Probe combination, kit and method for detecting genetic susceptibility gene of prostatic cancer

Abstract

The invention discloses a probe combination for detecting genetic susceptibility genes of prostatic cancer. The probe combination comprises at least one of sequences as shown in SEQ ID NO 1-89. The invention also discloses a kit comprising the probe combination and a method for detecting the genetic susceptibility gene of the prostatic cancer by using the kit. According to the present invention, the prostatic cancer genetic susceptibility related genes such as ATM, ATR, BRCA1, BRCA2, BRIP1, CHEK2, EPCAM, FANCA, HOXB13, MLH1, MRE11A, MSH2, MSH6, NBN, PALB2, PMS2, RAD51C, RAD51D and TP53 can be detected at one time by using the hybrid capture method; the probe provided by the invention is wide in coverage, high in sequencing depth and capable of detecting embryonic line variation of all exon regions of a target gene. All related diseases of the related genes of the genetic susceptibility of the prostatic cancer can be reported, genetic modes corresponding to the diseases are provided, and the application potential and the market value are large.

Description

Technical Field

[0001] The present invention relates to the technical field of multi-gene detection, and in particular to a probe combination, a kit and a method for detecting prostate cancer genetic susceptibility genes. Background Art

[0002] Prostate cancer is an epithelial malignant tumor that occurs in the prostate and is one of the most common malignant tumors in the male urogenital system. Its incidence and mortality rates rank 2nd and 5th respectively in the global male malignant tumor incidence and death spectra, ranking first and 3rd respectively among men in European and American countries, and ranking 6th and 7th respectively among Chinese men. Although the incidence of prostate cancer in China is much lower than that in European and American countries, it has been showing an increasing trend in recent years. In China, more than 2 / 3 of the patients are in the advanced or locally advanced stage at the first diagnosis, missing the best treatment opportunity. With the increasingly widespread application of NGS technology in the diagnosis and treatment of prostate cancer, more and more patients can benefit from the precision treatment of prostate cancer. In addition, NGS detection also plays an important role in the assessment of the tumor occurrence risk of the family members of patients carrying germline gene mutations.

[0003] Family history is a definite risk factor for prostate cancer. It is estimated that about 40%-50% of prostate cancers are related to genetic factors. At present, germline mutations in multiple DNA damage repair genes have been confirmed to be related to prostate cancer genetic susceptibility. DNA damage repair genes represented by BRCA1 and BRCA2 are the most well-known prostate cancer susceptibility genes so far. Other DNA damage repair genes, such as ATM, PALB2, CHEK2, and mismatch repair genes (MLH1, MSH2, MSH6, and PMS2), are also considered to be associated with an increased risk of prostate cancer. Some studies have found that the risk of prostate cancer in healthy men carrying germline mutations in mismatch repair genes is 2-5 times higher than that of non-carriers. Other genes that may be related to hereditary prostate cancer also include genes such as HOXB13, EPCAM, RAD51C, and RAD51D. Epidemiological studies have shown that if a direct relative has prostate cancer, the risk of the person himself having prostate cancer will increase by more than 1 time; if 2 or more direct relatives have prostate cancer, the relative risk will increase to 5-11 times. The diagnosis age of patients with a family history of prostate cancer is about 6-7 years earlier than that of patients without a family history.

[0004] Therefore, it is necessary to detect the genetic susceptibility genes of prostate cancer, and it has important clinical application value and social significance to develop a prostate cancer susceptibility gene detection panel that can comprehensively and deeply cover all relevant pathogenic sites.

[0005] Probe capture sequencing is one of the commonly used multi-locus detection schemes for target genes. In this scheme, effective specific probes are designed and synthesized to hybridize with genomic DNA. After capturing and enriching the target region sequences, high-throughput sequencing is performed using mainstream sequencing platforms. The probe hybridization capture efficiency and the coverage depth of genomic regions are affected by various factors. The development and validation of disease-related target gene panels and key loci rely on the study of target regions in a large number of samples. Only probes with high sequence accuracy can specifically bind to the target region sequences, greatly improving the capture efficiency of the probes and achieving the purpose of enriching target fragments. Summary of the Invention

[0006] The object of the present invention is to provide a multi-gene panel for detecting prostate cancer genetic susceptibility-related genes, which can detect 19 prostate cancer genetic susceptibility genes. It has strong specificity and can detect the variations in the whole exon regions of 19 genes in a test sample at one time under the same system, with a wide coverage area and can detect all germline variations in the exon regions.

[0007] The technical solution adopted by the present invention is:

[0008] A probe combination for detecting prostate cancer genetic susceptibility genes, wherein the probe combination includes at least one of the sequences shown in SEQ ID NO 1 to 88.

[0009]

[0010]

[0011]

[0012]

[0013]

[0014]

[0015]

[0016] Preferably, the probe combination includes all of the sequences shown in SEQ ID NO 1 to 88.

[0017] The prostate cancer genetic susceptibility genes detected by the probe combination of SEQ ID NOs: 1-88 include at least one of the following 19 genes: ATM, ATR, BRCA1, BRCA2, BRIP1, CHEK2, EPCAM, FANCA, HOXB13, MLH1, MRE11A, MSH2, MSH6, NBN, PALB2, PMS2, RAD51C, RAD51D, TP53.

[0018] The above-mentioned included related genes are from multiple databases, relevant guidelines and expert consensus, covering the genetic susceptibility related genes with high penetrance in prostate cancer. The designed capture probe combination covers the entire coding region sequences of the genes involved.

[0019] The genetic patterns of related diseases include: autosomal dominant inheritance, autosomal recessive inheritance.

[0020] According to the 2015 ACMG guidelines, the clinical significance of germline variants is classified, and the pathogenic information of variants can be accurately discriminated. The clinical significance of germline variants is classified as: pathogenic (P), likely pathogenic (LP), variant of uncertain significance (VUS), benign (B), likely benign (LB) variants.

[0021] Another object of the present invention is to provide a kit for detecting prostate cancer genetic susceptibility genes, and the kit includes the probe combination.

[0022] The kit may further include genomic DNA extraction reagents, DNA library construction reagents, capture DNA library construction reagents, etc., and corresponding commercially available kits can be used. For example, for genomic DNA extraction reagents, Tiangen Blood Genomic DNA Extraction Kit can be used; for DNA library construction reagents, Twist Library Preparation Enzymatic Fragmentation Kit 2.0 from Twist Company can be used; for capture DNA library construction reagents, the liquid hybridization capture kit from Twist Company can be used. It may also include instruments or reagents required for sequencing (such as Illumina high-throughput sequencing platform and human genome sequence).

[0023] The present invention also provides the application of the kit for detecting prostate cancer genetic susceptibility genes in detecting prostate cancer genetic susceptibility genes. Further, the application is to detect the germline variants of prostate cancer genetic susceptibility genes, and the germline variants are classified as pathogenic (P), likely pathogenic (LP), variant of uncertain significance (VUS), benign (B), likely benign (LB) variants.

[0024] Further, the kit for detecting prostate cancer genetic susceptibility genes can also be used to detect related diseases and corresponding genetic patterns.

[0025] The present invention also provides a method for detecting prostate cancer genetic susceptibility genes using the said kit, and the method includes the following steps:

[0026] (1) Extract DNA from the sample to be tested;

[0027] (2) Construct a DNA library;

[0028] Constructing a DNA library generally includes DNA fragmentation, end repair, adding a poly-A tail, ligating universal adapters, PCR amplification, and finally purification to obtain a DNA library;

[0029] (3) Use a probe combination for detecting prostate cancer genetic susceptibility genes for hybridization capture and PCR amplification to obtain a library after hybridization capture;

[0030] (4) Sequence the library after hybridization capture, perform bioinformatics analysis on the sequencing data for detection, obtain the germline variation situation of prostate cancer genetic susceptibility genes, and provide relevant diseases and corresponding inheritance patterns.

[0031] Compared with the prior art, the technical solution of the present invention has the following advantages:

[0032] Existing genetic susceptibility detections are mostly based on PCR and first-generation sequencing technologies. Such methods can only detect partial hotspot variations of the selected genes and cannot detect unknown variations. The present invention is based on next-generation sequencing and adopts a hybridization capture method, which can achieve the detection of variations in the whole exon regions of 19 genes in a test sample at one time in the same system, with a wide coverage area and the ability to detect all germline variations in the exon regions.

[0033] The kit designed by the present invention is targeted at a single cancer type and can detect all genes with clear genetic susceptibility to prostate cancer at one time, with strong specificity for prostate cancer patients. Compared with multi-tumor genetic susceptibility detections, it can reduce costs and increase efficiency.

[0034] The determination of gene variations in the present invention is based on the ACMG guidelines, and there is a standard annotation process for variations not included in the ClinVar database, effectively avoiding missed detections. The annotation sources of related diseases of genes are extensive and do not rely solely on a single database. The present invention has manually sorted out multiple databases, relevant guidelines, and expert consensuses, and can output all related diseases and provide the inheritance patterns corresponding to the diseases.

[0035] The capture probes designed by the present invention cover the entire coding region sequences of the genes involved, and the coverage of the target region can reach 100%, and the average sequencing depth is as high as 100x.

[0036] The present invention can detect germline variations in all exon regions of target genes, report all related diseases of genes associated with genetic susceptibility to prostate cancer, and provide the corresponding inheritance patterns of diseases, having great application potential and market value. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 It is the fragment analysis result of the library pool after hybridization capture constructed. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0038] The technical solutions of the present invention will be further described in detail below in conjunction with the drawings and specific embodiments, but the protection scope of the present invention is not limited thereto.

[0039] Example 1: Probe Design and Synthesis of Genes Associated with Genetic Susceptibility to Prostate Cancer

[0040] The genes associated with genetic susceptibility to prostate cancer are screened by the following methods:

[0041] By manually reading the National Comprehensive Cancer Network (NCCN) guidelines, the information on genes associated with genetic susceptibility in the guidelines is sorted, screened, and summarized.

[0042] The relationship between phenotypes and genes is obtained through the Online Mendelian Inheritance in Man (OMIM) database.

[0043] Pathogenic / possibly pathogenic variations of relevant genes are screened through the ClinVar database, and these variations need to be included in probe design.

[0044] Finally, the following 19 genes are selected: ATM, ATR, BRCA1, BRCA2, BRIP1, CHEK2, EPCAM, FANCA, HOXB13, MLH1, MRE11A, MSH2, MSH6, NBN, PALB2, PMS2, RAD51C, RAD51D, TP53.

[0045] II. Probe Design and Synthesis

[0046] According to the 19 relevant genes, the hybridization capture probe sequences are designed as shown in Table 1 below:

[0047] Table 1

[0048]

[0049]

[0050]

[0051]

[0052]

[0053]

[0054] III. Detection Method of the Designed Probes

[0055] 1. Genomic DNA Extraction

[0056] Extract the genomic DNA from the peripheral blood of the subject to be tested.

[0057] 2. Construction of DNA Library

[0058] Use Twist Library Preparation Enzymatic Fragmentation Kit 2.0 (lot number: 34000002908) from Twist Bioscience to construct a DNA library for the genomic DNA obtained in step 1 above. The specific library construction process is as follows:

[0059] 1) DNA fragmentation, end repair, and dA tailing. The reaction system is shown in Table 2.

[0060] Table 2 Reaction System

[0061]

[0062]

[0063] After preparing the system, pipette and mix well, then place it in a PCR instrument for incubation. Table 3 shows the incubation program for DNA fragmentation, end repair, and dA tailing.

[0064] Table 3 Reaction Program

[0065] 37℃ 20 min 65℃ 30 min 4℃ Hold

[0066] 2) Ligation of Twist Universal Adapter. The reaction system is shown in Table 4.

[0067] Table 4 Reaction System

[0068] Name Dosage DNA fragmentation, end repair and dA tailing products 50ul Twist universal adapters 5ul Ligation Master Mix 20ul Total 75ul

[0069] After preparing the system, pipette and mix well, then place it in a PCR instrument for incubation. Table 5 shows the incubation program for adapter ligation.

[0070] Table 5 Incubation Program for Adapter Ligation

[0071] 20℃ 15 min

[0072] 3) Purification

[0073] a. Add 60 μl of DNAPurification Beads (0.8 times the sample volume) that have been equilibrated to room temperature and thoroughly mixed to the sample from the previous step. Pipette up and down 30 times to mix well and incubate at room temperature for 5 min;

[0074] b. Centrifuge briefly and place on the magnetic stand for 5 min. Discard the supernatant;

[0075] c. Add 200 μl of freshly prepared 80% ethanol, let stand at room temperature for 1 min, and discard the supernatant;

[0076] d. Repeat step c;

[0077] e. Centrifuge briefly to collect the residual liquid at the bottom of the tube. Place on the magnetic stand and then discard the residual liquid. Air dry at room temperature for 3 - 5 min until the alcohol has completely evaporated;

[0078] f. Add 18 μl of Nuclease-free water, pipette up and down to mix well, and let stand at room temperature for 5 min;

[0079] g. Centrifuge briefly and place on the magnetic stand for 2 min until the liquid is clear. Transfer 15 μl of the supernatant to a new PCR tube for PCR amplification. Table 6 shows the reaction system.

[0080] Table 6 Reaction System

[0081] Name Dosage Adapter-ligated DNA 15ul Equinox Library Amp Mix (2X) 25ul UDI primers (Twist Bioscience) 10ul Total 50ul

[0082] After preparing the system, pipette up and down to mix well and place in a PCR instrument for incubation. Table 7 shows the PCR program.

[0083] Table 7

[0084]

[0085] Purification

[0086] a. Add 50 μl of DNAPurification Beads that have been equilibrated to room temperature and thoroughly mixed to the PCR product from the previous step. Pipette up and down 30 times to mix well and incubate at room temperature for 5 min;

[0087] b. Centrifuge briefly and place on the magnetic stand for 5 min. Discard the supernatant;

[0088] c. Add 200 μl of freshly prepared 80% ethanol, let stand at room temperature for 1 min, and discard the supernatant;

[0089] d. Repeat step c;

[0090] e. Centrifuge briefly to collect the residual liquid at the bottom of the tube. Place on the magnetic stand and then discard the residual liquid. Air dry at room temperature for 3 - 5 min until the alcohol has completely evaporated;

[0091] f. Add 24 μl of nuclease-free water, pipette up and down to mix well, and let it stand at room temperature for 5 min;

[0092] g. Briefly centrifuge, place on the magnetic stand for 2 min until the liquid becomes clear, transfer 22 μl of the supernatant to a new centrifuge tube to obtain the DNA library, and mix well by shaking;

[0093] Take 2 μl of the above DNA library, dilute it 10-fold, and then quantify it using the Qubit dsDNA HS Assay Kit, and record the library concentration; Take 1 μl of the DNA library diluted 10-fold in the previous step and use the Agilent 2100 Bioanalyzer system (Agilent DNA 1000 Kit) to measure the library fragment length, and the library length is approximately between 220 - 320 bp.

[0094] 3. Library Pooling, Hybridization Capture, PCR Amplification and Purification

[0095] 3.1 Library Pooling (1 - 8 samples)

[0096] a. The total amount of each library pool is 1.5 - 4.0 μg, and calculate the pooling volume of each library according to the library concentration; b. Mix each library in a 1.5 ml centrifuge tube, gently shake, and centrifuge;

[0097] 3.2 Library Hybridization and Capture

[0098] 3.2.1 a. Concentrate the library, and the system is shown in Table 8;

[0099] Table 8 Concentration System

[0100] Name Dosage Pooled library pool X (volume of samples 1 - 8 added together) Universal Blockers (Twist Bioscience) 8ul Blocking Solution (Twist Bioscience) 5ul Probe (probe sequences are SEQ ID NO 1 - 88) 4ul

[0101] b. Place the reaction tube in a vacuum dryer and dry it at 45 °C until completely dry.

[0102] 3.2.2 Library Hybridization

[0103] a. After drying, add 20 μl of FastHybridizationMix (do not restore to room temperature) to the centrifuge tube, gently flick to mix well, let it stand at room temperature for 5 min, and then transfer it to a new PCR tube;

[0104] b. Add 30 μl of Hyb Enhancer to the surface of the liquid in the PCR tube, briefly centrifuge to remove air bubbles, and then place it in a PCR instrument. The hybridization reaction program is shown in Table 9;

[0105] Table 9 Hybridization Reaction System

[0106] 95℃ 5 min 60℃ 2 h - 16 h (usually 2 h) 60℃ Hold

[0107] 3.2.3 Library Capture

[0108] a. Pipette 100 uL of Streptavidin Binding Beads into a 1.5 mL tube (which has been placed at room temperature for 30 min and shaken well).

[0109] b. Add 200 uL of FastBinding Buffer, mix well, place on a magnetic stand for 1 min, discard the supernatant, and repeat this washing step 2 more times for a total of 3 times. Then add 200 uL of FastBinding Buffer and resuspend by shaking to ensure thorough mixing.

[0110] c. After hybridization, open the lid of the PCR tube on the PCR instrument, quickly transfer all the hybridization solution to the magnetic bead solution, and immediately pipette to mix well.

[0111] d. Place the centrifuge tube on a rotator at room temperature, centrifuge at 1200 rpm for 30 min. Quickly centrifuge the centrifuge tube, place on a magnetic stand for 1 min, and discard the supernatant.

[0112] e. Add 200 uL of preheated FastWashBuffer 1 (do not remove from the metal bath), mix gently, and quickly incubate at 66 °C for 5 min.

[0113] f. Place on a magnetic stand for 1 min, discard the supernatant, then add 200 uL of preheated FastWashBuffer 1 again, mix gently, and quickly incubate at 66 °C for 5 min.

[0114] g. Transfer all the liquid in the tube to a new 1.5 mL tube, place on a magnetic stand for 1 min, and discard the supernatant.

[0115] h. Add 200 uL of preheated WashBuffer 2 (do not remove from the metal bath), mix gently, and quickly incubate at 48 °C for 5 min. Place on a magnetic stand for 1 min, discard the supernatant, and repeat the washing with WashBuffer 2 2 more times for a total of 3 times. I. After a brief centrifugation, place on a magnetic stand and use a 10 uL pipette tip to aspirate all the residual liquid.

[0116] J. Add 45 uL of H2O, pipette to mix well, and incubate on ice (Note: If PCR is not performed immediately, store the magnetic bead mixture at -20 °C).

[0117] 3.2.4 PCR Amplification, the reaction system is shown in Table 10, and the reaction program is shown in Table 11.

[0118] Table 10 Reaction System

[0119] Name Dosage Magnetic bead mixture 22.5ul KAPA HiFi HotStart ReadyMix 25ul Amplification Primers 2.5ul Total 50ul

[0120] After preparing the reaction system, pipette to mix well and place on the PCR instrument for reaction.

[0121] Table 11 PCR Procedure

[0122]

[0123] 3.2.5 Purification

[0124] a. Add 90 μl of DNAPurification Beads that have been equilibrated to room temperature and thoroughly mixed to the PCR product of the previous step. Pipette and mix 30 times, and incubate at room temperature for 5 min;

[0125] b. Briefly centrifuge and place on the magnetic stand for 5 min, then discard the supernatant;

[0126] c. Add 200 μl of freshly prepared 80% ethanol, let stand at room temperature for 1 min, then discard the supernatant;

[0127] d. Repeat step c;

[0128] e. Briefly centrifuge to collect the residual liquid at the bottom of the tube, place on the magnetic stand, then discard the residual liquid, and air dry at room temperature for 5 - 7 min until the alcohol has completely evaporated;

[0129] f. Add 34 μl of Nuclease-free water, pipette and mix, and let stand at room temperature for 5 min;

[0130] g. Briefly centrifuge and place on the magnetic stand for 2 min until the liquid is clear, then transfer 32 μl of the supernatant to a new centrifuge tube.

[0131] Obtain the library after hybridization capture and mix well by shaking;

[0132] Take 2 μl of the amplified and captured DNA library above and quantify it using the Qubit dsDNA HS Assay Kit, record the library concentration, which is approximately 1 - 15 ng / μl; take 1 μl of the amplified and captured DNA library above and determine the library fragment length using the Agilent 2100 Bioanalyzer system (Agilent DNA 1000 Kit), and the library length is approximately between 300 - 450 bp.

[0133] 4. Sequencing

[0134] Sequence the amplified and captured DNA library obtained in step 3 above using the Illumina Nextseq 2000.

[0135] Example 2: Detection of Clinical Samples

[0136] One full blood sample was taken from each of 2 prostate cancer patients, and the variant information of genetic susceptibility genes in these 2 samples was detected. First, the genomic DNA of the whole blood of the 2 prostate cancer patients was extracted separately, and then library construction was carried out according to the method described in Example 1. An amplified and captured DNA library was successfully constructed. The amplified and captured DNA library was sequenced on an Illumina high-throughput sequencer, and the sequencing parameters were PE: 2×150. Then, after quality control of the downloaded data, bioinformatics analysis was performed on the data using relevant software such as BWA / GATK, and relevant databases such as gnomAD / Clinvar / OMIM were annotated. According to the ACMG guidelines, the clinical significance of germline variants was classified as: pathogenic (P), likely pathogenic (LP), variant of unknown significance (VUS), benign (B), likely benign (LB) variants. Finally, each locus was verified by Sanger sequencing, and the coincidence rate was 100%.

[0137] The fragment analysis results of the constructed hybrid capture library pool are as Figure 1 shown. The sequencing quality is shown in Table 12, the test results of Sample 1 are shown in Table 13, and the test results of Sample 2 are shown in Table 14:

[0138] Table 12

[0139] Sample Target region coverage Average depth of target region Whether qualified Sample 1 100.0％ 202.69 Qualified Sample 2 100.0％ 235.17 Qualified

[0140] Test results of Sample 1:

[0141]

[0142] Test results of Sample 2:

[0143]

[0144] It can be seen from the test results that the probe coverage of the kit described in the present invention can reach 100%, the average depth of the target region is more than 100x, and the quality control is qualified. The test results of conventional samples show that the probes, methods and kits described in the present invention can accurately detect the variant conditions of genes related to genetic susceptibility to prostate cancer and provide tips for related diseases.

Claims

1. A probe combination for detecting prostate cancer genetic susceptibility genes, characterized in that The probe combination includes at least one of the sequences shown in SEQ ID NOs: 1 to 88; 2. The probe combination according to claim 1, wherein The probe combination includes all the sequences shown in SEQ ID NOs: 1 to 88.

3. The probe combination according to claim 1, wherein The prostate cancer genetic susceptibility genes detected by the probe combination of SEQ ID NOs: 1 to 88 include at least one of the following 19 genes: ATM, ATR, BRCA1, BRCA2, BRIP1, CHEK2, EPCAM, FANCA, HOXB13, MLH1, MRE11A, MSH2, MSH6, NBN, PALB2, PMS2, RAD51C, RAD51D, TP53.

4. A kit for detecting prostate cancer genetic susceptibility genes, comprising the probe combination according to claim 1 or 2.

5. Use of the kit for detecting prostate cancer genetic susceptibility genes according to claim 4 in detecting prostate cancer genetic susceptibility genes.

6. The application according to claim 5, wherein The application is to detect germline variations of prostate cancer genetic susceptibility genes, and the germline variations are classified as pathogenic, likely pathogenic, of uncertain clinical significance, benign, likely benign variations.

7. A method for detecting prostate cancer genetic susceptibility genes, characterized in that Using the kit for detecting prostate cancer genetic susceptibility genes according to claim 4, the method comprises the following steps: (1) Extracting DNA from the sample to be tested; (2) Constructing a DNA library; (3) Performing hybridization capture and PCR amplification using the probe combination for detecting prostate cancer genetic susceptibility genes to obtain a library after hybridization capture; (4) Sequencing the library after hybridization capture, and performing bioinformatics analysis on the sequencing data to detect the germline variation situation of prostate cancer genetic susceptibility genes, and providing relevant diseases and corresponding inheritance patterns.

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