CN106755399B - A kind of I type neurofibromatosis pathogenic mutation gene and the Etiologic reagent based on this mutated gene - Google Patents
A kind of I type neurofibromatosis pathogenic mutation gene and the Etiologic reagent based on this mutated gene Download PDFInfo
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Abstract
The present invention relates to a kind of mutation of I type neurofibromatosis Disease-causing gene and Etiologic reagents based on this gene mutation.Neurofibroma cause of disease diagnostic reagent provided by the invention includes point mutation analysis kit, and the kit includes 1) Total RNAs extraction system reagent box: tested individual peripheric venous blood Total RNAs extraction；2) RNA reverse transcription and cDNA amplification system kit；3) system reagent box is sequenced in cDNA；System reagent box is sequenced in the cDNA: carrying out PCR amplification to the cDNA that reverse transcription obtains with five primer pairs shown in SEQ ID NO.7-16, with primer pair shown in SEQ ID NO.17-60 while nested PCR amplification and sequencing are carried out to obtained five large fragments of cDNA of amplification again, and it is compared with former NF1 gene DNA sequences encoding, it is I type neurofibroma patient in the presence of c.7106G > A in amplified production, p.W2369X mutation.Etiologic reagent of the invention is time saving, accurate and testing cost is low.
Description
Technical field
It causes a disease and dashes forward the present invention relates to molecular biology and genetic test field more particularly to a kind of I type neurofibromatosis
Become gene and the Etiologic reagent based on this mutated gene.
Background technique
I type neurofibromatosis (Neurofibromatosis type I, NF1) by von Recklinghausen in
It reports for the first time within 1882, therefore also known as von Recklinghausen is sick.It is aobvious that I type neurofibromatosis is generally autosome
Property heredity, partially to distribute.Clinically it is mainly shown as skin coffee spot, multiple cutaneous fibroma molle；Non-exposed position
The freckle and iris melanin hamartoma that (such as armpit, groin) occurs；Also multiple organs and system be can be involved, such as eye, bone
Bone, blood vessel, endocrine system, maincenter and peripheral neverous system etc., and can with intellectual development obstacle, congenital dysplasia,
Extremely rare canceration person.
The disease is caused by the mutation of NF1 gene.The NF1 assignment of genes gene mapping includes outside 57 compositions in chromosome 17q11.2
Aobvious son and 3 alternative splicing exons, overall length 283kb, having 3 sizes is the mature transcript of 11~13kb, encodes opposite point
Nerve fibre (Neurofibromin) albumen that protonatomic mass is 327000, is made of 2818 amino acid, great expression is in mind
Through member, astroglia, oligodendroglia, adrenal cells, gonadal cell and leucocyte.NF1 gene is tumor suppression
Gene, it is many research shows that NF1 gene mutation can be by the cAMP signal transduction pathway of interference Ras circulation to cause tumour.
NF1 gene spontaneous mutation rate is about 1/10000, is one of highest gene loci of human mutation rate, and about 50% patient is
New mutation still has catastrophe point not yet explicitly so far, but has found some mutation concentrated areas.Its Exon 4b, 7,10b,
13,15,20,28,29,31 may be mutantional hotspot, and the mutation of 32 exons may be Chinese's NF1 gene mutation hot spot
One of region.Presently found gene mutation is more than 700 kind, conversion, transversion including base, insertion, missing, frameshit
Mutation, splicing mutation and translocation etc..In these mutation, there are about 82% mutation to cause nerve fiber protein
It truncates, the truncation of albumen generates important influence to the functional structure of albumen.
Currently, for I type neurofibromatosis high incidence detection demand increasingly increase, design studies it is scientific and reasonable and
Accurately detection kit is very necessary.Since NF1 genetic fragment is larger, catastrophe is complicated, easily causes missing inspection.And the disease
The clinical manifestation of different patients is different, and the different patients with clinical manifestations of same family are also different, considerably increase clinical diagnosis
Difficulty, therefore establish fast and accurately diagnostic techniques, capture the diagnosis problem of I type neurofibromatosis, its clinic is controlled
It treats and scientific research all has important value.
But the mutantional hotspot found at present be not enough to solve clinically I type neurofibromatosis gene screening, diagnosis
Problem.New pathogenic mutation is found, the NF1 neurofibromatosis spectrum of mutation is enriched, it will be clinically conveniently screening and to examine
Disconnected I type neurofibroma patient provides important target spot and theoretical foundation, this is always that this field researcher is diligent
Probe into field.
Since NF1 gene includes 58 composition exons and 3 alternative splicing exons, traditional is directed to outside each
Showing sub sequencing analysis method, time-consuming, and each sequencing sample, which completes detection, generally to be needed six, seven days.And other detection methods are such as
NGS, FISH or MLPA etc., need to be equipped with expensive large-scale instrument and equipment, and experiment condition requires height, causes testing cost big
It is big to increase.
In addition, the mutation type of NF1 gene is in addition to point mutation, there is also large stretch of breakthrough to lose, one or more exons
The multiple types such as repetition/missing.It detects NF1 gene mutation by single method to be easy to miss inspection, this is also current NF1 Disease-causing gene
Abrupt climatic change perplexs the problem of clinical genetic diagnosis personnel.
In conclusion find to diagnosing effective new pathogenic mutation, and develop time saving, accurate and experiment condition requirement compared with
Low diagnostic reagent is the basic method for solving I type neurofibromatosis diagnosis problem to NF1 gene mutation complete detection.
Summary of the invention
In view of this, to capture problem in the prior art, the present inventor has found that one kind can be examined effectively by hardships research
The pathogenic mutation gene of disconnected I type neurofibromatosis, and develop the Etiologic reagent based on this mutated gene.The examination
Agent can obtain diagnostic result in a short time, and experiment condition requirement is low, and diagnostic result is accurate.
The present invention discloses a kind of I type neurofibromatosis pathogenic mutation gene, and the gene is NF1 gene, the mutation
Guanine positioned at the 48th exon region of NF1 gene, coding the 7106th replaces with adenine so as to cause the 2369th
The tryptophan of position sports terminator codon (c.7106G > A, p.W2369X).
I type neurofibromatosis pathogenic mutation gene disclosed by the invention and I type neurofibromatosis are highly relevant, this is prominent
Becoming (c.7106G > A, p.W2369X) makes nerve fiber protein truncate and lose normal function, causes autosomal dominant inheritance
The I type neurofibromatosis of disease.
The present invention also provides the neurofibroma cause of disease diagnosis based on above-mentioned I type neurofibromatosis pathogenic mutation gene
Reagent, the diagnostic reagent include point mutation analysis kit, and the kit includes 1) Total RNAs extraction system reagent box: quilt
Survey individual peripheric venous blood Total RNAs extraction；2) RNA reverse transcription and cDNA amplification system kit；3) system examination is sequenced in cDNA
Agent box；System reagent box is sequenced in the cDNA: the cDNA obtained to reverse transcription is with five primer pairs shown in SEQ ID NO.7-16
PCR amplification is carried out, then obtained five large fragments of cDNA of amplification with primer pair shown in SEQ ID NO.17-60 while being carried out
Nested PCR amplification is simultaneously sequenced, and compares with former NF1 gene DNA sequences encoding, as c.7106G > A in amplified production,
It is I type neurofibroma patient in the presence of p.W2369X mutation.
Further, the diagnostic reagent further includes NF1 gene extron deletion analysis kit: with reverse transcription acquisition
NF1 gene cDNA be template, with primer pair shown in SEQ ID NO.61-70 respectively to the exon 3 of NF1,11,25,34,
58 carry out real-time fluorescence quantitative PCR amplification, with 2-ΔΔCtMethod carries out data analysis, and the Relative gene of five exons is calculated
Content, when the Relative gene content of five exons 0.5 or so when be Exon deletion carrier, and prompt NF1 gene
Missing or excalation.
Further, the diagnostic reagent further includes NF1 full genome large fragment deletion assay kit, and the kit includes
1) DNA extraction system kit: tested individual peripheric venous blood extracting genome DNA；2) Microsatellite polymorphism mark point in gene
Analyse kit: using the genomic DNA as template, with three couples of primer pair 27AC28.4 shown in SEQ ID NO.1-6,
Tri- polymorphic sites of 27CAGT, 38TG53.0 carry out PCR amplification, and polyacrylamide gel electrophoresis detects PCR product, works as PCR
NFI gene a possibility that there are full genome large fragment deletions when the segment of three primer pair amplifies is homozygote in product.
Further, when progress PCR amplification five large fragments of acquisition to cDNA, primer annealing temperature is 60 DEG C；To five
When a large fragment carries out nested PCR amplification, primer annealing temperature is 62 DEG C.
Further, the real-time fluorescence quantitative PCR amplification synthetic primer segment is no more than 350bp.
Further, the size of the 27CAGT amplified fragments is 201bp or so, and the size of 27AC28.4 amplified fragments is
The size of 214bp or so, 38TG53.0 amplified fragments is 479bp or so.
The present invention also provides the application method of above-mentioned Etiologic reagent, include the following steps: 1) external with tested
All venous blood is sample, to include c.7106G > A in the point mutation analysis kit assay sample, p.W2369X mutation
Point mutation whether there is, and such as exist for I type neurofibroma patient, and such as there is no carry out following step；2) with the full base of NF1
Because large fragment deletion assay kit analysis sample in full genome large fragment deletion there are a possibility that, such as there is heterozygote, then
A possibility that excluding full genome large fragment deletion；Such as it is homozygote, then is mutated patient suspected of full genome large fragment deletion, into
Row following step；3) whether it is Exon deletion carrier with NF1 gene extron deletion analysis kit assay, lacks in this way
It loses carrier and is then diagnosed as patient, be other gene mutations or normal individual if not being.
The present invention also provides the primer pair groups for being used to detect NF1 gene mutation in Etiologic reagent, including for examining
The primer pair of NF1 gene large deletion is surveyed, nucleotide sequence is as shown in SEQ ID NO.1-6；It is obtained for expanding cDNA
The primer pair of five large fragments is obtained, nucleotide sequence is as shown in SEQ ID NO.7-16；For five large fragments to cDNA
The primer pair of nested PCR amplification is carried out, nucleotide sequence is as shown in SEQ ID NO.17-60；For detecting outside NF1 gene
The primer pair of aobvious son missing, nucleotide sequence is as shown in SEQ ID NO.61-70.
The preparation method of above-mentioned Etiologic reagent includes the following steps: 70 in the primer pair group are single-stranded
After DNA is individually packed, it is packaged in same reagent box at least one of following substances: PCR reaction buffer, DNA
Polymerase and 4 kinds of dNTP.
It realizes specific experiment method of the invention and steps are as follows:
1. the design and synthesis of amplimer
Obtain NF1 gene nucleic acid sequence from ncbi database, using Primer-BLAST software (http:// www.ncbi.nlm.nih.gov/tools/primer-blast/) design cDNA housing primer, cDNA nested primer, STR mark
Will primer, Real time PCR primer are 35 pairs total, and all sequences see attached list 1.All sequences are from Shanghai bio-engineering corporation
It orders, PAGE purifying.
2. sample pre-treatments (nucleic acid extraction)
The extraction (RNAisoTMPlus, Japanese TaKaRa) of 2.1 total serum IgEs
2.1.1 peripheral blood is extracted, takes 100 μ l peripheral bloods in 1.5ml EP pipe.
2.1.2 1ml RNAiso Plus is added, blows and beats born of the same parents repeatedly and is thoroughly cracked to cell.
2.1.3 200 μ l chloroforms are added, sufficiently vibrating 15sec keeps solution fully emulsified, is stored at room temperature 5min.
2.1.4 after 4 DEG C of centrifugation 15min of 12000rpm, supernatant is transferred in new 1.5ml centrifuge tube (it is divided into three layers,
Under being sure not in sucking two layers).
2.1.5 isometric isopropanol is added, after being sufficiently mixed, is stored at room temperature 10min.
2.1.6 after 12000rpm, 4 DEG C of centrifugation 10min, liquid is discarded supernatant, precipitating is stayed.
2.1.7 75% ethanol solution of 1ml, washing precipitating is added.12000rpm, 4 DEG C of centrifugation 5min.
2.1.8 it exhausts after supernatant, drying at room temperature 30min (is carried out) in superclean bench.
2.1.9 20 μ l DEPC water are added, 55-60 DEG C of water-bath 10min dissolves RNA.
2.1.10 RNA concentration is measured, is stored in spare in -80 DEG C of refrigerators.
2.2 whole blood DNAs extract (Tiangeng poba gene group DNA extraction kit-centrifugal column method)
2.2.1 600 μ l cell pyrolysis liquid CL are added in the sample, are mixed by inversion for the anticoagulated whole blood for taking 300 μ l,
10000rpm is centrifuged 1min, sucks supernatant, leaves nucleus precipitating.
2.2.2 it repeats the above steps primary.
2.2.3 add 200 μ l buffer GS into the nucleus precipitating being collected by centrifugation, oscillation is mixed to thorough.
2.2.4 20 μ l Proteinase K solution are added, mix.
2.2.5 200 μ l buffer GB are added, are sufficiently mixed by inversion, 56 DEG C of placement 10min are mixed by inversion for several times therebetween,
Until solution answer it is limpid.
2.2.6 plus 200 μ l dehydrated alcohols, sufficiently it is mixed by inversion, at this time it is possible that flocculent deposit.
2.2.7, previous step acquired solution and flocculent deposit be all added in an adsorption column CB3 (adsorption column CB3 is put into 2ml
In collecting pipe), 12,000rpm centrifugation 30sec outwell the waste liquid in collecting pipe, adsorption column CB3 are put into new 2ml collecting pipe
In.
2.2.8 500 μ l buffer GD are added into adsorption column CB3,12,000rpm are centrifuged 30sec, outwell in collecting pipe
Waste liquid, then adsorption column CB3 is put into new 2ml collecting pipe.
2.2.9 600 μ l rinsing liquid PW are added into adsorption column CB3,12,000rpm are centrifuged 30sec, outwell in collecting pipe
Waste liquid, then adsorption column CB3 is put into new 2ml collecting pipe.
2.2.10 repetitive operation step 9.
2.2.11 12,000rpm is centrifuged 2min, outwells waste liquid.Adsorption column CB3 is placed in and is placed at room temperature for several minutes, with thorough
Dry rinsing liquid remaining in adsorbent material in bottom.
2.2.12 adsorption column CB3 is transferred in 1.5ml centrifuge tube, 100 μ l elution is vacantly added dropwise to adsorbed film middle position
Buffer TB, is placed at room temperature for 2-5min, and solution is collected into centrifuge tube by 12,000rpm centrifugation 2min.
2.2.13 1 μ l DNA is taken to measure OD260nm/280nm ratio, remaining DNA in Nanodrop ultraviolet specrophotometer
It is stored in 4 DEG C of refrigerators, is used for follow-up study.
3. nucleic acid amplification
3.1 using Microsatellite polymorphism mark analysis (27AC28.4,38TG53.0,27CAGT) in three genes, and investigation is complete
A possibility that gene large deletion.
3.1.1 polyacrylamide gel (8%) 130ml is prepared:
3.1.2 the liquid prepared above is poured into plastic plate, after gel polymerisation, using 1 × TBE as electrophoretic buffer,
PCR product is carefully added in corresponding gel pore after prerunning half an hour, 100V electrophoresis about 4-5h.
3.1.3 it is a kind of sensitive and simple means that DNA molecular is detected with the method for silver staining.It can be with core using silver ion
The characteristic that acid combines, silver ion reduction makes DNA band in gel develop the color under formaldehyde effect.Its operating process is as follows:
1. the complete polyacrylamide gel of electrophoresis is impregnated 5 minutes with 10% alcohol.
2. abandoning alcohol, 1%HNO is added3, 3 minutes.
3. abandoning HNO3, cleaned twice with clear water.
4. AgNO is added3, dye 20 minutes.
5. using ddH2O cleaning is primary.
6. developer solution is added, until the DNA electrophoretic band of visible clear wash rice.
7. abandoning developer solution, 10% acetic acid fixing 5 minutes is added.
8. abandoning acetic acid, impregnated 5 minutes or more in clear water.
9. observation analysis and scanning achieve with glassine paper encapsulated drying.
3.2RNA reverse transcription and cDNA amplification, RT-PCR
3.2.1 reverse transcription PCR
Reverse transcription PCR reaction system is following (PrimeScriptTM RT reagent Kit, Japanese TaKaRa):
Reverse transcription PCR reaction condition is as follows:
37℃ 15min
85℃ 5s
- 20 DEG C of product save backup.
3.2.2cDNA it is sequenced:
First totally 5 large fragments, these large fragments cover the overall length of gene to amplification NF1 gene cDNA 1,2,3,4,5.
Reaction system: 30ul
PCR reaction cycle condition:
Nido sequencing:
cDNA1-1,1-2,1-3,1-4；cDNA2-1,2-2,2-3,2-4,2-5；cDNA3-1,3-2,3-3,3-4, 3-5；
cDNA4-1,4-2,4-3,4-4；CDNA5-1,5-2,5-3,5-4 totally 22 pairs of primers.
PCR reaction cycle condition: 30ul
* Exon31 is variable sheer, so there are two bands by cDNA3-2,3-3.
3.2.3Real time PCR
Selected in the primer synthesized eligible clip size be no more than 350bp (optimum clip size 300bp with
Under) and 60 DEG C of annealing temperature have exons 3,11,25,34,58 carry out Real time PCR.Reaction is in ABI
It is carried out on 7500real-time PCR system (Applied Biosystem, Foster City, CA) instrument, DNA sample
Concentration dilution to 5ng/ μ l, reaction system does 3 multiple holes referring to following preparations, every part of sample each reaction.
Reaction system and condition:
Reaction condition is as follows:
Data analysis:
Experiment data analysing method used is 2-ΔΔCtMethod.Ct value is exactly in fluorescent PCR amplification procedure, when amplification produces
The fluorescence signal of object reaches the amplification cycles number passed through when the threshold value of setting.There is linear for its copy number with template
Relationship, starting copy number is more, and the recurring number of process is fewer, and Ct value is also just smaller, and vice versa.Average Ct (mCt) is every
The average value of part three multiple holes Ct values of sample.Δ Ct is the difference of NF1 gene extron and reference gene (β-globin) Ct value
Value.The calculation formula of Δ Δ Ct are as follows: Δ Δ Ct=[mCtNF1exon(to be measured)-mCtβ-globin(to be measured)]-[mCtNF1 exonIt is (right
According to)-mCtβ-globin(control)].Each pair of primer chooses 3 normal females as control.According to the method be calculated it is each to
The Relative gene content of test sample sheet, 0.5 or so indicates normal individual for missing carrier, 1 or so.
Method currently used for detecting gene has Sanger PCR sequencing PCR, two generation PCR sequencing PCRs, multiple linking probe amplification MLPA
(multiple ligation-dependent probe amplification, MLPA) method, fluorescence in situ hybridization FISH
(Fluorescence in situ hybridization, FISH) method etc., however this detection method otherwise it is time-consuming excessive or
It need to be equipped with expensive large-scale instrument and equipment, testing cost is caused to increase significantly.The present invention uses the sequencing after nested amplification
And technique of polyacrylamide gel electrophoresis greatly shortens detection time, testing cost substantially reduces.
Traditional Sanger sequencing approach is only capable of detection point mutation, small fragment insertion and missing, cannot detect Exon deletion
With the missing of entire NF1 gene or repeat etc..The present invention is further marked using STR after the point mutation detection that nido is sequenced
Will analysis and Real time PCR, which combine, carries out fast and accurately complete detection to the full fragment deletion of NF1 gene.Practice card
Bright STR MARKER ASSAYS method can effectively apply to the detection of the missing of NF1 whole gene.Real time PCR it is highly sensitive,
The characteristics of high specific and accurate quantification, missing to NF1 whole gene or can repeat Accurate Diagnosis.
Compared with prior art, the beneficial effects of the present invention are:
1. I type neurofibromatosis Disease-causing gene mutation disclosed by the invention is highly relevant with I type neurofibromatosis, fortune
More than ten patients are successfully detected with the method.Wherein mutation (c.7106G > A, p.W2369X) cuts nerve fiber protein
It is short and lose normal function, cause the I type neurofibromatosis of autosomal dominant inherited disease.
2. the present invention greatly shortens detection time using the sequencing after nested amplification, testing cost is substantially reduced.
3. the present invention judges that gene is big using the method that STR MARKER ASSAYS is mutually proved with Real time PCR analysis
The presence of fragment deletion, result are more accurate.
4. the present invention combine Sanger be sequenced, the multiple technologies such as Real time PCR and STR MARKER ASSAYS method it is respective excellent
Point for the point mutation of NF1 gene, frameshift mutation, splicing mutation, large fragment deletion and repeats fast and accurately complete
Face detection.
5. primer pair group provided by kit of the present invention, can be quick, quasi- using cheap common archaeal dna polymerase
Really, it efficiently identifies neurofibroma genetic defect, obtains testing result.
6. diagnostic reagent of the present invention be based on nucleic acid amplification technologies and develop design, with equipment requirement is low, detection time is short,
The features such as accuracy rate is high, omission factor is extremely low, convenient and efficient.
It, can be into one 7. the accurate detection of NF1 gene mutation of the present invention provides possibility for pre-natal diagnosis and subsequent supplementary reproduction
Step prevention birth defect, improves newborn population quality.
8. the present invention provides reference for the Molecular screening of neurofibroma genetic defect gene.
Detailed description of the invention
Fig. 1: the amplification of DNA is carried out using nucleotide sequence primer pair as shown in SEQ ID NO.3-4；
In figure letter A represent 27CAGT label title, the subsequent digital representation STR label each allelotype,
According to amplified fragments size incremental arrangement.The one of segment of A1, A2 or A3 occur is homozygote, is occurred in which any
Combination A1/A2, A1/A3, A2/A3 of two segments are heterozygote.
A1 is CA repeat number 14 in glue figure, and such as detecting the one of segment of A2 or A3 is homozygote.
Fig. 2: the amplification of DNA is carried out using nucleotide sequence primer pair as shown in SEQ ID NO.1-2；
Letter D represents 27AC28.4 label title, each allele of subsequent digital representation STR label in figure
Type, according to amplified fragments size incremental arrangement.The one of segment of D1, D2 or D3 occur is homozygote, occurs in which and appoints
Combination D1/D2, D1/D3, D2/D3 of two segments of anticipating are heterozygote.
D1 is AC repeat number 15 in glue figure, and such as detecting mono- segment of D3 is homozygote.
Fig. 3: the amplification of DNA is carried out using nucleotide sequence primer pair as shown in SEQ ID NO.5-6；
Letter C represents 38TG53.0 label title, each allele of subsequent digital representation STR label in figure
Type, according to amplified fragments size incremental arrangement.The one of segment of C1, C2 or C3 occur is homozygote, occurs in which and appoints
Combination C1/C2, C1/C3, C2/C3 of two segments of anticipating are heterozygote.
C1 is 9 repeat numbers in glue figure, and such as detecting mono- segment of C2 is homozygote.
Fig. 4: amplification five large fragments of cDNA are carried out using nucleotide sequence primer pair as shown in SEQ ID NO.7-16
Amplification；
Fig. 5: the amplification of nest-type PRC is carried out using nucleotide sequence primer pair as shown in SEQ ID NO.17-60；
Fig. 6: Sanger sequencing result；
Fig. 7: diagnostic reagent overhaul flow chart of the present invention.
Specific embodiment
Embodiment 1
The discovery of I type neurofibromatosis NF1 gene mutation
Inventor has carried out gene mutation analysis to 13 patients, and detailed step is as follows:
1) detected using mutation analysis reagent: the reagent includes 1) Total RNAs extraction system reagent；2) RNA is inverted
Record and cDNA amplification system reagent；3) system reagent is sequenced in cDNA.
2) suspected patient and normal control sample EDTA each 2ml of anticoagulation cirumferential blood sample are extracted, the extraction of total serum IgE is carried out,
Reverse transcription PCR, cDNA amplification obtain totally 5 large fragments of cDNA1,2,3,4,5 (result is shown in Fig. 4)；To 5 sections of cDNA large fragments into
Row nest-type PRC, totally 22 pairs of primers, PCR primer agarose electrophoresis result (Fig. 5) and sequencing result (Fig. 6).
I type neurofibromatosis is autosomal dominant inheritance, this research combines family actual conditions and NF1 gene high
The characteristics of spontaneous mutation rate, find to have in 13 patients 1 there are NF1 gene c.7106G > A, p.W2369X is mutated (Fig. 6).
It is expected that the mutation makes protein truncation and loses normal function, the result be consistent with clinical diagnosis (patient's second sample from certain three
First hospital, 8 or more skin cafe au lait macules have armpit or groin freckle, there is 3 neurofibromas, tentative diagnosis 1
Type neurofibroma).3 phenotypes do not find above-mentioned mutation normally per capita.It can tentatively infer that said gene sports this as a result,
Patient's pathogenic mutation.Remaining patient is other types mutation, specific as follows:
1) NF1 genetic test result is that c.2835delT, p.Phe945Leufsx10 mutation is positive
2) NF1 gene heterozygosity loss (result of other detection methods detection other than sequencing)
3) NF1 gene sequencing testing result is c.2447G > A p.R816Q and c.7352delC p.Pro2451Leufs*
17 heterozygous mutants are positive
4) NF1 genetic test result is that c.6772C > T p.R2258X heterozygous mutant is positive
5) NF1 genetic test result is that p.T1923K heterozygous mutant is positive
6) NF1 genetic test result is that c.989C > T P.A330V mutation is positive
7) NF1 genetic test result is that c.4786C > T P.Q1596X heterozygous mutant is positive
8) it is positive to be detected as c.3826C > T P.R1276X heterozygous mutant by NF1 gene exon28
9) NF1 genetic test result is exon28c.3721C > T, and p.R1241X heterozygous mutant is positive
10) NF1 genetic test result is that c.1019_1020delCT, p.Ser340Cysfsx12 heterozygous mutant is positive
11) NF1 genetic test result is that c.989C-T p.A330V heterozygous mutant is positive
12) patient's methyl because testing result be gene large deletion (other than sequencing other detection methods detection knot
Fruit)
Embodiment 2
The application method and application examples diagnosed using kit of the present invention.
One, it obtains clinical sample and experimental group and control group is set
1) patient's first
Patient's first, male, sample come from certain Grade A hospital, and 6 skin cafe au lait macules have armpit or groin freckle,
There are 9 neurofibromas, tentative diagnosis is 1 type neurofibroma.
2) patient's second
Patient's second sample comes from certain Grade A hospital, and 8 or more skin cafe au lait macules have armpit or groin sparrow
Spot, there is 3 neurofibromas, and tentative diagnosis is 1 type neurofibroma.
3) control group
Choose 3 normal artificial control groups.
Two, experimental program and step
Detected using point mutation analysis kit of the present invention: the kit includes 1) Total RNAs extraction system reagent
Box；2) RNA reverse transcription and cDNA amplification system kit；3) system reagent box is sequenced in cDNA.
Concrete operations are as follows:
1. extracting suspected patient first, patient second and each 2ml of normal control sample EDTA anticoagulation cirumferential blood sample.
2. the extraction of total serum IgE:
2.1 extract person under test's new blood 2ml, take in the blood and 1.5mlEP pipe of 100 μ l.
2.2 are added 1ml RNAiso Plus, are blown and beaten repeatedly to thorough cracking.
2.3 are added 200 μ l chloroforms, and sufficiently vibrating 15sec keeps solution fully emulsified, are stored at room temperature 5min.
After 2.4 4 DEG C of 12000rpm centrifugation 15min, supernatant is transferred in new 1.5ml centrifuge tube and (is divided into three layers, cuts
Under in sucking two layers).
2.5 are added isometric isopropanol, after being sufficiently mixed, are stored at room temperature 10min.
After 2.6 12000rpm, 4 DEG C of centrifugation 10min, liquid is discarded supernatant, precipitating is stayed.
2.7 are added 75% ethanol solution of 1ml, washing precipitating.12000rpm, 4 DEG C of centrifugation 5min.
After 2.8 exhaustion supernatants, drying at room temperature 30min (is carried out) in superclean bench.
2.9 are added 20 μ l DEPC water, and 55-60 DEG C of water-bath 10min dissolves RNA.
2.10 measurement RNA concentration, suspected patient group: 133ng/ μ l；Normal group: 165ng/ μ l is stored in -80 DEG C of ice
Case.
3, reverse transcription PCR
Taking the 1 μ g of total serum IgE of patient group and Normal group is that template carries out reverse transcription.
Reverse transcription PCR reaction system is as follows:
Reverse transcription PCR reaction condition is as follows:
37℃ 15min
85℃ 5s
- 20 DEG C of cDNA product saves backup.
4, cDNA amplification (of the present invention kit cDNA housing primer, cDNA nested primer sequence see attached list 1)
The cDNA of 4.1 pairs of suspected patient groups and Normal group carries out PCR amplification, amplification obtain NF1 gene cDNA 1,2,
3,4,5 totally 5 large fragments.
Reaction system: 30ul
PCR reaction condition:
4.2 pairs of 5 large fragment PCR products carry out agarose electrophoresis (result is shown in Fig. 4).
4.3 nest-type PRCs: nest-type PRC is carried out to 5 sections of cDNA large fragments.(cDNA nested primer sequence of the present invention is seen attached list
1cDNA1-1,1-2,1-3,1-4；cDNA2-1,2-2,2-3,2-4,2-5；cDNA3-1,3-2,3-3, 3-4,3-5；cDNA4-
1,4-2,4-3,4-4；CDNA5-1,5-2,5-3,5-4 totally 22 pairs of primers)
PCR reaction cycle condition: 30ul
4.4PCR product agarose electrophoresis result (Fig. 5) and sequencing result (Fig. 6).
Interpretation of result: as shown, the sequencing detection of patient first NF1 58 exon regions of gene does not find to be mutated:
C.7106G > A, p.W2369X.The sequencing detection discovery mutation of patient second NF1 58 exon regions of gene: c.7106G > A,
P.W2369X, it is contemplated that the mutation makes protein truncation and loses normal function, which is consistent with clinical diagnosis.3 it is normal per capita
Above-mentioned mutation is not found.Patient's second can be diagnosed as I type neurofibroma patient.
Embodiment 3
Abovementioned steps are substantially the same manner as Example 2, institute the difference is that, to be not detected mutation: c.7106G > A,
Patient's first of p.W2369X and 3 normal persons continue with detection.
It is detected using NF1 full genome large fragment deletion assay kit of the present invention, the kit includes 1) DNA
Extract system reagent box；2) Microsatellite polymorphism mark assay kit in gene.
Concrete operations are as follows:
5, extracting suspected patient first and normal control peripheral blood DNA using genome DNA extraction kit (ensures to extract
DNA stores concentration > 50ng/ μ l):
5.1 take the anticoagulated whole blood of 300 μ l, and 600 μ l cell pyrolysis liquid CL are added in the sample, are mixed by inversion, 10 000rpm
It is centrifuged 1min, sucks supernatant, leaves nucleus precipitating.
5.2 repeat the above steps once.
5.3 into the nucleus precipitating being collected by centrifugation plus 200 μ l buffer GS, oscillation are mixed to thorough.
5.4 are added 20 μ l Proteinase K solution, mix.
5.5 are added 200 μ l buffer GB, are sufficiently mixed by inversion, and 56 DEG C of placement 10min are mixed by inversion for several times therebetween, until
Solution is answered limpid.
5.6 add 200 μ l dehydrated alcohols, are sufficiently mixed by inversion, at this time it is possible that flocculent deposit.
5.7 previous step acquired solution and flocculent deposit are all added in an adsorption column CB3 (adsorption column CB3 is put into 2ml and receives
In collector), 12 000rpm are centrifuged 30sec, outwell the waste liquid in collecting pipe, adsorption column CB3 is put into new 2ml collecting pipe
In.
5.8 500 μ l buffer GD, 12 000rpm are added into adsorption column CB3 is centrifuged 30sec, outwells useless in collecting pipe
Liquid, then adsorption column CB3 is put into new 2ml collecting pipe.
5.9 600 μ l rinsing liquid PW, 12 000rpm are added into adsorption column CB3 is centrifuged 30sec, outwells useless in collecting pipe
Liquid, then adsorption column CB3 is put into new 2ml collecting pipe.
5.10 repetitive operation steps 9.
5.11 12 000rpm are centrifuged 2min, outwell waste liquid.Adsorption column CB3 is placed in and is placed at room temperature for several minutes, with thorough
Dry rinsing liquid remaining in adsorbent material.
5.12 are transferred to adsorption column CB3 in 1.5ml centrifuge tube, and it is slow that 100 μ l elution is vacantly added dropwise to adsorbed film middle position
Fliud flushing TB, is placed at room temperature for 2-5min, and 12 000rpm are centrifuged 2min, solution is collected into centrifuge tube.
5.13 measurement DNA concentrations, suspected patient group: 117ng/ μ l；Normal group: 102ng/ μ l is stored in -80 DEG C of ice
Case.
6, using Microsatellite polymorphism mark analysis in three genes, (NF1 gene STR mark primer sequence of the present invention is seen attached list
1)
30 normal control samples (statistics heterozygosis rate is dedicated) DNA of 6.1 pairs of suspected patient groups and Normal group is carried out
PCR (primer sequence of 227AC28.4,38TG53.0,27CAGT see attached list 1).According to the site experimental result 27CAGT heterozygosis
Ziren number accounts for total number of persons 47.2%, and the site 27AC28.4 heterozygote number accounts for the site 51%, 38TG53.0 heterozygote number and accounts for
40%.Using above-mentioned 27AC28.4,38TG53.0 and 27CAGT tri- to primer amplification, when three pairs of primer experimental results are pure
When zygote, a possibility that judging NFI gene there are whole gene loss of heterozygosity.
(1) Intron36-IVS27AC28.4:
Reaction condition
(2)Intron36-IVS27CAGT
Reaction condition
(3)Intron47-IVS38TG53.0
Reaction condition
6.2PCR product carries out PAGE electrophoresis: polyacrylamide gel (8%) 130ml is prepared
6.3 using 1 × TBE is carefully added in corresponding gel pore as electrophoretic buffer, by PCR product, and 100V electrophoresis is about
4-5h。
6.4 silver staining
1. the complete polyacrylamide gel of electrophoresis is impregnated 5 minutes with 10% alcohol.
2. abandoning alcohol, 1%HNO is added3, 3 minutes.
3. abandoning HNO3, cleaned twice with clear water.
4. AgNO is added3, dye 20 minutes.
5. using ddH2O cleaning is primary.
6. developer solution is added, until the DNA electrophoretic band of visible clear wash rice.
7. abandoning developer solution, 10% acetic acid fixing 5 minutes is added.
8. abandoning acetic acid, impregnated 5 minutes or more in clear water.
9. saving result (see Fig. 1-3).
Interpretation of result: three polymorphic marks of patient's first are homozygote, and there are whole gene loss of heterozygosity for NFI gene
A possibility that.Three polymorphic marks of 3 normal persons are heterozygote.Judge that three normal persons do not suffer from I type neurofibroma
Disease.
Embodiment 4
Abovementioned steps are substantially the same manner as Example 3, institute the difference is that, to gene mutation is not detected: c.7106G >
A, p.W2369X, and there is a possibility that patient's first of whole gene loss of heterozygosity and continue with detection.
It is detected using NF1 gene extron deletion analysis kit of the present invention, concrete operations are as follows:
8,1) Real time PCR:(Real time PCR primer sequence of the present invention is seen attached list
The exons 3,11,25,34,58 of 8.1 couples of NF1 carries out Real time PCR, extremely by DNA sample concentration dilution
5ng/ μ l is template, and for reaction system referring to following preparations, every part of sample is tested every time does 3 multiple holes.
Reaction system and condition:
Reaction condition is as follows:
8.2 data analysis: analysis method 2-ΔΔCtAs a result method sees attached list 2.
The analysis of subordinate list 2:Real time PCR data
* the Relative gene content of sample to be tested, 0.5 or so indicates normal individual for missing carrier, 1 or so.
Interpretation of result: in conclusion patient's first is the half of normal control in the segment of exon3,11,25,34 and 58 reading
Number judge patient's first accordingly for full genome missing carrier, this with Microsatellite polymorphism mark analyzes it, and there are NF1 genetic heterozygosis
Property missing (Loss of Heterozygosity, LOH) possibility result mutually prove.So far, patient's first is diagnosed as I type
Neurofibroma patient.
Subordinate list 1:NF1 gene STR indicates primer, cDNA housing primer, cDNA nested primer sequence, Real time PCR
Primer sequence is 35 pairs total, and all sequences are ordered from Shanghai bio-engineering corporation, PAGE purifying.
Embodiment 5
300 clinical normal control screenings do not find c.7106G > A, and the presence of p.W2369X mutation shows that the mutation exists
It is rare in normal population.
The above embodiments are merely illustrative of the technical scheme of the present invention and are not intended to be limiting thereof, although referring to above-described embodiment pair
The present invention is described in detail, and those of ordinary skill in the art still can be to a specific embodiment of the invention
It is modified or replaced equivalently, and these are without departing from any modification of spirit and scope of the invention or equivalent replacement,
Within the scope of the claims of the invention pending application.
SEQUENCE LISTING
<110>Co., Ltd, Hangzhou Ai Nuo medical test institute
<120>a kind of I type neurofibromatosis Disease-causing gene mutation and the Etiologic reagent based on this gene mutation
<130> 2016
<160> 70
<170> PatentIn version 3.5
<210> 1
<211> 21
<212> DNA
<213>artificial sequence
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gttctcaact taaatgtaag t 21
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gaacattaac aacaagtacc 20
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taacaattgt ggaactgcag caattatt 28
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cccataccta gttcttaaag tctgt 25
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gagaacagcc tgggcaactt gc 22
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ccaaccccaa ttagatctcc atttt 25
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atggccgcgc acaggccggt ggaat 25
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tgacaggaac ttctatctgc ctgctta 27
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atggtgaaac taattcatgc agat 24
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tgtcaaattc tgtgccttg 19
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atggaagcag tagtttcact t 21
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taggactttt gttcgctctg ctga 24
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ggagtacacc aagtatcatg ag 22
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tatacggaga ctatctaaag tatgcag 27
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atggaggcat gcatgagaga tattc 25
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tctgcacttg gcttgcggat 20
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gcgcacaggc cggtggaat 19
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tgctgtttcc ttcaggagtc gtttt 25
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accagcatgc agctgaactt cgg 23
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tcctccatgg ccagcaagag ct 22
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acgtaaagca gcagtttggc ca 22
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tgctgggtgt gctccacaac c 21
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tggtggccta agattgatgc tgtgt 25
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tgacaggaac ttctatctgc ctgct 25
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tcatgcagat ccaaagctct tgct 24
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ccgctgcatc ctgctgcact 20
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acggggtagg atgtgatatt ccttc 25
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tctatggatc ctcctccact caca 24
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gatggcactg ctgaggcgca 20
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gtgcccttgg gggagtgtgc 20
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catctcttgg caaaatgaga ggtca 25
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ggtcaggtat gttcgtgtgc ttggg 25
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gtcaaattct gtgccttgtt gaagg 25
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agcagtagtt tcacttctag ctggt 25
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agagtctgca tggagtctgc ca 22
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tgtggttcct tgttctcagt ggga 24
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acggtgagac aatggcagga 20
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cttcgaagtg tgtgccactg t 21
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gtgctctgga ggacccaggt 20
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gcaactttga tgcagcacgc agg 23
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aagcgattgc taggcccggt 20
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agctgggact tccaaagctg gga 23
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aggacttttg ttcgctctgc tga 23
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atgagcggct gctgactggc 20
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ggcacacaga agattatagg cagct 25
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gcccgactct atcccccaac aca 23
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gcagtatctg ccatcacctc agc 23
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tggggaagcc ttgggcagat 20
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ccaggagaga atgacctgtc ccgg 24
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acttgctgtt tggcattagc aaagt 25
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tgcaggtttt gttcaagaag tgcgg 25
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acgtgcaagt ggctggacca 20
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gcaggtgaag gatgcctgta ccc 23
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ggagtggcac tgcaagcaaa tgg 23
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gggggtgttg tgatccctga 20
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gcctggacat ggggcaacct 20
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acggtcctgc aaaccgccac 20
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accacagatg agtttgatca acgaa 25
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ttggcttgcg gatccgtggc 20
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tggtagcaga aagtgaaact a 21
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ataggactgt cctcttggtc cac 23
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cttagtgttt tttttttaaa ctttcta 27
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atttttttag taatttagca atacc 25
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ttaaacggag agtgttcact atc 23
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cagtcctgga aggaaaagaa gaagt 25
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tctggcaaca tggcaaaccg g 21
Claims (8)
1. a kind of Etiologic reagent based on the mutation of I type neurofibromatosis Disease-causing gene, which is characterized in that the mutation
Guanine positioned at the 48th exon region of NF1 gene, coding the 7106th replaces with adenine so as to cause the 2369th
The tryptophan of position sports terminator codon (c.7106G > A, p.W2369X)；The diagnostic reagent includes point mutation analysis reagent
Box, the kit include 1) Total RNAs extraction system reagent box: tested individual peripheric venous blood Total RNAs extraction；2) RNA is inverted
Record and cDNA amplification system kit；3) system reagent box is sequenced in cDNA；System reagent box is sequenced in the cDNA: obtaining to reverse transcription
The cDNA obtained carries out PCR amplification with five primer pairs shown in SEQ ID NO.7-16, then to five sheets of cDNA that amplification obtains
Section carries out nested PCR amplification and sequencing simultaneously with primer pair shown in SEQ ID NO.17-60, and with former NF1 gene coding DNA sequence
Column compare, and are I type neurofibroma patient in the presence of c.7106G > A in amplified production, p.W2369X mutation.
2. Etiologic reagent described in accordance with the claim 1, which is characterized in that the diagnostic reagent further includes NF1 gene
Exon deletion assay kit: the NF1 gene cDNA obtained using the reverse transcription is template, with SEQ ID NO.61-70 institute
Show that primer pair carries out real-time fluorescence quantitative PCR amplification to the exon 3 of NF1,11,25,34,58 respectively, with 2-ΔΔCtMethod is counted
According to analysis, the Relative gene content of five exons is calculated, when the Relative gene content of five exons is 0.5 or so
When be Exon deletion carrier.
3. according to Etiologic reagent described in claim 2, which is characterized in that the diagnostic reagent further includes NF1 full genome
Large fragment deletion assay kit, the kit include 1) DNA extraction system kit: tested individual peripheric venous blood gene
Group DNA is extracted；2) Microsatellite polymorphism mark assay kit in gene: using the genomic DNA as template, with SEQ ID
Three couples of primer pairs 27AC28.4,27CAGT, 38TG53.0, tri- polymorphic sites progress PCR amplifications shown in NO.1-6, poly- third
Acrylamide detected through gel electrophoresis PCR product, the NFI gene when the segment of three primer pair amplifies in PCR product is homozygote
A possibility that there are full genome large fragment deletions.
4. Etiologic reagent described in accordance with the claim 1, which is characterized in that described to carry out PCR amplification acquisition to cDNA
When five large fragments, primer annealing temperature is 60 DEG C；When carrying out nested PCR amplification to five large fragments, primer annealing temperature is
62℃。
5. Etiologic reagent according to claim 2, which is characterized in that the real-time fluorescence quantitative PCR amplification is closed
It is no more than 350bp at primer segments.
6. Etiologic reagent described in accordance with the claim 3, which is characterized in that the size of the 27CAGT amplified fragments is
The size of 201bp or so, 27AC28.4 amplified fragments is 214bp or so, and the size of 38TG53.0 amplified fragments is 479bp left
It is right.
7. according to, for detecting the primer pair group of NF1 gene mutation, feature exists in Etiologic reagent described in claim 3
In including the primer pair for detecting NF1 gene large deletion, nucleotide sequence is as shown in SEQ ID NO.1-6；For
The primer pair that cDNA obtains five large fragments is expanded, nucleotide sequence is as shown in SEQ ID NO.7-16；For to cDNA's
Five large fragments carry out the primer pair of nested PCR amplification, and nucleotide sequence is as shown in SEQ ID NO.17-60；For detecting
The primer pair of NF1 gene extron missing, nucleotide sequence is as shown in SEQ ID NO.61-70.
8. according to claim 1,2 or the preparation method of the 3 Etiologic reagents, which comprises the steps of:
After 70 single stranded DNAs in the primer pair group are individually packed, it is packaged in at least one of following substances same
In kit: PCR reaction buffer, archaeal dna polymerase and 4 kinds of dNTP.
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| CN110184275B (en) * | 2019-06-19 | 2021-06-04 | 中国人民解放军陆军军医大学第一附属医院 | NF1 new mutation pathogenic gene, application and kit |
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| CN104745697A (en) * | 2015-03-24 | 2015-07-01 | 济南艾迪康医学检验中心有限公司 | Method and primers for detecting 31st-34th whole exons of NF1 gene |
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| CN104450885A (en) * | 2014-10-29 | 2015-03-25 | 百世诺（北京）医疗科技有限公司 | Kit for detecting neurofibromatosis 1 (NF1)-related gene mutation |
| CN104745697A (en) * | 2015-03-24 | 2015-07-01 | 济南艾迪康医学检验中心有限公司 | Method and primers for detecting 31st-34th whole exons of NF1 gene |
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| Identification of forty‐five novel and twenty‐three known NF1 mutations in Chinese patients with neurofibromatosis type 1;Ming-Jen Lee等;《Human Mutation》;20061231;第27卷(第8期);832 * |
| Ion Torrent 测序技术检测Ⅰ型神经纤维瘤病患者NF1基因突变;朱艳慧等;《临床检验杂志》;20131130;第31卷(第11期);821-824 * |
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