Illustration of the AluYb8 insertion in <i>MUTYH</i> gene and splicing analysis on this variant.

<p>(A) Schematic representation of the genomic structure of <i>MUTYH</i> from exon 15 to 16 indicates the position of the AluYb8 insertion in intron 15, depicted as a deep blue arrow. Exons are shown by boxes. The genotyping primers are represented by black arrowheads, and the lengths of their PCR products are also depicted. (B) A schematic illustration of the alternative splicing pattern between exons 14 and 16 of the <i>MUTYH</i> gene. Scores (MaxEnt score) of splice sites for the three exons were recorded in red. An alternatively spliced cassette exon within intron 15 was inferred from aligning two ESTs (BM679345.1 and AW518294.1) with human genomic DNA, and the putative cassette exon in the variant intron 15 is shown. (C) Splicing assays were performed in healthy adults. A representative ethidium bromide-stained agarose gel separating the RT-PCR products spanning <i>MUTYH</i> exon 14 to exon 16 are shown. PCR products were confirmed by sequencing (the bottom of the agarose gel electrophoresis). (D) The constructed wild-type (wt) and mutant minigene plasmids were transfected into 293 T cells, respectively, total RNA was extracted, and splicing products were separated on a 2% agarose gel after RT-PCR analysis. PCR products were sequenced. Similar results were obtained in two independent experiments.</p

Analysis of the association between the <i>AluYb8MUTYH</i> and MUTYH expression.

<p>(A) Transcriptional analysis by qrtPCR of <i>MUTYH</i> targets in PBMCs from healthy adult individuals. The abundance of <i>MUTYH</i> targeted transcripts normalized to <i>GAPDH</i> gene are shown, and the values are expressed as fold changes relative to mean mRNA levels of each type of targeted transcripts in the <i>A/A</i> genotype group. Error bars indicate the standard error of the mean. (B) Representative immunoblot for anti-MUTYH antibody (BS2535, Bioworld Technology, Inc.) showing the protein expression of MUTYH and GAPDH in PBMCs whole cell extracts from seven healthy adult individuals. This panel shows the altered pattern of MUTYH expression in PBMCs from the homozygous <i>P/P</i> individuals. The two major MUTYH types, type 1 and type 2, are indicated. GAPDH was used as a protein loading control. The individual IDs are shown. (C) MUTYH quantification. Densitometric quantification of the bands was performed with Image J software (NIH) and normalized to the GAPDH signal. Type 1 MUTYH protein levels are expressed as fold values relative to GAPDH expression. Solid circles represent the checked individuals in the protein expression analysis. <i>P</i>-values are indicated, by Kruskal-Wallis test.</p

The Polymorphic AluYb8 Insertion in the <i>MUTYH</i> Gene is Associated with Reduced Type 1 Protein Expression and Reduced Mitochondrial DNA Content

<div><p>The human <i>mutY homolog</i> (<i>MUTYH</i>) participates in base excision repair (BER), which is critical for repairing oxidized DNA bases and maintaining DNA replication fidelity. The polymorphic AluYb8 insertion in the 15^th intron of the <i>MUTYH</i> gene (<i>AluYb8MUTYH</i>) has been shown to associate with an aggregated 8-hydroxy-2′-deoxyguanosine (8-OH-dG) lesion in genomic DNA and to serve as a risk factor for age-related diseases. In this work, we demonstrate that this variant is associated with a significant reduction of the type 1 MUTYH protein that localizes to mitochondria. Notably, this variant affects mitochondrial DNA (mtDNA) maintenance and functional mitochondrial mass in individuals homozygous for the <i>AluYb8MUTYH</i> variant. These findings provide evidence for an association between the <i>AluYb8MUTYH</i> variant and decreased mitochondrial homeostasis and, consequently, contribute to elucidating the roles of the <i>AluYb8MUTYH</i> variant in impairing the mitochondrial base excision repair (mtBER) system and increasing the risk of acquiring an age-related disease.</p></div

The alteration of mitochondria phenotypes associated with the <i>AluYb8MUTYH</i>.

<p>(A, B) The leukocytic mtDNA content was associated with the <i>AluYb8MUTYH</i> variant. The relative amounts of mt-CO 1 and mt-tRNA^Leu content in the newborn, young, middle-aged and aged groups are shown. The mean mtDNA/nDNA ratio in the <i>A/A</i> genotype group of healthy newborn infants was set to 1. Error bars indicate the standard error of the mean. The number of participants is shown in brackets. <i>P</i>-values are indicated (multinomial logistic regression). (C) The respiring mitochondria mass in cultured cells (passage number 5) of C:2, C:3 and C:5 was detected by flow cytometry. The results were confirmed in triplicate, and similar results were obtained when other cultured fibroblast-like cells with different <i>AluYb8MUTYH</i> genotypes were used. Error bars represent the mean ± SD with n = 3. The geometric mean value of fluorescence intensity in the <i>A/A</i> cells was set to 1. <i>P</i>-values are indicated (2-sided t-test). (D) The respiring mitochondria mass were confirmed in cultured cells by confocal microscopy. Representative images from independent experiments are shown. DAPI (4′, 6-diamidino-2-phenylin-dole, Sigma) nuclear staining is shown. Scale bar, 20 µm. Five images of each fibroblast-like cell culture, with the indicated genotype, from the same experiment were quantified. Fluorescence was quantified with Image J software (NIH). A plot of the relative values of the respiring mitochondria levels is displayed in the lower right corner. The geometric mean of the fluorescence intensity in the <i>A/A</i> cells was set to 1. The values are presented as the mean ± SD (n = 5). <i>P</i>-values are indicated (2-sided t-test). The result was confirmed in triplicate. (E) Relative oxygen consumption capacity of intact cells (<i>P/P</i> and <i>A/A</i> cells). The oxygen consumption rate in <i>A/A</i> cells was set to 1. The data are the mean ± SD from at least two independent experiments. <i>P</i>-value is indicated (2-sided t-test).</p

Expression of type 1 MUTYH protein in the mitochondria of cultured fibroblast-like cells.

<p>Mitochondrial fractions were prepared from cultured fibroblast-like cells (passage number 4). The genotypes of the cultured cells and their corresponding cell IDs are indicated. The MUTYH signal for anti-MUTYH antibody (BS2535) was normalized to COX IV loading control by Image J software (NIH), and the mitochondrial MUTYH percentage (Rel. MUTYH) is indicated at the bottom of each column. COX IV, cytochrome c oxidase subunit IV. HSP 60, heat shock protein 60. PCNA, proliferating cell nuclear antigen.</p

MUTYH protein expression detected by immunoblot analysis with MUTYH antibody (sc-25169).

<p>The two major MUTYH proteins, type 1 and type 2, are indicated. GAPDH was used as a protein loading control. The <i>AluYb8MUTYH</i> genotypes and individual IDs are shown, respectively.</p

The relative amplification of long-range targets among the cells with different <i>AluYb8MUTYH</i> genotypes.

<p>The long-range PCR was performed for both the mitochondrial DNA (A, open bars) and nuclear β-globin fragments (B, solid bars) among the healthy individuals aged 22 to 44 with different <i>AluYb8MUTYH</i> genotypes. The boxes cover the 25^th to 75^th percentiles, and the minimal and maximal values are shown by the ends of the bars. Relative amplification is presented relative to average of <i>A/A</i> group values, n = 18. <i>P</i>-values are indicated, multinomial logistic regression.</p