散发性结直肠癌1号染色体等位基因杂合缺失精细定位研究

目的抑癌基因的杂合缺失（LOH）被认为是结直肠癌形成的通路之一。本实验通过对1号染色体1p36．33～36．31、1q31．1～32、1区域进行杂合缺失精细定位分析，以发现更精确的高频杂合缺失区域。方法在1p36．33～36．31、1q31．1～32．1区域分别选择7个、6个荧光标记微卫星引物与83例结直肠癌的肿瘤和正常组织进行聚合酶链反应（PCR）反应。产物在电泳后进行LOH分析。LOH结果与临床病理参数之间的关系比较采用X^2检验。结果1p36．33～36．31区域平均杂合缺失率是31．47％，以D1S243位点最高，为47．22％（34／72），最低是D1S1347，为7．35％（5／68）。存在两个高频杂合缺失区域：D1S243位点（1p36．33）以及D1S468-D1S2660区域（1p36．32～36．31）。1q31．1～32．1区域平均杂合缺失率是22．98％，以D1S2622位点最高，为36．73％（18／49），最低是D1S412，为16．42％（11／67）。更精确的缺失范围定位在D1S413和D1S2622之间（1q31．3—32．1），大约2cm的遗传距离范围内。1p36．33～36．31、1q31．1～32．1区域各位点的杂合缺失率与性别、年龄、肿瘤大小、生长方式以及Dukes分期无显著相关。提示该区域上的杂合缺失现象普遍存在于各种类型的散发性结直肠癌中。结论1号染色体上存在3个高频杂合缺失区域，D1S243位点（1cm）、D1S468和D1S2660位点之间（3cm）以及D1S413和D1S2622之间（2cm），提示在这些区域存在与结直肠癌相关的抑癌基因。     

散发性结直肠癌相关半胱氨酸甘氨酸富集蛋白1抑癌基因的筛选

目的 本课题组前期研究对染色体1q31.1-32.1区域进行杂合缺失精细定位,发现D1S413-D1S2622区域存在高频杂合缺失现象,提示可能有抑癌基因的存在.本研究在此基础上,对该区域与散发性结直肠癌发生相关的抑癌基因开展筛选研究.方法 构建包含上述区域基因的基因芯片,对19例散发性结直肠癌标本进行基因芯片扫描,并与其临床病理特征进行统计学分析,筛选该区域与结直肠癌相关的未知抑癌基因,然后对筛选出的候选基因采用Real-time PCR进行初步验证.结果 根据前期实验结果,通过检索,挑选了25个基因进行散发性结直肠癌相关基因的筛选.结果发现半胱氨酸甘氨酸富集蛋白1 (cysteine and glycine-rich protein 1,CSRP1)、LMOD1 、PPP1R12B和CFHL3 4个基因表达显著下调.这4个基因表达情况与结直肠癌患者的临床病理特征无关.通过生物信息学分析,推测CSRP1基因可能是该区域中与结直肠癌相关的抑癌基因.通过Real-time PCR验证结果也发现CSRP1基因在结直肠癌组织中表达显著下调,与芯片结果相符.结论 CSRP1基因可能是一个与结直肠癌发生相关的新的抑癌基因.     

散发性结直肠癌染色体7q21-22区杂合性缺失精细定位

目的 研究散发性结直肠癌7号染色体杂合性缺失,对7q21-22区精细定位,寻找新的结直肠癌抑癌基因.方法 采用15对微卫星DNA标记7号染色体,在高频杂合缺失区另取5对微卫星标记对83例结直肠癌病例的肿瘤和正常组织进行PCR反应.PCR产物在ABI Prism 377自动荧光测序仪进行电泳3 h,以GeneScan3.1和Genotyper 2.1软件进行基因分型.结果 在7号染色体上发现1个高频杂合缺失区即7q21-22区.对该区再用5对微卫星标记引物行精细定位,界定了1个跨越D7S657、D7S646位点精细的高频杂合缺失区域.结论 通过精细杂合缺失作图的研究,在7号染色体发现了1个跨越D7S657、D7S646位点的精细杂合缺失区,该区很可能存在1个或多个与结直肠癌相关的新的抑癌基因.     

胃癌染色体1q43区域等位基因杂合缺失精细定位研究

目的 对胃癌1号染色体1q43区域的微卫星位点进行杂合缺失(LOH)研究,为筛选此区域内可能存在的胃癌相关抑癌基因提供依据.方法 4对荧光标记的微卫星引物(D1S1594、D1S2785、D1S304、D1S321)覆盖1q43区域与96例胃癌患者的肿瘤组织及正常组织进行多重聚合酶链反应(PCR).产物经毛细管电泳后进行LOH分析.结果 该区域所测位点平均杂合缺失率17.9%,其中D1S1594位点最高,杂合缺失率为26.5%;D1S2785位点杂合缺失率最低为7.7%.1q43区域各位点的杂合缺失率与性别、年龄、肿瘤大小及TNM分期无明显相关.结论 在1q43区域内发现一个高频LOH区域,即D1S1594及D1S2785位点之间约1 cm区域,提示该区域内存在与胃癌相关的抑癌基因.     

散发性结直肠癌22q13区域杂合缺失的精细定位分析

目的在染色体高频杂合缺失区22q13精细定位，以筛查可能与结直肠癌相关的肿瘤抑制基因。方法荧光标记的微卫星引物与83例结直肠癌的肿瘤和正常组织进行PCR反应。产物在ABI Prism 377自动荧光测序仪进行电泳、扫描以及杂合缺失分析。其结果与临床病理因素进行相关性检验。结果8个位点平均杂合缺失率为35．6％。发现两个高频缺失区域：一个在D22S1171和D22S274之间，约2．7厘摩（cM）；另一个在D22S1160和D22S1149位点之间，约1．8cM。D22S1171位点与肿瘤发生部位显著相关（P=0．020）；D22S114位点与肝转移显著相关（P=0．008）；D22S1160位点与淋巴结转移显著相关（P=0．016）；其余位点与临床病理因素无显著相关性（P〉0．05）。筛选发现ARHGAP8基因和PPARA基因可能是肿瘤抑制基因。结论散发性结直肠癌22q13区域存在两个高频杂合缺失区，分别约2．7cM及1．8cM。ARHGAP8基因和PPARA基因可能是22q13区域与散发性结直肠癌相关的肿瘤抑制基因。     

散发性结直肠癌患者18号染色体高频杂合缺失的研究

目的：探讨散发性结直肠癌患者18号染色体上抑癌基因相关的杂合缺失（LOH）情况，并探索新的抑癌基因位点。方法：对83例散发性结直肠癌患者基因组DNA用14个不同荧光标记的高度多态性微卫生引物，扩增相应的微卫星位点，平均距离为10厘摩（centi-morgan,cM）。用ABI PRISM377测序仪进行基因扫描，统计各位点杂合缺失率。结果：在12个获得有效数据的微卫星位点中，平均杂合缺失率为36．78％,18p中最高为D18S53（38．09％），18q中最高为D18S474（55．74％）。4位患者的18号染色体所有杂合位点都存在缺失，30位患者的杂合缺失位点不少于50％（平均6个／人）；缺失位点少于50％的有53人（平均1个／人）。结论：结直肠癌患者18号染色体存在高频的LOH，并以整体缺失为特点。存在高频LOH的区域定位有转化生长因子（TGF）信号传导相关基因、结直肠癌缺失基因（DCC）、Rb结合蛋白8(RbBP8），特别是TGF信号传导相关基因MADH2、4、转化生长因子－β1反应元件（TGF－β1）等的缺失可能对结直肠癌的发生有重要影响。18p也有存在未知抑癌基因的可能。     

散发性结直肠癌染色体4p15等位基因杂合缺失的精细定位研究

目的通过在染色体4p15精细定位高频杂合缺失区域的范围．为筛选高频杂合缺失区内存在的散发性结直肠癌相关肿瘤抑制基因提供依据。方法7个荧光标记的微卫星引物与83例散发性结直肠癌的肿瘤和正常组织进行聚合酶链反应。微卫星之间的的平均遗传距离是1．02cM（centi—Morgon，里摩）。产物进行电泳、扫描及杂合缺失分析，并与临床、病理因素进行相关性检验。结果染色体4p15的平均杂合缺失率为21，34％，最高的是D4S3103位点（35．62％）；最低的是D4S2933位点（12．50％）。可能的肿瘤抑制基因的范围在D4S3017-D4S2933之间约1．7cM的遗传距离内，该区域内有PPARGC1A和GBA3两个基因。D4S1546位点杂合缺失与肿瘤直径显著相关（P〈0．05），其余位点与临床病理因索均无显著相关（P〉0．05）。结论染色体4p15精细定位后高频杂合缺失区域的范围限定在D4S3017-D4S2933之间约1．7cM的范围内。该区域内PPARGC1A和GBA3两个基因可能是散发性结直肠癌相关的肿瘤抑制基因。     

散发性结直肠癌染色体20q11-13区杂合性缺失精细定位

目的 研究散发性结直肠癌20号染色体杂合性缺失情况,并对20q11-13区进行精细定位.方法 收集1998年至1999年上海市第一人民医院83例结直肠癌患者的肿瘤组织和对应的正常黏膜组织,采用10个微卫星标记的引物对20号染色体进行杂合性缺失分析,在20q11-13区域另取10个微卫星标记的引物并对标本进行PCR分析.以Genescan 3.1和Genotyper 2.1软件进行基因分型和精确定位.结果 在20号染色体上发现一个高频杂合性缺失区即20q11-13区.进一步的精细定位,界定了两个高频杂合性缺失区:20q11.2、20q12,并在该杂合性缺失区发现了抑癌基因E2F1、PMP24和MAFB.结论 20号染色体有两个高频精细杂合性缺失区,该区很可能存在一个或多个与结直肠癌相关的新的抑癌基因.     

散发性结直肠癌4号染色体等位基因杂合缺失的研究

目的 通过在4号染色体寻找杂合缺失区域，为定位、筛选高频杂合缺失区存在的散发性结直肠癌相关肿瘤抑制基因提供依据。方法 20个荧光标记的微卫星引物与83例结、直肠癌的肿瘤和正常组织进行聚合酶链反应。微卫星的平均遗传距离是10．4里摩（cm01）。产物进行电泳、扫描及杂合缺失分析，并与临床、病理因素进行相关性检验。结果 短臂（4p）、长臂（4q）的平均杂合缺失率为24．25％、28．56％，可见3个最小的高频缺失区域（Region）：R1：在D4S405和D4s3013（4p14—15．2）之间；R2：在D4s3000和D4s2915位点之间（4q12—21．1）；R3：在D4S407和IMS2939位点之间（4q25—31．1）。D4S1534位点与肝脏转移有关（P〈0．05），其余位点与临床病理因素均无显著相关（P〉0．05）。结论 4号染色体的3个高频杂合缺失区域4p14—15．2、4q12—21．1、4q25—31．1存在散发性结直肠癌发生、发展相关的肿瘤抑制基因。     

散发性结肠直肠癌肿瘤分化及转移相关基因杂合缺失分析

目的：探讨散发性结肠直肠癌患者2号染色体上可能的肿瘤转移相关基因位点。方法：以2号染色体上30个不同荧光标记的高度多态性微卫星引物对83例散发性结肠直肠癌患者基因组DNA扩增相应的微卫星位点,用ABI PRISM 377测序仪进行基因扫描,检测各位点杂合缺失率,比较与肿瘤分期、分化的关系。结果：24个位点获得有效数据,平均遗传距离为11厘摩（cM),杂合缺失率平均为15．16％,较高的有2个们点：D2S206（2q33-37)的32．08％和D2S364（2q24.2)、31．03％,其余位点的杂合缺失率均小于20．00％;D2S142（2q24.1)、D2S126（2q35)、D2S2211（2q24.2)、D2S305（2q23.3)的杂合缺失率随着肿瘤恶性程度的增加而增高,后2个位点间的缺失有相关性。结论：已知几个错配修复基因位点附近的微卫星位点并无高频杂合缺失发生,D2S2305（2q23.3)到D2S2211（2q24.2)之间区域为整体性缺失,此区域和D2S142（2q24.1)、D2S126（2q35)2个位点与肿瘤的恶性程度相关,提示存在未知的肿瘤分化和转换相关基因的可能。     

膀胱移行细胞癌9q的杂合性丢失及抑癌基因位点的定位

目的研究膀胱移行细胞癌的９号染色体长臂（９ｑ）杂合性丢失并对抑癌基因位点进行定位。方法通过应用２５对高密度多态性微小卫星标记经ＰＣＲ扩增，６％变性聚丙烯酰胺凝胶电泳，检测膀胱移行细胞癌的９ｑ杂合性丢失，寻找膀胱移行细胞癌相关抑癌基因的位点。结果２５例病人约有９２％的肿瘤至少有一个以上位点的杂合性丢失，最常见的丢失区域为９ｑ１２～９ｑ２１、９ｑ２２和９ｑ３４，最常见的丢失位点是ＤＢＨ４４．０％、Ｄ９Ｓ１５２２７％、Ｄ９Ｓ１８１５２２７％、Ｄ９Ｓ１７６１２．０％、Ｄ９Ｓ１８３１１６．０％。绘制出膀胱移行细胞癌９ｑ杂合性丢失的染色体图谱，９ｑ杂合性丢失与肿瘤的分级、分期无相关性。结论膀胱移行细胞癌９ｑ的杂合性丢失是肿瘤发生的早期事件之一，在９ｑ３４的ＤＢＨ位点及其附近可能有与膀胱肿瘤相关的抑癌基因存在，并与肿瘤的发生有关。     

Refined mapping of allele loss at chromosome 10q23-26 in prostate cancer

BACKGROUND: Allele loss of at least two segments in 10q, one mapping to the PTEN gene and one more distal were described in prostate cancer, with loss more frequent in advanced prostate cancer. METHODS: A 63 cM region from 10q23 to q26 was studied for allele loss (LOH) in 59 prostate cancer samples using a dense map of microsatellite markers. RESULTS: LOH of at least one marker in 10q was observed in 13/59 tumors. LOH increased with grade and stage. Detailed deletion mapping identified three regions of allele loss. The first region mapped to the site of the PTEN gene, the second is defined by loss of one marker, D10S1692, in one tumor, and the third is defined between markers D10S1757 and D10S587, including DMBT, with a subregion of approximately 1.2 Mb mapping between markers D10S209 and D10S1679, lost in one tumor. CONCLUSIONS: LOH at the PTEN gene is frequent but mutations in the remaining allele were not detected by SSCP-screening. There may be more than two tumor suppressor (TS) genes mapping more distal of PTEN. The site for these putative TS genes can now be mapped with a dense set of precisely localized markers in a larger series of advanced tumors.     

Loss of heterozygosity on chromosome 2 in Japanese patients with prostate cancer

BACKGROUND: Loss of heterozygosity (LOH) on chromosome 2 is thought to occur only occasionally in prostate cancer (PCa), but allelic losses in this region are frequent in other types of human cancer, such as lung, thyroid, head and neck, and cervix. Here, we show a high-resolution deletion map of markers on chromosome 2 in Japanese patients with PCa. METHODS: Tissue samples were obtained from 66 patients with PCa. DNA from normal, tumor, or metastatic tissue was used as the template for polymerase chain reaction amplification for LOH using 24 microsatellite markers on human chromosome 2. RESULTS: Nineteen of the 66 cases (29%) showed LOH for at least one locus on chromosome 2. LOH on 2p was observed more frequently in cancer death cases than in organ confined and regional diseases (P < 0.001). Paired DNAs were available from both primary and metastatic tumors in the eight cases of cancer death; among those pairs, we detected LOH on 2p in four primary tumors, and in all metastatic foci (P < 0.05). Detailed deletion mapping in these tumors identified four distinct commonly deleted regions on 2p 16.3, 2p 12-cent, 2q 21.3, and 2q 23.1-2q 32.1. CONCLUSIONS: These results suggest that inactivation of putative tumor suppressor genes on chromosome 2 that may play an important role in the progression of Japanese patients with PCa.     

Comprehensive loss of heterozygosity analysis and identification of a novel hotspot at 3p21 in salivary gland neoplasms.

OBJECTIVES: We sought to assess loss of heterozygosity (LOH) profiles of 3p, 6q, 8q, 10q, 12q, 13q, and 17p and to identify the tumor suppressor genes involved in salivary gland neoplasms. STUDY DESIGN: LOH analysis was performed using 26 microsatellite markers by polymerase chain reaction-polyacrylamide gel electrophoresis method in 20 benign and 6 malignant salivary gland tumors. RESULTS: Overall, LOH was detected in at least one informative locus in 18 of 20 (90%) of benign tumors and in all of 6 cases of malignant tumors. High LOH frequencies were revealed at the loci D3S1307 (22%, 3p26), D3S966 (41%, 3p21), D6S255 (27%, 6q25), D8S166 (25%, 8q12), D8S199 (21%, 8q24), and D10S1765 (28%, 10q23) in benign tumors, defining the hotspot regions for putative tumor suppressor genes. CONCLUSIONS AND SIGNIFICANCE: The hotspot regions defined by the present study suggest that new tumor suppressor genes related to the development of salivary gland tumors may reside at several chromosomal loci, including loci at 3p, 6q, 8q and 10q.     

Steroid-resistant nephrotic syndrome and congenital anomalies of kidneys: evidence of locus on chromosome 13q

BACKGROUND: Steroid-resistant nephrotic syndrome (SRNS) and congenital anomalies of kidney and urinary tract (CAKUT) are major causes of renal dysfunction in children. Although a few patients with 13q deletion have been previously reported with renal anomalies, the association of SRNS with 13q has not been reported and critical regions associated with CAKUT have not been identified. We present the results of deletion mapping studies to identify the critical regions. METHODS: Cytogenetic and deletion mapping studies were performed on DNA obtained from peripheral blood of two children with renal anomalies and interstitial deletion of 13q as well as their parents. Twenty eight microsatellite markers with a spacing of 1-8 Mb (1-3 cM) were utilized. RESULTS: The patients (both males, 5 and 10 years old) had varying severity of developmental delay and other neurologic disorders. The renal involvement included hydronephrosis, ureterocele, renal dysplasia, and mesangioproliferative SRNS. Our studies imply existence of at least two critical regions in the 13q area that are linked to CAKUT. The first is a 7 Mb region defined by markers D13S776 and D13S891 shared by both patients. The second is a much larger region extending at least 33 Mb above D13S776 seen in one patient with severe renal malformations and SRNS. CONCLUSION: We report an association of chromosome 13q with CAKUT as well as SRNS. Our studies suggest the presence of more than one gene in this region that is likely to be involved in renal development and function.     

Linkage and association mapping of a chromosome 1q21-q24 type 2 diabetes susceptibility locus in northern European Caucasians

We have identified a region on chromosome 1q21-q24 that was significantly linked to type 2 diabetes in multiplex families of Northern European ancestry and also in Pima Indians, Amish families, and families from France and England. We sought to narrow and map this locus using a combination of linkage and association approaches by typing microsatellite markers at 1.2 and 0.5 cM densities, respectively, over a region of 37 cM (23.5 Mb). We tested linkage by parametric and nonparametric approaches and association using both case-control and family-based methods. In the 40 multiplex families that provided the previous evidence for linkage, the highest parametric, recessive logarithm of odds (LOD) score was 5.29 at marker D1S484 (168.5 cM, 157.5 Mb) without heterogeneity. Nonparametric linkage (NPL) statistics (P = 0.00009), SimWalk2 Statistic A (P = 0.0002), and sib-pair analyses (maximum likelihood score = 6.07) all mapped to the same location. The one LOD CI was narrowed to 156.8-158.9 Mb. Under recessive, two-point linkage analysis, adjacent markers D1S2675 (171.5 cM, 158.9 Mb) and D1S1679 (172 cM, 159.1 Mb) showed LOD scores >3.0. Nonparametric analyses revealed a second linkage peak at 180 cM near marker D1S1158 (163.3 Mb, NPL score 3.88, P = 0.0001), which was also supported by case-control (marker D1S194, 178 cM, 162.1 Mb; P = 0.003) and family-based (marker ATA38A05, 179 cM, 162.5 Mb; P = 0.002) association studies. We propose that the replicated linkage findings actually encompass at least two closely spaced regions, with a second susceptibility region located telomeric at 162.5-164.7 Mb.