Circadian Regulation of Cardiac Arrhythmias and Electrophysiology.

Circadian rhythms in physiology and behavior are ≈24-hour biological cycles regulated by internal biological clocks (ie, circadian clocks) that optimize organismal homeostasis in response to predictable environmental changes. These clocks are present in virtually all cells in the body, including cardiomyocytes. Many decades ago, clinicians and researchers became interested in studying daily patterns of triggers for sudden cardiac death, the incidence of sudden cardiac death, and cardiac arrhythmias. This review highlights historical and contemporary studies examining the role of day/night rhythms in the timing of cardiovascular events, delves into changes in the timing of these events over the last few decades, and discusses cardiovascular disease-specific differences in the timing of cardiovascular events. The current understanding of the environmental, behavioral, and circadian mechanisms that regulate cardiac electrophysiology is examined with a focus on the circadian regulation of cardiac ion channels and ion channel regulatory genes. Understanding the contribution of environmental, behavioral, and circadian rhythms on arrhythmia susceptibility and the incidence of sudden cardiac death will be essential in developing future chronotherapies.

Characterization of gene expression in the kidney of renal tubular cell-specific NFAT5 knockout mice.

Nuclear factor of activated T cells 5 (NFAT5; also called TonEBP/OREBP) is a transcription factor that is activated by hypertonicity and induces osmoprotective genes to protect cells against hypertonic conditions. In the kidney, renal tubular NFAT5 is known to be involved in the urine concentration mechanism. Previous studies have suggested that NFAT5 modulates the immune system and exerts various effects on organ damage, depending on organ and disease states. Pathophysiological roles of NFAT5 in renal tubular cells, however, still remain obscure. We conducted comprehensive analysis by performing transcription start site (TSS) sequencing on the kidney of inducible and renal tubular cell-specific NFAT5 knockout (KO) mice. Mice were subjected to unilateral ureteral obstruction to examine the relevance of renal tubular NFAT5 in renal fibrosis. TSS sequencing analysis identified 722 downregulated TSSs and 1,360 upregulated TSSs, which were differentially regulated ≤-1.0 and ≥1.0 in log2 fold, respectively. Those TSSs were annotated to 532 downregulated genes and 944 upregulated genes, respectively. Motif analysis showed that sequences that possibly bind to NFAT5 were enriched in TSSs of downregulated genes. Gene Ontology analysis with the upregulated genes suggested disorder of innate and adaptive immune systems in the kidney. Unilateral ureteral obstruction significantly exacerbated renal fibrosis in the renal medulla in KO mice compared with wild-type mice, accompanied by enhanced activation of immune responses. In conclusion, NFAT5 in renal tubules could have pathophysiological roles in renal fibrosis through modulating innate and adaptive immune systems in the kidney.NEW & NOTEWORTHY TSS-Seq analysis of the kidney from renal tubular cell-specific NFAT5 KO mice uncovered novel genes that are possibly regulated by NFAT5 in the kidney under physiological conditions. The study further implied disorders of innate and adaptive immune systems in NFAT5 KO mice, thereby exacerbating renal fibrosis at pathological states. Our results may implicate the involvement of renal tubular NFAT5 in the progression of renal fibrosis. Further studies would be worthwhile for the development of novel therapy to treat chronic kidney disease.

Feeding behavior modifies the circadian variation in RR and QT intervals by distinct mechanisms in mice.

Rhythmic feeding behavior is critical for regulating phase and amplitude in the ≈24-h variation of heart rate (RR intervals), ventricular repolarization (QT intervals), and core body temperature in mice. We hypothesized changes in cardiac electrophysiology associated with feeding behavior were secondary to changes in core body temperature. Telemetry was used to record electrocardiograms and core body temperature in mice during ad libitum-fed conditions and after inverting normal feeding behavior by restricting food access to the light cycle. Light cycle-restricted feeding modified the phase and amplitude of 24-h rhythms in RR and QT intervals, and core body temperature to realign with the new feeding time. Changes in core body temperature alone could not account for changes in phase and amplitude in the ≈24-h variation of the RR intervals. Heart rate variability analysis and inhibiting ß-adrenergic and muscarinic receptors suggested that changes in the phase and amplitude of 24-h rhythms in RR intervals were secondary to changes in autonomic signaling. In contrast, changes in QT intervals closely mirrored changes in core body temperature. Studies at thermoneutrality confirmed that the daily variation in QT interval, but not RR interval, primarily reflected daily changes in core body temperature (even in ad libitum-fed conditions). Correcting the QT interval for differences in core body temperature helped unmask QT interval prolongation after starting light cycle-restricted feeding and in a mouse model of long QT syndrome. We conclude feeding behavior alters autonomic signaling and core body temperature to regulate phase and amplitude in RR and QT intervals, respectively.NEW & NOTEWORTHY We used time-restricted feeding and thermoneutrality to demonstrate that different mechanisms regulate the 24-h rhythms in heart rate and ventricular repolarization. The daily rhythm in heart rate reflects changes in autonomic input, whereas daily rhythms in ventricular repolarization reflect changes in core body temperature. This novel finding has major implications for understanding 24-h rhythms in mouse cardiac electrophysiology, arrhythmia susceptibility in transgenic mouse models, and interpretability of cardiac electrophysiological data acquired in thermoneutrality.

FGF9 variant in 46,XY DSD patient suggests a role for dimerization in sex determination.

46,XY gonadal dysgenesis (GD) is a Disorder/Difference of Sex Development (DSD) that can present with phenotypes ranging from ambiguous genitalia to complete male-to-female sex reversal. Around 50% of 46,XY DSD cases receive a molecular diagnosis. In mice, Fibroblast growth factor 9 (FGF9) is an important component of the male sex-determining pathway. Two FGF9 variants reported to date disrupt testis development in mice, but not in humans. Here, we describe a female patient with 46,XY GD harbouring the rare FGF9 variant (missense mutation), NM_002010.2:c.583G > A;p.(Asp195Asn) (D195N). By biochemical and cell-based approaches, the D195N variant disrupts FGF9 protein homodimerisation and FGF9-heparin-binding, and reduces both Sertoli cell proliferation and Wnt4 repression. XY Fgf9D195N/D195N foetal mice show a transient disruption of testicular cord development, while XY Fgf9D195N/- foetal mice show partial male-to-female gonadal sex reversal. In the general population, the D195N variant occurs at an allele frequency of 2.4 × 10-5 , suggesting an oligogenic basis for the patient's DSD. Exome analysis of the patient reveals several known and novel variants in genes expressed in human foetal Sertoli cells at the time of sex determination. Taken together, our results indicate that disruption of FGF9 homodimerization impairs testis determination in mice and, potentially, also in humans in combination with other variants.

The role of the cardiomyocyte circadian clocks in ion channel regulation and cardiac electrophysiology.

Daily variations in cardiac electrophysiology and the incidence for different types of arrhythmias reflect ≈24 h changes in the environment, behaviour and internal circadian rhythms. This article focuses on studies that use animal models to separate the impact that circadian rhythms, as well as changes in the environment and behaviour, have on 24 h rhythms in heart rate and ventricular repolarization. Circadian rhythms are initiated at the cellular level by circadian clocks, transcription-translation feedback loops that cycle with a periodicity of 24 h. Several studies now show that the circadian clock in cardiomyocytes regulates the expression of cardiac ion channels by multiple mechanisms; underlies time-of-day changes in sinoatrial node excitability/intrinsic heart rate; and limits the duration of the ventricular action potential waveform. However, the 24 h rhythms in heart rate and ventricular repolarization are primarily driven by autonomic signalling. A functional role for the cardiomyocyte circadian clock appears to buffer the heart against perturbations. For example, the cardiomyocyte circadian clock limits QT-interval prolongation (especially at slower heart rates), and it may facilitate the realignment of the 24 h rhythm in heart rate to abrupt changes in the light cycle. Additional studies show that modifying rhythmic behaviours (including feeding behaviour) can dramatically impact the 24 h rhythms in heart rate and ventricular repolarization. If these mechanisms are conserved, these studies suggest that targeting endogenous circadian mechanisms in the heart, as well as modifying the timing of certain rhythmic behaviours, could emerge as therapeutic strategies to support heart function against perturbations and regulate 24 h rhythms in cardiac electrophysiology.

Pentafluoroethylation of Carbonyl Compounds Using HFC-125 in a Flow Microreactor System.

The protocol of micro-flow nucleophilic pentafluoroethylation using pentafluoroethane (HC2F5, HFC-125), a nontoxic, inexpensive, and commercially available greenhouse gas, is described. The micro-flow pentafluoroethylation by HFC-125 proceeded smoothly at room temperature or at -10 °C in DMF or toluene in the presence of a potassium base, namely, t-BuOK or KHMDS. A broad range of ketones, aldehydes, and chalcones with various substituted benzene rings were successfully converted to the corresponding pentafluoroethyl carbinols instantly with good to high yields.

Pentafluoroethylation of Carbonyl Compounds by HFC-125 via the Encapsulation of the K Cation with Glymes.

A simple protocol to overcome the explosive pentafluoroethylation of carbonyl compounds by HFC-125 is described. The use of potassium (K) bases with triglyme or tetraglyme as a solvent safely yields the pentafluoroethylation products in good to high yields. The experimental results suggest that an encapsulation of the K cation by glymes as K(glyme)2 inhibits the contact between the K cation and the reactive anionic pentafluoroethyl counterion, preventing their transformation into KF and explosive tetrafluoroethylene (TFE). The generation of sterically demanding [K(G3)2]+ and [K(G4)2]+ is an effective way as an unstable pentafluoroethyl anion reservoir.

[Asymptomatic Congenital Left Ventricular Aneurysm Treated with Endocardial Linear Infarct Exclusion Technique( ELIET):Report of a Case].

Surgical repair of asymptomatic congenital left ventricular aneurysm is poorly reported. A 30-yearold man presented with an asymptomatic abnormal electrocardiogram. Computed tomography (CT) and angiography revealed a congenital left ventricular aneurysm, and surgical repair was conducted with endocardial linear infarct exclusion technique (ELIET). His postoperative course was uneventful. Postoperative CT showed an elliptical cardiac shape with no recurrence of aneurysm. ELIET would serve as a surgical procedure for congenital left ventricular aneurysm.

Synthesis of trifluoromethyl ketones by nucleophilic trifluoromethylation of esters under a fluoroform/KHMDS/triglyme system.

A straightforward method that enables the formation of biologically attractive trifluoromethyl ketones from readily available methyl esters using the potent greenhouse gas fluoroform (HCF3, HFC-23) was developed. The combination of fluoroform and KHMDS in triglyme at -40 °C was effective for this transformation, with good yields as high as 92%. Substrate scope of the trifluoromethylation procedure was explored for aromatic, aliphatic, and conjugated methyl esters. This study presents a straightforward trifluoromethylation process of various methyl esters that convert well to the corresponding trifluoromethyl ketones. The tolerance of various pharmacophores under the reaction conditions was also explored.

Factors associated with sleep duration among pupils.

BACKGROUND: Sleep shortage has been pervasive among pupils. METHODS: Multiple regression analysis was used to analyze 2,722 questionnaires obtained from grade 5 to 12 pupils, to determine factors associated with sleep duration. RESULTS: Significant regression formulae for sleep duration were obtained for all school types: adjusted R2 / P value were 0.14/<0.001 for elementary school; 0.11/<0.001 for junior high school; 0.06/<0.001 for high school. Longer after-school activities (standardized regression coefficient/ P value were -0.22/<0.001 for elementary school; -0.10/<0.01 for junior high school; -0.18/<0.001 for high school) and more sleepiness (-0.09/<0.001 for elementary school; -0.07/<0.05 for junior high school; -0.07/<0.05 for high school) were significantly associated with reduced sleep duration for all school types. In both elementary and junior high schools, the higher grade (-0.53/<0.001 for elementary school; -0.10/<0.01 for junior high school), and longer weekday screen time (-0.15/<0.001 for elementary school; -0.19/<0.001 for junior high school) were also significantly associated with sleep loss. In elementary school, irregular dinner (-0.07/<0.05), breakfast skipping (-0.11/<0.001), longer weekend screen time (-0.09/<0.05) and better self-reported academic performance (0.07/<0.05) also revealed significant associations with sleep loss. In high school, reduction of sleep duration was also significantly associated with higher standardized body mass index (-0.08/<0.05). CONCLUSIONS: Excessive after-school activity might be considered in association with pupils' sleep reduction.

Impact of Magnesium Stearate Content: Modeling of Drug Degradation Using a Modified Arrhenius Equation.

To accelerate drug development, the pharmaceutical industry is working to shorten and improve studies on stability. The Accelerated Stability Assessment Program (ASAP) incorporating the humidity-corrected Arrhenius equation as an accelerated methodology has been proposed for both drug substances and drug products. In this study, the effect of magnesium stearate (MgSt) content on the chemical stability of acetylsalicylic acid was evaluated as a model system of drug-excipient compatibility studies using ASAP. In the acetylsalicylic acid powder blends, temperature and humidity showed a first-order linear response to the natural logarithm of the reaction rate constant, and MgSt content also showed a first-order linear response. A polynomial model was built in which temperature, humidity, and MgSt content were independent each other. The fitting index of the model, the coefficient of determination, was 0.9567, which was a good fit. In the long-term stability study (25 °C/60% relative humidity, 6 months), there was good agreement in total between measured values and model-predicted values. Using this model, we inferred that the degradation rates were depended on MgSt content at the fixed temperature and humidity because the micro-environmental pH of the excipient was catalytically affected. Applying this model equation can significantly reduce the duration of formulation design and stability studies and save time and costs in drug development.

Degradation Pathway of a Taxane Derivative DS80100717 Drug Substance and Drug Product.

The degradation pathway of a taxane derivative and anticancer agent, DS80100717, was investigated. Several degradants were generated under acidic, basic, and oxidative stress conditions in solution. The chemical structures of eight degradants of DS80100717 were elucidated using MS and NMR. The major degradant of the DS80100717 drug substance derived by heating in solid-state was the N-oxide form via oxidation and C2'-epimer of the side chain via acid hydrolysis. We proposed previously unreported degradation pathways of DS80100717 with taxane derivatives such as paclitaxel, docetaxel, and cabazitaxel.

[Septal Myectomy for Obstructive Hypertrophic Cardiomyopathy Due to Noonan Syndrome;Report of a Case].

A 15-year-old boy with Noonan syndrome, who had been diagnosed with hypertrophic cardiomyopathy (HCM) at 4 and treated by drugs, was referred to our hospital because of progression of left ventricular outflow tract obstruction (LVOTO). Transthoracic echocardiogram (TTE) revealed left ventricular hypertrophy( LVH), LVOTO( systolic gradient:102 mmHg), and systolic anterior motion of the mitral valve( SAM) with mild mitral regurgitation(MR). We performed septal myectomy by transaortic and transapical approaches. Postoperative TTE revealed significant reduction in left ventricular outflow tract (LVOT) gradient (9 mmHg), resolution of SAM, and reduction in MR grade. The postoperative course was uneventful except for transient atrial tachyarrhythmia. Myectomy for HCM in a patient with Noonan syndrome is rare. This case suggests that myectomy is useful for the patients with Noonan syndrome.

Addition of a ß1-Blocker to Milrinone Treatment Improves Cardiac Function in Patients with Acute Heart Failure and Rapid Atrial Fibrillation.

BACKGROUND: Tachycardia worsens cardiac performance in acute decompensated heart failure (ADHF). We investigated whether heart rate (HR) optimization by landiolol, an ultra-short-acting ß1-selective blocker, in combination with milrinone improved cardiac function in patients with ADHF and rapid atrial fibrillation (AF). METHODS AND RESULTS: We enrolled9 ADHF patients (New York Heart Association classification IV; HR, 138 ± 18 bpm; left ventricular [LV] ejection fraction, 28 ± 8%; cardiac index [CI], 2.1 ± 0.3 L/min-1/m-2; pulmonary capillary wedge pressure [PCWP], 24 ± 3 mm Hg), whose HRs could not be reduced using standard treatments, including diuretics, vasodilators, and milrinone. Landiolol (1.5-6.0 µg/kg-1/min-1, intravenous) was added to milrinone treatment to study its effect on hemodynamics. The addition of landiolol (1.5 µg/kg-1/min-1) significantly reduced HR by 11% without changing systolic blood pressure (BP) and resulted in a significant decrease in PCWP and a significant increase in stroke volume index (SVI), suggesting that HR reduction restores incomplete LV relaxation. Administration of more than 3.0 µg/kg-1/min-1 of landiolol decreased BP, CI, and SVI. CONCLUSION: The addition of landiolol at doses of <3.0 µg/kg/min to milrinone improved cardiac function in decompensated chronic heart failure with rapid atrial fibrillation by selectively reducing HR.

Insufficiency of urinary acid excretion of overweight or obese patients with chronic kidney disease and its involvement with renal tubular injury.

AIM: Metabolic acidosis occurs due to insufficient urinary ammonium excretion as chronic kidney disease (CKD) advances. Because obese subjects tend to have excessive consumption of protein and sodium chloride, they are prone to chronic acid loading and may therefore be predisposed to acid-induced kidney injury. We investigated the involvement of obesity in ammoniagenesis within damaged kidneys. METHODS: In the clinical study, urinary ammonium excretion was compared between 13 normal-weight and 15 overweight/obese CKD outpatients whose creatinine clearance was higher than 25 mL/min. For animal experiments, NH4 Cl was loaded to KKAy/TaJcl (KKAy), a metabolic syndrome model, and control BALB/c mice for 20 weeks. Kidney injury was evaluated through histological analysis and the expression of proinflammatory markers. RESULTS: Urinary ammonium excretion was lower in overweight/obese patients than in normal-weight patients, while intakes of protein and sodium chloride were higher in overweight/obese patients, implying that subclinical metabolic acidosis occurs in overweight/obese patients. The increase in urinary ammonium excretion induced by NH4 Cl loading was attenuated in KKAy mice after 16 weeks, whereas the increase was maintained in BALB/c mice throughout the study period. Histological study and real-time polymerase chain reaction analysis showed proximal tubular injury and enhanced expression levels of neutrophil gelatinase-associated lipocalin (NGAL) protein and messenger RNA, respectively, in KKAy mice but not in BALB/c mice. Finally, urinary NGAL concentration was higher in overweight/obese patients than in normal-weight patients in the early stage of CKD. CONCLUSION: Obesity could facilitate the induction of subclinical metabolic acidosis and acid accumulation in the kidney, which may potentially exacerbate kidney injury in CKD patients.

Insufficient sleep syndrome: An unrecognized but important clinical entity.

BACKGROUND: A sleep clinic for adults and children was established in the Tokyo Bay Urayasu Ichikawa Medical Centre, in August 2012. Given that few sleep clinics are available in Japan specifically for children, this clinic provides the opportunity to provide data on child patients with sleep problems. METHODS: Records of patients who visited the sleep clinic at the Tokyo Bay Urayasu Ichikawa Medical Centre aged ≤20 years at the first visit were retrospectively examined, along with the initial and final diagnoses. RESULTS: Of 2,157 patients who visited the sleep clinic at Tokyo Bay Urayasu Ichikawa Medical Centre between August 2012 and March 2017, 181 were ≤20 years old. In these 181 patients, the most frequent final diagnosis was insufficient sleep syndrome (ISS), n = 56, followed by circadian rhythm sleep-wake disorder, n = 28; insomnia, n = 28; and sleep-related movement disorder, n = 15. CONCLUSIONS: Insufficient sleep produces various brain dysfunctions in both adults and children, and is associated with behavioral, cognitive and physical problems, as well as with atypical early development. Insufficient sleep has also been reported to cause obesity. Insufficient sleep-induced obesity is often associated with the occurrence of metabolic syndrome. More effort is needed to ensure that children are receiving sufficient sleep.

FGFR2 mutation in 46,XY sex reversal with craniosynostosis.

Patients with 46,XY gonadal dysgenesis (GD) exhibit genital anomalies, which range from hypospadias to complete male-to-female sex reversal. However, a molecular diagnosis is made in only 30% of cases. Heterozygous mutations in the human FGFR2 gene cause various craniosynostosis syndromes including Crouzon and Pfeiffer, but testicular defects were not reported. Here, we describe a patient whose features we would suggest represent a new FGFR2-related syndrome, craniosynostosis with XY male-to-female sex reversal or CSR. The craniosynostosis patient was chromosomally XY, but presented as a phenotypic female due to complete GD. DNA sequencing identified the FGFR2c heterozygous missense mutation, c.1025G>C (p.Cys342Ser). Substitution of Cys342 by Ser or other amino acids (Arg/Phe/Try/Tyr) has been previously reported in Crouzon and Pfeiffer syndrome. We show that the 'knock-in' Crouzon mouse model Fgfr2c(C342Y/C342Y) carrying a Cys342Tyr substitution displays XY gonadal sex reversal with variable expressivity. We also show that despite FGFR2c-Cys342Tyr being widely considered a gain-of-function mutation, Cys342Tyr substitution in the gonad leads to loss of function, as demonstrated by sex reversal in Fgfr2c(C342Y/-) mice carrying the knock-in allele on a null background. The rarity of our patient suggests the influence of modifier genes which exacerbated the testicular phenotype. Indeed, patient whole exome analysis revealed several potential modifiers expressed in Sertoli cells at the time of testis determination in mice. In summary, this study identifies the first FGFR2 mutation in a 46,XY GD patient. We conclude that, in certain rare genetic contexts, maintaining normal levels of FGFR2 signaling is important for human testis determination.

Lower body weight and BMI at birth were associated with early adiposity rebound in 21-hydroxylase deficiency patients.

21-hydroxylase deficiency (21-OHD) is the most common type of congenital adrenal hyperplasia. In addition to the clinical problems caused by adrenal insufficiency and androgen excess, a risk for obesity and metabolic syndrome during young adulthood is a major ramification of the disease. Although glucocorticoid therapy is very likely to be one of the contributory factors, the precise causes of the metabolic status of adult 21-OHD patients remain to be clarified. Previously we reported that 21-OHD patients developed early onset AR, a condition which might create a risk for obesity and metabolic syndrome in adulthood. In order to elucidate the association between the onset of AR and factors during the fetal period to early infancy, we conducted a retrospective longitudinal analysis of 29 21-OHD patients (male: 14 cases, female: 15 cases, salt wasting type: 16, simple virilizing type: 13), who were identified by newborn screening and followed up at least until the age 10 years. Body size at birth, lower body weight, and lower BMI were found to precipitate the timing of AR. On the other hand, no significant association was observed between the timing of AR and sex, gestational age, treatment regimen (including cumulative dose of HDC), and disease severity (the type of the disease, the value of DHEA-S and 17-OHP). There are two points to consider: first, in 21-OHD patients treated with glucocorticoid substitution therapy, the risk for early AR cannot be reduced by adjusting the dose of glucocorticoid; second, fetal factors might affect the metabolic status of 21-OHD patients.

Characterization and Quality Control of Pharmaceutical Cocrystals.

Recent active research and new regulatory guidance on pharmaceutical cocrystals have increased the rate of their development as promising approaches to improve handling, storage stability, and bioavailability of poorly soluble active pharmaceutical ingredients (APIs). However, their complex structure and the limited amount of available information related to their performance may require development strategies that differ from those of single-component crystals to ensure their clinical safety and efficacy. This article highlights current methods of characterizing pharmaceutical cocrystals and approaches to controlling their quality. Different cocrystal regulatory approaches between regions are also discussed. The physical characterization of cocrystals should include elucidating the structure of their objective crystal form as well as their possible variations (e.g., polymorphs, hydrates). Some solids may also contain crystals of individual components. Multiple processes to prepare pharmaceutical cocrystals (e.g., crystallization from solutions, grinding) vary in their applicable ingredients, scalability, and characteristics of resulting solids. The choice of the manufacturing method affects the quality control of particular cocrystals and their formulations. In vitro evaluation of the properties that govern clinical performance is attracting increasing attention in the development of pharmaceutical cocrystals. Understanding and mitigating possible factors perturbing the dissolution and/or dissolved states, including solution-mediated phase transformation (SMPT) and precipitation from supersaturated solutions, are important to ensure the bioavailability of orally administrated lower-solubility APIs. The effect of polymer excipients on the performance of APIs emphasizes the relevance of formulation design for appropriate use.

Clinical, biochemical, and genetic features of non-classical 21-hydroxylase deficiency in Japanese children.

Non-classical 21-hydroxylase deficiency (NC21-OHD) is a mild form of 21-hydroxylase deficiency lacking apparent symptoms of androgen excess at birth. Most NC21-OHD cases are diagnosed after the onset of puberty, while a substantial number of patients are not diagnosed during childhood. Previous studies have reported ethnic differences in the prevalence of NC21-OHD. To date, the clinical features of NC21-OHD in Japanese children have not been systemically reported. Thus, we performed 3 independent analyses: retrospective analyses of newborn screening in 2 major Japanese cities (Sapporo and Niigata) and a national surveillance collecting clinical information from pediatric endocrinologists throughout the country. During the last 10 years, one case of NC21-OHD was diagnosed by newborn screening in each city, resulting in incidences of 2.0 (95% confidence interval = 0.0-5.9) and 2.1 (0.0-6.2) per 1,000,000 in Sapporo and Niigata, respectively. We collected information from 85% of the 135 Councilors of Japanese Society of Pediatric Endocrinology. Fifteen NC21-OHD patients were diagnosed during childhood, resulting in the estimated prevalence of 0.58 (0.28-1.1) per 1,000,000. Eleven patients were discovered by newborn screening, 7 patients developed hyperandrogenism symptoms (2-8 years of age, median 7), and 9 patients were treated with hydrocortisone at the time of the survey. Ten out of 13 patients showed compound heterozygosity for the P30L mutation of CYP21A2. Our study suggests that the prevalence/incidence of NC21-OHD is lower than that in Western countries, and that the age for initial onset of androgen excess symptoms varies during the prepubertal period.