The gustin (CA6) gene polymorphism, rs2274333 (A/G), as a mechanistic link between PROP tasting and fungiform taste papilla density and maintenance

Taste sensitivity to PROP varies greatly among individuals and is associated with polymorphisms in the bitter receptor gene TAS2R38, and with differences in fungiform papilla density on the anterior tongue surface. Recently we showed that the PROP non-taster phenotype is strongly associated with the G variant of polymorphism rs2274333 (A/G) of the gene that controls the salivary trophic factor, gustin. The aims of this study were 1) to investigate the role of gustin gene polymorphism rs2274333 (A/G), in PROP sensitivity and fungiform papilla density and morphology, and 2) to investigate the effect of this gustin gene polymorphism on cell proliferation and metabolic activity. Sixty-four subjects were genotyped for both genes by PCR techniques, their PROP sensitivity was assessed by scaling and threshold methods, and their fungiform papilla density, diameter and morphology were determined. In vitro experiments examined cell proliferation and metabolic activity, following treatment with saliva of individuals with and without the gustin gene mutation, and with isolated protein, in the two iso-forms. Gustin and TAS2R38 genotypes were associated with PROP threshold (p=0.0001 and p=0.0042), but bitterness intensity was mostly determined by TAS2R38 genotypes (p<0.000001). Fungiform papillae densities were associated with both genotypes (p<0.014) (with a stronger effect for gustin; p=0.0006), but papilla morphology was a function of gustin alone (p<0.0012). Treatment of isolated cells with saliva from individuals with the AA form of gustin or direct application of the active iso-form of gustin protein increased cell proliferation and metabolic activity (p<0.0135). These novel findings suggest that the rs2274333 polymorphism of the gustin gene affects PROP sensitivity by acting on fungiform papilla development and maintenance, and could provide the first mechanistic explanation for why PROP super-tasters are more responsive to a broad range of oral stimul

Statistical issues in Mendelian randomization: use of genetic instrumental variables for assessing causal associations

Mendelian randomization is an epidemiological method for using genetic variation to estimate the causal effect of the change in a modifiable phenotype on an outcome from observational data. A genetic variant satisfying the assumptions of an instrumental variable for the phenotype of interest can be used to divide a population into subgroups which differ systematically only in the phenotype. This gives a causal estimate which is asymptotically free of bias from confounding and reverse causation. However, the variance of the causal estimate is large compared to traditional regression methods, requiring large amounts of data and necessitating methods for efficient data synthesis. Additionally, if the association between the genetic variant and the phenotype is not strong, then the causal estimates will be biased due to the “weak instrument” in finite samples in the direction of the observational association. This bias may convince a researcher that an observed association is causal. If the causal parameter estimated is an odds ratio, then the parameter of association will differ depending on whether viewed as a population-averaged causal effect or a personal causal effect conditional on covariates. We introduce a Bayesian framework for instrumental variable analysis, which is less susceptible to weak instrument bias than traditional two-stage methods, has correct coverage with weak instruments, and is able to efficiently combine gene–phenotype–outcome data from multiple heterogeneous sources. Methods for imputing missing genetic data are developed, allowing multiple genetic variants to be used without reduction in sample size. We focus on the question of a binary outcome, illustrating how the collapsing of the odds ratio over heterogeneous strata in the population means that the two-stage and the Bayesian methods estimate a population-averaged marginal causal effect similar to that estimated by a randomized trial, but which typically differs from the conditional effect estimated by standard regression methods. We show how these methods can be adjusted to give an estimate closer to the conditional effect. We apply the methods and techniques discussed to data on the causal effect of C-reactive protein on fibrinogen and coronary heart disease, concluding with an overall estimate of causal association based on the totality of available data from 42 studies.This work was supported by the U.K. Medical Research Council [grant number U.1052.00.001]

An Average-Case Sublinear Exact Li and Stephens Forward Algorithm

Hidden Markov models of haplotype inheritance such as the Li and Stephens model allow for computationally tractable probability calculations using the forward algorithms as long as the representative reference panel used in the model is sufficiently small. Specifically, the monoploid Li and Stephens model and its variants are linear in reference panel size unless heuristic approximations are used. However, sequencing projects numbering in the thousands to hundreds of thousands of individuals are underway, and others numbering in the millions are anticipated. To make the Li and Stephens forward algorithm for these datasets computationally tractable, we have created a numerically exact version of the algorithm with observed average case O(nk^{0.35}) runtime in number of genetic sites n and reference panel size k. This avoids any tradeoff between runtime and model complexity. We demonstrate that our approach also provides a succinct data structure for general purpose haplotype data storage. We discuss generalizations of our algorithmic techniques to other hidden Markov models

Almost exact Mendelian randomization

Mendelian randomization (MR) is a natural experimental design based on the random transmission of genes from parents to offspring. However, this inferential basis is typically only implicit or used as an informal justification. As parent-offspring data becomes more widely available, we advocate a different approach to MR that is exactly based on this natural randomization, thereby formalizing the analogy between MR and randomized controlled trials. We begin by developing a causal graphical model for MR which represents several biological processes and phenomena, including population structure, gamete formation, fertilization, genetic linkage, and pleiotropy. This causal graph is then used to detect biases in population-based MR studies and identify sufficient confounder adjustment sets to correct these biases. We then propose a randomization test in the within-family MR design using the exogenous randomness in meiosis and fertilization, which is extensively studied in genetics. Besides its transparency and conceptual appeals, our approach also offers some practical advantages, including robustness to misspecified phenotype models, robustness to weak instruments, and elimination of bias arising from population structure, assortative mating, dynastic effects, and horizontal pleiotropy. We conclude with an analysis of a pair of negative and positive controls in the Avon Longitudinal Study of Parents and Children. The accompanying R package can be found at https://github.com/matt-tudball/almostexactmr.Comment: 41 pages, 10 figure

Genetic variation and differential expression of p21 (WAF1/CIP1) in the context of HIV-1 control

A dissertation submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Master of Science (Med) Virology. Johannesburg, 2016A recent study has shown variable p21 expression levels linked to individuals displaying different levels of HIV-1 control, with elite controllers (ECs) and viraemic controllers (VCs) exhibiting higher p21 expression when compared to both healthy HIV-1 negative individuals and HIV-1-infected progressors. The role of p21 in HIV-1 control in a sub-Saharan African population has not been established. Polymorphisms in the regulatory regions of p21, as well as in the microRNAs (miRNAs) that affect p21 regulation can contribute to differential p21 expression. In this study we developed real-time PCR assays to genotype the p21 exonic rs1801270 and 3‘UTR rs1059234 SNPs, in addition to the p21-associated miRNA (miR-106b) rs999885 SNP. We determined their allelic and genotypic frequencies in Black South African HIV-1 negative individuals (n=72), HIV-1 controllers (HICs) (n=52) further subdivided into ECs (n=11), VCs (n=30) and high viral load long term non-progressors (HVL LTNPs) (n=11), and HIV-1 infected progressors (n=74). We sequenced a region of the p21 5‘UTR and 3‘UTR in a subset of these individuals (HICs: n=52, progressors: n=44) to identify variants that may be modulating p21 expression. We compared levels of p21 mRNA, a marker for p21 expression, in a smaller group of individuals (n=50) with similar clinical phenotypes to determine if p21 upregulation was associated with natural control of HIV-1. Lastly, we developed a real-time PCR assay to genotype a p21 5‘UTR SNP, rs733590, that alone, and together with HLA-B*2705, was recently shown to directly impact on p21 expression in Caucasians. This SNP was genotyped and analysed in the individuals with p21 mRNA expression data. The p21 rs1801270 and rs1059234 SNPs were found to occur in partial linkage disequilibrium (LD) (r2=0.61). Although ECs had markedly less representation of the 3‘UTR rs1059234 mutant allele (T) and heterozygosity (CT) compared to progressors (T allele: 9.1% ECs vs. 25% progressors; CT genotype: 18.2% ECs vs. 42% progressors), this did not reach significance (p=0.11, OR=3.33; p=0.19, OR=3.49, respectively). Interestingly, HIV-1 controllers with <400 HIV-1 RNA copies/ml (<400 HICs) also had less representation of the CT genotype when compared to progressors (20% vs. 42%, respectively; p=0.11, OR=2.91). In silico analysis of this 3‘UTR SNP suggested that there are functional implications in terms of miRNA regulation, however when p21 mRNA expression was analysed with respect to this SNP, no effect was seen. The role of this 3‘UTR SNP on p21 expression and/or function and HIV-1 control requires further investigation. The p21 exonic rs1801270 SNP showed no difference in representation among the clinical phenotypic groups and no effect was seen on p21 mRNA expression. When comparing HIV-1 controllers with >400 HIV-1 RNA copies/ml (>400 HICs) to progressors, the >400 HICs had significantly lower representation of the minor allele (A) of the miR-106b rs999885 SNP (p=0.04, OR=2.28). In addition, heterozygosity for this SNP (GA) was found in a much lower frequency in >400 HICs when compared to progressors (p=0.05; OR=2.56). Stratification of individuals according to their miR-106b rs999885 SNP genotype and p21 mRNA expression revealed the GA genotype to be associated with a trend to higher p21 mRNA expression (p=0.066). A role for the miR-106b rs999885 SNP in HIV-1 control in individuals with higher viraemia needs to be validated in larger cohorts. Characterisation of the p21 regulatory regions, namely a region of the 5‘UTR and the 3‘UTR, identified 19 polymorphisms (18 SNPs and one indel) and 12 SNPs in the respective regions. A prevalent, previously uncharacterised 11-SNP haplotype (LD: r2=1) was detected in the p21 promoter region at a frequency of 39.42% in the HIV-1 controllers and 48.86% in the progressor cohort. In addition, a 2-SNP haplotype was identifed and was found to be in moderate LD with the 11-SNP haplotype (r2=0.67). The ECs were found to have a trend of less representation of the 2-SNP haplotype minor allele when compared to progressors (p=0.08, OR=2.83). Other than the rs1059234 SNP, no other SNPs in the 3‘UTR were differentially represented in any of our studied groups. p21 mRNA expression analysis showed significant correlations between p21 mRNA expression and markers of disease progression (HIV-1 viral load: r=0.69, p<0.0001 and CD4+ T cell count: r=-0.53, p=0.0005). In our study, ECs and VCs had significantly lower p21 mRNA expression compared to progressors (p=0.002 and p=0.001, respectively). Furthermore, in our Black South African population (n=50), the p21 5‘UTR rs733590 SNP CT and TT genotypes were not associated with higher p21 mRNA expression as has been shown in Caucasians. This, together with the absence of HLA-B*2705 in our Black South African population, points to host genetic differences as the likely contributors to the different results seen in our study with respect to p21 expression and HIV-1 control when compared to reported literature. Future work with larger sample sizes and varied population groups will be highly informative in determining the role of p21 and natural control of HIV-1 in the Black South African population.MB201

Recombination between heterologous human acrocentric chromosomes

The short arms of the human acrocentric chromosomes 13, 14, 15, 21 and 22 (SAACs) share large homologous regions, including ribosomal DNA repeats and extended segmental duplications1,2. Although the resolution of these regions in the first complete assembly of a human genome—the Telomere-to-Telomere Consortium’s CHM13 assembly (T2T-CHM13)—provided a model of their homology3, it remained unclear whether these patterns were ancestral or maintained by ongoing recombination exchange. Here we show that acrocentric chromosomes contain pseudo-homologous regions (PHRs) indicative of recombination between non-homologous sequences. Utilizing an all-to-all comparison of the human pangenome from the Human Pangenome Reference Consortium4 (HPRC), we find that contigs from all of the SAACs form a community. A variation graph5 constructed from centromere-spanning acrocentric contigs indicates the presence of regions in which most contigs appear nearly identical between heterologous acrocentric chromosomes in T2T-CHM13. Except on chromosome 15, we observe faster decay of linkage disequilibrium in the pseudo-homologous regions than in the corresponding short and long arms, indicating higher rates of recombination6,7. The pseudo-homologous regions include sequences that have previously been shown to lie at the breakpoint of Robertsonian translocations8, and their arrangement is compatible with crossover in inverted duplications on chromosomes 13, 14 and 21. The ubiquity of signals of recombination between heterologous acrocentric chromosomes seen in the HPRC draft pangenome suggests that these shared sequences form the basis for recurrent Robertsonian translocations, providing sequence and population-based confirmation of hypotheses first developed from cytogenetic studies 50 years ago9.Our work depends on the HPRC draft human pangenome resource established in the accompanying Article4, and we thank the production and assembly groups for their efforts in establishing this resource. This work used the computational resources of the UTHSC Octopus cluster and NIH HPC Biowulf cluster. We acknowledge support in maintaining these systems that was critical to our analyses. The authors thank M. Miller for the development of a graphical synopsis of our study (Fig. 5); and R. Williams and N. Soranzo for support and guidance in the design and discussion of our work. This work was supported, in part, by National Institutes of Health/NIDA U01DA047638 (E.G.), National Institutes of Health/NIGMS R01GM123489 (E.G.), NSF PPoSS Award no. 2118709 (E.G. and C.F.), the Tennessee Governor’s Chairs programme (C.F. and E.G.), National Institutes of Health/NCI R01CA266339 (T.P., L.G.d.L. and J.L.G.), and the Intramural Research Program of the National Human Genome Research Institute, National Institutes of Health (A.R., S.K. and A.M.P.). We acknowledge support from Human Technopole (A.G.), Consiglio Nazionale delle Ricerche, Italy (S.B. and V.C.), and Stowers Institute for Medical Research (T.P., L.G.d.L., B.R. and J.L.G.).Peer Reviewed"Article signat per 13 autors/es: Andrea Guarracino, Silvia Buonaiuto, Leonardo Gomes de Lima, Tamara Potapova, Arang Rhie, Sergey Koren, Boris Rubinstein, Christian Fischer, Human Pangenome Reference Consortium, Jennifer L. Gerton, Adam M. Phillippy, Vincenza Colonna & Erik Garrison " Human Pangenome Reference Consortium: "Haley J. Abel, Lucinda L. Antonacci-Fulton, Mobin Asri, Gunjan Baid, Carl A. Baker, Anastasiya Belyaeva, Konstantinos Billis, Guillaume Bourque, Silvia Buonaiuto, Andrew Carroll, Mark J. P. Chaisson, Pi-Chuan Chang, Xian H. Chang, Haoyu Cheng, Justin Chu, Sarah Cody, Vincenza Colonna, Daniel E. Cook, Robert M. Cook-Deegan, Omar E. Cornejo, Mark Diekhans, Daniel Doerr, Peter Ebert, Jana Ebler, Evan E. Eichler, Jordan M. Eizenga, Susan Fairley, Olivier Fedrigo, Adam L. Felsenfeld, Xiaowen Feng, Christian Fischer, Paul Flicek, Giulio Formenti, Adam Frankish, Robert S. Fulton, Yan Gao, Shilpa Garg, Erik Garrison, Nanibaa’ A. Garrison, Carlos Garcia Giron, Richard E. Green, Cristian Groza, Andrea Guarracino, Leanne Haggerty, Ira Hall, William T. Harvey, Marina Haukness, David Haussler, Simon Heumos, Glenn Hickey, Kendra Hoekzema, Thibaut Hourlier, Kerstin Howe, Miten Jain, Erich D. Jarvis, Hanlee P. Ji, Eimear E. Kenny, Barbara A. Koenig, Alexey Kolesnikov, Jan O. Korbel, Jennifer Kordosky, Sergey Koren, HoJoon Lee, Alexandra P. Lewis, Heng Li, Wen-Wei Liao, Shuangjia Lu, Tsung-Yu Lu, Julian K. Lucas, Hugo Magalhães, Santiago Marco-Sola, Pierre Marijon, Charles Markello, Tobias Marschall, Fergal J. Martin, Ann McCartney, Jennifer McDaniel, Karen H. Miga, Matthew W. Mitchell, Jean Monlong, Jacquelyn Mountcastle, Katherine M. Munson, Moses Njagi Mwaniki, Maria Nattestad, Adam M. Novak, Sergey Nurk, Hugh E. Olsen, Nathan D. Olson, Benedict Paten, Trevor Pesout, Adam M. Phillippy, Alice B. Popejoy, David Porubsky, Pjotr Prins, Daniela Puiu, Mikko Rautiainen, Allison A. Regier, Arang Rhie, Samuel Sacco, Ashley D. Sanders, Valerie A. Schneider, Baergen I. Schultz, Kishwar Shafin, Jonas A. Sibbesen, Jouni Sirén, Michael W. Smith, Heidi J. Sofia, Ahmad N. Abou Tayoun, Françoise Thibaud-Nissen, Chad Tomlinson, Francesca Floriana Tricomi, Flavia Villani, Mitchell R. Vollger, Justin Wagner, Brian Walenz, Ting Wang, Jonathan M. D. Wood, Aleksey V. Zimin & Justin M. Zook"Postprint (published version

Full-length de novo viral quasispecies assembly through variation graph construction

International audienceMotivation: Viruses populate their hosts as a viral quasispecies: a collection of genetically related mutant strains.Viral quasispecies assembly refers to reconstructing the strain-specific haplotypes from read data, and predicting their relative abundances within the mix of strains, an important step for various treatment-related reasons. Reference-genome-independent ("de novo") approaches have yielded benefits over reference-guided approaches, because reference-induced biases can become overwhelming when dealing with divergent strains. While being very accurate, extant de novo methods only yield rather short contigs. It remains to reconstruct full-length haplotypes together with their abundances from such contigs. Method: We first construct a variation graph, a recently popular, suitable structure for arranging and integrating several related genomes, from the short input contigs, without making use of a reference genome. To obtain paths through the variation graph that reflect the original haplotypes, we solve a minimization problem that yields a selection of maximal-length paths that is optimal in terms of being compatible with the read coverages computed for the nodes of the variation graph. We output the resulting selection of maximal length paths as the haplotypes, together with their abundances. Results: Benchmarking experiments on challenging simulated data sets show significant improvements in assembly contiguity compared to the input contigs, while preserving low error rates. As a consequence, our method outperforms all state-of-the-art viral quasispecies assem-blers that aim at the construction of full-length haplotypes, in terms of various relevant assembly measures. Our tool, Virus-VG, is publicly available at https://bitbucket.org/jbaaijens/ virus-vg