論文

Geroderma osteodysplastica (GO) is a subtype of cutis laxa syndrome characterized by congenital wrinkly skin, a prematurely aged face, extremely short stature, and osteoporosis leading to recurrent fractures. GO exhibits an autosomal recessive inheritance pattern and is caused by loss-of-function mutations in GORAB, which encodes a protein important for Golgi-related transport. Using whole exome sequencing, we identified novel compound heterozygous nonsense mutations in the GORAB in a GO patient. The patient was a 14-year-old Japanese boy. Wrinkled skin and joint laxity were present at birth. At 1 year of age, he was clinically diagnosed with cutis laxa syndrome based on recurrent long bone fractures and clinical features, including wrinkled skin, joint laxity, and a distinctive face. He did not show retarded gross motor and cognitive development. At 11 years of age, he was treated with oral bisphosphonate and vitamin D owing to recurrent multiple spontaneous fractures of the vertebral and extremity bones associated with a low bone mineral density (BMD). Bisphosphonate treatment improved his BMD and fracture rate. Whole exome sequencing revealed two novel compound heterozygous nonsense mutations in the GORAB gene (p. Arg60* and p. Gln124*), and the diagnosis of GO was established. GO is a rare connective tissue disorder. Approximately 60 cases have been described to date, and this is the first report of a patient from Japan. Few studies have reported the effects of bisphosphonate treatment in GO patients with recurrent spontaneous fractures. Based on this case study, we hypothesize that oral bisphosphonate and vitamin D are effective and safe treatment options for the management of recurrent fractures in GO patients. It is important to establish a precise diagnosis of GO to prevent recurrent fractures and optimize treatment plans. (C) 2017 Elsevier Masson SAS. All rights reserved.

NR0B1 is the causative gene for X-linked adrenal hypoplasia congenita, characterized by adrenal insufficiency, hypogonadotropic hypogonadism, and infertility. We identified an NR0B1 frameshift mutation in a boy with precocious puberty who had no signs of adrenal insufficiency. Blood examination revealed elevated testosterone levels and gonadotropin hyperresponses to gonadotropin releasing hormone (GnRH) stimulation, together with normal adrenal hormone levels. GnRH analog treatment partially ameliorated his clinical features. Molecular analysis identified a p.Glu3fsAla*16 in NR0B1. These results expand the clinical manifestations of NR0B1 mutations to include central precocious puberty without adrenal insufficiency. NR0B1 mutations likely underlie androgen overproduction via GnRH-dependent and -independent mechanisms.

Lay Summary Pathogenic variants in POLE are linked to a novel form of infantile-onset inflammatory bowel disease with Mott cell infiltration. In this novel form of inflammatory bowel disease, hematopoietic cell transplantation induces mucosal healing, suggesting a distinct disease mechanism and therapeutic potential.

The potential effect of artificial oocyte activation (AOA) following intracytoplasmic sperm injection (ICSI) on neurodevelopment remains uncertain. Since AOA does not fully replicate the physiological Ca²⁺ oscillations essential for fertilization, concerns persist regarding its safety. This study evaluates neurodevelopmental outcomes in children conceived via ICSI-AOA compared to conventional ICSI. A multicentre, cross-sectional study was conducted at Keio University Hospital and eight affiliated institutions, assessing 158 children (ICSI: n = 81, ICSI-AOA: n = 77) aged 12–60 months using the Japanese Ages and Stages Questionnaire, 3rd Edition (J-ASQ-3), covering communication, gross motor, fine motor, problem-solving, and personal-social skills. No significant differences were found in J-ASQ-3 subdomain scores across age groups, and fully adjusted models showed no significant differences in children scoring below the monitoring zone. Subgroup analyses compared differences between AOA protocols (A23187: n = 59; ionomycin: n = 15). AOA protocols were not significantly associated with score variations. Neurodevelopmental outcomes in children conceived via ICSI-AOA were comparable to those conceived via conventional ICSI, suggesting that AOA does not adversely affect early childhood neurodevelopment. These findings support the safety of AOA in assisted reproduction, though further longitudinal research is needed to assess long-term outcomes.

Selecting optimal therapeutic interventions for febrile urinary tract infection (f-UTI) is crucial to prevent complications such as kidney scarring. While current clinical guidelines provide risk-stratified imaging recommendations, they are largely based on Western populations and lack specific predictors for which children will ultimately require therapeutic interventions. This study aimed to establish risk stratification criteria for East Asian children with first-episode f-UTI. This retrospective single-center study analyzed patients aged 2–24 months with first-episode f-UTI. All patients underwent a standardized diagnostic and management protocol, including kidney–bladder ultrasound (KBUS) and voiding cystourethrography (VCUG), to ensure uniform evaluation. The primary outcome was “requirement for therapeutic intervention,” defined as one or more of the following: (1) urological surgery (2) antimicrobial prophylaxis (for vesicoureteral reflux grade ≥III) and (3) antimicrobial treatment for recurrent f-UTI. Multivariate logistic regression was performed to identify independent predictors associated with the interventions. A total of 216 patients were included (median age: 4 months). Overall, 59 patients required therapeutic interventions. Non-Escherichia coli infection (OR 3.3, 95% CI 1.3–8.7) and abnormal KBUS findings (OR 5.3, 95% CI 2.7–10.6) were identified as independent predictors. The sensitivity and specificity of the factors for predicting therapeutic intervention were 64.4% and 73.2%, respectively. This study identified non-E. coli infection and abnormal KBUS findings as key predictors for therapeutic interventions in East Asian children with first-episode f-UTI. These findings suggest that a more targeted approach based on these factors may optimize risk stratification and patient selection for VCUG, improving clinical decision-making.

Growth failure is one of the most common late complications in children undergoing hematopoietic stem cell transplantation (SCT). The present report describes a qualitative method of evaluating height growth after SCT, using a growth chart. The patients were divided into three groups according to the shape of their growth chart: the normal growth chart group, the early-onset growth retardation group (E-group), in which a decreased growth rate was seen during the first year after SCT, and the late-onset growth retardation group (L-group), in which a decreased growth rate was seen more than 1 yr after the SCT. In the E-group, total body irradiation and prolonged steroid therapy were thought to contribute to the growth failure, whereas in the L-group, impaired pubertal development was thought to be responsible. The growth pattern in the L-group may, therefore, be of particular clinical importance, because the final stature of the subjects in this group can be improved by pharmacological adjustment of pubertal onset. Although limited by the small size and heterogeneous nature of the sample, our results suggest that growth-chart-based evaluation may provide important information to stratify subjects showing inadequate growth after SCT into two groups whose follow-up and treatment should be individualized.

Recently, germline mutations in SAMD9 and SAMD9L were increasingly found in children with monosomy 7. We report the outcomes in 2 infants with the SAMD9/SAMD9L variant, who presented with anemia and thrombocytopenia (patient 1), and neutropenia and nonsymptomatic white-matter-encephalopathy (patient 2). Both patients received cord blood transplantation and experienced critical post-cord blood transplantation adverse events; patients 1 and 2 developed fulminant engraftment syndrome and life-threatening graft-versus-host disease, respectively. Of note, selective loss of chromosome 7 in bone marrow-derived CD34+ cells was inferred.

Congenital hypothyroidism due to thyroid dysgenesis (TD), presented as thyroid aplasia, hypoplasia or ectopia, is one of the most prevalent rare diseases with an isolated organ malformation. The pathogenesis of TD is largely unknown, although a genetic predisposition has been suggested. We performed a genome-wide association study (GWAS) with 142 Japanese TD cases and 8380 controls and found a significant locus at 2q33.3 (top single nucleotide polymorphism, rs9789446: P = 4.4 × 10-12), which was replicated in a German patient cohort (P = 0.0056). A subgroup analysis showed that rs9789446 confers a risk for thyroid aplasia (per allele odds ratio = 3.17) and ectopia (3.12) but not for hypoplasia. Comprehensive epigenomic characterization of the 72-kb disease-associated region revealed that it was enriched for active enhancer signatures in human thyroid. Analysis of chromosome conformation capture data showed long-range chromatin interactions of this region with promoters of two genes, FZD5 and CCNYL1, mediating Wnt signaling. Moreover, rs9789446 was found to be a thyroid-specific quantitative trait locus, adding further evidence for a cis-regulatory function of this region in thyroid tissue. Specifically, because the risk rs9789446 allele is associated with increased thyroidal expression of FDZ5 and CCNYL1 and given the recent demonstration of perturbed early thyroid development following overactivation of Wnt signaling in zebrafish embryos, an enhanced Wnt signaling in risk allele carriers provides a biologically plausible TD mechanism. In conclusion, our work found the first risk locus for TD, exemplifying that in rare diseases with relatively low biological complexity, GWAS may provide mechanistic insights even with a small sample size.

Rationale:Paraganglioma (PGL), an extra-adrenal pheochromocytoma, is a rare tumor, especially in children. While hypersecretion of catecholamines causes the classic triad of headaches, palpitations, and profuse sweating, prompt diagnosis is still challenging.Patient concerns:For 7 months, an 8-year-old boy complained of polyuria and weight loss, followed by proteinuria and headache for 1 month prior to admission. He was admitted to our hospital due to an afebrile seizure.Diagnosis:His blood pressure remained markedly elevated even after cessation of the convulsion. Magnetic resonance imaging of the brain revealed posterior reversible encephalopathy syndrome. Abdominal computed tomography showed a mass lesion encasing the left renal artery, measuring 41 mm in length along its major axis. The plasma and urine levels of normetanephrine were elevated. Additionally, iodine-123-metaiodobenzylguanidine scintigraphy showed an abnormal uptake in the abdominal mass with no evidence of metastasis. Based on these findings, we tentatively diagnosed him with PGL.Intervention:Substantial alpha-and beta-blocking procedures were performed, followed by a tumor resection and an extended left nephrectomy on day 31 of hospitalization. Pathological findings confirmed the diagnosis of PGL.Outcome:The postoperative course was uneventful, and his blood pressure normalized without the use of antihypertensive agents. Genetic testing revealed a known SDHB germline mutation. The same mutation was also detected on his father and paternal grandfather without any history of hypertension or malignant tumor.Lesson:It remains challenging to diagnose pheochromocytoma/paraganglioma (PPGL) promptly because PPGL can present with a variety of symptoms. Preceding symptoms of the presented case might be caused by PGL. Although PPGL is a rare disease, especially in children, it should be considered in differential diagnosis when various unexplained symptoms persist.

For salt-wasting 21-hydroxylase deficiency (21OHD), fludrocortisone (FC) is usually supplemented at 0.05–0.2 mg/d dose. To date, no report has described 21OHD neonates requiring > 0.4 mg/d of FC. Our female 21OHD patient was lethargic and experienced weight loss with hyponatremia (133 mEq/L), hyperkalemia (6.5 mEq/L), and elevated active renin concentration (ARC, 1942.2 pg/mL) at 6 days of life. Hydrocortisone and FC replacement were initiated. FC dose was gradually increased to 0.4 mg/d at 21 days of life, but her hyperkalemia (6.4 mEq/L) and high ARC (372.3 pg/mL) persisted. We increased FC to 0.6 mg/d and used a low-potassium and high-sodium formula. Hyperkalemia subsequently improved. At 33 days of life, the ARC decreased to 0.6 pg/mL and FC dosage was gradually decreased. At 3 months of age, the low-potassium and high-sodium formula was discontinued, but the serum potassium level was normal and ARC remained low at 0.1 mg/d of FC. We speculated that severe mineralocorticoid resistance was the reason why her hyperkalemia persisted even with 0.4 mg/d of FC; however, the pathophysiology of transiently severe resistance to FC in this patient is unknown. In conclusion, 21OHD neonates may show severe salt-wasting that transiently require > 0.4 mg/d of FC.

SOX9, a transcription factor, is expressed in the undifferentiated XX and XY gonads. SRY induces significant upregulation of SOX9 expression in XY gonads. Loss-of-function SOX9 variants cause testicular dysgenesis in 46,XY patients, while duplication of the total gene or the upstream regulatory region results in testicular development in 46,XX patients. However, gain-of-function (GoF) SOX9 variants have not been reported previously. We report the case of a 16-year-old female patient with a 46,XX karyotype who had masculinized external genitalia and unilateral ovotestis. Next-generation sequencing-based genetic screening for disorders of sex development led to the identification of a novel SOX9 variant (p.Glu50Lys), transmitted from the phenotypically normal father. Expression analysis showed that E50K-SOX9 enhanced transactivation of the luciferase reporter containing the testis enhancer sequence core element compared with that containing the wildtype-SOX9. This GoF activity was not observed in the luciferase reporter containing Amh, the gene for anti-Müllerian hormone. We genetically engineered female mice (Sox9E50K/E50K), and they showed no abnormalities in the external genitalia or ovaries. In conclusion, a novel SOX9 variant with a promoter-specific GoF activity was identified in vitro; however, the disease phenotype was not recapitulated by the mouse model. At present, the association between the GoF SOX9 variant and the ovotestis phenotype remains unclear. Future studies are needed to verify the possible association.

Context: Inactivating variants of thyrotropin (thyroid-stimulating hormone; TSH) receptor (TSHR) cause congenital hypothyroidism. More than 60 such variants have been reported so far, most of which were located in the extracellular or transmembrane domain. Objective: We report the identification and characterization of a frameshift TSHR variant in the intracytoplasmic C-tail region. Methods: Sequencing of TSHR was performed in a patient with congenital hypothyroidism. The functionality of the identified variants was assessed by expressing TSHR in HEK293 cells and measuring TSH-dependent activation of the cAMP-response element-luciferase reporter. A series of systematic mutagenesis experiments were performed to characterize the frameshifted amino acid sequence. Results: The proband was heterozygous for a known TSHR variant (p.Arg519His) and a novel frameshift TSHR variant (p.Val711Phefs∗18), which removed 54 C-terminal residues and added a 17-amino acid frameshifted sequence. The loss of function of Val711Phefs∗18-TSHR was confirmed in vitro, but the function of Val711∗-TSHR was found to be normal. Western blotting showed the low protein expression of Val711Phefs∗18-TSHR. Fusion of the frameshift sequence to green fluorescent protein or luciferase induced inactivation of them, indicating that the sequence acted as a degron. A systematic mutagenesis study revealed that the density of hydrophobic residues in the frameshift sequence determined the stability. Eight additional frameshift TSHR variants that covered all possible shifted frames in C-tail were created, and another frameshift variant (Thr748Profs∗27) with similar effect was found. Conclusions: We characterized a naturally occurring frameshift TSHR variant located in C-tail, and provided a unique evidence that hydrophobicity in the C-terminal region of the receptor affects protein stability.

BACKGROUND: Silver-Russell syndrome (SRS) is characterized by growth failure and dysmorphic features. Major (epi)genetic causes of SRS are loss of methylation on chromosome 11p15 (11p15 LOM) and maternal uniparental disomy of chromosome 7 (upd(7)mat). However, IGF2, CDKN1C, HMGA2, and PLAG1 mutations infrequently cause SRS. In addition, other imprinting disturbances, pathogenic copy number variations (PCNVs), and monogenic disorders sometimes lead to SRS phenotype. This study aimed to clarify the frequency and clinical features of the patients with gene mutations among etiology-unknown patients with SRS phenotype. RESULTS: Multigene sequencing was performed in 92 out of 336 patients referred to us for genetic testing for SRS. The clinical features of the patients were evaluated based on the Netchine-Harbison clinical scoring system. None of the patients showed 11p15 LOM, upd(7)mat, abnormal methylation levels for six differentially methylated regions (DMRs), namely, PLAGL1:alt-TSS-DMR on chromosome 6, KCNQ1OT1:TSS-DMR on chromosome 11, MEG3/DLK1:IG-DMR on chromosome 14, MEG3:TSS-DMR on chromosome 14, SNURF:TSS-DMR on chromosome 15, and GNAS A/B:TSS-DMR on chromosome 20, PCNVs, or maternal uniparental disomy of chromosome 16. Using next-generation sequencing and Sanger sequencing, we screened four SRS-causative genes and 406 genes related to growth failure and/or skeletal dysplasia. We identified four pathogenic or likely pathogenic variants in responsible genes for SRS (4.3%: IGF2 in two patients, CDKN1C, and PLAG1), and five pathogenic variants in causative genes for known genetic syndromes presenting with growth failure (5.4%: IGF1R abnormality (IGF1R), SHORT syndrome (PIK3R1), Floating-Harbor syndrome (SRCAP), Pitt-Hopkins syndrome (TCF4), and Noonan syndrome (PTPN11)). Functional analysis indicated the pathogenicity of the CDKN1C variant. The variants we detected in CDKN1C and PLAG1 were the second and third variants leading to SRS, respectively. Our patients with CDKN1C and PLAG1 variants showed similar phenotypes to previously reported patients. Furthermore, our data confirmed IGF1R abnormality, SHORT syndrome, and Floating-Harbor syndrome are differential diagnoses of SRS because of the shared phenotypes among these syndromes and SRS. On the other hand, the patients with pathogenic variants in causative genes for Pitt-Hopkins syndrome and Noonan syndrome were atypical of these syndromes and showed partial clinical features of SRS. CONCLUSIONS: We identified nine patients (9.8%) with pathogenic or likely pathogenic variants out of 92 etiology-unknown patients with SRS phenotype. This study expands the molecular spectrum of SRS phenotype.

Digital clubbing is characterized by bulbous enlargement of the terminal segments of the fingers. Hypotheses including hypoxia have been proposed for the pathogenesis of digital clubbing, but the exact pathogenesis of digital clubbing is still uncertain. Lysinuric protein intolerance (LPI) is caused by pathogenic variants in SLC7A7 and is often associated with interstitial lung disease. Previously two patients of LPI with digital clubbing but without hypoxia have been reported. It is unclear whether digital clubbing in LPI is secondary to hypoxia or directly related to SLC7A7 deficiency. Here we report a 6-year-old Japanese boy presented with digital clubbing without hypoxia. He had episodic vomiting, each episode consisting of a single vomiting event occurring once a month, and his growth had been delayed. He had interstitial lung disease and hepatomegaly. He had compound heterozygous pathogenic variants in the SLC7A7, leading to the diagnosis of LPI. Together with the two previously reported patients mentioned above, we conclude that digital clubbing can occur in the absence of hypoxia. Digital clubbing in the absence of hypoxia has been observed in two genetic disorders related to prostaglandin (PG) E2, HPGD and SLCO2A1. PGE2 synthesis is primarily regulated by the cyclooxygenase 2, which plays a critical role in the control of inflammation. A high urine PGE level in the patient was compatible with the notion that PGE2 production may be increased in LPI. The occurrence of digital clubbing in the absence of hypoxia in LPI patients with SLC7A7 may be attributed to the mechanism of increased PGE2 production.

Background: Congenital hypothyroidism (CH) is caused by mutations in cysteine residues, including Cys655 and Cys825 that form disulfide bonds in thyroid peroxidase (TPO). It is highly likely that these disulfide bonds could play an important role in TPO activity. However, to date, no study has comprehensively analyzed cysteine mutations that form disulfide bonds in TPO. In this study, we induced mutations in cysteine residues involved in disulfide bonds formation and analyzed their effect on subcellular localization, degradation, and enzyme activities to evaluate the importance of disulfide bonds in TPO activity. Methods: Vector plasmid TPO mutants, C655F and C825R, known to occur in CH, were transfected into HEK293 cells. TPO activity and protein expression levels were measured by the Amplex red assay and Western blotting. The same procedure was performed in the presence of MG132 proteasome inhibitor. Subcellular localization was determined using immunocytochemistry and flow cytometry. The locations of all disulfide bonds within TPO were predicted using in silico analysis. All TPO mutations associated with disulfide bonds were induced. TPO activity and protein expression levels were also measured in all TPO mutants associated with disulfide bonds using the Amplex red assay and Western blotting. Results: C655F and C825R showed significantly decreased activity and protein expression compared with the wild type (WT) (p < 0.05). In the presence of the MG132 proteasome inhibitor, the protein expression level of TPO increased to a level comparable with that of the WT without increases in its activity. The degree of subcellular distribution of TPO to the cell surface in the mutants was lower compared with the WT TPO. Twenty-four cysteine residues were involved in the formation of 12 disulfide bonds in TPO. All TPO mutants harboring an amino acid substitution in each cysteine showed significantly reduced TPO activity and protein expression levels. Furthermore, the differences in TPO activity depended on the position of the disulfide bond. Conclusions: All 12 disulfide bonds play an important role in the activity of TPO. Furthermore, the mutations lead to misfolding, degradation, and membrane insertion.

Background: The annual administration of the influenza vaccine is the most effective method for preventing influenza. We have evaluated the effectiveness of the inactivated influenza vaccine in children aged 6 months to 15 years across the seasons from 2013/2014 to 2022/2023. This study aims to investigate the effectiveness of the inactivated influenza vaccine in the 2023/2024 season, the first year following the easing of strict COVID-19 measures, and possibly the last season when only the inactivated vaccine is available on the market. Methods: Adjusted vaccine effectiveness for the 2023/2024 season was assessed using a test-negative case-control design, with results based on polymerase chain reaction and rapid influenza diagnostic tests. Vaccine effectiveness was calculated by influenza type and patient hospitalization/outpatient status. Results: A total of 1832 children were recruited. The inactivated influenza vaccine was effective in preventing both symptomatic influenza A and B in both inpatient and outpatient settings. Overall vaccine effectiveness for influenza A was 51% (95% confidence interval [CI], 23%–69%, n = 930) in inpatient settings and 54% (95%CI, 27%–71%, n = 559) in outpatient settings. For influenza B, effectiveness was 60% (95%CI, 22%–79%, n = 859) in inpatient settings and 56% (95%CI, 26%–74%, n = 558) in outpatient settings. Analysis suggested that administering two doses enhanced effectiveness specifically against influenza B. Conclusions: This is the first study to demonstrate influenza vaccine effectiveness in children after the relaxation of strict COVID-19 measures in Japan (2023/2024). We recommend the current inactivated vaccine for preventing both influenza A and B in children, with consideration for the potential use of two doses to enhance effectiveness against influenza B.

BACKGROUND: Fetal goiter is only rarely observed in pregnant women without autoimmune thyroid disorders, and there is no epidemiological data on its pathophysiology. Dual oxidase maturation factor 2 (DUOXA2), together with dual oxidase 2, serves pivotal roles in thyroid hormone biosynthesis. To date, all reported patients with DUOXA2 mutations were diagnosed postnatally through newborn screening for congenital hypothyroidism. CASE REPORT: The mother of a male fetus presented at 33 + 4 gestational weeks (GW) with a fetal goiter and polyhydramnios. Cordocentesis revealed fetal hypothyroidism (TSH 253.4 mU/L, FT4 0.29 ng/dL). Intra-amniotic levothyroxine injections were performed at GW 34 + 3 and 35 + 3. The patient was born after spontaneous vaginal delivery at 35 + 6 GW without obstetrical complications. He was treated with levothyroxine until the age of 6 years when reevaluation of his thyroid functions showed normal results (TSH 1.32 mU/L, FT4 1.81 ng/dL). Eleven causative genes of CH, including DUOXA2, were analyzed with use of a next-generation sequencing technique. RESULTS: A next-generation sequencing-based mutation screen led us to find that he was compound heterozygous for 2 previously reported nonsense DUOXA2 mutations (p.[Tyr138*];[Tyr246*]). CONCLUSION: The present case not only illustrates the phenotypic diversity of DUOXA2 mutation carriers but also implies that DUOXA2 is important in prenatal thyroid hormone production.

Background: Individuals carrying a heterozygous inactivating PAX8 mutation are affected by congenital hypothyroidism (CH), although heterozygous Pax8 knockout mice are not. It has remained unclear whether CH in PAX8 mutation carriers is caused by haploinsufficiency or a dominant negative mechanism. Objective: To report clinical and molecular findings of four novel PAX8 mutations, including one early-truncating frameshift mutation. Subjects and methods: Four probands were CH patients. Two had family history of congenital or childhood hypothyroidism. Three probands were diagnosed in the frame of newborn screening for CH, while one had a negative result in screening but was diagnosed subsequently. Three had thyroid hypoplasia and one had a slightly small thyroid with low echogenicity. For these probands and their family members, we sequenced PAX8 using a standard PCR-based method. Pathogenicity of identified mutations was verified in vitro. Results: We found four novel heterozygous PAX8 mutations in the four probands: L16P, F20S, D46SfsX24, and R133Q. Family studies showed four additional mutation carriers, who were confirmed to have high serum TSH levels. Expression experiments revealed that three mutations (L16P, F20S, and R133Q) had defects in target DNA binding, while D46fs had protein instability that was rescued by the proteasome inhibitor MG132. All four mutations had reduced transactivation on the thyroglobulin promoter, supporting that they were inactivating mutations. Conclusion: D46fs is the first PAX8 mutation with confirmed protein instability. Our clinical and in vitro findings together suggest that pure PAX8 haploinsufficiency can cause CH in humans.