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Objective: Pseudohypoparathyroidism type 1B (PHP1B) caused by methylation defects of differentially methylated regions (DMRs) on the GNAS locus can be categorized into groups according to etiologies and methylation defect patterns of the DMRs. The aim of this study was to clarify the clinical characteristics of each group., Design: Comprehensive molecular analyses consisting of methylation, copy number, and microsatellite analyses., Methods: Eighty-four patients with PHP1B were included in this study. We classified them into 5 groups, namely, autosomal dominant inheritance-PHP1B (Group 1, G1), sporadic-PHP1B (G2), and atypical-PHP1B (G3-G5), based on the methylation defect patterns in 4 DMRs on the GNAS locus and etiologies and evaluated the clinical findings in each group and compared them among the groups., Results: G2 had the youngest age and the highest serum intact parathyroid hormone levels among the 5 groups at the time of diagnosis. The most common symptoms at the time of diagnosis were tetany in G1, and seizures or loss of consciousness in G2. Albright's hereditary osteodystrophy and PHP-suggestive features were most frequently observed in the G2 proband. Nine patients had neurodevelopmental disorders (NDs) consisting of mild to borderline intellectual disability and/or developmental delay. There were no significant correlations between the average methylation ratios of 7 CpG sites in the GNAS-A/B:TSS-DMR and hormonal and biochemical findings., Conclusion: This study revealed the differences in some clinical characteristics, particularly clinical features, and ages at the time of diagnosis between G2 and other groups and detailed NDs observed in some patients with PHP1B., Competing Interests: Conflict of interest: The authors report no conflicts of interest., (© The Author(s) 2023. Published by Oxford University Press on behalf of European Society of Endocrinology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Objectives: The concept of locomotive syndrome (LS) and its evaluation method, the LS risk test, have been applied in an integrated manner to capture the decline in mobility resulting from musculoskeletal disorders. The purpose of this study was to evaluate the impact of total knee arthroplasty (TKA) in the elderly with knee osteoarthritis, a common disorder found in LS., Methods: A total of 111 patients were registered prior to TKA and postoperatively followed up for 1 year. Three components of the LS risk test (the two-step test, stand-up test, and Geriatric Locomotive Function Scale-25) were assessed pre- and postoperatively., Results: After surgery, all three components of the test showed significant improvements from the baseline. The ratio of Stage 3 LS patients (progressed stage of decrease in mobility) reduced from 82.3% to 33.9% postoperatively. There was no significant difference in the degree of change in the scores between the younger (60-74 years) and older (≥75 years) age groups., Conclusions: We found that TKA has a major impact in preventing the progression of LS in patients with knee osteoarthritis. The LS risk test is a feasible tool for the longitudinal evaluation of patients with musculoskeletal diseases of varying severity and with multiple symptoms., (© Japan College of Rheumatology 2022. Published by Oxford University Press.)

Context: We have previously reported that a specific "AGATC" haplotype in a >34 kb tight linkage disequilibrium (LD) block within ESR1 is strongly associated with cryptorchidism and hypospadias in Japanese boys., Objective: We aimed to determine the true susceptibility factor for cryptorchidism and hypospadias linked to the "AGATC" haplotype., Methods: We performed various molecular studies in hitherto unreported 230 Italian boys (80 with cryptorchidism and 150 with normal genitalia) and previously reported and newly recruited 415 Japanese boys (149 with cryptorchidism, 141 with hypospadias, and 125 with normal genitalia). We also performed ESR1 expression analyses using breast cancer-derived MCF-7 cells., Results: Haplotype analysis revealed the LD block and positive association of the "AGATC" haplotype with cryptorchidism in Italian boys. Whole genome sequencing identified an identical 2249-bp microdeletion (ΔESR1) generated by a microhomology-mediated replication error in both Japanese and Italian boys with the specific haplotype. ΔESR1 was found to be strongly associated with cryptorchidism and hypospadias by Cochran-Armitage trend test and was revealed to show nearly absolute LD with the "AGATC" haplotype. ESR1 expression was upregulated in MCF-7 cells with a homozygous deletion encompassing ΔESR1 and those with a homozygous deletion involving a CTCF-binding site within ΔESR1., Conclusion: The results reveal that ΔESR1, which has been registered as "DEL_6_75504" in gnomAD SVs v2.1, is the true susceptibility factor for cryptorchidism and hypospadias. It appears that ΔESR1 was produced in a single ancestral founder of modern humans and has been maintained within the genome of multiple ethnic groups by selection., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Loss of methylation (LOM) at GNAS-A/B:TSS-differentially methylated regions (DMRs) in the GNAS locus is observed in pseudohypoparathyroidism type 1B (PHP1B). Many PHP1B cases are sporadic, but autosomal dominant-PHP1B has a deletion involving NESP55 expressed from the maternal allele or STX16 located upstream of the GNAS locus on the maternal allele. We report the possible first familial PHP1B cases with retrotransposon insertion in the GNAS locus on the maternal allele. To our knowledge, they are the possible first cases with imprinting disorders caused by retrotransposon insertion. The two sibling cases experienced tetany and/or cramps from school age and had hypocalcemia and an increased serum intact parathyroid hormone (PTH) level together with overweight, round face, and normal intellectual levels. Methylation analysis for DMRs in the GNAS locus showed only LOM of the GNAS-A/B:TSS-DMR. Copy number abnormalities at STX16 and the GNAS locus were not detected by array comparative genomic hybridization. Whole-genome sequencing and Sanger sequencing revealed an approximately 1000-bp SVA retrotransposon insertion upstream of the first exon of A/B on the GNAS locus in these siblings. Whole-genome methylome analysis by Enzymatic Methyl-Seq in the siblings showed normal methylation status in the region surrounding the insertion site and mild LOM of the GNAS-A/B:TSS-DMR. We conducted transcriptome analysis using mRNA from skin fibroblasts and induced pluripotent stem cells (iPSCs) derived from the siblings and detected no aberrant NESP55 transcripts. Quantitative reverse-transcriptase PCR (qRT-PCR) analysis in skin fibroblasts showed increased A/B expression in the patients and no NESP55 expression, even in a control. qRT-PCR analysis in iPSCs showed decreased NESP55 expression with normal methylation status of the GNAS-NESP:TSS-DMR in the patients. The retrotransposon insertion in the siblings likely caused decreased NESP55 expression that could lead to increased A/B expression via LOM of the GNAS-A/B:TSS-DMR, subsequent reduced Gsα expression, and finally, PHP1B development. © 2022 American Society for Bone and Mineral Research (ASBMR)., (© 2022 American Society for Bone and Mineral Research (ASBMR).)

We analyzed the local control (LC) of cervical squamous cell carcinoma treated by computed tomography (CT)-based image-guided brachytherapy (IGBT) using central shielding (CS). We also examined the value of tumor diameter before brachytherapy (BT) as a factor of LC. In total, 97 patients were analyzed between April 2016 and March 2020. Whole-pelvic (WP) radiotherapy (RT) with CS was performed, and the total pelvic sidewall dose was 50 or 50.4 Gy; IGBT was delivered in 3-4 fractions. The total dose was calculated as the biologically equivalent dose in 2 Gy fractions, and distribution was modified manually by graphical optimization. The median follow-up period was 31.8 months (6.3-63.2 months). The 1- and 2-year LC rates were 89% and 87%, respectively. The hazard ratio was 10.11 (95% confidence interval: 1.48-68.99) for local recurrence in those with a horizontal tumor diameter ≥ 4 cm compared to those with < 4 cm before BT. In CT-based IGBT for squamous cell carcinoma, favorable LC can be obtained in patients with a tumor diameter < 4 cm before BT. However, if the tumor diameter is ≥ 4 cm, different treatment strategies such as employing interstitial-BT for dose escalation may be necessary., (© The Author(s) 2022. Published by Oxford University Press on behalf of The Japanese Radiation Research Society and Japanese Society for Radiation Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Context: Children born small-for-gestational-age with short stature (SGA-SS) is associated with (epi)genetic defects, including imprinting disorders (IDs), pathogenic copy number variants (PCNVs), and pathogenic variants of genes involved in growth. However, comprehensive studies evaluating these 3 factors are very limited., Objective: To clarify the contribution of PCNVs and candidate pathogenic variants to SGA-SS., Design: Comprehensive molecular analyses consisting of methylation analysis, copy number analysis, and multigene sequencing., Methods: We enrolled 140 patients referred to us for genetic testing for SGA-SS. Among them, we excluded 42 patients meeting Netchine-Harbison clinical scoring system criteria for Silver-Russell syndrome and 4 patients with abnormal methylation levels of the IDs-related differentially methylated regions. Consequently, we conducted copy number analysis and multigene sequencing for 86 SGA-SS patients with sufficient sample volume. We also evaluated clinical phenotypes of patients with PCNVs or candidate pathogenic variants., Results: We identified 8 (9.3%) and 11 (12.8%) patients with PCNVs and candidate pathogenic variants, respectively. According to the American College of Medical Genetics standards and guidelines, 5 variants were classified as pathogenic and the remaining 6 variants were classified as variants of unknown significance. Genetic diagnosis was made in 12 patients. All patients with PCNVs or candidate pathogenic variants did not correspond perfectly to characteristic clinical features of each specific genetic cause., Conclusion: We clarified the contribution of PCNVs and pathogenic variants to SGA-SS without IDs. Comprehensive molecular analyses, including copy number analysis and multigene sequencing, should be considered for patients with unknown SGA-SS etiology., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Context: Aromatase excess syndrome (AEXS) is a very rare disorder characterized by prepubertal gynecomastia, bone age acceleration, and early growth arrest. Heterozygote submicroscopic rearrangements within the promotor of CYP19A1 result in overexpression of aromatase and enhanced aromatization of androgens., Objective: The objective was to study long-term treatment effects of an aromatase inhibitor., Methods: Data from 7 boys with AEXS were retrospectively collected. Genetic analysis revealed upstream of CYP19A1 a 165 901 bp deletion in 4 German cousins, a 198 662 bp deletion in 2 Japanese brothers, and a 387 622 bp tandem duplication in a Japanese boy., Results: All boys developed prepubertal gynecomastia, at median 9.0 years of age (range: 7.0-11.0). Height was +1.20 standard deviation score (SDS) (-0.24 to +1.98); predicted adult height was -1.29 SDS (-3.29 to +1.09). Four boys were treated with 1.0 mg of anastrozole daily, while 3 reached adult height untreated. Treatment with anastrozole was stopped after 5.6 years (4.0-6.8). Three treated boys exceeded their prognosis by 2.4, 6.9, and 8.1 cm, while 1 untreated boy fell below the prognosis by 8.6 cm. One treated with a low dose and 2 untreated reached their prognosis. Adult heights were -0.91 SDS with anastrozole (-2.86 to -0.29) and -0.15 SDS without (-2.31 to -0.03). Distance to target height was -0.22 SDS with anastrozole (-1.72 to +0.52) and +0.54 SDS without (+0.23 to +1.30)., Conclusion: Spontaneous growth in AEXS varied, even in the same family. Our data suggest that early started, long-term inhibition by anastrozole promotes adult height in boys with AEXS., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: (Epi)genetic disorders associated with small-for-gestational-age with short stature (SGA-SS) include imprinting disorders (IDs). Silver-Russell syndrome (SRS) is a representative ID in SGA-SS and has heterogenous (epi)genetic causes., Subjects and Methods: To clarify the contribution of IDs to SGA-SS and the molecular and phenotypic spectrum of SRS, we recruited 269 patients with SGA-SS, consisting of 103 and 166 patients referred to us for genetic testing for SGA-SS and SRS, respectively. After excluding 20 patients with structural abnormalities detected by comparative genomic hybridization analysis using catalog array, 249 patients were classified into 3 subgroups based on the Netchine-Harbison clinical scoring system (NH-CSS), SRS diagnostic criteria. We screened various IDs by methylation analysis for differentially methylated regions (DMRs) related to known IDs. We also performed clinical analysis., Results: These 249 patients with SGA-SS were classified into the "SRS-compatible group" (n = 148), the "non-SRS with normocephaly or relative macrocephaly at birth group" (non-SRS group) (n = 94), or the "non-SRS with relative microcephaly at birth group" (non-SRS with microcephaly group) (n = 7). The 44.6% of patients in the "SRS-compatible group," 21.3% of patients in the "non-SRS group," and 14.3% in the "non-SRS with microcephaly group" had various IDs. Loss of methylation of the H19/IGF2:intergenic-DMR and uniparental disomy chromosome 7, being major genetic causes of SRS, was detected in 30.4% of patients in the "SRS-compatible group" and in 13.8% of patients in the "non-SRS group.", Conclusion: We clarified the contribution of IDs as (epi)genetic causes of SGA-SS and the molecular and phenotypic spectrum of SRS. Various IDs constitute underlying factors for SGA-SS, including SRS., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Endocrine Society.)

Mammalian male meiosis requires homologous recombination between the X and Y chromosomes. In humans, such recombination occurs exclusively in the short arm pseudoautosomal region (PAR1) of 2.699 Mb in size. Although it is known that complete deletion of PAR1 causes spermatogenic arrest, no studies have addressed to what extent male meiosis tolerates PAR1 size reduction. Here, we report two families in which PAR1 partial deletions were transmitted from fathers to their offspring. Cytogenetic analyses revealed that a ∼400-kb segment at the centromeric end of PAR1, which accounts for only 14.8% of normal PAR1 and 0.26% and 0.68% of the X and Y chromosomes, respectively, is sufficient to mediate sex chromosomal recombination during spermatogenesis. These results highlight the extreme recombinogenic activity of human PAR1. Our data, in conjunction with previous findings from animal studies, indicate that the minimal size requirement of mammalian PARs to maintain male fertility is fairly small., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

A mouse single taste bud contains 10-100 taste bud cells (TBCs) in which the elongated TBCs are classified into 3 cell types (types I-III) equipped with different taste receptors. Accordingly, differences in the cell numbers and ratios of respective cell types per taste bud may affect taste-nerve responsiveness. Here, we examined the numbers of each immunoreactive cell for the type II (sweet, bitter, or umami receptor cells) and type III (sour and/or salt receptor cells) markers per taste bud in the circumvallate and foliate papillae and compared these numerical features of TBCs per taste bud to those in fungiform papilla and soft palate, which we previously reported. In circumvallate and foliate taste buds, the numbers of TBCs and immunoreactive cells per taste bud increased as a linear function of the maximal cross-sectional taste bud area. Type II cells made up approximately 25% of TBCs irrespective of the regions from which the TBCs arose. In contrast, type III cells in circumvallate and foliate taste buds made up approximately 11% of TBCs, which represented almost 2 times higher than what was observed in the fungiform and soft palate taste buds. The densities (number of immunoreactive cells per taste bud divided by the maximal cross-sectional area of the taste bud) of types II and III cells per taste bud are significantly higher in the circumvallate papillae than in the other regions. The effects of these region-dependent differences on the taste response of the taste bud are discussed., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Objective: IGF2 is a paternally expressed growth-promoting gene. Here, we report five cases with IGF2 mutations and review IGF2 mutation-positive patients described in the literature. We also compare clinical features between patients with IGF2 mutations and those with H19/IGF2:IG-DMR epimutations., Results: We recruited five cases with IGF2 mutations: case 1 with a splice site mutation (c.-6-1G>C) leading to skipping of exon 2 and cases 2-5 with different missense mutations (p.(Cys70Tyr), p.(Cys71Arg), p.(Cys33Ser), and p.(Cys45Ser)) affecting cysteine residues involved in the S-S bindings. All the mutations resided on the paternally inherited allele, and the mutation of case 5 was present in a mosaic condition. Clinical assessment revealed Silver-Russell syndrome (SRS) phenotype with Netchine-Harbison scores of ≥5/6 in all the apparently nonmosaic 14 patients with IGF2 mutations (cases 1-4 described in this study and 10 patients reported in the literature). Furthermore, compared with H19/IGF2:IG-DMR epimutations, IGF2 mutations were associated with low frequency of hemihypoplasia, high frequency of feeding difficulty and/or reduced body mass index, and mild degree of relative macrocephaly, together with occasional development of severe limb malformations, high frequency of cardiovascular anomalies and developmental delay, and low serum IGF-II values., Conclusions: This study indicates that IGF2 mutations constitute a rare but important cause of SRS. Furthermore, while both IGF2 mutations and H19/IGF2:IG-DMR epimutations lead to SRS, a certain degree of phenotypic difference is observed between the two groups, probably due to the different IGF2 expression pattern in target tissues., (© Endocrine Society 2019. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

A randomized phase III trial commenced in Japan in February 2018. Definitive chemoradiotherapy (CRT) with cisplatin plus 5-fluorouracil is the current standard treatment for locally advanced unresectable esophageal carcinoma. The purpose of this study is to confirm the superiority of induction chemotherapy with docetaxel plus cisplatin and 5-fluorouracil (DCF) followed by conversion surgery or definitive CRT over definitive CRT alone for overall survival (OS) in patients with locally advanced unresectable squamous-cell carcinoma of thoracic esophagus. A total of 230 patients will be accrued from 47 Japanese institutions over 4.5 years. The primary endpoint is OS, and the secondary endpoints are progression-free survival, complete response rate of CRT, response rate of DCF, adverse events of DCF and CRT, late adverse events and surgical complications. This trial has been registered at the Japan Registry of Clinical Trials as jRCTs031180181., (© The Author(s) 2019. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Study Question: Do monosomy rescue (MR) and trisomy rescue (TR) in preimplantation human embryos affect other developmental processes, such as X-chromosome inactivation (XCI)?, Summary Answer: Aneuploid rescue precedes XCI and increases the incidence of XCI skewness by reducing the size of the embryonic progenitor cell pools., What Is Known Already: More than half of preimplantation human embryos harbor aneuploid cells, some of which can be spontaneously corrected through MR or TR. XCI in females is an indispensable process, which is predicted to start at the early-blastocyst phase., Study Design, Size, Duration: We examined the frequency of XCI skewness in young females who carried full uniparental disomy (UPD) resulting from MR or TR/gamete complementation (GC). The results were statistically analyzed using a theoretical model in which XCI involves various numbers of embryonic progenitor cells., Participants/materials, Setting, Methods: We studied 39 children and young adults ascertained by imprinting disorders. XCI ratios were determined by DNA methylation analysis of a polymorphic locus in the androgen receptor gene. We used Bayesian approach to assess the probability of the occurrence of extreme XCI skewness in the MR and TR/GC groups using a theoretical model of 1-12 cell pools., Main Results and the Role of Chance: A total of 12 of 39 individuals (31%) showed skewed XCI. Extreme skewness was observed in 3 of 15 MR cases (20%) and 1 of 24 TR/GC cases (4.2%). Statistical analysis indicated that XCI in the MR group was likely to have occurred when the blastocyst contained three or four euploid embryonic progenitor cells. The estimated size of the embryonic progenitor cell pools was approximately one-third or one-fourth of the predicted size of normal embryos. The TR/GC group likely had a larger pool size at the onset of XCI, although the results remained inconclusive., Limitations, Reasons for Caution: This is an observational study and needs to be validated by experimental analyses., Wider Implications of the Findings: This study provides evidence that the onset of XCI is determined by an intrinsic clock, irrespectively of the number of embryonic progenitor cells. Our findings can also be applied to individuals without UPD or imprinting disorders. This study provides a clue to understand chromosomal and cellular dynamics in the first few days of human development, their effects on XCI skewing and the possible implications for the expression of X-linked diseases in females., Study Funding/competing Interest(s): This study was supported by the Grants-in-aid for Scientific Research on Innovative Areas (17H06428) and for Scientific Research (B) (17H03616) from Japan Society for the Promotion of Science (JSPS), and grants from Japan Agency for Medical Research and Development (AMED) (18ek0109266h0002 and 18ek0109278h0002), National Center for Child Health and Development and Takeda Science Foundation. The authors declare no conflict of interest., Trial Registration Number: Not applicable., (© The Author(s) 2019. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Context: Maternal uniparental disomy for chromosome 20 [UPD(20)mat], resulting in aberrant expression of imprinted transcripts at the GNAS locus, is a poorly characterized condition. These patients manifested a phenotype similar to that of Silver-Russell syndrome (SRS) and small for gestational age-short stature (SGA-SS); however, the etiological relationship between UPD(20)mat and SRS/SGA-SS remains unclear. Moreover, no report has described endocrinological assessment of UPD(20)mat patients, although paternal UPD(20), the mirror image entity of UPD(20)mat, is known to cause multiple hormone resistance reflecting reduced α-subunit of the stimulatory G protein expression., Participants: Patients 1 to 5 showed nonmosaic heterodisomy and/or isodisomy for the entire chromosome 20. Patients 1 to 3 and 4 were identified through UPD(20)mat screening for 55 patients with etiology-unknown SRS and 96 patients with SGA-SS, respectively. Patient 5 was identified through molecular analysis for patients with developmental defects. Patients 1 to 5 manifested postnatal growth failure and feeding problems, with or without developmental delay, and other clinical features. Patients 1 to 4 were born SGA. Patients 4 and 5 exhibited hypercalcemia and low or low-normal parathyroid hormone levels. Patient 1 showed constantly decreased thyroid-stimulating hormone (TSH) levels after 12 years of age, although she had a normal TSH level at 5.2 years of age., Conclusion: The results suggest that UPD(20)mat underlies growth failure and feeding problems with additional features and could account for >5% of etiology-unknown SRS and small percentages of SGA-SS. Most important, this study provides an indication that UPD(20)mat can be associated with hypersensitivity of hormone receptors, which may gradually develop with age.

Objective: To confirm the morphine to hydromorphone conversion ratio for hydromorphone (DS-7113b) immediate-release tablets in cancer patients who achieved pain control with oral morphine., Methods: This was a multicenter, active-controlled, randomized, double-blind, parallel-group, comparative study (July 2013 to December 2014) at 39 Japanese sites. Seventy-one patients (aged >20 years) who had achieved pain control with morphine 60 mg/day and 90 mg/day were randomly allocated 1:1 to hydromorphone immediate-release tablets at a dose converted at a hydromorphone:morphine ratio of 1:5 or 1:8, respectively, and treated for up to 5 days. The efficacy was evaluated as the pain control ratio., Results: The pain control ratio in the full analysis set was 83.3% (25/30) in the conversion ratio 1:5 group and 95.0% (38/40) in the conversion ratio 1:8 group, and both groups demonstrated highly successful pain control. The incidence of adverse events was 46.7% (14/30) in the conversion ratio 1:5 group and 58.5% (24/41) in the 1:8 group; the difference was not clinically relevant. Frequently observed adverse events (incidence ≥5%) were nausea, vomiting, diarrhea, somnolence and dyspnea., Conclusions: A high pain control ratio was maintained by a switch at either conversion ratio, and no notable difference was observed in the incidence of adverse events. A switch from morphine to hydromorphone is effective at a dose converted at ratios of 1:5 and 1:8.

Expressed sequence tag analyses revealed that two marine Chlorophyceae green algae, Chlamydomonas sp. W80 and Chlamydomonas sp. HS5, contain genes coding for chloroplastic class IIA aldolase (fructose-1, 6-bisphosphate aldolase: FBA). These genes show robust monophyly with those of the marine Prasinophyceae algae genera Micromonas, Ostreococcus and Bathycoccus, indicating that the acquisition of this gene through horizontal gene transfer by an ancestor of the green algal lineage occurred prior to the divergence of the core chlorophytes (Chlorophyceae and Trebouxiophyceae) and the prasinophytes. The absence of this gene in some freshwater chlorophytes, such as Chlamydomonas reinhardtii, Volvox carteri, Chlorella vulgaris, Chlorella variabilis and Coccomyxa subellipsoidea, can therefore be explained by the loss of this gene somewhere in the evolutionary process. Our survey on the distribution of this gene in genomic and transcriptome databases suggests that this gene occurs almost exclusively in marine algae, with a few exceptions, and as such, we propose that chloroplastic class IIA FBA is a marine environment-adapted enzyme. This hypothesis was also experimentally tested using Chlamydomonas W80, for which we found that the transcript levels of this gene to be significantly lower under low-salt (that is, simulated terrestrial) conditions. Expression analyses of transcriptome data for two algae, Prymnesium parvum and Emiliania huxleyi, taken from the Sequence Read Archive database also indicated that the expression of this gene under terrestrial conditions (low NaCl and low sulfate) is significantly downregulated. Thus, these experimental and transcriptome data provide support for our hypothesis.

SHOX deficiency is the most frequent genetic growth disorder associated with isolated and syndromic forms of short stature. Caused by mutations in the homeobox gene SHOX, its varied clinical manifestations include isolated short stature, Léri-Weill dyschondrosteosis, and Langer mesomelic dysplasia. In addition, SHOX deficiency contributes to the skeletal features in Turner syndrome. Causative SHOX mutations have allowed downstream pathology to be linked to defined molecular lesions. Expression levels of SHOX are tightly regulated, and almost half of the pathogenic mutations have affected enhancers. Clinical severity of SHOX deficiency varies between genders and ranges from normal stature to profound mesomelic skeletal dysplasia. Treatment options for children with SHOX deficiency are available. Two decades of research support the concept of SHOX as a transcription factor that integrates diverse aspects of bone development, growth plate biology, and apoptosis. Due to its absence in mouse, the animal models of choice have become chicken and zebrafish. These models, therefore, together with micromass cultures and primary cell lines, have been used to address SHOX function. Pathway and network analyses have identified interactors, target genes, and regulators. Here, we summarize recent data and give insight into the critical molecular and cellular functions of SHOX in the etiopathogenesis of short stature and limb development.

Context: Pseudohypoparathyroidism type 1b (PHP-1b) results from methylation defects at the G protein stimulatory α subunit (GNAS) exon A/B-differentially methylated region (DMR). Although microduplications in the GNAS region were recently identified in two PHP-1b patients, genetic information on these patients remained fragmentary., Case Description: A 20-year-old Japanese male and his mother presented with hypocalcemia and elevated blood levels of intact PTH. The proband had a maternal uncle who was previously diagnosed with PHP-1b. Methylation-specific multiplex ligation-dependent probe amplification, array-based comparative genomic hybridization, pyrosequencing, fluorescence in situ hybridization, and whole-genome sequencing were performed for this family. The proband, mother, and uncle carried maternally derived approximately 133-kb duplication-triplication-duplication rearrangements at 20q13.32 involving NESP55, NESPAS, XLαs, and exon A/B-DMR but not STX16 or the Gsα coding region. These individuals exhibited partial methylation defects of NESP55-, NESPAS-, and XLαs-DMRs, which were ascribable to the increased copy numbers of these regions retaining the maternally derived methylation pattern and loss of methylation of exon A/B-DMR, which was inexplicable by the copy-number alterations. Fusion junctions of the rearrangement resided within non-repeat sequences and were accompanied by short-templated insertions., Conclusions: Our results indicate that maternally derived copy-number gains in the GNAS region mediated by nonhomologous end-joining and/or by break-induced replication can underlie autosomal dominant PHP-1b. These rearrangements likely affect methylation of exon A/B-DMR by disconnecting or disrupting its cis-acting regulator(s). This study provides a novel example of human disorders resulting from functional disturbance in the cis-regulatory machinery of DNA methylation.

Context: Loss of function (LoF) mutations in more than 20 genes are now known to cause isolated GnRH deficiency (IGD) in humans. Most causal IGD mutations are typically private, ie, limited to a single individual/pedigree. However, somewhat paradoxically, four IGD genes (GNRH1, TAC3, PROKR2, and GNRHR) have been shown to harbor LoF founder mutations that are shared by multiple unrelated individuals. It is not known whether similar founder mutations occur in other IGD genes., Objective: The objective of the study was to determine whether shared deleterious mutations in IGD-associated genes represent founder alleles., Setting: This study was an international collaboration among academic medical centers., Methods: IGD patients with shared mutations, defined as those documented in three or more unrelated probands in 14 IGD-associated genes, were identified from various academic institutions, the Human Gene Mutation Database, and literature reports by other international investigators. Haplotypes of single-nucleotide polymorphisms and short tandem repeats surrounding the mutations were constructed to assess genetic ancestry., Results: A total of eight founder mutations in five genes, GNRHR (Q106R, R262Q, R139H), TACR3 (W275X), PROKR2 (R85H), FGFR1 (R250Q, G687R), and HS6ST1 (R382W) were identified. Most founder alleles were present at low frequency in the general population. The estimated age of these mutant alleles ranged from 1925 to 5600 years and corresponded to the time of rapid human population expansion., Conclusions: We have expanded the spectrum of founder alleles associated with IGD to a total of eight founder mutations. In contrast to the approximately 9000-year-old PROKR2 founder allele that may confer a heterozygote advantage, the rest of the founder alleles are relatively more recent in origin, in keeping with the timing of recent human population expansion and any selective heterozygote advantage of these alleles requires further evaluation.

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 Author 2015. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

The purpose of this study was to perform dosimetry analyses comparing high-dose-rate brachytherapy (HDR-BT) with simulated stereotactic body radiotherapy (SBRT). We selected six consecutive patients treated with HDR-BT monotherapy in 2010, and a CyberKnife SBRT plan was simulated for each patient using computed tomography images and the contouring set used in the HDR-BT plan for the actual treatment, but adding appropriate planning target volume (PTV) margins for SBRT. Then, dosimetric profiles for PTVs of the rectum, bladder and urethra were compared between the two modalities. The SBRT plan was more homogenous and provided lower dose concentration but better coverage for the PTV. The maximum doses in the rectum were higher in the HDR-BT plans. However, the HDR-BT plan provided a sharper dose fall-off around the PTV, resulting in a significant and considerable difference in volume sparing of the rectum with the appropriate PTV margins added for SBRT. While the rectum D5cm(3) for HDR-BT and SBRT was 30.7 and 38.3 Gy (P < 0.01) and V40 was 16.3 and 20.8 cm(3) (P < 0.01), respectively, SBRT was significantly superior in almost all dosimetric profiles for the bladder and urethra. These results suggest that SBRT as an alternative to HDR-BT in hypofractionated radiotherapy for prostate cancer might have an advantage for bladder and urethra dose sparing, but for the rectum only when proper PTV margins for SBRT are adopted., (© The Author 2014. Published by Oxford University Press on behalf of The Japan Radiation Research Society and Japanese Society for Radiation Oncology.)

Astrocytes are the most abundant cell type in the mammalian brain and are important for the functions of the central nervous system. Although previous studies have shown that the STAT signaling pathway or its regulators promote the generation of astrocytes from multipotent neural precursor cells (NPCs) in the developing mammalian brain, the molecular mechanisms that regulate the astrocytic fate decision have still remained largely unclear. Here, we show that the high mobility group nucleosome-binding (HMGN) family proteins, HMGN1, 2, and 3, promote astrocyte differentiation of NPCs during brain development. HMGN proteins were expressed in NPCs, Sox9(+) glial progenitors, and GFAP(+) astrocytes in perinatal and adult brains. Forced expression of either HMGN1, 2, or 3 in NPCs in cultures or in the late embryonic neocortex increased the generation of astrocytes at the expense of neurons. Conversely, knockdown of either HMGN1, 2, or 3 in NPCs suppressed astrocyte differentiation and promoted neuronal differentiation. Importantly, overexpression of HMGN proteins did not induce the phosphorylation of STAT3 or activate STAT reporter genes. In addition, HMGN family proteins did not enhance DNA demethylation and acetylation of histone H3 around the STAT-binding site of the gfap promoter. Moreover, knockdown of HMGN family proteins significantly reduced astrocyte differentiation induced by gliogenic signal ciliary neurotrophic factor, which activates the JAK-STAT pathway. Therefore, we propose that HMGN family proteins are novel chromatin regulatory factors that control astrocyte fate decision/differentiation in parallel with or downstream of the JAK-STAT pathway through modulation of the responsiveness to gliogenic signals., (© 2014 AlphaMed Press.)

Context: C-type natriuretic peptide-natriuretic peptide receptor B (NPR-B) signaling is critical for endochondral ossification, which is responsible for longitudinal growth in limbs and vertebrae. Biallelic NPR2 mutations cause acromesomelic dysplasia, type Maroteaux, which is bone dysplasia characterized by severe short stature and short limbs. A monoallelic NPR2 mutation has been suggested to mildly impair long bone growth., Objective: The goal of this study was to identify and characterize NPR2 mutations among Japanese patients with short stature., Subjects and Methods: We enrolled 101 unrelated Japanese patients with short stature. NPR2 and NPPC were sequenced, and the identified variants were characterized in vitro., Results: In two subjects, we identified two novel heterozygous NPR2 mutations (R110C and Q417E) causing a loss of C-type natriuretic peptide-dependent cGMP generation capacities and having dominant-negative effects. R110C was defective in trafficking from the endoplasmic reticulum to the Golgi apparatus. In contrast, Q417E showed clear cell surface expression., Conclusions: We identified heterozygous NPR2 mutations in 2% of Japanese patients with short stature. Our in vitro findings indicate that NPR2 mutations have a dominant negative effect, and their dominant-negative mechanisms vary corresponding to the molecular pathogenesis of the mutations.

Context: Although AP2S1 has recently been shown to be a causative gene for familial hypocalciuric hypercalcemia type 3 (FHH3), knowledge about FHH3 remains poor., Objective: Our objective was to report AP2S1 mutation and effects of low calcium formula in a patient with hypercalcemia and hypercalciuria., Patient: This Japanese female infant was found to have hypercalcemia by a routine laboratory test for poor weight gain on breast feeding. At 49 days of age, serum calcium (adjusted by Payne's formula) was 13.1 mg/dL, intact PTH 27 pg/mL, and urinary calcium-to-creatinine ratio 1.29 mg/mg. There was no evidence for hyperparathyroidism, PTHrP-producing neoplasm, and vitamin D excess. These data, except for hypercalciuria, appeared to be consistent with defective calcium-sensing receptor-mediated signaling. With use of low calcium formula containing 2.6 mg/dL of calcium, she showed catch-up growth, and serum calcium was decreased, as was urinary calcium-to-creatinine ratio. Furthermore, feeding with a mixture of low calcium formula and standard formula with a 2:1 ratio maintained serum calcium ∼12 mg/dL without markedly increasing serum PTH., Results: Although no pathologic mutation was detected in CASR or GNA11, a presumably de novo heterozygous mutation (p.Arg15Leu), a previously reported causative mutation for FHH3, was identified in AP2S1 of this patient., Conclusions: The results imply that lack of hypocalciuria does not necessarily argue against the presence of AP2S1 mutations. The early infantile age of this patient would have played a certain role in the occurrence of hypercalciuria, and low calcium formula is worth attempting in infants with FHH.

Context: Genomic rearrangements at 15q21 have been shown to cause overexpression of CYP19A1 and resultant aromatase excess syndrome (AEXS). However, mutation spectrum, clinical consequences, and underlying mechanisms of these rearrangements remain to be elucidated., Objective: The aim of the study was to clarify such unsolved matters., Design, Setting, and Methods: We characterized six new rearrangements and investigated clinical outcome and local genomic environments of these rearrangements and of three previously reported duplications/deletions., Results: Novel rearrangements included simple duplication involving exons 1-10 of CYP19A1 and simple and complex rearrangements that presumably generated chimeric genes consisting of the coding region of CYP19A1 and promoter-associated exons of neighboring genes. Clinical severities were primarily determined by the copy number of CYP19A1 and the property of the fused promoters. Sequences at the fusion junctions suggested nonallelic homologous recombination, nonhomologous end-joining, and replication-based errors as the underlying mechanisms. The breakpoint-flanking regions were not enriched with GC content, palindromes, noncanonical DNA structures, or known rearrangement-associated motifs. The rearrangements resided in early-replicating segments., Conclusions: These results indicate that AEXS is caused by duplications involving CYP19A1 and simple and complex rearrangements that presumably lead to the usage of cryptic promoters of several neighboring genes. Our data support the notion that phenotypes depend on the dosage of CYP19A1 and the characteristics of the fused promoters. Furthermore, we show that the rearrangements in AEXS are generated by both recombination- and replication-mediated mechanisms, independent of the known rearrangement-inducing DNA features or late-replication timing. Thus, AEXS represents a unique model for human genomic disorders.

Although mastermind-like domain containing 1 (MAMLD1) (CXORF6) on human chromosome Xq28 has been shown to be a causative gene for 46,XY disorders of sex development with hypospadias, the biological function of MAMLD1/Mamld1 remains to be elucidated. In this study, we first showed gradual and steady increase of testicular Mamld1 mRNA expression levels in wild-type male mice from 12.5 to 18.5 d postcoitum. We then generated Mamld1 knockout (KO) male mice and revealed mildly but significantly reduced testicular mRNA levels (65-80%) of genes exclusively expressed in Leydig cells (Star, Cyp11a1, Cyp17a1, Hsd3b1, and Insl3) as well as grossly normal testicular mRNA levels of genes expressed in other cell types or in Leydig and other cell types. However, no demonstrable abnormality was identified for cytochrome P450 17A1 and 3β-hydroxysteroid dehydrogenase (HSD3B) protein expression levels, appearance of external and internal genitalia, anogenital distance, testis weight, Leydig cell number, intratesticular testosterone and other steroid metabolite concentrations, histological findings, in situ hybridization findings for sonic hedgehog (the key molecule for genital tubercle development), and immunohistochemical findings for anti-Müllerian hormone (Sertoli cell marker), HSD3B (Leydig cell marker), and DEAD (Asp-Glu-Ala-Asp) box polypeptide 4 (germ cell marker) in the KO male mice. Fertility was also normal. These findings imply that Mamld1 deficiency significantly reduces mRNA expression levels of multiple genes expressed in mouse fetal Leydig cells but permits normal genital and reproductive development. The contrastive phenotypic findings between Mamld1 KO male mice and MAMLD1 mutation positive patients would primarily be ascribed to species difference in the fetal sex development.

To develop an easy independent patient-specific quality assurance (QA) method using a benchmark plan for high-dose-rate intracavitary brachytherapy for cervix cancer, we conducted benchmark treatment planning with various sizes and combinations of tandem-ovoid and tandem-cylinder applications with 'ideal' geometry outside the patient. Two-dimensional-based treatment planning was conducted based on the Manchester method. We predicted the total dwell time of individual treatment plans from the air kerma strength, total dwell time and prescription dose of the benchmark plan. In addition, we recorded the height (dh), width (dw) and thickness (dt) covered with 100% isodose line. These parameters were compared with 169 and 29 clinical cases for tandem-ovoid or tandem-cylinder cases, respectively. With regard to tandem-ovoid cases, differences in total dwell time, dh, dt and dw between benchmark and individual plans were on average -0.2% ± 3.8%, -1.0 mm ± 2.6 mm, 0.8 mm ± 1.3 mm and -0.1 mm ± 1.5 mm, respectively. With regard to tandem-cylinder cases, differences in total dwell time, dh(front) (the distance from tandem tip to tandem ring), dt and dw between benchmark and individual plans were on average -1.5% ± 3.1%, -1.5 mm ± 4.9 mm, 0.1 mm ± 1.0 mm and 0.2 mm ± 0.8 mm, respectively. Of two cases, more than 13% differences in total dwell time were observed between benchmark plans and the clinical cases, which turned out to be due to the use of the wrong source position setting. These results suggest that our method is easy and useful for independent verification of patient-specific treatment planning QA.

A family of eight genes with homology to mammalian glutathione peroxidase (GPX) isoenzymes, designated AtGPX1-AtGPX8, has been identified in Arabidopsis thaliana. In this study we demonstrated the functional analysis of Arabidopsis AtGPX8 with peroxidase activity toward H(2)O(2) and lipid hydroperoxides using thioredoxin as an electron donor. The transcript and protein levels of AtGPX8 in Arabidopsis were up-regulated coordinately in response to oxidative damage caused by high-light (HL) stress or treatment with paraquat (PQ). Furthermore, the knockout Arabidopsis mutants of AtGPX8 (KO-gpx8) exhibited increased sensitivity to oxidative damage caused by PQ treatment in root elongation compared with the wild-type plants. In contrast, transgenic lines overexpressing AtGPX8 (Ox-AtGPX8) were less sensitive to oxidative damage than the wild-type plants. The levels of oxidized proteins in the KO-gpx8 and Ox-AtGPX8 lines were enhanced and suppressed, respectively, compared with the wild-type plants under HL stress or PQ treatment. The fusion protein of AtGPX8 tagged with green fluorescent protein was localized in the cytosol and nucleus of onion epidermal cells. In addition, the AtGPX8 protein was detected in the cytosolic and nuclear fractions prepared from leaves of Arabidopsis plants using the AtGPX8 antibody. Oxidative DNA damage under treatment with PQ increased in the wild-type and KO-gpx8 plants, while it decreased in the OX-AtGPX8 plants. These results suggest that AtGPX8 plays an important role in the protection of cellular components including nuclear DNA against oxidative stress.

We investigated an electronic portal image device (EPID)-based method to see whether it provides effective and accurate relative dose measurement at abutment leaves in terms of positional errors of the multi-leaf collimator (MLC) leaf position. A Siemens ONCOR machine was used. For the garden fence test, a rectangular field (0.2 20 cm) was sequentially irradiated 11 times at 2-cm intervals. Deviations from planned leaf positions were calculated. For the nongap test, relative doses at the MLC abutment region were evaluated by sequential irradiation of a rectangular field (2 20 cm) 10 times with a MLC separation of 2 cm without a leaf gap. The integral signal in a region of interest was set to position A (between leaves) and B (neighbor of A). A pixel value at position B was used as background and the pixel ratio (A/B 100) was calculated. Both tests were performed at four gantry angles (0, 90, 180 and 270°) four times over 1 month. For the nongap test the difference in pixel ratio between the first and last period was calculated. Regarding results, average deviations from planned positions with the garden fence test were within 0.5 mm at all gantry angles, and at gantry angles of 90 and 270° tended to decrease gradually over the month. For the nongap test, pixel ratio tended to increase gradually in all leaves, leading to a decrease in relative doses at abutment regions. This phenomenon was affected by both gravity arising from the gantry angle, and the hardware-associated contraction of field size with this type of machine.

Context: Acrodysostosis is a rare autosomal dominant disorder characterized by short stature, peculiar facial appearance with nasal hypoplasia, and short metacarpotarsals and phalanges with cone-shaped epiphyses. Recently, mutations of PRKAR1A and PDE4D downstream of GNAS on the cAMP-mediated G protein-coupled receptor (GPCR) signaling cascade have been identified in acrodysostosis with and without hormone resistance, although functional studies have been performed only for p.R368X of PRKAR1A., Objective: Our objective was to report a novel PRKAR1A mutation and its functional consequence in a Japanese female patient with acrodysostosis and hormone resistance., Patient: This patient had acrodysostosis-compatible clinical features such as short stature and brachydactyly and mildly elevated serum PTH and TSH values., Results: Although no abnormality was detected in GNAS and PDE4D, a novel de novo heterozygous missense mutation (p.T239A) was identified at the cAMP-binding domain A of PRKAR1A. Western blot analysis using primary antibodies for the phosphorylated cAMP-responsive element (CRE)-binding protein showed markedly reduced CRE-binding protein phosphorylation in the forskolin-stimulated lymphoblastoid cell lines of this patient. CRE-luciferase reporter assays indicated significantly impaired response of protein kinase A to cAMP in the HEK293 cells expressing the mutant p.T239A protein., Conclusions: The results indicate that acrodysostosis with hormone resistance is caused by a heterozygous mutation at the cAMP-binding domain A of PRKAR1A because of impaired cAMP-mediated GPCR signaling. Because GNAS, PRKAR1A, and PDE4D are involved in the GPCR signal transduction cascade and have some different characters, this would explain the phenotypic similarity and difference in patients with GNAS, PRKAR1A, and PDE4D mutations.

Background: There is an increased incidence of rare imprinting disorders associated with assisted reproduction technologies (ARTs). The identification of epigenetic changes at imprinted loci in ART infants has led to the suggestion that the techniques themselves may predispose embryos to acquire imprinting errors and diseases. However, it is still unknown at what point(s) these imprinting errors arise, or the risk factors., Methods: In 2009 we conducted a Japanese nationwide epidemiological study of four well-known imprinting diseases to determine any association with ART. Using bisulfite sequencing, we examine the DNA methylation status of 22 gametic differentially methylated regions (gDMRs) located within the known imprinted loci in patients with Beckwith-Wiedemann syndrome (BWS, n=1) and also Silver-Russell syndrome (SRS, n= 5) born after ART, and compared these with patients conceived naturally., Results: We found a 10-fold increased frequency of BWS and SRS associated with ART. The majority of ART cases showed aberrant DNA methylation patterns at multiple imprinted loci both maternal and paternal gDMRs (5/6), with both hyper- and hypomethylation events (5/6) and also mosaic methylation errors (5/6). Although our study may have been limited by a small sample number, the fact that many of the changes were mosaic suggested that they occurred after fertilization. In contrast, few of the patients who were conceived naturally exhibited a similar pattern of mosaic alterations. The differences in methylation patterns between the patients who were conceived naturally or after ART did not manifest due to the differences in the disease phenotypes in these imprinting disorders., Conclusion: A possible association between ART and BWS/SRS was found, and we observed a more widespread disruption of genomic imprints after ART. The increased frequency of imprinting disorders after ART is perhaps not surprising given the major epigenetic events that take place during early development at a time when the epigenome is most vulnerable.

Background: The clinical differential diagnosis of classic 21-hydroxylase deficiency (C21OHD) and cytochrome P450 oxidoreductase deficiency (PORD) is sometimes difficult, because both deficiencies can have similar phenotypes and high blood concentrations of 17α-hydroxyprogesterone (17OHP). The objective of this study was to identify biochemical markers for the differential diagnosis of C21OHD, PORD, and transient hyper 17α-hydroxyprogesteronemia (TH17OHP) in Japanese newborns. We established a 2-step biochemical differential diagnosis of C21OHD and PORD., Methods: We recruited 29 infants with C21OHD, 9 with PORD, and 67 with TH17OHP, and 1341 control infants. All were Japanese and between 0 and 180 days old; none received glucocorticoid treatment before urine sampling. We measured urinary pregnanetriolone (Ptl), the cortisol metabolites 5α- and 5β-tetrahydrocortisone (sum of these metabolites termed THEs), and metabolites of 3 steroids, namely dehydroepiandrosterone, androstenedione (AD4), and 11β-hydroxyandrostenedione (11OHAD4) by GC-MS., Results: At a cutoff of 0.020, the ratio of Ptl to THEs differentiated C21OHD and PORD from TH17OHP and controls with no overlap. Among metabolites of DHEA, AD4, and 11OHAD4, only 11β-hydroxyandrosterone (11HA), a metabolite of 11OHAD4, showed no overlap between C21OHD and PORD at a cutoff of 0.35 mg/g creatinine., Conclusions: A specific cutoff for the ratio of Ptl to THEs can differentiate C21OHD and PORD from TH17OHP and controls. Additionally, the use of a specific cutoff of 11HA can distinguish between C21OHD and PORD.

The term 'sake yeast' is generally used to indicate the Saccharomyces cerevisiae strains that possess characteristics distinct from others including the laboratory strain S288C and are well suited for sake brewery. Here, we report the draft whole-genome shotgun sequence of a commonly used diploid sake yeast strain, Kyokai no. 7 (K7). The assembled sequence of K7 was nearly identical to that of the S288C, except for several subtelomeric polymorphisms and two large inversions in K7. A survey of heterozygous bases between the homologous chromosomes revealed the presence of mosaic-like uneven distribution of heterozygosity in K7. The distribution patterns appeared to have resulted from repeated losses of heterozygosity in the ancestral lineage of K7. Analysis of genes revealed the presence of both K7-acquired and K7-lost genes, in addition to numerous others with segmentations and terminal discrepancies in comparison with those of S288C. The distribution of Ty element also largely differed in the two strains. Interestingly, two regions in chromosomes I and VII of S288C have apparently been replaced by Ty elements in K7. Sequence comparisons suggest that these gene conversions were caused by cDNA-mediated recombination of Ty elements. The present study advances our understanding of the functional and evolutionary genomics of the sake yeast.

Context: POR (cytochrome P450 oxidoreductase) is a ubiquitously expressed gene encoding an electron donor to all microsomal P450 enzymes and several non-P450 enzymes. POR mutations cause an autosomal recessive disorder characterized by skeletal dysplasia, adrenal dysfunction, and disorders of sex development. Although recent studies have indicated the presence of a CpG-rich region characteristic of housekeeping genes around the untranslated exon 1 (exon 1U) and a tropic effect of thyroid hormone on POR expression via thyroid hormone receptor-β, detailed regulatory mechanisms for the POR expression remain to be clarified., Objective: Our objective was to report a pivotal element of the proximal promoter of POR., Results: We first studied three patients (cases 1-3) with POR deficiency due to compound heterozygosity with an p.R457H mutation and transcription failure of an apparently normal allele, by oligoarray comparative genomic hybridization and serial direct sequencing of the deletion fusion points. Consequently, a 2,487-bp microdeletion involving exon 1U was identified in case 1 and an identical 49,604-bp deletion involving exon 1U and exon 1 was found in cases 2 and 3. We next analyzed the 2,487-bp region commonly deleted in cases 1-3 by in silico analysis, DNA binding analysis, luciferase assays, and methylation analysis. The results showed a critical function of the evolutionally conserved SP1 binding sites just upstream of exon 1U, especially the binding site at the position -26/-17, in the transcription of POR., Conclusions: The results suggest that the SP1 binding sites constitute an essential element of the POR proximal promoter.

Bonner sphere spectrometer (BSS) is used in radiation protection measurement because of its wide energy range (thermal to MeV) and easy operation. Mitsubishi Heavy Industries (MHI) has used BSS to obtain neutron spectrum and has used the neutron spectrum to estimate neutron dose or induced activity. Calibration of BSS is important to estimate precise neutron dose or induced activity. MHI BSS was calibrated at National Institute of Advanced Industrial Science and Technology (AIST). The calibration results at AIST are in good agreement with calculation results.

Context: Aromatase excess syndrome (AEXS) is a rare autosomal dominant disorder characterized by gynecomastia. Although cryptic inversions leading to abnormal fusions between CYP19A1 encoding aromatase and its neighboring genes have been identified in a few patients, the molecular basis remains largely unknown., Objective: The objective of the study was to examine the genetic causes and phenotypic determinants in AEXS., Patients: Eighteen affected males from six families participated in the study., Results: We identified three types of heterozygous genomic rearrangements, i.e. a 79,156-bp tandem duplication involving seven of 11 noncoding CYP19A1 exons 1, a 211,631-bp deletion involving exons 2-43 of DMXL2 and exons 5-10 of GLDN, and a 165,901-bp deletion involving exons 2-43 of DMXL2. The duplicated exon 1 functioned as transcription start sites, and the two types of deletions produced the same chimeric mRNA consisting of DMXL2 exon 1 and CYP19A1 coding exons. The DMXL2 exon 1 harbored a translation start codon, and the DMXL2/CYP19A1 chimeric mRNA was identified in only 2-5% of CYP19A1-positive transcripts. This was in contrast to the inversion-mediated chimeric mRNA that had no coding sequence on the fused exon 1 and accounted for greater than 80% of CYP19A1-positive transcripts. CYP19A1 was expressed in a limited number of tissues, whereas its neighboring genes involved in the chimeric mRNA formation were expressed widely., Conclusions: This study provides novel mechanisms leading to gain of function of CYP19A1. Furthermore, it appears that clinical severity of AEXS is primarily determined by the tissue expression pattern of relevant genes and by the structural property of promoter-associated exons of chimeric mRNA.

Context: Short stature (SS) is a multifactorial developmental condition with a significant genetic component. Recent studies have revealed that rare deleterious mutations in the GH-secretagogue receptor type 1A (GHSR1A) gene could be a cause of familial SS or GH deficiency., Objective: The aim of this study was to evaluate the contribution of GHSR1A mutations to the molecular mechanism underlying SS in Japanese subjects., Methods: We performed mutational screening of the GHSR1A gene in 127 unrelated Japanese SS patients diagnosed with either isolated GH deficiency or idiopathic SS. Identified mutations were analyzed in 188 control subjects, and their functional properties were examined in a heterologous expression system., Results: Four novel heterozygous GHSR1A mutations were identified (ΔQ36, P108L, C173R, and D246A). Expression studies demonstrated that these mutations had varying functional consequences: 1) all mutations showed a loss-of-function effect on the constitutive signaling activity of GHSR1A, but the degree of loss varied widely; 2) C173R caused intracellular retention of the mutated protein, resulting in total loss of receptor function; 3) P108L resulted in a large decrease in binding affinity to ghrelin, without affecting its surface expression; 4) D246A uniquely impaired agonist- and inverse agonist-stimulated receptor signaling; and 5) ΔQ36 showed only a subtle reduction in constitutive activity. The cumulative frequency of these putative functional mutations was significantly higher in the patient group than in controls (4.72 vs. 0.53%; P = 0.019; odds ratio = 9.28; 95% confidence interval, 1.10-78.0)., Conclusions: Our results suggest that GHSR1A mutations contribute to the genetic etiology of SS in the Japanese population.

We previously showed that carbon ion irradiation can inhibit the expression of the anillin (ANLN) gene, which is regulated by the activation of the phosphatidylinositol-3-kinase (PI3K)/Akt signaling pathway associated with metastasis. The purpose of this study is to compare the effects of carbon ion irradiation on the PI3K/Akt signaling pathway to those of photon irradiation. Our study showed that carbon ion irradiation of human lung adenocarcinoma cells A549 decreased their invasion more effectively than photon irradiation did. We found that carbon ion irradiation reduced the nuclear localization of ANLN at lower dose, but did not affect its expression. Low-dose carbon ion irradiation also reduced the level of phosphorylated Akt compared to untreated controls, whereas photon irradiation did not. These results suggest that carbon ion irradiation effectively suppresses the metastatic potential of A549 cells by suppressing the PI3K/Akt signaling pathway.

Aim: To construct a self-cloning brewer's yeast that can minimize the unfavourable flavours caused by oxidation and certain kinds of sulfur compounds., Methods and Results: DNA fragments of a high-expression promoter from the TDH3 gene originating from Saccharomyces cerevisiae were integrated into the promoter regions of the S. cerevisiae-type and Saccharomyces bayanus-type SSU1 genes of bottom-fermenting brewer's yeast. PCR and sequencing confirmed the TDH3 promoter was correctly introduced into the SSU1 regions of the constructed yeasts, and no foreign DNA sequences were found. Using the constructed yeasts, the concentration of sulfite in fermenting wort was higher when compared with the parent strain. In addition, the concentrations of hydrogen sulfide, 3-methyl-2-buten-1-thiol (MBT) and 2-mercapto-3-methyl-1-butanol (2M3MB) were lower when compared with the parent strain., Conclusion: We successfully constructed a self-cloning brewer's yeast with high SSU1 expression that enhanced the sulfite-excreting ability and diminished the production ability of hydrogen sulfide, MBT and 2M3MB., Significance and Impact of the Study: The self-cloning brewer's yeast with high SSU1 expression would contribute to the production of superior quality beer with a high concentration of sulfite and low concentrations of hydrogen sulfide, MBT and 2M3MB., (© 2010 The Authors. Journal of Applied Microbiology © 2010 The Society for Applied Microbiology.)

Plastomes of the peridinin-containing dinoflagellates are composed of a limited number of genes, which are carried individually on small circular molecules, termed 'minicircles'. Although the prevalent plastid chromosome of most algae and plants has only a single copy of each gene, our previous study showed that low copy numbers of multiple variants of the gene psbA co-exist with the 'ordinary' gene encoding the D1 protein in minicircles of Alexandrium tamarense. Although none of the psbA variants encoded the entire protein, they persisted in culture. In this study, we compared the distribution and structure of psbA and psbD variants in two species of Alexandrium to characterize DNA rearrangement within these genes. In addition to four previously reported psbA variants, three psbD variants were found in A. tamarense minicircles. The ordinary psbA and psbD genes also co-existed with variants in another species, A. catenella. The sequences of the ordinary genes were virtually identical in the two species. All the variants comprised insertion or deletion mutations, with no base substitutions being identified. Duplicated parts of the coding sequences were contained in most of the insertions. Short direct repeats (4-14 bp) and/or adenine + thymine-rich motifs were present in all mutation regions, although the position and/or the sequence of each DNA rearrangement was unique to each variant. The results indicated that replication-based repeat-mediated recombination was responsible for generation of the variants.

Context: Mutations of multiple transcription factor genes involved in pituitary development have been identified in a minor portion of patients with combined pituitary hormone deficiency (CPHD). However, copy number aberrations involving such genes have been poorly investigated in patients with CPHD., Objective: We aimed to report the results of mutation and gene copy number analyses in patients with CPHD., Subjects and Methods: Seventy-one Japanese patients with CPHD were examined for mutations and gene copy number aberrations affecting POU1F1, PROP1, HESX1, LHX3, LHX4, and SOX3 by PCR-direct sequencing and multiplex ligation-dependent probe amplification. When a deletion was indicated, it was further studied by fluorescence in situ hybridization, oligoarray comparative genomic hybridization, and serial sequencing for long PCR products encompassing the deletion junction., Results: We identified a de novo heterozygous 522,009-bp deletion involving LHX4 in a patient with CPHD (GH, TSH, PRL, LH, and FSH deficiencies), anterior pituitary hypoplasia, ectopic posterior pituitary, and underdeveloped sella turcica. We also identified five novel heterozygous missense substitutions (p.V201I and p.H387P in LHX4, p.T63M and p.A322T in LHX3, and p.V53L in SOX3) that were assessed as rare variants by sequencing analyses for control subjects and available parents and by functional studies and in silico analyses., Conclusions: The results imply the rarity of abnormalities affecting the six genes in patients with CPHD and the significance of the gene copy number analysis in such patients.

Context: Although recent studies have suggested a positive role of OTX2 in pituitary as well as ocular development and function, detailed pituitary phenotypes in OTX2 mutations and OTX2 target genes for pituitary function other than HESX1 and POU1F1 remain to be determined., Objective: We aimed to examine such unresolved issues., Subjects: We studied 94 Japanese patients with various ocular or pituitary abnormalities., Results: We identified heterozygous p.K74fsX103 in case 1, p.A72fsX86 in case 2, p.G188X in two unrelated cases (3 and 4), and a 2,860,561-bp microdeletion involving OTX2 in case 5. Clinical studies revealed isolated GH deficiency in cases 1 and 5; combined pituitary hormone deficiency in case 3; abnormal pituitary structures in cases 1, 3, and 5; and apparently normal pituitary function in cases 2 and 4, together with ocular anomalies in cases 1-5. The wild-type Orthodenticle homeobox 2 (OTX2) protein transactivated the GNRH1 promoter as well as the HESX1, POU1F1, and IRBP (interstitial retinoid-binding protein) promoters, whereas the p.K74fsX103-OTX2 and p.A72fsX86-OTX2 proteins had no transactivation functions and the p.G188X-OTX2 protein had reduced ( approximately 50%) transactivation functions for the four promoters, with no dominant-negative effect. cDNA screening identified positive OTX2 expression in the hypothalamus., Conclusions: The results imply that OTX2 mutations are associated with variable pituitary phenotype, with no genotype-phenotype correlations, and that OTX2 can transactivate GNRH1 as well as HESX1 and POU1F1.

Although the use of small segments in step and shoot IMRT provides better dose distribution, extremely small segments decrease treatment accuracy. The purpose of this study was to determine the optimum minimum segment size (MSS) in two-step optimization in prostate step and shoot IMRT with regard to both planning quality and dosimetric accuracy. The XiO treatment planning system and Oncor Impression Plus were used. Results showed that the difference in homogeneity index (HI), defined as the ratio of maximum to minimum doses for planning target volume, between the MSS 1.0 cm and 1.5 cm plans, and 2.0 cm plans, was 0.1%, and 9.6%, respectively. With regard to V107 of PTV, the volume receiving 107% of the prescribed dose of the PTV, the difference between MSS 1.0 cm and 1.5 cm was 2%. However, the value of the MSS 2.0 cm or greater plans was more than 2.5-fold that of the MSS 1.0 cm plan. With regard to maximum rectal dose, a significant difference was seen between the MSS 1.5 cm and 2.0 cm plans, whereas no significant difference was seen between the MSS 1.0 cm and 1.5 cm plans. Composite plan verification revealed a greater than 5% dose difference between planned and measured dose in many regions with the MSS 1.0 cm plan, but in only limited regions in the MSS 1.5 cm plan. Our data suggest that the MSS should be determined with regard to both planning quality and dosimetric accuracy.

Superb biological effectiveness and dose conformity represent a rationale for heavy-ion therapy, which has thus far achieved good cancer controllability while sparing critical normal organs. Immediately after irradiation, heavy ions produce dense ionization along their trajectories, cause irreparable clustered DNA damage, and alter cellular ultrastructure. These ions, as a consequence, inactivate cells more effectively with less cell-cycle and oxygen dependence than conventional photons. The modes of heavy ion-induced cell death/inactivation include apoptosis, necrosis, autophagy, premature senescence, accelerated differentiation, delayed reproductive death of progeny cells, and bystander cell death. This paper briefly reviews the current knowledge of the biological aspects of heavy-ion therapy, with emphasis on the authors' recent findings. The topics include (i) repair mechanisms of heavy ion-induced DNA damage, (ii) superior effects of heavy ions on radioresistant tumor cells (intratumor quiescent cell population, TP53-mutated and BCL2-overexpressing tumors), (iii) novel capacity of heavy ions in suppressing cancer metastasis and neoangiogenesis, and (iv) potential of heavy ions to induce secondary (especially breast) cancer.

Aim: To determine the structure of the chimeric chromosome X of bottom-fermenting yeasts., Methods and Results: Eight cosmid clones carrying DNA from chromosome X of bottom-fermenting yeasts were selected by end-sequencing. Four of the cosmid clones had Saccharomyces cerevisiae (SC)-type and Saccharomyces bayanus (SB)-type chimeric ends, two had SC-type ends and two had SB-type ends. Sequencing revealed that the bottom-fermenting yeast strains in this study had four types of chromosome X: SC-SC, SC-SB, SB-SC and SB-SB. The translocation site in the chimeric chromosome is conserved among bottom-fermenting yeast strains, and was created by homologous recombination within a region of high sequence identity between the SC-type sequence and the SB-type sequence., Conclusions: Existing bottom-fermenting yeast strains share a common ancestor in which the chimeric chromosome X was generated., Significance and Impact of the Study: The knowledge gained in this study sheds light on the evolution of bottom-fermenting yeasts.

To determine whether two HorC molecules coordinately form a single unit, the functional properties of covalently linked dimers of HorC encoded by tandemly fused horC genes were studied. Lactobacillus brevis introduced with the fused horC genes and a single horC gene exhibited same degree of resistance to hop compounds and cetyltrimethylammonium bromide. This suggests that HorC functions as a homodimer.

Unlabelled: CONTEX: Although GH values measured by an immunoassay usually reflect GH bioactivities, discrepancy exists between immunoactivity and bioactivity in a rare condition known as 'bioinactive GH'., Objective: To report an immunologically anomalous but considerably bioactive GH., Methods: We performed mutational and functional analyses of GH1 in a 7-year-old Japanese boy with short stature (-3.0 s.d.) in whom serum GH values measured with a Tosoh immunoassay kit were all undetectable in three provocation tests, whereas urine GH value measured with a Hitachi immunoassay kit was within the normal range. Serum IGF-1 was at a low-normal range, and IGF-binding protein-3 was below the normal range., Results: Mutation analysis showed a missense GH produced by a novel GH1 mutation (p.D116E) of paternal origin and a frameshift mutation (p.Q68fsX106) of maternal origin. Genotype-phenotype correlations in this family and in vitro functional studies indicated that the p.D116E-GH was immeasurable with the Tosoh kit but was measurable, though maybe not precise, with a Daiichi kit, and had a reduced in vivo bioactivity. The p.Q68fsX106 yielded no GH protein., Conclusions: The results suggest that the p.D116E affects the GH epitope primarily recognized by the Tosoh kit but not by the Hitachi or the Daiichi kits, thereby producing an immunologically anomalous but considerably bioactive GH. The presence of such a hormone discordant for immunoactivity and bioactivity should be kept in mind, to allow for an appropriate assessment of endocrine data.

Context: Cytochrome P450 oxidoreductase (POR) deficiency is a rare autosomal recessive disorder characterized by skeletal dysplasia, adrenal dysfunction, disorders of sex development (DSD), and maternal virilization during pregnancy. Although multiple studies have been performed for this condition, several matters remain to be clarified, including the presence of manifesting heterozygosity and the underlying factors for clinical variability., Objective: The objective of the study was to examine such unresolved matters by detailed molecular studies and genotype-phenotype correlations., Patients: Thirty-five Japanese patients with POR deficiency participated in the study., Results: Mutation analysis revealed homozygosity for R457H in cases 1-14 (group A), compound heterozygosity for R457H and one apparently null mutation in cases 15-28 (group B), and other combinations of mutations in cases 29-35 (group C). In particular, FISH and RT-PCR sequencing analyses revealed an intragenic microdeletion in one apparent R457H homozygote, transcription failure of apparently normal alleles in three R457H heterozygotes, and nonsense mediated mRNA decay in two frameshift mutation-positive cases examined. Genotype-phenotype correlations indicated that skeletal features were definitely more severe, and adrenal dysfunction, 46,XY DSD, and pubertal failure were somewhat more severe in group B than group A, whereas 46,XX DSD and maternal virilization during pregnancy were similar between two groups. Notable findings also included the contrast between infrequent occurrence of 46,XY DSD and invariable occurrence of 46,XX DSD and pubertal growth pattern in group A mimicking that of aromatase deficiency., Conclusions: The results argue against the heterozygote manifestation and suggest that the residual POR activity reflected by the R457H dosage constitutes the underlying factor for clinical variability in some features but not other features, probably due to the simplicity and complexity of POR-dependent metabolic pathways relevant to each phenotype.

The pathway from beta-carotene to astaxanthin is a crucial step in the synthesis of astaxanthin, a red antioxidative ketocarotenoid that confers beneficial effects on human health. Two enzymes, a beta-carotene ketolase (carotenoid 4,4'-oxygenase) and a beta-carotene hydroxylase (carotenoid 3,3'-hydroxylase), are involved in this pathway. Cyanobacteria are known to utilize the carotenoid ketolase CrtW and/or CrtO, and the carotenoid hydroxylase CrtR. Here, we compared the catalytic functions of CrtW ketolases, which originated from Gloeobacter violaceus PCC 7421, Anabaena (also known as Nostoc) sp. PCC 7120 and Nostoc punctiforme PCC 73102, and CrtR from Synechocystis sp. PCC 6803, Anabaena sp. PCC 7120 and Anabaena variabilis ATCC 29413 by complementation analysis using recombinant Escherichia coli cells that synthesized various carotenoid substrates. The results demonstrated that the CrtW proteins derived from Anabaena sp. PCC 7120 as well as N. punctiforme PCC 73102 (CrtW148) can convert not only beta-carotene but also zeaxanthin into their 4,4'-ketolated products, canthaxanthin and astaxanthin, respectively. In contrast, the Anabaena CrtR enzymes were very poor in accepting either beta-carotene or canthaxanthin as substrates. By comparison, the Synechocystis sp. PCC 6803 CrtR converted beta-carotene into zeaxanthin efficiently. We could assign the catalytic functions of the gene products involved in ketocarotenoid biosynthetic pathways in Synechocystis sp. PCC 6803, Anabaena sp. PCC 7120 and N. punctiforme PCC 73102, based on the present and previous findings. This explains why these cyanobacteria cannot produce astaxanthin and why only Synechocystis sp. PCC 6803 can produce zeaxanthin.

Specific PCR primers were designed based on the 16S rRNA genes of recently proposed beer-spoilage species, Pectinatus haikarae, Megasphaera sueciensis, and M. paucivorans, and two sets of our previously reported multiplex PCR methods for Pectinatus spp. and beer-spoilage cocci were reconstructed. Each modified multiplex PCR method was found specifically to detect beer-spoilage species of Pectinatus and cocci, including new species.