Severe pathogenic variants of intestinal sucrase-isomaltase interact avidly with the wild type enzyme and negatively impact its function and trafficking.

Sucrase-isomaltase (SI) is the major disaccharidase of the small intestine, exhibiting a broad α-glucosidase activity profile. The importance of SI in gut health is typified by the development of sucrose and starch maldigestion in individuals carrying mutations in the SI gene, like in congenital sucrase-isomaltase deficiency (CSID). Common and rare defective SI gene variants (SIGVs) have also been shown to increase the risk of irritable bowel syndrome (IBS) with symptoms and clinical features similar to CSID and also in symptomatic heterozygote carriers. Here, we investigate the impact of the most abundant and highly pathogenic SIGVs that occur in heterozygotes on wild type SI (SIWT) by adapting an in vitro system that recapitulates SI gene heterozygosity. Our results demonstrate that pathogenic SI mutants interact avidly with SIWT, negatively impact its enzymatic function, alter the biosynthetic pattern and impair the trafficking behavior of the heterodimer. The in vitro recapitulation of a heterozygous state demonstrates potential for SIGVs to act in a semi-dominant fashion, by further reducing disaccharidase activity via sequestration of the SIWT copy into an inactive form of the enzymatic heterodimer. This study provides novel insights into the potential role of heterozygosity in the pathophysiology of CSID and IBS.

Differential Effects of Sucrase-Isomaltase Mutants on Its Trafficking and Function in Irritable Bowel Syndrome: Similarities to Congenital Sucrase-Isomaltase Deficiency.

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.

A mutation map for human glycoside hydrolase genes.

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.

Heterozygotes Are a Potential New Entity among Homozygotes and Compound Heterozygotes in Congenital Sucrase-Isomaltase Deficiency.

Congenital sucrase-isomaltase deficiency (CSID) is an autosomal recessive disorder of carbohydrate maldigestion and malabsorption caused by mutations in the sucrase-isomaltase (SI) gene. SI, together with maltase-glucoamylase (MGAM), belongs to the enzyme family of disaccharidases required for breakdown of -glycosidic linkages in the small intestine. The effects of homozygote and compound heterozygote inheritance trait of SI mutations in CSID patients have been well described in former studies. Here we propose the inclusion of heterozygote mutation carriers as a new entity in CSID, possibly presenting with milder symptoms. The hypothesis is supported by recent observations of heterozygote mutation carriers among patients suffering from CSID or patients diagnosed with functional gastrointestinal disorders. Recent studies implicate significant phenotypic heterogeneity depending on the character of the mutation and call for more research regarding the correlation of genetics, function at the cellular and molecular level and clinical presentation. The increased importance of SI gene variants in irritable bowel syndrome (IBS) or other functional gastrointestinal disorders FGIDs and their available symptom relief diets like fermentable oligo-, di-, mono-saccharides and polyols FODMAPs suggest that the heterozygote mutants may affect the disease development and treatment.

Isolation and Quantification of Sphingosine and Sphinganine from Rat Serum Revealed Gender Differences.

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.

Congenital Lactase Deficiency: Mutations, Functional and Biochemical Implications, and Future Perspectives.

Congenital lactase deficiency (CLD) is a severe autosomal recessive genetic disorder that affects the functional capacity of the intestinal protein lactase-phlorizin hydrolase (LPH). This disorder is diagnosed already during the first few days of the newborn's life due to the inability to digest lactose, the main carbohydrate in mammalian milk. The symptoms are similar to those in other carbohydrate malabsorption disorders, such as congenital sucrase-isomaltase deficiency, and include severe osmotic watery diarrhea. CLD is associated with mutations in the translated region of the LPH gene that elicit loss-of-function of LPH. The mutations occur in a homozygote or compound heterozygote pattern of inheritance and comprise missense mutations as well as mutations that lead to complete or partial truncations of crucial domains in LPH, such as those linked to the folding and transport-competence of LPH and to the catalytic domains. Nevertheless, the identification of the mutations in CLD is not paralleled by detailed genotype/protein phenotype analyses that would help unravel potential pathomechanisms underlying this severe disease. Here, we review the current knowledge of CLD mutations and discuss their potential impact on the structural and biosynthetic features of LPH. We also address the question of whether heterozygote carriers can be symptomatic for CLD and whether genetic testing is needed in view of the severity of the disease.

Methods to Study Lipid Alterations in Neutrophils and the Subsequent Formation of Neutrophil Extracellular Traps.

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-ß-cyclodextrin (MßCD) to induce lipid alterations in the cells. Using HPTLC and HPLC, we have shown that MßCD treatment of the cells leads to lipid alterations associated with a significant reduction in the cholesterol content of the cell. At the same time, MßCD 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.

Formation of Neutrophil Extracellular Traps under Low Oxygen Level.

Since their discovery, neutrophil extracellular traps (NETs) have been characterized as a fundamental host innate immune defense mechanism. Conversely, excessive NET-release may have a variety of detrimental consequences for the host. A fine balance between NET formation and elimination is necessary to sustain a protective effect during an infectious challenge. Our own recently published data revealed that stabilization of hypoxia-inducible factor 1α (HIF-1α) by the iron chelating HIF-1α-agonist desferoxamine or AKB-4924 enhanced the release of phagocyte extracellular traps. Since HIF-1α is a global regulator of the cellular response to low oxygen, we hypothesized that NET formation may be similarly increased under low oxygen conditions. Hypoxia occurs in tissues during infection or inflammation, mostly due to overconsumption of oxygen by pathogens and recruited immune cells. Therefore, experiments were performed to characterize the formation of NETs under hypoxic oxygen conditions compared to normoxia. Human blood-derived neutrophils were isolated and incubated under normoxic (21%) oxygen level and compared to hypoxic (1%) conditions. Dissolved oxygen levels were monitored in the primary cell culture using a Fibox4-PSt3 measurement system. The formation of NETs was quantified by fluorescence microscopy in response to the known NET-inducer phorbol 12-myristate 13-acetate (PMA) or Staphylococcus (S.) aureus wild-type and a nuclease-deficient mutant. In contrast to our hypothesis, spontaneous NET formation of neutrophils incubated under hypoxia was distinctly reduced compared to control neutrophils incubated under normoxia. Furthermore, neutrophils incubated under hypoxia showed significantly reduced formation of NETs in response to PMA. Gene expression analysis revealed that mRNA level of hif-1α as well as hif-1α target genes was not altered. However, in good correlation to the decreased NET formation under hypoxia, the cholesterol content of the neutrophils was significantly increased under hypoxia. Interestingly, NET formation in response to viable S. aureus wild-type or nuclease-deficient strain was retained under hypoxia. Our results lead to the conclusion that hypoxia is not the ideal tool to analyze HIF-1α in neutrophils. However, the data clearly suggest that neutrophils react differently under hypoxia compared to normoxia and thereby highlight the importance of the usage of physiological relevant oxygen level when studying neutrophil functions.