Probiotic Yeast from Miso Ameliorates Stress-Induced Visceral Hypersensitivity by Modulating the Gut Microbiota in a Rat Model of Irritable Bowel Syndrome

Background/Aims: Recent studies indicate that probiotics, which have attracted attention as a treatment for irritable bowel syndrome, affect intestinal homeostasis. In this study, we investigated whether Zygosaccharomyces sapae (strain I-6), a probiotic yeast isolated from miso (a traditional Japanese fermented food), could improve irritable bowel syndrome symptoms. Methods: Male Wistar rats were exposed to water avoidance stress (WAS). The number of defecations during WAS and the visceral hypersensitivity before and after WAS were evaluated using colorectal distension. Tight junction changes were assessed by Western blotting. Some rats were fed with strain I-6 or β-glucan from strain I-6. Changes in the intestinal microbiota were analyzed. The effect of fecal microbiota transplantation after WAS was evaluated similarly. Caco-2 cells were stimulated with interleukin-1β and tight junction changes were investigated after coculture with strain I-6. Results: The increased number of stool pellets and visceral hypersensitivity induced by WAS were suppressed by administering strain I-6. The decrease in tight junction protein occludin by WAS was reversed by the administration of strain I-6. β-Glucan from strain I-6 also suppressed those changes induced by WAS. In the rat intestinal microbiota, treatment with strain I-6 altered the β-diversity and induced changes in bacterial occupancy. Upon fecal microbiota transplantation, some symptoms caused by WAS were ameliorated. Conclusions: These results suggest that traditional fermented foods such as miso in Japan are valuable sources of probiotic yeast candidates, which may be useful for preventing and treating stress-induced visceral hypersensitivity

NUDT15 polymorphism influences the metabolism and therapeutic effects of acyclovir and ganciclovir

Nucleobase and nucleoside analogs (NNA) are widely used as anti-viral and anti-cancer agents, and NNA phosphorylation is essential for the activity of this class of drugs. Recently, diphosphatase NUDT15 was linked to thiopurine metabolism with NUDT15 polymorphism associated with drug toxicity in patients. Profiling NNA drugs, we identify acyclovir (ACV) and ganciclovir (GCV) as two new NNAs metabolized by NUDT15. NUDT15 hydrolyzes ACV and GCV triphosphate metabolites, reducing their effects against cytomegalovirus (CMV) in vitro. Loss of NUDT15 potentiates cytotoxicity of ACV and GCV in host cells. In hematopoietic stem cell transplant patients, the risk of CMV viremia following ACV prophylaxis is associated with NUDT15 genotype (P = 0.015). Donor NUDT15 deficiency is linked to graft failure in patients receiving CMV-seropositive stem cells (P = 0.047). In conclusion, NUDT15 is an important metabolizing enzyme for ACV and GCV, and NUDT15 variation contributes to inter-patient variability in their therapeutic effects

Germline genetic variation in ETV6 and risk of childhood acute lymphoblastic leukaemia: a systematic genetic study.

BackgroundHereditary predisposition is rarely suspected for childhood acute lymphoblastic leukaemia (ALL). Recent reports of germline ETV6 variations associated with substantial familial clustering of haematological malignancies indicated that this gene is a potentially important genetic determinant for ALL susceptibility. Our aims in this study were to comprehensively identify ALL predisposition variants in ETV6 and to determine the extent to which they contributed to the overall risk of childhood ALL.MethodsWhole-exome sequencing of an index family with several cases of ALL was done to identify causal variants for ALL predisposition. Targeted sequencing of ETV6 was done in children from the Children's Oncology Group and St Jude Children's Research Hospital front-line ALL trials. Patients were included in this study on the basis of their enrolment in these clinical trials and the availability of germline DNA. ETV6 variant genotypes were compared with non-ALL controls to define ALL-related germline risk variants. ETV6 variant function was characterised bioinformatically and correlated with clinical and demographic features in children with ALL.FindingsWe identified a novel non-sense ETV6 variant (p.Arg359X) with a high penetrance in an index family. Subsequent targeted sequencing of ETV6 in 4405 childhood ALL cases identified 31 exonic variants (four non-sense, 21 missense, one splice site, and five frameshift variants) that were potentially related to ALL risk in 35 cases (1%). 15 (48%) of 31 ALL-related ETV6 variants clustered in the erythroblast transformation specific domain and were predicted to be highly deleterious. Children with ALL-related ETV6 variants were significantly older at leukaemia diagnosis than those without (10·2 years [IQR 5·3-13·8] vs 4·7 years [3·0-8·7]; p=0·017). The hyperdiploid leukaemia karyotype was highly over-represented in ALL cases harbouring germline ETV6 risk variants compared with the wild-type group (nine [64%] of 14 cases vs 538 [27%] of 2007 cases; p=0·0050).InterpretationOur findings indicated germline ETV6 variations as the basis of a novel genetic syndrome associated with predisposition to childhood ALL. The development of recommendations for clinical interventions and surveillance for individuals harbouring ALL-related ETV6 variants are needed.FundingUS National Institutes of Health and American Lebanese Syrian Associated Charities

<i>NUDT15</i> polymorphisms alter thiopurine metabolism and hematopoietic toxicity

Widely used as anti-cancer and immunosuppressive agents, thiopurines have narrow therapeutic indices due to frequent toxicities, partly explained by TPMT genetic polymorphisms. Recent studies identified germline NUDT15 variation as another critical determinant of thiopurine intolerance, but the underlying molecular mechanisms and its clinical implications remain unknown. In 270 children enrolled in clinical trials for acute lymphoblastic leukemia in Guatemala, Singapore, and Japan, we identified 4 NUDT15 coding variants (p.Arg139Cys, p.Arg139His, p.Val18Ile, p.Val18_Val19insGlyVal) that resulted in 74.4%–100% loss of nucleotide diphosphatase activity. Loss-of-function NUDT15 diplotypes were consistently associated with thiopurine intolerance across three cohorts (P=0.021, 2.1×10(−5), and 0.0054, respectively; meta-analysis P=4.45×10(−8), allelic effect size=−11.5). Mechanistically, NUDT15 inactivated thiopurine metabolites and decreased its cytotoxicity in vitro, and patients with defective NUDT15 alleles showed excessive thiopurine active metabolites and toxicity. Taken together, our results indicate that a comprehensive pharmacogenetic model integrating NUDT15 variants may inform personalized thiopurine therapy

Massively parallel variant characterization identifies NUDT15 alleles associated with thiopurine toxicity

As a prototype of genomics-guided precision medicine, individualized thiopurine dosing based on pharmacogenetics is a highly effective way to mitigate hematopoietic toxicity of this class of drugs. Recently, NUDT15 deficiency was identified as a genetic cause of thiopurine toxicity, and NUDT15-informed preemptive dose reduction was quickly adopted in clinical settings. To exhaustively identify pharmacogenetic variants in this gene, we developed massively parallel NUDT15 function assays to determine the variants' effect on protein abundance and thiopurine cytotoxicity. Of the 3,097 possible missense variants, we characterized the abundance of 2,922 variants and found 54 hotspot residues at which variants resulted in complete loss of protein stability. Analyzing 2,935 variants in the thiopurine cytotoxicity-based assay, we identified 17 additional residues where variants altered NUDT15 activity without affecting protein stability. We identified structural elements key to NUDT15 stability and/or catalytical activity with single amino acid resolution. Functional effects for NUDT15 variants accurately predicted toxicity risk alleles in patients treated with thiopurines with far superior sensitivity and specificity compared to bioinformatic prediction algorithms. In conclusion, our massively parallel variant function assays identified 1,152 deleterious NUDT15 variants, providing a comprehensive reference of variant function and vastly improving the ability to implement pharmacogenetics-guided thiopurine treatment individualization.This article is available to RD&E staff via NHS OpenAthens. Click on the Publisher URL, and log in with NHS OpenAthens if prompted.R01 CA096670/CA/NCI NIH HHS/United States R25 CA023944/CA/NCI NIH HHS/United States P30 CA021765/CA/NCI NIH HHS/United States U10 CA098543/CA/NCI NIH HHS/United States U10 CA180899/CA/NCI NIH HHS/United States R01 GM118578/GM/NIGMS NIH HHS/United States U10 CA180886/CA/NCI NIH HHS/United States P50 GM115279/GM/NIGMS NIH HHS/United States U10 CA098413/CA/NCI NIH HHS/United States U10 CA095861/CA/NCI NIH HHS/United Statespublished version, accepted version (6 month embargo)