鼻咽癌遗传易感性及其易感基因的初步研究
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摘要
【鼻咽癌遗传易感性及其易感基因研究进展】
人类基因组计划的启动和发展，大大推动了遗传性疾病相关基因定位和克隆研究进程，对于单基因遗传病已产生了一整套行之有效的研究方法体系，目前染色体上已定位的疾病基因已达一千二百多个。多基因遗传病易感基因的定位和遗传分析由于其意义重大和机制复杂成为近年来医学遗传学研究的热点和难点。近年来，随着分子生物学、生物信息学的发展，越来越多的复杂疾病易感基因也被定位和克隆，但是仍然有许多具有遗传易感性的疾病，特别是遗传模式复杂的疾病，还有待深入研究，鼻咽癌就是其中之一。
流行病学研究显示鼻咽癌具有明显的区域和人种聚集性，同时又具有明显的家族聚集性，以上结果提示鼻咽癌易感基因的存在，且在鼻咽癌发生发展中起着重要作用。
目前鼻咽癌致病基因的定位克隆研究主要通过两种途径，表型克隆策略和定位克隆策略。
表型克隆策略主要通过对比鼻咽癌与正常组织之间在不同层面上的差异从而寻找鼻咽癌相关基因。本研究室通过对鼻咽癌进行全基因组微卫星等位基因不平衡分析和比较基因组杂交，发现染色体3p、9p及6q等染色体区域部分位点等位基因不平衡在鼻咽癌中最为常见，很可能是鼻咽癌发生的早期事件，在这些区域中极可能存在鼻咽
博士学位论文 摘要
癌致病的关键基因，且该发现与多家实验室结果基本吻合。同时通过
表型克隆策略，本研究室还克隆了一大批鼻咽癌相关基因，已有基因
功能分析研究结果表明它们在鼻咽癌发病过程中可能起着重要作用，
它们是否影响着鼻咽癌的遗传易患性还需相应遗传学分析证实。
定位克隆策略则通过大量采集鼻咽癌遗传资源，通过各种遗传学
分析手段将鼻咽癌易感基因定位于染色体特定区域，然后进行克隆。
中山医科大学曾益新教授领导的课题组通过对20个操广州方言的鼻
咽癌家系进行全基因组扫描，连锁分析，发现了4号染色体与鼻咽癌
连锁的显著性证据，并将鼻咽癌易感基因定位于4p．l—4qlZ之间。
但由于鼻咽癌存在明显的遗传异质性，并不清楚是否能把以上结果推
广到其他人群，对其他人群进行研究还十分有必要。
【本丈研究思路］
鉴于以上分析，本文通过基于家系的连锁分析策略对3p、gP。
6q等鼻咽癌高频等位基因不平衡区域，以及曾益新等发现的 4o 5．l
一4q12广东家族性鼻咽癌连锁区域在我们采集的湖南鼻咽癌家系中
进行连锁分析，以期定位湖南家族性鼻咽癌的遗传易感基因；通过基
于病例一对照的关联分析策略，在本研究室新克隆，功能研究较深入
的 NGX6、UBAPI和 NORI等基因，以及位于鼻咽癌 9p21最小共同
缺失区，大量研究结果提示可能与鼻咽癌密切相关的己知抑瘤基因
CDKNZA内部选取单核昔酸多态（single nucleotide polymorphism，
SNP）位点在较大样本的散发鼻咽癌病例及对照组正常人群间进行关
V
g 一
g 联分析，以期确定它们与鼻咽癌遗传易感性的关系。
g［染色体3P部分位点的连锁分析】
＼我们首先使用在全基因组扫描中等位基因不平衡频率高于25％
g＿的10个位点D3S1297、D351304、D3S1263。D3S1489、D351266。
f D3S1568、D351289、D351300、D3S1285和 D353681在 12 个家系中
i 进行基因分型，8个位点得到可靠的分型结果，LINKAGE软件进行
g 两点参数连锁分析发现位于染色体3p2．31处的D3S1568在重组频率
g 为 0．00时，得到了最高 L皿值 3．2以P—5．25 X 10”‘），提示该位点与鼻
Z 咽癌发病紧密连锁。此外，与D3S1568相邻的D3S1289（位于染色体
g
g 3pZI．2义OD值亦达到 1．26（功刀5卜 GENEHUNTER软件进行多点连
g 锁分析，在 D351581 位点处 LOD值为 3．72卜l．91 10－‘），D351289
g 为 2．60 （＜．51 10”’），非参数连锁分析 NPL值最高为 1．14（－0．08）。
Z 提示该区域可能与我们的鼻咽癌家系连锁。
。随后，我们又收集到鼻咽癌家系6个，对上述位点在新采集家系
。中进行了基因分型，整合18个家系的资料进行分析。两点连锁分析
。LOD值最高为3．77卜l＊98 X 10“），NPL值最高为2．96（P＝＝＊X 10“），
。多点连锁分析 LOD值最高为 3．57（＜．692 XIO－‘），NPL值最高为 2．88
l 汐 ＝＝ 5刀 XIO”勺，该区域与鼻咽癌的连锁得到了进一步确证。
、在 D351568 位点附近增加 D353727、D351582、D353560。
－D351298． D353624、D353564和 D353553等 7个微卫星位点，进行
＿精细扫描，集中 18个家系 15个位点的全部资料进行分析。LINKAGE
VI
一
软件参数两点连锁分析D3 SIS 68位点L皿值仍然最高O．77），其附
近 D351289、D353564、D353624等 3个?
1. Progress of studies on genetic susceptibility and susceptible genes of nasopharyngeal carcinoma
Benefited form the launching and progress of Human Genome Project (HGP), the past two decades have witnessed an explosion in the identification, largely by positional cloning, of genes associated with Mendelian diseases. The roughly 1,200 genes that have been characterized, have clarified our understanding of the molecular basis of human genetic disease. Because of their large social and economic value, location susceptible genes of poly genie inheritance disorders and analysis their genetic susceptibility become a more and more important and difficult research field. At the present time, some complex disease genes have been cloned, but for many multifactorial inheritance disorders, especially which have complicated inheritance model, including nasopharyngeal carcinoma (NPC), their susceptibility gene or genes are still unknown.
Epidemiological investigation shows that NPC has remarkable geographical and racial differentiations. It occurs with high frequency in South China and South-East Asia. In areas with high incidence, NPC clusters in families. These observations have suggested the
existence of genetic susceptibility in at least a proportion of NPC patients, and the susceptible gene or genes may play influence the pathogenesis of NPC.
At present, there are two ways to study the gene or genes pause of NPC, phenotypic cloning strategy and positional cloning strategy. Phenotypic cloning strategy means to search NPC associated genes by compare difference of NPC and normal tissues. Our laboratory has performed a genome-wide scan of microsatellite allelic imbalance (AI) and comparative genomic hybridization (CGH), finding that the AI of chromosome regions 3p, 9p, and 6q are common in NPC and may be the early event of NPC, there are some key genes of NPC located in these regions. Some similar studies performed by other labs also obtain the same result. Using this strategy, our lab have cloned many NPC associated genes, functional analysis have proved that these genes may play important role in start and progress of NPC, but it is necessary to prove whether them influence the genetic susceptibility of NPC.
Positional cloning strategy can locate NPC susceptible genes on certain chromosome regions by perform a serial of genetic analysis using large numbers of NPC genetic resource, and then clone them. A group of researchers leaded by professor Yi-xin Zeng reported a results of a genome-wide search carried out in families at high risk
of NPC from Guangdong Province, China. Their findings provide evidence of a major susceptibility locus for NPC on chromosome 4p15.1 - 4q12 in a subset of families. Because there is genetic heterogeneity among different NPC families, we dose not know whether this result is still true in other people, it is necessary to perform parallel test in other ethnic group.
2. The purpose of this paper
As reasons of above-mentioned, a family-based linkage analysis strategy was used to look forward to locate susceptible genes of familial NPC of Hunan province. We have collected 18 NPC families in Hunan province, and selected a serial of microsatellite loci in high frequency allele imbalance chromosome region 3p, 9p, 6q, and 4p15.1 -4ql2, which linked with Guangdong familial NPC in Yi-xin Zeng's paper, genotyped these loci in NPC families and then performed linkage analysis. On the other hand, a case - control - based association analysis strategy was used for confirm the relationship between NPC associated genes and NPC genetic susceptibility. We selected three genes, NGX6, UBAP1, NOR1, which cloned by our lab and have functional evidence on NPC progress, and a suppressor gene, CDKN2A, which located in chromosome 9p21 NPC deletion region, search some single nucleotide polymorphisms (SNP) in these genes,
genotyped these SNP loci in case and control subjects and performed association analysis.
3. Linkge analysis of short arm of chromosome 3 and NPC
10 loci, D3S129
人类基因组计划的启动和发展，大大推动了遗传性疾病相关基因定位和克隆研究进程，对于单基因遗传病已产生了一整套行之有效的研究方法体系，目前染色体上已定位的疾病基因已达一千二百多个。多基因遗传病易感基因的定位和遗传分析由于其意义重大和机制复杂成为近年来医学遗传学研究的热点和难点。近年来，随着分子生物学、生物信息学的发展，越来越多的复杂疾病易感基因也被定位和克隆，但是仍然有许多具有遗传易感性的疾病，特别是遗传模式复杂的疾病，还有待深入研究，鼻咽癌就是其中之一。
流行病学研究显示鼻咽癌具有明显的区域和人种聚集性，同时又具有明显的家族聚集性，以上结果提示鼻咽癌易感基因的存在，且在鼻咽癌发生发展中起着重要作用。
目前鼻咽癌致病基因的定位克隆研究主要通过两种途径，表型克隆策略和定位克隆策略。
表型克隆策略主要通过对比鼻咽癌与正常组织之间在不同层面上的差异从而寻找鼻咽癌相关基因。本研究室通过对鼻咽癌进行全基因组微卫星等位基因不平衡分析和比较基因组杂交，发现染色体3p、9p及6q等染色体区域部分位点等位基因不平衡在鼻咽癌中最为常见，很可能是鼻咽癌发生的早期事件，在这些区域中极可能存在鼻咽
博士学位论文 摘要
癌致病的关键基因，且该发现与多家实验室结果基本吻合。同时通过
表型克隆策略，本研究室还克隆了一大批鼻咽癌相关基因，已有基因
功能分析研究结果表明它们在鼻咽癌发病过程中可能起着重要作用，
它们是否影响着鼻咽癌的遗传易患性还需相应遗传学分析证实。
定位克隆策略则通过大量采集鼻咽癌遗传资源，通过各种遗传学
分析手段将鼻咽癌易感基因定位于染色体特定区域，然后进行克隆。
中山医科大学曾益新教授领导的课题组通过对20个操广州方言的鼻
咽癌家系进行全基因组扫描，连锁分析，发现了4号染色体与鼻咽癌
连锁的显著性证据，并将鼻咽癌易感基因定位于4p．l—4qlZ之间。
但由于鼻咽癌存在明显的遗传异质性，并不清楚是否能把以上结果推
广到其他人群，对其他人群进行研究还十分有必要。
【本丈研究思路］
鉴于以上分析，本文通过基于家系的连锁分析策略对3p、gP。
6q等鼻咽癌高频等位基因不平衡区域，以及曾益新等发现的 4o 5．l
一4q12广东家族性鼻咽癌连锁区域在我们采集的湖南鼻咽癌家系中
进行连锁分析，以期定位湖南家族性鼻咽癌的遗传易感基因；通过基
于病例一对照的关联分析策略，在本研究室新克隆，功能研究较深入
的 NGX6、UBAPI和 NORI等基因，以及位于鼻咽癌 9p21最小共同
缺失区，大量研究结果提示可能与鼻咽癌密切相关的己知抑瘤基因
CDKNZA内部选取单核昔酸多态（single nucleotide polymorphism，
SNP）位点在较大样本的散发鼻咽癌病例及对照组正常人群间进行关
V
g 一
g 联分析，以期确定它们与鼻咽癌遗传易感性的关系。
g［染色体3P部分位点的连锁分析】
＼我们首先使用在全基因组扫描中等位基因不平衡频率高于25％
g＿的10个位点D3S1297、D351304、D3S1263。D3S1489、D351266。
f D3S1568、D351289、D351300、D3S1285和 D353681在 12 个家系中
i 进行基因分型，8个位点得到可靠的分型结果，LINKAGE软件进行
g 两点参数连锁分析发现位于染色体3p2．31处的D3S1568在重组频率
g 为 0．00时，得到了最高 L皿值 3．2以P—5．25 X 10”‘），提示该位点与鼻
Z 咽癌发病紧密连锁。此外，与D3S1568相邻的D3S1289（位于染色体
g
g 3pZI．2义OD值亦达到 1．26（功刀5卜 GENEHUNTER软件进行多点连
g 锁分析，在 D351581 位点处 LOD值为 3．72卜l．91 10－‘），D351289
g 为 2．60 （＜．51 10”’），非参数连锁分析 NPL值最高为 1．14（－0．08）。
Z 提示该区域可能与我们的鼻咽癌家系连锁。
。随后，我们又收集到鼻咽癌家系6个，对上述位点在新采集家系
。中进行了基因分型，整合18个家系的资料进行分析。两点连锁分析
。LOD值最高为3．77卜l＊98 X 10“），NPL值最高为2．96（P＝＝＊X 10“），
。多点连锁分析 LOD值最高为 3．57（＜．692 XIO－‘），NPL值最高为 2．88
l 汐 ＝＝ 5刀 XIO”勺，该区域与鼻咽癌的连锁得到了进一步确证。
、在 D351568 位点附近增加 D353727、D351582、D353560。
－D351298． D353624、D353564和 D353553等 7个微卫星位点，进行
＿精细扫描，集中 18个家系 15个位点的全部资料进行分析。LINKAGE
VI
一
软件参数两点连锁分析D3 SIS 68位点L皿值仍然最高O．77），其附
近 D351289、D353564、D353624等 3个?
1. Progress of studies on genetic susceptibility and susceptible genes of nasopharyngeal carcinoma
Benefited form the launching and progress of Human Genome Project (HGP), the past two decades have witnessed an explosion in the identification, largely by positional cloning, of genes associated with Mendelian diseases. The roughly 1,200 genes that have been characterized, have clarified our understanding of the molecular basis of human genetic disease. Because of their large social and economic value, location susceptible genes of poly genie inheritance disorders and analysis their genetic susceptibility become a more and more important and difficult research field. At the present time, some complex disease genes have been cloned, but for many multifactorial inheritance disorders, especially which have complicated inheritance model, including nasopharyngeal carcinoma (NPC), their susceptibility gene or genes are still unknown.
Epidemiological investigation shows that NPC has remarkable geographical and racial differentiations. It occurs with high frequency in South China and South-East Asia. In areas with high incidence, NPC clusters in families. These observations have suggested the
existence of genetic susceptibility in at least a proportion of NPC patients, and the susceptible gene or genes may play influence the pathogenesis of NPC.
At present, there are two ways to study the gene or genes pause of NPC, phenotypic cloning strategy and positional cloning strategy. Phenotypic cloning strategy means to search NPC associated genes by compare difference of NPC and normal tissues. Our laboratory has performed a genome-wide scan of microsatellite allelic imbalance (AI) and comparative genomic hybridization (CGH), finding that the AI of chromosome regions 3p, 9p, and 6q are common in NPC and may be the early event of NPC, there are some key genes of NPC located in these regions. Some similar studies performed by other labs also obtain the same result. Using this strategy, our lab have cloned many NPC associated genes, functional analysis have proved that these genes may play important role in start and progress of NPC, but it is necessary to prove whether them influence the genetic susceptibility of NPC.
Positional cloning strategy can locate NPC susceptible genes on certain chromosome regions by perform a serial of genetic analysis using large numbers of NPC genetic resource, and then clone them. A group of researchers leaded by professor Yi-xin Zeng reported a results of a genome-wide search carried out in families at high risk
of NPC from Guangdong Province, China. Their findings provide evidence of a major susceptibility locus for NPC on chromosome 4p15.1 - 4q12 in a subset of families. Because there is genetic heterogeneity among different NPC families, we dose not know whether this result is still true in other people, it is necessary to perform parallel test in other ethnic group.
2. The purpose of this paper
As reasons of above-mentioned, a family-based linkage analysis strategy was used to look forward to locate susceptible genes of familial NPC of Hunan province. We have collected 18 NPC families in Hunan province, and selected a serial of microsatellite loci in high frequency allele imbalance chromosome region 3p, 9p, 6q, and 4p15.1 -4ql2, which linked with Guangdong familial NPC in Yi-xin Zeng's paper, genotyped these loci in NPC families and then performed linkage analysis. On the other hand, a case - control - based association analysis strategy was used for confirm the relationship between NPC associated genes and NPC genetic susceptibility. We selected three genes, NGX6, UBAP1, NOR1, which cloned by our lab and have functional evidence on NPC progress, and a suppressor gene, CDKN2A, which located in chromosome 9p21 NPC deletion region, search some single nucleotide polymorphisms (SNP) in these genes,
genotyped these SNP loci in case and control subjects and performed association analysis.
3. Linkge analysis of short arm of chromosome 3 and NPC
10 loci, D3S129
引文
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