Citations

Background : Zn is implicated as having a role in the formation of calcium oxalate (CaOx) kidney stones. Our studies indicate that Slc39a10 (Zip10) may be important in this process. Zip10 has identifiable sequences in human, dog and fly ( Drosophila ). As we are interested in a translational model of kidney stones, we examined Zip10 from all 3 species. Methods: Zip10 cDNAs were cloned from human & dog (kidney) and fly (whole fly) using gene specific primers and RT‐PCR. Clones were put into a Xenopus oocyte expression vector so that Zip10 function could be measured. Three days post‐injection, oocytes were pre‐incubated in ND96, followed by 30 min in ND96 + 10 μM ZnCl + the PET isotope 63 Zn, with varying pHs and ion concentrations. 63 Zn uptake was measured in each oocyte using a gamma counter. Zip10 oocytes were also checked using electrophysiology (ion selective electrodes & voltage clamping). Results: Zip10 mRNA & protein are found in gut and kidney. In mouse kidney, Zip10 antibodies stained the apical membrane of proximal tubules. All clones transported 63 Zn (nmol/oocyte/hr): hZip10 = 70±8; cZip10= 44 ±7; dZip10= 64 ±8; water = 0.4 ±0.1. These clones seem to be most active at pH 7.5 as uptakes decreased at 5.5 and 8.5. Replacement of either all Cl ‐ (with HCO 3 舉 ) or all Na + (with choline) reduced Zn‐uptake. Electrophysiology revealed no evidence of I or V changes with addition of Zn (electroneutral). Conclusions: Our results indicated that all three Zip10 clones transport Zn 2+ . Understanding the detailed mechanisms of these Zn transporters in oocytes presents the opportunity to better use our Drosophila model in the study of CaOx stone formation and absorption. Grant Funding Source : Supported by DK83007, DK92408

Abstract Background Across complex traits, common variants explain only a modest amount of variance, with SNP-heritability consistently below heritability estimates from close relatives. Here, we examined the contribution of rare variant to tobacco use risk in up to 26,000 individuals of European ancestry in the Trans-Omics for Precision Medicine (TOPMed) program with whole genome sequence (WGS;~30X coverage).Method We grouped about 35 million genetic variants by their minor allele frequencies (MAF) and linkage disequilibrium (LD) and estimated SNP-heritability for age of smoking initiation (N = 14,747), cigarettes smoked per day (N = 15,425), smoking cessation (N = 17,871) and initiation (N = 26,340) using linear mixed model. Rare variant population structure is detected and adjusted for by permutation procedure. We estimated an upper bound for narrow-sense heritability for tobacco use using available pedigrees consisting of close relatives in TOPMed.Results Rare variants with MAF 0.1–0.01%, mostly from non-protein altering region, accounted for 26% of variation in age of initiation and 15% for cessation. Follow-up analysis indicated that about one-third of these rare variants contribtion is potentially confounded with rare variants structure even after adjusting for principal components. After further conservative adjustment of population structure, we estimated SNP-based heritability to be 0.21 (SE = 0.08) for age of initiation, 0.15 (0.06) for cigarettes per day, 0.21 (0.09) for cessation, and 0.24 (0.07) for initiation, 1.8–4.5 times higher than previous SNP-based estimates. Our pedigree-based upper-bound for SNP-based heritability ranged from 0.18–0.35.Conclusion The substantial contribution of rare variants for several smoking phenotypes sheds light on the missing heritability and genetic etiology of tobacco use. It also informs fine-mapping strategies since the majority of the rare variant contribution was located in non-coding regulatory regions.

AbstractPhenylketonuria (PKU) is the most common inborn error of metabolism of the liver, and results from mutations of both alleles of the phenylalanine hydroxylase gene (PAH). As such, it is a suitable target for gene therapy via gene delivery with a recombinant adeno‐associated virus (AAV) vector. Here we use the synthetic AAV vector Anc80 via systemic administration to deliver a functional copy of a codon‐optimized human PAH gene, with or without an intron spacer, to the Pahenu2 mouse model of PKU. Dose‐dependent transduction of the liver and expression of PAH mRNA were present with both vectors, resulting in significant and durable reduction of circulating phenylalanine, reaching near control levels in males. Coat color of treated Pahenu2 mice reflected an increase in pigmentation from brown to the black color of control animals, further indicating functional restoration of phenylalanine metabolism and its byproduct melanin. There were no adverse effects associated with administration of AAV up to 5 × 1012 VG/kg, the highest dose tested. Only minor and/or transient variations in some liver enzymes were observed in some of the AAV‐dosed animals which were not associated with pathology findings in the liver. Finally, there was no impact on cell turnover or apoptosis as evaluated by Ki‐67 and TUNEL staining, further supporting the safety of this approach. This study demonstrates the therapeutic potential of AAV Anc80 to safely and durably cure PKU in a mouse model, supporting development for clinical consideration.

Abstract The utility of the chronic lymphocytic leukemia-international prognostic index (CLL-IPI) in predicting outcomes of individuals with Rai 0 stage CLL and monoclonal B-cell lymphocytosis (MBL) is unclear. We identified 969 individuals (415 MBL and 554 Rai 0 CLL; median age, 64 years; 65% men) seen at Mayo Clinic between 1 January 2001 and 1 October 2018, and ascertained time to first therapy (TTFT) and overall survival (OS). After a median follow up of 7 years, the risk of disease progression needing therapy was 2.9%/y for MBL (median, not reached) and 5%/y for Rai 0 CLL (median, 10.4 years). Among patients with low, intermediate, and high/very high-risk CLL-IPI risk groups, the estimated 5-year risk of TTFT was 13.5%, 30%, and 58%, respectively, P< .0001 (c-statistic = 0.69); and the estimated 5-year OS was 96.3%, 91.5%, and 76%, respectively, P< .0001 (c-statistic = 0.65). In a multivariable analysis of absolute B-cell count with individual factors of the CLL-IPI, the absolute B-cell count was associated with shorter TTFT (hazard ratio [HR] for each 10 × 109/L increase: 1.31; P< .0001) and shorter OS (HR: 1.1; P = .02). The OS of the entire cohort was similar to that of the age- and sex-matched general population of Minnesota (P = .17), although Rai 0 CLL patients with high and very high-risk CLL-IPI score had significantly shorter OS (P= .01 and P= .0001, respectively). The results of this study demonstrate the ability of CLL-IPI to predict time from diagnosis to first treatment (an end point not affected by therapy) in a large cohort of patients whose only manifestation of disease is a circulating clonal lymphocyte population.

Abstract Background: There is little prospective, longitudinal data on the health outcomes of low count MBL in the U.S. adult population. We screened for MBL in asymptomatic adults with normal blood counts using highly sensitive flow-cytometry in a clinic-based cohort of participants from the Mayo Clinic Biobank and explored the relationship between MBL and risk of hospitalization with infection. Methods: The Mayo Clinic Biobank is a large scale biorepository of 50,000 adults (age>18) seen in primary care-based clinics. All consented participants completed a health history questionnaire and provided blood samples. For the present study, we focused on the study participants from Olmsted County, Minnesota, 40 years of age or older who had stored PBMC. We used these PBMC to screen for MBL using an 8 color (CD38, CD45, Kappa, Lambda, CD19, CD23, CD5 and CD20) flow cytometry assay validated to consistently detect clonal B-cell events to the 0.005% level (1/20,000 events). The sensitivity of this approach of collecting 500,000 events from isolated PBMC is 2-3-fold higher than collecting 500,000 events from whole blood due to the increased concentration of lymphocytes in these samples. Infections associated with hospitalization were considered serious or life-threatening infections. Every hospitalization at both Mayo Clinic and Olmsted Medical Center hospitals in Olmsted County, MN following the sample collection was reviewed to document hospitalization with infection. Data on hospitalization with infection was abstracted from clinical records using our previously published approach (Moriera, Leukemia 27:136). This approach allows medical record validation and review of detailed clinical and treatment information related to infection. For this analysis, follow-up began at date of sample collection and ended at the earliest of death, migration from Olmsted County, or 12/31/2016. The individual abstracting information on hospitalization for infection was blinded to the results of MBL screening. Chi-square tests were used to assess associations between MBL status and demographic characteristics. Cox regression models were used to determine association with risk of hospitalization with infection accounting for the competing risk of death using Fine Gray models. Results : We screened for MBL in 1045 Olmsted County residents age 40 and older. Of these, 984 (94%) had interpretable results. 119 of these 984 (12%) had a clonal B-cell population detected including 106 (11%) with CLL phenotype (CD5, CD19, CD20 [dim], CD23, kappa or lambda light chain restriction [dim]).This analysis focused on the 106 individuals with CLL-like MBL clones (mean age 69 years, 55% male) and the 865 individuals with normal immunophenotype (mean age 61 years, 40% male). The presence of CLL phenotype MBL was strongly associated with age. MBL was detected in 3% of patients age 40-49 years, 7% of those 50-59, 10% of those 60-69, 20% of those 70-79, and 27% of those age 80+ (p<0.001). MBL was also more common among men (women 8%; men 14%; p=0.003). After a median follow-up of 7 years from biobank enrollment, 76 of the 971 participants were hospitalized with infection in Olmsted County at least once. The estimated cumulative incidence of infections at 7 years was 19% for LC MBL and 9% for those without MBL (Figure). The most frequent site of infections for which these individuals were first hospitalized were pneumonia (14 individuals; 18%), cellulitis (20 individuals; 26%), urinary tract infection (21 individuals; 26%), and blood stream infection (6 individuals; 8%). An association between LC MBL and hospitalization with infection was observed with the unadjusted HR for hospitalization with infection among the 106 Olmsted County residents with LC MBL relative to the 865 Olmsted County residents with normal immunophenotype is 2.41 (95%CI: 1.39-4.19; p=0.002). After adjusting for age and sex, the HR is 1.65 (95% CI: 0.95-2.88; p=0.08). Conclusions: We report one of the first studies to systematically explore the relationship between LC MBL and the risk of serious infection. It appears that individuals with LC MBL may be at increased risk for serious infection. If this observation is confirmed, it would suggest that 5-10% of adults over age 40 (6-12 million U.S. adults) have a largely unstudied condition with potentially serious health implications. Figure 1 Figure 1. Disclosures Shanafelt: Hospira: Research Funding; AbbVie: Research Funding; Celgene: Research Funding; Pharmacyclics: Research Funding; Jannsen: Research Funding; GlaxoSmithKline: Research Funding; Genentech: Research Funding. Kay: Gilead: Research Funding; Agios: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Research Funding; Tolero Corporation: Research Funding. Parikh: Pharmacyclics: Research Funding; AstraZeneca: Honoraria; Pharmacyclics: Honoraria.

OBJECTIVE The use of intrawound vancomycin powder in spine surgery has been shown to decrease the rate of surgical site infections; however, the optimal dose is unknown. High-dose vancomycin inhibits osteoblast proliferation in vitro and may decrease the rate of solid arthrodesis. Bone marrow–derived mesenchymal stem cells (BMSCs) are multipotent cells that are a source of osteogenesis in spine fusions. The purpose of this study was to determine the effects of vancomycin on rat BMSC viability and differentiation in vitro. METHODS BMSCs were isolated from the femurs of immature female rats, cultured, and then split into two equal groups; half were treated to stimulate osteoblastic differentiation and half were not. Osteogenesis was stimulated by the addition of 50 µg/mL l-ascorbic acid, 10 mM β-glycerol phosphate, and 0.1 µM dexamethasone. Vancomycin was added to cell culture medium at concentrations of 0, 0.04, 0.4, or 4 mg/mL. Early differentiation was determined by alkaline phosphatase activity (4 days posttreatment) and late differentiation by alizarin red staining for mineralization (9 days posttreatment). Cell viability was determined at both the early and late time points by measurement of formazan colorimetric product. RESULTS Viability within the first 4 days decreased with high-dose vancomycin treatment, with cells receiving 4 mg/mL vancomycin having 40%–60% viability compared to the control. A gradual decrease in alizarin red staining and nodule formation was observed with increasing vancomycin doses. In the presence of the osteogenic factors, vancomycin did not have deleterious effects on alkaline phosphatase activity, whereas a trend toward reduced activity was seen in the absence of osteogenic factors when compared to osteogenically treated cells. CONCLUSIONS Vancomycin reduced BMSC viability and impaired late osteogenic differentiation with high-dose treatment. Therefore, the inhibitory effects of high-dose vancomycin on spinal fusion may result from both reduced BMSC viability and some impairment of osteogenic differentiation.

Abstract Intratumoral genomic heterogeneity in glioblastoma (GBM) is a barrier to overcoming radiation (RT) resistance. To discover genotype-independent mediators of RT resistance, we correlated RT resistance with the concentration of approximately 700 metabolites across 23 GBM cell lines. Purine metabolites, especially those containing the base guanine, were most correlated with RT resistance. Similarly, increased abundance of tumor purines was associated with decreased survival in GBM patients treated with RT. This relationship is causal. Purine supplementation protected RT-sensitive GBMs from RT and promoted the repair of RT-induced double strand DNA breaks (DSBs). In vitro and in vivo stable isotope tracing confirmed that GBM cell lines and orthotopic patient-derived xenografts primarily generated purines through the de novo synthetic pathway. RT treatment further increased de novo purine synthesis in GBM through signaling via the DNA damage response. Inhibition of de novo GTP synthesis with mycophenolic acid (MPA) sensitized multiple GBM cell lines and neurospheres to RT by slowing the repair of RT-induced DSBs. MPA-induced radiosensitization was GTP-dependent as it was rescued by nucleoside supplementation. Modulating pyrimidine metabolism affected neither RT resistance nor DSB repair, suggesting these GTP-specific effects are due to active signaling rather than its ability to act as a physical substrate for DNA repair and candidate signaling molecules have been identified. These results were recapitulated in vivo with mycophenolate mofetil (MMF), the orally bioavailable FDA-approved prodrug of MPA. MMF potentiated RT efficacy, reduced tumor guanylates and slowed the repair of RT-induced DSBs across multiple models. Because de novo purine synthesis is activated by many of the oncogenic alterations that drive GBM, its inhibition is a promising genotype-independent strategy to overcome GBM RT resistance. We have now begun a clinical trial to determine whether combining MMF and RT is safe and potentially efficacious in patients with GBM.

Abstract Coronary artery disease (CAD) is a leading cause of death, yet its genetic determinants are not fully elucidated. We report a multi-ethnic genome-wide association study of CAD involving nearly a quarter of a million cases, incorporating the largest cohorts to date of Whites, Blacks, and Hispanics from the Million Veteran Program with existing studies including CARDIoGRAMplusC4D, UK Biobank, and Biobank Japan. We verify substantial and nearly equivalent heritability of CAD across multiple ancestral groups, discover 107 novel loci including the first nine on the X-chromosome, identify the first eight genome-wide significant loci among Blacks and Hispanics, and demonstrate that two common haplotypes are largely responsible for the risk stratification at the well-known 9p21 locus in most populations except those of African origin where both haplotypes are virtually absent. We identify 15 loci for angiographically derived burden of coronary atherosclerosis, which robustly overlap with the strongest and earliest loci reported to date for clinical CAD. Phenome-wide association analyses of novel loci and externally validated polygenic risk scores (PRS) augment signals from the insulin resistance cluster of risk factors and consequences, extend previously established pleiotropic associations of loci with traditional risk factors to include smoking and family history, and confirm a substantially reduced transferability of existing PRS to Blacks. Downstream integrative genomic analyses reinforce the critical role of endothelial, fibroblast, and smooth muscle cells within the coronary vessel wall in CAD susceptibility. Our study highlights the value of a multi-ethnic design in efficiently characterizing the genetic architecture of CAD across all human populations.