A novel splice-site variant of the base excision repair gene MYH is associated with production of an aberrant mRNA transcript encoding a truncated MYH protein not localized in the nucleus
Hong Tao1,
Kazuya Shinmura1,
Tomoyuki Hanaoka2,
Syusuke Natsukawa3,
Kozo Shaura4,
Yoichi Koizumi5,
Yoshio Kasuga6,
Takachika Ozawa7,
Toshimasa Tsujinaka8,
Zhongyou Li1,
Satoru Yamaguchi9,
Jun Yokota9,
Haruhiko Sugimura1,10 and
Shoichiro Tsugane2
1 First Department of Pathology, Hamamatsu University School of Medicine, 1-20-1 Handayama, Hamamatsu, Shizuoka 431-3192, Japan, 2 Epidemiology and Prevention Division, Research Center for Cancer Prevention and Screening, National Cancer Center, Tokyo 104-0045, Japan, 3 Saku General Hospital, 197 Usuda, Usudamachi, Minamisakugun, Nagano 384-1301, Japan, 4 Hokushin General Hospital, 1-5-63 Nishi, Nagano 383-0022, Japan, 5 Shinonoi General Hospital, 666-1 Shinonoiai, Nagano 388-8004, Japan, 6 Nagano Matsushiro General Hospital, 183 Matsushiro, Nagano 381-1231, Japan, 7 Department of Pathology, Hamamatsu Medical Center, 328 Tomitsuka-cho, Hamamatsu, Shizuoka 432-8580, Japan, 8 Department of Surgery, Osaka National Hospital, 2-1-14, Hoenzaka, Chuo-ku, Osaka 540-0006, Japan and 9 Biology Division, National Cancer Center Research Institute, 1-1, Tsukiji 5-chome, Chuo-ku, Tokyo 104-0045, Japan
10 To whom correspondence should be addressed Email: hsugimur{at}hama-med.ac.jp
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Abstract
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The MYH gene encodes a DNA glycosylase involved in the excision repair of adenines paired with 8-hydroxyguanines, a major component of oxidative DNA damage, and bi-allelic germline MYH mutations have been reported to predispose individuals to multiple colorectal adenomas and carcinoma. To determine whether the MYH gene is involved in gastric carcinogenesis, we examined blood specimens from 20 Japanese familial gastric cancer (GC) patients for MYH mutations by polymerase chain reactionsingle-strand conformation polymorphism (PCRSSCP) analysis followed by direct sequencing. Bi-allelic germline MYH mutations were not found in any of the specimens, but in addition to four known variants, a novel splice-site variant, IVS10-2A > G (c.892-2A > G), was found in two patients as its heterozygote. Reverse transcriptionPCR analysis revealed that the IVS10-2A > G variant caused the production of an aberrant mRNA transcript encoding a truncated MYH protein. Immunofluorescence analysis showed that the wild-type MYH protein, but not the variant-type, is localized in the nucleus. We then searched for the IVS10-2A > G variant in 128 digestive tract cancer patients by PCR with confronting two-pair primers, and eight cancers from six patients with the IVS10-2A/G genotype were identified. However, no other germline MYH mutations or inactivation of the remaining wild-type allele was detected. We next tested the presumed correlation of the IVS10-2G allele with GC risk in a case-control study of 148 GC cases and 292 controls, but no significant difference in the distribution of the IVS10-2A > G variant was found between the cases and controls. Interestingly, the homozygote for the IVS10-2G allele was found in one GC case, but not in any controls. These results suggested that the ability to repair 8-hydroxyguanine in nuclear DNA may differ among Japanese individuals due to the splicing abnormality based on the MYH IVS10-2A > G variant, and that the bi-allelic IVS10-2A > G variation may be responsible for the occurrence of GC.
Abbreviations: GC, gastric cancer; LOH, loss of heterozygosity; NLS, nuclear localization signal; oh8G, 8-hydroxyguanine; OR, odds ratio; PBGD, porphobilinogen deaminase; PCR, polymerase chain reaction; PCRCTPP, PCR with confronting two-pair primers; QRTPCR, quantitative real-timePCR; RTPCR, reverse transcriptionPCR; SSCP, single-strand conformation polymorphism
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Introduction
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8-Hydroguanine (oh8G) is one of the most mutagenic products of oxidative DNA damage, and it can pair with adenine in double-stranded DNA during DNA replication. If the mispairing is not repaired, it leads to a G:C to T:A transversion mutation in cells. A base excision repair (BER) pathway is involved in the repair of oh8G to prevent such mutations, and the DNA glycosylases encoded by the OGG1 gene and the MYH gene initiate this repair pathway by recognizing and removing oh8G and the adenine paired with oh8G, respectively, by hydrolyzing the N-glycosidic bond (14). It was recently discovered that the bi-allelic germline mutations of the MYH gene lead to autosomal recessive colorectal adenomatous polyposis and very high colorectal cancer risk in a Caucasian population (510), and colorectal tumors from the affected individuals displayed a significant excess of somatic G:C to T:A mutations in the APC gene. Based on these findings regarding colorectal tumors, we hypothesized: (i) that bi-allelic MYH mutations are involved in the pathogenesis of tumors in other organs in Japanese individuals and (ii) that the MYH variants associated with low repair activity act as low penetrance susceptibility alleles, as shown in the APC and CHK2 gene variants (11,12).
The incidence of gastric cancer (GC) is high in Japan, and familial aggregation of GC is not very rare, however, the deterministic genetic cause has not been identified clearly. Although E-cadherin germline mutations have been reported to be associated with the occurrence of the diffuse-type GC in GC families of New Zealand Maori, European and African-American origins (13,14), only the missense mutations of the E-cadherin gene have been found in Japanese familial GC cases (1517). The gastric mucosa, in particular, is exposed to oxidative stresses, including inflammation induced by sodium chloride, Helicobacter pylori infection and smoking (1820), and the huge amounts of oxidative DNA damage may play an important role in carcinogenesis and a predisposition to GC. As the MYH gene is involved in the repair of oh8G, an oxidatively damaged mutagenic base, the MYH gene appeared to be a good candidate for study of the cause of familial and sporadic GCs in a Japanese population.
In this study we searched for MYH mutations in familial GC cases in Japan and identified a novel splice-site variant of IVS10-2A > G (c.892-2A > G). We demonstrated that the IVS10-2A > G variant generates an aberrant mRNA transcript encoding a truncated MYH protein. We also demonstrated by immunofluorescence analysis that, in contrast to the wild-type protein, the variant-type MYH protein is not localized in the nucleus, suggesting insufficient ability of the variant-type protein to repair nuclear DNA. In addition, we investigated the possible involvement of this variant in human gastric carcinogenesis.
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Materials and methods
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Samples
Blood specimens from Japanese familial GC patients and cancer-free Australian Caucasians had been collected previously (17,21). Familial cases were selected from unrelated families and the absence of germline E-cadherin mutations had been demonstrated in a previous experiment (17). Esophageal, gastric and colorectal cancers were obtained from Hamamatsu University Hospital, Shizuoka, Japan. Blood samples of cancer-free Japanese were obtained from Yokufukai Hospital, Tokyo, Japan. The gastric and colorectal cancer cell lines used in this study were: AGS, KATO-III, MKN1, MKN28, MKN45, MKN74, TMK1, HT29, SW480 and SW620 (22,23). DNA was purified by standard SDSproteinase K digestion and phenolchloroform extraction. This study was approved by the Institutional Review Board (IRB) of Hamamatsu University School of Medicine (12-11, 12-13, 12-14).
Microdissection
Cancerous cell clusters in paraffin sections of the digestive tract cancers were examined and microdissected under an inverted microscope (Olympus IX71; Olympus, Tokyo, Japan) equipped with a microdissection device (MicroDissector; Eppendorf AG, Hamburg, Germany). Areas of interest were microdissected with an ultrasonically oscillating needle, and the tissue particles obtained were aspirated into a pipette tip with a piezo-driven micropipette. DNA was extracted from the collected sample with a DNeasy Tissue Kit (QIAGEN, Valencia, CA).
Polymerase chain reaction (PCR)single-strand conformation polymorphism (SSCP) followed by direct sequencing
The 10 fragments covering entire coding exons of the MYH gene and their boundary regions were amplified by PCR with HotStarTaq DNA polymerase (QIAGEN). Information on the PCR primers and conditions is available on request. The restriction enzyme digestion for the PCR product was performed before SSCP in order to adjust the size to <250 bp. For the SSCP analysis, the PCR products were diluted with two volumes of loading solution and applied to 8% polyacrylamide gels with and without 5% glycerol. The products were electrophoresed at room temperature and 4°C and detected by silver staining according to the method described previously (24). PCR products showing the abnormally shifted band in SSCP were directly sequenced in both directions with a BigDye Terminator Cycle Sequencing Reaction Kit (Applied Biosystems, Japan) and the ABI 3100 Genetic Analyzer (Applied Biosystems).
RNA isolation and reverse transcription (RT)PCR
Total RNA was extracted with an RNeasy Mini Kit (QIAGEN) and converted to first-strand cDNA with a SuperScript First-Strand Synthesis System for RTPCR (Invitrogen, Carlsbad, CA) according to supplier's protocol. The primers were: 5'-GGA GAT TTC AAC CAA GCA GCC-3'/5'-GGA AGT TGA CCA CTC CCA GG-3' for the MYH transcript and 5'-CCA AGG TCA TCC ATG ACA AC-3'/5'-CAC CCT GTT GCT GTA GCC A-3' for the GAPDH transcript. A 1-µl vol of the cDNA conversion mixture was amplified by PCR in a 20 µl reaction mixture containing 1x PCR buffer, 200 µM of each dNTP, 0.25 µM of each primer and 0.5 U of HotStartTaq DNA polymerase (QIAGEN) under the following conditions: 30 s at 94°C, 30 s at 60°C and 60 s at 72°C for 25 or 35 cycles, followed by 10 min at 72°C. PCR products were fractionated by agarose gel electrophoresis, and each band was directly sequenced.
Quantitative real-time (QRT)PCR
Expression of the MYH mRNA transcript was measured by QRTPCR with a LightCycler instrument (Roche, Palo Alto, CA). PCR amplification was performed with a QuantiTect SYBR Green PCR kit (QIAGEN) and the following sets of PCR primers: 5'-AGC AGC TCT GGG GTC TAG CC-3' and 5'-TTC CTG CTC CAC TCT CTG GC-3' for the wild-type MYH transcript; 5'-GGA AGG GGC AGT GAG AAG TC-3' and 5'-CCA GTG TTG GGA GCA CAC TC-3' for the variant-type MYH transcript; and 5'-GTC TGG TAA CGG CAA TGC GG-3' and 5'-TCC CCT GTG GTG GAC ATA GC-3' for the transcript of a control housekeeping gene, porphobilinogen deaminase (PBGD). As standards for absolute quantification of the transcripts, a plasmid vector containing each PCR product was constructed, and the copy number of the standard DNA molecule was calculated using the following formula: (plasmid DNA concentration/[plasmid length in base pairs x 660]) x 6.022 x 1023. The standard was then used as a basis for determining the copy number of each mRNA transcript by QRTPCR. The amount of each MYH transcript was then standardized by that of the PBGD transcript to adjust for the variation in amount of cDNA input.
Construction of expression vectors
The wild-type MYH expression vector with a FLAG tag was constructed previously by inserting the wild-type MYH (type 2) cDNA with the FLAG sequence at the C-terminus into pcDNA3.1(+) plasmid vector (Invitrogen) (25). To construct the variant-type MYH (type 2) expression vector with a FLAG tag at the C-terminus, a cDNA fragment was amplified from cDNA derived from the non-cancerous gastric mucosa with the IVS10-2A/G genotype using a set of primers (F: 5'-CGC GGA TCC GCG GGC GGG AAC GCG GGG CCT CCG TGT TCT GC-3' and R: 5'-CCG GAA TTC CGG TCA CTT GTC ATC GTC GTC CTT GTA GTC CTG CCC CTT CCC CAG TAG GCT TAC TC-3'). The PCR product was cloned into the BamHIEcoRI site of the pcDNA3.1(+) vector. Each construct was confirmed by sequencing.
Immunofluorescence analysis
The expression vector was transfected into the NCI-H1299 human lung cancer cell line cultured on the slide glass with the LipofectAMINE 2000 reagent (Invitrogen) according to the supplier's recommendations. After 24 h, the cells were washed with PBS () and fixed with 4% paraformaldehyde at 4°C. After microwave treatment and permeabilization, the cells were incubated with anti-FLAG M2 antibody (SIGMA, St Louis, MO), 40 µg/ml, at room temperature for 1 h. Indirect immunofluorescence labeling was performed with a fluorescein 5-iso-thiocyanate (FITC)-conjugated anti-mouse IgG second antibody (Cappel, Aurora, OH) at room temperature for 30 min, and the nuclei were stained with propidium iodide (PI) (Vysis, Downers Grove, IL). The slides were examined under a fluorescence microscope (Olympus BX-50-FL; Olympus) equipped with epifluorescence filters and a photometric CCD camera (Quantix 1400; Roper Scientific, Tucson, AZ). The images captured were digitized and stored in the image analysis program (IPlab Spectrum; Scanalytics, Fairfax, VA).
PCR with confronting two-pair primers (PCRCTPP)
PCRCTPP is a genotyping method that can be applied to most single-nucleotide variations (26). The amplification of allele-specific bands of different lengths by using the four primers below enables genotyping by electrophoresis without other steps. As shown in Figure 3A, the four primers consist of F1 and R1 for the amplification of one allele, and F2 and R2 for the amplification of the other allele. F1 and R2 produce a common PCR product that is independent of the difference in alleles. F2 and R1 confront each other at the 3' end with the base specific to the allele. The primers designed to detect the IVS10-2A > G variant were: F1 (5'-GGA GAT TTC AAC CAA GCA GCC-3'), R1 (5'-AGC TGT TCC TGC TCC ACC C-3'), F2 (5'-ACT CAA CCC TGT GCC TCT CA-3') and R2 (5'-CTT CTG ACT GGG CCA GGA AG-3'), and the 20 µl reaction mixture consisted of 1x PCR buffer, 200 µM of each dNTP, 0.375 µM of each primer, 0.5 U of HotStartTaq DNA polymerase (QIAGEN) and 50 ng genomic DNA. Amplification was achieved by 15 min of initial denaturation at 95°C followed by 30 cycles of 30 s at 94°C, 30 s at 61°C and 1 min at 72°C and a 10-min final extension at 72°C. PCR products were fractionated by electrophoresis with a 2.5% agarose gel and stained with ethidium bromide.

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Fig. 3. Genotyping of the MYH IVS10-2A > G variant by PCRCTPP. (A) PCRCTPP for the IVS10-2A > G variant. Primers are shown with the horizontal arrows below the schema representing exonintron structure. The nucleotide at the 3'-end is the adenine of the F2 primer and the cytosine of the R1 primer. The position of the IVS10-2A > G variation is indicated by a vertical arrow. The agarose gel electrophoresis panel represents the PCR amplification of the samples with IVS10-2A/A, A/G and G/G genotypes using the primers indicated below. The size of each band is shown beside it. (B) Sequencing analysis of the PCR product with the F1 and R2 primers.
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A case-control study
A multi-center, hospital-based case-control study of gastric and colorectal cancer was conducted at four hospitals in Nagano Prefecture, Japan between October 1998 and March 2002. The newly diagnosed eligible patients ranging in age from 20 to 74 years old were identified during the survey at those hospitals. Two controls were matched for each case by sex, age (±3 years) and area of residence during the same period in the same hospitals. The controls were selected from persons who came to the hospitals for routine health examinations, and they were confirmed not to have cancer. As a result, we collected 153 cases and 303 controls (27), and we used the 148 cases and 292 controls that met the criteria for inclusion in this study. PCRCTPP was used for the genotyping of the IVS10-2A > G variant. This study was approved by the IRB of the National Cancer Center (G14-04).
Statistical analysis
Odds ratios (ORs), 95% confidence intervals (CIs) and P values for trends in the case-control study were obtained by conditional logistic regression analysis to assess the association between genotype and risk of GC. We calculated both crude and adjusted ORs. ORs for GC were adjusted for smoking status, family history of GC, salt intake, CagA antibody and membership in the Japan Agricultural Cooperative (JA). As no other factors altered the risk estimates, we did not include them. All analyses were conducted by using the SAS (version 8.2) program (SAS Institute, Cary, NC).
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Results
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Identification of the novel IVS10-2A > G variation of the MYH gene in Japanese familial GC patients
Blood specimens from 20 primary GC patients with a familial history of GC were tested for MYH mutations by PCRSSCP and subsequent sequencing analyses. No bi-allelic germline MYH mutations were found in any of the cases, but five MYH variants, including one missense (c.972C > G: Gln324His) and one splice-site (IVS10-2A > G) change, were detected (Table I). In regard to the Gln324His variant, we demonstrated previously the absence of any clear difference in the DNA repair activities between Gln324-type and His324-type proteins (28). On the other hand, the effect of the IVS10-2A > G variant on the MYH gene structure has not been examined previously, as it was a novel one. Two of the cases in our first set exhibited the IVS10-2A/G genotype, but no cases with the G/G genotype were found (Table II). These results indicated that the familial GC in this study was not attributable to the bi-allelic germline MYH mutations.
Effect of the IVS10-2A > G variation on pre-mRNA splicing
Interestingly, the IVS10-2A > G variant is located in the splice acceptor site at intron 10, and it is well known that variants in the splice acceptor site induce a splicing abnormality (29). Thus, in order to determine whether the IVS10-2A > G variant affected pre-mRNA splicing, we investigated the MYH mRNA transcript in the blood specimens from the patients with IVS10-2A/A and IVS10-2A/G genotypes by RTPCR analysis using primers located in exons 10 and 12, as shown in Figure 1A. A larger band than the band corresponding with the transcript by normal splicing was specifically detected in the case with the IVS10-2A/G genotype (Figure 1A), and sequencing of the band revealed that it was an mRNA transcript retaining the full intron 10 sequence (Figure 1B), leading to generation of the premature stop codon. RTPCR analyses with two other sets of primers yielded essentially the same results (data not shown). Next, to assess the efficiency of production of the variant-type transcript corresponding to the band (II) transcript in Figure 1A resulting from the IVS10-2A > G variant, we used QRTPCR to quantitatively measure the levels of expression of wild-type and variant-type MYH transcripts in the cases shown in Figure 1A. The primer set that specifically amplified each type of transcript was prepared (Figure 1C upper right and left), and the amount of each mRNA transcript was calculated by QRTPCR with a plasmid vector containing each PCR product as a standard. The amounts of each of the MYH transcripts standardized by that of the PBGD transcript are summarized at the lower left of Figure 1C. The amount of the variant-type transcript was much lower than that of the wild-type transcript in the cases with the IVS10-2A/A genotype, but similar to that of wild-type transcript in the case with the IVS10-2A/G genotype (Figure 1C, lower left and right). At first glance, the amount of variant-type transcript shown in Figure 1C seems to be different from the results shown in Figure 1A, however, we consider the difference to be attributable to the fact that shorter PCR products are more effectively amplified than longer PCR products, when amplified with the same primer set in the same tube at the same time. These results indicate that the IVS10-2G allele was associated with the efficient production of the variant-type transcript.

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Fig. 1. Detection and measurement of the aberrant mRNA transcript in a patient with the MYH IVS10-2A/G genotype compared with that of the IVS10-2A/A genotype. (A) RTPCR analysis for the MYH (upper panel) and GAPDH (lower panel) mRNA transcripts in three cases. Genotypes of the IVS10-2A > G variant are shown on the panel. The MYH transcripts are indicated by arrowheads (I) and (II). The locations of the PCR primers are indicated on the right by two opposite arrows. (B) Sequencing analysis of PCR bands (I) and (II) shown in (A). Parts of the intron 10 sequence are represented by a dotted line. (C) Measurement of the expression level of the wild-type and variant-type transcript by QRTPCR with a Light Cycler instrument. Primer sets that specifically amplify each independent transcript were selected, and production of a single PCR band was confirmed by agarose gel electrophoresis of the reaction mixture used for QRTPCR with the primer sets (upper left). Template 1, cDNA derived from Case 3 in (A); templates 2 and 3, plasmid DNA containing a wild-type transcript and variant-type transcript, respectively; template 4, genomic DNA from Case 3. The locations of the PCR primers are indicated by arrows (upper right). The dotted lines represent sequences not contained in the primers. The bar graph (lower left) shows the amount of each MYH transcript standardized by that of PBGD transcript in the cases in (A). The ratio of the expression level of the variant-type transcript to the expression level of the wild-type transcript was calculated in each case (lower right).
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Distinct subcellular localization of wild- and variant-type MYH protein
The read-through transcript of intron 10 detected in the case with the IVS10-2A/G genotype encodes a truncated MYH protein without a nuclear localization signal (NLS) and the binding sites for APE1 and PCNA proteins, as shown in Figure 2A (30,31). As the MYH type 2 protein has been reported to be localized in the cell nucleus (25,32) and the variant-type MYH protein encoded by the IVS10-2G lost the NLS, we investigated whether the structural change actually affects the subcellular localization. Both types of MYH type 2 cDNA with the FLAG sequence at the C-terminus inserted into the pcDNA3.1 mammalian expression vector were prepared, and after transiently transfecting them into the NCI-H1299 cell line, we examined the fluorescence microscopic images of the transfected cells for exogenous MYH-FLAG proteins. Consistent with the previous results (32), wild-type MYH-FLAG protein was localized in the nucleus, as shown in Figure 2B (a), however, the variant-type MYH-FLAG protein was localized in the cytoplasms, as shown in Figure 2B (b). These results indicated that the variant-type truncated MYH protein lacking an NLS is localized in the cytoplasm, in contrast to the nuclear localization of the wild-type MYH type 2 protein, suggesting that the excisional repair ability of the variant-type MYH protein for oh8G in nuclear DNA is impaired.

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Fig. 2. Differences in structure and subcellular localization between the wild- and variant-type MYH protein. (A) Schematic diagrams showing the structures of the wild-type MYH type 2 protein (upper) and the truncated MYH protein predicted from the abnormal transcript due to the IVS10-2A > G variant (lower). In the truncated protein the 238 aa of the C-terminus of the wild-type MYH protein are replaced by 8 unrelated aa. MYH domains and proteins binding with the MYH protein are shown. HhH-GPD, helixhairpinhelix structural element followed by a glycine/proline-rich loop and a conserved aspartic acid; APE1, apurinic/apyrimidinic endonuclease 1; PCNA, proliferating cell nuclear antigen; MutS , MSH2/MSH6 complex (MutS-homologous heterodimer MSH2-MSH6). (B) The distinct subcellular localization of the wild- and variant-type of MYH protein. The wild-type (a) and variant-type (b) MYH with the FLAG epitope tag at the C-terminus were expressed in NCI-H1299 cells and stained with anti-FLAG M2 as the first antibody and FITC-conjugated anti-mouse IgG as the second antibody. The immunofluorescence microscopic image of FITC (green)-stained cells showed the localization of exogenous MYH protein. The nuclei were counterstained with PI (red). The merged FITC and PI stained images showed overlapping yellow signals in the nuclei of the wild-type, but not in the variant-type, MYH transfected cells.
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MYH gene status in digestive tract cancer cases with the IVS10-2A/G genotype
As no GC surgical specimens from the familial GC cases were available, we were unable to evaluate the status of the second MYH allele in the GC of the cases with the IVS10-2A/G genotype. We therefore searched for digestive tract cancer cases with the IVS10-2A/G genotype and investigated whether somatic mutations or chromosomal deletions at the MYH gene locus were present in such cancers. A search was also made for cases with the IVS10-2G/G genotype. PCRCTPP was successfully used for genotyping of the IVS10-2A > G variant (Figure 3). Cases with the IVS10-2A/G genotype were detected at a frequency of 6.3% in the primary GC patients without a familial history of GC, 8.3% in the primary esophageal patients and 3.6% in the colorectal cancer patients (Table II). No cases with the G/G genotype were found. Cancer tissue from a total of eight carcinomas obtained from six patients was microdissected with a microdissection device, and DNA was extracted from them. PCRSSCP analysis covering all the MYH exons did not detect any MYH somatic mutations (Table III). Loss of heterozygosity (LOH) was also assessed using the IVS10-2A > G variant (Table III). Two cancers showed LOH; however, the IVS10-2G allele, not the IVS10-2A allele, was deleted in both of them. Thus, these results indicated that the digestive tract cancers in the cases with the IVS10-2A/G genotype were not attributable to the additional inactivation of the second allele of the MYH gene.
Evaluation of the IVS10-2G allele in GC risk in a case-control study
When healthy individuals from Japanese and Australian Caucasian population were searched for the IVS10-2A > G variant, the individuals with the IVS10-2A/G genotype were detected at a frequency of 2.1 and 0%, respectively (Table II). As these frequencies in healthy individuals were lower than the frequencies determined from the analysis of cancer patients, we tested the presumed correlation between the IVS10-2G allele and GC risk. Genotyping in the case-control study was successfully performed in 148 GC cases and 292 controls, but no significant difference in the distribution of the IVS10-2A > G variant was found between the cases and controls (Table IV). It is noteworthy that the homozygote for the IVS10-2G allele was found in one GC case, but not in any controls. Unfortunately, however, we were unable to obtain DNA from the GC tissue of the one GC case that was homozygous for the variant allele, blood specimens from the patient's family, or information on the history of cancer in the family, because this sample set was collected for a case-control study. These results suggested that the heterozygote for the MYH-IVS10-2G allele and wild-type allele is unassociated with the risk of GC. It was also suggested that the bi-allelic IVS10-2A > G variant may be responsible for the occurrence of GC.
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Discussion
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No bi-allelic germline MYH mutations were found in Japanese GC patients with a family history of GC in this study. However, a total of five different types of MYH variants were detected, and one of them, a novel splice-site variation of the IVS10-2A > G, caused a splicing abnormality that led to the production of an aberrant mRNA transcript encoding a truncated MYH protein. In contrast to the wild-type MYH protein, immunofluorescence analysis showed that the variant-type MYH protein was localized in the cytoplasm, not in the nucleus, suggesting that the excision repair ability of this variant-type MYH protein for nuclear DNA was impaired. However, no inactivation of the remaining wild-type allele by somatic mutation or LOH was detected in the digestive tract carcinomas of patients with the IVS10-2A/G genotype, and the distribution of genotypes of the IVS10-2A > G variation did not significantly differ between the GC cases and controls. The homozygote for the IVS10-2G allele was found in one GC case, but not in any non-cancer controls. These results indicate that oh8G repair activity may differ among Japanese individuals due to the MYH-IVS10-2A > G variant, that the heterozygote for the IVS10-2G and wild-type allele is unassociated with risk of GC, and that bi-allelic IVS10-2A > G variation may be responsible for the occurrence of GC.
The IVS10-2A > G variant was found at a frequency of 4.8% in the control group in the Japanese GC case-control study. Interestingly, as this variant has not been reported in previous papers and has not been registered in the Entrez SNP homepage of the NCBI web site, it is a novel SNP. As shown in Figure 2A, the truncated MYH protein due to the IVS10-2A > G variant lacks an NLS and binding sites for APE1 and PCNA proteins. The NLS is likely to be needed for the repair function of MYH protein for nuclear DNA. Moreover, the APE1 protein is required for both the short- and long-patch BER pathways (33), and the PCNA protein acts as a molecular adaptor coordinating and regulating the actions of DNA replication, DNA repair and cell cycle control (34). In this study, the variant-type truncated MYH protein was experimentally shown not to be localized in the nucleus, which is consistent with the loss of NLS. This suggested that the IVS10-2A > G change impaired the BER function of the MYH protein for oh8G in the chromosomal DNA in the nucleus. A low level of oh8G repair capacity in the cells with the IVS10-2A > G variant may lead to an increase in their mutation rate, and the effect would be substantial in organs exposed to severe oxidative stress, such as the stomach and colorectum. Thus, although no clear association between this variant and GC risk was detected in our analysis of small numbers of cases and controls, a comparative analysis of the MYH variant in a larger number of healthy individuals and patients with a variety of cancers should be carried out to assess the involvement of the MYH gene in human carcinogenesis.
Interestingly, the Tyr165Cys and Gln382Asp mutations have been reported to be common among patients with multiple colorectal adenomas due to bi-allelic MYH mutations in European Caucasians. The frequency of the Tyr165Cys and Gln382Asp mutations in a series of Finnish colorectal cancer patients was 0.2 and 0.4%, respectively (9). We searched for these mutations in 50 Japanese GC patients, but none of them had either mutation (data not shown). We also failed to detect them in 55 Japanese lung cancer patients (35), indicating that the frequency of the MYH Tyr165Cys and Gln382Asp mutations differs among ethnic groups.
Although the genetic factors resulting in the familial aggregation of GC in Japan have been unclear, the results of the present study indicate that bi-allelic germline MYH mutations were not responsible for the clustering. However, a homozygote for the IVS10-2G allele was only found in one of 148 GC cases (frequency: 0.67%), but not in any of the controls in the case-control set used in the present study. Moreover, the results of the present study suggested that the truncated protein arising from the IVS10-2G allele was associated with the reduction in the ability to repair nuclear DNA, suggesting that bi-allelic IVS10-2A > G variation is a significant factor in gastric carcinogenesis. However, since no GC tissue from the index case, blood specimens from the family, or information on the history of cancer in the family was obtained, it has not been determined that correlation between the bi-allelic IVS10-2A > G variation and the occurrence of GC in family members, and the effect of the bi-allelic MYH variation on mutation frequency in tumor DNA. Thus, it remains unclear whether the bi-allelic IVS10-2A > G variation in the Japanese is really responsible for the occurrence of GC. We would like to continue to investigate this matter. As demonstrated in the MYH-deficient multiple colorectal adenomas in patients of European origin, one manifestation of bi-allelic germline MYH mutations in Japanese may be multiple colorectal adenomas and carcinoma, although no evidence of this has been found at present. Therefore, we are also currently examining the MYH gene status of patients with colorectal tumors.
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Acknowledgments
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The authors would like to thank Mr H.Igarashi of the Hamamatsu University School of Medicine, Japan, for technical assistance concerning the immunofluorescence analysis. This work was supported in part by a Grant-in-Aid from the Ministry of Health, Labour and Welfare (15-5 and 1522), from the Ministry of Education, Culture, Sports, Science and Technology of Japan for Scientific Research on Priority Area and the 21st century COE program Medical Photonics, from the Aichi Cancer Research Foundation, from the Smoking Research Foundation, and from the Foundation for Promotion of Cancer Research.
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Received February 12, 2004;
accepted May 27, 2004.