Development of genotyping method for functionally relevant variants of cytochromes P450 in cynomolgus macaques.

In cynomolgus macaques (Macaca fascicularis), widely used in drug metabolism studies, CYP2C9, CYP2C76, CYP2D6, CYP3A4, and CYP3A5, important drug-metabolizing enzymes, are abundantly expressed in liver and metabolize cytochrome P450 substrates. CYP2C9 (c.334A>C), CYP2C76 (c.449TG>A), CYP2D6 (c.891A>G), CYP3A4 (IVS3 + 1G>del), and CYP3A5 (c.625A>T) substantially influence metabolic activity of enzymes, and thus are important variants in drug metabolism studies. In this study, a real-time PCR method was developed for genotyping these variants. The validity of the methods was verified by genotyping two wild type, two heterozygous, and two homozygous DNAs and was used to genotype 41 cynomolgus macaques (from Cambodia, Indonesia, the Philippines, or Vietnam) for the five variants, along with another important variant CYP2C19 (c.308C>T). The CYP2C9 and CYP2C19 variants were found only in Cambodian and Vietnamese animals, while the CYP2C76 and CYP2D6 variants were found only in Indonesian and Philippine animals. The CYP3A4 and CYP3A5 variants were not found in any of the animals analyzed. Mauritian animals, genotyped using next-generation sequencing data for comparison, possessed the CYP2C19 and CYP2D6 variants, but not the other variants. These results indicated differences in prevalence of these important variants among animal groups. Therefore, the genotyping tool developed is useful for drug metabolism studies using cynomolgus macaques.

A novel rare variant R292H in RTN4R affects growth cone formation and possibly contributes to schizophrenia susceptibility.

Reticulon 4 receptor (RTN4R) plays an essential role in regulating axonal regeneration and plasticity in the central nervous system through the activation of rho kinase, and is located within chromosome 22q11.2, a region that is known to be a hotspot for schizophrenia (SCZ) and autism spectrum disorder (ASD). Recently, rare variants such as copy-number variants and single-nucleotide variants have been a focus of research because of their large effect size associated with increased susceptibility to SCZ and ASD and the possibility of elucidating the pathophysiology of mental disorder through functional analysis of the discovered rare variants. To discover rare variants with large effect size and to evaluate their role in the etiopathophysiology of SCZ and ASD, we sequenced the RTN4R coding exons with a sample comprising 370 SCZ and 192 ASD patients, and association analysis using a large number of unrelated individuals (1716 SCZ, 382 ASD and 4009 controls). Through this mutation screening, we discovered four rare (minor allele frequency <1%) missense mutations (R68H, D259N, R292H and V363M) of RTN4R. Among these discovered rare mutations, R292H was found to be significantly associated with SCZ (P=0.048). Furthermore, in vitro functional assays showed that the R292H mutation affected the formation of growth cones. This study strengthens the evidence for association between rare variants within RTN4R and SCZ, and may shed light on the molecular mechanisms underlying the neurodevelopmental disorder.

Rare genetic variants in CX3CR1 and their contribution to the increased risk of schizophrenia and autism spectrum disorders.

CX3CR1, a G protein-coupled receptor solely expressed by microglia in the brain, has been repeatedly reported to be associated with neurodevelopmental disorders including schizophrenia (SCZ) and autism spectrum disorders (ASD) in transcriptomic and animal studies but not in genetic studies. To address the impacts of variants in CX3CR1 on neurodevelopmental disorders, we conducted coding exon-targeted resequencing of CX3CR1 in 370 Japanese SCZ and 192 ASD patients using next-generation sequencing technology, followed by a genetic association study in a sample comprising 7054 unrelated individuals (2653 SCZ, 574 ASD and 3827 controls). We then performed in silico three-dimensional (3D) structural modeling and in vivo disruption of Akt phosphorylation to determine the impact of the detected variant on CX3CR1-dependent signal transduction. We detected a statistically significant association between the variant Ala55Thr in CX3CR1 with SCZ and ASD phenotypes (odds ratio=8.3, P=0.020). A 3D structural model indicated that Ala55Thr could destabilize the conformation of the CX3CR1 helix 8 and affect its interaction with a heterotrimeric G protein. In vitro functional analysis showed that the CX3CR1-Ala55Thr mutation inhibited cell signaling induced by fractalkine, the ligand for CX3CR1. The combined data suggested that the variant Ala55Thr in CX3CR1 might result in the disruption of CX3CR1 signaling. Our results strengthen the association between microglia-specific genes and neurodevelopmental disorders.

High-resolution copy number variation analysis of schizophrenia in Japan.

Recent schizophrenia (SCZ) studies have reported an increased burden of de novo copy number variants (CNVs) and identified specific high-risk CNVs, although with variable phenotype expressivity. However, the pathogenesis of SCZ has not been fully elucidated. Using array comparative genomic hybridization, we performed a high-resolution genome-wide CNV analysis on a mainly (92%) Japanese population (1699 SCZ cases and 824 controls) and identified 7066 rare CNVs, 70.0% of which were small (<100 kb). Clinically significant CNVs were significantly more frequent in cases than in controls (odds ratio=3.04, P=9.3 × 10-9, 9.0% of cases). We confirmed a significant association of X-chromosome aneuploidies with SCZ and identified 11 de novo CNVs (e.g., MBD5 deletion) in cases. In patients with clinically significant CNVs, 41.7% had a history of congenital/developmental phenotypes, and the rate of treatment resistance was significantly higher (odds ratio=2.79, P=0.0036). We found more severe clinical manifestations in patients with two clinically significant CNVs. Gene set analysis replicated previous findings (e.g., synapse, calcium signaling) and identified novel biological pathways including oxidative stress response, genomic integrity, kinase and small GTPase signaling. Furthermore, involvement of multiple SCZ candidate genes and biological pathways in the pathogenesis of SCZ was suggested in established SCZ-associated CNV loci. Our study shows the high genetic heterogeneity of SCZ and its clinical features and raises the possibility that genomic instability is involved in its pathogenesis, which may be related to the increased burden of de novo CNVs and variable expressivity of CNVs.

Divergence of repetitive DNA sequences in the heterochromatin of medaka fishes: Molecular cytogenetic characterization of constitutive heterochromatin in two medaka species: Oryzias hubbsi and O. celebensis (Adrianichthyidae, Beloniformes).

The large biarmed chromosomes of Oryzias celebensis [2n = 36, fundamental arm number (FN) = 48] are considered to have resulted from fusions of acrocentric chromosomes in the ancestral karyotype of Oryzias, which is retained in O. hubbsi (2n = 48, FN = 48). To understand the molecular evolution of heterochromatin associated with karyotype reorganization in medaka fishes, we cloned 3 and 6 novel families of heterochromatin-related repetitive DNA sequences from O. hubbsi and O. celebensis, respectively, and characterized them using molecular cytogenetics. Two AT-rich repetitive sequences isolated from the genomic DNA of O. hubbsi, a 164-bp satellite DNA (OHU-RsaI-Scen) and a 177-bp telomere-specific repeat (OHU-RsaI-Stelo), were shown to be major components of the constitutive heterochromatin of centromeres and telomeres, respectively. A GC-rich 326-bp sequence, named OHU-AluI-M1, was colocalized with the 18S-28S ribosomal RNA gene cluster to a single autosomal pair of chromosomes and the W chromosome. In O. celebensis, 2 major satellite DNA sequences (the AT-rich 157-bp OCE-AluI-Scen sequence and the 186-bp OCE-HinfI-Scen sequence) were identified in the centromeric regions of almost all chromosomes. The 197-bp OCE-HinfI-S6 sequence was located in the centromeric and distal and/or interstitial heterochromatin of almost all chromosomes, and the 191-bp OCE-HinfI-S8 sequence was located in 6 pairs of chromosomes. Constitutive heterochromatin on the short arm of large submetacentric chromosome 5 was composed of at least 3 different repetitive sequences: the 171-bp OCE-AluI-S18 sequence, the 197-bp OCE-HinfI-S6 sequence and the 172-bp OCE-HinfI-S11 sequence. All families of repeated sequences showed no nucleotide sequence similarity with each other and high species-specificity among 7 different species. These results suggest that the heterochromatin of O. hubbsi and O. celebensis consists of various types of repetitive sequence and that the sequences evolved independently and were then amplified site-specifically in each lineage after karyotype reorganization occurred in the ancestral karyotype.

Homoeologous chromosomes of Xenopus laevis are highly conserved after whole-genome duplication.

It has been suggested that whole-genome duplication (WGD) occurred twice during the evolutionary process of vertebrates around 450 and 500 million years ago, which contributed to an increase in the genomic and phenotypic complexities of vertebrates. However, little is still known about the evolutionary process of homoeologous chromosomes after WGD because many duplicate genes have been lost. Therefore, Xenopus laevis (2n=36) and Xenopus (Silurana) tropicalis (2n=20) are good animal models for studying the process of genomic and chromosomal reorganization after WGD because X. laevis is an allotetraploid species that resulted from WGD after the interspecific hybridization of diploid species closely related to X. tropicalis. We constructed a comparative cytogenetic map of X. laevis using 60 complimentary DNA clones that covered the entire chromosomal regions of 10 pairs of X. tropicalis chromosomes. We consequently identified all nine homoeologous chromosome groups of X. laevis. Hybridization signals on two pairs of X. laevis homoeologous chromosomes were detected for 50 of 60 (83%) genes, and the genetic linkage is highly conserved between X. tropicalis and X. laevis chromosomes except for one fusion and one inversion and also between X. laevis homoeologous chromosomes except for two inversions. These results indicate that the loss of duplicated genes and inter- and/or intrachromosomal rearrangements occurred much less frequently in this lineage, suggesting that these events were not essential for diploidization of the allotetraploid genome in X. laevis after WGD.

Mushroom body-preferential expression of proteins/genes involved in endoplasmic reticulum Ca(2+)-transport in the worker honeybee (Apis mellifera L.) brain.

To identify the molecular characteristics specific to the mushroom body (MB, a higher processing centre) neurones in the honeybee brain, we previously used proteomics to identify proteins that are preferentially expressed in these MBs. Here we continued our proteomic analysis to show that reticulocalbin, which is involved in endoplasmic reticulum (ER) Ca(2+) transport, is also preferentially expressed in the MBs in the honeybee brain. Gene expression analysis revealed that reticulocalbin is preferentially expressed in the large-type Kenyon cells, which are MB-intrinsic neurones. In addition, the gene for the ryanodine receptor, which is also involved in ER Ca(2+) transport, was also preferentially expressed in the large-type Kenyon cells. In contrast, the expression of three other ER-related genes, protein disulphide isomerase, sec61 and erp60, was not enriched in the MBs. These findings further support the notion that the function of ER Ca(2+)-signalling, but not the mere intracellular density of ER, is specifically enhanced in the large-type Kenyon cells in the honeybee brain.

Quantitative autistic traits ascertained in a national survey of 22 529 Japanese schoolchildren.

OBJECTIVE: Recent epidemiologic studies worldwide have documented a rise in prevalence rates for autism spectrum disorders (ASD). Broadening of diagnostic criteria for ASD may be a major contributor to the rise in prevalence, particularly if superimposed on an underlying continuous distribution of autistic traits. This study sought to determine the nature of the population distribution of autistic traits using a quantitative trait measure in a large national population sample of children. METHOD: The Japanese version of the Social Responsiveness Scale (SRS) was completed by parents on a nationally representative sample of 22 529 children, age 6-15. RESULTS: Social Responsiveness Scale scores exhibited a skewed normal distribution in the Japanese population with a single-factor structure and no significant relation to IQ within the normal intellectual range. There was no evidence of a natural 'cutoff' that would differentiate populations of categorically affected children from unaffected children. CONCLUSION: This study provides evidence of the continuous nature of autistic symptoms measured by the SRS, a validated quantitative trait measure. The findings reveal how paradigms for diagnosis that rest on arbitrarily imposed categorical cutoffs can result in substantial variation in prevalence estimation, especially when measurements used for case assignment are not standardized for a given population.

Quantification of interferon, interleukin, and Toll-like receptor 7 mRNA in quail splenocytes using real-time PCR.

Japanese quail (Coturnix japonica) are farmed worldwide as poultry. Quail have been used as experimental animals in various scientific fields, but their immunological characteristics have not been well characterized. In this study, to develop a method for analyzing the innate immune response of quail to infectious pathogens, we determined the nucleotide sequences of major interleukins (IL) and Toll-like receptor (TLR)-7 of quail and developed quantitative real-time PCR assays. The nucleotide sequences of quail IL-1ß, IL-4, IL-6, IL-8, IL-10, IL-12a, IL-12b, IL-13, IL-18, and TLR-7 were determined based on the sequences of the chicken genes. Specific primers for each of these genes and previously reported interferon (IFN)-α, IFN-γ, and IL-2 genes were designed for quantitative real-time PCR. Standard curves for quantification were established using serial dilutions of external standard plasmids containing real-time PCR products. Then, real-time PCR was performed to monitor the kinetics of quail immune-related gene expression induced in splenocytes stimulated with concanavalin A. After amplification, the r(2) values of the standard curves for all target genes were above 0.980. Melting analysis of real-time PCR revealed specific amplification of each gene that could be visualized clearly as a single peak of melting temperature in a melt peak chart. These data show that the mRNA expressions of quail immune-related genes can be accurately quantified using this real-time PCR assay. In this study, we showed the nucleotide sequences of several quail cytokine mRNA and constructed the quantitative real-time PCR for quail immune-related genes.

The ZW micro-sex chromosomes of the Chinese soft-shelled turtle (Pelodiscus sinensis, Trionychidae, Testudines) have the same origin as chicken chromosome 15.

The Chinese soft-shelled turtle (Pelodiscus sinensis, Trionychidae, Testudines) has ZZ/ZW-type micro-sex chromosomes where the 18S-28S ribosomal RNA genes (18S-28S rDNA) are located. The W chromosome is morphologically differentiated from the Z chromosome by partial deletion and amplification of 18S-28S rDNA and W-specific repetitive sequences. We recently found a functional gene (TOP3B) mapped on the P. sinensis Z chromosome, which is located on chicken (Gallus gallus, GGA) chromosome 15. Then we cloned turtle homologues of 4 other GGA15-linked genes (GIT2, NF2, SBNO1, SF3A1) and localized them to P. sinensis chromosomes. The 4 genes all mapped on the Z chromosome, and 2 of them (SBNO1, SF3A1) were also localized to the W chromosome. Our mapping data suggest that at least one large inversion occurred between GGA15 and the P. sinensis Z chromosome, and that there are homologous regions in the distal portions of both the short and long arms between the Z and W chromosomes. W chromosomal differentiation in P. sinensis probably proceeded by the deletion of the proximal chromosomal region followed by 18S-28S rDNA amplification, after a paracentric inversion occurred at the breakpoints between the distal region of 18S-28S rDNA and the proximal region of SBNO1 on the Z chromosome.

Karyological characterization of the butterfly lizard (Leiolepis reevesii rubritaeniata, Agamidae, Squamata) by molecular cytogenetic approach.

Karyological characterization of the butterfly lizard (Leiolepis reevesii rubritaeniata) was performed by conventional Giemsa staining, Ag-NOR banding, FISH with the 18S-28S and 5S rRNA genes and telomeric (TTAGGG)n sequences, and CGH. The karyotype was composed of 2 distinct components, macrochromosomes and microchromosomes, and the chromosomal constitution was 2n = 2x = 36 (L(4)(m) + L(2)(sm) + M(2)(m) + S(4)(m) + 24 microchromosomes). NORs and the 18S-28S rRNA genes were located at the secondary constriction of the long arm of chromosome 1, and the 5S rRNA genes were localized to the pericentromeric region of chromosome 6. Hybridization signals of (TTAGGG)n sequences were observed at the telomeric ends of all chromosomes and interstitially at the same position as the 18S-28S rRNA genes, suggesting that in the Leiolepinae tandem fusion probably occurred between chromosome 1 and a microchromosome where the 18S-28S rRNA genes were located. CGH analysis, however, failed to identify sex chromosomes, suggesting that this species may have a TSD system or exhibit GSD with morphologically undetectable cryptic sex chromosomes. Homologues of 6 chicken Z-linked genes (ACO1/IREBP, ATP5A1, CHD1, DMRT1, GHR, RPS6) were all mapped to chromosome 2p in the same order as on the snake chromosome 2p.

Cynomolgus monkey CYPs: a comparison with human CYPs.

Characteristics of twelve cytochromes P450 (CYPs) from cynomolgus monkeys were compared with those of human CYPs that play an important role in drug metabolism. Eleven members of CYP1A, CYP2A, CYP2C, CYP2D, CYP2E, and CYP3A subfamilies from cynomolgus monkeys exhibited a high degree of homologies (more than 90%) in cDNA and amino acid sequences with corresponding human CYPs, and catalysed typical reactions of corresponding human CYPs. One member of the cynomolgus monkey CYP2C subfamily, CYP2C76, exhibited a lower homology (around 70%) in amino acid sequences with other cynomolgus monkey and human CYP2C subfamilies. CYP2C76 catalysed typical CYP2C substrates with low activities, and has not been found in humans. CYPs identified in cynomolgus monkeys were similar to CYP1A1, CYP1A2, CYP2A6, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4, and CYP3A5 in humans. These results indicate that cynomolgus monkeys express CYPs similar to human CYPs that are important in drug metabolism.

Genomic structure and transcript variants of the bovine DAZL gene.

The Deleted in AZoospermia Like (DAZL) gene is a member of the DAZ family and encodes an RNA-binding protein that is expressed in prenatal and postnatal germ cells of males and females. In the human, there are five highly-related members in the DAZ family, four (DAZ1-4) on the Y chromosome and one (DAZL) on an autosome (HSA3). Mutations in these genes have been linked to severe spermatogenic failure and infertility in men. In the present study, we have cloned and characterized the bovine DAZL (bDAZL) gene. The full-length bDAZL cDNA is predicted to encode a protein of 295 amino acids with an RNA recognition motif. The deduced protein sequence of bDAZL is 96 and 97% similar to human and mouse DAZL, respectively. Fluorescence in situ hybridization (FISH) maps bDAZL to the distal region on BTA1q. The bDAZL gene consists of 11 exons and 10 introns. A bDAZL pseudogene was identified on BTA16. Expression analysis of bDAZL in 13 different tissues by RT-PCR shows that two transcripts, variant 1 (2,996 bp) and variant 2 (1,373 bp), of the bDAZL gene are detected only in testis mRNA. The variants probably result from alternative RNA splicing as variant 1 contains an additional 1,623-bp insertion in the 3' UTR. Our results lay the groundwork for possible single nucleotide polymorphism (SNP) and functional studies of the DAZL gene in cattle.

CYP2C76-mediated species difference in drug metabolism: a comparison of pitavastatin metabolism between monkeys and humans.

The monkey is often used to predict metabolism of drugs in humans since it generally shows a metabolic pattern similar to humans. However, metabolic profiles different from humans are occasionally seen in monkeys for some drugs including pitavastatin. Recently, we have successfully identified a monkey-specific cytochrome P450 (CYP) 2C76, which possibly accounts for a species difference between monkeys and humans because of its sequence and functional uniqueness. The present study on the role of CYP2C76 and other monkey CYP2Cs in pitavastatin metabolism, as an example, has revealed that CYP2C76 is important for the metabolism of the lactone form, indicating a major role of CYP2C76 for the difference in the metabolism of pitavastatin and possibly other drugs between monkeys and humans. The current investigation on the involvement of CYP2C76 in the metabolism of other drugs is expected to reveal further the further importance of this monkey-specific drug-metabolizing enzyme.