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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.

Abstract 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.

Insufficient thyroid hormone production in newborns is referred to as congenital hypothyroidism. Multinodular goiter (MNG), characterized by an enlarged thyroid gland with multiple nodules, is usually seen in adults and is recognized as a separate disorder from congenital hypothyroidism. Here we performed a linkage analysis of a family with both nongoitrous congenital hypothyroidism and MNG and identified a signal at 15q26.1. Follow-up analyses with whole-genome sequencing and genetic screening in congenital hypothyroidism and MNG cohorts showed that changes in a noncoding TTTG microsatellite on 15q26.1 were frequently observed in congenital hypothyroidism (137 in 989) and MNG (3 in 33) compared with controls (3 in 38,722). Characterization of the noncoding variants with epigenomic data and in vitro experiments suggested that the microsatellite is located in a thyroid-specific transcriptional repressor, and its activity is disrupted by the variants. Collectively, we presented genetic evidence linking nongoitrous congenital hypothyroidism and MNG, providing unique insights into thyroid abnormalities. Noncoding variants in a TTTG microsatellite on 15q26.1 are identified in Japanese patients with childhood and adult-onset thyroid abnormalities. Functional analyses suggest that these variants affect the role of the microsatellite as a potential regulator of thyroid cell growth.

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.

Membrane-less organelles (MLOs) are formed by biomolecular liquid–liquid phase separation (LLPS). Proteins with charged low-complexity domains (LCDs) are prone to phase separation and localize to MLOs, but the mechanism underlying the distributions of such proteins to specific MLOs remains poorly understood. Recently, proteins with Arg-enriched mixed-charge domains (R-MCDs), primarily composed of R and Asp (D), were found to accumulate in nuclear speckles via LLPS. However, the process by which R-MCDs selectively incorporate into nuclear speckles is unknown. Here, we demonstrate that the patterning of charged amino acids and net charge determines the targeting of specific MLOs, including nuclear speckles and the nucleolus, by proteins. The redistribution of R and D residues from an alternately sequenced pattern to uneven blocky sequences caused a shift in R-MCD distribution from nuclear speckles to the nucleolus. In addition, the incorporation of basic residues in the R-MCDs promoted their localization to the MLOs and their apparent accumulation in the nucleolus. The R-MCD peptide with alternating amino acids did not undergo LLPS, whereas the blocky R-MCD peptide underwent LLPS with affinity to RNA, acidic poly-Glu, and the acidic nucleolar protein nucleophosmin, suggesting that the clustering of R residues helps avoid their neutralization by D residues and eventually induces R-MCD migration to the nucleolus. Therefore, the distribution of proteins to nuclear speckles requires the proximal positioning of D and R for the mutual neutralization of their charges.

Somatic activating GNAS mutations cause McCune-Albright syndrome (MAS). Owing to low mutation abundance, mutant-specific enrichment procedures, such as the peptide nucleic acid (PNA) method, are required to detect mutations in peripheral blood. Next generation sequencing (NGS) can analyze millions of PCR amplicons independently, thus it is expected to detect low-abundance GNAS mutations quantitatively. In the present study, we aimed to develop an NGS-based method to detect low-abundance somatic GNAS mutations. PCR amplicons encompassing exons 8 and 9 of GNAS, in which most activating mutations occur, were sequenced on the MiSeq instrument. As expected, our NGS-based method could sequence the GNAS locus with very high read depth (approximately 100,000) and low error rate. A serial dilution study with use of cloned mutant and wildtype DNA samples showed a linear correlation between dilution and measured mutation abundance, indicating the reliability of quantification of the mutation. Using the serially diluted samples, the detection limits of three mutation detection methods (the PNA method, NGS, and combinatory use of PNA and NGS [PNA-NGS]) were determined. The lowest detectable mutation abundance was 1% for the PNA method, 0.03% for NGS and 0.01% for PNA-NGS. Finally, we analyzed 16 MAS patient-derived leukocytic DNA samples with the three methods, and compared the mutation detection rate of them. Mutation detection rate of the PNA method, NGS and PNA-NGS in 16 patient-derived peripheral blood samples were 56%, 63% and 75%, respectively. In conclusion, NGS can detect somatic activating GNAS mutations quantitatively and sensitively from peripheral blood samples. At present, the PNA-NGS method is likely the most sensitive method to detect low-abundance GNAS mutation.

GATA2 deficiency, a syndrome caused by heterozygous loss-of-function variants in the gene, is characterized by immunodeficiency, bone marrow failure, and predisposition to myeloid neoplasms. Its clinical presentation is highly variable, making early diagnosis challenging. Although GATA2 deficiency has been linked to systemic inflammation, gastrointestinal involvement mimicking inflammatory bowel disease (IBD) is extremely rare. This report presented the case of two adolescent boys with no family history of novel heterozygous frameshift variants. Notably, Patient 1 initially presented with clinical and endoscopic features strongly suggestive of Crohn's disease, including weight loss, perianal abscess, and characteristic intestinal ulcers, before developing acute myeloid leukemia with monosomy 7. This is a rare presentation of GATA2 deficiency manifesting initially with Crohn's disease-like symptoms. Patient 2 presented with intractable cutaneous warts and pancytopenia, later diagnosed as myelodysplastic syndrome with der(1;7)(q10;p10). Both patients harbored novel frameshift variants predicted to eliminate the DNA-binding domain, suggesting a loss-of-function mechanism. These cases expand the phenotypic spectrum of GATA2 deficiency and highlight that atypical IBD-like symptoms, including Crohn's disease-like presentations, may cause an initial manifestation. GATA2 deficiency should be considered in patients with IBD-like symptoms, refractory skin disorders, and hematological abnormalities. Early genetic testing and family screening are essential to ensuring timely diagnosis and curative hematopoietic stem cell transplantation before progression to advanced myeloid disease.

MIRAGE syndrome, a congenital multisystem disorder due to pathogenic SAMD9 variants, describes a constellation of clinical features including 46,XY disorders of sex development (DSD), small for gestational age (SGA) and adrenal insufficiency (AI). It is poorly understood whether SAMD9 variants underlie 46,XY DSD patients born SGA (46,XY DSD SGA) without AI. This study aimed to define the frequency and phenotype of SAMD9 variants in 46,XY DSD SGA without AI. Forty-nine Japanese patients with 46,XY DSD SGA (Quigley scale, 2 to 6; gestational age-matched birth weight percentile, <10) without history of AI were enrolled. The single coding exon of SAMD9 was PCR-amplified and sequenced for each patient. Pathogenicity of an identified variant was verified in vitro. Placenta tissues were obtained from the variant-carrying patient, as well as from another previously described patient, and were analyzed histologically. In one 46,XY DSD SGA patient, a novel heterozygous SAMD9 variant, p.Phe1017Val, was identified. Pathogenicity of the mutant was experimentally confirmed. In addition to DSD and SGA, the patient had neonatal thrombocytopenia, severe postnatal grow restriction, chronic diarrhea and susceptibility to infection, all features consistent with MIRAGE, leading to premature death at age 14 months. The patient did not have any manifestations or laboratory findings suggesting AI. Placenta tissues of the two variant-carrying patients were characterized by maldevelopment of distal villi without other findings of maternal underperfusion. MIRAGE syndrome is a rare cause of 46,XY DSD SGA without AI. This study exemplifies that AI is a common feature of MIRAGE syndrome but that the absence of AI should not rule out a diagnosis of the syndrome.

Abstract Disclosure: H. Iwasaki: None. H. Suwanai: None. K. Kanekura: None. N. Satoshi: None. F. Yakou: None. H. Sakai: None. K. Ishii: None. N. hara: None. R. Suzuki: None. 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 disulfide bonds play an important role in TPO activity. However, no study has comprehensively analyzed cysteine mutations that form disulfide bonds in TPO. In this study, we induced mutations in cysteine residues involved in the formation of disulfide bonds and analyzed their effect on subcellular localization, degradation, and enzyme activities to evaluate the importance of disulfide bonds in TPO. Methods: Vector plasmid TPO mutants C655F and C825R in CH patients were transfected into HEK293 cells. TPO activity and protein expression levels were measured by Amplex red assay and western blotting. The same procedure was performed in the presence of proteasome inhibitor MG132. Subcellular localization was determined by immunocytochemistry and flow cytometry. The location of all disulfide bonds within TPO was predicted by 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 to the wild-type (P < 0.05). In the presence of a proteasome inhibitor MG132, the protein expression level of TPO was increased to the level comparable with that of the wild-type but its activity did not. The degree of subcellular distribution of TPO to the cell surface in the mutant was lower than that in the wild-type TPO. Twenty-four cysteine residues were involved in the formation of 12 disulfide bonds in TPO, and 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. Presentation: 6/2/2024