ALG1-CDG: Clinical and Molecular Characterization of 39 Unreported Patients

Abstract

Congenital disorders of glycosylation (CDG) arise from pathogenic mutations in over 100 genes leading to impaired protein or lipid glycosylation. ALG1 encodes a β1,4 mannosyltransferase that catalyzes the addition of the first of nine mannose moieties to form a dolichol-lipid linked oligosaccharide intermediate required for proper N-linked glycosylation. ALG1 mutations cause a rare autosomal recessive disorder termed ALG1-CDG. To date 13 mutations in 18 patients from 14 families have been described with varying degrees of clinical severity. We identified and characterized 39 previously unreported cases of ALG1-CDG from 32 families and add 26 new mutations. Pathogenicity of each mutation was confirmed based on its inability to rescue impaired growth or hypoglycosylation of a standard biomarker in an alg1-deficient yeast strain. Using this approach we could not establish a rank order comparison of biomarker glycosylation and patient phenotype, but we identified mutations with a lethal outcome in the first two years of life. The recently identified protein-linked xeno-tetrasaccharide biomarker, NeuAc-Gal-GlcNAc2 , was seen in all 27 patients tested. Our study triples the number of known patients and expands the molecular and clinical correlates of this disorder.

Keywords: CDG; asparagine-linked glycosylation protein 1; carbohydrate-deficient transferrin; xeno-tetrasaccharide.

Figure 1

Pathway showing the enzymatic step requiring ALG1 mannosyltransferase activity and schematic showing mutations identified within ALG1. (A) Schematic showing only the early N-linked glycosylation pathway on the cytoplasmic facing side of the ER with the ALG1-dependent step highlighted with a red (X) over the arrow. (B) Schematic showing exon location of all the ALG1 mutations identified in this study. The mutations identified in this study are highlighted in red. For splicing mutations, nucleotide numbering for cDNA uses +1 as the A of the ATG translation initiation codon in the reference sequence.

Figure 2

Yeast complementation assay using the Δ alg1 deficient yeast strain. The alg1 deficient yeast strain was transformed with the indicated pYES2.1 construct expressing either human wild-type ALG1 or the indicated missense variants and allowed to grow for 96 hours at both permissive (26°C) and restrictive temperatures (37°C). Western blot analysis of carboxypeptidase Y (CPY) glycosylation was performed as previously described [Grubenmann et al., 2004]. Both CPY glycosylation and growth complementation were performed in triplicate with representative data presented.

Figure 3

Phenotype summary from the thirty-nine ALG1-CDG cases. Phenotypic data were provided for each individual and summarized as a percentage of patients affected. The number of deceased individuals is further broken down by the age at which the individual passed away. (*) Indicates that not all individuals could be assessed for intellectual disability, due to early death or young age.

Figure 4. Kaplan-Meier survival curves for ALG1-CDG patients

(A) Probability of survival for all thirty-nine ALG1-CDG cases “Group 1”. (B) Probability of survival for all thirty-nine ALG1-CDG cases separated by genotype. Group 2 are individuals homozygous for the p.Ser258Leu (n=6), Group 3 are individuals compound heterozygous for the p.Gln50Arg (n=5) and Group 4 comprises the remaining individuals (n=28). In order to see the effect of the p.Ser258Leu and p.Gln50Arg we made the time scale limit 100 months, however it should be noted that several individuals survived pass 100 months and this is denoted using ().