November/December 2000 . Vol. 2 . No. 6

Congenital disorders of : Have you encountered them? Vibeke Westphal, PhD, Geetha Srikrishna, PhD, and Hudson H. Freeze, PhD

Protein glycosylation creates hundreds of sugar structures complete absence of any glycosylation seems inconsequen- that serve a spectrum of functions. So it is not surprising that tial.1,7J1,1zIt is impossible to predict whether the presence of a one percent of transcribed produce or recognize sugar glycan or a unique structure on a sugar chain will ultimately chains. Given this substantial investment, it is also not surpris- affect a function. Like phosphorylation, glycosylation ing that defects in sugar chain biosynthesis have substantial is a post-translational event and is not read from the genetic consequences for development, growth, and survival. In the template. Instead, an amino acid consensus sequence generates past few years, mutations in seven of these genes have been a potential glycosylation site. Many of them are used, but oth- shown to cause congenital disorders of glycosylation (CDG). ers are not. Furthermore, a single glycosylation site can house a Their pathologies, like glycosylation itself, are diverse and af- series of structurally related sugar chains at this same loca- fect multiple systems. The CDG are almost certainly underdi- ti~n.~~~~%Iycosylationcan be diverse, heterogenous, and yet agnosed in the community, so increased clinical awareness of specific. It is maleable, adaptable, yet indispensible for survival, the many presentations of CDG is important. Simple diagnos- from yeast to h~mans.~,Il,~5 tic tests, and possible dietary therapy for some ofthese patients, Much ofour understanding of glycan biosynthesis and func- make it important to consider and rule out altered glycosyla- tion emerged from metabolic labeling, analysis of mutant cell tion in unexplained cases of psychomotor retardation, hypo- lines, and effects of specific inhibitors on glyco~ylation.~~-~~ tonia, coagulopathy, hepatic fibrosis, protein-losing enteropa- Recently, the ability to delete genes coding for glycosyltrans- thy, feeding difficulties, and failure to thrive. ferases in various biosynthetic pathways of the mouse has shown unsuspected connections to signal transduction,3 im- mune regulati~n,~),~~ and human di~ease.'~ THE SETTING Several types of linkages occur between sugars and . An overview of the field of glycobiology can be found in a The two major ones are called N- and 0-linkages. N-linked recent comprehensive textbook.' Protein glycosylation can be glycans are attached through nitrogen of an asparagine and as simple as adding a single sugar (monosaccharide). More 0-linked glycans through the oxygen of serine or threonine. often, it is complex. Mammalian cells select nine different Since the types of CDG discovered so far are primarily disor- monosaccharides and join them to proteins and/or to each ders of N-glycosylation, we will focus on that in this review. In other, forming hundreds of different structures (glycans). this pathway, a 14-sugar residue oligosaccharide is first trans- Branched sugar chains composed of various monosaccharides ferred from a transient lipid carrier to nascent proteins in the decorate cell surfaces, extracellular matrices, and secreted pro- lumen of the endoplasmic reticulum (ER). The sugar chain is teins. About 1% of the transcribed genome is devoted to the trimmed, sometimes removing most of the original glycan, synthesis and recognition of sugar chains.' What are their and then selectively extended with different sugars in various functions? There are many, including proper nascent protein branching patterns (Fig. 1).At least 50 known genes contribute folding, imparting protease resistance, intracellular trafficking, to the synthesis of glycans typically found on plasma proteins. cell-substratum and cell-cell specific interactions, leukocyte In the past few years, mutations in seven of these genes have trafficking, and growth regulation, to name a fe~.l~-l-~~A blan- been found to cause human disease. Their clinical presenta- ket statement of glycan function is not possible, because there tions are, like glycosylation itself, diverse and heterogenous, are so many structures. In addition, the function of a glycan affecting multiple functions and organ systems. varies with oligosaccharide presentation, regulated expression of proteins in specific cell types, expression and specificities of glycosidases and glycosyl and microheterogeneity N-LINKED GLYCOSYLATION of glycan structures on individual proteins. In some cases, a Nearly all cell surface and secreted proteins are N-glycosy- highly specific glycan structure is essential, while in others, lated as they pass through the ER during or shortly after they enter the ER lumen. A dolichol pyrophosphate (Dol-P linked ]*-sugar oligosaccharide (LLO) is transferred rrs; from the lipid carrier to asparagine (Asn) residues in the con- text Asn-Xaa-Ser or Asn-Xaa-Thr, where Xaa can be any amino acid except proline. The 14-sugar unit is a tri-branched oligosaccharide composed of 3 (Glc), 9 Westphal et a/.

(Man), and 2 N-acetylglucosamine (GlcNAc) residues, anno- cause it is poorly transferred to the protein by the oligosac- tated as Glc,Man,GlcNAc, (Normal Lipid-Linked Precursor, charyltransferase complex. Fig. 1). It is sequentially synthesized using a specific glycosyl- Several types of CDG specifically affect only the N-linked to add each sugar. Initially, the growing sugar chain pathway, while others affect the synthesis of early precursors faces the cytoplasmic side of the ER where UDP-GlcNAc con- such as GDp-Man or Dol-P-Man, which are used in multiple tributes GlcNAc-1-P, and a second GlcNAc, and GDP-Man pathways. For instance, CDG-Ia (OMIM 212066) is causedby contributes the first 5 Man units. The molecule then "flips" to mutations in the PMM2 located on 16. The the lumen of the ER where the remaining 4 Man and 3 Glc, are gene encodes the phosphomannomutase involved in the con- attached in specific linkages using hexose-phosphate-dolichol version from Man-6-P to Man-1-P. Mutations in this gene donors, Dol-P-Man and Dol-P-Glc, respectively. The oligosac- reduce the size of GDP-Man pool and produces insufficient charide is then transferred to the nascent polypeptide chain by LLO for full glycosylation.'7,'8 A second PMM gene, PMMl, is the oligosaccharyl transferase complex located in the ER mem- located on chromosome 22. However, this gene encodes an brane near the translocation complex. The GlcNAc residue with a slightly different enzymatic activity and is not that was directly linked to the Dol-PP lipid is now bound to the involved in the disorder."^^^ CDG-Ib (OMIM 602579)3L-33re- amide N of Asn. After the transfer, specific glycosidases trim sults from mutations in the MPIl gene on chromosome 15, the Glc,Man,GlcNAc, chain, removing Glc and some Man in encoding phosphomannose isomerase (PMI) that converts the ER. Additional Man is often removed in the Golgi and further trimming of some chains is followed by the addition of fructose-6-P to Man-6-P. Loss of this enzyme also reduces the 2-4 branches composed of GlcNAc, Gal, and a terminal sialic size of the GDP-Man pool, but the clinical pictures of CDG-Ib acid (Sia), to form complex-type sugar chains. The restructur- and CDG-Ia patients are quite different (see below). Mutations ing of these chains is called oligosaccharide processing. Con- in DPMl (chromosome 20), the catalytic subunit of Dol-P- genital disorders of glycosylation are caused by defective Man synthase, cause CDG-Ie (OMIM 603503). The truncated monosaccharide activation or interconversion or by defective LLO (Man,GlcNAc,-PP-Dol) is inefficiently transferred to addition or removal of the sugar units to the chain. This defect pr~tein.'~,~~Effects of these mutations on other types of glyco- leads to insufficient or abnormal N-linked sugar chains on sylation have not been systematically examined. some proteins and the pathology seen in CDG patients. CDG-Ic (OMIM 603147), -Id (OMIM 601110), -1Ia (OMIM 212066), and -1Ib are all specific to the N-linked gly- cosylation pathway. Mutations in the human homolog of ALG6 (chromosome 11, which encodes an a-1,3-glucosyl- CONGENITAL DISORDERS OF GLYCOSYLATION transferase used to add the first Glc to the LLO precursor, cause Congenital disorders of glycosylation were previously called CDG-IC.'~-'~CDG-Id patients are deficient in an a-mannosyl carbohydrate deficient glycoprotein syndromes (CDGS). transferase encoded by the NOT56 1 gene (). Types were biochemically defined by abnormal isoelectric fo- Mutations in this gene affect the function of the mannosyl- cusing (IEF) patterns of serum transferrin (Tf).'5 The current transferase that transfers mannose from dolichol-phosphate nomenclature for CDG is based on the identification ofspecific mannose (Dol-P-Man) on to the LLO intermediate gene defects.26 Group I disorders affect the biosynthesis of the Man,GlcNAc,-PP-Do1 resulting in an accumulation of the dolichol-linked precursor oligosaccharide and its transfer to LLO intermediate.', CDG-IIb results from the failure of a-glu- proteins. Group I1 includes defects in N-linked oligosaccharide cosidase I to remove the terminal Glc from the oligosaccharide processing and/or the addition of other kinds ofsugar chains to after its transfer from the lipid carrier to protein. CDG-IIa proteins. The type is defined within the group by lower case results from defects in MGAT2 (chromosome 14), a GlcNAc- letters based on the chronological order of discovery of the transferase required for the synthesis of complex sugar chains defective gene. CDG patients with an unknown defect are des- with two or more Sia-containing branche~.~~,~~Leukocyte ad- ignated CDG-x. This nomenclature will continue to evolve as hesion deficiency type I1 (LAD-11, OMIM 116920) results from more defects in other glycosylation biosynthetic pathways are impaired import of GDP-Fucose into the Golgi from the cyto- identified. plasm." This disorder may also be caused by defects in the conversion of GDP-Man to GDP-Fuc.4' Since the defective genes have not been identified, it is classified as "CDG-x." BIOCHEMICAL OVERVIEW OF CDG Recent estimates of the frequency of CDG-Ia is 1/20,000 in Table 1 lists the defects identified in CDG, and Figure 1 the European populations. This estimation is based on the de- shows their position in the biosynthetic pathway. All are auto- termination of the frequency ofthe most common mutation in soma1 recessive disorders. Carriers have normal Tf patterns PMM2 (G422A -+ R 14 1H) believed to originate from Scandi- and are asymptomatic. CDG is divided into two major groups. navia..'"" However, this mutation has never been found in a In Group I disorders (CDG I-a to I-e), there is an absence of homozygous state.444hThe geographical origin and the fie- entire oligosaccharide chains on some proteins either because quency of the known PMM2 mutations have been reviewed of an insufficient amount of precursor oligosaccharide or be- recently .4ha Have you encountered CDG?

GDP- A (Golgi) 0-1 -P Dol-P

GDP-A GDP-0 (cytosol)

0

UDP-W \O/ \O/

+-

DO~-P-P DO~-P-P DO~-P-P DO~-P-P Do l * A I A A I I A A I I 00 0 00 0 I I I

0 0 \o/

Dol-P-P Dol-P-P

o0 O i i

Flg. 1 N-linked oligosaccharide biosynthesis and congenital disorders of glycosylation (CDG).This condensed pathway outlines the biosynthesis and processing of N-linked oligosac- charides. Steps indicated by an asterisk are deficient in various forms of CDG. Symbols are used to denote monosaccharides: (A) glucose. (-1 fructose. (0)mannose. (@) galactose, (A) fucose, (*) sialic acid, and (W) N-acetylglucosamine. Starting at the upper left, mannose (0)delivered through the mannose transporter can be used directly for glycoprote~nsynthesis by conversion to Man-6-P (0-6-P)using hexokinase and ATP. Alternatively, fructose-6-P (0-6-P)from glucose (A)is converted to Man-6-P using phosphomannose isomerase (PMI, the defective enzyme in CDG-lb). Phosphomannomutase (PMM) converts Man-6-P into Man-I-P (0-I-P).PMM2 is defective in CDG-la. Man-I-P and GTP form GDP-Man (GDP-0)via GDP-Man pyrophosphorylase. GDP-Man has several fates. It can be incorporated into glycoproteins directly or converted into dolichol-P-Man (Dol-P- 0)or into GDP-Fuc (GDP-A). Dol-P-Man can be incorporated into glycoproteins or glycophospholipid anchors and into C-mannosylated pr~teins.~~.~~Dolichol, the lipid carrier for N-linked oligosaccharides is converted to dolichol-P (Dol-P) prior to addition of GlcNAc- I-P (W-I-P) from UDP-GlcNAc (UDP-W),to form Dol-P-P-GlcNAc, the first step In synthesis of the precursor for N-linked chains. A second GlcNAc residue is added to form GlcNAc-GlcNAc core. Five Man are added from GDP-Man, each one using a separate mannosyl transferase. An additional 4 a-mannosyl transferases are required to add the final 4 Man residues derived from Dol-P-Man. Mutations in the first of these four a-mannosyl transferases causes CDG-Id. Defective production of Dol-P-Man causes CDG-le. Three separate a-glucosyl transferases use Dol-P-Glc (Dol-P- A) to add three tandem Glc residues to the lipid-bound sugar chain. Addit~onof the first Glc residue is deficient In CDG-lc. The normal lipid linked precursor oligosaccharide (LLOI chain composed of 2 GlcNAc. 9 Man, and 3 Glc is transferred to Asn-X-ThrISer (NXSIT) sequons on proteins in the endoplasmic reticulum. Processing of the high mannose-type N-linked oligosaccharide beglnswith the removal ofall Glc by a set ofhvo a-glucosidases. Defects in the first a-glucosidase cause CDG-lib. This is followed by removal of a portion of the Man residues using two different a-mannosidases. A GlcNAc residue is transferred to the cham followed by removal of two more Man units and this is followed by the addition of a GlcNAc using GlcNAc transferase I. A second GlcNAc is added using GlcNAc transferase I1 (MGAT2,which is deficient in CDG-Ila). Further build-up of the chain continues by the addition of Gal (@) and finally Sia (*I residues to generate a two-branched chain. Different branching patterns and other chain extensions can occur on other proteins, but the final structure shown is typical of plasma glycoproteins synthesized by the liver. LAD-I1 is caused by a defect in the import of GDP-Fuc (GDP-A) into the Golgi from the cytoplasm. Like GDP-Man, GDP-A is a donor for other types of glycosylat~onpathways besides the N-linked pathway. Westphal et a/.

Table 1 Types of congenital disorders of glycosylation NO. of Year Name Defective enzymelgene Reaction patients identified Reference

CDG-Ia Phosphomannomutase (PMM2) OMIM 212066 Man-6-P a Man-1-P CDG-Ib Phosphomannose isomerase (MPII) OMIM 602579 Fru-6-P a Man-6-P

CDG-Ic a1,3 (ALG6) OMIM 603147 M,GN2-Do1 + GM,GN,-Do1

CDG-Id a1,3 (ALG3) OMIM 601 110 M,GN,-Do1 + M,GN,-Do1

CDG-Ie Dol-P-Man synthase (DPMI) OMIM 603503 Dol-P + GDP-Man + Dol-P-Man

CDG-IIa GlcNAc transferase 2 (MGAT2) OMIM 212066 GNlM,GN2 + GN,M,GN,

CDG-IIb Glucosidase I (GLSJ ) G,M9GNI --* G,M9GN, LADII (CDG-x) GDP-fucose transporter (genetic defect unknown) OMIM 116920 GDP-Fuc transport into the Golgi CDG-X Genetic defect unknown GDP-Man GDP-Fuc l? 1998 43,87

LABORATORY DIAGNOSIS OF CDG proteins is altered in CDG patient^,'^."-^' as is p-trace protein in cerebrospinal The serum Tf IEF is the best way to Many glycoproteins are misglycosylated in CDG patients, detect nearly all CDG cases.25However, the abnormal forms but serum Tf is widely used as the best biochemical indicator of were reported to not appear in patients until a few weeks after CDG.25Normal Tf has 2 N-linked complex type chains. Each birth.5' Also, Tf IEF does not indicate the specific defect. Pa- chain usually has two branches each one terminating in a neg- tients with different defects and clinical features can have iden- atively charged sialic acid (Sia),which contributes to the charge tical abnormal patterns. Uncontrolled fructosemia, galac- on the protein. The extent of sialylation determines the mobil- tosemia, and heavy alcohol consumption also produce ity of the protein in electrophoretic separations, such as IEF. abnormal patterns similar to those seen in CDG patients and The great majority of normal serum Tf contains four sialic should be excluded while making a differential diagnosis of acids and is said to be tetrasialylated. Tf molecules lacking one CDG.54-57A few types of CDG cannot be detected by Tf IEF. or both sugar chains, or chains with an abnormal underlying Even with these limitations, any patient suspected of having a structure have fewer Sia and higher isoelectric points (Fig. 2). CDG should be tested for altered Tf glycosylation. They are easily resolved from normally glycosylated species. The hypoglycosylation of Tf observed in the CDG patients Electrospray ionization-mass ~pectrometry4~,"has also been can be caused by reduced activity of various . To test, used to analyze the glycosylation status of Tf. Glycosylation of the specific defect cells from the patient are needed. Fibroblasts a-1 antitrypsin, haptoglobin, AT-111, and many other plasma or leukocytes can easily be tested for reduced PMM or PMI activity, and fibroblasts are useful for studying the composi- tion of the LLO by metabolic labeling. CDG Patients Most plasma proteins contain complex-type oligosaccha- ride chains, and AT-111, protein C, protein S, and Factor XI are frequently underglycosylated in CDG patients leading to co- agulopathy, which possibly contributes to the stroke-llke epi- sodes in some patient~.~Voagulationtests such as prothrom- bin time and prolonged partial thromboplastin time as well as analysis of individual coagulation factors, therefore, are rec- ommended especially if liver biopsies and other invasive pro- cedures are to be considered. Prenatal diagnosis of CDGIa have been possible using chorionic villus samples and cultured am- ni~cytes,~',"but prenatal transferrin IEF is not a reliable te~t.'~,~' Fie;.- 2 Examplea of transferrin isoelectr~cfocusing patterns in controls and congenital disorder ~fglyco~ylation(CDG) patients. The isoelectric focusing (IEF) patterns of trans- ferrin are currently the best way to b~ochemicallyconfirm CDG. Each transferrin mole- cule has two N-g~ycosy~at~ons~tes, and each one is normally occupied by a sugar chain CLINICAL FEATURES OF CM; containing two negatively charged sial~cacids to yield "tetrasialotransferrin." This is marked "4" in thc figure. Mutations that lead to the absence ofoneor both chainsgenerate Table 2 lists the most common clinical features in various--..-. molecules with two or zero sialic acids, respectively. These correspond to di- and asialo types of CDG.6'-63Many organ systems are affected to variable forms oftransferrln and have different isoelectric points (2 and 0 in the figure). Even when both chaln~are added, defects in assembly or processing of the chain can also affect the degrees, but it is stress the clinical heterogeneity final addltlonof sialic acids and the isoelectric point. even within a single known defe~t.~~,~~P~ychom~t~~ retarda-

332 ~e;h&tcsIN Medicine Have you encountered CDG?

Table 2 arrow on the metabolic chart (Fig. 1 ). The CDG-Ib patients do Clinical features of the congenital disorders of glycosylation (CDG) not have mental or psychomotor retardation or neuropathy, spectrum but exhibit coagulopathy (e.g., low AT-III), protein losing en- Type Features "teropathy, hypoglycemia, hepatic fibrosis, cyclic vomiting, and Variable psychomotor retardation, hypotonia, peripheral diarrhea. Many of these symptoms are treatable as discussed neuropathy, stroke-like episodes, seizures, cerebellar below. hypoplasia, internal strabismus, abnormal eye movements. subcutaneous fat distribution. inverted nipples, LADII patients are mentally retarded, have failure to thrive, cardiomyopathy, prote~nuria and suffer from recurrent serious infections. In these patients, Normal development, hypoglycemia, coagulopathy. neutrophil extravasation is greatly reduced due to the absence hepatomegaly, protein-losing enteropathy, hepatic fibrosis, of fucosylated oligosaccharides, which are critical for neutro- cyclic vomiting, diarrhea phi1 rolling prior to extravasation..'? Hypotonia, psychomotor retardation, internal strabismus, The constellation of clinical manifestations associated with feeding problems, coagulopathy, seizures, normal cerebellar the disease calls for the attention and care of a multidisci- development plinary team of pediatricians and pediatric neurologists, hema- Hypotonia, intractable seizures, severe psychomotor retardation, tologists, gastroenterologists, and endocrinologists. microcephaly, reduced responsiveness, optic atrophy, adducted thumbs, high-arched palate An informal survey of 60 parents of CDG children in the United States conducted by our lab indicated that about 25% Hypotonia, intractable seizures, delayed myelination, cortical blindness, severe psychomotor retardation, high-arched palate of children are correctly diagnosed early on, but most were initially considered to have a "metabolic defect," "multisys- Hypotonia, severe psychomotor retardation, frequent infections, normal cerebellum, coarse facies, widely spaced nipples, low temic genetic disorder," or "cerebral palsy." The heterogeneity set ears, ventricular septa1 defect of patients with these disorders underscores the need to test Hypotonia, generilized edema, hypoventilation, apnea, more broadly than is currently being done. The disorder has hepatomegaly, demyelinating polyneuropathy almost certainly been underdiagnosed so far. Increased aware- Elevated peripheral leukocytes, absence of CD15, Bombay blood ness of CDG is evidenced by the growing number of CDG group phenotype, failure to thrive, hypotonla, psychomotor papers and recognition of specific defects (Fig. 3). retardation, short arms and legs, simian crease The majority of CDG patients diagnosed so far are type Ia (Table 1).The mortality rate in CDG-Ia children is about 20% during the first few years.62 Throughout childhood CDG-Ia tion ranging from mild to severe (reported in Ia, Ic, Id, Ie, IIa, patients may be severely delayed in achieving developmental and LADII patients) and hypotonia (Ia, Ic, Id, IIa, and IIb) are milestones, but they tend to stabilize after childhood and have the most consistent features of CDG. Other neurological find- stable intellectual statush9in adolescence and adulthood.70Ad- ings include ataxia (Ia and Ic), seizures, and stroke-like epi- olescence and adulthood are characterized by progressive mo- sodes. CT and MRI are recommended in patients with psy- tor neuron weakness associated with atrophy of lower limbs. chomotor retardation since they have revealed cerebellar Adolescent CDG patients are extrovert, and communicate well hypoplasia (Ia, Ic), delayed myelination (Ie, IIa, IIb), micro- despite dysarthria. Few CDG adults are known and some men- cephaly, and atrophy of the cerebrum (Ia, Ic, Id, Ie). The cog- tally retarded adults have been diagnosed in their second de- nitive deficiency can be mild." Nearly all patients have feeding cade. Some adult patients will do well in assisted living envi- problems, and fail to thrive, probably reflecting the great de- ronments. Since CDG-Ia patients tend to survive and stabilize mand for cellular proliferation and synthesis of the abundant glycoconjugates.66 Coagulopathies are frequently seen, and strabismus is also present in most patients. Other ocular find- ings include cortical blindness, retinal degeneration and re- duction in retinal vascularization. Cutaneous and subcutane- ous abnormalities include peau d'orange of the skin, abnormal fat distribution, and retracted nipples. Dysmorphic features such as adducted thumbs, dysplastic ears, highly arched palate, and thoracic deformities have also been reported (Id, Ie, IIa). Hypothyroidism is seen in a majority of Ia patient^.^' Thus, idiopathic neonatal hypothyroidism or euthyroid status with low thyroxine-binding globulin call for investigation of CDG. Male patients with CDG-Ia undergo puberty and develop sec- ondary sexual characteristics, but female patients generally do Fig. 3 Congen~tald~sorder of glycosvlation lCDGI puhllcdtions dnd ident~fiiationof not.68 Hypocortisolism has also been observed in CDG-Ic. spec~ficdefects. In the paht two years, the number of ident~fiedCDG defects has increased shdrply along w~thpuhl~shed papcrstn the field. The trend IS llkely to continue as addl- The presentations of CDG-Ib patients are dramatically dif- tiondl defects are he~nguncovered by Idhoratories that interface glycohiology and clinic~l ferent from CDG-Ia patients, even though their Tf IEF patterns medicine. Expanded awdreness of CDG w~lldeepen and broaden the appreciation of are identical(Fig. 2), and their defects are separated by a single many roles glycosylat~onplays in illnlcal medliine.

N- N- No. 6 Westphal et a/. after childhood, it is very likely that many adult CDG patients chomotor improvement was also observed in this child, remain undiagnosed. probably due to the more stable clinical status. Only a few cases of CDG-Ib, -Id, -Ie, -Ha, and -1Ib are cur- rently known (Table l). CDG-Ic was discovered 2 years ago, and may be more common than the number in Table 1 indi- TESTING FOR CDG cates, since 7 mutations have already been found in the defec- When is it appropriate to test for CDG? Certainly patients tive gene encoding an a-1,3 glucosyltransferase.37~70a~71~7Z showing variable psychomotor retardation, hypotonia, and the classic dysmorphic features of inverted nipples, fat pads, stra- bismus, and decreased AT-111 are likely candidates. However, THERAPY unexplained feeding problems, failure to thrive, hepatic fibro- Most CDG patients significantly benefit from typical pallia- sis, gastrointestinal abnormalities, coagulopathy, cerebellar tive therapies. The only effective treatment therapy is oral ataxia, hypothyroidism and hypoglycemia could also be due to mannose for CDG-Ib. This therapy works because mannose CDG. Even in the absence of psychomotor retardation, severe can be transported into cells by a mannose-specific transporter neurological disease, and dysmorphism, CDG should be ruled and then converted to Man-6-P via hexokinase, effectively by- out. Again, Tf IEF will detect most ofthese cases. Since CDG-Ib passing the impaired fructose-6-P 224 Man-6-P pathway and is treatable, Tf IEF is imperative in cases of unexplained hepatic replenishing the depleted GDP-Man Four doses of dysfunction, protein-losing enteropathy, coagulopathy, and 0.1-0.15 g/kg per day mannose reverses hypoglycemia and de- hypoglycemia. ficient AT-111 within a few weeks, and within 1-2 months Neither LAD-I1 nor CDG-IIb, the al,2glucosidase defi- plasma protein levels are normal and protein-losing enterop- ciency, can be detected by the Tf IEF test. The latter was de- athy disappear~.33,~3.~-'A few patients have now been on man- tected by the accumulation of a tetrasaccharide in the urine. nose supplemented diets for several years with only minor This molecule resulted from an endo-a-mannosidase cleavage side-effects (bloating, loose stools). Excessive dosing can in- of the sugar chains shortly after their addition to protein. crease HbAlc levels, which return to normal when mannose LAD-I1 was identified by the absence of fucosylated glycan an- intake is reduced.75 tigen CD15, and presence of Bombay blood group phenotype, Few foods contain free mannose, except for mllk (50-100 which indicates the absence of the fucosylated H-antigen.83 pM), although plant and animal glycoproteins contain avail- Tf IEF analysis to identify CDG patients is available at sev- able mannose. Plant products such as locust bean gum are eral locations including Mayo Medical Laboratories, Univer- composed of gala~tomannan,~~but most of the mannose is sity of California San Diego Biochemical Genetics Laboratory, probably unavailable because it occurs in a beta-linkage, rather and at Laboratory for Molecular Diagnosis, University Hospi- than the alpha linkage seen in most oligosaccharides. Carob tal Leuven-Gasthuisberg, Belgium. Enzymatic analysis for powder, which is made from this gum contains a small amount Types-Ia and I-b are available in San Diego and Leuven. Add- of free mannose, but it should not be considered as a therapeu- tional molecular analysis and/or identification of other CDG tic mannose substitute. defects are also available at research laboratories affiliated with Early studies in cultured fibroblasts from CDG-Ia patients these centers. showed that adding 200-500 pM mannose to the growth me- In 1996, parents of CDG children in the United States dium corrects inadequate glyc~sylation~~by replenishing the founded The CDG Family Network, Inc. By publishing a news- deficient GDP-Man pool. Mannose is efficiently absorbed letter, arranging annual family conferences, through the Inter- from the gut and multiple oral doses can maintain plasma lev- net page www.cdgs.com, and email correspondence els at several fold higher than the normal average of 55 pM, [email protected]~n,this nonprofit organization provides although CDG-Ia patients have lower steady state levels (5-40 information and support for the CDG community. Similar PM).~~Short-79.80 and longer-terms1 therapy studies to date groups in Scandinavia (hq://~ww.emu.lu.se/cdg/indexen~. have not shown verifiable improvement in the clinical condi- shtml) and Germany (http:Nwww.cdg-syndrom.dd provide tion or biochemical markers for the CDG-Ia patients. Some information in Europe. mannose therapy studies are still under way. Another thera- peutic alternative would be a hydrophobic "prodrug" deriva- IDENTIFICATION OF MORE PATIENTS AND NEW tive of Man-1-P that could enter cells and generate the parent DEFECTS compound using resident cytosolic esterases. This approach is being explored. Gene replacement therapy has not been Based on current population and birth rates, Matthijs esti- attempted. mates that over 100 CDG-la patients are born each year in the One LAD-I1 patient has also shown a positive response to United States and a similar or higher number in Europe (Mat- monosaccharide therapy.82 Fucose therapy immediately nor- thijs, personal communication). Because < 100 CDG patients malized the neutrophil levels, and over time partially restored of all types have been identified in the United States, the cur- synthesis of the fucosylated oligosaccharides. Fevers and recur- rent spectrum of CDG is highly underdiagnosed. Additional rent infections disappeared, prophylactic antibiotics were dis- defects are being identified in the N-linked pathway and many continued, and the patient gained weight. Significant psy- of them affect the addition of glycan chains to proteins and

Gen-4% 'CS IN Medicine Have you encountered CDG?

should be apparent from Tf IEF analysis. However, it is impor- 12. Dwek RA. Biological importance of glycosylation. Dev Biol Starrd 1998;96:43-47. tant to point out that CDG-IIb and LAD-I1 Tf patterns are 13. Prydz K. Dalen KT. Synthesis and sorting of proteoglycans. I Cell SCI2000;113(Pt ?):193-205. normal; therefore, other methods to detect altered glycosyla- 14. Lis H, Sharon N. Prote~nglycosylation. Structural and funct~onalaspects. EurlBro- tion, e.g., lectins or carbohydrate specific antibodies need to be chem 1993;218:1-27. developed. Mice ablated in specific show 15. Dennis RP.A review of the biolog~cals~gn~ficance of carbohydrates on glycoproteins and methods for the~ranalysis. Ada Exp Med B~ol1995;376:1-11. altered lectin binding patterns in blood,",'.' but animal exper- 16. Varkr A. Radioactive tracer techniques in the sequencing of glycoprotein oligosac- iments often strive for complete loss of function and give a charides. FASEB 1 1991;5:226-235. clearly altered lectin binding picture. CDG mutations that are 17. Stanley P, loffe E. mutants: key to new insights in glycobiology. FASEB 1 1995;9: 1436-1444. compatible with life appear to be leaky and lectin or antibody 18. Jacob GS. Glycosylation inhibitors in biology and medicine. Curr Oprfr Strrrct Biol probes may show less dramatic changes than those seen in 1995;5:605-61 1. knockout mice. 19. Lehle L. Tanner W. The specific site of tunrcamycin inh~brtionin the formation of dolichol-bound N-acetylglucosamine derivatives. FEBS Lett 1976;71:167-170. Furthermore, genetic background and environmental 20. Priatel 11. Chur D. Hiraoka N, Simmons CI, Richardson KB, Page DM. Fukuda M. stresses, such as infections, fever, and nutritional state could be Varkl Nh4, Marth ID. The ST3Gal-I controls CD8+ T lymphocyte important determinants in the severity of CDG expression. homeostasis by modulating 0-glycan biosynthes~s.Imnrlrmty 2000;12:273-283. 21. Hennet T. Chu~D. Paulson IC. Marth ID. Immune regulation by the ST6Gal sialyl- Heightened awareness of glycosylation as a cause of inherited transferase. Proc Natl Acad Scr USA 1998;95:4504 -4509. disease, appreciation of the diversity of CDG patients, and 22. Campbell RM. Metzler M, Granovsky M, Dennls IW. Marth ID. Complex asparag- broader-based testing for these disorders will all contribute to ine-linked oligosaccharides in Mgatl-null embryos. Glycobrology 1995;5:535-543. 23. Shafi R, lyer SP, Ell~esLC. O'Donnell N. Marek KW. Chui D. Hart GW. Marth. I. D. the diagnosis of new and previously overlooked patients, the The 0-GlcNAc transferase gene resides on the X chromosome and is essential for discovery of new types of CDG, and the development of poten- embryon~cstem cell vlabdity and mouse ontogeny. Proc Natl Acad So USA ?000;97: tial therapies for them. 5735-5739. 24. Chu~D. Oh-Eda M, Liao YF. Panneerselvam K. Lal A. Marek KW. Freeze HH. Moremen KW. Fukuda MN, Marth ID. Alpha-mannosidase-I1 deficiency results in Acknowledgments dyserythropo~eslsand unveils an alternate pathway in ol~gosacchar~debiosynthesis. This work was supported by grants from the NIH (RO 1 Cell 1997;90:157-167. 25. Stibler H, Holzbach U, Kristiansson B. lsoforms and levelsoftransferrin, antithrom- DK55695), March of Dimes (FY99-205), and the Cure Autism bin, alpha( I )-antitrypsin and thyroxine-brnding globulin in 48 patients with carbo- Now Foundation. VW was supported by grant 99,0059120 hydrate- defic~entglycoprotein syndrome type I. Sci~ndICIinLabinvest 1998;58:55- from the Carlsberg Foundation. We thank Sandra Peterson 61. 26. Part~c~pantsat "First International Workshop on CDGS". L.. Belgium. November and Angelica Romero for technical help and the latter for co- 12-13. 1999. Carbohydrate-deficient glyioprote~nsyndrome become congen~tal ordinating the informal survey data from the parents. Donna d~sordersof glycosylation: an updated nomenclature for CDG. Glycobrolo~y2000; Krasnewich is thanked for reviewing the clinical aspects of this l0:iii-v. 27. Rush IS, Panneerselvam K. Waechter CJ. Freeze HH. Mannose supplementat~on manuscript. We are indebted to the past members ofthe Freeze corrects GDP-mannose deficiency in cultured fibroblasts from some patients with laboratory who have made significant contributions to the congenital disorders of glycosylatron (CDG). Glycobiology 2000;10:829-835. studies reviewed here, especially Krishnasamy Panneerselvam, 28. Krasnewich DM. Holt GD, Brantly M. Skovby F, Redwine 1. Gahl WA. Abnormal synthes~sof dolichol-linked oligosaccharides In carbohydrate-defic~entglycopro- Gordon Alton, Jim Etchison, and Soohyun Kim. We apologize tein syndrome. Glycobrology 1995;5:503-510. to contributors whose work could not be cited because of space 29. Matthlis G. Schollen E. Pirard M, BudarfML. Van Schaftiigen E, Casslman 11. PMM limitations. (PMMI ), the human homologue of SEC53 or yeast phosphomannomutase, IS lo- callzed on chromosome 22q13. GC~~O~IIISS1997;40:41-47. 30. Plrard M. Achour~Y. CoUet IF, Schollen E. Matthils G. Schaftlngen EV. Klnetic References properties and trssular distribution of mammalian phosphomannomutase 1. Varki A, Cummings R. Esko I, Freeze H. Hart G, Marth J, editors. Essent~alsof rsozymes. Bluchcnr /1999;339(Pt 1 ):201-207. glycobiology. 1st ed. Cold Spring Harbor. New York: Spring Harbor Labordtory 31, laeken I. Matthijs G, Saudubray IM. Dionisi-Vici C. Bertini E, de Lonlay P. Henri H. Press. 1999. Carchon H. Schollen E, Van Schaftingen E. Phosphomannose isomerase deficiency: 2. Varlu A, Marth JD. Oligosacchar~desIn vertebrate development. Semin Dev Biol a carbohydrate-deficient glycoprotein syndrome with hepatic-intestinal presenta- 1995;6:127-1 38. tion [letter].Am I Hlrrit Gef~et1998;6?:1535-1539. 3. Granovsky M. Fata J. Pawling I. Muller WI. Khokha R, Dennis IW. Suppress~onof 32. de Kon~ngTI. Dorland L. van Diggelen OP. Boonman AM, de long GI, van Noort tumor growth and metastasis in Mgat5-deficient mlce. Nat Med 2000;6:306-3 I?. WL. De Schryver I. Duran M, van den Berg IE. Genvig GJ. Berger R. Poll-The BT. A 4. Ellgaard L, Mollnari M, Helenius A. Setting the standards: qudl~tycontrol in the novel dlsorder of N-glyiosylat~ondue to phosphomannose isomerase deficiency. secretory pathway. Science 1999;286:188?-1888. B~ocl~cnrBluphys Res Comin~rrr1998;?45:38-42. 5. Thor G, Brian AA. Glycosylation variants of murine interleukin-4: evidence for 33. Niehues R. Hasilik M, Alton G, Korner C. Schiebe-Sukumar M. Koch HG. Zimmer K. different functional properties. Imm~rnology1992;75:143-149. KP, Wu R, Harms E, Re~terK, von Figurd Freeze HH. Harms HK. Mdrquardt T. 6. Kornfeld S. Structure and function of the mannose 6-phosphatelinsulinlike growth Carbohydrate-deficient glycoprotein syndrome type Ih. Phosphomannose isomer- factor 11 receptors. Annlr Rn,Blocl~em 1992;61:307-330. ase deficiency and mannose therdpy. I Clrr~li~l,~st 1998;101:1414-1420. 7. Varki A. Biolog~calroles of oligosaccharides: all of the theories are correct. Glymhr- 34. lmbach T, Schenk B. Schollen E. Burda P. Stutz A. Grnewald S. Bailie NM. King ology 1993;3:97-130. MD, laeken I. Matthris G. Berger EG. Aeb~M. Hennet T. Deficiency of dolichol- 8. Rudd PM. Wormald MR, Stanfield RL. Huang M. Mattsson N. Speir IA. D~Gennaro phmphatc-mannose synthase- l causer congenital disorder of glycosylation type le. JA, Fetrow IS, Dwek RA. Wilson IA. Roles for glycosylation of cell surface receptors I CIIII11rvest 1000;105:233-239. involved in cellular immune recognition. I Mol Biol 1999;293:351-66. 35. Kim S. Miestphal V. Srikrishna G. Mehta DP. Peterson S. F~lianoI. Karnes PS, 9. Gahmberg CG, Tolvanen M. Why mammalian cell surface proteins are glycopro- Patterson MC. Freeze HH. Dol~cholphorphate mannose synthdse (DPMI) muta- teins. Trends Biochem Sci 1996;21:308-31 I. tions define congenital disorder ofglycorylation Ie (CDG-Ie).ICII~I Iirt~est2000;105: 10. Springer TA. Traffic signals on endothelium for lymphocyte recirculat~onand leu- 191-198. kocyte emigration. In: Paul LC. Issekutz TB, editors. Adhesion molecules in health 36. Korner C, Knauer R. Holzbach U. Hanefeld F. Lehle L, von Figura K. Carbohydrate- and disease. New York: Marcel Dekker. Inc.. 1997:l-54. defic~ent glycoprotein syndrome type V: deficiency of dolichyl-P-Glc: I I. Gagneux P. Varki A. Evolutionary considerat~onsin relatlng oligosacchar~dediver- Man9GlcNAc2-PP-dol~chyl glucosyltransferase. Pro< Natl Acad Scr USA 1998;95: sity to biological function. Glycobiolo~y1999;9:747-755. 13200-13205.

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Have you encountered CDG?

81. Marquardt T, Hasilik M, Niehues R, Herting M, Muntau A, Holzbach U, et al. 86. De Praeter CM, Gerwig GI, Bause E, Nuytinck LK, Vliegenthart JF. Breuer W. Mannose therapy in carbohydrate-deficient glycoprotein syndrome type 1: first re- Kamerling JP. Espeel MF, Martin Jl, De Paepe AM. Chan NW. Ddcremont GA, sults from a German multicenter study. Amino acids 1997;12:389. Van Coster RN. A novel dlsorder caused by defective biosynthesis of N-linked 82. Marquardt T, Luhn K, Srikrishna G. Freeze HH. Harms E. Vestweber D. Correction oligosaccharides due to glucosidase I deficiency. Atn IHutn Genet 2000;66:1744- of leukocyte adhesion deficiency type I1 with oral fucose. Blood 1999;94:3976-3985, 1756. 83. Marquardt T. Brune T. Luhn K, Zimmer KP, Korner C. Fabritz L, van der Werft N. 87. Sturla L. Etzioni A, Bisso A, Zanardi D. De Flora G. Silengo L. De Flora A, Tonetti M. Vormoor I. Freeze HH. Louwen F, Biermann B, Harms E, von Figura K. Vestweber Defective intracellular actlvlty of GDP-D-mannose-46dehydratase in leukocyte D,Koch HG. Leukocyte adhesion deficiency I1 syndrome, a generalized defect in adhes~ondeficiency type 11 syndrome. FEBS Lett 1998;429:274-278. fucose metabolism. I Pediatr 1999;134:681-688. 88. Krieg I. Hartmann S, Vicent~niA. Glasner W, Hess D, Hofsteenge J. Recognition 84. Van Schaftingen E, laeken 1. Phosphomannomutase deficiency is a cause of carbo- signal for C-mannosylation of Trp-7 In RNase 2 consists of sequence Trp-x-x-Trp. hydrate-deficient glycoprotein syndrome type I. FEBS Lett 1995;377:318-320. Mol Biol Cell 1998;9:301-309. 85. Burda P. Borsig 1 de Rijk-van Andel I, Wevers R, laeken 1, Carchon H. Berger EG, Aehi 89. Doucey MA, Hess D, Cacan R, Hofsteenge J. Protein C-mannosylation is enzyme- M. A novel carbohydrate-deficient glycoprotein syndrome characterized by a deficiency catalysed and uses dolichyl-phosphate-mannose as a precursor. Mol Biol Cell 1998; in glucosylationof the dolichol-linked oligosaccharide.I Clin Illvest 1998;102:647-652. 9:291-300.

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