CN110317876A - Application of the unstable variation of one group chromosome in preparation diagnosis Huppert's disease, the reagent or kit of assessing prognosis - Google Patents
Application of the unstable variation of one group chromosome in preparation diagnosis Huppert's disease, the reagent or kit of assessing prognosis Download PDFInfo
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Abstract
The present invention relates to biotechnologys and area of medical diagnostics, application of the specifically group chromosome unstable region in the reagent or kit of preparation diagnosis Huppert's disease, it includes following 7 that the chromosome instability, which determines region: 1p, 1q, 6q, 11q13.3,13q, 14q, 17p.The present invention collects the multiple myeloma patients of clinical definite and the sample of Healthy People, pass through genome sequencing comparison of tumor patient and the chromosomal abnormalities that compare, illustrate chromosome instability distribution characteristics specific to Huppert's disease, it discloses 7 common chromosome instabilities of multiple myeloma patients and determines region, total carrying rate is up to 89.3%, this has a very big significance the diagnosis of clinically Huppert's disease, treatment, monitoring meter prognosis evaluation, is early diagnosed and is formulated individualized treatment scheme for next step and provide scientific basis.
Description
Technical field
The present invention relates to biotechnologys and medical diagnostic techniqu field, specifically, being that one group of Huppert's disease is related
Chromosome instability variation and these unstability variation preparation diagnosis Huppert's disease, assess prognosis reagent
Or the application in kit.
Background technique
Huppert's disease (Multiple Myeloma, MM) is a kind of pernicious characterized by gathering thick liquid cell in marrow
Tumour can lead to destruction of bone and marrow failure.According to statistics, MM accounts for the 10% of the 1% of all cancers, malignant hematologic disease, and 2018
Share 30770 new hair MM year, 12770 patients MM are dead.International Myeloma working group (Internastional in 2014
Myeloma Working Group, IMWG) it modifies to MM diagnostic criteria, the disease development of MM is divided into three phases, point
It Wei not meaning monoclonal immunoglobulin blood sick (MGUS), type of smoldering myeloma (SMM) and the activity myeloma run after fame.It is multiple
Property myeloma have that apparent cytogenetics is heterogeneous, cytogenetic exception is to the risk assessment of patient MM, treatment, in advance
After have an impact[5][6][7].MM cytogenetics pathogenesis includes the easy of No. 14 chromosome coding immunoglobulin heavy chain locus
The amplification and missing of position, odd number chromosome hyperdiploid and chromosome arm.Multiple academic articles, which report, loses these cells
The research for learning variation is passed, and its risk is classified according to its influence to treatment and prognosis.
Chromosome instability is fixed usually related to tumour, specifically includes the missing of whole chromosome or chromosome segment copy
Or amplification.Tumour is often containing the amplification and missing that relevant chromosome or chromosome segment occur to tumour, and institute spy occurs
Have, the detection to tumor staining body unstable region, research tumour is occurred and exploitation diagnosing tumor technology is all to pass
It is important.There is the method using Fluorescence in situ hybridization on Present clinical, unstability detection subregional for chromosome top, but
The distribution characteristics for lacking multiple myeloma patients whole gene group level chromosome instability, has more fully been instructed multiple
The diagnosis and prognosis of property myeloma.
Summary of the invention
It is special that present invention aims at the distributions of chromosome instability specific to research China's Huppert's disease (MM)
Sign, the detailed process and chromosome instability for disclosing full-length genome two generations sequencing approach determine region, it was found that 7 and multiple bone
Common chromosomal variation region occurs for myeloma, and total incidence accounts for 89.3% in Huppert's disease, is clinical early in next step
Phase diagnosis and formulation individualized treatment scheme provide scientific basis.
The first aspect of the present invention provides the chromosome instability often occurred in one group of China's multiple myeloma patients and determines area
Domain, including following 7: 1p, 1q, 6q, 11q13.3,13q, 14q, 17p.
Wherein 1q amplification may cause proto-oncogene MCL1 high expression, promote tumour progression;13q loss leads to tumor suppressor gene
RB1 low expression;17p missing leads to TP53 gene low expression, promotes tumour progression;The regions such as 1p, 6q, 11q13.3,14q are lost
It loses or amplification loss also leads to suppressor genes correlation low expression, promote tumour progression.
The second aspect of the present invention provides a group chromosome unstable region in the reagent of preparation diagnosis Huppert's disease
Or the application in kit, the chromosome instability determine region include following 7: 1p, 1q, 6q, 11q13.3,13q, 14q,
17p。
Further, the chromosome instability includes the missing or expansion of whole chromosome or chromosome segment copy surely
Increase.
Third aspect present invention provides a group chromosome unstable region in preparation treatment Huppert's disease drug
Using it includes following 7 that the chromosome instability, which determines region: 1p, 1q, 6q, 11q13.3,13q, 14q, 17p.
Fourth aspect present invention provides a group chromosome unstable region in the examination of preparation Huppert's disease assessment prognosis
Application in agent or kit, the chromosome instability determine region include following 7: 1p, 1q, 6q, 11q13.3,13q,
14q、17p。
Fifth aspect present invention provides a group chromosome unstable region in the examination of preparation Huppert's disease early screening
Application in agent or kit, the chromosome instability determine region include following 7: 1p, 1q, 6q, 11q13.3,13q,
14q、17p。
Sixth aspect present invention provides a group chromosome unstable region in the examination for preparing the monitoring of myelomatosis multiplex feelings
Application in agent or kit, the chromosome instability determine region include following 7: 1p, 1q, 6q, 11q13.3,13q,
14q、17p。
Seventh aspect present invention provides a kind of Huppert's disease screening, diagnosis, the examination of prognosis evaluation or disease surveillance
Agent box includes in kit: 1) detecting one or more reagents that 7 chromosome instabilities as described above determine region;2) it is selected from
One or more components of the following group: specification, positive reference product, negative reference product, buffer, enzyme, library connector, detectable mark
Label, nucleic acid extracting reagent, nucleic acid purification reagent.
Detection kit of the invention has the operation instructions of kit, includes how to carry out experiment behaviour in specification
Make, how according to data result to judge how whether above-mentioned 7 chromosomal regions occur unstable variation and result to multiple
The diagnosis of property myeloma, treatment, prognosis evaluation etc. are illustrated.
Using kit of the invention, above-mentioned 7 can be detected by various methods (including but not limited to) selected from the group below
Chromosomal region: digital pcr, Fluorescence in situ hybridization, nucleic acid probe hybridization method etc..
In addition, the kit also include clinically for the screening of Huppert's disease, diagnosis, therapeutic scheme selection,
Other reagents of monitoring and prognosis evaluation, to assist or be verified the detection obtained result of above-mentioned 7 chromosomal regions.This
Field those of ordinary skill carries out conventional selection according to specific needs.
Using kit of the invention, selected using the screening, diagnosis, therapeutic scheme that following methods carry out Huppert's disease
It selects, monitor and prognosis evaluation: 1) obtaining sample to be tested from object;2) sample to be tested is contacted with using detection reagent of the invention;
3) above-mentioned 7 chromosomal regions of sample to be tested are detected;4) screening of Huppert's disease is carried out according to testing result, is diagnosed, is controlled
Treat Scheme Choice, monitoring and prognosis evaluation.
Eighth aspect present invention provides a kind of unique library connector with specific label for the sequencing of two generations, is used for
Exclude when machine is gone up in other types library and library of the present invention simultaneously the mistake because caused by library index sequence is same or similar.
Further, the nucleotide sequence of the library connector with specific label is respectively as follows:
Connector -1 (SEQ ID NO.1):
5'-CGTGATAGATCGGAAGAGCACACGTCTGAACTCCAGTC-3';
Connector -2 (SEQ ID NO.2):
5’-ACACTCTTTCCCTACACGACGCTCTTCCGATCTATCACGT-3’。
Ninth aspect present invention provides a kind of library connector with specific label for the sequencing of two generations as described above
Application in the kit of preparation diagnosis Huppert's disease.
The technical solution adopted in the present invention:
The sequencing of two generations, analysis are carried out to 347 multiple myeloma patients of definitive pathological diagnosis, the nucleic acid of 176 Healthy Peoples
It counts tumor patient and control group and threshold value delimited, to reach higher by ROC curve in the difference of the fixed aspect of chromosome instability
Sensitivity and specificity.
The method that the present invention uses is sequenced for genome sequencing or target area targeted capture, passes through low depth high pass
Sequence is measured, chromosomal copy number is analyzed, identification occurs chromosome instability and determines region.This method is easy to operate, accuracy
Height, the chromosome instability that can be detected 0.1% are determined.Due to the high sensitivity of this method, it can be widely used for various clinical sample classes
Type, such as body fluid, tissue, cell.
Target area catching method includes but is not limited to probe hybrid method, PCR amplification method.
We are interrupted human genome using physics or enzymatic cleavage methods, or directly use the Cell-free DNA of fragmentation,
It repaired by end, add A, the connector of specific label is had in connection, by amplification plus for distinguishing sample index, obtained literary
Library.Then library is quantified, upper machine sequencing.
Upper machine data analysis is completed in four steps: (a) sequence alignment examines genome sequence to ginseng;(b) to coverage
Carry out G/C content and the correction of other weighting coefficients;(c) relative cover Z-score is calculated, the counting of test sample subtracts ginseng
According to sample counting, then the standard deviation covered divided by the sample for reference section;(d) Z-score is more than given threshold (such as 3), fixed
Justice is that target area expands, and Z-score is lower than given threshold (such as -3), is defined as target area loss.
It can be the reference sequences that NCBI or UCSC are provided that genome is referred in step a.Comparison software is any software, packet
Include freeware BWA, SOAPaligner, Bowtie etc., including business software OmicSoft etc..
In step b, weighting coefficient is the possible correction coefficient for calculating error of sequencing sequence feature, and copy number calculates
When (Z-score) be the factor for influencing copy number and calculating, including G/C content, if repetitive sequence etc., chromosome translocation (B-
To influence the factor that dystopy calculates when Score) calculating, simple repeated sequence and length are included whether, if genome is conservative
Sequence, if include STR sequence etc..
In step c, this preferred case, sample for reference (no less than 10) is healthy sample for reference.
By aforesaid operations, it was found that with the very high 7 common chromosome abnormality regions of Huppert's disease correlation, packet
Containing 1p, 1q, 6q, 11q13.3,13q, 14q, 17p.Wherein 1q amplification may cause proto-oncogene MCL1 high expression, promote tumour
Progress;13q loss leads to tumor suppressor gene RB1 low expression;17p missing leads to TP53 gene low expression, promotes tumour progression;1p,
The regions such as 6q, 11q13.3,14q or loss or amplification, which are lost, also leads to suppressor genes correlation low expression, promotes tumour progression.
From the point of view of carrying frequency, 7 chromosome instabilities determine region and are up to 89.3% in multiple myeloma patients, amount to
310 patients carry above-mentioned chromosome instability and determine region.Wherein unstable most commonly seen with 1q, carrying rate is up to 59.7%;
13q, 17p exception occurrence frequency are only second to 1q, are 40.6% and 27.7%;Other chromosome abnormality incidences are respectively 1p
(20.3%), 6q (15.2%), 11q13.3 (12.6%), 14q (15.8%).
By aforesaid operations, it was found that the included chromosome instability of aforementioned present invention is determined region and do not examined in normal healthy controls
It is abnormal out.
The invention has the advantages that:
The present invention collects the multiple myeloma patients of clinical definite and the sample of Healthy People, passes through genome sequencing ratio
Compared with tumor patient and the chromosomal abnormalities compareed, it is special to illustrate the distribution of chromosome instability specific to Huppert's disease
Sign, discloses 7 common chromosome instabilities of multiple myeloma patients and determines region, and total carrying rate is up to 89.3%, this is to facing
The diagnosis, treatment of Huppert's disease, monitoring meter prognosis evaluation have a very big significance on bed, to be early diagnosed in next step
Scientific basis is provided with individualized treatment scheme is formulated.
Detailed description of the invention
Fig. 1 chromosome instability setting analysis result figure;A:1p-, 1q+, 6q- (part 6p+);B:11q13.3+, 13q-,
14q-;C:17p-.
Fig. 2 detection method ROC curve figure.
Specific embodiment
It elaborates below with reference to embodiment to specific embodiment provided by the invention.
Embodiment 1: the preparation of library connector
Via third company's synthetic linker sequence:
Connector -1 (SEQ ID NO.1):
5'-CGTGATAGATCGGAAGAGCACACGTCTGAACTCCAGTC-3';
Connector -2 (SEQ ID NO.2):
5’-ACACTCTTTCCCTACACGACGCTCTTCCGATCTATCACGT-3’。
5 ' ends of connector -1 carry out phosphorylation modification, carry out thiophosphoric acid modification between 3 ' end G and T of connector -2.
The dry powder of connector -1 and connector -2 is diluted to 20 μM respectively using annealing buffer (Annealing buffer),
The two dilution equal proportion is mixed, by mixed liquor as in PCR instrument, runs program such as the following table 1:
Table 1
Product is subjected to agarose electrophoresis, electrophoretic band magnitude range is between 80-100bp.
Embodiment 2:
Use material:
Human gene group DNA
DNA enzymatic, which is cut, interrupts reagent (Enzymatics)
The end DNA is repaired plus A, adjunction head enzyme reaction system (NEB)
Pcr amplification reaction system (Kapa Biosystems)
It purifies magnetic bead (Beckman)
Operating procedure:
1, genomic fragment
20ng genomic DNA is taken, using endonuclease reaction system, reaction system is configured according to table 2, is reacted according to table 3.
Is needed not move through by genomic fragment, is directly entered down such as the cfDNA in blood for cell-free dissociative DNA
Step operation.
Table 2
DNA interrupts reaction | System |
Genomic DNA | xμL |
Interrupt enzyme | 3μL |
Buffer Buffer | 7μL |
Seedless sour water | 25-xμL |
Total volume | 35μL |
Oscillation mixes, runs following procedure in PCR instrument after centrifugation (avoiding bubble):
Table 3
2, end, which is repaired, adds A
Add A reaction system such as the following table 4 with set terminal reparation:
Table 4
Oscillation mixes, runs following procedure in PCR instrument after centrifugation (avoiding bubble):
Table 5
3, adjunction head and product purification
Connector is connected into premixed liquid (30 μ L/ sample), connector link enhancement agent (1 μ L/ sample) according to sample detected
After number absorption volume appropriate is mixed, the end-o f-pipe -control reaction mixture that 15.5 μ L mixed liquors are added to previous step is drawn
In, the connector prepared in 2.5 μ L/ embodiments 1 is drawn again after oscillation mixing, centrifugation is added in above-mentioned reaction solution, oscillation mixing,
Centrifugation.Ultimately form the reaction system in the following table 6.
Table 6
Above-mentioned system is placed in PCR instrument, run program: 20 DEG C, 30min, heat lid is closed.
Purification step after connection reaction:
A, library is taken out from magnetic bead kit in advance and purify magnetic bead, be stored at room temperature at least 30min, need to mix before use.
B, the connector connection reaction solution in above-mentioned steps is transferred to the 1.5mL centrifuge tube accordingly numbered, 86 μ L weight is added
Outstanding library purifies magnetic bead, is at the uniform velocity inhaled and is made a call to 20 times using the pipettor of appropriate range, is incubated at room temperature 5min.
C, centrifuge tube is placed on magnetic frame, after solution clarification, abandons supernatant liquor.
D, the ethyl alcohol of 200 μ L 80% of fresh configuration is added thereto, after standing 30s, abandons supernatant liquor.
E, it is primary to repeat step d.
F, centrifuge tube, brief centrifugation 3sec are removed on magnetic frame, centrifuge tube is put back on magnetic frame, and suction discards remaining 80%
Ethyl alcohol is careful not to be drawn onto magnetic bead.Pipe lid is opened, room temperature dries 2-10min.
G, to magnetic bead at matt color, 16 μ L Low TE buffers (or nuclease-free water) are added in centrifuge tube, it is slight to shake
Swinging is resuspended magnetic bead, is incubated at room temperature 5min.
H, centrifuge tube is placed on magnetic frame, stands 2min.After solution clarification, 15 μ L supernatants is taken to remain to expand in next step
Reaction.
4, PCR amplification and product purification
According to the following table 7, corresponding reagent is added into PCR pipe:
Table 7
The PCR pipe mixed is put into PCR instrument, following procedure is run.
Table 8
PCR product purifying:
A, library is taken out from magnetic bead kit in advance and purify magnetic bead, be stored at room temperature at least 30min, need to mix before use.
B, the connector connection reaction solution in above-mentioned steps is transferred to the 1.5mL centrifuge tube accordingly numbered, 22.5 μ L is added
The library of resuspension purifies magnetic bead, is at the uniform velocity inhaled and is made a call to 20 times using the pipettor of appropriate range, is incubated at room temperature 5min.
C, centrifuge tube is placed on magnetic frame, after solution clarification, abandons supernatant liquor.
D, the ethyl alcohol of 200 μ L 80% of fresh configuration is added thereto, after standing 30s, abandons supernatant liquor.
E, it is primary to repeat step d.
F, centrifuge tube, brief centrifugation 3sec are removed on magnetic frame, centrifuge tube is put back on magnetic frame, and suction discards remaining 80%
Ethyl alcohol is careful not to be drawn onto magnetic bead.Pipe lid is opened, room temperature dries 2-10min.
G, to magnetic bead at matt color, 31 μ L Low TE buffers (or nuclease-free water) are added in centrifuge tube, it is slight to shake
Swinging is resuspended magnetic bead, is incubated at room temperature 5min.
H, centrifuge tube is placed on magnetic frame, stands 2min.After solution clarification, 30 μ L supernatants is taken to remain to expand in next step
Reaction.
5, machine is quantitatively gone up in library
Above-mentioned purified library analyzes clip size using Agilent BioAnalyzer, uses
DsDNA HS Assay Kit measures Library Quality concentration, and it is dense to calculate library mole by mass concentration and clip size
Degree, is sequenced according to upper sequenator specification using Illumina Hiseq X-ten.
Interpretation of result discussion:
After upper machine sequencing data data analysing method described in this patent, chromosome sequencing depth profile is obtained, such as
Shown in Fig. 1.Analysis result is made of three parts: depth distribution, dyeing is sequenced in chromosome numbers, chromosome subregion and chromosome
Body be sequenced depth distribution region orbicular spot be chromosome zonule copy number distribution situation, when its in longitudinal axis scores value-3-
Determine to be judged to expanding when its scores value is greater than 3, sentence when its scores value is less than -3 without unstable generation when between 3
It is set to missing, the region for occurring to expand or lack is distinguished using gray background and normal region.By analyzing result
Check whether that above-mentioned chromosome instability occurs surely, if being determined as the positive, if not being determined as feminine gender.In two generations, were analyzed
As a result it is compared with pathological examination, calculates the sensitivity and specificity of this analysis method, obtain ROC curve, as shown in Figure 2.
The AUC area of the method for the present invention ROC curve reaches 0.958, illustrates unstable using 1p, 1q, 6q, 11q13.3,13q, 14q, 17p
It is set for identifying for analysis, diagnosis Huppert's disease, which is sequenced, by two generations has very high sensitivity and specificity.
The preferred embodiment of the present invention has been described in detail above, but the invention be not limited to it is described
Embodiment, those skilled in the art can also make various equivalent on the premise of not violating the inventive spirit of the present invention
Variation or replacement, these equivalent variation or replacement are all included in the scope defined by the claims of the present application.
Sequence table
<110>Suzhou Hong Yuan Biotechnology Co., Ltd
The unstable variation of<120>one group chromosomes is in preparation diagnosis Huppert's disease, the reagent or kit of assessing prognosis
Application
<130> /
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 38
<212> DNA
<213>artificial sequence (Artificial)
<400> 1
cgtgatagat cggaagagca cacgtctgaa ctccagtc 38
<210> 2
<211> 40
<212> DNA
<213>artificial sequence (Artificial)
<400> 2
acactctttc cctacacgac gctcttccga tctatcacgt 40
Claims (9)
1. application of the group chromosome unstable region in the reagent or kit of preparation diagnosis Huppert's disease, described
It includes following 7 that chromosome instability, which determines region: 1p, 1q, 6q, 11q13.3,13q, 14q, 17p.
2. reagent or examination of the group chromosome unstable region according to claim 1 in preparation diagnosis Huppert's disease
Application in agent box, which is characterized in that the chromosome instability includes that whole chromosome or chromosome segment copy surely
Missing or amplification.
3. application of the group chromosome unstable region in preparation treatment Huppert's disease drug, the chromosome instability
Determining region includes following 7: 1p, 1q, 6q, 11q13.3,13q, 14q, 17p.
4. application of the group chromosome unstable region in the reagent or kit of preparation Huppert's disease assessment prognosis, institute
It includes following 7 that the chromosome instability stated, which determines region: 1p, 1q, 6q, 11q13.3,13q, 14q, 17p.
5. application of the group chromosome unstable region in the reagent or kit of preparation Huppert's disease early screening, institute
It includes following 7 that the chromosome instability stated, which determines region: 1p, 1q, 6q, 11q13.3,13q, 14q, 17p.
6. application of the group chromosome unstable region in the reagent or kit for preparing the monitoring of myelomatosis multiplex feelings, institute
It includes following 7 that the chromosome instability stated, which determines region: 1p, 1q, 6q, 11q13.3,13q, 14q, 17p.
7. a kind of Huppert's disease screening, Diagnosis of Multiple Myeloma, Huppert's disease prognosis evaluation or multiple bone
The kit of myeloma disease surveillance includes: 1) detecting one or more reagents that 7 chromosome instabilities determine region;2) it is selected from down
One or more components of group: specification, positive reference product, negative reference product, buffer, enzyme, library connector, detectable mark
Label, nucleic acid extracting reagent, nucleic acid purification reagent;7 chromosome instabilities determine region and include: 1p, 1q, 6q, 11q13.3,
13q、14q、17p。
8. a kind of library connector with specific label for the sequencing of two generations, which is characterized in that described has specific label
Library connector nucleotide sequence as shown in SEQ ID NO.1 or SEQ ID NO.2.
9. a kind of library connector with specific label as claimed in claim 8 is in the reagent of preparation diagnosis Huppert's disease
Application in box.
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RU2771933C1 (en) * | 2021-03-15 | 2022-05-13 | Федеральное государственное бюджетное учреждение науки "Кировский научно-исследовательский институт гематологии и переливания крови Федерального медико-биологического агентства" | Method for identifying chromosomal abnormalities by the fish method in multiple myeloma |
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RU2771933C1 (en) * | 2021-03-15 | 2022-05-13 | Федеральное государственное бюджетное учреждение науки "Кировский научно-исследовательский институт гематологии и переливания крови Федерального медико-биологического агентства" | Method for identifying chromosomal abnormalities by the fish method in multiple myeloma |
CN113025721A (en) * | 2021-04-28 | 2021-06-25 | 苏州宏元生物科技有限公司 | Prostate cancer diagnosis and prognosis evaluation kit |
RU2789782C1 (en) * | 2022-05-04 | 2023-02-09 | Федеральное государственное бюджетное учреждение науки "Кировский научно-исследовательский институт гематологии и переливания крови" Федерального медико-биологического агентства" | Method for determining hyperdiploidy using morphometric analysis in multiple myeloma |
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