二代測(cè)序技術(shù)在家族性非髓樣甲狀腺癌遺傳易感基因篩查中的應(yīng)用
發(fā)布時(shí)間：2018-08-14 13:18
【摘要】：目的:家族性非髓樣甲狀腺癌(familial non-medullary thyroid carcinoma,FNMTC)包括甲狀腺乳頭狀癌、甲狀腺濾泡癌、甲狀腺未分化癌,其特異性致病基因尚不清楚。隨著測(cè)序技術(shù)發(fā)展革新,二代測(cè)序技術(shù)以其高通量、平行測(cè)序及經(jīng)濟(jì)高效的特點(diǎn)得到廣泛的應(yīng)用推廣。本研究通過(guò)篩選與甲狀腺癌遺傳易感性相關(guān)的基因,制備適用于非髓樣甲狀腺癌易感性篩查的二代測(cè)序試劑盒探針,應(yīng)用于非髓樣甲狀腺癌遺傳易感基因的篩查,探討二代測(cè)序技術(shù)對(duì)非髓樣甲狀腺癌遺傳易感性篩查的價(jià)值。方法:以甲狀腺癌相關(guān)的31個(gè)基因的全外顯子以及RET基因的部分內(nèi)含子為目標(biāo)區(qū)域制備二代測(cè)序試劑盒探針。收集47例FNMTC及16例散發(fā)性非髓樣甲狀腺癌(sporadic non-medullary thyroid carcinoma,SNMTC)患者外周血,提取DNA樣本,利用探針,與全基因組DNA雜交,富集目標(biāo)區(qū)域,再利用Illumina MiSeq第二代測(cè)序儀進(jìn)行測(cè)序。測(cè)序數(shù)據(jù)分析采用BWA aligner 0.7.10,DNA易位分析采用Tophat2和Factera 1.4.3完成;用ExAC和ClinVar數(shù)據(jù)庫(kù)分析、過(guò)濾,篩出高質(zhì)量的突變位點(diǎn),采用Sanger測(cè)序法驗(yàn)證突變位點(diǎn)。利用SPSS19.0軟件對(duì)FNMTC和SNMTC兩組患者的突變比例、突變組和未突變組患者的臨床病理資料進(jìn)行統(tǒng)計(jì)學(xué)分析,以P0.05作為差異有統(tǒng)計(jì)學(xué)意義。結(jié)果:1.在基于二代測(cè)序的FNMTC易感性檢測(cè)中,每個(gè)樣本的產(chǎn)生的總測(cè)序數(shù)據(jù)量約為1.3~2.9兆(M)測(cè)序讀段(reads),平均為2.5M測(cè)序讀段,平均98.9%的讀段在基因組,54.3%的讀段分布在二代測(cè)序的目標(biāo)區(qū)域內(nèi);對(duì)于FNMTC檢測(cè)的二代測(cè)序目標(biāo)區(qū)域,平均測(cè)序深度為500×,最小和最大測(cè)序深度分別為157×和1505×;所有樣本中,超過(guò)96%的目標(biāo)測(cè)序區(qū)域的平均測(cè)序深度大于200×(測(cè)序深度是測(cè)序得到的堿基總量與基因組大小的比值,是評(píng)價(jià)測(cè)序量的指標(biāo)之一)。通過(guò)不同樣本間每個(gè)目標(biāo)區(qū)域覆蓋深度的關(guān)聯(lián)性對(duì)探針捕獲效率可重復(fù)性的評(píng)估顯示,大多數(shù)成對(duì)的樣本間關(guān)聯(lián)系數(shù)大于0.8。2.在63例非髓樣甲狀腺癌患者中共發(fā)現(xiàn)45個(gè)高質(zhì)量的雜合性胚系突變,其中包括38個(gè)單核苷酸變異,1個(gè)缺失突變,1個(gè)剪接供體變異,和5個(gè)框內(nèi)插入或缺失突變;47例FNMTC患者共攜帶37個(gè)胚系突變,14例SNMTC患者共攜帶8個(gè)胚系突變。FNMTC患者中基因突變攜帶者的比率高于散發(fā)組(61.7%vs.37.5%,P=0.092);最常見的胚系突變基因是MSH6,其次為APC;在8個(gè)FNMTC家系中發(fā)現(xiàn)同一家系的患者攜帶相同的胚系突變位點(diǎn),分別為APC L292F、A2778S,MSH6 G355S、A36V,MSH2 L719F,BRAF D22N,MEN1 G508D,BRCA1 SS955S,BRCA2 G2508S和GNAS P459PDAPADPDSGAAR。3.根據(jù)突變結(jié)果將非髓樣甲狀腺癌患者分為兩組:突變組(n=29)和未突變組(n=18)。對(duì)患者的臨床病理特征進(jìn)行比較分析發(fā)現(xiàn),突變組患者中央?yún)^(qū)淋巴結(jié)轉(zhuǎn)移率較未突變組明顯增高(68.6%vs.30.8%,P=0.003)。且在具有親子關(guān)系的FNMTC患者中,子代患者的中央?yún)^(qū)淋巴結(jié)轉(zhuǎn)移率高于親代患者(100%vs.42.9%,P=0.019)。結(jié)論:本研究中的FNMTC易感性探針捕獲效率穩(wěn)定,得到的突變數(shù)據(jù)經(jīng)全面質(zhì)量控制(堿基變異分布、最小等位基因頻數(shù)分布等)符合胚系突變單核苷酸多態(tài)性變異的典型特征,數(shù)據(jù)可靠性高。二代測(cè)序技術(shù)在FNMTC樣本易感性基因篩查中的應(yīng)用,幫助我們發(fā)現(xiàn)了與FNMTC相關(guān)的一些新突變位點(diǎn);同時(shí)提示我們不同的FNMTC家系攜帶的胚系突變基因可能不同。攜帶有胚系突變的FNMTC患者更易于出現(xiàn)中央?yún)^(qū)淋巴結(jié)的轉(zhuǎn)移,對(duì)其進(jìn)行預(yù)防性中央?yún)^(qū)淋巴結(jié)清掃是必要的。因此,利用FNMTC易感性探針檢測(cè)胚系基因突變,對(duì)FNMTC患者治療方案的制定具有指導(dǎo)意義。
[Abstract]:Objective: The specific pathogenic genes of familial non-medullary thyroid carcinoma (FNMTC) including papillary thyroid carcinoma, follicular thyroid carcinoma and undifferentiated thyroid carcinoma are unknown. In this study, by screening genes related to genetic susceptibility to thyroid cancer, a second-generation sequencing kit probe for non-medullary thyroid cancer susceptibility screening was prepared, which was used to screen genes susceptible to non-medullary thyroid cancer, and to explore the second-generation sequencing technology for non-medullary thyroid cancer genetic susceptibility screening. Methods: DNA samples were extracted from peripheral blood of 47 FNMTC patients and 16 sporadic non-medullary thyroid carcinoma (SNMTC) patients. DNA samples were extracted from the peripheral blood of the patients with FNMTC and 16 sporadic non-medullary thyroid carcinoma (SNMTC). Genome-wide DNA hybridization was performed to enrich the target region, and then sequenced using Illumina MiSeq second-generation sequencer. BWA aligner 0.7.10 was used to analyze the sequence data. Tophat 2 and Factera 1.4.3 were used to analyze the DNA translocation. ExAC and CLINVAR databases were used to analyze, filter and screen out high-quality mutation sites. Sanger sequencing was used to verify the mutation sites. Results: 1. In FNMTC susceptibility testing based on second-generation sequencing, the total amount of sequencing data produced by each sample was about 1.3-2.9 trillion (M) measurements. The average reads were 2.5M, 98.9% in genome and 54.3% in the target region of the second-generation sequencing. For the second-generation sequencing target region detected by FNMTC, the average sequencing depth was 500 x, and the minimum and maximum sequencing depth were 157 X and 1505 x, respectively. The average sequencing depth is more than 200 * (the ratio of the total number of bases to the genome size is one of the criteria for evaluating the sequencing quantity). The correlation between the coverage depth of each target area among different samples shows that the correlation coefficient between most pairs of samples is greater than 0.8. 2. A total of 45 high-quality heterozygous embryoline mutations were found in 63 patients with non-medullary thyroid carcinoma, including 38 single nucleotide mutations, 1 deletion mutation, 1 splice donor mutation, and 5 intra-frame insertion or deletion mutations; 47 patients with FNMTC carried 37 embryoline mutations, and 14 patients with SNMTC carried 8 embryoline mutations. The frequency of gene mutation carriers was higher than that of sporadic group (61.7% vs. 37.5%, P = 0.092); the most common embryonic line mutation gene was MSH6, followed by APC; the same embryonic line mutation sites were found in eight FNMTC families, namely APC L292F, A2778S, MSH6 G355S, A36V, MSH2 L719F, BRAF D22N, MEN1 G508D, BRCA1 SS955S, BRCA2 2050, BRCA2 G355S 8S and GNAS P459 PDAPAD PDSGAAR.3. Patients with non-medullary thyroid carcinoma were divided into two groups according to the mutation results: mutation group (n=29) and non-mutation group (n=18). A comparative analysis of the clinical and pathological characteristics showed that the central lymph node metastasis rate of the mutation group was significantly higher than that of the non-mutation group (68.6% vs. 30.8%, P = 0.003). The central lymph node metastasis rate in the offspring of FNMTC patients was higher than that in the parental group (100% vs. 42.9%, P = 0.019). Conclusion: The FNMTC susceptibility probe capture efficiency in this study was stable, and the mutation data obtained by total quality control (base mutation distribution, minimum allele frequency distribution, etc.) conformed to the single nucleotide polymorphism of embryonic line mutation. The application of second-generation sequencing technology in FNMTC sample susceptibility gene screening has helped us find some new mutation sites related to FNMTC, and suggested that different FNMTC families may carry different mutation genes of embryonic line. Preventive central lymph node dissection is necessary for central lymph node metastasis. Therefore, the detection of embryonic gene mutation by FNMTC susceptibility probe is of guiding significance for the treatment of FNMTC patients.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：R736.1
本文編號(hào)：2182975
[Abstract]:Objective: The specific pathogenic genes of familial non-medullary thyroid carcinoma (FNMTC) including papillary thyroid carcinoma, follicular thyroid carcinoma and undifferentiated thyroid carcinoma are unknown. In this study, by screening genes related to genetic susceptibility to thyroid cancer, a second-generation sequencing kit probe for non-medullary thyroid cancer susceptibility screening was prepared, which was used to screen genes susceptible to non-medullary thyroid cancer, and to explore the second-generation sequencing technology for non-medullary thyroid cancer genetic susceptibility screening. Methods: DNA samples were extracted from peripheral blood of 47 FNMTC patients and 16 sporadic non-medullary thyroid carcinoma (SNMTC) patients. DNA samples were extracted from the peripheral blood of the patients with FNMTC and 16 sporadic non-medullary thyroid carcinoma (SNMTC). Genome-wide DNA hybridization was performed to enrich the target region, and then sequenced using Illumina MiSeq second-generation sequencer. BWA aligner 0.7.10 was used to analyze the sequence data. Tophat 2 and Factera 1.4.3 were used to analyze the DNA translocation. ExAC and CLINVAR databases were used to analyze, filter and screen out high-quality mutation sites. Sanger sequencing was used to verify the mutation sites. Results: 1. In FNMTC susceptibility testing based on second-generation sequencing, the total amount of sequencing data produced by each sample was about 1.3-2.9 trillion (M) measurements. The average reads were 2.5M, 98.9% in genome and 54.3% in the target region of the second-generation sequencing. For the second-generation sequencing target region detected by FNMTC, the average sequencing depth was 500 x, and the minimum and maximum sequencing depth were 157 X and 1505 x, respectively. The average sequencing depth is more than 200 * (the ratio of the total number of bases to the genome size is one of the criteria for evaluating the sequencing quantity). The correlation between the coverage depth of each target area among different samples shows that the correlation coefficient between most pairs of samples is greater than 0.8. 2. A total of 45 high-quality heterozygous embryoline mutations were found in 63 patients with non-medullary thyroid carcinoma, including 38 single nucleotide mutations, 1 deletion mutation, 1 splice donor mutation, and 5 intra-frame insertion or deletion mutations; 47 patients with FNMTC carried 37 embryoline mutations, and 14 patients with SNMTC carried 8 embryoline mutations. The frequency of gene mutation carriers was higher than that of sporadic group (61.7% vs. 37.5%, P = 0.092); the most common embryonic line mutation gene was MSH6, followed by APC; the same embryonic line mutation sites were found in eight FNMTC families, namely APC L292F, A2778S, MSH6 G355S, A36V, MSH2 L719F, BRAF D22N, MEN1 G508D, BRCA1 SS955S, BRCA2 2050, BRCA2 G355S 8S and GNAS P459 PDAPAD PDSGAAR.3. Patients with non-medullary thyroid carcinoma were divided into two groups according to the mutation results: mutation group (n=29) and non-mutation group (n=18). A comparative analysis of the clinical and pathological characteristics showed that the central lymph node metastasis rate of the mutation group was significantly higher than that of the non-mutation group (68.6% vs. 30.8%, P = 0.003). The central lymph node metastasis rate in the offspring of FNMTC patients was higher than that in the parental group (100% vs. 42.9%, P = 0.019). Conclusion: The FNMTC susceptibility probe capture efficiency in this study was stable, and the mutation data obtained by total quality control (base mutation distribution, minimum allele frequency distribution, etc.) conformed to the single nucleotide polymorphism of embryonic line mutation. The application of second-generation sequencing technology in FNMTC sample susceptibility gene screening has helped us find some new mutation sites related to FNMTC, and suggested that different FNMTC families may carry different mutation genes of embryonic line. Preventive central lymph node dissection is necessary for central lymph node metastasis. Therefore, the detection of embryonic gene mutation by FNMTC susceptibility probe is of guiding significance for the treatment of FNMTC patients.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：R736.1
【相似文獻(xiàn)】
相關(guān)期刊論文 前10條
1 吳雙濱，，潘志貴;近代人類胚系突變：從乙型血友病患者的推斷[J];國(guó)外醫(yī)學(xué).遺傳學(xué)分冊(cè);1995年06期
2 孟曉明;金鵬;盛劍秋;陸曉娟;付蕾;范如英;李恕軍;李世榮;;散發(fā)性大腸癌患者中錯(cuò)配修復(fù)基因胚系突變篩查策略[J];胃腸病學(xué)和肝病學(xué)雜志;2010年07期
3 劉曉蓉,單祥年,Friedl W,Uhlhaas S,Propping P,王亞平;家族性腺瘤樣息肉病中APC基因的胚系突變分析(英文)[J];中華醫(yī)學(xué)遺傳學(xué)雜志;2005年03期
4 劉曉蓉,單祥年,FriedlW,UhlhaasS,李金田,ProppingP,王亞平;應(yīng)用蛋白截短技術(shù)檢測(cè)APC基因胚系突變[J];遺傳學(xué)報(bào);2005年09期
5 焦益群,宮恩聰,孟桂霞,劉叔平,鄂文,丁怡;家族性腺瘤性息肉病患者APC基因胚系突變的檢測(cè)[J];北京醫(yī)科大學(xué)學(xué)報(bào);1997年03期
6 周建農(nóng);陳森清;張曉梅;周欣;朱明;馮波;李金田;馬國(guó)建;張?jiān)f;;家族性腺瘤性息肉病家系中一種新的APC基因的胚系突變[J];中華醫(yī)學(xué)遺傳學(xué)雜志;2006年04期
7 許焱;艾斌;;非小細(xì)胞肺癌EGFR基因胚系突變研究現(xiàn)狀[J];中國(guó)實(shí)用醫(yī)藥;2013年20期
8 王振軍,嚴(yán)仲瑜,萬(wàn)遠(yuǎn)廉,劉玉村,呂有勇,徐文懷,黃儦庭;黑斑息肉病LKB1基因胚系突變和腫瘤易感性研究[J];中國(guó)胃腸外科雜志;2000年02期
9 潘鍵;陳春燕;李玫;張曉梅;朱明;劉炯;;兒童Peutz-Jeghers綜合征家系的基因突變[J];醫(yī)學(xué)研究生學(xué)報(bào);2012年09期
10 張?jiān)f,李金田,朱明,張曉梅,吳曉柳,王亞平;E-Cadherin(CDH1)基因胚系突變與胃癌風(fēng)險(xiǎn)的研究[J];癌變.畸變.突變;2005年06期
相關(guān)博士學(xué)位論文 前1條
1 周建農(nóng);家族性腺瘤性息肉病家系中一種新的APC基因的胚系突變[D];南京醫(yī)科大學(xué);2006年
相關(guān)碩士學(xué)位論文 前5條
1 楊曉晨;基于高通量測(cè)序的中國(guó)人群中具有遺傳傾向乳腺癌家系中易感基因的篩查[D];復(fù)旦大學(xué);2014年
2 陳青偉;基于MLPA和DHPLC技術(shù)平臺(tái)的家族性腺瘤息肉病家系致病性APC基因胚系突變篩查及突變分子機(jī)制分析[D];南京醫(yī)科大學(xué);2015年
3 董莉;二代測(cè)序技術(shù)在家族性非髓樣甲狀腺癌遺傳易感基因篩查中的應(yīng)用[D];天津醫(yī)科大學(xué);2016年
4 陳春燕;家族性Peutz-Jeghers綜合征患者LKB1基因胚系突變的分析[D];南京大學(xué);2012年
5 李聯(lián)杰;Peutz-Jeghers綜合征STK11基因胚系突變及STK11、TGFβ1蛋白表達(dá)研究[D];南方醫(yī)科大學(xué);2009年
本文編號(hào)：2182975
本文鏈接：http://sikaile.net/kejilunwen/jiyingongcheng/2182975.html
最近更新
教材專著
