Single nucleotide polymorphism-based validation of exonic splicing enhancers

Because deleterious alleles arising from mutation are filtered by natural selection, mutations that create such alleles will be underrepresented in the set of common genetic variation existing in a population at any given time. Here, we describe an approach based on this idea called VERIFY (variant elimination reinforces functionality), which can be used to assess the extent of natural selection acting on an oligonucleotide motif or set of motifs predicted to have biological activity. As an application of this approach, we analyzed a set of 238 hexanucleotides previously predicted to have exonic splicing enhancer (ESE) activity in human exons using the relative enhancer and silencer classification by unanimous enrichment (RESCUE)-ESE method. Aligning the single nucleotide polymorphisms (SNPs) from the public human SNP database to the chimpanzee genome allowed inference of the direction of the mutations that created present-day SNPs. Analyzing the set of SNPs that overlap RESCUE-ESE hexamers, we conclude that nearly one-fifth of the mutations that disrupt predicted ESEs have been eliminated by natural selection (odds ratio = 0.82 +/- 0.05). This selection is strongest for the predicted ESEs that are located near splice sites. Our results demonstrate a novel approach for quantifying the extent of natural selection acting on candidate functional motifs and also suggest certain features of mutations/SNPs, such as proximity to the splice site and disruption or alteration of predicted ESEs, that should be useful in identifying variants that might cause a biological phenotype.     

Exome Sequencing of Only Seven Qataris Identifies Potentially Deleterious Variants in the Qatari Population

The Qatari population, located at the Arabian migration crossroads of African and Eurasia, is comprised of Bedouin, Persian and African genetic subgroups. By deep exome sequencing of only 7 Qataris, including individuals in each subgroup, we identified 2,750 nonsynonymous SNPs predicted to be deleterious, many of which are linked to human health, or are in genes linked to human health. Many of these SNPs were at significantly elevated deleterious allele frequency in Qataris compared to other populations worldwide. Despite the small sample size, SNP allele frequency was highly correlated with a larger Qatari sample. Together, the data demonstrate that exome sequencing of only a small number of individuals can reveal genetic variations with potential health consequences in understudied populations.     

High proportions of deleterious polymorphisms in constrained human genes

Previous studies on human mitochondrial genomes showed that the ratio of intra-specific diversities at nonsynonymous-to-synonymous positions was two to ten times higher than the ratio of interspecific divergences at these positions, suggesting an excess of slightly deleterious nonsynonymous polymorphisms. However, such an overabundance of nonsynonymous single nucleotide polymorphisms (SNPs) was not found in human nuclear genomes. Here, genome-wide estimates using >14,000 human-chimp nuclear genes and 1 million SNPs from four human genomes showed a significant proportion of deleterious nonsynonymous SNPs (～ 15%). Importantly, this study reveals a negative correlation between the magnitude of selection pressure and the proportion of deleterious SNPs on human genes. The proportion of deleterious amino acid replacement polymorphisms is 3.5 times higher in genes under high purifying selection compared with that in less constrained genes (28% vs. 8%). These results are explained by differences in the extent of contribution of mildly deleterious mutations to diversity and substitution.     

Natural selection affects multiple aspects of genetic variation at putatively neutral sites across the human genome

A major question in evolutionary biology is how natural selection has shaped patterns of genetic variation across the human genome. Previous work has documented a reduction in genetic diversity in regions of the genome with low recombination rates. However, it is unclear whether other summaries of genetic variation, like allele frequencies, are also correlated with recombination rate and whether these correlations can be explained solely by negative selection against deleterious mutations or whether positive selection acting on favorable alleles is also required. Here we attempt to address these questions by analyzing three different genome-wide resequencing datasets from European individuals. We document several significant correlations between different genomic features. In particular, we find that average minor allele frequency and diversity are reduced in regions of low recombination and that human diversity, human-chimp divergence, and average minor allele frequency are reduced near genes. Population genetic simulations show that either positive natural selection acting on favorable mutations or negative natural selection acting against deleterious mutations can explain these correlations. However, models with strong positive selection on nonsynonymous mutations and little negative selection predict a stronger negative correlation between neutral diversity and nonsynonymous divergence than observed in the actual data, supporting the importance of negative, rather than positive, selection throughout the genome. Further, we show that the widespread presence of weakly deleterious alleles, rather than a small number of strongly positively selected mutations, is responsible for the correlation between neutral genetic diversity and recombination rate. This work suggests that natural selection has affected multiple aspects of linked neutral variation throughout the human genome and that positive selection is not required to explain these observations.     

Negative linkage disequilibrium between amino acid changing variants reveals interference among deleterious mutations in the human genome

Evolutionary forces like Hill-Robertson interference and negative epistasis can lead to deleterious mutations being found on distinct haplotypes. However, the extent to which these forces depend on the selection and dominance coefficients of deleterious mutations and shape genome-wide patterns of linkage disequilibrium (LD) in natural populations with complex demographic histories has not been tested. In this study, we first used forward-in-time simulations to predict how negative selection impacts LD. Under models where deleterious mutations have additive effects on fitness, deleterious variants less than 10 kb apart tend to be carried on different haplotypes relative to pairs of synonymous SNPs. In contrast, for recessive mutations, there is no consistent ordering of how selection coefficients affect LD decay, due to the complex interplay of different evolutionary effects. We then examined empirical data of modern humans from the 1000 Genomes Project. LD between derived alleles at nonsynonymous SNPs is lower compared to pairs of derived synonymous variants, suggesting that nonsynonymous derived alleles tend to occur on different haplotypes more than synonymous variants. This result holds when controlling for potential confounding factors by matching SNPs for frequency in the sample (allele count), physical distance, magnitude of background selection, and genetic distance between pairs of variants. Lastly, we introduce a new statistic H_R^(j) which allows us to detect interference using unphased genotypes. Application of this approach to high-coverage human genome sequences confirms our finding that nonsynonymous derived alleles tend to be located on different haplotypes more often than are synonymous derived alleles. Our findings suggest that interference may play a pervasive role in shaping patterns of LD between deleterious variants in the human genome, and consequently influences genome-wide patterns of LD.     

Mutation, selection and the future of human evolution

Several recent analyses provide growing evidence of the influence of positive selection acting in the ancestors of modern humans. Additionally, the best way to explain current fluctuations in neutral variation across the genome is by including negative selection against a high rate of deleterious mutants. We suggest that explaining these predicted high deleterious mutation rates in humans could require the inclusion of additional factors, such as inbreeding and prezygotic selection, in addition to rank-order selection and fitness interactions among mutations. We also suggest that some forms of selection, rather than being relaxed in modern humans, are probably still acting and might intensify in the near future, and make some predictions about the next several millennia of human evolution.     

Current relaxation of selection on the human genome: Tolerance of deleterious mutations on olfactory receptors

Knowledge and understanding about the selective pressures that have shaped present human genetic diversity have dramatically increased in the last few years in parallel with the availability of large genomic datasets. The release of large datasets composed of millions of SNPs across hundreds of genomes by HAPMAP, the Human Genome Diversity Panel, and other projects has led to considerable effort to detect selection signals across the nuclear genome (Coop et al., 2009, Lopez Herraez et al., 2009, Sabeti et al., 2006, Sabeti et al., 2007, Voight et al., 2006). Most of the research has focused on positive selection forces although other selective forces, such as negative selection, may have played a substantive role on the shape of our genome. Here we studied the selective strengths acting presently on the genome by making computational predictions of the pathogenicity of nonsynonymous protein mutations and interpreting the distribution of scores in terms of selection. We could show that the genetic diversity for all the major pathways is still constrained by negative selection in all 11 human populations studied. In a single exception, we observed a relaxation of negative selection acting on olfactory receptors. Since a decreased number of functioning olfactory receptors in human compared with other primates had already been shown, this suggests that the role of olfactory receptors for survival and reproductive success has decreased during human evolution. By showing that negative selection is still relaxed, the present results imply that no plateau of minimal function has yet been reached in modern humans and therefore that olfactory capability might still be decreasing. This is a first clue to present human evolution.     

Evidence for hitchhiking of deleterious mutations within the human genome

Deleterious mutations present a significant obstacle to adaptive evolution. Deleterious mutations can inhibit the spread of linked adaptive mutations through a population; conversely, adaptive substitutions can increase the frequency of linked deleterious mutations and even result in their fixation. To assess the impact of adaptive mutations on linked deleterious mutations, we examined the distribution of deleterious and neutral amino acid polymorphism in the human genome. Within genomic regions that show evidence of recent hitchhiking, we find fewer neutral but a similar number of deleterious SNPs compared to other genomic regions. The higher ratio of deleterious to neutral SNPs is consistent with simulated hitchhiking events and implies that positive selection eliminates some deleterious alleles and increases the frequency of others. The distribution of disease-associated alleles is also altered in hitchhiking regions. Disease alleles within hitchhiking regions have been associated with auto-immune disorders, metabolic diseases, cancers, and mental disorders. Our results suggest that positive selection has had a significant impact on deleterious polymorphism and may be partly responsible for the high frequency of certain human disease alleles.     

High level of functional polymorphism indicates a unique role of natural selection at human immune system loci

Several studies have shown that immune system proteins have on average a higher rate of amino acid evolution between different species of mammals than do most other proteins. To test whether immune-system-expressed loci show a correspondingly elevated rate of within-species nonsynonymous (amino acid altering) polymorphism, we examined gene diversity (heterozygosity) at 4,911 single nucleotide polymorphism (SNP) sites at 481 protein-coding loci. At loci with nonimmune functions, gene diversity at nonsynonymous SNP sites was typically lower than that at silent SNP sites (those not altering the amino acid sequence) in the same gene, a pattern that is an evidence of purifying selection acting to eliminate slightly deleterious variants. However, this pattern was not seen at nonsynonymous SNPs causing conservative amino acid replacements in immune system proteins, indicating that the latter are subject to a reduced level of functional constraint. Similarly, immune system genes showed higher gene diversities in their 5′ noncoding regions than did other proteins. These results identified certain immune system loci that are likely to be subject to balancing selection that acts to maintain polymorphism in either coding or regulatory regions. Electronic Supplementary Material Supplementary material is available for this article at .     

Evolutionary Processes Acting on Candidate cis-Regulatory Regions in Humans Inferred from Patterns of Polymorphism and Divergence

Analysis of polymorphism and divergence in the non-coding portion of the human genome yields crucial information about factors driving the evolution of gene regulation. Candidate cis-regulatory regions spanning more than 15,000 genes in 15 African Americans and 20 European Americans were re-sequenced and aligned to the chimpanzee genome in order to identify potentially functional polymorphism and to characterize and quantify departures from neutral evolution. Distortions of the site frequency spectra suggest a general pattern of selective constraint on conserved non-coding sites in the flanking regions of genes (CNCs). Moreover, there is an excess of fixed differences that cannot be explained by a Gamma model of deleterious fitness effects, suggesting the presence of positive selection on CNCs. Extensions of the McDonald-Kreitman test identified candidate cis-regulatory regions with high probabilities of positive and negative selection near many known human genes, the biological characteristics of which exhibit genome-wide trends that differ from patterns observed in protein-coding regions. Notably, there is a higher probability of positive selection in candidate cis-regulatory regions near genes expressed in the fetal brain, suggesting that a larger portion of adaptive regulatory changes has occurred in genes expressed during brain development. Overall we find that natural selection has played an important role in the evolution of candidate cis-regulatory regions throughout hominid evolution.     

Synonymous SNPs Provide Evidence for Selective Constraint on Human Exonic Splicing Enhancers

The human SNP database was used to detect selection on 238 hexamers previously identified as exonic splicing enhancers (ESEs). We compared the distribution of the 238 putative ESEs in biallelic and triallelic SNPs within five different functional categories of the SNP database: synonymous, nonsynonymous, introns, UTRs, and nongenic SNPs. Since true ESEs do not function outside of exons, SNPs that disrupt ESE motifs were expected to be more common in nonexonic portions of the genome. Our results supported this expectation: ESEs were least prevalent within synonymous SNPs and most common in nongenic SNPs. There were ∼11% fewer ESEs within synonymous biallelic SNPs than expected under no selective constraint. We also compared the frequency of neutral SNPs, those where neither allele was an ESE, with deleterious SNPs, those where one or more alleles was an ESE, across the five different functional classes of SNPs. In comparison with the other functional classes of SNPs, synonymous SNPs contained an excess of neutral variants (+1.64% and +6.04% for biallelic and triallelic SNPs, respectively) and a dearth of deleterious variants (−13.11% and −52.39% for biallelic and triallelic SNPs, respectively). The observed patterns were consistent with purifying selection on the 238 hexamers to maintain their function as ESEs. However, in contrast to previous work, we did not find evidence for selection to maintain ESE function at nonsynonymous SNPs because selection at the protein level probably obscured any difference at the level of ESE function.     

A neutral theory predicts multigenic aging and increased concentrations of deleterious mutations on the mitochondrial and Y chromosomes

Population genetic forces have molded the constitution of the human genome over evolutionary time, and some of the most important parameters are the initial frequency of the allele, p, the effective population size, Ne, and the selection coefficient, s. There is considerable agreement among evolutionary gerontologists that the amplitude of -s is small for alleles that are Deleterious In Late Life (DILL), and thus DILL traits are effectively neutral and should be fixed in the human population in relationship to Ne and p. Even higher rates of fixation of deleterious mutations are predicted to occur in the two nonrecombinant genomes in humans, i.e., the Y chromosome and the mitochondrial genome, as a consequence of their lower Ne than autosomes, and the predicted higher rate of fixation of deleterious alleles on the Y may explain the reduced average life span of males vs. females. The high probability of fixation of neutral and mildly deleterious mutations in the mitochondrial genome explains in part its fast rate of evolution, the high observed frequency of mitochondrial disease in relationship to this genome's small size, and may be the underlying reason for the transfer of mitochondrial genes over evolutionary time to the nucleus. The predicted higher concentration of deleterious mutations on the mitochondrial genome could have some leverage to cause more dysfunction than that predicted by mitochondrial gene number alone, because of the essential role of mitochondrial gene function in multisubunit complexes, the coupling of mitochondrial functions, the observation that some mtDNA sequences facilitate somatic mutation, and the likelihood of deleterious mutations either increasing the production of or the sensitivity to mitochondrial ROS.     

Combined sequence and sequence-structure-based methods for analyzing RAAS gene SNPs: a computational approach

Abstract

The renin–angiotensin–aldosterone system (RAAS) plays a key role in the regulation of blood pressure (BP). Mutations on the genes that encode components of the RAAS have played a significant role in genetic susceptibility to hypertension and have been intensively scrutinized. The identification of such probably causal mutations not only provides insight into the RAAS but may also serve as antihypertensive therapeutic targets and diagnostic markers. The methods for analyzing the SNPs from the huge dataset of SNPs, containing both functional and neutral SNPs is challenging by the experimental approach on every SNPs to determine their biological significance. To explore the functional significance of genetic mutation (SNPs), we adopted combined sequence and sequence-structure-based SNP analysis algorithm. Out of 3864 SNPs reported in dbSNP, we found 108 missense SNPs in the coding region and remaining in the non-coding region. In this study, we are reporting only those SNPs in coding region to be deleterious when three or more tools are predicted to be deleterious and which have high RMSD from the native structure. Based on these analyses, we have identified two SNPs of REN gene, eight SNPs of AGT gene, three SNPs of ACE gene, two SNPs of AT1R gene, three SNPs of CYP11B2 gene and three SNPs of CMA1 gene in the coding region were found to be deleterious. Further this type of study will be helpful in reducing the cost and time for identification of potential SNP and also helpful in selecting potential SNP for experimental study out of SNP pool.     

Single nucleotide polymorphism (SNP) discovery in porcine expressed genes

High-throughput genotyping of swine populations is a potentially efficient method for establishing animal lineage and identification of loci important to animal health and efficient pork production. Markers were developed based upon single nucleotide polymorphisms (SNPs), which are abundant and amenable to automated genotyping platforms. The focus of this research was SNP discovery in expressed porcine genes providing markers to develop the porcine/human comparative map. Locus specific amplification (LSA) and comparative sequencing were used to generate PCR products and allelic information from parents of a swine reference family. Discovery of 1650 SNPs in 403 amplicons and strategies for optimizing LSA-based SNP discovery using alternative methods of PCR primer design, data analysis, and germplasm selection that are applicable to other populations and species are described. These data were the first large-scale assessment of frequency and distribution of porcine SNPs.     

Shifting paradigm of association studies: value of rare single-nucleotide polymorphisms

Currently, single-nucleotide polymorphisms (SNPs) with minor allele frequency (MAF) of >5% are preferentially used in case-control association studies of common human diseases. Recent technological developments enable inexpensive and accurate genotyping of a large number of SNPs in thousands of cases and controls, which can provide adequate statistical power to analyze SNPs with MAF <5%. Our purpose was to determine whether evaluating rare SNPs in case-control association studies could help identify causal SNPs for common diseases. We suggest that slightly deleterious SNPs (sdSNPs) subjected to weak purifying selection are major players in genetic control of susceptibility to common diseases. We compared the distribution of MAFs of synonymous SNPs with that of nonsynonymous SNPs (1) predicted to be benign, (2) predicted to be possibly damaging, and (3) predicted to be probably damaging by PolyPhen. Our sources of data were the International HapMap Project, ENCODE, and the SeattleSNPs project. We found that the MAF distribution of possibly and probably damaging SNPs was shifted toward rare SNPs compared with the MAF distribution of benign and synonymous SNPs that are not likely to be functional. We also found an inverse relationship between MAF and the proportion of nsSNPs predicted to be protein disturbing. On the basis of this relationship, we estimated the joint probability that a SNP is functional and would be detected as significant in a case-control study. Our analysis suggests that including rare SNPs in genotyping platforms will advance identification of causal SNPs in case-control association studies, particularly as sample sizes increase.     

Comparative population genomics identified genomic regions and candidate genes associated with fruit domestication traits in peach

Crop evolution is a long‐term process involving selection by natural evolutionary forces and anthropogenic influences; however, the genetic mechanisms underlying the domestication and improvement of fruit crops have not been well studied to date. Here, we performed a population structure analysis in peach (Prunus persica) based on the genome‐wide resequencing of 418 accessions and confirmed the presence of an obvious domestication event during evolution. We identified 132 and 106 selective sweeps associated with domestication and improvement, respectively. Analysis of their tissue‐specific expression patterns indicated that the up‐regulation of selection genes during domestication occurred mostly in fruit and seeds as opposed to other organs. However, during the improvement stage, more up‐regulated selection genes were identified in leaves and seeds than in the other organs. Genome‐wide association studies (GWAS) using 4.24 million single nucleotide polymorphisms (SNPs) revealed 171 loci associated with 26 fruit domestication traits. Among these loci, three candidate genes were highly associated with fruit weight and the sorbitol and catechin content in fruit. We demonstrated that as the allele frequency of the SNPs associated with high polyphenol composition decreased during peach evolution, alleles associated with high sugar content increased significantly. This indicates that there is genetic potential for the breeding of more nutritious fruit with enhanced bioactive polyphenols without disturbing a harmonious sugar and acid balance by crossing with wild species. This study also describes the development of the genomic resources necessary for evolutionary research in peach and provides the large‐scale characterization of key agronomic traits in this crop species.     

Effects of natural selection on interpopulation divergence at polymorphic sites in human protein-coding Loci

To develop new strategies for searching for genetic associations with complex human diseases, we analyzed 2784 single-nucleotide polymorphisms (SNPs) in 396 protein-coding genes involved in biological processes relevant to cancer and other complex diseases, with respect to gene diversity within samples of individuals representing the three major historic human populations (African, European, and Asian) and with respect to interpopulation genetic distance. Reduced levels of both intrapopulation gene diversity and interpopulation genetic distance were seen in the case of SNPs located within the 5'-UTR and at nonsynonymous SNPs, causing radical changes to protein structure. Reduction of gene diversity at SNP loci in these categories was evidence of purifying selection acting at these sites, which in turn causes a reduction in interpopulation divergence. By contrast, a small number of SNP sites in these categories revealed unusually high genetic distances between the two most diverged populations (African and Asian); these loci may have historically been subject to divergent selection pressures.     

Slow evolution of sex‐biased genes in the reproductive tissue of the dioecious plant Salix viminalis

The relative rate of evolution for sex‐biased genes has often been used as a measure of the strength of sex‐specific selection. In contrast to studies in a wide variety of animals, far less is known about the molecular evolution of sex‐biased genes in plants, particularly in dioecious angiosperms. Here, we investigate the gene expression patterns and evolution of sex‐biased genes in the dioecious plant Salix viminalis. We observe lower rates of sequence evolution for male‐biased genes expressed in the reproductive tissue compared to unbiased and female‐biased genes. These results could be partially explained by the lower codon usage bias for male‐biased genes leading to elevated rates of synonymous substitutions compared to unbiased genes. However, the stronger haploid selection in the reproductive tissue of plants, together with pollen competition, would also lead to higher levels of purifying selection acting to remove deleterious variation. Future work should focus on the differential evolution of haploid‐ and diploid‐specific genes to understand the selective dynamics acting on these loci.     

Do essential genes evolve slowly?

Approximately two thirds of all knockouts of individual mouse genes give rise to viable fertile mice. These genes have thus been termed 'non-essential' in contrast to 'essential' genes, the knockouts of which result in death or infertility. Although non-essential genes are likely to be under selection that favours sequence conservation [1], it is predicted that they are less subject to such stabilising selection than essential genes, and hence evolve faster [2]. We have addressed this issue by analysing the molecular evolution of 108 non-essential and 67 essential genes that have been sequenced in both mouse and rat. On preliminary analysis, the non-essential genes appeared to be faster evolving than the essential ones. We found, however, that the non-essential class contains a disproportionate number of immune-system genes that may be under directional selection (that is, selection favouring change) because of host-parasite coevolution. After correction for this bias, we found that the rate at which genes evolve does not correlate with the severity of the knockout phenotype. This was corroborated by the finding that, whereas neuron-specific genes have significantly lower rates of change than other genes, essential and non-essential neuronal genes have comparable rates of evolution. Our findings most probably reflect strong selection acting against even very subtle deleterious phenotypes, and indicate that the putative involvement of directional selection in host-parasite coevolution and gene expression within the nervous system explains much more of the variance in rates of gene evolution than does the knockout phenotype.