Abstract
Objective To perform gene mutation splicing analysis on 12 suspected pathogenic MMUT/MUT gene mutation sites to verify the pathogenicity of the mutation.
Methods Wild-type and mutant minigenes were inserted into pcMINI vector, and total 5 wild-type recombinant vectors and 12 mutant recombinant vectors were constructed. The total RNA in 293T cells was extracted after the recombinant vectors were transfected into 293T cell line. Then PCR products were detected by agarose gel electrophoresis and analyzed by sequencing.
Results RT-PCR and sequencing results showed that among 12 mutations, 9 mutations (c.419T>C, 469G>T, c.470T>A, c.626dupC, c.693C>G, c.976A>G, c.1009T>C, c.1777G>T and c.1874A>C) did not affect the gene splicing, and the other 3 mutations (c.454C>T, c.421G>A and c.2125-3C>G) all affected mRNA splicing.
Conclusion In recent case reports of MMUT/MUT gene mutation sites, variant of uncertain significance (VUS) variation is very common. In this study, the pathogenicity of three mutation sites is confirmed by the mini-gene method.
Competing Interest Statement
The authors have declared no competing interest.
