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Obesity is a major complication in children with 21-hydroxylase deficiency (21-OHD). There is evidence to show that higher body mass index (BMI) during infancy and early childhood is associated with an increased risk for the subsequent development of obesity in the general population; however, limited information is currently available on this issue in 21-OHD patients. Additionally, despite the frequent use of supraphysiological dosages of hydrocortisone in 21-OHD, the association between BMI and hydrocortisone dosage during these periods remains largely unclear; therefore, we retrospectively investigated BMI at approximately 1 and 3 years old and its association with hydrocortisone dosage in 56 children with 21-OHD. The median BMI-standard deviation score (SDS) was 0.28 (Interquartile range [IQR]: -0.53 to 1.09) and 0.39 (IQR: -0.44 to 1.14) at approximately 1 and 3 years old, respectively, and no association was observed between hydrocortisone dosage and BMI-SDS at either time-point; however, multivariate analysis revealed that hydrocortisone dosage at approximately 1 year old was positively associated with changes in BMI (β = 0.57, p = 0.013) and BMI-SDS (β = 0.59, p = 0.011) between approximately 1 and 3 years old after adjustment for age, sex, and changes in hydrocortisone dosage during the same period. The average dosage of hydrocortisone between approximately 6 months and 1 year old also showed similar results. These results indicate that a higher dosage of hydrocortisone during late infancy is associated with a higher BMI at approximately 3 years old, which may lead to the development of obesity later in life in children with 21-OHD.

Objectives Emerging evidence suggests a fat depot-specific relationship with bone mineral density (BMD) in children, particularly in those who are overweight/obese. However, this has not yet been investigated in detail in children with Prader–Willi syndrome (PWS), a genetic syndrome characterized by a decreased lean mass (LM) and increased fat mass (FM). The objective of this study is to investigate the relationships of LM and FM, particularly fat distribution, with bone mineral parameters. Methods This is a retrospective and cross-sectional study. Forty-seven prepubertal Japanese children with PWS (22 males, mean age: 6.86 years) were included. No subjects had type 2 diabetes mellitus or osteoporotic medications. LM, FM, and BMD and bone mineral content in the total body less head and the lumbar spine were measured using dual-energy x-ray absorptiometry, in addition to subcutaneous/visceral adipose tissue (SAT/VAT), and the ratio of VAT to SAT (V/S) by computed tomography at the umbilical level. Bone mineral apparent density was calculated to correct for bone size. Results LM positively correlated with bone mineral parameters after controlling for age, sex, growth hormone (GH) treatment, and FM. Although FM did not correlate with bone mineral parameters, compartment-specific analysis revealed that SAT positively and V/S negatively correlated with bone mineral parameters after controlling for age, sex, GH treatment and LM. Conclusions A compartment-specific effect of FM on bone mineral parameters was noted such that SAT was a positive predictor for BMD independent of LM in prepubertal children with PWS.

The pathogenesis of gonadotropin-independent precocious puberty (PP) includes both congenital and acquired forms, the latter of which may be associated with neoplasms, such as sex-steroid hormone-producing tumors. Beta-human chorionic gonadotropin (β-hCG)-producing tumors also cause gonadotropin-independent PP by stimulating the production of testosterone in Leydig cells. Germ cell tumors and hepatoblastoma both produce β-hCG; however, there is limited evidence to show that gonadotropin-independent PP is caused by other β-hCG-producing tumors. We herein report the first case of β-hCG-producing neuroblastoma associated with the development of gonadotropin-independent PP. A 2-year-old boy presented with an increased penile length, enlargement of the testes, pigmentation of the external genitalia, and growth acceleration. Imaging, blood, and urinary examinations revealed the presence of neuroblastoma in the right adrenal region. Decreased levels of luteinizing hormone and follicle-stimulating hormone with an increased testosterone level were indicative of gonadotropin-independent PP. Since serum β-hCG was elevated, β-hCG-producing neuroblastoma was suspected. Histological findings of the resected tumor were compatible with neuroblastoma. An immunohistochemical analysis using serial sections revealed staining for β-hCG in synaptophysin-positive cells. Furthermore, immunofluorescence showed the co-staining of β-hCG with neuron-specific enolase. These results suggested that β-hCG was produced by tumor cells. Surgical removal of the tumor promptly normalized serum β-hCG and testosterone levels. In conclusion, we propose the addition of neuroblastoma to the list of differential diagnoses of gonadotropin-independent PP with β-hCG positivity in serum that includes germ cell tumors and hepatoblastoma.

Objective Investigate efficacy, safety, and tolerability of 3 once-weekly somapacitan doses compared with daily growth hormone (GH) administration in short children born small for gestational age (SGA). Design Randomised, multi-centre, open-label, controlled phase 2 study comprising a 26-week main phase and a 4-year extension (NCT03878446). The study was conducted at 38 sites across 12 countries. 26-week main phase results are presented here. Sixty-two GH treatment-naïve, prepubertal short children born SGA were randomised and exposed; 61 completed the main phase. Three somapacitan doses (0.16 [n = 12], 0.20 [n = 13], 0.24 [n = 12] mg/kg/week) and 2 daily GH doses (0.035 [n = 12], 0.067 [n = 13] mg/kg/day) were administered subcutaneously. Results After 26 weeks of treatment, the estimated mean annualised height velocity (HV) was 8.9, 11.0, and 11.3 cm/year for somapacitan 0.16, 0.20, and 0.24 mg/kg/week, respectively, compared to 10.3 and 11.9 cm/year for daily GH 0.035 and 0.067 mg/kg/day. Changes from baseline in HV standard deviation score (SDS), height SDS, and insulin-like growth factor I (IGF-I) SDS showed similar dose-dependent responses. Exposure-response modelling indicated the greatest efficacy correlated with the highest somapacitan exposure. Similar safety and tolerability were demonstrated for all weekly somapacitan and daily GH doses. Conclusions Based on the totality of data on improvements in height-based parameters combined with exposure-response analyses, somapacitan 0.24 mg/kg/week appears most efficacious, providing similar efficacy, safety, and tolerability as daily GH 0.067 mg/kg/day in short children born SGA after 26 weeks of treatment.

BACKGROUND Children born small for gestational age (SGA), particularly when associated with an extremely low birthweight (ELBW), have a higher risk of renal dysfunction. Growth hormone (GH) treatment is used to treat short-statured children born SGA; however, its effects on renal function remain elusive, especially in those born SGA with ELBW. METHODS Short-statured children born SGA (N = 42) were included. Subjects were subdivided into two groups based on their birthweight: the ELBW group (N = 15) with a birthweight of

Gain-of-function variants in SAMD9, which resides on chromosome 7, cause MIRAGE syndrome that is associated with congenital adrenal insufficiency and gonadal dysgenesis. We previously reported a Japanese patient with MIRAGE syndrome carrying a de novo heterozygous SAMD9 variant (p.Ala1479Ser). In this study, we confirmed the pathogenicity of Ala1479Ser-SAMD9 in vitro. Genetic study results revealed an atypically low variant allele frequency (26%) and we suspected of genomic rearrangement(s) involving chromosome 7. Single nucleotide polymorphism (SNP) array and short tandem repeat analysis showed presence of mosaic maternal isodisomic uniparental disomy 7 (UPD7). Deep sequencing using DNA samples obtained at 0, 6, 10, and 25 mo of age revealed that the percentage of cells with UPD7 increased constantly from 6% to 82% over 25 mo, and this increase coincided with a decrease in the percentage of cells with p.Ala1479Ser from 94% to nearly undetectable levels. We further screened for low-allele-frequency and rare SAMD9 variants in eight patients with Silver-Russel syndrome and maternal UPD7; however, none of the patients harbored such a variant. In conclusion, our case demonstrates that genetic findings can vary considerably in patients with MIRAGE syndrome and that a comprehensive diagnostic approach, including SNP array and deep sequencing, is important in such cases.

The effectiveness of growth hormone (GH) treatment for height gain in short-stature children born small for gestational age (SGA) with extremely low birthweight (ELBW; birthweight1,000 g) remains largely unknown.In study 1, 35 prepubertal Japanese children born SGA with ELBW were categorized into three groups based on the presence or absence of catch-up growth by age 3 (CU(+) and CU(-), respectively) and GH treatment (GH(+) and GH(-), respectively). Height standard deviation (SD) scores (HT-SDS) in the CU-/GH+ group (n = 19) were compared with those in the age-matched CU+/GH- (n = 9) and CU-/GH- groups (n = 7). In study 2, 66 prepubertal Japanese SGA children treated with GH were divided into three groups by birthweight:1,000 g (n = 19), 1,000-2,000 g (n = 20), and2,000 g (n = 27). Changes in HT-SDS during the initial 3 years of GH treatment were compared among the three groups.In study 1, the mean HT-SDS in the CU-/GH+ group (-1.15 SD) was similar to that in the CU+/GH- group (-1.39 SD) but higher than that in the CU-/GH- group (-2.24 SD). In study 2, GH achieved a height gain of +1.62 SD in the ELBW group, which was similar to that in the other groups (1,000-2,000 g: +1.46 SD,2,000 g: +1.53 SD).Growth hormone treatment in short-stature children born SGA with ELBW increased HT-SDS, which was similar to that in SGA children born with a birthweight ≥1,000 g. These results indicate that GH treatment may be an effective approach to promote adequate growth recovery for short-stature children born SGA with ELBW.

The circadian clock network is an evolutionarily conserved system that regulates systemic metabolism, such as glucose homeostasis. Intestinal tissue is a pivotal organ for the regulation of glucose metabolism, mainly via glucose absorption into the circulation; however, the significance of the intestinal circadian clock network for glucose metabolism remains largely unclear. We herein utilized a mouse model in which Bmal1, a core clock gene, was deleted in an intestine-specific manner (Bmal1Int–/– mice) and demonstrated a rhythmic expression of Sglt1 with its peak at zeitgeber time (ZT) 10.7 ± 2.8 in control mice, whereas this was lost in Bmal1Int–/– mice. Mechanistically, chromatin immunoprecipitation analysis revealed rhythmic binding of CLOCK to the E-box elements in the Sglt1 gene in control mice; however, this was absent in Bmal1Int–/– mice. Accordingly, SGLT1 protein levels were decreased during the dark phase in Bmal1Int–/– mice and this was associated with impaired glucose absorption, leading to a decline in hepatic glycogen levels at ZT4, which was restored by ingestion of high-sucrose water. Additionally, when mice were starved from ZT0, greater expression of the lipolysis-related gene Pnpla2 was observed in adipose tissue of Bmal1Int–/– mice, and this was not noted when glycogen storage was restored by high-sucrose water prior to fasting, suggesting that higher Pnpla2 expression in Bmal1Int–/– mice was likely caused by lower glycogen storage. These results indicate that disruption of the intestinal circadian clock system impairs glucose absorption in the intestine and affects systemic glucose homeostasis.

As evidenced by the intact histology of the testes during infancy, testicular differentiation during the prenatal period occurs normally in individuals with 5 alpha-reductase type 2 deficiency (5αRD); however, a majority of these individuals suffer from azoospermia or oligospermia during adulthood, indicating that impaired spermatogenesis occurs postnatally. Although the accompanying cryptorchidism may be partly responsible for this process, the underlying mechanisms remain largely unknown. To address this issue, we retrospectively compared the histological findings of descended testes in a 3-mo-old patient and undescended testes in an 18-yr-old patient with 5αRD. In the latter, testicular histology was compared to that of cryptorchid testes obtained from five adolescent patients without endocrinological abnormalities. Histological findings of a 3-mo-old patient revealed normal number of germ cells with intact seminiferous tubules. In contrast, an 18-yr-old patient showed marked reduction in germ cell number and atrophic seminiferous tubules. The findings were very similar to those observed in cryptorchid testes without endocrinological abnormalities. These findings suggest that the decrease in germ cells in 5αRD patients may be at least partly caused by accompanying cryptorchidism. As the number of germ cells did not decrease during the infantile period, early orchiopexy is recommended to prevent a decrease in germ cell number and preserve fertility.

A 4-year-old girl was admitted to our hospital because of precocious puberty. Radiologic findings revealed a fist-sized solid tumor in the left ovary without ascites, peritoneal dissemination, and distant metastasis. The patient underwent left salpingo-oophorectomy without spillage. The size of the excised tumor was 10.0×9.0×4.8 cm. On pathologic examination, the tumor was diagnosed as an ovarian steroid cell tumor, not otherwise specified. In the present case, although the diameter of the tumor (7 cm) and three mitoses per 10 high-power fields represented some potential for malignancy, we opted for careful observation without chemotherapy as the tumor was of clinical stage Ia.

Existing guidelines recommend long-term follow-up of childhood cancer survivors (CCS). However, in Japan, transitional care for CCS has not been established. To ascertain the current status in Japan, and to cultivate a better understanding, a questionnaire survey was conducted on transitional care in CCS, and adolescent and young adult (AYA) cancer survivors. Questionnaires were distributed to 183 councilors (137 institutions) of the Japanese Society for Pediatric Endocrinology. A total of 131 responses, representative of 174 councilors, were obtained. The response rate was 95%. Among the respondents, 91% had experience in medical care for cancer patients, while 63% had experience in transitional care; however, the number of patients referred to adult clinics was small. Further, 89% acknowledged the availability of adult endocrinologists who were willing to accept these patients; although their numbers were insufficient. Pediatric endocrinologists highlighted difficulties in medical examinations concerning infertility, obesity, pregnancy/delivery, and gonadal dysfunction, in that order. Staff and time shortages were listed as some of the challenges faced by medical staff, while multisystem morbidity was listed for patients. This nationwide questionnaire survey revealed that Japanese pediatric endocrinologists require cooperation between related departments and collaborative infrastructure to develop transitional care for cancer survivors.

In females, endogenous estrogen secretion increases gradually before pubertal development. The benefits of low-dose estrogen therapy in patients with Turner syndrome were originally discussed by Ross et al. and Quigley et al. These seminal studies used ethinyl estradiol (EE2), starting at a dose of 25 ng/kg/d. We hypothesized that the initial dosage of estrogen could be titrated to more closely mimic physiological increments of endogenous estrogen. Therefore, our recent study initiated EE2 treatment at a dosage of 1–2 ng/kg/d, an ultra-low-dose estrogen therapy in pediatric patients with Turner syndrome. The ultra-low-dose estrogen therapy in this syndrome produced a good final height outcome but achieved suboptimal bone mineral density (BMD). In the present review, we have explained our findings to clarify the merits and demerits of this new therapy and to promote further discussion and research. This type of ultra-low-dose estrogen therapy, initiated at an early age, could be ideal for estrogen replacement in female patients with hypogonadism, such as Turner syndrome.

MIRAGE syndrome is a recently identified disorder characterized by myelodysplasia, infection, restriction of growth, adrenal hypoplasia, genital phenotypes, and enteropathy. It is caused by a gain-of-function variant in the SAMD9 gene, but there is limited knowledge regarding the genotype–phenotype correlation. We herein report a Japanese patient with MIRAGE syndrome carrying a novel de novo heterozygous missense variant in the SAMD9 gene (c.4435 G > T; p.Ala1479Ser).

For preterm and very low birthweight infants, the mother’s own milk is the best nutrition. Based on the latest information for mothers who give birth to preterm and very low birthweight infants, medical staff should encourage and assist mothers to pump or express and provide their own milk whenever possible.If the supply of maternal milk is insufficient even though they receive adequate support, or the mother’s own milk cannot be given to her infant for any reason, donor human milk should be used.Donors who donate their breast milk need to meet the Guideline of the Japan Human Milk Bank Association.Donor human milk should be provided according to the medical needs of preterm and very low birthweight infants, regardless of their family’s financial status.In the future, it will be necessary to create a system to supply an exclusive human milk‐based diet (EHMD), consisting of human milk with the addition of a human milk‐derived human milk fortifier, to preterm and very low birthweight infants.

Nutritional intervention for maintaining an appropriate body composition is central to the management of Prader-Willi syndrome (PWS). Despite evidence that visceral adipose tissue (VAT) is associated with increased metabolic risks, the effects of nutritional intervention on fat distribution have not been evaluated for PWS children. We herein investigated fat distribution in 20 genetically diagnosed PWS children (9 males and 11 females); 17 of which received nutritional intervention with or without growth hormone (GH) treatment [GH-treated group (n = 8), GH-untreated group (n = 9)]. GH treatment continued for median of 4.9 years. GH treatment significantly increased height standard deviation score (SDS) whereas body weight SDS and body mass index SDS were not affected in GH-treated group. In GH-untreated group, height SDS significantly decreased during approximately 5 years of follow-up. Fat distribution was evaluated at the median age of 6.93 years in GH-treated group and 7.01 years in GH-untreated group. VAT was maintained within the reference range in both groups. Subcutaneous adipose tissue (SAT) was elevated in GH-untreated groups compared to reference values whereas it was not in GH-treated group. The remaining three subjects, who had never received nutritional intervention or GH treatment, showed increased VAT and SAT. In conclusion, nutritional intervention is beneficial in maintaining VAT within the reference range during childhood, although excessive nutritional intervention may cause unfavorable effect on linear growth.

Osteoporosis is one of the clinical features of women with Turner syndrome (TS). The reasons for low bone mineral density (BMD) and increased bone fragility are multifactorial, including estrogen deficiency, X-chromosome abnormalities, and environmental factors. Few, large-scale studies on bone mineral density in either adolescents or adults with TS have been done in Japan. The goal of the present study was to investigate spinal BMD in women with TS, assess its relationship with clinical parameters, especially estrogen replacement therapy, and investigate its longitudinal changes. The spinal BMD and clinical data of 149 Japanese women with TS aged 15 to 49 years who were followed at the four participating hospitals were retrospectively analyzed. The BMD Z-scores of the women with TS ranged from -5.30 to +1.89. Women with TS aged 15-39 years had lower BMD than healthy Japanese women (p < 0.01) while women with spontaneous menstruation had a significantly higher BMD Z-score than those without spontaneous menstruation (-0.73 ± 1.11 vs. -1.67 ± 1.18, p < 0.01). In women without spontaneous menstruation, BMD Z-scores correlated with the duration of their estrogen therapy (r = 0.167, p < 0.01). Women aged 15-39 years with TS had low BMD, which was associated with primary amenorrhea and short estrogen replacement therapy duration.

The circadian clock system is an evolutionarily conserved system by which organisms adapt their metabolic activities to environmental inputs, including nutrient availability. The disruption of this system has been pathogenically linked to the disintegration of metabolic homeostasis, leading to the development of metabolic complications, including obesity. Lifestyle factors that disrupt this system have recently been emerging and associated with the development of obesity, which is most evidenced by the finding that shift workers are at an increased risk of developing various disorders, such as obesity and obesity-related complications. Lifestyle factors that contribute to a misalignment between the internal clock system and environmental rhythms have also been identified in the pediatric field. A short sleep duration and breakfast skipping are prevalent in children and there is mounting evidence that these lifestyle factors are associated with an increased risk of pediatric obesity; however, the underlying mechanisms have not yet been elucidated in detail. Our current understanding of the impact of lifestyle factors that cause a misalignment between the internal clock system and environmental rhythms on the development of pediatric obesity is summarized herein with a discussion of potential mechanistic insights.

OBJECTIVE Insulin growth factor-1 (IGF-1) is used to evaluate growth hormone (GH) sufficiency and is decreased in children with Prader-Willi syndrome (PWS). Although IGF-1 is negatively affected by body size and nutritional status, both of which are impaired in PWS children, these variables are typically not considered when assessing IGF-1 levels in these subjects. Here, we compared IGF-1 levels in PWS children to controls matched for age, sex, anthropometric parameters, and nutritional status. DESIGN/PATIENTS/MEASUREMENTS The retrospective analysis included genetically diagnosed PWS subjects (n = 65, median age; 14.0 months) and controls (n = 111, 14.3 months) matched for age, sex, anthropometric parameters (height-standard deviation score [SDS], weight-SDS, body mass index-SDS), and serum albumin levels, a marker for nutritional status. IGF-1 SDS was compared between PWS subjects and controls after adjustment for confounding variables. The GH provocation test was performed in 29 PWS subjects, and IGF-1 SDS was compared between GH-sufficient (n = 20) and GH-deficient (n = 9) subjects. Spearman's rank correlation coefficient was performed to investigate the association between age and IGF-1 SDS. None had received GH or levothyroxine treatment. RESULTS After adjustment for confounding variables, IGF-1 SDS was significantly lower in PWS subjects than controls (-1.56 vs. -1.01, p = .003), while it was not different between GH-sufficient and GH-deficient PWS subjects. Correlation analysis failed to show an association between age and IGF-1 SDS both in control and PWS groups. CONCLUSIONS IGF-1 SDS is lower in very young children with PWS independent of anthropometric parameters and nutritional status, suggesting the presence of hypothalamic dysfunction of GH secretion.

Treatment of persistent short stature in children born small for gestational age (SGA) requires daily growth hormone (GH) injections that can be burdensome for patients and caregivers. Once-weekly somapacitan is a long-acting GH currently in phase 3 development for replacement therapy in children with GH deficiency. We report the 26-week results of the first phase 2, multinational, randomized, open-label, controlled, dose-finding trial (NCT03878446) investigating the efficacy and safety of somapacitan as treatment for short stature in children born SGA compared with daily GH (Norditropin®, Novo Nordisk A/S). A total of 62 (35.5% female) GH-treatment-naïve, prepubertal children received 0.16, 0.20 or 0.24 mg/kg/week subcutaneous (s.c.) somapacitan, or 0.035 or 0.067 mg/kg/day s.c. daily GH for 26 weeks (main phase). All children are subsequently included in an extension to this trial. The primary outcome was annualized height velocity (HV). At week 26, the estimated mean HV was: 8.9, 11.0 and 11.3 cm/year for 0.16 mg/kg/week (n=12), 0.20 mg/kg/week (n=13) and 0.24 mg/kg/week (n=12) somapacitan, respectively, versus 10.3 and 11.9 cm/year for 0.035 mg/kg/day (n=12) and 0.067 mg/kg/day (n=13) daily GH, respectively. A dose-dependent response in the estimated mean HV was observed with both somapacitan and daily GH. The effect of somapacitan on HV was not statistically significantly different compared with daily GH. A similar pattern was observed for change from baseline in height standard deviation score (SDS) and change from baseline in HV SDS. Consistent dose-dependent increases were observed in insulin-like growth factor-I (IGF-I) SDS with both somapacitan and daily GH. Exposure-response modeling of somapacitan exposure versus HV and IGF-I SDS vs. HV indicated an exposure-dependent increase in HV for both these parameters. Somapacitan was well tolerated at all doses investigated, with no safety or local tolerability issues identified. There were no clinically relevant findings with respect to glucose metabolism, and no detection of anti-drug antibodies in any of the dose groups. In conclusion, all investigated doses of weekly somapacitan were efficacious and well tolerated throughout the study period. Based on the totality of data on improvements in height-based parameters combined with exposure-response analyses, a somapacitan dose of 0.24 mg/kg/week appears as the most efficacious dose, providing similar efficacy, safety and tolerability to daily GH 0.067 mg/kg/day after 26 weeks of treatment. Presentation: Monday, June 13, 2022 12:30 p.m. - 2:30 p.m., Monday, June 13, 2022 12:51 p.m. - 12:56 p.m.

Hypophosphatasia (HPP) is highly variable in clinical expression and is generally classified into six subtypes. Although it would be beneficial to be able to predict the clinical course from the ALPL genotype, studies on this issue are limited. Here, we aimed to clarify the features of Japanese HPP and the relationships between genotype and clinical manifestations. We analyzed 98 unrelated Japanese patients to investigate the percentage of each clinical form, frequently detected mutations, and the relationship between the genotype and phenotype. Some of the identified mutants were characterized by transfection experiments. Perinatal severe form was the most frequent (45.9%), followed by perinatal benign form (22.4%). Among the 196 alleles, p.Leu520ArgfsX86 (c.1559delT) was detected in 89 alleles, and p.Phe327Leu (c.979T>C) was identified in 23 alleles. All of the homozygotes for p.Leu520ArgfsX86 were classified into perinatal severe form, and patients carrying p.Phe327Leu in one of the alleles were classified into perinatal benign or odonto HPP. Twenty of the 22 patients with perinatal benign HPP were compound heterozygous for p.Phe327Leu and another mutation. Most patients with odonto HPP were found to be monoallelic heterozygotes for dominant-negative mutations or compound heterozygotes with mutants having residual activity. The high prevalence of p.Leu520ArgfsX86 and p.Phe327Leu mutations might underlie the high rate of perinatal severe and perinatal benign forms, respectively, in Japanese HPP. Although ALPL genotyping would be beneficial for predicting the clinical course to an extent, the observed phenotypical variability among patients sharing the same genotypes suggests the presence of modifiers.

Noonan syndrome (NS) is a heterogeneous disorder with multiple congenital malformations. Recent advances in molecular and genetic approaches have identified a number of responsible genes for NS, most of which are components of the RAS/MAPK signaling pathway, and genotype-phenotype correlation analyses have been extensively performed; however, analysis of Japanese NS patients is limited. Here, we evaluated clinical characteristics in genetically diagnosed NS patients and their relationships to genotypes. A total of 48 clinically diagnosed NS were included, and responsible mutations were identified in 39 patients (81.3%) with PTPN11 mutations being the most prevalent followed by SOS1 mutations. Cardiac anomalies including pulmonary stenosis and hypertrophic cardiomyopathy were most prevalent (87.2%), and the prevalence of hypertrophic cardiomyopathy was greater in patients without PTPN11 mutations than in those with PTPN11 mutations. Short stature was the second-most prevalent (69.2%) characteristic, and present height SD score was significantly associated with height SD score at 1 year old. Patients with SOS1 mutations had greater present height SD score and better growth during infancy. These findings suggest the presence of a genotype-phenotype correlation in Japanese patients with NS, which enables us to use genetic information to predict the clinical course and may allow for genotype-based medical interventions.

Serum inorganic phosphate (Pi) levels are higher in children than in adults; however, the underlying mechanisms remain unclear. Therefore, we herein attempted to elucidate the mechanisms altering Pi metabolism from youth to adulthood using 4-week-old (young) and 12-week-old (adult) mice. Despite higher serum Pi levels, serum fibroblast growth factor 23 (FGF23) levels were lower in young mice, and the amount of FGF23 in bone tended to increase from youth to adulthood. Increases in serum FGF23 levels during growth were associated with the up- and down-regulation of the renal expression of Cyp24a1 encoding vitamin D-24-hydroxylase and Slc34a3 encoding the type IIc sodium/phosphate (Na

Natriuretic peptide receptor B (NPRB) produces cyclic guanosine monophosphate (cGMP) when bound by C-type natriuretic peptide (CNP). Activating mutations in NPRB cause a skeletal overgrowth disorder, which has been named epiphyseal chondrodysplasia, Miura type (ECDM; OMIM #615923). Here we explored the cellular and molecular mechanisms for the skeletal overgrowth in ECDM using a mouse model in which an activating mutant NPRB is specifically expressed in chondrocytes. The mutant mice (NPRB[p.V883M]-Tg) exhibited postnatal skeletal overgrowth and increased cGMP in cartilage. Both endogenous and transgene-derived NPRB proteins were localized at the plasma membrane of hypertrophic chondrocytes. The hypertrophic zone of growth plate was thickened in NPRB[p.V883M]-Tg. An in vivo BrdU-labeling assay suggested that some of the hypertrophic chondrocytes in NPRB[p.V883M]-Tg mice continued to proliferate, although wild-type (WT) chondrocytes stopped proliferating after they became hypertrophic. In vitro cell studies revealed that NPRB activation increased the phosphorylation of cyclic AMP-responsive element binding protein (CREB) and expression of cyclin D1 in matured chondrocytes. Treatment with cell-permeable cGMP also enhanced the CREB phosphorylation. Inhibition of cyclic adenosine monophosphate (cAMP)/protein kinase A pathway had no effects on the CREB phosphorylation induced by NPRB activation. In immunostaining of the growth plates for the proliferation marker Ki67, phosphorylated CREB and cyclin D1, most signals were similarly observed in the proliferating zone in both genotypes, but some cells in the hypertrophic zone of NPRB[p.V883M]-Tg were also positively stained. These results suggest that NPRB activation evokes its signal in hypertrophic chondrocytes to induce CREB phosphorylation and make them continue to proliferate, leading to the skeletal overgrowth in ECDM.

OBJECTIVE Abnormalities in the hypothalamic-pituitary-thyroid (HPT) axis have been implicated in Prader-Willi syndrome (PWS); however, limited information is currently available on age-dependent alterations in the HPT axis. We herein investigated age-dependent differences in thyroid hormone levels in PWS children. DESIGN/PATIENTS/MEASUREMENTS Free T4 (FT4), free T3 (FT3) and thyroid-stimulating hormone (TSH) concentrations were retrospectively compared between genetically confirmed PWS children (N = 43, median age: 11.2 months) and controls (N = 85, median age: 14.5 months) matched for age, sex, body weight-SD score (SDS), height-SDS, body mass index-SDS and serum albumin level, a marker of the nutritional status. Subjects were subdivided into two groups based on their age: an infant group aged between 1 and 11 months (PWS: N = 22, controls: N = 30) and a toddler group aged between 12 and 47 months (PWS: N = 21, controls: N = 55). None of the subjects had ever been treated with growth hormone or levothyroxine. RESULTS After adjustments for confounding variables, in the infant group, FT4 levels (pmol/L) were significantly lower in PWS (11.24 in PWS vs 14.32 in controls, P = .0002), whereas no significant differences were observed in FT3 or TSH levels. In the toddler group, no significant differences were noted in FT4 (12.23 in PWS vs 15.31 in controls, P = .10), FT3 or TSH levels. The FT3/FT4 ratio was significantly increased in PWS in both groups. FT4 levels were positively correlated with age in PWS. CONCLUSIONS Infants with PWS had lower FT4 levels, but FT3 levels were normal, indicating that the levothyroxine replacement therapy may not need to be routinely performed.

Background Children born small for gestational age (SGA) with catch-up growth are at high risk for developing obesity; however, the characteristics of body composition, especially fat distribution, before and after growth hormone (GH) treatment in SGA children without catch-up growth remains largely unknown. Methods Anthropometric characteristics, body composition by dual-energy X-ray absorption, and fat distribution by computed tomography at the umbilical level were examined in 27 prepubertal short-stature children born SGA before and 1 year after GH treatment. Results Before GH treatment, short-stature SGA children had lean phenotypes, and both visceral adipose tissue (VAT) and subcutaneous adipose tissue (SAT) were significantly lower than the age- and sex-matched Japanese reference values. Growth hormone treatment significantly increased height standard deviation scores (SDS), without affecting body mass index SDS. Percentage fat mass decreased with GH treatment; however, fat mass was not altered. Both VAT and SAT were significantly lower than the reference values after GH treatment. The ratio of VAT over SAT significantly increased by GH treatment. Conclusions Both VAT and SAT were within or below the age- and sex-matched Japanese reference values in short-stature children born SGA before and after GH treatment, indicating that GH treatment may not have unfavorable effects on adiposity in short-stature children born SGA, although it may alter fat distribution.

In mammals, phosphate balance is maintained by influx and efflux via the intestines, kidneys, bone, and soft tissue, which involves multiple sodium/phosphate (Na+/Pi) cotransporters, as well as regulation by several hormones. Alterations in the levels of extracellular phosphate exert effects on both skeletal and extra-skeletal tissues, and accumulating evidence has suggested that phosphate itself evokes signal transduction to regulate gene expression and cell behavior. Several in vitro studies have demonstrated that an elevation in extracellular Piactivates fibroblast growth factor receptor, Raf/MEK (mitogen-activated protein kinase/ERK kinase)/ERK (extracellular signal-regulated kinase) pathway and Akt pathway, which might involve the type III Na+/Picotransporter PiT-1. Excessive phosphate loading can lead to various harmful effects by accelerating ectopic calcification, enhancing oxidative stress, and dysregulating signal transduction. The responsiveness of mammalian cells to altered extracellular phosphate levels suggests that they may sense and adapt to phosphate availability, although the precise mechanism for phosphate sensing in mammals remains unclear. Unicellular organisms, such as bacteria and yeast, use some types of Pitransporters and other molecules, such as kinases, to sense the environmental Piavailability. Multicellular animals may need to integrate signals from various organs to sense the phosphate levels as a whole organism, similarly to higher plants. Clarification of the phosphate-sensing mechanism in humans may lead to the development of new therapeutic strategies to prevent and treat diseases caused by phosphate imbalance.

GH therapy in pediatric patients with Prader-Willi syndrome (PWS) improves body composition, but discontinuation of GH after achieving adult height has been implicated in its deterioration. Although there is evidence for the deleterious effects of visceral adipose tissue (VAT) rather than subcutaneous adipose tissue (SAT) on the development of obesity-related complications, the effects of GH discontinuation on fat distribution in adults with PWS has not been fully investigated. Therefore, we utilized dual-energy X-ray absorptiometry (DEXA) and abdominal computed tomography (CT) to compare the fat distribution between before and 6 months or 12 months after the cessation of GH therapy in 7 adult PWS patients. GH therapy was initiated at a mean age of 4.1 ± 1.4 years and discontinued at a mean age of 18.9 ± 1.8 years. Serum IGF-1 levels were decreased by discontinuation of GH therapy. Fat mass was significantly increased 6 and 12 months after GH cessation, whereas muscle mass and bone mineral density were unchanged during both study periods. Abdominal CT analysis revealed that elevations in fat mass were due to increases in VAT rather than SAT. Circulating low-density lipoprotein (LDL) cholesterol levels were significantly elevated 6 months after GH cessation. In conclusion, discontinuation of GH therapy caused rapid increases in visceral adipose tissue and LDL cholesterol levels. These findings indicate that continuation of GH therapy may be a therapeutic option to maintain body composition; however, further studies regarding the long-term benefits and adverse effects of GH therapy in adults with PWS are required.

Selenium (Se) is an essential trace element that is involved in numerous biological processes in the form of a selenoprotein such as iodothyronine deiodinase (DIO). Se deficiency may prevent the conversion of T4 to T3 through reducing DIO expression and thereby affecting thyroid hormone status. However, this has not been well documented in humans. In this study, to clarify the association between Se and thyroid hormone status, we investigated the thyroid hormone levels in patients with severe Se deficiency (< 2 µg/dl). Severe Se deficiency was associated with increases in free T4 levels, but not with decreases and increases in free T3 and thyroid stimulating hormone (TSH) levels, respectively. Increases in free T4 levels during Se deficiency were reduced with Se supplementation; however, neither free T3 nor TSH levels were affected. Taken together, these findings indicate that free T4 may be a useful biomarker for Se status when serum Se levels are severely low.

Multiple actions of extracellular Pi on the skeletal cells are likely to be partly mediated by type III sodium/phosphate (Na+/Pi) cotransporters Pit1 and Pit2, although the details are not fully understood. In the current study, to determine the roles of Pit1 and Pit2 in osteoblasts, we generated Pit1-knockout (KO) and Pit2-KO osteoblastic cells by applying CRISPR/Cas9 genome editing to an osteoblastic cell line MC3T3-E1 subclone 4. The extracellular Pi level was increased in the Pit1-KO and Pit2-KO clones due to the reduced Pi uptake. Interestingly, in vitro mineralization was accelerated in the Pit1-KO and Pit2-KO clones, although the induction of the expression of osteogenic marker genes was suppressed. In the cells before mineralization, extracellular levels of pyrophosphate (PPi) and adenosine triphosphate (ATP) were increased in the Pit1-KO and Pit2-KO clones, which might be attributable to the reduced expression and activity of tissue-nonspecific alkaline phosphatase (TNSALP). A 24-h treatment with high Pi reduced the expression and activity of TNSALP, suggesting that the suppression of TNSALP in the Pit1-KO and Pit2-KO clones was caused by the increased availability of extracellular Pi. Lentiviral gene transfer of Pit1 and Pit2 restored the changes observed in Pit1-KO and Pit2-KO clones, respectively. The expressions of P2Y2 and P2X7 which encode receptors for extracellular ATP were altered in the Pit1-KO and Pit2-KO clones, suggesting an influence on purinergic signaling. In mineralized cells after long-term culture, intracellular levels of PPi and ATP were higher in the Pit1-KO and Pit2-KO clones. Taken together, ablation of Pit1 or Pit2 in this osteoblastic cell model led to accelerated mineralization, suppressed TNSALP and altered the levels of extracellular and intracellular PPi and ATP, which might be partly mediated by changes in the availability of extracellular Pi.

Dentin matrix protein 1 (Dmp1) is an extracellular matrix protein involved in phosphate metabolism and biomineralization, and its expression markedly increases during the maturation of osteoblasts into osteocytes. We previously reported that an increased level of inorganic phosphate (Pi) in media up-regulated the expression of Dmp1 in primary osteocytes isolated from mouse bones. In the present study, we found that elevated extracellular Pi strongly induced the expression of Dmp1 in osteoblasts and explored its underlying mechanism of action. In an osteoblastic cell line MC3T3-E1, increases in extracellular Pi induced the phosphorylation of ERK1/2 and up-regulated the expression of Dmp1, fibroblast growth factor 2 (Fgf2), and Fgf receptor 1 (Fgfr1). A co-treatment with the MEK inhibitor U0126 abolished the increase in the expression of Dmp1 and Fgfr1 by elevated Pi, suggesting the involvement of the MEK/ERK pathway in this up-regulation. Elevated extracellular Pi also resulted in the phosphorylation of FGF receptor substrate 2α (FRS2α), which was diminished by knockdown of Slc20a1 encoding Pit1 sodium-phosphate co-transporter. The co-treatment with an inhibitor against FGFR (SU5402) abolished the up-regulation of Dmp1 induced by elevated extracellular Pi. In primary osteoblasts, a treatment with 4 mM Pi transiently increased the expression of early growth response 1 (Egr1) before the up-regulation of Dmp1. These results indicate that FGFR mediates the direct effects of extracellular Pi on the expression of Dmp1 in osteoblasts and enhance the close relationship between the signaling evoked by elevated extracellular Pi and FGF/FGFR signaling. J. Cell. Biochem. 118: 1151-1163, 2017. © 2016 Wiley Periodicals, Inc.