A haplotype variation affecting the mitochondrial transportation of hMYH protein could be a risk factor for colorectal cancer in Chinese

Base excision repair (BER) is a major mechanism for the repair of DNA base damage by reactive oxygen species (ROS)[1]. The most stable product of oxidative DNA damage, 8-oxo-guanine (8-oxoG), tends to mispaire with adenine, which would lead to a transversion of G:C to T:A [2, 3]. The MutY DNA glycosylase initiates the repair pathway by recognizing and removing the adenine misincorporated with 8-oxoG [4]. A series of biochemical studies demonstrate that the E. coli strain lacking MutY is a mutator for G:C to T:A transversions [5, 6]. Moreover, it is recently discovered that the germline mutations of the human MutY homolog (hMYH) increase the susceptibility to develop colorectal cancers (CRC) associated with adenomatous polyposis [7, 8].

Our previous study detected 2 heterozygous base pair substitutions in Chinese, c.53C>T and c.74G>A, in hMYH gene [9]. Further cloning-sequencing showed that the mutations occurred at the same allele (haplotype T/A variation). The frequency of variant allele in suspected hereditary gastric cancer patients was significantly higher than that in the control group, which indicated that the T/A haplotype might form a partial genetic basis for the familial GC susceptibility in Chinese population. Interestingly, similar mutants have only been described in East Asian region. Shinmura et al. [10] has reported the 2 somatic mutations of MYH gene from lung cancer tissues from Japanese. However, no more analysis was made to clarify whether these two occurred on a same allele. Kim et al. [11] identified the germline haplotype T/A variation in patients with familial adenomatous polyposis (FAP) and showed tentative association with the development of FAP in Korean population.

On the other hand, germline mutations of hMYH have been extensively studied as risk factors for sporadic CRC in Caucasian populations [7, 12, 13]. In China, CRC has remained the fifth most common cancer and its morbidity has risen rapidly in recent years [14]. Based on the association regarding hMYH mutation and colorectal tumours, we therefore hypothesized that the haplotype T/A variation might be related with the pathogenesis of CRC in Chinese.

In addition, amino acid sequence analysis of hMYH protein illustrates that the haplotype T/A substitutions is predicted to generate missense mutations of p.Pro18Leu and p.Gly25Asp, respectively, and then mapped near to the functional N-terminal mitochondrial targeting sequences (MTS) domain [15, 16]. This targeting sequence has been widely studied with a focus on mitochondrial transportation of protein, which is required for maintenance of the mitochondrial DNA repair capacity and genome stability [17, 18]. Increasing evidences have suggested that mitochondrial oxidative damage contributed to human diseases, such as Alzheimer's disease, diabetes and cancer [19‐22]. To elucidate the functional consequence of the haplotype T/A variation of hMYH, we constructed the recombinant cDNA with mutations, expressed them in cultured cos-7 cells and checked the mutant protein subcellular localization, and thereby investigated whether the haplotype variant of hMYH gene was associated with human colorectal carcinogenesis.

ROS is the most prevalent source of DNA lesions in aerobic organisms, and mammalian cells have developed several repair pathways to cope with the resultant oxidative DNA damage [28]. The BER protein hMYH is essential in protecting against such damage and inherited defects within the protein lead to predisposition to carcinogenesis in humans [1, 29]. Our previous study have demonstrated that a haplotype variation of hMYH (c.53C>T-c.74G>A, T/A) might be a genetic factor for the gastric cancer susceptibility in Chinese [9].

CRC is one of the major causes for cancer mortality in the world, and is becoming more prevalent in Asian countries [14]. In China, the incidence of CRC was initially low, but in recent years, the rate is increasing due to the changes of life style and nutritional habits. According to the report of the Health Ministry of China in 2002, the incidence rate of CRC is approx 0.037% and is the fifth leading cause of cancer mortality [30]. CRC is a collective term for cancers of the colon and rectum. Most of CRC occurs at colon in Caucasian, while the rectal cancer appears a higher frequency in Chinese. In the present study, the percentage of cancer was 11.6% in the proximal colon, 31.5% in the distal colon, and 56.5% in the rectum, respectively. Although the issue whether colon and rectal cancer should be considered as a single or two distinct entities is still being debated [31], cancers of the colon and rectum share many common features [32] and show little different in our results, and thus were discussed together in this study.

The heterozygous T/A haplotype variant configuration was discovered in 6/138 (4.35%) sporadic CRC cases and 3/343 (0.87%) controls. The frequency of variant T/A haplotype in patients was significantly higher than that in the control group (P = 0.019). It suggested a correlation between such hMYH variation and sporadic CRC susceptibility in Chinese. As oxidative DNA damage plays an important role in the carcinogenesis, we proposed that the variation of the BER gene hMYH might be commonly associated with the processes. It should be pointed out that we could not formally exclude the possibility that selection bias contributed to the observed differences between the two groups, due to a low frequency of this variant allele found.

Eukaryotic cells have nuclear and mitochondrial genomes and thus the cells have necessarily to develop either two distinct repair enzymes or a transport system to deliver the same enzymes into separate organelles [25, 33]. The human MutY homolog comprises the entire MutY sequence flanked by extended N- and C-terminal domains, which are involved in subcellular targeting of hMYH: e.g. MTS and nuclear localization sequence (NLS) to the N- and C-terminus, respectively (Figure 2A) [34]. It suggests that the same hMYH protein presents differential subcellular localizations in human cells.

Several lines of evidence have shown that hMYH protein is a nuclear protein, preventing mutations from oxidative damage in the nuclei [4, 35, 36]. Takao et al. [17] detected that the C-terminus of hMYH was a functional NLS, which could target the heterologous hMYH protein into the nucleus. However, the full-length hMYH (type 1) with both NLS and MTS displayed rather a mitochondrial distribution. An alternative spliced form of hMYH (type 2) missing the first exon, pointed to MTS at N-terminus of hMYH, presented a nuclear localization [37]. It suggested that the MTS could be functionally dominant over the NLS in hMYH transportation. Tsai-Wu et al. [36] once proposed that hMYH was a shuttling enzyme with a steady-state nuclear distribution. Upon induction, presumably elevated oxidative potential in the mitochondria, MTS of hMYH presented its dominant function, and some unidentified mitochondrial factors would recognize this domain and transport hMYH from the nuclei to the mitochondria.

As it has been described above, the type 1 transcription of hMYH is concentrated in the mitochondria in cos-7 cells and recombinant hMYH has essentially similar activities to the partially purified protein [36]. This allows us to analyze the role of variation on MTS domain in the cos-7 cell model. We found that the hMYH protein with both missense mutations, that we detected previously, was dually localized in nucleus and mitochondria, and not focused in the mitochondria any more [38]. It indicated that the mutation affected the ability of hMYH protein to be transported into mitochondria. One rational explanation for it is that the p.Pro18Leu – p.Gly25Asp missense mutation in the N-terminus of hMYH influences the function of MTS domain, and then the MTS domain is difficult to be recognized. As a result, a proportion of the mutative hMYH protein would not be transported into mitochondria, and display the nuclear distribution instead [36]. Interestingly, immunofluorescence showed that the hMYH proteins with single missense mutation, p.Pro18Leu or p.Gly25Asp, remained in the mitochondria, similar to the wild-type protein. It seems that the effect of the mutations are additive and either one of the missense mutations in the haplotype is not enough to impair the function of MTS domain.

Mitochondrial oxidative energy metabolism is the major intracellular source of ROS and mitochondrial biomolecules including mitochondrial DNA are constantly exposed to a high level of ROS [39]. Almost all the studies performed to date have found increased oxidative damage in mitochondrial DNA and it is consistent with the observation that mitochondrial DNA mutations accumulate [40]. Mitochondrial DNA mutations have been reported in 10% to 70% of CRC and the presence of tumour mitochondrial DNA mutations seems to be a prognostic marker and a relevant predictive factor of CRC [21]. On the other hand, mitochondrial DNA repair enzymes involved in BER system play an important role in mitochondrial genome stability and hMYH protein is the requisite enzymes in this system [18, 41, 42]. Thus, it is possible that the haplotype variant allele of hMYH impairs the ability of oxidative damage repair in mitochondria and increases the instability of the mitochondrial genome, thus contributes to the carcinogenesis, including CRC. However, the glycolsylase activation of hMYH protein in mitochondria has not been directly analyzed in this study. Another point should be mentioned that, as no homozygous variation was detected in the population investigated, the impact of the heterozygous variant genotype on the hMYH gene will be explored in the following study.