APOBEC mediated mutagenesis drives genomic heterogeneity in endometriosis.

Endometriosis is a benign gynecologic condition, acting as a precursor of certain histological subtypes of ovarian cancers. The epithelial cells of endometriotic tissues and normal uterine endometrium accumulated somatic mutations in cancer-associated genes such as phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) and Kirsten rat sarcoma (KRAS) proto-oncogene. To determine the genomic characteristic of endometriotic epithelial cells and normal uterine endometrium and to identify the predominant mutational process acting on them, we studied the somatic mutation profiles obtained from whole exome sequencing of 14 endometriotic epithelium and 11 normal uterine endometrium tissues and classified them into mutational signatures. We observed that single base substitutions 2/13 (SBS), attributed to Apolipoprotein B mRNA Editing Enzyme Catalytic Subunit (APOBEC) induced mutagenesis, were significant in endometriotic tissues, but not in the normal uterine endometrium. Additionally, the larger number and wider allele frequency distribution of APOBEC signature mutations, compared to cancer-associated driver mutations in endometriotic epithelium suggested APOBEC mutagenesis as an important source of mutational burden and heterogeneity in endometriosis. Further, the relative risk of enriched APOBEC signature mutations was higher in endometriosis patients who were carriers of APOBEC3A/3B germline deletion, a common polymorphism in East Asians which involves the complete loss of APOBEC3B coding region. Our results illustrate the significance of APOBEC induced mutagenesis in driving the genomic heterogeneity of endometriosis.

AMBRA1 p.Gln30Arg Mutation, Identified in a Cowden Syndrome Family, Exhibits Hyperproliferative Potential in hTERT-RPE1 Cells.

Cowden syndrome (CS) is a rare autosomal dominant disorder associated with multiple hamartomatous and neoplastic lesions in various organs. Most CS patients have been found to have germline mutations in the PTEN tumor suppressor. In the present study, we investigated the causative gene of CS in a family of PTEN (phosphatase and tensin homolog deleted on chromosome 10) -negative CS patients. Whole exome sequencing analysis revealed AMBRA1 (Autophagy and Beclin 1 Regulator 1) as a novel candidate gene harboring two germline variants: p.Gln30Arg (Q30R) and p.Arg1195Ser (R1195S). AMBRA1 is a key regulator of the autophagy signaling network and a tumor suppressor. To functionally validate the role of AMBRA1 in the clinical manifestations of CS, we generated AMBRA1 depletion and Q30R mutation in hTERT-RPE1 (humanTelomerase Reverse Transcriptase-immortalized Retinal Pigmented Epithelial cells) using the CRISPR-Cas9 gene editing system. We observed that both AMBRA1-depleted and mutant cells showed accumulation in the S phase, leading to hyperproliferation, which is a characteristic of hamartomatous lesions. Specifically, the AMBRA1 Q30R mutation disturbed the G1/S transition of cells, leading to continuous mitotic entry of mutant cells, irrespective of the extracellular condition. From our analysis of primary ciliogenesis in these cells, we speculated that the mitotic entry of AMBRA1 Q30R mutants could be due to non-functional primary cilia that lead to impaired processing of extracellular sensory signals. Additionally, we observed a situs inversus phenotype in ambra1-depleted zebrafish, a developmental abnormality resulting from dysregulated primary ciliogenesis. Taken together, we established that the AMBRA1 Q30R mutation that we observed in CS patients might play an important role in inducing the hyperproliferative potential of cells through regulating primary ciliogenesis.

Nonequilibrium Neutral Theory for Hitchhikers.

Selective sweep is a phenomenon of reduced variation at presumably neutrally evolving sites (hitchhikers) in the genome that is caused by the spread of a selected allele at a linked focal site, and is widely used to test for action of positive selection. Nonetheless, selective sweep may also provide an unprecedented opportunity for studying nonequilibrium properties of the neutral variation itself. We have demonstrated this possibility in relation to ancient selective sweep for modern human-specific changes and ongoing selective sweep for local population-specific changes.

Coevolution of Siglec-11 and Siglec-16 via gene conversion in primates.

BACKGROUND: Siglecs-11 and -16 are members of the sialic acid recognizing Ig-like lectin family, and expressed in same cells. Siglec-11 functions as an inhibitory receptor, whereas Siglec-16 exhibits activating properties. In humans, SIGLEC11 and SIGLEC16 gene sequences are extremely similar in the region encoding the extracellular domain due to gene conversions. Human SIGLEC11 was converted by the nonfunctional SIGLEC16P allele, and the converted SIGLEC11 allele became fixed in humans, possibly because it provides novel neuroprotective functions in brain microglia. However, the detailed evolutionary history of SIGLEC11 and SIGLEC16 in other primates remains unclear. RESULTS: We analyzed SIGLEC11 and SIGLEC16 gene sequences of multiple primate species, and examined glycan binding profiles of these Siglecs. The phylogenetic tree demonstrated that gene conversions between SIGLEC11 and SIGLEC16 occurred in the region including the exon encoding the sialic acid binding domain in every primate examined. Functional assays showed that glycan binding preference is similar between Siglec-11 and Siglec-16 in all analyzed hominid species. Taken together with the fact that Siglec-11 and Siglec-16 are expressed in the same cells, Siglec-11 and Siglec-16 are regarded as paired receptors that have maintained similar ligand binding preferences via gene conversions. Relaxed functional constraints were detected on the SIGLEC11 and SIGLEC16 exons that underwent gene conversions, possibly contributing to the evolutionary acceptance of repeated gene conversions. The frequency of nonfunctional SIGLEC16P alleles is much higher than that of SIGLEC16 alleles in every human population. CONCLUSIONS: Our findings indicate that Siglec-11 and Siglec-16 have been maintained as paired receptors by repeated gene conversions under relaxed functional constraints in the primate lineage. The high prevalence of the nonfunctional SIGLEC16P allele and the fixation of the converted SIGLEC11 imply that the loss of Siglec-16 and the gain of Siglec-11 in microglia might have been favored during the evolution of human lineage.

Long-lived dichotomous lineages of the proteasome subunit beta type 8 (PSMB8) gene surviving more than 500 million years as alleles or paralogs.

On an evolutionary time scale, polymorphic alleles are believed to have a short life, persisting at most tens of millions of years even under long-term balancing selection. Here, we report highly diverged trans-species dimorphism of the proteasome subunit beta type 8 (PSMB8) gene, which encodes a catalytic subunit of the immunoproteasome responsible for the generation of peptides presented by major histocompatibility complex (MHC) class I molecules, in lower teleosts including Cypriniformes (zebrafish and loach) and Salmoniformes (trout and salmon), whose last common ancestor dates to 300 Ma. Moreover, phylogenetic analyses indicated that these dimorphic alleles share lineages with two shark paralogous genes, suggesting that these two lineages have been maintained for more than 500 My either as alleles or as paralogs, and that conversion between alleles and paralogs has occurred at least once during vertebrate evolution. Two lineages termed PSMB8A and PSMB8F show an A(31)F substitution that would probably affect their cleaving specificity, and whereas the PSMB8A lineage has been retained by all analyzed jawed vertebrates, the PSMB8F lineage has been lost by most jawed vertebrates except for cartilaginous fish and basal teleosts. However, a possible functional equivalent of the PSMB8F lineage has been revived as alleles within the PSMB8A lineage at least twice during vertebrate evolution in the amphibian Xenopus and teleostean Oryzias species. Dynamic evolution of the PSMB8 polymorphism through long-term persistence, loss, and regaining of dimorphism and conversion between alleles and paralogs implies the presence of strong selective pressure for functional polymorphism of this gene.

Dimorphisms of the proteasome subunit beta type 8 gene (PSMB8) of ectothermic tetrapods originated in multiple independent evolutionary events.

The proteasome subunit beta type 8 gene (PSMB8) encodes one of the beta subunits of the immunoproteasome responsible for the generation of peptides presented by major histocompatibility complex class I molecules. Dimorphic alleles of the PSMB8 gene, termed A and F types, based on the deduced 31st amino acid residue of the mature protein have been reported from various vertebrates. Phylogenetic analysis revealed the presence of dichotomous ancient lineages, one comprising the F-type PSMB8 of basal ray-finned fishes, and the other comprising the A-type PSMB8 of these animals and both the F- and A-type PSMB8 of Xenopus and acanthopterygians, indicating that evolutionary history of the PSMB8 dimorphism was not straightforward. We analyzed the PSMB8 gene of five reptile and one amphibian species and found both the A and F types from all six. Phylogenetic analysis indicated that the PSMB8 F type was apparently regenerated from the PSMB8 A type at least five times independently during tetrapod evolution. Genomic typing of wild individuals of geckos and newts indicated that the frequencies of the A- and F-type alleles are not highly biased in these species. Phylogenetic analysis of each exon of the reptile PSMB8 gene suggested interallelic sequence homogenization as a possible evolutionary mechanism for the apparent recurrent regeneration of PSMB8 dimorphism in tetrapods. An extremely strong balancing selection acting on PSMB8 dimorphism was implicated in an unprecedented pattern of allele evolution.

Detection of APP gene recombinant in human blood plasma.

The pathogenesis of Alzheimer's disease (AD) is believed to involve the accumulation of amyloid-ß in the brain, which is produced by the sequential cleavage of amyloid precursor protein (APP) by ß-secretase and γ-secretase. Recently, analysis of genomic DNA and mRNA from postmortem brain neurons has revealed intra-exonic recombinants of APP (gencDNA), which have been implicated in the accumulation of amyloid-ß. In this study, we computationally analyzed publicly available sequence data (SRA) using probe sequences we constructed to screen APP gencDNAs. APP gencDNAs were detected in SRAs constructed from both genomic DNA and RNA obtained from the postmortem brain and in the SRA constructed from plasma cell-free mRNA (cf-mRNA). The SRA constructed from plasma cf-mRNA showed a significant difference in the number of APP gencDNA reads between SAD and NCI: the p-value from the Mann-Whitney U test was 5.14 × 10-6. The transcripts were also found in circulating nucleic acids (CNA) from our plasma samples with NGS analysis. These data indicate that transcripts of APP gencDNA can be detected in blood plasma and suggest the possibility of using them as blood biomarkers for Alzheimer's disease.

Highly divergent dimorphic alleles of the proteasome subunit beta type-8 (PSMB8) gene of the bichir Polypterus senegalus: implication for evolution of the PSMB8 gene of jawed vertebrates.

The proteasome subunit beta type-8 (PSMB8) gene encodes a catalytic subunit of the immunoproteasome, which is involved in the generation of peptides presented by MHC class I molecules. To date, highly diverged dichotomous alleles of PSMB8 have been reported in Oryzias species (actinopterygian teleosts) and Xenopus species (sarcopterygian amphibians). These dimorphic alleles share a similar substitution (A/V(31)F/Y) at the 31st position of the mature protein, which is most probably involved in formation of the S1 pocket. This substitution likely confers different cleavage specificities on the dimorphic PSMB8s. In addition, two paralogous PSMB8 genes possessing the A and F residues at the 31st position have been reported in sharks. Phylogenetic analysis indicated that the two types of PSMB8 of Oryzias, Xenopus, and sharks arose by independent evolutionary events. Here, we identified another pair of dimorphic alleles of PSMB8, which have the A and F residues at the 31st position of the mature protein, from bichir, Polypterus senegalus, a basal actinopterygian. The sequences of the mature proteins-encoding region of the dimorphic alleles of bichir PSMB8, the A and F types, showed only 72.7% and 77.5% identities at the nucleotide and the deduced amino acid levels, respectively. Their intronic sequences show almost no similarity, indicating that the dimorphic alleles of bichir PSMB8 have a very ancient origin. However, phylogenetic analysis showed that the dimorphisms of PSMB8 of bichir, Xenopus, and Oryzias arose by independent evolutionary events, suggesting the presence of a strong selective pressure for possessing the dimorphism.

Detection of Ancient Viruses and Long-Term Viral Evolution.

The COVID-19 outbreak has reminded us of the importance of viral evolutionary studies as regards comprehending complex viral evolution and preventing future pandemics. A unique approach to understanding viral evolution is the use of ancient viral genomes. Ancient viruses are detectable in various archaeological remains, including ancient people's skeletons and mummified tissues. Those specimens have preserved ancient viral DNA and RNA, which have been vigorously analyzed in the last few decades thanks to the development of sequencing technologies. Reconstructed ancient pathogenic viral genomes have been utilized to estimate the past pandemics of pathogenic viruses within the ancient human population and long-term evolutionary events. Recent studies revealed the existence of non-pathogenic viral genomes in ancient people's bodies. These ancient non-pathogenic viruses might be informative for inferring their relationships with ancient people's diets and lifestyles. Here, we reviewed the past and ongoing studies on ancient pathogenic and non-pathogenic viruses and the usage of ancient viral genomes to understand their long-term viral evolution.

Genomic Variation and Recent Population Histories of Spotted (Strix occidentalis) and Barred (Strix varia) Owls.

Spotted owls (SOs, Strix occidentalis) are a flagship species inhabiting old-growth forests in western North America. In recent decades, their populations have declined due to ongoing reductions in suitable habitat caused by logging, wildfires, and competition with the congeneric barred owl (BO, Strix varia). The northern spotted owl (S. o. caurina) has been listed as "threatened" under the Endangered Species Act since 1990. Here, we use an updated SO genome assembly along with 51 high-coverage whole-genome sequences to examine population structure, hybridization, and recent changes in population size in SO and BO. We found that potential hybrids identified from intermediate plumage morphology were a mixture of pure BO, F1 hybrids, and F1 × BO backcrosses. Also, although SO underwent a population bottleneck around the time of the Pleistocene-Holocene transition, their population sizes rebounded and show no evidence of any historical (i.e., 100-10,000 years ago) population decline. This suggests that the current decrease in SO abundance is due to events in the past century. Finally, we estimate that western and eastern BOs have been genetically separated for thousands of years, instead of the previously assumed recent (i.e., <150 years) divergence. Although this result is surprising, it is unclear where the ancestors of western BO lived after the separation. In particular, although BO may have colonized western North America much earlier than the first recorded observations, it is also possible that the estimated divergence time reflects unsampled BO population structure within central or eastern North America.

Lower promoter activity of the ST8SIA2 gene has been favored in evolving human collective brains.

ST8SIA2 is an important molecule regulating expression of the phenotype involved in schizophrenia. Lowered promoter activity of the ST8SIA2 gene is considered to be protective against schizophrenia by conferring tolerance to psychosocial stress. Here, we examined the promoter-type composition of anatomically modern humans (AMHs) and archaic humans (AHs; Neanderthals and Denisovans), and compared the promoter activity at the population level (population promoter activity; PPA) between them. In AMHs, the TCT-type, showing the second lowest promoter activity, was most prevalent in the ancestral population of non-Africans. However, the detection of only the CGT-type from AH samples and recombination tracts in AH sequences showed that the CGT- and TGT-types, exhibiting the two highest promoter activities, were common in AH populations. Furthermore, interspecies gene flow occurred into AMHs from AHs and into Denisovans from Neanderthals, influencing promoter-type compositions independently in both AMHs and AHs. The difference of promoter-type composition makes PPA unique in each population. East and Southeast Asian populations show the lowest PPA. This results from the selective increase of the CGC-type, showing the lowest promoter activity, in these populations. Every non-African population shows significantly lower PPA than African populations, resulting from the TCT-type having the highest prevalence in the ancestral population of non-Africans. In addition, PPA reduction is also found among subpopulations within Africa via a slight increase of the TCT-type. These findings indicate a trend toward lower PPA in the spread of AMHs, interpreted as a continuous adaptation to psychosocial stress arising in migration. This trend is considered as genetic tuning for the evolution of collective brains. The inferred promoter-type composition of AHs differed markedly from that of AMHs, resulting in higher PPA in AHs than in AMHs. This suggests that the trend toward lower PPA is a unique feature in AMH spread.

Two-dimensional site frequency spectrum for detecting, classifying and dating incomplete selective sweeps.

The two-dimensional site frequency spectrum (2D SFS) was investigated to describe the intra-allelic variability (IAV) maintained within a derived allele (D) group that has undergone an incomplete selective sweep against an ancestral allele group. We observed that recombination certainly muddles the ancestral relationships of allelic lineages between the two allele groups; however, the 2D SFS reveals intriguing signatures of recombination as well as the genealogical structure of the D group, particularly the size of a mutation and the time to the most recent common ancestor (TMRCA). Coalescent simulations were performed to achieve powerful and robust 2D SFS-based statistics with special reference to accurate evaluation of IAV, significance of recombination effects, and distinction between hard and soft selective sweeps. These studies were extended to a case wherein an incomplete selective sweep is no longer in progress and ceased in the recent past. The 2D SFS-based method was applied to 100 intronic linkage disequilibrium regions randomly chosen from the East Asian population of modern humans to examine the P value distributions of the summary statistics under the null hypothesis of neutrality in a nonequilibrium demographic model. We argue that about 96% of intronic variants are non-adaptive with a 10% false discovery rate. Furthermore, this method was applied to six genomic regions in Eurasian populations that were claimed to have experienced recent selective sweeps. We found that two of these genomic regions did not have significant signals of selective sweeps, but the remaining four had undergone hard and soft sweeps and were dated, in terms of TMRCA, after the major out-of-Africa dispersal of modern humans.

A new inference method for detecting an ongoing selective sweep.

A simple method was developed to detect signatures of ongoing selective sweeps in single nucleotide polymorphism (SNP) data. Based largely on the traditional site frequency spectrum (SFS), the method additionally incorporates linkage disequilibrium (LD) between pairs of SNP sites and uniquely represents both SFS and LD information as hierarchical "barcodes." This barcode representation allows the identification of a hitchhiking genomic region surrounding a putative target site of positive selection, or a core site. Sweep signals at linked neutral sites are then measured by the proportion (Fc) of derived alleles within the hitchhiking region that are linked in the derived allele group defined at the core site. In measuring Fc or intra-allelic variability in an informative way, certain conditions for derived allele frequencies are required, as illustrated with the human ST8SIA2 locus. Coalescent simulators with and without positive selection are used to assess the false-positive and false-negative rates of the Fc statistic. To demonstrate its power, the method was further applied to the LCT, OCA2, EDAR, SLC24A5 and ASPM loci, which are known to have undergone positive selection in human populations. Overall, the method is powerful and can be used to identify core sites responsible for ongoing selective sweeps.

Positive selection on schizophrenia-associated ST8SIA2 gene in post-glacial Asia.

A number of loci are associated with highly heritable schizophrenia and the prevalence of this mental illness has had considerable negative fitness effects on human populations. Here we focused on one particular schizophrenia-associated gene that encodes a sialyltransferase (ST8SIA2) and is expressed preferentially in the brain with the level being largely determined by three SNPs in the promoter region. It is suggested that the expression level of the ST8SIA2 gene is a genetic determinant of schizophrenia risk, and we found that a geographically differentiated non-risk SNP type (CGC-type) has significantly reduced promoter activity. A newly developed method for detecting ongoing positive selection was applied to the ST8SIA2 genomic region with the identification of an unambiguous sweep signal in a rather restricted region of 18 kb length surrounding the promoter. We also found that while the CGC-type emerged in anatomically modern humans in Africa over 100 thousand years ago, it has increased its frequency in Asia only during the past 20-30 thousand years. These findings support that the positive selection is driven by psychosocial stress due to changing social environments since around the last glacial maximum, and raise a possibility that schizophrenia extensively emerged during the Upper Paleolithic and Neolithic era.

Evolution of the thioester-containing proteins (TEPs) of the arthropoda, revealed by molecular cloning of TEP genes from a spider, Hasarius adansoni.

The thioester-containing protein (TEP) family of genes, found in most Eumetazoan genomes, is classified into two subfamilies: the alpha-2-macroglobulin (A2M) subfamily and the C3 subfamily. Many A2M subfamily members, including insect TEP (iTEP), have been reported from the Arthropoda, whereas the C3 subfamily members have been reported only from two horseshoe crab species thus far. To elucidate the evolution of these genes among the Arthropoda, TEP genes were isolated from a spider, Hasarius adansoni (Chelicerata), by reverse transcription polymerase chain reaction (RT-PCR) amplification using universal degenerate primers specific for the thioester region. Four different TEP genes were identified. Phylogenetic analysis using the entire amino acid sequences of these and various other TEP sequences from the Eumetazoa indicated that two of the spider genes are type C3 (HaadC3-1 and HaadC3-2), one is type A2M (HaadA2M) and the other is closely related to iTEP (HaadiTEP). These results suggest that the common ancestor of the Arthropoda possessed at least three TEP genes, C3, A2M and iTEP and that they were lost differentially in the Crustacean and Hexapodan lineages.

Evolution of thioester-containing proteins revealed by cloning and characterization of their genes from a cnidarian sea anemone, Haliplanella lineate.

To elucidate the evolutionary origin of genes encoding thioester-containing proteins (TEPs), TEP genes were isolated from a cnidarian, a sea anemone, Haliplanella lineate. Phylogenetic tree analysis of the four identified cnidarian TEP genes and various TEP genes of many metazoa, indicated that they could be classified into two subfamilies: the alpha-2-macroglobulin (A2M) subfamily encodining A2M, CD109 and insect TEPs, and the C3 subfamily encoding complement C3, C4 and C5. Two of the four cnidarian TEP genes belonged to the A2M subfamily, showing a close similarity to human A2M and CD109, respectively and thus were termed HaliA2M and HaliCD109. The other two genes belonged to the C3 subfamily, and were termed HaliC3-1 and HaliC3-2. Cnidarian TEPs retained the basic domain structure and functionally important residues for each molecule, and their mRNA were detected at different parts of the sea anemone body. These results suggest that gene duplication and subsequent functional differentiation among C3, A2M and CD109 were very ancient events predating the divergence of the cnidaria and bilateria.