Abstract
Pathogenic variants in specific complement-related genes lead to atypical hemolytic uremic syndrome (aHUS). Some reports have indicated that patients with digenic variants in these genes might present severer phenotypes. Upon detecting novel intronic variants, transcriptional analysis is necessary to prove pathogenicity; however, when intronic variants are located in intron 1 and, as a result, no transcript is produced, no appropriate method had been established to reveal the pathogenicity. Recently, the minigene assay was used to assess the pathogenicity of intronic variants. Here, we report an infantile case of aHUS caused by digenic mutations in two different complement-related genes, C3 and MCP. Targeted sequencing detected a known variant in C3 and a novel variant in the intron 1 splicing donor site of MCP. To assess the pathogenicity of this intronic variant, we conducted functional splicing assay using a minigene construct and quantitative PCR analysis of the MCP transcript, revealing the pathogenicity of the intronic variant. In conclusion, the minigene assay revealed the pathogenicity of the intron 1 splicing donor site variant for the first time. This case showed a severe phenotype of infantile-onset aHUS associated with digenic variants in two complement-related genes.
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References
Bresin E, Rurali E, Caprioli J, Sanchez-Corral P, Fremeaux-Bacchi V, Rodriguez de Cordoba S, et al. Combined complement gene mutations in atypical hemolytic uremic syndrome influence clinical phenotype. J Am Soc Nephrol. 2013;24:475–86.
Zhang T, Lu J, Liang S, Chen D, Zhang H, Zeng C, et al. Comprehensive analysis of complement genes in patients with atypical hemolytic uremic syndrome. Am J Nephrol. 2016;43:160–9.
Malone AF, Funk SD, Alhamad T, Miner JH. Functional assessment of a novel COL4A5 splice region variant and immunostaining of plucked hair follicles as an alternative method of diagnosis in X-linked Alport syndrome. Pediatr Nephrol. 2017;32:997–1003.
Nakanishi K, Nozu K, Hiramoto R, Minamikawa S, Yamamura T, Fujimura J, et al. A comparison of splicing assays to detect an intronic variant of the OCRL gene in Lowe syndrome. Eur J Med Genet. 2017;60:631–4.
Tsuji Y, Nozu K, Sofue T, Hara S, Nakanishi K, Yamamura T, et al. Detection of a splice site variant in a patient with glomerulopathy and fibronectin deposits. Nephron. 2018;138:166–71.
Yamamura T, Nozu K, Miyoshi Y, Nakanishi K, Fujimura J, Horinouchi T, et al. An in vitro splicing assay reveals the pathogenicity of a novel intronic variant in ATP6V0A4 for autosomal recessive distal renal tubular acidosis. BMC Nephrol. 2017;18:353.
Nozu K, Iijima K, Kawai K, Nozu Y, Nishida A, Takeshima Y, et al. In vivo and in vitro splicing assay of SLC12A1 in an antenatal salt-losing tubulopathy patient with an intronic mutation. Hum Genet. 2009;126:533–8.
Thi Tran HT, Takeshima Y, Surono A, Yagi M, Wada H, Matsuo M. A G-to-A transition at the fifth position of intron-32 of the dystrophin gene inactivates a splice-donor site both in vivo and in vitro. Mol Genet Metab. 2005;85:213–9.
Tran VK, Takeshima Y, Zhang Z, Habara Y, Haginoya K, Nishiyama A, et al. A nonsense mutation-created intraexonic splice site is active in the lymphocytes, but not in the skeletal muscle of a DMD patient. Hum Genet. 2007;120:737–42.
Yoshida Y, Miyata T, Matsumoto M, Shirotani-Ikejima H, Uchida Y, Ohyama Y, et al. A novel quantitative hemolytic assay coupled with restriction fragment length polymorphisms analysis enabled early diagnosis of atypical hemolytic uremic syndrome and identified unique predisposing mutations in Japan. PLoS ONE. 2015;10:e0124655.
Tran VK, Takeshima Y, Zhang Z, Yagi M, Nishiyama A, Habara Y, et al. Splicing analysis disclosed a determinant single nucleotide for exon skipping caused by a novel intraexonic four-nucleotide deletion in the dystrophin gene. J Med Genet. 2006;43:924–30.
Bergmann C, Frank V, Kupper F, Schmidt C, Senderek J, Zerres K. Functional analysis of PKHD1 splicing in autosomal recessive polycystic kidney disease. J Hum Genet. 2006;51:788–93.
Malone AF, Funk SD, Alhamad T, Miner JH. Functional assessment of a novel COL4A5 splice region variant and immunostaining of plucked hair follicles as an alternative method of diagnosis in X-linked Alport syndrome. Pediatr Nephrol. 2016;32:997–1003.
Takeuchi Y, Mishima E, Shima H, Akiyama Y, Suzuki C, Suzuki T, et al. Exonic mutations in the SLC12A3 gene cause exon skipping and premature termination in Gitelman syndrome. J Am Soc Nephrol. 2015;26:271–9.
Acknowledgements
We thank Edanz Group (www.edanzediting.com/ac) for editing a draft of this manuscript.
Funding
This study was supported by a grant from the Ministry of Health, Labour and Welfare of Japan for Research on Rare Intractable Diseases in the Kidney and Urinary Tract [H24-nanchitou (nan)-ippan-041 to Kazumoto Iijima] in the “Research on Measures for Intractable Diseases” Project, Grants-in-Aid for Scientific Research (KAKENHI) from the Ministry of Education, Culture, Sports, Science and Technology of Japan (subject ID: 15K09691 to Kandai Nozu and 17H04189 to Kazumoto Iijima), and AMED (Grant Number 7930006 to Kandai Nozu and Kazumoto Iijima).
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Yamamura, T., Nozu, K., Ueda, H. et al. Functional splicing analysis in an infantile case of atypical hemolytic uremic syndrome caused by digenic mutations in C3 and MCP genes. J Hum Genet 63, 755–759 (2018). https://doi.org/10.1038/s10038-018-0436-9
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DOI: https://doi.org/10.1038/s10038-018-0436-9
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