A patient-based medaka alg2 mutant as a model for hypo-N-glycosylation.

Defects in the evolutionarily conserved protein-glycosylation machinery during embryonic development are often fatal. Consequently, congenital disorders of glycosylation (CDG) in human are rare. We modelled a putative hypomorphic mutation described in an alpha-1,3/1,6-mannosyltransferase (ALG2) index patient (ALG2-CDG) to address the developmental consequences in the teleost medaka (Oryzias latipes). We observed specific, multisystemic, late-onset phenotypes, closely resembling the patient's syndrome, prominently in the facial skeleton and in neuronal tissue. Molecularly, we detected reduced levels of N-glycans in medaka and in the patient's fibroblasts. This hypo-N-glycosylation prominently affected protein abundance. Proteins of the basic glycosylation and glycoprotein-processing machinery were over-represented in a compensatory response, highlighting the regulatory topology of the network. Proteins of the retinal phototransduction machinery, conversely, were massively under-represented in the alg2 model. These deficiencies relate to a specific failure to maintain rod photoreceptors, resulting in retinitis pigmentosa characterized by the progressive loss of these photoreceptors. Our work has explored only the tip of the iceberg of N-glycosylation-sensitive proteins, the function of which specifically impacts on cells, tissues and organs. Taking advantage of the well-described human mutation has allowed the complex interplay of N-glycosylated proteins and their contribution to development and disease to be addressed.

Targeted Proteomics Reveals Quantitative Differences in Low-Abundance Glycosyltransferases of Patients with Congenital Disorders of Glycosylation.

Protein glycosylation is an essential post-translational modification in all domains of life. Its impairment in humans can result in severe diseases named congenital disorders of glycosylation (CDGs). Most of the glycosyltransferases (GTs) responsible for proper glycosylation are polytopic membrane proteins that represent challenging targets in proteomics. We established a multiple reaction monitoring (MRM) assay to comprehensively quantify GTs involved in the processes of N-glycosylation and O- and C-mannosylation in the endoplasmic reticulum. High robustness was achieved by using an enriched membrane protein fraction of isotopically labeled HEK 293T cells as an internal protein standard. The analysis of primary skin fibroblasts from eight CDG type I patients with impaired ALG1, ALG2, and ALG11 genes, respectively, revealed a substantial reduction in the corresponding protein levels. The abundance of the other GTs, however, remained unchanged at the transcript and protein levels, indicating that there is no fail-safe mechanism for the early steps of glycosylation in the endoplasmic reticulum. The established MRM assay was shared with the scientific community via the commonly used open source Skyline software environment, including Skyline Batch for automated data analysis. We demonstrate that another research group could easily reproduce all analysis steps, even while using different LC-MS hardware.

Quantification of Dolichyl Phosphates Using Phosphate Methylation and Reverse-Phase Liquid Chromatography-High Resolution Mass Spectrometry.

Dolichyl monophosphates (DolPs) are essential lipids in glycosylation pathways that are highly conserved across almost all domains of life. The availability of DolP is critical for all glycosylation processes, as these lipids serve as membrane-anchored building blocks used by various types of glycosyltransferases to generate complex post-translational modifications of proteins and lipids. The analysis of DolP species by reverse-phase liquid chromatography-mass spectrometry (RPLC-MS) remains a challenge due to their very low abundance and wide range of lipophilicities. Until now, a method for the simultaneous qualitative and quantitative assessment of DolP species from biological membranes has been lacking. Here, we describe a novel approach based on simple sample preparation, rapid and efficient trimethylsilyl diazomethane-dependent phosphate methylation, and RPLC-MS analysis for quantification of DolP species with different isoprene chain lengths. We used this workflow to selectively quantify DolP species from lipid extracts derived of Saccharomyces cerevisiae, HeLa, and human skin fibroblasts from steroid 5-α-reductase 3- congenital disorders of glycosylation (SRD5A3-CDG) patients and healthy controls. Integration of this workflow with global lipidomics analyses will be a powerful tool to expand our understanding of the role of DolPs in pathophysiological alterations of metabolic pathways downstream of HMG-CoA reductase, associated with CDGs, hypercholesterolemia, neurodegeneration, and cancer.

Phenotypic diversity, disease progression, and pathogenicity of MVK missense variants in mevalonic aciduria.

Mevalonic aciduria (MVA) and hyperimmunoglobulinemia D syndrome (MKD/HIDS) are disorders of cholesterol biosynthesis caused by variants in the MVK gene and characterized by increased urinary excretion of mevalonic acid. So far, 30 MVA patients have been reported, suffering from recurrent febrile crises and neurologic impairment. Here, we present an in-depth analysis of the phenotypic spectrum of MVA and provide an in-silico pathogenicity model analysis of MVK missense variants. The phenotypic spectrum of 11 MVA patients (age range 0-51 years) registered in the Unified European Registry for Inherited Metabolic Disorders database was systematically analyzed using terms of the Human Phenotype Ontology. Biochemical, radiological as well as genetic characteristics were investigated. Six of eleven patients have reached adulthood and four have reached adolescence. One of the adolescent patients died at the age of 16 years and one patient died shortly after birth. Symptoms started within the first year of life, including episodic fever, developmental delay, ataxia, and ocular involvement. We also describe a case with absence of symptoms despite massive excretion of mevalonic acid. Pathogenic variants causing MVA cluster within highly conserved regions, which are involved in mevalonate and ATP binding. The phenotype of adult and adolescent MVA patients is more heterogeneous than previously assumed. Outcome varies from an asymptomatic course to early death. MVK variants cluster in functionally important and highly conserved protein domains and show high concordance regarding their expected pathogenicity.

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 Man5 GlcNAc2 -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.

A rapid and simple procedure for the isolation and cultivation of fibroblast-like cells from medaka and zebrafish embryos and fin clip biopsies.

In many human diseases, the molecular pathophysiological mechanisms are not understood, which makes the development and testing of new therapeutic approaches difficult. The generation and characterization of animal models such as mice, rats, fruit flies, worms or fish offers the possibility for in detail studies of a disease's development, its course and potential therapies in an organismal context, which considerably minimizes the risk of therapeutic side effects for patients. Nevertheless, due to the high numbers of experimental animals used in research worldwide, attempts to develop alternative test systems will help in reducing their count. In this regard, the cell culture system displays a suitable option due to its potential of delivering nearly unlimited material and the good opportunities for high-throughput studies such as drug testing. Here, we describe a quick and simple method to isolate and cultivate vital fibroblast-like cells from embryos and adults of two popular teleost model organisms, the Japanese rice fish medaka (Oryzias latipes) and the zebrafish (Danio rerio).

Fatal outcome after heart surgery in PMM2-CDG due to a rare homozygous gene variant with double effects.

Variants in Phosphomannomutase 2 (PMM2) lead to PMM2-CDG, the most frequent congenital disorder of glycosylation (CDG). We here describe the disease course of a ten-month old patient who presented with the classical PMM2-CDG symptoms as cerebellar hypoplasia, retinitis pigmentosa, seizures, short stature, hepato- and splenomegaly, anaemia, recurrent vomiting and inverted mamillae. A severe form of tetralogy of Fallot was diagnosed and corrective surgery was performed at the age of 10 months. At the end of the cardiopulmonary bypass, a sudden oedematous reaction of the myocardium accompanied by biventricular pump failure was observed immediately after heparin antagonization with protamine sulfate. The patient died seven days after surgery, since myocardial function did not recover on ECMO support. We here describe the first patient carrying the homozygous variant g.18313A > T in the PMM2 gene (NG_009209.1) that either can lead to c.394A > T (p.I132F) or even loss of 100 bp due to exon 5 skipping (c.348_447del; p.G117Rfs*4) which is comparable to a null allele. Proliferation and doubling time of the patient's fibroblasts were affected. In addition, we show that the induction of cellular stress by elevating the cell culture temperature to 40 °C led to a decrease of the patients' PMM2 transcript as well as PMM2 protein levels and subsequently to a significant loss of residual activity. We assume that metabolic stressful processes occurring after cardiac surgery led to the drop of the patient's PMM activity below a life-sustaining niveau which paved the way for the fatal outcome.