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 Both patients were shown to carry previously unpublished hALG12-mutations.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.     

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.     

Clinical and molecular characterization of the first adult congenital disorder of glycosylation (CDG) type Ic patient

Congenital disorder of glycosylation (CDG) type Ic, the second largest subtype of CDG, is caused by mutations in human ALG6 (hALG6). This gene encodes the alpha1,3-glucosyltransferase that catalyzes transfer of the first glucose residue to the lipid-linked oligosaccharide precursor for N-linked glycosylation. In this report, we describe the first adult patient diagnosed with CDG-Ic, carrying two previously unknown mutations. The first is a three base deletion (897-899delAAT) leading to the loss of I299, the second is an intronic mutation (IVS7 + 2T > G) that causes aberrant splicing. Wildtype hALG6, delivered by a lentiviral vector into patient's fibroblasts, clearly improves the biochemical phenotype, which confirms that the mutations are disease-causing. Striking clinical findings include limb deficiencies in the fingers, resembling brachydactyly type B, a deep vein thrombosis, pseudotumor cerebri, and endocrine disturbances with pronounced hyperandrogenism and virilization. However, even in adulthood, this patient shows normal magnetic resonance imaging of the brain.     

Congenital disorders of glycosylation: an update on defects affecting the biosynthesis of dolichol-linked oligosaccharides

Defects in the biosynthesis of the oligosaccharide precursor for N-glycosylation lead to decreased occupancy of glycosylation sites and thereby to diseases known as congenital disorders of glycosylation (CDG). In the last 20 years, approximately 1,000 CDG patients have been identified presenting with multiple organ dysfunctions. This review sets the state of the art by listing all mutations identified in the 15 genes (PMM2, MPI, DPAGT1, ALG1, ALG2, ALG3, ALG9, ALG12, ALG6, ALG8, DOLK, DPM1, DPM3, MPDU1, and RFT1) that yield a deficiency of dolichol-linked oligosaccharide biosynthesis. The present analysis shows that most mutations lead to substitutions of strongly conserved amino acid residues across eukaryotes. Furthermore, the comparison between the different forms of CDG affecting dolichol-linked oligosaccharide biosynthesis shows that the severity of the disease does not relate to the position of the mutated gene along this biosynthetic pathway. Hum Mutat 30:1–14, 2009. © 2009 Wiley-Liss, Inc.     

Improved diagnostics lead to identification of three new patients with congenital disorder of glycosylation-Ip

Congenital disorders of glycosylation (CDG) comprise a clinically and biochemically heterogeneous group of monogenetic-inherited, multisystemic diseases that affect the biosynthesis of N- and/or O-glycans linked to glycoconjugates. Recently, we identified the first patient with a defect in the cytosolic-orientated GDP-mannose:Man(3-4) GlcNAc(2)-PP-dolichol alpha-1,2-mannosyltransferase (ALG11), who presented an accumulation of shortened dolichol-linked oligosaccharides leading to CDG-Ip (ALG11-CDG). Here we describe an improved metabolic labeling method that allowed the identification of three new CDG-Ip cases that were missed so far in routine diagnostics. Although all CDG-Ip patients carry different mutations in the ALG11 gene, they share a variety of clinical syndromes like an unremarkable prenatal period followed by developmental delay, psychomotor, and mental retardation, strabismus convergens and seizures occurring in the first year of life.     

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.     

Severe ALG8-CDG (CDG-Ih) associated with homozygosity for two novel missense mutations detected by exome sequencing of candidate genes

Posttranslationally glycosylated proteins are important in many biological processes in humans and Congenital disorders of glycosylation (CDGs) are associated with a broad range of phenotypes. Type I CDGs are a group of rare autosomal recessive conditions. To date 17 subtypes have been enzymatically and molecularly characterized. Impaired function of the enzyme dolichyl pyrophosphate Glc₁Man₉GlcNAc₂ alpha-1,3-glucosyltransferase encoded by the ALG8 gene, causes ALG8-CDG (CDG-Ih, OMIM #608104). This enzyme facilitates the transfer of a second glucose molecule to a growing lipid-linked oligosaccharide chain, a process that transpires in the endoplasmic reticulum (ER). We present a female patient of consanguineous parents, with pre- and postnatal growth retardation, dysmorphic features, significant developmental delay, visual impairment and an electrophoretic serum transferrin pattern indicative of a type I CDG. Type I CDG subgroup was determined by exome sequencing facilitated by homozygosity analysis. The patient was homozygous for two variants, nine nucleotides apart, in exon 8 of ALG8; c.799T > C [p.Ser267Pro] and c.808T > C [p.Phe270Leu]. Both missense mutations are predicted to affect a conserved region of an intraluminal ER loop of dolichyl pyrophosphate Glc₁Man₉GlcNAc₂ alpha-1,3-glucosyltransferase. To our knowledge, the current report describes the ninth published case of ALG8-CDG, contributing to the further delineation of this rare and variable disorder.     

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.     

CDG-Id caused by homozygosity for an ALG3 mutation due to segmental maternal isodisomy UPD3(q21.3-qter)

We report on a patient with a congenital disorder of glycosylation type Id (CDG-Id) caused by a homozygous mutation in the ALG3 gene, which results from a de novo mutation in combination with a segmental maternal uniparental isodisomy (UPD). The patient presented with severe psychomotor delay, primary microcephaly, and opticus atrophy, compatible with a severe form of CDG. Isoelectric focusing of transferrin showed a type I pattern and lipid-linked oligosaccharide analysis showed an accumulation of dol-PP-GlcNAc2Man5 in patient's fibroblasts suggesting a defect in the ALG3 gene. A homozygous ALG3 missense mutation p.R266C (c.796C > T) was identified. Further evaluation revealed that neither the mother nor the father were carrier of the p.R266C mutation. Marker analysis revealed a segmental maternal isodisomy for the chromosomal region 3q21.3-3qter. UPD for this region has not been described before. More important, the combination of UPD with a de novo mutation is an exceptional coincidence and an extraordinary observation.     

Novel variants and clinical symptoms in four new ALG3‐CDG patients,review of the literature,and identification of AAGRP‐ALG3 as a novel ALG3 variant with alanine and glycine‐rich N‐terminus

ALG3‐CDG is one of the very rare types of congenital disorder of glycosylation (CDG) caused by variants in the ER‐mannosyltransferase ALG3. Here, we summarize the clinical, biochemical, and genetic data of four new ALG3‐CDG patients, who were identified by a type I pattern of serum transferrin and the accumulation of Man₅GlcNAc₂‐PP‐dolichol in LLO analysis. Additional clinical symptoms observed in our patients comprise sensorineural hearing loss, right‐descending aorta, obstructive cardiomyopathy, macroglossia, and muscular hypertonia. We add four new biochemically confirmed variants to the list of ALG3‐CDG inducing variants: c.350G>C (p.R117P), c.1263G>A (p.W421*), c.1037A>G (p.N346S), and the intron variant c.296+4A>G. Furthermore, in Patient 1 an additional open‐reading frame of 141 bp (AAGRP) in the coding region of ALG3 was identified. Additionally, we show that control cells synthesize, to a minor degree, a hybrid protein composed of the polypeptide AAGRP and ALG3 (AAGRP‐ALG3), while in Patient 1 expression of this hybrid protein is significantly increased due to the homozygous variant c.160_196del (g.165C>T). By reviewing the literature and combining our findings with previously published data, we further expand the knowledge of this rare glycosylation defect.     

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.     

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.     

Deficiency of UDP-GlcNAc:Dolichol Phosphate N-Acetylglucosamine-1 Phosphate Transferase (DPAGT1) causes a novel congenital disorder of Glycosylation Type Ij

Defects in the assembly of dolichol-linked oligosaccharide or its transfer to proteins result in severe, multi-system human diseases called Type I congenital disorders of glycosylation. We have identified a novel CDG type, CDG-Ij, resulting from deficiency in UDP-GlcNAc: dolichol phosphate N-acetyl-glucosamine-1 phosphate transferase (GPT) activity encoded by DPAGT1. The patient presents with severe hypotonia, medically intractable seizures, mental retardation, microcephaly, and exotropia. Metabolic labeling of cultured dermal fibroblasts from the patient with [2-(3)H]-mannose revealed lowered incorporation of radiolabel into full-length dolichol-linked oligosaccharides and glycoproteins. In vitro enzymatic analysis of microsomal fractions from the cultured cells indicated that oligosaccharyltransferase activity is normal, but the GPT activity is reduced to approximately 10% of normal levels while parents have heterozygous levels. The patient's paternal DPAGT1 allele contains a point mutation (660A>G) that replaces a highly conserved tyrosine with a cysteine (Y170C). The paternal allele cDNA produces a full-length protein with almost no activity when over-expressed in CHO cells. The maternal allele makes only about 12% normal mature mRNA, while the remainder shows a complex exon skipping pattern that shifts the reading frame encoding a truncated non-functional GPT protein. Thus, we conclude that the DPAGT1 gene defects are responsible for the CDG symptoms in this patient. Hum Mutat 22:144-150, 2003.     

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.     

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.     

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.     

CDG-Id in two siblings with partially different phenotypes

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. (c) 2007 Wiley-Liss, Inc.     

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.     

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.