A genotyping detection kit based on the rs4254535 site of the GKN1 gene and its application

Abstract

The present invention provides a genotyping detection kit based on the rs4254535 site of the GKN1 gene, which is used to detect the genotype of the rs4254535 site of the GKN1 gene of a cancer patient, and has the following characteristics: a primer set for performing PCR on the whole blood genome of the cancer patient, the primer set comprising a first upstream primer and a first downstream primer, wherein the nucleotide sequence of the first upstream primer is as shown in SEQ NO:1 in the sequence list, and the nucleotide sequence of the first downstream primer is as shown in SEQ NO:2 in the sequence list.

Description

Technical Field

[0001] The invention belongs to the field of cancer research, and specifically relates to a genotyping detection kit based on the rs4254535 site of the GKN1 gene and an application thereof. Background Art

[0002] Lung cancer is the most common cancer and the leading cause of cancer death in the world. With the remarkable achievements in surgical treatment, immunotherapy, chemoradiotherapy and molecular targeted therapy in the treatment of lung cancer, although the treatment of lung cancer has made great progress, the 5-year survival rate of lung cancer patients in my country is still <20%. The prognosis of lung cancer patients is affected by a variety of environmental factors, and it is difficult to predict. Therefore, finding effective predictive prognostic markers is crucial for predicting the prognosis of lung cancer patients, individualizing treatment for lung cancer patients and improving the 5-year survival rate of lung cancer patients. A large amount of research evidence shows that the occurrence and development of lung cancer is affected by multiple factors such as environment and individual genetics. Gene polymorphism is the most common gene variation in the human body, which has been shown to affect gene expression and predict the prognosis of patients with various cancers, including lung cancer. Summary of the invention

[0003] The present invention is made to solve the above problems, and aims to provide a genotyping detection kit based on the rs4254535 site of the GKN1 gene and its application.

[0004] The present invention provides a genotyping detection kit based on the rs4254535 site of the GKN1 gene, which is used to detect the genotype of the rs4254535 site of the GKN1 gene of a cancer patient, and has the following characteristics: a primer set for performing PCR on the whole blood genome of the cancer patient, the primer set comprising a first upstream primer and a first downstream primer, wherein the nucleotide sequence of the first upstream primer is as shown in SEQ NO:1 in the sequence list, and the nucleotide sequence of the first downstream primer is as shown in SEQ NO:2 in the sequence list.

[0005] The genotyping detection kit based on the GKN1 gene rs4254535 site provided by the present invention may also have the following characteristics: wherein the genotype is any one of the CC genotype, the CT genotype and the TT genotype.

[0006] The genotyping detection kit based on the rs4254535 site of the GKN1 gene provided by the present invention may also have the following characteristics, including: a 5' universal probe, a 3' universal probe, a site recognition sequence, a qPCR universal primer, a 5' specific ligation probe and a 3' specific ligation probe.

[0007] The genotyping detection kit based on the GKN1 gene rs4254535 site provided by the present invention may also have the following characteristics: wherein the cancer is lung cancer.

[0008] The genotyping detection kit based on the GKN1 gene rs4254535 site provided by the present invention may also have the following characteristics: wherein the lung cancer is non-small cell lung cancer.

[0009] The present invention provides an application of a genotyping detection kit based on the rs4254535 site of a GKN1 gene in predicting the prognosis and survival of lung cancer patients.

[0010] Functions and Effects of the Invention

[0011] According to the genotyping detection kit based on the rs4254535 site of the GKN1 gene involved in the present invention, because the kit includes a primer set, the primer set includes a first upstream primer and a first downstream primer, and the whole blood genome of the cancer patient can be PCR amplified by the primer set, and the genotype of the rs4254535 site of the GKN1 gene of the cancer patient can be determined according to the amplification result, and the prognosis and survival of the cancer patient can be predicted according to the typing result of the genotype. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 It is a schematic diagram of the process of genotyping detection in Example 2 of the present invention. DETAILED DESCRIPTION

[0013] In order to make the technical means, creative features, objectives and effects of the present invention easy to understand, the following examples and drawings specifically describe a genotyping detection kit based on the rs4254535 site of the GKN1 gene and its application.

[0014] The Qiagen Blood DNA Extraction Kit (Qiagen, Hilden, Germany), Cell Counting Kit-8, and Annexin V-FITC Cell Apoptosis Detection Kit used in the following examples are all commercially available kits.

[0015] <Example 1>

[0016] This embodiment provides a genotyping detection kit based on the rs4254535 site of the GKN1 gene, the kit comprising: a primer set for PCR of the whole blood genome of a cancer patient, a 5' universal probe, a 3' universal probe, a site recognition sequence, a qPCR universal primer, a 5' specific ligation probe and a 3' specific ligation probe.

[0017] The primer set includes a first upstream primer and a first downstream primer. The nucleotide sequence of the first upstream primer is shown in SEQ NO: 1 in the sequence list, and the nucleotide sequence of the first downstream primer is shown in SEQ NO: 2 in the sequence list.

[0018] The concentrations of the first upstream primer and the first downstream primer were both 10 mM.

[0019] The 5' universal probe, 3' universal probe, site recognition sequence, qPCR universal primer, 5' specific ligation probe and 3' specific ligation probe were all purchased from Shanghai Tianhao Biotechnology Co., Ltd., and the concentration and usage conditions were the same as those of the 2×48-Plex SNP scan TM kit of Shanghai Tianhao Biotechnology Co., Ltd.

[0020] By using the above-mentioned genotyping detection kit based on the rs4254535 site of the GKN1 gene to perform PCR amplification on the whole blood genome of the cancer patient, it can be determined whether the cancer patient has a gene mutation at the rs4254535 site of the GKN1 gene, and three genotyping detection results are obtained based on the gene mutation at the rs4254535 site of the GKN1 gene: CC genotype, CT genotype and TT genotype (where site C is a mutant type). In other words, it can be detected whether the genotype of the rs4254535 site of the GKN1 gene of the cancer patient belongs to CC genotype, CT genotype and TT genotype.

[0021] <Example 2>

[0022] Example 2 provides an application of the genotyping detection kit based on the rs4254535 site of the GKN1 gene in Example 1 in predicting the prognosis and survival of lung cancer patients. The specific process is as follows:

[0023] A total of 888 patients with primary lung cancer were enrolled from January to November 2009, including 536 patients from Changhai Hospital affiliated to Naval Medical University (Second Military Medical University) and 352 patients from Taizhou Health Science Institute of Fudan University. Inclusion criteria: newly diagnosed lung cancer patients diagnosed by pathological histology, without a history of malignant tumors in other organs, and without age or gender restrictions. Clinical data were obtained from medical records, and follow-up data were obtained from telephone interviews. All patients were Chinese. This study was approved by the Ethics Committee of the School of Life Sciences of Fudan University, and informed consent was obtained from the subjects for the collection of epidemiological survey data and blood samples.

[0024] 2.1 Use the genotyping detection kit based on the GKN1 gene rs4254535 site of Example 1 to detect the genotype of the patient's GKN1 gene rs4254535 site

[0025] 2.1.1 Blood sample collection

[0026] All patients gave informed consent and donated 5 mL of blood.

[0027] 2.1.2 DNA extraction

[0028] Genomic DNA was extracted from blood using Qiagen Blood DNA Extraction Kit (Qiagen, Hilden, Germany).

[0029] 2.1.3 Detection of genomic DNA using the genotyping detection kit based on the rs4254535 site of the GKN1 gene in Example 1

[0030] Figure 1 It is a schematic diagram of the process of genotyping detection in Example 2 of the present invention.

[0031] like Figure 1 As shown, the high specificity of the ligase ligation reaction is used to realize the recognition of the SNP site allele, and then by introducing non-specific sequences of different lengths at the end of the ligation probe and obtaining ligation products of different lengths corresponding to the site through the ligation reaction, the ligation products are PCR amplified using fluorescently labeled universal primers.

[0032] The PCR amplification reaction system included: 2×Es Taq MasterMix (Beijing Biolab Technology Co., Ltd.), primer set (0.5 mM each), and 100 ng human whole blood genomic DNA.

[0033] The PCR cycle conditions were: 94°C for 30 s, 60°C for 30 s, and 72°C for 30 s per cycle, for a total of 25 cycles.

[0034] The amplified products were separated by fluorescent capillary electrophoresis, and finally the genotypes of each SNP site were obtained by analyzing the GeneMapper software to determine the gene mutation status of the rs4254535 site of the GKN1 gene and to determine which of the three genotypes (CC genotype, CT genotype and TT genotype (site C is a mutant type)) it belongs to.

[0035] 2.2 Statistical analysis of patients’ clinical data

[0036] The basic clinical data of the patients were collected, including gender, age, smoking history, family history of malignant tumors, lung cancer pathological type, and TNM stage.

[0037] 2.3 Inferring the prognosis and survival of lung cancer patients based on statistical conclusions

[0038] Based on the genotyping test results and clinical information of lung cancer patients, the prognosis and mortality risk of lung cancer patients were predicted according to our statistical analysis conclusions.

[0039] 2.4 Statistical analysis

[0040] R v3.6.2 software was used for statistical analysis. The demographic variation values, smoking status, family cancer history, allele frequency differences between the control group and the case group, and Hardy-Weinberg equilibrium (HWE) test were analyzed using the χ2 test. Cox regression analysis and Kaplan-Meier method were used to draw survival curves using the allele model, genotype model, dominant model, and recessive model, and the hazard ratio (HR) and 95% confidence interval (CI) were calculated. Age and gender were used for correction. The various models were further analyzed according to age, gender, smoking status, family history, and lung cancer histological type to evaluate the relationship between gene polymorphism and lung cancer prognosis. P < 0.05 indicated statistically significant differences.

[0041] 2.5 Experimental Results

[0042] 2.5.1 Demographic and clinical characteristics of patients

[0043] The follow-up period was from January 2009 to November 15, 2019. Due to incomplete clinical data, 49 patients were excluded, and the data of 839 patients were analyzed. The study samples were Han nationality, 668 cases (79.6%) died, 103 cases (12.3%) survived for more than 5 years, and 68 cases (8.1%) were lost to follow-up. Among them, 610 cases (72.7%) were male, 524 cases (62.5%) were aged ≥ 60 years, 582 cases (69.4%) had a history of smoking, and 302 cases (36%) had a family history of malignant tumors. In terms of tumor subtypes, there were 367 cases (43.7%) of adenocarcinoma, 282 cases (33.6%) of squamous cell carcinoma, 72 cases (8.6%) of SCLC, and 118 cases (14.1%) of other types. These included adenosquamous carcinoma (ASC), large cell carcinoma (LCC), carcinosarcoma (CS), and mucoepidermoid carcinoma (MEC). 154 patients (18.4%) were diagnosed with stage I and II disease, and 625 patients (74.5%) were diagnosed with stage III and IV disease (Table 1).

[0044] 2.5.2 Relationship between patient characteristics and lung cancer prognosis

[0045] Table 1 Characteristics and prognostic analysis of lung cancer patients in China (n = 839)

[0046]

[0047]

[0048] #Other cancers include adenosquamous carcinoma, large cell carcinoma, carcinosarcoma, and mucoepidermoid carcinoma

[0049] As shown in Table 1, the median survival time of male patients was significantly lower than that of female patients (male, 34.27 months; female, 40.17 months; P = 0.01). The median survival time of patients aged < 60 years was significantly higher than that of patients aged ≥ 60 years (40.87 months, 33.20 months; P = 0.003). The median survival time of non-smoking patients was significantly higher than that of smoking patients (41.03 months, 33.90 months; P < 0.001). In addition, compared with patients with early tumors, the median survival time of patients with advanced tumors was significantly shortened (29.4 months, 113.93 months; P < 0). There was no significant statistical difference in the correlation between patient characteristics in different hospitals and the prognosis of lung cancer.

[0050] Table 2 Association analysis between GKN1 rs4254535 polymorphism and prognosis of lung cancer patients

[0051]

[0052]

[0053] a Adjusted for age and sex

[0054] As shown in Table 2 , among the overall patients, patients with CC genotype had a lower risk of prognostic death compared with those with TT genotype (adjusted HR=0.70; 95% CI: 0.52–0.95, P=0.022; log-rank P=0.053).

[0055] Table 3 Association analysis between GKN1 rs4254535 upper allele polymorphism and prognosis of Chinese lung cancer patients

[0056]

[0057]

[0058] a Adjusted for age and sex

[0059] Table 4 Association analysis between genotype polymorphism of GKN1 rs4254535 and prognosis of Chinese lung cancer patients

[0060]

[0061]

[0062] a Adjusted for age and sex

[0063] Table 5 Association analysis between dominant gene polymorphisms at GKN1 rs4254535 and prognosis of lung cancer patients in China

[0064]

[0065]

[0066] a Adjusted for age and sex

[0067] Table 6 Association analysis between recessive gene polymorphisms at GKN1 rs425455 and prognosis of lung cancer patients in China

[0068]

[0069] a Adjusted for age and sex

[0070] As shown in Tables 3 to 6, in female patients, compared with the TT genotype, patients with CC genotype had a lower risk of prognostic death (adjusted HR=0.51, 95%CI:0.29-0.89, P=0.018); compared with TT+TC, patients with recessive model CC had a lower risk of prognostic death (adjusted HR=0.49, 95%CI:0.28-0.84, P=0.009); in non-smoking patients, compared with the TT genotype, CC patients had a lower risk of prognostic death (adjusted HR=0.50, 95%CI:0.28-0.86, P=0.013; log-rank P=0.080); in patients with no family history of malignant tumors, compared with the TT genotype, CC patients had a lower risk of prognostic death (adjusted HR=0.61, 95%CI:0.41-0.93, P=0.020; log-rank In patients with advanced lung cancer (stage III+IV), CC patients had a lower risk of death compared with TT genotype (adjusted HR=0.65, 95%CI: 0.47–0.91, P=0.012); compared with TT+TC, CC patients in the recessive model had a lower risk of death (adjusted HR=0.63, 95%CI: 0.46–0.88, P=0.006).

[0071] Overall, the results of log-rank test analysis showed that the GKN1 rs4254535 mutation genotype CC had a protective effect on the prognosis and survival of lung cancer patients who were female (P=0.018), had no smoking history (P=0.013), had no family history of malignant tumors (P=0.020), and had advanced lung cancer (P=0.012). The GKN1 site SNP is closely related to the environment, and the site SNP can be used as a functional survival prognosis predictor to predict the risk and prognosis of cancer patients.

[0072] The above-mentioned embodiments are preferred examples of the present invention and are not intended to limit the protection scope of the present invention. Sequence Listing <110> Fudan University <120> A genotyping detection kit based on the rs4254535 site of the GKN1 gene and its application <160> 2 <170> SIPOSequenceListing 1.0 <210> 1 <211> twenty one <212> DNA <213> Artificial Sequence <400> 1 gtcaccggaa ccatcatgag a 21 <210> 2 <211> twenty one <212> DNA <213> Artificial Sequence <400> 2 aggcctgcaa aggactaaac t 21

Claims

1. An application of a genotyping detection kit based on the rs4254535 site of the GKN1 gene in the preparation of a product for predicting the prognosis and survival of lung cancer patients, characterized in that: The genotyping detection kit based on the rs4254535 site of the GKN1 gene is used to detect the genotype of the rs4254535 site of the GKN1 gene of a lung cancer patient, and comprises: A primer set for performing PCR on the whole blood genome of the lung cancer patient, the primer set comprising a first upstream primer and a first downstream primer, The nucleotide sequence of the first upstream primer is shown in SEQ NO: 1 in the sequence list, and the nucleotide sequence of the first downstream primer is shown in SEQ NO: 2 in the sequence list. Genotype test results include: TT genotype, TC genotype and CC genotype. Compared with the TT + TC genotype, individuals with the CC genotype had a lower risk of death.

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