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Glycoside hydrolases (GHs) are found in all domains of life, and at least 87 distinct genes encoding proteins related to GHs are found in the human genome. GHs serve diverse functions from digestion of dietary polysaccharides to breakdown of intracellular oligosaccharides, glycoproteins, proteoglycans and glycolipids. Congenital disorders of GHs (CDGHs) represent more than 30 rare diseases caused by mutations in one of the GH genes. We previously used whole-exome sequencing of a homogenous Danish population of almost 2000 individuals to probe the incidence of deleterious mutations in the human glycosyltransferases (GTs) and developed a mutation map of human GT genes (GlyMAP-I). While deleterious disease-causing mutations in the GT genes were very rare, and in many cases lethal, we predicted deleterious mutations in GH genes to be less rare and less severe given the higher incidence of CDGHs reported worldwide. To probe the incidence of GH mutations, we constructed a mutation map of human GH-related genes (GlyMAP-II) using the Danish WES data, and correlating this with reported disease-causing mutations confirmed the higher prevalence of disease-causing mutations in several GH genes compared to GT genes. We identified 76 novel nonsynonymous single-nucleotide variations (nsSNVs) in 32 GH genes that have not been associated with a CDGH phenotype, and we experimentally validated two novel potentially damaging nsSNVs in the congenital sucrase-isomaltase deficiency gene, SI. Our study provides a global view of human GH genes and disease-causing mutations and serves as a discovery tool for novel damaging nsSNVs in CDGHs. [ABSTRACT FROM AUTHOR]

Congenital sucrase-isomaltase deficiency (CSID) is a rare metabolic intestinal disorder with reduced or absent activity levels of sucrase-isomaltase (SI). Interestingly, the main symptoms of CSID overlap with those in irritable bowel syndrome (IBS), a common functional gastrointestinal disorder with unknown etiology. Recent advances in genetic screening of IBS patients have revealed rare SI gene variants that are associated with IBS. Here, we investigated the biochemical, cellular and functional phenotypes of several of these variants. The data demonstrate that the SI mutants can be categorized into three groups including immature, mature but slowly transported, and finally mature and properly transported but with reduced enzymatic activity. We also identified SI mutant phenotypes that are deficient but generally not as severe as those characterized in CSID patients. The variable effects on the trafficking and function of the mutations analyzed in this study support the view that both CSID and IBS are heterogeneous disorders, the severity of which is likely related to the biochemical phenotypes of the SI mutants as well as the environment and diet of patients. Our study underlines the necessity to screen for SI mutations in IBS patients and to consider enzyme replacement therapy as an appropriate therapy as in CSID. [ABSTRACT FROM AUTHOR]

Sphingolipids are an important group of lipids that play crucial roles in living cells, facilitating cell recognition, signal transduction and endocytosis. The concentration of sphingosine and some of its derivatives like sphinganine may serve as a biomarker for the diagnosis of sphingolipidoses or be used for further research into similar diseases. In this study, a sphingolipid extraction and a high resolution detection method specific for sphingosine and sphinganine was adapted and tested. Lipids were extracted from rats' serum, coupled to o-phthalaldehyde and detected with a fluorescence detector after running through a silica gel column in a high performance liquid chromatography system. With this method, we analysed 20 male and 20 female rat serum samples and compared the concentrations of sphingosine and sphinganine. The results showed a significant difference between the sphingosine concentrations in the male and female rats. The sphingosine concentration in female rats was 805 ng/mL (standard deviation, SD ± 549), while that in males was significantly lower at (75 ng/mL (SD ± 40)). Furthermore, the sphingosine:sphinganine ratio was almost 15-fold higher in the females' samples. The method presented here facilitates the accurate quantification of sphingosine and sphinganine concentrations down to 2.6 ng and 3.0 ng, respectively, and their ratio in small amounts of rat serum samples to study the sphingolipid metabolism and its potential modulation due to gene mutations or the effect of prevalent toxins. [ABSTRACT FROM AUTHOR]

Congenital sucrase-isomaltase deficiency (CSID) is a rare metabolic intestinal disorder with reduced or absent activity levels of sucrase-isomaltase (SI). Interestingly, the main symptoms of CSID overlap with those in irritable bowel syndrome (IBS), a common functional gastrointestinal disorder with unknown etiology. Recent advances in genetic screening of IBS patients have revealed rare SI gene variants that are associated with IBS. Here, we investigated the biochemical, cellular and functional phenotypes of several of these variants. The data demonstrate that the SI mutants can be categorized into three groups including immature, mature but slowly transported, and finally mature and properly transported but with reduced enzymatic activity. We also identified SI mutant phenotypes that are deficient but generally not as severe as those characterized in CSID patients. The variable effects on the trafficking and function of the mutations analyzed in this study support the view that both CSID and IBS are heterogeneous disorders, the severity of which is likely related to the biochemical phenotypes of the SI mutants as well as the environment and diet of patients. Our study underlines the necessity to screen for SI mutations in IBS patients and to consider enzyme replacement therapy as an appropriate therapy as in CSID.

Lipid analysis performed by high performance thin layer chromatography (HPTLC) is a relatively simple, cost-effective method of analyzing a broad range of lipids. The function of lipids (e.g., in host-pathogen interactions or host entry) has been reported to play a crucial role in cellular processes. Here, we show a method to determine lipid composition, with a focus on the cholesterol level of primary blood-derived neutrophils, by HPTLC in comparison to high performance liquid chromatography (HPLC). The aim was to investigate the role of lipid/cholesterol alterations in the formation of neutrophil extracellular traps (NETs). NET release is known as a host defense mechanism to prevent pathogens from spreading within the host. Therefore, blood-derived human neutrophils were treated with methyl-β-cyclodextrin (MβCD) to induce lipid alterations in the cells. Using HPTLC and HPLC, we have shown that MβCD treatment of the cells leads to lipid alterations associated with a significant reduction in the cholesterol content of the cell. At the same time, MβCD treatment of the neutrophils led to the formation of NETs, as shown by immunofluorescence microscopy. In summary, here we present a detailed method to study lipid alterations in neutrophils and the formation of NETs.