Research Paper Volume 11, Issue 3 pp 933—949

AluYb8 insertion polymorphism in the MUTYH gene impairs mitochondrial DNA maintenance and affects the age of onset of IPF

Figure 3. Association of the AluYb8 insertion with altered protein expression patterns. (A) Schematic representation of the genomic structure of the MUTYH gene. The AluYb8 element is inserted into existing AluSx1 sequences in the MUTYH 15th intron and is clearly marked by a red arrow. AluSx1 sequences are depicted with a bright green arrow. Exons are shown with black boxes. (B) Representative immunoblotting result showing the altered MUTYH protein expression pattern in human lung tissue cells with the mutant genotype (P/P). The two major MUTYH isoforms (MUTYH 1 and MUTYH 2) are indicated. β-actin was used as a protein loading control. The case IDs and their genotypes are shown. (C) AluYb8MUTYH genotyping of experimental cultured cell lines. (D) Schematic representation of the pEGFP cloned constructs with different 5’ exon (1st exon) sequences from the MUTYH gene. The 5’ exon sequences of the α, β and γ MUTYH transcripts are depicted as blue, gray and black boxes, respectively. (EH) Representative immunoblotting results for GFP expression in the reporter gene system. The GFP reporter protein of the recombinant protein with the 5’ exon from the α MUTYH transcript was observed in the A549 (A/A genotype) and HELF (A/P genotype) human cell lines and the C2C12 (mouse myoblasts) cell line but not in human cells (HEK293T) with the mutant (P/P) genotype. GFP reporters from the recombinant vector with the 5’ exon of the β and γ MUTYH transcripts were expressed in all cultured cells. β-actin was used as a protein loading control.