Genetic determinants of response to aspirin and warfarin and development of silicon nanowire based genotyping

PhD ThesisChronic diseases such as cardiovascular diseases and colorectal cancer are the leading cause of mortality worldwide. Commonly used drugs such as aspirin and warfarin are shown to effective at reducing the risk of chronic diseases but have a narrow therapeutic window and are associated with adverse drug reactions, particularly, hemorrhage. Identification of pharmacogenetic markers such as single nucleotide polymorphisms (SNPs) that could help deliver personalized dose could help improve the risk-benefit ratio. Furthermore, development of a rapid point of care genotyping device consisting of a pharmacogenetic SNP panel for aspirin and warfarin could help implement personalized medicine in the clinical setting. Analysis of candidate SNPs in aspirin’s pharmacokinetic and pharmacodynamic pathways was carried out to explain variation in aspirin’s colorectal chemopreventive efficacy using two large population based case-control datasets. Associations and interactions were tested using logistic regression models and meta-analysis of the 2 datasets. A novel sitespecific association for rs1799853 (OR=0.73, 95% CI=0.60-0.90, P=0.003) and rs1105879 (OR=1.16, 95% CI=1.02-1.32, P=0.03) with colon cancer risk was observed. Furthermore, stratification by aspirin use showed increased risk of colorectal cancer in aspirin users but not in non-users carrying variant allele of the SNPs rs4936367 and rs7112513 in PAFAH1B2 gene and rs2070959 and rs1105879 in UGT1A6 gene (Pinteraction<0.05 for all). These results provide insight into aspirin’s differential chemopreventive efficacy and the neoplastic transformation of cells in colon and rectum. Utility of clinically validated pharmacogenetic dosing algorithms consisting of three warfarin dose associated SNPs from the European population needs to tested in the Gujarati Indians, an Indian sub-population. Dose prediction accuracy of the algorithms was compared between Gujarati Indian and European population. Mean squared difference of both pharmacogenetic algorithms was higher in Gujarati Indian compared to European population (Klein et al 2009, 216.3 v/s 160.7, P=0.05; Gage et al 2008, 170.6 v/s 143.2, P=0.07). Poor prediction accuracy could be explained by the presence of study subjects requiring dose for target INR range 2.5-3.5 and low frequency of the VKORC1 rs9923231 variant, which is the most important genetic determinant of warfarin dosing in Europeans. Therefore, the SNP panel and dosing algorithms developed from European populations cannot be assumed to have utility in the Gujarati Indian population. Finally, to help develop a rapid, point-of care, silicon nanowire (SiNW) based SNP genotyping device, a panel of isothermal melting probes were designed to genotype three warfarin dose associated SNPs. Testing of hybridization and washing conditions to have optimal hybridization kinetics between the probe and target DNA and high target sequence specificity was carried out using custom designed microarray platform. Accurate genotype calls for all 3 SNPs in 2 anonymised samples using empirically optimized hybridization and washing conditions was carried out successfully. Current work highlighted associations between probe characteristics and hybridization parameters, which would be useful in designing and testing probes on the SiNW platform. Identification, validation and testing of clinical utility of population specific pharmacogenetic markers along with development and deployment of ultra-rapid point of care genotyping technologies could help deliver personalized risk-benefit ratio for aspirin and warfarin

Cancer prevention with aspirin in hereditary colorectal cancer (Lynch syndrome), 10-year follow-up and registry-based 20-year data in the CAPP2 study: a double-blind, randomised, placebo-controlled trial

Background Lynch syndrome is associated with an increased risk of colorectal cancer and with a broader spectrum of cancers, especially endometrial cancer. In 2011, our group reported long-term cancer outcomes (mean follow-up 55.7 months [SD 31.4]) for participants with Lynch syndrome enrolled into a randomised trial of daily aspirin versus placebo. This report completes the planned 10-year follow-up to allow a longer-term assessment of the effect of taking regular aspirin in this high-risk population. Methods In the double-blind, randomised CAPP2 trial, 861 patients from 43 international centres worldwide (707 [82%] from Europe, 112 [13%] from Australasia, 38 [4%] from Africa, and four [ Findings Between January, 1999, and March, 2005, 937 eligible patients with Lynch syndrome, mean age 45 years, commenced treatment, of whom 861 agreed to be randomly assigned to the aspirin group or placebo; 427 (50%) participants received aspirin and 434 (50%) placebo. Participants were followed for a mean of 10 years approximating 8500 person-years. 40 (9%) of 427 participants who received aspirin developed colorectal cancer compared with 58 (13%) of 434 who received placebo. Intention-to-treat Cox proportional hazards analysis revealed a significantly reduced hazard ratio (HR) of 0.65 (95% CI 0.43-0.97; p= 0. 035) for aspirin versus placebo. Negative binomial regression to account for multiple primary events gave an incidence rate ratio of 0.58 (0.39-0.87; p=0.0085). Per-protocol analyses restricted to 509 who achieved 2 years' intervention gave an HR of 0 .56 (0 .34-0 .91; p=0 .019) and an incidence rate ratio of 0.50 (0.31-0.82; p=0.0057). Non-colorectal Lynch syndrome cancers were reported in 36 participants who received aspirin and 36 participants who received placebo. Intention-to-treat and per-protocol analyses showed no effect. For all Lynch syndrome cancers combined, the intention-to-treat analysis did not reach significance but per-protocol analysis showed significantly reduced overall risk for the aspirin group (HR=0.63, 0 .43-0 .92; p=0.018). Adverse events during the intervention phase between aspirin and placebo groups were similar, and no significant difference in compliance between intervention groups was observed for participants with complete intervention phase data; details reported previously. Interpretation The case for prevention of colorectal cancer with aspirin in Lynch syndrome is supported by our results. Copyright (C) 2020 The Author(s). Published by Elsevier Ltd.Peer reviewe

A novel panel of short mononucleotide repeats linked to informative polymorphisms enabling effective high volume low cost discrimination between mismatch repair deficient and proficient tumours

<div><p>Somatic mutations in mononucleotide repeats are commonly used to assess the mismatch repair status of tumours. Current tests focus on repeats with a length above 15bp, which tend to be somatically more unstable than shorter ones. These longer repeats also have a substantially higher PCR error rate, and tests that use capillary electrophoresis for fragment size analysis often require expert interpretation. In this communication, we present a panel of 17 short repeats (length 7–12bp) for sequence-based microsatellite instability (MSI) testing. Using a simple scoring procedure that incorporates the allelic distribution of the mutant repeats, and analysis of two cohort of tumours totalling 209 samples, we show that this panel is able to discriminate between MMR proficient and deficient tumours, even when constitutional DNA is not available. In the training cohort, the method achieved 100% concordance with fragment analysis, while in the testing cohort, 4 discordant samples were observed (corresponding to 97% concordance). Of these, 2 showed discrepancies between fragment analysis and immunohistochemistry and one was reclassified after re-testing using fragment analysis. These results indicate that our approach offers the option of a reliable, scalable routine test for MSI.</p></div

A de novo paradigm for male infertility

De novo mutations are known to play a prominent role in sporadic disorders with reduced fitness. We hypothesize that de novo mutations play an important role in severe male infertility and explain a portion of the genetic causes of this understudied disorder. To test this hypothesis, we utilize trio-based exome sequencing in a cohort of 185 infertile males and their unaffected parents. Following a systematic analysis, 29 of 145 rare (MAF < 0.1%) protein-altering de novo mutations are classified as possibly causative of the male infertility phenotype. We observed a significant enrichment of loss-of-function de novo mutations in loss-of-function-intolerant genes (p -value = 1.00 × 10 −5) in infertile men compared to controls. Additionally, we detected a significant increase in predicted pathogenic de novo missense mutations affecting missense-intolerant genes (p -value = 5.01 × 10 −4) in contrast to predicted benign de novo mutations. One gene we identify, RBM5, is an essential regulator of male germ cell pre-mRNA splicing and has been previously implicated in male infertility in mice. In a follow-up study, 6 rare pathogenic missense mutations affecting this gene are observed in a cohort of 2,506 infertile patients, whilst we find no such mutations in a cohort of 5,784 fertile men (p -value = 0.03). Our results provide evidence for the role of de novo mutations in severe male infertility and point to new candidate genes affecting fertility. Germline de novo mutations can impact individual fitness, but their role in human male infertility is understudied. Trio-based exome sequencing identifies many new candidate genes affecting male fertility, including an essential regulator of male germ cell pre-mRNA splicing

Interaction between polymorphisms in aspirin metabolic pathways, regular aspirin use and colorectal cancer risk: A case-control study in unselected white European populations

Regular aspirin use is associated with reduced risk of colorectal cancer (CRC). Variation in aspirin’s chemoprevention efficacy has been attributed to the presence of single nucleotide polymorphisms (SNPs). We conducted a meta-analysis using two large population-based case-control datasets, the UK-Leeds Colorectal Cancer Study Group and the NIH-Colon Cancer Family Registry, having a combined total of 3325 cases and 2262 controls. The aim was to assess 42 candidate SNPs in 15 genes whose association with colorectal cancer risk was putatively modified by aspirin use, in the literature. Log odds ratios (ORs) and standard errors were estimated for each dataset separately using logistic regression adjusting for age, sex and study site, and dataset-specific results were combined using random effects meta-analysis. Meta-analysis showed association between SNPs rs6983267, rs11694911 and rs2302615 with CRC risk reduction (All P<0.05). Association for SNP rs6983267 in the CCAT2 gene only was noteworthy after multiple test correction (P = 0.001). Site-specific analysis showed association between SNPs rs1799853 and rs2302615 with reduced colon cancer risk only (P = 0.01 and P = 0.004, respectively), however neither reached significance threshold following multiple test correction. Meta-analysis of SNPs rs2070959 and rs1105879 in UGT1A6 gene showed interaction between aspirin use and CRC risk (Pinteraction = 0.01 and 0.02, respectively); stratification by aspirin use showed an association for decreased CRC risk for aspirin users having a wild-type genotype (rs2070959 OR = 0.77, 95% CI = 0.68–0.86; rs1105879 OR = 0.77 95% CI = 0.69–0.86) compared to variant allele cariers. The direction of the interaction however is in contrast to that published in studies on colorectal adenomas. Both SNPs showed potential site-specific interaction with aspirin use and colon cancer risk only (Pinteraction = 0.006 and 0.008, respectively), with the direction of association similar to that observed for CRC. Additionally, they showed interaction between any non-steroidal anti-inflammatory drugs (including aspirin) use and CRC risk (Pinteraction = 0.01 for both). All gene x environment (GxE) interactions however were not significant after multiple test correction. Candidate gene investigation indicated no evidence of GxE interaction between genetic variants in genes involved in aspirin pathways, regular aspirin use and colorectal cancer risk

Colorectal cancer incidences in Lynch syndrome: a comparison of results from the prospective lynch syndrome database and the international mismatch repair consortium

Objective To compare colorectal cancer (CRC) incidences in carriers of pathogenic variants of the MMR genes in the PLSD and IMRC cohorts, of which only the former included mandatory colonoscopy surveillance for all participants. Methods CRC incidences were calculated in an intervention group comprising a cohort of confirmed carriers of pathogenic or likely pathogenic variants in mismatch repair genes (path_MMR) followed prospectively by the Prospective Lynch Syndrome Database (PLSD). All had colonoscopy surveillance, with polypectomy when polyps were identified. Comparison was made with a retrospective cohort reported by the International Mismatch Repair Consortium (IMRC). This comprised confirmed and inferred path_MMR carriers who were first- or second-degree relatives of Lynch syndrome probands. Results In the PLSD, 8,153 subjects had follow-up colonoscopy surveillance for a total of 67,604 years and 578 carriers had CRC diagnosed. Average cumulative incidences of CRC in path_MLH1 carriers at 70 years of age were 52% in males and 41% in females; for path_MSH2 50% and 39%; for path_MSH6 13% and 17% and for path_PMS2 11% and 8%. In contrast, in the IMRC cohort, corresponding cumulative incidences were 40% and 27%; 34% and 23%; 16% and 8% and 7% and 6%. Comparing just the European carriers in the two series gave similar findings. Numbers in the PLSD series did not allow comparisons of carriers from other continents separately. Cumulative incidences at 25 years were < 1% in all retrospective groups. Conclusions Prospectively observed CRC incidences (PLSD) in path_MLH1 and path_MSH2 carriers undergoing colonoscopy surveillance and polypectomy were higher than in the retrospective (IMRC) series, and were not reduced in path_MSH6 carriers. These findings were the opposite to those expected. CRC point incidence before 50 years of age was reduced in path_PMS2 carriers subjected to colonoscopy, but not significantly so

Colorectal cancer incidences in Lynch syndrome : a comparison of results from the prospective lynch syndrome database and the international mismatch repair consortium

Objective: To compare colorectal cancer (CRC) incidences in carriers of pathogenic variants of the MMR genes in the PLSD and IMRC cohorts, of which only the former included mandatory colonoscopy surveillance for all participants. Methods: CRC incidences were calculated in an intervention group comprising a cohort of confirmed carriers of pathogenic or likely pathogenic variants in mismatch repair genes (path_MMR) followed prospectively by the Prospective Lynch Syndrome Database (PLSD). All had colonoscopy surveillance, with polypectomy when polyps were identified. Comparison was made with a retrospective cohort reported by the International Mismatch Repair Consortium (IMRC). This comprised confirmed and inferred path_MMR carriers who were first- or second-degree relatives of Lynch syndrome probands. Results: In the PLSD, 8,153 subjects had follow-up colonoscopy surveillance for a total of 67,604 years and 578 carriers had CRC diagnosed. Average cumulative incidences of CRC in path_MLH1 carriers at 70 years of age were 52% in males and 41% in females; for path_MSH2 50% and 39%; for path_MSH6 13% and 17% and for path_PMS2 11% and 8%. In contrast, in the IMRC cohort, corresponding cumulative incidences were 40% and 27%; 34% and 23%; 16% and 8% and 7% and 6%. Comparing just the European carriers in the two series gave similar findings. Numbers in the PLSD series did not allow comparisons of carriers from other continents separately. Cumulative incidences at 25 years were < 1% in all retrospective groups. Conclusions: Prospectively observed CRC incidences (PLSD) in path_MLH1 and path_MSH2 carriers undergoing colonoscopy surveillance and polypectomy were higher than in the retrospective (IMRC) series, and were not reduced in path_MSH6 carriers. These findings were the opposite to those expected. CRC point incidence before 50 years of age was reduced in path_PMS2 carriers subjected to colonoscopy, but not significantly so.publishedVersionPeer reviewe

Simplifying Revenue Management

In this thesis, we study three revenue management problems where we propose simple algorithms with provable guarantees. While online marketplaces provide retailers with tremendous flexibility, they are often large, noisy, have multiple stakeholders, and could be more challenging to characterize. These complexities give rise to a preference for simple, interpretable policies. Further, traditional marketplaces such as brick-and-mortar stores cannot always leverage tools designed for online environments due to physical constraints, higher latency, etc. With these motivations in mind, we develop algorithms for assortment optimization and pricing that are easy to implement in practice and have theoretical justifications for their performance. In Chapter 1, we consider a dynamic assortment optimization problem where the seller has a fixed inventory of multiple substitutable products to sell over a fixed time horizon. We consider two modifications to the traditional problem. First, we simplify the assortment planning by restricting assortment changes to "product retirements". When a product is retired, it becomes unavailable to all future customers. Second, we assume the seller has flexibility regarding which customers to approach. In each period, the seller chooses which subset of products to retire and selects a customer to visit. The selected customer then receives an option to purchase one of the available products, i.e., non-retired products with positive remaining inventory. We provide two policies for this problem. Our first policy guarantees a constant fraction of the best possible revenue. Our second policy is near-optimal but requires the problem to have a specific structure. In Chapter 2, we study the fundamental joint pricing and inventory management problem. The optimal policy for the model we consider is known to be an (s, S, p) policy: when the inventory level drops to s units, the seller immediately places an order to replenish the inventory to S units. Specifically, the optimal pricing policy p has a different price for every inventory state. We proposed simple policies requiring no more than three prices and prove that these policies are near-optimal compared to optimal policies which require more prices and are less robust. In particular, when orders cannot be backlogged, we show that a single price is sufficient for good performance. In Chapter 3, we analyze assortment optimization and pricing with opaque products. An opaque product is one for which only partial information is available to the buyer at the time of purchase. When a customer selects the opaque product, the seller can fulfill the purchase using any of the offered products. Opaque products can help sellers boost total sales. We propose simple policies for assortment optimization with provable constant factor guarantees, which are near-optimal in numerical experiments. We also provide upper bounds for the advantage of selling opaque products