Evidence of pathogenic role an intronic variant in PAX6 gene using functional analysis

Congenital aniridia (OMIM #106210) (ВА) is a Mendelian autosomal dominant panocular disorder with complete penetrance and variable expressivity. The incidence of aniridia is 1 in 45,000-100,000 births. Aniridia is characterized by congenital absence of the iris with foveal hypoplasia and other eye abnormalities. The most cases of aniridia is caused by heterozygous mutations in the PAX6 gene or chromosome 11p13 rearrangements. Molecular analysis (including Sanger sequencing as well as MLPA analysis and the loss of heterozygosity analysis) of 110 patients referred with congenital aniridia from 84 unrelated families identifies causative PAX6 mutations in all except 3 patients (81/84). A significant proportion of point PAX6 mutations affecting canonical splicing sites and deeper intronic sequences (13,6%) (11 different mutations in 18 patients) have been revealed. Intron sequence variants pathogenic status is established in accordance with ACMG recommendations for interpretation of sequence variants. The manuscript gives consideration to the functional evidence of a revealed in patient with congenital aniridia VUS (variant of unknown clinical significance) NG_008679.1(PAX6_v001):c.142-14C>G effect on the splicing pattern. We used a minigene system to determine the effect of investigated mutation in vitro in human cell line HEK293. Our analysis has shown that this single nucleotide substitution leads to a splicing disruption due to the use of the new acceptor-site of intron 5. That leads to exon 6 elongation by 13 nucleotides, open reading frame shifting (NM_000280.4(PAX6_v001):c.141_142insTTCCCCTATGCAG, p.Val48PhefsTer12) and aberrant mRNA degradation by NMD (nonsense-mediated mRNA decay) mechanism. Thus, the intron sequence variant produces a null allele, leading to a haploinsufficiency of the PAX6 -function and the development of disease.

врожденная аниридия, гаплонедостаточность, PAX6, мутации, приводящие к нарушению сплайсинга, функциональный анализ, клонирование, система экспрессии минигенов in vitro, ОТ-ПЦР, HEK293, congenital aniridia, PAX6, haploinsufficiency, splicing mutations, variant of unknown clinical significance, functional evidence, cloning, in vitro mini-gene expression system, RT-PCR, HEK293

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