Genetic defects in the human glycome

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1 Nature Reviews Genetics AOP, published online 6 June 2006; doi: /nrg1894 REVIEWS Genetic defects in the human glycome Hudson H. Freeze Abstract The spectrum of all glycan structures the glycome is immense. In humans, its size is orders of magnitude greater than the number of proteins that are encoded by the genome, one percent of which encodes proteins that make, modify, localize or bind sugar chains, which are known as glycans. In the past decade, over 30 genetic diseases have been identified that alter glycan synthesis and structure, and ultimately the function of nearly all organ systems. Many of the causal mutations affect key biosynthetic enzymes, but more recent discoveries point to defects in chaperones and Golgi-trafficking complexes that impair several glycosylation pathways. As more glycosylation disorders and patients with these disorders are identified, the functions of the glycome are starting to be revealed. Burnham Institute for Medical Research, North Torrey Pines Road, La Jolla, California 92037, USA. hudson@burnham.org doi: /nrg1894 Published online 6 June 2006 Genomes and proteomes now share the molecular stage with their younger, larger sibling, the glycome 1. Defined as all the sugar chains (glycans) that an organism makes, the glycome is estimated to be times larger than the proteome, depending on the species. Glycosylation the addition of glycans to proteins and lipids is not template driven, so a single carrier can have a bewildering array of glycoforms. Some glycans have known, varied or context-specific functions, but the functions of many others await discovery. Several biochemical pathways synthesize glycans, and genetic defects that affect these pathways cause a range of diseases. Nearly 30 such genetic disorders have been identified in the past decade, mainly affecting N-linked and O-linked pathways 2 4. Patients have a broad range of phenotypes that cut across many medical specialties, which has contributed to an under-recognition of these disorders. However, a recent increase in awareness of the importance of glycosylation in disease, combined with improved diagnostic testing, has uncovered the existence of even more disorders of this type. Further developments promise to increase this number and to indicate how many people might be affected a number that is likely to be far greater than is currently appreciated. Here, I outline the glycan biosynthesis pathways in which genetic defects have been identified that lead to human disease. For each class of disease, I highlight the examples that have provided the greatest insights towards understanding the consequences of genetic defects of glycosylation. A better understanding of these disorders will improve diagnosis and counselling, might lead to the development of new therapies and, ultimately, is hoped to provide important insights into why we need a glycome. Biosynthetic pathways and glycan functions In eukaryotes, 11 biosynthetic pathways link glycans to proteins and lipids 5. I focus here on six pathways for which genetic disorders are known (summarized in FIG. 1) and outline the main functions of the glycans that are produced (TABLE 1). N-linked protein glycosylation. N-glycosylation begins with the formation of an amide linkage between N-acetylglucosamine (GlcNAc) and asparagine on target proteins. First, a 14-sugar precursor lipid-linked oligosaccharide (LLO) that contains 2 GlcNAc, 9 mannose and 3 glucose units is constructed on a lipid carrier by a series of glycosyltransferases 6. The glycan is then transferred to nascent proteins with an available asparagine in a suitable context 7,8 (FIG. 1). Three glucose units and up to six mannose units are then removed from many glycans, and GlcNAc, galactose, sialic acid and fucose residues can be added to create multiple branches. The order of sugar addition and deletion is prescribed, but not template driven, and several enzymes can compete for the same substrate, which yields a range of N-glycan structures 6. The specific target protein, and the cellspecific expression and organization of this biosynthetic factory determine glycan structure. Nearly all proteins that travel through the endoplasmic reticulum (ER) Golgi conduit are N-glycosylated, and this modification can determine or influence protein folding, stability, trafficking, localization and oligomerization, with important implications for cell cell interactions and intracellular signalling. The importance of modifications of this type is shown by the fact that a lack of all N-glycans is lethal in species ranging from yeast to mammals. NATURE REVIEWS GENETICS ADVANCE ONLINE PUBLICATION 1

2 Ribosome N-GlcNAc O-mannose O-xylose O-GalNAc GPI GSL Lipid Nascent protein ETNP ETNP ER ETNP S S S S Golgi ETNP ETNP ETNP Plasma membrane S S S S Sialic acid Galactose GalNAc Mannose Glucose GlcNAc Glucuronic acid Xylose Figure 1 Overview of glycan biosynthetic pathways. The six glycan biosynthetic pathways in which diseasecausing genetic defects have been identified are shown, indicating the subcellular location of the various steps. The type of glycan that is produced is indicated above each pathway. Dotted lines are used to separate the pathways, rather than to indicate that they are located in separate compartments. It should also be noted that proteins contain multiple types of glycan, although one type per protein is shown here for simplicity. The N-linked pathway begins in the endoplasmic reticulum (ER) with transfer of a preformed glycan to glycosylation sites soon after the nascent protein emerges from the ribosome into the ER lumen. Glycan remodelling begins in the ER, but mostly occurs in the Golgi. The first step of O-mannose-linked glycan assembly occurs in the ER and is then completed in the Golgi, whereas all of the O-xylose and O-GalNAc (O-linked N-acetylgalactosamine) pathway steps occur in the Golgi. GPI (glycophospholipid) anchors are preassembled in the ER. They are added near the C termini of specific proteins soon after their synthesis. The synthesis of glycosphingolipids (GSLs) begins on the cytoplasmic side of the ER, after which the nascent glycan flips into the lumen and is extended with additional sugars in the Golgi. After leaving the Golgi, glycan-decorated proteins and lipids traffic to the cell surface, lysosome or other vesicles; trafficking to the plasma membrane only is shown here for simplicity. GPI-linked proteins and GSLs both cluster in similar regions of the plasma membrane as both are associated with lipid rafts. A substantial number of glycan-carrying proteins are subsequently secreted into the extracellular matrix. ETNP, ethanolamine phosphate; GlcNAc, N-acetylglucosamine; S, sulphate. 2 ADVANCE ONLINE PUBLICATION

3 Table 1 Glycan classes: functions and biosynthesis Glycan Type Linkage Synthesis Functions Protein N-Linked GlcNAc-βasparagine Added in ER, modified in Golgi Protein folding and stability; complex formation; signalling; cell cell recognition O-Linked Mannose-α-serine/ threonine Begins in ER, completed in Golgi Stability and function of dystrophin glycoprotein complex Xylose-β-serine Golgi Mechanical cushioning; establishment of growth-factor and morphogen gradients Lipid Glycosphingolipid GPI anchor GalNAc-α-serine/ threonine Glucuronic acid-βceramide N-glucuronic acidinositol Golgi Begins in ER, completed in Golgi Made in ER and transferred to proteins ER, endoplasmic reticulum; GalNAc, N-acetylgalactosamine; GlcNAc, N-acetylglucosamine Lubrication; barrier against pathogens; leukocyte/lymphocyte trafficking Lipid raft component; signalling; glycanmediated cell cell recognition Lipid raft component; haematopoeisis; protein trafficking O-linked protein glycosylation. O-linked protein glycosylation involves initial linkage steps between serine or threonine and mannose, xylose or N-acetylgalactosamine (GalNAc) (FIGS 1,2). In the case of O-mannose glycans, GlcNAc, galactose and sialic acid are subsequently added 9. Complex branched glycans of this type can be produced, which indicates that the biosynthetic pathway is also complex 10. About a third of all O-linked chains in the brain are built on O-mannose, although α-dystroglycan which has a crucial role in skeletal muscle cells is the only identified carrier of these glycans. After the addition of O-αGalNAc to serine or threonine, any of four sugars are added to form disaccharides, and many can be extended by sequential addition of galactose, GlcNAc, fucose and sialic acid to generate linear or multi-branched chains 11. These glycans are mainly found on secreted or surface-bound mucins of epithelial cells, where they serve as lubricants and barriers against pathogens. Some are ligands for selectins during leukocyte extravasation and lymphocyte recirculation. Glycans that are O-linked through xylose to serine are known as glycosaminoglycans (GAGs), and include heparan sulphate, heparin, chondroitin sulphate and dermatan sulphate 12. After addition of the first sugar to serine, two galactose molecules and one glucuronic acid molecule (GlcA) are added, followed by extension of this core with repeating disaccharides of GlcA- GalNAc (when the first sugar is chondroitin sulphate or dermatan sulphate) or GlcA-GlcNAc (for heparin or heparan sulphate). Some sugars are then modified by epimerization and sulphation. Proteins that carry chondroitin sulphate occur in the extracellular matrix (ECM), where they provide physical integrity and cushioning, whereas heparan sulphate chains that are present on cell membrane proteins function as co-receptors for growth factors, such as those of the fibroblast growth factor (FGF) family. Heparan sulphate also binds cytokines and morphogens during development to establish gradients of these proteins 13. Addition of glycans to lipids. Glycosphingolipids (GSLs) are characterized by an O-linkage between glucose (sometimes galactose) and a ceramide, forming glucosyl ceramide, which is extended to lactosyl ceramide. This core can then be extended to form more complex GSLs 14. Sialylated versions are called gangliosides, and are especially abundant in the brain and peripheral nervous system. GSLs occur in lipid rafts, and their glycans bind to each other 15 or to proteins such as integrins, through which they affect signalling. Glycophospholipid (GPI) anchor glycans contain mannose and glucosamine (not GlcNAc), which are assembled in the ER on a phosphatidylinositol backbone. The entire glycolipid is transferred to C-terminal regions of proteins and anchors them into membranes. GPI anchors also have roles in membrane diffusion, intracellular protein sorting 16 and signalling 17. Disorders of N-linked glycosylation the CDGs The largest number of known glycosylation disorders affect N-glycosylation, with around 600 patients currently identified 3,18. The term congenital disorders of glycosylation (CDGs) was coined in 1999 to accommodate one rapidly expanding group of these disorders 19, which were previously called carbohydrate deficient glycoprotein syndromes 20. The identification of patients who are affected by CDGs was aided by diagnostic tests for many such disorders (BOX 1), of which there are now 19 known types. Mortality is about 20% in the first 5 years of life, but then decreases. As a result, many adults with CDGs probably remain undiagnosed because these disorders were unknown at the time when the parents of these patients would have sought a diagnosis. The CDGs are divided into two groups, I and II, with lower case letters indicating different subtypes in chronological order of identification of the defective gene (FIG. 3; TABLES 2,3). All known CDGs are autosomal recessive. NATURE REVIEWS GENETICS ADVANCE ONLINE PUBLICATION 3

4 Hypomorphic allele A mutation that causes a partial decrease in the activity of the gene product. Heterosis Describes situations in which individuals who are heterozygous for a specific pair of alleles have a fitness advantage over those who carry either homozygous genotype. a O-mannose b O-xylose S/T S/T S/T S/T S/T 2S 6S 2S NS 6S 2S Dolichol phosphate Xylose 3S UDP UDP 4S NS UDP UDP Chondroitin sulphate/dermatan sulphate Heparan sulphate/heparin Galactose Mannose Type I CDGs. Hypoglycosylated proteins occur in all 12 type I CDGs because of the synthesis of insufficient and/or incomplete LLO glycan precursors that are inefficiently transferred to proteins All these defects lead to the same outcome: unoccupied glycosylation sites on multiple proteins. It might therefore be assumed that all CDG-I patients would have similar phenotypes, but this is not the case. There is also considerable variation within these disorders, which indicates that genetic modifiers are important in determining the specific phenotypic outcome. Alternatively, inappropriate proteinbound glycans, accumulated metabolic intermediates, or CMP CMP Link O-serine Glucuronic acid GlcNAc Sialic acid Iduronic acid GalNAc Nucleotide-sugar transporter Figure 2 O-mannose and O-xylose biosynthetic pathways. a Biosynthesis of O-mannose-linked glycans begins with the activity of the POMT1 POMT2 mannosyltransferase complex, which uses dolichol-p-mannose to add α-mannose to serine (S) or threonine (T) residues. POMGnT1 then uses UDP-GlcNAc (N-acetylglucosamine) to add a β1,2glcnac residue to the mannose. The O-mannose structure is completed by the addition of a β1,4-galactose and 2,3-sialic acid residues. The specific transferases have not been identified for these steps. Glucuronic acid, sulphate and other branches can also be added. b Synthesis of a generic glycosaminoglycan chain. The linkage-region and chondroitin sulphate/dermatan sulphate and heparan sulphate/heparin structures are shown. The common tetrasaccharide core linkage region is assembled, and then a specific N-acetyl hexosamine transferase determines whether elongation proceeds towards chondroitin sulphate/dermatan sulphate (GalNAc (N-acetylgalactosamine) transferase) or heparan sulphate/heparin (GlcNAc transferase). Separate co-polymerases then carry out stepwise additions of either glucuronic acid-galnac (chondroitin sulphate/dermatan sulphate) or glucuronic acid-glcnac (heparan sulphate/heparin). These glycan backbones are modified in several ways that include epimerization of glucuronic acid to iduronic acid residues, N-de-acetylation/sulphation (heparan sulphate/heparin) and an array of 3 -phosphoadenosine-5 -phosphosulphate-dependent sulphation reactions generate enormous sequence diversity. NS, 2S, 3S, 4S and 6S represent 2-N-, 2-O-, 3-O-, 4-O- and 6-O-sulphate, in that order. truncated LLOs themselves might cause the pathology. In type I CDGs, the range of substitutions, insertions, microdeletions and occasional larger deletions in coding regions and splice junctions leads to loss-of-function and hypomorphic alleles rather than complete knockout, which is likely to be lethal. CDG-Ia is by far the most prevalent CDG, with more than 500 patients worldwide and an estimated incidence of up to 1/20,000 (REF. 22). It is caused by mutations in PMM2, one of two genes that encode phosphomannomutases, which convert mannose-6-phosphate to mannose-1-phosphate. This molecule is the immediate precursor for GDP-mannose, the activated donor for mannose addition. Mutations reduce intracellular GDP-mannose and LLO levels, generating unoccupied glycosylation sites on many different proteins. PMM2 is highly conserved (57% identity between human and yeast) and is expressed in all human tissues. Mice that lack Pmm2 die 2 3 days after fertilization (C. Körner, personal communication). By contrast, the homologous gene PMM1 is mostly expressed in brain and lung, and no patients with CDG have mutations in this gene. The PMM1 protein has in vitro enzymatic activity, but it clearly cannot substitute for PMM2, which leaves its biosynthetic role unclear. There is a large amount of variation among CDG-Ia patients in terms of the range of symptoms they display. Patients with the classic form have severe psychomotor retardation, seizures, cerebellar hypoplasia and coagulopathy 23, whereas a minority of patients have much milder presentations General genotype phenotype correlations have been elusive 28. Nearly 90 mutations have been discovered in PMM2, with the resulting mutated enzymes showing increased thermolability 29,30, poor substrate binding or complete loss of activity. Some patients with mild phenotypes have a higher residual PMM2 activity (up to 30%) 24,31, indicating a correlation with modestly reduced enzyme function, but such relationships between enzyme activity and phenotypic severity are seen only in some cases. One strong genotype phenotype correlation is seen for the most prevalent mutation in PMM2 in Northern European patients, which causes an Arg141His amino-acid substitution that almost completely inactivates the enzyme. No homozygous patients have been identified, probably indicating lethality, but the carrier frequency of 1/80 in northern Europe indicates positive heterosis 32. The wide phenotypic variation in CDG-Ia and the lack of solid genotype phenotype correlations indicates that the genetic background has an important role in disease expressivity. In support of this, a frequent non-pathological mutation in asparagine-linked glycosylation 6 (ALG6; which is the causal mutation in CDG-Ic) occurs twice as often in severely affected CDG-Ia patients as in those with a mild or moderate phenotype 33. With fewer affected patients, less is known about the other type I CDGs, which range in severity and phenotype. Patients with CDG-Ib, which is caused by mutations in phosphomannose isomerase (MPI) have a milder phenotype than those with CDG-Ia, without developmental delay or psychomotor retardation, and 4 ADVANCE ONLINE PUBLICATION

5 Box 1 Identifying CDG and the underlying genetic defects Glycosylation status and glycan analysis A blood test for the glycosylation status of transferrin is the most popular indicator of congenital disorders of glycosylation (CDGs). Many serum glycoproteins are affected in these disorders, but transferrin is the most sensitive marker. Its glycosylation status can be investigated by various methods that include isoelectric focusing, ion-exchange highperformance liquid chromatography and capillary electrophoresis. Electrospray-ionization mass spectrometry (ESI-MS) is the most informative method because it distinguishes between the loss of entire glycans and the loss of monosaccharides. Occasionally, patients have combinations of defects that are undetectable with other methods. Although analysing transferrin status remains the best method, it has some problems. Alcoholism, uncontrolled galactosaemia, and fructose intolerance also alter transferrin glycosylation. Moreover, transferrin glycosylation seems normal in patients with CDG-IIb, CDG-IIc and CDG-IIf, and even some proven cases of other CDGs do not always show abnormal transferrin. Glycosylation of other serum proteins, such as α1-antitrypsin, are sometimes used to substantiate CDGs. Type I CDGs have also been diagnosed using protein chip technologies. In addition, although analysis of serum transferrin can detect altered glycosylation, it cannot pinpoint the defect. Glycan analysis helps to focus the search. Fibroblasts and/or leukocytes are assayed for phosphomannomutase and phosphomannose isomerase activity to eliminate or confirm CDG-Ia and CDG-Ib, followed by metabolic radiolabelling with sugars (usually mannose) to analyse glycosylation intermediates. The size of lipid-linked oligosaccharide (LLO) glycans and their transfer efficiency are especially important. Accumulation of incomplete LLO often pinpoints a candidate gene, and sequencing can reveal the causal mutations. The initial identification of the defects that underlie CDG-IIa, CDG-IIc and CDG-IIf relied on glycan analysis of transferrin, selected blood cell glycoproteins or total serum glycans. However, these analyses are not always informative; for example, in CDG-IIe the defect lies in a Golgi-associated cytoplasmic protein that transports and organizes the glycosylation machinery in cells. A growing number of patients with as yet unclassified CDG-II defects might have similar trafficking mutations that affect multiple glycosylation pathways, and in these cases glycan analysis alone is unlikely to pinpoint the defect. Complementation assays Yeast or mammalian cells with functional defects in the genes that encode gylcosylation-pathway proteins provide a complementation system to confirm the pathology of specific mutations. The use of Saccharomyces cerevisiae mutants in this way has made important contributions to understanding the CDGs, especially the type I disorders. The early stages of the N-linked glycosylation pathway, including precursor activation, LLO assembly, glycan transfer to protein and initial trimming steps are nearly identical to those in humans, with a few important exceptions. Yeast cell lines with individual or multiple genetic glycosylation deficiencies with measurable, sometimes lethal, effects on glycoprotein synthesis provided an ideal complementation system to test the effect of putative pathological mutations in the human orthologues. The normal human alleles restore growth or rescue impaired glycosylation of a marker protein (usually carboxypeptidase Y), whereas the mutant allele does not. Even mild human mutations can be characterized. Where the yeast pathway differs from that in humans, equally robust mutant mammalian cell lines, usually Chinese Hamster Ovary (CHO) cells, are available. These have been used to identify CDG-Ie, CDG-If and CDG-Ij, and have provided important insights into all of the CDG-II defects, as yeast lack most of these steps. Screening for CDGs An effort is underway in the United States to include CDG transferrin glycosylation in the next generation of infant screening tests. ESI-MS methods can be applied to infant blood cards to detect at least 17 different types of CDG. This should begin to provide information on the incidence of CDG disorders, but determining the specific gene that is involved in each case will be more difficult and labour intensive, even with efficient advanced sequencing methods. Isoelectric focusing A method that is used to separate proteins within an electric field on the basis of their isoelectric points. Electrospray ionization An ionization technique that is used in the mass-spectrometric analysis of large biomolecules. Charged droplets of analyte solution are used to produce gas-phase ions for analysis. respond to dietary therapy 34. It is possible that maternal compensation contributes to this milder phenotype, and such compensation has actually been demonstrated for another type I CDG: in CDG-Id, a 19-week-old affected foetus with an unaffected mother had normal glycosylation of several indicator plasma glycoproteins, but chorion cells synthesized truncated LLO species 35. This indicates maternal compensation, at least for fetal liverderived plasma glycoproteins. The causal genetic defects for many cases of CDG-I remain unsolved. These CDG-Ix patients might turn out to carry mutations that lead to defective oligosaccharyltransferase activity or dolichol biosynthesis and recycling, but this has yet to be confirmed. Type II CDGs. Whereas type I CDGs involve defects in LLO synthesis and transfer, the genetic defects that cause type II CDGs alter the processing of protein-bound sugar chains. These disorders seem to be less frequent than type I disorders, but this might reflect a lack of awareness rather than the actual prevalence. The various genetic defects that underlie type II CDGs impair specific steps of the glycosylation pathway. Although this might be expected to result in phenotypic patterns that could help to link clinical features to causal genetic defects, this has not generally been the case. Instead, structural analyses of serum glycans have helped to pinpoint the genetic defects in CDG-IIa and CDG-IId (REFS 36 38) (BOX 1), whereas for CDG-IIc and CDG-IIf a combination of glycan analysis and knowledge of specific phenotypes has provided clues to the underlying defects 39,40. Mutations in the Golgi-localized nucleotide sugar transporters for GDP-fucose and CMP-sialic acid cause CDG-IIc and CDG-IIf, respectively, and in both cases synthesis of the O-linked glycan sialyl LeX is NATURE REVIEWS GENETICS ADVANCE ONLINE PUBLICATION 5

6 Thrombocytopaenia Decreased number of blood platelets. Neutropaenia A type of leukopaenia that mainly affects neutrophils. Complex-type glycans N-glycans that contain multiple GlcNAc-based branches, usually terminated with sialic acid or galactose. Dysmorphology A morphological defect that results from an abnormal developmental process. eliminated In CDG-IIc this elevates circulating leukocytes, even in the absence of infection 43, whereas in CDG-IIf it produces giant platelets with abnormal membranes, thrombocytopaenia and moderate neutropaenia 44. The splice-site mutation that underlies CDG-IIf disrupts the production of sialylated glycans on the surface of haematopoietic progenitor and mature cells, but does not alter sialylation of serum glycoproteins, which possibly indicates differential penetrance of the splice mutation in different tissues 40. CDG-IIe is the first example of what will probably turn out to be a range of new CDG-II defects. A splicesite mutation in the COG7 gene disrupts several glycosylation pathways by impeding the normal trafficking of multiple glycosyltransferases and nucleotide-sugar transporters 45,46. COG7 is part of the 8-subunit COG (conserved oligomeric Golgi) complex, which is thought to have multiple roles in trafficking within the Golgi, and between this and other compartments 47,48. Mammalian cells that are deficient in COG1, COG2, COG3 or COG5 also show various degrees of altered glycosylation 49 51, indicating that patients with defects in these and the other subunits will be found among the growing number of as yet unclassified CDG-II patients who show defects in multiple pathways Indeed, several patients were recently identified with defects in COG1 and COG8 (REFS 55,56). As well as affecting N-linked glycans, these COG mutations are likely to affect the synthesis of GAG chains and the normal assembly of the ECM. Their discovery indicates that mutations in other trafficking-related protein complexes might also affect glycosylation 57. There are several CDG-IIx disorders for which identification of the underlying genetic defects is awaited. Some cases selectively affect the liver 58, whereas others have abnormalities in the ECM 53. As with the CDG-Ix disorders, a better understanding of these conditions will require a broader and more thorough analysis of multiple glycosylation pathways to determine which are affected. Mouse models of CDGs. Most CDG cases result from hypomorphic alleles rather than complete loss of gene function. Consistent with this, there are few useful systemic knockout mouse models, as mice that are null for the genes that are mutated in CDGs usually die early in development. Creating disease models for many types of CDGs could be achieved by introducing proven pathological point mutations into the mouse orthologues of the defective human genes. Another option would be tissue-specific conditional ablation, but this is less useful as few patients carry two null mutations. However, there are already several successful CDG-like mouse models. Mice that are null for Mgat2, which encodes the GlcNAc-transferase II enzyme that is required for the synthesis of complex-type glycans, rarely survive beyond weaning (1%) and none survive beyond 4 weeks. However, the few that do survive up to this point resemble patients with CDG-IIa which is caused by mutations in the human orthologue of this gene in nearly all respects, from their glycan profile to their dysmorphology and other pathologies 59. Mice that are null for Tsta3 (also known as Fx), which encodes an enzyme that converts GDP-mannose to GDP-fucose, have a phenotype that is similar to that of patients with CDG-IIc. In some genetic backgrounds, rare Tsta3-null mice that survive (<1%) produce offspring whose survival and phenotype respond to fucose in drinking water 60. Fucose is converted to GDP-fucose through fucose-1-phospate, bypassing the TSTA3 block, and without fucose the mice die within a few weeks. This is similar to the fucose therapy 41 that is used in patients with CDG-IIc, who lack the GDP-fucose transporter 39,61. The specific genes that can compensate for the lack of TSTA3 function have not been identified, but they probably allow more efficient salvage of fucose or higher fucose-kinase activity for GDP-fucose synthesis. The possibility of identifying such factors shows the potential of mouse models to provide insights into the phenotypic diversity of patients with these disorders. Emerging themes from the CDGs. The wide clinical variation between CDGs and within each type is remarkable, and probably results from a combination of the severity of the mutations, genetic modifiers, and loss or substitution of specific glycans with their normal counterparts. Nevertheless, some phenotypic patterns have emerged. Defects in N-linked glycans tend to affect those organs and cells with high glycosylation demands or rapid turnover; for example, hepatocytes, enterocytes and leukocytes 62. They also tend to affect secreted proteins (coagulation factors) and proteins that probably have roles in growth and cell cell interactions. Developmental delay, particularly that which affects brain development, is one of the most consistent findings in CDG. Although the specific proteins or processes that are responsible have not been identified, this is consistent with the failure of appropriate cell cell interactions that are needed to establish a normal nervous system during development. Further mouse models that allow these developmental phenotypes to be studied should prove useful in pinpointing the specific proteins and pathways that are affected. Other disorders that affect N-glycans Disorders other than the CDGs also affect N-glycan synthesis, and their discovery actually predates that of the CDGs. In some cases the affected genes and/or proteins have not been identified. Nevertheless, these disorders have provided insights into how defects in N-linked glycoslyation can affect specific pathways or cell types. For example, in contrast to the global impairment of N-glycan synthesis in CDGs, mucolipidosis II (ML-II or I-cell disease) and a milder version, ML-III (pseudo- Hurler polydystrophy), specifically affect glycans on lysosomal enzymes. These enzymes are mistargeted because they lack a recognition marker, mannose-6- phosphate, which is required for binding to receptors in the Golgi that mediate the delivery of these enzymes to the lysosome 63. Patients with ML-II and ML-III carry 6 ADVANCE ONLINE PUBLICATION

7 Cytoplasm GDP 1P 6P Fructose-6P Glycolysis CDG-Ia CDG-Ib PMM2 MPI CTP CDP UDP GDP CDG-Ie DPM1 CDG-If CDG-IL ALG9 CDG-Ig ALG12 UDP CDG-If MPDU1 CDG-Ij DPAGT1 CDG-Ik CDG-Ii ALG1 ALG2 Cholesterol biosynthesis CDG-Id ALG3 CDG-Ic ALG6 ER Golgi Hybrid Complex High mannose ALG8 CDG-IIf SLC35A1 CDG-IId CDG-IIc CDG-IIa CDA-II Nascent CDG-IIb B4GALT1 SLC35C1 MGAT2? protein GLS1 CDG-Ih Dolichol phosphate Galactose GlcNAc NeuAc Fucose Animal model Glucose Mannose MPDU1 CMP-sialic acid GDP-fucose transporter Known CDG Figure 3 Sites of genetic defects in the N-linked glycan biosynthetic pathway. During N-linked glycan biosynthesis, monosaccharides of GlcNAc (N-acetylglucosamine) and mannose are activated or interconverted and used to build the lipid-linked oligosaccharide precursor (LLO), which is assembled on dolichol, a polyisoprenoid that is derived from the cholesterol pathway. Dolichol in the enoplasmic reticulum (ER) membrane is activated to dolichol phosphate by a kinase. UDP-GlcNAc provides GlcNAc-1-phosphate and GlcNAc, and GDP-mannose provides five mannose residues that are added on the cytoplasmic side of the ER. The resulting mannose 5 -P-P-dolichol flips into the ER lumen, where 4 mannose and 3 glucose residues are added using dolichol-p-mannose and dolichol-p-glucose donors, respectively. An oligosaccharyltransferase complex delivers the glycan to asparagine residues in the nascent protein core. The protein-bound glycan is trimmed to mannose 8 -protein in the ER and is trimmed further in the Golgi. One GlcNAc residue is added to the trimmed mannose 5 structure, and this GlcNAc can be extended with galactose and sialic acid to form a hybrid glycan or, by adding more GlcNAc, branching can continue to form complex-type oligosaccharides. Nucleotide sugar transporters in the Golgi provide substrates for glycan extension. Steps in the pathway at which genetic disorders occur are indicated, with the associated genes underneath, as are steps at which an animal model is available. MPDU1 encodes a protein that enables the utilization of dolichol-p-mannose and dolichol-pglucose, but does not catalyse the reactions. mutations in the transferase complex that is involved in the first of two steps in the addition of mannose-6- phosphate 64,65. Other diseases might also be caused by N-glycosylation defects with phenotypes that affect specific cell types. For example, autosomal recessive type II congenital dyserythropoietic anaemia impairs erythropoiesis. An N-glycan-processing defect might underlie the disorder, but genetic confirmation of this has yet to be obtained 66,67. NATURE REVIEWS GENETICS ADVANCE ONLINE PUBLICATION 7

8 Table 2 Human diseases caused by genetic defects in N-glycosylation pathways Disorder Gene Enzyme OMIM Key Features CDG-Ia PMM2 Phosphomannomutase II Mental retardation, hypotonia, esotropia, lipodystrophy, cerebellar hypoplasia, stroke-like episodes, seizures CDG-Ib MPI Phosphomannose isomerase Hepatic fibrosis, protein-losing enteropathy, coagulopathy, hypoglycaemia CDG-Ic ALG6 Glucosyltransferase I Dol-P- Glc: Man 9 -P-P-Dol glucosyltransferase Moderate mental retardation, hypotonia, esotropia, epilepsy CDG-Id ALG3 Dol-P-Man:Man 5 -P-P-Dol mannosyltransferase CDG-Ie DPM1 Dol-P-Man synthase I GDP-Man: Dol-P-mannosyltransferase Profound psychomotor delay, optic atrophy, acquired microcephaly, iris colobomas, hypsarrhythmia Severe mental retardation, epilepsy, hypotonia, mild dysmorphism, coagulopathy CDG-If MPDU1 Man-P-Dol utilization 1/Lec Short stature, icthyosis, psychomotor retardation, pigmentary retinopathy CDG-Ig ALG12 Dol-P-Man:Man 7 P-P-Dol mannosyltransferase CDG-Ih ALG8 Glucosyltransferase II Dol-P-Glc: Glc 1 -Man 9 -P-P-Dol glucosyltransferase CDG-Ii ALG2 Mannosyltransferase II GDP- Man: Man 1 -P-P-Dol mannosyltransferase CDG-Ij DPAGT1 UDP-GlcNAc: Dol-P-GlcNAc-P transferase CDG-Ik ALG1 Mannosyltransferase I GDP- Man: GlcNAc 2 -P-P-Dol mannosyltransferase CDG-Il ALG9 Mannosyltransferase Dol-P-Man: Man 6 - and Man 8 -P-P-Dol mannosyltransferase Hypotonia, facial dysmorphism, psychomotor retardation, acquired microcephaly, frequent infections Hepatomegaly, protein-losing enteropathy, renal failure, hypoalbuminaemia, oedema, ascites Normal at birth; mental retardation, hypomyelination, intractable seizures, iris colobomas, hepatomegaly, coagulopathy Severe mental retardation, hypotonia, seizures, microcephaly, exotropia Severe psychomotor retardation, hypotonia, acquired microcephaly, intractable seizures, fever, coagulopathy, nephrotic syndrome, early death Severe microcephaly, hypotonia, seizures, hepatomegaly CDG-IIa MGAT2 GlcNAc transferase Mental retardation, dysmorphism, stereotypies, seizures CDG-IIb GLS1 Glucosidase I Dysmorphism, hypotonia, seizures, hepatomegaly, hepatic fibrosis; death at 2.5 months CDG-IIc SLC35C1/ FUCT1 GDP-fucose transporter Recurrent infections, persistent neutrophilia, mental retardation, microcephaly, hypotonia; normal transferrin CDG-IId B4GALT1 β1,4 galactosyltransferase Hypotonia (myopathy), spontaneous haemorrhage, Dandy Walker malformation CDG-IIe COG7 Conserved oligomeric Golgi complex subunit Fatal in early infancy; dysmorphism, hypotonia, intractable seizures, hepatomegaly, progressive jaundice, recurrent infections, cardiac failure CDG-IIf SLC35A1 CMP-sialic acid transporter Thrombocytopaenia, no neurological symptoms; normal transferrin, abnormal platelet glycoproteins CDG-II/COG1 COG1 Conserved oligomeric Golgi complex subunit 1 Mucolipidosis II and III GNPTA UDP-GlcNAc: lysosomal enzyme, GlcNAc-P transferase Hypotonia, growth retardation, progressive microcephaly, hepatosplenomegaly, mild mental retardation Coarsening features, organomegaly, joint stiffness, dysostosis, median neuropathy at the wrist; MLIII is less severe than MLII, which presents in infancy Congenital dyserythropoietic anaemia (CDA II) Unknown Unknown Anaemia, jaundice, splenomegaly, gall bladder disease CDG, congenital disorder of glycosylation; Dol, dolichol; Glc, glucose; GlcNAc, N-acetylglucosamine; Man, mannose. Galactosaemia provides an example in which a genetic defect that does not directly affect glycan biosynthesis might cause disease through secondary affects on N-glycosylation. This condition is caused by an inability to metabolize galactose, which results in decreased UDP-galactose levels or the accumulation of potentially toxic metabolites 68. Most cases are caused by galactose-1- phosphate uridyltransferase (GALT) deficiency, with mutations in UDP-galactose-4 epimerase (GALE) and galactokinase 1 (GALK1) underlying other cases. However, some of the pathology that is seen in galactosaemia might be caused by defective protein glycosylation. 8 ADVANCE ONLINE PUBLICATION

9 Table 3 Human diseases caused by genetic defects in O-glycosylation and glycolipid synthesis pathways Disorder Gene Enzyme OMIM Key Features Defects in O-glycosylation pathways Walker Warburg syndrome Fukuyama muscular dystrophy Congenital muscular dystrophy type 1C (MDC1C) Congenital muscular dystrophy type 1D (MDC1D) Hereditary inclusionbody myopathy-ii (IBM2) POMT1/ POMT2 FCMD FKRP O-mannosyltransferase Type II lissencephaly, cerebellar malformations, ventriculomegaly, anterior chamber malformations, severe delay; death in infancy Fukutin, a putative glycosyltransferase Fukutin-related protein, a putative glycosyltransferase Cortical dysgenesis, myopia, weakness and hypotonia; 40% have seizures Hypotonia, impaired motor development, respiratory muscle weakness LARGE Putative glycosyltransferase Muscular dystrophy with profound mental retardation GNE UDP-GlcNAc epimerase/kinase Adult onset with progressive distal and proximal muscle weakness; spares quadriceps Ehlers Danlos syndrome B4GALT7 β1,4-galactosyltransferase Progeroid Ehlers-Danlos syndrome; macrocephaly, joint hyperextensibility Heriditary multiple exostosis Chondrodysplasias Spondyloepimetaphyseal dysplasia Macular corneal dystrophy types I and II Familial tumoral calcinosis EXT1/ EXT2 DTDST/ SLC26A2 ATPSK2 Glucuronyltransferase/GlcNAc transferase Sulphate anion transporter phosphoadenosine- 5 -phosphosulphate synthase Multiple exostoses (diaphyseal, juxtaepiphyseal) Diastrophic dysplasia: airway collapse, early death in severe cases, adults reported. Achondrogenesis Ib: usually stillborn or early death of respiratory failure. Atelosteogenesis II: pulmonary hypoplasia, fatal in infants Abnormal skeletal development and linear growth CHST6 Keratan sulphate Corneal clouding and erosions, painful photophobia sulphotransferase GALNT3 GalNAc transferase Massive calcium deposits in skin and tissue Tn syndrome COSMC Chaperone of β1,3galt Anaemia, leukopaenia, thrombocytopaenia (somatic mutation) Defects in glycolipid synthesis Paroxysmal nocturnal PIGA PI-GlcNAcT Complement-mediated haemolysis (somatic mutation) haemoglobinuria Amish infantile epilepsy SIAT9 Sia2,3Galβ1,4Glc-Cer synthase Tonic-clonic seizures, arrested development, neurological decline CDG, congenital disorder of glycosylation; Cer, ceramide; Gal, galactose; GalNAc, N-acetylgalactosamine; Glc, glucose; GlcNAc, N-acetylglucosamine; Sia, sialic acid. Interferon A pro-inflammatory cytokine that is produce by T cells in response to antigenic or mitogenic stimuli. Fibroblasts from patients with GALT who are exposed to galactose accumulate substantial intracellular galactose-1- phosphate 69, which might alter the concentration of nucleotide sugars. Recent studies identified several patients with GALT who were not controlling dietary galactose intake and had unoccupied glycosylation sites on serum transferrin and altered N-glycan structures in total serum glycoprotein samples 70. These features are reversed when galactose is removed from the diet. Nevertheless, even patients with GALT who carefully control their dietary galactose intake consistently show mental retardation, apraxic speech and ovarian failure in females. Incomplete sialylation of follicle-stimulating hormone indicates that glycoproteins might be affected 71, but it is uncertain whether other effects are caused by accumulation of toxic metabolites such as galactitol or by improper glycosylation 72. Mice with GALT deficiency do not recapitulate the human phenotype when given massive amounts of galactose, indicating that subsequent metabolism of galactose-1-phosphate might be limiting in humans but not in mice 68. Too much N-glycosylation? Not all available N-glycosylation sites on proteins that pass through the ER and Golgi are occupied 73,74 and some are variably occupied in different tissues 75. Nevertheless, the location of N-glycan sites is conserved in most proteins. Point mutations that create novel glycosylation sites could cause protein misfolding and rapid degradation 76. Alternatively, the survival of such proteins could have even more severe effects if the presence of the glycan impaired formation of a signalling complex or multimeric protein. For example, one mutation that can cause Marfan syndrome creates an N-linkage site in fibrillin 1 (FBN1) that alters the processing and proper assembly of monomers of this protein 77,78. Recently, several patients with heightened susceptibility to mycobacterial infections were found to carry a pathological point mutation that creates a novel glycosylation site in the interferon receptor IFNγR2 (REF. 79). This mutation does not impair folding of the protein or its surface localization, but its function is dramatically decreased. Inhibitors of N-glycosylation, or enzymatic NATURE REVIEWS GENETICS ADVANCE ONLINE PUBLICATION 9

10 Reichert s membrane The first basement membrane that is formed during mouse embryonic development; it is not present in humans. Variable expressivity The variability in severity of a disease-causing genetic trait. removal of the additional glycan, restored activity to IFNγR2, showing that addition of an N-glycan at that site destroyed the function of the receptor. The same study also identified function-compromising mutations that generate new glycosylation sites in other immunerelated proteins 79. Furthermore, the authors carried out a survey of 577 missense mutations that affect proteins that traverse the ER Golgi pathway and showed that 13% of them create inappropriate glycosylation sites. This surprisingly high number might mean that protein over-glycosylation actually has a greater effect on function than loss of glycosylation. It should not be surprising that randomly inserting bulky glycans into proteins such as IFNγR2 has negative effects; even a properly placed glycan with the wrong structure can affect the ability to form signalling complexes 80. Variable glycosylation-site occupancy might be a physiological way to control the formation of such complexes. Disorders of O-linked glycosylation Disease-causing defects in O-glycan biosynthesis occur primarily in the O-mannose and O-xylose pathways, and more patients are affected by these disorders than those with N-glycan defects. Their clinical presentations are also usually distinct from those that are seen in N-glycosylation disorders: the O-mannose-based disorders all cause muscular dystrophy, an uncommon finding in N-glycan defects, whereas the O-xylose-based defects usually cause bone, cartilage and other ECM abnormalities. Defects in O-mannose glycans: congenital muscular dystrophies. Disorders that result from defects in O-mannose-based glycans were originally known by other names, and their link to glycosylation is a recent development. Mutations in the glycosylation machinery that adds O-mannose-based glycans to α-dystroglycan have recently been shown to cause at least five types of congenital muscular dystrophy (CMD) (TABLES 2,3), and other glycosylation-related defects will probably be identified for CMDs with as yet unknown aetiologies 4, The clinically assigned categories of these disorders are now being revised on the basis of knowledge of the defective genes, as different mutations in the same gene produce phenotypes that were previously considered to characterize distinct disorders 83. Identifying the disease-causing genes also raises new questions about how their protein products affect glycosylation, as some do not have proven catalytic activity but are able to correct multiple glycosylation defects in cells and mouse models of CMD. α-dystroglycan is one of the two subunits of the dystrophin glycoprotein complex (DGC), which connects the ECM to the cytoskeleton in many tissues. Both subunits α-dystroglycan and β-dystroglycan are derived from a single gene, DAG1. In muscle, cytoskeletal actin is linked to β-dystroglycan, which spans the cell membrane. The extracellular domain of β-dystroglycan binds to α-dystroglycan, which in turn binds laminin 2 in the ECM through its glycan-containing extracellular domain 81. The degree and types of α-dystroglycan glycosylation vary between different tissues, but the presence of sialic acid in the most commonly added glycan is required for laminin-2 binding. The use of monoclonal antibodies against glycans has been important in identifying glycosylation-related defects that affect α-dystroglycan, similar to the use of transferrin for N-glycosylation defects (BOX 1). Although no pathological mutations have been found in α-dystroglycan itself, the name α-dystroglycanopathy has been suggested to describe these glycosylation disorders as it is the only protein that has been proved to carry the glycans that are affected in the CMDs 82. Walker-Warburg syndrome (WWS) is the most severe of the CMDs that are caused by defective α-dystroglycan glycosylation, with a short life span (mean <1 year), multiple brain abnormalities and severe muscular dystrophy. About 20% of the cases have mutations in POMT1, which encodes the glycosyltransferase that is required to add O-mannose to α-dystroglycan. Similar to DAG1- null mice, POMT-null animals die at E , and both models fail to synthesize Reichert s membrane, supporting the functional connection between the two genes 85. A POMT1 POMT2 complex in the ER is required for transferase activity 86, and mutations in POMT2 are also seen in WWS cases 87. A few clinically defined patients with WWS have defects in the genes that encode Fukutin and Fukutin-related protein (FKRP) 83,84, which are also thought to function in this glycosylation pathway. Mutations in fukutin and FKRP also cause other, milder forms of CMD (see below), which emphasizes phenotypic dependence on both the specific gene and the mutation. Linkage-based mapping studies of WWS consanguineous families indicate that at least two other genes also carry WWS-causing mutations 83. The POMGNT1 gene encodes the transferase that catalyses the step in the pathway after initial O-mannose addition 88, and mutations in this gene cause muscleeye-brain disease (MEB), which is characterized by symptoms that are similar to those of WWS. This disease has a variable expressivity: the most severely affected patients die during the first years of life, but towards the mild end of the spectrum most survive to adulthood. The more severely affected WWS-like patients have mutations at the 5 end of the gene, but none of the mutated proteins have enzymatic activity, indicating that other factors determine severity 89. These might include higher expression of like-glycosyltransferase (LARGE), which can override some of the DGC mutations, as I discuss below. POMGnT1-null mice are viable, but have multiple defects in muscle, eye and brain 90. For some CMDs, the mutated gene has been identified but the function of the protein that is encoded is unknown. For example, Fukuyama muscular dystrophy (FCMD) is caused by a 3-kb 3 retrotransposon insertion in fukutin, which partially reduces the stability of the mrna, making it a relatively mild mutation. The products of fukutin are expressed in muscle and other tissues, and co-localize with α-dystroglycan in various regions of the adult mouse brain. The protein has a putative glycosyltransferase signature (Asp-X-Asp, where X is any amino acid) and localizes to the cis-golgi, but no 10 ADVANCE ONLINE PUBLICATION

11 Haploinsufficiency The inability of the remaining wild-type allele to compensate for a heterozygous loss-offunction mutation. transferase activity has been demonstrated. Similarly, congenital muscular dystrophy type Ic (MDCIC) is a relatively mild disorder and is caused by mutations in FKRP 91. Again, the protein is mainly Golgi-localized and has an Asp-X-Asp glycosyltransferase signature, but no proven transferase activity. Studies of one type of CMD, MDCID, have provided insights into potentially important features of glycosylation pathways 84. Patients with this disorder carry a mutation in LARGE, which was originally described in myodystrophic mice (myd, now called Large myd ). The protein contains two glycosytransferase signatures in different domains, which indicates the possibility of a bi-functional enzyme. No enzymatic activity has been demonstrated, but mutating these domains suppresses the ability of ectopic LARGE expression to rescue defective glycosylation 92. LARGE resides in the Golgi and seems to recognize an N-terminal region of α-dystroglycan, which is subsequently proteolyzed, leaving the glycosylated mucin domain. Overexpression of LARGE results in α-dystroglycan hyperglycosylation, which indicates that it is a rate-limiting step 92. LARGE overexpression phenotypically rescues the Large myd mouse, and prevents the development of muscular dystrophy without increasing the expression of the other dystroglycan-complex proteins 93. Most surprisingly, LARGE can also rescue defective glycosylation in myoblasts from patients with FCMD and WWS, and fibroblasts from patients with MEB. This is not due to an increase in the activity of the enzymes that are defective in these disorders; glycosylation is restored by an unknown compensatory mechanism. These remarkable results are significant because they indicate that ectopic expression of LARGE might be a potential therapy for CMD. LARGE is not alone in this mode of action. Overexpression of a specific cytotoxic T-cell βgalnac transferase 94 can also create a novel glycan on α-dystroglycan and prevent the onset of pathology in the mdx mouse, which is deficient in dystrophin, the protein that is defective in Duchenne muscular dystrophy. So, formation of an effective extracellular complex can override and substitute for the loss of the intracellular link to actin. The mechanism seems to involve increased binding to utrophin, a dystrophin homologue that is not normally present at the muscle membrane in sufficient levels without the booster gene, which creates an artificial sweetener that overrides these deficiencies 95,96. Defects in O-xylose glycosaminoglycans. Mutations that affect various stages in GAG synthesis cause several human disease phenotypes (TABLES 2,3). For example, the ECM defects that cause bone and cartilage abnormalities in the progeriod variant of Ehlers Danlos syndrome result from mutations in B4GALT7, which encodes the enzyme that is responsible for adding the first galactose residue to xylose 97.The best-understood genetic defects of this type affect the formation of heparan sulphate and cause hereditary multiple exostosis (HME), which is an autosomal dominant disease with a prevalence of about 1:50,000 (REF. 98). HME is characterized by bony outgrowths (exostoses), usually at the growth plates of the long bones, and osteosarcoma also develops in 1 2% of patients 99. Studies of this disease have provided insights into the role of GAGs in signalling and development. HME is caused by missense or frameshift mutations in 3 genes, exostoses 1 (EXT1), EXT2 and EXT3, all of which are involved in heparan sulphate synthesis 98. Most HME mutations occur in EXT1 (60 70%) and EXT2 (30 40%). These proteins are thought to exist as a complex in the Golgi and both are required for polymerizing the alternating residues of GlcNAcα1,4 and GlcAβ1,3 that define heparan sulphate 98. The partial loss of one allele of either gene seems to be sufficient to cause MHE, which indicates that haploinsufficiency decreases the amount of heparan sulphate and that Ext activity is rate limiting for heparan sulphate biosynthesis. The mechanism of HME pathology might be rooted in a disruption of the normal distribution of heparansulphate-binding growth factors, which include FGF and morphogens such as Hedgehog, Wingless and Decapentaplegic 13. Consistent with this theory, lack of heparan sulphate disrupts hedgehog, wingless and decapentaplegic pathways in Drosophila. Mice that are null for either Ext1 or Ext2 are embryonic lethal and fail to gastrulate 100 ; however, Ext2-heterozygous animals are viable and about one third develop a single visible exostosis on the ribs 101. No exostoses develop on the long bones of these animals, in contrast to HME, but subtle chondrocyte-growth abnormalities are seen in the growth plates of these bones. These defects are not due to defective Hedgehog signalling, as the distribution of this protein is not affected. Ext1 heterozygotes also develop exostoses, although the penetrance depends on the mouse strain. In addition, they show reduced signalling of FGF18 through FGF receptor 3 (FGFR3), and combining the Ext1 mutation with an Fgfr3 +/ background increases penetrance and severity (J. Esko, personal communication), which indicates that this pathway is affected by impaired heparan sulphate synthesis. Other CDGs of as yet unknown genetic origin that affect trafficking of glycosylation machinery might also affect GAG-chain synthesis and alter skeletal development by affecting FGF signalling. Many properties of GAGs depend on sulphation, and several autosomal recessive chondrodysplasias cause abnormal skeletal development (TABLES 2,3) because of defects in sulphate-anion transport into cells 102 or its activation to 3 -phosphoadenosine-5 -phosphosulphate, the universal sulphate donor 103. Defects in O-GalNAc glycans. Familial tumoral calcinosis is a severe autosomal recessive metabolic disorder that involves phosphataemia and massive calcium deposits in the skin and subcutaneous tissues. Linkage analysis identified bi-allelic mutations in GALNT3, one of more than 20 known homologues that encode GalNAc transferases, which initiate O-glycosylation 104. Mutations in O-glycosylated FGF23 also cause phosphataemia 105, which indicates a link between GALNT3 and FGF23 function 106, although this remains to be confirmed. NATURE REVIEWS GENETICS ADVANCE ONLINE PUBLICATION 11