purification of ligation product, amplification and library purification for multi-gene snp detection in gastric cancer

使用 Ion Torrent 测序的 SNP 指导的胃癌 5-FU 疗法

Gastric cancer is a heavy burden in the field of global public health. According to the Global Cancer Statistics 2020, published by the International Agency for Research on Cancer (IARC), gastric cancer is the fifth most diagnosed cancer and the fourth leading cause of cancer-related death. Worldwide, the incidence of age-standardized rate in East Asia is the highest in both males and females1. The occurrence of gastric cancer is insidious, which means that patients often do not have any obvious and specific symptoms in the early stage. Among all gastric cancer patients, in countries without routine screening, 80%-90% of patients are either diagnosed at an advanced stage when the tumor cannot be operated on or relapses within 5 years after the operation2.
For advanced or metastatic gastric cancer, chemotherapy is the main treatment, which can improve the survival rate and quality of life of patients. For the initial therapy of patients with metastatic gastric cancer, a platinum-fluoropyrimidine regimen is the principal choice for a first-line chemotherapeutic regimen3. Fluoropyrimidine mainly includes 5-fluorouracil (5-FU) and oral fluoropyrimidine derivatives, such as capecitabine and tegafur. The main target of 5-FU is folate metabolism-related enzymes, which inhibit DNA synthesis and slow the growth of tumor tissue. Adverse drug reactions limit their utility, with diarrhea, mucositis, myelosuppression, and hand-foot syndrome among the most frequent side effects. It has been reported that therapeutic response and adverse drug reactions are closely related to factors in the folate metabolic pathway. Notably, the homozygous mutation of rs1801131 has been identified as an indicator for hand-foot syndrome (p = 4.1 x 10-6, OR =9.99, 95% CI: 3.84-27.8)4. Although fluoropyrimidines are extensively used in anticancer chemotherapy, their chemoresistance is a common emergency, causing therapeutic failure in gastric cancer treatment. For instance, the overall response rate is only 10%-15% among patients with advanced colorectal cancer treated with 5-FU only5. Also, fluoropyrimidines have toxicity that cannot be ignored. The toxicity reactions induced by 5-FU mainly include diarrhea, hand-foot syndrome, stomatitis, neutropenia, thrombocytopenia, neurotoxicity, and even death6. Severe treatment-related toxicity occurs in 10%-30% of patients treated with fluoropyrimidines, and fatal toxicity occurs in 0.5%-1% of these patients7.
A quality-of-life study of patients with advanced gastric cancer found that the response rate was less than 50% for those who received 5-FU-based chemotherapy8. Therefore, understanding the factors related to the sensitivity of 5-FU-based chemotherapy is particularly important for precise treatment to maximize the response rate and effectiveness while minimizing toxicity. Considering that 5-FU is closely related to folate metabolism, the genetic variants of enzymes in folate metabolic pathway may be one of the factors. About 90% of human sequence variation is attributed to single base mutations in DNA, known as single nucleotide polymorphisms (SNPs)9. When SNPs change the enzyme properties of folate metabolism, it may lead to individual differences in efficacy, toxicity and chemoresistance to fluorouracil in gastric cancer patients.
Methylenetetrahydrofolate reductase (MTHFR) is mainly used to convert 5,10-methylenetetrahydrofolate (5,10-MTHF) into 5-methyltetrahydrofolate. 5-FdUMP,a metabolite of 5-FU, forms inactive ternary with 5,10-MTHF and thymidylate synthase (TS), inhibiting activity of TS and leading to deficiency of dTMP10. Accumulation of 5,10-MTHF can enhance the inhibition effect of 5-FU on TS, which is correlated with the activity of MTHFR. MTHFR rs1801131 and rs1801133 are the most common polymorphisms, which are related to lower enzyme activity (a decrease of 75% for rs1801133 and 30% for rs181131) and accumulation of 5,10-MTHF11.
Dihydrofolate reductase (DHFR) is the key enzyme in folate metabolism and DNA synthesis. DHFR reduces dihydrofolate, using NADPH, to tetrahydrofolate (THF) which is used to carry one-carbon unit. SNPs of DHFR may affect its expression, change the activity and abundance of THF, and further affect folate metabolism and sensitivity of 5-FU. DHFR rs1650697 point mutation occurs in the major promoter of DHFR gene, which increases the DHFR expression12. A study found that rs442767 is associated with the efficacy and toxicity of antifolate antitumor drugs, such as pemetrexed and methotrexate. Regarding the SNP rs442767, a GT genotype signifies the inheritance of a G allele and a T allele at the same locus on the homologous chromosomes from each parent. Similarly, the genotypes GG and TT denote the inheritance of either two G alleles or two T alleles, correspondingly. Compared with genotype GT+TT, GG is related to decreased event-free survival and increased risk13. This suggests that rs442767 may lead to certain potential influence on 5-FU.
Methionine synthase (MTR) catalyzes the re-methylation of homocysteine to methionine, which plays an important role in folate metabolism. MTR rs1805087 is the most common polymorphism of MTR gene. MTR rs1805087 substitutes glycine for aspartic acid at the potentially functional site of protein, which may decrease the activity of MTR. Subjects with the G allele had increased plasma folate level and decreased plasma homocysteine level14. On the contrary, a study showed that rs1805087 has no statistically significant association with the efficacy of 5-FU. But this study focused on colorectal cancer and the sample size was small. The relationship between rs1805087 and the efficacy of 5-FU in gastric cancer patients remains to be explored15.
Gamma-glutamyl hydrolase (GGH) is a lysosomal enzyme that regulates intracellular folate concentrations. Pteroylglutamic acid is the synonym of folic acid that is made up of pterin, p-aminomethylbenzoic acid and glutamic acid. There are two forms of folic acid in organisms, monoglutamate folate and polyglutamated folate. THF-polyglutamate is enzymatically converted to monoglutamic folate by GGH, successively releasing either mono-Glutamate (mono-Glu) or di-Glutamate (di-Glu)16. A study about GGH expression in patients with locally advanced gastric cancer, showed high GGH expression could reduce 5,10-MTHF and TS, which means that only a small dosage of 5-FU is needed to achieve the TS inhibitory effect in these patients17. GG is a prognostic biomarker in patients with locally advanced gastric cancer treated with postoperative adjuvant chemotherapy with S-1, a prodrug of 5-FU, and plays an important role in maintaining intracellular homeostasis of folic acid18. GGH rs11545078 is missense variant and alters Thr-127 to Ile-127. A study focusing on substrate specificity of GGH suggests that rs11545078, compared to wild-type, results in higher Km and lower catalytic efficiency for methotrexate and the structure is similar to folic acid19. Together, exploring the relationship between rs11545078 and clinical outcomes of 5-FU is a promising strategy to understand drug resistance.
Solute carrier family 19 member 1 (SLC19A1), also named reduced folate transporter, is a typical facilitative transmembrane protein that imports reduced folates that mammalian cells lack the ability to de novo synthesize, which is recognized to estimate the response of tumor to 5-FU20. However, only a few studies have been performed regarding the association between 5-FU and SLC19A1 polymorphism21. In patients with non-small cell lung cancer receiving pemetrexed which is a folate analog, the rs1051298 on the SLC19A1 gene contributed to increase the risk of all adverse drug reaction and decrease overall survival22,23. SLC19A1 rs1051298, a 3&#39;untranslated region variant about folate metabolism, may help explain some of the individual differences about 5-FU therapy. Here the aim is to evaluate the association between rs1051298 and 5-FU resistance among patients with gastric cancer.
A kit, based on semiconductor sequencing, is used for qualitative gene detection in vitro (Figure 1), which can detect 7 selected SNPs in 5 genes, the rs1801131 and rs1801133 mutations of MTHFR gene, rs1650697 and rs442767 mutations of DHFR gene, rs1805087 mutation of MTR gene, rs11545078 mutation of GGH gene and rs1051298 of SLC19A1 gene in tumor tissue samples of gastric cancer patients. Firstly, the sample nucleic acid was extracted, and the target fragment was specifically amplified by PCR. A universal sequencing adapter was added at both ends of the DNA fragment to construct a library that can be used for sequencing. Then amplification of the sequencing library by PCR to form a sequencing template was done. The positive template was enriched to meet the sequencing requirements. Using the semiconductor sequencing system, by fixing the DNA strand in the tiny hole of the semiconductor chip. DNA polymerase takes the single-stranded DNA as the template and synthesizes the complementary DNA strand according to the principle of complementary base pairing. Each time a base is extended in a DNA chain, a proton will be released, causing local pH changes. An ionic sensor detects pH changes and converts chemical signals into digital signals, so that bases can be interpreted in real time, and finally the base sequence of each DNA segment can be obtained. Bioinformatics analysis was used to match these sequences to the reference map of the human genome. When gastric cancer related genes mutate, their corresponding DNA base sequences will change, so as to obtain the mutation information of related genes.
The result can display gene mutation status and provide reference for clinicians to select appropriate types and dosages of chemotherapy drugs and predict the drug resistance for gastric cancer patients. However, the test results are only for clinical reference and are not recommended to be the only basis for individualized treatment of patients. Clinicians should make a comprehensive judgment based on the patient&#39;s condition, drug indications, treatment reactions and other laboratory test indicators.

作者聚焦：胃癌氟尿嘧啶反应的遗传分析

基于离子半导体测序平台的胃癌多基因单核苷酸多态性检测

Our research team is devolving into the elements that dictate the sensitivity and resistance to 5-fluorouracil, a key chemotherapeutic agent for gastric cancer, emphasizing genetic inferences. We are dedicated to uncovering how genetic differences impact drug reactions and assessing the feasibility of tailoring treatment to individual genetic profiles. This protocol is tailored to detect single-nucleotide polymorphisms in patients with gastric cancer, elucidating the correlation between genetic mutations and their sensitivity to fluorouracil chemotherapy.This breakthrough enables the prediction of patients'responses to fluorouracil, facilitating the customization of treatment strategies. This protocol stands out by offering high sensitivity for various sample types, including blood, reducing invasiveness with multiple samples. Utilizing semiconductor sequencing simplifies operations and consequently time by directly detecting ion changes by passing fluorouracils.

连接产物的纯化、扩增和文库纯化在胃癌多基因SNP检测中的应用

首先，从 2 至 8 摄氏度的冰箱中取出 DNA 纯化磁珠。混合后将珠子以 2， 500G 离心 10 秒。将接头连接的反应产物转移到 1.5 毫升低吸收微量离心管中。向每个试管中加入 45 微升 DNA 纯化的磁珠。涡旋组件后，将试管离心 10 秒钟，然后将试管放在磁性架上 3 分钟。小心地丢弃上清液，不要移液珠子。现在将 300 微升 75% 新鲜制备的乙醇转移到每个微量离心管中。轻轻地将管子旋转 180 度四次。溶液澄清后，立即丢弃上清液，同时避免移出珠子。然后，从磁铁架上取下试管后，短暂离心试管。将试管放在磁铁架上，然后吸取任何剩余的液体。打开 1.5 毫升微量离心管的盖子，让珠子在室温下干燥 5 分钟。解冻并涡旋PCR相关试剂后，将试剂离心10秒。从磁铁架上取出1.5毫升微量离心管，加入PCR试剂。短暂离心涡旋管，以避免液滴落在管壁和管盖上。然后将PCR产物转移到新的PCR管中。在热循环仪上孵育，并运行DNA和CDNA样品的扩增程序。孵育后，将PCR管离心10秒，然后将产物转移到新的1.5毫升低吸收微量离心管中。向每个试管中加入 25 微升 DNA 纯化的磁珠，并通过涡旋充分混合。将试管低速离心，并在室温下孵育五分钟。然后将试管放在磁性架上三分钟，直到溶液澄清。将上清液转移到新的微量离心管中，避免移出珠子。将 300 微升 75% 新鲜制备的乙醇转移到试管中，并使用磁架洗涤珠子两次，如前所述。短暂离心试管后，将试管放在架子上并移出任何剩余的液体。打开 1.5 毫升管的盖子，在室温下干燥珠子 5 分钟。将 50 微升洗脱液移液到试管中。涡旋试管后进行短暂的离心。将试管放在磁性架上三分钟，直到溶液变得透明。小心地将液体转移到新管中，并在其上贴上文库名称。

purification-ligation-product-amplification-library-purification-for

Research

JoVE Journal

Medicine

Purification of Ligation Product, Amplification and Library Purification for Multi-Gene SNP Detection in Gastric Cancer

233 次观看.  Nanfang Hospital, Southern Medical University. 首先，从 2 至 8 摄氏度的冰箱中取出 DNA 纯化磁珠。混合后将珠子以 2， 500G 离心 10 秒。将接头连接的反应产物转移到 1.5 毫升低吸收微量离心管中。向每个试管中加入 45 微升 DNA 纯化的磁珠。涡旋组件后，将试管离心 10 秒钟，然后将试管放在磁性架上 3 分钟。小心地丢弃上清液，不要移液珠子。现在将 300 微升 75% 新鲜制备的乙醇转移到每个微量离心管中。轻轻地...

视频: 连接产物的纯化、扩增和文库纯化在胃癌多基因SNP检测中的应用

Multi-Gene Single Nucleotide Polymorphism Detection in Gastric Cancer Based on Ion Semiconductor Sequencing Platform

Gastric cancer is a common heterogeneous tumor. Most patients have advanced gastric cancer at the time of diagnosis and often need chemotherapy. Although 5-fluorouracil (5-FU) is widely used for treatment, its therapeutic sensitivity and drug tolerance still need to be determined, which emphasizes the importance of individualized administration. Pharmacogenetics can guide the clinical implementation of individualized treatment. Single nucleotide polymorphisms (SNPs), as a genetic marker, contribute to the selection of appropriate chemotherapy regimens and dosages. Some SNPs are associated with folate metabolism, the therapeutic target of 5-FU. Methylenetetrahydrofolate reductase (MTHFR) rs1801131 and rs1801133, dihydrofolate reductase (DHFR) rs1650697 and rs442767, methionine synthase (MTR) rs1805087, gamma-glutamyl hydrolase (GGH) rs11545078 and solute carrier family 19 member 1 (SLC19A1) rs1051298 have been investigated in different kinds of cancers and antifolate antitumor drugs, which have potential forecasting and guiding significance for application of 5-FU. The ion torrent next-generation semiconductor sequencing technology can rapidly detect gastric cancer-related SNPs. Each time a base is extended in a DNA chain, an H+ will be released, causing local pH changes. The ionic sensor detects pH changes and converts chemical signals into digital signals, achieving sequencing by synthesis. This technique has low sample requirement, simple operation, low cost, and fast sequencing speed, which is beneficial for guiding individualized chemotherapy by SNPs.

This protocol proposes holistic laboratory procedures required to detect single nucleotide polymorphisms in gastric cancer samples based on an ion semiconductor sequencing platform. The target sequences, ligating adapters, library amplification and purification, and quality control criteria are also described in detail.

Gastric cancer is a common heterogeneous tumor. Most patients have advanced gastric cancer at the time of diagnosis and often need chemotherapy. Although ...