Molecular, cell biological and genetic aspects of diseases

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1 Molecular, cell biological and genetic aspects of diseases Glycosylation associated diseases Background: Glycosylation associated disorders: The Glycogenomics Paradigma 1

2 Glycosylation The most common modification of cellular proteins and lipids Glycolipids Up to 5 X 10 5 different glycan structures estimated to exist > 50% of all cellular proteins are glycosylated important The Glycocalyx: glycans attached to cell surface proteins and lipids Functions of glycans: Folding, transport, sorting, protein stability RECOGNITION: Interactions between cells, cells and ECM, and pathogens GLYCANS ARE ESSENTIAL FOR MULTICELLULAR LIFE What they do? OR what do they not do? Glycans have many flavors: different sugars their order different linkages 2

3 Glycan diversity and biotechnology applications: Differences between: Species Individuals (e.g. ABO antigens) Tissues Cells Glycan changes in cancers, inflammation, type II diabetes: generally not considered as inherited disorders 3

4 Congenital disorders of glycosylation (CDGs): Represent a constellation of diseases that result from defects in the synthesis of glycans and in the attachment of glycans to other compounds. Involve the N linked and O linked glycosylation pathways, GPI anchor synthesis, biosynthesis of proteoglycans as well as lipid glycosylation pathways. Figure 1. Glycosylation associated disorders In 2013 alone, a genetically proven glycosylation disorder was reported, on average, every 17 days. Main organelles for glycosylation in an eukaryotic cell: rer > High mannose type N glycans Golgi >N and O linked glycans, GAGs, Glycolipids Plasma membrane > Hyaluronan Cytoplasm > O linked GlNAc 4

5 Glycan synthesis (basic principle): Nucleotidesugars Specific Transporters Glycosyltransferases Glycosidases Acceptor (amino acid, sugar, lipid) No template! Synthesis of nucleotide sugars: Glucose and fructose (and galactose) are taken up from nutrients Other nucleotide sugars are made form these in the cytoplasm Involves multiple steps, including Phosphorylation Epimerization Acetylation Regulated by feed back and energy metabolism/state 5

6 Nucletide sugar transporters: Act as antiporters Import nucleotide sugars Export monophosphates Facilitate concentration of nucleotide sugars (K m for transferases ~2 mm > 50 x concentration needed) Glycosyltransferases (and Glycosidases) > 250 distinct enzymes Type 2 membrane proteins Substrate, acceptor, and linkage type specific Current view: act as complexes Cat Stem TMD N terminus 6

7 Live cell FRET measurements FRET principle < 10 nm Em 480 Ex 430 nm FRET Signal ( nm) homomers heteromers GnTI GnTI Man II? Man II? GnTII GnTII Other GnTs Other GnTs GalTI GalTI ST6Gal I ST6Gal I Other known GTase heteromers SO3 Kellokumpu et al (review) 7

8 How glycans are normally made: N glycans How glycans are normally made: Glycosylphosphatidylinositolanchored proteins (GPI anchor): Lipid precursor synthesis in the ER membrane Attachment close to the C terminus of a protein precursor Upon arrival in the Golgi, accumulate in lipid rafts GPI anchored proteins typically function in cell signalling, have some targeting function and enable escape from immune surveillance 8

9 How glycans are normally made: O glycans: Mucins: most common Synthesis begins in the cis Golgi GalNAc is attached to ser or thr Extension to: 1. Core structures Core extension (more variable) How glycans are normally made: Proteoglycans and glycosaminoglycans: Common in extracellular matric proteins Xylose (+Gal Gal) as a linking trisaccharide sugar Glycan chains consist of GlcA GlcNAc or GlcA GalNAc repeats Sulphated to variable extent 9

10 How glycans are normally made: Glyco(spingo)lipids: Glykosfingolipids: Ceramide precursor (ER) Synthesis proceeds typically via forming glucosylceramide Important components of for cellular membranes and signaling cascades (Gangliosides) Congenital disorders of glycosylation (CDGs) Rare diseases (~1:20 000) some prevalent locally Most associated with defects in N linked glycan synthesis Defects in other pathways also exist Divided into two classes: CDG type I (ER) CDG type II (Golgi) 10

11 General Clinical features: CDGs Symptoms begin in infancy Biochemically and clinically heterogeneous: however, most types have been described in only a few individuals, and thus understanding of the phenotypes is limited. Most commonly, they include psychomotor retardation, low muscle tone, incomplete brain development, visual problems, coagulation defects, and endocrine abnormalities (mimic other inherited multisystemic metabolic disorders (e.g. mitochondrial) Main causes: Insufficient amounts of sugar nucleotides (and/or their precursors) Activity or expression changes in glycosidases and glycosyltransferases Changes in proteins that control organelle homeostasis or trafficking of the glycosylation enzymes Due to hypomorphic alleles (why?) The phenotype can vary even when the mutated gene is the same (e.g. siblings)? Possible reasons: residual enzymatic activity genetic background context dependent (compensatory pathways may exist) dietary and environmental impacts 11

12 Diagnosis of N linked CDG patients: Altered glycosylation of serum transferrin is the most reliable indicator for N linked CDG (Type I and II) Changes can be identified using isoelectric focusing (IEF) IEF pattern allows detection of only differentially sialylated glycoforms of transferrin Not all CDGs* can be detected with transferrin IEF analysis: Instead: IEF, MS or loss of lectin binding to specific glycoconjugates present in the urine *CDG IIb, CDG IIc, muscle eye brain disease, Walker Warburg syndrome, hereditary multiple exostoses, progeria variant of Ehlers Danlos syndrome and HEMPAS Type I CDGs (ER): Disorder Gene Enzyme CDG Ia PMM2 phosphomannomutase II CDG Ib MPI phosphomannose isomerase CDG Ic ALG6 glucosyltransferase I; Dol P Glc: Man 9 GlcNAc 2 PP Dol glucosyltransferase CDG Id ALG3 Dol P Man: Man 5 GlcNAc 2 PP Dol mannosyltransferase CDG Ie DPM1 Dol P Man synthase I GDP Man: Dol Pmannosyltransferase Dolichol kinase deficiency DK1 CTP+ dolichol dolichol P +CDP, activation of dolichol CDG Ig ALG12 Dol P Man: Man 7 GlcNAc 2 PP Dol mannosyltransferase CDG Ih ALG8 glucosyltransferase II Dol P Glc: Glc 1 Man 9 GlcNAc 2 PP Dol glucosyltransferase CDG Ii ALG2 mannosyltransferase II GDP Man: Man 1 GlcNAc2 PP Dol mannosyltransferase CDG Ij DPAGT1 UDP GlcNAc: dolichol phosphate N acetylglucosamine 1 phosphate transferase CDG Ik ALG1 mannosyltransferase I GDP Man: GlcNAc 2 PP Dol mannosyl transferase CDG IL ALG9 mannosyltransferase Dol P Man: Man 6 and Man 8 GlcNAc 2 PP Dol mannosyltransferase RFT1(flippase) deficiency RFT1 thought to be needed to flip LLO donor to ER lumen Oligosaccharyl transferase subunit TUSC3 subunit of the OST complex Oligosaccharyl transferase subunit IAP subunit of OST complex found on X chromosome 12

13 Type II CDGs (Golgi): CDG IIa MGAT2 GlcNAc transferase 2 (GnT II) CDG IIb GLS1 α1 2 glucosidase I CDG IIc SLC35C1/FUCT1 GDP fucose transporter CDG IId B4GALT1 β1 4 galactosyltransferase CDG IIf SLC35A1 CMP sialic acid transporter Type II CDGs: sc. multipathway disorders COG7 CDG (CDG IIe) COG7 COG complex subunit 7 COG1 CDG (CDG IIg) COG1 COG complex subunit 1 COG8 CDG (CDG IIh) COG8 COG complex subunit 8 COG5 CDG (CDG IIi) COG5 COG complex subunit 5 COG4 CDG (CDG IIj) COG4 COG complex subunit 4 COG6 CDG (CDG IIL) COG6 COG complex subunit 6 TMEM165 CDG (CDG IIk) TMEM165 Transmembrane protein 165 (Golgi ph) Vacuolar ATPase ATP6V0A2 Golgi ph (trafficking): Cutis laxa 13

14 Key clinical features (N linked CDGs) Disorder Gene Key clinical features CDG Ia PMM2 Mental retardation (MR), hypotonia, esotropia, lipodystrophy, cerebellar hypoplasia, stroke like episodes, seizures CDG Ib MPI Hepatic fibrosis, protein losing enteropathy, coagulopathy, hypoglycemia: OBS! Treatment with mannose diet CDG Ic ALG6 Moderate MR, hypotonia, esotropia, epilepsy CDG Id ALG3 Profound psychomotor delay, optic atrophy, acquired microcephaly, iris colobomas, hypsarrhythmia CDG Ie DPM1 Severe MR, epilepsy, hypotonia, mild dysmorphism, coagulopathy CDG If MPDU1 Short stature, icthyosis, psychomotor retardation, pigmentary retinopathy CDG Ig ALG12 Hypotonia, facial dysmorphism, psychomotor retardation, acquired microcephaly, frequent infections CDG Ih ALG8 Hepatomegaly, protein losing enteropathy, renal failure, hypoalbuminemia, edema, ascites CDG Ii ALG2 Normal at birth, mental retardation, hypomyelination, intractable seizures, iris colobomas, hepatomegaly, coagulopathy CDG Ij DPAGT1 Severe MR, hypotonia, seizures, microcephaly, exotropia CDG Ik ALG1 Severe psychomotor retardation, hypotonia, acquired microcephaly, seizures, fever, coagulopathy, nephrotic syndrome, early death CDG IL ALG9 Severe microcephaly, hypotonia, seizures, hepatomegaly RFT1 deficiency RFT1 Developmental delay, hypotonia, seizures, hepatomegaly Oligosaccharyltransf. subunit TUSC3 Nonsyndromic mental retardation; abnormal glycosylation not proven; does not affect serum protein glycans Oligosaccharyltransf. subunit IAP X linked nonsyndromic mental retardation; abnormal glycosylation not proven; does not affect serum protein glycans Dolichol kinase deficiency DK1 Dry, thin skin, ichthyosis, baldness, hypotonia, dialyated cardiomyopathy, hypoketotic hypoglycemia CDG IIa MGAT2 MR, dysmorphism, seizures, dysmorphism, hypotonia, hepatomegaly, hepatic fibrosis (death at 2.5 months) CDG IIb GLS1 CDG IIc SLC35C1/FUCT1 Recurrent infections, persistent neutrophilia, MR, microcephaly, hypotonia (normal Tf) CDG IId B4GALT1 Hypotonia (myopathy), spontaneous hemorrhage, Dandy Walker malformation CDG IIe COG7 Fatal in early infancy, dysmorphism, hypotonia, seizures, hepatomegaly, progressive jaundice, recurrent infections, cardiac failure CDG IIf SLC35A1 Thrombocytopenia, no neurologic symptoms, normal Transferrin, abnormal platelet glycoproteins Examples: CDG 1a PMM2 CDG (CDG Ia) is the most common CDG (> 800 cases identified worldwide) moderate to severe developmental and motor deficits, hypotonia, dysmorphic features, failure to thrive, liver dysfunction, coagulopathy, and abnormal endocrine functions. More than 100 mutations in phosphomannomutase 2 (PMM2) Impairs conversion of Man 6 P to Man 1 P, a precursor for the synthesis of GDP mannose (GDP Man) and dolichol P mannose (Dol P Man) and the synthesis of Glc 3 Man 9 GlcNAc 2 P P Dol Due to hypomorphic alleles (complete loss is lethal). There are currently no therapeutic options for PMM2 CDG patients. 14

15 CDG Ib Caused by mutations in mannose 6 phosphate isomerase. This enzyme interconverts fructose 6 P and mannose 6 P (Man 6 P). Patients have impaired growth, hypoglycemia, coagulopathy, severe vomiting and diarrhea, proteinlosing enteropathy, and hepatic fibrosis. However, no intellectual disability or developmental abnormalities Mannose diet: Man 6 P can be generated directly by hexokinase catalyzed phosphorylation of mannose Corrects coagulopathy, hypoglycemia, protein losing enteropathy, and intermittent gastrointestinal problems, also normalizes the glycosylation of plasma transferrin and other serum glycoproteins. GALACTOSEMIA Clinical features: GALT deficient infants/patients fail to thrive and have enlarged liver, jaundice, and cataracts. Main causes: Accumulation of intracellular galactose and gal 1 phosphate and insufficient synthesis and availability of UDP Gal Accumulation of hypogalactosylated glycans is secondary to a general metabolic abnormality Mutations in three genes encoding enzymes involved in galactose metabolism: Classical galactosemia: Defective galactose 1 phosphate uridyltransferase (GALT): Rare: Defects in UDP galactose 4 epimerase (GALE) or galactokinase (GALK). Therapy: Lactose free diet helps relieve acute symptoms (reduces galactose accumulation): However, does not prevent the appearance of cognitive disability, ataxia, growth retardation, and ovarian dysfunction. How Gal(1 P) accumulation leads to the complications in the patients is unclear! 15

16 Congenital Muscular Dystrophies (CMDs) Autosomal recessive diseases with muscle weakness and joint deformities that slowly progress after birth Causes: mutations that alter normal alpha dystrolglycan O mannosylation (other defects also exist) O mannosylation pathway of alpha dystroglycan: ER: POMT1/POMT2 (a heterodimeric enzyme) adds mannose in α1 O linkage to ser/thr (Dol P Man donor) Golgi: POMGNT1 (a pathway specific O mannoside β1,2 GlcNAc transferase) Galactose and sialic acid are then added by as yet unspecified transferases αdg is the only known carrier of these glycans Why O mannosylation of α Dystroglycan (DG) is important? α DG is a vital component of the dystrophin glycoprotein complex (DGC) on the sarcolemma of skeletal muscle cells DGC connects the ECM to themusclecellcytoskeleton. In muscle, actin in the cytoskeleton is linked to βdg, which spans the cell membrane. The extracellular domain of βdg binds to αdg, which in turn binds laminin 2 in the ECM via O mannosylated glycans in its extracellular domain. Sialylated form important for laminin binding! Diagnosis: DG kda vs. 90 Kda in CMD 16

17 Known mutations that cause congenital muscular dystrophy (CMD): Walker Warburg syndrome (WWS) Is the most severe form of the MDC caused by defective αdg glycosylation. Patients have a short life span (less than 1 year on average), multiple brain abnormalities, and severe muscular dystrophy. About 20% of patients have mutations in POMT1 and a few have mutations in POMT2. A few clinically defined WWS patients have defects in the fukutin and FKRP (fukutin related protein) genes. Mutations in fukutin and FKRP also cause other, milder forms of CMD (see below). Fukuyama muscular dystrophy (FCMD) Caused by a single 3 kb 3 retrotransposon insertional event into the fukutin gene years ago. Carrier frequency in Japan is 1/188. Reduces the stability of the mrna, making it a relatively mild mutation. Fukutin has a typical glycosyltransferase domain motif (DXD) and localizes to the cis Golgi; yet no transferase activity has been demonstrated. Fukutin null mice die by E9.5 in embryogenesis: chimeric mice with less than 50% loss of fukutin activity show typical muscular dystrophy symptoms 17

18 Disorders associated with O GalNAc linked glycans: Familial tumoral calcinosis: Severe autosomal recessive metabolic disorder shows phosphatemia and massive calcium deposits in the skin and subcutaneous tissues The disorder is due to mutations in GALNT3, one of the O GalNAc transferases in mucin type O glycosylation Mutations in the O glycosylated fibroblast growth factor 23 (FGF23) also cause phosphatemia, suggesting that GALNT3 modifies FGF23 Tn polyagglutination syndrome: An autoimmune disorder characterized by the polyagglutination of red blood cells by naturally occurring anti Tn antibodies following exposure of the Tn antigen on the surface of erythrocytes. Caused by somatic mutations in the X linked gene COSMC. Encodes a highly specific chaperone for core 1 synthase (β1 3 galactosyltransferase C1GalT1). Required for the proper folding and normal activity of the C1GalT1 Mutations in COSMC result in the expression of the Tn antigen on blood cells, thrombocytes and platelets, and increases their polyagglutination and eventually results in anemia, thrombocytopenia and leukopenia Tn polyagglutinability is sometimes associated with leukemia and may predispose to leukemia 18

19 Disorders caused by defects in the synthesis of proteoglycans Disorder Gene Entsyme Ehlers Danlos syndrome (progeroid) B4GALT7 β1 4 galactosyltransferase 7 Hereditary multiple exostosis EXT1/EXT2 glucuronyltransferase/n acetylglucosaminyltransferase Chondrodysplasias DTDST/SLC26A2 sulfate transporter Spondyloepimetaphyseal dysplasia ATPSK2 PAPS synthase Molecular corneal dystrophy type I and II CHST6 Keratan sulfate 6 0 sulfotransferase Schneckenbecken dysplasia SLC35D1 UDP GlcA/UDP GalNAc transporter deficiency Ehlers Danlos Syndrome (Progeroid Type) A connective tissue disorder characterized by failure to thrive, loose skin, skeletal abnormalities, hypotonia, and hypermobile joints, together with delayed motor development and delayed speech. Caused by defective synthesis of the core region of xylose based GAG chains especially in Decorin, a dermatan sulfate proteoglycan that binds to collagen fibrils Due to inactivity (5% left) of Galactosyltransferase I (B4GALT7 ), an enzyme that adds galactose to xylosyl serine has only of normal activity. The inactivity of galactosyltransferase II (20% left), the enzyme responsible for adding the second galactose residue to the GAG chain core > formation or stability of a biosynthetic complex involving several GAG chain biosynthetic enzymes. Why only decorin affected? 19

20 Hereditary multiple exostosis (HME) An autosomal dominant disease with a prevalence of ~1:50,000 Clinical features: bony outgrowths at the growth plates of long bones (made of disorganized cartilagenous material with chrondrocytes); about 1 2% of patients develop osteosarcoma Patients have abnormal heparin sulfate (HS) GAG chains Caused by mutations in two genes EXT1 (60 70%) and EXT2 (30 40%). A partial loss of one allele of either gene appears sufficient to cause HME. These enzymes are thought to exist as a complex in the Golgi and both are required for polymerizing GlcNAcα1 4 and GlcAβ1 3 into HS. The HME pathology is likely due to disruption of the normal distribution of HS binding growth factors (FGF and morphogens such as hedgehog, Wnt, and members of the TGF β family) DEFECTS IN GLYCOSPHINGOLIPID SYNTHESIS Amish infantile epilepsy syndrome: Key clinical features include: seizures, arrested development, neurological decline Analysis of plasma glycosphingolipids shows the accumulation of multiple nonsialylated glycolipids An autosomal recessive syndrome that is caused by defective sialyltransferase (SIAT9 ) required for the synthesis of the ganglioside GM3 (Siaα2 3Galβ1 4Glcceramide) from lactosylceramide (Galβ1 4Glc ceramide). Due to nonsense mutation that truncates the protein and abolishes GM3 synthase activity. Mice strains that are null for the SIAT9 and an N acetylgalactosaminyltransferase needed for the synthesis of complex gangliosides also develop seizures, suggesting that the absence of more complex gangliosides may be the underlying cause of the syndrome. 20

21 Congenital Dyserythropoietic Anemia Type II, CDA II (or HEMPAS = hereditary erythroblastic multinuclearity with a positive acidified serum lysis test) An autosomal recessive disorder (~200 cases known) Patients generally have a normal life span, although complications may develop with age, including an enlarged liver, jaundice, gallstones, or diabetes Patients suffer from anemia and morphological abnormalities of erythroblasts in the bone marrow due to abnormal erythropoiesis In erythroid cells, poly N acetyllactosamines on glycosphingolipids are greatly increased, a loss of complex N glycans and an increase in hybrid forms The cause of this disorder is still unknown Defects in GPI anchor synthesis Disorder Gene Enzyme Clinical features Paroxysmal nocturnal PIGA PI GlcNAcT complement mediated hemolysis, anemia hemoglobinuria Autosomal recessive GPI anchor deficiency (promoter mutated) PIGM promoter first mannosyltransferase in GPI anchor synthesis venous thrombosis and seizures 21

22 Genetic defects in degradation of glycans: Lysosomal storage diseases (LSDs) ~50 inherited diseases that are associated with impaired lysosomal degradation of macromolecules (prevalence ~1:8000 births) Caused by mutations in lysosomal enzymes, lysosomal integral membrane proteins, and proteins involved in the post translational modification and trafficking of lysosomal proteins. Result in the accumulation of degradation substrate molecules in the cells/tissues and their (or related fragments) appearance in urine of patients Despite of known genetic mutations behind these disorders, the cellular mechanisms by which accumulated products disrupt normal cellular processes remain unclear: they may involve secondary metabolites or downstream cellular pathways that become dysfunctional An early onset is the most severe later onset have milder symptoms > different phenotypes: Lysosomal degradation pathways for glycans Normally, most glycans are degraded in lysosomes by highly ordered and specific pathways employing endo and exoglycosidases (50 60 different) sometimes with the help of certain noncatalytic proteins Exoglycosidases cleave the glycosidic linkage of terminal sugars with a specific anomeric linkage type from the nonreducing end of the glycan (i.e., the residue at the outermost end of the molecule). They act on a broad range of substrates. For full activity, all hydroxyl groups of the terminal sugar must be unmodified (acetate, sulfate, or phosphate groups are removed by esterases, sulfatases, and phosphatases) Endoglycosidases cleave internal glycosidic linkages of larger chains, and can also cope with sugars with modified hydroxyl groups Each linkage break theoretically requires one anomer specific glucosidase. Their number is close to the known number of enzymes in each degradation pathway. However, other enzymes outside of this group are also needed in some cases 22

23 Lysosomal storage diseases: mucopolysaccharidoses Caused by accumulation of undegradedglycans The pathology likely depends on the cell type and the cellular balance of synthesis and turnover rates Dermatan sulfate (DS), a GAG, predominates in connective tissue, which might explain the bone, joint, and skin problems in mucopolysaccharidosis (MPS) I, II, VI, and VII. Keratan sulphate (KS), another GAG, is present in cartilage; therefore, MPS IV is largely a skeletal disease. Gangliosides are most abundant in neurons; therefore, gangliosidoses are predominantly brain disorders. Therapy: reducing synthesis by the use of an inhibitor may help to retard the accumulation of undegraded material and reduces the pathology of some diseases. Oligosaccharidoses/Mucosaccharidoses substrate Disorder Defect Glycoprotein Glycolipid Clinical symptoms α Mannosidosis (type I and II) α mannosidase major none type I: infantile onset, progressive mental retardation, hepatomegaly, death between 3 and 12 years type II: juvenile/adult onset, milder, slowly progressive β Mannosidosis β mannosidase major none severe quadriplegia, death by 15 months in most severe cases; mild cases have mental retardation, angiokeratoma, facial dysmorphism Aspartylglucosaminuria aspartyl glucosaminidase major none progressive, coarse facies, mental retardation Sialidosis (mucolipidosis I) sialidase major minor progressive, severe mucopolysaccharidosis like features, mental retardation Schindler (types I and II) α N acetylgalactosaminidase yes? type I: infantile onset, neuroaxonal dystrophy. severe psychomotor and mental retardation, cortical blindness, neurodegeneration type II: mild intellectual impairment, angiokeratoma, corpus diffusum Galactosialidosis protective major minor coarse facies, skeletal dysplasia, early death protein/cathepsin A Fucosidosis α fucosidase major minor spectrum of severities includes psychomotor retardation, coarse facies, growth retardation GM1 gangliosidosis β galactosidase minor major progressive neurological disease and skeletal dysplasia in severe infantile form GM2 gangliosidosis β hexosaminidase minor major severe form: neurodegeneration with death by 4 years less severe form: slower onset of symptoms and variable symptoms, all relating to various parts of the central nervous system 23

24 Number Common name Enzyme deficiency Glycosaminoglycan affected Clinical symptoms MPS I H Hurler/Scheie α L iduronidase DS, HS Hurler: corneal clouding, organomegaly, heart disease, mental retardation, death in childhood Hurler/Scheie and Scheie: less severe, individuals survive longer MPS II Hunter iduronate 2 sulfatase DS, HS severe: organomegaly, no corneal clouding, mental retardation, death before 15 years less severe: normal intelligence, short stature, survival age MPS III A Sanfilippo A heparan N sulfatase HS profound mental deterioration, hyperactivity, relatively mild somatic manifestations MPS III B Sanfilippo B α N acetylglucosaminidase HS similar to III A MPS III C Sanfilippo C acetyl CoA: α glucosaminide acetyltransferase HS similar to III A MPS III D Sanfilippo D N acetylglucosamine 6 sulfatase HS similar to III A MPS IV A Morquio A galactose 6 sulfatase KS, CS distinctive skeletal abnormalities, corneal clouding, odontoid hypoplasia, milder forms known to exist MPS IV B Morquio B β galactosidase KS same as IV A MPS VI Maroteaux Lamy N acetylgalactosamine 4 sulfatase DS corneal clouding, normal intelligence, survival to teens in severe form; milder forms known to exist MPS VII Sly β glucuronidase DS, HS, CS wide spectrum of severity, including hydrops fetalis and neonatal form multiple sulfatase deficiency sulfatase modifying factor; converts cysteine formyl glycine all sulfated glycans hypotonia, retarded psychomotor development, quadriplegia Defects in the degradation of glycolipids ( sfingolipidoses ) Disease name Enzyme or protein deficiency Clinical symptoms Tay Sachs β hexosaminidase A severe: neurodegeneration, death by 4 years less severe: slower onset of symptoms, variable symptoms of the nervous system Sandhoff β hexosaminidase A and B as above GM1 gangliosidosis β galactosidase progressive neurological disease and skeletal dysplasia in severe infantile form Sialidosis sialidase progressive, severe mucopolysaccharidosis like features, mental retardation Fabry α galactosidase severe pain, angiokeratoma, corneal opacities, death from renal or cerebrovascular disease Gaucher s β glucoceramidase severe: childhood or infancy onset, hepatosplenomegaly, neurodegeneration mild: child/adult onset, no neurodegenerative course Krabbe β galactoceramidase early onset with progression to severe mental and motor deterioration Metachromatic leukodystrophy arylsulfatase A (cerebroside sulfatase) infantile, juvenile, and adult forms can include mental regression, peripheral neuropathy, seizures, dementia Saposin deficiency saposin precursor (helper protein) similar to Gaucher or Tay Sachs and Sandhoff 24

25 I cell disease (Mucolipidosis II, ML II) The disease results from a defective Golgi localized GlcNAc phosphotransferase that transfers phosphate to mannose residues on lysosomal enzyme proteins In normal cells, the man P serves as a marker for their targeting to lysosomes: lysosome Without this marker, lysosomal enzymes are secreted outside the cell and found in high concentrations in the blood. Not yet clear whether they cause any problem in the circulation Mucolipidosis II (ML II) is a particularly severe form that resembles another mucopolysaccharidosis disease termed Hurler syndrome: The only difference is the presence of specific lipids, GAG's and carbohydrates in the blood of I Cell patient while in Hurlers only GAG chains are present. Affected children often fail to grow (dwarfism). Delays in the development of their motor skills, have corneal problems, have recurrent respiratory tract infections, middle ear infections, and bronchitis. The patients die before their seventh year of life, often as a result of heart failure or recurrent respiratory tract infections. 25

26 Salla disease: One of tree sialic acid storage disorders characterized by the abnormal accumulation of sialic acid in various cells and tissues of the body. Three subtypes: Infantile free sialic acid storage disease (ISSD; most severe form) Salla disease (mildest form) Intermediate Salla disease All the disorders are characterized by some degree of degeneration of nerve cells (neurodegeneration) and cognitive impairment. Caused by mutations of the SLC17A5 gene (sialin) and inherited in an autosomal recessive fashion. Low levels or inactivity of sialin transport protein leads to the abnormal accumulation (storage) of free sialic acid in the tissues of affected individuals. Sialin normally helps transport sialic acid out of lysosomes. Current therapies for LSDs Substrate reduction therapy (SRT): Reducing the synthesis of a glycan may help symptoms: mouse model of Sandhoff disease by using N butyldeoxynojirimycin (an inhibitor of glucosylceramide synthase), the amount of glycosphingolipids fell 50 70% in all tissues without obvious pathological effects, the accumulation of GM2 in the brain was blocked and the amount of stored ganglioside was reduced. However, in clinical trials, the drug appeared to be only slightly effective, and the treatment caused significant side effects. Enzyme enhancement therapy (EET). Enzyme inhibitors used as molecular chaperones, to stabilize the mutated enzymes and prevent their misfolding and proteasomal degradation in the ER. Inhibitor stabilized glucocerebrosidase was transported to lysosomes where it becomes active yet the overall increase in activity is small, but sufficient enough in mice (clinical trials on going) Treatment of Gaucher s disease by injecting glucocerebrosidase carrying mannose terminated N glycans targets the enzyme to the macrophage/monocytes i.e. main cells of substrate accumulation. The added enzyme improves patients clinical features with minimal side effects. However, the cost of treatment is quite substantial, and even high doses do not improve visceral and hematological features. In addition, patients sometimes develop antibodies against the injected human protein. Gene therapy (when works): CRISPR CAS9 system 26

27 Presentations (pick one/pair): klo a 20min Spondylocostaldysostosis (Jarcho Levin syndrome) CGD type Ia (PMM2 CDG) Autosomal recessive Cutis Laxa (type 2a) I cell disease 27