Molecular and clinical description of the first US patients with congenital disorder of glycosylation Ig

In this report we describe the first two US patients with congenital disorder of glycosylation type Ig (CDG-Ig). Both patients presented with symptoms indicating CDG, including developmental delay, hypotonia and failure to thrive, and tested positive for deficient glycosylation of transferrin. Labeling of the patients' lipid-linked oligosaccharides suggested mutations in the hALG12 gene, encoding a mannosyltransferase. Both patients were shown to carry previously unpublished hALG12-mutations. Patient 1 has one allele with a deletion of G29, resulting in a premature stop codon, and another allele with an 824G>A mutation yielding an S275N amino acid change. Patient 2 carries two heterozygous mutations (688T>G and 931C>T), resulting in two amino acid exchanges, Y230D and R311C. An adenoviral vector expressing wild type hALG12 corrects the abnormal lipid-linked oligosaccharide pattern of the patients' cells. In addition to common CDG symptoms, these patients also presented with low IgG and genital hypoplasia, symptoms previously described in CDG-Ig patients. We therefore conclude that a combination of developmental delay, low IgG, and genital hypoplasia should prompt CDG testing.     

Analysis of multiple mutations in the hALG6 gene in a patient with congenital disorder of glycosylation Ic

Congenital disorder of glycosylation Ic is caused by mutations in the hALG6 gene that encodes an alpha-1,3 glucosyltransferase. This enzyme is required for the addition of the first glucose residue to the lipid-linked oligosaccharide precursor for N-linked glycosylation. Here we describe the biochemical and molecular analysis of a patient with three mutations in the hALG6 gene. The maternal allele has an intronic G --> A mutation resulting in skipping of exon3 (IVS3 + 5G > A). This produces a nonfunctional enzyme as shown by its inability to restore normal glycosylation in a Saccharomyces cerevisiae strain lacking a functional ALG6. The paternal allele has two mutations. One is a deletion of three bases (895-897delATA) leading to an in-frame deletion of isoleucine 299 (delI299) located in a transmembrane domain. The second mutation on the same allele 911T > C causes a F304S change. When expressed in the ALG6 deficient yeast strain, this allele restores glycosylation but the mRNA is unstable or inefficiently transcribed, contributing to the impaired glycosylation in the patient.     

Deficiency of the first mannosylation step in the N-glycosylation pathway causes congenital disorder of glycosylation type Ik

Defects of N-linked glycosylation represent diseases with multiple organ involvements that are classified as congenital disorders of glycosylation (CDG). In recent years, several CDG types have been attributed to defects of dolichol-linked oligosaccharide assembly in the endoplasmic reticulum. The profiling of [3H]mannose-labeled lipid-linked oligosaccharides was instrumental in identifying most of these glycosylation disorders. However, this method is poorly suited for the identification of short lipid-linked oligosaccharide biosynthesis defects. To adequately resolve deficiencies affecting the first steps of lipid-linked oligosaccharide formation, we have used a non-radioactive procedure employing the fluorescence detection of 2-aminobenzamide-coupled oligosaccharides after HPLC separation. By applying this method, we have detected the accumulation of dolichylpyrophosphate-GlcNAc2 in a previously untyped CDG patient. The accumulation pattern suggested a deficiency of the ALG1 beta1,4 mannosyltransferase, which adds the first mannose residue to lipid-linked oligosaccharides. This was supported by the finding that this CDG patient was compound heterozygous for three mutations in the ALG1 gene, leading to the amino acid substitutions S150R and D429E on one allele and S258L on the other. The detrimental effect of these mutations on ALG1 protein function was demonstrated in a complementation assay using alg1 Saccharomyces cerevisiae yeast mutants. The ALG1 mannosyltransferase defect described here represents a novel type of CDG, which should be referred to as CDG-Ik.     

Novel ALG8 mutations expand the clinical spectrum of congenital disorder of glycosylation type Ih

Congenital disorders of glycosylation (CDG) are an expanding group of inherited disorders caused by defects in the N- or O-Glycosylation of proteins and lipids. Several CDG subtypes have been described so far, including CDG type Ih which is caused by a deficiency of the dolichyl-P-Glc:Glc₁Man₉GlcNAc₂-PP-dolichyl α1,3-glucosyltransferase (hALG8). The defect leads to an accumulation of Dol-PP-GlcNAc₂Man₉ and Dol-PP-GlcNAc₂Man₉Glc₁ in the endoplasmic reticulum of patients’ fibroblasts that can be detected by analyzing the lipid-linked oligosaccharyl intermediates. Five patients with CDG-Ih have been described so far. The clinical presentation of four of these patients was severe with death in early infancy. In this report, we describe two mildly affected siblings with CDG-Ih caused by two novel mutations.While one mutation (c.1434delC) causes a frame shift resulting in a premature termination codon (p.485X), the point mutation of the other allele (c.845C>T, p.A282V) causes an amino acid replacement in a highly conserved region of the hALG8 gene. The two siblings show similar symptoms, including pseudo-gynecomastia, epicanthus, muscular hypotonia, mental retardation and ataxia, expanding the genetic and clinical spectrum of CDG-Ih.     

ALG11‐CDG syndrome: Expanding the phenotype

ALG11‐Congenital Disorder of Glycosylation (ALG11‐CDG, also known as congenital disorder of glycosylation type Ip) is an inherited inborn error of metabolism due to abnormal protein and lipid glycosylation. We describe two unrelated patients with ALG11‐CDG due to novel mutations, review the literature of previously described affected individuals, and further expand the clinical phenotype. Both affected individuals reported here had severe psychomotor disabilities and epilepsy. Their fibroblasts synthesized truncated precursor glycan structures, consistent with ALG11‐CDG, while also showing hypoglycosylation of a novel biomarker, GP130. Surprisingly, one patient presented with normal transferrin glycosylation profile, a feature that has not been reported previously in patients with ALG11‐CDG. Together, our data expand the clinical and mutational spectrum of ALG11‐CDG.     

A frequent mild mutation in ALG6 may exacerbate the clinical severity of patients with congenital disorder of glycosylation Ia (CDG-Ia) caused by phosphomannomutase deficiency

Single nucleotide polymorphisms occur throughout the human genome. A gene that causes one of the congenital disorders of glycosylation (CDG) has a mutation (911T-->C ) that changes a phenylalanine to serine at position 304 (F304S) of the alpha 1,3 glucosyl transferase. We show that this change reduces the ability of the gene product to rescue defective glycosylation of an alg6-deficient strain of Saccharomyces cerevisiae during rapid growth. This finding suggested that the mutation might affect glycosylation in humans. We therefore compared the frequency of this variant in 301 controls and in 101 CDG patients who carry known mutations in other genes involved in CDG, i.e. PMM2 (CDG-Ia; 91 patients) and MPI (CDG-Ib; 10 patients). The variant allele frequency is identical in both CDG patients (0.30) and controls (0.28). Importantly, the F304S genotype frequency in 55 CDG-Ia patients classified as mild/moderate (n = 28), or severe (n = 27) was significantly higher in severely affected patients (0.41) than in mild/moderately affected patients (0.21). Mortality (n = 9) was higher when F304S was present (n = 6). Severely affected patients with the PMM2 mutations F119L/R141H (n = 22) carry the F304S mutation more often (0.36) than mildly affected patients (0.18, n = 11) with this mutation. Clinical severity of mildly affected sibs with the same PMM2 mutations did not correlate with F304S genotype. Thus, the presence of the F304S allele may exacerbate the clinical outcome, especially in severely affected CDG patients. We speculate that this type of variant may be implicated in other multi-factorial disorders that involve N-glycosylation.     

Therapeutic approaches in Congenital Disorders of Glycosylation (CDG) involving N-linked glycosylation: an update

Congenital disorders of glycosylation (CDG) are a group of clinically and genetically heterogeneous metabolic disorders. Over 150 CDG types have been described. Most CDG types are ultrarare disorders. CDG types affecting N-glycosylation are the most common type of CDG with emerging therapeutic possibilities. This review is an update on the available therapies for disorders affecting the N-linked glycosylation pathway. In the first part of the review, we highlight the clinical presentation, general principles of management, and disease-specific therapies for N-linked glycosylation CDG types, organized by organ system. The second part of the review focuses on the therapeutic strategies currently available and under development. We summarize the successful (pre-) clinical application of nutritional therapies, transplantation, activated sugars, gene therapy, and pharmacological chaperones and outline the anticipated expansion of the therapeutic possibilities in CDG. We aim to provide a comprehensive update on the treatable aspects of CDG types involving N-linked glycosylation, with particular emphasis on disease-specific treatment options for the involved organ systems; call for natural history studies; and present current and future therapeutic strategies for CDG.     

Oligosaccharyltransferase complex‐congenital disorders of glycosylation: A novel congenital disorder of glycosylation

Congenital disorders of glycosylation (CDG) are metabolic disorders that affect the glycosylation of proteins and lipids. Since glycosylation affects all organs, CDG show a wide spectrum of phenotypes. We present a patient with microcephaly, dysmorphic facies, congenital heart defect, focal epilepsy, infantile spasms, skeletal dysplasia, and a type 1 serum transferrin isoelectrofocusing due to a novel CDG caused by a homozygous variant in the oligosaccharyltransferase complex noncatalytic subunit (OSTC) gene involved in glycosylation and confirmed by serum transferrin electrophoresis.     

Expanding the Molecular and Clinical Phenotype of SSR4‐CDG

Congenital disorders of glycosylation (CDG) are a group of mostly autosomal recessive disorders primarily characterized by neurological abnormalities. Recently, we described a single CDG patient with a de novo mutation in the X‐linked gene, Signal Sequence Receptor 4 (SSR4). We performed whole‐exome sequencing to identify causal variants in several affected individuals who had either an undifferentiated neurological disorder or unsolved CDG of unknown etiology based on abnormal transferrin glycosylation. We now report eight affected males with either de novo (4) or inherited (4) loss of function mutations in SSR4. Western blot analysis revealed that the mutations caused a complete loss of SSR4 protein. In nearly all cases, the abnormal glycosylation of serum transferrin was only slightly above the accepted normal cutoff range.     

Single‐center experience of N‐linked Congenital Disorders of Glycosylation with a Summary of Molecularly Characterized Cases in Arabs

Congenital disorders of glycosylation (CDG) represent an expanding group of conditions that result from defects in protein and lipid glycosylation. Different subgroups of CDG display considerable clinical and genetic heterogeneity due to the highly complex nature of cellular glycosylation. This is further complicated by ethno‐geographic differences in the mutational landscape of each of these subgroups. Ten Arab CDG patients from Latifa Hospital in Dubai, United Arab Emirates, were assessed using biochemical (glycosylation status of transferrin) and molecular approaches (next‐generation sequencing [NGS] and Sanger sequencing). In silico tools including CADD and PolyPhen‐2 were used to predict the functional consequences of uncovered mutations. In our sample of patients, five novel mutations were uncovered in the genes: MPDU1, PMM2, MAN1B1, and RFT1. In total, 9 mutations were harbored by the 10 patients in 7 genes. These are missense and nonsense mutations with deleterious functional consequences. This article integrates a single‐center experience within a list of reported CDG mutations in the Arab world, accompanied by full molecular and clinical details pertaining to the studied cases. It also sheds light on potential ethnic differences that were not noted before in regards to CDG in the Arab world.     

Mutations in TRAPPC11 are associated with a congenital disorder of glycosylation

Congenital disorders of glycosylation (CDG) are a heterogeneous and rapidly growing group of diseases caused by abnormal glycosylation of proteins and/or lipids. Mutations in genes involved in the homeostasis of the endoplasmic reticulum (ER), the Golgi apparatus (GA), and the vesicular trafficking from the ER to the ER–Golgi intermediate compartment (ERGIC) have been found to be associated with CDG. Here, we report a patient with defects in both N‐ and O‐glycosylation combined with a delayed vesicular transport in the GA due to mutations in TRAPPC11, a subunit of the TRAPPIII complex. TRAPPIII is implicated in the anterograde transport from the ER to the ERGIC as well as in the vesicle export from the GA. This report expands the spectrum of genetic alterations associated with CDG, providing new insights for the diagnosis and the understanding of the physiopathological mechanisms underlying glycosylation disorders.     

Reduced heparan sulfate accumulation in enterocytes contributes to protein-losing enteropathy in a congenital disorder of glycosylation

Intestinal biopsy in a boy with gastroenteritis-induced protein-losing enteropathy (PLE) showed loss of heparan sulfate (HS) and syndecan-1 core protein from the basolateral surface of the enterocytes, which improved after PLE subsided. Isoelectric focusing analysis of serum transferrin indicated a congenital disorder of glycosylation (CDG) and subsequent analysis showed three point mutations in the ALG6 gene encoding an alpha1,3-glucosyltransferase needed for the addition of the first glucose to the dolichol-linked oligosaccharide. The maternal mutation, C998T, causing an A333V substitution, has been shown to cause CDG-Ic, whereas the two paternal mutations, T391C (Y131H) and C924A (S308R) have not previously been reported. The mutations were tested for their ability to rescue faulty N:-linked glycosylation of carboxypeptidase Y in an ALG6-deficient Saccharomyces cerevisiae strain. Normal human ALG6 rescues glycosylation and A333V partially rescues, whereas the combined paternal mutations (Y131H and S308R) are ineffective. Underglycosylation resulting from each of these mutations is much more severe in rapidly dividing yeast. Similarly, incomplete protein glycosylation in the patient is most severe in rapidly dividing enterocytes during gastroenteritis-induced stress. Incomplete N:-linked glycosylation of an HS core protein and/or other biosynthetic enzymes may explain the selective localized loss of HS and PLE.     

Identification of a frequent variant in ALG6, the cause of Congenital Disorder of Glycosylation-Ic

Congenital Disorder of Glycosylation (CDG) type Ic is caused by mutations in ALG6. This gene encodes an alpha1,3 glucosyltransferase used for synthesis of the lipid linked oligosaccharide (LLO) precursor of the protein N-glycosylation pathway. CDG-Ic patients have moderate to severe psychomotor retardation, seizures, hypotonia, strabismus, and feeding difficulties. We previously identified a typical patient with a heterozygous point mutation, c.391T>C (p.Tyr131His) in ALG6. Using complementation analysis of ALG6-deficient yeast, we show that this alteration is as severe as the most common disease-causing mutation, c998C>T (p. Ala333Val), which occurs in over half of all known CDG-Ic patients. The frequency of c.391T>C (p.Tyr131His) in the US population, is 0.0214, suggesting that homozygotes would occur at a rate of& tilde;1:2,200. We identified one patient with typical CDG-Ic symptoms and a homozygous p.Tyr131His alteration in ALG6. However, in contrast to most CDG patients, her LLO and plasma transferrin glycosylation appeared normal. Thus, it is unclear whether c.391T>C causes CDG-Ic or contributes to the symptoms. Genotyping additional patients with CDG-like symptoms will be required to resolve this issue.     

Identification of the first COG-CDG patient of Indian origin

Mutations in the Conserved Oligomeric Golgi (COG) complex give rise to type II congenital disorders of glycosylation (CDG). Thus far, mutations have been identified in 6 of the 8 COG subunits. Here we present data identifying a previously reported CDG-IIx case from Singapore as a new COG4 patient with 2 novel mutations leading to p.E233X and p.L773R; with p.E233X being a de novo mutation. As a result, COG4 protein expression was dramatically reduced, while expression of the other subunits remained unaffected. Analysis of serum N-glycans revealed deficiencies in both sialylation and galactosylation. Furthermore, patient fibroblasts have impaired O-glycosylation. Importantly, patient fibroblasts exhibited a delay in Brefeldin A (BFA) induced retrograde transport, a common characteristic seen in COG deficiencies.     

Neurological presentation of a congenital disorder of glycosylation CDG‐Ia: Implications for diagnosis and genetic counseling

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. © 2001 Wiley‐Liss, Inc.     

Pharmacological Chaperoning: A Potential Treatment for PMM2‐CDG

The congenital disorder of glycosylation (CDG) due to phosphomannomutase 2 deficiency (PMM2‐CDG), the most common N‐glycosylation disorder, is a multisystem disease for which no effective treatment is available. The recent functional characterization of disease‐causing mutations described in patients with PMM2‐CDG led to the idea of a therapeutic strategy involving pharmacological chaperones (PC) to rescue PMM2 loss‐of‐function mutations. The present work describes the high‐throughput screening, by differential scanning fluorimetry, of 10,000 low‐molecular‐weight compounds from a commercial library, to search for possible PCs for the enzyme PMM2. This exercise identified eight compounds that increased the thermal stability of PMM2. Of these, four compounds functioned as potential PCs that significantly increased the stability of several destabilizing and oligomerization mutants and also increased PMM activity in a disease model of cells overexpressing PMM2 mutations. Structural analysis revealed one of these compounds to provide an excellent starting point for chemical optimization since it passed tests based on a number of pharmacochemical quality filters. The present results provide the first proof‐of‐concept of a possible treatment for PMM2‐CDG and describe a promising chemical structure as a starting point for the development of new therapeutic agents for this severe orphan disease.     

ALG12 mannosyltransferase defect in congenital disorder of glycosylation type lg

In the endoplasmic reticulum (ER) of eukaryotes, N-linked glycans are first assembled on the lipid carrier dolichyl pyrophosphate. The GlcNAc(2)Man(9)Glc(3) oligosaccharide is transferred to selected asparagine residues of nascent polypeptides. Defects along the biosynthetic pathway of N-glycans are associated with severe multisystemic syndromes called congenital disorders of glycosylation. Here, we describe a deficiency in the ALG12 ER alpha1,6-mannosyltransferase resulting in a novel type of glycosylation disorder. The severe disease was identified in a child presenting with psychomotor retardation, hypotonia, growth retardation, dysmorphic features and anorexia. In the patient's fibroblasts, the biosynthetic intermediate GlcNAc(2)Man(7) oligosaccharide was detected both on the lipid carrier dolichyl pyrophosphate and on newly synthesized glycoproteins, thus pointing to a defect in the dolichyl pyrophosphate-GlcNAc(2)Man(7)-dependent ALG12 alpha1,6 mannosyltransferase. Analysis of the ALG12 cDNA in the CDG patient revealed compound heterozygosity for two point mutations that resulted in the amino acid substitutions T67M and R146Q, respectively. The impact of these mutations on ALG12 protein function was investigated in the Saccharomyces cerevisiae alg12 glycosylation mutant by showing that the yeast ALG12 gene bearing the homologous mutations T61M and R161Q and the human mutant ALG12 cDNA alleles failed to normalize the growth defect phenotype of the alg12 yeast model, whereas expression of the normal ALG12 cDNA complemented the yeast mutation. The ALG12 mannosyltransferase defect defines a new type of congenital disorder of glycosylation, designated CDG-Ig.     

RFT1 deficiency in three novel CDG patients

The medical significance of N‐glycosylation is underlined by a group of inherited human disorders called Congenital Disorders of Glycosylation (CDG). One key step in the biosynthesis of the Glc₃Man₉GlcNAc₂‐PP‐dolichol precursor, essential for N‐glycosylation, is the translocation of Man₅GlcNAc₂‐PP‐dolichol across the endoplasmic reticulum membrane. This step is facilitated by the RFT1 protein. Recently, the first RFT1‐deficient CDG (RFT1‐CDG) patient was identified and presented a severe N‐glycosylation disorder. In the present study, we describe three novel CDG patients with an RFT1 deficiency. The first patient was homozygous for the earlier reported RFT1 missense mutation (c.199C>T; p.R67C), whereas the two other patients were homozygous for the missense mutation c.454A>G (p.K152E) and c.892G>A (p.E298 K), respectively. The pathogenic character of the novel mutations was illustrated by the accumulation of Man₅GlcNAc₂‐PP‐dolichol and by reduced recombinant DNase 1 secretion. Both the glycosylation pattern and recombinant DNase 1 secretion could be normalized by expression of normal RFT1 cDNA in the patients' fibroblasts. The clinical phenotype of these patients comprised typical CDG symptoms in addition to sensorineural deafness, rarely reported in CDG patients. The identification of additional RFT1‐deficient patients allowed to delineate the main clinical picture of RFT1‐CDG and confirmed the crucial role of RFT1 in Man₅GlcNAc₂‐PP‐dolichol translocation. Hum Mutat 30:1–7, 2009. © 2009 Wiley‐Liss, Inc.     

A deletion–insertion mutation in the phosphomannomutase 2 gene in an African American patient with congenital disorders of glycosylation‐Ia

Congenital disorders of glycosylation (CDG) are a group of metabolic disorders with multisystemic involvement characterized by abnormalities in the synthesis of N‐linked oligosaccharides. The most common form, CDG‐Ia, resulting from mutations in the gene encoding the enzyme phosphomannomutase (PMM2), manifests with severe abnormalities in psychomotor development, dysmorphic features and visceral involvement. While this disorder is panethnic, we present the first cases of CDG‐Ia identified in an African American family with two affected sisters. The proband had failure to thrive in infancy, hypotonia, ataxia, cerebellar hypoplasia and developmental delay. On examination, she also exhibited strabismus, inverted nipples and an atypical perineal fat distribution, all features characteristic of CDG‐Ia. Direct sequencing demonstrated that the patient had a unique genotype, T237M/c.565‐571 delAGAGAT insGTGGATTTCC. The novel deletion–insertion mutation, which was confirmed by subcloning and sequencing of each allele, introduces a stop codon 11 amino acids downstream from the site of the deletion. The presence of this deletion–insertion mutation at cDNA position 565 suggests that this site in the PMM2 gene may be a hotspot for chromosomal breakage. Published 2002 Wiley‐Liss, Inc.