CN109321653A - It is a kind of for diagnosing the probe combinations and its application of PRCC-MITF transposition kidney - Google Patents
It is a kind of for diagnosing the probe combinations and its application of PRCC-MITF transposition kidney Download PDFInfo
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Abstract
The invention discloses a kind of for diagnosing the probe combinations and its application of PRCC-MITF transposition kidney.It is a kind of for diagnosing the probe combinations of PRCC-MITF transposition kidney, which is combined into BAC cloning probe RP11-867E4 and BAC cloning probe RP11-963H1.Wherein RP11-867E4 is positioned at the centromere PRCC side, labeled as the fluorescence of any one color；RP11-963H1 is positioned at MITF telomere side, labeled as the different fluorescence of the color marked from the centromere PRCC side.Probe of the present invention is preparing the application in MITF transposition Diagnosis of Renal Cell Carcinoma reagent.The specificity and sensibility of probe combinations diagnosis have reached 100%.
Description
Technical field
The invention belongs to fluorescence in situ hybridization probe application fields, are related to a kind of for diagnosing PRCC-MITF transposition kidney
Fluorescence in situ hybridization (FISH) the fusion probe combinations of cancer and its preparation PRCC-MITF transposition Diagnosis of Renal Cell Carcinoma reagent in
Application.
Background technique
New edition WHO tumor of kidney Pathological cassification has increased a kind of clear-cell carcinoma type: MiT family transposition newly within 2016
Property clear-cell carcinoma.MiT family is microphthalmia transcription factor family (microphthalmia-associated
Transcription factor family) abbreviation, which includes MITF, TFE3, TFEB and TFEC gene.
The MiT family transposition clear-cell carcinoma having now been found that includes Xp11.2 transposition/TFE3 Gene Fusion correlation
Clear-cell carcinoma and t (6；11)(p21；Q12) transposition/TFEB Gene Fusion correlation kidney.MITF and TFEC group translocation swells
Tumor is worldwide temporarily without document report.
MiT family transposition clear-cell carcinoma pathogenesis clear and definite, MiT family member gene (TFE3/TFEB) it is easy
Position is its key pathogenetic factor: this kind of tumour all refers to MiT family member gene with other chromosome translocations and its caused formation
Fusion, by promoter transformation to high expression TFE3/TFEB fusion protein, and TFE3/TFEB as a transcription because
Son, by combining with special DNA structure, in transcriptional control body, several genes expression is final causes a disease.At least 10 kinds at present
Different transposition companions and fusion are reported, including ASPL-TFE3, PRCC-TFE3, SFPQ-TFE3, NONO-TFE3,
CLTC-TFE3, LUC7L3-TFE3, KHSRP-TFE3, PARP14-TFE3, DVL2-TFE3, RBM10-TFE3 and MALAT1-
TFEB etc., every tumour is interior, and there is only single translocated forms.
MiT family transposition clear-cell carcinoma is a rare tumor types, and it is thin that TFE3 group translocation kidney accounts for about all kidneys
The 1.6%-4% of born of the same parents' cancer, TFEB group translocation kidney are relatively more rare.But the disease is gently prominent special with age of onset
Sign, accounts for the 40% of renal cell carcinoma in preschool children, and cause extremely heavy family and burden on society.In addition, there is positive evidence to show MiT
The patient of family's transposition clear-cell carcinoma is to vascular endothelial growth factor receptor (vascular endothelial growth
Factor receptor, VEGFR) or mammal rapamycin (mammalian target of rapamycin, mTOR)
Molecular targeted therapy is sensitive.Separately some researches show that MET tyrosine kinase is the target gene of ASPL-TFE3 fusion, and is expected to
Therapy target as TFE3 transposition tumour.Therefore, the such tumour of Precise Diagnosis seems particularly significant.
Team of the present invention meets MiT family transposition clear-cell carcinoma spy by high throughput sequencing technologies, in an example morphology
PRCC-MITF fusion is detected in the case of sign, which is to find for the first time, both at home and abroad without report, and this
It is international discovery MITF group translocation associated renal cell carcinoma for the first time.Due to the detection hand in the past without being directed to the group translocation
Section, can guess, previous MITF group translocation associated renal cell carcinoma all by mistaken diagnosis or is failed to pinpoint a disease in diagnosis.
High-flux sequence is the detection means that can uniquely specify unknown transposition site at present, however high-flux sequence expense
High, detection cycle is long, and detection platform is rare, requires height to sample quality, is unfavorable for popularizing, Most patients are come
It says nor preferred detection means.
Chromosome fluorescence in-situ hybridization (FISH) starts from the combination of traditional cytogenetics and DNA technique, rapid sensitive,
Specificity is good, can detecte concealment or small chromosome aberration and complex karyotype；A variety of fluorescent markers can also be used, are shown
Show the relative position and direction between DNA fragmentation and gene, space orientation is accurate.In addition, the method for FISH, it can be in paraffin packet
Retrospective study is carried out on the sample buried, greatly reduces the requirement to research sample.Currently, utilizing fluorescence in situ hybridization
(FISH) method of PRCC-MITF fusion is detected both at home and abroad without report.
Summary of the invention
It is a kind of for diagnosing the probe groups of MITF transposition kidney the purpose of the present invention is providing in view of the above technical problems
It closes.
Another object of the present invention is to provide above-mentioned probe combinations and is preparing answering in MITF transposition Diagnosis of Renal Cell Carcinoma reagent
With.
Further object of the present invention is to provide the diagnostic kit containing above-mentioned probe combinations.
The purpose of the present invention is what is realized by following technical proposal:
Team of the present invention meets MiT family transposition clear-cell carcinoma spy by high throughput sequencing technologies, in an example morphology
PRCC-MITF fusion is detected in the case of sign, it is found that the gene merges shape by PRCC exon 5 and MITF exon 4
At.MITF, PRCC gene order come from GeneBank, sequential version number GRCh38.p7, PRCCtranscript_id=" XM_
005245313.1 ", MITF transcript_id=" NM_198159.2 " ".
The present invention devises diagnosis PRCC-MITF transposition for the new fusion of the MiT family transposition clear-cell carcinoma
The probe combinations of property kidney.
It is a kind of for diagnosing the probe combinations of PRCC-MITF transposition kidney, which is combined into BAC cloning probe RP11-
867E4 and BAC cloning probe RP11-963H1.
The probe combinations, wherein BAC cloning probe RP11-867E4 is positioned at the centromere PRCC side, is labeled as
The fluorescence of any one color；BAC cloning probe RP11-963H1 is positioned at MITF telomere side, is labeled as and the centromere PRCC
The different fluorescence of the color of side label.
The probe combinations, wherein RP11-867E4, which is preferably marked, is, RP11-963H1 preferably mark for
Red fluorescence；The fluorescence color of label can be interchanged.
Probe of the present invention is preparing the application in MITF transposition Diagnosis of Renal Cell Carcinoma reagent.
A kind of MITF transposition Diagnosis of Renal Cell Carcinoma kit includes probe combinations of the present invention.
It is technical solution of the present invention detailed description below:
The probe combinations that the present invention uses detect PRCC-MITF fusion using FISH method for the first time to be domestic and international, are
Point based on the different BAC cloning probe binding sites to PRCC gene centromere side on chromosome and MITF gene telomere side
The analysis of situations such as cloth position, size and the preferred embodiment taken.
In the present invention, the centromere PRCC side BAC cloning probe is RP11-867E4 (fragment length 222kb), the end MITF
Grain side BAC cloning probe is RP11-963H1 (fragment length 196kb), these segments are bacterial artificial chromosome
(Bacterial artificial chromosome, BAC) clone, the positioning on human chromosomal have disclosed, respectively
For RP11-867E4 is positioned at No. 1 chromosome 156414562-156636320, and RP11-963H1 is positioned at No. 3 chromosomes
70151790-70348243.PRCC gene is positioned as No. 1 chromosome 156737274-156770609.The positioning of MITF gene
For No. 3 chromosome 69788586-70017488.BAC cloning probe and the sequence that links of PRCC gene are PRCC, RP11-
867E4, No. 1 chromosome centromere；The sequence that links of BAC cloning probe and MITF gene is No. 3 chromosome centromeres, MITF,
RP11-963H1。
BAC cloning probe upper fluorescence of label in conjunction with the sequence of corresponding position on chromosome, piece fragment position and PRCC,
Keep certain distance without overlapping (present invention in distance be up to 134kb, minimum 101kb) between MITF gene, thus
The fracture rearrangement of PRCC, MITF gene internal will not influence BAC cloned sequence in conjunction with chromosome corresponding position.In addition, MITF
Gene telomere side and the control of PRCC gene centromere side BAC cloning probe maximum distance are within 1500kb.In this way, in non-MITF
When PRCC-MITF fusion is not present in transposition kidney, red green two kinds of fluorescence from it is distant, without amplification when observation
See the farther away red green two kinds of signals of separation；When MITF transposition kidney is there are when PRCC-MITF fusion, red green two kinds glimmering
Depend alone closer, when observation occur red green fusion signal (show as it is red it is green be connected or yellow signal point), it is easy to observe.
Select this 2 kinds of BAC cloning probes to have the considerations of the following aspects: 1. 2 BAC cloning probe sizes are close, band
Come benefit be: the fluorescence intensity of every one end is consistent, prevent one end too strong and the other end it is excessively weak and influence observation；In addition may be used
Make being consistent property of in situ hybridization condition.2. between BAC cloning probe position and PRCC, MITF gene keep certain distance without
Overlapping, thus the fracture rearrangement of PRCC, MITF gene internal will not influence BAC cloning probe in conjunction with chromosome corresponding position.
3. the gene telomere side MITF and PRCC gene centromere side BAC cloning probe maximum distance can be made to control within 1500kb, when
Fusion signal (such as showing as red green connected or yellow signal point) is presented when two Gene Fusions, and in MITF gene and PRCC gene
There is no when fusion, two kinds of fluorescence color separation are distant, it is easy to observe.Otherwise, if the gene telomere side MITF and PRCC base
Because centromere side BAC cloning probe maximum distance is excessive, such as even more big more than 1500kb, then no matter whether two genes are merged,
It observes two kinds of fluorescence being clearly separated, is difficult to judge whether there is PRCC-MITF fusion.
Beneficial effects of the present invention:
The characteristics of present invention is according to MITF transposition kidney, design be incorporated in the gene telomere side MITF and PRCC gene silk
The fluorescent label DNA probe combinations of grain side carry out in situ hybridization on the basis of paraffin-embedded tissue slice, detection fusion and point
From signal, it is greatly improved the accuracy rate for diagnosing such tumour.Foundation is provided for diagnosis typing and molecular targeted therapy.According to me
Experimental result, the specificity and sensibility of probe combinations diagnosis reached 100%, and operation object is only needed in stone
It is carried out on wax investing tissue slice, the time is only two working days.Detection MITF is carried out using probe combinations provided by the invention
Transposition clear-cell carcinoma not only easily and fast, reliably but also success rate is high can be used for preparing MITF transposition clear-cell carcinoma and examine
Disconnected kit provides new tool for the fast and accurately diagnosis of MITF transposition clear-cell carcinoma.
Detailed description of the invention
Fig. 1: BAC cloning probe station-keeping mode figure.
Fig. 2: RT-PCR method detects the fusion of MITF transposition kidney.Sequencing result shows No. 1 chromosome and 3
There are transpositions for number chromosome, are formed PRCC-MITF fusion (PRCC exon 5 is connected with MITF exon 4)；
Fig. 3: PRCC-MITF fusion probe FISH testing result shows that tumour has fusion signal, is denoted as positive findings.
Fig. 4: control group hyaline cell cancerous tissue FISH detection is shown with PRCC-MITF3 fusion probe, is not present in tissue
Signal is merged, negative findings are denoted as.
Fig. 5: shown with PRCC-MITF fusion probe normal tissue FISH detection, there is no fusion signal in tissue, be denoted as
Negative findings.
Specific embodiment
The present invention will be further explained by examples below.
Probe as described in the examples, that is, BAC cloned sequence, can also be BAC cloning probe.
The preparation of embodiment 1:DNA probe combinations:
Selection can be separately connected in No. 3 gene telomere sides chromosome MITF and No. 1 chromosome PRCC gene centromere side
2 BAC cloned sequences, control maximum distance between the probe of both ends and keep certain within 1500kb, between BAC cloned sequence
Distance is not overlapped, and clip size is close.Cloned sequence source is that the mankind BAC of EmpireGenomics company clones center
(http://www.empiregenomics.com/helixhq/clonecentral/search/human).The centromere PRCC
Side BAC cloned sequence is RP11-867E4 (fragment length 222kb), and MITF telomere side BAC cloned sequence is RP11-
963H1 (fragment length 196kb), these segments are bacterial artificial chromosome (Bacterial artificial
Chromosome, BAC) it clones, the positioning on human chromosomal has disclosed, and respectively, RP11-867E4 is positioned at No. 1
Chromosome 156414562-156636320), RP11-963H1 be positioned at No. 3 chromosome 70151790-70348243.PRCC base
Cause is positioned as No. 1 chromosome 156737274-156770609.MITF gene is positioned as No. 3 chromosome 69788586-
70017488.BAC cloning probe and the sequence that links of PRCC gene are PRCC, RP11-867E4, No. 1 chromosome centromeres；BAC
The sequence that links of cloning probe and MITF gene is No. 3 chromosome centromeres, MITF, RP11-963H1.The positioning of probe combinations
Structure is as shown in Figure 1.The BAC cloning probe of MITF telomere side is marked as red fluorescence, the BAC of the centromere PRCC side is cloned
Probe is marked as marking green fluorescence with MITF telomere side；The fluorescence color of both ends label also can be interchanged.These methods are
(providing these above-mentioned services by EmpireGenomics company of the U.S.) well known to those skilled in the art.The centromere PRCC side
BAC cloning probe is a green florescent signal under fluorescence microscope, represents PRCC gene centromere side.MITF telomere side
BAC cloning probe is a red fluorescent under fluorescence microscope, represents MITF gene telomere side.The fluorescence of both ends label
Color can be interchanged.Red and green signals separate under normal circumstances, in tumour there are when PRCC-MITF group translocation, then observe and melt
Close signal.
Further, the probe combinations of preparation can be used fluorescence in-situ hybridization method, in MITF transposition clear-cell carcinoma, non-
Its positioning is verified in MITF transposition clear-cell carcinoma and swollen peri- tumorous normal tissues and/or whether diagnosis effect is reliable.
Embodiment 2: fluorescence in situ hybridization process:
(the Fig. 2, with this of MITF transposition kidney 1 is made a definite diagnosis by high-flux sequence and RT-PCR detection fusion genetic results
Experimental result corresponds to each other the reliability for better reflecting this experiment), and by two veteran pathologists referring to WHO
2016 uropoiesis and male reproductive system classification standard collect Nanjing General Hospital, Nanjing Military Area Command, PLA's Diagnosis of renal cell carcinoma 30 as control
Group.
3 μ m-thick of wax stone slice, after dewaxing, is sequentially placed into each 2min in 100%, 85%, 70% ethyl alcohol, is subsequently dipped to
In ionized water, 100 DEG C of water-bath 15min.Histotomy is put into pepsin K solution (0.1g pepsin, 40ml 0.01M
HCL) 37 DEG C, 15min；2 × SSC (sodium chloride, sodium citrate) is rinsed 2 times, each 5min, and slice is placed in room in 0.1mol/L HCl
Temperature impregnates 10min, is rinsed 2 times with 2 × SSC, each 5min；It is respectively dehydrated 2min through 70%, 85%, 100% ethyl alcohol, is done in air
It is dry；In tissue regions plus 10 μ l probe mixed liquors, (wherein each 1 μ l of each probe, totally 2 μ l, another plus 8 μ l hybridization are slow for 2 probes
Fliud flushing, hybridization buffer are provided when buying probe by EmpireGenomics company, include mankind Cot1DNA), cover glass
Piece, with rubber adhesive edge；It is put into 88 DEG C of denaturation 6min in situ hybridization instrument (GeneAmp In Situ PCR System 1000)
37 DEG C overnight (16h) afterwards；Coverslip is removed, slide is placed in 0.4 × SSC (sodium chloride, sodium citrate, 0.3%NP-40) solution
In, 69 DEG C of rinsing 1min；1min, 70% ethyl alcohol are rinsed in 2 × SSC (sodium chloride, sodium citrate, 0.1%NP-40) solution
3min, room temperature dark place are dry；10 μ l 4', 6- diamidinos -2-phenylindone (4', 6-diamidino-2- is added dropwise in target region
Phenylindole DAPI), after coverslip mounting, then with fluorescence microscope result.
Result judgement:
Visible 2 danger signals of normal cell and 2 greens.All signals are independent signal (each cell
It is diploid, so being exactly normally two red two green).
The tumour cell of PRCC-MITF transposition occurs, it is seen that the abnormal signal of a pair of red green fusion and red green each 1 open country
Raw type signal.
To exclude false positive and false negative, 100 cells of each sample counting, only in the presence of 4 fluorescence signals are equal,
Just be included in count target (no matter normal and tumour we can all see 4 mono signals (two green and two is red), only it is normal carefully
Born of the same parents' red and green signals are separation, it appears that are four mono signal compositions.A fusion signal is seen in tumour and a pair is red green
Isolated mono signal, seemingly 3 signals, but practical or be made of 4 mono signals).Between red green fluorescence signal
Distance less than a signal width when be calculated as fusion signal.The positive is denoted as when abnormal signal is greater than 10%.Result above is sentenced
Disconnected method is according to the judgment criteria that most similar commercialization probes are exercised on the market, such as Vysis company and Dako company
Probe application method.
As a result:
We detect 31 kidneys, and wherein MITF transposition kidney 1, TFE3 transposition kidney 6, TFEB are easy
Position property kidney 4, clear cell carcinoma 10, papillary renal carcinoma 10.As a result 1 MITF transposition kidney detects positive fusion
Signal, positive cell number range normal kidney tissue by 85%, remaining control group tumor tissues and cancer do not detect to merge
Signal (Fig. 3-5 give the representative positive picture of MITF transposition kidney, remaining tumour such as clear cell carcinoma negative picture,
The negative picture of normal kidney tissue).Illustrate using probe in detecting MITF transposition kidney specificity and sensibility be all
100%
Sensibility=true positives/all MITF transposition renal cancer tumor patient's number × 100%；
Specificity=true negative/all non-MITF transposition renal cancer tumor patient's number × 100%；
Evaluation:
The cloned sequence that this group of probe uses is relatively fewer, and cloned sequence size is relatively uniform.Consider in design
Economy has been arrived it is also contemplated that the consistency of in situ hybridization condition.In addition, the fluorescence in situ hybridization probe in this experiment
Specificity and sensibility have reached 100%.Using FISH technology, visited using the double-colored fusion fluorescence in situ hybridization of PRCC-MITF
Needle come diagnose MITF transposition clear-cell carcinoma quickly, reliable and success rate it is high, be one for diagnosing MITF transposition clear-cell carcinoma
New technology is worthy to be popularized.
Claims (5)
1. a kind of for diagnosing the probe combinations of PRCC-MITF transposition kidney, it is characterised in that by BAC cloning probe RP11-
867E4 and BAC cloning probe RP11-963H1 composition.
2. probe combinations according to claim 1, it is characterised in that the BAC cloning probe RP11-867E4 is positioned at
The centromere PRCC side, labeled as the fluorescence of any one color；The BAC cloning probe RP11-963H1 is positioned at MITF
Telomere side, labeled as the fluorescence of the different colours marked with the centromere PRCC side.
3. probe combinations according to claim 1, it is characterised in that the BAC cloning probe RP11-867E4 is labeled as
Green fluorescence, the BAC cloning probe RP11-963H1 are labeled as red fluorescence；The fluorescence color of label can be interchanged.
4. probe combinations of any of claims 1-3 are preparing the application in MITF transposition Diagnosis of Renal Cell Carcinoma reagent.
5. a kind of MITF transposition Diagnosis of Renal Cell Carcinoma kit, it is characterised in that the kit includes any one of claim 1-3
The probe combinations.
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