Identification of recurrent pathogenic alleles using exome sequencing data: Proof-of-concept study of Russian subjects.

Whole exome sequencing (WES) is a powerful tool for the cataloguing of population-specific genetic diseases. Within this proof-of-concept study we evaluated whether analysis of a small number of individual exomes is capable of identifying recurrent pathogenic alleles. We considered 106 exomes of subjects of Russian origin and revealed 13 genetic variants, which occurred more than twice and fulfilled the criteria for pathogenicity. All these alleles turned out to be indeed recurrent, as revealed by the analysis of 1045 healthy Russian donors. Eight of these variants (NAGA c.973G>A, ACADM c.985A>C, MPO c.2031-2A>C, SLC3A1 c.1400T>C, LRP2 c.6160G>A, BCHE c.293A>G, MPO c.752T>C, FCN3 c.349delC) are non-Russian-specific, as their high prevalence was previously demonstrated in other European populations. The remaining five disease-associated alleles appear to be characteristic for subjects of Russian origin and include CLCN1 c.2680C>T (myotonia congenita), DHCR7 c.453G>A (Smith-Lemli-Opitz syndrome), NUP93 c.1162C>T (steroid-resistant nephrotic syndrome, type 12), SLC26A2 c.1957T>A (multiple epiphyseal dysplasia) and EIF3F c.694T>G (mental retardation). These recessive disease conditions may be of particular relevance for the Russian Federation and other countries with a significant Slavic population.

Silencing of α-N-acetylgalactosaminidase in the gastric cancer cells amplified cell death and attenuated migration, while the multidrug resistance remained unchanged.

Although the elevated level of the α-N-acetylgalactosaminidase enzyme (encoded by the NAGA gene) is a well-recognized feature of cancer cells; little research works have been undertaken on the cancer malignancy mechanisms. The effects of NAGA gene downregulation on cancer cells' features such as drug resistance, impaired programmed cell death, and migration were analyzed in this study. The cells grew exponentially with a doubling time of 30 h in an optimal condition. Toxicity of daunorubicin chemotherapy drug on NAGA-transfected EPG85.257RDB cells was evaluated in comparison to control cells and no significant change was recorded. Quantitative transcript analyses and protein levels revealed that the MDR1 pump almost remained unchanged during the study. Moreover, the NAGA gene downregulation enhanced the late apoptosis rate in EPG85.257RDB cells at 24 h posttransfection. The investigated expression level of genes and proteins involved in the TNFR2 signaling pathway, related to cancer cell apoptosis, showed considerable alterations after NAGA silencing as well. MAP3K14 and CASP3 genes were downregulated while IL6, RELA, and TRAF2 experienced an upregulation. Also, NAGA silencing generally diminished the migration ability of EPG85.257RDB cells and the MMP1 gene (as a critical gene in metastasis) expression decreased significantly. The expression of the p-FAK protein, which is located in the downstream of the α2 ß1 integrin signaling pathway, was reduced likewise. It could be concluded that despite drug resistance, NAGA silencing resulted in augmentative and regressive effects on cell death and migration.

The EngCP endo α-N-acetylgalactosaminidase is a virulence factor involved in Clostridium perfringens gas gangrene infections.

Clostridium perfringens is the causative agent of human clostridial myonecrosis; the major toxins involved in this disease are α-toxin and perfringolysin O. The RevSR two-component regulatory system has been shown to be involved in regulating virulence in a mouse myonecrosis model. Previous microarray and RNAseq analysis of a revR mutant implied that factors other than the major toxins may play a role in virulence. The RNAseq data showed that the expression of the gene encoding the EngCP endo α-N-acetylgalactosaminidase (CPE0693) was significantly down-regulated in a revR mutant. Enzymes from this family have been identified in several Gram-positive pathogens and have been postulated to contribute to their virulence. In this study, we constructed an engCP mutant of C. perfringens and showed that it was significantly less virulent than its wild-type parent strain. Virulence was restored by complementation in trans with the wild-type engCP gene. We also demonstrated that purified EngCP was able to hydrolyse α-dystroglycan derived from C2C12 mouse myotubes. However, EngCP had little effect on membrane permeability in mice, suggesting that EngCP may play a role other than the disruption of the structural integrity of myofibres. Glycan array analysis indicated that EngCP could recognise structures containing the monosaccharide N-acetlygalactosamine at 4C, but could recognise structures terminating in galactose, glucose and N-acetylglucosamine under conditions where EngCP was enzymatically active. In conclusion, we have obtained evidence that EngCP is required for virulence in C. perfringens and, although classical exotoxins are important for disease, we have now shown that an O-glycosidase also plays an important role in the disease process.

Prospecting for microbial α-N-acetylgalactosaminidases yields a new class of GH31 O-glycanase.

α-Linked GalNAc (α-GalNAc) is most notably found at the nonreducing terminus of the blood type-determining A-antigen and as the initial point of attachment to the peptide backbone in mucin-type O-glycans. However, despite their ubiquity in saccharolytic microbe-rich environments such as the human gut, relatively few α-N-acetylgalactosaminidases are known. Here, to discover and characterize novel microbial enzymes that hydrolyze α-GalNAc, we screened small-insert libraries containing metagenomic DNA from the human gut microbiome. Using a simple fluorogenic glycoside substrate, we identified and characterized a glycoside hydrolase 109 (GH109) that is active on blood type A-antigen, along with a new subfamily of glycoside hydrolase 31 (GH31) that specifically cleaves the initial α-GalNAc from mucin-type O-glycans. This represents a new activity in this GH family and a potentially useful new enzyme class for analysis or modification of O-glycans on protein or cell surfaces.

Blind prediction of deleterious amino acid variations with SNPs&GO.

SNPs&GO is a machine learning method for predicting the association of single amino acid variations (SAVs) to disease, considering protein functional annotation. The method is a binary classifier that implements a support vector machine algorithm to discriminate between disease-related and neutral SAVs. SNPs&GO combines information from protein sequence with functional annotation encoded by gene ontology (GO) terms. Tested in sequence mode on more than 38,000 SAVs from the SwissVar dataset, our method reached 81% overall accuracy and an area under the receiving operating characteristic curve of 0.88 with low false-positive rate. In almost all the editions of the Critical Assessment of Genome Interpretation (CAGI) experiments, SNPs&GO ranked among the most accurate algorithms for predicting the effect of SAVs. In this paper, we summarize the best results obtained by SNPs&GO on disease-related variations of four CAGI challenges relative to the following genes: CHEK2 (CAGI 2010), RAD50 (CAGI 2011), p16-INK (CAGI 2013), and NAGLU (CAGI 2016). Result evaluation provides insights about the accuracy of our algorithm and the relevance of GO terms in annotating the effect of the variants. It also helps to define good practices for the detection of deleterious SAVs.

A genome-wide survey of CD4(+) lymphocyte regulatory genetic variants identifies novel asthma genes.

BACKGROUND: Genome-wide association studies have yet to identify the majority of genetic variants involved in asthma. We hypothesized that expression quantitative trait locus (eQTL) mapping can identify novel asthma genes by enabling prioritization of putative functional variants for association testing. OBJECTIVE: We evaluated 6706 cis-acting expression-associated variants (eSNPs) identified through a genome-wide eQTL survey of CD4(+) lymphocytes for association with asthma. METHODS: eSNPs were tested for association with asthma in 359 asthmatic patients and 846 control subjects from the Childhood Asthma Management Program, with verification by using family-based testing. Significant associations were tested for replication in 579 parent-child trios with asthma from Costa Rica. Further functional validation was performed by using formaldehyde-assisted isolation of regulatory elements (FAIRE) quantitative PCR and chromatin immunoprecipitation PCR in lung-derived epithelial cell lines (Beas-2B and A549) and Jurkat cells, a leukemia cell line derived from T lymphocytes. RESULTS: Cis-acting eSNPs demonstrated associations with asthma in both cohorts. We confirmed the previously reported association of ORMDL3/GSDMB variants with asthma (combined P = 2.9 × 10(-8)). Reproducible associations were also observed for eSNPs in 3 additional genes: fatty acid desaturase 2 (FADS2; P = .002), N-acetyl-α-D-galactosaminidase (NAGA; P = .0002), and Factor XIII, A1 (F13A1; P = .0001). Subsequently, we demonstrated that FADS2 mRNA is increased in CD4(+) lymphocytes in asthmatic patients and that the associated eSNPs reside within DNA segments with histone modifications that denote open chromatin status and confer enhancer activity. CONCLUSIONS: Our results demonstrate the utility of eQTL mapping in the identification of novel asthma genes and provide evidence for the importance of FADS2, NAGA, and F13A1 in the pathogenesis of asthma.

α-N-acetylgalactosaminidase from infant-associated bifidobacteria belonging to novel glycoside hydrolase family 129 is implicated in alternative mucin degradation pathway.

Bifidobacteria inhabit the lower intestine of mammals including humans where the mucin gel layer forms a space for commensal bacteria. We previously identified that infant-associated bifidobacteria possess an extracellular membrane-bound endo-α-N-acetylgalactosaminidase (EngBF) that may be involved in degradation and assimilation of mucin-type oligosaccharides. However, EngBF is highly specific for core-1-type O-glycan (Galß1-3GalNAcα1-Ser/Thr), also called T antigen, which is mainly attached onto gastroduodenal mucins. By contrast, core-3-type O-glycans (GlcNAcß1-3GalNAcα1-Ser/Thr) are predominantly found on the mucins in the intestines. Here, we identified a novel α-N-acetylgalactosaminidase (NagBb) from Bifidobacterium bifidum JCM 1254 that hydrolyzes the Tn antigen (GalNAcα1-Ser/Thr). Sialyl and galactosyl core-3 (Galß1-3/4GlcNAcß1-3(Neu5Acα2-6)GalNAcα1-Ser/Thr), a major tetrasaccharide structure on MUC2 mucin primarily secreted from goblet cells in human sigmoid colon, can be serially hydrolyzed into Tn antigen by previously identified bifidobacterial extracellular glycosidases such as α-sialidase (SiaBb2), lacto-N-biosidase (LnbB), ß-galactosidase (BbgIII), and ß-N-acetylhexosaminidases (BbhI and BbhII). Because NagBb is an intracellular enzyme without an N-terminal secretion signal sequence, it is likely involved in intracellular degradation and assimilation of Tn antigen-containing polypeptides, which might be incorporated through unknown transporters. Thus, bifidobacteria possess two distinct pathways for assimilation of O-glycans on gastroduodenal and intestinal mucins. NagBb homologs are conserved in infant-associated bifidobacteria, suggesting a significant role for their adaptation within the infant gut, and they were found to form a new glycoside hydrolase family 129.

Use of a modified alpha-N-acetylgalactosaminidase in the development of enzyme replacement therapy for Fabry disease.

A modified alpha-N-acetylgalactosaminidase (NAGA) with alpha-galactosidase A (GLA)-like substrate specificity was designed on the basis of structural studies and was produced in Chinese hamster ovary cells. The enzyme acquired the ability to catalyze the degradation of 4-methylumbelliferyl-alpha-D-galactopyranoside. It retained the original NAGA's stability in plasma and N-glycans containing many mannose 6-phosphate (M6P) residues, which are advantageous for uptake by cells via M6P receptors. There was no immunological cross-reactivity between the modified NAGA and GLA, and the modified NAGA did not react to serum from a patient with Fabry disease recurrently treated with a recombinant GLA. The enzyme cleaved globotriaosylceramide (Gb3) accumulated in cultured fibroblasts from a patient with Fabry disease. Furthermore, like recombinant GLA proteins presently used for enzyme replacement therapy (ERT) for Fabry disease, the enzyme intravenously injected into Fabry model mice prevented Gb3 storage in the liver, kidneys, and heart and improved the pathological changes in these organs. Because this modified NAGA is hardly expected to cause an allergic reaction in Fabry disease patients, it is highly promising as a new and safe enzyme for ERT for Fabry disease.

Molecular cloning, sequence identification and tissue expression profile of three novel sheep (Ovis aries) genes - BCKDHA, NAGA and HEXA.

The complete coding sequences of three sheep genes- BCKDHA, NAGA and HEXA were amplified using the reverse transcriptase polymerase chain reaction (RT-PCR), based on the conserved sequence information of the mouse or other mammals. The nucleotide sequences of these three genes revealed that the sheep BCKDHA gene encodes a protein of 313 amino acids which has high homology with the BCKDHA gene that encodes a protein of 447 amino acids that has high homology with the Branched chain keto acid dehydrogenase El, alpha polypeptide (BCKDHA) of five species chimpanzee (93%), human (96%), crab-eating macaque (93%), bovine (98%) and mouse (91%). The sheep NAGA gene encodes a protein of 411 amino acids that has high homology with the alpha-N-acetylgalactosaminidase (NAGA) of five species human (85%), bovine (94%), mouse (91%), rat (83%) and chicken (74%). The sheep HEXA gene encodes a protein of 529 amino acids that has high homology with the hexosaminidase A(HEXA) of five species bovine (98%), human (84%), Bornean orangután (84%), rat (80%) and mouse (81%). Finally these three novel sheep genes were assigned to GenelDs: 100145857, 100145858 and 100145856. The phylogenetic tree analysis revealed that the sheep BCKDHA, NAGA, and HEXA all have closer genetic relationships to the BCKDHA, NAGA, and HEXA of bovine. Tissue expression profile analysis was also carried out and results revealed that sheep BCKDHA, NAGA and HEXA genes were differentially expressed in tissues including muscle, heart, liver, fat, kidney, lung, small and large intestine. Our experiment is the first to establish the primary foundation for further research on these three sheep genes.

Molecular cloning, sequence identification and tissue expression profile of three novel sheep (Ovis aries) genes - BCKDHA, NAGA and HEXA

The complete coding sequences of three sheep genes- BCKDHA, NAGA and HEXA were amplified using the reverse transcriptase polymerase chain reaction (RT-PCR), based on the conserved sequence information of the mouse or other mammals. The nucleotide sequences of these three genes revealed that the sheep BCKDHA gene encodes a protein of 313 amino acids which has high homology with the BCKDHA gene that encodes a protein of 447 amino acids that has high homology with the Branched chain keto acid dehydrogenase El, alpha polypeptide (BCKDHA) of five species chimpanzee (93 percent), human (96 percent), crab-eating macaque (93 percent), bovine (98 percent) and mouse (91 percent). The sheep NAGA gene encodes a protein of 411 amino acids that has high homology with the alpha-N-acetylgalactosaminidase (NAGA) of five species human (85 percent), bovine (94 percent), mouse (91 percent), rat (83 percent) and chicken (74 percent). The sheep HEXA gene encodes a protein of 529 amino acids that has high homology with the hexosaminidase A(HEXA) of five species bovine (98 percent), human (84 percent), Bornean orangután (84 percent), rat (80 percent) and mouse (81 percent). Finally these three novel sheep genes were assigned to GenelDs: 100145857, 100145858 and 100145856. The phylogenetic tree analysis revealed that the sheep BCKDHA, NAGA, and HEXA all have closer genetic relationships to the BCKDHA, NAGA, and HEXA of bovine. Tissue expression profile analysis was also carried out and results revealed that sheep BCKDHA, NAGA and HEXA genes were differentially expressed in tissues including muscle, heart, liver, fat, kidney, lung, small and large intestine. Our experiment is the first to establish the primary foundation for further research on these three sheep genes.

Three dimensional structural studies of alpha-N-acetylgalactosaminidase (alpha-NAGA) in alpha-NAGA deficiency (Kanzaki disease): different gene mutations cause peculiar structural changes in alpha-NAGAs resulting in different substrate specificities and clinical phenotypes.

BACKGROUND: Kanzaki disease (OMIM#104170) is attributable to a deficiency in alpha-N-acetylgalactosaminidase (alpha-NAGA; E.C.3.2.1.49), which hydrolyzes GalNAcalpha1-O-Ser/Thr. Missense mutations, R329W or R329Q were identified in two Japanese Kanzaki patients. Although they are on the same codon, the clinical manifestation was more severe in R329W because an amino acid substitution led to protein instability resulting in structural change, which is greater in R329W than in R329Q. OBJECTIVE: To examine whether the different clinical phenotypes are attributable to the two mutations. METHODS: Plasma alpha-NAGA activity and urinary excreted glycopeptides were measured and three-dimensional models of human alpha-NAGA and its complexes with GalNAcalpha1-O-Ser and GalNAcalpha1-O-Thr were constructed by homology modeling. RESULTS: Residual enzyme activity was significantly higher in the R329Q- than the R329W mutant (0.022+/-0.005 versus 0.005+/-0.001 nmol/h/ml: p<0.05); the urinary ratios of GalNAcalpha1-O-Ser:GalNAcalpha1-O-Thr were 2:10 and 8:10, respectively. GalNAcalpha1-O-Ser/Thr fit tightly in a narrow space of the active site pocket of alpha-NAGA. GalNAcalpha1-O-Thr requires a larger space to associate with alpha-NAGA because of the side chain (CH3) of the threonine residue. CONCLUSION: Our findings suggest that the association of alpha-NAGA with its substrates is strongly affected by the amino acid substitution at R329 and that the association with GalNAcalpha1-O-Thr is more highly susceptible to structural changes. The residual mutant enzyme in R329W could not associate with GalNAcalpha1-O-Thr and GalNAcalpha1-O-Ser. However, the residual mutant enzyme in R329Q catalyzed GalNAcalpha1-O-Ser to some extent. Therefore, the urinary ratio of GalNAcalpha1-O-Ser:GalNAcalpha1-O-Thr was lower and the clinical phenotype was milder in the R329Q mutation. Structural analysis revealed biochemical and phenotypic differences in these Kanzaki patients with the R329Q and R329W mutation.