SARS-CoV-2 ORF3c impairs mitochondrial respiratory metabolism, oxidative stress, and autophagic flux.

Coronaviruses encode a variable number of accessory proteins that are involved in host-virus interaction, suppression of immune responses, or immune evasion. SARS-CoV-2 encodes at least twelve accessory proteins, whose roles during infection have been studied. Nevertheless, the role of the ORF3c accessory protein, an alternative open reading frame of ORF3a, has remained elusive. Herein, we show that the ORF3c protein has a mitochondrial localization and alters mitochondrial metabolism, inducing a shift from glucose to fatty acids oxidation and enhanced oxidative phosphorylation. These effects result in increased ROS production and block of the autophagic flux. In particular, ORF3c affects lysosomal acidification, blocking the normal autophagic degradation process and leading to autolysosome accumulation. We also observed different effect on autophagy for SARS-CoV-2 and batCoV RaTG13 ORF3c proteins; the 36R and 40K sites are necessary and sufficient to determine these effects.

Autoinflammation in Syndromic Hidradenitis Suppurativa: The Role of AIM2.

BACKGROUND: AIM2 is a key cytoplasmatic pathogen-sensor that detects foreign DNA from viruses and bacteria; it can also recognize damaged or anomalous presence of DNA, promoting inflammasome assembly and activation with the secretion of IL-1ß, thus sustaining a chronic inflammatory state, potentially leading to the onset of autoinflammatory skin diseases. Given the implication of the IL-1ß pathway in the pathogenesis of syndromic hidradenitis suppurativa (HS), an autoinflammatory immune-mediated skin condition, the potential involvement of AIM2 was investigated. METHODS: Sequencing of the whole coding region of the AIM2 gene, comprising 5'- and 3' UTR and a region upstream of the first exon of ~800 bp was performed in twelve syndromic HS patients. RESULTS: Six out of twelve syndromic HS patients carried a heterozygous variant c.-208 A ≥ C (rs41264459), located on the promoter region of the AIM2 gene, with a minor allele frequency of 0.25, which is much higher than that reported in 1000 G and GnomAD (0.075 and 0.094, respectively). The same variant was found at a lower allelic frequency in sporadic HS and isolated pyoderma gangrenosum (PG) (0.125 and 0.065, respectively). CONCLUSION: Our data suggest that this variant might play a role in susceptibility to develop syndromic forms of HS but not to progress to sporadic HS and PG. Furthermore, epigenetic and/or somatic variations could affect AIM2 expression leading to different, context-dependent responses.

Simplexviruses Successfully Adapt to Their Host by Fine-Tuning Immune Responses.

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.

The substitution spectra of coronavirus genomes.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has triggered an unprecedented international effort to sequence complete viral genomes. We leveraged this wealth of information to characterize the substitution spectrum of SARS-CoV-2 and to compare it with those of other human and animal coronaviruses. We show that, once nucleotide composition is taken into account, human and most animal coronaviruses display a mutation spectrum dominated by C to U and G to U substitutions, a feature that is not shared by other positive-sense RNA viruses. However, the proportions of C to U and G to U substitutions tend to decrease as divergence increases, suggesting that, whatever their origin, a proportion of these changes is subsequently eliminated by purifying selection. Analysis of the sequence context of C to U substitutions showed little evidence of apolipoprotein B mRNA editing catalytic polypeptide-like (APOBEC)-mediated editing and such contexts were similar for SARS-CoV-2 and Middle East respiratory syndrome coronavirus sampled from different hosts, despite different repertoires of APOBEC3 proteins in distinct species. Conversely, we found evidence that C to U and G to U changes affect CpG dinucleotides at a frequency higher than expected. Whereas this suggests ongoing selective reduction of CpGs, this effect alone cannot account for the substitution spectra. Finally, we show that, during the first months of SARS-CoV-2 pandemic spread, the frequency of both G to U and C to U substitutions increased. Our data suggest that the substitution spectrum of SARS-CoV-2 is determined by an interplay of factors, including intrinsic biases of the replication process, avoidance of CpG dinucleotides and other constraints exerted by the new host.

Alternation between taxonomically divergent hosts is not the major determinant of flavivirus evolution.

Flaviviruses display diverse epidemiological and ecological features. Tick-borne and mosquito-borne flaviviruses (TBFV and MBFV, respectively) are important human pathogens that alternate replication in invertebrate vectors and vertebrate hosts. The Flavivirus genus also includes insect-specific viruses (ISFVs) and viruses with unknown invertebrate hosts. It is generally accepted that viruses that alternate between taxonomically different hosts evolve slowly and that the evolution of MBFVs and TBFVs is dominated by strong constraints, with limited episodes of positive selection. We exploited the availability of flavivirus genomes to test these hypotheses and to compare their rates and patterns of evolution. We estimated the substitution rates of CFAV and CxFV (two ISFVs) and, by taking into account the time-frame of measurement, compared them with those of other flaviviruses. Results indicated that CFAV and CxFV display relatively different substitution rates. However, these data, together with estimates for single-host members of the Flaviviridae family, indicated that MBFVs do not display relatively slower evolution. Conversely, TBFVs displayed some of lowest substitution rates among flaviviruses. Analysis of selective patterns over longer evolutionary time-frames confirmed that MBFVs evolve under strong purifying selection. Interestingly, TBFVs and ISFVs did not show extremely different levels of constraint, although TBFVs alternate among hosts, whereas ISFVs do not. Additional results showed that episodic positive selection drove the evolution of MBFVs, despite their high constraint. Positive selection was also detected on two branches of the TBFVs phylogeny that define the seabird clade. Thus, positive selection was much more common during the evolution of arthropod-borne flaviviruses than previously thought. Overall, our data indicate that flavivirus evolutionary patterns are complex and most likely determined by multiple factors, not limited to the alternation between taxonomically divergent hosts. The frequency of both positive and purifying selection, especially in MBFVs, suggests that a minority of sites in the viral polyprotein experience weak constraint and can evolve to generate new viral phenotypes and possibly promote adaptation to new hosts.

Kinetochore proteins and microtubule-destabilizing factors are fast evolving in eutherian mammals.

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.

Antigenic variation of SARS-CoV-2 in response to immune pressure.

Analysis of the bat viruses most closely related to SARS-CoV-2 indicated that the virus probably required limited adaptation to spread in humans. Nonetheless, since its introduction in human populations, SARS-CoV-2 must have been subject to the selective pressure imposed by the human immune system. We exploited the availability of a large number of high-quality SARS-CoV-2 genomes, as well as of validated epitope predictions, to show that B cell epitopes in the spike glycoprotein (S) and in the nucleocapsid protein (N) have higher diversity than nonepitope positions. Similar results were obtained for other human coronaviruses and for sarbecoviruses sampled in bats. Conversely, in the SARS-CoV-2 population, epitopes for CD4+ and CD8+ T cells were not more variable than nonepitope positions. A significant reduction in epitope variability was instead observed for some of the most immunogenic proteins (S, N, ORF8 and ORF3a). Analysis over longer evolutionary time frames indicated that this effect is not due to differential constraints. These data indicate that SARS-CoV-2 evolves to elude the host humoral immune response, whereas recognition by T cells is not actively avoided by the virus. However, we also found a trend of lower diversity of T cell epitopes for common cold coronaviruses, indicating that epitope conservation per se is not directly linked to disease severity. We suggest that conservation serves to maintain epitopes that elicit tolerizing T cell responses or induce T cells with regulatory activity.

Past and ongoing adaptation of human cytomegalovirus to its host.

Cytomegaloviruses (order Herpesvirales) display remarkable species-specificity as a result of long-term co-evolution with their mammalian hosts. Human cytomegalovirus (HCMV) is exquisitely adapted to our species and displays high genetic diversity. We leveraged information on inter-species divergence of primate-infecting cytomegaloviruses and intra-species diversity of clinical isolates to provide a genome-wide picture of HCMV adaptation across different time-frames. During adaptation to the human host, core viral genes were commonly targeted by positive selection. Functional characterization of adaptive mutations in the primase gene (UL70) indicated that selection favored amino acid replacements that decrease viral replication in human fibroblasts, suggesting evolution towards viral temperance. HCMV intra-species diversity was largely governed by immune system-driven selective pressure, with several adaptive variants located in antigenic domains. A significant excess of positively selected sites was also detected in the signal peptides (SPs) of viral proteins, indicating that, although they are removed from mature proteins, SPs can contribute to viral adaptation. Functional characterization of one of these SPs indicated that adaptive variants modulate the timing of cleavage by the signal peptidase and the dynamics of glycoprotein intracellular trafficking. We thus used evolutionary information to generate experimentally-testable hypotheses on the functional effect of HCMV genetic diversity and we define modulators of viral phenotypes.

Recent Out-of-Africa Migration of Human Herpes Simplex Viruses.

Herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) are ubiquitous human pathogens. Both viruses evolved from simplex viruses infecting African primates and they are thus thought to have left Africa during early human migrations. We analyzed the population structure of HSV-1 and HSV-2 circulating strains. Results indicated that HSV-1 populations have limited geographic structure and the most evident clustering by geography is likely due to recent bottlenecks. For HSV-2, the only level of population structure is accounted for by the so-called "worldwide" and "African" lineages. Analysis of ancestry components and nucleotide diversity, however, did not support the view that the worldwide lineage followed early humans during out-of-Africa dispersal. Although phylogeographic analysis confirmed an African origin for both viruses, molecular dating with a method that corrects for the time-dependent rate phenomenon indicated that HSV-1 and HSV-2 migrated from Africa in relatively recent times. In particular, we estimated that the HSV-2 worldwide lineage left the continent in the 18th century, which corresponds to the height of the transatlantic slave trade, possibly explaining the high prevalence of HSV-2 in the Americas (second highest after Africa). The limited geographic clustering of HSV-1 makes it difficult to date its exit from Africa. The split between the basal clade, containing mostly African sequences, and all other strains was dated at ∼5,000 years ago. Our data do not imply that herpes simplex viruses did not infect early humans but show that the worldwide distribution of circulating strains is the result of relatively recent events.

Possible European Origin of Circulating Varicella Zoster Virus Strains.

Varicella zoster virus (VZV) is the causative agent of chickenpox and shingles. The geographic distribution of VZV clades was taken as evidence that VZV migrated out of Africa with human populations. We show that extant VZV strains most likely originated in Europe and not in Africa. Europe was also identified as the ancestral location for most internal nodes of the VZV phylogeny, including the ancestor of clade 5 strains. We also show that strains from clades 1, 2, 3, and 5 derived a major proportion of their ancestry from each of 4 ancestral populations. Conversely, viruses from other clades displayed variable levels of admixture. Some low-level admixture was also observed for clade 5 genomes, but only for non-African viruses. This pattern indicates that the clade 5 VZV strains do not represent recent introductions from Africa due to migratory fluxes. These data have also relevance for the definition and classification of VZV clades.

Arenavirus genomics: novel insights into viral diversity, origin, and evolution.

Next-generation sequencing technologies have revolutionized our knowledge of virus diversity and evolution. In the case of arenaviruses, which are the focus of this review, metagenomic/metatranscriptomic approaches identified reptile-infecting and fish-infecting viruses, also showing that bi-segmented genomes are not a universal feature of the Arenaviridae family. Novel mammarenaviruses were described, allowing inference of their geographic origin and evolutionary dynamics. Extensive sequencing of Lassa virus (LASV) genomes revealed the zoonotic nature of most human infections and a Nigerian origin of LASV, which subsequently spread westward. Future efforts will likely identify many more arenaviruses and hopefully provide insight into the ultimate origin of the family, the pathogenic potential of its members, as well as the determinants of their geographic distribution.

Analysis of Reptarenavirus genomes indicates different selective forces acting on the S and L segments and recent expansion of common genotypes.

Reptarenaviruses, a genus of snake-infecting viruses belonging to the family Arenaviridae, have bi-segmented genomes. The long (L) segment encodes the Z and L (RNA polymerase) proteins, whereas the short (S) segment codes for the glycoprotein precursor (GPC) and for the nucleoprotein (NP). Presently, reptarenaviruses have only been described in captive snakes. In these animals, mixed infections are common and most infected reptiles harbor multiple S and/or L segment genotypes. Within single animals, L segments are more genetically diverse than S segments and one S segment genotype (S6) was detected in the majority of snakes. Whether the unbalanced L to S segment ratio is due to stochastic events, to distinct replication/packaging efficiencies, or to differential selective pressure is presently unknown. We addressed these open questions by analyzing the ancient and recent evolutionary history of reptarenavirus genomes. Results indicated that purifying selection shaped the bulk of reptarenavirus coding sequences, although selective constraint was stronger for NP and L compared to GPC. During the divergence of reptarenavirus genomes, episodic positive selection contributed to the evolution of the viral polymerase, an observation that parallels those on mammarenaviruses. Population genetics analyses indicated that the most common S and L segment genotypes (including S6) display markedly negative Tajima's D values, but not low nucleotide diversity, suggesting recent population expansion. In conclusion, our data indicate that the selective pressures were stronger for the L segment than for the S segment, at least during reptarenavirus genotype divergence. More recently, the population sizes of some L and S segment genotypes expanded, suggesting that they out-competed the other genotypes, which show D values consistent with constant or decreasing population size. Competition among segments may have driven the disappearance of some S segment genotypes from wild and/or captive snake populations, eventually leading to the observed L to S imbalance.

Evolutionary rates of mammalian telomere-stability genes correlate with karyotype features and female germline expression.

Telomeres protect the ends of eukaryotic chromosomes and are essential for cell viability. In mammals, telomere dynamics vary with life history traits (e.g. body mass and longevity), suggesting differential selection depending on physiological characteristics. Telomeres, in analogy to centromeric regions, also represent candidate meiotic drivers and subtelomeric DNA evolves rapidly. We analyzed the evolutionary history of mammalian genes implicated in telomere homeostasis (TEL genes). We detected widespread positive selection and we tested two alternative hypotheses: (i) fast evolution is driven by changes in life history traits; (ii) a conflict with selfish DNA elements at the female meiosis represents the underlying selective pressure. By accounting for the phylogenetic relationships among mammalian species, we show that life history traits do not contribute to shape diversity of TEL genes. Conversely, the evolutionary rate of TEL genes correlates with expression levels during meiosis and episodes of positive selection across mammalian species are associated with karyotype features (number of chromosome arms). We thus propose a telomere drive hypothesis, whereby (sub)telomeres and telomere-binding proteins are engaged in an intra-genomic conflict similar to the one described for centromeres.

Evolutionary Analysis Provides Insight Into the Origin and Adaptation of HCV.

Hepatitis C virus (HCV) belongs to the Hepacivirus genus and is genetically heterogeneous, with seven major genotypes further divided into several recognized subtypes. HCV origin was previously dated in a range between ∼200 and 1000 years ago. Hepaciviruses have been identified in several domestic and wild mammals, the largest viral diversity being observed in bats and rodents. The closest relatives of HCV were found in horses/donkeys (equine hepaciviruses, EHV). However, the origin of HCV as a human pathogen is still an unsolved puzzle. Using a selection-informed evolutionary model, we show that the common ancestor of extant HCV genotypes existed at least 3000 years ago (CI: 3192-5221 years ago), with the oldest genotypes being endemic to Asia. EHV originated around 1100 CE (CI: 291-1640 CE). These time estimates exclude that EHV transmission was mainly sustained by widespread veterinary practices and suggest that HCV originated from a single zoonotic event with subsequent diversification in human populations. We also describe a number of biologically important sites in the major HCV genotypes that have been positively selected and indicate that drug resistance-associated variants are significantly enriched at positively selected sites. HCV exploits several cell-surface molecules for cell entry, but only two of these (CD81 and OCLN) determine the species-specificity of infection. Herein evolutionary analyses do not support a long-standing association between primates and hepaciviruses, and signals of positive selection at CD81 were only observed in Chiroptera. No evidence of selection was detected for OCLN in any mammalian order. These results shed light on the origin of HCV and provide a catalog of candidate genetic modulators of HCV phenotypic diversity.

Ancient Evolution of Mammarenaviruses: Adaptation via Changes in the L Protein and No Evidence for Host-Virus Codivergence.

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.

Genetic susceptibility to infectious diseases: Current status and future perspectives from genome-wide approaches.

Genome-wide association studies (GWASs) have been widely applied to identify genetic factors that affect complex diseases or traits. Presently, the GWAS Catalog includes >2800 human studies. Of these, only a minority have investigated the susceptibility to infectious diseases or the response to therapies for the treatment or prevention of infections. Despite their limited application in the field, GWASs have provided valuable insights by pinpointing associations to both innate and adaptive immune response loci, as well as novel unexpected risk factors for infection susceptibility. Herein, we discuss some issues and caveats of GWASs for infectious diseases, we review the most recent findings ensuing from these studies, and we provide a brief summary of selected GWASs for infections in non-human mammals. We conclude that, although the general trend in the field of complex traits is to shift from GWAS to next-generation sequencing, important knowledge on infectious disease-related traits can be still gained by GWASs, especially for those conditions that have never been investigated using this approach. We suggest that future studies will benefit from the leveraging of information from the host's and pathogen's genomes, as well as from the exploration of models that incorporate heterogeneity across populations and phenotypes. Interactions within HLA genes or among HLA variants and polymorphisms located outside the major histocompatibility complex may also play an important role in shaping the susceptibility and response to invading pathogens.

Evolutionary analysis of Old World arenaviruses reveals a major adaptive contribution of the viral polymerase.

The Old World (OW) arenavirus complex includes several species of rodent-borne viruses, some of which (i.e., Lassa virus, LASV and Lymphocytic choriomeningitis virus, LCMV) cause human diseases. Most LCMV and LASV infections are caused by rodent-to-human transmissions. Thus, viral evolution is largely determined by events that occur in the wildlife reservoirs. We used a set of human- and rodent-derived viral sequences to investigate the evolutionary history underlying OW arenavirus speciation, as well as the more recent selective events that accompanied LASV spread in West Africa. We show that the viral RNA polymerase (L protein) was a major positive selection target in OW arenaviruses and during LASV out-of-Nigeria migration. No evidence of selection was observed for the glycoprotein, whereas positive selection acted on the nucleoprotein (NP) during LCMV speciation. Positively selected sites in L and NP are surrounded by highly conserved residues, and the bulk of the viral genome evolves under purifying selection. Several positively selected sites are likely to modulate viral replication/transcription. In both L and NP, structural features (solvent exposed surface area) are important determinants of site-wise evolutionary rate variation. By incorporating several rodent-derived sequences, we also performed an analysis of OW arenavirus codon adaptation to the human host. Results do not support a previously hypothesized role of codon adaptation in disease severity for non-Nigerian strains. In conclusion, L and NP represent the major selection targets and possible determinants of disease presentation; these results suggest that field surveys and experimental studies should primarily focus on these proteins.

REST, a master regulator of neurogenesis, evolved under strong positive selection in humans and in non human primates.

The transcriptional repressor REST regulates many neuronal genes by binding RE1 motifs. About one third of human RE1s are recently evolved and specific to primates. As changes in the activity of a transcription factor reverberate on its downstream targets, we assessed whether REST displays fast evolutionary rates in primates. We show that REST was targeted by very strong positive selection during primate evolution. Positive selection was also evident in the human lineage, with six selected sites located in a region that surrounds a VNTR in exon 4. Analysis of expression data indicated that REST brain expression peaks during aging in humans but not in other primates. Because a REST coding variant (rs3796529) was previously associated with protection from hippocampal atrophy in elderly subjects with mild cognitive impairment (MCI), we analyzed a cohort of Alzheimer disease (AD) continuum patients. Genotyping of two coding variants (rs3796529 and rs2227902) located in the region surrounding the VNTR indicated a role for rs2227902 in modulation of hippocampal volume loss, indirectly confirming a role for REST in neuroprotection. Experimental studies will be instrumental to determine the functional effect of positively selected sites in REST and the role of REST variants in neuropreservation/neurodegeneration.

Susceptibility to type 2 diabetes may be modulated by haplotypes in G6PC2, a target of positive selection.

BACKGROUND: The endoplasmic reticulum enzyme glucose-6-phosphatase catalyzes the common terminal reaction in the gluconeogenic/glycogenolytic pathways and plays a central role in glucose homeostasis. In most mammals, different G6PC subunits are encoded by three paralogous genes (G6PC, G6PC2, and G6PC3). Mutations in G6PC and G6PC3 are responsible for human mendelian diseases, whereas variants in G6PC2 are associated with fasting glucose (FG) levels. RESULTS: We analyzed the evolutionary history of G6Pase genes. Results indicated that the three paralogs originated during early vertebrate evolution and that negative selection was the major force shaping diversity at these genes in mammals. Nonetheless, site-wise estimation of evolutionary rates at corresponding sites revealed weak correlations, suggesting that mammalian G6Pases have evolved different structural features over time. We also detected pervasive positive selection at mammalian G6PC2. Most selected residues localize in the C-terminal protein region, where several human variants associated with FG levels also map. This region was re-sequenced in ~560 subjects from Saudi Arabia, 185 of whom suffering from type 2 diabetes (T2D). The frequency of rare missense and nonsense variants was not significantly different in T2D and controls. Association analysis with two common missense variants (V219L and S342C) revealed a weak but significant association for both SNPs when analyses were conditioned on rs560887, previously identified in a GWAS for FG. Two haplotypes were significantly associated with T2D with an opposite effect direction. CONCLUSIONS: We detected pervasive positive selection at mammalian G6PC2 genes and we suggest that distinct haplotypes at the G6PC2 locus modulate susceptibility to T2D.