Association of <Emphasis Type="Italic">ENPP1</Emphasis>gene polymorphisms with hand osteoarthritis in a Chuvasha population

Periarticular calcification is a common attendant symptom of generalized arterial calcification of infancy, a rare Mendelian disorder caused by mutations of the gene coding for ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1). This prompted us to perform a family-based association study to test the hypothesis that genetic variation at the ENPP1 locus is involved in the etiology of osteoarthritis of the hand. The study population comprised 126 nuclear families with 574 adult individuals living in small villages in the Chuvasha and Bashkirostan autonomies of the Russian Federation. The extent of osteoarthritis was determined by analyzing plain hand radiographs. The outcome of a principal component analysis of osteoarthritis scores of a total of 28 joints of both hands was used as a primary phenotype in this study. Maximum likelihood estimates of the variance component analysis revealed a substantial contribution of genetic factors to the overall trait variance of about 25% in this homogeneous population. Three short tandem repeat (STR) polymorphisms – one intragenic and two flanking markers – and four single-nucleotide polymorphisms were tested. The markers tagged the ENPP1 locus at nearly equal intervals. We used three different transmission disequilibrium tests and obtained highly significant association signals. Alleles of the upstream microsatellite marker as well as several single-nucleotide polymorphism haplotypes consistently revealed the association. Thus, our data highlights variability of ENPP1 as an important genetic factor in the pathogenesis of idiopathic osteoarthritis.     

Glycogen debranching enzyme 6 (AGL), enolase 1 (ENOSF1), ectonucleotide pyrophosphatase 2 (ENPP2_1), glutathione S-transferase 3 (GSTM3_3) and mannosidase (MAN2B2) metabolism computational network analysis between chimpanzee and human left cerebrum

We identified significantly higher expression of the genes glycogen debranching enzyme 6 (AGL), enolase 1 (ENOSF1), ectonucleotide pyrophosphatase 2 (ENPP2_1), glutathione S-transferase 3 (GSTM3_3) and mannosidase (MAN2B2) from human left cerebrums versus chimpanzees. Yet the distinct low- and high-expression AGL, ENOSF1, ENPP2_1, GSTM3_3 and MAN2B2 metabolism networks between chimpanzee and human left cerebrum remain to be elucidated. Here, we constructed low- and high-expression activated and inhibited upstream and downstream AGL, ENOSF1, ENPP2_1, GSTM3_3 and MAN2B2 metabolism network between chimpanzee and human left cerebrum in GEO data set by gene regulatory network inference method based on linear programming and decomposition procedure, under covering AGL, ENOSF1, ENPP2_1, GSTM3_3 and MAN2B2 pathway and matching metabolism enrichment analysis by CapitalBio MAS 3.0 integration of public databases, including Gene Ontology, KEGG, BioCarta, GenMapp, Intact, UniGene, OMIM, etc. Our results show that the AGL, ENOSF1, ENPP2_1, GSTM3_3 and MAN2B2 metabolism network has more activated and less inhibited molecules in chimpanzee, but less activated and more inhibited in the human left cerebrum. We inferred stronger carbohydrate, glutathione and proteoglycan metabolism, ATPase activity, but weaker base excision repair, arachidonic acid and drug metabolism as a result of inducing cell growth in low-expression AGL, ENOSF1, ENPP2_1, GSTM3_3 and MAN2B2 metabolism network of chimpanzee left cerebrum; whereas stronger lipid metabolism, amino acid catabolism, DNA repair but weaker inflammatory response, cell proliferation, glutathione and carbohydrate metabolism as a result of inducing cell differentiation in high-expression AGL, ENOSF1, ENPP2_1, GSTM3_3 and MAN2B2 metabolism network of human left cerebrum. Our inferences are consistent with recent reports and computational activation and inhibition gene number patterns, respectively.     

ENPP1 variants in patients with GACI and PXE expand the clinical and genetic heterogeneity of heritable disorders of ectopic calcification

Pseudoxanthoma elasticum (PXE) and generalized arterial calcification of infancy (GACI) are clinically distinct genetic entities of ectopic calcification associated with differentially reduced circulating levels of inorganic pyrophosphate (PPi), a potent endogenous inhibitor of calcification. Variants in ENPP1, the gene mutated in GACI, have not been associated with classic PXE. Here we report the clinical, laboratory, and molecular evaluations of ten GACI and two PXE patients from five and two unrelated families registered in GACI Global and PXE International databases, respectively. All patients were found to carry biallelic variants in ENPP1. Among ten ENPP1 variants, one homozygous variant demonstrated uniparental disomy inheritance. Functional assessment of five previously unreported ENPP1 variants suggested pathogenicity. The two PXE patients, currently 57 and 27 years of age, had diagnostic features of PXE and had not manifested the GACI phenotype. The similarly reduced PPi plasma concentrations in the PXE and GACI patients in our study correlate poorly with their disease severity. This study demonstrates that in addition to GACI, ENPP1 variants can cause classic PXE, expanding the clinical and genetic heterogeneity of heritable ectopic calcification disorders. Furthermore, the results challenge the current prevailing concept that plasma PPi is the only factor governing the severity of ectopic calcification.     

Loss-of-Function ENPP1 Mutations Cause Both Generalized Arterial Calcification of Infancy and Autosomal-Recessive Hypophosphatemic Rickets

The analysis of rare genetic disorders affecting phosphate homeostasis led to the identification of several proteins that are essential for the renal regulation of phosphate homeostasis; for example, fibroblast growth factor 23 (FGF23), which inhibits renal phosphate reabsorption and 1,25-dihydroxyvitamin D synthesis. Here, we report presumable loss-of-function mutations in the ENPP1 gene (ectonucleotide pyrophosphatase/phosphodiesterase) in members of four families affected with hypophosphatemic rickets. We provide evidence for the conclusion that ENPP1 is the fourth gene—in addition to PHEX, FGF23, and DMP1—that, if mutated, causes hypophosphatemic rickets resulting from elevated FGF23 levels. Surprisingly, ENPP1 loss-of-function mutations have previously been described in generalized arterial calcification of infancy, suggesting an as yet elusive mechanism that balances arterial calcification with bone mineralization.     

Identification of Ectonucleotide Pyrophosphatase/Phosphodiesterase 3 (ENPP3) as a Regulator of N-Acetylglucosaminyltransferase GnT-IX (GnT-Vb)

Our previous studies on a β1,6-N-acetylglucosaminyltransferase, GnT-IX (GnT-Vb), a homolog of GnT-V, indicated that the enzyme has a broad GlcNAc transfer activity toward N-linked and O-mannosyl glycan core structures and that its brain-specific gene expression is regulated by epigenetic histone modifications. In this study, we demonstrate the existence of an endogenous inhibitory factor for GnT-IX that functions as a key regulator for GnT-IX enzymatic activity in Neuro2a (N2a) cells. We purified this factor from N2a cells and found that it is identical to ectonucleotide pyrophosphatase/phosphodiesterase 3 (ENPP3), as evidenced by mass spectrometry and by the knockdown and overexpression of ENPP3 in cultured cells. Kinetic analyses revealed that the mechanism responsible for the inhibition of GnT-IX caused by ENPP3 is the ENPP3-mediated hydrolysis of the nucleotide sugar donor substrate, UDP-GlcNAc, with the resulting generation of UMP, a potent and competitive inhibitor of GnT-IX. Indeed, ENPP3 knockdown cells had significantly increased levels of intracellular nucleotide sugars and displayed changes in the total cellular glycosylation profile. In addition to chaperones or other known regulators of glycosyltransferases, the ENPP3-mediated hydrolysis of nucleotide sugars would have widespread and significant impacts on glycosyltransferase activities and would be responsible for altering the total cellular glycosylation profile and modulating cellular functions.     

Cole Disease Results from Mutations in ENPP1

The coexistence of abnormal keratinization and aberrant pigmentation in a number of cornification disorders has long suggested a mechanistic link between these two processes. Here, we deciphered the genetic basis of Cole disease, a rare autosomal-dominant genodermatosis featuring punctate keratoderma, patchy hypopigmentation, and uncommonly, cutaneous calcifications. Using a combination of exome and direct sequencing, we showed complete cosegregation of the disease phenotype with three heterozygous ENPP1 mutations in three unrelated families. All mutations were found to affect cysteine residues in the somatomedin-B-like 2 (SMB2) domain in the encoded protein, which has been implicated in insulin signaling. ENPP1 encodes ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1), which is responsible for the generation of inorganic pyrophosphate, a natural inhibitor of mineralization. Previously, biallelic mutations in ENPP1 were shown to underlie a number of recessive conditions characterized by ectopic calcification, thus providing evidence of profound phenotypic heterogeneity in ENPP1-associated genetic diseases.     

Autosomal-Recessive Hypophosphatemic Rickets Is Associated with an Inactivation Mutation in the ENPP1 Gene

Human disorders of phosphate (Pi) handling and hypophosphatemic rickets have been shown to result from mutations in PHEX, FGF23, and DMP1, presenting as X-linked recessive, autosomal-dominant, and autosomal-recessive patterns, respectively. We present the identification of an inactivating mutation in the ecto-nucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) gene causing autosomal-recessive hypophosphatemic rickets (ARHR) with phosphaturia by positional cloning. ENPP1 generates inorganic pyrophosphate (PPi), an essential physiologic inhibitor of calcification, and previously described inactivating mutations in this gene were shown to cause aberrant ectopic calcification disorders, whereas no aberrant calcifications were present in our patients. Our surprising result suggests a different pathway involved in the generation of ARHR and possible additional functions for ENPP1.     

Association of common polymorphisms in known susceptibility genes with rheumatoid arthritis in a Slovak population using osteoarthritis patients as controls

Introduction  

Both genetic and environmental factors contribute to rheumatoid arthritis (RA), a common and complex autoimmune disease. As well as the major susceptibility gene HLA-DRB1, recent genome-wide and candidate-gene studies reported additional evidence for association of single nucleotide polymorphism (SNP) markers in the PTPN22, STAT4, OLIG3/TNFAIP3 and TRAF1/C5 loci with RA. This study was initiated to investigate the association between defined genetic markers and RA in a Slovak population. In contrast to recent studies, we included intensively-characterized osteoarthritis (OA) patients as controls.     

DD genotype of ace gene I/D polymorphism is associated in a turkish study population with osteoarthritis

This study was conducted in Turkish osteoarthritis patients to determine the frequency of I/D polymorphism genotypes of angiotensin converting enzyme gene, and to examine the role of this polymorphism in osteoarthritis development. Genomic DNA obtained from 200 persons (135 patients with osteoarthritis and 65 healthy controls) was used in the study. DNA was multiplied by polymerase chain reaction using I and D allele-specific primers. Polymerase chain reaction products were assessed with CCD camera by being exposed to 2% agarose gel electrophoresis. There was statistically significant difference between the groups with respect to genotype distribution (P < 0.001). The D allele frequency was indicated as 69% and I allele was as 31% in the patients, whereas it was 55–45% in the control group. Consequently, in this study, we may assert that ACE gene I/D polymorphism DD genotype determination is significant criteria for identifying patients who are likely to develop osteoarthritis in east population of Turkey.     

A Novel Highly Potent Autotaxin/ENPP2 Inhibitor Produces Prolonged Decreases in Plasma Lysophosphatidic Acid Formation In Vivo and Regulates Urethral Tension

Autotaxin, also known as ectonucleotide pyrophosphatase/phosphodiesterase 2 (ENPP2), is a secreted enzyme that has lysophospholipase D activity, which converts lysophosphatidylcholine to bioactive lysophosphatidic acid. Lysophosphatidic acid activates at least six G-protein coupled recpetors, which promote cell proliferation, survival, migration and muscle contraction. These physiological effects become dysfunctional in the pathology of cancer, fibrosis, and pain. To date, several autotaxin/ENPP2 inhibitors have been reported; however, none were able to completely and continuously inhibit autotaxin/ENPP2 in vivo. In this study, we report the discovery of a highly potent autotaxin/ENPP2 inhibitor, ONO-8430506, which decreased plasma lysophosphatidic acid formation.The IC₅₀ values of ONO-8540506 for lysophospholipase D activity were 6.4–19 nM for recombinant autotaxin/ENPP2 proteins and 4.7–11.6 nM for plasma from various animal species. Plasma lysophosphatidic acid formation during 1-h incubation was almost completely inhibited by the addition of >300 nM of the compound to human plasma. In addition, when administered orally to rats at a dose of 30 mg/kg, the compound demonstrated good pharmacokinetics in rats and persistently inhibited plasma lysophosphatidic acid formation even at 24 h after administration.Smooth muscle contraction is a known to be promoted by lysophosphatidic acid. In this study, we showed that dosing rats with ONO-8430506 decreased intraurethral pressure accompanied by urethral relaxation. These findings demonstrate the potential of this autotaxin/ENPP2 inhibitor for the treatment of various diseases caused by lysophosphatidic acid, including urethral obstructive disease such as benign prostatic hyperplasia.     

Search for hand osteoarthritis susceptibility locus on chromosome 6p12.3-p12.1

The existence of osteoarthritis susceptibility loci on chromosome 6 for individuals suffering from hip and knee osteoarthritis has been suggested. We determined whether radiographic hand osteoarthritis in a demographically homogeneous population of European origin can be linked to loci on chromosome 6p12.3-p12.1. Nine single nucleotide polymorphisms (SNPs) were genotyped in 764 individuals (members of 189 nuclear and more complex two- or three-generation families). Radiographic hand osteoarthritis was characterized by two traits: (1) the total individual osteoarthritis score (PC1-OA) and (2) the osteophytes score (PC1-OS), obtained from the principal components analysis of sums of the Kellgren and Lawrence grade and of the osteophyte grades, respectively, for 14 joints on each hand. The contribution of genetic and environmental factors and of covariates such as age and body mass index to hand osteoarthritis was evaluated by variance components analysis. The association between the studied traits and selected DNA markers was evaluated by three types of transmission disequilibrium tests. The parent-offspring and sib-sib correlations were statistically significant for all studied traits. The additive genetic effects for PC1-OA and PC1-OS were estimated to be 43% and 37.9%, respectively. Transmission disequilibrium tests consistently revealed a statistically significant association (p values ranged from 0.017 to 0.030) between SNP rs1508632 and PC1-OS. In the tested cohort the putative genetic factors are influential enough to determine interindividual differences regarding the extent of hand osteoarthritis. SNP rs1508632 lies in immediate proximity to the TINAG gene, implicating it as a possible hand osteoarthritis susceptibility gene.     

筛选地中海拟无枝酸菌无痕基因敲除菌株的简便方法

将抗生素抗性基因作为标记筛选无痕基因敲除菌株比较费时,因而建立筛选无痕基因敲除菌株的简便方法。通过敲除茄红素生物合成途径中第一个反应的酶编码基因dxs(1-脱氧-D-木酮糖-5-磷酸合酶基因),获得白色地中海拟无枝酸菌突变菌株,以此菌株为受体菌,对S-丙二酰转移酶基因(mtf)进行无痕敲除。针对菌落本身携带颜色的地中海拟无枝酸菌(橘红色),利用茄红素合成酶基因dxs无痕敲除获得了白色菌株,在此基础上进行mtf的无痕敲除。以茄红素生物合成途径中任意一个反应的酶编码基因作为标记,很容易筛选得到无痕基因敲除的突变菌株。     

ISOLATION AND CHARACTERIZATION OF THE PHYTOENE DESATURASE GENE AS A POTENTIAL SELECTIVE MARKER FOR GENETIC ENGINEERING OF THE ASTAXANTHIN‐PRODUCING GREEN ALGA CHLORELLA ZOFINGIENSIS (CHLOROPHYTA)1

Phytoene desaturase (PDS) is a rate‐limiting enzyme in carotenoid biosynthesis. Algal PDS is inhibited by some herbicides, leading to the bleaching of the cells due to destruction of chl. Specific point mutations in PDS confer resistance to the herbicide norflurazon, suggesting that mutated PDS could be used as a dominant selectable marker for genetic engineering of algae, for which very few selective markers are available. In this study, we report the isolation and characterization of the PDS gene from the astaxanthin‐producing green alga Chlorella zofingiensis Dönz. The open reading frame (ORF) of this PDS gene, interrupted by six introns, encoded a polypeptide of 558 amino acid residues. The deduced protein sequence showed significant homology to phytoene desaturases of algae, cyanobacteria, and higher plants. Expression of the PDS gene in Escherichia coli demonstrated that the enzyme was able to convert phytoene to ζ‐carotene. The PDS gene in Chlorella was shown to be up‐regulated by high light and glucose treatment. With a single amino acid change (L516R), the mutated PDS‐L516R was still active and exhibited ～36‐fold greater resistance to the bleaching herbicide norflurazon than the unaltered enzyme. Thus, the modified PDS gene could be a useful tool for genetic engineering of carotenoid biosynthesis in C. zofingiensis and perhaps also in other algae.     

Genetic and biochemical studies on the conversion of flavanones to dihydroflavonols in flowers of Petunia hybrida

Summary Chemogenetic investigations and precursor experiments on flowers of Petunia hybrida suggest that recessive alleles of the gene An3 block the biosynthetic pathway of flavonols and anthocyanins between the flavanone and dihydroflavonol step. In confirmation of this hypothesis, activity of the enzyme flavanone 3-hydroxylase, which catalyses the conversion of flavanones to dihydroflavonols, was readily demonstrated in enzyme preparations from flowers of lines with the dominant allele An3, whereas no or very low activity could be found in extracts from lines with recessive alleles (an3an3). A second genetic factor is described which clearly reduces the amount of flavonols in the flowers but not the amount of anthocyanins. Crossing experiments revealed that this factor represents a third allele of the An3 gene. It is referred to as an3-1. As expected, a residual flavanone 3-hydroxylase activity of about 10% could be found in enzyme extracts from plants with the an3-1 allele. The decreased level of dihydroflavonol formed under this condition is obviously still sufficient for anthocyanin formation but not for flavonol synthesis.Similar to flavanone 3-hydroxylases from other plants, the enzyme of Petunia is a soluble enzyme and belongs according to its cofactor requirements to the 2-oxoglutarate-dependent dioxygenases. The residual flavanone 3-hydroxylase activity found in plants with the an3-1 allele is identical to the activity extracted from An3-genotypes with regard to cofactors, substrate specificity and most of the inhibitors. The difference observed in the respective pH-optima and the genetic data suggest that the mutation providing the an3-1 phenotype is localized in the structural gene for flavanone 3-hydroxylase.     

Genetic flux between h1 and h2 haplotypes of the 17q21.31 inversion in European population

The chromosome 17q21.31 inversion is a 900-kb common structural polymorphism found primarily in European population.Although the genetic flux within inversion region was assumed to be considerable suppressed, it is still unclear about the details of genetic exchange between the H1(non-inverted sequence)and H2(inverted sequence) haplotypes of this inversion.Here we describe a refined map of genetic exchanges between pairs of gene arrangements within the 17q21.31 region.Using HapMap phase II data of 1,546 single nucleotide polymorphisms, we successfully deduced 96 H1 and 24 H2 haplotypes in European samples by neighbor-joining tree reconstruction. Furthermore, we identified 15 and 26 candidate tracts with reciprocal and non-reciprocal genetic exchanges, respectively.In all 15 regions harboring reciprocal exchange, haplotypes reconstructed by clone sequencing did not support these exchange events, suggesting that such signals of exchange between two sister chromosomes in certain heterozygous individual were caused by phasing error regions.On the other hand, the finished clone sequencing across 4 of 26 tracts with non-reciprocal genetic flux confirmed that this kind of genetic exchange was caused by gene conversion.In summary, as crossover between pairs of gene arrangements had been considerably suppressed, gene conversion might be the most important mechanism for genetic exchange at 17q21.31.     

Multifactor dimensionality reduction reveals a strong gene–gene interaction between STC1 and COL11A1 genes as a possible risk factor of knee osteoarthritis

Articular cartilage is an avascular tissue with a structure that allows it to support and cushion the overload of the surfaces in contact. It maintains its metabolic functions due to the contribution of different signaling pathways. However, several factors play a role in its deterioration, allowing to the development of osteoarthritis (OA), and one of the major factors is genetic. Our goal was to identify gene–gene interactions (epistasis) between five signaling pathways involved in the articular cartilage metabolism as possible indicators of OA risk. We applied the Multifactor-Dimensionality Reduction (MDR) method to identify and characterize the epistasis between 115 SNPs located in 73 genes related to HIF-1α, Wnt/β-catenin, cartilage extracellular matrix metabolism, oxidative stress, and uric acid transporters. Ninety three patients diagnosed with primary knee OA and 150 healthy controls were included in the study. Genotyping was performed with the OpenArray system, the statistical analysis was carried out with the STATA software v14, and epistasis was analyzed with the MDR software v3.0.2. The MDR analysis revealed that the best interaction model was between polymorphisms rs17786744 of the STC1 gene and rs2615977 of the COL11A1 gene, with an entropy value of 4.44%, CVC 8/10, OR 5.60, 95% CI 3.27–9.59, p?<?0.0001. Under this interaction model, we identified high and low risk genotypes involved in OA development. Our results suggest complex interactions between STC1 and COL11A1 genes that might have an impact on genetic susceptibility to develop OA. Further studies are required to confirm it.

     

Ectonucleotide pyrophosphatase/phosphodiesterase-1 (ENPP1) protein regulates osteoblast differentiation

ENPP1 (ectonucleotide pyrophosphatase/phosphodiesterase-1) is an established regulator of tissue mineralization. Previous studies demonstrated that ENPP1 is expressed in differentiated osteoblasts and that ENPP1 influences matrix mineralization by increasing extracellular levels of inorganic pyrophosphate. ENPP1 is also expressed in osteoblastic precursor cells when stimulated with FGF2, but the role of ENPP1 in preosteoblastic and other precursor cells is unknown. Here we investigate the function of ENPP1 in preosteoblasts. We find that ENPP1 expression is critical for osteoblastic differentiation and that this effect is not mediated by changes in extracellular concentration levels of phosphate or pyrophosphate or ENPP1 catalytic activity. MC3T3E1(C4) preosteoblastic cells, in which ENPP1 expression was suppressed by ENPP1-specific shRNA, and calvarial cells isolated from Enpp1 knock-out mice show defective osteoblastic differentiation upon stimulation with ascorbate, as indicated by a lack of cellular morphological change, a lack of osteoblast marker gene expression, and an inability to mineralize matrix. Additionally, MC3T3E1(C4) cells, in which wild type or catalytic inactive ENPP1 expression was increased, exhibited an increased tendency to differentiate, as evidenced by increased osteoblast marker gene expression and increased mineralization. Notably, treatment of cells with inorganic phosphate or pyrophosphate inhibited, as opposed to enhanced, expression of multiple genes that are expressed in association with osteoblast differentiation, matrix deposition, and mineralization. Our results indicate that ENPP1 plays multiple and distinct roles in the development of mineralized tissues and that the influence of ENPP1 on osteoblast differentiation and gene expression may include a mechanism that is independent of its catalytic activity.     

Replication of Type 2 Diabetes Candidate Genes Variations in Three Geographically Unrelated Indian Population Groups

Type 2 diabetes (T2D) is a syndrome of multiple metabolic disorders and is genetically heterogeneous. India comprises one of the largest global populations with highest number of reported type 2 diabetes cases. However, limited information about T2D associated loci is available for Indian populations. It is, therefore, pertinent to evaluate the previously associated candidates as well as identify novel genetic variations in Indian populations to understand the extent of genetic heterogeneity. We chose to do a cost effective high-throughput mass-array genotyping and studied the candidate gene variations associated with T2D in literature. In this case-control candidate genes association study, 91 SNPs from 55 candidate genes have been analyzed in three geographically independent population groups from India. We report the genetic variants in five candidate genes: TCF7L2, HHEX, ENPP1, IDE and FTO, are significantly associated (after Bonferroni correction, p<5.5E−04) with T2D susceptibility in combined population. Interestingly, SNP rs7903146 of the TCF7L2 gene passed the genome wide significance threshold (combined P value = 2.05E−08) in the studied populations. We also observed the association of rs7903146 with blood glucose (fasting and postprandial) levels, supporting the role of TCF7L2 gene in blood glucose homeostasis. Further, we noted that the moderate risk provided by the independently associated loci in combined population with Odds Ratio (OR)<1.38 increased to OR = 2.44, (95%CI = 1.67–3.59) when the risk providing genotypes of TCF7L2, HHEX, ENPP1 and FTO genes were combined, suggesting the importance of gene-gene interactions evaluation in complex disorders like T2D.