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 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

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

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

Aberrant EXT2 splicing transcripts with premature termination codon.

<p>(A) Screenshot of a CRYP-SKIP output. The input EXT2 sequences included exon 4 (upper) and the flanking intronic sequences (lower). The table on the left lists the summarized values of the predictor variables used in CRYP-SKIP (PESS, putative exonic splicing silencers; NN 5'ss, neural network 5' splice sites; SF2/ASF, the most important SR protein for aberrant splice-site activation, FAS-ESS, ESSs discovered by a fluorescence-activated screen; EIE, exon and intron identity element). PCR-E (shown in light blue) for the mutated sequence is 0.23 in favor of exon skipping. The red vertical mark in the sequence indicates the predicted cryptic donor splice site. The blue vertical mark indicates the predicted acceptor site. (B) BDGP prediction. The red vertical mark indicates the authentic splice sites. The vertical mark indicates the predicted cryptic 5' splice sites with scores >0.90. The blue vertical mark shows the decoy splice site that was confirmed by PCR. (C) Electrophoresis of RT-PCR products derived from the proband, affected individuals, and the controls. Lane 1: DNA marker; Lanes 2, 4, 6: proband and cases; Lanes 3, 5: controls. (D) Direct sequencing of RT-PCR products. Arrow shows the heterozygous insertion of one cryptic splice site 5 bp downstream of the original splice donor site. (E) Clone sequencing of RT-PCR products. The mutant mRNA sequence (c.743+1G>A) has an ATAAG insertion (arrow) compared with the wild-type (WT). (F) Schematic representation of wild-type and aberrant mRNA transcripts. In the wild-type sequence (WT) splicing occurred at the authentic splice sites. In the mutant mRNAs, splicing occurred at the decoy splice site in intron 4 the five additional nucleotides (ATAAG) of intron4 were inserted, which generated the premature termination codon UAA.</p

Mutation analysis and identification of EXT2.

<p>(A) DNA sequences of the EXT2 gene. Arrows indicate the heterozygous G to A transition site in intron 4 of EXT2 from the proband (upper) and one affected individuals (lower). The mutation was not detected in normal family members or in the healthy controls of the same ethnic origin (control, middle). (B) Alignment of EXT2 gene sequences from 43 species. Conservative character analysis indicated that the G residue (shown in red) at the first position of intron 4 was a highly conserved splicing donor site.</p

Decay of mutant EXT2 mRNA and protein.

<p>(A) Levels of EXT2 mRNA by RT-PCR. The mRNA levels were higher in the patients than that in the controls (<i>P</i> = 0.016 in a two-sided Student's t-test). (B) Clone sequencing of wild-type and mutant transcripts. Among the 32 randomly picked clones, 27 (84.4%) were identified as wild-type by direct sequencing, while only five (15.6%) were identified as mutant transcript. (C) Western blots of wild-type and mutant proteins. The band for the predicted truncated protein could not be detected in the HME patients. (D) Comparison of EXT2 protein levels in HME patients and controls. The levels were significantly lower in the patients compared with the controls (<i>P</i> = 0.006). (E) Comparison of EXT1 mRNA expression levels in HME patients and controls. Real-time PCR revealed that EXT1 mRNA was more highly expressed in the patients compared with the controls (<i>P</i> = 0.024). (F) Comparison of EXT1 protein levels in HME patients and controls. The EXT1 levels higher in the patients compared with the controls (<i>P</i> = 0.003). (G) Comparison of HS proteoglycan expression in HME patients and controls. A group of HS proteoglycans around 70 kDa (upper box) were detected in patients and controls; while two HS proteoglycans patterns around 40 kDa and 25–30 kDa (middle and lower boxes) were detected only in the patients.</p