CN110241198A - A kind of genome recombination fingerprint and its identification method characterizing hHRD HR defective - Google Patents
A kind of genome recombination fingerprint and its identification method characterizing hHRD HR defective Download PDFInfo
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
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- G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
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- G—PHYSICS
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- G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
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- G16B20/50—Mutagenesis
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Abstract
The present invention relates to a kind of novel identification methods of HR defective and application thereof.It is analyzed more particularly it relates to resurvey sequence, bioinformatic analysis and correlation statistically using high-throughput genome, the relevant characteristic gene group of the hHRD type homologous recombination repair defect parsed recombinates fingerprint.This feature hHRD recombinates fingerprint caused by the functional defect of specific homologous recombination repair mechanism (i.e. the mono- ubiquitination access of CRL4WDR70-H2B), sum frequency comprising genome recombination in single sample, the composition and locus specificity of chromosomal structural variation type copy number variation, infectious disease or tumour with its identification with this feature mutation fingerprint, and for instructing the targeted drug of PARP inhibitor class to treat, including but not limited to have the breast cancer of this category feature, oophoroma, endometrium (sample) cancer, clear cell carcinoma of ovary, prostate cancer, cancer of pancreas, cutaneum carcinoma and gastric cancer, and hepatitis b virus infected or relevant liver fibrosis cirrhosis, the method and purposes of liver cancer and cholangiocellular carcinoma.
Description
1, technical field
Present invention relates in general to as the genome recombination fingerprint and life for identifying a kind of special homologous recombination repair defect
Object informatics identification method, for identifying the virus infection, tumour or the intermediate pathological state (such as cirrhosis) that have this feature.
Specifically, the present invention relates to for identifying the cell, body fluid or the biopsy that have hHRD type gene group recombination feature.
More particularly, the present invention relate to identify since hepatitis B infection virus infection or cellular genome are mutated caused hHRD
The relevant characteristic gene group of HR defective recombinates fingerprint, and the sum frequency comprising full-length genome recombination, chromosome structure becomes
The composition and high frequency locus specificity of foreign peoples's type copy number variation, identify homologous recombination repair defect related disease with it, also may be used
With instruct this kind of disease targeted drug treat therapeutic effect evaluation, including but not limited to this category feature breast cancer,
Oophoroma, endometrium (sample) cancer, clear cell carcinoma of ovary, prostate cancer, cancer of pancreas, cutaneum carcinoma and gastric cancer, it is especially B-mode
Hepatitis virus (Hepatitis Virus B, hereinafter referred to as HBV) infection or relevant liver fibrosis cirrhosis, liver cancer and gallbladder
The method and purposes of solencyte cancer.
2, background technique
More and more researchs confirm that the disease of homologous recombination repair defect type occupies comparable ratio in all kinds of tumours
Example.Wherein, the mutation of mastocarcinoma gene BRCA1 and BRCA2 or abnormal expression are the prototype genes of homologous recombination repair defect
(proto-type).These mutation are the Important cause of disease for leading to breast cancer, oophoroma, cancer of pancreas, prostate cancer and incidence gastric cancer
(ref), such disease is collectively referred to as the tumour (BRCAness) of BRCA series.Moreover, the mutation of BRCA gene is also weight
The drug target wanted, or the index (ref) used for judging targeted drug.But with BRCA1 and BRACA2 detection in Gene Mutation
Clinical classification and clinical decision as HR defective disease type have significant ASIC limitation.Its reason is: 1) existing
Gene diagnosis is confined to the limited mutation site of specific gene, these mutational sites have specific biological significance, such as nonsense
It is mutated (non-sense) and missing (truncation) etc., can indicate whether that the mutation causes the forfeiture or attenuating of protein function.So
And this method can not mutational site indefinite to biological significance or not yet studying report or mutant form make and effectively commenting
Estimate, as there are a large amount of VUS (Variants of Uncertain Significance, with reference to BMC in breast cancer
Cancer.2015；It is 15:936) that the clinical diagnosis of HR defective causes very big obstacle；2) can not efficient diagnosis go out not
The HR defective case for BRCA1 and the BRCA2 mutation that can be detected with the prior art, such as a kind of Rucaparib (PARP
Inhibitor) the test of 2 phase of clinic in, have been surprisingly found that drug to the oophoroma cases of certain BRCA1 wild types generate compared with treatment
Effect, later period prove that tumour cell BRCA gene shows loss of heterozygosity in these cases.For another example BRCA1/BRCA2 gene can
Can be due to promoter mutation, promoter methylation is abnormal or (gene order does not occur in this case for large fragment chromosome indexing
Lose or change), the prior art is difficult to that these genome signatures are quantitatively evaluated or changes the influence to BRCA related gene, therefore
It cannot effectively be contacted with HR defective itself；3) increasingly complex, homologous recombination repair defect not only due to
The mutation of BRCA1/BRCA2 or dysfunction cause, it is also possible to be related to more gene mutations or abnormal expression, such as SWI/SNF
The component subunit (ARID1A, ARID1B and SMARCD1) of protein complexes is the key factor for regulating and controlling homologous recombination repair, and
The gene mutation (ARID1A, ARID1B, ARID2, PBRM1, SMARCA4 and SMARCB1 etc.) of SWI/SNF component is present in
In about 20% Oncogenome, part or all of these mutation can cause homologous recombination repair defect, further make same
The clinical diagnosis of source defective recombinant complicates.
It can be seen that the existing skill for carrying out clinical diagnosis to HR defective based on BRCA1/BRAC2 gene mutation
Art be it is unreliable, incomplete, all HR defective cases cannot be covered.Homologous recombination proposed in the present invention is repaired
The genome signature mutation fingerprint of multiple defect can have wider detection range and more reliable judgement in clinical detection
Standard.In addition, guidance is up to the present diagnosed or treated to disease according to full genome mutated and structure variation fingerprint
Technology there is no precedent.
By to 7042 different types of tumours in the graduate Cosmic project research of Britain Camb mulberry lattice in 2013
Sample carries out deep sequencing, analyzes its genome signature, and cluster parses 30 kinds of different SNV essential characteristic labels
(Signatures).Wherein, only label 3 defines and mesh associated with HR defective caused by BRCA1/BRCA2 mutation
The preceding HR defective point mutation feature uniquely parsed is (with reference to Nat, Genet.2017Oct；49 (10): 1476-
1486.).Further, sequence scientific discovery BRCA1 and BRCA2 gene mutation is resurveyed by the genome of developed recently to cause extensively
General genome structure variation (Structural variation, SV) or recombination (Rearrangement), and spy can be passed through
Determine algorithm and summarizes characteristic recombination fingerprint.This feature point mutation label related to high-frequency HR defective
(Signature 3) together forms the characteristic base of the breast cancer of HR defective type, oophoroma and prostate cancer
Because of a group variation fingerprint, totally it is defined as HR defective (HRD) index of breast cancer (with reference to Nat Med.2017 Apr；
23 (4): 517-525).Up to the present, only only breast cancer has been characterized relatively accurate HRD index.
The research work discovery of the present inventor, HBV virus infection or genome conformity will cause the homologous recombination of liver cell
Defect is repaired, mechanism is that the early stage of homologous recombination repair is blocked by the mono- ubiquitination of interference host cell CRL4WDR70-H2B
DNA processing, the process are related to multiple regulatory pathway in addition to BRCA1/BRCA2 and SWI/SNF protein complexes, but and mammary gland
Cancer and oophoroma difference, HBV interference homologous recombination repair are not related to the mutation of BRCA1/BRCA1 gene.HBV passes through dry as a result,
The coordination for disturbing the function interference histone modification and homologous recombination repair machine of host protein complex slows down the reparation of DNA break
Speed, to cause genome mutation rate increase in reproduction process, chromosome instability and genome structure recombination.
Therefore, because HBV virus infection causes liver cell homologous recombination repair defect, will cause and breast cancer, oophoroma
Wait the similar mutation fingerprint of BRCA panel of tumor, the TMB including full-length genome higher level, point mutation pedigree (such as label 3)；But
Simultaneously as the mechanism of action of HBV is not only by BRCA1 and BRCA2 gene mutation, therefore HBV infection or associated hepatocellular carcinoma are made
At genome mutation feature again it is totally different with the breast cancer of BRCA gene mutation.
Therefore, the present invention recombinates fingerprint to logical with the mono- ubiquitination signal of CRL4WDR70-H2B by genome signature
Road functional defect or gene mutation carry out the prediction of hHRD homologous recombination repair defect but without the disease of BRCA gene mutation
Or identification.The hHRD genome recombination fingerprint includes the sum frequency of full-length genome recombination, the group of chromosomal structural variation type
Number variation (Copy Number Variation, CNV) is copied at high frequency locus specificity.The hHRD recombination fingerprint is in HBV
It is accounted in associated hepatocellular carcinoma by obvious specific gravity, and is not showed in HBV negative HCC, there is significant statistical difference.The hHRD
The disease of type includes HBV infection related disease and similar disorder type, as breast cancer, oophoroma, endometrium (sample) cancer,
Clear cell carcinoma of ovary, cancer of pancreas, prostate cancer, cutaneum carcinoma and gastric cancer etc..Not yet discovery utilizes genome signature weight at present
Group fingerprint characterizes the identification method of such disease.
3, summary of the invention
Study discovery for the first time the present invention is based on the present inventor: it is same that HBV virus infection or genome conformity can lead to liver cell
Source recombinantal repair defect；Homologous recombination repair defect caused by HBV causes the characteristic genome of liver cell and chromosome instability
It is fixed, the specific sum frequency comprising full-length genome recombination, high frequency locus specificity copy number variation, chromosomal structural variation or recombination
Composition, these features collectively constitute the genome recombination fingerprint (such as Fig. 1-2) of hHRD.In the genome recombination fingerprint and document
Report the recombination fingerprint significant difference of BRCA series:
1. the sum frequency of full-length genome recombination it is higher (single sample is 89-602 event in embodiment, average 292,
It is 228 in BRCA1/2 deficiency mammary gland, 88) (refers to Nat Med.2017 Apr in oophoroma；23 (4): 517-525).
2. the composition of chromosomal structural variation type: having a high proportion of interchromosomal in the genome structure variation of hHRD
Indexable (average proportions: 72.8%)；Low ratio missing (average proportions: 13.3%), the tandem repetitive sequence of extremely low ratio
(5.7%), the ratio that these make a variation and in breast cancer (refers to Nature.2016 Jun 2 in phenomenon is reversed；534 (7605):
47-54.)。
Experiment of the invention also demonstrates, inhibits the gene expression (WDR70) of CRL4WDR70 complex in cell, and
Occurs high frequency locus specificity copy in expression HBV coding oncogene (HBx) or in vivo the mouse liver genome of silencing WDR70
Number variation and apparent Chromosome recombination, and most of recombination event is interchromosomal indexing (in clinical samples and mouse model
50%) interchromosomal indexing ratio is all larger than, consistent with the variation features of HBV associated hepatocellular carcinoma genome.In conjunction with these data with
And HBx interferes the fact that the assembling of CRL4WDR70 protein complexes (not issue data), HBx is by destroying CRL4WDR70 activity
Lead to the generation of the characteristic fingerprint of hHRD.
In the present invention program, the HBV infection related disease refers to various diseases caused by HBV virus infection, including
But it is not limited to HBV virus infection or carrier, further preferably HBV infection correlation oxyhepatitis, chronic hepatitis, liver fibrosis, liver
Hardening, liver cancer and cholangiocellular carcinoma further include physically different caused by HBV infection, such as physical signs is abnormal, immunosupress,
Circulatory system symptom and gastrointestinal symptom etc..
In the present invention, it further includes similar that the homologous recombination repair deficiency disorders, which include but is not limited to HBV infection,
The genes such as RNF20/40, histone H2B ubiquitination, CUL4-DDB1-WDR70-19S proteasome and related deubiquitinating enzymes are prominent
The HRD genome recombination type for becoming and being different from BRCA series caused by the reasons such as protein function obstacle.
In the present invention, the homologous recombination repair deficiency disorders further include the function of drug-induced above-mentioned signal transduction pathway
The DNA of disorder repairs exception, and including but not limited to demethylase, deacetylase inhibitor etc. can induce hHRD characteristic
The drug treatment regimes of genome recombination fingerprint and combination.
It should be pointed out that although genome signature genome recombination fingerprint of the present invention is the homologous of hHRD type
A part of the characteristic finger-print of recombination group, can be logical because of the mono- ubiquitination signal of CRL4WDR70-H2B with characterization
Oncogenome trace feature caused by the dysfunction of road.
The present invention provide it is a kind of using full-length genome weight sequencing technologies and bioinformatic analysis identification hHRD homologous recombination
The genome recombination fingerprint that defect is formed.The present inventor is found especially that height of the hHRD fingerprint in HBV infection and related disease
Repeatability performance makes the fingerprint characteristic can be applied to disease of the identification with hHRD fingerprint characteristic from tissue and body fluid (special
Be not HBV infection related disease) generation and progress.In addition, such index applies also for targeted drug (PARP inhibitor
Deng) to the medication guide of the disease with hHRD indication and the evaluation of therapeutic effect.
It is a further object of this invention to one or more combinations based on genome recombination fingerprint characteristic provided by the invention,
Same high-throughput genome can be resurveyed into sequence technology and be applied to Circulating tumor DNA in detection HBV infection person's blood sample
(ctDNA), it is identified applied to the early stage blood serum of hHRD tumor types.
A yet further object of the present invention is one or more groups based on genome recombination fingerprint characteristic provided by the invention
It closes, during oncotherapy, resurveys sequence and ctDNA technology in conjunction with genome, largely discharged after Quantitative Monitoring death of neoplastic cells
Characteristic hHRD segment, to evaluate the therapeutic effect of anti-tumor drug；
In another aspect, the tumour that newest homologous recombination targeted drug such as PARP inhibitor is mutated BRCA1/2 have it is good
Good therapeutic effect.Existing drug includes: Olaparib, BGB-290, Wei Lipani, Ni Lapani, Lu Kapani, E7449,
The neutrality or salt derivative of Talazoparib and BGP-15 etc. and these drugs.The present inventor studies discovery PARP class suppression
Preparation is to HBV associated hepatocellular carcinoma, or to the hHRD type with the mono- ubiquitination signal path defect of CRL4WDR70-H2B or mutation
Cell, group are woven with apparent targeting killing effect.Therefore, a yet further object of the present invention is based on genome weight provided by the invention
One or more combinations of group fingerprint, can be used as the PARP inhibitor for treating method for hHRD type HR defective
With identification of indicator, for instructing the clinical use of such drug, especially HBV infection and the relevant liver cancer of HBV and bile duct thin
Born of the same parents' cancer, and part have breast cancer, oophoroma, endometrium (sample) cancer, clear cell carcinoma of ovary, pancreas of hHRD feature
Cancer, gastric cancer, cutaneum carcinoma and prostate cancer.
In the present invention program, wherein the hHRD genome recombination fingerprint can pass through cell, fresh pathological tissue, ice
The biomaterial for freezing the sources such as tissue, paraffin fixing organization, blood, urine or saliva is identified.
In the present invention program, wherein the hHRD genome recombination fingerprint can resurvey sequence, polymerase by genome
The nucleic acid analysis techniques such as chain reaction (PCR), nucleic acid hybridization and genetic chip are identified.
In the present invention program, wherein the hHRD genome recombination can be with the biology of other diagnosing tumors, drug evaluation
Marker or fingerprint use in conjunction.
On the other hand, the present invention provides a kind of gene identified or diagnose specific mechanism homologous recombination repair defect (hHRD)
The method of group or its disease, comprising the following steps:
1) by vitro body fluid or tissue through gene sequencing；
2) data for obtaining sequencing are parsed using delly software or are extracted using the operation of controlfree software；
3) genomic map is obtained after software is handled；
4) finger-print and genome recombination fingerprint comparison of the invention obtained step 3), or
5) genome is compared with genome recombination marker of the invention.
Detailed description of the invention
The hot spot region that copy number changes in Fig. 1, HBV associated hepatocellular carcinoma genome.
The CNV event of Controlfreec software analysis HBV associated hepatocellular carcinoma.No. 1 region chromosome 1p36.33 as the result is shown
There are the hot spot regions (0-2579999bp) of copy number variation.White camber line arrow instruction chromosomal copy number increases area in figure
Domain (copy number >=3), black camber line arrow show copy number absent region (copy number=0,1).All samples as seen from the figure
All occur significantly copying number variation in the region 1p36.33.1p36.33 copy number variation hot spot region is referred to by black line arrow
Show.
Fig. 2, a high proportion of interchromosomal indexing are the genome recombination features of HBV associated hepatocellular carcinoma.
(including interchromosomal turns for recombination that A.delly software analyzes 16 HBV associated hepatocellular carcinoma full-length genomes and structure variation
Position, missing, inversion and tandem repetitive sequence) total number of events, grey histogram shows that individual gene group detects recombination event, model
Enclose 89-602 every samples.
B.16 in example sample, above-mentioned different types of recombination event proportion.Wherein, interchromosomal indexing event is in institute
There is proportion in sample maximum, single sample accounting range 51-91.5%.Tandem repetitive sequence institute in overall recombination event
Accounting example: less than 12% (range are as follows: 2.6-11.5%)；Chromosome inversion proportion is less than 25% (range are as follows: 1.4-
22.8%), chromosome deficiency proportion range are as follows: 2-40.6%.SV: chromosomal structural variation, CTX: interchromosomal indexing,
DEL: large fragment deletion, INV: inversion, DUP: large fragment repeats.
The genome recombination feature of Fig. 3 HBV negative HCC
A. it takes delly software to analyze the genome recombination feature of 4 HBV negative tumours, finds its structure variation quantity
Low (the every sample of 8-200), and it is indexable (19-29%) to lack a high proportion of interchromosomal.Abscissa indicates sample number, ordinate
Indicate the quantity and composition ratio of each sample structure variation.The CNV thing of C.Controlfreec software analysis HBV negative HCC
Part finds that there are the hot spot regions that copy number changes in its shortage region 1p36.33.
The correlation analysis of Fig. 4 HBV associated hepatocellular carcinoma hHRD genome recombination fingerprint
A-B.hHRD genome recombination feature is related to HBV and the correlation of negative HCC, and interchromosomal turns as the result is shown
The indexs such as mean value and HBV associated hepatocellular carcinoma are highly relevant between position (A) and the sample of chromosomal structural variation quantity (B), and negative with HBV
Property liver cancer performance statistics difference (P < 0.001 or P=0.004).
C.1p36.33 the copy number change frequency in region.Analysis shows that the region copy number changes ratio in HBV associated hepatocellular carcinoma
Example is 13/16, and quantity is 0 in HBV negative HCC, it is therefore apparent that there are significant differences for the two.
Fig. 5, HBx transgenic mice and the genome recombination of WDR70 functional defect type mouse liver accumulation hHRD type refer to
Line.
A. the transgenic mice of liver-specific expression HBx measures liver full-length genome recombination feature after birth 6 months.Greatly
Part recombination event is that interchromosomal is indexable (accounting 53.1%).
There is high-frequency Chromosome recombination in the mouse liver genome of B.C57/B6-HBx and internal silencing WDR70,
Most of recombination event is that interchromosomal is indexable (accounting 83.3%), consistent with the variation features of HBV associated hepatocellular carcinoma genome.This
Group data illustrate HBx expression and WDR70 afunction is the principal element for pushing hHRD fingerprint characteristic to generate.In conjunction with these data
And HBx interferes the fact that the assembling of CRL4WDR70 protein complexes (not issue data), HBx is living by destroying CRL4WDR70
Property leads to the generation of the characteristic fingerprint of hHRD.
15, specific embodiment
Following non-limiting embodiment is used to illustrate selected embodiment of the invention.It should be understood that shown component
Ratio variation and alternative elements will be apparent to those skilled in the art, therefore be to fall into embodiment of the present invention
In the range of.
C57/B6 (SPF grades, female derives from Sichuan University's Experimental Animal Center), C57/B6-HBx in the following example
Transgenic mice (building of You Saiye company provides), C57 background WDR70 gene silencing mouse is voluntarily to construct, and only makees the present invention
Middle experiment is used, not as other purposes.HBV associated hepatocellular carcinoma sample used in embodiment comes from People's Hospital of Deyang City.Gene
It is according to pertinent literature basic information, independently that group credit analysis and inspection software, which are all genome signature alanysis process frames,
Research and development.Various reagents box and chemical reagent are commercial products, are used to test in the present invention, not as other purposes.
It is the fingerprint characteristic side hHRD that 1 HBV associated hepatocellular carcinoma genome medium-high frequency locus specificity of embodiment, which copies number variation,
Method: operation sampling fresh HCC and blood tissues, sample volume > 1cm3, blood sample is 5 milliliters of anticoagulations, and is centrifuged extraction blood
Cell.Sample liquid nitrogen flash freezer is placed in independent sealed package, prevents from polluting between sample, and basic clinical letter is marked on packing container
Breath.Sample, which is stored in -80 DEG C and was built in dry ice in two weeks, to be transported to sequencing company and carries out sample quality inspection and nucleic acid extraction and survey
Sequence.Qubit, 1%AGE is used to detect sample breakdown situation, concentration >=20ng/ul, total amount >=500ng, sample after genome purification
This can be used for downstream analysis sequencing without obvious degradation band.
Microarray dataset uses illumina2000 sequenator, uses Hiseq based on illumina matching technology microarray dataset
The bis- ends X-ten (paireed-End) bridge-type PCR sequencing PCR carries out full-length genome and resurveys sequence.Using independent research Soft Roll XTenseq-
(liver cancer)-LC--quality control carries out Quality Control.All sample Q30 >=80%.
Sequencing strategy defers to following below scheme:
1. sequencing library constructs.The total DNA ultrasound of independent sample is fractured into 350bp segment.Further, fragment ends are mended
It is flat, the end of segment 3 ' plus A, in addition joint sequence.Further, PCR method expands library, and amplified production AMPure XP system is pure
Change.Further, 2100 Bioanalyzer of Agilent analyzes fragment size distribution situation in library, and real-time quantitative PCR is fixed
It measures library concentration (3Nm) further, completes library DNA fasciation using HiSeq X PE Cluster Kit V2.5 kit
At process.Finally, complete fasciation at DNA library be available on the machine sequencing.Sequencing uses bridge amplification, finally reads segment (read)
Size is 150bp.
2. valid data screening analysis.Sequencing initial data is stored as FASTQ format.Data are then carried out to carry out except weight,
Screening Treatment.Screening strategy is removing 1) joint sequence, 2) read sequence in uncertain Sequence, 3) removal sequence in
Low-quality Sequence, 4) remove unrecognizable Sequence in data.3. comparing and genome mutation monitor plan
Slightly.Effective sequencing data is compared by Burrows-Wheeler Aligner (BWA), with reference to genome UCSC
hg19.Comparison result is with the generation of BAM format.SAM tools, Picard (http://broadinstitute.github.io/
Picard/), and GATK software is ranked up BAM file, repeating label, local directed complete set, and base calibration is further given birth to
At available BAM document, and detect sequencing depth and coverage.
With the CNV event on controlfree software detection HBV associated hepatocellular carcinoma genome.We refer to
Parameter guide on the official website controlfree, the first step first determine copy number diversity (CNP,
Copy-number polymorphism) initial count, wherein program calculates not duplicate reads number window ginseng
Number is set as 50000bp；Second step carries out CNP framework standard, and the key point in the step is that, referring to setting, this project is real
It applies in example using the blood tissues of corresponding individual as reference standard.Third step we in second step data carry out segmentation criteria
Processing.The variance predicted in model calculating is reduced to mention using lasso trick algorithm according to the method that Harchaoui et al. is proposed
Height is predicted accurate.The change of copy number in genome range is finally predicted according to lasso trick algorithm CNP reduced model obtained
Change (CNV), setting range is usually in 1kb to 3MB.
As a result: controlfreec software in all HBV related gene groups on No. 1 chromosome analysis shows that have identical
CNV hot spot accumulation regions.Thus we define the area as one of hHRD molecular fingerprint feature in HBV associated hepatocellular carcinoma genome.
Further analyze the sequence signature of CNV hot spot aggregation zone.We define more fine CNV hot spot site: 1p36.33
(0-2579999 base) (Fig. 1, partial data).It is presumed that the CNV variation within the scope of fixed area is just because of HBV correlation
A wide range of Genomic Imprinting caused by HR defective in liver cancer.This feature is defined as together with genome recombination feature
HHRD molecular fingerprint feature.The parameter is not found or reports in liver cancer and other tumours before this.
High proportion interchromosomal indexing event in 2 HBV associated hepatocellular carcinoma genome of embodiment is hHRD molecular fingerprint feature
One of
Method: it is further, thing is recombinated with the full-length genome of delly software definition statistics HBV associated hepatocellular carcinoma genome
Part.Software is based on soft-clipped principle.When lacking on genome, indexing, the structure variations such as insertion are caused across scarce
When unsceptered point reads is compared to genome, a reads is cut into two sections, is matched to different region (soft-clipped
Reads), for identifying chromosomal structural variation and exogenous array integration.The soft-clipped of at least three covering breaking points
The event that reads and soft-clipped reads is covered on joint sequence end can be screened out.It is pairs of for cancer
Sample (Tumor-Normal) carries out SV abrupt climatic change, obtains the SV systematic mutation data of tumour itself.
As a result: delly software is analysis shows that HBV associated hepatocellular carcinoma genome individual gene group detects recombination event, range 89-
702 every samples, average 292 every samples.High-caliber interchromosomal translocation mutant (CTX, averagely 149 with generality
A/sample), it is mutated in all SV event interchromosomal translocation mutants much higher than other types, this is HBV associated hepatocellular carcinoma genome
Significant characterization of molecules (Fig. 2) in sample.High-caliber indexing event shows in HBV associated hepatocellular carcinoma genome exist with general
The large area genetic mutation fingerprint that HR defective driving all over property meaning generates, this kind of fingerprint control homologous recombination targeting
Treating has direct Clinical significance of MG.The parameter is not found or reports in liver cancer and other tumours before this.
The genome recombination feature of 3 HBV negative HCC of embodiment
Method: ibid operation samples the fresh HCC of 4 HBV feminine genders and the blood tissues of corresponding individual (it is thin to extract blood
Born of the same parents), illumina microarray dataset and the bis- end (paireed- of Hiseq X-ten are based on using illumina2000 sequenator
End) bridge-type PCR sequencing PCR carries out full-length genome and resurveys sequence；Using the genome recombination feature of delly software analysis tumour, take
Controlfreec software analyzes CNV event.
As a result: A.delly software analysis finds that the genome structure variation quantity of four HBV negative HCCs is low, lacks high
The interchromosomal indexing of ratio.Abscissa indicates sample number, and ordinate indicates the quantity and ratio of components of each sample structure variation
Example.B.Controlfreec software analysis finds that there are copy number variations in four HBV negative HCCs its shortage region 1p36.33.
The correlation analysis of 4 HBV associated hepatocellular carcinoma hHRD genome recombination feature of embodiment
Method: using delly software analysis hHRD genome recombination feature in HBV correlation and negative HCC structure variation
Accounting value respectively.Calculate the copy number variation that the region 1p36.33 in HBV associated hepatocellular carcinoma and single sample of HBV negative HCC is deposited
Appearance ratio, mean value between chromosomal structural variation quantity and the sample of interchromosomal indexing, and it is significant using t check analysis
Sex differernce.
As a result: the copy number change frequency in the display region 1p36.33 is 13/16 in HBV associated hepatocellular carcinoma, and in HBV yin
Property liver cancer in quantity be 0, it is therefore apparent that there are significant differences for the two.Chromosomal structural variation quantity and interchromosomal indexing
Sample between the indexs such as mean value and HBV associated hepatocellular carcinoma it is highly relevant, and show with HBV negative HCC statistically significant significant
Sex differernce (P < 0.001 or P=0004).
The genome recombination fingerprint of 5 HBx transgenosis of embodiment and WDR70 gene silencing mouse liver accumulation hHRD type
Method: in order to further verify the hHRD fingerprint characteristic observed in liver cancer sample at us, we are constructed
The sequencing of two class animal models is further verified.One is liver specificities to express (albumin gene promoter) hepatitis B cancer
The mouse model of gene HBx, another kind are homologous recombination repair GAP-associated protein GAP WDR70 functional defect types identical with cell model
Mouse model.The building of transgenosis and gene silencing mouse is as follows:
1. the HBx transgenic mice that liver specificity expresses (albumin gene promoter) carries out structure by Guangzhou Sai Ye company
It builds.
2.WDR70 gene silencing mouse carries out gene silencing using internal perturbation technique.To interfere WDR70 gene expression
And with internal small fragment siRNA, ((siG1117130927,5 '-CUGCCAGAAUGGAAGCAUA-3 ') are by the auspicious rich public affairs in Guangzhou for control
Take charge of (Ribobio, Guangzhou, China) synthesis.SiRNA and internal transfection reagent (Biotool, B45215) mixing rear molding are quiet
Arteries and veins injects 4-6 week old C57 mouse.Injection is primary weekly, and continuous injection 3 months.
3. all model mices take liver region up to or so 4 monthly ages and control group mouse of the same age, genome sequencing is carried out.
All mouse samples send survey mode and detection, sequencing, and analysis platform is consistent with the above.Mutant mice genomic data with mark
Quasi- genome (GCF_000001635.26_GRCm38.p6_genomic.fna) is compared.
As a result: parsing the genome of HBx expression or WDR70 gene silencing mouse model respectively with delly software, as a result
Showing has the recombination variation features similar with HBV associated hepatocellular carcinoma: the ratio of interchromosomal indexing event in two class model mouse
Highest (HBx expression mouse: 56.3%；WDR70 gene silencing mouse: 83.3%) (Fig. 3).The recombination feature of this hHRD type
The expression of HBx is shown with the high similarity of HBV associated hepatocellular carcinoma and WDR70 functional defect is driving hepatitis B phase in vivo
The same mechanism for closing liver cancer chromosomal variation, ultimately causes the imprinting signature being identical in genomic level.Two class mouse moulds
The HBV associated hepatocellular carcinoma homologous recombination machinery that the hHRD type gene group recombination feature strong support occurred in type is predicted before this lacks
It falls into；The fingerprint characteristic of this kind of hHRD is different from the BRCA1/2 mutation HR defective feature being currently known, and is CRL4WDR70
Function lowers or the peculiar characterization of molecules of the Oncogenome variation of HBx expression driving.
Claims (17)
1. one kind has for identifying the genome recombination fingerprint of specific mechanism homologous recombination repair defect (hHRD) as selected from figure
Fingerprint characteristic shown in an at least figure in 1-2.
2. a kind of genome recombination marker relevant to hHRD type homologous recombination repair defect, has such as claim 1 institute
The genome recombination fingerprint stated is used to identify cell, gene or the related disease for having hHRD characteristic fingerprint.
3. genome recombination marker as claimed in claim 2, the genome recombination fingerprint includes in the swollen of independent sample
There is at least one following core feature: A. interchromosomal indexing ratio proportion in recombination event in tumor gene group
50-92%；B.1p36.33 copy number change occurs for 0-2579999 base range and in the region 1p36.33, including on No. 1 chromosome
Strange happening part, the event include missing copy number=1 or 0, and copy number amplification >=3 or/and range of variation are 1kb-3MB；C. it uses
Delly software detects the genome structure variation or recombination 89-602 event of quantitative range of single Oncogenome, and goes here and there
Joining repetitive sequence, proportion is less than 12% in overall recombination event, and chromosome inversion proportion is less than 25%.
4. genome recombination marker as claimed in claim 3, the structure variation or recombination quantity of the Oncogenome are adopted
It is parsed with improved delly software, the copy number variation is extracted using the operation of controlfree software.
5. the genome recombination marker as described in claim 2-3, the genome recombination fingerprint can be used for distinguishing due to BRCA1
With BRCA2 gene mutation, the genome recombination fingerprint that expression is lowered or other function is extremely caused.
6. the genome recombination marker as described in claim 2-3, the hHRD type homologous recombination repair defect, including spy
Determine caused by mechanism with the mono- ubiquitination signal path dysfunction of CRL4WDR70-H2B, the obstacle include CUL4A, DDB1,
WDR70 and/or the relevant gene mutation of the mono- ubiquitination of histone H2B or protein function lower, and due to hepatitis B
CRL4WDR70 afunction caused by the oncogene HBx expression of poison coding.
7. the genome recombination marker as described in claim 2-3, the hHRD HR defective, including it is drug-induced on
State the exception of homologous recombination repair caused by the dysfunction of signal transduction pathway.
8. genome recombination marker as claimed in claim 7, the drug includes demethylation enzyme inhibitor or removes acetyl
Change enzyme inhibitor.
9. genome recombination fingerprint described in claim 1, as application of the indentifying substance in medical diagnosis on disease, the disease is
The relevant disease of homologous recombination repair defect.
10. the utilization of genome recombination fingerprint as claimed in claim 9, the disease of the homologous recombination repair defect, including
But it is not limited to hepatitis b virus infected related disease, with BRCA1/BRCA2 mutation, gene expression attenuating or dysfunction
Cancer, or without BRCA1/BRCA2 mutation, gene expression attenuating or dysfunction but still performance HR defective
Cancer.
11. the utilization of genome recombination fingerprint as claimed in claim 10, the hepatitis b virus infected related disease are
Physiology caused by oxyhepatitis, chronic hepatitis, liver fibrosis, cirrhosis, liver cancer, cholangiocellular carcinoma and hepatitis B virus infection
Indexes Abnormality, circulatory system symptom, immunosupress and gastrointestinal symptom, preferably hepatitis b virus infected correlation Acute Hepatic
Inflammation, chronic hepatitis, liver fibrosis, cirrhosis, liver cancer or cholangiocellular carcinoma.
12. the utilization of genome recombination fingerprint as claimed in claim 10, the tumour includes breast cancer, oophoroma, uterus
Inner membrance (sample) cancer, clear cell carcinoma of ovary, cutaneum carcinoma, gastric cancer, cancer of pancreas or prostate cancer.
13. the utilization of the genome recombination fingerprint of any one of claim 8-11, including provide for above-mentioned disease type homologous heavy
Group repairs the auxiliary identification of defect, provides medication foundation for the treatment of homologous recombination targeted drug, is homologous with such hHRD
The prophylactic surgery excision of the oophoroma of defective recombinant, breast cancer and prostate cancer provides operation foundation.
14. the utilization of genome recombination fingerprint as claimed in claim 13, the homologous recombination targeted drug is PARP inhibition
The use of agent or its combining with other anticancer drugs etc., further include said medicine and other radiation and chemotherapy drug (preferably platinum
Class) use in conjunction.
15. the utilization of genome recombination fingerprint as claimed in claim 14, the PARP inhibitor includes including but is not limited to
Phthalazines ketone compounds and 2- [4- ((3S) -3- piperidyl) phenyl] -2H- indazole -7- formamide, BGB-290, Wei Lipani,
Ni Lapani, Lu Kapani, E7449, Talazoparib and BGP-15 or its pharmaceutical salts.
16. genetic recombination fingerprint or marker and other hHRD genome signatures mutation fingerprint connection that above-mentioned all authority requires
It closes and uses, identify the HR defective and its related disease of hHRD type.
17. a kind of method for the genome for identifying specific mechanism homologous recombination repair defect (hHRD), comprising the following steps:
1) by vitro body fluid or tissue through gene sequencing；
2) data for obtaining sequencing are parsed using delly software or are extracted using the operation of controlfree software；
3) genomic map is obtained after software is handled；
4) the genome recombination fingerprint comparison of the finger-print and claim 1 that obtain step 3), or
5) genome is compared with the genome recombination marker of claim 2.
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