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Research Notes: Congenital Disorders of Glycosylation (CDGs) and Related Disorders
Am J Med Genet A. 2007 Sep 1. We present two siblings with a previously undescribed congenital disorder of glycosylation (CDG). The first child died in utero with severe hydrops fetalis and the second presented following preterm delivery with respiratory insufficiency, generalised edema and a protein-losing enteropathy. Both had a similar pattern of facial dysmorphism and joint contractures. The diagnosis of CDG-I was made following the birth of the second child based on the serum transferrin isoform pattern. CDG-Ia and -Ib were excluded by specific enzyme analysis. Joint contractures are a relatively uncommon finding in CDG, although fetal hydrops (CDG-Ia) and protein-losing enteropathy (CDG-Ib) are well recognized. CDG must be considered in the differential diagnosis of hydrops fetalis, congenital hypoproteinemia and death in early infancy, particularly when associated with dysmorphic features. J Neurol. 2007 Aug 15. Cerebellar ataxia can have many genetic causes among which are the congenital disorders of glycosylation type I (CDG-I). In this group of disorders, a multisystem phenotype is generally observed including the involvement of many organs, the endocrine, hematologic and central nervous systems. A few cases of CDG-Ia have been reported with a milder presentation, namely cerebellar hypoplasia as an isolated abnormality. To identify patients with a glycosylation disorder, isofocusing of plasma transferrin is routinely performed. Here, we describe two CDG-Ia patients, who presented with mainly ataxia and cerebellar hypoplasia and with a normal or only slightly abnormal transferrin isofocusing result. Surprisingly, the activity of the corresponding enzyme phosphomannomutase was clearly deficient in both leucocytes and fibroblasts. Therefore, in patients presenting with apparently recessive inherited ataxia caused by cerebellar hypoplasia and an unknown genetic aetiology after proper diagnostic work-up, we recommend the measurement of phosphomannomutase activity when transferrin isofocusing is normal or inconclusive. Immunity. 2007 Aug. Autoimmune diseases are prevalent and often life-threatening syndromes, yet the pathogenic triggers and mechanisms involved remain mostly unresolved. Protein asparagine linked- (N-) glycosylation produces glycan structures that substantially differ among the extracellular compartments of evolutionarily divergent organisms. Alpha-mannosidase-II (alphaM-II) deficiency diminishes complex-type N-glycan branching in vertebrates and induces an autoimmune disease in mice similar to human systemic lupus erythematosus. We found that disease pathogenesis provoking glomerulonephritis and kidney failure was nonhematopoietic in origin, independent of complement C3 and the adaptive immune system, mitigated by intravenous administration of immunoglobulin-G, and linked to chronic activation of the innate immune system. N-glycans produced in alphaM-II deficiency bear immune-stimulatory mannose-dependent ligands for innate immune lectin receptors, disrupting the phylogenic basis of this glycomic recognition mechanism. Thus, mammalian N-glycan branching safeguards against the formation of an endogenous immunologic signal of nonself that can provoke a sterile inflammatory response in the pathogenesis of autoimmune disease. J Clin Neurosci. 2007 Jul. We report 3 siblings (1 male and 2 female) recently diagnosed with congenital disorder of glycosylation type Ia (CDG-Ia) in their mid-20s. They experience mild mental retardation but manage to function independently in society. Their professions are library assistant, professional artistic painter and secretarial work. All three siblings have cerebellar hypoplasia and ataxia, but are able to ambulate easily. Two of the siblings have required strabismus surgical repairs. All have antithrombin III deficiency, osteoporosis, and mild dysmorphic features. Hypergonadotrophic hypogonadism was a feature of the two female siblings. A type 1 sialotransferrin pattern and phosphomannomutase (PMM) deficiency have been demonstrated. They are compound heterozygotes for R141H and L32R mutations in the PMM2 gene. While there is clinical heterogeneity in CDG-Ia, we believe that our patients are among the mildest of intellectually affected CDG-Ia patients reported to date. Am J Med Genet A. 2007 Jun 15. In this report, we describe a brother and sister who presented at birth with short-limb skeletal dysplasia, polyhydramnios, prematurity, and generalized edema. Dysmorphic features included broad nose, thick ears, thin lips, micrognathia, inverted nipples, ulnar deviation at the wrists, spatulate fingers, fifth finger camptodactyly, nail hypoplasia, and talipes equinovarus. Other features included short stature, microcephaly, psychomotor retardation, B-cell lymphopenic hypogammaglobulinemia, sensorineural deafness, retinal detachment and blindness, intestinal malrotation with poor gastrointestinal motility, persistent hyponatremia, intermittent hypoglycemia, and thrombocytopenia. Cardiac anomalies included PDA, VSD, hypertrophic cardiomyopathy, and arrhythmias. The brother had a small penis with hypospadias, hypoplastic scrotum, and non-palpable testes. Skeletal findings included absent ossification of cervical vertebral bodies, pubic bones, knee epiphyses, and tali. Both sibs died before age 2 years, one of overwhelming sepsis and the other of cardiorespiratory failure associated with her cardiomyopathy. Metabolic studies showed a type 1 pattern of abnormal serum transferrin glycosylation. Fibroblasts synthesized truncated LLOs, primarily Man(7)GlcNAc(2), suggestive of CDG-Ig. Both sibs were compound heterozygotes for a novel 301 G > A (G101R) mutation and a previously described 437 G > A (R146Q) mutation in ALG12. Congenital disorders of glycosylation should be considered for children with undiagnosed multi-system disease including neurodevelopmental delay, skeletal dysplasia, immune deficiency, male genital hypoplasia, and cardiomyopathy. Am J Med Genet A. 2007 Jun 5. We present two sibs with congenital disorder of glycosylation (CDG) type Id. Each shows severe global delay, failure to thrive, seizures, microcephaly, axial hypotonia, and disaccharidase deficiency. One sib has more severe digestive issues, while the other is more neurologically impaired. Each is compound heterozygous for a novel point mutation and an already known mutation in the ALG3 gene that leads to the synthesis of a severely truncated oligosaccharide precursor for N-glycans. The defect is corrected by introduction of a normal ALG3 cDNA. CDG should be ruled out in all patients with severe seizures and failure to thrive. Curr Mol Med. 2007 Jun. The Congenital Disorders of Glycosylation (CDG) are a collection of over 20 inherited diseases that impair protein N-glycosylation. The clinical appearance of CDG patients is quite diverse making it difficult for physicians to recognize them. A simple blood test of transferrin glycosylation status signals a glycosylation abnormality, but not the specific defect. An abnormal trasferrin glycosylation pattern suggests that the defect is in either genes that synthesize and add the precursor glycan (Glc(3)Man(9)GlcNAc(2)) to proteins (Type I) or genes that process the protein-bound N-glycans (Type II). Type I defects create unoccupied glycosylation sites, while Type II defects give fully occupied sites with abnormally processed glycans. These types are expected to be mutually exclusive, but a group of patients is now emerging who have variable coagulopathy and hypoglycemia together with a combination of Type I and Type II transferrin features. This surprising finding makes identifying their defects more challenging, but the defects and associated clinical manifestations of these patients suggest that the N-glycosylation pathway has some secrets left to share. Eur J Hum Genet. 2007 Jun. We describe the clinical and biochemical characteristics in three patients from two different families diagnosed with Congenital Disorder of Glycosylation type IIe owing to a defect in Conserved Oligomeric Golgi complex (COG)7; one of the eight subunits of the COG. The siblings and an unrelated single child of consanguineous parents presented with growth retardation, progressive, severe microcephaly, hypotonia, adducted thumbs, feeding problems by gastrointestinal pseudo-obstruction, failure to thrive, cardiac anomalies, wrinkled skin and episodes of extreme hyperthermia. A combined disorder in the biosynthesis of N- and O-linked glycosylation with hyposialylation was detected. Western blot analysis showed a severe reduction in the COG5 and 7 subunits of the COG. A homozygous, intronic splice site mutation (c.169+4A>C) of the COG7 gene was identified in all patients. The phenotype is similar to that previously described in two patients of North African ethnicity with the same mutation, except for the lack of skeletal anomalies and only a mild liver involvement in our patients. We suggest performing protein glycosylation studies and Western blot for the different COG subunits in patients with progressive microcephaly, growth retardation, hypotonia, adducted thumbs and cardiac defects, especially in association with skin anomalies or episodes of hyperthermia. The presence of the characteristic phenotype might warrant direct DNA analysis. Mol Genet Metab. 2007 Jun. Mutations in the N-linked glycosylation pathway cause rare autosomal recessive defects known as Congenital Disorders of Glycosylation (CDG). A previously reported mutation in the Conserved Oligomeric Golgi complex gene, COG7, defined a new subtype of CDG in a Tunisian family. The mutation disrupted the hetero-octomeric COG complex and altered both N- and O-linked glycosylation. Here we present clinical and biochemical data from a second family with the same mutation. J Inherit Metab Dis. 2007 Jun. Hereditary fructose intolerance (HFI) is caused by a deficiency of aldolase B due to mutations of the ALDOB gene. The disease poses diagnostic problems because of unspecific clinical manifestations. We report three cases of HFI all of whom had a chronic disease with neurological, nephrological or gastroenterological symptoms, whereas nutritional fructose intolerance, the pathognomonic sign of HFI, was apparent only in retrospect. In all patients a hypoglycosylated pattern of transferrin isoforms was found but was misinterpreted as a sign of CDG Ix. The correct diagnosis was achieved with marked delay (26, 36 and 24 months, respectively) by sequencing of the ALDOB gene two common mutations were identified on both alleles or on one (A150P/A175D, A150P/-, and A150P/A175D). The diagnosis was further supported by normalization of transferrin isoforms on a fructose-free diet. Data available in two patients showed that following the fructose restriction the type I pattern of carbohydrate-deficient transferrin detectable on fructose-containing diet disappeared after 3-4 weeks. These cases illustrate that in the first years of life HFI may show misleading variability in clinical presentation and that protein glycosylation analysis such as transferrin isofocusing may give important diagnostic clues. However, care should be taken not to misinterpret the abnormal results as CDG Ix as well as to remember that a normal profile does not exclude HFI due to the possibility of spontaneous fructose restriction in the diet. The presented data also emphasize the usefulness of ALDOB mutation screening for diagnosis of HFI. Hum Mol Genet. 2007 Apr 1. The hetero-octameric conserved oligomeric Golgi (COG) complex is essential for the structure/function of the Golgi apparatus through regulation of membrane trafficking. Here, we describe a patient with a mild form of a congenital disorder of glycosylation type II (CDG-II), which is caused by a homozygous nonsense mutation in the hCOG8 gene. This leads to a premature stop codon resulting in a truncated Cog8 subunit lacking the 76 C-terminal amino acids. Mass spectrometric analysis of the N- and O-glycan structures identified a mild sialylation deficiency. We showed that the molecular basis of this defect in N- and O-glycosylation is caused by the disruption of the Cog1-Cog8 interaction due to truncation. As a result, Cog1 deficiency accompanies the Cog8 deficiency, preventing assembly of the intact, stable complex and resulting in the appearance of smaller subcomplexes. Moreover, levels of beta1,4-galactosytransferase were significantly reduced. The defects in O-glycosylation could be fully restored by transfecting the patient's fibroblasts with full-length Cog8. The Cog8 defect described here represents a novel type of CDG-II, which we propose to name as CDG-IIh or CDG caused by Cog8 deficiency (CDG-II/Cog8). Hum Mol Genet. 2007 Apr 1. We describe a new Type II congenital disorder of glycosylation (CDG-II) caused by mutations in the conserved oligomeric Golgi (COG) complex gene, COG8. The patient has severe psychomotor retardation, seizures, failure to thrive and intolerance to wheat and dairy products. Analysis of serum transferrin and total serum N-glycans showed normal addition of one sialic acid, but severe deficiency in subsequent sialylation of mostly normal N-glycans. Patient fibroblasts were deficient in sialylation of both N- and O-glycans, and also showed slower brefeldin A (BFA)-induced disruption of the Golgi matrix, reminiscent of COG7-deficient cells. Patient fibroblasts completely lacked COG8 protein and had reduced levels and/or mislocalization of several other COG proteins. The patient had two COG8 mutations which severely truncated the protein and destabilized the COG complex. The first, IVS3 + 1G > A, altered the conserved splicing site of intron 3, and the second deleted two nucleotides (1687-1688 del TT) in exon 5, truncating the last 47 amino acids. Lentiviral-mediated complementation with normal COG8 corrected mislocalization of other COG proteins, normalized sialylation and restored normal BFA-induced Golgi disruption. We propose to call this new disorder CDG-IIh or CDG-II/COG8. J Med Genet. 2007 Apr. There is a growing awareness that inborn errors of metabolism can be a cause of non-immune hydrops fetalis. The association between congenital disorders of glycosylation (CDG) and hydrops fetalis has been based on one case report concerning two sibs with hydrops fetalis and CDG-Ik. Since then two patients with hydrops-like features and CDG-Ia have been reported. Two more unrelated patients with CDG-Ia who presented with hydrops fetalis are reported here, providing definite evidence that non-immune hydrops fetalis can be caused by CDG-Ia. The presence of congenital thrombocytopenia and high ferritin levels in both patients was remarkable. These might be common features in this severe form of CDG. Both patients had one severe mutation in the phosphomannomutase 2 gene, probably fully inactivating the enzyme, and one milder mutation with residual activity, as had the patients reported in literature. The presence of one severe mutation might be required for the development of hydrops fetalis. CDG-Ia should be considered in the differential diagnosis of hydrops fetalis and analysis of PMM activity in chorionic villi or amniocytes should also be considered. J Inherit Metab Dis. 2007 Feb. CDG Ia (phosphomannomutase deficiency) has a wide clinical spectrum with the most severe affected patients having multisystemic disease in addition to severe nervous system involvement. We report a patient with CDG Ia and an intermediate phenotype due to mild neurological impairment and borderline cognitive abilities despite the occurrence of typical extraneurological symptoms. These included liver involvement, coagulopathy and failure to thrive with enteropathy. Genotype analyses showed that he was compound heterozygous for T237R/C241S mutations. This observation underlines that the CDG Ia clinical spectrum may include intraindividual variability that might reflect different degrees of glycosylation abnormalities among distinct body compartments. CDG Ia should be considered in cases of unexplained liver involvement and/or enteropathy in patients with mild developmental delay and subtle neurological signs. Clin Chem. 2007 Feb. Background: Apolipoprotein C-III (apoC-III) isoelectric focusing (IEF) can be used to detect abnormalities in the biosynthesis of core 1 mucin-type O-glycans. Methods: We studied plasma samples from 55 patients with various primary defects in N- and/or O-glycosylation, 21 patients with secondary N-glycosylation defects, and 6 patients with possible glycosylation abnormalities. Furthermore, we analyzed 500 plasma samples that were sent to our laboratory for selective screening for inborn errors of metabolism. Results: Plasma samples from patients with congenital disorders of glycosylation (CDG) types -IIe and -IIf showed a hypoglycosylated apoC-III isoform profile, as did plasma samples from 75% of the patients with an unspecified CDG type II. Hyposialylated O-glycan profiles were also seen in plasma from 2 patients with hemolytic-uremic syndrome. In the 500 plasma samples from the selective screening, 3 patients were identified with a possible isolated defect in the biosynthesis of core 1 mucin-type O-glycans. Conclusions: To our knowledge this is the first study in which use of a plasma marker protein has identified patients in whom only O-glycan biosynthesis might be affected. The primary defect(s) remain as yet unknown. Plasma apoC-III IEF is complementary to transferrin isofocusing. In conjunction both tests identify biosynthesis defects in N-glycan and mucin-type core 1 O-glycan biosynthesis. The apoC-III IEF assay is likely to help metabolic laboratories to identify and unravel further subtypes of inborn errors of glycan biosynthesis. Bioorg Med Chem Lett. 2007 Jan 1. An efficient and convergent method for the synthesis of mannose-1-phosphate prodrugs is described as a potential therapy for congenital disorders of glycosylation-Ia (CDG-Ia). The key feature of the proposed approach is the silver assisted nucleophilic substitution of 2,3,4,6-tetra-O-protected-alpha-d-mannopyranosyl bromides with various silver phosphate salts to afford mono, di, and tri-mannopyranosyl phosphates. A preliminary biological evaluation of the synthesized phosphate prodrugs has been carried out. Biochem Biophys Res Commun. 2006 Dec 1. The recent identification of mutations in genes encoding demonstrated or putative glycosyltransferases has revealed a novel mechanism for congenital muscular dystrophy. Hypoglycosylated alpha-dystroglycan (alpha-DG) is commonly seen in Fukuyama-type congenital muscular dystrophy (FCMD), muscle-eye-brain disease (MEB), Walker-Warburg syndrome (WWS), and Large(myd) mice. POMGnT1 and POMTs, the gene products responsible for MEB and WWS, respectively, synthesize unique O-mannose sugar chains on alpha-DG. The function of fukutin, the gene product responsible for FCMD, remains undetermined. Here we show that fukutin co-localizes with POMGnT1 in the Golgi apparatus. Direct interaction between fukutin and POMGnT1 was confirmed by co-immunoprecipitation and two-hybrid analyses. The transmembrane region of fukutin mediates its localization to the Golgi and participates in the interaction with POMGnT1. Y371C, a missense mutation found in FCMD, retains fukutin in the ER and also redirects POMGnT1 to the ER. Finally, we demonstrate reduced POMGnT1 enzymatic activity in transgenic knock-in mice carrying the retrotransposal insertion in the fukutin gene, the prevalent mutation in FCMD. From these findings, we propose that fukutin forms a complex with POMGnT1 and may modulate its enzymatic activity. Pediatr Res. 2006 Dec. The congenital disorders of N-glycosylation (CDG), a steadily increasing group of multi-systemic disorders, have severe clinical implications in infancy and early childhood. The various inborn errors responsible adversely affect N-glycosylation of lysosomal proteins because of either failing assembly of lipid-linked (LL) oligosaccharides (OS) in the endoplasmic reticulum, CDG Type I, or faulty processing of the asparagines (N)-linked OS in the ER and in the Golgi, CDG Type II. The overlap of phenotypes precludes specific clinical delineation. Isoelectric focusing (IEF) of plasma transferrin remains a valuable, albeit imperfect, screening tool. IEF of plasma ApoC-III protein, introduced O-glycosylation defects that delineated some new CDGs due to mutations of both N- and O-glycosylation. Only CDG-Ib is amenable to treatment with free mannose supplementation. Hence, early specific diagnosis of any one entity is crucial for genetic counseling and elective preventive measures. J Chromatogr A. 2006 Oct 20. Capillary zone electrophoresis (CZE) with a dynamic double coating based on the new CEofix reagents is shown to provide high-resolution separations of serum transferrin (Tf) isoforms, a prerequisite for the monitoring of unusual and complex Tf patterns, including those seen with genetic variants and disorders of glycosylation. A 50 microm I.D. fused-silica capillary of 60 cm total length, an applied voltage of 20 kV and a capillary temperature of 30 degrees C results in 15 min CZE runs of high assay precision and thus provides a robust approach for the determination of carbohydrate-deficient transferrin (CDT, sum of asialo-Tf and disialo-Tf in relation to total Tf) in human serum. Except for selected samples of patients with severe liver diseases and sera with high levels of paraproteins, interference-free Tf patterns are detected. Compared with the use of the previous CEofix reagents for CDT under the same instrumental conditions, the resolution between disialo-Tf and trisialo-Tf is significantly higher (1.7 versus 1.4). The CDT levels of reference and patient sera are comparable, suggesting that the new assay can be applied for screening and confirmation analyses. The high-resolution CZE assay represents an attractive alternative to HPLC and can be regarded as a candidate of a reference method for CDT. J Inherit Metab Dis. 2006 Aug. Congenital disorder of glycosylation Ia is the most common defect of glycosylation and is due to mutations in phosphomannomutase 2. This leads to aberrant N-linked oligosaccharides. The phenotype of CDG Ia reflects the essential nature of glycosylation and patients typically present with multiple organs affected, with hypotonia, developmental delay, inverted nipples and abnormal fat pads. Later features include retinitis pigmentosa, stroke, cerebellar atrophy and malabsorption. Approximately 20% of patients die in the first year of life and infection is the most common cause of death. Immunological function has not previously been investigated in these patients and the critical role of oligosaccharides on adhesion molecules suggested that haematopoietic cell migration and communication could be disrupted by mutations in phosphomannomutase 2. We characterized the clinical features, performed standard immunological evaluations, and performed specific analyses of neutrophil adhesion molecules on two patients to address this question. Patient neutrophils had diminished chemotaxis but expressed comparable levels of adhesion molecules and rolled on artificial endothelium equivalently to control neutrophils. The most significant feature of the patients' immunological function was poor vaccine responses. These two affected patients were begun on intravenous immunoglobulin with some improvement in their infections. Mov Disord. 2006 Jun. Congenital disorders of glycosylation (CDG) are a recently described, underrecognized group of syndromes characterized biochemically by abnormal glycosylation of serum and cellular glycoproteins. We report a previously undiagnosed adult male who presented with early-onset cerebellar ataxia in the context of mental impairment, peripheral neuropathy, retinopathy, body dysmorphism, cardiomyopathy, and hypogonadism. Newly available screening and genetic testing confirmed the diagnosis as CDG type Ia. This case emphasizes that CDG should be considered as a differential diagnosis for adults with early-onset cerebellar ataxia, particularly in those persons with the aforementioned features, and that undiagnosed cases of childhood ataxia may require reassessment now that testing is available. NeuroRx. 2006 Apr. The congenital disorders of glycosylation (CDG) are a rapidly expanding group of metabolic syndromes with a wide symptomatology and severity. They all stem from deficient N-glycosylation of proteins. To date the group contains 18 different subtypes: 12 of Type I (disrupted synthesis of the lipid-linked oligosaccharide precursor) and 6 of Type II (malfunctioning trimming/processing of the protein-bound oligosaccharide). Main features of CDG involve psychomotor retardation; ataxia; seizures; retinopathy; liver fibrosis; coagulopathies; failure to thrive; dysmorphic features, including inverted nipples and subcutaneous fat pads; and strabismus. No treatment currently is available for the vast majority of these syndromes (CDG-Ib and CDG-IIc are exceptions), even though attempts to synthesize drugs for the most common subtype, CDG-Ia, have been made. In this review we will discuss the individual syndromes, with focus on their neuronal involvement, available and possible treatments, and future directions. Proteomics. 2006 Apr. A method for the diagnosis of the congenital disorders of glycosylation type I (CDG-I) by SELDI-TOF-MS of serum transferrin immunocaptured on protein chip arrays is described. The underglycosylation of glycoproteins in CDG-I produces glycoforms of transferrin with masses lower than that of the normal fully glycosylated transferrin. Immobilisation of antitransferrin antibodies on reactive-surface protein chip arrays (RS100) selectively enriched transferrin by at least 100-fold and allowed the detection of patterns of transferrin glycoforms by SELDI-TOF-MS using approximately 0.3 microL of serum/plasma. Abnormal patterns of immunocaptured transferrin were detected in patients with known defects in glycosylation (CDG-Ia, CDG-Ib, CDG-Ic, CDG-If and CDG-Ih) and in patients in whom the basic defect has not yet been identified (CDG-Ix). The correction of the N-glycosylation defect in a patient with CDG-Ib after mannose therapy was readily detected. A patient who had an abnormal transferrin profile by IEF but a normal profile by SELDI-TOF-MS analysis was shown to have an amino acid polymorphism by sequencing transferrin by quadrupole-TOF MS. Complete agreement was obtained between analysis of immunocaptured transferrin by SELDI-TOF-MS and the IEF profile of transferrin, the clinical severity of the disease and the levels of aspartylglucosaminidase activity (a surrogate marker for the diagnosis of CDG-I). SELDI-TOF-MS of transferrin immunocaptured on protein chip arrays is a highly sensitive diagnostic method for CDG-I, which could be fully automated using microtitre plates and robotics. Proc Natl Acad Sci U S A. 2006 Mar 7. The conserved oligomeric Golgi (COG) complex is a heterooctameric complex that regulates intraGolgi trafficking and the integrity of the Golgi compartment in eukaryotic cells. Here, we describe a patient with a mild form of congenital disorder of glycosylation type II (CDG-II) that is caused by a deficiency in the Cog1 subunit of the complex. This patient has a defect in both N- and O-glycosylation. Mass spectrometric analysis of the structures of the N-linked glycans released from glycoproteins from the patient's serum revealed a reduction in sialic acid and galactose residues. Peanut agglutinin (PNA) lectin staining revealed a decrease in sialic acids on core 1 mucin type O-glycans, indicating a combined defect in N- and O-glycosylation. Sequence analysis of the COG1 cDNA and gene identified a homozygous insertion of a single nucleotide (2659-2660insC), which is predicted to lead to a premature translation stop and truncation of the C terminus of the Cog1 protein by 80 amino acids. This mutation destabilizes several other COG subunits and alters their subcellular localization and hence the overall integrity of the COG complex. This results in reduced levels and/or altered Golgi localization of alpha-mannosidase II and beta-1,4 galactosyltransferase I, which links it to the glycosylation deficiency. Transfection of primary fibroblasts of this patient with the full length hemagglutinin-tagged Cog1 indeed restored beta-1,4 galactosyltransferase Golgi localization. We propose naming this disorder CDG-II/Cog1, or CDG-II caused by Cog1 deficiency. Eur J Ophthalmol. 2006 Jan-Feb. PURPOSE: Several types of inborn errors of the O-glycan biosynthesis are known, leading to clinically very distinct phenotypes. Children with O-mannosyl glycan biosynthesis defects commonly present as a severe form of congenital muscular dystrophy with decreased alpha-dystroglycan staining, congenital eye anomalies, and brain migration defects. Alpha-dystroglycan is an O-mannosylated glycoprotein with additional mucin type O-glycans. METHODS: Based on overlapping clinical features with O-mannosyl glycan defects, especially with muscle-eye-brain disease, the authors performed a muscle biopsy in a child with severe congenital hypotonia, high myopia, partial pachygyria, mental retardation, cutis laxa, and an inborn error affecting the biosynthesis of both mucin type O-glycans and N-linked glycans. RESULTS: The histology showed no signs of muscle dystrophy, but a mild myopathy with slight increase in the muscle fiber diameter variability and type I fiber predominance. No significant decrease in the alpha-dystroglycan staining was detected; therefore, in spite of the phenotypic similarities the authors could not confirm the role of abnormal dystroglycan in the etiology of the muscle weakness and the developmental anomalies. CONCLUSIONS: High myopia, muscle weakness, and cortical neuronal migration abnormalities are common in disorders of O-mannosylation and also observed in the authors' patient. However, compared to the severe generalized defect observed in mannosyl glycan defects, in this child the cerebral white matter and cerebellum were spared, and no muscle dystrophy could be confirmed. This is the first description of high myopia in cutis laxa syndrome in combination with congenital disorders of glycosylation. Glycobiology. 2005 Dec. Defects in the biosynthesis of N- and core 1 O-glycans may be found by isoelectric focusing (IEF) of plasma transferrin and apolipoprotein C-III (apoC-III). We hypothesized that IEF of transferrin and apoC-III in combination with sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of apoC-III may provide a classification for congenital disorders of glycosylation (CDG) patients. We analyzed plasma from 22 patients with eight different and well-characterized CDG subtypes and 19 cases with unsolved CDG. Transferrin IEF (TIEF) has been used to distinguish between N-glycan assembly (type 1 profile) and processing (type 2 profile) defects. We differentiated two different CDG type 2 TIEF profiles: The "asialo profile" characterized by elevated levels of asialo- and monosialotransferrin and the "disialo profile" characterized by increased levels of disialo- and trisialotransferrin. ApoC-III IEF gave two abnormal profiles ("apoC-III(0)" and "apoC-III(1)" profiles). The results for the eight established CDG forms exactly matched the theoretical expectations, providing a validation for the study approach. The combination of the three electrophoretic techniques was not additionally informative for the CDG-Ix patients as they had normal apoC-III IEF patterns. However, the CDG-IIx patients could be further subdivided into six biochemical subgroups. The robustness of the methodology was supported by the fact that three patients with similar clinical features ended in the same subgroup and that another patient, classified in the "CDG-IIe subgroup," turned out to have a similar defect. Dividing the CDG-IIx patients in six subgroups narrows down drastically the options of the primary defect in each of the subgroups and will be helpful to define new CDG type II defects. Glycobiology. 2005 Nov. Patients with Type I congenital disorders of glycosylation (CDG-I) make incomplete lipid-linked oligosaccharides (LLO). These glycans are poorly transferred to proteins resulting in unoccupied glycosylation sequons. Mutations in phosphomannomutase (PMM2) cause CDG-Ia by reducing the activity of PMM, which converts mannose (Man)-6-P to Man-1-P before formation of GDP-Man. These patients have reduced Man-1-P and GDP-Man. To replenish intracellular Man-1-P pools in CDG-Ia cells, we synthesized two hydrophobic, membrane permeable acylated versions of Man-1-P and determined their ability to normalize LLO size and N-glycosylation in CDG-Ia fibroblasts. Both compounds, compound I (diacetoxymethyl 2,3,4,6-tetra-O-acetyl-alpha-D-mannopyranosyl phosphate) (C-I) and compound II (diacetoxymethyl 2,3,4,6-tetra-O-ethyloxycarbonyl-alpha-D-mannopyranosyl phosphate) (C-II), contain two acetoxymethyl (CH2OAc) groups O-linked to phosphorous. C-I contains acetyl esters and C-II contains ethylcarbonate (CO2Et) esters on the Man residue. Both C-I and C-II normalized truncated LLO, but C-II was about 2-fold more efficient than C-I. C-II replenished the GDP-Man pool in CDG-Ia cells and was more efficiently incorporated into glycoproteins than exogenous Man at low concentrations (25-75 mM). In a glycosylation assay of DNaseI in CDG-Ia cells, C-II restored glycosylation to control cell levels. C-II also corrected impaired LLO biosynthesis in cells from a Dolichol (Dol)-P-Man deficient patient (CDG-Ie) and partially corrected LLO in cells from an ALG12 mannosyltransferase-deficient patient (CDG-Ig), whereas cells from an ALG3-deficient patient (CDG-Id) and from an MPDU1-deficient patient (CDG-If) were not corrected. These results validate the general concept of using pro-Man-1-P substrates as potential therapeutics for CDG-I patients. Rinsho Shinkeigaku. 2005 Nov. Fukuyama congenital muscular dystrophy (FCMD), Walker-Warburg syndrome (WWS), and muscle-eye-brain (MEB) disease are similar disorders characterized by congenital muscular dystrophy, brain and eye anomalies. We previously identified the genes for FCMD and MEB, which encode fukutin and POMGnT1. Recent studies have revealed that posttranslational modification of alpha-dystroglycan is associated with congenital muscular dystrophy with brain malformations. Since hypoglycosylation of alpha-dystroglycan is common amongst several other disorders, a new clinical entity called alpha-dystroglycanopathy is proposed. However, only POMGnT1 (MEB) and POMT1 (WWS) are shown to have a definite enzymatic activity, and no enzymatic activity has been detected in fukutin. We show positive interactions between fukutin and POMGnT1. Fukutin may form a protein complex with POMGnT1 and modulate POMGnT1's enzymatic activity. Through cDNA microarray, we also show aberrant neuromuscular junction formation and delayed muscle fiber maturation in alpha-dystroglycanopathies, suggesting a new pathomechanism. J Biol Chem. 2005 Sep 23. The conserved oligomeric Golgi (COG) complex is an eight-subunit (Cog1-8) peripheral Golgi protein involved in Golgi-associated membrane trafficking and glycoconjugate synthesis. We have analyzed the structure and function of COG using Cog1 or Cog2 null Chinese hamster ovary cell mutants, fibroblasts from a patient with Cog7-deficient congenital disorders of glycosylation, and stable Cog5-deficient HeLa cells generated by RNA interference. Although the dilation of some Golgi cisternae in Cog5-deficient cells resembled that observed in Cog1- or Cog2-deficient cells, their global glycosylation defects (less severe) and intracellular processing and function of low density lipoprotein receptors (essentially normal) differed from Cog1- and Cog2-deficient cells. Immunoblotting, gel filtration, and immunofluorescence microscopy analyses of the COG-deficient cells and cell extracts indicated that 1) Cog2-4 and Cog5-7 form stable subcomplexes, 2) Cog1 mediates Golgi association of a Cog2-4 plus Cog8 subcomplex, 3) Cog8 associates stably with both Cog5-7 and Cog1-4 subcomplexes, and thus 4) Cog8 helps assemble the Cog1-4 and Cog5-7 subcomplexes into the complete COG complex. This model of the subunit organization of COG is in excellent agreement with in vitro data presented in an accompanying paper (Ungar, D., Oka, T., Vasile, E., Krieger, M., and Hughson, F. M. (2005) J. Biol. Chem. 280, 32729-32735). Only one or two of the seven Cog1- or Cog2-dependent Golgi membrane proteins called GEARs are also sensitive to Cog5 or Cog7 deficiency, indicating that the COG subunits play distinctive roles in controlling Golgi structure and function. Biochim Biophys Acta. 2005 Jun 30. Based on our preliminary observation of abnormal glycosylation in a cutis laxa patient, nine cutis laxa patients were analyzed for congenital defects of glycosylation (CDG). Isoelectric focusing of plasma transferrin and apolipoproteinC-III showed that three out of nine patients had a defect in the biosynthesis of N-glycans and core 1 mucin type O-glycans, respectively. Mass spectrometric N-glycan analyses revealed a relative increase of glycans lacking sialic acid and glycans lacking sialic acid and galactose residues. Mutation analysis of the fibulin-5 gene (FBLN5), which has been reported in cases of autosomal recessive cutis laxa, revealed no mutations in the patients' DNA. Evidence is presented that extracellular matrix (ECM) proteins of skin are likely to be highly glycosylated with N- and/or mucin type O-glycans by using algorithms for predicting glycosylation. The conclusions in this study were that the clinical phenotype of autosomal recessive cutis laxa seen in three patients is not caused by mutations in the FBLN5 gene. Our findings define a novel form of CDG with cutis laxa and neurological involvement due to a defect in the sialylation and/or galactosylation of N- and O-glycans. Improper glycosylation of ECM proteins of skin may form the pathophysiological basis for the cutis laxa phenotype. Acta Neuropathol (Berl). 2005 Apr. Congenital disorders of glycosylation (CDG) represent a newly delineated group of inherited multisystem disorders characterized by defective glycoprotein biosynthesis. In the present study we report and discuss the clinical and neuropathological findings in a newborn with CDG type Ia (CDG-Ia). The patient presented mild dysmorphic facial features, inverted nipples, progressive generalized edema, hypertrophic cardiomyopathy, hepatosplenomegaly, muscular hypotonia and had severe hypoalbuminemia. Deficiency of phosphomannomutase (PMM)-2 activity was detected. Molecular analysis showed V231M/T237R mutations of the PMM2 gene. Muscular biopsy, disclosed myopathic alterations with myofibrillar disarray by electron microscopy. The patient died at 1 month of age of circulatory and respiratory failure. Autopsy showed liver fibrosis and renal abnormalities. Neuropathological abnormalities were mainly confined to the cerebellum. Histological and immunocytochemical examination of cerebellar tissue showed partial atrophy of cerebellar folia with severe loss of Purkinje cells, granular cell depletion and various morphological changes in the remaining Purkinje cells and their dendritic arborization. Autopsy findings confirm the complexity of the CDG-Ia syndrome, and indicate that CDG-Ia is a distinct disease entity, which can be differentiated from other neurological disorders and other types of CDG, not only clinically, but also based on unique pathological findings. The data proved useful in determining the underlying disease process associated with a defective N-glycosylation pathway. Eur J Hum Genet. 2005 Apr. Congenital cutis laxa is a genetically heterogeneous condition presenting with loose and redundant skin folds, decreased elasticity of the skin, connective tissue involvement and a highly variable spectrum of associated features. The most common forms are inherited in an autosomal recessive or dominant fashion. Fibulin 5 and elastin mutations were detected in a limited number of patients, but in most cases the etiology is not known. Based on a previous observation of an abnormal transferrin isoelectric focusing pattern in a patient with cutis laxa indicating an N-glycosylation defect, we performed a screening for disorders of protein glycosylation in unrelated children with cutis laxa syndrome, including a recently developed test for defective O-glycosylation. Here, we describe five patients from consanguineous marriages with a cutis laxa syndrome with skeletal and joint involvement, developmental delay and neurological findings. Three of these five children have an inborn error of glycan biosynthesis affecting the synthesis of both N- and O-linked glycans. Two patients had normal glycosylation patterns. All known causes of secondary glycosylation disorders were excluded in the children. No mutations were found in the FBLN5 gene. In conclusion, we have identified a new combined glycosylation defect with a distinct clinical phenotype. Our results suggest that a combined defect of glycosylation might be a causative factor in congenital cutis laxa. This is the first report where abnormal N- and O-linked glycosylation is implicated in the etiology of cutis laxa syndrome. No To Hattatsu. 2005 Mar. Congenital muscular dystrophy (CMD) is a group of heterogeneous disorders characterized clinically by delayed milestones due to generalized muscle weakness and dystrophic muscle pathology. The discovery of fukutin, responsible gene for Fukuyama CMD (FCMD) and defective glycosylation in its muscle biopsy has lead significant advances in CMD researches, especially disorders with glycosylation defects to a dystroglycan (alphaDG). The highly glycosylated a DG is one of the major dystrophin-associated proteins anchored a basement membrane protein, laminin 2 to the dystrophin molecule. The disorders with the defective glycosylation are now categorized as a dystroglycanopathies which include FCMD, muscle-eye-brain (MEB) disease, Walker-Warburg syndrome (WWS) and diseases with mutations in fukutin-related protein (FKRP) and LARGE genes. Among them, MEB and WWS were proven to have mutations in the glycosyltransferase genes, POMGnT1 (protein O-mannose beta 1,2-N-acetylglucosaminyl/transferase 1) and POMT1 (protein O-mannosyltransferase 1), respectively, though others are still unknown how the glycosylation defect is induced. Although the disease with FKRP mutation has variable phenotypes from CMD to limb-girdle muscular dystrophy, others with defective to decreased a DG show CMD, central nervous system involvement with migration disorder (polymicrogyria) and ocular abnormalities. J Inherit Metab Dis. 2005. Congenital disorders of glycosylation (CDG) represent a group of inherited multiorgan diseases caused by defects in the biosynthesis of glycoproteins. We report on two dysmorphic siblings with severe liver disease who died at the age of a few weeks. Increased activities of lysosomal enzymes in plasma were found, though total sialic acid in plasma was strongly decreased. Isoelectric focusing of serum sialotransferrins showed a type 2-like CDG pattern. Some of the known CDG subtypes were excluded. O-Glycosylation was investigated by isoelectric focusing of apolipoprotein C-III, which showed increased fractions of hyposialylated isoforms. In a consecutive study a defect in the conserved oligomeric Golgi complex was established at the level of subunit COG-7, leading to disruption of multiple glycosylation functions of the Golgi. This report on patients with a new variant of CDG, due to a multiple Golgi defect, emphasizes in addition to sialotransferrins the importance of analysis of a serum O-linked glycoprotein, e.g. apolipoprotein C-III, in unclassified CDG-X cases. Med Sci Monit. 2004 Aug. Background: The features of Joubert syndrome include hypotonia, ataxia, characteristic neuro-imaging findings, episodic hypoventilation, psychomotor retardation, and abnormal eye movements. Common symptoms in congenital disorders of glycosylation (CDG) type Ia are muscle hypotonia, cerebellar hypoplasia, ataxia, mental retardation, ophthalmologic involvement, failure to thrive, abnormal fat distribution, and hepatopathy. It has been postulated that some Joubert syndrome patients might have an underlying disorder of protein glycosylation. Material/methods: Screening for disorders of glycosylation was performed in five children diagnosed with Joubert syndrome. Data were retrospectively collected from clinical charts, the patients were reexamined by clinical geneticists, and available neuro-imaging data were also reanalyzed. Diagnoses were established based on results of serum transferrin isoelectric focusing, phosphomannomutase enzyme activity measurements, and DNA mutation analysis. Results: We confirmed the diagnoses of CDG type Ia in two of the five children originally diagnosed with Joubert syndrome. The symptoms of the two syndromes were clearly distinguishable. Conclusions: Syndromic patients with congenital vermis malformations should be screened for congenital disorders of glycosylation. Glycobiology. 2004 Jun. The structural variations among extracellular N-glycans reflect the activity of glycosyltransferases and glycosidases that operate in the Golgi apparatus. More than other types of vertebrate glycans, N-glycans are highly branched oligosaccharides with multiple antennae linked to an underlying mannose core structure. The branching patterns of N-glycans consist of three types, termed high-mannose, hybrid, and complex. Though most extracellular mammalian N-glycans are of the complex type, some cells variably express hybrid and high-mannose forms. Nevertheless, a requirement for hybrid and complex N-glycan branching exists in embryonic development and postnatal function among mice and humans inheriting defective Mgat1 or Mgat2 alleles. The resulting defects in formation N-glycan branching patterns cause multiple abnormalities, including neurologic defects, and have inferred the presence of distinct functions for hybrid and complex N-glycan branches among different cell lineages. We have further explored N-glycan structure-function relationships in vivo by using Cre-loxP conditional mutagenesis to abolish hybrid and complex N-glycan branching specifically among neuronal cells. Our findings show that hybrid N-glycan branching is an essential posttranslational modification among neurons. Loss of Mgat1 resulted in a unique pattern of neuronal glycoprotein deficiency concurrent with caspase 3 activation and apoptosis. Such animals exhibited severe locomotor deficits, tremors, paralysis, and early postnatal death. Unexpectedly, neuronal Mgat2 deletion resulting in the loss of complex but not hybrid N-glycan branching was well tolerated without phenotypic markers of neuronal or locomotor dysfunction. Structural features associated with hybrid N-glycan branching comprise a requisite posttranslational modification to neuronal glycoproteins that permits normal cellular function and viability. From the full text article: Asparagine (N)-linked oligosaccharides (N-glycans) are among the most abundant posttranslational modifications to cellular proteins. N-glycans make up a diverse repertoire of polymeric branched structures produced in the Golgi apparatus and destined for secretion or presentation on the cell surface. There has been considerable forethought given to the likely roles of glycan variation in controlling important biologic processes (Kobata, 1992; Rademacher et al., 1988; Varki, 1993). These deductions have provided strong incentive for the development of approaches to investigate the physiologic functions of glycans among intact organisms. In the past decade, genetic deconstruction of glycan synthetic and diversification in vivo has been suggested as one approach to determining glycan function (Marth, 1994). Moreover, humans bearing various and informative genetic biosynthetic defects in N-glycan synthesis have been identified (Jaeken et al., 1991, 2001). Novel functions of mammalian glycans and additional pathways in N-glycan formation have been thereby discovered with evidence that different glycan linkages play cell type–specific roles in physiology and disease (reviewed in Lowe and Marth, 2003). After N-glycan processing and protein folding in the endoplasmic reticulum, transport to the Golgi occurs, where N-glycan structures are branched and diversified prior to localization among extracellular compartments. Three subtypes of secreted and cell surface N-glycans exist and are termed high-mannose, hybrid, and complex. Their expression and relative abundance is controlled by the sequential actions of specific glycotransferases and glycosidases (Kornfeld and Kornfeld, 1985). The conversion of high-mannose to hybrid and complex structures is initiated by the action of the GlcNAcT-I glycosyltransferase (E.C. 2.4.1.101) encoded by the Mgat1 gene (Kumar and Stanley, 1989; Kumar et al., 1990; Sarkar et al., 1991). The conversion of hybrid to complex structures is controlled subsequently by the Mgat2-encoded GlcNAcT-II glycosyltransferase (E.C. 2.4.1.143; Bendiak and Schachter, 1987; D'Agostaro et al., 1995; Schachter, 1991). The study of N-glycan deficiency in mice and humans have indicated multiple and essential roles for hybrid and complex branch structures. Lethality among all embryos lacking Mgat1 function, and hence both hybrid and complex N-glycans, was accompanied by several morphologic and developmental abnormalities (Ioffe and Stanley, 1994; Metzler et al., 1994). Although high-mannose N-glycans are insufficient for normal ontogeny, hybrid-type branching structures represent the minimal N-glycan repertoire needed to complete embryogenesis. Nevertheless, restricting N-glycan branching to the formation of only hybrid structures by Mgat2 inactivation results in very low frequencies of survival to adulthood and disease occurrence with a postnatal phenotype in the mouse that is similar to human CDG-IIa (Jaeken et al., 1994; Wang et al., 2001). The predominance of early lethality due to the inheritance of defective Mgat1 or Mgat2 alleles has restricted the ability to discriminate among the cellular functions of mammalian N-glycan branches. The ability to ablate glycosyltransferase expression systemically and compared with conditional mutagenesis approaches could provide information pertaining to distinct roles of different N-glycan structures among specific cell lineages (Marth, 1996). Glycans in the mammalian brain have been previously analyzed, and results indicate the presence of unique structures as well as a greater abundance of high mannose N-glycans as compared with other tissues (Chen et al., 1998; Clark et al., 1998; Krusius and Finne, 1977; Zamze et al., 1998). It seemed possible that neurons may be less reliant than other cell types on the formation of hybrid and complex N-glycans. We pursued this possibility by eliminating Mgat1 and Mgat2 genes specifically in neuronal cell types using Cre-loxP mutagenesis. Our data reveal that hybrid N-glycans are essential for neuronal and postnatal viability in mice, whereas complex N-glycans appear dispensable in this cell lineage. Med Clin (Barc). 2004 May 15. Congenital disorders of glycosylation (CDG) are a group of inherited disorders caused by defects in the synthesis and processing of the linked glycans of glycoproteins and other molecules. The first patients with CDG were described in 1980. Fifteen years later, phosphomannomutase was found to be the basis of the most frequent type: CDG-Ia. Over the last years, several novel types have been identified related to the N-glycosylation pathway, affecting enzymes or transporters of the cytosol, endoplasmic reticulum or the Golgi compartment. CDGs are multisystemic disorders, mainly affecting the central nervous system. Yet CDG-Ib and Ih are mainly hepato-intestinal diseases. Recently, several defects involving the O-glycosylation pathways have been described, indicating that some congenital muscular dystrophies and neuronal migration disorders are caused by congenital disorders of glycosylation. Nat Med. 2004 May. The congenital disorders of glycosylation (CDG) are characterized by defects in N-linked glycan biosynthesis that result from mutations in genes encoding proteins directly involved in the glycosylation pathway. Here we describe two siblings with a fatal form of CDG caused by a mutation in the gene encoding COG-7, a subunit of the conserved oligomeric Golgi (COG) complex. The mutation impairs integrity of the COG complex and alters Golgi trafficking, resulting in disruption of multiple glycosylation pathways. These cases represent a new type of CDG in which the molecular defect lies in a protein that affects the trafficking and function of the glycosylation machinery. J Pediatr Gastroenterol Nutr. 2004 Mar. OBJECTIVES: The typical signs and symptoms of congenital disorders of glycosylation (CDG) include dysmorphy, failure to thrive, and neurologic abnormalities. However, more and more children diagnosed at a young age are not dysmorphic and do not have neurologic involvement. The authors studied the gastrointestinal and other clinical manifestations of CDG type Ia, Ib, and Ic. METHODS: As of January 2003, 17 children were identified with CDG at the authors' institution. The medical records of the patients were reviewed. RESULTS: Five children had CDG Ia, three children CDG Ib, and nine children CDG Ic. Age at diagnosis ranged from 2 months to 15 years. Failure to thrive was present in 80% of patients with CDG Ia, in 66% of those with CDG Ib, and in 11% of those with CDG Ic. Five children had protein-losing enteropathy (two CDG Ia, two CDG Ib, and one CDG Ic). Hepatomegaly was present in 40% of patients with CDG Ia, in 66% of those with CDG Ib, and in 11% of those with CDG Ic. In CDG Ic, hepatomegaly was transient. In CDG Ia, histologic analysis of the liver showed swollen hepatocytes, steatosis, and fibrosis. In CDG Ib, hamartomatous collections of bile ducts were seen. In one patient with CDG Ib, the clinical picture was restricted to congenital hepatic fibrosis for more than a decade. CONCLUSIONS: The study confirms the heterogeneity of the clinical picture in children with CDG type Ia, Ib, and Ic. Children with protein-losing enteropathy should be tested for CDG. Protein-losing enteropathy can be caused, not only by CDG Ia and Ib, but also by type Ic. Children with congenital hepatic fibrosis should be tested for CDG, even in the absence of other symptoms. In CDG Ib, histologic analysis of the liver showed hamartomatous collections of bile ducts (Meyenburg complex). Clin Chem. 2004 Jan. BACKGROUND: Congenital disorders of glycosylation (CDG) are usually diagnosed by isoelectric focusing (IEF) of serum transferrin (Tf). The aim of this study was to evaluate capillary zone electrophoresis (CZE) as a diagnostic alternative to IEF. METHODS: We performed 792 CZE analyses of Tf, using the CEofix(TM)-CDT (carbohydrate-deficient transferrin) assay. Peak identification was based on relative migration times (RMTs) to reduce migration variability. RESULTS: Tf profiles comprised three main groups (A-C). Groups A and B were characterized by one or two dominant tetrasialo-Tf peaks, whereas group C showed a widely variable Tf isoform composition. Group A was composed of four subgroups: a major group with a typical Tf profile (considered as reference group), two minor groups with decreased or moderately increased trisialo-Tf isoform, and a group showing the presence of unknown compounds with RMTs similar to mono- and disialo-Tf. However, these compounds were absent on IEF. Group C contained all profiles from patients with confirmed as well as putative CDG. From the reference group, 99% confidence intervals were calculated for the RMTs of the Tf isoforms, and percentiles representing the Tf isoform distributions were defined. CONCLUSIONS: All patients with abnormal IEF results and confirmed CDG were identified by CZE; thus, this method can be used as a diagnostic alternative to IEF in a manner suitable for automation. Because whole serum is analyzed, it should be kept in mind that CZE profiles can show substances other than Tf. J Inherit Metab Dis. 2004. Congenital disorders of glycosylation (CDG) represent a family of genetic diseases with broad clinical presentation. Initial diagnosis is currently mainly based on the identification of hyposialylated serum transferrin (TF) by isoelectric focusing (IEF). To improve the diagnosis of known CDG types and to identify so far unknown CDG cases, additional glycoproteins, alpha1-antitrypsin titrypsin (alpha1-AT) and alpha1-antichymotrypsin (alpha1-ACT), were studied. According to the patterns of transferrin, enzyme assays and mutation analysis, 16 patients with various clinical symptoms suspicious for CDG were divided into three groups: group A (n = 6) with confirmed CDG; group B (n = 4) with clear abnormal TF-IEF patterns of unknown origin (all known CDG types were excluded) and group C (n = 6) with borderline TF-IEF patterns; 164 samples served as a control group. Automated IEF of TF, alpha1-AT and alpha1-ACT was carried out using a PhastSystem. CDG patients with glycosylation defects of known origin (group A) and patients with abnormal TF-IEF patterns due to glycosylation defects of as yet unknown origin (group B) showed abnormal IEF patterns of all three glycoproteins. These results confirmed generalized defects of glycosylation. Furthermore, the IEF pattern of alpha1-ACT seems to allow a differentiation between CDG Ia and CDG Ic. However, patients with borderline TF-IEF pattern (group C) showed a normal alpha1-AT-IEF pattern. Four of these six patients also showed a normal alpha1-ACT-IEF pattern; this constellation suggests that CDG can most likely be excluded. In the two remaining patients of group C with a borderline TF-IEF pattern an abnormal pattern of alpha1-ACT-IEF was obtained which needs further investigations. We conclude that the combined investigation of three glycoproteins provides additional information in the diagnostic work-up of patients with possible CDG. The suspicion of CDG in patients with apparent glycosylation defects of unknown origin or borderline TF-IEF pattern can be either substantiated or weakened. Glycoconj J. 2004. The dystrophin glycoprotein complex (DGC) is an assembly of proteins spanning the sarcolemma of skeletal muscle cells. Defects in the DGC appear to play critical roles in several muscular dystrophies due to disruption of basement membrane organization. O -mannosyl oligosaccharides on alpha-dystroglycan, a major extracellular component of the DGC, are essential for normal binding of alpha-dystroglycan to ligands (such as laminin) in the extracellular matrix and subsequent signal transmission to actin in the cytoskeleton of the muscle cell. Muscle-Eye-Brain disease (MEB) and Walker-Warburg Syndrome (WWS) have mutations in genes encoding glycosyltransferases needed for O -mannosyl oligosaccharide synthesis. Myodystrophic myd mice and humans with Fukuyama Congenital Muscular Dystrophy (FCMD), congenital muscular dystrophy due to defective fukutin-related protein (FKRP) and MDC1D have mutations in putative glycosyltransferases. These human congenital muscular dystrophies and the myd mouse are associated with defective glycosylation of alpha-dystroglycan. It is expected other congenital muscular dystrophies will prove to have mutations in genes involved in glycosylation. Hum Mol Genet. 2003 Oct 15. Recently, post-translational modification of proteins has been defined as a new area of focus for muscular dystrophy research by the identification of a group of disease genes that encode known or putative glycosylation enzymes. Walker-Warburg Syndrome (WWS) and muscle-eye-brain disease (MEB) are caused by mutations in two genes involved in O-mannosylation, POMT1 and POMGnT1, respectively. Fukuyama muscular dystrophy (FCMD) is due to mutations in fukutin, a putative phospholigand transferase. Congenital muscular dystrophy type 1C and limb girdle muscular dystrophy type 2I are allelic, both being due to mutations in the gene-encoding fukutin-related protein (FKRP). Finally, the causative gene in the myodystrophy (myd) mouse is a putative bifunctional glycosyltransferase (Large). WWS, MEB, FCMD and the myd mouse are also associated with neuronal migration abnormalities (often type II lissencephaly) and ocular or retinal defects. A deficiency in post-translational modification of alpha-dystroglycan is a common feature of all these muscular dystrophies and is thought to involve O-glycosylation pathways. This abnormally modified alpha-dystroglycan is deficient in binding to extracellular matrix ligands, including laminin and agrin. Selective deletion of dystroglycan in the central nervous system (CNS) produces brain abnormalities with striking similarities to WWS, MEB, FCMD and the myd mouse. Thus, impaired dystroglycan function is strongly implicated in these diseases. However, it is unlikely that these five glycosylation enzymes only have a role in glycosylation of alpha-dystroglycan and it is important that other protein targets are identified. J Chromatogr A. 2003 Sep 26. Capillary zone electrophoresis (CZE) with a dynamic double coating permits the simultaneous, individual, quantitative determination of transferrin (Tf) isoforms in human serum and thus carbohydrate-deficient transferrin (CDT), the most specific marker available today for the detection of chronic, excessive alcohol intake. CZE of serum Tf was carefully evaluated using the P/ACE MDQ with fused-silica capillaries of 50 microm I.D. and 60.2 cm total length, the CEofix CDT kit and the instrumental conditions recommended by the kit manufacturer. The precision performance assessed over a 20-day period according to the internationally accepted NCCLS EP5-A guidelines revealed the CZE assay as being highly reproducible with within-run and total precision being dependent on the Tf isoform level and RSD values ranging between 2.2 and 17.6%. Inter-day RSD values for asialo-Tf were noted to be between 9.8 and 11.5% and for disialo-Tf between 3.8 and 8.6%, whereas those for CDT levels of 0.87 and 4.31% of total Tf were determined to be 8.6 and 3.4%, respectively. The RSD values for trisialo-Tf, tetrasialo-Tf, pentasialo-Tf and hexasialo-Tf were found to be between 0.4 and 4.1%. Tf patterns are recognized and identified via detection times of Tf isoforms (intra-day and inter-day RSD values < 1.0% and < 1.7%, respectively), immunosubtraction of Tf and enzymatic sequential cleavage of sialic acid residues. Furthermore, heterozygous Tf BC and Tf CD variants are assigned via spiking with a known mixture of Tf isoforms (e.g. the serum of a healthy Tf C homozygote). Among the non-Tf peaks monitored, the CRP peak detected shortly before disialo-Tf was identified by immunosubtraction and peak magnitudes were found to correlate well with immunochemically determined CRP serum levels. The CZE assay with dynamic double coating could thereby be shown to be sensitive enough to determine elevated CRP levels in human serum. Furthermore, unusual peaks in the gamma-region were identified by customary serum protein CZE, immunosubtraction CZE and immunofixation. Glycobiology. 2003 Sep. The fundamental importance of correct protein glycosylation is abundantly clear in a group of diseases known as congenital disorders of glycosylation (CDGs). In these diseases, many biological functions are compromised, giving rise to a wide range of severe clinical conditions. By performing detailed analyses of the total serum glycoproteins as well as isolated transferrin and IgG, we have directly correlated aberrant glycosylation with a faulty glycosylation processing step. In one patient the complete absence of complex type sugars was consistent with ablation of GlcNAcTase II activity. In another CDG type II patient, the identification of specific hybrid sugars suggested that the defective processing step was cell type-specific and involved the mannosidase III pathway. In each case, complementary serum proteome analyses revealed significant changes in some 31 glycoproteins, including components of the complement system. This biochemical approach to charting diseases that involve alterations in glycan processing provides a rapid indicator of the nature, severity, and cell type specificity of the suboptimal glycan processing steps; allows links to genetic mutations; indicates the expression levels of proteins; and gives insight into the pathways affected in the disease process. Arch Pediatr. 2003 Jul. Congenital disorders of glycosylation type I (GDG-I) is a class of genetic multisystem disorders characterised by defective glycosylation of glycoproteins. The characteristics and mechanisms of failure to thrive and intestinal diseases present in CDG-I are anectodal. Patients and methods: The aim of this study was to analyse 7 CDG-I (4 CDG-Ia, 2 CDG-Ib and 1 CDG-Ix) with important digestive symptoms and failure to thrive in order to characterise the mechanisms implied. Results: Four children had no skin abnormality or dysmorphia (1 CDG-Ia, 2 CDG-Ib, 1 CDG-Ix). An encephalopathy with cerebellar hypoplasia was present only in the 4 CDG-Ia. Failure to thrive and diarrhea were present during the first month of life in 6 and appeared at 5 years in one CDG-Ia associated to mild or severe hepatopathy in all patients. One CDG-Ia, 1 CDG-Ib, 1 CDG-Ix had an exsudative enteropathy. A positive steatorrhea was present in 3 patients. Five patients had an abnormal small bowel biopsy. Abnormalities were variable: moderate inflammation of the chorion without villous atrophy in 2, intra-enterocyte fat accumulation without villous atrophy in 2, and partial villous atrophy with lymphangectasia in 1. In 2 CDG-Ia the intestinal biopsy was normal. Enteral nutrition in 4 and parenteral nutrition in 2 were effective in 4 patients and 1 patient with an exsudative enteropathy respond to a free fat diet (CDG-Ix). Conclusion: The digestive symptoms with failure to thrive is a common feature of CDG-I and could be the first symptoms. The diagnostic should be suspected if no other cause is found. Mechanisms of the intestinal symptoms appear to be multiple such as inflammation, abnormal enterocyte lipid transport or intestinal permeability related to the abnormal glycosylation of intestinal mucosa glycoproteins. Trends Pharmacol Sci. 2003 Apr. Glycosylation is the most frequent modification of proteins and is important for many ligand-receptor interactions. Recently, defects in protein glycosylation have been linked to several forms of congenital muscular dystrophy that are frequently associated with brain abnormalities. Muscle-eye-brain disease and Walker-Warburg syndrome are caused by mutations in enzymes involved in O-mannosylation, whereas Fukuyama congenital muscular dystrophy and congenital muscular dystrophy type 1C are caused by mutations in genes that encode putative glycosyltransferases. The common factor in these disorders is defective processing and maturation of a protein called alpha-dystroglycan. This is thought to disrupt the link between alpha-dystroglycan and components of the extracellular matrix, and result in muscle disease and, in many cases, a neuronal-migration disorder. Neuropediatrics. 2003 Feb. Objective: Congenital disorders of glycosylation (CDG), formerly called carbohydrate-deficient glycoprotein syndromes, constitute a newly identified group of multisystem disorders characterized by defective glycosylation of N-glycosylated proteins. The objective of this work was to describe precisely neurological findings in patients with type Ia CDG (CDG-Ia) and to compare our results with the literature. Study design: We retrospectively reviewed neurological and neurodevelopmental, neuroimaging, and genetic features in ten patients with CDG-Ia who mainly presented with neurological abnormalities during childhood and therefore were referred to a neuropediatrician or a neurogeneticist. Results: Neurological manifestations had a static clinical course, dominated by mental retardation and cerebellar dysfunction, and acute episodes: stroke-like episodes and seizures. However, microcephaly, retinopathy, and polyneuropathy were progressive. All patients had severe global neurodevelopmental delay: only one was able to walk alone at ten years of age and only one could read. Marked heterogeneity in manifestations and delay of diagnosis was noted across the patients. Cerebellar hypoplasia was found by magnetic resonance imaging in all ten patients and olivopontocerebellar hypoplasia in four patients. As in the literature, there was no clear phenotype-mutation correlation. Conclusion: Our findings confirm the importance of a precise and complete description of the neurological and neuroradiological phenotype delineating the phenotype of CDG-Ia to increase the likelihood of diagnosing the disease. Cell Mol Life Sci. 2003 Feb. The gene mutated in the myodystrophy mouse, a model of muscular dystrophy, encodes a putative glycosyltransferase, Large. Mutations in genes encoding proteins thought to be involved in glycosylation have now been identified in six human forms of muscular dystrophy. Hereditary inclusion body myopathy and Nonaka myopathy result from defects in sialic acid production. Two forms of congenital muscular dystrophy, Fukuyama-type and MDC1C, result from mutations in members of the fukutin family. MDC1C and limb girdle muscular dystrophy type 2I are allelic, as they are both associated with mutations in the FKRP gene. Mutations in POMGnT, which encodes an enzyme involved in the synthesis of O-mannosyl glycans, result in muscle-eye-brain disease--another congenital form of muscular dystrophy. Abnormal alpha-dystroglycan has been reported in the myodystrophy mouse, and in the congenital and limb girdle muscular dystrophies. Recent data have shown that there is altered glycosylation of the protein and that this reduces its ability to bind to extracellular matrix ligands such as laminin and agrin. Cas Lek Cesk. 2003. Background: Congenital disorders of glycosylation (CDG syndrome) represent a newly delineated group of inherited diseases of glycoprotein synthesis. We present results of biochemical and molecular analyses in two Czech patients with CDG Ia syndrome. Methods and results: Serum concentrations of the nonglycosylated and hypoglycosylated transferrin were measured using turbidimetric immunoassay. In positive patients, the isoelectric focusing of serum transferrin and molecular analyses of the gene for phosphomannomutase 2 were performed. The disease manifested in both children in infancy with failure to thrive, inverted nipples, strabismus, epilepsy, muscle hypotonia, microcephaly, psychomotor retardation and hypoplasia of the cerebellum. The biochemical investigation revealed elevated liver enzymes, low concentration of factor XI and protein S. In one child lower concentration of the antithrombin III and protein C were found. Activities of arylsulfatase A and beta-glucuronidase in serum were higher and activity of alpha-mannosidase in leucocytes was lower in comparison with controls. Molecular analyses revealed that both children are compound heterozygotes for the mutation 422G > A and 357C > A in gene for phosphomanomutase 2. Both siblings are also homozygotes for polymorfism IVS5 + 19 C-->T and heterozygygotes for polymorfism IVS5 + 22 T-->A. Conclusions: The prognosis of children with CDG Ia is unfavourable. Enzymatic and/or molecular studies are necessary for genetic counselling and the prenatal diagnosis. J Inherit Metab Dis. 2003. Congenital disorders of glycosylation (CDGs) are due to defects in the synthesis of the glycan moiety of glycoproteins or other glycoconjugates. This review is devoted mainly to the clinical aspects of protein glycosylation defects. There are two main types of protein glycosylation: N-glycosylation and O-glycosylation. N-glycosylation generally consists of an assembly pathway (in cytosol and endoplasmic reticulum) and a processing pathway (in endoplasmic reticulum and Golgi). O-glycosylation lacks a processing pathway but is otherwise more complex. Sixteen disease-causing defects are known in protein glycosylation: 12 in N-glycosylation and four in O-glycosylation. The N-glycosylation defects comprise eight assembly defects (CDG-I) designated CDG-Ia to CDG-Ih, and four processing defects (CDG-II) designated CDG-IIa to CDG-IId. By far the most frequent is CDG-Ia (phosphomannomutase-2 deficiency). It affects the nervous system and many other organs. Its clinical expression varies from extremely severe to very mild (and thus probably underdiagnosed). The most interesting disease in this group is CDG-Ib (phosphomannose isomerase deficiency) because it is so far the only efficiently treatable CDG (mannose treatment). It has a hepatic-intestinal presentation. The O-glycosylation defects comprise two O-xylosylglycan defects (a progeroid variant of Ehlers-Danlos syndrome and the multiple exostoses syndrome) and two O-mannosylglycan defects (Walker-Warburg syndrome and muscle-eye-brain disease). All known CDGs have a recessive inheritance except for multiple exostoses syndrome, which is dominantly inherited. There is a rapidly growing group of putative CDGs with a large spectrum of clinical presentations (CDG-x). Serum transferrin iso-electrofocusing remains the cornerstone of the screening for N-glycosylation defects associated with sialic acid deficiency. Abnormal patterns can be grouped in to type 1 and type 2. However, a normal pattern does not exclude these defects. Screening for the other CDGs is much more difficult, particularly when the defect is organ- or system-restricted. The latter group promises to become an important new chapter in CDG. It is concluded that CDGs will eventually cover the whole clinical spectrum of paediatric and adult disease manifestations. Ann Pharm Fr. 2003. Congenital disorders of glycosylation (CDG) is a fast growing group of autosomal recessive inherited diseases caused by defects in glycosylation. The biosynthesis of the glycans is a pathways which occurs in the endoplasmic reticulum and Golgi complex thanks to highly specific enzymes: glycosidases and glycosyltransferases. The sequential addition of monosaccharides needs precursors which are nucleotide sugars or dolichyl sugars. CDG are divided into two groups: CDG I composed of defects in enzymes involved in the assembly of dolicholpyrophosphate oligosaccharide and in the transfer of oligosaccharide from dolicholpyrophosphate to an Asn residue on nascent proteins; CDG II composed of defects in the processing of protein-bound glycans with alterations in enzymes or in the transporters of monosaccharides. Clinical symptoms are poorly specific and multisystemic, biochemistry provides the diagnosis: Isoelectrofocalisation and western blot of serum transferrin and some other glycoproteins; Measurement of enzyme activities; Research of gene mutations. Today, thirteen CDG are identified, the most frequent is CDG Ia due to a defect in the phosphomannomutase activities and CDG Ib due to a defective phosphomannose isomerase, is the only CDG which is successfully treated with mannose. J Inherit Metab Dis. 2003. We report a 7-year-old girl with hyperinsulinaemic hypoglycaemia and hepatomegaly due to congenital disorder of glycosylation (CDG) Ib without gastrointestinal symptoms. Oral mannose therapy produced clinical and biochemical normalization after 2 years of treatment. Biochim Biophys Acta. 2002 Dec 19. The GlcNAc(beta)1,2Man(alpha)- moiety can be synthesized by at least two mammalian glycosyltransferases, UDP-GlcNAc:alpha-3-D-mannoside beta1,2-N-acetylglucosaminyltransferase I (GnT I, EC 2.4.1.101) and UDP-GlcNAc:alpha-D-mannoside beta1,2-N-acetylglucosaminyltransferase I.2 (GnT I.2). GnT I adds a GlcNAc residue in beta1,2 glycosidic linkage to the Man(alpha)1,3 arm of the N-glycan core to initiate the biosynthesis of hybrid and complex N-glycans. GnT I.2 can add GlcNAc in beta1,2 linkage to any alpha-linked terminal Man residue but has a strong preference for the Man(alpha)1-O-Thr- moiety which occurs in alpha-dystroglycan and other O-mannosylated glycoproteins. Mouse embryos lacking a functional GnT I gene (MgatI) were unable to synthesize complex N-glycans and none survived past 10.5 days after fertilization. The embryos showed multisystemic defects in various morphogenic processes such as neural tube formation, vascularization and the determination of left-right body plan asymmetry. Six human patients with muscle-eye-brain disease (MEB) were recently shown to have point mutations in the gene encoding GnT I.2 (MGATI.2). MEB is an autosomal recessive disease characterized by congenital muscular dystrophy, ocular abnormalities, brain malformations and other multisystemic defects. Both the MGATI.2 gene and MEB disease have been mapped to chromosome 1p32-p34. At least one of the biochemical sites affected by the MGATI.2 mutations is probably the interaction between laminin in the extracellular matrix and the peripheral membrane glycoprotein alpha-dystroglycan since this interaction is believed to require the presence of the sialyl(alpha)2,3Gal(beta)1,4GlcNAc(beta)1,2Man(alpha)1-O-Ser/Thr moiety on alpha-dystroglycan. It can be concluded that the GlcNAc(beta)1,2Man(alpha)- moiety is important for mammalian development due to an essential role in two distinct biochemical pathways. Biochim Biophys Acta. 2002 Dec 19. Mice homozygous for a deletion of the Mgat2 gene encoding UDP-N-acetylglucosamine:alpha-6-D-mannoside beta1,2-N-acetylglucosaminyltransferase II (GlcNAcT-II, EC 2.4.1.143) have been reported. GlcNAcT-II is essential for the synthesis of complex N-glycans. The Mgat2-null mice were studied in a comparison with the symptoms of congenital disorder of glycosylation type IIa (CDG-IIa) in humans. Mutant mouse tissues were shown to be deficient in GlcNAcT-II enzyme activity and complex N-glycan synthesis, resulting in severe gastrointestinal, hematologic and osteogenic abnormalities. All mutant mice died in early post-natal development. However, crossing the Mgat2 mutation into a distinct genetic background resulted in a low frequency of survivors exhibiting additional and novel disease signs of CDG-IIa. Analysis of N-glycan structures in the kidneys of Mgat2-null mice showed a novel bisected hybrid N-glycan structure in which the bisecting GlcNAc residue was substituted with a beta1,4-linked galactose or the Lewis(x) structure. These studies suggest that some of the functions of complex N-glycan branches are conserved in mammals and that human disease due to aberrant protein N-glycosylation may be modeled in the mouse, with the expectation in this case of gaining insights into CDG-IIa disease pathogenesis. Further analyses of the Mgat2-deficient phenotype in the mouse have been accomplished involving cells in which the Mgat2 gene is dispensable, as well as other cell lineages in which a severe defect is present. Pre-natal defects appear in a significant number of embryos, and likely reflect a limited window of time in which a future therapeutic approach might effectively operate. J Chromatogr A. 2002 Dec 6. Serum transferrin (Tf) comprises several isoforms with up to two complex oligosaccharide chains containing zero to eight sialic acid residues and neutral sugars. The major glycoform, known as tetrasialo-Tf, contains four sialic acid residues and accounts for about 80% of whole Tf in human serum. Carbohydrate-deficient transferrin (CDT) encompasses isoforms that are deficient in carbohydrate chains and consequently in sialic acid residues (including asialo-, monosialo- and disialo-Tf) and is a well known marker for chronic alcohol abuse. Recently capillary zone electrophoresis (CZE) has been reported as a tool extremely effective for the simultaneous, individual, quantitative determination of CDT isoforms. Three CZE methods that feature different dynamic capillary coatings were evaluated and optimized for CDT determination in human serum of alcohol abusers and control subjects. CZE separation was performed in alkaline borate buffers after serum sample saturation with iron, electropherograms were detected at 200 nm, data were evaluated as % area of disialo-Tf in relation to tetrasialo-Tf and peak identification was accomplished via relative migration times to tetrasialo-Tf, immunosubtraction and enzymatic sequential cleavage of sialic acid residues. Dynamic capillary coatings with diaminobutane, spermine and a double coating produced by commercially available proprietary agents were investigated and found to be suitable for determination of CDT in human serum. For all three approaches, best results were obtained in 50 microm I.D. fused-silica capillaries of 50 cm effective length and a capillary cartridge temperature of 20-25 degrees C. Using 3 mM 1,4-diaminobutane or 0.02 mM spermine in a borate-based running buffer of pH 8.3 provided data of remarkable similarity with resolution of di-, tri-, tetra- and pentasialo-Tf within 15-18 min. With the double coating, asialo-Tf and Tf isoforms with two to six sialic acid residues were baseline separated. Compared to the two amine-based procedures, the run times were found to be somewhat shorter, the detector signals higher, the applied power level significantly lower and the reproducibility better. Lancet. 2002 Nov 2. CONTEXT: Over the past 15 years the causative genes of several inherited muscular dystrophies have been identified. These genes encode sarcolemmal, extracellular matrix, sarcomeric, and nuclear envelope proteins. Although the post-translational processing of muscle proteins has a significant role in their correct assembly and function, these processes have not been shown to be primarily involved in the pathogenesis of muscular dystrophies until recently. In the past 18 months, four different forms of inherited muscular dystrophy in human beings have been associated with mutations in genes encoding for putative glycosyltransferases. Aberrant glycosylation of alpha-dystroglycan, an external membrane protein expressed in muscle, brain, and other tissues, is a common feature in these disorders. alpha-dystroglycan is highly glycosylated, its sugar components varying in different tissues and controlling its interaction with extracellular matrix partners. Disrupted glycosylation of alpha-dystroglycan results in a loss of these interactions, giving rise to both progressive muscle degeneration and abnormal neuronal migration in the brain. STARTING POINT: Kevin Campbell and colleagues have recently demonstrated that patients with muscle-eye-brain disease (MEB) and Fukuyama congenital muscular dystrophy (FCMD), as well as the myodystrophy (myd) mouse, have an abnormally glycosyated form of alpha-dystroglycan (Nature 2002; 418: 417-22 and 422-25). The abnormally glycosylated protein did not bind to three of its extracellular matrix ligands, laminin alpha2 chain, agrin, and neurexin. The investigators also showed that a neuronal migration disorder occurs in both the myd mouse and in a brain-restricted alpha-dystroglycan knock-out mouse that is similar to that seen in patients with MEB and FCMD. These results identify alpha-dystroglycan as having an essential role in both muscle and brain development and function. WHERE NEXT: Emphasis is moving away from identifying the protein components of the muscle fibre that are involved in muscular dystrophies towards the post-translational processing of proteins and the enzymes involved in these modifications. This opens up new avenues of research. Abnormal glycosylation of alpha-dystroglycan may underlie other as yet uncharacterised forms of muscular dystrophy and neuronal migration disorders. Pediatr Res. 2002 Nov. Congenital disorders of glycosylation (CDGs) are a rapidly growing group of inherited disorders caused by defects in the synthesis and processing of the asparagine(ASN)-linked oligosaccharides of glycoproteins. The first CDG patients were described in 1980. Fifteen years later, a phosphomannomutase deficiency was found as the basis of the most frequent type, CDG-Ia. In recent years several novel types have been identified. The N-glycosylation pathway is highly conserved from yeast to human, and the rapid progress in this field can largely be attributed to the systematic application of the knowledge of yeast mutants. Up to now, eight diseases have been characterized, resulting from enzyme or transport defects in the cytosol, endoplasmic reticulum, or Golgi compartment. CDGs affect all organs and particularly the CNS, except for CDG-Ib, which is mainly a hepatic-intestinal disease. Pediatr Res. 2002 Nov. Congenital disorder of glycosylation Ia (CDG-Ia) is an autosomal recessive disease, characterized by the impaired biosynthesis of the N-linked oligosaccharide chains of proteins due to a deficiency of phosphomannomutase (PMM), the enzyme converting mannose-6-phosphate into mannose-1-phosphate. We investigated the consequences of the altered N-linked glycoprotein (GP) biosynthesis on the quantity and quality of glycosphingolipids (GSLs) in fibroblasts of CDG-Ia patients. First, we found that CDG-Ia fibroblasts contain an increased amount of total GSLs when compared with normal fibroblasts. Further, we assessed by metabolic labeling of CDG-Ia fibroblasts with radioactive sugar precursors, including galactose and N-acetylmannosamine, that a diminished biosynthesis of cellular GPs is antagonized by an increased biosynthesis of GSLs. An increased GSL biosynthesis was also observed by means of radiolabeled lipid precursors including sphingosine and lactosylceramide. Notably, also the degradation of GLSs is slowed down in CDG-Ia fibroblasts. Finally, when we labeled normal human fibroblasts and CHO cells with radioactive galactose in the presence and absence of deoxymannojirimycin (dMM), an inhibitor of N-glycan processing, we found that this cellular model mimics what occurs in CDG-Ia fibroblasts. Since an inverse relationship between GP expression and GSL content does exist, we assume that increased glycosphingolipid biosynthesis is secondary to protein hypoglycosylation. Altogether, our data suggest that the cell metabolic machinery may be able to partially re-equilibrate protein hypoglycosylation with increased biosynthesis of glycosphingolipids, possibly to preserve the overall physico-chemical equilibrium of the outer layer of the plasma membrane. Nervenarzt. 2002 Aug. Congenital disorders of glycosylation (CDG) are a group of hereditary multisystem diseases due to different defects of enzymes or transport molecules involved in the synthesis of glycoproteins. CDG-la is the most common subtype, with cerebellar ataxia as the main neurological symptom. Currently there is little information about CDG-la manifestation in adulthood. Here we present two sisters in whom the diagnosis of CDG-la was made in the fourth decade of life and who to our knowledge are the oldest known patients with the disorder in Germany. The clinical course of the disease was typical, although less severe than previously described. The carbohydrate-deficient transferrin (CDT) level was increased but lower than in other CDG patients. Isoelectric focusing of transferrin revealed changes typical of CDG, whereas those of alpha 1-antitrypsin were only moderately pathologic. This might be due to the milder manifestation of the disease in our patients or it could be indicative of a stabilization of the disease after puberty. The CDG should be included in the differential diagnostic workup of hereditary cerebellar ataxia in adults. Nature. 2002 Jul 25. Muscle eye brain disease (MEB) and Fukuyama congenital muscular dystrophy (FCMD) are congenital muscular dystrophies with associated, similar brain malformations. The FCMD gene, fukutin, shares some homology with fringe-like glycosyltransferases, and the MEB gene, POMGnT1, seems to be a new glycosyltransferase. Here we show, in both MEB and FCMD patients, that alpha-dystroglycan is expressed at the muscle membrane, but similar hypoglycosylation in the diseases directly abolishes binding activity of dystroglycan for the ligands laminin, neurexin and agrin. We show that this post-translational biochemical and functional disruption of alpha-dystroglycan is recapitulated in the muscle and central nervous system of mutant myodystrophy (myd) mice. We demonstrate that myd mice have abnormal neuronal migration in cerebral cortex, cerebellum and hippocampus, and show disruption of the basal lamina. In addition, myd mice reveal that dystroglycan targets proteins to functional sites in brain through its interactions with extracellular matrix proteins. These results suggest that at least three distinct mammalian genes function within a convergent post-translational processing pathway during the biosynthesis of dystroglycan, and that abnormal dystroglycan-ligand interactions underlie the pathogenic mechanism of muscular dystrophy with brain abnormalities. J Fr Ophtalmol. 2002 Apr. Introduction: Carbohydrate-deficient-glycoprotein syndromes are new described multisystemic diseases. We report here the case of a young boy who presented with CDG syndrome Ia associated with typical ocular disorders. Case report: This 18-month-old boy presented facial dysmorphism, ataxia, hypotonia and cerebellar hypoplasia. Ocular examination showed esotropia with nystagmoid movements and at fundoscopy signs of retinitis pigmentosa with foveal hypoplasia. Photopic and scotopic electroretinograms were altered. Discussion: CDG syndromes are metabolic disorders which affect N-glycoprotein synthesis. Clinical manifestations are various: hypotonia, cerebellar hypoplasia, developmental delay, pericardial effusion, etc. Four variants are described; each of them is associated with particular clinical disorders and prognosis. Ocular features are frequently associated (esotropia, retinitis pigmentosa) and especially in CDG syndrome Ia. Conclusion: Esotropia associated with fundus alterations appearing in a multisystemic disorder requires explorations to search for CDG syndrome. Neuropediatrics. 2002 Feb. A 1.5-year-old boy with macrocephaly due to a Dandy-Walker malformation presented with progressive hydrocephalus, extensive muscular hypotonia, transient cholestatic syndrome, extensive coagulation abnormalities and elevated creatine kinase indicating myopathy. Diagnostic work-up indicated a congenital disorder of glycosylation (CDG, formerly carbohydrate deficient glycoprotein syndrome). The serum transferrin pattern obtained by automated isoelectric focusing (IEF) showed an hitherto unreported pattern with strongly elevated tri-, di-, mono- and asialotransferrin bands, increasing in this order together with markedly decreased tetrasialotransferrin. Investigation of two additional glycoproteins, alpha(1)-antitrypsin and alpha(1)-antichymotrypsin, confirmed a generalised defect of glycosylation. All known glycosylation defects could be ruled out by enzymatic analyses in either leukocytes or fibroblasts or by the results obtained by IEF. SDS-electrophoresis demonstrated a marked difference in the molecular weight of transferrin, suggesting the lack of parts or of all oligosaccharide chains. The defect could be delineated to a deficiency of beta-1,4-galactosyltransferase (E.C.2.4.1.38) due to a homozygous insertion (1031 - 1032 insC). Details of the biochemical and molecular findings will be described elsewhere. Acta Paediatr. 2002. Phosphomannose isomerase (PMI) deficiency (CDG-Ib) is a newly recognized disorder of mannose and glycoprotein metabolism. PMI deficiency manifests itself mainly as a gastrointestinal disorder with protein-losing enteropathy and life-threatening intestinal bleeding. Hypoglycaemia is an additional prominent symptom. In contrast to phosphomannomutase deficiency (CDG-Ia), there are no neurological symptoms. PMI deficiency blocks the endogenous mannose formation from glucose. Exogenous oral mannose supply bypasses the enzymatic block and leads to the disappearance of all symptoms in the patient. The striking ultrastructural abnormalities of the rough endoplasmatic reticulum of the duodenal epithelial cells completely normalize and the hypoglycosylation disappears, as evidenced by the normal isoelectric focusing pattern of serum transferrin, the standard diagnostic procedure for recognition of CDG. This paper includes a detailed description of the clinical symptomatology of the first-ever diagnosed and treated patient with PMI deficiency and a 5-y follow-up study of mannose therapy. J Inherit Metab Dis. 2001 Dec. A male infant is described who presented with persistent hyperinsulinaemic hypoglycaemia, responding to diazoxide treatment. However, this therapy was discontinued because of seizures as a consequence of disturbed water and electrolyte balance. Glucose homeostasis could only be maintained by subtotal pancreatectomy, which was performed at 3 8/12 years of age. He developed a severe thrombosis, whereon a congenital disorder of glycosylation (CDG) was suspected. An abnormal transferrin isoelectric focusing pattern was found and the diagnosis of CDG Ia was confirmed by enzyme and molecular genetic analysis. This is the first patient with phosphomannomutase deficiency (McKusick 601785) described presenting with severe hyperinsulinaemic hypoglycaemia. Arch Dis Child. 2001 Oct. An Asian girl presented with failure to thrive, congenital hepatic fibrosis, protein losing enteropathy, and hypoglycaemia. Phosphomannose isomerase activity in skin fibroblasts was reduced. She is homozygous for a mutation, D131N, in the phosphomannose isomerase gene (PM1), consistent with the diagnosis of carbohydrate deficient glycoprotein syndrome type 1b. She responded to oral mannose treatment. J Paediatr Child Health. 2001 Oct. A patient with carbohydrate-deficient glycoprotein syndrome type 1b (CDGS1b) is reported. The patient presented at 5 months of age with failure to thrive, prolonged diarrhoea, hepatomegaly and elevated serum liver transaminases. Liver biopsy showed steatosis. A low serum albumin and elevated serum liver transaminases persisted throughout childhood during which he had repeated infectious illnesses. From the age of 10 years he had oesophageal and duodenal ulceration together with recurrent bacterial cholangitis. Liver biopsy demonstrated hepatic fibrosis. CDGS1b was suspected, supported by the finding of a protein-losing enteropathy and finally confirmed by showing a reduced phosphomannoseisomerase activity. This case illustrates a rare condition with a wide range of presentations. Am J Med Genet. 2001 Jun 1. The congenital disorders of glycosylation (CDG) constitute a new group of recessively inherited metabolic disorders that are characterized biochemically by defective glycosylation of proteins. Several types have been identified. CDG-Ia, the most frequent type, is a multisystemic disorder affecting the nervous system and numerous organs including liver, kidney, heart, adipose tissue, bone, and genitalia. A phosphomannomutase (PMM) deficiency has been identified in CDG-Ia patients and numerous mutations in the PMM2 gene have been identified in patients with a PMM deficiency. We report on a French family with 3 affected sibs, with an unusual presentation of CDG-Ia, remarkable for 1) the neurological presentation of the disease, and 2) the dissociation between intermediate PMM activity in fibroblasts and a decreased PMM activity in leukocytes. This report shows that the diagnosis of CDG-Ia must be considered in patients with non-regressive early-onset encephalopathy with cerebellar atrophy, and that intermediate values of PMM activity in fibroblasts do not exclude the diagnosis of CDG-Ia. Am J Hum Genet. 2001 Feb. Congenital disorders of glycosylation (CDGs) are a rapidly enlarging group of inherited diseases with abnormal N-glycosylation of glycoconjugates. Most patients have CDG-Ia, which is due to a phosphomannomutase (PMM) deficiency. In this article, we report that a significant portion (9 of 54) of patients with CDG-Ia had a rather high residual PMM activity in fibroblasts included in the normal range (means of the controls +/- 2 SD) and amounting to 35%-70% of the mean control value. The clinical diagnosis of CDG-Ia was made difficult by the fact that most (6 of 9) of these patients belong to a subgroup characterized by a phenotype that is milder than classical CDG-Ia. These patients lack some of the symptoms that are suggestive for the diagnosis, such as inverted nipples and abnormal fat deposition, and, as a mean, had higher residual PMM activities in fibroblasts (2.05+/-0.61 mU/mg protein, n=9; vs. controls 5.34+/-1.74 mU/mg protein, n=22), compared with patients with moderate (1.32+/-0.86 mU/mg protein, n=18) or severe (0.63+/-0.56 mU/mg protein, n=27, P<.001) cases. Yet they all showed mild mental retardation, hypotonia, cerebellar hypoplasia, and strabismus. All of them had an abnormal serum transferrin pattern and a significantly reduced PMM activity in leukocytes. Six of the nine patients with mild presentations were compound heterozygotes for the C241S mutation, which is known to reduce PMM activity by only approximately 2-fold. Our results indicate that intermediate PMM values in fibroblasts may mask the diagnosis of CDG-Ia, which is better accomplished by measurement of PMM activity in leukocytes and mutation search in the PMM2 gene. They also indicate that there is some degree of correlation between the residual activity in fibroblasts and the clinical phenotype. Proc Natl Acad Sci USA. 2001 Jan 30. Autoimmune diseases are among the most prevalent of afflictions, yet the genetic factors responsible are largely undefined. Protein glycosylation in the Golgi apparatus produces structural variation at the cell surface and contributes to immune self-recognition. Altered protein glycosylation and antibodies that recognize endogenous glycans have been associated with various autoimmune syndromes, with the possibility that such abnormalities may reflect genetic defects in glycan formation. We show that mutation of a single gene, encoding alpha-mannosidase II, which regulates the hybrid to complex branching pattern of extracellular asparagine (N)-linked oligosaccharide chains (N-glycans), results in a systemic autoimmune disease similar to human systemic lupus erythematosus. alpha-Mannosidase II-deficient autoimmune disease is due to an incomplete overlap of two conjoined pathways in complex-type N-glycan production. Lymphocyte development, abundance, and activation parameters are normal; however, serum immunoglobulins are increased and kidney function progressively falters as a disorder consistent with lupus nephritis develops. Autoantibody reactivity and circulating immune complexes are induced, and anti-nuclear antibodies exhibit reactivity toward histone, Sm antigen, and DNA. These findings reveal a genetic cause of autoimmune disease provoked by a defect in the pathway of protein N-glycosylation. From the full text article: Autoimmune diseases afflict an estimated 5% of the human population, yet inherited genetic susceptibilities and causes are for the most part unknown (1, 2). The immune system recognizes glycan-dependent features in self-/non-self-discrimination, and distinct changes in protein glycosylation have been reported in various autoimmune syndromes (3-7). The first autoantibodies to be discovered were the cold agglutinins that bind to glycan chains (termed I/i antigens) and appear to be responsible for approximately 20% of human autoimmune hemolytic anemia cases (3). Elevated levels of autoantibodies to glycolipids are noted in various neurologic disorders, including motor neuron disease (3). Altered glycosylation may also affect immune complex formation. Immunoglobulins with affinity for the Fc region of IgG molecules are found in rheumatoid arthritis, and the severity of the disease is associated with the extent of galactose-deficient N-glycans on Fc (8). Human IgA nephropathy has been associated with altered O-glycosylation of the IgA1 hinge region and Ig deposition in the kidney (9, 10). Another possible role for aberrant glycan production in autoimmune disease includes Tn syndrome, in which reduced transcription of the core 1 O-glycan 1-3 GalT enzyme occurs among hematopoietic compartments. This reduced transcription results in exposure of the Tn antigen on cell surfaces, and some patients suffer hemolytic anemia, thrombopenia, and leukopenia, likely because of the presence of anti-Tn antibodies found in normal serum (11). Glycan structures can clearly participate in pathogenic processes. Yet determining whether glycan recognition and production abnormalities are a cause of autoimmune disease or are secondary events induced by lesions in other metabolic pathways has awaited studies involving in vivo genetic modifications of the glycosylation program itself. Golgi-resident glycosidase and glycosyltransferase enzymes operating in the glycan synthesis pathways are thereby hypothetically promising targets of genetic studies aimed at gaining further insights into the pathogenesis of autoimmune disease. The alpha-mannosidase II enzyme is encoded by a single gene in mammals and resides in the Golgi apparatus, where it trims two mannose residues from hybrid N-linked oligosaccharides. This trimming of the mannose residues allows the subsequent addition of multiple glycan branches by glycosyltransferases, as required for the generation of complex N-glycansthe most prevalent and diverse forms found on mammalian cell surfaces (12-15). Nonerythroid cells from mice lacking a functional alpha-mannosidase II gene were unexpectedly found to compensate for this defect by the activity of another alpha-mannosidase defining an alternative pathway (Fig. 1 and ref. 14). In erythroid cells, glycoproteins were expressed normally at the cell surface, but their portfolio of attached carbohydrate structures was altered with a loss of complex N-glycan branching concurrent with an induction of hybrid N-glycan forms. These animals exhibit a non-life-threatening dyserythropoiesis similar to human congenital dyserythropoietic anemia type II (14). We have since observed an increased morbidity of aged mice lacking alpha-mannosidase II and have therefore attempted to determine whether the loss of alpha-mannosidase II in some tissues is not fully compensated for by the alternative pathway and leads to physiologic defects among nonerythroid cell types. Our findings herein have revealed that alpha-mannosidase II is essential for promoting complex N-glycan branching to varying degrees in different tissues and cell types and on subsets of glycoproteins. The resulting alteration of N-glycan branching provokes a systemic autoimmune disease, indicating that inheritance of an abnormal protein N-glycosylation pathway is an etiologic factor in the pathogenesis of autoimmunity. ... A systemic autoimmune disease was indicated on further immunological analyses of -mannosidase II-deficient mice. At any one time, more than 60% of -mannosidase II-deficient mice with hematuria exhibited anti-nuclear antibody reactivity toward nucleolar as well as nuclear envelope epitopes (Fig. 5A). Antibodies that bound histone, Sm antigen, double-stranded DNA, and single-stranded DNA were also detected (Fig. 5B). In addition, circulating immune complexes were frequently elevated, indicating that some fraction of the immune deposition in the kidney may reflect immune complex trapping. Autoantibodies to autologous protein from the kidney, liver, and lung were also elevated (Fig. 5C). The increased titers of autoantibody reactivity were not significantly affected by the removal of N-glycans from denatured protein with the use of PNGase F (Fig. 5D). Although N-glycan-dependent reactivity to native N-glycosylated glycoprotein conformations cannot be determined, our findings suggest that most autoantibody is produced against a wide range of intracellular and nuclear proteins induced, perhaps by increased phagocytosis and self-antigen presentation that commonly appears in systemic autoimmune disease (2). Taken together with the above spectrum of phenotypic findings, our results reveal a systemic autoimmune disease that is remarkably similar to human systemic lupus erythematosus. ... Discussion Currently identified causes of autoimmune disease encompass modifications of lymphocyte activation or development, chemical or pathogenic exposure, and changes in histocompatibility complex expression (1, 28, 29). We have found that an autosomal recessive genetic defect in the pathway of protein N-glycosylation is also a unique factor capable of inducing systemic autoimmune disease exhibiting symptoms found in human systemic lupus erythematosus, including hematological disorder, immunological disorder (anti-DNA or anti-Sm), renal disorder, and anti-nuclear antibody (30). Examples of single gene lesions that provoke systemic autoimmune disease involve SHP-1, CD22, CTLA-4, IL-2, IL-4, PD-1, transforming growth factor-beta, Fas, Fas ligand, the T cell antigen receptor, and the lyn tyrosine kinase. These defects overtly alter lymphocyte development, abundance, viability, or immune responses (2). The emergence of systemic autoimmune disease can also reflect the involvement of multiple genes. For example, the NZB and NZB/NZW F1 autoimmune mouse models result from defects in multiple genes and exhibit B lymphocyte immune hyperactivity. alpha-Mannosidase II deficiency does not similarly alter lymphoid development, abundance, or proliferation in response to antigen receptor activation and thus is more similar to human systemic autoimmune diseases that also occur without such developmental and immune response abnormalities (2). Although the immune system is obviously the source of increased self-reactivity and thereby involved in the disease process, we have shown that lymphocytes lacking alpha-mannosidase II continue to produce complex N-glycans at the cell surface and at close to normal levels (14). Our findings support the view that the lymphoid population exists without cell-intrinsic defects that easily explain the origin of this systemic autoimmune disease. Additional studies by adoptive transfer, conditional mutagenesis, and transplantation approaches can further address this possibility. Although histocompatibility molecules were expressed normally in mice lacking alpha-mannosidase II, autoimmune disease emergence and severity can be modulated by major histocompatibility haplotypes in some animal models (2). We do not yet know whether MHC haplotype or N-glycosylation plays a role in disease etiology. When N-glycosylation of MHC is completely abrogated by tunicamycin or by mutagenesis of asparagine residues, unglycosylated MHC infrequently reaches the cell surface, being retained in the endoplasmic reticulum; however, those that are found on the cell surface appear to function normally (31). Several hundred Golgi-resident glycosidase and glycosyltransferase enzymes orchestrate the repertoire of cell surface glycan structures. Growth and differentiation signals provided during normal and pathologic metabolism regulate the expression of these enzymes (32). The changing enzyme expression levels in the Golgi of a given cell can alter glycan structures present on the cell surface. Nutritional modifications in humans have also been found to influence the extracellular N-glycan repertoire (33, 34). Interestingly, ingestion of the alpha-mannosidase II inhibitor Swainsonine has been reported to alleviate tumor-induced immune suppression, to increase the propensity for lymphocyte activation, and to inhibit tumor growth and metastasis (35). Whether a clinical regime of alpha-mannosidase II inhibitors can lead to autoimmune disease is not known. Autoimmunity has been divided into systemic and organ-specific types with common mechanistic underpinnings indicated (36). Systemic autoimmunity can lead to organ-specific disease (37), whereas molecular mimicry, histocompatibility haplotypes, and lymphoid involvement may be crucial for the emergence and progression of both disease types. However, antigens that evoke systemic autoimmune disease and factors influencing disease progression are not well understood (2, 24). The previous association of autoimmune syndromes with the induction of anti-carbohydrate antibodies and with carbohydrate structure abnormalities indicated the possibility that glycan recognition or structural alterations might be pathogenic in some circumstances. We can conclude that it is the altered N-glycosylation of one or more glycoproteins that is the cause of systemic autoimmune disease with symptoms of lupus nephritis in the absence of alpha-mannosidase II. It is possible that alterations in N-glycan branching among some glycoproteins and tissues may result in the formation of unusual epitopes that do not fully participate in the immune determination of self. We find that the alternative pathway in complex N-glycan production fails to sufficiently overlap with alpha-mannosidase II function and results in the production of unusual and sometimes unique hybrid glycan N-glycan branches among a subset of glycoproteins and cell types. It is also possible that this abnormal N-glycan expression varies in an age-dependent manner by a developmental change in glycoprotein substrate production and the efficacy of the alternative pathway in complex N-glycan formation. By whatever means, the loss of alpha-mannosidase II alters N-glycan branching and clearly attenuates the immune system's ability to maintain self-tolerance. It is further intriguing to consider whether alpha-mannosidase II inhibition bestows its antitumorigenic effect by modulating the immune-autoimmune threshold. J Med Genet. 2001 Jan. Introduction: Congenital disorders of glycosylation (CDG), or carbohydrate deficient glycoprotein syndromes, form a new group of multisystem disorders characterised by defective glycoprotein biosynthesis, ascribed to various biochemical mechanisms. Methods: We report the clinical, biological, and molecular analysis of 26 CDG I patients, including 20 CDG Ia, two CDG Ib, one CDG Ic, and three CDG Ix, detected by western blotting and isoelectric focusing of serum transferrin. Results: Based on the clinical features, CDG Ia could be split into two subtypes: a neurological form with psychomotor retardation, strabismus, cerebellar hypoplasia, and retinitis pigmentosa (n=11), and a multivisceral form with neurological and extraneurological manifestations including liver, cardiac, renal, or gastrointestinal involvement (n=9). Interestingly, dysmorphic features, inverted nipples, cerebellar hypoplasia, and abnormal subcutaneous fat distribution were not consistently observed in CDG Ia. By contrast, the two CDG Ib patients had severe liver disease, enteropathy, and hyperinsulinaemic hypoglycaemia but no neurological involvement. Finally, the CDG Ic patient and one of the CDG Ix patients had psychomotor retardation and seizures. The other CDG Ix patients had severe proximal tubulopathy, bilateral cataract, and white matter abnormalities (one patient), or multiorgan failure and multiple birth defects (one patient). Conclusions: Owing to the remarkable clinical variability of CDG, this novel disease probably remains largely underdiagnosed. The successful treatment of CDG Ib patients with oral mannose emphasises the paramount importance of early diagnosis of PMI deficiency. Annu Rev Genomics Hum Genet. 2001. Congenital disorders of glycosylation (CDG) are a rapidly growing group of genetic diseases that are due to defects in the synthesis of glycans and in the attachment of glycans to other compounds. Most CDG are multisystem diseases that include severe brain involvement. The CDG causing sialic acid deficiency of N-glycans can be diagnosed by isoelectrofocusing of serum sialotransferrins. An efficient treatment, namely oral D-mannose, is available for only one CDG (CDG-Ib). In many patients with CDG, the basic defect is unknown (CDG-x). Glycan structural analysis, yeast genetics, and knockout animal models are essential tools in the elucidation of novel CDG. Eleven primary genetic glycosylation diseases have been discovered and their basic defects identified: six in the N-glycan assembly, three in the N-glycan processing, and two in the O-glycan (glycosaminoglycan) assembly. This review summarizes their clinical, biochemical, and genetic characteristics and speculates on further developments in this field. Clin Chim Acta. 2000 Dec. The activity of beta-hexosaminidase, determined with 4-methylumbelliferyl-beta-N-acetylglucopyranoside substrate, and of beta-D-mannosidase was significantly higher in the serum of patients with carbohydrate-deficient glycoprotein (CDG) syndrome type IA (phosphomannomutase deficiency) than in controls. No significant differences were observed in the activity of beta-hexosaminidase, determined using 4-methylumbelliferyl-beta-N-acetylglucopyranoside-6-sulphate as substrate, and the activity of alpha-D-mannosidase. Using DEAE-cellulose chromatography, a greater amount of hexosaminidase B than hexosaminidase A was detected in CDG serum. In CDG serum, hexosaminidase A was eluted in a more basic position in the salt gradient. An isoenzyme of alpha-D-mannosidase and beta-D-mannosidase was identified in control and CDG sera. alpha-D-Mannosidase isoenzyme was eluted in a slightly more basic position in CDG serum than in control serum, whereas beta-D-mannosidase isoenzyme was eluted in the same position. Clin Chem Lab Med. 2000 Oct. Congenital disorders of glycosylation (CDG) are genetic multisystemic diseases due to various defects in the biosynthesis or processing of glycoproteins. Our aim is to present our experience in the selective screening of CDG syndrome in a paediatric population (421 patients) with clinical suspicion of the disease, analysing serum carbohydrate-deficient transferrin (CDT) by radioimmunoassay and/or immunoturbidimetry. We established the normal values for our paediatric population. The abnormal results were confirmed and classified by isoelectric focusing of serum sialotransferrins, and by enzymatic and molecular studies. We found 14 patients (3.3%) with abnormal serum CDT; 11 of them were classified as CDG type Ia (CDG-Ia) and the other three showed altered isoelectrofocusing patterns but remain untyped and are under investigation. In conclusion, both CDT assays proved to be useful tools for CDG screening. Isoelectric focusing is a simple procedure but it requires specific instruments that are not always available. Since the immunoturbidimetric procedure is commonly used to monitor for recent excessive alcohol consumption in clinical laboratories and does not require special equipment, it may also be reliably used to screen for CDG in children under clinical suspicion. Glycobiology. 2000 Aug. Congenital Disorders of Glycosylation (CDG) are human deficiencies in glycoprotein biosynthesis. Previous studies showed that 1 mM mannose corrects defective protein N-glycosylation in cultured fibroblasts from some CDG patients. We hypothesized that these CDG cells have limited GDP-mannose (GDP-Man) and that exogenous mannose increases the GDP-Man levels. Using a well established method to measure GDP-Man, we found that normal fibroblasts had an average of 23.5 pmol GDP-Man/10(6) cells, whereas phosphomannomutase (PMM)-deficient fibroblasts had only 2.3-2.7 pmol/10(6) cells. Adding 1 mM mannose to the culture medium increased the GDP-Man level in PMM-deficient cells to approximately 15.5 pmol/10(6) cells, but had no significant effect on GDP-Man levels in normal fibroblasts. Similarly, mannose supplementation increased GDP-Man from 4.6 pmol/10(6) cells to 24.6 pmol/10(6) cells in phosphomannose isomerase (PMI)-deficient fibroblasts. Based on the specific activity of the GDP-[(3)H]Man pool present in [2-(3)H]mannose labeled cells, mannose supplementation also partially corrected the impaired synthesis of mannosylphosphoryldolichol (Man-P-Dol) and Glc(0)(-)(3)Man(9)GlcNAc(2)-P-P-Dol. These results confirm directly that deficiencies in PMM and PMI result in lowered cellular GDP-Man levels that are corrected by the addition of mannose. In contrast to these results, GDP-Man levels in fibroblasts from a CDG-Ie patient, who is deficient in Man-P-Dol synthase, were normal and unaffected by mannose supplementation even though mannose addition was found to correct abnormal lipid intermediate synthesis in another study (Kim et al. [2000] J. Clin. Invest., 105, 191-198). The mechanism by which mannose supplementation corrects abnormal protein N-glycosylation in Man-P-Dol synthase deficient cells is unknown, but this observation suggests that the regulation of Man-P-Dol synthesis and utilization may be more complex than is currently understood. Ann Neurol. 2000 Jun. We report on 8 patients with a recently described novel subtype of congenital disorder of glycosylation type Ic (CDG-Ic). Their clinical presentation was mainly neurological with developmental retardation, muscular hypotonia, and epilepsy. Several symptoms commonly seen in CDG-Ia such as inverted nipples, abnormal fat distribution, and cerebellar hypoplasia were not observed. The clinical course is milder overall, with a better neurological outcome, than in CDG-Ia. The isoelectric focusing pattern of serum transferrin in CDG-Ia and CDG-Ic is indistinguishable. Interestingly, beta-trace protein in cerebrospinal fluid derived from immunoblot analysis of the brain showed a less pronounced hypoglycosylation pattern in CDG-Ic patients than in CDG-Ia patients. Analysis of lipid-linked oligosaccharides revealed an accumulation of Man9GlcNAc2 intermediates due to dolichol pyrophosphate-Man9GlcNAc2 alpha-1,3 glucosyltransferase deficiency. All patients were homozygous for an A333V mutation. J Pediatr. 2000 May. A 6-year-old male patient presented with Budd-Chiari syndrome and glycoprotein abnormalities associated with carbohydrate deficient glycoprotein syndrome type I with yet unidentified molecular defect (type Ix). Budd-Chiari syndrome most likely developed after hepatic venous thrombosis caused by coagulation abnormalities resulting from hypoglycosylation and functional impairment of anticoagulant proteins. J Chromatogr B Biomed Sci Appl. 2000 Feb 28. Carbohydrate-deficient transferrin (CDT) is a reliable marker of chronic or repeated alcohol abuse. It indicates a group of isoforms of human transferrin (Tf), the main iron transport serum protein, deficient in sialic acid residues (asialo-, monosialo- and disialo-Tf) in comparison to the main isotransferrin which contains four sialic acid groups (tetrasialo-Tf). The aim of the present work was to develop a capillary electrophoretic method suitable for rapid determination of CDT components in serum. Serum samples (0.1 ml) were saturated with iron by incubation with 10 mM FeCl3 (2 microl) and 500 mM NaHCO3 (3 microl) for 30 min, then diluted 1:10 in water and injected by positive pressure (0.5 p.s.i. for 10 s). Separation was performed with a capillary zone electrophoretic method using bare fused-silica capillaries (57 cm x 20 microm I.D.) and a buffer composed of 100 mM sodium tetraborate adjusted with 6 M HCl to pH 8.3 added with 1.5 mM diaminobutane. Applied voltage was 20 kV and temperature 25 degrees C. Detection was by UV absorption at 200 nm wavelength. Under the described conditions, asialo-, monosialo-, disialo-, trisialo- and tetrasialo-transferrin were baseline separated. The limit of detection (signal-to-noise ratio of 2) was about 0.3% for disialo-Tf, and 0.5% of trisialo-Tf, expressed as percentages of the terasialo-Tf peak area. Day-to-day RSDs of relative migration times were < or = 0.2%. Quantitation showed day-to-day RDSs < or = 6.9% and < or = 10.9% for disialo- and trisialo-Tf, respectively. The results from 79 control subjects, including social drinkers, and 23 alcoholics showed disialo- and trisialo-Tf significantly increased in patients (P<0.0001 and <0.01, respectively). A clear interference from trisialo-Tf in an immunoassay for CDT was demonstrated. The present method is suitable for confirmation of CDT immunoassays by independent technique. J Pediatr. 1999 Dec. We describe clinical, biochemical, and molecular findings in a 2(1/2)-year-old girl with a phosphomannose isomerase deficiency who presented with severe and persistent hypoglycemia and subsequently developed protein-losing enteropathy, liver disease, and coagulopathy. Six months of therapy with mannose supplementation resulted in clinical improvement and partial correction of biochemical abnormalities. Biochim Biophys Acta. 1999 Oct 8. The carbohydrate-deficient glycoprotein or CDG syndromes (OMIM 212065) are a recently delineated group of genetic, multisystem diseases with variable dysmorphic features. The known CDG syndromes are characterized by a partial deficiency of the N-linked glycans of secretory glycoproteins, lysosomal enzymes, and probably also membranous glycoproteins. Due to the deficiency of terminal N-acetylneuraminic acid or sialic acid, the glycan changes can be observed in serum transferrin or other glycoproteins using isoelectrofocusing with immunofixation as the most widely used diagnostic technique. Most patients show a serum sialotransferrin pattern characterized by increased di- and asialotransferrin bands (type I pattern). The majority of patients with type I are phosphomannomutase deficient (type IA), while in a few other patients, deficiencies of phosphomannose isomerase (type IB) or endoplasmic reticulum glucosyltransferase (type IC) have been demonstrated. This review is an update on CDG syndrome type IA. Biochim Biophys Acta. 1999 Oct 8. The carbohydrate-deficient glycoprotein syndromes (CDGS) are a group of autosomal recessive multisystemic diseases characterized by defective glycosylation of N-glycans. This review describes recent findings on two patients with CDGS type II. In contrast to CDGS type I, the type II patients show a more severe psychomotor retardation, no peripheral neuropathy and a normal cerebellum. The CDGS type II serum transferrin isoelectric focusing pattern shows a large amount (95%) of disialotransferrin in which each of the two glycosylation sites is occupied by a truncated monosialo-monoantennary N-glycan. Fine structure analysis of this glycan suggested a defect in the Golgi enzyme UDP-GlcNAc:alpha-6-D-mannoside beta-1,2-N-acetylglucosaminyltransferase II (GnT II; EC 2.4.1.143) which catalyzes an essential step in the biosynthetic pathway leading from hybrid to complex N-glycans. GnT II activity is reduced by over 98% in fibroblast and mononuclear cell extracts from the CDGS type II patients. Direct sequencing of the GnT II coding region from the two patients identified two point mutations in the catalytic domain of GnT II, S290F (TCC to TTC) and H262R (CAC to CGC). Either of these mutations inactivates the enzyme and probably also causes reduced expression. The CDG syndromes and other congenital defects in glycan synthesis as well as studies of null mutations in the mouse provide strong evidence that the glycan moieties of glycoproteins play essential roles in the normal development and physiology of mammals and probably of all multicellular organisms. J Pediatr. 1999 Sep. We report the case of a patient with carbohydrate-deficient glycoprotein syndrome type Ib who developed normally until 3 months of age, when she was referred to the hospital for evaluation of hypoglycemia that was found to be related to hyperinsulinism. She also had vomiting episodes, hepatomegaly, and intractable diarrhea, which evoked the diagnosis of carbohydrate-deficient glycoprotein syndrome. Oral mannose treatment at a dose of 0.17 g/kg body weight 6 times/d was followed by a clinical improvement and normalization of blood glucose, aminotransferases, and coagulation factor levels. Hyperinsulinemic hypoglycemia should be considered as a leading sign of carbohydrate-deficient glycoprotein syndrome type Ib, especially when it is associated with enteropathy and abnormal liver tests. Neuropediatrics. 1999 Apr. A 10-month old girl is described with a serum transferrin isoform abnormality of the same kind as in two previously reported girls with carbohydrate-deficient glycoprotein syndrome type III. This patient presented with joint abnormalities and rapidly developing hypsarrhythmia, hypotonia, psychomotor delay and growth retardation. Fingers, toes, nails and local skin were dysmorphic. She had pale optic discs, thoracic syringomyelia and frontal lobe atrophy at three months. The CDT value in serum was greatly elevated. Several carbohydrate-deficient isoforms were found in transferrin (four), alpha1-antitrypsin (three), antithrombin (two) and thyroxine-binding globulin (four). Mutations in the CDGS 1-gene were excluded. The CDGS III glycoprotein abnormality most probably represents a distinct disorder of glycoprotein metabolism, and needs to be considered in unclear hypsarrhythmia with developmental delay. Dysmorphic features may be added to this syndrome. Ann Clin Biochem. 1999 Jan. The carbohydrate-deficient glycoprotein (CDG) syndromes (CDGS) are a series of autosomal recessive enzyme deficiencies which result in incomplete glycosylation of plasma proteins. CDGS types Ia and Ib have been related to deficiencies of phosphomannomutase and phosphomannose isomerase, respectively, while CDGS type II results from a deficiency of N-acetylglucosaminyltransferase II. Secondary CDG syndromes are associated with galactosaemia and hereditary fructose intolerance. The diagnosis of CDGS is most easily made by studying the glycoforms of suitable marker proteins using either electrophoresis or isoelectric focusing. This paper reviews the structure of the glycan chains of proteins and structural alterations in CDGS. It also outlines analytical techniques which are useful in the laboratory study of protein glycoforms and the diagnosis of CDGS. Ann Dermatol Venereol. 1998 Oct. BACKGROUND: Type I carbohydrate deficient glycoprotein (CDG) syndrome is an inborn hereditary error of metabolism with a broad clinical spectrum. It is characterized by partial N-glycan deficiency of glycoproteins. Skin features may be part of this syndrome in infancy. CASE REPORT: A male infant failed to thrive, presenting psychomotor retardation, liver disease and multiple biological abnormalities. Very suggestive prominent skin manifestations were noted including abnormal subcutaneous fat with lipoma-like pads on the lower back and buttocks, thickened orange-peel skin on the limbs, thinned proximal knuckles, inverted nipples. Deficient serum transferrin sialylation and phosphomannomutase deficiency were identified confirming type I CDG syndrome. DISCUSSION: Although inconstantly present, skin manifestations of type I CDG syndrome are very suggestive and may be the inaugural signs of the disease. Acta Paediatr. 1998 Aug. Carbohydrate-deficient glycoprotein syndrome type 1A (CDGS1A) is an inherited disorder with multisystemic abnormalities resulting from failure to generate sufficient lipid-linked oligosaccharide precursor or to transfer the sugar chain to many glycoproteins. Cultured fibroblasts from these patients have reduced incorporation of mannose into glycoproteins which can be corrected by adding D-mannose to the culture medium. Providing dietary mannose to elevate mannose concentrations in vivo therefore might remedy some of the underglycosylation in the patients. Five children with CDGS1A aged 15 months to 14 y completed a protocol of enteral supplementation with D-mannose 100 mg/kg every 3 h for 9 d. The mean S-mannose level increased from 32 microM (range 22-42 microM) to a trough value of 72 microM (range 39-103 microM). No serious side effects were observed. Surprisingly, the mean serum concentration of four glycoproteins (transferrin, alpha1-antitrypsin, antithrombin, and thyroxine-binding globulin) tended to decrease, and the mean serum concentration of carbohydrate-deficient transferrin (CDT) increased. Furthermore, the initially present abnormal isoforms of these glycoproteins and of protein C became more prominent and/or additional abnormal isoforms appeared. This short-term trial does not support a benefit of mannose to the deficient glycosylation of CDGS1A patients. Glycoconj J. 1998 May. In the carbohydrate deficient glycoprotein syndrome (CDGS) type 1 glycoproteins with less and shorter N-linked oligosaccharides are synthesized due to a deficiency of phosphomannomutase. Glucose deprivation or mannose addition are shown to partially or fully correct the size of oligosaccharides incorporated into lipid linked oligosaccharides and nascent glycoproteins in skin fibroblasts from CDGS type 1 patients with a phosphomannomutase defect. The corrective effect is ascribed to regulatory mechanisms and/or metabolic pathways that bypass phosphomannomutase. J Clin Invest. 1998 Apr 1. Phosphomannose isomerase (PMI) deficiency is the cause of a new type of carbohydrate-deficient glycoprotein syndrome (CDGS). The disorder is caused by mutations in the PMI1 gene. The clinical phenotype is characterized by protein-losing enteropathy, while neurological manifestations prevailing in other types of CDGS are absent. Using standard diagnostic procedures, the disorder is indistinguishable from CDGS type Ia (phosphomannomutase deficiency). Daily oral mannose administration is a successful therapy for this new type of CDG syndrome classified as CDGS type Ib. From the full text article: Carbohydrate-deficient glycoprotein syndromes (CDGS)1 are a group of hereditary multisystem disorders first recognized by Jaeken et al. (1). The clinical phenotype of all known CDG syndromes is dominated by severe psychomotor and mental retardation, as well as blood coagulation abnormalities presenting as thrombosis, bleedings, or stroke-like episodes. The characteristic biochemical abnormality of CDG syndromes is the hypoglycosylation of glycoproteins. Depending on the type of CDGS, the carbohydrate side chains of glycoproteins are either truncated or completely missing from the protein core. The hypoglycosylation is routinely determined by isoelectric focusing (IEF) of serum transferrin. Affected glycoproteins have altered isoelectric focusing patterns compared with normal due to missing or truncated sugar chains. Based on the IEF patterns and the clinical symptoms, four different types have been classified so far (2-5). The most common form, CDGS type Ia, is caused by phosphomannomutase (PMM) deficiency (6). In this paper, we describe the identification of a new type of CDG syndrome (CDGS type Ib). The clinical phenotype of the new disorder is fundamentally different from all other types of CDGS: no psychomotor or mental retardation is present. Instead, CDGS type Ib presents as a gastrointestinal disorder characterized by protein-losing enteropathy. Thrombosis as well as life-threatening bleeding can occur. We identified a deficiency of phosphomannose isomerase (PMI), a key enzyme in the metabolism of mannose, as the cause of the syndrome, analyzed the PMI1 gene for mutations and developed an effective therapy of oral administration of mannose. Mannose treatment corrected the clinical phenotype as well as the hypoglycosylation of serum glycoproteins. ... The boy was born at term with a normal birth weight. Clinical symptoms began at the age of 11 mo with diarrhoea and vomiting. The leading symptom in the following years was protein-losing enteropathy. Ultrastructural analysis of a small bowel biopsy showed lysosomal inclusion bodies as well as a dilated rough endoplasmic reticulum filled with prominent tubular bundles (Fig. 1). In addition to protein-losing enteropathy, recurrent thrombotic events occured at variable locations. Antithrombotic prophylaxis with vitamin K antagonists was started when thrombosis in both legs occurred by the end of the fifth year of life. Within the next months, several episodes of severe life-threatening gastrointestinal bleedings of diffuse origin appeared that continued even after the prophylaxis was stopped. Laboratory diagnostics repeatedly revealed severe hypoproteinemia, anemia, and markedly reduced antithrombin III (AT III) levels (10-30%). Low AT III levels initiated CDGS diagnostics (11). The IEF of serum transferrin, the standard diagnostic procedure for CDGS, showed the characteristic hypoglycosylation pattern of CDGS type Ia. The diagnosis of CDG syndrome, however, was considered unlikely as all known types of CDG syndrome show psychomotor and mental retardation, and these were absent in our patient. Further clinical details will be described elsewhere (Harms, H.K., K. Reiter, K.-P. Zimmer, K. Auberger, R.M. Bertele-Harms, S. Weidinger, H. Freeze, L. Niehues, M. Hasilik and T. Marquardt, manuscript in preparation). Carbohydrate structure of glycoproteins Since CDG syndrome seemed an unlikely diagnosis with regard to the clinical phenotype, a more detailed serum glycoprotein analysis was performed. The IEF pattern of serum transferrin of the patient was identical to the pattern observed in CDGS type Ia (Fig. 2 A, lane 2). In CDGS type Ia, complete carbohydrate side chains are absent from the protein core. Since IEF analysis gives no information on the size of the oligosaccharides, SDS-PAGE was also used. In SDS-PAGE, even small truncations of the carbohydrate side chains of glycoproteins can be detected (12). The SDS-PAGE banding pattern of the patient was identical to CDGS type Ia (Fig. 2 B, lane 6). It was concluded that the patient's transferrin lacked most or all of the complete carbohydrate chains. The carbohydrate structures released from the glycoproteins of the patient's fibroblasts were analyzed in detail. In CDGS type Ia, a portion of the carbohydrates released from lipid-linked oligosaccharide precursors and newly synthesized glycoproteins are truncated, but subsequent oligosaccharide processing occurs normally (13-15). In the patient, oligosaccharides released by mild acid hydrolysis from lipid-linked precursors or by PNGase F from newly synthesized glycoproteins were identical to controls based on Concanavalin A-Sepharose lectin affinity chromatography, ion-exchange chromatography, or amine adsorption HPLC analysis (see Methods in references 16 and 17). No structural abnormality of the carbohydrates was detected. Mannose incorporation CDGS type Ia is a disorder of mannose metabolism. Mannose incorporation into newly synthesized glycoproteins is considerably decreased (13-15). When fibroblasts of the patient were labeled with [2-3H]-mannose, the incorporation of mannose into newly synthesized glycoproteins was 4.4x the incorporation found in controls and 25.9x the incorporation found in CDGS type Ia cells. In addition, incorporation of the label into mannose derivatives used for the synthesis of the oligosaccharide precursor was also considerably higher than normal (Table I). One explanation for increased labeled mannose was that the size of the intracellular pools was decreased, thus increasing the specific activity of those pools. A possible defect could be in the metabolic pathway providing mannose 6-phosphate from glucose (Fig. 3). Enzymatic defect A deficiency of phosphomannose isomerase was identified as the specific enzymatic defect. The specific activity of PMI in the patient's fibroblasts was reduced to 7.4% of normal (Fig. 4). In leukocytes, the PMI activity was 3.3% of normal. In mixed lysates from the patient and a control, the activities gave the expected sums. The father and mother of the patient had PMI activities of 53.0 and 33.7%, respectively, in their leukocytes, indicating their heterozygous status (Table II). The PMI activity in the healthy brother was 47%. ... Therapy PMI deficiency causes a decreased synthesis of mannose 6-phosphate from glucose (Fig. 3). In this situation, mannose 6-phosphate can only be synthesized from external mannose originating from the diet or from glycoconjugate breakdown. Apparently, these pathways did not supply sufficient mannose. However, mammalian cells have mannose-specific transporters that under normal conditions provide the majority of mannose used for N-glycosylation (16). At the physiological mannose concentration in the blood (mean 55 µM) (7), this transporter operates at about half maximal rate. Therefore, increasing the extracellular mannose concentration should increase the amount of transported mannose available for N-glycosylation. When the boy was 6 yr of age, recurrent severe gastrointestinal bleedings that were refractory to medical and surgical treatment caused a life-threatening condition. At this time, the PMI deficiency was unknown. Based on studies showing that mannose could correct abnormal glycosylation in CDGS type Ia fibroblasts (15), mannose therapy was initiated and followed up for 11 mo. In accordance with previous human ingestion studies (19), mannose was given orally in an initial dose of 100 mg/kg body wt three times a day. 8 mo later, the dose was increased to 150 mg/kg five times a day. Serum mannose levels reached a maximum of 490 µM 90 min after oral ingestion of 150 mg mannose per kg body wt (Fig. 5). Gastrointestinal bleeding and the chronic diarrhoea disappeared within the first few weeks and did not reappear. In the past, severe bleedings could not be stopped by medical treatment and surgery was necessary on several occasions. Total serum protein and antithrombin III rose to normal levels and stayed in the normal range without further changes in the dosing regimen. No side effects were observed. After the initiation of mannose therapy, serum transferrin was reanalyzed several times by IEF, SDS-PAGE, and 2D electrophoresis. After the first 6 mo with a dose of three times daily 100 mg/kg, a partial correction in the IEF and SDS-PAGE patterns of serum transferrin was observed. The mannose dose was increased to achieve a more complete correction, and 11 mo after the initiation of therapy, a profound decrease of the abnormal isoforms and a shift towards a normal pattern were observed (Fig. 2 A, lanes 2-3). The same result was seen in SDS-PAGE (Fig. 2 B, lanes 6-7). 2D electrophoresis demonstrated that the improvement in the glycosylation of affected proteins was not restricted to transferrin (Fig. 6). Discussion PMI deficiency represents a new enzyme defect in humans causing an additional variant of CDG syndrome. For historical reasons, the current classification of CDG syndromes is based on the isoelectric focusing patterns of serum glycoproteins instead of on the underlying enzymatic defects. Since the IEF patterns in PMI deficiency are indistinguishable from the most common form of CDGS, PMM deficiency (CDGS type Ia), PMI deficiency is classified as CDGS type Ib. CDGS type Ib is caused by a genetic defect in the PMI1 gene. The Arg219 mutation of the paternal allele decreased the enzyme activity to a low level, but no mutation was detected in the maternal allele. Her low PMI activity (33% normal) suggests a decreased level of functional maternal transcripts. PMI deficiency abolishes or decreases the synthesis of mannose 6-phosphate from fructose 6-phosphate. Mannose provided by the diet or that released from cellular glycoconjugates becomes the only source of mannose 6-phosphate (Fig. 3). Whereas mannose uptake through a high affinity mannose-specific transporter has been demonstrated (16), the extent of reutilization is unknown. Mannose 6-phosphate supplies 9 of the 14 monosaccharides in the lipid-linked oligosaccharide (LLO) precursor required for protein N-glycosylation, can be a precursor for fucose, and is required for the synthesis of glycophospholipid-anchored glycoproteins. A decreased pool of mannose 6-phosphate results in a decreased amount of full-length LLO available for N-glycosylation. Hypoglycosylation of liver-derived serum glycoproteins was a prominent biochemical feature in the PMI-deficient patient and was as severe as in CDGS type Ia. Little is known about the quantity or bioavailability of diet-derived mannose. A regular diet apparently did not provide sufficient mannose for glycoprotein biosynthesis in the liver of our patient. By simply increasing the external supply of mannose, it was possible to correct the phenotype of PMI deficiency. The clinical symptoms disappeared within weeks after the initiation of mannose therapy and hypoglycosylation was nearly completely corrected after 11 mo. The apparently normal glycosylation of glycoproteins synthesized in cultured fibroblasts suggests that, under cell culture conditions in a medium lacking free mannose, the supply of mannose derived from breakdown of endogenous or internalized glycoconjugates is sufficient to ensure normal N-glycosylation. The same might be true for different organs in the patient. Phenotypic correction of the PMI deficiency in yeast was achieved by supplying 5 mM mannose together with glucose (20). It has to be mentioned that excessive mannose doses can be potentially toxic in PMI deficiency. However, this so-called "honeybee syndrome" (21) has no relevance for the oral mannose therapy in PMI-deficient patients, since the uptake capacity of the gut is a natural protection. Single mannose doses of > 200 mg/kg body wt induce osmotic diarrhea and limit the maximal serum concentrations that can be reached by oral uptake. The peak mannose concentrations in the patient's serum were 60-fold lower than the toxic dose in PMI-deficient tumor cells (22). Based on the four known types of CDG syndrome, it was generally assumed that CDG syndromes present as disorders with severe psychomotor and mental retardation. For this reason, CDGS diagnostics was restricted to children with neurological impairment. With the detection of PMI deficiency, this restriction is no longer prudent. Although the metabolic block in PMI deficiency is localized in the same biosynthetic pathway as in PMM deficiency, the clinical presentation of PMI deficiency is fundamentally different from all other types of CDGS. PMI deficiency is dominated by severe gastrointestinal symptoms and no neurological impairment was present in the patient. Only the occurrence of thrombosis and bleeding was a common clinical feature. The reason for the different clinical phenotypes is unknown, but could be related to the fact that mannose uptake from both mannose and glucose are blocked in PMM deficiency, whereas the uptake of exogenous mannose is unaffected by PMI deficiency. The PMI deficiency demonstrates that the screening for carbohydrate-deficient glycoproteins should be considerably extended if the complete spectrum of hypoglycosylation disorders in humans is to be detected. J Inherit Metab Dis. 1998 Apr. From 10 patients with carbohydrate-deficient glycoprotein (CDG) syndrome due to phosphomannomutase (PMM) deficiency, out of 10 lysosomal enzymes, 7 enzyme activities were measured in serum and 9 in leukocytes. In serum there was a 2-fold to 4-fold increase in activity of beta-glucuronidase, beta-hexosaminidase, beta-galactosidase, and arylsulphatase A. In leukocytes, however, several enzymes had reduced activity, particularly alpha-fucosidase, beta-glucuronidase and alpha-mannosidase. These abnormalities could result from missorting, defective reuptake and/or reduced stability of the enzymes due to the defective glycosylation. Glycobiology. 1998 Apr. The glycosylation of serum transferrin from galactosemic patients with a deficiency of galactose-1-phosphate uridyl transferase (EC 2.7.7.12) is abnormal but becomes normal after treatment with a galactose-free diet. To understand the structural and biochemical basis of the abnormal glycosylation, transferrin was purified from the serum of untreated and treated galactosemic patients and normal controls and the N-linked glycans analyzed by HPLC. The glycans from normal transferrin consisted predominantly (86%) of the disialylated biantennary complex type. The glycans from untreated galactosemic patients were more heterogeneous and contained four major truncated glycans in addition to a smaller amount (13%) of the disialylated biantennary complex type. The truncated glycans were deficient in galactose and sialic acid and their structures were consistent with a decrease in galactosyltransferase activity in hepatocytes, the probable cells of origin of the transferrin. This is postulated to be due to direct inhibition of the galactosyltransferase activity by the accumulated galactose-1-phosphate or to an effect on the formation of UDP-galactose, the donor substrate in the reaction. After treatment the proportion of the truncated glycans decreased and the proportion of the disialylated biantennary complex type increased, returning almost but never completely to normal, even after prolonged treatment in some cases. There was no clear relationship between the length of treatment and the normalization of glycosylation and the level of galactose-1-phosphate in red blood cells, the usual parameter for monitoring the treatment of galactosemics. It is suggested that the persistence of abnormally glycosylated proteins may contribute to the long-term complications in galactosemia. Scand J Clin Lab Invest. 1998 Feb. Carbohydrate-deficient glycoprotein syndrome type I (CDGS I) is an autosomal recessive disease with multiple organ manifestations. The diagnostic biochemical marker has been typical carbohydrate-deficient isoforms of transferrin (Tf). Many other glycoproteins in blood may show similar defects, but have not been systematically studied before. Forty-eight CDGS I patients and 22 controls were examined for total concentrations and isoform distribution of Tf, antithrombin (AT), alpha(1)-antitrypsin (alpha(1)-AT) and thyroxine-binding globulin (TBG), and for the level of carbohydrate-deficient transferrin (CDT). The absolute values varied with age. The most frequent persistent quantitative changes were reduced levels of AT (97%) and elevated CDT values (100%). Isoforms lacking one to eight of four to eight possible sialic acid residues were found in AT, TBG and Tf in all cases, with variable intensity and frequency, and in all except one patient in alpha(1)-AT. The isoform changes were most constant and pronounced in Tf. The other three glycoproteins showed more abnormal heterogeneity in the youngest than in the older patients. The results indicated that the biochemical defect stabilizes with age, and suggested partial hypoglycosylation rather than non-glycosylation of these glycoproteins. Analysis of Tf isoforms is still the safest diagnostic marker of CDGS I from full-term birth and over the ages. Crit Rev Oral Biol Med. 1998. Protein N-glycosylation is a metabolic process that has been highly conserved in evolution. In all eukaryotes, N-glycosylation is obligatory for viability. It functions by modifying appropriate asparagine residues of proteins with oligosaccharide structures, thus influencing their properties and bioactivities. N-glycoprotein biosynthesis involves a multitude of enzymes, glycosyltransferases, and glycosidases, encoded by distinct genes. The majority of these enzymes are transmembrane proteins that function in the endoplasmic reticulum and Golgi apparatus in an ordered and well-orchestrated manner. The complexity of N-glycosylation is augmented by the fact that different asparagine residues within the same polypeptide may be modified with different oligosaccharide structures, and various proteins are distinguished from one another by the characteristics of their carbohydrate moieties. Furthermore, biological consequences of derivatization of proteins with N-glycans range from subtle to significant. In the past, all these features of N-glycosylation have posed a formidable challenge to an elucidation of the physiological role for this modification. Recent advances in molecular genetics, combined with the availability of diverse in vivo experimental systems ranging from yeast to transgenic mice, have expedited the identification, isolation, and characterization of N-glycosylation genes. As a result, rather unexpected information regarding relationships between N-glycosylation and other cellular functions - including secretion, cytoskeletal organization, proliferation, and apoptosis - has emerged. Concurrently, increased understanding of molecular details of N-glycosylation has facilitated the alignment between N-glycosylation deficiencies and human diseases, and has highlighted the possibility of using N-glycan expression on cells as potential determinants of disease and its progression. Recent studies suggest correlations between N-glycosylation capacities of cells and drug sensitivities, as well as susceptibility to infection. Therefore, knowledge of the regulatory features of N-glycosylation may prove useful in the design of novel therapeutics. While facing the demanding task of defining properties, functions, and regulation of the numerous, as yet uncharacterized, N-glycosylation genes, glycobiologists of the 21st century offer exciting possibilities for new approaches to disease diagnosis, prevention, and cure. From the full text article: Because of their vital roles in so many biological processes, N-glycoproteins constitute an important group of proteins that are involved in various aspects of oral and craniofacial biology and pathology. They are likely to be critical to the development and homeostasis of oral tissues, such as the major and minor salivary glands, the gingiva, and the oral mucosa. In addition, the modification of proteins with N-linked glycans may affect the onset and progression of oral pathologies. Since the components of the immune system, such as immunoglobulins and selectins, are N-glycoproteins, N-linked glycans may function as potential regulators of oral immune responses, including mucosal immunity. Moreover, many N-glycoproteins, such as lactoferrin, participate directly in non-immune oral defenses. ... Acta Paediatr. 1997 Dec. Carbohydrate-deficient isoforms of transferrin (CDT) were examined in Guthrie cards from patients with galactosaemia before and during dietary treatment for up to 9 y. In untreated patients the CDT values were elevated due to abnormal asialo- and/or disialotransferrin. During treatment, the CDT levels were normal except on a few temporary occasions. Galactose or its close metabolites did not inhibit two relevant glycosyltransferases in vitro, and their levels were not correlated to the CDT values. The transferrin isoform changes in untreated patients were similar to, but less pronounced than in CDG syndrome type I. Glycobiology. 1997 Jul. One or more mevalonate derivatives of non-sterol type have been proposed to be of indispensable importance for cell growth. Conceivable mevalonate-dependent mechanisms involved in growth control are farnesylation of Ras proteins, regulation of c-myc expression, and N-linked glycosylation of the IGF-1 receptor. The latter mechanism might be rate-limited by dolichyl phosphate, which acts as a donor of oligosaccharides in glycoprotein synthesis in the endoplasmic reticulum. In order to study the significance for cell proliferation of the three aforementioned mevalonate-dependent processings, their inhibitory response due to mevalonate deprivation was explored and compared with the effect on DNA synthesis in the malignant melanoma cell line SK-MEL-2. We found that mevalonate depletion due to treatment with 3 microM lovastatin for 24 h, which efficiently growth-arrested the cells, hardly at all affected the expression of c-myc, and although Ras prenylation was inhibited by 50%, the most pronounced effect of lovastatin was seen on N-linked glycosylation of IGF-1 receptors, which was inhibited by more than 95%. The order and magnitude of the decreased IGF-1 receptor glycosylation, which was followed by a decreased expression of IGF-1 receptors at the cell membrane, correlated well with the inhibition of DNA synthesis. We investigated whether dolichol, and in particular dolichyl phosphate, through its participation in N-linked glycosylation, act as regulators of IGF-1 receptor expression. First, we could confirm that exogenous dolichol became phosphorylated and in this form took part in the glycosylation processing. Secondly, we showed that dolichyl phosphate, in a dose-dependent manner, could increase the number of IGF-1 receptors at the cell membrane, simultaneously as DNA synthesis was stimulated. Taken together, our results provide direct evidence for an important role of dolichyl phosphate as a regulator of cell growth through limiting N-linked glycosylation of the IGF-1 receptor. From the full text article: Mevalonate (MVA) has been demonstrated to be required for mammalian cell growth (Brown and Goldstein, 1980, 1994; Goldstein and Brown, 1990), as well as constituting the key metabolite in the biosynthesis of cholesterol and a variety of non-sterol isoprenoid derivatives. The production of MVA from 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) is catalyzed by HMG-CoA reductase, and represents the principal regulatory step in the pathway (Brown and Goldstein, 1980). Increasing evidence suggests that the MVA-derived product critical for cellular growth is an isoprene of non-sterol type (Brown and Goldstein, 1980, 1994; Goldstein and Brown, 1990). One MVA-derived product that may be involved in control of cellular growth is dolichyl phosphate, which acts as a carrier of oligosaccharides in the assembly of N-linked glycoproteins in the lumen of the endoplasmic reticulum (ER) (Hirschberg and Snider, 1987; Hart, 1992). It has been shown that proliferation of breast cancer cells is blocked specifically in Gl by HMG-CoA reductase inhibition (Larsson, 1993). This inhibition also resulted in a drastic depression of N-linked glycosylation. Following addition of MVA the depression of N-linked glycosylation was overcome and the cells subsequently initiated DNA synthesis (Larsson, 1993). However, if the MVA-induced increase in protein glycosylation was prevented by the N-linked glycosylation inhibitor tunicamycin (TM) (Heifetz et al, 1979), the cells were not able to proliferate. If instead TM was added 4 h after the addition of MVA, the cells synthesized DNA normally (Larsson, 1993). Similar stage-specific dependence of N-linked glycosylation in the Gl phase was found in 3T3-cells (Larsson, 1985) and in human fibroblasts (Carlberg et al., 1994). The latter study suggested that N-linked glycosylation of 90—240 kDa proteins in the prereplicative phase may be critical for induction of DNA replication (Carlberg et al., 1994). These high-molecular weight glycoproteins may include growth factor receptors. More recent studies have in fact shown that MVA is necessary for translocation of the IGF-1 receptor (IGF-IR) to the cell surface, a mechanism in turn critical for the subsequent initiation of DNA synthesis (Carlberg and Larsson, 1996; Carlberg et al., 1996). ... ...As shown, both the rate of N-linked glycosylation, protein prenylation and DNA synthesis was rapidly decreased following treatment with 3 (JLM lovastatin. After 24 h the levels of glycosylation, protein prenylation and DNA synthesis were reduced by 75%, 55%, and >95%, respectively. After repletion with MVA all three processings were restored within 24 h. ... ... As shown in Figure 2A, N-linked glycosylation of IGF-IR was reduced by 95% within 8 h. Addition of MVA after a 24 h depletion resulted in an increase in IGF-IR glycosylation. This increase was detected as soon as after 8 h, and after an additional 16 h N-linked glycosylation of IGF-IR was completely restored (Figure 2A). A considerably lower inhibition was seen regarding Ras prenylation (50% at 24 h) following MVA depletion (Figure 2A). This inhibition was likewise completely deleted after addition of MVA. Finally, lovastatin only slightly inhibited the expression of c-myc (Figure 2B). Thus, out of the three MVA-dependent processings analyzed in SK-MEL-2 cells, the most drastic effect of MVA depletion was seen on N-linked glycosylation of IGF-IR. In comparison with Figure 1, N-linked glycosylation of IGF-IR was more drastically affected by lovastatin (95% inhibition was obtained within 8 h) than overall N-linked glycosylation (60% inhibition was obtained within 8 h). These results might reflect a shorter half-life of IGF-IR and thus a higher turnover rate of its glycosylation than of the "average" N-linked glycoprotein. By performing Western blotting using an antibody against the a-subunit of the IGF-IR (N-20), it was shown that MVA depletion abolished the expression of membrane-bound IGF-IR in SK-MEL-2 cells (Figure 2C). A similar effect was seen in another cell line, WiDr, which is derived from human colon carcinoma (Figure 2C). Furthermore, immunocytostaining using a polyclonal antibody against the IGF-1R demonstrated that inhibition of MVA synthesis due to treatment with 3 u,M lovastatin led to a depletion of membrane-bound IGF-1R in SK-MEL-2 cells whereas the amount of intracellular ones remains unchanged (data not shown). We also confirmed that the specific N-linked glycosylation inhibitor TM and the IGF-1R antibody otIR-3 decreased IGF-1 binding and DNA synthesis substantially (by 75-90%) in SKMEL-2 cells which had been given both 3 uM lovastatin and 0.77 mM MVA (Figure 3). Thus, a growth-arresting effect can be obtained both by a lowered N-linked glycosylation of the IGF-1R, leading to an inhibited translocation and membrane expression, or by an antibody-mediated block of IGF-1 binding to the IGF-1R. From these data, N-linked glycosylation of IGF-1R emerges as pivotal in the regulation of cell growth. ... Discussion We have recently demonstrated that MVA is rate-limiting for translocation of de novo synthesized IGF-1R proteins to the cell surface and that this event is necessary for cell growth (Carlberg and Larsson, 1996; Carlberg et al., 1996). Our findings presented in this investigation provide strong evidence that dolichyl phosphate is the MVA-derived isoprene critical for cell growth through limiting N-linked glycosylation of IGF-IR proteins. Firstly, MVA deprivation due to treatment with a relatively low dose (3 uM) lovastatin drastically decreased N-linked glycosylation in SK-MEL-2 cells, especially of the IGF-IR, in close correlation with decreased DNA synthesis and growth arrest. On the contrary, lovastatin had a much less pronounced inhibitory effect on Ras prenylation, equal to the inhibition of total protein prenylation (which was not surprising since we found that the majority of prenylated proteins in SK-MEL-2 cells were of the same size as Ras). Furthermore, lovastatin hardly at all affected c-myc expression in these cells. Thus, out of the three proposed known MVA-dependent mechanisms involved in growth control, i.e., N-linked glycosylation of the IGF-IR (Carlberg and Larsson, 1996; Carlberg et al, 1996), Ras prenylation (Hancock et al, 1989; Schafer et al, 1989), and c-myc expression (Barbu and Dautry, 1990), N-linked glycosylation of the IGF-IR correlated closest to cellular growth. Secondly, addition of exogenous dolichyl phosphate, which was proven functional as an oligosaccharide donor, was demonstrated to increase de novo synthesis of membrane IGF-IR proteins in MVA-depleted SK-MEL-2. Dolichyl phosphate also in a dose-dependent manner increased 125I-IGF-1 binding (as did dolichol and MVA). In addition, the up-regulated 125IIGF-1 binding induced by dolichyl phosphate and MVA closely correlated to the stimulatory effect on DNA synthesis. Thus, these findings consist compelling evidence that MVA-dependent IGF-IR expression is regulated by the amount of dolichyl phosphate molecules, acting as oligosaccharide donors in the assembly of N-linked glycoproteins. Since the membrane expression of IGF-IR was dependent on dolichyl phosphate-stimulated glycosylation of receptor proteins, dolichyl phosphate appears crucial for growth control. Also in lovastatin-treated colon carcinoma cells (line WiDr) we found a decreased amount of membrane-bound IGF-IR. These findings indicate that dolichyl phosphate-dependent N-linked glycosylation of IGF-IR, necessary for its translocation to the cell surface, constitutes a widespread mechanism by which MVA regulates cell growth. Since lovastatin inhibits both farnesylation and geranylgeranylation, the relatively undramatic inhibitory effect on Ras prenylation could not be attributed to an unmeasured effect of any specific type of Ras prenylation. In addition, the Ras antibody used for immunoprecipitation in the present study, recognizes all kinds of mammalian Ras proteins (H-, K-, and N-Ras), why it can be excluded that our results are due to a lack of immunoreactivity with any specific Ras protein. Our results appear complementary to earlier findings that specific inhibition of Ras farnesylation using newly designed inhibitors of Ras famesyl transferase, is growth-inhibitory only in Ras-transformed cells (DeClue et al, 1991; James et al, 1994; Dalton et al, 1995; Danesi et al., 1996). Regarding Ras, it has also been shown that a very strong inhibition of HMG-CoA reductase activity is necessary to block its farnesylation (Leonard et al, 1990). In fact a 500-fold increase in lovastatin dose, as compared to that required for a 50% suppression of cholesterol synthesis, was required (Leonard et al, 1990). Moreover, the suppression of HMG-CoA reductase activity following treatment with 25-hydroxycholesterol was shown to be insufficient for this purpose (Leonard et aL, 1990). Taken together with several observations showing that growth inhibition can be obtained even with low or moderate doses of competitive inhibitors (Endo, 1985), as well as with oxysterols (Larsson and Zetterberg, 1986), not affecting Ras prenylation (Leonard et al., 1990), it appears that cell cycle arrest induced by HMG-CoA reductase inhibitors in mammalian cells is not primarily a result of suppressed farnesylation of p21ras. It has recently been reported that the farnesylation inhibitor BZA-5B, despite an effective inhibition of farnesylation of P21"1* and nuclear lamins, did not affect Ras membrane localization, lamin assembly, or growth of CH0-K1 cells (Dalton et al, 1995). Neither was NGF-mediated, Ras-dependent differentiation of PC-12 cells affected by BZA-5B (Dalton et al., 1995). The latter findings suggested that Ras function is independent of farnesylation (Dalton et al., 1995). Thus, Ras prenylation does not appear to be the most critical MVA-dependent growth regulatory mechanism. Instead, dolichyl phosphate-dependent N-linked glycosylation and cell surface expression of IGF-1R proteins might constitute a widespread MVA-regulated mechanism, obtained by MVA-mediated limitation of the number of dolichyl phosphate molecules. This matter must of course be investigated in other systems to examine the generality of our results. It would be interesting to study the role of dolichyl phosphate for expression of IGF-1R in an IGF-1R knockout cell line transfected with IGF-1R cDNA, for example. MVA has also been reported to affect c-fos and c-myc (Barbu and Dautry, 1990; Vincent et al., 1991). MVA deprivation before growth stimulation of quiescent human fibroblasts by serum decreased the gene expression of c-fos and c-myc (Barbu and Dautry, 1990). These effects seemed to take place at the transcriptional level and did not depend on the type of the initial events of signal transduction (Barbu and Dautry, 1990). A quite novel study shows that the protein prenylation inhibitor phenyl acetate also induces myc suppression (Danesiet aL, 1996), a result indicating that Ras prenylation is necessary for mediation of the molecular events leading to myc expression. The moderate inhibition of Ras prenylation and the unaffected myc expression presented in this study do not contradict this finding, assuming that the action of Ras precedes the action of myc and that downregulation of myc is very slow. It is also possible that despite a moderate inhibition of Ras prenylation, the remaining amount of prenylated Ras proteins are enough to maintain the expression of myc. Growth stimulation by for example PDGF includes activation of Ras (McCormick, 1994) and HMG-CoA reductase (Habenicht et al., 1980), induction of c-fos and c-myc RNA (Paulsson etal., 1987), IGF-1R transcripts (Rubini etal., 1994) and increased IGF-1 production (Clemmons, 1984). Recently, another set of genes, involved in the early steps of the dolichol pathway, have been demonstrated to be coordinately induced as the early genes (Lennon et aL, 1995). These genes are ALG7, ALG1, ALG2, and two genes encoding oligosaccharyl transferase units (Lennon et al., 1995). ALG7 encodes the first enzyme in the dolichol pathway of N-linked glycosylation, i.e., dolichol-P-dependent N-acetylglucosamine-1-P transferase (GPT) (Mota et aL, 1994). ALG7 is regulated at two major control points in the Gl phase of the S. cerevisiae cell cycle, G0/G1 and START (Pretel et al., 1995). Since growth stimulation requires an increased glycoprotein synthesis, due to the need for, e.g., IGF-1 receptors (Carlberg and Larsson, 1996; Carlberg et al., 1996), it seems reasonable that except for an increased production of MVA and dolichyl phosphate induced by the activation of HMG-CoA reductase, activation of the dolichol pathway enzymes is also included in the growth stimulatory response. It is certainly interesting to find out whether overexpression of the IGF-1R in tumor cells can be stimulated only by an increased dolichyl phosphate production, or if other genes like ALG7 also must be coordinately up-regulated. It appears however that cell cycle control is not at all points dependent of the dolichol pamway and N-linked glycosylation. For example, the PDGF receptor still binds its ligand PDGF and subsequently undergoes autophosphorylation even if under-glycosylated (Keating et al., 1989). Thus, it is reasonable that some pathways of growth signal transduction are not affected by an inhibited N-linked glycosylation. PDGF-induced myc expression, perhaps via Ras, might represent one such pathway, which would explain why the expression of myc was not affected by lovastatin. Pediatr Res. 1996 Nov. Isoelectrofocusing of serum sialotransferrins from patients with untreated hereditary fructose intolerance (HFI) shows a cathodal shift similar to that in carbohydrate-deficient glycoprotein (CDG) syndrome type I and in untreated galactosemia. This report is on serum lysosomal enzyme abnormalities in untreated HFI that are identical to those found in CDG syndrome type I but different from those in untreated galactosemia. CDG syndrome type I is due to phosphomannomutase deficiency, a defect in the early glycosylation pathway. It was found that fructose 1-phosphate is a potent competitive inhibitor (Ki congruent to 40 microM) of phosphomannose isomerase (EC 5.3.1.8), the first enzyme of the N-glycosylation pathway thus explaining the N-glycosylation disturbances in HFI. Am J Hum Genet. 1996 Oct. Carbohydrate-deficient glycoprotein syndrome (CDGS) type II is a multisystemic congenital disease with severe involvement of the nervous system. Two unrelated CDGS type II patients are shown to have point mutations (one patient having Ser-->Phe and the other having His-->Arg) in the catalytic domain of the gene MGAT2, encoding UDP-GlcNAc:alpha-6-D-mannoside beta-1,2-N- acetylglucosaminyltransferase II (GnT II), an enzyme essential for biosynthesis of complex Asn-linked glycans. Both mutations caused both decreased expression of enzyme protein in a baculovirus/insect cell system and inactivation of enzyme activity. Restriction-endonuclease analysis of DNA from 23 blood relatives of one of these patients showed that 13 donors were heterozygotes; the other relatives and 21 unrelated donors were normal homozygotes. All heterozygotes showed a significant reduction (33%-68%) in mononuclear-cell GnT II activity. The data indicate that CDGS type II is an autosomal recessive disease and that complex Asn-linked glycans are essential for normal neurological development. From the full text article: The two CDGS type II patients, a girl (patient A) and a boy (patient B), came from unrelated Iranian and Belgian families, respectively. Both patients had a similar facial dysmorphy, a ventricular septal defect, and severely retarded psychomotor development (Jaeken et al. 1993b, 1994). Isoelectrofocusing of serum transferrin showed that tetrasialotransferrin was nearly absent compared with that in controls and that there was a large increase of disialotransferrin carrying two truncated biantennary N-acetyllactosaminic glycans in each of which the trisaccharide element sialyl(ct2-6)Gal(P1-4)GlcNAc(P1-2) attached to the (al-6)-linked Man residue of the N-glycan core was missing (Jaeken et al. 1994). This is consistent with the finding that fibroblast extracts from the two CDGS type II patients A and B showed a reduction of >98% in GnT II (Jaeken et al. 1994) and that patient B had no detectable GnT II in his mononuclear-cell extract (Charuk et al. 1995). Thromb Haemost. 1996 Oct. CDG syndrome (CDGS) type I is the most frequent form of a group of metabolic disorders characterised by a defect of the carbohydrate moiety of glycoproteins. A large number of plasma glycoproteins, including clotting factors and inhibitors, are decreased and stroke-like episodes have been described in about half of the reported patients. We studied blood coagulation factors, inhibitors and D-dimer plasma levels in four subjects, aged 12-23 years, with CDGS type I. Factors VIII, XI, antithrombin III activity, antigen plasma levels of antithrombin III, free protein S and protein C were decreased whereas protein C as activity was normal. In addition two patients had reduction of factors II, V, VII, IX, X reflecting the phenotypic heterogeneity associated with CDGS type I. D-dimer plasma concentrations were elevated in all subjects. The hypercoagulable state as consequence of the combined deficiencies of coagulation inhibitors could contribute to the stroke-like phenomena in CDGS type I. J Biol Chem. 1996 Jul 19. Depletion of mevalonic acid (MVA), obtained by inhibition of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase using lovastatin, depressed the biosynthesis of dolichyl-phosphate and the rate of N-linked glycosylation and caused growth arrest in the melanoma cell line SK-MEL-2. The growth arrest was partially prevented by addition of high concentrations of insulin-like growth factor-1 (IGF-1) to the cells, indicating that MVA depletion may inhibit cell growth through decreasing the number of IGF-1 receptors (IGF-1R) at the cell surface. Such a decrease in receptor number might be a result of a lowered translocation of de novo synthesized receptors to the cell membrane which in turn might be a result of a decreased N-linked glycosylation of the receptor proteins. We could also demonstrate that IGF-1R became underglycosylated and that the amount of de novo synthesized IGF-1R proteins at the cell membrane was drastically decreased upon MVA depletion. Analysis of receptor proteins cross-linked with IGF-1, as well as binding assays and immunocytostaining confirmed that the number of functional membrane-bound IGF-1R was substantially reduced. The N-linked glycosylation and the expression of de novo synthesized IGF-1R proteins at the cell surface as well as the number of IGF-1 binding sites were completely restored upon replenishment of MVA. These effects of MVA were efficiently abrogated by the glycosylation inhibitor tunicamycin. The translocation of IGF-1R to the cell membrane was shown to take place just prior to initiation of DNA synthesis in arrested cells stimulated with MVA. Additionally, there was a clear correlation between IGF-1 binding and initiation of DNA synthesis with regard to the MVA dose requirement. It was confirmed that inhibition of HMG-CoA reductase activity and N-linked glycosylation also depressed the expression of functional IGF-1R in other cell types (i.e. hepatoblastoma cells and colon cancer cells). Our data suggest that this mechanism is involved in MVA-regulated cell growth. From the full text article: It is well known that MVA1 is required for growth of mammalian cells (1, 2, 3, 4) as well as constituting the key metabolite in the biosynthesis of cholesterol and a variety of nonsterol isoprenoid derivatives (e.g. dolichyl-phosphate, farnesyl pyrophosphate, isopentenyladenine, and ubiquinone). The formation of MVA from HMG-CoA, which is catalyzed by HMG-CoA reductase, is the principal regulatory step in the pathway (1). Increasing evidence suggests that the MVA-derived product critical for cell growth is an isoprene of nonsterol type (1, 2, 3, 4). A possible mechanism for MVA-regulated cell growth is the involvement of dolichyl-phosphate in N-linked glycosylation. De novo synthesized dolichyl-phosphate acts as a carrier of oligosaccharides in the assembly of glycoproteins in the lumen of the endoplasmic reticulum (ER) (5, 6). In a recent study we investigated the potential regulatory role of N-linked glycosylation in initiation of DNA synthesis in human fibroblasts stimulated by serum (7). Our results suggested that N-linked glycosylation of proteins of 90-240 kDa in the prereplicative phase may be critical for induction of DNA replication. These high molecular mass glycoproteins may include growth factor receptors. This raises the possibility that MVA may regulate the expression of growth factor receptors through limiting the biosynthesis of dolichyl-phosphate. The existence of such a mechanism would constitute a substantial link between HMG-CoA reductase and cell growth. The aim of the present study was to investigate this issue in detail. Our experiments provide evidence that MVA is critical for the translocation of insulin-like growth factor-1 receptor (IGF-1R) to the cell surface. ... The extracellular domains of growth factor receptors usually contain N-glycosidically linked carbohydrates (11, 26). Since inhibition of MVA synthesis leads to a decreased rate of N-linked glycosylation (cf. Fig. 1, A and B), the possibility is raised that MVA may be rate-limiting for the receptor function. For instance, MVA-regulated glycosylation might be required for the translocation of growth factor receptor proteins from ER to the cell membrane. If this is the case, a suppression of HMG-CoA reductase activity would reduce the number of growth factor receptors at the cell surface. This would lead to a decreased number of receptor-ligand interactions, which in turn would decrease receptor-mediated signaling events (i.e. tyrosine phosporylation) and finally lead to growth arrest. Hypothetically, this inhibitory response would be delayed if increased concentrations of the appropriate ligand were administered. In Table II it is shown that neither bombesin, EGF, platelet-derived growth factor nor transforming growth factor 1 could counteract the growth inhibitory effect following inhibition of HMG-CoA reductase. In contrast, both insulin and IGF-1 caused a substantial, dose-dependent, counteractive effect. ... ... It has been confirmed elsewhere that IGF-1R contains N-glycosidically linked carbohydrates (11). The alpha-subunit contains 11 possible glycosylation sites, whereas the beta-subunit contains only 5 sites (11). This means that the whole receptor, which is an alpha-beta-beta-alpha oligotetramer, may contain 32 glycosylation sites. ... Our present study on SK-MEL-2 cells demonstrates that regulation of IGF-1R expression at the cell surface constitutes a MVA-dependent mechanism. Inhibition of HMG-CoA reductase rapidly decreased the number of IGF-1R molecules at the cell membrane which occurred simultaneously with a decline in the rate of cell proliferation. Furthermore, initiation of DNA synthesis was preceded by a drastic increase in the expression of IGF-1R when arrested cells were stimulated with MVA. The importance of the IGF-1R for cell cycle progression of SK-MEL-2 cells was confirmed by blocking the IGF-1R binding sites with an antibody against the binding domain of IGF-1R. We provide evidence that MVA through regulating the rate of N-linked glycosylation is limiting for the translocation of IGF-1R from the intracellular compartment (ER and Golgi) to the cell surface. The mechanism by which MVA is limiting for N-linked glycosylation is that intermediates in the metabolism of MVA serve as precursors in the biosynthesis of dolichyl-phosphate. It was also confirmed in our experiments that inhibition of HMG-CoA reductase resulted in a rapid and substantial suppression of dolichyl-phosphate synthesis. Only newly synthesized dolichyl-phosphate has in fact been shown to be involved in N-linked glycosylation (37, 38). N-Linked glycosylation has been shown to be carried out on all proteins that are to be transported to the cell membrane (5, 39). The core structure of the oligosaccharide complex is commonly composed of two N-acetyl-GlcN molecules, nine mannose and three glucose residues. Dolichyl-phosphate acts as a membrane-bound carrier of this complex inside the ER and makes it available to an oligosaccharyl transferase, which couples it to asparagine residues of de novo synthesized proteins. Only sites containing Asn-X-Thr/Ser sequences (X is any amino acid with the exception of proline) are recognized by the transferase (6, 40). Since N-linked glycosylation occurs cotranslationally (6, 33, 34) only newly synthesized proteins are modified in this manner. In the present study we demonstrate that N-linked glycosylation of IGF-1R was efficiently blocked upon inhibition of HMG-CoA reductase, and also that the amount of de novo synthesized IGF-1R proteins at the cell surface was drastically decreased. In fact the expression of de novo synthesized IGF-1R proteins at the cell membrane correlated well with the MVA-dependent changes in IGF-1 binding. This strongly suggests that the MVA-controlled translocation of newly synthesized and glycosylated receptors plays a pivotal role in the expression of functional IGF-1 binding sites. In glycoproteins the carbohydrate moieties attached to the asparagine residues serve several and different specific purposes. For instance, N-linked glycosylation may prevent intracellular protein aggregation in the ER and may in addition be required for correct folding and cleavage of the precursor proteins. The N-linked oligosaccharides may also be needed for the direction of proteins through different compartments of the Golgi network in which they are further processed before the subsequent translocation to the cell surface (39). Deficiency in N-linked glycosylation at certain glycosylation sites may therefore result in incorrect protein folding, suppressed efficiency and decreased rate of intracellular glycoprotein transport, or impaired protein function. Experiments with glycosylation site mutations of transfected cDNA of the insulin receptor suggest that the first four glycosylation sites of the alpha subunit have to be glycosylated for the subsequent intracellular transport of de novo synthesized receptor proteins (26). When these four glycosylation sites, due to mutations, were not glycosylated, the proteins were retained in the ER (26). These data taken together with the fact that a large homology exists between insulin-R and IGF-1R (11) suggest that translocation-specific glycosylation sites also exist in IGF-1R. On the other hand, N-linked glycosylation is not critical for the translocation of all glycoproteins destined for the cell membrane. For example, glycosylation is not required for the export of the apoprotein B of the low density lipoprotein (41), the acetylcholine receptors (42), muscarinic receptors (43), and rhodopsin in retinal disk membranes (44). As was also shown in our experiments, inhibition of HMG-CoA reductase only caused a limited decrease in the total amount of de novo synthesized proteins located at the cell membrane. This indicates that glycoproteins on the whole are only slightly inhibited in their translocation to the cell surface. ... Eur J Pediatr. 1996 Apr. Blood Coagul Fibrinolysis. 1996 Mar. Carbohydrate-deficient glycoprotein (CDG) syndrome type I is an autosomal recessive disease with multisystemic manifestations. During childhood the patients may suffer from hemorrhages, which may be lethal, venous thromboses and stroke-like episodes. In this study 15 patients with CDG syndrome type I were examined from the levels and isoform patterns of coagulation factors and inhibitors and fibrinolysis parameters. The screening assays APTT and PTC were unaffected in most cases. In spite of this reduced levels were found particularly for factors II, V, X and XI and for antithrombin and protein C. Low values tended to be associated with elevated liver enzyme levels in serum. The values were at potential clinical risk levels for protein C and/or antithrombin in more than half of the patients, and for factor V and/or factor XI in one third of them. There were no current differences in values between patients who had previously displayed clinical symptoms of coagulation disturbance and those without such symptoms. Partially carbohydrate-deficient isoforms were demonstrated in antithrombin, protein C, protein S and in alpha 2-antiplasmin, but not in factors II, X and fibrinogen. Abnormal isoforms did not appear to reduce the functional activity of the respective glycoproteins. Analysis of individual hemostatic parameters is recommended in these patients in connection with clinical symptoms or elective surgery. The observed variability of the carbohydrate defect in glycoproteins in this disease may be a clue to its pathogenesis. J Clin Endocrinol Metab. 1995 Dec. Carbohydrate-deficient glycoprotein (CDG) syndrome is a newly recognized hereditary disorder that presents with psychomotor retardation, cerebellar ataxia, peripheral sensorimotor neuropathy, and, variably, skeletal abnormalities, lipodystrophy, and retinitis pigmentosa. These abnormalities appear to be produced by a defect that causes reduced carbohydrate content in glycoproteins. We studied seven patients with CDG type I belonging to five unrelated families. The concentration of serum TBG, a glycoprotein of hepatic origin, was measured by RIA and T4 saturation and was found to be below the normal range in three of the seven patients and normal in four of them. More than half of the total serum TBG had reduced sialic acid content and localized on isoelectric focusing (IEF) as two prominent bands cathodal to the three major bands of normal TBG. The latter two bands are responsible for the characteristic IEF pattern or CDG syndrome. TBG in patients with CDG had immunoreactivity indistinguishable from that of normal TBG and had normal affinity for T4, T3, and rT3. Serum total T4, T3, and rT3 were below the normal range in seven, five, and seven patients, respectively. The free T4 index was also below normal in four patients, but the free T4 concentration, measured by equilibrium dialysis at low dilution, and serum TSH were in the midnormal range. The serum total T4 and rT3 levels were disproportionately reduced relative to the serum TBG concentration and compared to the concentrations of these iodothyronines in matched subjects with inherited partial TBG deficiency. Chronic illness cannot explain these changes, because, contrary to patients with nonthyroidal illness, those with CDG had significantly higher serum total T3/T4 and lower rT3/T4 ratios. It is concluded that IEF of TBG is a rapid and simple method for the diagnosis of CDG type I and that the abnormal pattern can be detected as early as 5 days postpartum. Patients with CDG are chemically euthyroid, and it is postulated that the reduction in serum iodothyronine concentrations beyond that explained on the basis of low TBG levels may be due to the interference with binding to TBG by an unidentified substance. Thromb Res. 1994 Oct 15. The carbohydrate deficient glycoprotein (CDG) syndrome is a newly described disorder characterized by impaired glycosylated molecules. It has been reported that transient stroke-like episodes appear in half of the patients. We performed hemostatic studies on three CDG syndrome patients belonging to two unrelated families. The most characteristic findings were decreases in antithrombin III (AT III), protein C and alpha 2 plasmin inhibitor to nearly half normal levels. Protein S was reduced in two (siblings) patients. Isoelectric focusing of AT III in native plasma revealed decreased intensity of the major band and increased intensity of a minor cathodal band. These minor AT III molecules were considered to lack an oligosaccharide sidechain. A 12-year-old girl defective not only for AT III but also protein C and protein S developed disseminated intravascular coagulation accompanied by arterial thrombosis in her left hand following dyspnea associated with bronchial asthma. These findings suggest that thrombotic predisposition in patients with CDG syndrome is due to decreased levels of major coagulation inhibitors, particularly as a result of impaired glycosylation of AT III. J Neurol Neurosurg Psychiatry. 1994 May. A new group of recessively inherited metabolic disorders affecting glycoprotein metabolism has been identified--the carbohydrate-deficient-glycoprotein (CDG) syndromes. Here the course and clinical expression of CDG syndrome type I in 13 patients who have passed the age of 15 years are described. All presented with early onset psychomotor retardation, in most cases combined with slight facial dysmorphic features, some degree of hepatic dysfunction, and in one case, pericardial effusion. About half of the patients had subcutaneous lipodystrophy and comatose or stroke-like episodes during childhood. After the age of 15 the disease was mainly characterised by neurological symptoms consisting of non-progressive ataxia associated with cerebellar hypoplasia, stable mental retardation, variable peripheral neuropathy, and strabismus. One third of the patients had generalised seizures, usually sporadic, and all had retinal pigmentary degeneration. In all cases there was more or less pronounced thoracic deformity and no female had passed puberty. Also, the oldest female showed premature aging. Severe internal organ symptoms, which are common in pediatric patients, were absent. All patients had highly raised serum concentrations of the biochemical marker carbohydrate-deficient transferrin, which can be used to verify the diagnosis. It is concluded that after childhood, CDG syndrome type I is a largely non-progressive disease compatible with a socially functioning but dependent lifestyle. Pediatr Neurol. 1993 Jul-Aug. A new group of metabolic disorders, the carbohydrate-deficient glycoprotein (CDG) syndromes, is reviewed with emphasis on the key condition, the CDG syndrome type I. This disease, an autosomal-recessive multisystem condition, has now been diagnosed in 45 Scandinavian patients. It is characterized by carbohydrate deficiencies of a number of glycoproteins, including uniform changes in transferrin. The transferrin alterations provide a distinct biologic marker and a practical and simple laboratory diagnostic means employing analysis of serum or blood spots from Guthrie-type filter paper. The syndrome presents differently through various life periods. A four-stage grouping system by age has been constructed and is presented. During infancy, internal organ symptoms are dominant; some may be life-threatening. In later childhood and adolescence, static mental deficiency, cerebellar ataxia, slowly progressive lower limb neuropathy, and pigmentary retinal degeneration, as well as secondary skeletal deformities, are the most prominent findings. Two very recently described clinical and biologic variants, CDG syndromes II and III, are summarized and compared to CDG type I. Pediatr Res. 1993 May. The carbohydrate-deficient glycoprotein syndromes are a recently individualized group of genetic multisystemic disorders. A predominant feature is a severe involvement of the central and peripheral nervous system resulting in psychomotor retardation, seizures, ataxia, and, mostly after infancy, stroke-like episodes. The hallmark biochemical feature is a carbohydrate deficiency in a large number of serum glycoproteins. Because coagulation factors and inhibitors are also glycoproteins, we performed a systematic study of these factors and inhibitors in nine patients with carbohydrate-deficient glycoprotein syndrome. All showed a decreased activity of factor XI and of the coagulation inhibitors antithrombin III and protein C. In five of seven patients more than 1 y old, there was also a (less pronounced) decrease of protein S and of heparin cofactor II. This combined coagulation inhibitor deficiency could explain the stroke-like episodes occurring in these children. Acta Paediatr. 1993 Jan. Carbohydrate-deficient glycoprotein syndrome is a recently identified recessively inherited, multisystemic disease with severe nervous system involvement. It is characterized biochemically by carbohydrate-deficient serum glycoproteins, and can be diagnosed by analysis of abnormal isoforms of serum transferrin. Using stored, neonatally collected filter paper blood spots from such patients, it was shown that neonatal diagnosis was possible by immune-isoelectric focusing of transferrin eluted from up to 14-year-old samples. Freshly collected blood on filter paper was readily analyzed quantitatively for carbohydrate-deficient isotransferrins by a rapid microchromatographic assay, revealing highly elevated values in all patients. The presently described methods thus provide a means for early diagnosis of the carbohydrate-deficient glycoprotein syndrome in microliter volumes of capillary blood. Sampling on filter paper offers an important simplification in sample collection, storage and transport, and may make population studies possible. J Pediatr. 1993 Jan. We diagnosed the carbohydrate-deficient glycoprotein syndrome in five children who were seen during their first year of life with failure to thrive, feeding difficulties, psychomotor retardation, hypotonia, esotropia, inverted nipples, lipodystrophy, pericardial effusion, and hepatic dysfunction. Steatosis was observed in liver biopsy specimens, and cerebellar hypoplasia was present on computed tomography. The disorder is characterized by a complex carbohydrate deficiency in certain glycoproteins, notably transferrin, which can be used as a marker of the disease. The carbohydrate-deficient glycoprotein syndrome may be an important and easily identifiable cause of failure to thrive and neurologic dysfunction in infancy. The presence of the disorder in siblings of different gender and the finding of biochemical abnormalities in some unaffected parents suggest an autosomal recessive inheritance. J Inherit Metab Dis. 1993. The carbohydrate-deficient glycoprotein (CDG) syndromes are a newly recognized family of diseases with autosomal recessive inheritance. The basic defects are probably in the glycosylation pathway (endoplasmic reticulum, Golgi apparatus or post-Golgi). In the present state of our knowledge the central nervous system is always severely affected but nearly all other organs are involved to a variable degree. Like the peroxisomal disorders they also comprise dysmorphic features, the most typical being an abnormal distribution of subcutaneous adipose tissue. A reliable diagnostic test is isoelectric focusing of serum transferrin showing a cathodal shift as a consequence of the partial sialic acid deficiency. Prenatal diagnosis and heterozygote detection are not yet available. These diseases should be differentiated from secondary CDG syndromes such as classical galactosaemia. J Inherit Metab Dis. 1993. The carbohydrate-deficient glycoprotein (CDG) syndromes are a newly recognized group of inherited metabolic diseases. We report a Japanese brother and sister with a CDG syndrome. Both patients showed decreased activities of blood coagulation Factor XI and of the coagulation inhibitor protein C. In one of them there was also a somewhat decreased activity of Factor IX and of antithrombin III. Isoelectric focusing of antithrombin III revealed a decrease of negatively charged fractions and an increase of more cathodal bands. Furthermore, there was a discrepancy between activity and antigen level of Factor VIII and protein C. The patients had an incidental deficiency of factor XII. This is the first detailed report on blood coagulation systems in the CDG syndromes. These blood coagulation abnormalities may explain at least in part the thrombotic or haemorrhagic complications of the CDG syndromes. Tidsskr Nor Laegeforen. 1991 Apr 20. The carbohydrate-deficient glycoprotein syndrome is a newly described hereditary disorder which may be due to a defect in the glycoprotein metabolism. Predominant symptoms are mental retardation, epilepsy, cerebellar ataxia, polyneuropathy, squint, retinitis pigmentosa, retarded growth, hypothyroidism and liver steatosis. Increased serum glycoprotein-deficient transferrin is a marker of the disease and confirms the diagnosis. We describe four Norwegian children with this syndrome. Olivopontocerebellar degeneration was found upon examination of the brain in two patients who died. |
