Impact of ITPA gene polymorphism for predicting anemia and treatment outcome in HCV infected patients taking Sofosbuvir Ribavirin therapy.

BACKGROUND: Globally, 80 million people are suffering from chronic Hepatitis C virus (HCV) infection. Sofosbuvir ribavirin-based anti-HCV therapy is associated with anemia and other adverse effects. Polymorphisms of Inosine triphosphatase (ITPA) gene may cause functional impairment in the Inosine triphosphate pyrophosphatase enzyme, resulting in enhanced sustained viral response (SVR) and protection from ribavirin-associated anemia in patients on therapy. The study objective was to investigate the effect of Inosine triphosphatase gene polymorphism on SVR achievement, hemoglobin decline and ribavirin dose reduction in patients on therapy. METHODS: This prospective cohort study was of 170 hepatitis C infected patients received 6-month sofosbuvir ribavirin therapy. Patient viral load, reduction in ribavirin amount, liver function test, and complete blood count were noted monthly. Inosine triphosphatase variants rs1127354 and rs7270101 were assessed through the restriction fragment length polymorphism and confirmed using Sanger sequencing. The impact of polymorphism on cumulative reduction of ribavirin, and anti-HCV therapy outcome were studied. RESULTS: A total of 74.3% of patients had ITPA rs1127354 CC genotype, 25.7% were CA and AA 0%. The frequency of ITPA genotype rs7270101-AA was 95%, AC 5%, and CC was 0%. ITPA rs1127354-CA had a notably positive impact on SVR achievement with a zero-relapse rate. ITPA rs1127354-CA genotype was significantly (P ˂0.05) protective against ≥ 2 g/dl Hb reduction from baseline to 1st, 2nd and 6th months of therapy. During treatment, Hb reduction ≥ 10 g/dl was frequently observed in rs1127354-CC genotype and rs7270101-AA genotype patients. Ribavirin dose reduction was significantly (P ˂0.05) high in rs1127354-CC genotype as compared to genotype CA whereas no significant difference was observed in ribavirin dose reduction in rs7270101 AA and non-AA genotype. Patient baseline characteristics such as age, body mass index, rs1127354-CC genotype, and baseline Hb were significantly associated with significant Hb reduction. CONCLUSION: Pretreatment evaluation of ITPA polymorphism can be a diagnostic tool to find out patients at risk of anemia and improve treatment adherence. ITPA genotype rs1127354-CA contributes to improved compliance with ribavirin dose and protects against hemoglobin decline in HCV patients while taking ribavirin-based therapy. However, ITPA rs1127354, rs7270101 polymorphism have no significant impact on SVR achievement.

Structural and biochemical characterization of active sites mutant in human inorganic pyrophosphatase.

Inorganic pyrophosphatases (PPases) are enzymes that catalyze the conversion of inorganic pyrophosphate (PPi) into phosphate (Pi). Human inorganic pyrophosphatase 1 (Hu-PPase) exhibits high expression levels in a variety of tumors and plays roles in cell proliferation, apoptosis, invasion and metastasis, making it a promising prognostic biomarker and a target for cancer therapy. Despite its widespread presence, the catalytic mechanism of Hu-PPase in humans remains inadequately understood. The signature motif amino acid sequence (DXDPXD) within the active sites of PPases is preserved across different species. In this research, an enzymatic activity assay revealed that mutations led to a notable reduction in enzymatic function, although the impact of the four amino acids on the activity of the pocket varied. To investigate the influence of these residues on the substrate binding and enzymatic function of PPase, the crystal structure of the Hu-PPase-ED quadruple mutant (D116A/D118A/P119A/D121A) was determined at 1.69 Å resolution. The resulting structure maintained a barrel-like shape similar to that of the wild-type, albeit lacking Mg2+ ions. Molecular docking analysis demonstrated a decreased ability of Hu-PPase-ED to bind to PPi. Further, molecular dynamics simulation analysis indicated that the mutation rendered the loop of Mg2+ ion-binding residues less stable. Therefore, the effect on enzyme activity did not result from a change in the gross protein structure but rather from a mutation that abolished the Mg2+-coordinating groups, thereby eliminating Mg2+ binding and leading to the loss of enzyme activity.

The novel phosphatase NUDT5 is a critical regulator of triple-negative breast cancer growth.

BACKGROUND: The most aggressive form of breast cancer is triple-negative breast cancer (TNBC), which lacks expression of the estrogen receptor (ER) and progesterone receptor (PR), and does not have overexpression of the human epidermal growth factor receptor 2 (HER2). Treatment options for women with TNBC tumors are limited, unlike those with ER-positive tumors that can be treated with hormone therapy, or those with HER2-positive tumors that can be treated with anti-HER2 therapy. Therefore, we have sought to identify novel targeted therapies for TNBC. In this study, we investigated the potential of a novel phosphatase, NUDT5, as a potential therapeutic target for TNBC. METHODS: The mRNA expression levels of NUDT5 in breast cancers were investigated using TCGA and METABRIC (Curtis) datasets. NUDT5 ablation was achieved through siRNA targeting and NUDT5 inhibition with the small molecule inhibitor TH5427. Xenograft TNBC animal models were employed to assess the effect of NUDT5 inhibition on in vivo tumor growth. Proliferation, death, and DNA replication assays were conducted to investigate the cellular biological effects of NUDT5 loss or inhibition. The accumulation of 8-oxo-guanine (8-oxoG) and the induction of γH2AX after NUDT5 loss was determined by immunofluorescence staining. The impact of NUDT5 loss on replication fork was assessed by measuring DNA fiber length. RESULTS: In this study, we demonstrated the significant role of an overexpressed phosphatase, NUDT5, in regulating oxidative DNA damage in TNBCs. Our findings indicate that loss of NUDT5 results in suppressed growth of TNBC both in vitro and in vivo. This growth inhibition is not attributed to cell death, but rather to the suppression of proliferation. The loss or inhibition of NUDT5 led to an increase in the oxidative DNA lesion 8-oxoG, and triggered the DNA damage response in the nucleus. The interference with DNA replication ultimately inhibited proliferation. CONCLUSIONS: NUDT5 plays a crucial role in preventing oxidative DNA damage in TNBC cells. The loss or inhibition of NUDT5 significantly suppresses the growth of TNBCs. These biological and mechanistic studies provide the groundwork for future research and the potential development of NUDT5 inhibitors as a promising therapeutic approach for TNBC patients.

The dePARylase NUDT16 promotes radiation resistance of cancer cells by blocking SETD3 for degradation via reversing its ADP-ribosylation.

Poly(ADP-ribosyl)ation (PARylation) is a critical posttranslational modification that plays a vital role in maintaining genomic stability via a variety of molecular mechanisms, including activation of replication stress and the DNA damage response. The nudix hydrolase NUDT16 was recently identified as a phosphodiesterase that is responsible for removing ADP-ribose units and that plays an important role in DNA repair. However, the roles of NUDT16 in coordinating replication stress and cell cycle progression remain elusive. Here, we report that SETD3, which is a member of the SET-domain containing protein (SETD) family, is a novel substrate for NUDT16, that its protein levels fluctuate during cell cycle progression, and that its stability is strictly regulated by NUDT16-mediated dePARylation. Moreover, our data indicated that the E3 ligase CHFR is responsible for the recognition and degradation of endogenous SETD3 in a PARP1-mediated PARylation-dependent manner. Mechanistically, we revealed that SETD3 associates with BRCA2 and promotes its recruitment to stalled replication fork and DNA damage sites upon replication stress or DNA double-strand breaks, respectively. Importantly, depletion of SETD3 in NUDT16-deficient cells did not further exacerbate DNA breaks or enhance the sensitivity of cancer cells to IR exposure, suggesting that the NUDT16-SETD3 pathway may play critical roles in the induction of tolerance to radiotherapy. Collectively, these data showed that NUDT16 functions as a key upstream regulator of SETD3 protein stability by reversing the ADP-ribosylation of SETD3, and NUDT16 participates in the resolution of replication stress and facilitates HR repair.

ENPP1 inhibits the transcription activity of the hepatitis B virus pregenomic promoter by upregulating the acetylation of LMNB1.

Current therapies for hepatitis B virus (HBV) infection can slow disease progression but cannot cure the infection, as it is difficult to eliminate or permanently silence HBV covalently closed circular DNA (cccDNA). The interaction between host factors and cccDNA is essential for their formation, stability, and transcriptional activity. Here, we focused on the regulatory role of the host factor ENPP1 and its interacting transcription factor LMNB1 in HBV replication and transcription to better understand the network of host factors that regulate HBV, which may facilitate the development of new antiviral drugs. Overexpression of ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) in Huh7 cells decreased HBV pregenomic RNA (pgRNA) and hepatitis B core antigen (HBcAg) expression levels, whereas knockdown of ENPP1 increased them. A series of HBV promoter and mutant plasmids were constructed, and a luciferase reporter assay showed that overexpression of ENPP1 caused inhibition of the HBV promoter and its mutants. A DNA pull-down assay showed that lamin B1 (LMNB1), but not ENPP1, interacts directly with the HBV enhancer II/ basic core promoter (EnhII/BCP). ZDOCK and PyMOL software were used to predict the interaction of ENPP1 with LMNB1. Overexpression of LMNB1 inhibited the activity of the HBV promoter and its mutant. The acetylation levels at the amino acids 111K, 261K, and 483K of LMNB1 were reduced compared to the control, and an LMNB1 acetylation mutant containing 111R, 261Q, 261R, 483Q, and 483R showed increased promoter activity. In summary, ENPP1 together with LMNB1 increased the acetylation level at 111K and 261K, and LMNB1 inhibited the activity of HBV promoter and downregulated the expression of pregenomic RNA and HBcAg. Our follow-up studies will investigate the expression, clinical significance, and relevance of ENPP1 and LMNB1 in HBV patient tissues, explore the effect of LMNB1 on post-transcriptional progression, and examine whether ENPP1 can reduce cccDNA levels in the nucleus.

Measurement of the intracellular active metabolites of thiopurine drugs to evaluate the enzymatic activity of nudix hydrolase 15 in human blood samples.

Thiopurine is metabolized to 6-thio-(deoxy) guanosine triphosphate (6-thio-(d) GTP), which is then incorporated into DNA or RNA and causes cytotoxicity. Nudix hydrolase 15 (NUDT15) reduces the cytotoxic effects of thiopurine by converting 6-thio-(d) GTP to 6-thio-(d) guanosine monophosphate (6-thio-(d) GMP). NUDT15 polymorphisms like the Arg139Cys variant are strongly linked to thiopurine-induced severe leukocytopenia and alopecia. Therefore, measurement of NUDT15 enzymatic activity in individual patients can help predict thiopurine tolerability and adjust the dosage. We aimed to develop a quantitative assay for NUDT15 enzymatic activity in human blood samples. Blood samples were collected from donors whose NUDT15 genetic status was determined. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to assess the 6-thio-GTP metabolic activity in cell extracts. Because 6-thio-guanosine diphosphate (6-thio-GDP) and 6-thio-GMP were generated upon incubation of 6-thio-GTP with human blood cell extracts, the method detecting 6-thio-GTP, 6-thio-GDP, and 6-thio-GMP was validated. All three metabolites were linearly detected, and the lower limit of quantification (LLOQ) of 6-thio-GTP, 6-thio-GDP, and 6-thio-GMP were 5 µM, 1 µM, and 2 µM, respectively. Matrix effects of human blood cell extracts to detect 6-thio-GTP, 6-thio-GDP, and 6-thio-GMP were 99.0 %, 100.5 %, and 101.4 %, respectively, relative to the signals in the absence of blood cell extracts. The accuracy and precision of the method and the stability of the samples were also assessed. Using this established method, the genotype-dependent differences in NUDT15 activities were successfully determined using cell extracts derived from human blood cells with NUDT15 wild-type (WT) or Arg139Cys variant and 6-thio-GTP (100 µM) as a substrate (18.1, 14.9, and 6.43 µM/h/106 cells for WT, Arg139Cys heterozygous, and homozygous variant, respectively). We developed a method for quantifying intracellular NUDT15 activity in peripheral blood mononuclear cells (PBMCs), which we defined as the conversion of 6-thio-GTP to 6-thio-GMP. Although PBMCs preparation takes some time, its reproducibility in experiments makes it a promising candidate for clinical application. This method can tell the difference between WT and Arg139Cys homozygous blood samples. Even in patients with WT NUDT15, WT samples showed variations in NUDT15 activity, which may correlate with variations in thiopurine dosage.

Cartilage tissues regulate systemic aging via ectonucleotide pyrophosphatase/phosphodiesterase 1 in mice.

Aging presents fundamental health concerns worldwide; however, mechanisms underlying how aging is regulated are not fully understood. Here, we show that cartilage regulates aging by controlling phosphate metabolism via ectonucleotide pyrophosphatase/phosphodiesterase 1 (Enpp1). We newly established an Enpp1 reporter mouse, in which an EGFP-luciferase sequence was knocked-in at the Enpp1 gene start codon (Enpp1/EGFP-luciferase), enabling detection of Enpp1 expression in cartilage tissues of resultant mice. We then established a cartilage-specific Enpp1 conditional knockout mouse (Enpp1 cKO) by generating Enpp1 flox mice and crossing them with cartilage-specific type 2 collagen Cre mice. Relative to WT controls, Enpp1 cKO mice exhibited phenotypes resembling human aging, such as short life span, ectopic calcifications, and osteoporosis, as well as significantly lower serum pyrophosphate levels. We also observed significant weight loss and worsening of osteoporosis in Enpp1 cKO mice under phosphate overload conditions, similar to global Enpp1-deficient mice. Aging phenotypes seen in Enpp1 cKO mice under phosphate overload conditions were rescued by a low vitamin D diet, even under high phosphate conditions. These findings suggest overall that cartilage tissue plays an important role in regulating systemic aging via Enpp1.

ENPP1 in Blood and Bone: Skeletal and Soft Tissue Diseases Induced by ENPP1 Deficiency.

The enzyme ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) codes for a type 2 transmembrane glycoprotein that hydrolyzes extracellular ATP to generate pyrophosphate (PPi) and adenosine monophosphate, thereby contributing to downstream purinergic signaling pathways. The clinical phenotypes induced by ENPP1 deficiency are seemingly contradictory and include early-onset osteoporosis in middle-aged adults and life-threatening vascular calcifications in the large arteries of infants with generalized arterial calcification of infancy. The progressive overmineralization of soft tissue and concurrent undermineralization of skeleton also occur in the general medical population, where it is referred to as paradoxical mineralization to highlight the confusing pathophysiology. This review summarizes the clinical presentation and pathophysiology of paradoxical mineralization unveiled by ENPP1 deficiency and the bench-to-bedside development of a novel ENPP1 biologics designed to treat mineralization disorders in the rare disease and general medical population.

ENPP1 downregulation and FGF23 upregulation in growth-related calcification of the tibial tuberosity in rats.

During tibial tuberosity growth, superficial and deep portions can be observed; however, the deep portion is not observed after the growth period, as it develops into bone tissues. Calcification in vivo is known to be constitutively suppressed by ectonucleotide pyrophosphatase/phosphodiesterase-1 (ENPP1) but promoted by tissue-nonspecific alkaline phosphatase (TNAP). FGF23 promotes calcification of enthesis. Gene expression of FGF23 increased rapidly at 13W in this study. Therefore, the tibial tuberosity is speculated to develop via Enpp1 downregulation and Tnap upregulation; however, the understanding of these processes remains unclear. Hence, in the present study, we aimed to explore the age-related structural changes and underlying gene expression changes in the tibial tuberosity of rats. Male Wistar rats were divided into three groups (3-, 7-, and 13-week-old; eight each). The tibial tuberosity superficial and deep portions were clearly observed in 3- and 7-week-old rats, but the presence of the deep portion was not confirmed in 13-week-old rats. The extracellular matrix of hypertrophic chondrocytes was calcified. Furthermore, the Enpp1 expression was the highest in 3-week-old rats and decreased with growth. The TNAP expression did not differ significantly among the groups. The deep portion area was significantly lower in 3-week-old rats than in 7-week-old rats. Generally, the extracellular matrix of the immature chondrocytes is not calcified. Therefore, we speculated that the cartilaginous tibial tuberosity calcifies and ossifies with growth. The Enpp1 expression decreased with growth, whereas the Tnap expression remained unchanged. Thus, we surmise that the tibial tuberosity calcifies with growth and that this process involves Enpp1 downregulation and FGF23 upregulation. As Osgood-Schlatter disease is closely related to the calcification of the tibial tuberosity, these findings may help clarify the pathogenesis of this disease.

An ITPA Enzyme with Improved Substrate Selectivity.

Recent clinical data have identified infant patients with lethal ITPA deficiencies. ITPA is known to modulate ITP concentrations in cells and has a critical function in neural development which is not understood. Polymorphism of the ITPA gene affects outcomes for both ribavirin and thiopurine based therapies and nearly one third of the human population is thought to harbor ITPA polymorphism. In a previous site-directed mutagenesis alanine screen of the ITPA substrate selectivity pocket, we identified the ITPA mutant, E22A, as a gain-of function mutant with enhanced ITP hydrolysis activity. Here we report a rational enzyme engineering experiment to investigate the biochemical properties of position 22 ITPA mutants and find that the E22D ITPA has two- and four-fold improved substrate selectivity for ITP over the canonical purine triphosphates ATP and GTP, respectively, while maintaining biological activity. The novel E22D ITPA should be considered as a platform for further development of ITPA therapies.

Thiopurines exert harmful effects on spermatogenesis in Nudt15R138C knock-in mice.

BACKGROUND: The association between thiopurine use and testicular reproductive functions remains unclear. In this study, we investigated whether thiopurines affect testicular functions based on the NUDT15 genotypes using Nudt15R138C knock-in mice. METHODS: The male Nudt15R138C knock-in mice (9-12 weeks) were treated with mercaptopurine (MP: 0.5 mg/kg/day) for 4 or 12 weeks. To examine reversibility, some mice were maintained for a further 12 weeks under MP-free condition. RESULTS: After MP treatment for 4 weeks, Nudt15R138C/R138C mice exhibited a significant reduction of testis weight compared to Nudt15+/+ mice and Nudt15+/R138C mice. The epithelial height and diameter of seminiferous tubules were significantly reduced in Nudt15R138C/R138C mice compared to Nudt15+/+ and Nudt15+/R138C mice. Apoptotic cells were significantly increased in Nudt15R138C/R138C mice, and most of apoptotic cells were spermatogonia. There were no significant changes in sperm counts and sperm morphology in MP-treated Nudt15R138C/R138C mice after 4-week MP treatment. On the other hand, after MP treatment for 12 weeks, the Nudt15+/R138C mice, but not Nudt15+/+ mice, exhibited a significant reduction in the testis weight and atrophic changes of seminiferous tubules, but these changes disappeared after 12-week rearing under MP-free condition. Despite a significant increase in abnormal sperm rate, there were no changes in the ability to conceive. No differences in serum levels of follicle-stimulating hormone or testosterone were observed between MP-treated Nudt15+/R138C and Nudt15+/+ mice after 12-week MP treatment. CONCLUSIONS: Thiopurines exert harmful effects on testicular reproductive function according to host NUDT15 genotypes.

Genetic suppressor screen identifies Tgp1 (glycerophosphocholine transporter), Kcs1 (IP6 kinase), and Plc1 (phospholipase C) as determinants of inositol pyrophosphate toxicosis in fission yeast.

The inositol pyrophosphate signaling molecule 1,5-IP8 is an agonist of RNA 3'-processing and transcription termination in fission yeast that regulates the expression of phosphate acquisition genes pho1, pho84, and tgp1. IP8 is synthesized from 5-IP7 by the Asp1 N-terminal kinase domain and catabolized by the Asp1 C-terminal pyrophosphatase domain. asp1-STF mutations that delete or inactivate the Asp1 pyrophosphatase domain elicit growth defects in yeast extract with supplements (YES) medium ranging from severe sickness to lethality. We now find that the toxicity of asp1-STF mutants is caused by a titratable constituent of yeast extract. Via a genetic screen for spontaneous suppressors, we identified a null mutation of glycerophosphodiester transporter tgp1 that abolishes asp1-STF toxicity in YES medium. This result, and the fact that tgp1 mRNA expression is increased by >40-fold in asp1-STF cells, prompted discovery that: (i) glycerophosphocholine (GPC) recapitulates the toxicity of yeast extract to asp1-STF cells in a Tgp1-dependent manner, and (ii) induced overexpression of tgp1 in asp1+ cells also elicits toxicity dependent on GPC. asp1-STF suppressor screens yielded a suite of single missense mutations in the essential IP6 kinase Kcs1 that generates 5-IP7, the immediate precursor to IP8. Transcription profiling of the kcs1 mutants in an asp1+ background revealed the downregulation of the same phosphate acquisition genes that were upregulated in asp1-STF cells. The suppressor screen also returned single missense mutations in Plc1, the fission yeast phospholipase C enzyme that generates IP3, an upstream precursor for the synthesis of inositol pyrophosphates.IMPORTANCEThe inositol pyrophosphate metabolite 1,5-IP8 governs repression of fission yeast phosphate homeostasis genes pho1, pho84, and tgp1 by lncRNA-mediated transcriptional interference. Asp1 pyrophosphatase mutations that increase IP8 levels elicit precocious lncRNA termination, leading to derepression of the PHO genes. Deletions of the Asp1 pyrophosphatase domain result in growth impairment or lethality via IP8 agonism of transcription termination. It was assumed that IP8 toxicity ensues from dysregulation of essential genes. In this study, a suppressor screen revealed that IP8 toxicosis of Asp1 pyrophosphatase mutants is caused by: (i) a >40-fold increase in the expression of the inessential tgp1 gene encoding a glycerophosphodiester transporter and (ii) the presence of glycerophosphocholine in the growth medium. The suppressor screen yielded missense mutations in two upstream enzymes of inositol polyphosphate metabolism: the phospholipase C enzyme Plc1 that generates IP3 and the essential Kcs1 kinase that converts IP6 to 5-IP7, the immediate precursor of IP8.

Associations of ITPA gene polymorphisms with outcomes among chronic hepatitis C patients treated with Peg-interferon/ribavirin: A 5-year follow-up study.

To investigate associations between inosine triphosphatase (ITPA) gene polymorphisms and long-term outcomes among chronic hepatitis C (CHC) patients in Northeast China treated with Peg-interferon (IFN)/ribavirin (RBV). CHC patients who received Peg-IFN-2a/RBV treatment during between 2011 and 2013 at 5 hepatitis centers in Northeast China were enrolled. ITPA single nucleotide polymorphisms rs1127354 and rs7270101 from all patients were detected and their associations with 5-year outcomes were analyzed. A total of 635 patients, including 421 infected with hepatitis C virus (HCV) genotype 1 and 214 infected with non-genotype 1 were included. No significant differences were observed in the distribution frequencies of ITPA rs1127354 variants and ITPase activity between patients with HCV genotype 1 and non-genotype 1. In patients who received more than 80% of the planned RBV dose, the 5-year virological response rate and the improvement in liver fibrosis were higher in those with ITPA rs1127354 non-CC with ITPase activity <25% compared with these outcomes in patients with ITPA rs1127354 CC with 100% ITPase activity. Multiple regression analysis revealed that HCV genotype non-1, low baseline HCV ribose nucleic acid (RNA) levels (≤4 × 105 IU/mL), interleukin-28B rs12979860 CC genotype, low baseline liver fibrosis (Fibroscan 0-2), and ITPA rs1127354 non-CC genotype were independent predictors for a high long-term virological response rate, whereas interleukin-28B rs12979860 CC genotype, ITPA rs1127354 non-CC genotype, and low baseline liver fibrosis were independent predictors for improvement of liver fibrosis. ITPA rs1127354 polymorphisms is predictors of long-term outcomes in CHC patients treated with Peg-IFN/RBV.

Azathioprine dose tailoring based on pharmacogenetic information: Insights of clinical implementation.

Azathioprine is commonly used as an immunosuppressive antimetabolite in the treatment of acute lymphoblastic leukemia, autoimmune disorders (such as Crohn's disease and rheumatoid arthritis), and in patients receiving organ transplants. Thiopurine-S-methyltransferase (TPMT) is a cytoplasmic trans-methylase catalyzing the S-methylation of thiopurines. The active metabolites obtained from thiopurines are hydrolyzed into inactive forms by the Nudix hydrolase 15 (NUDT15). The TPMT*2 (defined by rs1800462), *3A (defined by rs1800460 and rs1142345), *3B (defined by rs1800460), *3C (defined by rs1142345), *6 (defined by rs75543815), and NUDT15 rs116855232 genetic variant have been associated, with the highest level of evidence, with the response to azathioprine, and, the approved drug label for azathioprine and main pharmacogenetic dosing guidelines recommend starting with reduced initial doses in TPMT intermediate metabolizer (IM) patients and considering an alternative treatment in TPMT poor metabolizer (PM) patients. This study aims to assess the clinical impact of azathioprine dose tailoring based on TPMT genotyping studying the azathioprine toxicity and efficacy, treatment starts, and dose adjustments during follow-up, comparing TPMT IM/PM and normal metabolizer (NM) patients. It also studied the association of NUDT15 rs116855232 with response to azathioprine in patients receiving a tailored treatment based on TPMT and characterized the TMPT and NUDT15 studied variants in our population. Results show that azathioprine dose reduction in TPMT IM patients (TPMT*1/*2, *1/*3A, or *1/*3C genotypes) is related to lower toxicity events compared to TPMT NM (TPMT *1/*1 genotype), and lower azathioprine dose adjustments during follow-up without showing differences in the efficacy. The results support the hypothesis of existing other genetic variants affecting azathioprine toxicity.

Activities, substrate specificity, and genetic interactions of fission yeast Siw14, a cysteinyl-phosphatase-type inositol pyrophosphatase.

IMPORTANCE: The inositol pyrophosphate signaling molecule 1,5-IP8 modulates fission yeast phosphate homeostasis via its action as an agonist of RNA 3'-processing and transcription termination. Cellular 1,5-IP8 levels are determined by a balance between the activities of the inositol polyphosphate kinase Asp1 and several inositol pyrophosphatase enzymes. Here, we characterize Schizosaccharomyces pombe Siw14 (SpSiw14) as a cysteinyl-phosphatase-family pyrophosphatase enzyme capable of hydrolyzing the phosphoanhydride substrates inorganic pyrophosphate, inorganic polyphosphate, and inositol pyrophosphates 5-IP7, 1-IP7, and 1,5-IP8. Genetic analyses implicate SpSiw14 in 1,5-IP8 catabolism in vivo, insofar as: loss of SpSiw14 activity is lethal in the absence of the Nudix-type inositol pyrophosphatase enzyme Aps1; and siw14∆ aps1∆ lethality depends on synthesis of 1,5-IP8 by the Asp1 kinase. Suppression of siw14∆ aps1∆ lethality by loss-of-function mutations of 3'-processing/termination factors points to precocious transcription termination as the cause of 1,5-IP8 toxicosis.

Investigation of the Association Between ITPA Gene 94C>A Sequence Variant and Kidney Transplant Rejection in Iranian Kidney Transplant Recipients.

OBJECTIVES: Thiopurine prodrugs are commonly used in kidney transplant recipients. Inosine triphosphate pyrophosphatase is an enzyme encoded by the ITPA gene. Alteration of ITPA gene is one of the pharmacogenetic sequence variants possibly involved in thiopurine metabolism. The ITPA 94C>A sequence variant (C-to-A substitution at nucleotide 94) is associated with an increased risk of adverse drug reactions in patients treated with the thiopurine drug. The aim of the present study was to investigate the effect of the ITPA 94C>A gene sequence variant in kidney transplant recipients. MATERIALS AND METHODS: The genotyping of the ITPA rs1127354 variant was performed by the polymerase chain reaction restriction fragment length polymorphism method in 140 kidney transplant recipients and in 100 control participants. Data were analyzed with SPSS statistical software. RESULTS: The results revealed a significant difference between control and nonrejection groups regarding the rs1127354 genotype and allele frequency. No significant difference was found between the rejection and nonrejection groups regarding the rs1127354 genotype and allele frequency. Also, a significant association was observed between the ageofthe control group and age of the rejection group. No significant differences between sex and underlying disease in patients with or without rejection were observed. CONCLUSIONS: We observed no significant differences between rejection and nonrejection transplant. Further studies are recommended, in a larger population and with different ethnicities.

Enzyme and Metabolic Engineering Strategies for Biosynthesis of α-Farnesene in Saccharomyces cerevisiae.

α-Farnesene, a type of acyclic sesquiterpene, is an important raw material in agriculture, aircraft fuel, and the chemical industry. In this study, we constructed an efficient α-farnesene-producing yeast cell factory by combining enzyme and metabolic engineering strategies. First, we screened different plants for α-farnesene synthase (AFS) with the best activity and found that AFS from Camellia sinensis (CsAFS) exhibited the most efficient α-farnesene production in Saccharomyces cerevisiae 4741. Second, the metabolic flux of the mevalonate pathway was increased to improve the supply of the precursor farnesyl pyrophosphate. Third, inducing site-directed mutagenesis in CsAFS, the CsAFSW281C variant was obtained, which considerably increased α-farnesene production. Fourth, the N-terminal serine-lysine-isoleucine-lysine (SKIK) tag was introduced to construct the SKIK∼CsAFSW281C variant, which further increased α-farnesene production to 2.8 g/L in shake-flask cultures. Finally, the α-farnesene titer of 28.3 g/L in S. cerevisiae was obtained by fed-batch fermentation in a 5 L bioreactor.

Mammalian Nudt15 hydrolytic and binding activity on methylated guanosine mononucleotides.

The Nudt15 enzyme of the NUDIX protein family is the subject of extensive study due to its action on thiopurine drugs used in the treatment of cancer and inflammatory diseases. In addition to thiopurines, Nudt15 is enzymatically active in vitro on several nucleotide substrates. It has also been suggested that this enzyme may play a role in 5'RNA turnover by hydrolyzing m7GDP, a product of mRNA decapping. However, no detailed studies on this substrate with Nudt15 are available. Here, we analyzed the enzymatic activity of Nudt15 with m7GDP, its triphosphate form m7GTP, and the trimethylated counterparts (m32,2,7GDP and m32,2,7GTP). Kinetic data revealed a moderate activity of Nudt15 toward these methylated mononucleotides compared to the dGTP substrate. However m7GDP and m32,2,7GDP showed a distinct stabilization of Nudt15 upon ligand binding, in the same range as dGTP, and thus these two mononucleotides may be used as leading structures in the design of small molecule binders of Nudt15.

The c.415C>T polymorphism in NUDT15 is more frequent than the polymorphisms in TPMT in Chilean patients who use thiopurine drugs.

Azathioprine (AZA) and 6-mercaptopurine (6-MP) are drugs widely used in the treatment of autoimmune diseases. Among the enzymes involved in the metabolism of AZA and 6-MP are thiopurine methyltransferase (TPMT) and nudix hydrolase 15 (NUDT15). The existence of single nucleotide polymorphisms in the genes that code for these enzymes could decreased enzymatic activity AND lead to severe myelosuppression. The most relevant polymorphism is NUDT15*3 (rs116855232), where the replacement of cytosine for thymine at position 415, which in turn leads to a loss of enzymatic activity. In a previous study, it was identified that together the polymorphisms in the TPMT gene reach an allelic frequency of 3.81%. There is no information regarding the rs116855232 polymorphism in the NUDT15 gene, so this corresponds to the objective of this report. Blood samples from Chilean adult patients with indications for the use of AZA or 6-MP for different pathologies and who had undergone a TPMT gene polymorphism study were retrospectively analyzed. A total of 253 blood samples were analyzed. Of the 253 patients, 47 presented the c.415C>T polymorphism in the NUDT15 gene, 3 being homozygous and 44 heterozygous. Four of the heterozygous patients for NUDT15 also had the *3A variant in the TPMT gene, also heterozygous. The allelic frequency of the minor T allele found (9.88%) was very similar to that found in patients of Asian origin, and much higher than that reported for the European Caucasian or Latin American population.