Oncogene (2004) 23, 6820–6822
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Genetic alterations of the MYH gene in gastric cancer Chang Jae Kim1, Yong Gu Cho1, Cho Hyun Park2, Su Young Kim1, Suk Woo Nam1, Sug Hyung Lee1, Nam Jin Yoo1, Jung Young Lee1 and Won Sang Park*,1 1
Department of Pathology, College of Medicine, The Catholic University of Korea, 505 Banpo-dong, Seocho-gu, Seoul 137-701, Korea; 2Department of Surgery, College of Medicine, The Catholic University of Korea, 505 Banpo-dong, Seocho-gu, Seoul 137-701, Korea
Increased oxygen free radicals produced in gastric mucosa by H. pylori induce DNA damage and lead to dyspalsia and gastric cancers. However, only a small percentage of individuals that carry H. pylori develop gastric cancer, indicating that other factors are involved. We have screened a set of 95 sporadic gastric cancers for mutations and allele loss of the DNA glycosylase MYH gene, which excises adenine misincorporated opposite unrepaired 8oxoG. Two of 95 cancers had bi-allelic mutations of the MYH gene with somatic missense mutation of one allele and loss of the remaining allele. The mutations were missense mutations, P391S and Q400R, in exon 13 encoding NUDIX hydrolase domain of the gene. The patients were H. pylori-positive and the tumors were of advanced intestinal-type gastric cancer with lymph node metastasis. In addition, 4 (17.4%) of 23 informative cases showed allele loss at MYH locus. Therefore, our data suggest that somatic mutations of base excision repair gene MYH contribute to the development of a sub-set of sporadic gastric cancers. Oncogene (2004) 23, 6820–6822. doi:10.1038/sj.onc.1207574 Published online 26 July 2004 Keywords: somatic mutation; stomach cancer; loss of heterozygosity; DNA repair
The gastrointestinal tract is constantly subject to traumas from ingested food particles and various noxious agents, including carcinogens, like H. pylori. It has been suggested that H. pylori infection is the single most important factor in determining oxidative DNA damage, as assessed as 8-hydroxydeoxyguanosine levels (Farinati et al., 1998). In addition, reactive oxygen species production with damage to macromeolcules, including DNA, may be important to gastric cancer development in the patients with H. pylori infection (Baik et al., 1996; Danese et al., 2001). The most stable product of oxidative DNA damage, 8-oxo-7,8-dihydro-20 -deoxyguanosine (8-oxoG), tends to mispair with adenine, which leads to an increased *Correspondence: WS Park; E-mail:
[email protected] Received 30 September 2003; revised 22 December 2003; accepted 19 January 2004; published online 26 July 2004
frequency of G:C-T:A transversion mutation in repair-deficient bacteria and yeast cells (Nghiem et al., 1988; Shibutani et al., 1991; Michaels and Miller, 1992; Moriya and Grollman, 1993). However, these transversion mutations caused by 8-oxoG are kept at low level in normal growing cells (Mo et al., 1991). In E. coli, the enzymes mutM, mutY and mutT function synergistically to protect cells from the deleterious effects of guanine oxidation. Of these, the DNA glycosylase mutY excises adenine misincorporated opposite unrepaired 8oxoG during replication (Lindahl, 1993). Interestingly, increased level of 8-oxoG was frequently seen in H. pylori-associated gastritis and several human cancers, including lung cancers, breast cancers and renal cell carcinoma (Malins and Haimanot, 1991; Jaruga et al., 1994; Okamoto et al., 1994; Noguchi et al., 2002). Thus, we hypothesized that the deficiencies of base excision repair pathway cause transversion mutations caused by reactive oxygen species and that they play an important role in the development of human cancer. Recently, Al-Tassan et al. (2002) found an autosomal recessive condition comprising multiple colorectal adenomas and cancers. They reported that three affected sibling from a single UK family carried two missense variants, Y165C and G382D, in the MYH gene. These mutations affect residues that are conserved in mutY of E. coli. Additionally, tumors from affected persons displayed excess somatic transversions of a G:C to T:A in the APC gene that is closely associated with familial adenomatous polyposis and sporadic colorectal cancers. Thus, it is possible that the repair defect of the MYH gene might lead to an increased susceptibility to human cancer. However, somatic inactivation of the MYH gene has not been found to date in sporadic cancers. In the present study, to investigate whether somatic changes of the MYH gene are involved in gastric carcinogenesis, we screened 95 sporadic gastric cancers for mutation and allelic loss of the MYH gene. A total of 95 methacarn-fixed paraffin-embedded gastric cancer specimens were obtained from the College of Medicine, The Catholic University of Korea in Seoul, Korea. Informed consent was obtained from every patient. No patient had a family history. We searched for potential mutations in all 16 exons for the MYH gene in 95 gastric cancer tissues by PCR-SSCP and sequencing analysis. Direct sequencing of aberrantly
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migrating band on SSCP gel led to the identification of mutation in two (2.1%) of the gastric cancer examined. The mutations were missense mutations, CCG-TCG (Pro-Ser) at codon 391 and CAG-CGG (Gln-Arg) at codon 400 in exon 13 encoding NUDIX (Nucleoside Diphosphate linked to some other moiety X) hydrolase domain (codon 366–497) of the gene (Figure 1). However, there was no mutation in corresponding normal DNAs of these cases, indicating that the mutations detected in the cancer cells had risen somatically. Interestingly, direct inspection of the SSCP gel showed clear loss of the wild-type MYH allele in both of the cases with mutations, indicating either a homozygous mutation or hemizygous mutation with allelic loss (Figure 1). Clinically, the patients with the mutation were of advanced intestinal-type gastric cancers with lymph node metastasis. Our patients with bi-allelic MYH mutations showed absence of polyp in colon and rectum. Unexpectedly, there was no sequence variation in codon 165, 324, 382 and 501 in our patients. To ensure the specificity of the results, we repeated the experiment three times including PCR-SSCP and
Figure 1 Representative results showing SSCP and sequencing analyses of the MYH gene in gastric cancer. We selectively procured tumor cells from H&E stained slides using a laser microdissection device (IONLMD, Biokhan Co., Seoul, Korea) and inflammatory or surrounding normal mucosal cells for corresponding normal DNAs from the same slides in all cases. DNA extraction was performed by a modified single-step DNA extraction method, as described previously (Lee et al., 1998). Genomic DNAs each from cancer cells and corresponding normal cells were amplified with 16 sets of primers covering the entire coding region (exons 1–16) of the MYH gene, which were described previously (Al-Tassan et al., 2002). Numbering of DNA of the MYH was carried out with respect to the ATG start codon according to the Slupska et al. (1996). Each PCR reaction was performed under standard conditions, as previously described (Lee et al., 1998). (a and b) SSCP of DNAs from tumors of case No. 30 and No. 69 shows only two aberrant bands without any wild-type bands as compared to SSCP from corresponding normal tissue. The mutations were missense mutations, CCG - TCG (Pro Ser) at codon 391 (a) and CAG - CGG (Gln - Arg) at codon 400 (b) in exon 13 of the gene
sequencing. We also performed direct sequencing with fresh PCR product and the data were consistent. We also analysed allelic loss of the MYH with microsatellite marker D1S2677, which is located B2.5 kb from the MYH locus. Of 95 gastric cancers, seven (7.4%) showed microsatellite instability at this marker (data not shown). In all, 23 (26.1%) of 88 cases without microsatellite instability were informative and four (17.4%) of them showed allelic loss (data not shown). One of the cases with MYH mutation showed LOH at this locus, suggesting bi-allelic inactivation through missense mutation of one allele and loss of remaining allele. Unfortunately, the other case was noninformative at this marker, but the absence of wildtype band on both SSCP gel and direct sequencing led us interpret the allelic status as either a homozygous mutation or hemizygous mutation with allelic loss. It is well known that H. pylori infection could induce DNA damage throughout the production of free radicals generated by polymophonuclear cells recruited at the mucosal site. When H. pylori is incubated with epithelial cells, direct damage to host-cell DNA occurs through the synthesis of oxygen species, as reflected by the formation of DNA adducts (Baik et al., 1996; Smoot et al., 2000). Especially, patients with cagA-positive H. pylori develop DNA damage in the gastric mucosa earlier in life, in association with more extensive gastric mucosal derangement (Maaroos et al., 1999), explaining the greater risk of gastric cancer in H. pylori infected patients. The enzyme encoded by the E. coli mutY gene is a component of base excision repair system and works together with OGG1 to correct A/G and A/C mismatches (McGoldrick et al., 1995). Mutations of the mutY and OGG1 genes lead to increased transversion of G:C to T:A (Nghiem et al., 1988; Thomas et al., 1997). Interestingly, it was recently discovered that biallelic mutations in the DNA glycosylase MYH lead to an autosomal recessive syndrome of adenomatous colorectal polyposis and high colorectal cancer risk (Cheadle and Sampson, 2003). In the present study, we found two missense mutations (2.1%), P391S and Q400R, which resulted in nonconservative amino-acid substitution of the MYH gene (Figure 1). Both of the mutations were somatic mutation and confined in the highly conserved NUDIX hydrolase domain of the gene (www.ensembl.org). Interestingly, the cases with the mutation showed only aberrant mutant bands on SSCP gel without wild-type allele (Figure 1), indicating bi-allelic inactivation of MYH by either a homozygous mutation or hemizygous mutation and loss of wild-type allele. Although we did not examine the adenine glycosylase activity of these mutant proteins, it is likely that the patients with the mutation of MYH might have defect in base excision repair after exposure to reactive oxygen species and may be susceptible to gastric cancers. Clinically, both of the tumors with MYH mutation were of advanced intestinal-type gastric cancer with lymph node metastasis. The serum level of antibody for H. pylori Immunoglobulin G in ELISA test showed Oncogene
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positivity for the H. pylori infection. Thus, it is possible that long-standing chronic gastritis by H. pylori infection produced oxygen free radicals and that the defect in DNA repair activity of the MYH accelerated the transformation of the gastritis to advanced gastric cancer. Recently, Halford et al. (2003) have reported that no somatic MYH mutations were found and mRNA and protein were expressed in colorectal cancer cell lines, suggesting that somatic inactivation of the DNA glycosylase does not involve in colorectal tumorigenesis. However, we found two somatic mutations in hydrolase domain of the MYH gene. Several observations support the possibility that defective MYH enzyme could result in the development of gastric cancer; (1) the gastrointestinal tract is constantly subject to traumas from ingested food particles and various noxious agents, including H. pylori; (2) H. pylori infection is the single most important factor in high frequency of oxidative DNA damage; (3) levels of 8-oxoG was significantly higher in H. pylori-positive gastric mucosa than in negative tissues (Noguchi et al., 2002); (4) inherited
defects of the MYH predispose to multiple colorectal adenomas and carcinoma (Jones et al., 2002; Sieber et al., 2003); (5) It has been expected that B1–3% of all colorectal cancers would result from MYH change (Halford et al., 2003). In this study, somatic mutations of the MYH were found in 2.1% of gastric cancer; and (6) Gastric cancer was developed in the patients with inherited mutation of MYH gene (Sampson et al., 2003). Even though the majority of sporadic gastric cancer in this study did not show mutations of the MYH, we conclude that somatic inactivation of the MYH contribute to the development of sub-set of gastric cancers. Additional studies of large populations and on other cancers are needed to verify these initial observation, and functional analysis of mutations identified in this study will broaden our understanding not only of the pathogenesis of gastric cancer but also of gastric mucosal protection mechanism from oxidative injury. Acknowledgements This work was supported by the Ministry of Health and Welfare of Korea (02-PJ1-PG10-20699-0004).
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