CN111593125A

CN111593125A - Human NPAS2 mutant gene and application thereof in liver cancer FOLFOX4 scheme chemotherapy drug resistance evaluation

Info

Publication number: CN111593125A
Application number: CN202010539082.2A
Authority: CN
Inventors: 陈平; 黄志清
Original assignee: Individual
Current assignee: Individual
Priority date: 2020-06-13
Filing date: 2020-06-13
Publication date: 2020-08-28

Abstract

The invention provides a g. [105054insGCCTA ] mutation form of a human NPAS2 gene, wherein the sequence of a fragment in which mutation is carried out is shown as SEQ ID NO: 2 is shown in the specification; hepatoma patients with g. [105054insGCCTA ] mutation in the NPAS2 gene were resistant to FOLFOX4 chemotherapy regimen. The NPAS2 mutant gene fragment with g. [105054insGCCTA ] mutation can be amplified and mutation detected using a kit containing 5'-TCACAAAGCCTTTGTGACGT-3' and 5'-ACACGGTGGTTTTGTCCATT-3' primers and a 5 '-VIC-AATGCCTAGTTCTCA-MGB-NFQ-3' probe or a method for constructing digital PCR or real-time fluorescence quantitative PCR based thereon. The g. [105054insGCCTA ] mutation form of the NPAS2 gene provided by the invention is not reported at present, and the mutation can be used as a gene marker of drug resistance of FOLFOX4 scheme chemotherapy of liver cancer, can be applied to prediction and evaluation of drug resistance, and has the advantages of accuracy, specificity, timeliness and effectiveness.

Description

Human NPAS2 mutant gene and application thereof in liver cancer FOLFOX4 scheme chemotherapy drug resistance evaluation

Technical Field

The invention belongs to the field of medical molecular biology, and particularly relates to a human NPAS2 mutant gene and application thereof in liver cancer FOLFOX4 scheme chemotherapy drug resistance evaluation.

Background

Liver cancer is the main cancer most frequently suffered and easily killed by Chinese people, according to the latest national cancer report (2019 edition) issued by the national cancer center, the incidence rate of liver cancer is ranked the fourth highest on malignant tumors, the death rate is the second highest on malignant tumors, and the current domestic new liver cancer cases and death cases account for more than half of the world. Early symptoms of liver cancer are not obvious, more than 60% of patients are diagnosed in middle and late stages, and the optimal chance of surgical treatment is lost. Chemotherapy is an important treatment mode for liver cancer in middle and late stages, however, liver cancer patients have different sensitivities to chemotherapeutic drugs, and the prognosis of the liver cancer patients resistant to chemotherapy is very poor; in addition, the cancer focus of the liver cancer has heterogeneity and is easy to generate new drug resistance mutation in the chemotherapy process, so secondary drug resistance often appears in the chemotherapy process, and the treatment scheme needs to be changed in time. At present, the response of liver cancer patients to chemotherapy drugs cannot be predicted and evaluated accurately in time clinically, so that most liver cancer chemotherapy patients continuously carry out experimental chemotherapy, the condition of the patients is delayed, and unnecessary drug toxic and side effects are borne. Therefore, it is important to effectively detect primary resistance before chemotherapy and to monitor whether secondary resistance occurs in time during chemotherapy. Currently, the clinical evaluation of the curative effect of liver cancer chemotherapy mainly depends on imaging means and serological indexes, however, certain lag often exists in the imaging means, the general specificity of serum markers (such as serum alpha-fetoprotein (AFP)) is not strong, and the two can not effectively and accurately reflect the molecular essence of cancer foci, so that the change trend of the cancer foci is difficult to analyze and predict, and the value of the therapy guidance is limited. Therefore, a need exists for finding specific and accurate molecular markers for liver cancer chemotherapy, predicting and evaluating the chemotherapy curative effect, and finding drug-resistant people and drug-resistant time nodes in time so as to effectively guide the selection and timely change of a chemotherapy scheme.

The FOLFOX4 chemotherapy scheme which is clinically used in the treatment of cancers at present and combines the advantages of common chemotherapeutics such as fluorouracil, oxaliplatin and the like is superior to the traditional chemotherapeutics (such as adriamycin) in terms of overall response rate, disease control rate, progression-free survival time and overall survival time, and has better tolerance and safety, so the FOLFOX4 scheme is recorded by the primary liver cancer diagnosis and treatment standard issued by the ministry of health of the original country since 2011 and is recommended to be used for treating the advanced primary liver cancer. The invention aims at primary liver cancer (hereinafter referred to as liver cancer) patients receiving FOLFOX4 chemotherapy treatment, blood samples before and during chemotherapy are collected, extracellular free DNA (cell-free DNA) of the patients is extracted, circulating tumor DNA (ctDNA) in the blood samples is analyzed by a second-generation DNA sequencing technology to monitor the cancer focus mutation change information, and the results of analysis of clinical efficacy evaluation information show that two specific mutations of NPAS2 (neural PAS domain protein 2) gene and two specific mutations of CNOT1 (CCR 4-NOT transcription complex x subbunit 1) gene can be used as a liver cancer chemotherapy-resistant marker of FOLFOX4 scheme, while the lack of effective chemotherapy efficacy and drug resistance specific evaluation molecular marker FOOX 4 scheme are a big problem in chemotherapy of liver cancer, so that the primary liver cancer (hereinafter referred to as liver cancer) patients are difficult to realize precise chemotherapy.

The protein encoded by the NPAS2 gene was first discovered to be present in brain cells, and in fact it has an expression profile in almost all surrounding tissues. The NPAS2 gene is necessary for organism to maintain normal biological rhythm, and its coding expressed NPAS2 protein participates in regulation of human body biological clock, and plays important role in cell growth, differentiation apoptosis, tumor growth inhibition, etc. When mutated or deleted, it causes disorders of the rhythm system. The research shows that when the NPAS2 gene is abnormally expressed, the incidence rate of diseases related to circadian rhythm such as hypertension, metabolic syndrome, tumor, cardiovascular and cerebrovascular diseases and the like is possibly increased. However, it is not clear what role the gene plays in the development of cancer and how it functions. The invention discovers two specific mutations of the NPAS2 gene for the first time, and applies the mutations to the prediction and evaluation of the chemotherapy resistance of the FOLFOX4 scheme of the liver cancer patient, which has significant significance to the selection and timely change of the chemotherapy scheme of the liver cancer patient, particularly the FOLFOX4 scheme, can effectively improve the benefit of the primary liver cancer patient on the FOLFOX4 scheme chemotherapy, and avoids ineffective chemotherapy and unnecessary toxic and side effects of chemotherapy; meanwhile, the method has important value for the research on the primary liver cancer FOLFOX4 scheme chemotherapy drug resistance mechanism and the research and development of effective chemotherapeutic drugs.

Disclosure of Invention

The invention provides two specific mutations of NPAS2 gene, a detection kit and a method thereof, wherein the two specific gene mutations can be used as molecular markers of primary liver cancer FOLFOX4 scheme chemotherapy resistance and applied to prediction and analysis of drug resistance.

The technical scheme adopted by the invention for solving the technical problems is as follows:

the object (1) of the present invention is to provide a human NPAS2 mutant gene with g. [ 105009-105012 delGCTT ] mutation, which is formed by deleting 4 nucleotides of GCTT at the 105009-bulphonate 105012 site of the wild-type NPAS2 gene, and the sequence within the 4989-bulphonate 181679 range in the sequence with NCBI accession number NG-023259.1 is the reference sequence of the NPAS2 wild-type gene, and the sequence of the fragment in which the g. [ 105009-105012 delGCTT ] mutation is located is shown as SEQ ID NO: 1, the rest of the sequences are referred to the NPAS2 wild-type gene reference sequence; hepatoma patients with g. [105009_105012delGCTT ] mutation in the NPAS2 gene were resistant to the FOLFOX4 chemotherapeutic regimen. The NPAS2 gene with g [105009_105012delGCTT ] mutation can be applied to the development of a liver cancer FOLFOX4 scheme chemotherapy drug resistance detection kit, a detection method and a therapeutic drug.

The object (2) of the present invention is to provide a human NPAS2 mutant gene in which g. [105054insGCCTA ] mutation occurs, which is formed by inserting 5 nucleotides of GCCTA into 105054 th site of wild-type NPAS2 gene, and the sequence in the range of 4989 and 181679 of the sequence with NCBI accession number NG _023259.1 is the reference sequence of NPAS2 wild-type gene, and the sequence of the fragment in which g. [105054insGCCTA ] mutation occurs is shown in SEQ ID NO: 2, the rest of the sequences are referred to the NPAS2 wild-type gene reference sequence; hepatoma patients with g. [105054insGCCTA ] mutation in the NPAS2 gene were resistant to FOLFOX4 chemotherapy regimen. The NPAS2 gene with g [105054insGCCTA ] mutation can be applied to the development of a liver cancer FOLFOX4 scheme chemotherapy drug resistance detection kit, a detection method and a therapeutic drug.

The object (3) of the present invention is to provide detection primers and probes for detecting g. [105009_105012delGCTT ] mutation of NPAS2 gene, wherein the sequence of the fragment in which g. [105009_105012delGCTT ] mutation of NPAS2 gene is shown as SEQ ID NO: 1, the rest of the sequences are referred to the NPAS2 wild-type gene reference sequence; the detection primers are 5'-TCACAAAGCCTTTGTGACGT-3' and 5'-ACACGGTGGTTTTGTCCATT-3', and the detection probe is 5 '-FAM-CAGACTCGAAACAAG-MGB-NFQ-3'; the detection primers and the probes can be used for constructing an NPAS2 gene g. [105009_105012delGCTT ] mutation detection kit and a detection method, and particularly used for constructing an NPAS2 gene g. [105009_105012delGCTT ] mutation detection method based on digital PCR or real-time fluorescence quantitative PCR.

The invention aims to provide detection primers and probes for detecting g. [105054insGCCTA ] mutation of NPAS2 gene, wherein the sequence of a fragment of g. [105054insGCCTA ] mutation of NPAS2 gene is shown as SEQ ID NO: 2, the rest of the sequences are referred to the NPAS2 wild-type gene reference sequence; the detection primers are 5'-TCACAAAGCCTTTGTGACGT-3' and 5'-ACACGGTGGTTTTGTCCATT-3', and the detection probe is 5 '-VIC-AATGCCTAGTTCTCA-MGB-NFQ-3'; the detection primer and the probe can be used for constructing a mutation detection kit and a detection method for the NPAS2 gene g. [105054insGCCTA ], and particularly can be used for constructing a mutation detection method for the NPAS2 gene g. [105054insGCCTA ] based on digital PCR or real-time fluorescence quantitative PCR.

Two mutation forms of g. [105009_105012delGCTT ] and g. [105054insGCCTA ] of the NPAS2 gene provided by the invention are not reported at present, and the two mutations can be used as gene markers of drug resistance of FOLFOX4 scheme chemotherapy of liver cancer, so that the two mutations can be applied to prediction and evaluation of drug resistance and have the advantages of accuracy, specificity and timeliness and effectiveness; in addition, primers and probes for specific detection of mutations are provided. The method has obvious significance for the selection and timely change of a chemotherapy scheme of a liver cancer patient, particularly a FOLFOX4 scheme, can effectively improve the benefit of the FOLFOX4 scheme chemotherapy of the liver cancer patient, and avoids ineffective chemotherapy and unnecessary toxic and side effects of chemotherapy; meanwhile, the method has important value for the research on the drug resistance mechanism of FOLFOX4 scheme chemotherapy of the liver cancer and the research and development of effective chemotherapeutic drugs.

Drawings

FIG. 1 is a schematic diagram of specific mutations in exon 3 of the human NPAS2 gene. The middle white box represents exon 3, and the two black boxes represent introns; the left and right arrows point to the occurrence of the g. [ 105009-105012 delGCTT ] mutation and g. [105054insGCCTA ] mutation, respectively.

Detailed Description

The resistance of the current clinical chemotherapy scheme mainly depends on the evaluation on imaging, and the chemotherapy effect is reflected by the change of the size, the size and the position of a cancer focus. In fact, cancer patients contain circulating tumor DNA (ctDNA) released from cancer foci in their blood, and more studies have confirmed that genetic mutation information of cancer foci, which is an intrinsic driving factor of the development and change of cancer foci, can be reflected by analyzing ctDNA. Therefore, by dynamic analysis and comparative analysis of blood sample ctDNA in the chemotherapy process of liver cancer FOLFOX4 chemotherapy regimen resistance and beneficial patient population, and combined with clinical curative effect evaluation, the FOLFOX4 chemotherapy regimen resistance gene marker can be researched.

The invention is further illustrated below with reference to specific examples.

Example 1 screening of liver cancer FOLFOX4 protocol chemotherapy drug resistance Gene markers

(1) Basic information of patients in group

15 cases of primary liver cancer patients diagnosed by clinical pathology in the period from 2017, 1 month to 2017, 12 months are selected, and are treated by adopting a chemotherapy scheme of FOLFOX4 (no intervention treatment, biological treatment and other treatment in the period), and the patient numbers, basic information and chemotherapy curative effect evaluation information are shown in table 1, wherein patients 1-5 are primary drug resistant groups (chemotherapy ineffective groups), patients 6-10 are secondary drug resistant groups (chemotherapy ineffective groups), and patients 11-15 are non-drug resistant groups (chemotherapy beneficial groups). The primary drug resistant group shows continuous progress without remission in the chemotherapy process, and usually has chemotherapy drug resistant mutation due to the existence of cancer focus before chemotherapy; the secondary drug resistance group means that the early stage of chemotherapy is remission, and the later stage of chemotherapy is changed into progress, and usually because a cancer focus has chemotherapy drug resistance mutation in the chemotherapy process; the non-drug resistant group means that only remission and no progress occur in the equivalent process of chemotherapy, and usually the reason is that a cancer focus does not exist before chemotherapy and the cancer focus does not have chemotherapy drug resistant mutation in the chemotherapy process.

The FOLFOX4 chemotherapy reference protocol was: 5% glucose injection 250ml is added with oxaliplatin 85mg/m²2h d1 intravenous drip, 250ml of normal saline is added with 200mg/m calcium folinate²2h d1-2, Fluorouracil (5-FU) 400mg/m for intravenous drip²D1-2, 5-FU 600mg/m for intravenous injection²Continuous intravenous pumping 22h d1-2, 14d for 1 chemotherapy cycle. The patient is subjected to not less than 2 chemotherapy cycles each time,and continue until the patient develops disease progression or an intolerable toxic response occurs. The patients are evaluated for curative effect and adverse reaction after every 1 or 2 chemotherapy cycles, and 10-20ml of blood samples are extracted at the same time (on the premise that the patients are fully informed and meet clinical diagnosis and treatment requirements).

The size of the tumor before and after chemotherapy is detected according to means such as CT or MRI and the like, the maximum diameter of the tumor body is determined, and the recent curative effect evaluation standard adopts the RECIST 1.1 standard of 2009 edition. Complete Remission (CR): all target lesions disappear; partial Remission (PR): the sum of the longest diameters of the target lesions is reduced by at least 30% compared with the baseline; progression (PD): the sum of the longest diameter of the target lesion is compared with the smallest sum of the longest diameter of the target lesion recorded after the treatment is started, the relative increase is 20 percent, and the absolute value of the sum is increased by at least 5mm or a new lesion appears; stable (SD): there was either a reduction in baseline lesion length but no PR or an increase but no PD. The treatment effect evaluation is based on the previous cancer focus or the cancer focus before treatment, and the patient is continuously followed during and after the treatment process.

Table 115 basic information of primary liver cancer chemotherapy patients and FOLFOX4 protocol chemotherapy curative effect evaluation information

Note: data representing no efficacy assessment without chemotherapy.

(2) Capture sequencing of ctDNA

Before chemotherapy and on the day of evaluation of curative effect of each chemotherapy, 10ml-20ml of venous anticoagulation blood of a patient is collected at the same time, and plasma and mononuclear cells are separated within 2 hours, wherein a plasma sample contains ctDNA which can reflect the information of cancer focus gene mutation, and the mononuclear cells contain normal genome DNA which can be used for eliminating individual gene polymorphism. DNA samples were sent to wara genes on dry ice for whole exome capture and second generation DNA sequencing. Whole Exome Sequencing (WES) refers to a genome analysis method of high-throughput Sequencing after capturing and enriching exome region DNA of a Whole genome using a probe capture technology. Given the high cost of second-generation DNA sequencing, each patient sent only one blood sample (corresponding to the bold-labeled node in table 1) for whole exome sequencing, with the refractory drug sent the blood sample for the last clinical assessment of progress, i.e., PD, and the non-refractory drug sent the blood sample collected at the end of the first chemotherapy.

Blood sample ctDNA and genome DNA are respectively extracted by using a QIAamp Circulating Nucleic Acid Kit and a QIAamp DNAafter Mini Kit of Germany Qiagen company, and are captured by using a SureSelect Human All Exon V6+ UTR whole exome liquid phase capture chip (75 Mb in the capture Region of Agilent company in the United states), wherein the chip is a liquid phase targeting sequence capture system based on oligonucleotide synthesis technology, provides higher sequencing efficiency, and completely covers up-to-date gene databases including RefSeq, CCDS, GENCODE, miRBase, TCGA and UCSC, the exome Region and a non-translated Region (Untranstrained Region) therein, ensures high capture uniformity, and can effectively reduce sequencing cost.

The Hiseq4000 platform of Illumnia company in USA is adopted for sequencing, the average sequencing depth requirement of a normal genome DNA sample from a monocyte is more than 100 times, and the average sequencing depth requirement of a plasma ctDNA sample is more than 1000 times. Adopting Hiseq4000 matched analysis software to carry out preliminary analysis on a sequencing result, mainly filtering out adapters, low-quality bases and undetected bases from original off-line data, then adopting sequence comparison software BWA to compare an original sequencing sequence of each sample with a human reference genome sequence (hg 19), adopting a Samtools software package to position and compare the compared reads sequence in a genome, adopting variation detection software Varscan2 to carry out variation detection on the sample, and adopting a variation annotation tool Annovar to carry out annotation; and taking the sequencing result of the normal genome DNA sample of the blood cells as a reference, and then filtering the polymorphic sites of the population by using a thousand human genome database, an SNP database, a COSMIC (version 64) database and the like to finally obtain the mutation site information (including SNP, InDel, CNV and the like) of the ctDNA sample of the blood plasma.

(3) Comprehensive analysis of results

The second generation DNA sequencing results of 15 blood samples showed that the mean sequencing depth of ctDNA was 1214X. In total, 46 mutations were detected in all samples with a content of more than 1%, belonging to 17 different genes including TP53, CYP1a1, axi, ALDH2, CNOT1, NPAS2, etc. The search for drug-resistance related gene markers focuses on high-frequency gene mutation and gene mutation with difference in drug-resistant groups and non-drug-resistant groups. Further analysis combined with clinical information revealed that 2 high-frequency mutant genes, namely the CNOT1 gene and the NPAS2 gene, were detected only in the drug-resistant group (including the primary drug-resistant group and the secondary drug-resistant group) and not in the non-drug-resistant group, and the results are shown in Table 2 (the mutations were confirmed by Sanger sequencing). The samples of 4 patients 3, 4, 7 and 9 contained specific mutations of NPAS2 gene, 2 of 3 and 7 contained g. [105009 — 105012delGCTT ] mutation of NPAS2 gene, and 2 of 4 and 9 contained g. [105054 inspgccta ] mutation of NPAS2 gene, both of which were located in exon 3 (see fig. 1). The sequence in the range of 4989-. Further, the g. [105009_105012delGCTT ] and g. [105054insGCCTA ] mutations of NPAS2 gene were verified by Sanger sequencing, wherein the g. [105009_105012delGCTT ] mutation of NPAS2 gene was in the sequence as shown in SEQ ID NO: 1 (GCTT deletion mutation is originally located at 129 th site to 132 th site of the sequence of SEQ ID NO: 1 before deletion, and the rest sequences are shown in a NPAS2 wild-type gene reference sequence), and the sequence of the [105054insGCCTA ] mutation is shown as the sequence of SEQ ID NO: 2 (GCCTA insertion mutation is from position 174 to position 178 of the sequence of SEQ ID NO: 2, and the rest sequences are shown in the reference sequence of NPAS2 wild-type gene).

In addition to the NPAS2 gene mutation, the CNOT1 gene mutation was contained in the samples of 6 patients No. 1, No. 2, No. 5, No. 6, No. 8 and No. 10, wherein 4 patients No. 1, No. 2, No. 6 and No. 8 contained the g. [42415INSATCA ] mutation of CNOT1 gene, and 2 patients No. 5 and No. 10 contained the g. [42481_42482delAG ] mutation of CNOT1 gene (the sequences within 58519951 and 58629826 range among the sequences shown by NCBI accession No. NC-000016.10 are the reference sequences of CNOT1 wild-type gene, and the length is 109876 bp).

TABLE 2 liver cancer FOLFOX4 chemotherapy regimen drug-resistant group specific high-frequency gene mutation information

Patient numbering	Mutant genes	Mutation site	Content of mutation
				1	CNOT1	g.[42415insATCA]	12.3%
2	CNOT1	g.[42415insATCA]	15.5%
				3	NPAS2	g.[105009_105012delGCTT]	20.4%
4	NPAS2	g.[105054insGCCTA]	13.5%
				5	CNOT1	g.[42481_42482delAG]	8.1%
6	CNOT1	g.[42415insATCA]	4.7%
				7	NPAS2	g.[105009_105012delGCTT]	9.4%
8	CNOT1	g.[42415insATCA]	11.9%
				9	NPAS2	g.[105054insGCCTA]	6.3%
10	CNOT1	g.[42481_42482delAG]	12.8%

From the above analysis, the specific mutations of NPAS2 and CNOT1 genes are specific to FOLFOX4 chemotherapy-resistant drug group liver cancer patients, but are not detected in non-drug-resistant group patients. In addition, NPAS2 and CNOT1 gene specific mutations exist in primary drug-resistant group patients, and are detected in drug-resistant node samples of secondary drug-resistant group patients after chemotherapy, and the patients are likely to have drug resistance due to NPAS2 and CNOT1 gene specific mutations in the process of chemotherapy. Therefore, preliminary results show that specific mutations of NPAS2 and CNOT1 genes are related to drug resistance of liver cancer FOLFOX4 scheme chemotherapy, and the effectiveness and specificity of the specific mutations serving as drug resistance gene markers need to be further verified.

Example 2 validation of markers for liver cancer FOLFOX4 protocol chemotherapy resistance genes

If the specific mutations of NPAS2 and CNOT1 genes are related to primary liver cancer FOLFOX4 chemotherapy resistance, the blood samples collected in the chemotherapy process of liver cancer patients in example 1 can be used for analyzing the dynamic change information thereof, and clinical efficacy evaluation information is combined to deeply verify and reveal the specific association between the two. Blood ctDNA samples of 15 primary liver cancer patients before and during chemotherapy as shown in Table 1 were extracted by the method described in example 1. Specific mutations of NPAS2 and CNOT1 genes, specifically g. [ 105009-105012 delGCTT ] mutation and g. [105054insGCCTA ] mutation of NPAS2 gene, and g. [42415insATCA ] mutation and g. [ 42481-42482 delAG ] mutation of CNOT1 gene, were quantitatively analyzed by digital PCR (Quantstrudio 3D Digital PCR System and matched 20K chip kit V2 kit of Life Tech, USA) according to the instruction method, wherein special primers and probes (Primer V5.0 software) are designed. All samples were tested for copy number of all 4 specific mutations of the NPAS2 and CNOT1 genes. Since the g. [105009 — 105012delGCTT ] mutation and g. [105054insGCCTA ] mutation of the NPAS2 gene are located in the No. 3 exon of the gene at short intervals (less than 60bp, see FIG. 1), the same amplification primers were used, and the amplification primer probes, systems and conditions thereof are shown in Table 3, wherein the primer concentration is 0.4. mu. mol/l and the probe concentration is 0.2. mu. mol/l. Primers and probes are also designed, a consistent method is adopted to detect CNOT1 gene mutation, final result analysis shows that the CNOT1 gene mutation is also related to drug resistance of FOLFOX4 scheme chemotherapy, and related results are displayed in the place where treatment of another scheme is temporarily stopped.

TABLE 3 amplification information for two specific mutations of the NPAS2 Gene

Note: FAM and VIC are fluorescence labeling groups, are modified on the 5' end base of the probe and are used for fluorescence detection of amplified fragments; NFQ is a non-fluorescence quenching group of ABI patent, and can absorb fluorescence emitted by FAM or VIC when used in a probe state; the MGB is a DNA minor groove binder, is beneficial to the combination of the probe and the amplification template, and improves the Tm value of the probe and the amplification template.

The dPCR test results of NPAS2 gene mutation show (see table 4) that only 4 patients, No. 3, No. 4, No. 7 and No. 9, contained the specific mutation of NPAS2 gene, wherein the g. [105009_105012delGCTT ] mutation of NPAS2 gene was detected in the 2 patient samples, No. 3 and No. 7, and the g. [105054insGCCTA ] mutation of NPAS2 gene was detected in the 2 patient samples, and the two specific mutations of NPAS2 gene were not detected in the samples of the other patients, which are consistent with the results of the second-generation DNA sequencing in example 1 and can be mutually verified.

Two specific mutations of the NPAS2 gene were detected only in patients in the drug resistant group, and both primary (patients 3 and 4) and secondary (patients 7 and 9) resistant patients contained both specific mutations of the NPAS2 gene, which were not detected in the samples from patients in the non-drug resistant group. Further analysis revealed that the NPAS2 gene mutation was detected in both primary resistance drugs prior to chemotherapy, specifically 12.2 copies/. mu.l in patient 3 and 7.9 copies/. mu.l in patient 4 (Table 4). In addition, the content of the detected NPAS2 gene specific mutation is gradually increased along with the disease progress in the chemotherapy process of the primary drug-resistant group patients, which is consistent with the trend of clinical disease change, so that the NPAS2 gene specific mutation can be used as a drug-resistant gene marker for selection and change of the FOLFOX4 chemotherapy scheme, and is more timely and accurate compared with the conventional curative effect evaluation mode.

Taking patient 4 as an example, the curative effect of FOLFOX4 chemotherapy cannot be predicted before conventional clinical chemotherapy, and the g. [105009_105012delGCTT ] mutation concentration of NPAS2 gene before chemotherapy is detected to be 7.9copies/μ l, so that the patient can be judged to be drug-resistant, i.e. FOLFOX4 chemotherapy is ineffective for the patient. Furthermore, patient 4 evaluated for the first and second chemotherapy with SD showed no significant increase in cancer foci, and it was still difficult to clinically determine the efficacy of FOLFOX4 and predict the subsequent trend of cancer foci, while the concentration of the mutant fragment rapidly increased to 27.5copies/μ l, and the clinical efficacy was evaluated for SD but was not effective for FOLFOX4 based on the information of the mutant fragment. If PD had been found to progress at the end of the third chemotherapy, the mutant concentration had continued to increase to 95.4 copies/. mu.l. The results of the analysis were similar for patient 3.

For patients with secondary drug resistance (patients No. 7 and 9), no specific mutation of the NPAS2 gene is detected before chemotherapy, but is detected in the chemotherapy process, and the content of the detected specific mutation of the NPAS2 gene is gradually increased along with the disease progress in the chemotherapy process of the patients with secondary drug resistance, which is consistent with the clinical disease change trend, so that the NPAS2 gene can be used as a drug resistance gene marker for selecting and changing the FOLFOX4 chemotherapy scheme, and is more timely and accurate compared with the conventional curative effect evaluation mode. In the case of patient 9, no specific mutation of the NPAS2 gene was detected in the blood samples from the patient before and during the first two chemotherapy treatments, and the clinical efficacy was evaluated as PR, i.e. local remission and SD, i.e. stability. Although SD was evaluated as clinical efficacy at 3 rd time, the g. [105054insGCCTA ] mutant fragment of NPAS2 gene was detected to be 7.7copies/μ l, at which time it could be predicted that a drug resistance mutation had occurred, i.e., the FOLFOX4 regimen had failed to work for the patient; however, at this time (the third chemotherapy, evaluated as SD), the follow-up efficacy cannot be ascertained and predicted in routine clinical practice, and chemotherapy continues to be performed using the original regimen. If the 4 th chemotherapy was switched to PD, the detected concentration of the mutant fragment increased to 61.6 copies/. mu.l. The results of the analysis were similar for patient 7.

TABLE 4 dPCR detection results of NPAS2 gene mutation

Note: the concentration units are copies/. mu.l, i.e.copy number/. mu.l.

From the above, g. [105009_105012delGCTT ] and g. [105054insGCCTA ] mutations of NPAS2 genes can be used as drug resistance gene markers to be applied to selection and change of FOLFOX4 chemotherapy schemes of primary liver cancer patients, and compared with a conventional chemotherapy effect evaluation mode, the method has the advantages of timeliness, accuracy, specificity and effectiveness.

The NPAS2 gene has homology with clock (circumdial Locomotor Output cycle Kaput) gene in biological clock gene, and belongs to transcription factor family bHLH-PAS (basic helix-loop-helix/PAS structural domain); the NPAS2 protein encoded and expressed by the protein not only participates in the regulation of human body biological clock, but also plays an important role in the aspects of cell growth, differentiation apoptosis, tumor growth inhibition and the like. When the mutation or deletion of the polypeptide is caused, the disorder of the rhythm system can be caused, and the incidence rates of tumors, cardiovascular and cerebrovascular diseases and the like can be increased. However, it is not clear what role the gene plays in the development of cancer and how it functions. The g. [105009_105012delGCTT ] and g. [105054insGCCTA ] mutations of the NPAS2 gene disclosed by the invention occur in the No. 3 exon of the gene, and the occurrence of the two mutations is shown by mRNA corresponding to a compared mutant gene to cause the abnormal transcription and translation of the NPAS2 gene, thereby influencing the function of the NPAS2 protein in cells.

Example 3 application value of liver cancer FOLFOX4 protocol chemotherapy drug resistance gene marker

The sample size is further enlarged to verify the accuracy and specificity of the NPAS2 gene specific mutation for evaluating the drug resistance of FOLFOX 4in liver cancer treatment and the application value of the NPAS2 gene specific mutation. 50 cases of clinically and pathologically confirmed primary liver cancer patients from 2018 to 2019 are treated by FOLFOX 4in a chemotherapy scheme (in which no intervention treatment, biological treatment and the like are performed). 10-20ml of venous anticoagulation blood was collected before chemotherapy and at the time of evaluation of the efficacy of chemotherapy, and g. [ 105009-105012 delGCTT ] and g. [105054insGCCTA ] mutation concentrations of NPAS2 gene in blood samples were determined by the method described in example 2, and the results are shown in Table 5. Judging the drug resistance condition through two specific mutations of NPAS2 gene, if the mutation is detected before chemotherapy and the blood sample concentration is not less than 5 copies/mul, judging the drug resistance condition as primary drug resistance; if the mutation is detected at a certain time in the chemotherapy process and the blood sample concentration is not lower than 5 copies/mu l, the secondary drug resistance is judged. For the determination of drug resistance by conventional clinical methods, see example 1.

The data in table 5 show that 7 patients before chemotherapy detected two specific mutations in NPAS2 gene and blood sample concentrations both exceeded 5copies/μ l (4 of g. [105009 — 105012delGCTT ] and 4 of g. [105054insGCCTA ] mutations), and these 7 patients were identified as primary drug resistance according to clinical efficacy assessment, but the results based on gene detection assessment were on average 3.0 months earlier than the conventional clinical efficacy assessment, and the difference was significant. Two specific mutations of NPAS2 gene are not detected in the early stage of 11 patients in the chemotherapy process until two specific mutations of NPAS2 gene are suddenly detected at one time (5 of g. [ 105009-105012 delGCTT ] mutation and 6 of g. [105054insGCCTA ] mutation), and the 11 patients are identified as secondary drug resistance according to clinical efficacy evaluation, but the result based on gene detection judgment is 5.8 months ahead of the conventional clinical efficacy evaluation (the mutation is detected at the evaluation node before the clinical development), and the two are very different. Therefore, the detection of g. [105009_105012delGCTT ] and g. [105054insGCCTA ] mutations of NPAS2 gene indicates that the FOLFOX4 regimen chemotherapy of liver cancer can appear or already appear drug resistance, and can be used as a gene marker of drug resistance.

TABLE 5 analysis of specific mutation in NPAS2 Gene and its concordance with assessment of clinical efficacy

Mutation detection node	Number of mutation detection cases	Clinical efficacy assessment, number of cases and consistency	Mean time earlier for gene detection than for clinical efficacy assessment
				Before chemotherapy	7 examples of	Primary drug resistance, 7 cases, 100%	3.0 month
In the course of chemotherapy	11 examples of	Secondary drug resistance, 11 cases, 100%	5.8 months

In conclusion, the invention firstly discloses two new specific mutations of NPAS2 gene, namely g. [105009_105012delGCTT ] mutation and g. [105054insGCCTA ] mutation, and the detection of the two mutations indicates that the FOLFOX4 scheme chemotherapy of the liver cancer can appear or has appeared to be resistant to drugs, so the invention can be applied to the prediction and evaluation of the resistance of the FOLFOX4 scheme chemotherapy of the liver cancer, and has the advantages of accuracy, specificity and timeliness and effectiveness. The method has obvious significance for the selection and timely change of a chemotherapy scheme of a liver cancer patient, particularly a FOLFOX4 scheme, can effectively improve the benefit of the FOLFOX4 scheme chemotherapy of the liver cancer patient, and avoids ineffective chemotherapy and unnecessary toxic and side effects of chemotherapy; meanwhile, the method has important value for the research on the drug resistance mechanism of FOLFOX4 scheme chemotherapy of the liver cancer and the research and development of effective chemotherapeutic drugs.

Sequence listing

<110> Chenping

<120> human NPAS2 mutant gene and application thereof in liver cancer FOLFOX4 scheme chemotherapy drug resistance evaluation

<160>2

<170>SIPOSequenceListing 1.0

<210>1

<211>286

<212>DNA

<213> Intelligent (Homo sapiens)

<400>1

ctgaaagagt tttttgataa gatagatgat cacaatagaa tgtgctctaa tagaaagtgc 60

ttttgtttca tcacaaagcc tttgtgacgt ggaatgttcc agtaacctgc tcgttttgtg 120

tttacagact cgaaacaagt ctgagaagaa gcgtcgggac cagttcaatg ttctcatcaa 180

agagctcagt tccatgctcc ctggcaacac gcggaaaatg gacaaaacca ccgtgttgga 240

aaaggtcatc ggatttttgc agaaacacaa tggtaaaggt caccct 286

<210>2

<211>295

<212>DNA

<213> Intelligent (Homo sapiens)

<400>2

ctgaaagagt tttttgataa gatagatgat cacaatagaa tgtgctctaa tagaaagtgc 60

ttttgtttca tcacaaagcc tttgtgacgt ggaatgttcc agtaacctgc tcgttttgtg 120

tttacagagc ttctcgaaac aagtctgaga agaagcgtcg ggaccagttc aatgcctagt 180

tctcatcaaa gagctcagtt ccatgctccc tggcaacacg cggaaaatgg acaaaaccac 240

cgtgttggaa aaggtcatcg gatttttgca gaaacacaat ggtaaaggtc accct 295

Claims

1. A human NPAS2 mutant gene is characterized in that the NPAS2 mutant gene is formed by inserting 5 nucleotides of GCCTA into the 105054 th site of a wild-type NPAS2 gene, namely, g. [105054insGCCTA ] mutation occurs, a sequence in the range of 4989 and 181679 in a sequence with an NCBI accession number of NG _023259.1 is a reference sequence of the NPAS2 wild-type gene, and the sequence of a fragment in which the g. [105054insGCCTA ] mutation exists is shown as SEQ ID NO: 2, the rest of the sequences are referred to the NPAS2 wild-type gene reference sequence; hepatoma patients with g. [105054insGCCTA ] mutation in the NPAS2 gene were resistant to FOLFOX4 chemotherapy regimen.

2. The human NPAS2 mutant gene of claim 1, which corresponds to mRNA and encodes a protein.

3. The use of the human NPAS2 mutant gene according to claim 1, wherein the NPAS2 mutant gene is used in a liver cancer FOLFOX4 regimen chemotherapy resistance detection kit, a detection method and a therapeutic drug development.

4. The assay kit of claim 1, wherein the assay kit comprises 5'-TCACAAAGCCTTTGTGACGT-3' and 5'-ACACGGTGGTTTTGTCCATT-3' primers and 5 '-VIC-AATGCCTAGTTCTCA-MGB-NFQ-3' probe.

5. The method of claim 1, wherein the method of detecting human NPAS2 mutant gene comprises the steps of constructing a digital PCR or real-time fluorescent quantitative PCR-based method using 5'-TCACAAAGCCTTTGTGACGT-3' and 5'-ACACGGTGGTTTTGTCCATT-3' primers and 5 '-VIC-AATGCCTAGTTCTCA-MGB-NFQ-3' probe to amplify and detect mutation of the g. [105054insGCCTA ] mutated NPAS2 gene fragment.