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Functional reassessment of PAX6 single nucleotide variants by in vitro splicing assay

European Journal of Human Genetics (2018) | Download Citation


Nucleotide variants that disrupt normal splicing might be the cause of a large number of diseases. Nevertheless, because of the complexity of splicing regulation, it is not always possible to accurately predict the effect of nucleotide sequence changes on splicing events and mRNA structure. Thereby, a number of newly identified nucleotide variants are falsely classified as VUS (a variant of uncertain significance). In the present study we used the minigene assay to analyze the functional consequences of six intronic (c.142−5T>G, c.142−14C>G, c.142−64A>C, c.141+4A>G, c.1032+ 6T>G, c.682+4delA), one missense (c.140A>G) and one synonymous (c.174C>T) variants in the PAX6 gene found in patients with congenital aniridia. We revealed that all except one (c.142−64A>C) variants lead to the disruption of normal splicing patterns resulting in premature termination codon formation followed by mRNA degradation through the nonsense mediated decay pathway. This produces a null allele of the PAX6 gene. That allowed us to reclassify the analyzed variants as loss-of-function and to establish their functional role.

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We appreciate and thank the patients and their families for participating in this study. We are also grateful to Galina Gening, the President of “Interregional Support Center for Patients with Aniridia “Iris” for cooperation and coordination of most patients’ efforts. This study was supported by Russian Foundation for Basic Research (RFBR) grant 17-04-00475.

Author information


  1. Research Centre for Medical Genetics, Moscow, Russian Federation

    • Alexandra Yu. Filatova
    • , Tatiana A. Vasilyeva
    • , Andrey V. Marakhonov
    • , Rena A. Zinchenko
    •  & Mikhail Yu. Skoblov
  2. Moscow Institute of Physics and Technology (State University), Moscow, Russian Federation

    • Andrey V. Marakhonov
    •  & Mikhail Yu. Skoblov
  3. Cheboksary branch of S. Fyodorov Eye Microsurgery Federal State Institution, Cheboksary, Russian Federation

    • Anna A. Voskresenskaya
  4. Pirogov Russian National Research Medical University, Moscow, Russian Federation

    • Rena A. Zinchenko


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The authors declare that they have no conflict of interest.

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Correspondence to Alexandra Yu. Filatova.

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