Complex Haplotypes of GSTM1 Gene Deletions Harbor Signatures of a Selective Sweep in East Asian Populations.

The deletion of the metabolizing Glutathione S-transferase Mu 1 (GSTM1) gene has been associated with multiple cancers, metabolic and autoimmune disorders, as well as drug response. It is unusually common, with allele frequency reaching up to 75% in some human populations. Such high allele frequency of a derived allele with apparent impact on an otherwise conserved gene is a rare phenomenon. To investigate the evolutionary history of this locus, we analyzed 310 genomes using population genetics tools. Our analysis revealed a surprising lack of linkage disequilibrium between the deletion and the flanking single nucleotide variants in this locus. Tests that measure extended homozygosity and rapid change in allele frequency revealed signatures of an incomplete sweep in the locus. Using empirical approaches, we identified the Tanuki haplogroup, which carries the GSTM1 deletion and is found in approximately 70% of East Asian chromosomes. This haplogroup has rapidly increased in frequency in East Asian populations, contributing to a high population differentiation among continental human groups. We showed that extended homozygosity and population differentiation for this haplogroup is incompatible with simulated neutral expectations in East Asian populations. In parallel, we found that the Tanuki haplogroup is significantly associated with the expression levels of other GSTM genes. Collectively, our results suggest that standing variation in this locus has likely undergone an incomplete sweep in East Asia with regulatory impact on multiple GSTM genes. Our study provides the necessary framework for further studies to elucidate the evolutionary reasons that maintain disease-susceptibility variants in the GSTM1 locus.

Complex evolution of the GSTM gene family involves sharing of GSTM1 deletion polymorphism in humans and chimpanzees.

BACKGROUND: The common deletion of the glutathione S-transferase Mu 1 (GSTM1) gene in humans has been shown to be involved in xenobiotic metabolism and associated with bladder cancer. However, the evolution of this deletion has not been investigated. RESULTS: In this study, we conducted comparative analyses of primate genomes. We demonstrated that the GSTM gene family has evolved through multiple structural variations, involving gene duplications, losses, large inversions and gene conversions. We further showed experimentally that the GSTM1 was polymorphically deleted in both humans and also in chimpanzees, through independent deletion events. To generalize our results, we searched for genic deletions that are polymorphic in both humans and chimpanzees. Consequently, we found only two such deletions among the thousands that we have searched, one of them being the GSTM1 deletion and the other surprisingly being another metabolizing gene, the UGT2B17. CONCLUSIONS: Overall, our results support the emerging notion that metabolizing gene families, such as the GSTM, NAT, UGT and CYP, have been evolving rapidly through gene duplication and deletion events in primates, leading to complex structural variation within and among species with unknown evolutionary consequences.

A limit to the divergent allele advantage model supported by variable pathogen recognition across HLA-DRB1 allele lineages.

Genetic diversity in human leukocyte antigen (HLA) molecules is thought to have arisen from the co-evolution between host and pathogen and maintained by balancing selection. Heterozygote advantage is a common proposed scenario for maintaining high levels of diversity in HLA genes, and extending from this, the divergent allele advantage (DAA) model suggests that individuals with more divergent HLA alleles bind and recognize a wider array of antigens. While the DAA model seems biologically suitable for driving HLA diversity, there is likely an upper threshold to the amount of sequence divergence. We used peptide-binding and pathogen-recognition capacity of DRB1 alleles as a model to further explore the DAA model; within the DRB1 locus, we examined binding predictions based on two distinct phylogenetic groups (denoted group A and B) previously identified based on non-peptide-binding region (PBR) nucleotide sequences. Predictions in this study support that group A allele and group B allele lineages have contrasting binding/recognition capacity, with only the latter supporting the DAA model. Furthermore, computer simulations revealed an inconsistency in the DAA model alone with observed extent of polymorphisms, supporting that the DAA model could only work effectively in combination with other mechanisms. Overall, we support that the mechanisms driving HLA diversity are non-exclusive. By investigating the relationships among HLA alleles, and pathogens recognized, we can provide further insights into the mechanisms on how humans have adapted to infectious diseases over time.

Comparative analysis of human masculinity.

To study rapidly evolving male specific Y (MSY) genes we retrieved and analyzed nine such genes. VCY, HSFY and RBMY were found to have functional X gametologs, but the rest did not. Using chimpanzee orthologs for XKRY, CDY, HSFY, PRY, and TSPY, the average silent substitution is estimated as 0.017 +/- 0.006/site and the substitution rate is 1.42 x 10(-9)/site/year. Except for VCY, all other loci possess two or more pseudogenes on the Y chromosome. Sequence differences from functional genes show that BPY2, DAZ, XKRY, and RBMY each have one pseudogene for each one that is human specific, while others were generated well before the human-chimpanzee split, by means of duplication, retro-transposition or translocation. Some functional MSY gene duplication of VCY, CDY and HSFY, as well as X-linked VCX and HSFX duplication, occurred in the lineage leading to humans; these duplicates have accumulated nucleotide substitutions that permit their identification.

Comparative analysis of human masculinity

To study rapidly evolving male specific Y (MSY) genes we retrieved and analyzed nine such genes. VCY, HSFY and RBMY were found to have functional X gametologs, but the rest did not. Using chimpanzee orthologs for XKRY, CDY, HSFY, PRY, and TSPY, the average silent substitution is estimated as 0.017 +/- 0.006/site and the substitution rate is 1.42 x 10(-9)/site/year. Except for VCY, all other loci possess two or more pseudogenes on the Y chromosome. Sequence differences from functional genes show that BPY2, DAZ, XKRY, and RBMY each have one pseudogene for each one that is human specific, while others were generated well before the human-chimpanzee split, by means of duplication, retro-transposition or translocation. Some functional MSY gene duplication of VCY, CDY and HSFY, as well as X-linked VCX and HSFX duplication, occurred in the lineage leading to humans; these duplicates have accumulated nucleotide substitutions that permit their identification.

Evidence for natural selection in the HAVCR1 gene: high degree of amino-acid variability in the mucin domain of human HAVCR1 protein.

The family of genes encoding T-cell immunoglobulin and mucin-domain containing proteins (Tim), which are cell-surface molecules expressed in CD4(+) T helper cells, has important roles in the immune system. Here, we report three unusual patterns of genetic variation in the human hepatitis A virus cellular receptor 1 gene (HAVCR1) that are similar to patterns observed in major histocompatibility complex loci. First, levels of polymorphism in exon 4 of HAVCR1 were exceptionally high in humans (nucleotide diversity (pi)=45.45 x 10(-4)). Second, nonsynonymous substitutions and insertion/deletion variants were more frequent than synonymous substitutions in that exon (10 out of 12 variants). The rate of the mean number of nucleotide substitutions at nonsynonymous sites to synonymous sites at HAVCR1-exon 4 is >1 (P(A)/P(S)=1.92 and pi(A)/pi(S)=2.23). Third, levels of divergence among human, chimp, and gorilla sequences were unusually high in HAVCR1-exon 4 sequences. These features suggest that patterns of variation in HAVCR1 have been shaped by both positive and balancing natural selection in the course of primate evolution. Evidence that the effects of natural selection are largely restricted to the mucin domain of HAVCR1 suggests that this region may be of particular evolutionary and epidemiological interest.

Chemically synthesized pathogen-associated molecular patterns increase the expression of peptidoglycan recognition proteins via toll-like receptors, NOD1 and NOD2 in human oral epithelial cells.

Peptidoglycan recognition proteins (PGRPs), a novel family of pattern recognition molecules (PRMs) in innate immunity conserved from insects to mammals, recognize bacterial cell wall peptidoglycan (PGN) and are suggested to act as anti-bacterial factors. In humans, four kinds of PGRPs (PGRP-L, -Ialpha, -Ibeta and -S) have been cloned and all four human PGRPs bind PGN. In this study, we examined the possible regulation of the expression of PGRPs in oral epithelial cells upon stimulation with chemically synthesized pathogen-associated molecular patterns (PAMPs) in bacterial cell surface components: Escherichia coli-type tryacyl lipopeptide (Pam3CSSNA), E. coli-type lipid A (LA-15-PP), diaminopimelic acid containing desmuramyl peptide (gamma-D-glutamyl-meso-DAP; iE-DAP), and muramyldipeptide (MDP). These synthetic PAMPs markedly upregulated the mRNA expression of the four PGRPs and cell surface expression of PGRP-Ialpha and -Ibeta, but did not induce either mRNA expression or secretion of inflammatory cytokines, in oral epithelial cells. Suppression of the expression of Toll-like receptor (TLR)2, TLR4, nucleotide-binding oligomerization domain (NOD)1 and NOD2 by RNA interference specifically inhibited the upregulation of PGRP mRNA expression induced by Pam3CSSNA, LA-15-PP, iE-DAP and MDP respectively. These PAMPs definitely activated nuclear factor (NF)-kappaB in the epithelial cells, and suppression of NF-kappaB activation clearly prevented the induction of PGRP mRNA expression induced by these PAMPs in the cells. These findings suggested that bacterial PAMPs induced the expression of PGRPs, but not proinflammatory cytokines, in oral epithelial cells, and the PGRPs might be involved in host defence against bacterial invasion without accompanying inflammatory responses.

The distribution of the ancestral haplotype in finite stepping-stone models with population expansion.

Recent extensive analyses of human DNA polymorphism reveal that the ancestral haplotype at various genetic loci occurs almost exclusively in African samples. We develop a coalescence-based simulation method in stepping-stone models with population expansion and examine the probability (P(A)) that the ancestral haplotype is found in African samples and the probability (Q(A)) that the most recent common ancestor of sampled genes occurs in Africa. These probabilities and other summary statistics are used to infer the human demographic history. It is shown that the high observed P(A) value cannot be explained simply by sampling bias. Rather, it suggests that the African population has been more strongly subdivided and isolated from each other than the non-African population and that there must have been some African populations which were not directly involved in the Out-of-Africa expansion in the late Pleistocene.

Comparison of DNA and protein polymorphisms between humans and chimpanzees.

To examine the nucleotide diversity at silent (synonymous + intron + untranslated) and non-silent (nonsynonymous) sites in chimpanzees and humans, genes at six nuclear loci from two chimpanzees were sequenced. The average silent diversity was 0.19%, which was significantly higher than that in humans (0.05%). This observation suggests a significantly larger effective population size and a higher extent of neutral polymorphism in chimpanzees than in humans. On the other hand, the non-silent nucleotide diversity is similar in both species, resulting in a larger fraction of neutral mutations at non-silent sites in humans than in chimpanzees. Other types of polymorphism data were collected from the literature or databases to examine whether or not they are consistent with the nuclear DNA sequence polymorphism observed here. The nucleotide diversity at both silent and non-silent sites in mitochondrial (mt) DNA genes was compatible with that of the nuclear genes. Microsatellite loci showed a similar high extent of heterozygosity in both species, perhaps due to the combined effect of a high mutation rate and a recent population expansion in humans. At protein loci, humans are more heterozygous than chimpanzees, and the estimated fraction of neutral alleles in humans (0.84) is much larger than that in chimpanzees (0.26). These data show that the neutral fraction in non-silent changes is relatively large in the human population. This difference may be due to a relaxation of the functional constraint against proteins in the human lineage. To evaluate this possibility, it will be necessary to examine nucleotide sequences in relation to the physiological or biochemical properties of proteins.

Alu-mediated inactivation of the human CMP- N-acetylneuraminic acid hydroxylase gene.

Inactivation of the CMP-N-acetylneuraminic acid hydroxylase gene has provided an example of human-specific genomic mutation that results in a widespread biochemical difference between human and nonhuman primates. We have found that, although a region containing a 92-bp exon and an AluSq element in the hydroxylase gene is intact in all nonhuman primates examined, the same region in the human genome is replaced by an AluY element that was disseminated at least one million years ago. We propose a mechanistic model for this Alu-mediated replacement event, which deleted the 92-bp exon and thus inactivated the human hydroxylase gene. It is suggested that Alu elements have played potentially important roles in genotypic and phenotypic evolution in the hominid lineage.

Integrative study on chromosome evolution of mammals, ants and wasps based on the minimum interaction theory.

There is well-known evidence that in many eukaryotes, different species have different karyotypes (e.g. n=1-47 in ants and n=3-51 in mammals). Alternative (fusion and fission) hypotheses have been proposed to interpret this chromosomal diversity. Although the former has long been accepted, accumulating molecular genetics evidence seems to support the latter. We investigated this problem from a stochastic viewpoint using the Monte Carlo simulation method under the minimum interaction theory. We found that the results of simulations consistently interpreted the chromosomal diversity observed in mammals, ants and wasps, and concluded that chromosome evolution tends to evolve as a whole toward increasing chromosome numbers by centric fission. Accordingly, our results support the fission hypothesis. We discussed the process of chromosome evolution based on the latest theory of the molecular structure of chromosomes, and reconfirmed that the fission burst is the prime motive force in long-term chromosome evolution, and is effective in minimizing the genetic risks due to deleterious reciprocal translocations and in increasing the potential of genetic divergence. Centric fusion plays a biological role in eliminating heterochromatin (C-bands), but is only a local reverse flow in contrast to the previously held views.

Testing multiregionality of modern human origins.

In order to examine the possibility of multiple founding populations of anatomically modern Homo sapiens, we collected DNA sequence data from 10 X-chromosomal regions, 5 autosomal regions, and 1 Y-chromosomal region, in addition to mitochondrial DNA. Except for five regions which are genealogically uninformative and two other regions for which chimpanzee orthologs are not available, the ancestral sequence and population for each of the remaining regions were successfully inferred. Of these 10 ancestral sequences, 9 occurred in Africa and only 1 occurred in Asia during the Pleistocene. Computer simulation was carried out to quantify the multiregional hypothesis based solely on the premise that there was more than one founding population in the Pleistocene. Allowing the breeding size to vary among the founding populations, the hypothesis may account for the observed African ancestry in 90% of the genomic regions. However, it is required that the founding population in Africa was much larger than that outside Africa. Likelihood estimates of the breeding sizes in the founding populations were more than 9,000 in Africa and less than 1,000 in outside of Africa, although these estimates can be much less biased at the 1% significance level. If the number of African ancestral sequences further increases as more data accumulate in other genomic regions, the conclusion of a single founding population of modern H. sapiens is inevitable.

The implications of intergenic polymorphism for major histocompatibility complex evolution.

A systematic survey of six intergenic regions flanking the human HLA-B locus in eight haplotypes reveals the regions to be up to 20 times more polymorphic than the reported average degree of human neutral polymorphism. Furthermore, the extent of polymorphism is directly related to the proximity to the HLA-B locus. Apparently linkage to HLA-B locus alleles, which are under balancing selection, maintains the neutral polymorphism of adjacent regions. For these linked polymorphisms to persist, recombination in the 200-kb interval from HLA-B to TNF must occur at a low frequency. The high degree of polymorphism found distal to HLA-B suggests that recombination is uncommon on both sides of the HLA-B locus. The least-squares estimate is 0.15% per megabase with an estimated range from 0.02 to 0.54%. These findings place strong restrictions on possible recombinational mechanisms for the generation of diversity at the HLA-B.

Mhc class II B gene evolution in East African cichlid fishes.

A distinctive feature of essential major histocompatibility complex (Mhc) loci is their polymorphism characterized by large genetic distances between alleles and long persistence times of allelic lineages. Since the lineages often span several successive speciations, we investigated the behavior of the Mhc alleles during or close to the speciation phase. We sequenced exon 2 of the class II B locus 4 from 232 East African cichlid fishes representing 32 related species. The divergence times of the (sub)species ranged from 6,000 to 8.4 million years. Two types of evolutionary analysis were used to elucidate the pattern of exon 2 sequence divergence. First, phylogenetic methods were applied to reconstruct the most likely evolutionary pathways leading from the last common ancestor of the set to the extant sequences, and to assess the probable mechanisms involved in allelic diversification. Second, pairwise comparisons of sequences were carried out to detect differences seemingly incompatible with origin by nonparallel point mutations. The analysis revealed point mutations to be the most important mechanism behind allelic divergences, with recombination playing only an auxiliary part. Comparison of sequences from related species revealed evidence of random allelic (lineage) losses apparently associated with speciation. Sharing of identical alleles could be demonstrated between species that diverged 2 million years ago. The phylogeny of the exon was incongruent with that of the flanking introns, indicating either a high degree of convergent evolution at the peptide-binding region-encoding sites, or intron homogenization.

Highly divergent sequences of the pollen self-incompatibility (S) gene in class-I S haplotypes of Brassica campestris (syn. rapa) L.

Self-incompatibility (SI) enables flowering plants to discriminate between self- and non-self-pollen. In Brassica, SI is controlled by the highly polymorphic S locus. The recently identified male determinant, termed SP11 or SCR, is thought to be the ligand of S receptor kinase, the female determinant. To examine functional and evolutionary properties of SP11, we cloned 14 alleles from class-I S haplotypes of Brassica campestris and carried out sequence analyses. The sequences of mature SP11 proteins are highly divergent, except for the presence of conserved cysteines. The phylogenetic trees suggest possible co-evolution of the genes encoding the male and female determinants.

DNA archives and our nearest relative: the trichotomy problem revisited.

Ever since Thomas H. Huxley correctly identified the chimpanzee and the gorilla as the two closest relatives of the human, the problem of the relationship among the three species ("the trichotomy problem") has remained unresolved. Comparative morphology and other classical methods of biological investigation have failed to answer definitively whether the chimpanzee or the gorilla is the closest relative of the human species. DNA sequences, both mitochondrial and nuclear, too, have provided equivocal solutions, depending on the region of the genome analyzed. Random sorting of ancestral allelic lineages, sequence convergence, and sequence exchanges between alleles or duplicated loci have been identified as likely factors confounding the interpretation of the interrelationships among the three species. In the present study most of these difficulties are overcome by identifying evolutionary causes that might potentially provide misleading information. Altogether, 45 loci consisting of 46, 855 bp are analyzed. About 60% of the loci and approximately the same proportion of phylogenetically informative sites support the human-chimpanzee clade. The remaining 40% of loci and sites support the two alternatives equally. It is demonstrated that, while incompatibility between loci can be explained by random sorting of allelic lineages, incompatibility within loci must be attributed largely to the joint effect of recombination and genetic drift. The trichotomy problem can be properly addressed only within this framework.

Sequence and structural diversity of the S locus genes from different lines with the same self-recognition specificities in Brassica oleracea.

Self-incompatibility (SI) is a mechanism for preventing self-fertilization in flowering plants. In Brassica, it is controlled by a single multi-allelic locus, S, and it is believed that two highly polymorphic genes in the S locus, SLG and SRK, play central roles in self-recognition in stigmas. SRK is a putative receptor protein kinase, whose extracellular domain exhibits high similarity to SLG. We analyzed two pairs of lines showing cross-incompatibility (S(2) and S(2-b); S(13) and S(13-b)). In S(2) and S(2-b), SRKs were more highly conserved than SLGs. This was also the case with S(13) and S(13-b). This suggests that the SRKs of different lines must be conserved for the lines to have the same self-recognition specificity. In particular, SLG(2-b) showed only 88. 5% identity to SLG(2), which is comparable to that between the SLGs of different S haplotypes, while SRK(2-b) showed 97.3% identity to SRK(2) in the S domain. These findings suggest that the SLGs in these S haplotypes are not important for self-recognition in SI.

Molecular clock and recombination in primate Mhc genes.

To set an accurate chronological framework to the evolution of primate class I and II genes in the major histocompatibility complex (Mhc), the rate of silent nucleotide substitutions in exons and introns is examined for various cDNA and genome sequences currently available. The rate is sensitive to the GC content and correlates negatively with increased GC biases at the third codon positions of Mhc genes. The intergenic recombination rate in the HLA region is estimated from the synonymous nucleotide differences at 37 linked loci. Any HLA subregion is recombined more or less at the ordinary rate of 1 cM per 1 Mb, although the rate may be reduced in some subregions. This information is used to discuss HLA haplotypes when they are applied to studies of human demography. The unusual polymorphism in the alpha-helix of HLA-DRB1 is also revisited in relation to intragenic recombination, but the molecular mechanism and the evolutionary cause both remain enigmatic.