Search

Background A variety of dietary supplements are commercially available. However, the efficacy and safety of dietary supplement use in children are not well established. Understanding dietary supplement use is important for developing public health policy regarding dietary supplements. This study aimed to investigate the types of dietary supplements used and characteristics of dietary supplement users among Japanese elementary school children. Method We conducted a cross-sectional web-based questionnaire study. Dietary supplement use, socio-demographics, and health-related behaviors were assessed through mother-reported questionnaire. Types of dietary supplements were identified based on ingredient using product barcodes and brand names. Multivariate logistic regression analysis was conducted to investigate the socio-demographics and health-related behaviors associated with supplement use. Results Among 4933 children, 333 (6.8%) were identified as dietary supplement users. The most common supplement was amino acids or protein (1.4%), followed by n–3 fatty acids or fish oil (1.0%), probiotics (1.0%), multivitamins (0.9%), multivitamin-minerals (0.8%), and botanicals (0.8%). Overall, any dietary supplement use was significantly associated with the highest frequency of sports participation (odds ratio [OR], 2.58; 95% confidence interval [CI], 1.65–4.02), highest household income (OR, 1.87; 95% CI, 1.13–3.10), highest maternal educational level (OR, 1.82; 95% CI, 1.31–2.52), and male sex (OR, 1.38; 95% CI, 1.09–1.75). The highest frequency of sports participation was significantly associated with higher odds of use of amino acids or protein (OR, 6.06; 95% CI, 1.78–20.6) and multivitamins (OR, 3.56; 95% CI, 1.11–11.5), compared to the lowest frequency of sports participation. Conclusion This study showed that Japanese children primarily use non-vitamin, non-mineral supplements. Non-vitamin, non-mineral supplements should thus be included in future studies aimed at monitoring dietary supplement use. We also found that dietary supplement use in children was associated with sports participation. Guidelines for dietary supplement use for children, in particular sport participants, are needed.

DNA aneuploidy, the altered DNA content of a cell, is a common feature of canine tumors. However, it is unclear whether aneuploid DNA in canine tumor cells show centrosome amplification (CA), which contributes to numerical and structural chromosome aberrations that result in DNA aneuploidy. Here, we evaluated whether DNA aneuploidy and CA occur concurrently in canine tumor cell lines. Centrosome numbers were evaluated in 18 canine tumor cell lines by immunocytochemistry with anti-γ-tubulin antibody, and DNA content was evaluated by flow cytometry using propidium iodide. A total of 15 cell lines showed DNA aneuploidy, and CA was observed in 5 of these 15 cell lines. Together, our results suggest that DNA aneuploidy in canine tumor cells might be explained at least in part by CA. In addition, cell lines with CA may be useful tools to examine the detailed relationship between CA and DNA aneuploidy and the molecular mechanism of CA in canine tumor.

Objective Ultra-sensitive hormone assays have detected slight sex differences in blood estradiol (E2) levels in young children before adrenarche. However, the origin of circulating E2 in these individuals remains unknown. This study aimed to clarify how E2 is produced in young girls before adrenarche. Design This is a satellite project of the Japan Environment and Children’s Study organized by the National Institute for Environmental Studies. Methods We collected blood samples from healthy 6-year-old Japanese children (79 boys and 71 girls). Hormone measurements and data analysis were performed in the National Institute for Environmental Studies and the Medical Support Center of the Japan Environment and Children’s Study, respectively. Results E2 and follicle stimulating hormone (FSH) levels were significantly higher in girls than in boys, while dehydroepiandrosterone sulfate (DHEA-S) and testosterone levels were comparable between the two groups. Girls showed significantly higher E2/testosterone ratios than boys. In children of both sexes, a correlation was observed between E2 and testosterone levels and between testosterone and DHEA-S levels. Moreover, E2 levels were correlated with FSH levels only in girls. Conclusions The results indicate that in 6-year-old girls, circulating E2 is produced primarily in the ovary from adrenal steroids through FSH-induced aromatase upregulation. This study provides evidence that female-dominant E2 production starts several months or years before adrenarche. The biological significance of E2 biosynthesis in these young children needs to be clarified in future studies.

SRY-negative 46,XX testicular disorders of sex development (DSD) are very rare conditions. Recently, we identified a novel heterozygous NR5A1 mutation, p.Arg92Trp (c.274C>T, p.R92W), in 2 unrelated cases of 46,XX testicular/ovotesticular DSD. We report the clinical course from infancy to puberty in a Japanese male with SRY-negative 46,XX testicular DSD, carrying this p.Arg92Trp mutation in NR5A1. The patient naturally acquired the development of a penis and pubic hair during puberty. However, hypergonadotropic hypogonadism subsequently developed. More clinical cases will be needed to fully understand the effects of the p.Arg92Trp mutation on the ability to maintain testosterone secretion in 46,XX testicular DSD.

Several genome-wide association studies (GWASs) have reported the association between genetic variants and the habitual consumption of foods and drinks, however, no association data are available regarding the consumption of black tea. The present study aimed to identify genetic variants associated with black tea consumption in 12,258 Japanese participants. Data on black tea consumption were collected by a self-administered questionnaire, and genotype data were obtained from a single nucleotide polymorphism array. In the discovery GWAS, two loci met suggestive significance (p &lt, 1.0 &times, 10&minus, 6). Three genetic variants (rs2074356, rs144504271, and rs12231737) at 12q24 locus were also significantly associated with black tea consumption in the replication stage (p &lt, 0.05) and during the meta-analysis (p &lt, 5.0 &times, 8). The association of rs2074356 with black tea consumption was slightly attenuated by the additional adjustment for alcohol drinking frequency. In conclusion, genetic variants at the 12q24 locus were associated with black tea consumption in Japanese populations, and the association is at least partly mediated by alcohol drinking frequency.

Deep learning has rapidly been filtrating many aspects of human lives. In particular, image recognition by convolutional neural networks has inspired numerous studies in this area. Hardware and software technologies as well as large quantities of data have contributed to the drastic development of the field. However, the application of deep learning is often hindered by the need for big data and the laborious manual annotation thereof. To experience deep learning using the data compiled by us, we collected 2429 constrained headshot images of 277 volunteers. The collection of face photographs is challenging in terms of protecting personal information; we therefore established an online procedure in which both the informed consent and image data could be obtained. We did not collect personal information, but issued agreement numbers to deal with withdrawal requests. Gender and smile labels were manually and subjectively annotated only from the appearances, and final labels were determined by majority among our team members. Rotated, trimmed, resolution-reduced, decolorized, and matrix-formed data were allowed to be publicly released. Moreover, simplified feature vectors for data sciences were released. We performed gender and smile recognition by building convolutional neural networks based on the Inception V3 model with pre-trained ImageNet data to demonstrate the usefulness of our dataset.

The spindle assembly checkpoint (SAC) is a surveillance mechanism that prevents unequal segregation of chromosomes during mitosis. Abnormalities in the SAC are associated with chromosome instability and resultant aneuploidy. This study was performed to evaluate the SAC competence in canine malignant melanoma (CMM) using four aneuploid cell lines (CMeC1, CMeC2, KMeC, and LMeC). After treatment with nocodazole, a microtubule disrupting agent, CMeC1, KMeC, and LMeC cells were arrested in M phase, whereas CMeC2 cells were not arrested, and progressed into the next cell cycle phase without cytokinesis. Chromosome spread analysis revealed a significantly increased rate of premature sister chromatid separation in CMeC2 cells. Expression of the phosphorylated form of the SAC regulator, monopolar spindle 1 (Mps1), was lower in CMeC2 cells than in the other CMM cell lines. These results indicate that the SAC is defective in CMeC2 cells, which may partially explain aneuploidy in CMM. Thus, CMeC2 cells may be useful for further studies of the SAC mechanism in CMM and in determining the relationship between SAC incompetence and aneuploidy.

The human genome encodes ~750 G‐protein‐coupled receptors (GPCRs), including prokineticin receptor 2 (PROKR2) involved in the regulation of sexual maturation. Previously reported pathogenic gain‐of‐function mutations of GPCR genes invariably encoded aberrant receptors with excessive signal transduction activity. Although in vitro assays demonstrated that an artificially created inactive mutant of PROKR2 exerted paradoxical gain‐of‐function effects when co‐transfected with wild‐type proteins, such a phenomenon has not been observed in vivo. Here, we report a heterozygous frameshift mutation of PROKR2 identified in a 3.5‐year‐old girl with central precocious puberty. The mutant mRNA escaped nonsense‐mediated decay and generated a GPCR lacking two transmembrane domains and the carboxyl‐terminal tail. The mutant protein had no in vitro signal transduction activity; however, cells co‐expressing the mutant and wild‐type PROKR2 exhibited markedly exaggerated ligand‐induced Ca2+ responses. The results indicate that certain inactive PROKR2 mutants can cause early puberty by enhancing the functional property of coexisting wild‐type proteins. Considering the structural similarity among GPCRs, this paradoxical gain‐of‐function mechanism may underlie various human disorders.

Recently, a heterozygous missense mutation in NR5A1, p.R92W, was identified as a cause of 46,XX testicular/ovo-testicular disorders of sexual development (DSD). We report a sibling pair with 46,XX DSD due to an NR5A1 mutation with distinct phenotypes, including external and internal genitalia and gonads, for whom different rearing sexes were selected. Thus, the phenotypes of p.R92W vary, even within a family. The father of the patients showed oligozoospermia with the p.R92W mutation, suggesting that in 46,XY individuals, the mutation would cause various gonadal phenotypes. We review and discuss the general role of the R92W mutation in sexual development.

Background Studies on genetic effects of coffee consumption are scarce for Asian populations. We conducted a genome-wide association study (GWAS) of habitual coffee consumption in Japan using a self-reporting online survey. Results Candidate genetic loci associated with habitual coffee consumption were searched within a discovery cohort (N = 6,264) and confirmed in a replication cohort (N = 5,975). Two loci achieved genome-wide significance (P

Background Japan is traditionally a country with one of the highest levels of fish consumption worldwide, although the westernization of the Japanese diet has resulted in the reduction of fish consumption. A recent meta-analysis of genome-wide association studies (GWASs) on Western populations has identified a single nucleotide polymorphism (SNP) associated with fish intake frequency. Here, we examined the genetic basis for fish intake frequency among Japanese individuals. Results We conducted a meta-analysis of a GWAS including 12,603 Japanese individuals and identified a susceptibility locus for fish intake frequency at 12q24 (lead variant was rs11066015, P = 5.4 × 10−11). rs11066015 was in a strong linkage disequilibrium with rs671, a well-known SNP related to alcohol metabolism. When adjusted for alcohol drinking, the association between rs11066015 and fish intake frequency was substantially attenuated. Subgroup analysis revealed that the effect of the 12q24 variant on fish intake frequency was stronger in males than in females (P for interaction = 0.007) and stronger in the older subgroup than in the younger subgroup (P for interaction = 0.006). Conclusions Our findings suggest that the 12q24 locus is associated with fish intake frequency via alcohol drinking. This study can help contribute to personalized nutrition information, suggesting that fish intake should be promoted to consumers who have the rs11066015 minor allele, which is genetically linked to low fish intake frequency, especially in male and older individuals. Electronic supplementary material The online version of this article (10.1186/s12263-019-0646-6) contains supplementary material, which is available to authorized users.

Endogenous and exogenous androgens induce masculinization of external genitalia through binding to the androgen receptor (AR). The target genes of androgens in external genitalia remain to be determined, although previous studies have shown that the apolipoprotein D gene (APOD) was significantly upregulated by dihydrotestosterone (DHT), the most potent androgen in humans. In the present study, we performed microarray analysis for genital skin fibroblasts obtained from four boys with buried penis (the control individuals) and a patient with partial androgen insensitivity syndrome (PAIS) due to a hypomorphic mutation in AR (the PAIS patient). We identified 24 transcripts that were upregulated or downregulated by DHT in all samples of control individuals and, to a lesser extent, in the sample of the PAIS patient. Differences between DHT-treated and -untreated samples were small; the results of 24 transcripts did not reach statistical significance. The 24 transcripts included CYP1B1, a gene possibly involved in the development of genital tubercle in mice, and APOD, as well as several genes that have been reported as androgen targets in prostate or other tissues. The results of this study indicate that androgen-mediated masculinization of external genitalia is unlikely to depend on massive transcriptional changes in specific AR target genes. Rather, minor transcriptional changes of several genes, and/or a complex molecular network may play a major role in penile development. Importantly, our data suggest the possible involvement of CYP1B1 in human genital development and confirm the clinical importance of APOD as a biomarker for AR function.

The role of monogenic mutations in the development of 46,XX testicular/ovotesticular disorders of sex development (DSD) remains speculative. Although mutations in NR5A1 are known to cause 46,XY gonadal dysgenesis and 46,XX ovarian insufficiency, such mutations have not been implicated in testicular development of 46,XX gonads. Here, we identified identical NR5A1 mutations in two unrelated Japanese patients with 46,XX testicular/ovotesticular DSD. The p.Arg92Trp mutation was absent from the clinically normal mothers and from 200 unaffected Japanese individuals. In silico analyses scored p.Arg92Trp as probably pathogenic. In vitro assays demonstrated that compared with wildtype NR5A1, the mutant protein was less sensitive to NR0B1-induced suppression on the SOX9 enhancer element. Other sequence variants found in the patients were unlikely to be associated with the phenotype. The results raise the possibility that specific mutations in NR5A1 underlie testicular development in genetic females. This article is protected by copyright. All rights reserved

We investigated the frequency of copy number variations (CNVs) in the Y chromosome of Japanese children with hypospadias. We analyzed the copy number of the azoospermia factor (AZF) region and SRY, using multiplex ligation-dependent probe amplification. Four AZF-linked CNVs, including one novel simple duplication, were identified in 39 of 89 patients, at a frequency comparable to that of those in unaffected individuals. SRY-linked CNVs were absent in our patients. The results imply that CNVs in the AZF region and SRY are not associated with the risk of hypospadias in the Japanese population, although the pathogenicity of the AZF-linked simple duplication remains to be elucidated.

SRY-negative 46,XX testicular disorders of sex development (DSD) are very rare conditions. Recently, we identified a novel heterozygous NR5A1 mutation, p.Arg92Trp (c.274CT, p.R92W), in 2 unrelated cases of 46,XX testicular/ovotesticular DSD. We report the clinical course from infancy to puberty in a Japanese male with SRY-negative 46,XX testicular DSD, carrying this p.Arg92Trp mutation in NR5A1. The patient naturally acquired the development of a penis and pubic hair during puberty. However, hypergonadotropic hypogonadism subsequently developed. More clinical cases will be needed to fully understand the effects of the p.Arg92Trp mutation on the ability to maintain testosterone secretion in 46,XX testicular DSD.

Recently, a heterozygous missense mutation in NR5A1, p.R92W, was identified as a cause of 46,XX testicular/ovo-testicular disorders of sexual development (DSD). We report a sibling pair with 46,XX DSD due to an NR5A1 mutation with distinct phenotypes, including external and internal genitalia and gonads, for whom different rearing sexes were selected. Thus, the phenotypes of p.R92W vary, even within a family. The father of the patients showed oligozoospermia with the p.R92W mutation, suggesting that in 46,XY individuals, the mutation would cause various gonadal phenotypes. We review and discuss the general role of the R92W mutation in sexual development.

SOX9 haploinsufficiency underlies campomelic dysplasia (CD) with or without testicular dysgenesis. Current understanding of the phenotypic variability and mutation spectrum of SOX9 abnormalities remains fragmentary. Here, we report three patients with hitherto unreported SOX9 abnormalities. These patients were identified through molecular analysis of 33 patients with 46,XY disorders of sex development (DSD). Patients 1–3 manifested testicular dysgenesis or regression without CD. Patients 1 and 2 carried probable damaging mutations p.Arg394Gly and p.Arg437Cys, respectively, in the SOX9 C‐terminal domain but not in other known 46,XY DSD causative genes. These substitutions were absent from ~120,000 alleles in the exome database. These mutations retained normal transactivating activity for the Col2a1 enhancer, but showed impaired activity for the Amh promoter. Patient 3 harbored a maternally inherited ~491 kb SOX9 upstream deletion that encompassed the known 32.5 kb XY sex reversal region. Breakpoints of the deletion resided within nonrepeat sequences and were accompanied by a short‐nucleotide insertion. The results imply that testicular dysgenesis and regression without skeletal dysplasia may be rare manifestations of SOX9 abnormalities. Furthermore, our data broaden pathogenic SOX9 abnormalities to include C‐terminal missense substitutions which lead to target‐gene‐specific protein dysfunction, and enhancer‐containing upstream microdeletions mediated by nonhomologous end‐joining.

The common phenotype of nuclear receptor superfamily 5, group A, member 1 (NR5A1) gene mutations in 46,XY is gonadal dysgenesis without adrenal deficiency. Though the phenotype of gonadal dysgenesis is variable, ranging from complete female to normal male genitalia, an asymptomatic 46,XY male is rare. Preserved fertility has so far been described in only three affected 46,XY males with different mutations, but no functional analysis of these mutations has been performed. Here, we report on male siblings with hypospadias and their asymptomatic father in whom we identified a heterozygous NR5A1 mutation of c.910G>A, p.E304K. Western blotting and subcellular localization revealed no significant difference between the wild type (WT) and E304K. Electrophoretic mobility shift assay experiments showed that E304K abrogated DNA-binding ability. E304K reduced transactivation and had no dominant negative effect. In conclusion, we report on a novel hypomorphic NR5A1 mutation, which may be associated with the phenotype of the family.

Submicroscopic duplications involving SOX3 and/or its flanking regions have been identified in 46,XX individuals both with and without disorders of sex development, raising the question whether SOX3 overdosage is sufficient to induce testicular development in genetically female individuals. Here, we report a mother-daughter pair with female phenotypes and random X inactivation. The individuals carry complex X chromosomal rearrangements leading to a copy number gain of genomic regions involving SOX3 and its upstream region. The amplified DNA fragments were detected at Xq27. These results provide evidence that SOX3 overdosage permits normal sex development in 46,XX individuals with random X inactivation.

The human genome encodes more than 700 G-protein-coupled receptors (GPCRs), many of which are involved in hormone secretion. To date, more than 100 gain-of-function (activating) mutations in at least ten genes for GPCRs, in addition to several loss-of-function mutations, have been implicated in human endocrine disorders. Previously reported gain-of-function GPCR mutations comprise various missense substitutions, frameshift mutations, intragenic inframe deletions and copy-number gains. Such mutations appear in both germline and somatic tumour cells, and lead to various hormonal abnormalities reflecting excessive receptor activity. Phenotypic consequences of these mutations include distinctive endocrine syndromes, as well as relatively common hormonal abnormalities. Such mutations encode hyperfunctioning receptors with increased constitutive activity, broadened ligand specificity, increased ligand sensitivity and/or delayed receptor desensitization. Furthermore, recent studies proposed a paradoxical gain-of-function mechanism caused by inactive GPCR mutants. Molecular diagnosis of GPCR activating mutations serves to improve the clinical management of mutation-positive patients. This review aims to introduce new aspects regarding gain-of-function mutations in GPCR genes associated with endocrine disorders.

Nuclear receptor subfamily 5, group A, member 1 (NR5A1) is a nuclear receptor involved in gonadal and adrenal development. We identified a novel C-terminally truncating NR5A1 mutation, p.Leu423Trpfs*7, in dizygotic twins with 46,XY disorders of sex development. Our results highlight the functional importance of C-terminal region of NR5A1 and indicate that NR5A1 mutations can be associated with intrafamilial phenotypic variations, progressive testicular dysfunction, hypogonadotropic hypogonadism, and borderline adrenal dysfunction.

We report four patients with SRY-positive 46,XX testicular disorders of sex development (46,XX-TDSD) (cases 1-4). Case 1 exhibited underdeveloped external genitalia with hypospadias, case 2 manifested micropenis and cases 3 and 4 showed normal external genitalia. The Xp;Yp translocations occurred between the X- and the Y-differential regions in case 1, between PRKX and inverted PRKY in case 2 and between the X-chromosomal short arm pseudoautosomal region and the Y-differential regions in cases 3 and 4. The distance of the Yp breakpoint from SRY was ~0.75 Mb in case 1, ~6.5 Mb in case 2, ~2.3 Mb in case 3 and ~72 kb in case 4. The Xp;Yp translocation occurred within an 87-bp homologous segment of PRKX and PRKY in case 2, and between non-homologous regions with addition of an 18-bp sequence of unknown origin in case 4. X-inactivation analysis revealed random inactivation in cases 1-4. The results argue against the notion that undermasculinization in 46,XX-TDSD is prone to occur when translocated Yp materials are small (100 kb of the Y-differential region), and imply that the Xp;Yp translocations result from several mechanisms including non-allelic homologous recombination and non-homologous end joining.

Obesity is highly associated with colon cancer development. Whereas it is generally attributed to pro-tumorigenic effects of high fat diet (HFD), we here show that a common genetic basis for predisposition to obesity and colon cancer might also underlie the close association. Comparison across multiple rat strains revealed that strains prone to colon tumorigenesis initiated by a dietary carcinogen amino-1-methyl-6-phenylimidazo [4,5-b] pyridine (PhIP) tended to develop obesity. Through transcriptome and extensive immunoblotting analyses, we identified the basal level of activated AKT in colonic crypts as a biomarker for the common predisposition. Notably, PhIP induced activation of AKT, which could persist for several weeks under a low fat diet (LFD), but not under HFD. On the other hand, PhIP and HFD independently induced Wnt pathway activation and inhibited apoptosis, through distinct mechanisms involving GSK-3β, caspase 3 and poly-ADP ribose polymerase (PARP). Taken together, these observations provide mechanistic insights into how PhIP-induced activation of AKT might cooperate with HFD at multiple levels toward development of colon cancer.

Combined pituitary hormone deficiency (CPHD), isolated hypogonadotropic hypogonadism (IHH), Kallmann syndrome (KS), and septo-optic dysplasia (SOD) are genetically related conditions caused by abnormal development of the anterior midline in the forebrain. Although mutations in the fibroblast growth factor receptor 1 (FGFR1) gene have been implicated in the development of IHH, KS, and SOD, the relevance of FGFR1 abnormalities to CPHD remains to be elucidated. Here, we report a Japanese female patient with CPHD and FGFR1 haploinsufficiency. The patient was identified through copy-number analyses and direct sequencing of FGFR1 performed for 69 patients with CPHD. The patient presented with a combined deficiency of GH, LH and FSH, and multiple neurological abnormalities. In addition, normal TSH values along with a low free T4 level indicated the presence of central hypothyroidism. Molecular analyses identified a heterozygous ~ 8.5 Mb deletion involving 56 genes and pseudogenes. None of these genes except FGFR1 have been associated with brain development. No FGFR1 abnormalities were identified in the remaining 68 patients, although two patients carried nucleotide substitutions (p.V102I and p.S107L) that were assessed as benign polymorphism by in vitro functional assays. These results indicate a possible role of FGFR1 in anterior pituitary function and the rarity of FGFR1 abnormalities in patients with CPHD.

Forkhead-box (FOX) family proteins, involved in cell growth and differentiation as well as embryogenesis and longevity, are DNA-binding proteins regulating transcription and DNA repair. The focus of this review is on the mechanisms of FOX-related human carcinogenesis. FOXA1 is overexpressed as a result of gene amplification in lung cancer, esophageal cancer, ER-positive breast cancer and anaplastic thyroid cancer and is point-mutated in prostate cancer. FOXA1 overexpression in breast cancer and prostate cancer is associated with good or poor prognosis, respectively. Single nucleotide polymorphism (SNP) within the 5'-UTR of the FOXE1 (TTF2) gene is associated with thyroid cancer risk. FOXF1 overexpression in breast cancer is associated with epithelial-to-mesenchymal transition (EMT). FOXM1 is overexpressed owing to gene amplification in basal-type breast cancer and diffuse large B-cell lymphoma (DLBCL), and it is transcriptionally upregulated owing to Hedgehog-GLI, hypoxia-HIF1α or YAP-TEAD signaling activation. FOXM1 overexpression leads to malignant phenotypes by directly upregulating CCNB1, AURKB, MYC and SKP2 and indirectly upregulating ZEB1 and ZEB2 via miR-200b downregulation. Tumor suppressor functions of FOXO transcription factors are lost in cancer cells as a result of chromosomal translocation, deletion, miRNA-mediated repression, AKT-mediated cytoplasmic sequestration or ubiquitination-mediated proteasomal degradation. FOXP1 is upregulated as a result of gene fusion or amplification in DLBCL and MALT lymphoma and also repression of miRNAs, such as miR-1, miR-34a and miR-504. FOXP1 overexpression is associated with poor prognosis in DLBCL, gastric MALT lymphoma and hepatocellular carcinoma but with good prognosis in breast cancer. In neuroblastoma, the entire coding region of the FOXR1 (FOXN5) gene is fused to the MLL or the PAFAH1B gene owing to interstitial deletions. FOXR1 fusion genes function as oncogenes that repress transcription of FOXO target genes. Whole-genome sequencing data from tens of thousands of human cancers will uncover the mutational landscape of FOX family genes themselves as well as FOX-binding sites, which will be ultimately applied for cancer diagnostics, prognostics, and therapeutics.

Nuclear receptor subfamily 5, group A, member 1 (NR5A1) is a nuclear receptor involved in gonadal and adrenal development. We identified a novel C-terminally truncating NR5A1 mutation, p.Leu423Trpfs*7, in dizygotic twins with 46,XY disorders of sex development. Our results highlight the functional importance of C-terminal region of NR5A1 and indicate that NR5A1 mutations can be associated with intrafamilial phenotypic variations, progressive testicular dysfunction, hypogonadotropic hypogonadism, and borderline adrenal dysfunction.

We developed a novel flow injection assay for cortisol based on competitive immunologic reactions, magnetic separation, and electrochemical measurement. The proposed flow assay system was composed ...

A novel immunologic method using antigen-antibody reactions and electrochemical measurement to monitor the oocyte maturation-inducing hormone 17, 20β-dihydroxy-4-pregnen-3-one (DHP) in fish was dev...

The present study was conducted to assess involvement of oxidative stress in lung adeno-carcinogenesis, using mice deficient in the 8-hydroxyguanine DNA glycosylase 1 (Ogg1) gene encoding an enzyme that repairs an oxidative DNA injury 8-oxoguanine (8-oxoG). Furthermore, for comparison with the human case, mutations of mouse epidermal growth factor receptor (Egfr) and K-ras genes were examined. The homo- and heterozygously Ogg1 gene-deficient and wild-type mice (C57BL6/J origin), 6 weeks old, were administered 4-(N-hydroxymethylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) by continuous subcutaneous infusion using an osmotic pump at a total dose of 6 mg/mouse for 1 week, then treated with one of 4 antioxidants (phenyl N-tert-butyl nitrone 0.13% in drinking water, resveratrol 20 ppm in diet, lactoferrin 2% in diet and bilberry powder 2% in diet) or no supplement for 33 weeks. Development of lung adenomas and preneoplastic atypical hypreplasias was significantly enhanced by the homo- and heterozygous Ogg1 gene deficiency only in female mice with intralesion accumulation of 8-oxoG. All antioxidants tended to inhibit enhanced adeno-carcinogenesis. The Egfr and K-ras gene mutations were detected at sites also found in human lung cancers with low incidences, while the Egfr gene mutation was detected for the first time in chemical lung carcinogenesis of animals. It is indicated that the Ogg1 gene deficiency enhances lung adeno-carcinogenesis in mice by virtue of accelerated oxidative stress. The presently utilized Ogg1 gene-deficient mice model may be useful to draw mechanism-based strategies to control human lung adenocarcinomas, especially in women.

An aromatic fatty acid, phenylacetate (PA), has been shown to have cytostatic, antitumor and cell differentiation-inducing effects on various kinds of tumors. Previously, we have demonstrated cell growth inhibition, malignant phenotype reduction and cell differentiation effects of sodium phenylacetate (NaPA) treatment in a canine mammary tumor cell line. To clarify the molecular mechanism of these effects, we examined the expression of Ras/MAPK signaling pathway-related molecules in human and canine breast cancer cell lines, and found that the level of c-Raf-1 protein was reduced by 5, 10 and 20 mM of NaPA treatments, though Ras activation was maintained. Dephosphorylation of c-Raf-1 at Serine (Ser) 259, Ser 338, and Ser 621 were also seen in NaPA-treated cells. Downstream factors in the pathway, such as mitogen-activated protein kinase/ERK kinase (MEK)1/2 and ERK1/2, showed decreased activity, and accordingly, expressions of cyclinD1, c-myc, and inactivation of p90 ribosomal S6 kinase (RSK), which are MAPK targets, were reduced. We also observed the reduction of cell-cycle-promoted molecules, such as cdc1/cdk2, cdk4, PCNA cyclin A, and cyclin B, and the increased expression of p27kip1. Furthermore, expression of an epithelial marker, E-cadherin, was increased by NaPA treatment. These results suggest that one of the molecular targets of NaPA treatment was the reduction of c-Raf-1 protein, and that its reduction results in the decrease of malignant characteristics of tumor cells through blockage of the Ras/MAPK signaling pathway.

Carcinogenic risk and molecular mechanisms underlying the liver tumor-promoting activity of copper gluconate, an additive of functional foods, were investigated using a rat medium-term liver carcinogenicity bioassay protocol (Ito test) and a 2-week short-term administration experiment. In the medium-term liver bioassay, Fischer 344 male rats were given a single i.p. injection of N-nitrosodiethylamine at a dose of 200 mg/kg b.w. as a carcinogenic initiator. Starting 2 weeks thereafter, rats received 0, 10, 300 or 6,000 ppm of copper gluconate in diet for 6 weeks. All rats underwent 2/3 partial hepatectomy at the end of week 3, and all surviving rats were killed at the end of week 8. In the short-term experiment, rats were given 0, 10, 300 or 6,000 ppm of copper gluconate for 2 weeks. Numbers of glutathione S-transferase placental form (GST-P) positive lesions, single GST-P-positive hepatocytes and 8-oxoguanine-positive hepatocytes, and levels of cell proliferation and apoptosis in the liver were significantly increased by 6,000 ppm of copper gluconate in the medium-term liver bioassay. Furthermore, hepatic mRNA expression of genes relating to the metal metabolism, inflammation and apoptosis were elevated by 6,000 ppm of copper gluconate both in the medium-term liver bioassay and the short-term experiments. These results indicate that copper gluconate possesses carcinogenic risk toward the liver at the high dose level, and that oxidative stress and inflammatory and pro-apoptotic signaling statuses may participate in its underlying mechanisms.

In this study we investigated the Ser33 phosphorylation status of beta-catenin protein in relation to genomic mutations in lung (pre)neoplastic lesions induced by N-nitrosobis(2-hydroxypropyl)amine (BHP) in male Fischer 344 rats. Six-week-old animals received 2000 ppm of BHP in the drinking water for 8 weeks and were sacrificed 12 weeks thereafter. Histopathologically, 69 of 75 rats demonstrated multiple lung (pre)neoplastic lesions, classified into 27 slight and 33 advanced hyperplasias (preneoplasms) and 61 neoplasms, including adenomas, adenocarcinomas, and adenosquamous carcinomas. Nucleotide mutation analysis of the beta-catenin gene detected a total of 33 mutations in 12 assessed lung (pre)neoplastic lesions. The mutations tended to accumulate in positions near the phosphorylation region of the gene, between codons 33 and 45. Immunohistochemical analysis showed beta-catenin protein expression to be increased and its localization changed from the cell membrane to the cytoplasm and finally the nuclei with advancing malignancy of the lung lesions. In contrast, the expression of phosphorlyated beta-catenin protein at Ser33 was weakened in lung (pre)neoplastic lesions compared to normal lung tissues. These results suggest that BHP-induced mutation of the beta-catenin gene results in amino acid conversions in its product protein, which in turn lead to inhibition of phosphorylation of the protein and escape from protein degradation. These phenomena might contribute to the malignant progression of the lung (pre)neoplastic lesions, which start from the relatively early stage in lung carcinogenesis.

Study question What percentage of cases with non-syndromic hypospadias can be ascribed to mutations in known causative/candidate/susceptibility genes or submicroscopic copy-number variations (CNVs) in the genome? Summary answer Monogenic and digenic mutations in known causative genes and cryptic CNVs account for >10% of cases with non-syndromic hypospadias. While known susceptibility polymorphisms appear to play a minor role in the development of this condition, further studies are required to validate this observation. What is known already Fifteen causative, three candidate, and 14 susceptible genes, and a few submicroscopic CNVs have been implicated in non-syndromic hypospadias. Study design, size, duration Systematic mutation screening and genome-wide copy-number analysis of 62 patients. Participants/materials, setting, methods The study group consisted of 57 Japanese and five Vietnamese patients with non-syndromic hypospadias. Systematic mutation screening was performed for 25 known causative/candidate/susceptibility genes using a next-generation sequencer. Functional consequences of nucleotide alterations were assessed by in silico assays. The frequencies of polymorphisms in the patient group were compared with those in the male general population. CNVs were analyzed by array-based comparative genomic hybridization and characterized by fluorescence in situ hybridization. Main results and the role of chance Seven of 62 patients with anterior or posterior hypospadias carried putative pathogenic mutations, such as hemizygous mutations in AR, a heterozygous mutation in BNC2, and homozygous mutations in SRD5A2 and HSD3B2. Two of the seven patients had mutations in multiple genes. We did not find any rare polymorphisms that were abundant specifically in the patient group. One patient carried mosaic dicentric Y chromosome. Limitations, reasons for caution The patient group consisted solely of Japanese and Vietnamese individuals and clinical and hormonal information of the patients remained rather fragmentary. In addition, mutation analysis focused on protein-altering substitutions. Wider implications of the findings Our data provide evidence that pathogenic mutations can underlie both mild and severe hypospadias and that HSD3B2 mutations cause non-syndromic hypospadias as a sole clinical manifestation. Most importantly, this is the first report documenting possible oligogenicity of non-syndromic hypospadias. Study funding/competing interests This study was funded by the Grant-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology; by the Grant-in-Aid from the Japan Society for the Promotion of Science; by the Grants from the Ministry of Health, Labour and Welfare, from the National Center for Child Health and Development and from the Takeda Foundation. The authors have no competing interests to disclose. Trial registration number Not applicable.

The present study was conducted as a preliminary evaluation of toxicologic and carcinogenic risks of copper gluconate and to determine its optimal dose for use in an upcoming study utilizing the medium-term multi-organ carcinogenesis protocol. Male BrlHan:WIST@Jcl (GALAS) rats were initially treated with N-nitrosodiethylamine, N-methylnitrosourea, 1,2-dimethylhydrazine, N-butyl-N-(4-hydroxybutyl)-nitrosamine and 2,2'-dihydroxy-di-n-propylnitrosamine for a total multiple initiation period of 4 weeks. In addition, rats were fed a diet containing copper gluconate at 0, 1000, 3000, 4800, 6000, or 12000 ppm from the commencement for 13 weeks. While no systemic toxicity was detected, hepatic granulomas were observed at copper gluconate doses of 6000 ppm, or greater. Hepatic copper accumulation and metallothionein induction were demonstrated at doses of 3000 ppm and 1000 ppm, respectively, or greater. Putative preneoplastic lesions in the liver appeared to increase at a dose of 12000 ppm, and 8-hydroxydeoxyguanosine formation was enhanced at doses of 6000 ppm, or greater. These results suggest that copper gluconate exerts effects on the liver at the relatively low dietary dose of 1000 ppm and causes hepatic injury at 6000 ppm. The hepatotoxicity and possible hepatocarcinogenicity of copper gluconate may be attributable to oxidative stress due to the impairment of hepatic copper metabolism.

MAMLD1 is a causative gene for disorders of sex development. Several MAMLD1 mutations have been shown to cause hypospadias by generating dysfunctional proteins and/or unstable mRNAs. Here, we identified an intronic mutation of MAMLD1 (g.IVS4−2A>G) in 1 of 180 hypospadias patients. RT-PCR of the patient's skin sample showed normal expression of full-length MAMLD1 and markedly reduced expression of a known splice variant lacking exon 4. A hitherto unreported splice variant that lacks exon 5 was similarly identified in samples of the patient and control individuals. The full-length transcript of the patient contained mutant mRNA lacking the first 10 nucleotides of exon 5 (c.1822_1831delACTCATGTAG, p.K609fsX1070). In vitro assays using cells expressing the full-length wild-type and mutant proteins revealed reduced expression of the mutant. The expression of the wild-type and mutant MAMLD1 showed parallel changes upon treatment with a proteasome inhibitor and a translation inhibitor. The mutant-expressing cells exerted low transactivation activity for the Hes3 promoter, which reflected limited expression of the mutant protein. These results imply that the pathogenic events resulting from MAMLD1 mutations include splice errors. Furthermore, this study raises the possibility of translation failure of MAMLD1 mutants, which deserves further investigation.

The common phenotype of nuclear receptor superfamily 5, group A, member 1 (NR5A1) gene mutations in 46,XY is gonadal dysgenesis without adrenal deficiency. Though the phenotype of gonadal dysgenesis is variable, ranging from complete female to normal male genitalia, an asymptomatic 46,XY male is rare. Preserved fertility has so far been described in only three affected 46,XY males with different mutations, but no functional analysis of these mutations has been performed. Here, we report on male siblings with hypospadias and their asymptomatic father in whom we identified a heterozygous NR5A1 mutation of c.910GA, p.E304K. Western blotting and subcellular localization revealed no significant difference between the wild type (WT) and E304K. Electrophoretic mobility shift assay experiments showed that E304K abrogated DNA-binding ability. E304K reduced transactivation and had no dominant negative effect. In conclusion, we report on a novel hypomorphic NR5A1 mutation, which may be associated with the phenotype of the family.

The present study was conducted with the original purpose of investigating the possibility that α- naphthylisothiocyanate (ANIT) might induce intrahepatic cholangiocellular neoplasms in rats after appropriate carcinogenesis-initiating treatments, based on its known effect of causing intrahepatic bile duct proliferation. Fischer 344 rats (6 weeks old) were given 3 weekly intraperitoneal administrations of vehicle (female and male), N-nitrosobis(2- oxopropyl)amine (BOP) (20 mg/kg body weight, female and male) or N-nitrosodimethylamine (DMN) (10 mg/kg body weight, male only), and fed a basal diet with or without 200 ppm of ANIT from the commencement for up to 24 weeks. Animals were sequentially sacrificed at the ends of weeks 8, 16 and 24 to examine morphological changes in the liver. ANIT caused proliferation of intrahepatic bile duct epithelial cells with no atypia when administered alone or in combination with BOP (female and male) or DMN (male only), while neither BOP nor DMN caused bile duct proliferation per se or altered the magnitude of the effect of ANIT. The degree of bile duct proliferation caused by ANIT was greater in females than in males. No hepatocellular or liver (pre)neoplastic changes were observed. These results indicate that although ANIT does not induce any neoplastic changes in the liver even after initiation with BOP (female and male) or DMN (male), it causes non-neoplastic intrahepatic bile duct proliferation with a clear sex difference. (J Toxicol Pathol 2004; 17: 205-210)

Aberrant biceps brachii muscles (BB); the right aberrant head, the left aberrant head, and the left aberrant bundle, were observed bilaterally in a 94-year-old Japanese female cadaver during a regional anatomy of the upper extremities. We examined the nerve supply as well as scrutinizing these aberrant BB. The branches to the right aberrant head and the right brachialis muscle arose as a common nerve trunk from the musculocutaneous nerve, and both branches entered each muscle from the ventral aspect. The branches to the superior portion of the left aberrant bundle and the short head of the BB arose as a common nerve trunk from the musculocutaneous nerve, and both branches entered each muscle from the dorsal aspect. The branches to the inferior portion of the left aberrant bundle and the left aberrant head arose separately from the musculocutaneous nerve, but they were found to be closely related after peeling off the epineurium. From these observations, we speculated that the right aberrant head could have differentiated from the brachialis muscle, the left aberrant head from the long head of the BB, and the left aberrant bundle aberrant head from the short and long head of the BB, based on the formation of the common nerve trunk and the close relation of the nerve fibers. Our suggested muscular differentiation agreed with the entering aspect (ventral/dorsal aspect) of the innervating nerve.

Introducing a point mutation is a fundamental method used to demonstrate the roles of particular nucleotides or amino acids in the genetic elements or proteins, and is widely used in in vitro experiments based on cultured cells and exogenously provided DNA. However, the in vivo application of this approach by modifying genomic loci is uncommon, partly due to its technical and temporal demands. This leaves many in vitro findings un-validated under in vivo conditions. We herein applied the CRISPR/Cas9 system to generate mice with point mutations in their genomes, which led to single amino acid substitutions in proteins of interest. By microinjecting gRNA, hCas9 mRNA and single-stranded donor oligonucleotides (ssODN) into mouse zygotes, we introduced defined genomic modifications in their genome with a low cost and in a short time. Both single gRNA/WT hCas9 and double nicking set-ups were effective. We also found that the distance between the modification site and gRNA target site was a significant parameter affecting the efficiency of the substitution. We believe that this is a powerful technique that can be used to examine the relevance of in vitro findings, as well as the mutations found in patients with genetic disorders, in an in vivo system.