應用毛細管電泳法分析hMSH2 及MDM2之基因多型性、K-ras 點突變 及α型海洋性貧血基因缺失之研究

Study on Single Nucleotide Polymorphism of hMSH2 and MDM2 Gene, Point Mutations of K-ras Gene and Gene Deletion of α-thalassemia by Capillary Electrophoresis

陳彥伶(Yen-Ling Chen)

指導教授 ： 吳秀梅

高雄醫學大學/藥學院/藥學研究所/博士(2008年)

摘要

近年來於基因體學中，研究特定點突變對於疾病的相關聯性以及判定遺傳性疾病基因型成為一重要的方向。本研究之主要目的為開發毛細管電泳法針對數種特定基因 (hMSH2 基因，MDM2 基因，K-ras 基因) 之單核苷酸基因多型性 (single nucleotide polymorphism, SNP) 進行測定並評估其 SNP 對於數種癌症的相關聯性。另外，利用所開發之毛細管電泳法針對α型海洋性貧血症缺失基因型進行測定，希望此分析法能做為臨床上診斷的依據。而本研究主要的研究成果如下:
首先針對單核苷酸差異之辨別利用構形差異毛細管電泳法
(conformation-sensitive capillary electrophoresis, CSCE) 做為基因多型性之直接分析法，其原理為於分離緩衝溶液中添加適當比例之化學變性試劑造成野生型與突變型DNA 因部分變性而造成構形之差異，以達到分離的效果。本實驗以hMSH2 基因啟動子上之基因多型性 (C/C 同型合子，T/T 同型合子及C/T 異型合子) 為分析對象。hMSH2 基因為人類錯誤配對修復基因，其功用為辨識及修補錯誤配對的核苷酸，此一基因之多型性已知與遺傳性非息肉性大腸癌(hereditary nonpolyposis colorectal cancer, HNPCC)
具有關聯性。經由聚合酶連鎖反應 (polymerase chain reaction, PCR) 所得的PCR 產物，不經前處理直接以電動取樣方式注入毛細管。CE 分析條件為1×TBE (Tris-borate-EDTA) 緩衝液含有3% (w/v) 羥乙基纖維素(hydroxyethyl cellulose, HEC) (MW: 250,000) 和6 M 尿素，分離電壓設定在-5 kV，分離溫度設定於15℃。本CSCE 法應用於204 位健康志願受試者及13 位大腸直腸癌 (colorectal cancer, CRC) 病患之基因型測定，所得
之結果與定序法所得結果一致，可知CSCE 分析法對基因分析具有方便性及準確性，對臨床檢體大量篩檢甚具應用潛力。

有鑑於利用CSCE 分析方法進行基因多型性之測定時，在某些條件上會遭遇限制而導致野生型與突變型DNA 無法區分，故於第二個研究中利用另一分析法-單鏈構象多態性毛細管電泳法 (single strand conformation polymorphism-capillary electrophoresis, SSCP-CE) 進行MDM2 基因多型性測定，先將PCR 產物於注入毛細管電泳儀前進行變性，使雙股DNA 變性成單鏈DNA，並利用突變型與野生型之單鏈DNA 於毛細管電泳中呈現不同的構形，以達到分離效果。Two-in-one PCR 則是利用fluorescence-labeled nonhuman-sequence primer 以及fragment-oriented primer 兩種不同功能之引子於同一試管中進行PCR，所獲得PCR 產物具有螢光基團以利偵測。此一研究利用SSCP-CE 搭配two-in-one PCR 針對MDM2 基因中啟動子309位置上之SNP 進行分析，其所呈現之基因型分為T/T 同型合子，G/G 同型合子及T/G 異型合子三種。Two-in-one PCR 產物，經變性前處理後，以電動取樣的方式注入毛細管。CE 分析條件為1×TBE 緩衝液含有1.5% (w/v)HEC，分離電壓設定在-10 kV，分離溫度設定於25℃。本SSCP-CE 法應用於43 位患有慢性骨髓性白血病 (chronic myeloid leukemia, CML)，123位罹患CRC 及138 位健康自願受試進行MDM2 SNP309 之基因型測定，所得之結果與定序法所得結果一致，可知此分析法對基因分析具有方便性及準確性，並經測定結果發現CML 與MDM2 SNP309 之基因多型性具有相關聯性。
由於fluorescence-labeled nonhuman-sequence primer 適用於任何欲研究的基因放大反應，故接下來的研究同樣利用SSCP-CE 搭配two-in-one PCR針對K-ras 基因中codons 12 及13 位置上之點突變進行分析。K-ras 基因為一原致癌基因，其codons 12 及13 中鹼基突變與多種癌症具有相關性。目前常見的突變基因型有七種分別為CGT，GTT，TGT，GCT，AGT，GAT於codon 12 以及GAC 於codon 13。Two-in-one PCR 產物，經變性前處理後，以電動取樣的方式注入毛細管。CE分析條件為1×TBE 緩衝液含有1.5% (w/v) HEC，分離電壓設定在-10 kV。第一階段分析分離溫度設定於15℃，可判定CGT，GTT，TGT，GAC 突變型。第二階段分析分離溫度設定於30℃，可解析出TGT，GCT，AGT，GAT，GAC 突變型。本SSCP-CE 法應用於105 位罹患CRC 病患其K-ras 基因中codons 12 及13 位置上之基因型測定，所得之結果與定序法所得結果一致，並測得之CRC 患者其K-ras基因中codons 12 及13 突變率為25%。
針對基因缺失偵測，本實驗室利用毛細管膠體電泳法 (capillary gel electrophoresis, CGE) 藉由聚合物所形成的分子網篩，以分離不同片段長度之DNA。此研究主要目的為利用不同種類的聚合物，分別為HEC，羥丙基甲基纖維素 (hydroxypropylmethyl cellulose, HPMC) (MW: 90,000) 以及聚環氧乙烷 (poly(ethylene oxide), PEO) (MW: 8,000,000)等聚合物，探討其對於分離200-bp DNA ladder的影響。並經實驗評估後，所得之最佳化條件為1×TBE緩衝液含有0.6% (w/v) PEO和1 μM YO-PRO-1，分離電壓設定在-10
kV，分離溫度設定於25℃。此分析條件搭配多重聚合酶連鎖反應 (multiplex PCR) 應用於分析5種α型海洋性貧血基因缺失型之PCR產物，分別為右端缺失型 (-α3.7 )，左端缺失型 (-α4.2 )，東南亞型 (Southeast Asian, --SEA)，菲律賓型 (Filipino, --FIL) 以及泰國型 (Thai, --THAI)。21個α型海洋性貧血DNA檢品經分析後，其基因型與臨床診斷結果一致。
另一分析法， 金奈米填充毛細管電泳法(gold nanoparticle-filled capillary electrophoresis, G-NFCE) 為一添加金奈米粒子於聚合物緩衝溶液中，可改變待測物通過聚合物網篩時的能力，增加待測物間的分離效果。此研究利用G-NFCE 分析法，搭配multiplex PCR 針對5 種α 型海洋性貧血症中缺失基因型進行測定。為了獲得最佳化的CE 分離條件，分別針對使用毛細管的種類，聚合物濃度，金奈米 (gold nanoparticles, GNPs) 粒子大小，GNPs 濃度，緩衝液濃度，緩衝液pH 值，分離溫度及分離電壓針對200-bp DNA ladder (片段長度為0.2 kb-3.0 kb) 進行分析討論。最後，所得
之最佳化條件為利用DB-17 毛細管，於GNP32nm 溶液及glycine (25 mM, pH 9.0) 的金奈米混合溶液 (80:20 v/v) 內含有0.6% (w/v) PEO 和1 μM YO-PRO-1 中進行分析，分離電壓為-10 kV，分離的溫度設定於25℃。將此分析條件應用於5 種α 型海洋性貧血症基因缺失型之PCR 產物，並實際利用G-NFCE 分析法檢測21 位患有α 型海洋性貧血的患者，所測得之基因型與臨床診斷結果一致。

關鍵字

毛細管電泳法 ； hMSH2 ； MDM2 ； K-ras ； α型海洋性貧血 ； 基因多型性 ； 點突變 ； 基因缺失

並列摘要

Genomics becomes an important research area in recent years, especially for identifying single nucleotide polymorphism (SNP) of special gene and genotyping of hereditary disease. The aims of our studies were to establish several kinds of DNA analytical methods by capillary electrophoresis (CE) for detecting the SNPs of hMSH2 gene, MDM2 gene and K-ras gene, and exploring the relationship between the SNPs and cancers. Besides, the
genotyping of α-thalassemia deletions by CE was also studied for clinical research. Several works have been done, including:
CE allows for highly reproducible analysis of DNA fragments which can be used to detect point mutations in DNA. We have utilized a simple and direct CE analysis method for SNP analysis called conformation-sensitive capillary electrophoresis (CSCE), based on the principle of single nucleotide difference to produce conformational changes in the mildly denaturing solvent system.This method was applied to analyze the mutation in the promoter region of the hMSH2 gene. This gene belongs to the human DNA mismatch repair system, which is responsible for recognizing and repairing mispaired nucleotides, and
mutations in the hMSH2 gene are known to cause hereditary nonpolyposis colorectal cancer (HNPCC). Polymerase chain reaction (PCR) fragments generated from the promoter region of the hMSH2 gene, displaying either a C/C homozygote, C/T heterozygote, or T/T homozygote genotypes, did not require further pretreatment before electrokinetic injection. The CE separation,using a 1× TBE buffer containing 3% (w/v) hydroxyethyl cellulose (HEC) (MW: 250,000) and 6 M urea, was performed under reverse polarity with a separation temperature of 15°C. The genotypes of 204 healthy volunteers and 13 colorectal cancer (CRC) patients were determined by using this method, and the results were confirmed by DNA sequencing. Those separations were shown
to be highly reproducible and sensitive. After screening our samples, no correlation was observed between cancer patients and this hMSH2 gene polymorphism.
SNP309 in the promoter region of the murine double minute 2 (MDM2) gene plays an important role in human tumorigenesis. We established a simple and effective CE method for SNP detection in MDM2 gene. We designed one
universal fluorescence-based nonhuman-sequence primer and one fragment-oriented primer, which were combined in one tube, and proceeded the PCR.The amplicons were analyzed by capillary electrophoresis using single strand conformation polymorphism (SSCP) method. PCR fragments generated from
this two-in-one PCR displayed either T/T or G/G homozygote, or T/G heterozygote. Running buffer was 1×TBE buffer containing 1.5% (w/v) HEC ; applied voltage was -10 kV (detector at anode side) and separation temperature
was at 25°C. Total 304 samples were blindly genotyped using this developed method, which included the DNA from 138 healthy volunteers, 43 chronic myeloid leukemia (CML) patients, and 123 CRC patients. The results were confirmed by DNA sequencing and showed good agreement. The SSCP-CE
method was feasible for SNP screening of MDM2 in large populations.
Mutations of K-ras gene in codons 12 and 13 were frequently observed in a variety of cancers. We established a simple and effective CE method for multiple SNPs detection in codons 12 and 13 of K-ras gene. One universal
fluorescence-based nonhuman-sequence primer and one fragment-oriented primer were combined in one tube, and proceeded the PCR. The amplicons were analyzed by CE using SSCP method. PCR fragments generated from this two-in-one PCR displayed wild type (GGT in codon 12，GGC in codon 13) and seven different point mutations at codons 12 (CGT， GTT， TGT， GCT，AGT，GAT) and 13 (GAC) of K-ras gene. Two steps of the CE separation,using a TBE buffer containing 1.5 % (w/v) HEC, was performed under reverse polarity with a separation temperature of 15℃ at first run and 30℃ at second run. Total 105 CRC patients were blindly genotyped using this developed method. All results were confirmed by DNA sequencing and showed good agreement. The SSCP-CE method was feasible for SNPs screening of K-rasgene populations.
Capillary gel electrophoresis (CGE) was the most common analytical method for DNA separation. In order to evaluate the polymer effect, different kinds of polymer were used for analyzing 200-bp DNA ladder. By optimized CE condition, the CE separation was performed by DB-17 capillary. Running
buffer was 1×TBE buffer containing 0.6% (w/v) poly(ethylene oxide) (PEO)(MW: 8,000,000) and 1 μM YO-PRO-1 ; applied voltage was -10 kV (detector at anode side) and separation temperature was at 25°C. Under these optimal conditions, 15 DNA fragments with their sizes ranging from 0.2 kb to 3.0 kb
were resolved within 11.5 min and the RSD of migration time were less than 0.55% (n=3). This method combining with three multiplex PCR was applied in five α-thalassemia deletions detection, including -α 3.7, -α 4.2, --SEA, --FIL and --THAI. Total 21 patients that diagnoses of α-thalassemia were analyzed using this developed method and all results were showed good agreements with clinical diagnosis.
Gold nanoparticle-filled CE (G-NFCE) combining with three multiplex PCR was first established for multiple diagnosis of five common α- thalassemia deletions. Gold anoparticles (GNPs) were used as pseudostationary phase to improve separation resolutions between DNA fragments in a low-viscosity polymer. In order to achieve best CE separation, several parameters were evaluated for optimizing separation condition, such as kinds of coated capillary,the concentrations of polymer sieving matrix, the sizes and concentrations of GNPs, the buffer concentrations and pH values. The CE separation, used for detecting 200-bp DNA ladder and α-thalassemia deletions. Sieving matrix was
0.6% (w/v) PEO prepared in a mixture of GNP32nm solution and glycine (25 mM, pH 9.0) (80:20 v/v) that containing 1 μM YO-PRO-1; applied voltage was -10 kV (detector at anode side) and separation temperature was at 25°C. Under
these optimal conditions, 15 DNA fragments with their sizes ranging from 0.2 kb to 3.0 kb were resolved within 11.5 min and the RSD of migration time were less than 2.81% (n=3). Total 21 patients that diagnosis of α-thalassemia
deletions were analyzed using this developed method and all results were showed good agreements with clinical diagnosis.

並列關鍵字

Capillary Electrophoresis ； hMSH2 ； MDM2 ； K-ras ； Single Nucleotide Polymorphism ； Point Mutations ； Gene Deletion

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