −iduronidase deficiency)

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3,5 =MPS I (excretion of dermatan sulphate/DS and heparan sulphate/HS, see arrows), 4 = neonatal control (traces of HS and DS1), 1,2,6 = controls Urine GAG ELFO in patients with MPS II, and its comparison with MPS I a MPS III

Urinary excretion of dermatan sulphate (DS1,2) and heparan sulphate (HS)

MPS II patients

KO = control

MPS II: iduronate-2-sulphate sulphatase deficiency, X – linked disorder MPS IVA deficiency of GalNAc-6-sulphate sulfatase (excretion of keratan sulfate, chondroitin-6-sulphate)

2,6 = MPS IVA (see arrows) 4 = MPS I 1,3,5,7 = controls Urine in glycoproteinoses and related disorders OLS (oligosaccharides) – low mol.w. glycoconjugates reflecting incomplete degradation of

GM1 gangliosidosis…………….. βgal-OLS α-mannosidosis…………………..αmann- OLS β-mannosidosis ………………….βmann- OLS α-Fucosidosis ………………….. αfuco - OLS Sialidosis ……………………….. sialyl - OLS Galactosialidosis ……………… gal- and sialyl – OLS AGU ……………………………… aspartylglucosamine (+ other glycoasparagines) ISSD (SALLA dis.) ……………... free sialic acid Schindler disease ……………. sialyl – OLS

GSD II ………………….………… occasionally (Glc)4 * * Glycoproteinoses – HPTLC of urinary oligosacharides

P1, P2 = susp.sialidosis confirmed later by enzymology (sample applied in two concentrations), Sial=sialidosis(archived pathologic control), GM1=GM1 gangliosidosis, Fuc= fucosidosis, Sch=Schindler disease, NANA= N-acetylneuraminic acid (standard), Ko=control urine patient AGU

ninhydrine detection – urine in AGU Urine in other LSDs (negative or unsignificant findings)

NPCs negative NCLs negative (or SCMAS) Danon dis. not studied ML IV phospholipids Cystinosis generalized AAU Clinical findings : dysmorphia; dysostosis neurology; visceromegaly; corneal clauding (absent in MPS II) heart valvular disease; storage vacuoles in lymphocytes incl. Alder-Reily granules

Urine analysis: • GAGs • OLS • free silaic acid • AGU LYSOSOMAL STORAGE DISORDERS IN THE CZECH and SLOVAK REPUBLICS 1975-2005 (n=525)

GSD II GP ML 3% 2% 1% NCL 16%

MPS LIPIDOSES 26% 53% LIPIDOSES IN THE CZECH AND SLOVAK REPUBLICS 1975-2005 (n=279)

GM2 other GM1 Gaucher 4% 2% 20% Krabbe 5% 8%

CESD 6%

MLD

9% NPC NPA, B 18% 10% Fabry IIMD, 2005 18% MUCOPOLYSACCHARIDOSESMUCOPOLYSACCHARIDOSES 19751975 -- 20052005 (n=103)(n=103)

VI IV B VII I IV A 2% 1% 1% 20% 15% III D 2%

III C 4%

III B 3%

III A II 27% 25% IIMD, 2005 Postnatal and prenatal diagnoses of LSDs in our Institute (1960 - 2005)

40 35 30 25 20 15 10 diagnosed in in diagnosed particular year particular 5 0 Number of patients patients of Number

0 6 9 2 5 1 7 0 6 9 2 5 6 6 6 7 8 8 9 9 9 0 97 00 19 196319 19 1 19 197819 198419 19 199319 19 2 20 Year No. of live births (A), postnatal and prenatal diagnoses of LSDs (B) in the Czech and Slovak Republics (1960 - 2003) 350000 300000 A 250000

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50000 30 0 1: 6000 25 20 B 15 10 5 0 6 8 1 0 3 6 9 2 1960 1963 196 1969 1972 1975 197 198 1984 1987 199 199 199 199 200 Institute of Inherited Metabolic Disorders Prague, March 2004 The diagnostic procedures

decisions at the decisions at the tissue level decisions at the clinical level biochemical/molecular (phenotype analysis) storage cell analysis (biopsy) results of urine analysis levels (48 entities !!) Intermediate auxiliary (directing) step enzyme deficiency (and its mechanism!) lys. noncatalytic membrane team work protein dysfunction Selected syndromes suggestive of LSD

systemic disorder in childhood mainly dysmorphy + dysostosis + corneal clouding + valvular heart disease + neurology (MPS, GP, GM1, PSD) adolescence - adulthood progressive nephropathy + cardiomyopathy + angiokeratomas + neuropathy (sensitive) early childhood progressive neurol. disturbance + retinopathy (blindness) neuronal ceroidlipofuscinoses childhood - adolescence neurological disturbance + VSO + splenomegaly (even mild) Niemann-Pick type C selected syndromes suggestive of LSD cont. adulthood (adolescence)

isolated splenomegaly + hypersplenism splenomegaly + unexplained femoral head necrosis Gaucher´s disease ( deficiency)

myoclonous epilepsy + cherry red spot ML I (sialidase deficiency)

isolated hypertrophic CMP cave! Fabry disease (αGal deficiency) (inter alia)

isolated hepatomegaly with slightly altered LFTs serum cholesterol increased in LDL (decreased in HDL) risk of accelerated atherosclerosis CESD (acid lipase deficiency) LSDs project into majority of clinical disciplins (only significant involvement is included) ophthalmology (NCL, GM1-2, NPA, Fabry, MPS, GP, ML IV) neurology (GM1-2, NCL1,2, NCL3,5,6,8, NPCs, Krabbe, MLD, MPS, GP, GD) psychiatry (adult neurolopidoses) pneumology (Gaucher, NPA/B, NPC2) cardiology (Fabry, MPS, GP, GSD II) angiology (Fabry, CESD, MPS) hepatology (CESD, NPC infantile, MPS, GSD II, GP) nephrology (Fabry, nephrosialidosis) dermatology (Fabry, Fuco-, βMannosidosis, PSD, Farber) hematology (Gaucher, NPA/B, NPC, ect) orthopedy/osteology (MPS, GP, GD, ML II/III) stomatology (MPS, GP, ML II/III) ORL (Fabry, MPS, GP)

GP glycoproteinoses, CESD acid lipase deficiency, PSD polysulphatase deficiency, GSD II Pompe disease MPS mucopolysaccharidoses; GD Gaucher disease, Farber dis. = ceramidase deficiency The end

ACKNOWLEDGEMENT clinical analysis (division A) – E. Hrubá, Jahnová, E. Košťálová, S. Štastná. biochemical analyses (divisions A and B): J. Ledvinová, H. Poupětová, L. Berná, O. Martinová, E. Pospíšilová, M. Hřebíček DNA analysis (Div.B) : L. Dvořáková and her group histology, EM, histochemistry: M. Elleder, H. Hůlková (div. B)