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Abstract: TH-PO609

Functional Splicing Analysis in an Infantile Case of Atypical Hemolytic Uremic Syndrome Caused by Digenic Mutations in C3 and MCP Genes

Session Information

Category: Nephrology Education

  • 1302 Fellows and Residents Case Reports

Authors

  • Yamamura, Tomohiko, Kobe University Graduate School of Medicine, Kobe, Japan
  • Nozu, Kandai, Kobe University Graduate School of Medicine, Kobe, Japan
  • Nakanishi, Keita, Kobe University Graduate School of Medicine, Kobe, Japan
  • Fujimura, Junya, Kobe University Graduate School of Medicine, Kobe, Japan
  • Minamikawa, Shogo, Kobe University Graduate School of Medicine, Kobe, Japan
  • Ueda, Hiroaki, Osaka City General Hospital, Osaka, Japan
  • Fujimaru, Rika, Osaka City General Hospital, Osaka, Japan
  • Shima, Yuko, Wakayama Medical University, Wakayama City, Japan
  • Nakanishi, Koichi, Graduate School of Medicine, University of the Ryukyus, Nishihara-cho, Japan
  • Kaito, Hiroshi, Kobe University Graduate School of Medicine, Kobe, Japan
  • Iijima, Kazumoto, Kobe University Graduate School of Medicine, Kobe, Japan
Background

Atypical hemolytic uremic syndrome (aHUS) is a heterogeneous disease that is caused by defective complement regulation as reported in over 50% of cases. Up to now, pathogenic variants have been identified in various compliment related genes. Some reports also indicated that patients of aHUS with digenic inheritance of these genes might present more severe phenotype than monogenic inheritance.
In addition, generally, transcript analysis is necessary for variants located outside of the splicing consensus sequence to assess the biological effect. However, this technique is often difficult for the influence of nonsense-mediated mRNA decay (NMD) for the products of truncating variants and quantity of mRNA of sample.

Methods

Here we report an infantile case with aHUS from unrelated parents. Targeted resequencing detected a reported variant of C3 gene and a novel intronic variant of MCP gene (c.97 + 5 G>A, IVS1 + 5 G>A), on maternal and paternal alleles respectively. These variants were thought to be working digenically since there was early onset in this patient although the parents were asymptomatic. However, the pathogenicity of a variant in MCP gene was unknown because this is a novel variant and located outside apparent splice consensus sequence. To assess the pathogenicity of a novel intronic variant of MCP gene, we tried to detect abnormal splicing variant by standard transcriptional analysis using mRNA extracted from peripheral blood. However, we obtained only normal splicing variant from maternal allele and transcript from paternal allele was missing. Then, we conducted functional splicing assay using minigene construction to detect abnormal splicing caused by c.97 + 5 G>A variant and quantitative mRNA PCR to confirm the result. As a result, it was revealed that the paternal allele of MCP gene with c.97 + 5 G>A variant did not produce any transcript as confirmed by qPCR and minigene splicing assay. These results lead us to conclude MCP gene c.97 + 5 G>A was pathogenic.

Conclusion

A combination of minigene assay and quantitative analysis is non-invasive and useful as methods for functional splicing assay of inherited diseases even if standard transcriptional analysis could not detect abnormal splicing.