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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.)

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.)

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.

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.)