KR102112951B1 - Ngs method for the diagnosis of cancer - Google Patents

Abstract

The present invention relates to an NGS analysis method for the diagnosis of cancer, capable of detecting a single nucleotide variation of BRCA1/2 gene, the insertion and deletion of a small unit of a base, and large gene rearrangement including a 5′ promoter region at once. Since the NGS analysis method using a primer set of the present invention is capable of detecting a single nucleotide variation of BRCA1/2 gene, the insertion and deletion of a small unit of a base, and large gene rearrangement including a 5′ promoter region, a BRCA genetic test can be performed with one NGS test instead of a procedure that was performed by combining both Sanger and MLPA methods because it was not detectable by a conventional NGS analysis method.

Description

NGS METHOD FOR THE DIAGNOSIS OF CANCER

The present invention relates to an NGS analysis method for cancer diagnosis capable of detecting single gene mutations of BRCA1 / 2 gene, small base insertion and deletion, and large gene rearrangement including the 5` promoter region at once.

Even today, with the development of medicine, human cancer, especially the majority of solid tumors (other than blood cancer), has a 5-year survival rate of less than 50%. About two-thirds of all cancer patients are found in advanced stages, most of whom die within 2 years of diagnosis. This is because the treatment effect of the poor cancer is not only a problem of the treatment method, but it is not easy to diagnose the actual cancer early and to diagnose the advanced cancer accurately and follow up after treatment. The diagnosis of cancer in the current clinical trials is followed by a history taking, a physical examination, and a clinical pathology examination, and once suspected, it is performed by radiographic examination and endoscopy, and finally confirmed by biopsy. However, with the existing clinical test method, the number of cancer cells is 1 billion and the diameter of the cancer must be 1 cm or more to be diagnosed. In this case, the cancer cells already have metastatic ability, and more than half of the cancer cells have already metastasized. On the other hand, tumor markers that find substances in the blood that are produced by the cancer directly or indirectly are used for cancer screening, which is limited in accuracy and appears normal to about half even when there is cancer. Even in the absence of this, it often appears positive, causing confusion. In addition, in the case of an anticancer agent mainly used for the treatment of cancer, there is a problem that exhibits its effect only when the volume of the cancer is small.

As described above, it is difficult to diagnose and treat both cancers because there are many differences from normal cells, and they are very complex and diverse. Cancer continues to grow spontaneously in excess, free from death, continues to survive, invades surrounding tissues and spreads (metastasizes) to distal organs, causing human death. Survives immune attacks or chemotherapy, constantly evolves, and the cell group (clone) that is most favorable for survival selectively proliferates. Cancer cells are highly viable survivors caused by mutations in a number of genes. In order for one cell to turn into a cancer cell and develop into a malignant cancerous mass seen in clinical practice, mutations must occur in multiple genes. Therefore, it is necessary to approach at the genetic level in order to diagnose and treat cancer fundamentally.

Sanger-based sequencing, a method of analyzing existing sequencing, can accurately obtain sequencing information, but it is not applicable to research fields that require large-scale DNA sequencing information such as personal genome analysis. It is inefficient in terms of time, labor and cost to analyze. For this reason, there has been a demand for a new analysis method capable of obtaining a large amount of DNA sequence information with high efficiency and low cost. In the mid-2000s, next generation sequencing (NGS), which can rapidly generate short-sequence sequencing targeting template DNA, was introduced. Recently, as the next generation sequencing (NGS) has become popular, it has attracted a lot of people's attention, and the technology using the next generation sequencing has been rapidly developed, and the genotyping price using it has become cheaper. As the development of NGS equipment, the improvement of reagents, and the development of bioinformatics techniques, NGS analysis has been used in many research fields because it has several advantages to replace the existing Sanger-based method.

Next-generation genome sequencers (NGSs) that implement next-generation sequencing are typical examples of SOLiDs from Roche / 454, Illumina / Solexa, and Life Technologies (ABI). Roche / 454 and SOLiD adopt a method of amplifying a template in a sample to be analyzed by performing emulsion PCR (Emulsion PCR; emPCR) in a state where the template is complementarily bound to DNA on a solid support or a bead (Michael L Metzker, Aapplications of next-generation sequencing; Sequencing technologies the next generation, Nature Reviews Genetics, Vol. 11, pp31-46, January 2010). The latest model of the Roche / 454 is the GS FLX Titanium sequencer and the miniaturized GS Junior Titanium sequencer, which can read 80 million sequences in 7 hours. Due to the advancement of this technology, the next-generation sequencing method, which was previously used only for research due to the huge test cost, can be utilized in clinical clinical tests. However, in the large-scale sequencing data generated using the next-generation sequencing method, the length of the sequence is significantly shorter than the sequencing data generated by the conventional Sanger method. That is, the next-generation sequencing method has a disadvantage in that it is necessary to collect short sequences and construct a new genome sequence without reference, or to construct a genome sequence by referring to sequences of the same or similar species. have. In addition, the Roche / 454-based sequencer for next-generation sequencing is complementarily annealed to an amplified pre-amplified template, an amplicon, to DNA bound on a solid support or beads in an emulsion PCR process. After the amplification, the template is not amplified, but dimers of primers that are not consumed during the preparation of the amplicon are amplified and adversely affect the sequencing results. In particular, in addition, the conventional NGS analysis method cannot detect the CNV of the 5` promoter (5` UTR or 5` noncoding) region including Exon 1 in addition to the protein translation site of the BRCA1 / 2 gene, and the existing Sanger and MLPA (Multiplex ligation-dependent probe amplification) method should be used together, but there was a disadvantage that it is possible to accurately test.

The present invention aims to develop an NGS analysis method capable of detecting single gene mutations in BRCA1 / 2 gene, inserting and deleting bases in small units, and large gene rearrangement including the 5` promoter region at once.

To achieve the above object, the present invention provides a primer set for NGS analysis for the diagnosis of cancer.

In addition, the present invention provides a composition for diagnosis or prognosis of cancer.

In addition, the present invention provides a kit for diagnosis or prognosis of cancer.

In addition, the present invention provides an NGS analysis method for cancer diagnosis.

In addition, the present invention provides a method for providing information for diagnosis or prognosis of cancer.

According to the present invention, the NGS analysis method using the primer set of the present invention is capable of detecting a single base mutation of the BRCA1 / 2 gene, insertion and deletion of a small base unit, and large gene rearrangement including the 5` promoter region. It is not detectable by the NGS analysis method of. It has the effect of performing the BRCA gene test with one of the NGS tests, which was a combination of the Sanger method and the MLPA method.

1 is a view showing the design of the amplicon BRCA1 promoter / 5'UTR region for use in the NGS platform of the present invention.
FIG. 2 is a diagram showing BRCA1 5`UTR deletions that were not detected by the conventional NGS but were detected by the MLPA method:
A: MLPA analysis result of BRCA1 ;
B and C: Sanger sequence analysis result of BRCA1 gene; And
D: Amplification plot of BRCA1 mRNA (ΔRn vs number of cycles) (orange line: control, blue line: patient).
3 is a diagram showing LGRs revealed by the NGS platform of the present invention:
A: 6 BRCA1 LGRs; And
B: 1 BRCA2 LGRs.

Hereinafter, the present invention will be described in detail with reference to the accompanying drawings as an embodiment of the present invention. However, the following embodiments are presented as examples of the present invention, and when it is determined that a detailed description of a technique or configuration well known to those skilled in the art may unnecessarily obscure the subject matter of the present invention, the detailed description may be omitted. , Thereby, the present invention is not limited. The present invention is capable of various modifications and applications within the scope of the claims and the equivalents interpreted therefrom.

In addition, terms used in the present specification (terminology) are terms used to properly represent a preferred embodiment of the present invention, which may vary according to a user, an operator's intention, or customs in the field to which the present invention pertains. Therefore, definitions of these terms should be made based on the contents throughout the specification. Throughout the specification, when a part “includes” a certain component, it means that the component may further include other components, not to exclude other components, unless otherwise stated.

In one aspect, the present invention relates to a primer set for NGS (next generation sequencing) analysis for the diagnosis of cancer.

In one embodiment, the primer set is the primer set of SEQ ID NO: 1 and 2, the primer set of SEQ ID NO: 3 and 4, the primer set of SEQ ID NO: 5 and 6, the primer set of SEQ ID NO: 7 and 8, SEQ ID NO: 9 and 10 It may be any one or more selected from the group consisting of a primer set of.

In one embodiment, the cancer may be hereditary solid cancer, gastric cancer, breast cancer or ovarian cancer.

In one embodiment, the primer set of the present invention is a copy number variation or large genomic rearrangement (CVV) of a 5` promoter (5` UTR or 5` noncoding) region including Exon 1 in addition to a protein translation site among mutations of the BRCA1 / 2 gene. Can be detected by NGS analysis method.

In one embodiment, the primer set of the present invention can detect deletion of the 5′UTR region of the BRCA1 / 2 gene by NGS analysis method, and detect heterozygous deleletion of the 5′UTR region of the BRCA1 / 2 gene. can do.

In one aspect, the present invention relates to a composition for the diagnosis or prognosis of cancer comprising the primer set of the present invention.

In one embodiment, the composition for diagnosis or prognosis of cancer may be a composition for NGS analysis.

In one embodiment, the primer set is the primer set of SEQ ID NO: 1 and 2, the primer set of SEQ ID NO: 3 and 4, the primer set of SEQ ID NO: 5 and 6, the primer set of SEQ ID NO: 7 and 8, SEQ ID NO: 9 and 10 It may be any one or more selected from the group consisting of a primer set of.

In one embodiment, the cancer may be hereditary solid cancer, gastric cancer, breast cancer or ovarian cancer.

In one aspect, the present invention relates to a kit for diagnosis or prognosis of cancer comprising the composition of the present invention.

As used in the present invention, the term "NGS (Next Generation Sequencing) Analysis Method" is a new concept of sequencing capable of reading a large amount of sequencing for a sample to be analyzed in a short time and generating a large amount of sequencing data. Analysis techniques (Michael L. Metzker, Aapplications of next-generation sequencing; Sequencing technologies the next generation, Nature Reviews Genetics, Vol. 11, pp31-46, January 2010).

NGS (next generation sequencing) is single nucleotide variation, small insertion / deletion mutation and large genomic rearrangement (also known as copy number variation (CNV)) The detection is possible at the same time, but the region detectable by the NGS test method is a protein translation site (from Exon 2 for the BRCA1 gene, from Exon 2 for the BRCA2 gene). Therefore, the conventional NGS analysis method cannot detect CNV of a 5` promoter (5` UTR or 5` noncoding) region including Exon 1 in addition to the protein translation site of the BRCA1 / 2 gene.

The NGS analysis method using the primer set of the present invention is a method capable of detecting a single base mutation of a BRCA1 / 2 gene, inserting and deleting a base in a small unit, and large gene rearrangement including the 5` promoter region. That is, when applied to the clinical, BRCA gene test can be performed as a single NGS test using a procedure performed by combining the Sanger method and the MLPA method because it cannot be detected by a conventional NGS analysis method.

The term “primer,” as used herein, can serve as a starting point for template-directed DNA synthesis under suitable conditions (ie, four different nucleoside triphosphates and polymerases) in a suitable buffer at a suitable temperature. Single-stranded oligonucleotide. The suitable length of the primer varies depending on various factors, such as temperature and the use of the primer, but is typically 15-30 nucleotides. Short primer molecules generally require lower temperatures to form a sufficiently stable hybrid complex with the template. The sequence of the primer does not need to have a completely complementary sequence with some of the sequences of the template, and it is sufficient to have sufficient complementarity within a range capable of hybridizing with the template and working with the primer. Therefore, the primer in the present invention need not have a sequence perfectly complementary to the above-described nucleotide sequence as a template, and it is sufficient to have sufficient complementarity within a range capable of hybridizing to this gene sequence and acting as a primer. The design of such a primer can be easily carried out by a person skilled in the art with reference to the nucleotide sequence described above, for example, a primer design program (eg, PRIMER 3 program) can be used. The primer used in the present invention may be used by attaching a fluorescent dye or a separate adapter sequence, or may be used as a non-labeled primer.

In the present invention, “diagnosis” refers to determining the susceptibility of an object to a specific disease or condition, determining whether an object currently has a specific disease or condition, as long as it has a specific disease or condition Determining an object's prognosis (e.g., identification of a pre-metastatic or metastatic cancer state, determining the stage of the cancer or determining the responsiveness of the cancer to treatment), or terrametrics (e.g., for therapeutic efficacy) And monitoring the state of the object to provide information).

In the present invention, 'prognosis' refers to a prospect for future symptoms or progress determined by diagnosing a disease. In cancer patients, the prognosis usually refers to the recurrence of cancer or the metastasis or survival within a certain period after surgical procedure. Prognosis prediction (or diagnosis of prognosis) is a very important clinical task, as it provides clues on the direction of cancer treatment in the future, including chemotherapy in early cancer patients. Prognosis predictions also include patient response to disease treatments and predictions of treatment progress.

The term "amplification" used in the present invention means a reaction for amplifying a nucleic acid molecule. Various amplification reactions have been reported in the art, which are polymerase chain reactions (hereinafter referred to as PCR) (US Pat. Nos. 4,683,195, 4,683,202, and 4,800,159), reverse transcriptase-polymerase chain reactions (hereinafter referred to as RT-PCR) (Sambrook et al., Molecular Cloning.A Laboratory Manual, 3rd ed.Cold Spring Harbor Press (2001)), Miller, HI (WO 89/06700) and Davey, C. et al. (EP 329,822), ligase chain reaction ( ligase chain reaction (LCR) (17, 18), Gap-LCR (WO 90/01069), repair chain reaction (EP 439,182), transcription-mediated amplification (TMA) (19) ( WO 88/10315), self sustained sequence replication (20) (WO 90/06995), selective amplification of target polynucleotide sequences (US Pat. No. 6,410,276) , Consensus sequence primed polymerase chain reaction; CP-PCR (US Pat. No. 4,437,975), Arbitrarily primed polymerase chain reaction (AP-PCR) (US Pat. Nos. 5,413,909 and 5,861,245), nucleic acid sequence-based amplification (nucleic acid sequence) based amplification (NASBA) (US Pat. Nos. 5,130,238, 5,409,818, 5,554,517, and 6,063,603), strand displacement amplification and loop-mediated isothermal amplification; LAMP).

PCR is the most well-known nucleic acid amplification method, and many modifications and applications have been developed. For example, touchdown PCR, hotstart PCR, nested PCR and booster PCR have been developed by modifying traditional PCR procedures to enhance the specificity or sensitivity of PCR. In addition, multiplex PCR, real-time PCR, differential display PCR (D-PCR), rapid amplification of cDNA ends, RACE, and inverse polymerase PCR Chain reaction (IPCR), vectorette PCR, and TAIL-PCR (thermal asymmetric interlaced PCR) have been developed for specific applications. For more information on PCR, see McPherson, M.J., and Moller, S.G. PCR. BIOS Scientific Publishers, Springer Verlag New York Berlin Heidelberg, N.Y. (2000), the teachings of which are incorporated herein by reference.

As used in the present invention, the term "emulsion PCR (emPCR"), a DNA library of genes in a sample to be analyzed is spatially separated for each template and amplified in an emulsion state in an oil droplet, thereby cloning for a single template. As a technique to perform clonal amplification, a single template is captured by dropping beads and PCR amplification reagents (including DNA polymerase, dNTP, etc.) combined with one-way PCR primers (forward or reverse primers) in oil. Refers to a technique for performing PCR amplification after making an emulsion containing one bead and a PCR amplification reagent. In this emulsion PCR, since the primer in one direction is bound and fixed on the beads contained in the oil droplets, a single template is present in the amplified state on the surface of the beads after amplification, and subsequent sequencing is performed when the beads are recovered. You can do it.

In one aspect, the invention provides a primer set of SEQ ID NOs: 1 and 2, a primer set of SEQ ID NOs: 3 and 4, a primer set of SEQ ID NOs: 5 and 6, a primer set of SEQ ID NOs: 7 and 8, and a primer of SEQ ID NOS: 9 and 10. It relates to an NGS analysis method using any one or more primer sets selected from the group consisting of sets.

In one embodiment, the NGS analysis method may be an NGS analysis method for cancer diagnosis.

In one embodiment, the NGS analysis method of the present invention is a copy number variation or large genomic rearrangement of a 5` promoter (5` UTR or 5` noncoding) region including Exon 1 in addition to a protein translation site among mutations of the BRCA1 / 2 gene. ), And in particular, heterozygous deletion of the 5′UTR region of the BRCA1 / 2 gene.

In one aspect, the present invention relates to a method for providing information for diagnosis or prognosis of cancer, comprising performing NGS analysis using a sample isolated from a subject and a primer set of the present invention.

In one embodiment, the primer set is the primer set of SEQ ID NO: 1 and 2, the primer set of SEQ ID NO: 3 and 4, the primer set of SEQ ID NO: 5 and 6, the primer set of SEQ ID NO: 7 and 8, SEQ ID NO: 9 and 10 It may be any one or more primer sets selected from the group consisting of a primer set of.

In one embodiment, the cancer may be hereditary solid cancer, gastric cancer, breast cancer or ovarian cancer.

In one embodiment, as a result of performing NGS analysis using the primer set of the present invention, when a heterozygous deletion of the 5′UTR region of the BRCA1 / 2 gene is detected, the method may further include determining that the risk of cancer is high. Can be.

In the present invention, the sample isolated from the subject may be DNA, and the DNA may be isolated from a sample selected from the group comprising blood, semen, vaginal cells, hair, saliva, urine, tissue, and mixtures thereof. The DNA sample may be obtained through a conventional method known in the art, and the DNA sample may be composed of a single-source sample or a mixture of two or more sources.

The present invention will be described in more detail through the following examples. However, the following examples are only intended to materialize the contents of the present invention, and the present invention is not limited thereto.

Example 1. Patient selection and sample preparation

1-1. Patient selection and criteria

Peripheral blood samples admitted to the St. Mary's Hospital in Seoul from December 2014 to April 2018 were obtained from 108 breast cancer and / or ovarian cancer patients and one patient without disease. All subjects provided written informed consent, and the experiment was conducted in accordance with the Helsinki Declaration and was approved with the approval of the Institutional Review Board (IRB) / Ethics Committee (KC17SESI0623) of St. Mary's Hospital in Seoul. The high-risk subgroup for large genomic rearrangement (LGR) is Kim DH, et al. Identification of large genomic rearrangement of BRCA1 / 2 in high risk patients in Korea. It was defined as reported in BMC medical genetics. 2017; 18:38. The inclusion criteria for the individual medical history for the high-risk subgroup are 1) early onset of breast cancer (diagnosed at <36 years); 2) two primary breast cancers; 3) diagnosed with breast cancer at any age with close relatives (including 1st, 2nd or 3rd) of ≥1 with breast and / or epithelial ovarian cancer; 4) diagnosed with both breast cancer and epithelial ovarian cancer at all ages; And 5) patients with epithelial ovarian cancer with a close relative of> 1 with breast cancer and / or epithelial ovarian cancer.

1-2. DNA isolation

Genomic DNA was extracted from peripheral blood cells using a column-based DNA isolation technique (QIAamp DNA Blood mini kit, QIAGEN, Hilden, Germany). DNA concentration was measured with a Qubit fluorometer (Life Technologies, San Francisco, CA, USA) combined with the Qubit dsDNA HS assay kit, and DNA quality was confirmed by agarose gel electrophoresis.

Example 2. NGS sequencing comparison

2-1. Perform and compare two NGS analysis platforms

2-1-1. Illumina NGS analysis

For targeted next-generation sequencing analysis, oligo-probes specific to the BRCA1 / 2 gene and BRCA1 / 2 gene tailored target enrichment libraries were designed using Illumina Design Studio (Illumina, San Diego, CA, USA). The target region was designed by capturing upstream and downstream 20 nucleotides of all coding exons and exon-intron junctions. The final design consisted of 150 amplicons ranging from 210-280 bp in length and a cumulative target area of 7.6 kb. The amplicon sequence library was constructed from 150 to 250 ng of DNA per sample according to the TruSeq Amplicon protocol (Illumina Inc, San Diego, CA, USA). The pooled library was sequenced paired-end (2 × 150) with V2 chemistry on a MiSeq instrument (Illumina Inc, San Diego, CA, USA) in a micro low cell. Sequencing quality control and primary analysis were performed using Illumina Real Time Analysis Software (RTA). After demultiplexing and generation of FASTQ files, sequence alignment and sequence quality filtering to the reference genome were performed with MiSeq Reporter v2.3 software. Variant calling was performed on the VCF (variant call format) output file by evaluating the range (number of times the targeted variant was read during sequencing) and quality score (Q-score; expected probability of false basic detection). In particular, only non-synonymous exonic single-nucleotide variants (SNVs) were filtered according to the following quality control criteria: (1) a range of at least 50 ×; (2) Q-score≥30 (basic detection error rate of 1/1000); And (3) at least 40% of reads indicating variation (variation frequency, VF). VCF files created with MiSeq Reporter v2.3 software were further filtered and annotated using Illmina VariantStudio ver 2.2 (Illumina Inc, San Diego, CA, USA). The minimum reading depth to demonstrate the amplicon was set to 40 for each nucleotide, and the minimum percentage of mutated readings was set to 20% by default. The obtained sequence was aligned with Genome Analysis Toolkit (GATK) using the human reference sequence hg19 / GRCh37 using MiSeq Reporter software (Illumina, USA). Variants were classified according to Human Gene Mutation Database (HGMD), Universal Mutation Database (UMD) BRCA database, Breast International Consortium (BIC) database, or Leiden Open Variation Database (LOVD). In addition, CNV analysis of NGS data was performed by comparing region ranges in sample and control subjects using a range-based algorithm tool from NextGENe version 2.4.2.1 (Softgenetics, PA, USA).

In addition, Sanger sequencing was performed for comparative analysis with the NGS method. Specifically, a DNA sample was amplified by PCR using 68 pairs of PCR primers. Each primer pair was designed to amplify axons and adjacent intron sequences containing splice positions. The PCR product was purified with ExoSap (USB, Cleveland, Ohio, USA), and sequencing was performed with BigDye Terminator v3.1 using the same primers used for PCR. PCR amplicons were bidirectionally sequenced with ABI PRISM 3100 Genetic Analyzer (Applied Biosystems) using Big Dye terminator v 3.1 cycle Sequencing kit (Applied Biosystems, Foster City, CA, USA). Sequencing results were compared to BRCA1 (NM_007294.3) and BRCA2 (NM_000059.3) reference sequences and chromatograms were analyzed with Sequencher software version 5.0 (Gene Codes, Ann Arbor, MI, USA). To classify variations, American College of Medical Genetics and Genomics for the interpretation of sequence variants [Richards S_Genet Med. 2015], and all mutations were in five pathogenic groups (class 1: benign; class 2: likely benign; class 3: uncertain significance (VUS); class 4: likely pathogenic; and class 5; pathogenic ). To identify new mutations, ethnic-specific Korean genome databases containing 1244 alleles: Public data (HGMD, ExAC, including KRGDB (Korean Reference Genome Database; http://152.99.75.168/KRGDB/ ) 1000genomes, dbSNP) as well as LOVD (locus-specific databases) were searched. To assess whether substitution could potentially be pathogenic, In silico analysis of missense mutations was performed using Polyphen-2 (http: // genetics. Bwh.harvard.edu/pph2/), Sorting Intolerant From Tolerant (SIFT; which predicts whether protein substitutions are tolerated, http://sift.jcvi.org/), MutationTaster2 ( http://www.mutationtaster.org ) and Align-GVGD (agvgd.iarc.fr). In addition, by using Alamut software version 2.9 rev. 0 (Interactive Biosoftware, Rouen, France), amino acids affected by missense mutations were aligned with species-derived orthologs to confirm conservation.

As a result, an average of 4,385,195 readings were generated by paired-end NGS, of which 4,259,518 of 97% were mapped to the human gene (version GRCh37). The average range of target regions per patient was 13,538 (165 to 138,617), and the average range of nucleotides per gene was 7988 (476 to 18,076) and 36,313 (1,697 to 138,617) for BRCA1 and BRCA2, respectively. In addition, a total of 32 pathogenic mutations were detected in 45 patients (Table 1). 23 BRCA1 mutations were detected in 29 patients and 9 BRCA2 mutations were detected in 16 patients. Specifically, the most frequent BRCA1 gene mutation was c.5496_5506delGGTGACCCGAGinsA detected in 5 patients, followed by c.390C> A and c.5445G> A (p.Trp1815 *), respectively, in 2 patients. 17 different mutations were detected in one patient each. The frameshift mutation c.2345delG was new. In addition, the most frequent BRCA2 mutation was c.7480C> T detected in 5 patients, followed by c.1399A> T in 3 patients. c.3744_3747delTGAG mutation was detected in two patients and the other mutations were detected in one patient each. Frameshift mutation (c.2345delG; p.Ser782Ilefs * 10) was novel. As for the BRCA2 mutation, c.7480C> T (p.Arg2494 *) was confirmed in 5 patients, and c.1399A> T (p.Lys467 *) was detected in 3 patients. c.3744_3747delTGAG (p.Ser1248Argfs * 10) mutations were detected in 2 patients and all other mutations were detected in one patient each. Frameshift mutation c.3445_3446dupAT; p.Met1149Ilefs * 2 is new.

In addition, a total of 9 VUSs were detected in 11 patients (Table 2). Specifically, in both Sanger method and NGS method, mutation of BRCA1 gene, i.e., splicing position deletion mutation c.547 + 14delG, three missense mutations (c.3448C> T (p.Pro1150Ser), c.4729T > C (p.Ser1577Pro) and cc5339T> C (p.Leu1780Pro)) were detected. In addition, splicing position missense mutations in the BRCA2 gene c.426-92A> G and 4 missense mutations (c.623T> G (p.Val208Gly), c.5969A> C (p.Asp1990Ala), c .7691C> G (p.Thr2564Ser), and c.8092G> A (p.Ala2698Thr)) were detected.

2-1-2. Ion torrent NGS analysis

For another target NGS analysis, an Oncomine ™ BRCA Research Assay (Life Technologies, Tokyo, Japan) consisting of a pool of 265 primer pairs was used. Amplification of the library was performed by emulsion PCR using the Ion AmpliSeq IC 200 Kit (Thermo Fisher Scientifc). The prepared library was sequenced in Ion S5 XL Sequencer using Ion 520 Chip and 520 kit-Chef Kit (Thermo Fisher Scientifc). Torrent Suite software version 5.6 was used to demultiplex barcode readings and generate performance metrics including chip loading efficiency, range analysis, count readings and quality (Table 1).

2-1-3. Comparison of NGS and MLPA analysis results for large gene rearrangements (LGRs)

MLPA analysis was performed with genomic DNA of breast and / or ovarian cancer patients with negative Sanger sequencing results and belonging to the LGR high-risk subgroup. As one of these cases, genetic testing and counseling were performed for patients diagnosed with ovarian cancer at age 49. The patient was diagnosed with breast cancer on the left side at the age of 30 and underwent tumor resection for axillary lymph node resection. The characteristics of breast cancer are: invasive duct carcinoma, estrogen receptor, progesterone receptor and human epidermal growth factor receptor (HER2) negative, and T1N0M0 stage IA. Twelve years later, the patient recurred breast cancer with the same pathological characteristics as the first breast cancer on the left, followed by axillary lymph node dissection and AC (doxorubicin hydrochloride and cyclophosphamide) followed by Taxotel (docetaxel) chemotherapy. As a result of sequencing the BRCA1 and BRCA2 genes in the patient, the BRCA mutation was negative. Because the patient belongs to the LGR high risk group, MLPA analysis (MLPA®probemix P002-D1 BRCA1) was performed. Specifically, MLPA probe mixes P002 and P045 were used to screen LGR in BRCA1 and BRCA2, respectively, and P087 and P077 were used for identification (MRC-Holland, Amsterdam, Netherlands). MLPA data were analyzed with Genemarker v1.91 (Softgenetics, State College, PA). Peak heights were normalized and deletions and overlaps were defined as recommended by the manufacturer (Kim et al. BMC Medical Genetics (2017) 18:38). As a result, heterozygous deletion upstream of the A probe (dose: 0.629) was suspected, and as a result of performing MLPA analysis (MLPA®probemix P087 BRCA1), three probes were heterozygous (one BRCA1 upstream probe and two BRCA1 ex01a probes). Dogs) (Fig. 2A). In addition, in the family history of the patient, the first sister received gastrectomy at the age of 56 for stomach cancer, and the second and third sisters never had cancer. Therefore, BRCA1 MLPA analysis of the three sisters of the patient was performed. As a result, the same BRCA1 promoter region deletion was detected from a sister who had stomach cancer, and the other two sisters showed normal BRCA1 MLPA results. Thus, sequencing of BRCA1 cDNA and genomic DNA (gDNA) was performed to confirm the disappearance of alleles having a BRCA1 promoter region deletion. As a result, the patient and her first sister had a c.114G> A heterologous mutation in BRCA1 genomic DNA (gDNA). In addition, as a result of performing BRCA1 complementary DNA (cDNA) sequencing, c.114G> A mutation was not observed (FIGS. 2B and C). In addition, in order to check the BRCA1 mRNA level, total RNA was extracted using a High Pure RNA isolation kit (Roche Diagnostics, Mannheim, Germany). Transcriptor First Strand. After synthesizing cDNA with total RNA (1 ug) using cDNA Synthesis (Roche Diagnostics, Mannheim, Germany), for BRCA1 expression analysis, BRCA1 The TaqMan®Gene Expression Assays Kit (Applied Biosystems, Foster City, CA, USA ) Was used for combined reverse transcription real-time PCR, and ABI 7500 Fast Real-Time PCR System (Applied Biosystems) was used to quantify mRNA levels. Probes for the endogenous housekeeping gene GAPDH (Assay ID: Hs01556193_m1) and the test gene BRCA1 (Assay ID: Hs99999905_ml) were used. For reaction, 2.5 μL TaqMan Gene Expression Assay 20X (Applied Biosystems) and 3 μL with 19,5 μL water, 25 μL TaqMan Fast Advanced 2X Master Mix (Applied Biosystems), primers and probes specific to each gene A total of 50 uL of reactants were prepared to contain each cDNA sample. PCR was performed by performing 40 cycles of the protocol at 50 ° C for 2 minutes, 95 ° C for 10 minutes, 95 ° C for 15 seconds, and 60 ° C for 60 seconds. All samples were analyzed in duplicate and the average Ct value of the replicate performance for each sample was used for calculation. The Ct value was normalized to the endogenous control gene, and the average GAPDH was subtracted from the average BRCA1 Ct value to obtain a corresponding ΔCt value. The corresponding ΔΔCt value was obtained by subtracting ΔCt from the mRNA sample from ΔCt from the mRNA of the normal volunteer. Relative quantification in BRCA1 mRNA expression is presented as 2-ΔΔ. As a result, normalized levels of BRCA1 mRNA (0.78 and 0.52, respectively) of the patient and her first sister were lower than those of the second and third sister and healthy controls (1.00, 1.03, and 1.31, respectively) (Figure 2D). . MLPA analysis of the patient's genomic DNA revealed six BRCA1 and one BRCA2 large axon rearrangements (Table 3). However, in the above two NGS analyzes (Illumina and Ion torrent platforms), 6 of 7 LGRs were identified, but the 5′UTR region deletion of BRCA1 identified in the MLPA analysis was both of the two NGS analyzes (Illumina and Ion torrent platforms). Was not detected in (Table 4).

Accordingly, a new NGS platform was devised to detect all six LGRs of BRCA1 and one LGR of BRCA2 (FIG. 1 and Table 5).

2-2. Confirm LGR detection using new NGS analysis

In the above example, deletion of the 5′UTR region of BRCA1 detected by MLPA but not by two conventional NGS analysis methods was confirmed by the new NGS analysis method of the present invention. Specifically, NGS library preparation and sequencing-BRCAaccuTest (NGeneBio Co., Ltd), consisting of two pools of 151 primer pairs, was introduced to generate NGS libraries of BRCA1 (NM_007294.3) and BRCA2 (NM_000059.3) genes. Briefly, 10 ng of sample DNA was amplified by PCR for the target regions of the BRCA1 and BRCA2 genes. In addition, amplicons (promoter of BRCA1 gene, 5'UTR (exon1), 5 amplicons for analyzing CNV of intron1 region) were ligated with an adapter and the library was amplified in the final step. Quantitative and qualitative evaluation of the amplified library was confirmed by Qubit 3.0 Fluorometer (Thermo Fisher Scientific) and Tapestation 2200 (Agilent Technologies), respectively. Thereafter, the final library was normalized to 4 nM concentration and prepared for sequencing using Illumina MiSeqDx (Illumina) with MiSeq Reagent Nano Kit V2 (300 cycles). Sequence read results were generated as FASTQ files and uploaded to the NGeneAnalySys software. NGeneAnalySys' pipeline workflow is: data QC (quality control), adapter trimming, sorting, anomaly detection and annotation. Briefly generated sequence read results were aligned to the human hg19 reference genome using the BWA-MEM algorithm. Genetic variations including single nucleotide variants (SNV) and indels (short insertions / deletions) were confirmed by the GATK (v2.3) and FreeBayes (v0.9.10) methods. To detect large gene rearrangements of the BRCA1 / 2 gene, the ratio in each sample of total amplicon was calculated based on the normalized amplicon coverage. Amplicon specific cutoff values were defined based on amplicon specific ratio ranges from different samples within the same run.

As a result, it was confirmed that the remaining CNVs including the 5′UTR deletion of BRCA1 can be detected in accordance with the MLPA results (FIG. 3).

<110> THE CATHOLIC UNIVERSITY OF KOREA INDUSTRY-ACADEMIC COOPERATION FOUNDATION <120> NGS METHOD FOR THE DIAGNOSIS OF CANCER <130> 520180255 <160> 10 <170> KoPatentIn 3.0 <210> 1 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Amp-1 forward primer <400> 1 gtcccatcct ctcatacata ccag 24 <210> 2 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Amp-1 reverse primer <400> 2 acgtcggctg gtcatgaggt caggagt 27 <210> 3 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Amp-2 forward primer <400> 3 ctttcccggg actctactac ctttac 26 <210> 4 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Amp-2 reverse primer <400> 4 accttgattt cgtattctga gaggct 26 <210> 5 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Amp-3 forward primer <400> 5 tacgaaatca aggtacaatc agagga 26 <210> 6 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Amp-3 reverse primer <400> 6 ataggtagcg attctgacct tcgtac 26 <210> 7 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Amp-4 forward primer <400> 7 ggcaagtagt cttgtaaggt cagtgg 26 <210> 8 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Amp-4 reverse primer <400> 8 aagaagattg gctcttacca cttgtc 26 <210> 9 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Amp-5 forward primer <400> 9 catgtctgcg cactcgtagt tc 22 <210> 10 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Amp-5 reverse primer <400> 10 gacaaagaca gtaactagtc ccgt 24

Claims (12)

Primer set of SEQ ID NO: 1 and 2, primer set of SEQ ID NO: 3 and 4, primer set of SEQ ID NO: 5 and 6, primer set of SEQ ID NO: 7 and 8, primer set of SEQ ID NO: 9 and 10 A primer set for analyzing next generation sequencing (NGS) for diagnosis of cancer, comprising any one or more primer sets. delete The primer set for NGS analysis according to claim 1, wherein the cancer is at least one genetic solid cancer selected from the group consisting of gastric cancer, breast cancer, and ovarian cancer. A composition for diagnosis or prognosis of cancer comprising the primer set of claim 1. delete The composition of claim 4, wherein the cancer is any one or more hereditary solid cancers selected from the group consisting of gastric cancer, breast cancer, and ovarian cancer. A kit for diagnosis or prognosis of cancer, comprising the composition of claim 4. Primer set of SEQ ID NO: 1 and 2, primer set of SEQ ID NO: 3 and 4, primer set of SEQ ID NO: 5 and 6, primer set of SEQ ID NO: 7 and 8, primer set of SEQ ID NO: 9 and 10 NGS analysis method using any one or more primer sets. The method of claim 8, wherein it is possible to confirm whether the 5′UTR region of the BRCA1 / 2 gene is deleted. A method for providing information for diagnosis or prognosis of cancer, comprising performing an NGS analysis using a sample separated from a subject and the primer set of claim 1. The method of claim 10, wherein the cancer is any one or more hereditary solid cancers selected from the group consisting of gastric cancer, breast cancer, and ovarian cancer. 11. The method of claim 10, to determine whether the 5′UTR region of the BRCA1 / 2 gene is deleted, a method of providing information for diagnosis or prognosis of cancer.

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