Clinical characteristics and outcomes of immune-complex membranoproliferative glomerulonephritis and C3 glomerulopathy in Japanese children.

BACKGROUND: Membranoproliferative glomerulonephritis (MPGN) can be divided into immune-complex MPGN (IC-MPGN) and C3 glomerulopathy (C3G), which includes dense deposit disease (DDD) and C3 glomerulonephritis (C3GN). These conditions result from abnormalities in different complement pathways and may lead to different prognoses. However, there are limited studies describing the respective clinical courses. METHODS: In this study, Japanese pediatric patients diagnosed with MPGN based on kidney biopsies conducted between February 2002 and December 2022 were reclassified as having IC-MPGN or C3G (DDD or C3GN). We retrospectively analyzed the clinical characteristics and outcomes of these patients. RESULTS: Out of 25 patients with MPGN, three (12.0%) were diagnosed with DDD, 20 (80.0%) with C3GN, and two (8.0%) with IC-MPGN. There were 13 (65.0%) patients and one (33.3%) patient in remission after treatment for C3GN and DDD, respectively, and no patients with IC-MPGN achieved remission. The median follow-up period was 5.3 (2.5-8.9) years, and none of the patients in either group progressed to an estimated glomerular filtration rate < 15 ml/min/1.73 m2. Patients with C3GN presenting mild to moderate proteinuria (n = 8) received a renin-angiotensin system inhibitor (RAS-I) alone, and these patients exhibited a significant decrease in the urinary protein creatinine ratio and a notable increase in serum C3 levels at the last follow-up. CONCLUSIONS: Most patients with MPGN were diagnosed with C3GN. The remission rate for C3GN was high, and no patients developed kidney failure during the approximately 5-year follow-up. Additionally, patients with C3GN with mild to moderate proteinuria had good outcomes with RAS-I alone, but continued vigilance is necessary to determine long-term prognosis.

Current Status and Problems of Newborn Infection Management at a Regional Hospital in Japan.

PURPOSE: To clarify neonatal bacterial infection management in near term and term infants at a regional hospital in Japan. METHODS: Between 2018 and 2020, of 729 births, 236 patients who underwent blood examination at least twice by the age of 3 days, were included. Data from the medical records were analyzed retrospectively. RESULTS: Median gestational age was 39 weeks, with 116 boys (49.1%) and 202 vaginal deliveries (85.6%). There were 37 cases of maternal group B streptococcus, 24 cases of premature rupture of membranes for more than 24 hours (PROM group), and 107 cases of amniotic fluid turbidity at birth (AFT group). Comparing groups, C-reactive protein (CRP) was significantly lower in the cesarean section (C/S) group (median 0.22 mg/dL; p < 0.05), and higher in the AFT group (0.44 mg/dL; p < 0.05). There were 77 positive cultures, (p < 0.05). Antibiotics were administered more frequently in cesarean section (19 cases; p < 0.001) and less in the PROM group (2 cases; p < 0.01). CONCLUSIONS: There were no asymptomatic cases of CRP >2 mg/dL, and no cases of severe sepsis in normal neonatal deliveries. CRP levels were elevated in the AFT group, where culture was positive, but few antibiotics were administered. In the C/S group, antibiotics were administered if respiratory symptoms occurred unless the CRP level was high. Further, all patients in PROM group who received antibiotics had any symptoms, suggesting that routine blood tests may not be necessary.

Endothelial VEGFR2-PLCγ signaling regulates vascular permeability and antitumor immunity through eNOS/Src.

Endothelial phospholipase Cγ (PLCγ) is essential for vascular development; however, its role in healthy, mature, or pathological vessels is unexplored. Here, we show that PLCγ was prominently expressed in vessels of several human cancer forms, notably in renal cell carcinoma (RCC). High PLCγ expression in clear cell RCC correlated with angiogenic activity and poor prognosis, while low expression correlated with immune cell activation. PLCγ was induced downstream of vascular endothelial growth factor receptor 2 (VEGFR2) phosphosite Y1173 (pY1173). Heterozygous Vegfr2Y1173F/+ mice or mice lacking endothelial PLCγ (Plcg1iECKO) exhibited a stabilized endothelial barrier and diminished vascular leakage. Barrier stabilization was accompanied by decreased expression of immunosuppressive cytokines, reduced infiltration of B cells, helper T cells and regulatory T cells, and improved response to chemo- and immunotherapy. Mechanistically, pY1173/PLCγ signaling induced Ca2+/protein kinase C-dependent activation of endothelial nitric oxide synthase (eNOS), required for tyrosine nitration and activation of Src. Src-induced phosphorylation of VE-cadherin at Y685 was accompanied by disintegration of endothelial junctions. This pY1173/PLCγ/eNOS/Src pathway was detected in both healthy and tumor vessels in Vegfr2Y1173F/+ mice, which displayed decreased activation of PLCγ and eNOS and suppressed vascular leakage. Thus, we believe that we have identified a clinically relevant endothelial PLCγ pathway downstream of VEGFR2 pY1173, which destabilizes the endothelial barrier and results in loss of antitumor immunity.

Management of children with obesity at local hospital and impact of COVID-19 pandemic.

This study investigated the status of children with obesity before and after the COVID-19 pandemic, and the effects of lifestyle guidance on weight loss among children in Japan. We analysed the data of patients who visited our hospital after check-ups for obesity and evaluated the efficacy of lifestyle guidance. The patients were divided into groups A, B, and C (year 2011, 2019, and 2021, respectively). There were no differences in body weight, obesity index (OI), blood pressure, or alanine transaminase (ALT) levels between the groups; however, aspartate transaminase (AST) level was the highest in Group C. In Group C, only OI increased between the primary and secondary screenings; however, OI and body mass index (BMI) improved during the second screening and more children in the weight loss group followed lifestyle guidance. OI/BMI did not change over the past decade; however, short-term weight gain was significant owing to the COVID-19 pandemic, and simple guidance was effective in reducing weight. Future challenges include identifying methods to achieve long-term weight loss.

Long-term outcome of combination therapy with corticosteroids, mizoribine and RAS inhibitors as initial therapy for severe childhood IgA vasculitis with nephritis.

BACKGROUND: Patients with severe IgA vasculitis with nephritis (IgAVN) typically receive aggressive therapy as an initial approach. We have consistently performed combination therapy including corticosteroids and immunosuppressants as initial therapy for severe IgAVN over a 20-year-plus period, with only minor changes to the treatment protocol. This study seeks to reveal the efficacy of combination therapy for severe IgAVN. METHODS: We retrospectively studied 50 Japanese children diagnosed between 1996 and 2019 with clinicopathologically severe IgAVN who were defined as ISKDC classification grade IIIb-V and/or serum albumin < 2.5 g/dL. RESULTS: The median age at the onset of IgAVN was 8.0 years (IQR: 6.0-10.0). At biopsy, 44% of patients had nephrotic syndrome and 14% had kidney dysfunction. All patients were treated with combination therapy after biopsy. Abnormal proteinuria resolved after initial therapy in all 50 patients. However, eight patients (16%) had recurrence of proteinuria. Abnormal proteinuria was again resolved in three of these patients with additional treatment. At the last follow-up (median 59.5 months; IQR, 26.2-84.2), the median urine protein-to-creatine ratio was 0.08 g/gCr (IQR, 0.05-0.15), and only one patient had kidney dysfunction. CONCLUSIONS: Combination therapy provided good kidney outcomes for Japanese children with severe IgAVN. Even including recurrent cases, the degree of proteinuria was slight, and kidney function was good at the last follow-up. A higher resolution version of the Graphical abstract is available as Supplementary information.

Clinical and pathological investigation of oligomeganephronia.

BACKGROUND: Oligomeganephronia (OMN) is a rare congenital anomaly involving the kidney and urinary tract, characterized by decreased number and compensatory hypertrophy of the nephron. It is caused by abnormal kidney development during the embryonic period, especially in patients with low birth weight; however, the actual etiology and clinical features remain unknown. We aim to reveal the clinical and pathological characteristics, treatment, and outcome. METHODS: Ten patients diagnosed with OMN between 2013 and 2020 were retrospectively investigated. The data were presented as the median ± interquartile range, and statistical significance was set at p < 0.05. RESULTS: The age at diagnosis was 14.1 years, the male-to-female ratio was 6:4, and only four cases were born with low birth weight. The estimated glomerular filtration rate (eGFR) was 62.2 mL/min/1.73 m2. The glomerulus diameter of OMN patients was significantly larger (217 vs. 154 µm, p < 0.001) in OMN patients, and the number of glomeruli of OMN patients was lower (0.89 vs. 2.05/mm2, p < 0.001) than the control group. Eight of the ten cases were identified by urinary screening. Nine patients were treated with renin-angiotensin system (RAS) inhibitors, following which proteinuria successfully decreased or disappeared. Their median eGFR was also stable, 53.3 mL/min/1.73 m2. CONCLUSIONS: As few symptoms can lead to OMN discovery, most patients were found during urine screening at school. Kidney dysfunction was observed in all patients at the time of kidney biopsy. Proteinuria has been significantly reduced and the decline rate of eGFR might be improved by RAS inhibitors. "A higher resolution version of the Graphical abstract is available as Supplementary information".

Early predictive factors for progression to kidney failure in infants with severe congenital anomalies of the kidney and urinary tract.

BACKGROUND: Severe congenital anomalies of the kidney and urinary tract (CAKUT) progress to infantile kidney failure with replacement therapy (KFRT). Although prompt and precise prediction of kidney outcomes is important, early predictive factors for its progression remain incompletely defined. METHODS: This retrospective cohort study included patients with CAKUT treated at 12 centers between 2009 and 2020. Patients with a maximum serum creatinine level ≤ 1.0 mg/dL during the first 3 days, patients who died of respiratory failure during the neonatal period, patients who progressed to KFRT within the first 3 days, and patients lacking sufficient data were excluded. RESULTS: Of 2187 patients with CAKUT, 92 were finally analyzed. Twenty-five patients (27%) progressed to KFRT and 24 (26%) had stage 3-5 chronic kidney disease without replacement therapy during the median observation period of 52.0 (interquartile range, 22.0-87.8) months. Among these, 22 (24%) progressed to infantile KFRT. The kidney survival rate during the infantile period was significantly lower in patients with a maximum serum creatinine level during the first 3 days (Cr-day3-max) ≥ 2.5 mg/dL (21.8%) compared with those with a Cr-day3-max < 2.5 mg/dL (95.2%) (log-rank, P < 0.001). Multivariate analysis demonstrated Cr-day3-max (P < 0.001) and oligohydramnios (P = 0.025) were associated with higher risk of infantile KFRT. Eighty-two patients (89%) were alive at the last follow-up. CONCLUSIONS: Neonatal kidney function, including Cr-day3-max, was associated with kidney outcomes in patients with severe CAKUT. Aggressive therapy for severe CAKUT may have good long-term life outcomes through infantile dialysis and kidney transplantation. A higher resolution version of the Graphical abstract is available as Supplementary information.

Evaluation of Suspected Autosomal Alport Syndrome Synonymous Variants.

Background: Alport syndrome is an inherited disorder characterized by progressive renal disease, variable sensorineural hearing loss, and ocular abnormalities. Although many pathogenic variants in COL4A3 and COL4A4 have been identified in patients with autosomal Alport syndrome, synonymous mutations in these genes have rarely been identified. Methods: We conducted in silico splicing analysis using Human Splicing Finder (HSF) and Alamut to predict splicing domain strength and disruption of the sites. Furthermore, we performed in vitro splicing assays using minigene constructs and mRNA analysis of patient samples to determine the pathogenicity of four synonymous variants detected in four patients with suspected autosomal dominant Alport syndrome (COL4A3 [c.693G>A (p.Val231=)] and COL4A4 [c.1353C>T (p.Gly451=), c.735G>A (p.Pro245=), and c.870G>A (p.Lys290=)]). Results: Both in vivo and in vitro splicing assays showed exon skipping in two out of the four synonymous variants identified (c.735G>A and c.870G>A in COL4A4). Prediction analysis of wild-type and mutated COL4A4 sequences using HSF and Alamut suggested these two variants may lead to the loss of binding sites for several splicing factors, e.g., in acceptor sites and exonic splicing enhancers. The other two variants did not induce aberrant splicing. Conclusions: This study highlights the pitfalls of classifying the functional consequences of variants by a simple approach. Certain synonymous variants, although they do not alter the amino acid sequence of the encoded protein, can dramatically affect pre-mRNA splicing, as shown in two of our patients. Our findings indicate that transcript analysis should be carried out to evaluate synonymous variants detected in patients with autosomal dominant Alport syndrome.

Efficacy of combination therapy for childhood complicated focal IgA nephropathy.

BACKGROUND: Patients with immunoglobulin A nephropathy who present with focal mesangial proliferation (focal IgAN) can have a relatively good prognosis, and renin-angiotensin system inhibitor (RAS-i) is commonly used as the initial treatment. However, there are some complicated focal IgAN cases with resistance to RAS-i treatment or nephrotic-range proteinuria. Thus, combination therapy including corticosteroids is often used. This study aimed to evaluate the efficacy of combination therapy for complicated focal IgAN cases by comparing to diffuse mesangial proliferation (diffuse IgAN). METHODS: We conducted a multicenter retrospective study on 88 children who received 2-year combination therapy. The participants were classified based on pathological severity: focal IgAN (n = 26) and diffuse IgAN (n = 62). RESULTS: In total, 26 patients with focal IgAN and 52 with diffuse IgAN achieved proteinuria disappearance within 2 years (100 vs. 83.9%, P = 0.03). Moreover, the time to proteinuria disappearance was significantly shorter in the focal IgAN group than in the diffuse IgAN group (2.9 vs. 4.2 months, P < 0.01) and all patients with focal IgAN achieved proteinuria disappearance within 8 months. At the last observation (8.6 vs. 10.4 years, P = 0.13), only patients with diffuse IgAN (n = 12) had greater than stage 2 chronic kidney disease. In terms of irreversible adverse events, one patient exhibited cataracts. CONCLUSION: Combination therapy was significantly effective in patients with complicated focal IgAN. Moreover, the long-term prognosis was good, and the duration of combination therapy for complicated focal IgAN can be decreased to reduce adverse events.

Use of renin-angiotensin system inhibitors as initial therapy in children with Henoch-Schönlein purpura nephritis of moderate severity.

BACKGROUND: Cases of Henoch-Schönlein purpura nephritis (HSPN) with moderate severity were demonstrated to achieve good prognosis after treatment with renin-angiotensin system (RAS) inhibitors. However, some patients required additional treatment for recurrence after remission. This study aimed to clarify the effect of RAS inhibitors in HSPN cases with moderate severity, including the proportion of cases with recurrence and their response to additional treatment. METHODS: Among 126 patients diagnosed with HSPN between 1996 and 2019, 71 patients with clinicopathologically diagnosed HSPN of moderate severity, defined as ISKDC grade II-IIIa and serum albumin ≥ 2.5 g/dL, were investigated. RESULTS: Proteinuria became negative after RAS inhibitor treatment alone in all 71 cases. However, 16 (22.5%) had recurrence. Eleven recurrent cases achieved negative proteinuria again following additional treatment. At the last follow-up (median 46.5 months; IQR, 23.2-98.2), 5 patients had persistent mild proteinuria; no patients had estimated glomerular filtration rate < 90 mL/min/1.73 m2. The pathological findings in all recurrent cases were ISKDC grade IIIa. The 16 recurrent cases had significantly higher proportions of glomeruli with global/segmental sclerosis (25.0 vs. 0%, P < 0.001) and tubular atrophy/interstitial fibrosis (37.5 vs. 12.7%, P =0.0 24) than 55 cases without recurrence. CONCLUSIONS: Japanese childhood HSPN cases with moderate severity had good outcomes without need for corticosteroids or immunosuppressants, when prescribed RAS inhibitor treatment. Even in recurrent cases, abnormal proteinuria was transient, and prognosis was excellent. A higher resolution version of the Graphical abstract is available as Supplementary information.

Mycophenolate Mofetil after Rituximab for Childhood-Onset Complicated Frequently-Relapsing or Steroid-Dependent Nephrotic Syndrome.

BACKGROUND: Rituximab is the standard therapy for childhood-onset complicated frequently relapsing or steroid-dependent nephrotic syndrome (FRNS/SDNS). However, most patients redevelop FRNS/SDNS after peripheral B cell recovery. METHODS: We conducted a multicenter, randomized, double-blind, placebo-controlled trial to examine whether mycophenolate mofetil (MMF) administration after rituximab can prevent treatment failure (FRNS, SDNS, steroid resistance, or use of immunosuppressive agents or rituximab). In total, 39 patients (per group) were treated with rituximab, followed by either MMF or placebo until day 505 (treatment period). The primary outcome was time to treatment failure (TTF) throughout the treatment and follow-up periods (until day 505 for the last enrolled patient). RESULTS: TTFs were clinically but not statistically significantly longer among patients given MMF after rituximab than among patients receiving rituximab monotherapy (median, 784.0 versus 472.5 days, hazard ratio [HR], 0.59; 95% confidence interval [95% CI], 0.34 to 1.05, log-rank test: P=0.07). Because most patients in the MMF group presented with treatment failure after MMF discontinuation, we performed a post-hoc analysis limited to the treatment period and found that MMF after rituximab prolonged the TTF and decreased the risk of treatment failure by 80% (HR, 0.20; 95% CI, 0.08 to 0.50). Moreover, MMF after rituximab reduced the relapse rate and daily steroid dose during the treatment period by 74% and 57%, respectively. The frequency and severity of adverse events were similar in both groups. CONCLUSIONS: Administration of MMF after rituximab may sufficiently prevent the development of treatment failure and is well tolerated, although the relapse-preventing effect disappears after MMF discontinuation.

Epidemiological impact of universal varicella vaccination on consecutive emergency department visits for varicella and its economic impact among children in Kobe City, Japan.

INTRODUCTION: Previous studies reported a dramatic decline in the incidence of varicella and varicella-related deaths after implementing universal varicella vaccination (VarV). Although previous studies reported the effectiveness and economic impact of VarV, they were unknown in the emergency department (ED) setting. METHODS: To determine the effectiveness and economic impact of VarV in the ED, Kobe, Japan, we retrospectively reviewed the clinical database of consecutive patients younger than 16 years presenting to our primary ED from 2011 to 2019. RESULTS: Of the 265,191 children presenting to our ED, 3,092 patients were clinically diagnosed with varicella. The number of patients with varicella was approximately 500 annually, before introducing the universal two-dose VarV for children aged 1 to <3 years in October 2014, in the Japanese national immunization program, and decreased to approximately 200 in 2019. The number of patients with varicella younger than 1 year (ineligible for the vaccination) also decreased. Regarding the economic impact, the medical cost in our ED reduced after the introduction of VarV was JPY 4.1 million (US$ 40,049) annually. From the central data, approximately 95% of children were vaccinated after October 2014; however, a relatively large percentage of infected unvaccinated children (59.0%) presented to ED in this study. After the implementation of the universal VarV, infection was mainly observed in older children (i.e., the unvaccinated generation). CONCLUSIONS: Our data showed the effectiveness and economic impact of VarV in the ED setting. Additionally, our data suggested that the public vaccination program should include older unvaccinated children and other unvaccinated individuals.

Clinical features of autosomal recessive polycystic kidney disease in the Japanese population and analysis of splicing in PKHD1 gene for determination of phenotypes.

BACKGROUND: Autosomal recessive polycystic kidney disease (ARPKD) is caused by mutations in the PKHD1 gene. The clinical spectrum is often more variable than previously considered. We aimed to analyze the clinical features of genetically diagnosed ARPKD in the Japanese population. METHODS: We conducted a genetic analysis of patients with clinically diagnosed or suspected ARPKD in Japan. Moreover, we performed a minigene assay to elucidate the mechanisms that could affect phenotypes. RESULTS: PKHD1 pathogenic variants were identified in 32 patients (0-46 years). Approximately one-third of the patients showed prenatal anomalies, and five patients died within one year after birth. Other manifestations were detected as follows: chronic kidney disease stages 1-2 in 15/26 (57.7%), Caroli disease in 9/32 (28.1%), hepatic fibrosis in 7/32 (21.9%), systemic hypertension in 13/27 (48.1%), and congenital hypothyroidism in 3 patients. There have been reported that truncating mutations in both alleles led to severe phenotypes with perinatal demise. However, one patient without a missense mutation survived the neonatal period. In the minigene assay, c.2713C > T (p.Gln905Ter) and c.6808 + 1G > A expressed a transcript that skipped exon 25 (123 bp) and exon 41 (126 bp), resulting in an in-frame mutation, which might have contributed to the milder phenotype. Missense mutations in cases of neonatal demise did not show splicing abnormalities. CONCLUSION: Clinical manifestations ranged from cases of neonatal demise to those diagnosed in adulthood. The minigene assay results indicate the importance of functional analysis, and call into question the fundamental belief that at least one non-truncating mutation is necessary for perinatal survival.

Identification of novel OCRL isoforms associated with phenotypic differences between Dent disease-2 and Lowe syndrome.

BACKGROUND: Although Lowe syndrome and Dent disease-2 are caused by Oculocerebrorenal syndrome of Lowe (OCRL) mutations, their clinical severities differ substantially and their molecular mechanisms remain unclear. Truncating mutations in OCRL exons 1-7 lead to Dent disease-2, whereas those in exons 8-24 lead to Lowe syndrome. Herein we identified the mechanism underlying the action of novel OCRL protein isoforms. METHODS: Messenger RNA samples extracted from cultured urine-derived cells from a healthy control and a Dent disease-2 patient were examined to detect the 5' end of the OCRL isoform. For protein expression and functional analysis, vectors containing the full-length OCRL transcripts, the isoform transcripts and transcripts with truncating mutations detected in Lowe syndrome and Dent disease-2 patients were transfected into HeLa cells. RESULTS: We successfully cloned the novel isoform transcripts from OCRL exons 6-24, including the translation-initiation codons present in exon 8. In vitro protein-expression analysis detected proteins of two different sizes (105 and 80 kDa) translated from full-length OCRL, whereas only one protein (80 kDa) was found from the isoform and Dent disease-2 variants. No protein expression was observed for the Lowe syndrome variants. The isoform enzyme activity was equivalent to that of full-length OCRL; the Dent disease-2 variants retained >50% enzyme activity, whereas the Lowe syndrome variants retained <20% activity. CONCLUSIONS: We elucidated the molecular mechanism underlying the two different phenotypes in OCRL-related diseases; the functional OCRL isoform translated starting at exon 8 was associated with this mechanism.

Impact of the State of Emergency during the COVID-19 Pandemic in 2020 on Asthma Exacerbations among Children in Kobe City, Japan.

The coronavirus disease (COVID-19) pandemic altered environmental factors. We studied the impact of these changes on asthma exacerbation (AE) by comparing the AE-related environmental factors between COVID-19 (2020) and pre-COVID-19 (2011-2019) eras. Between 2011 and 2020, 278,465 children (<16 years old) visited our emergency department, and 7476 were diagnosed with AE. The number of patients showed spring and fall peaks in 2011-2019. Multivariate analyses showed significant positive relationships of the number of AE patients with the average temperature among all patients and 0-5-year-olds and with sulfur dioxide (SO2) levels in 2011-2019 among 0-5-year-olds. Although the spring peak in the number of patients was not observed in 2020 after declaration of a state of emergency, the fall peak was again observed after the state of emergency was lifted. No changes in average temperature were detected, but SO2 was significantly reduced following declaration of the state of emergency in 2020. Therefore, SO2 reduction might have contributed to the disappearance of the peak of AE. However, a fall peak was observed again in 2020, although SO2 levels continued to be low. These data suggest that person to person interaction seems to be associated with AE, presumably due to unknown viral infections.

Current management for foreign body and toxic agent ingestion in a paediatric primary emergency centre.

OBJECTIVES: Accidental foreign body ingestion (FBI) and toxic agent ingestion (TAI) are commonly encountered among children in primary emergency settings. Early detection and appropriate medical intervention are crucial to improve outcomes. Although many reports from tertiary institutions have shown improvements in therapy, data are still lacking from primary emergency facilities. METHODS: We performed a retrospective analysis based on medical records of FBI/TAI over 4 years at the Kobe Children's Primary Emergency Medical Center. We collected patient information, including age, sex, time between FBI/TAI occurrence and centre visit, provision of first aid, symptoms, type of FBI/TAI, examinations, treatments, and outcomes. RESULTS: A total of 580 children were enrolled. The median age was 1.3 years, and patients under 2 years old accounted for 70% of total cases. Cigarettes (17.5%) were the most common ingested foreign body, followed by medicines (15.3%), detergents (8.1%), in TAI, plastics (14.1%), metal (13.4%), batteries (9.0%) in FBI, and others (22.6%). A total of 42 patients were transferred to advanced hospitals; among these, 22 patients were hospitalised but the foreign body was removed in only 3 (0.9%) patients. Transferred patients were significantly older (P<0.05) in FBI and had a higher rate of any of symptoms (P<0.05) in FBI/TAI. CONCLUSIONS: This large-scale retrospective study of accidental FBI/TAI conducted at a primary emergency facility clarified current management, including treatment at a primary facility. Very few cases of FBI/TAI were treated, even when they were transferred to an advanced treatment hospital. Unified protocols should be established, to improve the management of FBI/TAI.

Examination of the predicted prevalence of Gitelman syndrome by ethnicity based on genome databases.

Gitelman syndrome is an autosomal recessive inherited salt-losing tubulopathy. It has a prevalence of around 1 in 40,000 people, and heterozygous carriers are estimated at approximately 1%, although the exact prevalence is unknown. We estimated the predicted prevalence of Gitelman syndrome based on multiple genome databases, HGVD and jMorp for the Japanese population and gnomAD for other ethnicities, and included all 274 pathogenic missense or nonsense variants registered in HGMD Professional. The frequencies of all these alleles were summed to calculate the total variant allele frequency in SLC12A3. The carrier frequency and the disease prevalence were assumed to be twice and the square of the total allele frequency, respectively, according to the Hardy-Weinberg principle. In the Japanese population, the total carrier frequencies were 0.0948 (9.5%) and 0.0868 (8.7%) and the calculated prevalence was 0.00225 (2.3 in 1000 people) and 0.00188 (1.9 in 1000 people) in HGVD and jMorp, respectively. Other ethnicities showed a prevalence varying from 0.000012 to 0.00083. These findings indicate that the prevalence of Gitelman syndrome in the Japanese population is higher than expected and that some other ethnicities also have a higher prevalence than has previously been considered.

Genotype-Phenotype Correlation in WT1 Exon 8 to 9 Missense Variants.

INTRODUCTION: WT1 missense mutation in exon 8 or 9 causes infantile nephrotic syndrome with early progression to end-stage kidney disease (ESKD), Wilms tumor, and 46,XY female. However, some patients with missense mutations in exon 8 or 9 progress to ESKD in their teens or later. Therefore, we conducted a systematic review and functional analysis of WT1 transcriptional activity. METHODS: We conducted a systematic review of 174 cases with WT1 exon 8 or 9 missense variants from our cohort (n=13) and previous reports (n=161). Of these cases, mild and severe genotypes were selected for further in vitro functional analysis using luciferase assay. RESULTS: The median age of developing ESKD was 1.17 years. A comparative study was conducted among three WT1 genotype classes: mutations of the DNA-binding site (DBS group), mutations outside the DNA-binding site but at sites important for zinc finger structure formation by 2 cysteines and 2 histidines (C2H2 group), and mutations leading to other amino acid changes (Others group). The DBS group showed the severest phenotype and the C2H2 group was intermediate, whereas the Others group showed the mildest phenotype (developing ESKD at 0.90, 2.00, and 3.92 years, respectively, with significant differences). In vitro functional analysis showed dominant-negative effects for all variants; in addition, the DBS and C2H2 mutations were associated with significantly lower WT1 transcriptional activity than the other mutations. CONCLUSION: Not only the DNA-binding site but also C2H2 zinc finger structure sites are important for maintaining WT1 transcriptional activity, and their mutation causes severe clinical symptoms.

X-chromosome inactivation patterns in females with Fabry disease examined by both ultra-deep RNA sequencing and methylation-dependent assay.

BACKGROUND: Fabry disease is an X-linked inherited lysosomal storage disorder caused by mutations in the gene encoding α-galactosidase A. Males are usually severely affected, while females have a wide range of disease severity. This variability has been assumed to be derived from organ-dependent skewed X-chromosome inactivation (XCI) patterns in each female patient. Previous studies examined this correlation using the classical methylation-dependent method; however, conflicting results were obtained. This study was established to ascertain the existence of skewed XCI in nine females with heterozygous pathogenic variants in the GLA gene and its relationship to the phenotypes. METHODS: We present five female patients from one family and four individual female patients with Fabry disease. In all cases, heterozygous pathogenic variants in the GLA gene were detected. The X-chromosome inactivation patterns in peripheral blood leukocytes and cells of urine sediment were determined by both classical methylation-dependent HUMARA assay and ultra-deep RNA sequencing. Fabry Stabilization Index was used to determine the clinical severity. RESULTS: Skewed XCI resulting in predominant inactivation of the normal allele was observed only in one individual case with low ⍺-galactosidase A activity. In the remaining cases, no skewing was observed, even in the case with the highest total severity score (99.2%). CONCLUSION: We conclude that skewed XCI could not explain the severity of female Fabry disease and is not the main factor in the onset of various clinical symptoms in females with Fabry disease.