6p21.3区域鼻咽癌相关基因及其单核苷酸多态性
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摘要
鼻咽癌的种族性、地域和家族聚集特征提示其发病具有遗传易感性。目前为止，尚未发现与NPC明确相关的基因。有研究表明：6p21.3HLAI类和Ⅲ类基因序列之间可能存在NPC易感基因。然而，NPCLOH与CGH调查均未发现该区域有明显的大片段缺失与扩增，推测可能存在单个核苷酸的变异(SNPs)。
本研究通过生物信息学分析6p21.3TNFα至微卫星标记D6S1624之间的基因，选定可能与NPC发生相关的基因中含有cSNP的序列。利用DHPLC检测PCR序列变异，并测序验证。关联分析法比较鼻咽癌和对照组SNPs杂合频率差异，以期筛选NPC相关基因。
PCR扩增分布于7个侯选基因中的10个片段，包括NPC组织、NPC外周血及对照外周血标本共2521份。DHPLC检出511个杂合子，其中4号片段(PPP1R10基因)鼻咽癌与对照组的杂合率分别为28.3％和10.1％，x~2=9.55、P=0.002，鼻咽癌组的相对风险是对照组的3.5倍(OR=3.5，P＜0.01；95％CI=1.53～7.99)。
测序证实该片段杂合子为PPP1R10基因15110位的一个新SNPs位点，为内含子中的A-G转换，理应不影响蛋白序列和结构。根据基因连锁原理，推测在其附近可能存在NPC遗传易感基因，以它为标记将有望克隆出该易感基因。
选择代表性的纯合与杂合子测序，发现了6个新的SNPs候选位点，其中4个位于内含子内，1个位于5'非翻译区，一个在外显子中，需要加大样本量确定它们的人群基因型频率分布。另外，证实5个已知SNP
黄华 巾沫e学D匕中色e 第一军医大学
位点及其基因型。
分析NPC组织与配对血结果，约48％NPC组织标本出现SNP位
点的点突变，提示NPC组织有基因组不稳定性。
本研究应用 DHPLC筛选印．3 MHC区域内 SNPS标记，部分结果
为克隆NPC易感基因打下了基础，同时为中华民族SNPs研究提供了有
用的数据。
Nasopharyngeal Carcinoma (NPC) is an endemic malignant tumor with an obvious ethnic aggregation and familial clustering. It has been speculated that some hereditary predispositions may be involved in NPC development. However, the effort to identify the specific NPC related genes have not been successful so far.
There have been reports suggesting NPC susceptibility genes to be located in the region between class-I and III of MHC, nearing the D6S1624 microsatellite marker. Because neither large DNA fragment deletion nor amplification was detected, it was held that single nucleotide variation in that region, such as Single Nucleotide Polymorphisms (SNPs), could be associated with the susceptibility to NPC.
In this study, the interesting genes being likely related to NPC and containing known cSNPs were picked out from those settling in the region between TNF a and D6S1624 by bio-informatics analysis. The DNA variations were detected through performance of DHPLC assay on PCR products of targeted fragments of interesting genes, and verified by sequencing. The statistical difference of the heterozygotic frequencies at a given locus between NPC matched and non-NPC peripheral blood samples was determined by association analysis.
10 PCR primers were designed according to the sequences of 10 fragments of DNA within interesting 7 genes respectively. Total 2521 PCR products from the genomic DNA of NPC tissues, NPC and non-NPC control peripheral blood samples were subjected to DHPLC assay and 511 heterozygotes were detected. Among the heterozygotes, the heterozygoties of sequence No. 4 (belonged to gene PPP1R10) in NPC and non-NPC blood
samples were 28.3% and 10.1% respectively with x2= 9.55,P=0.002, and OR = 3.5, P<0.01; 95%CI =1.53 ~ 7.99, meaning the risk of NPC in NPC patients is 3.5 times of that in control group.
The sequencing of the heterozygotes discovered a new SNP locus, an A=>G transition, at 15110 of gene PPP1R10. With locating in the sequence of an intron, the SNP locus should have no effect on the protein sequence, hence on thereof structure. According to the principle of genetic linkage, it may be deduced that a NPC susceptibility gene should near this SNP locus. Taking the locus as a marker, perhaps, the effort to clone the hypothetical NPC susceptibility gene will be successful.
Sequencing representatives of heterozygotes and homozygotes revealed 6 new SNP loci. Among them, 4 were located in intron region, while 1 in 5' untranslated region and 1 in an exon. However, the identification of their genotypes in the population studied needs more samples to be sequenced. The performance of sequencing also verified known SNP loci occurred in the analyzed fragments as reported by other studies.
Through comparing DHPLC and sequencing results of NPC biopsy samples with matched blood ones, point mutations at SNP loci were found in nearly 48% NPC samples. It is suggested that genomic instability is occurred in NPC cells.
In summary, the present work, as a part of the campaign to clone NPC related genes, has screened SNPs marker of the genes within 6p21.3 MHC region. In addition to identification of 6 new SNP loci with one of them associated with NPC, the result of this work may be used as a basis to clone NPC susceptibility gene in the future, and also provide some useful data to the SNP database of Chinese population.
本研究通过生物信息学分析6p21.3TNFα至微卫星标记D6S1624之间的基因，选定可能与NPC发生相关的基因中含有cSNP的序列。利用DHPLC检测PCR序列变异，并测序验证。关联分析法比较鼻咽癌和对照组SNPs杂合频率差异，以期筛选NPC相关基因。
PCR扩增分布于7个侯选基因中的10个片段，包括NPC组织、NPC外周血及对照外周血标本共2521份。DHPLC检出511个杂合子，其中4号片段(PPP1R10基因)鼻咽癌与对照组的杂合率分别为28.3％和10.1％，x~2=9.55、P=0.002，鼻咽癌组的相对风险是对照组的3.5倍(OR=3.5，P＜0.01；95％CI=1.53～7.99)。
测序证实该片段杂合子为PPP1R10基因15110位的一个新SNPs位点，为内含子中的A-G转换，理应不影响蛋白序列和结构。根据基因连锁原理，推测在其附近可能存在NPC遗传易感基因，以它为标记将有望克隆出该易感基因。
选择代表性的纯合与杂合子测序，发现了6个新的SNPs候选位点，其中4个位于内含子内，1个位于5'非翻译区，一个在外显子中，需要加大样本量确定它们的人群基因型频率分布。另外，证实5个已知SNP
黄华 巾沫e学D匕中色e 第一军医大学
位点及其基因型。
分析NPC组织与配对血结果，约48％NPC组织标本出现SNP位
点的点突变，提示NPC组织有基因组不稳定性。
本研究应用 DHPLC筛选印．3 MHC区域内 SNPS标记，部分结果
为克隆NPC易感基因打下了基础，同时为中华民族SNPs研究提供了有
用的数据。
Nasopharyngeal Carcinoma (NPC) is an endemic malignant tumor with an obvious ethnic aggregation and familial clustering. It has been speculated that some hereditary predispositions may be involved in NPC development. However, the effort to identify the specific NPC related genes have not been successful so far.
There have been reports suggesting NPC susceptibility genes to be located in the region between class-I and III of MHC, nearing the D6S1624 microsatellite marker. Because neither large DNA fragment deletion nor amplification was detected, it was held that single nucleotide variation in that region, such as Single Nucleotide Polymorphisms (SNPs), could be associated with the susceptibility to NPC.
In this study, the interesting genes being likely related to NPC and containing known cSNPs were picked out from those settling in the region between TNF a and D6S1624 by bio-informatics analysis. The DNA variations were detected through performance of DHPLC assay on PCR products of targeted fragments of interesting genes, and verified by sequencing. The statistical difference of the heterozygotic frequencies at a given locus between NPC matched and non-NPC peripheral blood samples was determined by association analysis.
10 PCR primers were designed according to the sequences of 10 fragments of DNA within interesting 7 genes respectively. Total 2521 PCR products from the genomic DNA of NPC tissues, NPC and non-NPC control peripheral blood samples were subjected to DHPLC assay and 511 heterozygotes were detected. Among the heterozygotes, the heterozygoties of sequence No. 4 (belonged to gene PPP1R10) in NPC and non-NPC blood
samples were 28.3% and 10.1% respectively with x2= 9.55,P=0.002, and OR = 3.5, P<0.01; 95%CI =1.53 ~ 7.99, meaning the risk of NPC in NPC patients is 3.5 times of that in control group.
The sequencing of the heterozygotes discovered a new SNP locus, an A=>G transition, at 15110 of gene PPP1R10. With locating in the sequence of an intron, the SNP locus should have no effect on the protein sequence, hence on thereof structure. According to the principle of genetic linkage, it may be deduced that a NPC susceptibility gene should near this SNP locus. Taking the locus as a marker, perhaps, the effort to clone the hypothetical NPC susceptibility gene will be successful.
Sequencing representatives of heterozygotes and homozygotes revealed 6 new SNP loci. Among them, 4 were located in intron region, while 1 in 5' untranslated region and 1 in an exon. However, the identification of their genotypes in the population studied needs more samples to be sequenced. The performance of sequencing also verified known SNP loci occurred in the analyzed fragments as reported by other studies.
Through comparing DHPLC and sequencing results of NPC biopsy samples with matched blood ones, point mutations at SNP loci were found in nearly 48% NPC samples. It is suggested that genomic instability is occurred in NPC cells.
In summary, the present work, as a part of the campaign to clone NPC related genes, has screened SNPs marker of the genes within 6p21.3 MHC region. In addition to identification of 6 new SNP loci with one of them associated with NPC, the result of this work may be used as a basis to clone NPC susceptibility gene in the future, and also provide some useful data to the SNP database of Chinese population.
引文
1．盖莱哈特、柯林斯、金斯伯格 著，孙开来等译。医学遗传学原理 科学出版社，北京2001：P239
2. Miyoshi E, Uozumi N, Noda K, et al. Expression of alphal-6 fucosyltransferase in rat tissues and human cancer cell lines. Int J Cancer, 1997, 72(6): 1117-1121.
3．曾益新主编．肿瘤学。人民卫生出版社，北京1999年：P27
4. Hildeshim A, et al. Etiology of nasophayngeal carcinoma: a review. Epidemiol. Rev. 1993；15(3): 446-485
5．姚开泰．鼻咽癌研究的回顾与展望。湖南医科大学学报。1995：20(2)：99—102
6．姚开泰。从死因回顾调查资料看湖南省鼻咽癌流行病学的一些特点并探索其发病机理。湖南医学院学报。1982：7(1)：10—16
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2. Miyoshi E, Uozumi N, Noda K, et al. Expression of alphal-6 fucosyltransferase in rat tissues and human cancer cell lines. Int J Cancer, 1997, 72(6): 1117-1121.
3．曾益新主编．肿瘤学。人民卫生出版社，北京1999年：P27
4. Hildeshim A, et al. Etiology of nasophayngeal carcinoma: a review. Epidemiol. Rev. 1993；15(3): 446-485
5．姚开泰．鼻咽癌研究的回顾与展望。湖南医科大学学报。1995：20(2)：99—102
6．姚开泰。从死因回顾调查资料看湖南省鼻咽癌流行病学的一些特点并探索其发病机理。湖南医学院学报。1982：7(1)：10—16
7．绍建永等。鼻咽癌分子遗传学研究进展 评述。癌症。2002，21(1)：1—10
8. Luo J, et al. Detection of c-myc gene expression in nasopharyngeal carcinoma by nonradioactive in situ hybridization and in immunohistochemistry. Chin Med J(Engl). 1997;110:229-232
9. Shen LF, et al. Analysis of bcl-2 expression in normal, inflamed, dysplastic nasopharyngeal epithelia, and nasopharyngeal carcinoma: association with p53 expression. Hum Pathol. 1997;28:556-562
10. Potter MJ, Field KE, Leung SF, et al. The detection of the c-myc and ras oncogenes in nasopharyngeal carcinoma by immunohistochemistry. Acta Otolaryngol. 114:105-109
11. Sun Y, Yao KT, et al. An infrequent point mution of the p53 gene in human nasopharyngeal carcinoma. Proc Natl Acad Sci USA. 1992;89:6516-6520
12．谢奕，姚开泰，胡维新。鼻咽癌、宫颈癌和肺癌中的p53基因突变和表达的对比关系。中华病理学杂志。1997；26(4)：229—232
13. Sun Y, Dong Z, Nakamura K, et al. Dosage-dependent dominance over wild-type p53 of a mutant p53 isolated from nasopharyngeal carcinoma. FASEB J1993;7(10): 944-50
14. Yang HJ, Cho YJ, Kim HS, et al. As sociation of p53 and BCL-2 expression with Epstein-Barr virus infection in the cancers of head and neck. Head Neck 2001 Aug;23(8):629-36
15. Sun Y, Hildesheim A, Li H, et al. No point mutation but a codon 31ser-->arg polymorphism of the WAF-1/CIP -1/p21 tumor suppressor gene in nasopharyngeal carcinoma (NPC): the polymorphism distinguishes Caucasians from Chinese. Cancer Epidemiol Biomarkers Prev 1995 Apr-May;4(3):261-7
16. Sun Y, Hildesheim A, Lanier AP, Cao Y, Yao KT, et al. No point mutation but decreased expression of the p16/MTS1 tumor suppressor gene in nasopharyngeal carcinoma. Oncogene. 1995;10:785-788
17. Sun Y, Hildesheim A,...KT Yao, et al. The von Hippel-Lindau(VHL)disease tumor-suppressor gene is not mutated in nasopharyngeal carcinoma. Int J Cancer. 1995;60:437-438
18. Sun Y, Hegamyer G, Colburn NH. Nasopharyngeal carcinoma shows no detectable retinoblastoma susceptibility gene alterations. Oncogene 1993;8(3):791-795
19. Shao JY, Wang HY, Huang XM, et al. Genome-wide alleotype analysis of sporadic primary nasopharyngeal carcinoma from Southern China. Iht J Oncol, 2000,17:1267-1275
20. Lo KW, Teo PM, Hui AB, et al. High resolution alleotype of microdissected primary nasopharyngeal carcinoma.[J] Cancer Res. 2000;60:3348-3353
21. Chan ASC, TO KF, Lo KW, et al. High frequency of chromosome 3p deletion in histologically normal nasopharyngeal epithelia from Southern Chinese. Cancer Res. 2000;60:5365-5370
22. Song NS, Zeng Y, Raab-Traub. Alteration on chromosome 3 in endemic and nonendemic nasopharyngeal carcinoma. Int J Cancer. 2000;86:244-250
23. Huang DP, Lo KW, Van Hasselt CA, et al. A region of homozygous deletion on chromosome 9p21-22 in primary nasopharyngeal carcinoma. Cancer Res 1994;54:4003-4006
24. Wang GL, Lo KW, Tsang KS, et al. Inhibiting of tumorigenic potential by restoration of p16 in nasopharyngeal carcinoma. Br J Cancer. 1999;81:1122-1126
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