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Primate herpes simplex viruses are species-specific and relatively harmless to their natural hosts. However, cross-species transmission is often associated with severe disease, as exemplified by the virulence of macacine herpesvirus 1 (B virus) in humans. We performed a genome-wide scan for signals of adaptation of simplexviruses to their hominin hosts. Among core genes, we found evidence of episodic positive selection in three glycoproteins, with several selected sites located in antigenic determinants. Positively selected noncore genes were found to be involved in different immune-escape mechanisms. The herpes simplex virus (HSV)-1/HSV-2 encoded product (ICP47) of one of these genes is known to down-modulate major histocompatibility complex class I expression. This feature is not shared with B virus, which instead up-regulates Human Leukocyte Antigen (HLA)-G, an immunomodulatory molecule. By in vitro expression of different ICP47 mutants, we functionally characterized the selection signals. Results indicated that the selected sites do not represent the sole determinants of binding to the transporter associated with antigen processing (TAP). Conversely, the amino acid status at these sites was sufficient to determine HLA-G up-regulation. In fact, both HSV-1 and HSV-2 ICP47 induced HLA-G when mutated to recapitulate residues in B virus, whereas the mutated version of B virus ICP47 failed to determine HLA-G expression. These differences might contribute to the severity of B virus infection in humans. Importantly, they indicate that the evolution of ICP47 in HSV-1/HSV-2 led to the loss of an immunosuppressive effect. Thus, related simplexviruses finely tune the balance between immunosuppressive and immunostimulatory pathways to promote successful co-existence with their primate hosts.

Centromeres have central functions in chromosome segregation, but centromeric DNA and centromere-binding proteins evolve rapidly in most eukaryotes. The selective pressure(s) underlying the fast evolution of centromere-binding proteins are presently unknown. An attractive possibility is that selfish centromeres promote their preferential inclusion in the oocyte and centromeric proteins evolve to suppress meiotic drive (centromere drive hypothesis). We analysed the selective patterns of mammalian genes that encode kinetochore proteins and microtubule (MT)-destabilizing factors. We show that several of these proteins evolve at the same rate or faster than proteins with a role in centromere specification. Elements of the kinetochore that bind MTs or that bridge the interaction between MTs and the centromere represented the major targets of positive selection. These data are in line with the possibility that the genetic conflict fuelled by meiotic drive extends beyond genes involved in centromere specification. However, we cannot exclude that different selective pressures underlie the rapid evolution of MT-destabilizing factors and kinetochore components. Whatever the nature of such pressures, they must have been constant during the evolution of eutherian mammals, as we found a surprisingly good correlation in dN/dS (ratio of the rate of nonsynonymous and synonymous substitutions) across orders/clades. Finally, when phylogenetic relationships were accounted for, we found little evidence that the evolutionary rates of these genes change with testes size, a proxy for sperm competition. Our data indicate that, in analogy to centromeric proteins, kinetochore components are fast evolving in mammals. This observation may imply that centromere drive plays out at multiple levels or that these proteins adapt to lineage-specific centromeric features.

Four endemic coronaviruses infect humans and cause mild symptoms. Because previous analyses were based on a limited number of sequences and did not control for effects that affect molecular dating, we re-assessed the timing of endemic coronavirus emergence. After controlling for recombination, selective pressure, and molecular clock model, we obtained similar tMRCA (time to the most recent common ancestor) estimates for the four coronaviruses, ranging from 72 (HCoV-229E) to 54 (HCoV-NL63) years ago. The split times of HCoV-229E and HCoV-OC43 from camel alphacoronavirus and bovine coronavirus were dated ~268 and ~99 years ago. The split times of HCoV-HKU1 and HCoV-NL63 could not be calculated, as their zoonoticic sources are unknown. To compare the timing of coronavirus emergence to that of another respiratory virus, we recorded the occurrence of influenza pandemics since 1500. Although there is no clear relationship between pandemic occurrence and human population size, the frequency of influenza pandemics seems to intensify starting around 1700, which corresponds with the initial phase of exponential increase of human population and to the emergence of HCoV-229E. The frequency of flu pandemics in the 19th century also suggests that the concurrence of HCoV-OC43 emergence and the Russian flu pandemic may be due to chance.

Filovirus infection is mediated by engagement of the surface-exposed glycoprotein (GP) by its cellular receptor, NPC1 (Niemann-Pick C1). Two loops in the C domain of NPC1 (NPC1-C) bind filovirus GP. Herein, we show that filovirus GP and NPC1-C evolve under mutual selective pressure. Analysis of a large mammalian phylogeny indicated that strong functional/structural constraints limit the NPC1 sequence space available for adaptive change and most sites at the contact interface with GP are under negative selection. These constraints notwithstanding, we detected positive selection at NPC1-C in all mammalian orders, from Primates to Xenarthra. Different codons evolved adaptively in distinct mammals, and most selected sites are located within the two NPC1-C loops that engage GP, or at their anchor points. In Homininae, NPC1-C was a preferential selection target, and the T419I variant possibly represents a human-specific adaptation to filovirus infection. On the other side of the arms-race, GP evolved adaptively during filovirus speciation. One of the selected sites (S142Q) establishes several atom-to-atom contacts with NPC1-C. Additional selected sites are located within epitopes recognized by neutralizing antibodies, including the 14G7 epitope, where sites selected during the recent EBOV epidemic also map. Finally, pairs of co-evolving sites in Marburgviruses and Ebolaviruses were found to involve antigenic determinants. These findings suggest that the host humoral immune response was a major selective pressure during filovirus speciation. The S142Q variant may contribute to determine Ebolavirus host range in the wild. If this were the case, EBOV/BDBV (S142) and SUDV (Q142) may not share the same reservoir(s).

The Mammarenavirus genus includes several pathogenic species of rodent-borne viruses. Old World (OW) mammarenaviruses infect rodents in the Murinae subfamily and are mainly transmitted in Africa and Asia; New World (NW) mammarenaviruses are found in rodents of the Cricetidae subfamily in the Americas. We applied a selection-informed method to estimate that OW and NW mammarenaviruses diverged less than ∼45,000 years ago (ya). By incorporating phylogeographic inference, we show that NW mammarenaviruses emerged in the Latin America-Caribbean region ∼41,400–3,300 ya, whereas OW mammarenaviruses originated ∼23,100–1,880 ya, most likely in Southern Africa. Cophylogenetic analysis indicated that cospeciation did not contribute significantly to mammarenavirus–host associations. Finally, we show that extremely strong selective pressure on the viral polymerase accompanied the speciation of NW viruses. These data suggest that the evolutionary history of mammarenaviruses was not driven by codivergence with their hosts. The viral polymerase should be regarded as a major determinant of mammarenavirus adaptation.

Plasmodium falciparum, the causative agent of the deadliest form of malaria, is a member of the Laverania subgenus, which includes ape-infecting parasites. P. falciparum is thought to have originated in gorillas, although infection is now restricted to humans. Laverania parasites display remarkable host-specificity, which is partially mediated by the interaction between parasite ligands and host receptors. We analyse the evolution of BSG (basigin) and GYPA (glycophorin A) in primates/hominins, as well as of their Plasmodium-encoded ligands, PfRH5 and PfEBA175. We show that, in primates, positive selection targeted two sites in BSG (F27 and H102), both involved in PfRH5 binding. A population genetics-phylogenetics approach detected the strongest selection for the gorilla lineage: one of the positively selected sites (K191) is a major determinant of PfRH5 binding affinity. Analysis of RH5 genes indicated episodic selection on the P. falciparum branch; the positively selected W447 site is known to stabilize the interaction with human basigin. Conversely, we detect no selection in the receptor-binding region of EBA175 in the P. falciparum lineage. Its host receptor, GYPA, shows evidence of positive selection in all hominid lineages; selected codons include glycosylation sites that modulate PfEBA175 binding affinity. Data herein provide an evolutionary explanation for species-specific binding of the PfRH5-BSG ligand-receptor pair and support the hypothesis that positive selection at these genes drove the host shift leading to the emergence of P. falciparum as a human pathogen.

Dietary shifts can drive molecular evolution in mammals and a major transition in human history, the agricultural revolution, favored carbohydrate consumption. We investigated the evolutionary history of nine genes encoding brush-border proteins involved in carbohydrate digestion/absorption. Results indicated widespread adaptive evolution in mammals, with several branches experiencing episodic selection, particularly strong in bats. Many positively selected sites map to functional protein regions (e.g., within glucosidase catalytic crevices), with parallel evolution at SI (sucrase-isomaltase) and MGAM (maltase-glucoamylase). In human populations, five genes were targeted by positive selection acting on noncoding variants within regulatory elements. Analysis of ancient DNA samples indicated that most derived alleles were already present in the Paleolithic. Positively selected variants at SLC2A5 (fructose transporter) were an exception and possibly spread following the domestication of specific fruit crops. We conclude that agriculture determined no major selective event at carbohydrate metabolism genes in humans, with implications for susceptibility to metabolic disorders.

OAS (2'-5'-oligoadenylate synthases) proteins and cyclic GMP-AMP synthase (cGAS, gene symbol: MB21D1) patrol the cytoplasm for the presence of foreign nucleic acids. Upon binding to double-stranded RNA or double-stranded DNA, OAS proteins and cGAS produce nucleotide second messengers to activate RNase L and STING (stimulator of interferon genes, gene symbol: TMEM173), respectively; this leads to the initiation of antiviral responses. We analyzed the evolutionary history of the MB21D1-TMEM173 and OAS-RNASEL axes in primates and bats and found evidence of widespread positive selection in both orders. In TMEM173, residue 230, a major determinant of response to natural ligands and to mimetic drugs (e.g., DMXAA), was positively selected in Primates and Chiroptera. In both orders, selection also targeted an α-helix/loop element in RNase L that modulates the enzyme preference for single-stranded RNA versus stem loops. Analysis of positively selected sites in OAS1, OAS2, and MB21D1 revealed parallel evolution, with the corresponding residues being selected in different genes. As this cannot result from gene conversion, these data suggest that selective pressure acting on OAS and MB21D1 genes is related to nucleic acid recognition and to the specific mechanism of enzyme activation, which requires a conformational change. Finally, a population genetics-phylogenetics analysis in humans, chimpanzees, and gorillas detected several positively selected sites in most genes. Data herein shed light into species-specific differences in infection susceptibility and in response to synthetic compounds, with relevance for the design of synthetic compounds as vaccine adjuvants.

In addition to high intelligence, humans evolved specialized social-cognitive skills, which are specifically affected in children with autism spectrum disorder (ASD). Genes affected in ASD represent suitable candidates to study the evolution of human social cognition. We performed an evolutionary analysis on 68 genes associated to neurodevelopmental disorders; our data indicate that genetic diversity was shaped by distinct selective forces, including natural selection and introgression from archaic hominins. We discuss the possibility that segregation distortion during spermatogenesis accounts for a subset of ASD mutations. Finally, we detected modern-human-specific alleles in DYRK1A and TCF4. These variants are located within regions that display chromatin features typical of transcriptional enhancers in several brain areas, strongly suggesting a regulatory role. These SNPs thus represent candidates for association with neurodevelopmental disorders, and await experimental validation in future studies.

The albuminoid gene family comprises vitamin D-binding protein (GC), alpha-fetoprotein (AFP), afamin (AFM), and albumin (ALB). Albumin is the most abundant human serum protein, and, as the other family members, acts as a transporter of endogenous and exogenous substances including thyroxine, fatty acids, and drugs. Instead, the major cargo of GC is 25-hydroxyvitamin D. We performed an evolutionary study of albuminoid genes and we show that ALB evolved adaptively in mammals. Most positively selected sites are located within albumin-binding sites for fatty acids and thyroxine, as well as at the contact surface with neonatal Fc receptor. Positive selection was also detected for residues forming the prostaglandin-binding pocket. Adaptation to hibernation/torpor might explain the signatures of episodic positive selection we detected for few mammalian lineages. Application of a population genetics–phylogenetics approach showed that purifying selection represented a major force acting on albuminoid genes in both humans and chimpanzees, with the strongest constraint observed for human GC. Population genetic analysis revealed that GC was also the target of locally exerted selective pressure, which drove the frequency increase of different haplotypes in distinct human populations. A search for known variants that modulate GC and 25-hydroxyvitamin D concentrations revealed linkage disequilibrium with positively selected variants, although European and Asian major GC haplotypes carry alleles with reported opposite effect on GC concentration. Data herein indicate that albumin, an extremely abundant housekeeping protein, was the target of pervasive and episodic selection in mammals, whereas GC represented a selection target during the recent evolution of human populations.

Humans possess a communication system based on spoken and written language. Other animals can learn vocalization by imitation, but this is not equivalent to human language. Many genes were described to be implicated in language impairment (LI) and developmental dyslexia (DD), but their evolutionary history has not been thoroughly analyzed. Herein we analyzed the evolution of ten genes involved in DD and LI. Results show that the evolutionary history of LI genes for mammals and aves was comparable in vocal-learner species and non-learners. For the human lineage, several sites showing evidence of positive selection were identified in KIAA0319 and were already present in Neanderthals and Denisovans, suggesting that any phenotypic change they entailed was shared with archaic hominins. Conversely, in FOXP2, ROBO1, ROBO2, and CNTNAP2 non-coding changes rose to high frequency after the separation from archaic hominins. These variants are promising candidates for association studies in LI and DD.

The complement system is an innate immunity effector mechanism; its action is antagonized by a wide array of pathogens and complement evasion determines the virulence of several infections. We investigated the evolutionary history of the complement system and of bacterial-encoded complement-interacting proteins. Complement components targeted by several pathogens evolved under strong selective pressure in primates, with selection acting on residues at the contact interface with microbial/viral proteins. Positively selected sites in CFH and C4BPA account for the human specificity of gonococcal infection. Bacterial interactors, evolved adaptively as well, with selected sites located at interaction surfaces with primate complement proteins. These results epitomize the expectation under a genetic conflict scenario whereby the host's and the pathogen's genes evolve within binding avoidance-binding seeking dynamics. In silico mutagenesis and protein-protein docking analyses supported this by showing that positively selected sites, both in the host's and in the pathogen's interacting partner, modulate binding.

Middle East respiratory syndrome-related coronavirus (MERS-CoV) spreads to humans via zoonotic transmission from camels. MERS-CoV belongs to lineage C of betacoronaviruses (betaCoVs), which also includes viruses isolated from bats and hedgehogs. A large portion of the betaCoV genome consists of two open reading frames (ORF1a and ORF1b) that are translated into polyproteins. These are cleaved by viral proteases to generate 16 nonstructural proteins (nsp1 to nsp16) which compose the viral replication-transcription complex. We investigated the evolution of ORF1a and ORF1b in lineage C betaCoVs. Results indicated widespread positive selection, acting mostly on ORF1a. The proportion of positively selected sites in ORF1a was much higher than that previously reported for the surface-exposed spike protein. Selected sites were unevenly distributed, with nsp3 representing the preferential target. Several pairs of coevolving sites were also detected, possibly indicating epistatic interactions; most of these were located in nsp3. Adaptive evolution at nsp3 is ongoing in MERS-CoV strains, and two selected sites (G720 and R911) were detected in the protease domain. While position 720 is variable in camel-derived viruses, suggesting that the selective event does not represent a specific adaptation to humans, the R911C substitution was observed only in human-derived MERS-CoV isolates, including the viral strain responsible for the recent South Korean outbreak. It will be extremely important to assess whether these changes affect host range or other viral phenotypes. More generally, data herein indicate that CoV nsp3 represents a major selection target and that nsp3 sequencing should be envisaged in monitoring programs and field surveys. IMPORTANCE Both severe acute respiratory syndrome coronavirus (SARS-CoV) and MERS-CoV originated in bats and spread to humans via an intermediate host. This clearly highlights the potential for coronavirus host shifting and the relevance of understanding the molecular events underlying the adaptation to new host species. We investigated the evolution of ORF1a and ORF1b in lineage C betaCoVs and in 87 sequenced MERS-CoV isolates. Results indicated widespread positive selection, stronger in ORF1a than in ORF1b. Several selected sites were found to be located in functionally relevant protein regions, and some of them corresponded to functional mutations in other coronaviruses. The proportion of selected sites we identified in ORF1a is much higher than that for the surface-exposed spike protein. This observation suggests that adaptive evolution in ORF1a might contribute to host shifts or immune evasion. Data herein also indicate that genetic diversity at nonstructural proteins should be taken into account when antiviral compounds are developed.

Middle East respiratory syndrome coronavirus (MERS-CoV) originated in bats and spread to humans via zoonotic transmission from camels. We analyzed the evolution of the spike (S) gene in betacoronaviruses (betaCoVs) isolated from different mammals, in bat coronavirus populations, as well as in MERS-CoV strains from the current outbreak. Results indicated several positively selected sites located in the region comprising the two heptad repeats (HR1 and HR2) and their linker. Two sites (R652 and V1060) were positively selected in the betaCoVs phylogeny and correspond to mutations associated with expanded host range in other coronaviruses. During the most recent evolution of MERS-CoV, adaptive mutations in the HR1 (Q/R/H1020) arose in camels or in a previous host and spread to humans. We determined that different residues at position 1020 establish distinct inter- and intra-helical interactions and affect the stability of the six-helix bundle formed by the HRs. A similar effect on stability was observed for a nearby mutation (T1015N) that increases MERS-CoV infection efficiency in vitro. Data herein indicate that the heptad repeat region was a major target of adaptive evolution in MERS-CoV-related viruses; these results are relevant for the design of fusion inhibitor peptides with antiviral function.

A-to-I RNA editing operated by ADAR enzymes is extremely common in mammals. Several editing events in coding regions have pivotal physiological roles and affect protein sequence (recoding events) or function. We analyzed the evolutionary history of the 3 ADAR family genes and of their coding targets. Evolutionary analysis indicated that ADAR evolved adaptively in primates, with the strongest selection in the unique N-terminal domain of the interferon-inducible isoform. Positively selected residues in the human lineage were also detected in the ADAR deaminase domain and in the RNA binding domains of ADARB1 and ADARB2. During the recent history of human populations distinct variants in the 3 genes increased in frequency as a result of local selective pressures. Most selected variants are located within regulatory regions and some are in linkage disequilibrium with eQTLs in monocytes. Finally, analysis of conservation scores of coding editing sites indicated that editing events are counter-selected within regions that are poorly tolerant to change. Nevertheless, a minority of recoding events occurs at highly conserved positions and possibly represents the functional fraction. These events are enriched in pathways related to HIV-1 infection and to epidermis/hair development. Thus, both ADAR genes and their targets evolved under variable selective regimes, including purifying and positive selection. Pressures related to immune response likely represented major drivers of evolution for ADAR genes. As for their coding targets, we suggest that most editing events are slightly deleterious, although a minority may be beneficial and contribute to antiviral response and skin homeostasis.

The non-coding portion of human genome is punctuated by a large number of multispecies conserved sequence (MCS) elements with largely unknown function. We demonstrate that MCSs are unevenly distributed in human introns with the majority of relatively short introns (< 9 kb long) displaying no or a few MCSs and that MCS density reaching up to 10% of total size in longer introns. After correction for intron length, MCSs were found to be enriched within genes involved in development and transcription, whereas depleted in immune response loci. Moreover, many central nervous system tissues show a preferential expression of MCS-rich genes and MCS enrichment significantly correlates with gene functional complexity in terms of distinct protein domains. Analysis of human-mouse orthologous pairs indicated a significant association between intronic MCS density and conservation of protein sequence, promoter regions and untranslated sequences. Moreover, MCS density correlates with the predicted occurrence of human-mouse conserved alternative splicing events. These observations suggest that evolution acts on human genes as integrated units of coding and regulatory capacity and that functional complexity might represent a major source of negative selection on non-coding sequences. To substantiate our result, we also searched previously experimentally identified intronic regulatory elements and indicate that about half of these sequences map to an MCS; in particular, support to the notion whereby mutations in MCSs can result in human genetic diseases is provided, because three previously identified intronic pathological variations were found to occur within MCSs, and human disease and cancer genes were found significantly enriched in MCSs.

Human pre-mRNAs contain a definite number of exons and several pseudoexons which are located within intronic regions. We applied a computational approach to address the question of how pseudoexons are neglected in favor of exons and to possibly identify sequence elements preventing pseudoexon splicing. A search for possible splicing silencers was carried out on a pseudoexon selection that resembled exons in terms of splice site strength and exon splicing enhancer (ESE) representation; three motifs were retrieved through hexamer composition comparisons. One of these functions as a powerful silencer in transfection-based splicing assays and matches a previously identified silencer sequence with hnRNP H binding ability. The other two motifs are novel and failed to induce skipping of a constitutive exon, indicating that they might act as weak repressors or in synergy with other unidentified elements. All three motifs are enriched in pseudoexons compared with intronic regions and display higher frequencies in intronless gene-coding sequences compared with exons. We consider that a subpopulation of pseudoexons might rely on negative regulators for splicing repression; this hypothesis, if experimentally verified, might improve our understanding of exonic splicing regulatory sequences and provide the identification of a novel mutation target for human genetic diseases.

AIM2-like receptors (ALRs) are a family of nucleic acid sensors essential for innate immune responses against viruses and bacteria. We performed an evolutionary analysis of ALR genes (MNDA, PYHIN1, IFI16, and AIM2) by analyzing inter- and intraspecies diversity. Maximum-likelihood analyses indicated that IFI16 and AIM2 evolved adaptively in primates, with branch-specific selection at the catarrhini lineage for IFI16. Application of a population genetics-phylogenetics approach also allowed identification of positive selection events in the human lineage. Positive selection in primates targeted sites located at the DNA-binding interface in both IFI16 and AIM2. In IFI16, several sites positively selected in primates and in the human lineage were located in the PYD domain, which is involved in protein-protein interaction and is bound by a human cytomegalovirus immune evasion protein. Finally, positive selection was found to target nuclear localization signals in IFI16 and the spacer region separating the two HIN domains. Population genetic analysis in humans revealed that an IFI16 genic region has been a target of long-standing balancing selection, possibly acting on two nonsynonymous polymorphisms located in the spacer region. Data herein indicate that ALRs have been repeatedly targeted by natural selection. The balancing selection region in IFI16 carries a variant with opposite risk effect for distinct autoimmune diseases, suggesting antagonistic pleiotropy. We propose that the underlying scenario is the result of an ancestral and still ongoing host-pathogen arms race and that the maintenance of susceptibility alleles for autoimmune diseases at IFI16 represents an evolutionary trade-off.

RIG-I-like receptors (RLRs) are nucleic acid sensors that activate antiviral innate immune response. These molecules recognize diverse non-self RNA substrates and are antagonized by several viral inhibitors. We performed an evolutionary analysis of RLR genes (RIG-I, MDA5, and LGP2) in mammals. Results indicated that purifying selection had a dominant role in driving the evolution of RLRs. However, application of maximum-likelihood analyses identified several positions that evolved adaptively. Positively selected sites are located in all domains of MDA5 and RIG-I, whereas in LGP2 they are confined to the helicase domain. In both MDA5 and RIG-I, the linkers separating the caspase activation and recruitment domain and the helicase domain represented preferential targets of positive selection. Independent selective events in RIG-I and LGP2 targeted the corresponding site (Asp421 and Asp179, respectively) within a protruding α-helix that grips the V-shaped structure formed by the pincer. Most of the positively selected sites in MDA5 are in regions unique to this RLR, including a characteristic insertion within the helicase domain. Additional selected sites are located at the contact interface between MDA5 monomers, in spatial proximity to a positively selected human polymorphism (Arg843His) and immediately external to the parainfluenza virus 5 V protein binding region. Structural analyses suggested that the positively selected His834 residue is involved in parainfluenza virus 5 V protein binding. Data herein suggest that RLRs have been engaged in host-virus genetic conflict leading to diversifying selection and indicate parallel evolution at the same site in RIG-I and LGP2, a position likely to be of central importance in antiviral responses. © 2013 Elsevier Ltd.

The antigenic repertoire presented by MHC molecules is generated by the antigen processing and presentation (APP) pathway. We analyzed the evolutionary history of 45 genes involved in APP at the inter- and intra-species level. Results showed that 11 genes evolved adaptively in mammals. Several positively selected sites involve positions of fundamental importance to the protein function (e.g. the TAP1 peptide-binding domains, the sugar binding interface of langerin, and the CD1D trafficking signal region). In CYBB, all selected sites cluster in two loops protruding into the endosomal lumen; analysis of missense mutations responsible for chronic granulomatous disease (CGD) showed the action of different selective forces on the very same gene region, as most CGD substitutions involve aminoacid positions that are conserved in all mammals. As for ERAP2, different computational methods indicated that positive selection has driven the recurrent appearance of protein-destabilizing variants during mammalian evolution. Application of a population-genetics phylogenetics approach showed that purifying selection represented a major force acting on some APP components (e.g. immunoproteasome subunits and chaperones) and allowed identification of positive selection events in the human lineage. We also investigated the evolutionary history of APP genes in human populations by developing a new approach that uses several different tests to identify the selection target, and that integrates low-coverage whole-genome sequencing data with Sanger sequencing. This analysis revealed that 9 APP genes underwent local adaptation in human populations. Most positive selection targets are located within noncoding regions with regulatory function in myeloid cells or act as expression quantitative trait loci. Conversely, balancing selection targeted nonsynonymous variants in TAP1 and CD207 (langerin). Finally, we suggest that selected variants in PSMB10 and CD207 contribute to human phenotypes. Thus, we used evolutionary information to generate experimentally-testable hypotheses and to provide a list of sites to prioritize in follow-up analyses., Author Summary Antigen-presenting cells digest intracellular and extracellular proteins and display the resulting antigenic repertoire on cell surface molecules for recognition by T cells. This process initiates cell-mediated immune responses and is essential to detect infections. The antigenic repertoire is generated by the antigen processing and presentation pathway. Because several pathogens evade immune recognition by hampering this process, genes involved in antigen processing and presentation may represent common natural selection targets. Thus, we analyzed the evolutionary history of these genes during mammalian evolution and in the more recent history of human populations. Evolutionary analyses in mammals indicated that positive selection targeted a very high proportion of genes (24%), and revealed that many selected sites affect positions of fundamental importance to the protein function. In humans, we found different signatures of natural selection acting both on regions that are expected to regulate gene expression levels or timing and on coding variants; two human selected polymorphisms may modulate the susceptibility to Crohn's disease and to HIV-1 infection. Therefore, we provide a comprehensive evolutionary analysis of antigen processing and we show that evolutionary studies can provide useful information concerning the location and nature of functional variants, ultimately helping to clarify phenotypic differences between and within species.

Activation of the contact system leads to the cleavage of kini nogen by plasma kallikrein resulting in kinin release and in the initiation of the intrinsic pathway of coagulation. Proteolysis of kininogen also generates antimicrobial peptides (AMPs) and can be induced by diverse pathogens. Thus, the contact system is regarded as a branch of innate immunity. We performed an evolutionary analysis of contact system genes by analyzing both inter- and intraspecies diversity. Results indicated that mammalian kininogen genes evolved adaptively. Positively selected sites are located in all protein domains with the exclusion of the bradykinin region and also involve AMP sequences (including the highly effective NAT26 peptide); positively selected sites also occur at alternative cleavage sites for neutrophil-released kinins. Population genetic analysis in humans indicated that a region of the kininogen gene (KNG1) has been a target of long-standing multiallelic balancing selection and that the coalescence time of the haplotype phylogeny dates back to the split between the humans and chimpanzees. No selection signature was detected in the Pan troglodytes KNG1 gene or in human genes encoding other components of the contact system. The selection targets in human KNG1 might be accounted for by variants with transcriptional regulatory activity. Results herein indicate a continuum in selective pressure acting on different timescales and targeting KNG1. This is in line with evidences suggesting a central role for kininogen in modulating of immune response and with its being a target of an extremely diverse array of pathogen species.

Cagliani, Rachele et al., The THBS4 gene encodes a glycoprotein involved in inflammatory responses and synaptogenesis. THBS4 is expressed at higher levels in the brain of humans compared with nonhuman primates, and the protein accumulates in β-amyloid plaques. We analyzed THBS4 genetic variability in humans and show that two haplotypes (hap1 and hap2) are maintained by balancing selection and modulate THBS4 expression in lymphocytes. Indeed, the balancing selection region covers a predicted transcriptional enhancer. In humans, but not in macaques and chimpanzees, THBS4 brain expression increases with age, and variants in the balancing selection region interact with sex in influencing THBS4 expression (pinteraction = 0.038), with hap1 homozygous females showing lowest expression. In Alzheimer disease (AD) patients, significant interactions between sex and THBS4 genotype were detected for peripheral gray matter (pinteraction = 0.014) and total gray matter (pinteraction = 0.012) volumes. Similarly to the gene expression results, the interaction is mainly mediated by hap1 homozygous AD females, who show reduced volumes. Thus, the balancing selection target in THBS4 is likely represented by one or more variants that regulate tissue-specific and sex-specific gene expression. The selection signature associated with THBS4 might not be related to AD pathogenesis, but rather to inflammatory responses. © 2013 Wiley Periodicals, Inc.

Molecules involved in leukocyte trafficking have a central role in the development of inflammatory and immune responses. We performed F(ST) analysis of the selectin cluster, as well as of SELPLG, ICAM1 and VCAM1. Peaks of significantly high population genetic differentiation were restricted to two regions in SELP and one in SELPLG. Resequencing data indicated that the region covering SELP exons 11-13 displays high nucleotide diversity in Africans and Europeans (CEU), and a high level of within-species diversity compared with inter-specific divergence. Analysis of inferred haplotypes revealed a complex phylogeny with two deeply separated clades that coalesce at ~3.5 million years (MY) plus a minor clade with a TMRCA (time to the most recent common ancestor) of ~2.2 MY. A splicing assay indicated no haplotype-specific effect on SELP exon 14 inclusion. These data are consistent with a model of multiallelic balancing selection; single-nucleotide polymorphism analysis indicated that the Val640Leu variant represents a likely selection target. In populations of Asian ancestry a distinct haplotype, possibly carrying regulatory variants, has been driven to high frequency by positive selection. No deviation from neutrality was observed for the SELPLG region. Resequencing of SELP in chimpanzees revealed a haplotype phylogeny with extremely deep basal branches, suggesting either long-standing balancing selection or ancestral population structure. Thus, SELP has experienced a complex selective history, possibly as a result of local adaptation. Variants in the gene have been associated with autoimmune and cardiovascular diseases. Association studies would benefit from both taking the complex SELP haplotype structure into account and from analysis of possible regulatory variants in the gene.

The human ZC3HAV1 gene encodes an antiviral protein. The longest splicing isoform of ZC3HAV1 contains a C-terminal PARP-like domain, which has evolved under positive selection in primates. We analyzed the evolutionary history of this same domain in humans and in Pan troglodytes. We identified two variants that segregate in both humans and chimpanzees; one of them (rs3735007) does not occur at a hypermutable site and accounts for a nonsynonymous substitution (Thr851Ile). The probability that the two trans-specific polymorphisms have occurred independently in the two lineages was estimated to be low (P = 0.0054), suggesting that at least one of them has arisen before speciation and has been maintained by selection. Population genetic analyses in humans indicated that the region surrounding the shared variants displays strong evidences of long-standing balancing selection. Selection signatures were also observed in a chimpanzee population sample. Inspection of 1000 Genomes data confirmed these findings but indicated that search for selection signatures using low-coverage whole-genome data may need masking of repetitive sequences. A case–control study of more than 1,000 individuals from mainland Italy indicated that the Thr851Ile SNP is significantly associated with susceptibility to multiple sclerosis (MS) (odds ratio [OR] = 1.47, 95% confidence intervals [CI]: 1.08–1.99, P = 0.011). This finding was confirmed in a larger sample of 4,416 Sardinians cases/controls (OR = 1.18, 95% CI: 1.037–1.344, P = 0.011), but not in a population from Belgium. We provide one of the first instances of human/chimpanzee trans-specific coding variant located outside the major histocompatibility complex region. The selective pressure is likely to be virus driven; in modern populations, this variant associates with susceptibility to MS, possibly via the interaction with environmental factors.

The human TRIM5 genes encodes a retroviral restriction factor (TRIM5α). Evolutionary analyses of this gene in mammals have revealed a complex and multifaceted scenario, suggesting that TRIM5 has been the target of exceptionally strong selective pressures, possibly exerted by recurrent waves of retroviral infections. TRIM5 displays inter-individual expression variability in humans and high levels of TRIM5 mRNA have been associated with a reduced risk of HIV-1 infection. We resequenced TRIM5 in chimpanzees and identified two polymorphisms in intron 1 that are shared with humans. Analysis of the gene region encompassing the two trans-specific variants in human populations identified exceptional nucleotide diversity levels and an excess of polymorphism compared to fixed divergence. Most tests rejected the null hypothesis of neutral evolution for this region and haplotype analysis revealed the presence of two deeply separated clades. Calculation of the time to the most recent common ancestor (TMRCA) for TRIM5 haplotypes yielded estimates ranging between 4 and 7 million years. Overall, these data indicate that long-term balancing selection, an extremely rare process outside MHC genes, has maintained trans-specific polymorphisms in the first intron of TRIM5. Bioinformatic analyses indicated that variants in intron 1 may affect transcription factor-binding sites and, therefore, TRIM5 transcriptional activity. Data herein confirm an extremely complex evolutionary history of TRIM5 genes in primates and open the possibility that regulatory variants in the gene modulate the susceptibility to HIV-1.

Protozoa exert a strong selective pressure in humans. The selection signatures left by these pathogens can be exploited to identify genetic modulators of infection susceptibility. We show that protozoa diversity in different geographic locations is a good measure of protozoa-driven selective pressure; protozoa diversity captured selection signatures at known malaria resistance loci and identified several selected single nucleotide polymorphisms in immune and hemolytic anemia genes. A genome-wide search enabled us to identify 5180 variants mapping to 1145 genes that are subjected to protozoa-driven selective pressure. We provide a genome-wide estimate of protozoa-driven selective pressure and identify candidate susceptibility genes for protozoa-borne diseases.

BackgroundThe vasopressin receptor type 1b (AVPR1B) is mainly expressed by pituitary corticotropes and it mediates the stimulatory effects of AVP on ACTH release; commonAVPR1Bhaplotypes have been involved in mood and anxiety disorders in humans, while rodents lacking a functional receptor gene display behavioral defects and altered stress responses.ResultsHere we have analyzed the two exons of the gene and the data we present suggest thatAVPR1Bhas been subjected to natural selection in humans. In particular, analysis of exon 2 strongly suggests the action of balancing selection in African populations and Europeans: the region displays high nucleotide diversity, an excess of intermediate-frequency alleles, a higher level of within-species diversity compared to interspecific divergence and a genealogy with common haplotypes separated by deep branches. This relatively unambiguous situation coexists with unusual features across exon 1, raising the possibility that a nonsynonymous variant (Gly191Arg) in this region has been subjected to directional selection.ConclusionAlthough the underlying selective pressure(s) remains to be identified, we consider this to be among the first documented examples of a gene involved in mood disorders and subjected to natural selection in humans; this observation might add support to the long-debated idea that depression/low mood might have played an adaptive role during human evolution.

Familial Mediterranean Fever (FMF) is a recessively inherited systemic autoinflammatory disease caused by mutations in the MEFV gene. The frequency of different disease alleles is extremely high in multiple populations from the Mediterranean region, suggesting heterozygote advantage. Here, we characterize the sequence variation and haplotype structure of the MEFV 3' gene region (from exon 5 to the 3' UTR) in seven human populations. In non-African populations, we observed high levels of nucleotide variation, an excess of intermediate-frequency alleles, reduced population differentiation and a genealogy with two common haplotypes separated by deep branches. These features are suggestive of balancing selection having acted on this region to maintain one or more selected alleles. In line with this finding, an excess of heterozygotes was observed in Europeans and Asians, suggesting an overdominance regime. Our data, together with the earlier demonstration that the MEFV exon 10 has been subjected to episodic positive selection over primate evolution, provide evidence for an adaptive role of nucleotide variation in this gene region. Our data suggest that further studies aimed at clarifying the role of MEFV variants might benefit from the integration of molecular evolutionary and functional analyses.

Many human genes have adapted to the constant threat of exposure to infectious agents; according to the “hygiene hypothesis,” lack of exposure to parasites in modern settings results in immune imbalances, augmenting susceptibility to the development of autoimmune and allergic conditions. Here, by estimating the number of pathogen species/genera in a specific geographic location (pathogen richness) for 52 human populations and analyzing 91 interleukin (IL)/IL receptor genes (IL genes), we show that helminths have been a major selective force on a subset of these genes. A population genetics analysis revealed that five IL genes, including IL7R and IL18RAP, have been a target of balancing selection, a selection process that maintains genetic variability within a population. Previous identification of polymorphisms in some of these loci, and their association with autoimmune conditions, prompted us to investigate the relationship between adaptation and disease. By searching for variants in IL genes identified in genome-wide association studies, we verified that six risk alleles for inflammatory bowel (IBD) or celiac disease are significantly correlated with micropathogen richness. These data support the hygiene hypothesis for IBD and provide a large set of putative targets for susceptibility to helminth infections.

Analysis of the human beta defensin 1 promoter region in six human populations reveals a signature of balancing selection., Background Defensins, small endogenous peptides with antimicrobial activity, are pivotal components of the innate immune response. A large cluster of defensin genes is located on human chromosome 8p; among them the beta defensin 1 (DEFB1) promoterhas been extensively studied since discovery that specific polymorphisms and haplotypes associate with asthma and atopy, susceptibility to severe sepsis, as well as HIV and Candida infection predisposition. Results Here, we characterize the sequence variation and haplotype structure of the DEFB1 promoter region in six human populations. In all of them, we observed high levels of nucleotide variation, an excess of intermediate-frequency alleles, reduced population differentiation and a genealogy with common haplotypes separated by deep branches. Indeed, a significant departure from the expectation of evolutionary neutrality was observed in all populations and the possibility that this is due to demographic history alone was ruled out. Also, we verified that the selection signature is restricted to the promoter region and not due to a linked balanced polymorphism. A phylogeny-based estimation indicated that the two major haplotype clades separated around 4.5 million years ago, approximately the time when the human and chimpanzee lineages split. Conclusion Altogether, these features represent strong molecular signatures of long-term balancing selection, a process that is thought to be extremely rare outside major histocompatibility complex genes. Our data indicate that the DEFB1 promoter region carries functional variants and support previous hypotheses whereby alleles predisposing to atopic disorders are widespread in modern societies because they conferred resistance to pathogens in ancient settings.

Background Mammalian genomes consist of regions differing in GC content, referred to as isochores or GC-content domains. The scientific debate is still open as to whether such compositional heterogeneity is a selected or neutral trait. Results Here we analyze SNP allele frequencies, retrotransposon insertion polymorphisms (RIPs), as well as fixed substitutions accumulated in the human lineage since its divergence from chimpanzee to indicate that biased gene conversion (BGC) has been playing a role in within-genome GC content variation. Yet, a distinct contribution to GC content evolution is accounted for by a selective process. Accordingly, we searched for independent evidences that GC content distribution does not conform to neutral expectations. Indeed, after correcting for possible biases, we show that intron GC content and size display isochore-specific correlations. Conclusion We consider that the more parsimonious explanation for our results is that GC content is subjected to the action of both weak selection and BGC in the human genome with features such as nucleosome positioning or chromatin conformation possibly representing the final target of selective processes. This view might reconcile previous contrasting findings and add some theoretical background to recent evidences suggesting that GC content domains display different behaviors with respect to highly regulated biological processes such as developmentally-stage related gene expression and programmed replication timing during neural stem cell differentiation.

Different and contrasting models have been proposed to explain intron size evolution in mammals. Here, we demonstrate that intron and intergenic size per se has no adaptive role in gene expression regulation but reflects the need to preserve conserved intronic elements. Although the amount of non-coding functional elements explains the within-genome size variation of intergenic spacers, we show that an additional, additive pressure has been acting on highly expressed introns to reduce the cost of their transcription.

Constitutive and alternative splicing events are regulated, in higher eukaryotes, by the action of multiple weak cis-acting elements and trans-acting factors. In particular, several evidences have suggested that silencers might have a fundamental role in preventing pseudoexon inclusion in mature transcripts and in defining constitutive exons by suppressing nearby decoy splice sites. Moreover, silencer elements allow the recruitment of regulatory factors to alternatively spliced exons, therefore participating in the modulation of alternative splicing pathways. Here we focus on splicing repression mechanisms in mammals, with particular concern to both exonic and intronic silencer elements, secondary structure formation and role in human genetic disease.Recently, in addition to the availability of a growing number of sequence elements deriving from the analysis of individual regulated exons, approaches have been developed that allowed the systematic identification of splicing silencers. These methods and are briefly described, as well as the motifs they retrieved, and summary of silenced exons is provided.

The human DMD gene is the largest known to date, spanning > 2000 kb on the X chromosome. The gene size is mainly accounted for by huge intronic regions. We sequenced 190 kb of Fugu rubripes(pufferfish) genomic DNA corresponding to the complete dystrophin gene (FrDMD) and provide the first report of gene structure and sequence comparison among dystrophin genomic sequences from different vertebrate organisms. Almost all intron positions and phases are conserved between FrDMD and its mammalian counterparts, and the predicted protein product of the Fugu gene displays 55% identity and 71% similarity to human dystrophin. In analogy to the human gene, FrDMD presents several-fold longer than average intronic regions. Analysis of intron sequences of the human and murine genes revealed that they are extremely conserved in size and that a similar fraction of total intron length is represented by repetitive elements; moreover, our data indicate that intron expansion through repeat accumulation in the two orthologs is the result of independent insertional events. The hypothesis that intron length might be functionally relevant to the DMD gene regulation is proposed and substantiated by the finding that dystrophin intron gigantism is common to the three vertebrate genes.[Supplemental material is available online at www.genome.org.]

Background More than 2 billion individuals worldwide suffer from helminth infections. The highest parasite burdens occur in children and helminth infection during pregnancy is a risk factor for preterm delivery and reduced birth weight. Therefore, helminth infections can be regarded as a strong selective pressure. Results Here we propose that candidate susceptibility genes for parasitic worm infections can be identified by searching for SNPs that display a strong correlation with the diversity of helminth species/genera transmitted in different geographic areas. By a genome-wide search we identified 3478 variants that correlate with helminth diversity. These SNPs map to 810 distinct human genes including loci involved in regulatory T cell function and in macrophage activation, as well as leukocyte integrins and co-inhibitory molecules. Analysis of functional relationships among these genes identified complex interaction networks centred around Th2 cytokines. Finally, several genes carrying candidate targets for helminth-driven selective pressure also harbour susceptibility alleles for asthma/allergy or are involved in airway hyper-responsiveness, therefore expanding the known parallelism between these conditions and parasitic infections. Conclusions Our data provide a landscape of human genes that modulate susceptibility to helminths and indicate parasitic worms as one of the major selective forces in humans.