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Imafuku, Aya ; Nozu, Kandai ; Sawa, Naoki ; Nakanishi, Koichi ; Ubara, Yoshifumi
出版情報: Clinical and Experimental Nephrology.  24  pp.651-656,  2020-03-30.  Springer
URL: http://hdl.handle.net/20.500.12000/46978
概要: Both thin basement membrane nephropathy (TBMN) and autosomal dominant Alport syndrome (ADAS) are types of hereditary nep hritis resulting from heterozygous mutations in COL4A3 or COL4A4 genes. Although TBMN is characterized by hematuria and thinning of the glomerular basement membrane (GBM) with excellent renal prognosis, some patients develop end-stage renal disease (ESRD) later in life. In contrast, although AS is characterized by progressive nephropathy with lamellation of the GBM, there are some patients diagnosed with ADAS from a family history of ESRD but who only suffer from hematuria with GBM thinning. These findings indicate a limitation in distinction between TBMN and ADAS. Diagnosis of AS is significant because it facilitates careful follow-up and early treatment, whereas diagnosis of TBMN can underestimate the risk of ESRD. However, some experts are against using the term ADAS as the phenotypes of heterozygous variants vary from no urinary abnormality to ESRD, even between family members with the same mutations, indicating that unknown secondary factors may play a large role in the disease severity. These diagnostic difficulties result in significant confusion in clinical settings. Moreover, recent studies revealed that the number of patients with chronic kidney disease caused by these gene mutations is far higher than previously thought. The aim of this article is to review differing opinions regarding the diagnosis of heterozygous COL4A3 or COL4A4 variants, and to highlight the importance for nephrologists to recognize this disease, and the importance of the need to reclassify this disease to minimize the current confusion.
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2.

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Nagano, China ; Yamamura, Tomohiko ; Horinouchi, Tomoko ; Aoto, Yuya ; Ishiko, Shinya ; Sakakibara, Nana ; Shima, Yuko ; Nakanishi, Koichi ; Nagase, Hiroaki ; Iijima, Kazumoto ; Nozu, Kandai
出版情報: Scientific Reports.  10  2020-01-14.  Springer Nature
URL: http://hdl.handle.net/20.500.12000/46988
概要: Numerous disease-causing gene mutations have been identified in proteinuric diseases, such as nephrotic syndrome and glo merulosclerosis. This report describes the results of comprehensive genetic diagnosis of Japanese patients with severe proteinuria. In addition, the report describes the clinical characteristics of patients with monogenic disease-causing mutations. We conducted comprehensive gene screening of patients who had either congenital nephrotic syndrome, infantile nephrotic syndrome, steroid-resistant nephrotic syndrome, or focal segmental glomerular sclerosis. Using targeted next-generation sequencing, 60 podocyte-related genes were screened in 230 unrelated patients with proteinuria. A retrospective review of clinical data was conducted for these patients. We detected monogenic disease-causing mutations in 30% (69 of 230) of patients among 19 of the screened genes. Common genes with disease-causing mutations were WT1 (25%), NPHS1 (12%), INF2 (12%), TRPC6 (10%), and LAMB2 (9%). With various immunosuppressive or renoprotective therapies, remission of proteinuria in patients with unknown causative mutations was observed in 26% of patients, whereas only 5% of patients with monogenic disease-causing mutations exhibited complete remission. We assessed the genetic backgrounds of Japanese patients with severe proteinuria. The proportion of patients with gene defects was similar to that of other reports, but the disease-causing gene mutation frequency was considerably different.
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3.

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Horinouchi, Tomoko ; Yamamura, Tomohiko ; Minamikawa, Shogo ; Nagano, China ; Sakakibara, Nana ; Nakanishi, Koichi ; Shima, Yuko ; Morisada, Naoya ; Ishiko, Shinya ; Aoto, Yuya ; Nagase, Hiroaki ; Takeda, Hiroki ; Rossanti, Rini ; Ishimori, Shingo ; Kaito, Hiroshi ; Matsuo, Masafumi ; Iijima, Kazumoto ; Nozu, Kandai
出版情報: Molecular Genetics and Genomic Medicine.  8  2020.  Wiley Open Access
URL: http://hdl.handle.net/20.500.12000/46926
概要: Background: X-linked Alport syndrome (XLAS) is a progressive, hereditary glomerular nephritis of variable severity cause d 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.
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4.

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Horinouchi, Tomoko ; Nozu, Kandai ; Yamamura, Tomohiko ; Minamikawa, Shogo ; Nagano, China ; Sakakibara, Nana ; Nakanishi, Koichi ; Shima, Yuko ; Morisada, Naoya ; Ishiko, Shinya ; Aoto, Yuya ; Nagase, Hiroaki ; Takeda, Hiroki ; Rossanti, Rini ; Kaito, Hiroshi ; Matsuo, Masafumi ; Iijima, Kazumoto
出版情報: Scientific Reports.  9  2019-09-03.  Springer Nature
URL: http://hdl.handle.net/20.500.12000/45627
概要: 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.
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Nagano, China ; Sako, Mayumi ; Kamei, Koichi ; Ishikura, Kenji ; Nakamura, Hidefumi ; Nakanishi, Koichi ; Omori, Takashi ; Nozu, Kandai ; Iijima, Kazumoto
出版情報: BMC Nephrology.  20  2019-08-02.  Springer Nature
URL: http://hdl.handle.net/20.500.12000/45876
概要: Background: Eighty percent of children with idiopathic nephrotic syndrome respond well to steroid therapy, but up to 50% of patients with steroid-sensitive nephrotic syndrome exhibit frequently relapsing (FRNS) or steroid-dependent nephrotic syndrome (SDNS). Several studies identified the chimeric anti-CD20 monoclonal antibody rituximab as an effective treatment for patients with complicated FRNS/SDNS. Recent studies suggested that rituximab could also be a first-line treatment for early-stage uncomplicated FRNS/SDNS, although further studies are required to confirm its efficacy and safety. Methods/design: We are conducting a multicenter, double-blind, randomized placebo controlled trial to investigate the efficacy and safety of rituximab for the treatment of childhood-onset early-stage uncomplicated FRNS/SDNS. Patients will be allocated to receive two 375 mg/m(2) doses (maximum dose: 500 mg) of either rituximab or placebo. Investigators are permitted to request the disclosure of a subject's allocation code if he or she exhibits treatment failure. Additionally, if placebo-treated subjects display early relapse (a sign of treatment failure), they have the option to receive rituximab in an unblinded phase. The primary endpoint is relapse-free survival during the observation period. Discussion: The results will provide important data on the use of rituximab for patients with uncomplicated FRNS/SDNS. In the future, rituximab treatment will enable most patients with uncomplicated FRNS/SDNS to discontinue or reduce steroid therapy without relapse, and it is possible that rituximab could represent an immunosuppressive therapy for these diseases.
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6.

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Fujimura, Junya ; Nozu, Kandai ; Yamamura, Tomohiko ; Minamikawa, Shogo ; Nakanishi, Keita ; Horinouchi, Tomoko ; Nagano, China ; Sakakibara, Nana ; Nakanishi, Koichi ; Shima, Yuko ; Miyako, Kenichi ; Nozu, Yoshimi ; Morisada, Naoya ; Nagase, Hiroaki ; Ninchoji, Takeshi ; Kaito, Hiroshi ; Iijima, Kazumoto
出版情報: Kidney international reports.  4  pp.119-125,  2019-01.  Elsevier
URL: http://hdl.handle.net/20.500.12000/45950
概要: Introduction:Gitelman syndrome (GS) is a tubulopathy exhibited by salt loss. GS cases are most oftendiagnosed by chance blood test. Aside from that, some cases are also diagnosed from tetanic symptomsassociated with hypokalemia and/or hypomagnesemia or short stature. As for complications, thyroiddysfunction and short stature are known, but the incidence rates for these complications have not yet beenelucidated. In addition, no genotype–phenotype correlation has been identified in GS. Methods:We examined the clinical characteristics and genotype–phenotype correlation in geneticallyproven GS cases with homozygous or compound heterozygous variants inSLC12A3(n¼185). Results:In our cohort, diagnostic opportunities were by chance blood tests (54.7%), tetany (32.6%), orshort stature (7.2%). Regarding complications, 16.3% had short stature, 13.7% had experienced febrileconvulsion, 4.3% had thyroid dysfunction, and 2.5% were diagnosed with epilepsy. In one case, QT pro-longation was detected. Among 29 cases with short stature, 10 were diagnosed with growth hormone (GH)deficiency and GH replacement therapy started. Interestingly, there was a strong correlation in serummagnesium levels between cases with p.Arg642Cys and/or p.Leu858His and cases without these variants,which are mutational hotspots in the Japanese population (1.76 mg/dl vs. 1.43 mg/dl,P<0.001). Conclusion:This study has revealed, for thefirst time, clinical characteristics in genetically proven GScases in the Japanese population, including prevalence of complications. Patients with hypokalemiadetected by chance blood test should have gene tests performed. Patients with GS need attention fordeveloping extrarenal complications, such as short stature, febrile convulsion, thyroid dysfunction, epi-lepsy, or QT prolongation. It was also revealed for thefirst time that hypomagnesemia was not severe insome variants in SLC12A3.
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7.

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Yamamura, Tomohiko ; Nozu, Kandai ; Minamikawa, Shogo ; Horinouchi, Tomoko ; Sakakibara, Nana ; Nagano, China ; Aoto, Yuya ; Ishiko, Shinya ; Nakanishi, Koichi ; Shima, Yuko ; Nagase, Hiroaki ; Rossanti, Rini ; Ye, Ming J. ; Nozu, Yoshimi ; Ishimori, Shingo ; Morisada, Naoya ; Kaito, Hiroshi ; Iijima, Kazumoto
出版情報: Molecular Genetics & Genomic Medicine.  7  2019-09.  Wiley
URL: http://hdl.handle.net/20.500.12000/45970
概要: 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.
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8.

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Horinouchi, Tomoko ; Sako, Mayumi ; Nakanishi, Koichi ; Ishikura, Kenji ; Ito, Shuichi ; Nakamura, Hidefumi ; Oba, Saito Mari ; Nozu, Kandai ; Iijima, Kazumoto
出版情報: BMC Nephrology.  19  2018-11-01.  Springer Nature
URL: http://hdl.handle.net/20.500.12000/45851
概要: Background:Idiopathic nephrotic syndrome (INS) is the most common chronic glomerular disease in children. Approximately 80-90% of patients with childhood INS have steroid-sensitive nephrotic syndrome (SSNS), and can obtain remission with steroid therapy, while the remainder have steroid-resistant nephrotic syndrome (SRNS). Furthermore, approximately 50% of children with SSNS develop frequently-relapsing nephrotic syndrome (FRNS) or steroid-dependent nephrotic syndrome (SDNS). Children with FRNS/SDNS are usually treated with immunosuppressive agents such as cyclosporine, cyclophosphamide, or mizoribine in Japan. However, 10-20% of children receiving immunosuppressive agents still show frequent relapse and/or steroid dependence during or after treatment, which is defined as complicated FRNS/SDNS. Furthermore, 30% of SRNS patients who obtain remission after additional treatments such as cyclosporine also turn out to be complicated FRNS/SDNS. For such complicated FRNS/SDNS patients, rituximab (RTX) is currently used; however, recurrence after RTX treatment also remains an open issue. Because long-term use of existing immunosuppressive drugs has limitations, development of a novel treatment for maintenance therapy after RTX is desirable. Mycophenolate mofetil (MMF) is an immunosuppressive drug with fewer side effects than cyclosporine or cyclophosphamide. Importantly, recent studies have reported the efficacy of MMF in children with nephrotic syndrome.Methods:We conduct a multicenter, double-blind, randomized, placebo-controlled trial to evaluate the efficacy and safety of MMF after RTX therapy in children with complicated FRNS/SDNS. Patients are allocated to either RTX plus MMF treatment group, or RTX plus placebo treatment group. For the former group, MMF is administered at a dose of 1000-1200mg/m(2)/day (maximum 2g/day) twice daily for 17months after RTX treatment. The primary endpoint is time-to-treatment failure (development of frequent relapses, steroid dependence or steroid resistance).Discussion:The results will provide important data on the use of MMF as maintenance therapy after RTX to prevent complicated FRNS/SDNS patients from declining into treatment failure. In future, MMF in conjunction with RTX treatment may permit increased duration of remission in complicated' FRNS/SDNS cases.Trial registration:This trial was prospectively registered to UMIN Clinical Trials Registry on June 23, 2014 (UMIN Trial ID: UMIN000014347).
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9.

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Hama, Taketsugu ; Nakanishi, Koichi ; Ishikura, Kenji ; Ito, Shuichi ; Nakamura, Hidefumi ; Sako, Mayumi ; Saito-Oba, Mari ; Nozu, Kandai ; Shima, Yuko ; Iijima, Kazumoto ; Yoshikawa, Norishige
出版情報: BMC Nephrology.  19  2018-09-10.  Springer Nature
URL: http://hdl.handle.net/20.500.12000/45852
概要: Background: Eighty percent of children with steroid-sensitive nephrotic syndrome (SSNS) relapse within 2 years and 40-50 % patients show frequently-relapsing nephrotic syndrome (FRNS). Patients showing a relapse within 6 months after initial remission are at high risk of FRNS. Since frequent prednisolone treatment for FRNS induces severe prednisolone side effects, development of a treatment to prevent patients from shifting to FRNS is desirable. Mizoribine is an immunosuppressive drug with fewer side effects than prednisolone. Recent studies reported the efficacy of high-dose mizoribine in children with FRNS. Methods/design: We conduct a multicenter, open, randomized controlled trial to investigate the efficacy and safety of standard prednisolone plus high-dose mizoribine therapy in children with SSNS showing a relapse within 6 months after an initial remission. Patients are allocated to either standard prednisolone alone treatment group, or standard prednisolone plus high-dose mizoribine group. For the former group, mizoribine is administered at a dose of 10 mg/kg/day once daily and continued for 2 years. The primary endpoint is the duration to frequent relapse. Discussion: The results provide important data on use of high-dose mizoribine to prevent SSNS patients from shifting to FRNS. Since blood concentrations of mizoribine have not been investigated in detail until now, there is a possibility that mizoribine is underestimated in favor of other immunosuppressive drugs. In future, high-dose mizoribine therapy may lead to prevention of relapse in children at high risk of FRNS, and to decreased total dose of prednisolone.
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10.

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Yamamura, Tomohiko ; Nozu, Kandai ; Miyoshi, Yuya ; Nakanishi, Keita ; Fujimura, Junya ; Horinouchi, Tomoko ; Minamikawa, Shogo ; Mori, Nobuo ; Fujimaru, Rika ; Nakanishi, Koichi ; Ninchoji, Takeshi ; Kaito, Hiroshi ; Taniguchi-Ikeda, Mariko ; Morioka, Ichiro ; Matsuo, Masafumi ; Iijima, Kazumoto
出版情報: BMC Nephrology.  18  2017-12-04.  Springer Nature
URL: http://hdl.handle.net/20.500.12000/45877
概要: Background: Autosomal recessive distal renal tubular acidosis (dRTA) is a rare hereditary disease caused by pathogenic v ariants in the ATP6V0A4 gene or ATP6V1B1 gene, and characterized by hyperchloremic metabolic acidosis with normal anion gap, hypokalemia, hypercalciuria, hypocitraturia and nephrocalcinosis. Although several intronic nucleotide variants in these genes have been detected, all of them fell in the apparent splice consensus sequence. In general, transcriptional analysis is necessary to determine the effect on function of the novel intronic variants located out of splicing consensus sequences. In recent years, functional splicing analysis using minigene construction was used to assess the pathogenicity of novel intoronic variant in various field. Methods: We investigated a sporadic case of dRTA with a compound heterozygous mutation in the ATP6V0A4 gene, revealed by next generation sequencing. One variant was already reported as pathogenic; however, the other was a novel variant in intron 11 (c. 1029 + 5G > A) falling outside of the apparent splicing consensus sequence. Expression of ATP6V0A4 was not detected in peripheral leukocytes by RT-PCR analysis. Therefore, an in vitro functional splicing study using minigene construction was conducted to analyze the splicing pattern of the novel variant. Results: A minigene assay revealed that the novel intronic variant leads to a 104 bp insertion immediately following exon 11. In addition, this result was confirmed using RNA extracted from the patient's cultured leukocytes. Conclusion: These results proved the pathogenicity of a novel intronic variant in our patient. We concluded that the minigene assay is a useful, non-invasive method for functional splicing analysis of inherited kidney disease, even if standard transcriptional analysis could not detect abnormal mRNA.
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