Published papers

POU Class 1 Homeobox1 (POU1F1/Pou1f1) is a well-established pituitary-specific transcription factor, and causes, when mutated, combined pituitary hormone deficiency in humans and mice. POU1F1/Pou1f1 has 2 isoforms: the alpha and beta isoforms. Recently, pathogenic variants in the unique coding region of the beta isoform (beta domain) and the intron near the exon-intron boundary for the beta domain were reported, although their functional consequences remain obscure. In this study, we generated mice carrying the Pou1f1 c.143-83A>G substitution that recapitulates the human intronic variant near the exon-intron boundary for the beta domain. Homozygous mice showed postnatal growth failure, with an average body weight that was 35% of wild-type littermates at 12 weeks, which was accompanied by anterior pituitary hypoplasia and deficiency of circulating insulin-like growth factor 1 and thyroxine. The results of RNA-seq analysis of the pituitary gland were consistent with reduction of somatotrophs, and this was confirmed immunohistochemically. Reverse transcription polymerase chain reaction of pituitary Pou1f1 mRNA showed abnormal splicing in homozygous mice, with a decrease in the alpha isoform, an increase in the beta isoform, and the emergence of the exon-skipped transcript. We further characterized artificial variants in or near the beta domain, which were candidate positions of the branch site in pre-mRNA, using cultured cell-basis analysis and found that only c.143-83A>G produced transcripts similar to the mice model. Our report is the first to show that the c.143-83A>G variant leads to splicing disruption and causes morphological and functional abnormalities in the pituitary gland. Furthermore, our mice will contribute understanding the role of POU1F1/Pou1f1 transcripts in pituitary development.

Delayed and absent puberty and infertility in Turner syndrome (TS) are caused by primary hypogonadism. A majority of patients with TS who are followed at hospitals during childhood will not experience regular menstruation. In fact, almost all patients with TS need estrogen replacement therapy (ERT) before they are young adults. ERT in TS is administered empirically. However, some practical issues concerning puberty induction in TS require clarification, such as how early to start ERT. The present monograph aims to review current pubertal induction therapies for TS without endogenous estrogen production and suggests a new therapeutic approach using a transdermal estradiol patch that mimics incremental increases in circulating, physiological estradiol. Although evidence supporting this approach is still scarce, pubertal induction with earlier, lower-dose estrogen therapy more closely approximates endogenous estradiol secretion.

Background: Large datasets of detailed anthropometric measurements are scarce in children. The Japanese Standard Association 1978–1981 survey provides a rare opportunity to use high quality data from Japanese children. Aim: To construct inside leg length (ILL) and inside leg length to stature ratio (ILL/S) reference centile curves for Japanese children. Subjects and methods: The study sample consisted of 14,825 boys and 14,577 girls age 0–18 years for stature and weight measurements, and 9064 boys and 8796 girls age 0–12 years for ILL measurements, who participated in the 1978–1981 national survey on body sizes. LMS method was used to construct the reference centile curves. The reference centile curves for stature, weight, ILL, and ILL/S were compared to those of British children. Results: The L, M, and S reference values for Japanese children are presented for stature, weight, ILL, and ILL/S. Compared with British children of 0–12 years of age, Japanese children of 0–12 years of age had shorter median stature, shorter median ILL, and shorter median ILL/S. Conclusion: We present the first reference values for ILL and ILL/S in Japanese children. Japanese children had relatively shorter legs compared to British children from infancy.

Biallelic pathogenic variants in RMRP, the gene encoding the RNA component of RNase mitochondrial RNA processing enzyme complex, have been reported in individuals with cartilage hair hypoplasia (CHH). CHH is prevalent in Finnish and Amish populations due to a founder pathogenic variant, n.71A > G. Based on the manifestations in the Finnish and Amish individuals, the hallmarks of CHH are prenatal-onset growth failure, metaphyseal dysplasia, hair hypoplasia, immunodeficiency, and other extraskeletal manifestations. Herein, we report six Japanese individuals with CHH from four families. All probands presented with moderate short stature with mild metaphyseal dysplasia or brachydactyly. One of them had hair hypoplasia and the other immunodeficiency. By contrast, the affected siblings of two families showed only mild short stature. We also reviewed all previously reported 13 Japanese individuals. No n.71A > G allele was detected. The proportions of Japanese versus Finnish individuals were 0% versus 70% for birth length < −2.0 SD, 84% versus 100% for metaphyseal dysplasia and 26% versus 88% for hair hypoplasia. Milder manifestations in the Japanese individuals may be related to the difference of genotypes. The mildest form of CHH phenotypes is mild short stature without overt skeletal alteration or extraskeletal manifestation and can be termed “RMRP-related short stature”.

Context: Primary adrenal insufficiency (PAI) is a life-threatening condition characterized by the inability of the adrenal cortex to produce sufficient steroid hormones. E3 ubiquitin protein ligase zinc and ring finger 3 (ZNRF3) is a negative regulator of Wnt/β-catenin signaling. R-spondin 1 (RSPO1) enhances Wnt/β-catenin signaling via binding and removal of ZNRF3 from the cell surface. Objective: This work aimed to explore a novel genetic form of PAI. Methods: We analyzed 9 patients with childhood-onset PAI of biochemically and genetically unknown etiology using array comparative genomic hybridization. To examine the functionality of the identified single-exon deletions of ZNRF3 exon 2, we performed three-dimensional (3D) structure modeling and in vitro functional studies. Results: We identified various-sized single-exon deletions encompassing ZNRF3 exon 2 in 3 patients who showed neonatal-onset adrenal hypoplasia with glucocorticoid and mineralocorticoid deficiencies. Reverse-transcriptase polymerase chain reaction (RT-PCR) analysis showed that the 3 distinct single-exon deletions were commonly transcribed into a 126-nucleotide deleted mRNA and translated into 42-amino acid deleted protein (ΔEx2-ZNRF3). Based on 3D structure modeling, we predicted that interaction between ZNRF3 and RSPO1 would be disturbed in ΔEx2-ZNRF3, suggesting loss of RSPO1-dependent activation of Wnt/β-catenin signaling. Cell-based functional assays with the TCF-LEF reporter showed that RSPO1-dependent activation of Wnt/β-catenin signaling was attenuated in cells expressing ΔEx2-ZNRF3 as compared with those expressing wild-type ZNRF3. Conclusion: We provided genetic evidence linking deletions encompassing ZNRF3 exon 2 and congenital adrenal hypoplasia, which might be related to constitutive inactivation of Wnt/β-catenin signaling by ΔEx2-ZNRF3.

Current status and its background of Adult Turner Syndrome (TS) are not clarified well. Via a questionnaire survey of 492 adult women with TS, this study investigated the association between menstruation, Kaufmann therapy (menstrual induction therapy), social status (education, employment & marriage), complications, transition from pediatric to adult care, and sex chromosome karyotype using statistical methods. Spontaneous menarche occurred in 22.0% and more frequently among patients with the 45,X/46,XX karyotype. Over 60% of these subjects, menstruation did not persist regularly. Kauffmann therapy was performed in 69.4%; the most common formulation was a conjugated estrogen and progesterone combination. Marriage and higher education advancement rates were low in adults with TS, whereas their employment rate was similar to that of the age-matched general female population. Patients receiving Kauffmann therapy had higher complication rates, greater education length, and higher employment rates. The higher-education advancement rate was observed among patients with 45,X/46,X,Xi and 46,X,Xi karyotypes. Transition from pediatrician to adult specialist was not smooth, subjects were treated in pediatric departments (60.7%), gynecological department (21.4%), internal medicine departments (13.3%), and others. While reason is not clear, the largest number of TS patients are treated in general pediatrics and the percentage of receiving Kauffmann therapy and having complication were significantly lower than in pediatric and adult department of endocrinology (& metabolism). This Study revealed many novel findings of adult TS.

The Career Development for Women Pediatric Endocrinologists and Work-Life Balance Committee and Support Team for Women Doctors in Education and Training Committee investigated the current situation of women doctors in the Japanese Society for Pediatric Endocrinology (JSPE). The proportion of women doctors (PWD) was as follows. 1) Members of JSPE: 40.2% in fiscal 2018, versus 33.3% in fiscal 2010; 2) councilors: 21.6% from fiscal 2014 to 2017, versus 6.3% from fiscal 2008 to 2010; 3) board members: 13.6% from fiscal 2014 to 2017, versus 0% from fiscal 2008 to 2010; 4) board-certified endocrinologists (Pediatrics) and certified endocrine educators (Pediatrics): 31.7% and 25.4% in fiscal 2018, versus 22.4% and 15.3% in fiscal 2010, respectively; and 5) average value of first presenters and chairpersons in the Annual Scientific Meeting of JSPE was 41.4% and 22.3% from 2010 to 2019. These PWD figures for JSPE were higher than those of the Japan Pediatric Society and the Japan Endocrine Society, indicating a reducing gender gap in JSPE, although increases in the PWD of decision-making posts remains insufficient.

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.

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 < 130 mEq/L, K > 7 mEq/L or Na/K ratio < 20; except for one case, SSW developed in or after the second week of life. The serum concentrations of Na, K and Na/K were linearly correlated with age in days (R2 =.38,.25, and.34 respectively), suggesting that the risk of SSW increases linearly without a threshold. The age at which the regression lines reached Na < 130 mEq/L, K > 7 mEq/L and Na/K < 20 was approximately coincided, 11.1, 12.3 and 11.2 days, respectively. All SSW patients exhibited decreased body weight from birth in their second week of life. Conclusion: Our data revealed that the risk of developing SSW increases during the second week of life without a threshold, and for preventing SSW, early intervention, ideally during first week of life, is desirable. An increased body weight in the second week of life indicates the absence of SSW.

Herein, we report two girls with a neonatal screening (NS)-negative 21-hydroxylase deficiency (21-OHD) requiring treatment with hydrocortisone due to virilization that developed in late childhood. Patient 1 was born prematurely on the 30th gestational week with normal external genitalia at birth. She passed the NS for 21-OHD. At 6 yr of age, she was referred to a hospital for evaluation of premature pubarche and clitoromegaly. Her diagnosis was central precocious puberty, and GnRH agonist was initiated. However, her symptoms did not improve despite treatment for over 4 years. She was then referred to our hospital where she was diagnosed with 21-OHD. Although she was started on hydrocortisone therapy, her adult height reached only 140 cm (−3.4 SD). Patient 2 was delivered at 37 weeks of gestation and passed the NS for 21-OHD. She was referred to a hospital because of premature pubarche at the age of 6 yr. She was diagnosed with 21-OHD, and hydrocortisone replacement therapy was initiated. Her present height at 13 yr of age is 148 cm (−1.3 SD). These cases reminded us that the possibility of 21-OHD should be considered when patients show premature pubarche or precocious puberty, even if they passed the NS test for 21-OHD.

Context: Recently developed long-read sequencing (LRS) technology has been considered an option for CYP21A2 analysis. However, the clinical use of LRS for CYP21A2 analysis is limited. Objective: This study’s objective is to develop an efficient and low-cost LRS system for CYP21A2 screening. Methods: A DNA fragment library was prepared in a single polymerase chain reaction (PCR) that covers the entire CYP21A2 gene and all known junctions caused by TNXB gene structural rearrangements, yielding a single 8-kb product of CYP21A2 or CYP21A1P/CYP21A2 chimera. After barcoding, the PCR products were sequenced on a MinION-based platform with Flongle Flow Cell R9.4.1 and R10.4.1. Results: The reference genotypes of 55 patients with 21-hydroxylase deficiency (21OHD) were established using the conventional method with multiplex ligation-dependent probe amplification (MLPA) and nested PCR. LRS using Flongle Flow Cell R9.4.1 yielded consistent results. Additionally, the recently updated LRS “duplex” analysis with Flongle flow cell R10.4.1 was tested to reveal an advantage of accurately sequencing a variant located on the homopolymer region. By introducing a barcode system, the cost was reduced to be comparable to that of conventional analysis. A novel single-nucleotide variation was discovered at the acceptor site of intron 7, c.940-1G > C. We also identified a subtype of the classical chimeric junction CH2, “CH2a,” in the region from the latter part of intron 5 to exon 6. Conclusion: We successfully established a novel low-cost and highly accurate LRS system for 21OHD genetic analysis. Our study provides insight into the feasibility of LRS for diagnosing 21OHD and other genetic diseases caused by structural rearrangements.

Background: 17α-hydroxylase deficiency (17OHD) is a rare autosomal recessive disorder. Aldosterone levels are usually low in patients with 17OHD. However, among the approximately 150 cases of 17OHD reported to date, aldosterone levels were not low in all cases. Therefore, some 17OHD cases may have been misdiagnosed as primary aldosteronism (PA) cases. Often before puberty, 17OHD is diagnosed because of abnormal genital morphology and menstrual irregularities. However, we report a very rare case of 17OHD in an elderly patient with a high aldosterone/renin ratio (ARR) similar to that in PA. Case presentation: A 63-year-old Japanese woman was transferred to our medical facility for the evaluation of bilateral adrenal hypertrophy, which was incidentally discovered during an abdominal examination after cholecystectomy. The patient had hypokalemia and a high aldosterone/renin ratio. Her medical history included hypertension and right intracerebral capsular hemorrhage at the age of 30 years. Additional testing revealed low cortisol, high adrenocorticotropic hormone, and low testosterone and dehydroepiandrosterone sulfate, indicating congenital adrenal hyperplasia. Genetic analysis revealed a mutation in the CYP17A1 gene and a karyotype of 46, XY; hence, she was diagnosed with 17OHD. Conclusion: 17OHD can resemble PA. The combination of a high ARR and low cortisol level should trigger the consideration of 17OHD.

The occurrence of fetuses suspected of having ambiguous genitalia will likely increase in the future. Currently, the impact of prenatal genetic counseling on parents' understanding and psychological preparedness has not been addressed. We provided prenatal genetic counseling to parents of two fetuses suspected of ambiguous genitalia. Case 1: At 22 weeks of gestation, swelling of the labia majora, and a clitoris-like structure were noted despite 46,XY detected in amniotic fluid cells. Case 2: At 28 weeks of gestation, bladder exstrophy and a scrotum-like structure were noted. At 32 weeks (Case 1) and 37 weeks (Case 2) of gestation, we shared information with parents regarding the possible difficulty of legal sex assignment at birth, and a scenario for registration of the birth certificate. At birth, both babies presented with ambiguous genitalia. For both cases, the parents remained calm on seeing their baby's genitalia for the first time. After a month, we shared medical information with parents, including karyotype, testosterone production capacity, and surgical schedule. In both cases parents assigned their respective baby's legal sex as male. Several months later, parents were questioned on prenatal genetic counseling. Case 1: Mother, “I was prepared to address our baby's genitalia calmly.” Father, “I understood the procedure of legal sex assignment.” Case 2: Mother, “Without counseling, I would have been more upset and worried.” Father, “We were assured that multidisciplinary experts would support us.” Prenatal genetic counseling provides reassurance to parents, who remain informed and emotionally secure throughout the legal sex assignment of their child.

Objectives: The usefulness of continuous glucose monitoring (CGM) in infants with panhypopituitarism (PH) having hypoglycemia is yet to be explored. The potential adverse effects of growth hormone (GH) replacement therapy, such as hyperglycemia, cannot be comprehensively evaluated using the conventional measurement. Case presentation: A 2-month-old infant with PH, including severe GH deficiency, had hypoglycemia despite frequent feeding. Glucose levels were monitored using CGM before and after GH replacement therapy. The proportion of time for hypoglycemia decreased from 4.9 to 0% (p<0.017). Hyperglycemia did not increase. The CGM method did not contribute to any adverse events requiring intervention. Our patient only experienced minor bleeding and no episode of cellulitis. Conclusions: CGM is useful in controlling glucose levels in infants with hypoglycemia and PH.