中国人HNPCC家系中hMSH2基因新突变及其功能分析

目的:报道1个在中国人遗传性非息肉病性结直肠癌(HNPCC)家系中发现的基因突变,并对其功能进行分析.方法:抽提1组符合Amsterdam标准的HNPCC家系先证者和其他家系成员的基因组DNA,PCR扩增先证者hMLH1 19个外显子和hMSH2 16个外显子,利用变性高效液相技术(dHPLC)筛查,对异常峰型利用DNA测序方法检测基因突变.发现先证者hMSH2基因存在错义突变后,对家系中其他成员和50名散发性大肠癌患者和100名正常成年人进行相同位点的检测,以判定是单核苷酸多态性位点(SNP)还是突变.利用5对微卫星标记对该家系中的2例肿瘤进行微卫星不稳定分析,免疫组化检测蛋白表达,利用同源建模方法对发现的突变位点进行功能分析,以研究突变的病理意义. 结果:在该家系的2例结肠癌患者中均发现hMSH2基因第13外显子2 108位出现C-A的错义突变,导致703位Ser变异为Tyr,即C.2 108C>A(p.Ser703Tyr),微卫星结果显示2例肿瘤均为微卫星高度不稳定(MSI-H),肿瘤免疫组化结果显示hMSH1基因表达正常而hMSH2基因不表达.同源建模发现该位点与目前报道的hMSH2基因突变不同,突变位于第Ⅳ结构域,Ser突变为Tyr后空间位阻增大,影响了蛋白的正常折叠和功能.结论:Ser703Tyr是中国人HNPCC的一个新的病理性突变.     

遗传性非息肉病性结直肠癌患者hMLH1和hMSH2基因的突变规律

目的 探讨中国人遗传性非息肉病性结直肠癌(HNPCC)患者hMLH1与hMSH2基因的突变特点.方法 对76个HNPCC家系先证者的DNA样本用PCR法扩增其hMLH1与hMSH2基因的35个外显子,再进行测序以确定突变类型.结果 (1)25个样本发现hMLH1或hMSH2基因突变,总突变率为33%(25/76);(2)检测到的22种突变中:hMLH1基因突变16个,hMSH2基因突变6个;(3)突变类型包括移码突变、无义突变、剪接区突变、错义突变,其中错义突变较多见(hMLH1基因错义突变11个、hMSH2基因5个).结论 中国人HNPCC家系hMLH1和hMSH2突变谱广泛,突变类型多样,hMLH1突变较hMSH2突变多见.     

典型遗传性非息肉性大肠癌1例家系分析

目的 探讨遗传性非息肉性大肠癌(HNPCC)的临床特点、病理特点,提高早期诊断和治疗水平,提出HNPCC在临床和基因水平上的筛查策略.报道1例典型的HNPCC家族的临床资料.方法 提取肿瘤组织、正常组织DNA,进行微卫星不稳定性分析、免疫组织化学检测.检测1个家系3例HNPCC患者的肿瘤组织微卫星不稳定状态、错配修复基因hMSH2及hMLH1蛋白水平的表达变化.结果 3例先证者3个肿瘤组织均表现为高度微卫星不稳定性,3例均表现为hMSH2蛋白表达舁常.结论 典型的HNPCC病例中错配修复基因突变率较高,hMLH1、hMSH2蛋白免疫组化的检测可作为HNPCC可疑家系的临床筛选,以及DNA测序前的筛选手段.     

国人HNPCC临床病理及错配修复基因突变特点

目的检测和分析中国人遗传性非息肉性大肠癌(hereditary non-polyposis colorectal cancer, HNPCC)hMSH2和hMLH1基因突变和临床病理特点,并探索高效的检测方法。方法收集31个国人HNPCC家系,采用PCR及变性高效液相色谱分忻(DHPLC)筛查hMSH2和hMLH1基因的突变,对DHPLC图形异常的样本用377DNA测序仪测序。结果31个国人HNPCC家系132个病人中共发现180例恶性肿瘤,其中胃癌19例(10．6％)；同时性癌少见,仅占所有结直肠癌(colorectal cancer,CRC)的3．0％。8个家系携带hMSH2或hMLH1基因序列改变,其中包括第一个带有hMSH2基因突变的蒙古族家系。结论国人中胃痛是发病率仪次于CRC的HNPCC相关肿瘤；DHPLC是一种非常有效的筛选hMSH2和hMLH1基因突变的方法；在中国hMLH1基因尤其是其前9个外显子的突变较hMSH2基因的突变更为常见。     

遗传性非息肉病性结直肠癌患者的临床特点及hMSH2与hMLH1种系突变的筛查

目的　分析我国遗传性非息肉性结直肠癌 (HNPCC)患者的临床特点 ,报告hMSH2和hMLH1基因突变筛查结果。方法　共收集了 2 8个家系 ,其中 15个家系符合阿姆斯特丹Ⅰ标准 ,13个家系符合日本临床诊断标准。记录的数据包括患者性别 ,结直肠癌发生的部位 ,诊断年龄 ,是否具有同时和 /或异时结直肠癌及结肠外癌 ,肿瘤的组织病理特点等。通过PCR及变性高效液相色谱分析(DHPLC)筛查hMSH2和hMLH1基因的突变 ,然后对DHPLC图形异常的样本进行测序。结果　 12 6例患者共诊断 170例次恶性肿瘤 (2 3例患有多原发癌 )。 98例 (77 8% )的患者患有结直肠癌 ,且发病年龄早 (平均 4 5 9岁 ) ,右侧癌多见。共在 12个家系中发现 8种hMSH2或hMLH1基因序列改变 ,其中hMSH2基因的第 3个外显子的无义突变即G2 0 4X是发现的首例我国蒙古族家系错配修复 (MMR)基因突变。结论　HNPCC患者是恶性肿瘤 (尤其是结直肠癌 )的高发人群。DHPLC是一种非常有效的筛选hMSH2和hMLH1基因突变的方法。在我国hMLH1基因尤其是其前九个外显子的突变较hMSH2基因的突变更常见。     

多重连接依赖的探针扩增技术检测中国人遗传性非息肉病性结直肠癌错配修复基因大片段缺失

目的了解中国人遗传性非息肉病性结直肠癌(HNPCC)家系hMSH2和hMLH1基因大片段缺失特点。方法采用多重连接依赖的探针扩增(MLPA)技术和GeneMapper分析技术检测17个HNPCC家系先证者hMSH2和hM-LH1基因种系大片段缺失。结果在3个家系中分别发现hMSH2基因第8外显子、1~6外显子和1~7外显子3种大片段缺失类型,未发现hMLH1基因大片段缺失。大片段缺失占hMSH2和hMLH1基因总种系病理性突变的19%。结论中国人HNPCC错配修复(MMR)基因大片段缺失发生率较高,hMSH2基因缺失可能更为常见。在分子遗传学检测中有必要开展MMR基因大片段缺失的检测。     

青年大肠癌微卫星不稳定和hMLH1/hMSH2表达缺失在遗传性非息肉病性大肠癌初筛中的应用

目的 探讨青年大肠癌中微卫星不稳定发生率和hMLH1/hMSH2表达缺失率及其在遗传性非息肉病性大肠癌初步筛查中的作用.方法 对73例中国南方青年大肠癌患者(年龄≤40岁)进行微卫星不稳定和hMLH1/hMSH2蛋白免疫组化检测.结果 微卫星不稳定性发生率为56.16%,hMLH1和/或hMSH2表达缺失率为49.32%,二者皆随患者发病年龄的降低而迅速增加;二者对阳性病例的检出率相似.结论 中国人青年大肠癌DNA错配修复基因缺陷为频发事件,运用微卫星不稳定分析和hMLH1/hMSH2蛋白免疫组化检测可在青年大肠癌有效地进行HNPCC患者及家系的初步筛查.     

修订Bethseda标准筛选遗传性非息肉病性结直肠癌患者的队列研究

目的研究修订Bethseda标准筛选遗传性非息肉病性结直肠癌（HNPCC）的价值及在结直肠癌中的构成比。方法对2004年8月至2005年12月进行手术治疗的连续110例患者建立队列、多重荧光聚合酶链反应方法检测肿瘤的微卫星不稳定（MSI）状态,对于MSI结直肠癌患者检测hMSH2、hMLH1和hMSH6基因种系突变。结果110例患者中共检出MSI结直肠癌患者23例（20．9％）。在23例MSI结直肠癌患者中,共发现病理性突变7个（30．4％）,占所有结直肠癌6．4％;其中hMSH6基因种系突变3个,hMSH2基因突变3个,hMLH1基因突变1个。结论以修订Bethesda标准,MSI结直肠癌检出率为20．9％,HNPCC检出率6．4％;在中国人错配修复基因种系突变中hMSH2和hMSH6错义突变比较多见。     

遗传性非息肉病性结直肠癌8个家系临床及病理分析

目的分析遗传性非息肉病性结直肠癌（HNPCC）家系临床及病理特点。方法收集符合Amsterdam标准Ⅱ的8例HNPCC家系资料,绘制家系图谱。结果HNPCC发病率为1.59%,8个HNPCC家系总发病人数为31例,其中结直肠癌患者为25例,肠外相关肿瘤6例,8例先证者中6例为女性,2例为男性,4例发病年龄小于40岁,发病部位位于右半结肠为2例,左半结肠3例,直肠3例,组织学分型以中至低分化腺癌为主;均未出现淋巴结及远处转移;8例先证者错配修复（MMR）蛋白表达异常的为5例。结论HNPCC先证者发病年龄轻,组织学分型较好,家系成员发病率高,对其家族成员进行定期检查和及时治疗将能有效地预防其发生并降低死亡率;MMR基因突变检测对提高HNPCC的诊断起重要作用。     

中国人HNPCC家系的临床特征及M3胆碱能受体基因(A)8区突变检测

目的:分析中国人遗传性非息肉病性大肠癌(HNPCC)家系的临床特征并检测这些家系中M3胆碱能受体基因(A)8区的突变情况.方法:根据2003年4月杭州会议制定的中国人HNPCC家系标准收集HNPCC家系共15个,分析其临床特征;提取先证者的外周血基因组DNA,PCR扩增M3胆碱能受体基因第8外显子中一段长153 bp,包含有微卫星位点(A)8的基因片段,直接进行DNA测序.结果:15个家系共有恶性肿瘤患者55例,其中大肠癌患者41例,平均每个家系发生大肠癌2.73例,73%的大肠癌患者发病年龄<50岁,51%的病灶位于近端结肠,40%发生在直肠肛门,同时和异时多原发大肠癌总发生率为12%,2/3的家系属于Lynch Ⅱ型,共发生肠外恶性肿瘤14例(18个),其中胃癌最常见.15例先证者外周血中无1例检测到M3胆碱能受体基因(A)8区的突变.结论:M3胆碱能受体基因与中国人群中HPNCC的发病可能无密切关系.中国人HNPCC家系标准应在临床工作中推广应用,并进一步验证其合理性和科学性.     

可疑遗传性非息肉病性结直肠癌的hMLH1和hMSH2基因突变研究

目的比较分析符合国际诊断标准的遗传性非息肉病性结直肠癌（ＩＣＧＨＮＰＣＣ）家系和提出的可疑ＨＮＰＣＣ家系的分子特征的异同,建立可疑ＨＮＰＣＣ诊断标准并评价其应用价值。方法根据ＡｍｓｔｅｒｄａｍＨＮＰＣＣ诊断标准和作者提出的可疑ＨＮＰＣＣ诊断标准,分别收集得到２９个ＩＣＧ家系和３４个可疑家系。提取先证者的外周血基因组ＤＮＡ,应用ＰＣＲＳＳＣＰ和ＤＮＡ测序的方法进行ｈＭＬＨ１和ｈＭＳＨ２基因的突变筛选。结果２９个ＩＣＧ家系中有９个家系被检出含有ｈＭＬＨ１基因的种系突变,突变率为３１０％;３４个可疑家系中有１０个家系被检出含有ｈＭＬＨ１或ｈＭＳＨ２基因的突变,两个基因的突变率分别为２３５％和５９％。ＩＣＧ组和可疑组中两个基因总突变率之间差异无显著意义（Ｐ＞００５）。ｈＭＬＨ１基因同是两组家系中的主要相关基因。两组家系中,突变均较一致地分布于基因的后半部分,突变类型也极为相似,均以导致短缩蛋白的突变最为常见。结论ＩＣＧ组和可疑组在遗传背景上有着相似之处,对ＨＮＰＣＣ诊断标准有一定的临床应用价值,有助于ＨＮＰＣＣ家系的临床诊断。     

Novel hMLH1 and hMSH2 germline mutations in African Americans with colorectal cancer.

CONTEXT: Germline mutations of the DNA mismatch repair (MMR) genes hMLH1 and hMSH2 have been shown to cosegregate with the colorectal cancer phenotype in multiple hereditary nonpolyposis colorectal cancer (HNPCC) pedigrees. However, the frequency of these mutations among African American patients with colorectal cancer is unknown. OBJECTIVE: To investigate the frequency of germline alterations of the DNA MMR genes hMLH1 and hMSH2 among African Americans affected by HNPCC and early-age onset colorectal cancer. DESIGN, SETTING, AND PATIENTS: Forty unrelated African American HNPCC and early-age onset colorectal cancer patients (8 women, 3 men) were identified from the cancer registry at a National Cancer Institute-designated referral center, 11 of whom were available for and agreed to study participation from January 1997 to February 1998. The mean age of the subjects was 44 years. An additional 50 age- and sex-matched African Americans without personal or family history of colorectal, endometrial, ovarian, urinary tract, or upper gastrointestinal tract malignancy were also studied as a polymorphism control population. In all subjects, genomic DNA was amplified by polymerase chain reaction for all hMLH1 and hMSH2 exons and screened using single-strand conformation polymorphism (SSCP) analysis. Samples demonstrating significant SSCP shifts underwent automated nucleotide sequencing analysis. MAIN OUTCOME MEASURE: Frequency of hMLH1 and hMSH2 germline alterations in the affected and control subjects. RESULTS: Germline hMLH1 and hMSH2 mutations were detected in 3 (27%) of the African American colorectal cancer probands studied. Each mutation was novel. Two hMLH1 (an A-->T transversion at codon 26 and a GG-->AT substitution across codons 177 and 178) mutations and 1 hMSH2 mutation (a C-->T transition at codon 389) were identified in 3 female study subjects. Six other hMLH1 and hMSH2 alterations were detected but were presumed to be polymorphisms. Neither missense mutation (at codons 26 and 389) was detected in the control population. CONCLUSIONS: The results of our analysis support an association between the 3 mutations reported and predisposition to colorectal cancer. Further studies are needed to define DNA MMR gene-associated colorectal cancer in African Americans, an understudied population at increased risk of fatal colorectal cancer.     

Clinical features and hMSH2/hMLH1 germ-line mutations in Chinese patients with hereditary nonpolyposis colorectal cancer

Background At least five mismatch repair （MMR） genes, including hMSH2, hMLH1, hPMS, hPMS2, and hMSH6/GTBP, are associated with hereditary nonpolyposis colorectal cancer （HNPCC）. More than 90% of families with HNPCC harbor the hMSH2and hMLH1 gene mutations. We have analyzed the clinical features of HNPCC among Chinese patients and report the results of screening for mutations in the hMSH2 and hMLH1 genes.
Methods The data concerning gender, site of colorectal cancer （CRC）, age at diagnosis, history of synchronous and/or metachronous colorectal cancer, instance of extracolonic cancers, and histopathology of tumors for 126 patients from 28 independent families with HNPCC were collected. Fifteen of the families met the Amsterdam I criteria, and 13 met the Japanese clinical criteria for diagnosis. Genomic DNA was extracted from the peripheral lymphocytes. Polymerase chain reaction （PCR） and denaturing high-performance liquid chromatography （DHPLC） were used to screen the coding region of the hMSH2 and hMLH1 genes. Samples showing abnormal DHPLC profiles were sequenced.
Results One hundred and seventy malignant neoplasms were found in the 126 patients, of whom 23 had multiple cancers. Ninety-eight of the patients （77.8%） had colorectal cancers, with an average age at onset of 45.9 years and a right-sided predominance. Eight hMSH2 or hMLH1 gene sequence variations were found in 12 families, and a germ-line G204X nonsense mutation in the third exon of hMSH2 was found, representing the first mutation in an MMR gene ever found in people of Chinese Mongolian ethnicity.
Conclusions HNPCC is a typical autosomally dominant hereditary disease, characterized by early onset, a predominance of proximal colorectal cancer, and multiple synchronous and metachronous colorectal cancers. DHPLC is a powerful tool for detecting mutations in the hMSH2 and hMLH1 genes, Mutations in the first nine exons of the hMLH1 gene were more common in Chinese patients.     

Expression of Cyclooxygenase-2 and Its Relationship with Mismatch Repair and Microsatellite Instability in Hereditary Nonpolyposis Colorectal Cancer

Objective To investigate cyclooxygenase-2 （COX-2） expression and its relationship with mismatch repair （MMR） protein expression and microsatellite instability （MSI） in hereditary nonpolyposis colorectal cancer （HNPCC）. Methods A total of 28 cases of colorectal adenoma and 14 cases of colorectal carcinoma were collected between July 2003 and July 2007 from 33 HNPCC families. Sporadic colorectal adenoma （n=32） and carcinoma patients （n=24） served as controls. With samples of tumor tissues and normal colonic mucosa collected from the patients, the protein expressions of COX-2 and MMR （hMLH1, hMSH2, and hMSH6） were examined with immunohistochemical assay. Frequency of MSI in five standard MSI loci BAT25, BAT26, D2S123, D5S346, and D17S250 were analyzed by means of polymerase chain reaction. Results The rate of COX-2 high-expression was 53.6% （15/28） and 42.9% （6/14） in HNPCC adenoma and carcinoma; 62.5% （20/32） and 91.7% （22/24） in sporadic adenoma and carcinoma, respectively. That rate was lower in HNPCC carcinoma than in sporadic carcinoma （P〈0.05）. MMR-deletion rate and percentage of high-frequency MSI （MSI-H） in HNPCC carcinoma were higher than those in sporadic colorectal carcinoma [both 71.4% （10/14） vs. 12.5% （3/24）, both P〈0.01]. Among the 10 MMR-deficient HNPCC carcinoma patients, COX-2 low-expression was observed in 8 cases （80.0%）, while COX-2 high-expression was observed in all of the 4 MMR-positive HNPCC carcinoma cases （P〈0.05）. In comparison to MMR positive HNPCC carcinoma, HNPCC adenoma, and sporadic carcinoma, COX-2 expression was significantly lower in corresponding MMR-deficient cases （all P〈0.05）. The rates of COX-2 low-expression in HNPCC adenoma, HNPCC carcinoma, and sporadic carcinoma with MSI-H were significantly higher than those in the cases with microsatellite stability （all P〈0.05）. Conclusion COX-2 is expressed at a low level in HNPCC carcinoma, different from the high COX-2 expression in sporadic carcinoma.     

Interpretation of genetic test results for hereditary nonpolyposis colorectal cancer: implications for clinical predisposition testing.

CONTEXT: Genetic testing for cancer predisposition is evolving from purely research applications to affecting clinical management. OBJECTIVE: To determine how often genetic test results for hereditary nonpolyposis colorectal cancer (HNPCC) can be definitively interpreted and used to guide clinical management. DESIGN: Case-series study conducted in 1996 to 1998 in which a complete sequence analysis of hMSH2 and hMLH1 coding sequence and flanking intronic regions was performed. Mutations were categorized as protein truncating and missense. In the case of missense alterations, additional analyses were performed in an effort to assess pathogenicity. SETTING AND PARTICIPANTS: Families were identified by self-referral or health care provider referral to a cancer genetics program. Participants and kindreds were classified into 1 of 4 categories: (1) Amsterdam criteria for HNPCC, (2) modified Amsterdam criteria for HNPCC, (3) young age at onset, or (4) HNPCC-variant. In addition, each proband was classified according to the Bethesda guidelines for identification of individuals with HNPCC. MAIN OUTCOME MEASURE: Alterations of hMSH2 and hMLH1 genes. RESULTS: Twenty-seven alterations of hMSH2 and hMLH1 were found in 24 of 70 families (34.3%). Of these, deleterious mutations that could be used with confidence in clinical management were identified in 25.7% (18/70) of families. The rates of definitive results for families fulfilling Amsterdam criteria, modified Amsterdam criteria, young age at onset, HNPCC-variant, and Bethesda guidelines were 27 (39.3%), 13 (18.2%), 12 (16.7%), 11 (15.8%), and 21 (30.4%), respectively. The prevalence of missense mutations, genetic heterogeneity of the syndrome, and limited availability of validated functional assays present a challenge in the interpretation of genetic test results of HNPCC families. CONCLUSIONS: The identification of pathogenic mutations in a significant subset of families for whom the results may have marked clinical importance makes genetic testing an important option for HNPCC and HNPCC-like kindreds. However, for the majority of individuals in whom sequence analysis of hMSH2 and hMLH1 does not give a definitive result, intensive follow-up is still warranted.