Marker combination for detecting hereditary tumor susceptible syndrome and application thereof

Abstract

The invention discloses a marker combination for detecting a hereditary tumor susceptible syndrome and a probe composition for detecting the marker combination. The marker composition covers common genetic tumor susceptibility syndrome related genes of Chinese population. The detection method disclosed by the invention can be used for blood samples, oral swab scraped specimens and other normal tissue samples, and can be used for simultaneously detecting point mutation, small fragment insertion and deletion mutation (-15bp), gene level copy number variation and exon level large fragment rearrangement (gt; 1000 bp) and other embryonic line variation. The detection result provides reference for diagnosis, treatment and genetic risk prompt of the hereditary tumor susceptible syndrome.

Description

Technical Field

[0001] The present invention relates to the field of disease diagnosis, and in particular to a marker combination for detecting hereditary tumor susceptibility syndrome and application thereof. Background Art

[0002] Malignant tumors are one of the leading causes of morbidity and mortality worldwide. Hereditary Cancer Predisposition Syndromes (HCPS) refer to abnormal susceptibility to cancer due to genetic factors such as gene mutations or chromosomal abnormalities. Approximately 5-10% of cancer cases are related to HCPS.

[0003] HCPS is caused in part by a single gene mutation that inactivates the original gene function, mainly including 1. Genes that regulate cell cycle, division, differentiation and apoptosis, and control cell proliferation, such as TCS1 / 2NF1 / 2, PTEN, APC, TP53, etc.; 2. Genes that recognize and repair replication errors, such as mismatch repair genes (MLH1, MSH2 / 6, PMS2), BRCA1 / BRCA2, ATM, etc.; 3. Genes involved in oxidative metabolism regulation, such as FH, SDHB / SDHC / SDHD, etc.; these genes play a key role in maintaining normal cell growth and repairing DNA damage. It is characterized by an increased risk of individual cancer and early onset, and the presence of cancer case clustering in family history. In addition, some syndromes can affect multiple systems and organs, and there may be a risk of cancer in multiple organs, such as hereditary breast and ovarian cancer syndrome caused by BRCA1 and BRCA2 gene mutations, multiple breast cancers or ovarian cancers.

[0004] At present, more and more studies have found that the occurrence of many cancers has a complex pathogenic mechanism with multiple factors. Its genetic susceptibility is caused by the joint action of multiple gene mutations. The detection of only single genes or a few genes is prone to miss the diagnosis of other susceptibility gene carriers, resulting in inaccurate risk assessment, and then missing the opportunity for early intervention for individuals and family members, increasing the risk of disease. For example, in hereditary non-polyposis colorectal cancer syndrome, APC gene mutation is associated with familial adenomatous polyposis (FAP), Lynch syndrome (also known as hereditary non-polyposis colorectal cancer, HNPCC) is associated with MLH1, MSH2, MSH6, PMS2 and other gene mutations, and biallelic mutations of MUTYH gene are related to MUTYH-related adenomatous polyposis. For another example, in addition to the common BRCA1 and BRCA2 genes, mutations in other genes such as PALB2, RAD51C, RAD51D and BARD1 are also associated with increased risk in hereditary breast and ovarian cancer syndrome.

[0005] The National Comprehensive Cancer Network (NCCN), the American Society of Clinical Oncology (ASCO), the National Institute for Health and Care Excellence (NICE), and other medical professional organizations have developed clinical recommendations for molecular testing and genetic counseling for patients with breast, colon, thyroid, and other hereditary cancers. In addition, HCPS cancer patients can receive personalized treatment based on their genetic status. For example, identification of BRCA1 / BRCA2 pathogenic mutations may influence surgical decisions and affect their systemic therapy selection. In the long run, genetic testing of these susceptible populations is the basis for the selection of clinical observation strategies, which will improve the prevention of recurrence and secondary tumors.

[0006] In addition, the current methods and products for HCPS detection are basically based on the genomic DNA level. After identifying the germline variation in exons and splicing-related regions, the pathogenicity of the variation is judged by collecting relevant information. However, due to the incomplete functional research of many genes, it is impossible to obtain sufficient evidence for analyzing the changes in gene function caused by the variation, so that there is insufficient evidence to judge whether it is pathogenic during the interpretation process. Such sites are usually classified as variants of unknown significance (VUS). The incidence of VUS detected only at the DNA level can usually reach 35-48%, of which splicing-related variation accounts for 15-25%, that is, many sites cannot explain the individual's disease risk, and individuals with hereditary cancer risks are missed. With the maturity of RNA sequencing technology, on the basis of detecting germline variation at the DNA level, combined with RNA sequencing, it can provide functional evidence for the variation identified at the DNA level that may affect RNA splicing and expression, effectively reduce the VUS rate of variation that lacks functional explanation, improve the accuracy of variation interpretation, expand the identification of HCPS individuals, and increase the opportunity for personalized treatment and monitoring.

[0007] It is extremely important to design and apply HCPS detection tools that test multiple cancer types, multiple genes, and DNA / RNA. They can more comprehensively determine the possible cancer organs and discover more hidden or new susceptibility gene mutations, achieving tumor risk assessment accuracy that existing products cannot achieve. In turn, more reasonable and accurate early cancer screening, detection, and prevention plans can be formulated for individuals and family members to reduce the incidence of cancer. Therefore, the effective identification of HCPS with multiple cancer types and multiple genes and DNA / RNA can bring significant benefits to the risk identification of patients and their related relatives.

[0008] There are no similar products on the market in China. At present, there are some detection kits based on single genes and only targeting the DNA level, such as the human BRCA1 gene and BRCA2 gene mutation detection kit (reversible terminal termination sequencing method) of Xiamen Aide Biopharmaceutical Technology Co., Ltd. However, there are no approved detection kits for more hereditary tumors such as familial adenomatous polyposis (APC gene), neurofibromatosis (FN1, FN2), Li-Fraumeni syndrome (TP53), etc. This greatly limits the screening of hereditary tumors and the management of related high-risk populations, and cannot meet clinical needs. Summary of the invention

[0009] In order to solve the above technical problems, this application designs a set of DNA and RNA co-detection, hybrid capture combined with second-generation sequencing detection methods for all exon regions (CDS) of 79 genes related to HCPS and splicing-related regions of 20bp upstream and downstream. If the mutation type detected by DNA is a splicing-related variation, the RNA detection process will be further used; this method improves the accuracy of splicing-related variation interpretation on the basis of ensuring the accuracy of SNV / Indel detection, while taking into account the detection of CNV and LGR.

[0010] The present application provides a marker for detecting hereditary tumor susceptibility syndrome, and the marker includes the following genes and fragments thereof: APC, ARID1A, ATM, ATR, AXIN2, BAP1, BARD1, BLM, BMPR1A, BRCA1, BRCA2, BRIP1, CDH1, CDK12, CDKN1B, CDKN2A, CHEK1, CHEK2, DICER1, EMSY, EPCAM, ERCC3, FAM175A, FANCA, FANCC, FANCD2, FANCL, FANCM, FH, FLCN, GALNT12, GEN1, GREM1, HDAC2, HNF 1B, HOXB13, KDM5C, KDM6A, MCPH1, MEN1, MET, MITF, MLH1, MLH3, MRE11A, MSH2, MSH3, MSH6, MUTYH, NBN, NF1, NF2, PALB2, PBRM1, PMS1, PMS2, POLD1, PO LE, PPP2R2A, PTEN, RAD50, RAD51, RAD51B, RAD51C, RAD51D, RAD54L, RECQL, RET, SDHB, SDHC, SDHD, SMAD4, STK11, TP53, TSC1, TSC2, VHL, WT1, XRCC2.

[0011] The present application also provides a probe composition for detecting genetic mutations associated with hereditary tumor susceptibility syndrome, wherein the genes associated with hereditary tumor susceptibility syndrome include the following genes: APC, ARID1A, ATM, ATR, AXIN2, BAP1, BARD1, BLM, BMPR1A, BRCA1, BRCA2, BRIP1, CDH1, CDK12, CDKN1B, CDKN2A, CHEK1, CHEK2, DICER1, EMSY, EPCAM, ERCC3, FAM175A, FANCA, FANCC, FANCD2, FANCL, FANCM, FH, FLCN, GALNT12, GEN1, GREM1, H DAC2, HNF1B, HOXB13, KDM5C, KDM6A, MCPH1, MEN1, MET, MITF, MLH1, MLH3, MRE11A, MSH2, MSH3, MSH6, MUTYH, NBN, NF1, NF2, PALB2, PBRM1, PMS1, PMS2, POLD 1. POLE, PPP2R2A, PTEN, RAD50, RAD51, RAD51B, RAD51C, RAD51D, RAD54L, RECQL, RET, SDHB, SDHC, SDHD, SMAD4, STK11, TP53, TSC1, TSC2, VHL, WT1, XRCC2.

[0012] The present application also provides the use of the above-mentioned marker and probe combination in the preparation of early screening or diagnosis products for hereditary tumor susceptibility syndrome.

[0013] The present application also provides a kit for early screening or diagnosis of hereditary tumor susceptibility syndrome, wherein the kit comprises the above-mentioned probe composition.

[0014] The present application also provides a device for detecting mutations in hereditary tumor susceptibility syndromes, and the device for detecting mutations in genes related to hereditary tumor susceptibility syndromes includes the following modules: a sequencing module, a mutation information acquisition module, which is used to provide mutation information of target markers of samples to be tested, and the target markers are the above-mentioned markers for detecting hereditary tumor susceptibility syndromes; a mutation classification module, which is used to classify gene mutations based on the mutation information of target markers of samples to be tested and the functional data provided by RNA sequencing; a judgment module, which is used to judge whether the gene mutation is a positive mutation or a negative mutation based on the classification information; and a genetic interpretation module.

[0015] The present application also provides a computer-readable storage medium, which stores computer instructions. When the computer instructions are executed by a processor, a method for detecting gene mutations associated with hereditary tumor susceptibility syndrome is implemented, the method comprising: obtaining mutation information of a target marker of a sample to be tested and functional data provided by RNA sequencing, wherein the target marker is the above-mentioned marker for detecting hereditary tumor susceptibility syndrome; classifying gene mutations based on the mutation information of the target marker of the sample to be tested and the functional data provided by RNA sequencing; and judging whether the gene mutation is a positive mutation or a negative mutation based on the classification information.

[0016] The present application also provides an electronic terminal, which includes: a processor, a memory, an input / output interface and a communication port; the memory is used to store a computer program, and the processor is used to execute the computer program stored in the memory, so that the terminal executes the method for detecting genetic mutations related to hereditary tumor susceptibility syndrome as described in the above-mentioned computer-readable storage medium. The memory is used to store a computer program, and the processor is used to execute the computer program stored in the memory. This process realizes automatic splitting of high-throughput sequencing data, automatic analysis and obtaining of mutation information, and providing mutation interpretation information.

[0017] Compared with the prior art, the present invention has at least the following technical effects:

[0018] (1) The kit has a wide detection range and specifically covers 35 tumor-related susceptibility syndromes. It has more comprehensive clinical applications and has greater detection value for patients with similar phenotypes. Compared with whole genome sequencing, this kit focuses more on tumor susceptibility syndromes, does not have too many redundant results, and is more cost-effective. At the same time, it takes into account multiple variant types such as SNV, Indel, CNV at the exon level, and LGR related to gene inactivation or functional changes.

[0019] (2) This kit has excellent performance in practical applications and has been verified to have good performance in accuracy and repeatability. It can effectively detect pathogenic and potentially pathogenic variants in the target region.

[0020] (3) Supplementary RNA level detection can provide more accurate interpretation data of splicing-related variants, effectively reduce the number of sites of unknown significance, and reduce the missed diagnosis rate.

[0021] (4) Comprehensive detection of multiple genes combined with multiple phenotypes related to cancer can effectively reduce the missed detection rate and improve the accuracy of risk assessment. Testing patients with related tumor diseases and their first- and second-degree relatives can effectively screen out high-risk groups for tumors, assist clinical intervention as early as possible, formulate cancer screening, detection and prevention plans, and reduce the risk of disease in high-risk groups. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The present application will be further explained by way of exemplary embodiments, which will be described in detail with reference to the accompanying drawings.

[0023] Figure 1 The evaluation criteria are for judging the degree of benignity / pathogenicity based on the evidence shown in some embodiments of the present application.

[0024] Figure 2 This is a module diagram of a device for detecting hereditary tumor susceptibility syndrome-related variations according to some embodiments of the present application.

[0025] Figure 3 The present invention is a flowchart of a method for detecting mutations associated with hereditary tumor susceptibility syndrome according to some embodiments of the present application.

[0026] Figure 4 It is a schematic diagram of the architecture of the electronic terminal 400 according to some embodiments of the present application.

[0027] Figure 5 This is the system interface diagram for this application analysis.

[0028] Figure 6 The quality control information recorded in the report includes DNA quality and sequencing data quality display diagrams.

[0029] Figure 7 This is a graph showing the results of detecting mutation SNVs in the report.

[0030] Figure 8 Display graphs to explain the results in your report.

[0031] Fig. 9 The invention provides a basis for treating patients and cancer indicators return to normal values.

[0032] Fig.10 This is the genetic pedigree diagram of this group of example families.

[0033] Fig.11 This is a graph showing the copy number (large fragment rearrangement) detection results in the report.

[0034] Fig.12 This is a diagram for verifying the detection results of the present invention using the MLPA method.

[0035] Fig.13 IGV results showing functional data provided by RNA sequencing.

[0036] Fig.14 is the frequency of positive hereditary tumor susceptibility genes in the Chinese population. DETAILED DESCRIPTION

[0037] The present invention is further described below. The implementation cases introduced in this description are only exemplary and do not limit the scope of the present invention. It should be understood by those skilled in the art that without departing from the principles and methods of the present invention, the details and forms of the technical solution of the present invention may be partially modified or replaced, but such modification or replacement is within the scope of protection of the present invention.

[0038] As shown in this specification and claims, unless the context clearly indicates an exception, the words "a", "an", "an" and / or "the" do not refer to the singular and may also include the plural. Generally speaking, the terms "comprise" and "include" only indicate the inclusion of the steps and elements that have been clearly identified, and these steps and elements do not constitute an exclusive list. The method or device may also include other steps or elements.

[0039] Flowcharts are used in this specification to illustrate the operations performed by the system according to the embodiments of this specification. It should be understood that the preceding or following operations are not necessarily performed precisely in order. Instead, the steps may be processed in reverse order or simultaneously. At the same time, other operations may also be added to these processes, or one or more operations may be removed from these processes.

[0040] The present application provides a marker for detecting hereditary tumor susceptibility syndrome, and the marker includes the following genes and fragments thereof: APC, ARID1A, ATM, ATR, AXIN2, BAP1, BARD1, BLM, BMPR1A, BRCA1, BRCA2, BRIP1, CDH1, CDK12, CDKN1B, CDKN2A, CHEK1, CHEK2, DICER1, EMSY, EPCAM, ERCC3, FAM175A, FANCA, FANCC, FANCD2, FANCL, FANCM, FH, FLCN, GALNT12, GEN1, GREM1, HDAC2, HNF 1B, HOXB13, KDM5C, KDM6A, MCPH1, MEN1, MET, MITF, MLH1, MLH3, MRE11A, MSH2, MSH3, MSH6, MUTYH, NBN, NF1, NF2, PALB2, PBRM1, PMS1, PMS2, POLD1, PO LE, PPP2R2A, PTEN, RAD50, RAD51, RAD51B, RAD51C, RAD51D, RAD54L, RECQL, RET, SDHB, SDHC, SDHD, SMAD4, STK11, TP53, TSC1, TSC2, VHL, WT1, XRCC2.

[0041] In some embodiments, the marker may include all exon regions of the gene and splicing regions 20 bp upstream and downstream of the exon regions.

[0042] The present application also provides a probe composition for detecting genetic mutations associated with hereditary tumor susceptibility syndrome, wherein the genes associated with hereditary tumor susceptibility syndrome include the following genes: APC, ARID1A, ATM, ATR, AXIN2, BAP1, BARD1, BLM, BMPR1A, BRCA1, BRCA2, BRIP1, CDH1, CDK12, CDKN1B, CDKN2A, CHEK1, CHEK2, DICER1, EMSY, EPCAM, ERCC3, FAM175A, FANCA, FANCC, FANCD2, FANCL, FANCM, FH, FLCN, GALNT12, GEN1, GREM1, H DAC2, HNF1B, HOXB13, KDM5C, KDM6A, MCPH1, MEN1, MET, MITF, MLH1, MLH3, MRE11A, MSH2, MSH3, MSH6, MUTYH, NBN, NF1, NF2, PALB2, PBRM1, PMS1, PMS2, POLD 1. POLE, PPP2R2A, PTEN, RAD50, RAD51, RAD51B, RAD51C, RAD51D, RAD54L, RECQL, RET, SDHB, SDHC, SDHD, SMAD4, STK11, TP53, TSC1, TSC2, VHL, WT1, XRCC2.

[0043] The term "probe" refers to any molecule that can selectively bind to a target biomolecule (eg, a nucleic acid sequence that hybridizes to the probe). In some embodiments, the probe can be labeled, such as biotin labeling.

[0044] Each probe in the probe composition is designed for the exon region of a gene related to hereditary tumor susceptibility syndrome.

[0045] The reference genome is Hg19. "chr" and the following numbers (1-22, X, Y) represent specific chromosomes, "|" is an item separator symbol, and the numbers after "|" represent the base positions on the chromosome. Taking "chr1|17345344|17345464" as an example, it represents the base sequence between the 17345344th base to the 17345464th base on human chromosome 1. The probe composition can hybridize with genomic DNA, and after capturing and enriching the target region sequence, it can be sequenced using a sequencing platform with high throughput.

[0046] This application targets the CDS region of 79 genes related to HCPS and the target region related to splicing 20bp upstream and downstream, and designs hybridization capture probes by tiling method, that is, the designed probes are arranged along the target sequence on the genome to form a complete "tiled" coverage. The designed probe pool contains 2708 single-stranded DNA probes with a length of 120nt and carries a biotin marker. The probe is reverse complementary to the corresponding target region, binds to the target region by hybridization, and separates and enriches the target region sequence through the high affinity interaction between biotin-streptavidin, and performs high-throughput sequencing.

[0047] Mutation calling and annotation refers to the detection of gene mutation sites and mutation types, and annotating the clinical significance of mutation sites and mutation types according to the recognized interpretation standards and guidelines of gene mutations or the results predicted by the paradigmatic reverse time model. The recognized interpretation standards and guidelines of gene mutations are, for example, ACMG, etc.

[0048] The types of mutations mainly include SNV, Indel, CNV, and LGR. The clinical significance is divided into 5 categories: 5-pathogenic mutation, 4-likely pathogenic mutation, 3-unclear mutation, 2-likely benign mutation, and 1-benign mutation. Positive mutations mainly refer to pathogenic mutations and possibly pathogenic mutations. Negative mutations mainly refer to possibly benign mutations and benign mutations.

[0049] In some embodiments, the probe composition may include probes that hybridize to one or more of the following chromosomal loci:

[0050] chr1|17345344|17345464、chr1|17345364|17345484、chr1|17349103|17349223、chr1|17350458|17350578、chr1|17354241|17354361、chr1|17355102|17355222、chr1|17359527|17359647、chr1|17359547|17359667、chr1|17371259|17371379、chr1|17380418|17380538、chr1|27022874|27022994、chr1|27022991|27023111、chr1|27023108|27023228、chr1|27023226|27023346、chr1|27023343|27023463、chr1|27023461|27023581、chr1|27023578|27023698、chr1|27023696|27023816、chr1|27023813|27023933、chr1|27023930|27024050、chr1|27056128|27056248、chr1|27056247|27056367、chr1|27057629|27057749、chr1|27057748|27057868、chr1|27057868|27057988、chr1|27057988|27058108、chr1|27059164|27059284、chr1|27087346|27087466、chr1|27087467|27087587、chr1|27087859|27087979、chr1|27088622|27088742、chr1|27088710|27088830、chr1|27089443|27089563、chr1|27089559|27089679、chr1|27089676|27089796、chr1|27092691|27092811、chr1|27092757|27092877、chr1|27092942|27093062、chr1|27094265|27094385、chr1|27094385|27094505、chr1|27097593|27097713、chr1|27097713|27097833、chr1|27098996|27099116、chr1|27099282|27099402、chr1|27099378|27099498、chr1|27099816|27099936、chr1|27099887|27100007、chr1|27100079|27100199、chr1|27100280|27100400、chr1|27100799|27100919、chr1|27100915|27101035、chr1|27101031|27101151、chr1|27101147|27101267、chr1|27101263|27101383、chr1|27101379|27101499、chr1|27101495|27101615、chr1|27101611|27101731、chr1|27102072|27102192、chr1|27105493|27105613、chr1|27105611|27105731、chr1|27105729|27105849、chr1|27105847|27105967、chr1|27105965|27106085、chr1|27106083|27106203、chr1|27106201|27106321、chr1|27106319|27106439、chr1|27106438|27106558、chr1|27106556|27106676、chr1|27106674|27106794、chr1|27106792|27106912、chr1|27106910|27107030、chr1|27107028|27107148、chr1|27107146|27107266、chr1|45794983|45795103、chr1|45796148|45796268、chr1|45796833|45796953、chr1|45796906|45797026、chr1|45797099|45797219、chr1|45797312|45797432、chr1|45797421|45797541、chr1|45797666|45797786、chr1|45797817|45797937、chr1|45797882|45798002、chr1|45798051|45798171、chr1|45798242|45798362、chr1|45798410|45798530、chr1|45798550|45798670、chr1|45798745|45798865、chr1|45798916|45799036、chr1|45799064|45799184、chr1|45799175|45799295、chr1|4580006245800182、chr1|45805873|45805993、chr1|46714018|46714138、chr1|46714166|46714286、chr1|46715671|46715791、chr1|46724317|46724437、chr1|46724337|46724457、chr1|46725643|46725763、chr1|46726188|46726308、chr1|46726378|46726498、chr1|46726482|46726602、chr1|46726587|46726707、chr1|46726934|46727054、chr1|46733110|46733230、chr1|46733181|46733301、chr1|46736333|46736453、chr1|46738114|46738234、chr1|46738348|46738468、chr1|46739021|46739141、chr1|46739297|46739417、chr1|46739788|46739908、chr1|46740189|46740309、chr1|46740289|46740409、chr1|46743468|46743588、chr1|46743552|46743672、chr1|46743729|46743849、chr1|46743848|46743968、chr1|161284145|161284265、chr1|161293371|161293491、chr1|161298176|161298296、chr1|161310354|161310474、chr1|161326446|161326566、chr1|161326530|161326650、chr1|161332104|161332224、chr1|161332224|161332344、chr1|241661107|241661227、chr1|241661170|241661290、chr1|241663716|241663836、chr1|241663790|241663910、chr1|241665746|241665866、chr1|241667323|241667443、chr1|241667443|241667563、chr1|241669282|241669402、chr1|241669368|241669488、chr1|241671882|241672002、chr1|241671985|241672105、chr1|241675246|241675366、chr1|241675343|241675463、chr1|241676897|241677017、chr1|241680488|241680608、chr1|241682896|241683016、chr10|43572682|43572802、chr10|43595886|43596006、chr10|43595978|43596098、chr10|43596070|43596190、chr10|43597769|43597889、chr10|43597873|43597993、chr10|43597977|43598097、chr10|43600399|43600519、chr10|43600521|43600641、chr10|43601803|43601923、chr10|43601919|43602039、chr10|43604458|43604578、chr10|43604578|43604698、chr10|43606654|43606774、chr10|43606793|43606913、chr10|43607549|43607669、chr10|43608295|43608415、chr10|43609003|43609123、chr10|43609927|43610047、chr10|43610064|43610184、chr10|43612011|43612131、chr10|43612079|43612199、chr10|43613814|43613934、chr10|43614966|43615086、chr10|43615085|43615205、chr10|43615529|43615649、chr10|43617368|43617488、chr10|43619127|43619247、chr10|43620320|43620440、chr10|43622002|43622122、chr10|43622102|43622222、chr10|43623539|43623659、chr10|43623617|43623737、chr10|88635738|88635858、chr10|88635758|88635878、chr10|88649798|88649918、chr10|88649881|88650001、chr10|88651863|88651983、chr10|88651886|88652006、chr10|88659538|88659658、chr10|88659773|88659893、chr10|88671976|88672096、chr10|88672041|88672161、chr10|88676870|88676990、chr10|88676983|88677103、chr10|88678908|88679028、chr10|88679017|88679137、chr10|88679126|88679246、chr10|88681256|88681376、chr10|88681352|88681472、chr10|88683137|88683257、chr10|88683353|88683473、chr10|89623706|89623826、chr10|89623825|89623945、chr10|89623945|89624065、chr10|89624065|89624185、chr10|89624185|89624305、chr10|89653753|89653873、chr10|89653773|89653893、chr10|89685221|89685341、chr10|89685241|89685361、chr10|89690754|89690874、chr10|89690774|89690894、chr10|89692769|89692889、chr10|89692888|89693008、chr10|89711854|89711974、chr10|89711916|89712036、chr10|89717589|89717709、chr10|89717676|89717796、chr10|89720643|89720763、chr10|8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[0051] 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style=";text-align:left;direction:ltr">chr13|32905067|32905187、chr13|32906388|32906508、chr13|3290650 3|32906623、chr13|32906618|32906738、chr13|32906733|32906853、chr 13|32906848|32906968、chr13|32906963|32907083、chr13|32907078|32907198、chr13|32907193|32907313、chr13|32907308|32907428、chr13| 32907424|32907544,chr13|32910401|32910521,chr13|32910521|32910641,chr13|32910641|32910761,chr13|32910761|32910881,chr13|329 10882|32911002,chr13|32911002|32911122,chr13|32911122|32911242,chr13|32911243|32911363,chr13|32911363|32911483,chr13|329114 83|32911603、chr13|32911603|32911723、chr13|32911724|32911844、chr13|32911844|32911964、chr13|32911964|32912084、chr13|32912085| 32912205,chr13|32912205|32912325,chr13|32912325|32912445,chr13|32912446|32912566,chr13|32912566|32912686,chr13|32912686|329 12806,chr13|32912806|32912926,chr13|32912927|32913047,chr13|32913047|32913167,chr13|32913167|32913287,chr13|32913288|329134 08, chr13|32913408|32913528, chr13|32913528|32913648, chr13|32913649|32913769, chr13|32913769|32913889, chr13|32913889|32914009,<h2 style=";text-align:left;direction:ltr">chr13|32914009|32914129、chr13|32914130|32914250、chr13|3291425 0|32914370、chr13|32914370|32914490、chr13|32914491|32914611、chr 13|32914611|32914731、chr13|32914731|32914851、chr13|32914852|32914972、chr13|32914972|32915092、chr13|32915092|32915212、chr13| 32915212|32915332,chr13|32918672|32918792,chr13|32918692|32918812,chr13|32920928|32921048,chr13|32920948|32921068,chr13|329 28977|32929097,chr13|32929093|32929213,chr13|32929209|32929329,chr13|32929325|32929445,chr13|32930544|32930664,chr13|329306 46|32930766,chr13|32931858|32931978,chr13|32931966|32932086,chr13|32936639|32936759,chr13|32936730|32936850,chr13|32937313| 32937433,chr13|32937432|32937552,chr13|32937552|32937672,chr13|32944518|32944638,chr13|32944594|32944714,chr13|32945072|329 45192,chr13|32945137|32945257,chr13|32950807|32950927,chr13|32953433|32953553,chr13|32953552|32953672,chr13|32953866|329539 86, chr13|32953950|32954070, chr13|32954152|32954272, chr13|32968825|32968945, chr13|32968950|32969070, chr13|32971014|32971134,chr13|32971081|32971201, chr13|32972298|32972418, chr13|3297242 0|32972540, chr13|32972542|32972662, chr13|32972664|32972784, chr 13|32972787|32972907, chr14|45605214|45605334, chr14|45605321|4 5605441, chr14|45605428|45605548, chr14|45605535|45605655, chr14| 45605642|45605762、chr14|45606251|45606371、chr14|45606344|45606464、chr14|45609803|45609923、chr14|45609823|45609943、chr14|45618019|45618139、chr14|45618098|45618218、chr14|45620579|45620699、chr14|45620631|45620751、chr14|45623102|45623222、chr14|456231 55|45623275, chr14|45623879|45623999, chr14|45623925|45624045, chr14|45624548|45624668, chr14|45624568|45624688, chr14|45628278|45628398, chr14|45628383|45628503, chr14|45633545|45633665, chr14|45633664|45633784, chr14|45636143|45636263, chr14|45636263|456 36383, chr14|45639771|45639891, chr14|45639849|45639969, chr14|45642237|45642357, chr14|45642313|45642433, chr14|45644266|45644386, chr14|45644386|45644506, chr14|45644506|45644626, chr14|45644626|45644746, chr14|45644746|45644866, chr14|45644866|45644986,chr14|45644986|45645106、chr14|45645106|45645226、chr14|45645226|45645346、chr14|45645346|45645466、chr14|45645466|45645586、chr14|45645586|45645706、chr14|45645706|45645826、chr14|45645826|45645946、chr14|45645946|45646066、chr14|45646066|45646186、chr14| 45650609|45650729, chr14|45650629|45650749, chr14|45650803|45650923, chr14|45650823|45650943, chr14|45652980|45653100, chr14|45654399|45654519, chr14|45654476|45654596, chr14|45656963|45657083, chr14|45656990|45657110, chr14|45657985|45658105, chr14|456581 04|45658224, chr14|45658224|45658344, chr14|45658344|45658464, chr14|45658464|45658584, chr14|45665374|45665494, chr14|45665502|45665622, chr14|45665630|45665750, chr14|45667826|45667946, chr14|45667932|45668052, chr14|45668038|45668158, chr14|45669052|456 69172, chr14|45669111|45669231, chr14|68290242|68290362, chr14|68292177|68292297, chr14|68301794|68301914, chr14|68331727|68331847, chr14|68352565|68352685, chr14|68352605|68352725, chr14|68353717|68353837, chr14|68353821|68353941, chr14|68758588|68758708,chr14|68878132|68878252、chr14|68934867|68934987、chr14|68937184|68937304、chr14|68944302|68944422、chr14|68944322|68944442、chr14|68963778|68963898、chr14|68963798|68963918、chr14|69006856|69006976、chr14|69006876|69006996、chr14|69069323|69069443、chr14| 69077722|69077842、chr14|69077844|69077964、chr14|69149587|69149707、chr14|69149607|69149727、chr14|75483784|75483904、chr14|75485511|75485631、chr14|75485583|75485703、chr14|75489495|75489615、chr14|75489639|75489759、chr14|75489659|75489779、chr14|754972 25|75497345, chr14|75497305|75497425, chr14|75498766|75498886, chr14|75500097|75500217, chr14|75505018|75505138, chr14|75506605|75506725, chr14|75508300|75508420, chr14|75509070|75509190, chr14|75513058|75513178, chr14|75513176|75513296, chr14|75513295|755 13415, chr14|75513413|75513533, chr14|75513532|75513652, chr14|75513650|75513770, chr14|75513769|75513889, chr14|75513887|75514007, chr14|75514006|75514126, chr14|75514124|75514244, chr14|75514243|75514363, chr14|75514361|75514481, chr14|75514480|75514600,chr14|75514598|75514718、chr14|75514717|75514837、chr14|75514835|75514955、chr14|75514954|75515074、chr14|75515072|75515192、chr14|75515191|75515311、chr14|75515309|75515429、chr14|75515428|75515548、chr14|75515546|75515666、chr14|75515665|75515785、chr14| 75515783|75515903, chr14|75515902|75516022, chr14|75516020|75516140, chr14|75516139|75516259, chr14|75516258|75516378, chr14|95556814|95556934, chr14|95556900|95557020, chr14|95557338|95557458, chr14|95557358|95557478, chr14|95557519|95557639, chr14|955576 02|95557722、chr14|95560204|95560324、chr14|95560298|95560418、chr14|95560393|95560513、chr14|95562141|95562261、chr14|95562256|95562376、chr14|95562372|95562492、chr14|95562487|95562607、chr14|95562603|95562723、chr14|95562718|95562838、chr14|95562834|955 62954, chr14|95562949|95563069, chr14|95566096|95566216, chr14|95566172|95566292, chr14|95569662|95569782, chr14|95569778|95569898, chr14|95569895|95570015, chr14|95570012|95570132, chr14|95570129|95570249, chr14|95570246|95570366, chr14|95570362|95570482,chr14|95571387|95571507、chr14|95571483|95571603、chr14|95572007|95572127、chr14|95572357|95572477、chr14|95572460|95572580、chr 14|95573924|95574044、chr14|95573998|95574118、chr14|95574203|9 5574323、chr14|95574323|95574443、chr14|95574640|95574760、chr14| 95574740|95574860、chr14|95577663|95577783、chr14|95578476|9557 8596、chr14|95578496|95578616、chr14|95579434|95579554、chr14|955 81983|95582103、chr14|95582058|95582178、chr14|95582789|9558290 9、chr14|95582912|95583032、chr14|95583964|95584084、chr14|955905 29|95590649、chr14|95590648|95590768、chr14|95590768|95590888、c hr14|95590888|95591008、chr14|95592896|95593016、chr14|95592985| 95593105、chr14|95595788|95595908、chr14|95595869|95595989、chr1 4|95596374|95596494、chr14|95596429|95596549、chr14|95597850|955 97970、chr14|95598831|95598951、chr14|95598914|95599034、chr14|9 5599631|95599751、chr14|95599695|95599815、chr15|33022871|330229 91、chr15|33022989|33023109、chr15|33023108|33023228、chr15|3302 3227|33023347、chr15|33023346|33023466、chr15|40990939|40991059、chr15|40993270|40993390、chr15|40998373|40998493、chr15|41001208|41001328、chr15|41010989|41011109、chr15|41020905|41021025、chr 15|41021707|41021827、chr15|41022051|41022171、chr15|41023254|41023374、chr15|91290601|91290721、chr15|91290621|91290741、chr15| 91292587|91292707、chr15|91292706|91292826、chr15|91292826|91292946、chr15|91292946|91293066、chr15|91293066|91293186、chr15|912 93185|91293305、chr15|91294996|91295116、chr15|91295076|91295196、chr15|91298044|91298164、chr15|91303356|91303476、chr15|913034 09|91303529、chr15|91303803|91303923、chr15|91303919|91304039、chr15|91304035|91304155、chr15|91304152|91304272、chr15|91304268| 91304388, chr15|91304385|91304505, chr15|91306175|91306295, chr15|91306287|91306407, chr15|91308524|91308644, chr15|91310119|913 10239, chr15|91310153|91310273, chr15|91312351|91312471, chr15|91312647|91312767, chr15|91312716|91312836, chr15|91326031|913261 51, chr15|91326058|91326178, chr15|91328130|91328250, chr15|91328211|91328331, chr15|91333858|91333978, chr15|91333974|91334094,chr15|91337376|91337496、chr15|91337487|91337607、chr15|91341399 |91341519、chr15|91341467|91341587、chr15|91346730|91346850、chr1 5|91346850|91346970、chr15|91347376|91347496、chr15|91347489|913 47609、chr15|91352346|91352466、chr15|91352389|91352509、chr15|913 54415|91354535、chr15|91354535|91354655、chr15|91358311|91358431、chr15|91358409|91358529、chr16|2098212|2098332、chr16|2098567|2 098687、chr16|2098654|2098774、chr16|2100383|2100503、chr16|2103337|2103457、chr16|2104276|2104396、chr16|2104341|2104461、chr16|21 05401|2105521、chr16|2106160|2106280、chr16|2106647|2106767、chr16|2107082|2107202、chr16|2108750|2108870、chr16|2110650|2110770、 chr16|2110714|2110834、chr16|2111880|2112000、chr16|2112489|2112609、chr16|2112953|2113073、chr16|2114252|2114372、chr16|2114328|2 114448、chr16|2115517|2115637、chr16|2120457|2120577、chr16|2121503|2121623、chr16|2121764|2121884、chr16|2121835|2121955、chr16|2 122242|2122362、chr16|2122856|2122976、chr16|2124180|2124300、chr16|2124290|2124410、chr16|2125786|2125906、chr16|2126059|2126179、chr16|2126478|2126598、chr16|2127602|2127722、chr16|2129012|2129132、chr16|2129097|2129217、chr16|2129256|2129376、chr16|212932 9|2129449、chr16|2129553|2129673、chr16|2130152|2130272、chr16|2 130271|2130391、chr16|2131577|2131697、chr16|2131697|2131817、ch r16|2132410|2132530、chr16|2133696|2133816、chr16|2134228|2134348、chr16|2134350|2134470、chr16|2134473|2134593、chr16|2134596| 2134716、chr16|2134929|2135049、chr16|2135216|2135336、chr16|2136173|2136293、chr16|2136280|2136400、chr16|2136742|2136862、chr16 |2137842|2137962、chr16|2138034|2138154、chr16|2138216|2138336、chr16|2138426|2138546、chr16|2138511|2138631、chr16|23614765|23 614885、chr16|23614884|23615004、chr16|23619164|23619284、chr16|23619233|23619353、chr16|23625298|23625418、chr16|23625318|2362 5438、chr16|23632680|23632800、chr16|23634269|23634389、chr16|23634351|23634471、chr16|23635302|23635422、chr16|23635322|236354 42、chr16|23637536|23637656、chr16|23637618|23637738、chr16|23640500|23640620、chr16|23640955|23641075、chr16|23641075|23641195、chr16|23641195|23641315、chr16|23641315|23641435、chr16|2364143 5|23641555、chr16|23641555|23641675、chr16|23641675|23641795、chr 16|23646162|23646282、chr16|23646278|23646398、chr16|23646394|23646514、chr16|23646510|23646630、chr16|23646626|23646746、chr16| 23646742|23646862、chr16|23646858|23646978、chr16|23646974|23647094、chr16|23647090|23647210、chr16|23647206|23647326、chr16|236 47322|23647442、chr16|23647438|23647558、chr16|23647554|23647674、chr16|23649161|23649281、chr16|23649350|23649470、chr16|236493 70|23649490、chr16|23652394|23652514、chr16|68771282|68771402、chr16|68772196|68772316、chr16|68835564|68835684、chr16|68835684| 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7962846|17962966、chr2|17962966|17963086、chr2|17963086|17963206、chr2|47596622|47596742、chr2|47600595|47600715、chr2|47600946|47601066、chr2|47601067|47601187、chr2|47602335|47602455、chr2|47602355|47602475、chr2|47604114|47604234、chr2|47604134|47604254、chr2|47606071|47606191、chr2|47606093|47606213、chr2|47606888|47607008、chr2|47607007|47607127、chr2|47612256|47612376、chr2|47612276|47612396、chr2|47613661|47613781、chr2|47613681|47613801、chr2|47630316|47630436、chr2|47630435|47630555、chr2|47635519|47635639、chr2|47635594|47635714、chr2|47637212|47637332、chr2|47637311|47637431、chr2|47637411|47637531、chr2|47639532|47639652、chr2|47639599|47639719、chr2|47641387|47641507、chr2|47641457|47641577、chr2|47643441|47643561、chr2|47656860|47656980、chr2|47656980|47657100、chr2|47672666|47672786、chr2|47672696|47672816、chr2|47690149|47690269、chr2|47690193|47690313、chr2|47693776|47693896、chr2|47693847|47693967、chr2|47698082|47698202、chr2|47698102|47698222、chr2|47702163|47702283、chr2|47702289|47702409、chr2|47703488|47703608、chr2|47703607|47703727、chr2|47705410|47705530、chr2|47705538|47705658、chr2|47707814|47707934、chr2|47707910|47708030、chr2|47709897|47710017、chr2|47709988|47710108、chr2|48010372|48010492、chr2|48010512|48010632、chr2|48018045|48018165、chr2|48018162|48018282、chr2|48023012|48023132、chr2|48023102|48023222、chr2|48025729|48025849、chr2|48025846|48025966、chr2|48025963|48026083、chr2|48026081|48026201、chr2|48026198|48026318、chr2|48026315|48026435、chr2|48026433|48026553、chr2|48026550|48026670、chr2|48026668|48026788、chr2|48026785|48026905、chr2|48026902|48027022、chr2|48027020|48027140、chr2|48027137|48027257、chr2|48027254|48027374、chr2|48027372|48027492、chr2|48027489|48027609、chr2|48027607|48027727、chr2|48027724|48027844、chr2|48027841|48027961、chr2|48027959|48028079、chr2|48028076|48028196、chr2|48028193|48028313、chr2|48030538|48030658、chr2|48030631|48030751、chr2|48030724|48030844、chr2|48032047|48032167、chr2|48032731|48032851、chr2|48032751|48032871、chr2|48033322|48033442、chr2|48033397|48033517、chr2|48033570|48033690、chr2|48033690|48033810、chr2|48033888|48034008、chr2|48033908|48034028、chr2|58386847|58386967、chr2|58386867|58386987、chr2|58387208|58387328、chr2|58387228|58387348、chr2|58388636|58388756、chr2|58388673|58388793、chr2|58389971|58390091、chr2|58389991|58390111、chr2|58390116|58390236、chr2|58390136|58390256、chr2|58390540|58390660、chr2|58390560|58390680、chr2|58392838|58392958、chr2|58392909|58393029、chr2|58421315|58421435、chr2|58421335|58421455、chr2|58425685|58425805、chr2|58425705|58425825、chr2|58431252|58431372、chr2|58449066|58449186、chr2|58453820|58453940、chr2|58453840|58453960、chr2|58456908|58457028、chr2|58456928|58457048、chr2|58459147|58459267、chr2|58459167|58459287、chr2|58468340|58468460、chr2|128015177|128015297、chr2|128016851|128016971、chr2|128016924|128017044、chr2|128018802|128018922、chr2|128028910|128029030、chr2|128030428|128030548、chr2|128036730|128036850、chr2|128036849|128036969、chr2|128038002|128038122、chr2|128038107|128038227、chr2|128044258|128044378、chr2|128044375|128044495、chr2|128044493|128044613、chr2|128046218|128046338、chr2|128046337|128046457、chr2|128046892|128047012、chr2|128046977|128047097、chr2|128047272|128047392、chr2|128047764|128047884、chr2|128050184|128050304、chr2|128050303|128050423、chr2|128051071|128051191、chr2|128051191|128051311、chr2|128051583|128051703、chr2|190656541|190656661、chr2|190660474|190660594、chr2|190660577|190660697、chr2|190670373|190670493、chr2|190670492|190670612、chr2|190682722|190682842、chr2|190682806|190682926、chr2|190708687|190708807、chr2|190717360|190717480、chr2|190717403|190717523、chr2|190718644|190718764、chr2|190718708|190718828、chr2|190718944|190719064、chr2|190719059|190719179、chr2|190719175|190719295、chr2|190719291|190719411、chr2|190719406|190719526、chr2|190719522|190719642、chr2|190719638|190719758、chr2|190719753|190719873、chr2|190728468|190728588、chr2|190728590|190728710、chr2|190728712|190728832、chr2|190728834|190728954、chr2|190732504|190732624、chr2|190732555|190732675、chr2|190738201|190738321、chr2|190738282|190738402、chr2|190741977|190742097、<h2 style=";text-align:left;direction:ltr">chr2|190742062|190742182、chr2|215593386|215593506、chr2|215593 505|215593625、chr2|215593625|215593745、chr2|215595123|21559524 3、chr2|215609766|215609886、chr2|215609786|215609906、chr2|2156 10451|215610571、chr2|215617164|215617284、chr2|215632185|215632 305、chr2|215632278|215632398、chr2|215633925|215634045、chr2|21 5633945|215634065、chr2|215645278|215645398、chr2|215645398|2156 45518、chr2|215645518|215645638、chr2|215645638|215645758、chr2|2 15645758|215645878、chr2|215645878|215645998、chr2|215645998|215 646118、chr2|215646118|215646238、chr2|215657000|215657120、chr2 |215657069|215657189、chr2|215661742|215661862、chr2|215661762|2 15661882,chr2|215674115|215674235,chr2|215674193|215674313,chr22|29083869|29083989,chr22|29085102|29085222,chr22|29090002|29 090122,chr22|29091112|29091232,chr22|29091677|29091797,chr22|29091761|29091881,chr22|29092861|29092981,chr22|29092881|29093 001,chr22|29095815|29095935,chr22|29099453|29099573,chr22|29099473|29099593,chr22|29105950|29106070,chr22|29105970|29106090,<h2 style=";text-align:left;direction:ltr">chr22|29107890|29108010,chr22|29115357|29115477,chr22|29115377|29115497,chr22|29120873|29120993,chr22|29120992|29121112,chr2 2|29121210|29121330,chr22|29121255|29121375,chr22|29126411|29126531,chr22|29130370|29130490,chr22|29130489|29130609,chr22|291 30609|29130729,chr22|29999984|30000104,chr22|30032742|30032862,chr22|30035079|30035199,chr22|30038172|30038292,chr22|3005061 9|30050739,chr22|30051563|30051683,chr22|30054155|30054275,chr22|3005720030057320,chr22|30060945|30061065,chr22|30060965|3006 1085,chr22|30064318|30064438,chr22|30067815|30067935,chr22|30069246|30069366,chr22|30069366|30069486,chr22|30070817|30070937 chr22|30074188|30074308,chr22|30077407|30077527,chr22|30077490|30077610,chr22|30078966|30079086,chr22|30090705|30090825,chr3 |10070303|10070423、chr3|10070323|10070443、chr3|10074495|100746 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4384626|84384746、chr4|84384661|84384781、chr4|84388578|84388698 、chr4|84388598|84388718、chr4|84390184|84390304、chr4|84391335|8 4391455、chr4|84391449|84391569、chr4|84393337|84393457、chr4|843 93357|84393477、chr4|84397743|84397863、chr4|84397763|84397883、c hr4|84403281|84403401、chr4|84403301|84403421、chr4|84406121|844 06241、chr5|79950545|79950665、chr5|79950664|79950784、chr5|79952 229|79952349、chr5|79960952|79961072、chr5|79961071|79961191、chr 5|79965902|79966022、chr5|79966021|79966141、chr5|79968060|79968 180、chr5|79968558|79968678、chr5|79970781|79970901、chr5|7997084 7|79970967、chr5|79974725|79974845、chr5|79974812|79974932、chr5| 80021251|80021371、chr5|80021284|80021404、chr5|80024649|8002476 9、chr5|80024684|80024804、chr5|80037254|80037374、chr5|80037274| 80037394、chr5|80040319|80040439、chr5|80057370|80057490、chr5|80 063731|80063851、chr5|80063839|80063959、chr5|80064633|80064753、<h2 style=";text-align:left;direction:ltr">chr5|80064722|80064842,chr5|80071474|80071594,chr5|80071494|80071614,chr5|80074536|80074656,chr5|80083377|80083497,chr5|8008 8531|80088651,chr5|80088563|80088683,chr5|80109382|80109502,chr5|80109460|80109580,chr5|80149928|80150048,chr5|80150035|8015 0155、chr5|80160636|80160756、chr5|80168914|80169034、chr5|801690 06|80169126、chr5|80171565|80171685、chr5|112043394|112043514、ch r5|112043479|112043599,chr5|112074067|112074187,chr5|112090567|112090687,chr5|112090622|112090742,chr5|112101994|112102114,ch r5|112102014|112102134,chr5|112102866|112102986,chr5|112102986|112103106,chr5|112111305|112111425,chr5|112111334|112111454,c hr5|112116483|112116603,chr5|112128114|112128234,chr5|112128134|112128254,chr5|112136967|112137087,chr5|112151180|112151300,c hr5|112154662|112154782、chr5|112154791|112154911、chr5|11215492 1|112155041、chr5|112157570|112157690、chr5|112157590|112157710、 chr5|112158970|112159090,chr5|112162814|112162934,chr5|112163594|112163714,chr5|112163614|112163734,chr5|112164532|112164652,<h2 style=";text-align:left;direction:ltr">chr5|112164569|112164689,chr5|112170635|112170755,chr5|112170754|112170874,chr5|112173236|112173356,chr5|112173356|112173476, chr5|112173476|112173596,chr5|112173596|112173716,chr5|112173716|112173836,chr5|112173836|112173956,chr5|112173956|112174076, chr5|112174076|112174196、chr5|112174196|112174316、chr5|112174316|112174436、chr5|112174436|112174556、chr5|112174556|112174676、 chr5|112174676|112174796,chr5|112174796|112174916,chr5|112174916|112175036,chr5|112175036|112175156,chr5|112175156|112175276, chr5|112175276|112175396、chr5|112175396|112175516、chr5|112175516|112175636、chr5|112175636|112175756、chr5|112175756|112175876、 chr5|112175876|112175996、chr5|112175996|112176116、chr5|112176116|112176236、chr5|112176236|112176356、chr5|112176356|112176476、 chr5|112176476|112176596、chr5|112176596|112176716、chr5|112176716|112176836、chr5|112176836|112176956、chr5|112176956|112177076、 chr5|112177076|112177196、chr5|112177196|112177316、chr5|112177316|112177436、chr5|112177436|112177556、chr5|112177556|112177676、<h2 style=";text-align:left;direction:ltr">chr5|112177676|112177796,chr5|112177796|112177916,chr5|112177916|112178036,chr5|112178036|112178156,chr5|112178156|112178276, chr5|112178276|112178396,chr5|112178396|112178516,chr5|112178516|112178636,chr5|112178636|112178756,chr5|112178756|112178876, chr5|112178876|112178996、chr5|112178996|112179116、chr5|112179116|112179236、chr5|112179236|112179356、chr5|112179356|112179476、 chr5|112179476|112179596,chr5|112179596|112179716,chr5|112179716|112179836,chr5|131893020|131893140,chr5|131894947|131895067, chr5|131894967|131895087,chr5|131911448|131911568,chr5|131911520|131911640,chr5|131914988|131915108,chr5|131915094|131915214, chr5|131915536|131915656,chr5|131915655|131915775,chr5|131923233|131923353,chr5|131923282|131923402,chr5|131923595|131923715, chr5|131923681|131923801,chr5|131924358|131924478,chr5|131924472|131924592,chr5|131925306|131925426,chr5|131925425|131925545, chr5|131926895|131927015,chr5|131926998|131927118,chr5|131927548|131927668,chr5|131927626|131927746,chr5|131930540|131930660,chr5|131930636|131930756、chr5|131931263|131931383、chr5|1319313 83|131931503、chr5|131938971|131939091、chr5|131939081|131939201、 chr5|131939591|131939711、chr5|131939638|131939758、chr5|1319404 77|131940597、chr5|131940591|131940711、chr5|131944286|131944406、 chr5|131944317|131944437、chr5|131944784|131944904、chr5|1319448 04|131944924、chr5|131944954|131945074、chr5|131944988|131945108、 chr5|131951674|131951794、chr5|131951722|131951842、chr5|1319537 54|131953874、chr5|131953873|131953993、chr5|131972779|131972899、 chr5|131972799|131972919, chr5|131973752|131973872, chr5|131973815|131973935, chr5|131976370|131976490, chr5|131977849|131977969, chr5|131977956|131978076, chr6|114262166|114262286, chr6|114262186|114262306, chr6|114262830|114262950, ​​chr6|114262850|114262970, chr6|114264494|114264614, chr6|114264570|114264690, chr6|114265448|114265568, chr6|114266505|114266625, chr6|114266534|114266654, chr6|114267151|114267271, chr6|114267212|114267332, chr6|114270136|114270256, chr6|114270307|114270427, chr6|114270327|114270447,<h2 style=";text-align:left;direction:ltr">chr6|114274420|114274540、chr6|114274482|114274602、chr6|114277 185|114277305、chr6|114277759|114277879、chr6|114279811|1142799 31、chr6|114281049|114281169、chr6|114281082|114281202、chr6|114 291986|114292106、chr7|6013009|6013129、chr7|6013073|6013193、chr 7|6017200|6017320,chr7|6017319|6017439,chr7|6018206|6018326,chr7|6018227|6018347,chr7|6022434|6022554,chr7|6022522|6022642 chr7|6026369|6026489,chr7|6026480|6026600,chr7|6026592|6026712,chr7|6026704|6026824,chr7|6026815|6026935,chr7|6026927|6027 047,chr7|6027039|6027159,chr7|6027150|6027270,chr7|6029410|6029530,chr7|6029486|6029606,chr7|6031585|6031705,chr7|6035154| 6035274,chr7|6036945|6037065,chr7|6038718|6038838,chr7|6038806|6038926,chr7|6042077|6042197,chr7|6042197|6042317,chr7|60433 11|6043431,chr7|6043575|6043695,chr7|6043595|6043715,chr7|6045532|6045652,chr7|6048578|6048698,chr7|116339138|116339258,ch r7|116339258|116339378,chr7|116339378|116339498,chr7|116339498|116339618,chr7|116339618|116339738,chr7|116339738|116339858,<h2 style=";text-align:left;direction:ltr">chr7|116339858|116339978,chr7|116339978|116340098,chr7|116340098|116340218,chr7|116340218|116340338,chr7|116371701|116371821, chr7|116371813|116371933,chr7|116380010|116380130,chr7|116380885|116381005,chr7|116380979|116381099,chr7|116395388|116395508, chr7|116395469|116395589,chr7|116397470|116397590,chr7|116397493|116397613,chr7|116397671|116397791,chr7|116397728|116397848, chr7|116398492|116398612,chr7|116398574|116398694,chr7|116399370|116399490,chr7|116399444|116399564,chr7|116403093|116403213, chr7|116403212|116403332,chr7|116409689|116409809,chr7|116409809|116409929,chr7|116411531|116411651,chr7|116411608|116411728, chr7|116411882|116412002,chr7|116411943|116412063,chr7|116414930|116415050,chr7|116415049|116415169,chr7|116417412|116417532, chr7|116417432|116417552,chr7|116418809|116418929,chr7|116418911|116419031,chr7|116422021|116422141,chr7|116422051|116422171, chr7|116423337|116423457,chr7|116423423|116423543,chr7|116435716|116435836,chr7|116435939|116436059,chr7|116436059|116436179,<h2 style=";text-align:left;direction:ltr">chr7|152345726|152345846,chr7|152345846|152345966,chr7|152345966|152346086,chr7|152346087|152346207,chr7|152346207|15234632 7, chr7|152346328|152346448, chr7|152357756|152357876, chr7|152357776|152357896, chr7|152373084|152373204, chr8|6264139|6264259 ...96 hr8|6266775|6266895、chr8|6266795|6266915、chr8|6272284|6272404 、chr8|6288993|6289113、chr8|6289013|6289133、chr8|6293548|629366 8、chr8|6293583|6293703、chr8|6296453|6296573、chr8|6296517|62966 37、chr8|6299562|6299682、chr8|6299582|6299702、chr8|6301895|6302 015、chr8|6302014|6302134、chr8|6302134|6302254、chr8|6302254|63 02374、chr8|6302374|6302494、chr8|6302494|6302614、chr8|6302614|6 302734、chr8|6302734|6302854、chr8|6302854|6302974、chr8|6302973| 6303093、chr8|6312658|6312778、chr8|6335063|6335183、chr8|6335083 |6335203、chr8|6338214|6338334、chr8|6338297|6338417、chr8|635735 1|6357471、chr8|6478973|6479093、chr8|6479093|6479213、chr8|64953 81|6495501、chr8|6500482|6500602、chr8|26149278|26149398、chr8|26 150710|26150830、chr8|26150730|26150850、chr8|26151158|26151278、chr8|26196384|26196504、chr8|26196404|26196524、chr8|26211963|26212083、chr8|26212049|26212169、chr8|26217664|26217784、chr8|26217697|26217817、chr8|26218469|26218589、chr8|26218567|26218687、chr8|26220179|26220299、chr8|26220264|26220384、chr8|26221216|26221336、chr8|26221306|26221426、chr8|26223806|26223926、chr8|26223826|26223946、chr8|26227629|26227749、chr8|26227729|26227849、chr8|26227829|26227949、chr8|90947754|90947874、chr8|90947774|90947894、chr8|90949208|90949328、chr8|90949228|90949348、chr8|90955477|90955597、chr8|90958347|90958467、chr8|90958423|90958543、chr8|90960015|90960135、chr8|90960035|90960155、chr8|90965455|90965575、chr8|90965575|90965695、chr8|90965695|90965815、chr8|90965815|90965935、chr8|90967490|90967610、chr8|90967586|90967706、chr8|90967683|90967803、chr8|90970957|90971077、chr8|90976626|90976746、chr8|90982571|90982691、chr8|90982685|90982805、chr8|90983380|90983500、chr8|90983418|90983538、chr8|90990427|90990547、chr8|90990451|90990571、chr8|90992941|90993061、chr8|90993021|90993141、<h2 style=";text-align:left;direction:ltr">chr8|90993582|90993702,chr8|90993651|90993771,chr8|90994956|90995076,chr8|90996710|90996830,chr9|21968174|21968294,chr9|2196 8686|21968806,chr9|21970880|21971000,chr9|21970993|21971113,chr9|21971107|21971227,chr9|21974471|21974591,chr9|21974590|2197 4710, chr9|21974710|21974830, chr9|21994117|21994237, chr9|21994230|21994350, chr9|97863968|97864088, chr9|97864032|97864152, chr9 |97869329|97869449、chr9|97869449|97869569、chr9|97873457|97873577、chr9|97873527|97873647、chr9|97873724|97873844、chr9|97873819| 97873939,chr9|97876891|97877011,chr9|97879574|97879694,chr9|97887357|97887477,chr9|97888776|97888896,chr9|97897607|97897727 chr9|97897684|97897804,chr9|97912184|97912304,chr9|97912269|97912389,chr9|97933322|97933442,chr9|97933342|97933462,chr9|97934 298|97934418,chr9|97934329|97934449,chr9|98002907|98003027,chr9|98002927|98003047,chr9|98009685|98009805,chr9|98009705|98009 825, chr9|98011388|98011508, chr9|98011473|98011593, chr9|101569980|101570100, chr9|101570105|101570225, chr9|101570231|101570351<h2 style=";text-align:left;direction:ltr">chr9|101585517|101585637,chr9|101585607|101585727,chr9|101589013|101589133,chr9|101589123|101589243,chr9|101594033|101594153, chr9|101594139|101594259,chr9|101597529|101597649,chr9|101599233|101599353,chr9|101599330|101599450,chr9|101602289|101602409, chr9|101606374|101606494,chr9|101608238|101608358,chr9|101608305|101608425,chr9|101611213|101611333,chr9|101611274|101611394, chr9|135771601|135771721,chr9|135771711|135771831,chr9|135771821|135771941,chr9|135771931|135772051,chr9|135772041|135772161, chr9|135772550|135772670,chr9|135772632|135772752,chr9|135772789|135772909,chr9|135772897|135773017,chr9|135776102|135776222, chr9|135776970|135777090,chr9|135777971|135778091,chr9|135778074|135778194,chr9|135779017|135779137,chr9|135779104|135779224, chr9|135779759|135779879,chr9|135780947|135781067,chr9|135781066|135781186,chr9|135781186|135781306,chr9|135781306|135781426, chr9|135781426|135781546,chr9|135782109|135782229,chr9|135782652|135782772,chr9|135782672|135782792,chr9|135785958|135786078,chr9|135786384|135786504、chr9|135786837|135786957、chr9|135787648|135787768、chr9|135787744|135787864、chr9|135796726|135796846、chr9|135797185|135797305、chr9|135797260|135797380、chr9|135798714|135798834、chr9|135798779|135798899、chr9|135800953|135801073、chr9|135801026|135801146、chr9|135802579|135802699、chr9|135804146|135804266、chrX|44732777|44732897、chrX|44732858|44732978、chrX|44733141|44733261、chrX|44820508|44820628、chrX|44820537|44820657、chrX|44833865|44833985、chrX|44833885|44834005、chrX|44870164|44870284、chrX|44870184|44870304、chrX|44879834|44879954、chrX|44879875|44879995、chrX|44894132|44894252、chrX|44894152|44894272、chrX|44896846|44896966、chrX|44896866|44896986、chrX|44910930|44911050、chrX|44910950|44911070、chrX|44913076|44913196、chrX|44918239|44918359、chrX|44918481|44918601、chrX|44918601|44918721、chrX|44919273|44919393、chrX|44919833|44919953、chrX|44919909|44920029、chrX|44920556|44920676、chrX|44921871|44921991、chrX|44921893|44922013、chrX|44922646|44922766、chrX|44922751|44922871、chrX|44922856|44922976、chrX44922961|44923081、chrX|44928803|44928923、chrX|44928919|44929039、chrX|44929036|44929156、chrX|44929152|44929272、chrX|44929269|44929389、chrX|44929385|44929505、chrX|44929502|44929622、chrX|44935946|44936066、chrX|44937624|44937744、chrX|44937650|44937770、chrX|44938373|44938493、chrX|44938493|44938613、chrX|44941782|44941902、chrX|44941802|44941922、chrX|44941926|44942046、chrX|44941946|44942066、chrX|44942684|44942804、chrX|44942753|44942873、chrX|44945106|44945226、chrX|44948967|44949087、chrX|44949075|44949195、chrX|44949947|44950067、chrX|44950009|44950129、chrX|44966657|44966777、chrX|44969303|44969423、chrX|44969394|44969514、chrX|44970571|44970691、chrX|44970591|44970711、chrX|53205723|53205843、chrX|53220730|53220850、chrX|53221912|53222032、chrX|53222148|53222268、chrX|53222271|53222391、chrX|53222394|53222514、chrX|53222598|53222718、chrX|53222718|53222838、chrX|53222933|53223053、chrX|53223320|53223440、chrX|53223440|53223560、chrX|53223560|53223680、chrX53223680|53223800、chrX|53223800|53223920, chrX|53224121|53224241, chrX|53224392|53224512, chrX|53224492|53224612, chrX|532251 06|53225226, chrX|53225867|53225987, chrX|53225986|53226106, chrX|53226106|53226226, chrX|53226945|53227065, chrX|53227651|53227771, chrX|53227719|53227839, chrX|53227947|53228067, chrX|53228138|53228258, chrX|532282 40|53228360, chrX|53230711|53230831, chrX|53230826|53230946, chrX|53231035|53231155, chrX|53239575|53239695, chrX|53239658|53239778, chrX|53239837|53239957, chrX|53239939|53240059, chrX|53240658|53240778, chrX|532407 37|53240857, chrX|53240968|53241088, chrX|53243850|53243970, chrX|53243929|53244049, chrX|53244956|53245076, chrX|53245058|53245178, chrX|53245257|53245377, chrX|53246331|53246451, chrX|53246957|53247077, chrX|532470 48|53247168, chrX|53247458|53247578, chrX|53249999|53250119, chrX|53253901|53254021, chrX|53253971|53254091. ,

[0052] In some embodiments, each probe in the probe composition can be a single-stranded DNA probe.

[0053] In some embodiments, each probe in the probe composition may carry a label.

[0054] In some embodiments, each probe in the probe composition may carry a biotin label.

[0055] The present application also provides the use of the above-mentioned marker and probe combination in the preparation of early screening or diagnosis products for hereditary tumor susceptibility syndrome.

[0056] The present application also provides a kit for early screening or diagnosis of hereditary tumor susceptibility syndrome, wherein the kit comprises the above-mentioned probe composition.

[0057] In some embodiments, the kit further includes a positive control containing the above-mentioned marker.

[0058] In some embodiments, the kit may also include any one or more of the following: gDNA extraction reagents, RNA extraction reagents, DNA fragmentation reagents, RNA fragmentation and reverse transcription reagents, sequencing adapter connection reagents, pre-library amplification reagents, purification reagents, hybridization reaction reagents, library capture and cleaning reagents, final library amplification reagents, and at least one of DNA negative quality control products.

[0059] This test kit selects a total of 79 genes related to HCPS, involving a variety of tumor-related susceptibility syndromes and cancers, mainly based on tumor-related genetic syndrome-related genes reported by WHO, genetic tumor susceptibility genes recommended for detection by NCCN guidelines, and existing research literature reports, and includes important fusion genes reported in the above materials (Table 1). The detection of genetic tumors will be more comprehensive, and it will have a greater role in the diagnosis of patients with similar syndromic disease phenotypes exhibited by hereditary tumor syndromes.

[0060] Table 1 List of genes and diseases

[0061]

[0062]

[0063] The types of mutations that cause gene inactivation or functional changes include point mutations (SNVs), insertion-deletion mutations (Indels), copy number variations (CNVs), and large-scale rearrangements (LGRs), which can result in abnormal RNA splicing, truncated or nonfunctional protein products, or even no related gene expression. However, current studies have shown that there are no clear hotspots in genes for mutations that cause gene inactivation or functional changes. Therefore, in order to achieve comprehensive HCPS detection of multiple genes and multiple cancer types, it is necessary to design a set of DNA / RNA co-detection, hybrid capture, and next-generation sequencing detection methods for all exon regions (CDS) of 79 genes related to HCPS and the splicing-related regions of 20 bp upstream and downstream. This method improves the accuracy of interpretation of splicing-related variations while ensuring the accuracy of SNV / Indel detection, while taking into account the detection of CNV and LGR.

[0064] The present application also provides a method for detecting gene variations related to hereditary tumor susceptibility syndromes. The method includes the following steps: 1. extraction of genomic DNA (gDNA) and total RNA; 2. fragmentation of gDNA; 3. fragmentation and reverse transcription of RNA; 4. construction of a pre-library; 5. hybridization of a probe composition with the pre-library; 6. construction of a final library; 7. high-throughput sequencing; 8. data analysis.

[0065] The detection kit contains the relevant reagents for steps 3-6, mainly including RNA fragmentation reagents, reverse transcription reagents, sequencing adapter ligation reagents, pre-library amplification reagents, purification reagents, hybridization reagents, library capture and washing reagents, final library amplification reagents, etc. It is required that the total amount of extracted gDNA is greater than 50 ng, the total amount of RNA is greater than 100 ng, the concentration is greater than 15 ng / μL, and the average sequencing depth of the DNA / RNA library reaches more than 200x.

[0066] The data analysis in step 8 includes:

[0067] 1. SNV / INDEL analysis: Use BWA for sequence alignment, fastp for data quality control, Picard for marking duplicates, GATK4 realignment for sequence realignment, haplotypecaller for variant detection and preliminary filtering, use in-house data for re-filtering, ANNOVAR and SNPeff for annotation, and SpliceAI for predicting the impact of RNA splicing sites;

[0068] 2. CNV and LGR analysis: First, create a baseline for 50 peripheral blood leukocyte samples from normal individuals with the same sequencing platform, the same sequencing kit, and the same experimental environment. Then, for the sample to be tested, calculate the sequencing depth at each position, and then correct and normalize the GC content. Finally, calculate the copy number level of the sample to be tested based on the log2 ratio of the depth between the sample to be tested and the baseline (log2 ratio, hereinafter referred to as log2). In this algorithm, the genomic interval to be tested will be divided into small blocks for calculation, and different segmentation methods or no segmentation method can be selected according to the number of small blocks. At the same time, the filtering threshold is finely adjusted to improve the accuracy of the algorithm. Combine methods such as first-generation sequencing, digital PCR, and MLPA (Multiplex Ligation-dependent Probe Amplification) for verification, and finally determine the parameters: the threshold for a copy number of 0 is log2 ≤ -2, the threshold for a copy number of 1 is -2 < log2 ≤ -0.4, and the threshold for a copy number ≥ 3 is log2 ≥ -0.4.

[0069] 3. Capture RNA sequencing for alternative splicing analysis: RNA-seq data were aligned to the hg19 genome using STAR, and then alternative splicing events were detected and quantified using SUPPA2. The gene range detected was 79 genes covered by this panel. A self-developed algorithm was used to quantify the level of alternative splicing, in units of ASR (alternative splicing ratio), calculated as the number of reads supporting alternative splicing divided by the number of all reads covering the splicing event region. First, 300 healthy human whole blood samples were sequenced to construct a normal splicing event map. The same detection method was used to calculate the ASR of each alternative splicing event for the subject, and compared with the normal control, and the significance was calculated by a single-sample two-sided t-test. If the subject's splicing event ASR ≥ 5% and greater than the average ASR of the normal control, the p value < 0.05, and the number of all reads covering the splicing event region ≥ 50, the splicing event was considered to be significantly higher than the healthy control and was considered abnormal splicing.

[0070] Figure 2 FIG. 2 is a block diagram of a genetic mutation detection device 200 for hereditary tumor susceptibility syndrome according to some embodiments of the present application. Figure 2 As shown, the hereditary tumor susceptibility syndrome-related gene variation detection device 200 may include a variation information acquisition module 210 , a variation classification module 220 , and a judgment module 230 .

[0071] The variation information acquisition module 210 can be used to provide variation information of the target marker of the sample to be tested, wherein the target marker is the marker for detecting hereditary tumor susceptibility syndrome according to claim 1 or 2. In some embodiments, the variation information may include: gene name, variation site information, variation type, and variation allele frequency.

[0072] The variation classification module 220 can be used to classify gene variations based on the variation annotation information of the target marker of the sample to be tested and the functional data provided by RNA sequencing. In some embodiments, the variation classification module evaluates the population data, functional data (such as splicing site-related mutations, supplemented by RNA sequencing), and calculation prediction data of the variation site based on the variation classification standards specified in the "ACMG Genetic Variation Classification Standards and Guidelines" for evidence rating and summary. Among them, RNA sequencing is used to evaluate whether the variation affects gene splicing, and there are two prerequisites for use: the functional inactivation of the gene may lead to hereditary tumor susceptibility syndrome, and the gene has RNA expression in the blood. According to the ACMG guidelines, RNA sequencing results can be used as strong evidence of the pathogenicity or benignity of the variation. After the RNA data is analyzed and interpreted by professionals, it is summarized with all other evidence mentioned above to grade the pathogenicity or benignity of the gene variation. The gene variation is divided into one of the following five categories: benign, possibly benign, unclear significance, possibly pathogenic, and pathogenic, among which possibly pathogenic and pathogenic gene variations are judged as positive variations.

[0073] The judgment module 230 judges whether the gene variation is a positive variation or a negative variation based on the classification information.

[0074] The flowchart of the method described in the judgment module 230 is as follows Figure 3 As shown. In step S310, the variation information of the target marker of the sample to be tested and the functional data provided by RNA sequencing can be obtained, and the target marker is the above-mentioned marker for detecting hereditary tumor susceptibility syndrome. In some embodiments, the variation information includes: gene name, variation site, variation type and disease condition corresponding to the variation. In step S320, the gene variation can be classified based on the variation information of the target marker of the sample to be tested and the functional data provided by RNA sequencing. In some embodiments, the variation classification module is based on the variation classification standards specified in the "ACMG Genetic Variation Classification Standards and Guidelines", the population data of the variation site, the functional data (supplemented by RNA sequencing), the co-segregation data, the new data, the allele data, and the information of the calculated prediction data, and the pathogenicity rating of the gene variation. According to the classification of the gene variation, the gene variation is divided into 5 categories: benign, possibly benign, unclear significance, possibly pathogenic, and pathogenic, among which the possibly pathogenic and pathogenic gene variations are judged as positive variations, and the others are negative variations. In step S230, the gene variation can be judged as a positive variation or a negative variation based on the classified information.

[0075] The present application also provides an electronic terminal 400, such as Figure 4As shown, the electronic terminal 400 includes: a processor 410, a memory 420, an input / output interface 430 and a communication port 440; the memory 420 is used to store a computer program, and the processor 410 is used to execute the computer program stored in the memory, so that the terminal performs the method for detecting mutations associated with hereditary tumor susceptibility syndrome as described in the above-mentioned computer-readable storage medium. The terminal has been integrated into the molecular pathology operation process and data management software and has obtained the software copyright (registration number: 2020SR1197198). Figure 5 The analysis interface is shown below.

[0076] The processor 410 can execute computing instructions (program code) and perform the functions of the detection device 200 described in this application. The computing instructions may include programs, objects, components, data structures, processes, modules and functions (functions refer to specific functions described in this application). For example, the processor 410 can process instructions for detecting gene mutations in the genetic tumor susceptibility syndrome-related gene mutation detection device 200. In some embodiments, the processor 410 may include a microcontroller, a microprocessor, a reduced instruction set computer (RISC), an application-specific integrated circuit (ASIC), an application-specific instruction set processor (ASIP), a central processing unit (CPU), a graphics processing unit (GPU), a physical processing unit (PPU), a microcontroller unit, a digital signal processor (DSP), a field programmable gate array (FPGA), an advanced RISC machine (ARM), a programmable logic device, and any circuits and processors capable of performing one or more functions, etc., or any combination thereof. For illustration only, Figure 4 Only one processor 410 is described, but it should be noted that the present application may include multiple processors.

[0077] The memory 420 can store data / information obtained from any component in the genetic tumor susceptibility syndrome-related gene variation detection device 200. In some embodiments, the memory 420 may include a large-capacity memory, a removable memory, a volatile read and write memory, and a read-only memory (ROM), etc., or any combination thereof. Exemplary large-capacity memory may include a disk, an optical disk, and a solid-state drive, etc. The removable memory may include a flash drive, a floppy disk, an optical disk, a memory card, a USB flash drive, a compressed disk, and a mobile hard disk, etc. The volatile read and write memory may include a random access memory (RAM). RAM may include a dynamic RAM (DRAM), a double rate synchronous dynamic RAM (DDRSDRAM), a static RAM (SRAM), a thyristor RAM (T-RAM), and a zero capacitance (Z-RAM), etc. ROM may include a mask ROM (MROM), a programmable ROM (PROM), an erasable programmable ROM (PEROM), an electrically erasable programmable ROM (EEPROM), a compact disk ROM (CD-ROM), and a digital versatile disk ROM, etc.

[0078] The input / output interface 430 can be used to input or output signals, data or information. In some embodiments, the input / output interface 430 can be used to implement the interactive behavior between the user (for example, the person corresponding to the sample to be tested, the user of the genetic mutation detection device 200 associated with the hereditary tumor susceptibility syndrome, etc.) and the processor 410. In some embodiments, the user can input the characteristic information of the person corresponding to the sample to be tested through the input / output interface 430. In some embodiments, the input / output interface 430 may include an input device and an output device. Exemplary input devices may include a keyboard, a mouse, a touch screen, a microphone, etc., or any combination thereof. Exemplary output devices may include a display device, a speaker, a printer, a projector, etc., or any combination thereof. Exemplary display devices may include a liquid crystal display (LCD), a display based on a light emitting diode (LED), a flat panel display, a curved display, a television device, a cathode ray tube (CRT), etc., or any combination thereof.

[0079] The communication port 440 can be connected to a network for data communication. The connection can be a wired connection, a wireless connection, or a combination of the two. The wired connection can include an electric cable, an optical cable, or a telephone line, etc., or any combination thereof. The wireless connection can include Bluetooth, WiFi, WiMax, WLAN, ZigBee, a mobile network (e.g., 3G, 4G, or 5G, etc.), etc., or any combination thereof. In some embodiments, the communication port 440 can be a standardized port, such as RS232, RS485, etc. In some embodiments, the communication port 440 can be a specially designed port.

[0080] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are purchased from conventional biochemical reagent companies unless otherwise specified. The quantitative tests in the following examples are repeated three times, and the results are averaged.

[0081] Example 1 Detection of pathogenic sites of hereditary cancer-related susceptibility gene mutations

[0082] The detection technology solution of this embodiment mainly includes the following steps:

[0083] 1.1. Nucleic acid extraction

[0084] 1.1.1. gDNA extraction

[0085] There are 8 samples in group 1, and the sample types are human peripheral blood, oral swabs or other normal tissue samples. The blood / tissue genomic DNA extraction kit is used. Taking blood samples as an example, the volume is required to be greater than 200μL, and the nucleic acid extraction process is operated according to the extraction kit instructions (product name: QIAamp DNA Blood Mini Kit, item number: 51104). The extracted DNA is measured by Qubit fluorometer, and the extracted DNA yield must be greater than 50ng.

[0086] RNA extraction

[0087] There were 8 samples in group 1, and the sample type was human peripheral blood. The blood sample volume was greater than 200 μL. The nucleic acid extraction process was performed according to the instructions of the extraction kit (product name: QIAamp RNA Blood Mini Kit, catalog number: 52304). The concentration of the extracted RNA was measured using a Qubit fluorometer. The extracted RNA yield was required to be greater than 100 ng and the concentration was greater than 15 ng / μL.

[0088] 1.2. Fragmentation

[0089] DNA fragmentation

[0090] The process is to fragment gDNA into fragmented DNA suitable for Illumina high-throughput sequencing. The gDNA is fragmented according to the operating instructions of the Covaris M220 fragmentation instrument. The fragmentation conditions are shown in Table 2.

[0091] Table 2. DNA fragmentation conditions

[0092] parameter data unit peak power 50 Watt duty factor 20 percent cycles / burst 200 count avg.power 10 Watt Water temperature 6-10 ℃ Time 120-150 second

[0093] 1.2.2. RNA fragmentation and reverse transcription

[0094] The process is to fragment RNA and reverse transcribe it into cDNA suitable for Illumina high-throughput sequencing. 1.2.2.1. RNA fragmentation

[0095] Mix 100 ng of RNA with other RNA fragmentation reagent components according to Table 3.

[0096] Table 3. RNA fragmentation system

[0097] Components Input RNA 100g Fragmentation buffer (2×Frag / Prime Buffer) 8μL Nuclease-free water Make up to 16 μL

[0098] After mixing and centrifugation, the program in Table 4 was run in a PCR instrument with the heated lid maintained at 105°C.

[0099] Table 4. RNA fragmentation procedures

[0100] step Temperature(℃) Time (minutes) 1 94 5 3 4 Keep

[0101] 1.2.2.2. Reverse transcription (single-strand synthesis)

[0102] The RNA fragmentation product was mixed with the reverse transcription (single strand synthesis) reagent according to Table 5.

[0103] Table 5. One-chain synthesis system

[0104] Components Volume(uL) RNA fragmentation products 16 1st Strand Buffer3 7 1st Strand Enzyme Mix3 2 Total 25

[0105] After mixing and centrifugation, the program in Table 6 was run in a PCR instrument with the heated lid maintained at 105°C.

[0106] Table 6. One-strand synthesis program

[0107] step Temperature(℃) Time (minutes) 1 25 10 2 42 15 3 70 15 4 4 Keep

[0108] 1.2.2.3. Reverse transcription (second-strand synthesis)

[0109] The product of the completed one-strand synthesis is mixed with the reverse transcription (two-strand synthesis) reagent according to Table 7.

[0110] Table 7. Two-chain synthesis system

[0111] Components Volume(uL) One-chain synthesis product 25 2nd Strand Buffer 2 (with dUTP) 25 2nd Strand Enzyme Super Mix2 15 Total 65

[0112] After mixing and centrifugation, the program in Table 8 was run in a PCR instrument with the heated lid maintained at 105°C.

[0113] Table 8. Second-strand synthesis program

[0114] step Temperature(℃) Time (minutes) 1 16 30 2 65 15 3 4 Keep

[0115] 1.3. Prelibrary Construction

[0116] The process aims to convert the cDNA after reverse transcription of fragmented DNA and RNA into a dedicated library that can be used for the Illimuna high-throughput sequencing platform. The main process includes end repair, adapter ligation and purification, pre-library amplification and purification. The library construction kit used is KAPA HyperPrep Kit (Manufacturer: KAPA Biosystems, Catalog No.: KK8500).

[0117] 1.3.1. End repair

[0118] Mix 50 ng of fragmented DNA and 40 μL of reverse transcription product of each sample with other end repair components according to Table 9, centrifuge and run in a PCR instrument.

[0119] Table 9. End repair system

[0120]

[0121] Table 10 Program, where step 2 heated lid maintained at 85°C.

[0122] Table 10. End repair procedures

[0123] step Temperature(℃) Time (minutes) 1 20 30 2 65 30 3 10 Keep

[0124] 1.3.2. Adapter ligation and purification

[0125] The DNA repair product and RNA repair product of 1.3.1 were mixed with other adapter connection components according to Table 11, mixed and centrifuged, and then run in a PCR instrument at 20°C for 60 minutes. The hot cover temperature does not need to be maintained during the process. This step introduces the UDI (Unique Double Index) sequence required for data splitting, which can reduce library contamination due to adapter primers between different samples during data analysis. Different UDIs are used for DNA libraries and RNA libraries. The connection product is purified using 0.8 times purification magnetic beads of the repair system, and the elution volume of the purified product is 20μL.

[0126] Table 11. Connector connection system

[0127]

[0128]

[0129] 1.3.3. Prelibrary amplification and purification

[0130] The DNA connection purification product and RNA connection purification product of 1.3.2 were mixed with other amplification components according to Table 12, and the program of Table 13 was run on the PCR instrument after centrifugation, wherein the hot cover was kept at 105°C. The amplification primers used in this step were the I5 / I7 amplification primers commonly used in the Illumina high-throughput sequencing platform, in order to increase the library yield so that it meets the investment requirements of subsequent hybridization capture. The amplification product was purified using the 1x purification magnetic beads of the amplification system, and the elution volume of the purified product was 20 μL. After purification, the product was quantified using a Qubit fluorometer.

[0131] Table 12. Prelibrary amplification system

[0132] Components volume DNA / RNA ligation purification product 20μL PCR Master Mix (2X HiFi PCR Master Mix) 25μL Universal amplification primers (Amplification Primer Mix) 5μL Total volume 50μL

[0133] Table 13. Prelibrary amplification program

[0134]

[0135] 1.3.4. Hybridization of probe combination with pre-library

[0136] The DNA prelibraries of the eight samples were mixed at an equal mass of 500 ng, and the eight RNA prelibraries were mixed at an equal mass of 250 ng, and were mixed with 5 μL of Human Cot DNA and 2 μL of Universal blocker, respectively, and concentrated using a vacuum concentrator until no liquid was visible to the naked eye. 17 μL of the mixture prepared according to Table 14 was added to the DNA and RNA concentrated products, respectively, mixed and centrifuged, incubated at room temperature for 5 minutes, and then the program in Table 15 was run on the PCR instrument, hybridized at 65°C for 4 hours, with the hot cover maintained at 100°C.

[0137] Table 14. Hybridization mixture system

[0138] Components volume Hybridization Buffer (xGen 2X Hybridization Buffer) 8.5μL xGen Hybridization Buffer Enhancer 2.7μL Probe composition 4μL Nuclease-free water 1.8μL Total volume 17μL

[0139] Table 15. Hybridization program

[0140] step Temperature(℃) Time (minutes) 1 95 0.5 2 65 240 3 65 Keep

[0141] Hybridization capture probes were designed by tiling method for the CDS regions of 79 HCPS-related genes and the target regions related to splicing 20bp upstream and downstream, that is, the designed probes were arranged along the target sequence on the genome to form a complete "tiled" coverage. The designed probe pool contains 2708 single-stranded DNA probes with a length of 120nt and carries a biotin marker. The probes are reverse complementary to the corresponding target regions, bind to the target regions by hybridization, and separate and enrich the target region sequences through the high affinity interaction between biotin and streptavidin, and perform high-throughput sequencing.

[0142] The probe pool includes 2708 probes. The physical locations in the hg19 version of the human genome are as follows, including the chromosome number, start position, and end position of the region covered by each probe:

[0143] 1.3.5. Final library construction

[0144] This step aims to enrich the DNA fragments in the pre-library that have hybridized with the target probe and remove other DNA fragments. The main steps include target sequence capture, other sequence cleaning, and final library amplification and purification. The kit used is Hybridization and Wash Kit (Manufacturer: IDT, Part No.: 1080577)

[0145] 1.3.6. Target sequence capture

[0146] Take two 50 μL portions of streptavidin magnetic beads restored to room temperature, add 100 μL of magnetic beads washing buffer (Beads Wash Buffer) to each portion, mix well, adsorb on a magnetic rack, discard the supernatant, and repeat washing 3 times.

[0147] Resuspend the magnetic beads using 17 μL of Bead Resuspension Mix preheated to 65°C.

[0148] Add the resuspended streptavidin magnetic beads to the hybridized DNA and RNA samples respectively, mix and centrifuge, incubate at 65℃ for 45 minutes on the PCR plate, keep the heated cover at 70℃, and mix by pipetting quickly every 10 minutes.

[0149] 1.3.7. Other sequence cleaning

[0150] Add 100 μL of Wash Buffer 1 preheated to 65°C, mix well, adsorb on a magnetic rack, and discard the supernatant.

[0151] Then add 150 μL of Stringent Wash Buffer 4 preheated to 65°C, mix well, and place on a PCR instrument for incubation at 65°C for 5 minutes, with the hot cover maintained at 70°C. After the incubation is complete, adsorb on a magnetic rack and discard the supernatant. This step is repeated twice.

[0152] Then add 150 μL of Wash Buffer 1, Wash Buffer 2, and Wash Buffer 3, mix well, and incubate at room temperature for 2 minutes, then adsorb on a magnetic rack and discard the supernatant. After completing the room temperature washing with the three reagents, add 20 μL of nuclease-free water.

[0153] 1.3.8. Final library amplification and purification

[0154] The two cleaning products of 1.3.7 were mixed with other amplification components according to Table 16. After mixing and centrifugation, the program in Table 17 was run on the PCR instrument, wherein the hot cover was maintained at 105°C. The amplification primers used in this step were the I5 / I7 amplification primers commonly used in the Illumina high-throughput sequencing platform, in order to increase the library yield so that it meets the investment requirements for subsequent sequencing. The amplification product was purified using the 1x purification magnetic beads of the amplification system, and the elution volume of the purified product was 20 μL. After purification, the final library was quantified using a Qubit fluorometer.

[0155] Table 16. Final library amplification system

[0156]

[0157]

[0158] Table 17. Final library amplification program

[0159]

[0160] 1.3.9. High-throughput sequencing and data analysis

[0161] The two final libraries obtained were sequenced on the sequencing platform, and the data from the machine was greater than 150M for the sequencing data of the DNA library and RNA library of each sample. The sequencing results of the eight samples were all excellent in various performance indicators, meeting the conventional targeted capture sequencing quality control standards, such as the alignment rate of the DNA library was greater than 90%, the capture rate was greater than 50%, the average effective sequencing depth was greater than 200X, the coverage was greater than 99%, and the uniformity (greater than 0.2 times the average sequencing depth) was greater than 90%.

[0162] The detected variant sites are classified according to the ACMG interpretation principles. The interpretation principles and process can be found at (http: / / acmg.cbgc.org.cn / doku.php?id=start). In short, the population data, functional data (supplemented by RNA sequencing), co-segregation data, new data, allele data, and computational prediction data of the variant sites are collected, and the above evidence is graded into pathogenic or benign levels (see Figure 1 ), and then according to the grading of all evidence, the variant sites were divided into 5 categories (Table 18), benign (category 1), possibly benign (category 2), unclear significance (category 3), possibly pathogenic (category 4), and pathogenic (category 5), of which categories 4 and 5 were judged as positive variants.

[0163] Table 18 Joint standard rules for classification of genetic variations

[0164]

[0165]

[0166] The center's annotation database and public literature resources were used to annotate and interpret the sequencing results for SNV / Indel and CNV / LGR, and the mutations were rated according to the ACMG guidelines. People with a rating of 4-5 are at high risk for related hereditary tumors and need genetic counseling and take effective preventive measures according to the doctor's advice. Their families are also recommended to undergo testing for related gene loci.

[0167] The results of pathogenic site detection in the example family are shown in Table 19. The corresponding variant sites were detected in 6 of the 8 subjects, and the test results were consistent after Sanger sequencing, other NGS methods or MLPA verification.

[0168] Table 19 Example sample test results

[0169]

[0170]

[0171] Precision and accuracy verification results of Example 2

[0172] In 2018-2019, the laboratory began to select and screen genes related to genetic tumor susceptibility. Combined with the laboratory's DNA testing needs, a total of 79 genes were established as detection genes, and clinical samples were used to verify the precision, accuracy, and detection limit of gene variation (SNV, INDEL, CNV, LGR) detection. The results of the consistency study showed that (1) 3 positive clinical samples verified by MLPA, each repeated twice, had consistent test results between different batches, indicating that the precision of the self-built reagent method in this laboratory is good. (2) 44 clinical samples verified by Sanger sequencing / other NGS, including 36 SNVs and 8 INDELs. And 28 clinical samples verified by MLPA, including 16 negative variants and 12 positive variants. The results showed that the positive coincidence rate was 100%. Some of the test and verification results are shown in Table 20.

[0173] Table 20. Test and verification results of some cases

[0174]

[0175]

[0176] Example 3 Clinical report presentation of test results and their implications for treatment, prevention, and management of genetic syndromes.

[0177] This embodiment is derived from the detection information of a group of hereditary breast cancer-ovarian cancer syndrome family cases, and this example suggests the treatment of hereditary cancer by the present invention. The proband, female, 60 years old, had lower abdominal distension and pain for 4 months. She denied a family history of tumors. MRI showed a 15cm mass palpable in the adnexal area. Pathology of the puncture sample from another hospital suggested a malignant tumor. Neoadjuvant chemotherapy was performed first, followed by surgery. Peripheral blood was drawn from the patient, and steps such as nucleic acid extraction, fragmentation, reverse transcription, pre-library construction, final library construction, target sequence capture, high-throughput sequencing and data analysis were performed. Based on the analysis results, a clinical test report was generated. Figure 6 The quality control information of this sample is displayed, including DNA quality and sequencing data quality. The quality control of this case meets the requirements. The results of the germline mutation test of this sample are: Pathogenic or likely pathogenic mutations were detected in the DNA of the subject's peripheral blood nucleated cells: BRCA2 gene (NM_000059.3) exon 20 splicing site mutation c.8632+1_8632+3delinsTT, mutation abundance 47.1% ( Figure 7 ). The test results of this sample were interpreted for clinical significance ( Figure 8 ). Patients with BRCA2 germline mutations may be sensitive to the poly (ADP-ribosyltransferase) (PARP) inhibitor olaparib. In addition, the subjects may be at high risk for breast / ovarian cancer and related tumors. It is recommended to contact a specialist to develop a treatment plan or genetic counseling; and it is recommended that relevant family members undergo testing for this site.

[0178] The proband was treated with olaparib for maintenance therapy because of the detection of BRCA2 germline mutation. CA125 index decreased and stabilized to normal level ( Fig. 9 ). Because the proband was detected with a BRCA2 germline mutation, the patient's family is recommended to undergo mutation testing to clarify the genetic risk. The patient's youngest daughter was detected with the same mutation as the proband. At the age of 35, a breast tumor was found during the initial physical examination. Our hospital's ultrasound was 4B and the mammography target was 4B. The pathological results after surgery indicated invasive ductal carcinoma, grade III. Preventive bilateral salpingo-oophorectomy was required, and she was informed that she was currently young and could be closely followed up for ultrasound and tumor indicators. The BRCA gene test of the patient's eldest daughter and son was wild type. The genetic pedigree chart of this family drawn for this group of examples is shown in Fig.10 .

[0179] Example 4 - Presentation of clinical report of copy number (large fragment rearrangement) variation sample.

[0180] In this example, the patient was a patient with uterine fibroids, whose mother had a history of breast cancer and whose sister had ovarian cancer. The patient requested to undergo the test of the present invention to assess whether there were genetic risk factors. The patient was found to have a large deletion mutation of exons 5-6 of BRIP1 (NM_032043.2), and the results were further confirmed by MLPA method ( Fig.11 and Fig.12 ). The patient was 50 years old and strongly requested a risk-reducing salpingo-oophorectomy (RRSO). Postoperative pathological results showed that the right fallopian tube tissue sample showed p53-positive tubal intraepithelial lesions (STIL). This indicates an early type of precancerous lesions. This example demonstrates the importance of this test for genetic tumor risk indication and related patient management.

[0181] Example 6 - Classification of splicing gene variants using functional data provided by RNA sequencing

[0182] In this example, the variable splicing mutation c.-39-1_-39del in exon 2 of the BRCA2 gene (NM_000059.3) was detected at the DNA level, with a mutation abundance of 44%. Whether this mutation affects the splicing of exon 2 of BRCA2 is crucial to its functional rating. Calculated at the RNA level and observed with IGV, the functional data provided by RNA sequencing clearly show that there are transcripts supporting the untranscribed exon 2 of BRCA2, such as Fig.13 As shown in the red box, this mutation affects the splicing of BRCA2 exon 2. Based on this evidence, BRCA2 can be rated as a pathogenic variant.

[0183] Example 7-Display of the results of the detection of hereditary cancer-related susceptibility genes in the Chinese population

[0184] Furthermore, this application also detects 208 HCPS or suspected cases in the Chinese population from September 2020 to December 2022 in clinical practice, including 69 males and 139 females, with an age range of 18 to 76 years old (median age 44 years old). The total positive rate of the test was 31.2% (65 / 208). The positive cases detected involved hereditary leiomyomatosis and renal cell carcinoma syndrome, Lynch syndrome, Von Hippel-Lindau syndrome, tuberous sclerosis (TSC), hereditary medullary thyroid carcinoma, Cowden syndrome, neurofibromatosis type I, MUTYH-associated polyposis (MAP), familial adenomatous polyposis (FAP), and hereditary breast cancer and ovarian cancer, etc. The specific occurrence frequency is as follows Fig.14 shown.

[0185] The basic concepts have been described above. Obviously, for those skilled in the art, the above detailed disclosure is only for example and does not constitute a limitation of this specification. Although not explicitly stated here, those skilled in the art may make various modifications, improvements and corrections to this specification. Such modifications, improvements and corrections are suggested in this specification, so such modifications, improvements and corrections still belong to the spirit and scope of the exemplary embodiments of this specification.

[0186] At the same time, this specification uses specific words to describe the embodiments of this specification. For example, "one embodiment", "an embodiment", and / or "some embodiments" refer to a certain feature, structure or characteristic related to at least one embodiment of this specification. Therefore, it should be emphasized and noted that "one embodiment" or "an embodiment" or "an alternative embodiment" mentioned twice or more in different positions in this specification does not necessarily refer to the same embodiment. In addition, certain features, structures or characteristics in one or more embodiments of this specification can be appropriately combined.

[0187] In some embodiments, numbers describing the number of components and attributes are used. It should be understood that such numbers used in the description of the embodiments are modified by the modifiers "about", "approximately" or "substantially" in some examples. Unless otherwise specified, "about", "approximately" or "substantially" indicate that the numbers are allowed to vary by ±20%. Accordingly, in some embodiments, the numerical parameters used in the specification and claims are approximate values, which may change according to the required features of individual embodiments. In some embodiments, the numerical parameters should take into account the specified significant digits and adopt the general method of retaining digits. Although the numerical domains and parameters used to confirm the breadth of their range in some embodiments of this specification are approximate values, in specific embodiments, the setting of such numerical values ​​is as accurate as possible within the feasible range.

[0188] Finally, it should be understood that the embodiments described in this specification are only used to illustrate the principles of the embodiments of this specification. Other variations may also fall within the scope of this specification. Therefore, as an example and not a limitation, alternative configurations of the embodiments of this specification may be considered consistent with the teachings of this specification. Accordingly, the embodiments of this specification are not limited to the embodiments explicitly introduced and described in this specification.

Claims

1. A marker combination for detecting hereditary tumor susceptibility syndrome, the marker combination comprising the following genes and fragments thereof: APC, ARID1A, ATM, ATR, AXIN2, BAP1, BARD1, BLM, BMPR1A, BRCA1, BRCA2, BRIP1, CDH1, CDK12, CDKN1B, CDKN2A, CHEK1, CHEK2, DICER1, EMSY, EPCAM, ERCC3, FAM175A, FANCA, FANCC, FANCD2, FANCL, FANCM, FH, FLCN, GALNT12, GEN1, GREM1, HDAC2, HNF 1B, HOXB13, KDM5C, KDM6A, MCPH1, MEN1, MET, MITF, MLH1, MLH3, MRE11A, MSH2, MSH3, MSH6, MUTYH, NBN, NF1, NF2, PALB2, PBRM1, PMS1, PMS2, POLD1, PO LE, PPP2R2A, PTEN, RAD50, RAD51, RAD51B, RAD51C, RAD51D, RAD54L, RECQL, RET, SDHB, SDHC, SDHD, SMAD4, STK11, TP53, TSC1, TSC2, VHL, WT1, XRCC2.

2. The marker combination according to claim 1, characterized in that: The marker includes all exon regions of the gene and splicing regions of 20 bp upstream and downstream of the exon regions.

3. A probe composition for detecting hereditary tumor susceptibility syndrome, characterized in that: The probe composition is used to detect the marker combination according to claim 1 or 2.

4. The probe composition according to claim 3, characterized in that The probe composition includes probes capable of hybridizing to chromosomal loci on one or more of chromosomes 1-22.

5. The probe composition according to claim 3, characterized in that The probe is a single-stranded DNA probe, and / or the probe carries a label, preferably, the label is a biotin label.

6. A kit for early screening or diagnosis of hereditary tumor susceptibility syndrome, characterized in that: The kit comprises the probe composition according to any one of claims 2 to 5.

7. The kit according to claim 6, characterized in that The kit also includes a positive control for each marker in the marker composition according to any one of claims 1-2.

8. The kit according to claim 6, characterized in that The kit also includes any one or more of the following: gDNA extraction reagent, RNA extraction reagent, DNA fragmentation reagent, RNA fragmentation and reverse transcription reagent, sequencing adapter connection reagent, pre-library amplification reagent, purification reagent, hybridization reaction reagent, library capture and cleaning reagent, final library amplification reagent, and at least one of DNA negative quality control products.

9. Use of the probe composition according to any one of claims 3 to 5 or the kit according to any one of claims 6 to 7 in the preparation of a product for early screening or diagnosis of hereditary tumor susceptibility syndrome.

10. A detection device for genetic mutations associated with hereditary tumor susceptibility syndrome, characterized in that: The hereditary tumor susceptibility syndrome-related gene variation detection device comprises the following modules: a) a variation information acquisition module, used to provide variation information of a target marker of a sample to be tested, wherein the target marker is the marker combination for detecting a hereditary tumor susceptibility syndrome according to claim 1 or 2, and the test sample adopts the probe composition according to any one of claims 3 to 5 or the kit according to any one of claims 6 to 7; b) a variation classification module, which is used to classify gene variations based on the variation information of target markers of the sample to be tested and the functional data provided by RNA sequencing; c) a judgment module, used to judge whether the gene variation is a positive variation or a negative variation based on the classification information.

11. The detection device according to claim 10, characterized in that: The variation information includes: gene name, variation site, variation type and disease condition corresponding to the variation; the variation classification module is based on the variation classification standards specified in the "ACMG Genetic Variation Classification Standards and Guidelines", the population data of the variation site, functional data, co-segregation data, new data, allele data, and computational prediction data information, and grades the degree of pathogenicity or benignity of the gene variation, and then divides the gene variation into 5 categories according to the grading of the gene variation: benign, possibly benign, unclear significance, possibly pathogenic, and pathogenic, among which possibly pathogenic and pathogenic gene variation are judged as positive variation.

12. A computer-readable storage medium, characterized in that: The storage medium stores computer instructions. When the computer instructions are executed by the processor, a method for detecting a classification of gene mutations associated with hereditary tumor susceptibility syndrome is implemented. The method comprises: a) a variation information acquisition module, used to provide variation information of a target marker of a sample to be tested, wherein the target marker is the marker combination for detecting a hereditary tumor susceptibility syndrome according to claim 1 or 2, and the test sample adopts the probe composition according to any one of claims 3 to 5 or the kit according to any one of claims 6 to 7; b) a variation classification module, which is used to classify gene variations based on the variation information of target markers of the sample to be tested and the functional data provided by RNA sequencing; c) a judgment module, used to judge whether the gene variation is a positive variation or a negative variation based on the classification information.

13. The computer-readable storage medium according to claim 13, wherein: It also includes the following features: the variation information includes: gene name, variation site, variation type and disease condition corresponding to the variation; the variation classification module is based on the variation classification standards specified in the "ACMG Genetic Variation Classification Standards and Guidelines", the population data of the variation site, functional data, co-segregation data, new data, allele data, and computational prediction data information, and grades the pathogenicity or benignity of the gene variation, and then divides the gene variation into 5 categories according to the grading of the gene variation: benign, possibly benign, unclear significance, possibly pathogenic, and pathogenic, among which possibly pathogenic and pathogenic gene variation are judged as positive variation.

14. An electronic terminal analysis and reporting module, characterized in that: The electronic terminal analysis and reporting module includes: a processor, a memory, an input / output interface and a communication port; the memory is used to store a computer program, and the processor is used to execute the computer program stored in the memory. This process realizes automatic splitting, automatic analysis and obtaining of variation information, and providing variation interpretation information for high-throughput sequencing data. The terminal executes the method for detecting genetic variation associated with hereditary tumor susceptibility syndrome as described in any one of claims 13 to 14 in the computer-readable storage medium.

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