docCongenital Disorders of Glycosylation: Diagnostic steps
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Congenital Disorders of Glycosylation: Diagnostic steps
Congenital Disorders of Glycosylation: Diagnostic steps - - N ERNDIM Meeting, Basel 2009 Dirk J. Lefeber (D.Lefeber@neuro.umcn.nl) Nijmegen, The Netherlands O Dynamic glycosylation pathway Courtesy of Dr. T. Hennet (Zurich) General principle of Congenital Disorders of Glycosylation X Congenital Disorders of Glycosylation: Errors in the assembly line Glycoprotein Function • Protein stability, solubility and structure • Protection against proteases • Cell – cell interactions • Target – receptor interaction • Localization of proteins • Signal transduction • Bacterial adhesion •… > 50 % of human proteins are glycosylated > 1 % of our genes is involved in glycosylation N-glycosylation: multisystemic Liver (transferrin) Brain (MAG, P0, neurexin) Muscle (Dystroglycan, agrin) Glycoprotein hormones (LH/FSH/TSH) Coagulation (factor VII, IX, ATIII) N G M - - - N G N G G M M M N M M Asn Asn Clinical aspects of Congenital Disorders of Glycosylation Classical picture of CDG: • hypotonia/epilepsy/cerebellar atrophy, inverted nipples, fat pads, strabismus, feeding problems, ataxia, hypogonadism, mental retardation Control Patient When to perform transferrin isofocusing? • All patients with a suspicion of a metabolic disorder • Reason: • CDG: wide spectrum, mild isolated to severe multisystem • CDG-Ib/h, fructosemia • CDG-Ix with isolated myopathy, optic nerve atrophy, DCM, ichthyosis • New phenotypes in CDG-II: adducted • thumbs/microcephaly; cutis laxa; complex vertebral malformations CDG-II with liver pathology as main feature Ib Ia Fru-6-P Diagnostic approach Man-1-P Man-6-P UDP UDP - M If M Ij Ik Ie P P M P P M G M M M Id M M MM M M M IL M M MM M M M M Ig IL P M M M M M M M M M Ic G M M M M M M M M M G G G M M M M M M M M M Endoplasmatic Reticulum G G G M M M M M M M M OTase M G G G M M M M M M IIb M M Ih Im G G M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M UDP- G P M M M Cytoplasm M M M M M M M UDP G M Ii N-glycosylation GDP GDP - M CMP- GDP UDP IIf UDP IIc Median Golgi Trans Golgi Cis Golgi M M M M M M M IId M M M M IIa M M M M G = mannose = glucose = galactose = sialic acid M = fucose = COG complex M M M IIe/IIg/IIh = dolichol-PP = dolichol = GlcNAc M M M M M M M CDG patients with different profiles Type II; Golgi defects Type I; ER defects - 4 2 - 4 3 2 N N G G M M M 0 Asn control patient 86 CDG-I; solved 8 CDG-Ix; unsolved 1 0 Asn - -N N G G G M M M M M M Asn Asn patients 17 CDG-II; solved 29 CDG-IIx; unsolved Nomenclature changes in CDG • 1999: CDGS-I to VI to CDG-I(a-o) and CDG-II(a-h) • 2009: from CDG-I//II to PMM2-CDG PMM2-CDG MGAT2-CDG ALG6-CDG B4GALT1-CDG PMI-CDG POMT1-CDG CDG-I CDG-II Walker Warburg Syndrome (WWS) Keep CDG-I/II with gene name?? CDG-I(ALG3) Stage 1: Secondary causes & Type I/II determination Type I: 1. Galactosemia 2. Fructosemia 3. Alcohol abuse 1 2 2 4 2 0 Type II: 4. Haemolytic Uremic Syndrome (HUS) 4 5. Severe liver disease 0 6. Young age (<1-2 months) 7. Transferrin protein polymorphism 4 5 6 Transferrin protein polymorphism Lanes 1 - 4: No neuraminidase treatment Lanes 5 - 8: Neuraminidase treatment - + 1 2 3 4 5 6 7 8 0 1 2 3 4 5 -sialotransferrin 1/5: normal 2/6: The frequently occurring C1/C3 variant 3/7: protein polymorphism shifting towards anode (= B variants) 4/8: protein polymorphism shifting towards cathode ( D variants) Type I/II classification • In some cases, assignment of type I or type II is difficult • SDS-PAGE of transferrin might help SDS-PAGE TIEF 4 2 0 1a 1b 1c C 2 LC-MS of lane 2 Escape of the CDG-I vs CDG-II classification? 9 yr girl: cleft palate, dilated cardiomyopathy and chronic hepatitis Type I + Type II Stage 2: CDG-I diagnostic work-up Mevalonaat Short LLO: normal Dehydrodolichol-PP Ib Ia dolichol M-1-P GDP GDP - M UDP UDP M Ij Ik P Ie G P M P P P M G M M M M M M M M M M M Id IL M M M M M M M Ig M M M M M M M M IL UDP M M M M M M M M M Ic G M M M M M M M M M G GG M M M M M Ih M M M M P Endoplasmatic Reticulum G G G M M M M M M M M G G G M M M M M M M M OTase M G G M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M Cytoplasm M M M M M M M UDP- G If M M Ii Fr-6-P M-6-P = dolichol = GlcNAc M G = mannose = glucose Lipid-linked Oligosaccharide (LLO) analysis 16 Stage 2: CDG-II diagnostic work-up - - - M M M N-Glycan structural analysis M M M control patient UDP UDP CMP- GDP Golgi - - M Exit M M M M M M M M M M M M MGAT2 CDG-IIa M M M M M M M M M 10 0 1500 1782 1706 2000 2500 m/z 2936 2968 2607 2637 2690 2762 2794 2824 2593 2392 2432 2441 60 2158 70 2187 2239 2245 3000 3500 3982 3779 3809 3864 3634 3687 3417 3327 3213 3243 3197 3056 3605 2968 2824 2852 2762 2780 2607 2517 2433 2419 2145 2230 2246 2288 1970 40 2043 1956 1838 1766 1693 50 2013 2065 100 1984 CDG-IIa 1951 1796 1937 20 1609 10 1580 30 20 1579 Relative Abundance 2794 Control 100 90 80 70 60 0 90 80 50 40 30 4000 Glycan types • • N-glycosylation: amide (NH2) binding with Asparagine (ASN) O-glycosylation: hydroxy (OH) binding with Serine (Ser) or Threonine (Thr) N-glycan Neu5Ac 6Gal1 4GlcNAc1 Man1 Man1 Neu5Ac 6Gal1 4GlcNAc1 4GlcNAc1 Man1 O-glycan Neu5Ac2 3Gal1 3GalNAc1 4GlcNAc1 More options in the Golgi UDP Cytoplasm UDP CMP- GDP Golgi - - M Exit M CMP- Exit - - M M M M M M M M M M M M M M M M M M M M N UDP CMP CMP - UDP UDP UDP UDP O Isoelectric focusing of serum apolipoproteinC-III Core 1 mucin type O-glycan in position Thr-94 - pH 3.5 Apo CIII - 2 - Apo CIII - 1 - Apo CIII - 0 pH 5.0 Wopereis et al. Clin Chem 2003;49(11):1839-45. Profile types of ApoCIII in CDG type II patients Transferrin ApoCIII ApoCIII-0 profile ApoCIII-1 profile 4 3 2 2 1 1 0 0 control group 1 46 CDG-II: 15 N glycosylation 10 N+O glycosylation group 1 21 N+O glycosylation group 2 group 2 Wopereis, Glycobiology 2005 Options for a combined N+O glycosylation defect 2 6 steps UDP Cytoplasm UDP CMP- Golgi - - GDP 1 M Exit CMP- Exit - - M 3 M M M M M M M M M N UDP CMP CMP M M M M M M M M M M M - UDP 1 UDP UDP UDP O Option 4: Trafficking in the secretory pathway a. Indirect COG Golgi defects Conserved Oligomeric Golgi (COG) complex • Transport between Golgi vesicles Cutis laxa • ATPase defect influencing Golgi pH Group 1: COG defect in 5/10 patients COG7: • Microcephaly, adducted thumbs, growth retardation, VSD, episodes of hyperthermia, early fatal 4 3 2 1 2 1 0 0 group 1: ApoCIII-0 Morava J Hum Genet 2007 26 Model of COG complex cog2 2 cog3 cog3 cog1 cog8 1 8 cog4 X cog7 7 6 cog6 cog5 5 COG complex is required for recycling of glycosyltransferases Group 2: 14/21 patients with cutis laxa phenotype 4 2 3 1 2 1 0 group 2: ApoCIII-1 ATP6V0A2 and glycosylation? pH gradient 6.2 6.4 ATP6V0A2 • • <6 months of age: isolated ApoCIII -1 profile Patients with normal ApoCIII exist 4-5 Step by step diagnostic approach for CDG Stage 1: Interpretation of transferrin isofocusing gel Confirmation of generalized glycoprotein abnormality Exclude transferrin protein polymorphism Exclude secondary causes of N-glycan biosynthesis abnormalities Discriminate between CDG-I and CDG-II • • • • • Stage 2; CDG-I: PMM/PMI measurement; LLO analysis in fibroblasts Genetic tools & clinical information Stage 2; CDG II: Check O-glycan abnormalities and N-glycan structure Genetic tools & clinical information • • • •
