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Elrika A Wijaya, Purboyo Solek, Dzulfikar DL Hakim, Rini Rossanti, Ahmedz Widiasta, Dany Hilmanto Department of Child Health, Faculty of Medicine, Universitas Padjadjaran/Dr. Hasan Sadikin General Hospital, Bandung, West Java, IndonesiaCorrespondence: Dany Hilmanto, Tel +628122266879, Email danyhilmanto@yahoo.comPurpose: In children, chronic kidney disease (CKD) has been known to affect neurocognitive function which can impair the quality of life. This study aims to determine the factors and treatment modalities which might affect neurocognitive function in pediatric population with CKD.Patients and Methods: A systematic review was done using 3 electronic databases: PubMed, ScienceDirect, SpringerLink, and carried out based on PRISMA guidelines. Our review included articles published in the last 10 years (2011– 2021) in English, on children aged 0– 18 years with CKD. Factors affecting the children’s neurocognitive function were assessed.Results: Eight articles were included in this study. Three articles reported that parent’s education, especially maternal education affect the neurocognitive function of children with CKD. In relation with modalities, in general, children with CKD who had kidney transplant had a better neurocognitive outcome. A longer duration of hemodialysis (HD) was associated with poorer neurocognitive outcomes. Other factors that can affect the neurocognitive function included depression, a history of abnormal births, seizures, and hypertension.Conclusion: In children, CKD might cause neurocognitive function disorders through various complex and interconnected mechanisms. Further studies are needed to determine the mechanism and prevention of neurocognitive disorders, as well as the best choice of therapeutic modality to improve both kidney function and neurocognitive function in children with CKD.Keywords: chronic kidney disease, neurocognitive function, children

With breakthroughs in Natural Language Processing and Artificial Intelligence (AI), the usage of Large Language Models (LLMs) in academic research has increased tremendously. Models such as Generative Pre-trained Transformer (GPT) are used by researchers in literature review, abstract screening, and manuscript drafting. However, these models also present the attendant challenge of providing ethically questionable scientific information. Our study provides a snapshot of global researchers' perception of current trends and future impacts of LLMs in research. Using a cross-sectional design, we surveyed 226 medical and paramedical researchers from 59 countries across 65 specialties, trained in the Global Clinical Scholars' Research Training certificate program of Harvard Medical School between 2020 and 2024. Majority (57.5%) of these participants practiced in an academic setting with a median of 7 (2,18) PubMed Indexed published articles. 198 respondents (87.6%) were aware of LLMs and those who were aware had higher number of publications (p < 0.001). 18.7% of the respondents who were aware (n = 37) had previously used LLMs in publications especially for grammatical errors and formatting (64.9%); however, most (40.5%) did not acknowledge its use in their papers. 50.8% of aware respondents (n = 95) predicted an overall positive future impact of LLMs while 32.6% were unsure of its scope. 52% of aware respondents (n = 102) believed that LLMs would have a major impact in areas such as grammatical errors and formatting (66.3%), revision and editing (57.2%), writing (57.2%) and literature review (54.2%). 58.1% of aware respondents were opined that journals should allow for use of AI in research and 78.3% believed that regulations should be put in place to avoid its abuse. Seeing the perception of researchers towards LLMs and the significant association between awareness of LLMs and number of published works, we emphasize the importance of developing comprehensive guidelines and ethical framework to govern the use of AI in academic research and address the current challenges., Competing Interests: Declarations. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

The incidence of nephrolithiasis in children ranges from 5 to 10% in developing countries. Etiology of nephrolithiasis in children remains largely unknown, so metabolic evaluation is indicated in all case, because of potential morbidity and recurrence. We report a case of 2,8-Dihydroxyadenine nephrolithiasis present as bilateral staghorn stone in 11 years old boy with chronic kidney disease. 2,8-Dihydroxyadenine nephrolithiasis is the result of a metabolic abnormality due to the deficiency of the enzyme adenine phosphoribosyltransferase (APRT), it is not only promote stone formation, but also induced nephropathy. Early diagnosis ensure appropriate treatment and favorable prognosis for kidney function and stone management., Competing Interests: The authors report no declarations of interest., (© 2023 The Authors.)

Gitelman syndrome (GS) is a rare, autosomal recessive, salt-losing tubulopathy caused by loss of function in the SLC12A3 gene (NM&#95;000339.2), which encodes the natrium chloride cotransporter. The detection of homozygous or compound heterozygous SLC12A3 variants is expected in GS, but 18%-40% of patients with clinical GS carry only one mutant allele. Previous reports identified some pathogenic deep intronic variants in SLC12A3. Here, we report the screening of SLC12A3 deep intronic variants in 13 patients with suspected GS carrying one mutated SLC12A3 allele. Variant screening used the HaloPlex Target Enrichment System Kit capturing whole introns and the promotor region of SLC12A3, followed by SureCall variant analysis. Rare intronic variants (<1% frequency) were identified, and pathogenicity evaluated by the minigene system. Deep intronic variant screening detected seven rare SLC12A3 variants from six patients. Only one variant showed pathogenicity in the minigene system (c.602-16G>A, intron 4) through activation of a cryptic acceptor site. No variants were detected in the promotor region. Deep intronic screening identified only one pathogenic variant in patients with suspected GS carrying monoallelic SLC12A3 variants. Our results suggest that deep intronic variants partially explain the cause of monoallelic variants in patients with GS., (© 2022 Wiley Periodicals LLC.)

Nephronophthisis is an autosomal-recessive kidney disease that is caused by abnormalities in primary cilia. Nephronophthisis-related ciliopathies (NPHP-RCs) are a common cause of end-stage kidney disease (ESKD) in children and adolescents. NPHP-RCs are often accompanied by extrarenal manifestations, including intellectual disability, retinitis pigmentosa, or polydactyly. Although more than 100 causative genes have been identified, its diagnosis is difficult because the clinical features of each mutation often overlap. From September 2010 to August 2021, we performed genetic analysis, including next-generation sequencing (NGS), in 574 probands with kidney dysfunction and retrospectively studied cases genetically diagnosed with NPHP-RCs. RESULTS: We detected mutations related to NPHP-RCs in 93 patients from 83 families. Members of 60 families were diagnosed using NGS, and the mutations and the corresponding number of families are as follows: NPHP1 (24), NPHP3 (10), OFD1 (7), WDR35 (5), SDCCAG8 (4), BBS10 (3), TMEM67 (3), WDR19 (3), BBS1 (2), BBS2 (2), IFT122 (2), IFT140 (2), IQCB1 (2), MKKS (2), SCLT1 (2), TTC21B (2), ALMS1 (1), ANKS6 (1), BBS4 (1), BBS12 (1), CC2D2A (1), DYNC2H1 (1), IFT172 (1), and MAPKBP1 (1). A total of 39 cases (41.9%) progressed to ESKD at the time of genetic analysis, whereas 58 cases (62.3%) showed extrarenal manifestations, the most common being developmental delay, intellectual disability, and autism spectrum disorder in 44 patients. Comprehensive genetic analysis using NGS is useful for diagnosing patients with NPHP-RCs., (© 2022. The Author(s), under exclusive licence to The Japan Society of Human Genetics.)

Background: Approximately 30% of children with steroid-resistant nephrotic syndrome (SRNS) have causative monogenic variants. SRNS represents glomerular disease resulting from various etiologies, which lead to similar patterns of glomerular damage. Patients with SRNS mainly exhibit focal segmental glomerulosclerosis (FSGS). There is limited information regarding associations between histologic variants of FSGS (diagnosed using on the Columbia classification) and monogenic variant detection rates or clinical characteristics. Here, we report FSGS characteristics in a large population of affected patients., Methods: This retrospective study included 119 patients with FSGS, diagnosed using the Columbia classification; all had been referred to our hospital for genetic testing from 2016 to 2021. We conducted comprehensive gene screening of all patients using a targeted next-generation sequencing panel that included 62 podocyte-related genes. Data regarding patients' clinical characteristics and pathologic findings were obtained from referring clinicians. We analyzed the associations of histologic variants with clinical characteristics, kidney survival, and gene variant detection rates., Results: The distribution of histologic variants according to the Columbia classification was 45% ( n =53) FSGS not otherwise specified, 21% ( n =25) cellular, 15% ( n =18) perihilar, 13% ( n =16) collapsing, and 6% ( n =7) tip. The median age at end stage kidney disease onset was 37 years; there were no differences in onset age among variants. We detected monogenic disease-causing variants involving 12 of the screened podocyte-related genes in 34% (40 of 119) of patients. The most common genes were WT1 (23%), INF2 (20%), TRPC6 (20%), and ACTN4 (10%). The perihilar and tip variants had the strongest and weakest associations with detection of monogenic variants (83% and 0%, respectively; P <0.001)., Conclusions: We revealed the distributions of histologic variants of genetic FSGS and nongenetic FSGS in a large patient population. Detailed data concerning gene variants and pathologic findings are important for understanding the etiology of FSGS., Competing Interests: R. Hamada reports research funding from Chugai Pharmaceutical Co., Ltd., Kyowa Kirin Co., Ltd., Takeda Pharmaceutical Co., and Teijin Pharma Ltd.; and honoraria from Alexion Pharmaceuticals, Inc., Asahi Kasei Pharma Corporation, Chugai Pharmaceutical Co., Ltd., and Teijin Pharma Ltd. T. Horinouchi reports research funding from Otsuka Pharmaceutical Co., Ltd. K. Iijima reports consultancy for JCR Pharmaceuticals Co., Ltd., Kyowa Hakko Kirin Co., Ltd., Ono Pharmaceutical Co., Ltd., Sanofi K.K., Takeda Pharmaceutical Co., Ltd., and Zenyaku Kogyo Co., Ltd.; research funding from Air Water Medical, Inc., Astellas Pharma, Inc., Eisai Co., Ltd., JCR Pharmaceutical Co., Ltd., Mochida Pharmaceutical Co., Ltd., Nihon Pharmaceutical Co., Ltd., Otsuka Pharmaceutical Co., Ltd., Shionogi & Co., Ltd., and Zenyaku Kogyo Co., Ltd.; honoraria from Astellas Pharma, Inc., Chugai Pharmaceutical Co., Ltd., Integrated Development Associates Co., Ltd., Kyowa Hakko Kirin Co., Ltd., Shionogi & Co., Ltd., and Zenyaku Kogyo Co., Ltd.; a patent application on the development of antisense nucleotides for exon skipping therapy in Alport syndrome; and an advisory or leadership role for CJASN and Pediatric Nephrology (Editorial Board). S. Ito reports consultancy for Alexion Pharma, Takeda Pharmaceutical Company, Teijin Pharma, and Zenyaku Kogyo Co Ltd.; research funding from Asahi Kasei Pharma, Chugai Pharmaceutical Co., Ltd., CSL Behring, Kyowa Hakko Kirin Co., Ltd., Maruho Pharma, Mitsubishi Tanabe Pharma, and Teijin Pharma Ltd.; honoraria from Alexion Pharma, Teijin Pharma Ltd., and Zenyaku Kogyo Co., Ltd.; an advisory or leadership role for Clinical Experimental Nephrology, JMA Journal, and the Korean Journal of Pediatrics; and participation in a speakers’ bureau for AbbVie LLC, Alexion Pharma, Asahi Kasei Pharma, Astellas Pharma, Inc., Chugai Pharmaceutical Co., Ltd., CSL Behring, Daiichi Sankyo Co., Ltd., Mitsubishi Tanabe Pharma, Novartis Pharma K.K., Pfizer Japan, Inc., Sanofi Co., Ltd., Teijin Pharma Ltd., and Zenyaku Kogyo Co., Ltd. K. Kamei reports research funding from Astellas Pharma, Inc., Chugai Pharmaceutical Co., Ltd., Public Foundation of Vaccination Research Center, Taiju Life Social Welfare Foundation, Terumo Foundation for Life Sciences and Arts, Ono Pharmaceutical Co; Otsuka Pharmaceutical Co., Ltd., Teijin Pharma Ltd; Shionogi Co., Ltd.; and honoraria from Baxter Ltd., Tanabe Mitsubishi Pharma, and Zenyaku Kogyo Co., Ltd. K. Matsuoka reports participating in a speakers’ bureau for Chugai Pharmaceutical Co., Ltd. K. Nozu reports research funding from Chugai Pharmaceutical Company, Dainippon Sumitomo Pharmaceutical Company, Kyowa Kirin Pharmaceutical Company, and Shionogi, Inc.; patents or royalties from Daiichi Sankyo Pharmaceutical Company; an advisory or leadership role for Kyowa Kirin Pharmaceutical Company and Toa Eiyo Ltd.; and receiving lecture fees from Chugai Pharmaceutical Company, Chugai Pharmaceutical Co., Ltd., Daiichi Sankyo Company, Ltd., Kyowa Kirin Pharmaceutical Company, Novartis Pharma Co., Ltd., and Sumitomo Dainippon Pharma Co., Ltd. All remaining authors have nothing to disclose., (Copyright © 2022 by the American Society of Nephrology.)

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., (© 2021. The Author(s).)

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., (© The Author(s) 2021. Published by Oxford University Press on behalf of the ERA.)

Introduction: Autosomal dominant tubulointerstitial kidney disease (ADTKD)- MUC1 is predominantly caused by frameshift mutations owing to a single-base insertion into the variable number tandem repeat (VNTR) region in MUC1 . Because of the complexity of the variant hotspot, identification using short-read sequencers (SRSs) is challenging. Although recent studies have revealed the usefulness of long-read sequencers (LRSs), the prevalence of MUC1 variants in patients with clinically suspected ADTKD remains unknown. We aimed to clarify this prevalence and the genetic characteristics and clinical manifestations of ADTKD- MUC1 in a Japanese population using an SRS and an LRS., Methods: From January 2015 to December 2019, genetic analysis was performed using an SRS in 48 patients with clinically suspected ADTKD. Additional analyses were conducted using an LRS in patients with negative SRS results., Results: Short-read sequencing results revealed MUC1 variants in 1 patient harboring a cytosine insertion in the second repeat unit of the VNTR region; however, deeper VNTR regions could not be read by the SRS. Therefore, we conducted long-read sequencing analysis of 39 cases and detected MUC1 VNTR variants in 8 patients (in total, 9 patients from unrelated families). With the inclusion of family-affected patients ( n = 31), the median age at the development of end-stage kidney disease (ESKD) was 45 years (95% CI: 40-40 years)., Conclusion: In Japan, the detection rate of MUC1 variants in patients with clinically suspected ADTKD was 18.8%. More than 20% of patients with negative SRS results had MUC1 variants detected by an LRS., (© 2022 International Society of Nephrology. Published by Elsevier Inc.)

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., (© 2021. Japanese Society of Nephrology.)

Introduction: COL4A5 is a causative gene of X-linked Alport syndrome (XLAS). Male patients with XLAS with nonsense variants have the most severe phenotypes of early onset end-stage kidney disease (ESKD); those with splicing variants have middle phenotypes and those with missense variants have the mildest phenotypes. Therefore, genotyping for male patients with XLAS can be used to predict kidney prognosis. Single-base substitutions at the last nucleotide position in each exon are known to affect splicing patterns and could be splicing variants. Nevertheless, in XLAS, these variants are generally considered to be missense variants, without conducting a transcript analysis, which underestimates some patients as having mild phenotypes. This study aimed to investigate whether single-base substitutions at the last nucleotide position of COL4A5 exons cause aberrant splicing., Methods: In total, 20 variants were found in the Human Gene Mutation Database ( n  = 14) and our cohort ( n  = 6). We performed functional splicing assays using a hybrid minigene analysis and in vivo transcript analyses of patients' samples when available. Then, we investigated genotype-phenotype correlations for patients with splicing variants detected in this study by comparing data from our previous studies., Results: Among the 20 variants, 17 (85%) caused aberrant splicing. Male patients with splicing variants had more severe phenotypes when compared with those with missense variants. Findings from the in vivo analyses for 3 variants were identical to those from the minigene assay., Conclusion: Our study revealed that most single-base substitutions at the last nucleotide position of COL4A5 exons result in splicing variants, rather than missense variants, thereby leading to more severe phenotypes., (© 2021 International Society of Nephrology. Published by Elsevier Inc.)

Background: Pathogenic variants in single genes encoding podocyte-associated proteins have been implicated in about 30% of steroid-resistant nephrotic syndrome (SRNS) patients in children. However, LAMA5 gene biallelic variants have been identified in only seven patients so far, and most are missense variants of unknown significance. Furthermore, no functional analysis had been conducted for all but one of these variants. Here, we report three patients with LAMA5 gene biallelic truncating variants manifesting infantile nephrotic syndrome, and one patient with SRNS with biallelic LAMA5 missense variants., Methods: We conducted comprehensive gene screening of Japanese patients with severe proteinuria. With the use of targeted next-generation sequencing, 62 podocyte-related genes were screened in 407 unrelated patients with proteinuria. For the newly discovered LAMA5 variants, we conducted in vitro heterotrimer formation assays., Results: Biallelic truncating variants in the LAMA5 gene (NM&#95;005560) were detected in three patients from two families. All patients presented with proteinuria within 6 months of age. Patients 1 and 2 were siblings possessing a nonsense variant (c.9232C>T, p.[Arg3078*]) and a splice site variant (c.1282 + 1G>A) that led to exon 9 skipping and a frameshift. Patient 3 had a remarkable irregular contour of the glomerular basement membrane. She was subsequently found to have a nonsense variant (c.8185C>T, p.[Arg2720*]) and the same splice site variant in patients 1 and 2. By in vitro heterotrimer formation assays, both truncating variants produced smaller laminin α5 proteins that nevertheless formed trimers with laminin β1 and γ1 chains. Patient 4 showed SRNS at the age of 8 years, and carried compound heterozygous missense variants (c.1493C>T, p.[Ala498Val] and c.8399G>A, p.[Arg2800His])., Conclusions: Our patients showed clear evidence of biallelic LAMA5 truncating variants causing infantile nephrotic syndrome. We also discerned the clinical and pathologic characteristics observed in LAMA5 -related nephropathy. LAMA5 variant screening should be performed in patients with congenital/infantile nephrotic syndrome., Competing Interests: J. Miner reports having consultancy agreements with Alpha Insights, AstraZeneca, Bridge Bio, Deerfield Management, Janssen Biotech Inc., GLG Council, Kurma, Mantra Bio, National Institutes of Health, Retrophin, and The Planning Shop; reports receiving research funding from Chinook Therapeutics and Reneo Pharmaceuticals; reports receiving honoraria from Japanese Society of Pharmacology, NephCure Kidney International, Western Michigan University Medical School, and University of Kansas Medical Center; reports patents and inventions with Angion, Eli Lilly, Genentech, Kerafast, and Maze Therapeutics; reports being a scientific advisor or member of Journal of Clinical Investigation Consulting Editor, Kidney International Editorial Board, Matrix Biology Editorial Board, and Matrix Biology Plus Editorial Board; and reports other interests/relationships with the Alport Syndrome Foundation (Scientific Advisory Research Network) and the American Society for Matrix Biology (President-Elect). K. Iijima reports having consultancy agreements with JCR Pharmaceuticals Co., Kyowa Hakko Kirin Co., Ono Pharmaceutical Co., Sanofi K.K., Takeda Pharmaceutical Co., and Zenyaku Kogyo Co.; reports receiving research funding from Air Water Medical Inc., Astellas Pharma Inc., Eisai Co., JCR Pharmaceutical Co., Mochida Pharmaceutical Co., Nihon Pharmaceutical Co. Otsuka Pharmaceutical Co., Shionogi & Co., Zenyaku Kogyo Co.; reports receiving honoraria from Astellas Pharma Inc., Chugai Pharmaceutical Co., Integrated Development Associates Co., Kyowa Hakko Kirin Co., Shionogi & Co., and Zenyaku Kogyo Co.; reports being a scientific advisor or member of Clinical Journal of the American Society of Nephrology and the Pediatric Nephrology Editorial Board. K. Nozu reports receiving research funding from Chugai Pharmaceutical Company, Dainippon Sumitomo Pharmaceutical Company, Kyowa Kirin Pharmaceutical Company, and Shionogi Inc.; reports patents and inventions with Daiichi Sankyo Pharmaceutical Company; reports being a scientific advisor or member of Toa Eiyo Ltd.; and reports receiving lecture fees from Chugai Pharmaceutical Co., Daiichi Sankyo Company, Kyowa Kirin Pharmaceutical Co., Novartis Pharma Co., and Sumitomo Dainippon Pharma Co. K. Sekiguchi reports having an ownership interest in and receiving research funding from Matrixome, Inc.; and reports patents and inventions with Osaka University. T. Horinouchi reports receiving research funding from Otsuka Pharmaceutical Co. Y. Taniguchi reports being a Project Leader at Matrixome Inc. All remaining authors have nothing to disclose., (Copyright © 2021 by the American Society of Nephrology.)

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., Competing Interests: T. Horinouchi reports receiving research funding from Otsuka Pharmaceutical Co. Ltd.; K. Iijima reports receiving research funding from Air Water Medical Inc., Astellas Pharma Inc., Eisai Co. Ltd., JCR Pharmaceutical Co. Ltd., Mochida Pharmaceutical Co. Ltd., Nihon Pharmaceutical Co. Ltd., Otsuka Pharmaceutical Co. Ltd., Shionogi & Co. Ltd., and Zenyaku Kogyo Co. Ltd.; receiving consulting fees from Astellas Pharma Inc., Boehringer Ingelheim, Kyowa Kirin Co. Ltd., Ono Pharmaceutical Co. Ltd., and Takeda Pharmaceutical Co.; receiving honoraria from Astellas Pharma Inc., Chugai Pharmaceutical Co. Ltd., Integrated Development Associates Co. Ltd., Kyowa Hakko Kirin Co. Ltd., Shionogi & Co. Ltd., and Zenyaku Kogyo Co. Ltd.; serving as a scientific advisor for, or member of, CJASN and Pediatric Nephrology (on the editorial board); receiving grant support from Daiichi Sankyo Co. Ltd.; and having consultancy agreements with JCR Pharmaceuticals Co. Ltd., Kyowa Hakko Kirin Co. Ltd., Ono Pharmaceutical Co. Ltd., Sanofi K.K., Takeda Pharmaceutical Co. Ltd., and Zenyaku Kogyo Co. Ltd. K. Iijima and K. Nozu report having filed a patent application for the development of antisense nucleotides for exon skipping therapy in Alport syndrome. M. Matsuo reports serving as an advisor to Daiichi Sankyo Co. Ltd. (Japan) and JCR Pharma Co. Ltd. (Japan), and being a research professor at the Nucleic Acid Drug Discovery Department that is financially supported by KNC Laboratories Co. Ltd. Inc. (Japan). K. Nakanishi reports receiving honoraria from Asahi Kasei Corporation, Astellas Pharma Inc., AstraZeneca, Chugai Pharmaceutical Co. Ltd., Daiichi Sankyo Co. Ltd., JCR Pharmaceuticals Co. Ltd., Kyowa Hakko Kirin Co. Ltd., Miyarisan Pharmaceutical Co. Ltd., Novartis Pharma K.K., Ono Pharmaceutical Co. Ltd., Sanofi K.K., Taisho Toyama Pharmaceutical Co., and Teijin Pharma Ltd.; and receiving research funding from Asahi Kasei Corporation, Astellas Pharma Inc., Chugai Pharmaceutical Co. Ltd., CSL Behring, Daiichi Sankyo Co. Ltd., JCR Pharmaceuticals Co. Ltd., MSD K.K., Otsuka Pharmaceutical Co. Ltd., Pfizer Inc., Sanofi K.K., and Shionogi & Co. Ltd. K. Nozu reports receiving lecture fees from Chugai Pharmaceutical Co. Ltd., Daiichi Sankyo Co. Ltd., Novartis Pharma Co. Ltd., and Sumitomo Dainippon Pharma Co. Ltd.; having patents and inventions with Daiichi Sankyo Pharmaceutical Company; receiving consulting fees from Kyowa Kirin Co. Ltd.; and receiving lecture fees from Novartis Pharmaceuticals Corporation. K. Tamagaki reports receiving research funding from Baxter International Inc., Chugai Pharmaceutical Co. Ltd., Daiichi Sankyo Co. Ltd., Eli Lilly Japan K.K., JMS Co. Ltd., Kissei Pharmaceutical Co. Ltd., Kyowa Hakko Kirin Co. Ltd., Merck & Co. Inc., Mitsubishi Tanabe Pharma Corporation, Nippon Boehringer Ingelheim Co. Ltd., Otsuka Pharmaceutical Co. Ltd., Sanofi K.K., Takeda Pharmaceutical Co. Ltd., Teijin Pharma Ltd., and Torii Pharmaceutical Co. Ltd. All remaining authors have nothing to disclose., (Copyright © 2022 by the American Society of Nephrology.)

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., (© 2021. The Author(s).)

Introduction: Frasier syndrome (FS) is a rare inherited kidney disease caused by intron 9 splicing variants of WT1 . For wild-type WT1 , 2 active splice donor sites in intron 9 cause a mixture of 2 essential transcripts (with or without lysine-threonine-serine [+/KTS or -KTS]), and imbalance of the +KTS/-KTS ratio results in the development of FS. To date, 6 causative intron 9 variants have been identified; however, detailed transcript analysis has not yet been conducted and the genotype-phenotype correlation also remains to be elucidated., Methods: We conducted an in vitro minigene splicing assay for 6 reported causative variants and in vivo RNA sequencing to determine the +KTS/-KTS ratio using patients' samples. We also performed a systematic review of reported FS cases with a description of the renal phenotype., Results: The in vitro assay revealed that although all mutant alleles produced -KTS transcripts only, the wild-type allele produced both +KTS and -KTS transcripts at a 1:1 ratio. In vivo RNA sequencing showed that patients' samples with all heterozygous variants produced similar ratios of +KTS to -KTS (1:3.2-1:3.5) and wild-type kidney showed almost a 1:1 ratio (1:0.85). A systematic review of 126 cases clarified that the median age of developing ESKD was 16 years in all FS patients, and there were no statistically significant differences between the genotypes or sex chromosome karyotypes in terms of the renal survival period., Conclusion: Our study suggested no differences in splicing pattern or renal survival period among reported intron 9 variants causative of FS., (© 2021 International Society of Nephrology. Published by Elsevier Inc.)

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., (© 2021 International Society of Nephrology. Published by Elsevier Inc.)

The identification of monogenic causes in patients with proteinuria has revealed that the encoded proteins functionally participate in distinct cellular tasks and signaling pathways in the slit diaphragms of the glomerular basement membrane. FAT1 is a member of a small family of vertebrate-cadherin-like genes, which is a crucial component in slit diaphragms and has a vital role in tubular regeneration. Only 5 cases with glomerulonephritis having FAT1 gene biallelic variants have been reported. However, only one had the biallelic truncating variant, and others had missense variants. Therefore, we need further evidence of this gene being responsible for steroid-resistant nephrotic syndrome (SRNS) or glomerulonephritis. Here we describe a 5-year-old boy in who proteinuria was detected at the age of 3 years without any extrarenal symptom. The pathological findings were examined, and targeted exome sequencing was performed. We also conducted reviews for all previously-reported cases of glomerulonephritis possessing FAT1 biallelic gene variants. We found two novel truncating variants in FAT1 (NM&#95;005245.3), c.12867dup in exon 10, and, c.5480&#95;5483del in exon 25. Our case showed mild proteinuria compared to previously-reported cases who showed SRNS and extrarenal symptoms that might have been because the latter variant in our patient was located on out of cadherin domains; however, our follow up period is short and we further need careful follow up. Our findings corroborate the evidence that individuals with FAT1-truncating variants can show isolated mild proteinuria. Further studies are needed to investigate the genotype-phenotype correlation in this disease. Therefore, our case will provide vital information regarding this rare condition.

Early kidney failure in the hereditary type IV collagen disease, Alport syndrome, can be delayed by renin-angiotensin inhibitors. However, whether all patients and all different genotypes respond equally well to this kidney-protective therapy remains unclear. Here, we performed a retrospective study on 430 patients with male X-linked Alport syndrome to examine the relationships among kidney prognosis, genotype, and treatment effect in a large cohort of Japanese patients. We analyzed the clinical features, genotype-phenotype correlation, and kidney survival period for patients treated with or without renin-angiotensin inhibitors. As a result, the median kidney survival period of patients in this cohort was found to be at 35 years with a strong genotype-phenotype correlation. The median age at the onset of end stage kidney disease (ESKD) significantly differed between patients treated with and without renin-angiotensin inhibitors (over 50 years versus 28 years, respectively). Moreover, these drugs delayed the onset of ESKD in patients with truncating variants for 12 years, extending the median age from 16 years to 28 years. Thus, our results confirmed a strong genotype-phenotype correlation in patients with male X-linked Alport syndrome. Additionally, it was suggested that renin-angiotensin inhibitors could significantly delay ESKD progression. Despite these therapies, patients with truncating variants developed ESKD at the median age of 28 years., (Copyright © 2020 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

Background: X-linked Alport syndrome (XLAS) is a progressive, hereditary glomerular nephritis of variable severity caused by pathogenic COL4A5 variants. Currently, genetic testing is widely used for diagnosing XLAS; however, determining the pathogenicity of variants detected by such analyses can be difficult. Intronic variants or synonymous variants may cause inherited diseases by inducing aberrant splicing. Transcript analysis is necessary to confirm the pathogenicity of such variants, but it is sometimes difficult to extract mRNA directly from patient specimens., Methods: In this study, we conducted in vitro splicing analysis using a hybrid minigene assay and specimens from three XLAS patients with synonymous variants causing aberrant splicing, including previously reported pathogenic mutations in the same codon. The variants were c.876 A>T (p.Gly292=), c.2358 A>G (p.Pro786=), and c.3906 A>G (p.Gln1302=)., Results: The results from our hybrid minigene assay were sufficient to predict splicing abnormalities; c.876 A>T cause 17-bp del and 35-bp del, c.2358 A>G cause exon 29 skipping, c.3906 A>G cause exon 42 skipping, which are very likely to cause pathogenicity. Further, patients carrying c.2358 A>G exhibited a mild phenotype that may have been associated with the presence of both normal and abnormally spliced transcripts., Conclusion: The minigene system was shown to be a sensitive assay and a useful tool for investigating the pathogenicity of synonymous variants., (© 2020 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals LLC.)

Background: Autosomal recessive Alport syndrome (ARAS) is an inherited renal disorder caused by homozygous and compound heterozygous mutations in COL4A3 or COL4A4 , but the prognostic predictors for this disorder are not yet fully understood. Recently, the magnitude of the clinical spectrum of the COL4A3 and COL4A4 heterozygous state has attracted attention. This spectrum includes asymptomatic carriers of ARAS, benign familial hematuria, thin basement membrane disease, and autosomal dominant Alport syndrome., Methods: We retrospectively analyzed 49 patients with ARAS from 41 families with a median age of 19 years to examine the clinical features and prognostic factors of ARAS, including the associated genotypes., Results: The median age of patients with ARAS at ESKD onset was 27 years. There was no significant association between the presence or absence of hearing loss or truncating mutations and renal prognosis. However, there was a statistically significant correlation between renal prognosis and heterozygous variants that cause urinary abnormalities. Where the urinary abnormality-causing variant was absent or present in only one allele, the median age of ESKD onset was 45 years, whereas the same variant present on both alleles was associated with an age of onset of 15 years ( P <0.001)., Conclusions: This study was the first to demonstrate the clinical importance in ARAS of focusing on variants in COL4A3 or COL4A4 that cause urinary abnormalities in both the homozygous or heterozygous state. Although heterozygous mutation carriers of COL4A3 and COL4A4 comprise a broad clinical spectrum, clinical information regarding each variant is important for predicting ARAS prognosis., Competing Interests: K. Iijima and K. Nozu have filed a patent application on the development of antisense nucleotides for exon skipping therapy in Alport syndrome. K. Nozu has received lecture fees from Novartis Pharmaceuticals and corporation and consulting fees from Kyowa Kirin Co., Ltd. All remaining authors have nothing to disclose., (Copyright © 2020 by the American Society of Nephrology.)

Background: In recent years, the elucidation of splicing abnormalities as a cause of hereditary diseases has progressed. However, there are no comprehensive reports of suspected splicing variants in the CLCN5 gene in Dent disease cases. We reproduced gene mutations by mutagenesis, inserted the mutated genes into minigene vectors, and investigated the pathogenicity and onset mechanisms of these variants., Methods: We conducted functional splicing assays using a hybrid minigene for six suspected splicing variants (c.105G>A, c.105+5G>C, c.106-17T>G, c.393+4A>G, c.517-8A>G, c.517-3C>A) in CLCN5. We extracted information on these variants from the Human Gene Mutation Database. We reproduced minigene vectors with the insertion of relevant exons with suspected splicing variants. We then transfected these minigene vectors into cultured cells and extracted and analyzed the mRNA. In addition, we conducted in silico analysis to confirm our minigene assay results., Results: We successfully determined that five of these six variants are pathogenic via the production of splicing abnormalities. One showed only normal transcript production and was thus suspected of not being pathogenic (c.106-17T>G)., Conclusion: We found that five CLCN5 variants disrupted the original splice site, resulting in aberrant splicing. It is sometimes difficult to obtain mRNA from patient samples because of the fragility of mRNA or its low expression level in peripheral leukocytes. Our in vitro system can be used as an alternative to in vivo assays to determine the pathogenicity of suspected splicing variants.

Currently, there are no treatments for Alport syndrome, which is the second most commonly inherited kidney disease. Here we report the development of an exon-skipping therapy using an antisense-oligonucleotide (ASO) for severe male X-linked Alport syndrome (XLAS). We targeted truncating variants in exon 21 of the COL4A5 gene and conducted a type IV collagen α3/α4/α5 chain triple helix formation assay, and in vitro and in vivo treatment efficacy evaluation. We show that exon skipping enabled trimer formation, leading to remarkable clinical and pathological improvements including expression of the α5 chain on glomerular and the tubular basement membrane. In addition, the survival period was clearly prolonged in the ASO treated mice group. This data suggests that exon skipping may represent a promising therapeutic approach for treating severe male XLAS cases.

Pathogenic variants of paired box gene 2 (PAX2) cause autosomal-dominant PAX2-related disorder, which includes renal coloboma syndrome (RCS). Patients with PAX2-related disorder present with renal and ophthalmological pathologies, as well as with other abnormalities, including developmental problems and hearing loss. We sequenced PAX2 in 457 patients with congenital anomalies of the kidney and urinary tract or with renal dysfunction of unknown cause and identified 19 different pathogenic variants in 38 patients from 30 families (6.5%). Thirty-four patients had renal hypodysplasia or chronic kidney disease of unknown cause, and three had focal segmental glomerulosclerosis. Although no obvious genotype-phenotype correlation was observed, six of the seven patients who developed end-stage renal disease in childhood had truncating variants. Twenty-three patients had ocular disabilities, mostly optic disc coloboma. Non-renal and non-ophthalmological manifestations included developmental disorder, electrolyte abnormality, and gonadal abnormalities. Two unrelated patients had congenital cystic adenomatoid malformations in their lungs. Six of ten probands with PAX2 mutation identified by next-generation sequencing did not show typical RCS manifestations. We conclude that PAX2-related disorder has a variable clinical presentation and can be diagnosed by next-generation sequencing even in the absence of typical RCS manifestations.

X-linked Alport syndrome (XLAS) is a congenital renal disease caused by mutations in COL4A5. In XLAS cases suspected of being caused by aberrant splicing, transcript analysis needs to be conducted to determine splicing patterns and assess the pathogenicity. However, such analysis is not always available. We conducted a functional splicing assay using a hybrid minigene for seven COL4A5 intronic mutations: one was identified by us and six were found in the Human Gene Mutation Database. The minigene assay revealed exon skipping in four variants, exon skipping and a 10-bp insertion in one variant, and no change in one variant, which appeared not to be pathogenic. For one variant, our assay did not work. The results of all three cases for which transcript data were available were consistent with our assay results. Our findings may help to increase the accuracy of genetic test results and clarify the mechanisms causing aberrant splicing.

Background: Alport syndrome (AS) is a hereditary disease caused by mutations in COL4A3-5 genes. Recently, comprehensive genetic analysis has become the first-line diagnostic tool for AS. However, no reports comparing mutation identification rates between conventional sequencing and comprehensive screening have been published., Methods: In this study, 441 patients clinically suspected of having AS were divided into two groups and compared. The initial mutational analysis method involved targeted exome sequencing using next-generation sequencing (NGS) (n = 147, NGS group) or Sanger sequencing for COL4A3/COL4A4/COL4A5 (n = 294, Sanger group)., Results: In the NGS group, 126 patients (86%) were diagnosed with AS by NGS, while two had pathogenic mutations in other genes, NPHS1 and EYA1. Further, 239 patients (81%) were diagnosed with AS by initial analysis in the Sanger group. Thirteen patients who were negative for mutation detection in the Sanger group were analyzed by NGS; three were diagnosed with AS. Two had mutations in CLCN5 or LAMB2. The final variant detection rate was 90%., Discussion: Our results reveal that Sanger sequencing and targeted exome sequencing have high diagnostic ability. NGS also has the advantage of detecting other inherited kidney diseases and pathogenic mutations missed by Sanger sequencing., (© 2019 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals, Inc.)

Advances in molecular genetics have revealed that approximately 30% of cases with steroid-resistant nephrotic syndrome (SRNS) are caused by single-gene mutations. More than 50 genes are responsible for SRNS. One such gene is the nucleoporin, 93-KD (NUP93). Thus far, few studies have reported mutations of NUP93 in SRNS. Here, we describe an NUP93 biallelic mutation in a 9-year-old boy with focal segmental glomerular sclerosis (FSGS). Notably, one mutation comprised an intronic variant; we conducted in vivo and in vitro analysis to characterize this variant. We found two heterozygous mutations in NUP93: c.2137-18G>A in intron 19 and a novel nonsense mutation c.727A>T (p.Lys243*) in exon 8. We conducted RNA sequencing and in vitro splicing assays by using minigene construction, combined with protein expression analysis to determine the pathogenicity of the intronic variant. Both RNA sequencing and in vitro splicing assay showed exon 20-skipping by the intronic variant. In protein expression analysis, aberrant subcellular localization with small punctate vesicles in the cytoplasm was observed for the intronic variant. Taken together, we concluded that c.2137-18G>A was linked to pathogenicity due to aberrant splicing. NUP93 variants are quite rare; however, we have shown that even intronic variants in NUP93 can cause SRNS. This study provides a fundamental approach to validate the intronic variant, as well as new insights regarding the clinical spectrum of SRNS caused by rare gene variants.

Background: Recently, comprehensive genetic approaches for steroid-resistant nephrotic syndrome (SRNS) using next-generation sequencing (NGS) have been established, but causative gene mutations could not be detected in almost 70% of SRNS patients. Main reason for the low variant detection rate is that most of them are SRNS caused not by genetic but by immunological factors. But some of them are probably because of the difficulty of detecting copy number variations (CNVs) in causative genes by NGS., Methods: In this study, we performed two analytical methods of NGS data-dependent pair analysis and custom array comparative genomic hybridization (aCGH) in addition to NGS analysis in an infantile nephrotic syndrome case., Results: We detected only one known pathogenic heterozygous missense mutation in exon 7 of COQ6 c.782C > T, p.(Pro261Leu) by NGS. With pair analysis, heterozygous exon 1-2 deletion was suspected and was confirmed by custom aCGH. As a result, a small CNV was successfully detected in the COQ6 gene. Because we could detect variants in COQ6 and could start treatment by coenzyme Q10 (CoQ10) in his very early stage of SRNS, the patient achieved complete remission., Conclusions: These relatively novel methods should be adopted in cases with negative results in gene tests by NGS analysis. Especially, in cases with CoQ10 deficiency, it is possible to delay initiating dialysis by starting treatment at their early stages.