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

• Published in: European journal of human genetics : EJHG 2019-03, Vol.27 (3), p.488-493
• Main Authors: Filatova, Alexandra Yu, Vasilyeva, Tatiana A, Marakhonov, Andrey V, Voskresenskaya, Anna A, Zinchenko, Rena A, Skoblov, Mikhail Yu
• Format: Article
• Language: English
• Subjects: Aniridia; Aniridia - genetics; Aniridia - pathology; Brief Communication; Cell Line, Tumor; Genetic Testing - methods; HEK293 Cells; Humans; Loss of Function Mutation; mRNA; Nonsense mutation; Nucleotide sequence; Pax6 protein; PAX6 Transcription Factor - genetics; PAX6 Transcription Factor - metabolism; Polymorphism, Single Nucleotide; RNA Splicing; Splicing
• ISSN: 1018-4813; 1476-5438
• DOI: 10.1038/s41431-018-0288-y

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.