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Context There are limited reports on the detailed examination of steroid profiles for setting algorithms for 21-hydroxylase deficiency (21OHD) screening by liquid chromatography–tandem mass spectrometry (LC-MS/MS). Objective We aimed to define an algorithm for newborn screening of 21OHD by LC-MS/MS, measuring a total of 2077 dried blood spot samples in Tokyo. Methods Five steroids (17α-hydroxyprogesterone [17αOHP], 21-deoxycortisol [21DOF], 11-deoxycortisol [11DOF], androstenedione [4AD], and cortisol [F]) were included in the panel of LC-MS/MS. Samples from 2 cohorts were assayed: Cohort A, 63 “screening positive” neonates who were referred to an endocrinologist (n = 26 with 21OHD; n = 37 false-positive; obtained from 2015 to 2020); and Cohort B, samples (n = 2014) with 17αOHP values in the 97th percentile or above, in the first-tier test with 17αOHP ELISA from 2020 to 2021. Results Analysis of Cohort A revealed that the 3 indexes 21DOF, 11DOF/17αOHP, and (4AD + 17αOHP)/F had higher area under the curve (AUC) values (0.999, 0.997, 0.989, respectively), while the 17αOHP AUC was lower (0.970). Accordingly, in addition to 17αOHP, the 3 markers were included for defining the screening algorithm. The assay of Cohort B revealed that the new algorithm gave 92% of predicted positive predictive value without false-negative cases. We also determined the reference values for the 5 steroids at 4 to 7 days after birth, according to sex and gestational age (GA), revealing extremely low levels of 21DOF at any GA irrespective of sex differences. Conclusion Our study demonstrated the high relevance of 21DOF, (4AD + 17αOHP)/F, and 11DOF/17αOHP, rather than 17αOHP, for 21OHD screening.

RTL1 (also termed paternal expressed 11 (PEG11)) is considered the major imprinted gene responsible for the placental and fetal/neonatal muscle defects that occur in the Kagami–Ogata and Temple syndromes (KOS14 and TS14, respectively). However, it remains elusive whether RTL1 is also involved in their neurological symptoms, such as behavioral and developmental delay/intellectual disability, feeding difficulties, motor delay, and delayed speech. Here, we demonstrate that the mouse RTL1 protein is widely expressed in the central nervous system (CNS), including the limbic system. Importantly, two disease model mice with over‐ and under‐expression of Rtl1 exhibited reduced locomotor activity, increased anxiety, and impaired amygdala‐dependent cued fear, demonstrating that Rtl1 also plays an important role in the CNS. These results indicate that the KOS14 and TS14 are neuromuscular as well as neuropsychiatric diseases caused by irregular CNS RTL1 expression, presumably leading to impaired innervation of motor neurons to skeletal muscles as well as malfunction of the hippocampus‐amygdala complex. It is of considerable interest that eutherian‐specific RTL1 is expressed in mammalian‐ and eutherian‐specific brain structures, that is, the corticospinal tract and corpus callosum, respectively, suggesting that RTL1 might have contributed to the acquisition of both these structures themselves and fine motor skill in eutherian brain evolution., RTL1 was detected in most of the corpus callosum, comprising the neurons from cortex layers V and VI, in the neonatal brain. The corpus callosum is unique to eutherians; therefore, Rtl1 may play a role in the emergence of this structure as a eutherian‐specific gene.

In Japan, since the introduction of newborn screening for congenital adrenal hyperplasia in 1989, most patients with 21-hydroxylase deficiency (21OHD) have been treated with glucocorticoid from the neonatal period (1, 2). Although early initiation of glucocorticoid therapy from the neonatal period remarkably improved the outcomes of final height, the average final height in 21OHD patients is below the standard height observed in normal healthy adults by approximately –1 standard deviation (SD) (3, 4), and optimization of glucocorticoid therapy remains challenging (5,6,7, 8). Glucocorticoids have a strong potential for reducing linear growth, whereas undertreatment with glucocorticoids leads to androgen excess, precipitating pubertal development (5,6,7). Recent studies have revealed that the final height positively correlates with height at two years of age (9), and the lowest possible hydrocortisone (HC) dose for maintenance treatment during the first two years of life considered crucial for maximizing the final height of 21OHD patients. For maintenance therapy, 15–25 mg/m2 HC therapy is widely accepted (10). In contrast to maintenance HC therapy, the optimal HC dose for initial treatment has not been established. The consensus statement on 21OHD by the Lawson Wilkins Pediatric Endocrine Society and the European Society for Pediatric Endocrinology (ESPE) suggests an HC dose of up to 25 mg/m2 for treatment initiation (11). Conversely, the Japanese Society for Pediatric Endocrinology (JSPE) recommended a considerably higher HC dose of HC (100–200 mg/m2) for the initial treatment (12), and in the revised guideline published in 2014, the recommended dose was lowered to 25–100 mg/m2, which is still higher than that recommended by the ESPE (13). A high initial HC dose may help normalize the elevated adrenal androgen levels during the neonatal and early infantile periods, followed by sufficient control by a lower maintenance dose. Our previous study has revealed that the height at two years of age in patients with 21OHD treated with a higher HC dose during initial therapy could be compared with those receiving lower HC doses (14). However, detailed differences between patients with 21OHD treated with higher and lower doses during initial HC therapy have not been assessed. In the present study, we aimed to evaluate the effects of initial HC treatments on clinical profiles during infancy and early childhood in patients with 21OHD.

BACKGROUND One of the major purposes of newborn screening for 21-hydroxylase deficiency (21OHD) is preventing life-threatening adrenal crisis. However, the details of adrenal crisis in newborns are not precisely documented. AIM We aimed to clarify the clinical details of salt-wasting in newborn 21OHD patients. METHODS Based on the follow-up survey of the screening in Tokyo from 1989 to 2017, we retrospectively analysed the conditions of classical 21OHD neonates before the initiation of therapy. RESULTS One hundred classical 21OHD patients (55 male, 45 female) were analysed. The age at the first hospital visit was 0-20 days with sex difference (male: 9.0 ± 3.5 days; female: 6.2 ± 3.9 days). Thirty-seven (37.4%) patients exhibited severe salt-wasting (SSW), that is, Na 7 mEq/L or Na/K ratio 7 mEq/L and Na/K

In addition to malignant diseases, hematopoietic stem cell transplantation (HSCT) is also a vital option as a curative therapy for non-malignant diseases, such as immunodeficiency, and other hematological disorders. Not only for malignant diseases, but for non-malignant diseases, cytotoxic therapy of conditioning regimens are associated with high risks of adverse effects; however, clinical details regarding the long term outcomes of cytotoxic therapy for non-malignant diseases are not documented yet. To clarify the endocrinological consequences of pediatric HSCT for non-malignant disease patients, we conducted a retrospective analysis. From 1983 to 2014, 75 patients that underwent HSCT for non-malignant diseases were selected for this study. Of these, 23 patients (19 men, 4 women) were continuously followed up in our institute, with regular health check-ups for late effects. Based on a multiple linear regression analysis, the glucocorticoid treatment duration for chronic graft-versus-host disease (cGVHD) and the conditioning regimen were found to be independent predictors of growth retardation. All four female patients developed hypogonadism, and required hormone replacement therapy. The conditioning regimen for the four female patients with hypogonadism was based on the use of alkylating agents, and two female patients were treated with a reduced-intensity conditioning (RIC) regimen. Our study revealed that even the RIC regimen was toxic for the gonads in female patients, and that the survivors of both non-malignant and malignant diseases should be followed up carefully after pediatric HSCT.

Background The most severe forms of congenital hyperinsulinism (CHI) are caused by inactivating mutations of two KATP channel genes, KCNJ11 and ABCC8. Unresponsiveness to diazoxide and need for subtotal pancreatectomy can usually be predicted by genetic form, particularly biallelic mutations in KATP channel genes. A few reports indicated marked clinical heterogeneity in siblings with identical biallelic mutations in ABCC8. The clinical heterogeneity in biallelic KATP CHI was speculated to be caused by epigenetic and environmental factors or related to differences in splicing factor machinery. Objective To elucidate the clinical pathophysiology, especially heterogeneity, among three cases with CHI caused by a homogenous novel mutation. Patients and methods We report a case series that includes two siblings and one unrelated individual with CHI caused by a homogenous 1-bp deletion around the splice acceptor site at the exon 35 mutation of ABCC8, which exhibited markedly distinct phenotypes. To assess the effect of the mutation on splicing, we performed digital droplet polymerase chain reaction (ddPCR) on normal pancreas tissue and a patient's lymphocytes. Results ddPCR of ABCC8 cDNA revealed that expression of exon 35 and its upstream and downstream regions did not differ. These data suggested that clinical heterogeneity may not be caused by differences in splicing factor machinery. Conclusion The phenotypic variation in homozygotes could not be explained by splicing abnormalities. Though early genetic diagnosis of KATP CHI could contribute to selecting appropriate therapeutic options, more deliberate selection of therapeutic options in diffuse CHI due to biallelic ABCC8 mutations may be required.

Our understanding of the spatiotemporal regulation of cardiogenesis is hindered by the difficulties in modeling this complex organ currently by in vitro models. Here we develop a method to generate heart organoids from mouse embryonic stem cell-derived embryoid bodies. Consecutive morphological changes proceed in a self-organizing manner in the presence of the laminin-entactin (LN/ET) complex and fibroblast growth factor 4 (FGF4), and the resulting in vitro heart organoid possesses atrium- and ventricle-like parts containing cardiac muscle, conducting tissues, smooth muscle and endothelial cells that exhibited myocardial contraction and action potentials. The heart organoids exhibit ultrastructural, histochemical and gene expression characteristics of considerable similarity to those of developmental hearts in vivo. Our results demonstrate that this method not only provides a biomimetic model of the developing heart-like structure with simplified differentiation protocol, but also represents a promising research tool with a broad range of applications, including drug testing., Our understanding of the development of the heart has been limited by a lack of in vitro cellular models. Here, the authors treat mouse embryonic stem cell-derived embryoid bodies with laminin-entactin (to mimic the developing microenvironment) and FGF4 to form heart organoids, with atrial and ventricular-like parts.

For the understanding of the spatiotemporal regulation of cardiogenesis, it is important to generate in vitro model of hearts. This protocol introduces how to generate heart organoids from mouse embryonic stem cell-derived embryoid bodies (EB) in the presence of the laminin-entactin (LN/ET) complex and fibroblast growth factor 4 (FGF4) by three consecutive steps, 1) the culture of ESCs, 2) in vitro differentiation of ESCs into EBs and 3) in vitro culture of EBs for the generation of heart organoids. The generated heart organoids possess structural and functional capacity similar to atrial and ventricular parts of in vivo embryonic heart. This simplified protocol also provides a promising research tool with a broad range of applications, including drug testing.

Hematopoietic stem cell transplantation (HSCT) is a curative treatment for life-threatening malignancies and related diseases. Recently, the long-term prognosis of HSCT during childhood has greatly improved; however, the late adverse effects of HSCT have been found to cause substantial morbidity among long-term survivors. Although metabolic complications, such as diabetes mellitus (DM) and hyperlipidemia (HL), are the major late effects of pediatric HSCT, the clinical details are not clarified sufficiently. From 1983 to 2013, 75 participants underwent HSCT in our institute because of malignant or other related diseases. We retrospectively evaluated metabolic complications of eligible 22 participants (14 men and 8 women), and their clinical backgrounds. Among 22 participants, 4 and 9 participants developed DM and HL after HSCT, respectively, and all participants with DM developed HL. None of the participants with DM were obese, and all had substantial insulin resistance. Total body irradiation (TBI) was performed in 10 participants, including 4 participants with DM and 5 participants with HL, revealing that TBI is an independent risk factor for DM. The age at TBI for participants with DM was significantly lower than that for participants without DM (p = 0.01), and all participants with DM received TBI before the age of 6. Our data suggested that TBI was a risk factor for DM after HSCT, and TBI before the age of six increased the possibility of DM without obesity.

Epigenetic properties of cultured embryonic stem cells (ESCs), including DNA methylation imprinting, are important because they affect the developmental potential. Here, we tested a variety of culture media, including knockout serum replacement (KSR) and fetal bovine serum (FBS) with or without inhibitors of Gsk3β and Mek1/2 (2i) at various time points. In addition to the previously known passage-dependent global changes, unexpected dynamic DNA methylation changes occurred in both maternal and paternal differentially methylated regions: under the widely used condition of KSR with 2i, a highly hypomethylated state occurred at early passages (P1-7) as well as P10, but DNA methylation increased over further passages in most conditions, except under KSR with 2i at P25. Dramatic DNA demethylation under KSR+2i until P25 was associated with upregulated Tet1 and Parp1, and their related genes, whereas 2i regulated the expressions of DNA methyltransferase-related genes for the change in DNA methylation during the cumulative number of passages. Although DNA methylation imprinting is more labile under KSR with and without 2i, it can be more faithfully maintained under condition of cooperative FBS and 2i. Thus, our study will provide the useful information for improved epigenetic control of ESCs and iPSCs in applications in regenerative medicine.

10q26 deletion syndrome is caused by a rare chromosomal abnormality, and patients with this syndrome present with an extensive and heterogeneous phenotypic spectrum. Several genes, such as EMX2 and FGFR2, were identified as the cause genital anomalies and facial dysmorphism in 10q26 deletion syndrome. However, the critical region for 10q26 deletion syndrome is not determined and the precise relationships between the causative genes and the phenotypes are still controversial. WD repeat domain 11 (WDR11), located at 10q25–26, was recently identified as a causative gene in hypogonadotropic hypogonadism, but other clinical phenotypes caused by WDR11 variants have not been identified. In this study, we have identified a WDR11 missense mutation, NM_018117.11: c.2108G > A; p.(Arg703Gln); ClinVar accession SCV000852064, in a two-year-old boy with severe growth retardation, ventricular septal defect, and coloboma symptoms. The case suggests that WDR11 is partially responsible for the clinical features of 10q26 deletion syndrome and provides novel insights into the pathophysiology of this syndrome.