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

Abstract

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.

X‐linked hypohidrotic ectodermal dysplasia: clinical and molecular genetic analysis of a large Russian family with a synonymous p.Ser267= (c.801A>G) splice site mutation

“…2b). 4 The result confirmed that the EDA c.801A>G variant leads to exon 7 skipping. The absence of the exon 7 in the structure of mature EDA mRNA leads to a frame shift resulting in a truncated non-functional EDA protein p.(D265Gfs7*), lacking part of the conserved motifs in the TNF-related domain and the whole cysteine-rich C-terminal domain.…”

“…Only one non-compliant patient required a section due to LS-related scarring. 4 One patient developed LS in a perineal scar. Two women with newly diagnosed LS and who delivered by normal spontaneous vaginal delivery without problems have been reported.…”

“…Incidence of cutaneous adverse events after exposure to tenofovir-emtricitabine in HIVuninfected vs HIV-infected patients: pharmacovigilance within a large Midwestern U.S. patient population from the Research on Adverse Drug events And Reports program Editor Tenofovir disoproxil fumarate (TDF) combined with emtricitabine (FTC) for HIV pre-exposure prophylaxis (PrEP; FDA approval 07/2012) has been shown to dramatically decrease HIV acquisition rates in high-risk populations. [1][2][3][4] TDF-FTC is also used as part of multidrug combination antiretroviral (cART) regimens for treatment of HIV(+) patients (FDA approval 08/ 2004). When TDF-FTC is used as part of cART, cutaneous adverse events (cAEs), including serious cAEs, have been wellreported.…”

The Spectrum of PAX6 Mutations and Genotype-Phenotype Correlations in the Eye

“…The deletions or point mutations in non-translated 5' and 3'UTR of PAX6 have also been reported [75,97,98]. Minigene assays or in silico analysis revealed that most of these variants are likely to affect the normal splicing patterns, which results in the formation of PTCs and the consequent PAX6 haploinsufficiency [99].…”

A Family Case of Congenital Myasthenic Syndrome-22 Induced by Different Combinations of Molecular Causes in Siblings

“…To determine functional significance of the c.2094G>T (p.(Lys698Asn)) variant, we applied the minigene assay as previously described [9]. Exon-of-interest with about 300 bp of flanking intronic sequences was cloned into pSpl3-Flu2 vector.…”