Integrating mitochondrial and nuclear genomic data to decipher the evolutionary history of Eubranchipus species in Japan.

Understanding the genetic diversity and evolutionary history of species is crucial for their conservation and management. In this study, we investigated the genetic diversity and phylogenetic relationships among Eubranchipus species occurring in Japan. Phylogenetic analyses revealed that nuclear and mitochondrial data yield incompatible results. In E. uchidai, nuclear data support the monophyly of the Shimokita area, while mitochondrial data indicate a clustering of Higashidori2 individuals with Hokkaido (Ishikari and Wakkanai) E. uchidai. Similar incongruences were observed in E. hatanakai, where nuclear data favor the monophyly of the Chokai area, while mitochondrial data cluster some Chokai pool 3 individuals with Aizu individuals. These incompatibilities might be caused by mitochondrial gene flow. The findings emphasize the importance of considering both nuclear and mitochondrial data during phylogenetic studies and provide valuable insights into the complex dynamics of migration and genetic exchange in Eubranchipus species.

Identification and Characterization of Genes for the Allo A Lectins in Japanese Rhinoceros Beetle (Trypoxylus dichotomus [Allomyrina dichotoma]).

The Japanese rhinoceros beetle (Trypoxylus dichotomus [Allomyrina dichotoma]) produces the lectins allo A-I and allo A-II, which have strong N-acetyllactosamine (Galß1-4GlcNAc)-binding activity. It has been suggested that the two lectins are formed from three subunits (α, ß, and γ), with allo A-I comprising α and γ subunits and allo A-II comprising ß and γ subunits. Here, we determined the cDNA sequences of these subunits using both conventional polymerase chain reaction (PCR)-cloning-sequencing and transcriptome-sequencing analyses. For the α and ß subunits, one gene (locus) for each was predicted, whereas for the γ subunit, two types of cDNA sequences were obtained, which we named γ1 and γ2. These two types probably have distinct loci. Average nucleotide sequence identities among the subunits ranged from 87.6% (between α and γ1) to 92.6% (between γ1 and γ2), suggesting that they form a gene family. Although no homology was found between the sequences of allo A and other known lectin proteins in a protein database search, some unknown proteins containing the DUF3421 domain were identified. Those DUF3421 domain-encoding proteins are upregulated in the insect larval midgut. Thus, we infer that allo A genes also play an important role in larvae and that their lectin activity may have been obtained collaterally.

A founder haplotype of APOE-Sendai mutation associated with lipoprotein glomerulopathy.

Lipoprotein glomerulopathy (LPG) is a hereditary disease characterized by lipoprotein thrombi in the glomerulus, hyperlipoproteinemia, and a marked increase in serum apolipoprotein E (APOE). More than 12 APOE mutations have been identified as causes of LPG, and APOE-Sendai (Arg145Pro) mutation was frequently detected in patients from the eastern part of Japan including Yamagata prefecture. Recently, effective therapy with intensive lipid-lowering agents was established, and epidemiologic data are required for early diagnosis. We determined the haplotype structure of APOE-Sendai in 13 patients from 9 unrelated families with LPG, and found that the haplotype of all APOE-Sendai mutations was identical, suggesting that APOE-Sendai mutation is common in Japanese patients probably through a founder effect. We also studied the gene frequency of APOE-Sendai in 2023 control subjects and 418 patients receiving hemodialysis in Yamagata prefecture using the TaqMan method, but did not identify any subjects carrying the mutation, indicating that it is very rare in the general population even in the eastern part of Japan. In addition to APOE mutation, other genetic and/or epigenetic factors are considered to be involved in the pathogenesis of LPG because of its low penetrance. The patients did not have a common haplotype of the counterpart APOE allele, and some patients had the same haplotype of the counterpart APOE allele as the asymptomatic carriers. These results suggest that the counterpart APOE allele is not likely associated with the onset of LPG. Further study is required to clarify the pathogenesis of LPG.

The history of human populations in the Japanese Archipelago inferred from genome-wide SNP data with a special reference to the Ainu and the Ryukyuan populations.

The Japanese Archipelago stretches over 4000 km from north to south, and is the homeland of the three human populations; the Ainu, the Mainland Japanese and the Ryukyuan. The archeological evidence of human residence on this Archipelago goes back to >30 000 years, and various migration routes and root populations have been proposed. Here, we determined close to one million single-nucleotide polymorphisms (SNPs) for the Ainu and the Ryukyuan, and compared these with existing data sets. This is the first report of these genome-wide SNP data. Major findings are: (1) Recent admixture with the Mainland Japanese was observed for more than one third of the Ainu individuals from principal component analysis and frappe analyses; (2) The Ainu population seems to have experienced admixture with another population, and a combination of two types of admixtures is the unique characteristics of this population; (3) The Ainu and the Ryukyuan are tightly clustered with 100% bootstrap probability followed by the Mainland Japanese in the phylogenetic trees of East Eurasian populations. These results clearly support the dual structure model on the Japanese Archipelago populations, though the origins of the Jomon and the Yayoi people still remain to be solved.

Complete mitochondrial genomes of three fairy shrimps from snowmelt pools in Japan.

BACKGROUND: Fairy shrimps belong to order Anostraca, class Branchiopoda, subphylum Crustacea, and phylum Arthropoda. Three fairy shrimp species (Eubranchipus uchidai, E. asanumai, and E. hatanakai) that inhabit snowmelt pools are currently known in Japan. Whole mitochondrial genomes are useful genetic information for conducting phylogenetic analyses. Mitochondrial genome sequences for Branchiopoda members are gradually being collated. RESULTS: Six whole mitochondrial genomes from the three Eubranchipus species are presented here. Eubranchipus species share the anostracan pattern of gene arrangement in their mitochondrial genomes. The mitochondrial genomes of the Eubranchipus species have a higher GC content than those of other anostracans. Accelerated substitution rates in the lineage of Eubranchipus species were observed. CONCLUSION: This study is the first to obtain whole mitochondrial genomes for Far Eastern Eubranchipus species. We show that the nucleotide sequences of cytochrome oxidase subunit I and the 16S ribosomal RNA of E. asanumai presented in a previous study were nuclear mitochondrial DNA segments. Higher GC contents and accelerated substitution rates are specific characteristics of the mitochondrial genomes of Far Eastern Eubranchipus. The results will be useful for further investigations of the evolution of Anostraca as well as Branchiopoda.

A hypervariable STR polymorphism in the complement factor I (CFI) gene: Asian-specific alleles.

In this study, a short tandem repeat (STR) polymorphism in intron 7 of the human complement factor I (CFI) gene was studied in 637 DNA samples obtained from African, German, Thai, and Japanese populations and German and Japanese families. A total of 41 alleles were observed and classified into two groups, L and H, based on size differences. Group H, which consisted of 16 alleles, was observed only in Thai and Japanese populations at frequencies of 0.162 and 0.116, respectively, and was strongly associated with c.1217A in exon 11 (CFI*Ah). The heterozygosity values ranged from 0.89 in German to 0.93 in Thai populations. This STR would be a useful supplementary marker for forensic individualization.

Mitochondrial DNA analysis of Hokkaido Jomon skeletons: remnants of archaic maternal lineages at the southwestern edge of former Beringia.

To clarify the colonizing process of East/Northeast Asia as well as the peopling of the Americas, identifying the genetic characteristics of Paleolithic Siberians is indispensable. However, no genetic information on the Paleolithic Siberians has hitherto been reported. In the present study, we analyzed ancient DNA recovered from Jomon skeletons excavated from the northernmost island of Japan, Hokkaido, which was connected with southern Siberia in the Paleolithic period. Both the control and coding regions of their mitochondrial DNA (mtDNA) were analyzed in detail, and we confidently assigned 54 mtDNAs to relevant haplogroups. Haplogroups N9b, D4h2, G1b, and M7a were observed in these individuals, with N9b being the predominant one. The fact that all these haplogroups, except M7a, were observed with relatively high frequencies in the southeastern Siberians, but were absent in southeastern Asian populations, implies that most of the Hokkaido Jomon people were direct descendants of Paleolithic Siberians. The coalescence time of N9b (ca. 22,000 years) was before or during the last glacial maximum, implying that the initial trigger for the Jomon migration in Hokkaido was increased glaciations during this period. Interestingly, Hokkaido Jomons lack specific haplogroups that are prevailing in present-day native Siberians, implying that diffusion of these haplogroups in Siberia might have been after the beginning of the Jomon era, about 15,000 years before present.

Nutrient Imbalance of the Host Plant for Larvae of the Pale Grass Blue Butterfly May Mediate the Field Effect of Low-Dose Radiation Exposure in Fukushima: Dose-Dependent Changes in the Sodium Content.

The pale grass blue butterfly Zizeeria maha is sensitive to low-dose radioactive pollution from the Fukushima nuclear accident in the field but is also highly tolerant to radioactive cesium (137Cs) in an artificial diet in laboratory experiments. To resolve this field-laboratory paradox, we hypothesize that the butterfly shows vulnerability in the field through biochemical changes in the larval host plant, the creeping wood sorrel Oxalis corniculata, in response to radiation stress. To test this field-effect hypothesis, we examined nutrient contents in the host plant leaves from Tohoku (mostly polluted areas including Fukushima), Niigata, and Kyushu, Japan. Leaves from Tohoku showed significantly lower sodium and lipid contents than those from Niigata. In the Tohoku samples, the sodium content (but not the lipid content) was significantly negatively correlated with the radioactivity concentration of cesium (137Cs) in leaves and with the ground radiation dose. The sodium content was also correlated with other nutrient factors. These results suggest that the sodium imbalance of the plant may be caused by radiation stress and that this nutrient imbalance may be one of the reasons that this monophagous butterfly showed high mortality and morphological abnormalities in the field shortly after the accident in Fukushima.

Polyalanine expansion of PHOX2B in congenital central hypoventilation syndrome: rs17884724:A>C is associated with 7-alanine expansion.

With congenital central hypoventilation syndrome (CCHS), most patients have a de novo 5-13 polyalanine expansion mutation in PHOX2B. We reported previously that de novo polyalanine expansion mutations were of paternal origin and were derived from unequal sister chromatid exchange during spermatogenesis in six and four informative families, respectively. In this study, we analyzed the relationship between haplotypes and de novo polyalanine expansion in PHOX2B and found that haplotypes carrying rs17884724:A>C were detected frequently in 7-alanine expanded (27-alanine) mutant alleles, which are the most prevalent mutations in CCHS. The allele with rs17884724:A>C made fewer nucleotide mismatches in the misalignment at crossing-over than the allele without rs17884724:A>C. The high frequency of rs17884724:A>C in 7-alanine expansion (27-alanine) mutations also supported the unequal crossover mechanism for polyalanine expansion. We also confirmed the paternal origin of de novo polyalanine expansion mutation and unequal sister chromatid exchange association in three more patients. In spite of paternal bias, the paternal age effect on CCHS incidence was not observed. De novo polyalanine expansion mutations are mainly derived from unequal sister chromatid exchange during spermatogenesis because of replication and/or repair systems that are specific for spermatogenesis.

Mitochondrial DNA analysis of Jomon skeletons from the Funadomari site, Hokkaido, and its implication for the origins of Native American.

Ancient DNA recovered from 16 Jomon skeletons excavated from Funadomari site, Hokkaido, Japan was analyzed to elucidate the genealogy of the early settlers of the Japanese archipelago. Both the control and coding regions of their mitochondrial DNA were analyzed in detail, and we could securely assign 14 mtDNAs to relevant haplogroups. Haplogroups D1a, M7a, and N9b were observed in these individuals, and N9b was by far the most predominant. The fact that haplogroups N9b and M7a were observed in Hokkaido Jomons bore out the hypothesis that these haplogroups are the (pre-) Jomon contribution to the modern Japanese mtDNA pool. Moreover, the fact that Hokkaido Jomons shared haplogroup D1 with Native Americans validates the hypothesized genetic affinity of the Jomon people to Native Americans, providing direct evidence for the genetic relationships between these populations. However, probably due to the small sample size or close consanguinity among the members of the site, the frequencies of the haplogroups in Funadomari skeletons were quite different from any modern populations, including Hokkaido Ainu, who have been regarded as the direct descendant of the Hokkaido Jomon people. It appears that the genetic study of ancient populations in northern part of Japan brings important information to the understanding of human migration in northeast Asia and America.

HERC1 polymorphisms: population-specific variations in haplotype composition.

Human HERC1 is one of six HERC proteins and may play an important role in intracellular membrane trafficking. The human HERC1 gene is suggested to have been affected by local positive selection. To assess the global frequency distributions of coding and non-coding single nucleotide polymorphisms (SNPs) in the HERC1 gene, we developed a new simultaneous genotyping method for four SNPs, and applied this method to investigate 1213 individuals from 12 global populations. The results confirmed remarked differences in the allele and haplotype frequencies between East Asian and non-East Asian populations. One of the three common haplotypes observed was found to be characteristic of East Asians, who showed a relatively uniform distribution of haplotypes. Information on haplotypes would be useful for testing the function of polymorphisms in the HERC1 gene. This is the first study to investigate the distribution of HERC1 polymorphisms in various populations.

Cardiac ion channel gene mutations in sudden infant death syndrome.

Sudden infant death syndrome (SIDS) is multifactorial and may result from the interaction of a number of environmental, genetic, and developmental factors. We studied three major genes causing long QT syndrome in 42 Japanese SIDS victims and found five mutations, KCNQ1-K598R, KCNH2-T895M, SCN5A-F532C, SCN5A-G1084S, and SCN5A-F1705S, in four cases; one case had both KCNH2-T895M and SCN5A-G1084S. All mutations were novel except for SCN5A-F532C, which was previously detected in an arrhythmic patient. Heterologous expression study revealed significant changes in channel properties of KCNH2-T895M, SCN5A-G1084S, and SCN5A-F1705S, but did not in KCNQ1-K598R and SCN5A-F532C. Our data suggests that nearly 10% of SIDS victims in Japan have mutations of the cardiac ion channel genes similar to in other countries.

Allele frequencies of a SNP and a 27-bp deletion that are the determinant of earwax type in the ABCC11 gene.

Allele frequencies for a SNP (rs17822931) and a 27-bp deletion that are the determinant of earwax type in the ABCC11 gene were investigated in seven Japanese, one Korean, and one German populations. The SNP will be useful as one of ancestry information markers, because it showed marked difference in frequencies between Asian and European populations.

Brown hagfish from the northwest and east coasts of Honshu, Japan are genetically different.

The brown hagfish (Eptatretus atami) is one of several known hagfish species occurring in Japanese coastal waters. To date, there has been no research studying genetic polymorphisms in the species. In the present study, we analyzed differences in nucleotide sequences between two populations: one from Suruga Bay on the Pacific coast of Honshu, Japan, and the other from the Sea of Japan, off Akita on the northwest coast of Honshu. We sequenced part of the cytochrome oxidase subunit 1 gene (COX1) from the mitochondrial genome, and three G protein-coupled receptor genes from the nuclear genome. Phylogenetic networks of all four genes showed divergence between the two populations. Further, comparison of the COX1 data using a phylogenetic tree for a range of hagfish species indicated clear differences between the populations, suggesting that they differ at the species level. The numbers of their teeth, in particular of fused cusps (anterior/posterior multicusps), also supported these findings. Individuals of the Suruga Bay population had 3/3 fused cusps, as described for E. atami, whereas individuals of the Akita population had 3/2 fused cusps. These results suggest that the brown hagfish from the Sea of Japan, off the northwest coast of Honshu, is a distinct species from E. atami.

Japaneseplex: A forensic SNP assay for identification of Japanese people using Japanese-specific alleles.

It is sometimes necessary to determine whether a forensic biological sample came from a Japanese person. In this study, we developed a 60-locus SNP assay designed for the differentiation of Japanese people from other East Asians using entirely and nearly Japanese-specific alleles. This multiplex assay consisted of 6 independent PCR reactions followed by single nucleotide extension. The average number and standard deviation of Japanese-specific alleles possessed by an individual were 0.81 ±â€¯0.93 in 108 Koreans from Seoul, 8.87 ±â€¯2.89 in 103 Japanese from Tottori, 17.20 ±â€¯3.80 in 88 Japanese from Okinawa, and 0 in 220 Han Chinese from Wuxi and Changsha. The Koreans had 0-4 Japanese-specific alleles per individual, whereas the Japanese had 4-26 Japanese-specific alleles. Almost all Japanese were distinguished from the Koreans and other people by the factorial correspondence and principal component analyses. The Snipper program was also useful to estimate the degree of Japaneseness. The method described here was successfully applied to the differentiation of Japanese from non-Japanese people in forensic cases. This Japanese-specific SNP assay was named Japaneseplex.

Utility of haplogroup determination for forensic mtDNA analysis in the Japanese population.

Sequence analysis of the hypervariable regions (HVRs) of mitochondrial DNA (mtDNA) are routinely performed in forensic casework, however, there are still issues to be resolved, such as the existence of multiple errors in published databases or the limitations of individual discrimination in certain populations. Here, we analyzed the coding region of mtDNA in detail by examining 36 haplogroup (HG)-defining single nucleotide polymorphisms (SNPs) using amplified product-length polymorphisms (APLP) method in conjunction with sequence analysis of HVR1 and HVR2 to establish a methodology for forensically reliable and practical mtDNA testing. The mtDNAs from 217 unrelated Japanese were examined and could be classified into 27 haplogroups. By combining the data of the coding region with those of HVRs, genetic diversity was slightly increased from 0.9817 to 0.9888 for HVR1/HG and from 0.9967 to 0.9970 for HVR1/HVR2/HG, as compared to the results of HVRs only. Moreover, in most cases, reliability of the HVR data could be confirmed by haplogroup motif analysis. Our mtDNA profiling method can provide reliable data in a time and cost-saving way due to the rapid and economical nature of APLP analysis.

Origin and evolution of gene for prolactin-induced protein.

Prolactin-induced protein (PIP) is a small protein secreted into the fluid in several glands. We determined the PIP coding sequences of 5 hominoid species and estimated the numbers of synonymous and nonsynonymous substitutions for each branch of the mammalian PIP gene tree. The branch connecting hominoids and Old World monkeys showed significantly higher nonsynonymous than synonymous substitutions. These changes tended to be accumulated in the fibronectin-binding domain. Many other primate branches also showed higher nonsynonymous than synonymous substitutions, thus suggesting that the PIP genes of primates have experienced some kind of positive selection. We also considered the phylogenetic relationship of the PIP gene with the alpha-2-macroglobulin gene family. The results indicate that the PIP gene arose by partial gene duplication from a member of the alpha-2-macroglobulin gene family after the divergence between amphibians and other tetrapods.

Genotyping of the c.1423C>T (p.P475S) polymorphism in the ADAMTS13 gene by APLP and HRM assays: Northeastern Asian origin of the mutant.

ADAMTS13 is a von Willebrand factor-cleaving protease. The mutant types of p.P475S (c.1423C>T) polymorphism in ADAMTS13 have a reduced activity in comparison with the wild type. In the present study, we investigated the frequency of the C-to-T substitution in 2584 genomic DNA samples from 25 Asian, European, and African populations using APLP (amplified product length polymorphism) and/or HRM (high-resolution melting) assays. Allele T (ADAMTS13(∗)T) was detected only in Asian populations and its frequency was observed to decrease gradually from north to south in 24 East Asian populations. Almost all ADAMTS13(∗)T were associated with ABO(∗)O. These results suggested that ADAMTS13(∗)T had occurred on a chromosome with ABO(∗)O in a northern part of East Asia. This SNP is useful as an ancestry-informative marker, and the present genotyping techniques are applicable to the investigation of an association between this SNP and aortic dissection (Kobayashi et al., 2012).

Multiplex APLP System for High-Resolution Haplogrouping of Extremely Degraded East-Asian Mitochondrial DNAs.

Mitochondrial DNA (mtDNA) serves as a powerful tool for exploring matrilineal phylogeographic ancestry, as well as for analyzing highly degraded samples, because of its polymorphic nature and high copy numbers per cell. The recent advent of complete mitochondrial genome sequencing has led to improved techniques for phylogenetic analyses based on mtDNA, and many multiplex genotyping methods have been developed for the hierarchical analysis of phylogenetically important mutations. However, few high-resolution multiplex genotyping systems for analyzing East-Asian mtDNA can be applied to extremely degraded samples. Here, we present a multiplex system for analyzing mitochondrial single nucleotide polymorphisms (mtSNPs), which relies on a novel amplified product-length polymorphisms (APLP) method that uses inosine-flapped primers and is specifically designed for the detailed haplogrouping of extremely degraded East-Asian mtDNAs. We used fourteen 6-plex polymerase chain reactions (PCRs) and subsequent electrophoresis to examine 81 haplogroup-defining SNPs and 3 insertion/deletion sites, and we were able to securely assign the studied mtDNAs to relevant haplogroups. Our system requires only 1×10-13 g (100 fg) of crude DNA to obtain a full profile. Owing to its small amplicon size (<110 bp), this new APLP system was successfully applied to extremely degraded samples for which direct sequencing of hypervariable segments using mini-primer sets was unsuccessful, and proved to be more robust than conventional APLP analysis. Thus, our new APLP system is effective for retrieving reliable data from extremely degraded East-Asian mtDNAs.