We describe the second case of congenital disorder of glycosylation type IL (CDG-IL) caused by deficiency of the ALG9 a1,2 mannosyltransferase enzyme. The female infant's features included psychomotor retardation, seizures, hypotonia, diffuse brain atrophy with delayed myelination, failure to thrive, pericardial effusion, cystic renal disease, hepatosplenomegaly, esotropia, and inverted nipples. Lipodystrophy and dysmorphic facial features were absent. Magnetic resonance imaging of the brain showed volume loss in the cerebral hemispheres and cerebellum and delayed myelination. Laboratory investigations revealed low levels of multiple serum proteins including antithrombin III, factor XI, and cholesterol. Hypoglycosylation was confirmed by the typical CDG type 1 pattern of serum transferrin analyzed by isoelectric focusing. A defect in the ALG9 enzyme was suggested by the accumulation of the DolPP-GlcNAc2Man6 and DolPP-GlcNAc2Man8 in the patient's fibroblasts and confirmed by mutation analysis: the patient is homozygous for the ALG9 mutation p.Y286C. The causal effect of the mutation was shown by complementation assays in alg9 deficient yeast cells. The child described here further delineates the clinical spectrum of CDG-IL and confirms the significant clinical overlap amongst CDG subtypes.

Defects of lipid-linked oligosaccharide assembly lead to alterations of N-linked glycosylation known as "type I congenital disorders of glycosylation" (CDG). Dysfunctions along this stepwise assembly pathway are characterized by intracellular accumulation of intermediate lipid-linked oligosaccharides, the detection of which contributes to the identification of underlying enzymatic defects. Using this approach, we have found, in a patient with CDG, a deficiency of the ALG9 alpha1,2 mannosyltransferase enzyme, which causes an accumulation of lipid-linked-GlcNAc(2)Man(6) and -GlcNAc2Man8 structures, which was paralleled by the transfer of incomplete oligosaccharides precursors to protein. A homozygous point-mutation 1567G-->A (amino acid substitution E523K) was detected in the ALG9 gene. The functional homology between the human ALG9 and Saccharomyces cerevisiae ALG9, as well as the deleterious effect of the E523K mutation detected in the patient with CDG, were confirmed by a yeast complementation assay lacking the ALG9 gene. The ALG9 defect found in the patient with CDG-who presented with developmental delay, hypotonia, seizures, and hepatomegaly-shows that efficient lipid-linked oligosaccharide synthesis is required for proper human development and physiology. The ALG9 defect presented here defines a novel form of CDG named "CDG-IL."

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.

BackgroundLow-dose interleukin-2 (IL-2) therapy expands regulatory T cells (Treg) and provides clinical benefit for inflammatory diseases. AMG 592 is an investigational IL-2 mutein designed to expand Treg more selectively than recombinant IL-2 (aldesleukin). In a phase 1, double-blind, placebo (PBO)-controlled first-in-human (FIH) study, we investigated the safety and tolerability of AMG 592 and pharmacodynamic (PD) effects on Treg.ObjectivesWe recently presented FIH study results including summary of safety, PK and PD.1 Here we extend those findings by exploring phenotypes of AMG 592 expanded Foxp3+ Treg subsets using flow cytometry. We compared both analysis using predefined gates and unsupervised gating. Potential implications for dose selection and mechanism of action will be discussed.MethodsIn the FIH study, healthy subjects in multiple ascending dose cohorts received a single subcutaneous dose of AMG 592 (n=6 per cohort) or placebo (n=2 per cohort). Pharmacodynamic response was evaluated for 28 days after treatment. In addition to enumerating CD4+ Foxp3+ Treg we evaluated changes in Treg subsets after AMG 592 treatment. Changes from baseline were analysed with linear mixed effects models with visit, dose level, and baseline result as main effects. To identify cell subsets, independent of predefined gates the same data were also evaluated with unsupervised gating tools using raw data from days 1, 8, 15 and 22.ResultsWe observed a robust, dose-dependent expansion of Tregs that peaked at day 8 (~4–5 fold increase) and remained elevated above baseline up to day 29 for the highest doses. Expanded Tregs had increased levels of CD25 and Foxp3, and were enriched for CD31+recent thymic emigrants (RTE). Both naïve and memory Treg increases peaked at day 8, however, naïve Treg including RTE persisted at elevated levels through day 29 while memory Treg returned to normal levels earlier. The majority of Treg expressed the transcription factor Helios. Furthermore, expanded Tregs expressed higher proportions of PD-1. Unsupervised gating analysis identified several primary clusters of expanded Treg, one including naïve and RTE Treg and another including memory Treg with elevated levels of HLA-DR expression. Evaluation of these clusters over time suggests that both increase initially at day 8 followed by preferential persistence of cells in the naïve over memory Treg cluster by day 22.ConclusionsFoxp3+ Treg were expanded in a dose dependent fashion in healthy subjects treated with AMG 592. The phenotype of expanded Treg included elevation of CD25 and Foxp3 as well as enrichment for PD-1 positive subsets. Taken together the increase in Treg with an RTE phenotype and persistence of naïve Treg suggests that AMG 592 may increase diversity of the Treg pool as a possible mechanism of action in addition to effects on memory Treg.Reference[1] ASH 2017, AMG 592 Is an Investigational IL-2 Mutein That Induces Highly Selective Expansion of Regulatory T Cells, Nadia Tchao, Kevin S Gorski, Theresa Yuraszeck, Sue J Sohn, Katsuhiko Ishida, Hansen Wong and Kyong ParkDisclosure of InterestK. Gorski Shareholder of: Amgen, Inc., Employee of: Amgen, Inc., J. Stern Shareholder of: Amgen, Inc., Employee of: Amgen, Inc., Y.-H. Hsu Shareholder of: Amgen, Inc., Employee of: Amgen, Inc., A. Anderson Shareholder of: Amgen, Inc., Employee of: Amgen, Inc., M. Boedigheimer Shareholder of: Amgen, Inc., Employee of: Amgen, Inc., N. Tchao Shareholder of: Amgen, Inc., Employee of: Amgen, Inc.

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.

Deficiencies in the pathway of N-glycan biosynthesis lead to severe multisystem diseases, known as congenital disorders of glycosylation (CDG). The clinical appearance of CDG is variable, and different types can be distinguished according to the gene that is altered. In this report, we describe the molecular basis of a novel type of the disease in three unrelated patients diagnosed with CDG-I. Serum transferrin was hypoglycosylated and patients' fibroblasts accumulated incomplete lipid-linked oligosaccharide precursors for N-linked protein glycosylation. Transfer of incomplete oligosaccharides to protein was detected. Sequence analysis of the Lec35/MPDU1 gene, known to be involved in the use of dolichylphosphomannose and dolichylphosphoglucose, revealed mutations in all three patients. Retroviral-based expression of the normal Lec35 cDNA in primary fibroblasts of patients restored normal lipid-linked oligosaccharide biosynthesis. We concluded that mutations in the Lec35/MPDU1 gene cause CDG. This novel type was termed CDG-If.