中国人遗传性非息肉病性结直肠癌MSH6基因胚系突变的测序研究

目的探讨中国人遗传性非息肉病性结直肠癌(hereditary nonpolyposis colorectal cancer,HNPCC)家系中MSH6基因胚系突变。方法采用PCR-直接测序的方法检测39个无胚系MSH2及MLH1基因突变、符合不同临床标准的中国人HNPCC家系先证者MSH6基因各外显子胚系突变;对137名正常人胚系基因组DNA进行错义突变相应外显子的测序分析。应用Envision二步法检测有突变的先证者肿瘤组织MSH6蛋白表达。结果在39个HNPCC先证者中共发现6个MSH6基因的胚系突变,分别位于第4、6、9和第10外显子;突变类型为4个错义突变、1个无义突变、1个剪接区的插入突变;对4个错义突变的相应外显子的测序分析显示:137名正常人胚系基因组DNA5例具有第6外显子1163密码子处的c.3488A>T的错义突变,约占3.65%(5/137),为单核苷酸多态性(single nucleotide polymorphism,SNP);其余错义突变在正常人群中均未发现。在6例有MSH6基因胚系突变家系的肿瘤组织中免疫组化染色除1例为SNP的肿瘤组织MSH6蛋白阳性表达外,其余均为阴性表达。经过查询国际HNPCC突变数据库及SNP数据库证实上述突变中5个为国际上尚未报道的病理性突变,1个为新发现的SNP。结论MSH6基因胚系突变在符合不同临床标准的中国人HNPCC中均起一定作用,对无MSH2及MLH1基因胚系突变的先证者行MSH6基因胚系突变的测序分析对确诊HNPCC家系是必要的。     

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研究

目的了解中国人遗传性非息肉病性结直肠癌(hereditary nonpolyposis colorectal cancer．HNPCC)家系中MSH和MLH1基因大片段缺失情况及特点，以进一步完善中国人HNPCC家系遗传检测内容。方法取14个符合中国人HNPCC诊断标准的HNPCC家系肿瘤先证者外周血DNA，用荧光标记多重PCR技术结合GeneScan分析系统检测MSH2和MLH1基因大片段缺失。结果14例患者中有1例检测到MSH2基因第1～7外显子缺失，该家系另1例大肠癌患者和3个家系成员有同样的基因片段缺失。结论中国人HNPCC家系错配修复基因大片段缺失可能以MSH2比较常见。建议在中国人HNPCC家系遗传检测中常规包含错配修复基因大片段缺失检测。     

遗传性非息肉性结直肠癌家系的MLH1基因两个胚系新突变

目的初步评价遗传性非息肉性结直肠癌（HNPCC）胚系MLH1基因突变中新突变的病理性。方法收集符合AmsterdamⅡ标准的12个不同家系的12例患者外周血,用特异引物和耐热性逆转录酶特异地逆转录MLH1的mRNA;利用长模板PCR扩增酶扩增逆转录产物（cDNA）;测序分析扩增产物;利用PCR-Genescan技术和免疫组织化学染色分别检测有新突变患者肿瘤组织的5个微卫星位点（BAT26,BAT25,D5S346,D2S123和Mfd15）和MLH1蛋白的表达。结果在4例患者中检出4个MLH1突变,其中2个突变为第12外显子的第384密码子（1151bp处）GTT→GAT的突变,该突变是已报道的病理性突变;另外2个突变分别是第8外显子的第217密码子（649bp处）CGC→TGC突变和第16外显子的第581密码子（1742bp处）CC→CTG突变;后两者为尚未报道的新突变。2个新突变的患者肿瘤组织均呈高度微卫星不稳定性,两者的瘤组织MLH1蛋白均失表达。结论MLH1第8外显子的第217密码子突变和第16外显子的第581密码子的两个新突变很可能为病理性突变。     

两个遗传性非患肉性结直肠癌家系中MLH1和MSH2基因的突变检测

目的 确定两个遗传性非息肉性结直肠癌(hereditary nonpolyposis colorectal cancer,HNPCC)家系的致病基因,选择MLH1基因和MSH2基因进行突变检测.方法 采用聚合酶链反应结合DNA直接测序法,对两个遗传性非息肉性结直肠癌家系的患者进行MLH1基因和MSH2基因的突变检测;发现变异后,采用PCR-限制性片段长度多态性或直接测序法鉴定此变异是否属于突变.结果 在家系A的患者中发现了位于MLH1基因第3外显子内的新突变c.243_244 insA;在家系B的患者中发现了MSH2基因第7外显子内的c.1215_1218dupCCGA突变,这两个突变都导致了编码蛋白的提前终止.结论 MLH1基因的c.243_244insA突变和MSH2基因的c.1215_1218dupCCGA突变分别是导致家系A和家系B发生遗传性非息肉性结直肠癌的致病突变.     

中国人家族性结直肠癌错配修复基因大片段变异分析

目的分析中国人家庭性结直肠癌错配修复基因大片段变异的特点。方法 采用多重连接探针扩增技术，分析32例具有家庭史结直肠癌、20例散发性结直肠癌患者错配修复基因MSH2的16个外显子、MLH1的19个外显子及7个其它基因外显子的拷贝数。研究工作包括：（1）双盲法分析阴性和阳性对照样本，完成方法学可靠性检验；（2）分析结直肠癌患者外周血细胞DNA，筛查MSH2和MLH1基因大片段变异。结果 多重连接探针扩增技术分析系统稳定检出阳性对照样本的DNA大片段缺失；在3/32（9.4%）具有家庭聚集性结直肠癌患者中检出遗传性MSH2基因DNA大片段缺失。而在20例散发性结直肠癌患者未检出这类突变。结论 中国人家族性结直肠癌患者中错配修复基因的大片段变异是频发事件，对此类患者的遗传检测应包含错配修复基因大片段变异的筛查。     

一个遗传性非息肉性结直肠癌大家系调查及种系突变研究

目的探讨一个中国人遗传性非息肉性结直肠癌（heraditary nonpolyposis colorectal cancer，HNPCC）大家系的临床特点，报告基因突变筛查结果。方法调查一个HNPCC大家系，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。抽取家族成员外周血，采用聚合酶链反应和扩增产物直接测序进行基因检测。结果该家系符合阿姆斯特丹Ⅰ标准，4代31人中17例患者共诊断21例次恶性肿瘤。12例（70．6％）患者患有直肠癌，且发病年龄早（平均42．9岁），右半结肠癌多见。基因检测发现一种国内外尚未见报道的MSH2基因的新突变。该突变位于MSH2基因的第7外显子中，由于4个核苷酸（CCGA）的重复导致移码突变，形成截短蛋白。结论HNPCC患者是恶性肿瘤（尤其是结直肠癌）的高发人群。新的MSH2基因突变（MSH2：C．1215-1218dupCCGA）导致该家系遗传性非息肉性结直肠癌的发生。     

Analysis of CDH1 gene expression in hereditary diffuse gastric cancer

目的 研究在遗传性弥漫型胃癌家系中是否存在上皮型钙黏素基因(CDH1)以及基因表达的异常.方法 收集符合遗传性弥漫型胃癌(HDGC)诊断标准的1个家系中15例成员的外周血和组织标本.用免疫组化和Westernblot法检测组织CDH1蛋白表达;提取基因组DNA,通过PCR扩增DNA直接测序检测CDH1基因16个外显子突变.用克隆测序法,鉴定CDH1基因启动子区CpG位点甲基化状况.结果 先证者和另一胃癌患者(家系2号成员)的癌旁胃黏膜上皮细胞CDH1蛋白表达较正常胃黏膜减弱,两者肿瘤组织的蛋白表达几乎为阴性.包括先证者在内的11例家系成员第14外显子mRNA水平2 377位点存在一个C→T的单核苷酸多态性(SNP),但未检测到16个外显子的胚系突变.相对于正常胃黏膜,先证者和家系2号成员的胃癌组织均有CDH1基因启动子的高甲基化,其瘤旁黏膜也有高甲基化.结论 此HDGC家系中,CDH1基因外显子胚系突变不是其致病原因,基因启动子区的甲基化可能是导致基因失活的原因之一.     

一种DAX-1基因移码突变(428delG)引起的X-连锁先天性肾上腺发育不良

目的 研究1个X-连锁先天性肾上腺发育不良家系的临床特征,检测患者及其家属中是否存在相关DAx-1基因的突变.方法 收集1个临床诊断X-连锁先天性肾上腺发育不良的家系中2例患者及亲属的临床资料和外周血标本;提取基因组DNA,设计4对引物扩增DAX-1基因的2个外显子,PCR扩增DAX-1的全部外显子,扩增产物经纯化后进行直接测序.测序结果 在核苷酸序列数据库进行比较分析.结果 两例患者(表兄弟)DAX-1基因第1外显子处均存在428delG半合子移码突变.而患者的基因型与其临床表型并不一致.家系中有3例女性(他们的母亲及外祖母)为此突变的杂合子,正常人及家族中其他成员无该位点突变.结论 在1个中国人先天性肾上腺发育不良家系中发现DAX-1新的移码突变428delG.各种基因型与表现型之间并无相关性.     

定量多重PCR-高效液相色谱分析错配修复基因大片段缺失

目的 建立一种以多重PCR-高效液相色谱(high performance liquid chromatography，HPLC)分析为基础的错配修复基因DNA大片段缺失检测技术。方法 设计合成35对引物，分8个多重PCR反应扩增MSH2和MLHl基因的35个外显子，PCR产物经高效液相色谱半定量分析，确定各外显子拷贝数。(1)双盲法分析阴性和阳性对照样本，完成方法学可靠性检验。(2)分析14例遗传性非息肉性大肠癌患者外周血细胞DNA和13例散发性大肠癌患者癌组织细胞DNA样本，筛查MSH2和．MLHl基因DNA大片段缺失。结果 (1)稳定检出阳性对照样本的DNA大片段缺失；(2)在筛查样本检出2例新的MSH2基因DNA大片段缺失，分别为MSH2基因外显子7遗传性缺失和MSH2基因外显子1～6体细胞性缺失。结论 多重PCR—HPLC分析系统可以作为突变基因分析系统的一个重要补充，在基因DNA大片段缺失检测中发挥作用。     

一个单纯家族性嗜铬细胞瘤家系的VHL基因突变筛查

目的检测一个单纯家族性嗜铬细胞瘤家系的VHL基因突变情况。方法对一个单纯家族性嗜铬细胞瘤家系进行VHL基因突变检测，抽取该家系5例患者及15名血缘亲属外周血基因组DNA，对VHL基因3个外显子进行PCR，产物进行DNA测序。结果该家系5例患者均检测出VHL基因第2外显子上第587位核苷酸A—C突变，该突变导致第125位编码氨基酸由组氨酸（H）转变为脯氨酸（P）。15名家系成员中筛查出7名成员为该突变基因携带者，B超检查发现1例为双侧肾上腺肿瘤，1例为右肾囊肿。该突变为首次报道。结论该嗜铬细胞瘤家系中检测到可能的致病突变，VHL基因检测可早期发现致病基因携带者，建议对单纯家族性嗜铬细胞瘤患者常规进行VHL基因突变筛查。     

Four novel MSH2 / MLH1 gene mutations in portuguese HNPCC families

Hereditary non-polyposis colorectal cancer (HNPCC) is considered to be determined by germline mutations in the mismatch repair (MMR) genes, especially MSH2 and MLH1. While screening for mutations in these two genes in HNPCC portuguese families, 3 previously unreported MSH2 and 1 MLH1 mutations have been identified in families meeting strict Amsterdam criteria. Hum Mutat 15:116, 2000.     

Germline mutations in MLH1, MSH2 and MSH6 in Korean hereditary non-polyposis colorectal cancer families

Hereditary non-polyposis colorectal cancer (HNPCC), the most common hereditary colon cancer syndrome, is a dominant disorder caused by germline defects in mismatch repair (MMR) genes. Identification of MMR gene mutations can have direct clinical implications in counseling and management of HNPCC families. We screened 44 HNPCC and 97 suspected HNPCC Korean families for germline mutations in three MMR genes: MLH1, MSH2 and MSH6. We identified twelve novel mutations: nine in MLH1(c.632_633insT, c.808_811delACTT, c.845C>G, c.1625A>C, c.1730+1delG, c.1907T>C, c.1918C>T, c.2104-2A>G and c.2170T>A), two in MSH2 (c.1886A>G, c.1316_1318delCCT) and one in MSH6 (c.3488A>T). In addition, two statically significant cSNPs in MLH1: c.1128T>C ( p=0.008 in HNPCC and p=0.037 in early-onset CRC) and c.2168C>A ( p<0.001 in HNPCC). Interestingly, the most frequent mutation, c.1757_1758insC in MLH1, was a founder mutation inherited from a common Korean ancestor.     

Mononucleotide microsatellite instability and germline MSH6 mutation analysis in early onset colorectal cancer.

Germline mutations in the MSH2 and MLH1 mismatch repair genes account for most cases of hereditary non-polyposis colon cancer syndrome (HNPCC). In addition, germline MSH2 and MLH1 mutations have been detected in patients with non-HNPCC early onset colorectal cancer. Germline MSH6 mutations appear to be rare in classical HNPCC families, but their frequency in young colorectal cancer cases has not been studied previously. In a population based study of early onset colorectal cancer (<50 years) investigated for tumour microsatellite instability (MSI), we identified a subgroup of tumours with MSI for mono- but not dinucleotide repeat markers (m-MSI+ group). In contrast to tumours with classical MSI for dinucleotide markers (d-MSI+), the m-MSI+ group cancers were mainly left sided (6/7). As MSH6 mutations in yeast and human cell lines are associated with weak (and preferential mononucleotide) MSI, the complete MSH6 gene coding region was sequenced in blood DNA from the five m-MSI+ cases available for analysis. A germline nonsense mutation was identified in an isolated case of early onset colorectal cancer (age 43 years). These results support previous findings that germline MSH6 mutations may not be associated with classical MSI and suggest a role for germline MSH6 mutations in isolated early onset colorectal cancer.     

Partial duplications of the MSH2 and MLH1 genes in hereditary nonpolyposis colorectal cancer

Numerous reports have highlighted the contribution of MSH2 and MLH1 genomic deletions to hereditary nonpolyposis colorectal cancer (HNPCC) or Lynch's syndrome, but genomic duplications of these genes have been rarely reported. Using quantitative multiplex PCR of short fluorescent fragments (QMPSF), 962 and 611 index cases were, respectively, screened for MSH2 and MLH1 genomic rearrangements. This allowed us to detect, in 11 families, seven MSH2 duplications affecting exons 1-2-3, exons 4-5-6, exon 7, exons 7-8, exons 9-10, exon 11, and exon 15, and three MLH1 duplications affecting exons 2-3, exon 4 and exons 6-7-8. All duplications were confirmed by an independent method. The contribution of genomic duplications of MSH2 and MLH1 to HNPCC can therefore be estimated approximately to 1% of the HNPCC cases. Although this frequency is much lower than that of genomic deletions, the presence of MSH2 or MLH1 genomic duplications should be considered in HNPCC families without detectable point mutations.     

Mean age of tumor onset in hereditary nonpolyposis colorectal cancer (HNPCC) families correlates with the presence of mutations in DNA mismatch repair genes

Fourteen Italian families affected with hereditary nonpolyposis colorectal cancer (HNPCC) were screened for germline mutations at three DNA mismatch repair (MMR) genes, MSH2, MLH1, and GTBP, by using a combination of different methods that included an in vitro synthesized protein assay, single-strand conformation polymorphism analysis, and direct sequencing. DNA alterations were observed in six instances, including a single base deletion in MSH2 exon 14, an A-to-G transition in the splice donor site of MLH1 exon 6, and two missense mutations in MLH1 exons 5 and 9. A previously reported common mutation affecting the splice donor site of MSH2 exon 5 was identified in two families. No mutations were detected in the GTBP gene. In total, eight of 16 Italian HNPCC families (50%), including two previously reported kindreds, were found to carry a mutation in MMR genes. We compared the mean age of colorectal cancer onset in the index cases (three patients for each family) between the two groups of kindreds, those with identified mutation vs. those without, and found that the first had a significantly lower value (43.0 vs. 53.7 years, P = 0.014). This finding suggests that HNPCC families with a more advanced age of tumor onset are less likely to be associated with known MMR genes. Genes Chromsom. Cancer 19:135–142, 1997. © 1997 Wiley-Liss Inc.     

No association between MUTYH and MSH6 germline mutations in 64 HNPCC patients

Hereditary non-polyposis colorectal cancer (HNPCC) is an autosomal dominant tumour predisposition syndrome caused by germline mutations in mismatch repair (MMR) genes. In contrast to MLH1 and MSH2, germline mutations in MSH6 are associated with a milder and particularly variable phenotype. Based on the reported interaction of the MMR complex and the base excision repair protein MUTYH, it was hypothesised that MUTYH mutations serve as phenotypical modifiers in HNPCC families. Recently, a significantly higher frequency of heterozygosity for MUTYH mutations among MSH6 mutation carriers was reported. We examined 64 MSH6 mutation carriers (42 truncating mutations, 19 missense mutations and 3 silent mutations) of the German HNPCC Consortium for MUTYH mutations by sequencing the whole coding region of the gene. Monoallelic MUTYH mutations were identified in 2 of the 64 patients (3.1%), no biallelic MUTYH mutation carrier was found. The frequency of MUTYH mutations was not significantly higher than that in healthy controls, neither in the whole patient group (P=0.30) nor in different subgroups regarding mutation type. Our results do not support the association between MSH6 mutations and heterozygosity for MUTYH mutations.