CN109456978A - The mutation of marfan's syndrome Disease-causing gene and the diagnostic reagent based on this gene mutation - Google Patents

Abstract

The invention discloses a kind of relevant gene mutations of marfan's syndrome.The present invention has found that c.7785delC (p.Tyr2596Thrfs*86) mutation can lead to marfan's syndrome by two generation sequencing technologies for the first time.On the one hand research achievement of the invention can be used for early screening marfan's syndrome Disease-causing gene carriers of mutation, provide prenatal and postnatal care guidance, on the other hand can provide molecular diagnosis foundation for marfan's syndrome patient.

Description

The mutation of marfan's syndrome Disease-causing gene and the diagnostic reagent based on this gene mutation

Technical field

The invention belongs to area of medical diagnostics, it is related to a kind of marfan's syndrome Disease-causing gene mutation and based on this gene mutation Diagnostic reagent.

Background technique

Fibrillin-1 (FBN1) gene encodes big glycoprotein, these glycoprotein constitute large numbers of extracellular proteins, shape At the core of height tissue, the aggregation (microfibre) for extending and being generally distributed.Spontaneous and hereditary knot relevant to elastomer Form the significance that tissue disease spectrum highlights fibrillin microfibre.The main building component of old information model includes 47 A epidermal growth factor-like (EGF) structural domain, each structural domain are given birth to by 6 highly conserved cysteine residues and 7 conversions The long factor-β binding protein sample (TGFBP) structural domain composition, it is characterised in that there are 8 cysteine residues.It is tied in these EGF In structure domain, 43 are calcium mating type (cb EGF).In addition, being assembled with the old information model and ligand of remarkable effect to microfibre Interaction be to be mediated by cbEGF structural domain, and TGFBP structural domain takes part in the intermolecular interchain of each old information model The formation of disulfide bond.It interacts rich in fibrinous microfibre and basilar memebrane and elastomer, is provided for extracellular matrix Stability.

Marfan's syndrome (MFS) refers to since old information model gene (FBN1) heterozygous mutant is led on chromosome 15q21 The fibrous connective tissue autosome dominant disease of cause.The phenotypic characteristic of clinical diagnosis is allowed to occur mainly in bone, eye Eyeball and cardiovascular system.The Global prevalence rate of MFS is about every 100,000 people 2-3, is inclined to without gender or race.MFS is shown as Hypomegasoma, out-of-proportion long limb and toes, preceding chest wall deformity, slightly (especially to moderate arthrochalasis and frequent deformity of spine It is scoliosis and thoracic vertebrae lordosis) and aortic root is expanded and/or decomposition, phacometachoresis.

The application is identified using sequencing technologies to be specifically mutated present on the FBN1 gene for causing marfan's syndrome, to face Bed diagnosis marfan's syndrome provides new molecular marker.

Summary of the invention

Research Thinking of the invention is as follows: it is prominent to screen Disease-causing gene relevant to marfan's syndrome first with the sequencing of two generations Become, in order to avoid false positive results appearance, is verified later by Sanger sequencing, be finally obtained a marfan's syndrome Disease-causing gene mutation, mutation be FBN1 gene c.7785delC.

Marfan's syndrome patient and normal population can be distinguished and be separated in the Disease-causing gene mutation that the present invention screens, because This, Disease-causing gene mutation of the invention can be used as the biomarker of diagnosis marfan's syndrome.

Specifically, the gene mutation is FBN1 base the present invention provides a kind of relevant gene mutation of marfan's syndrome Because of upper mutation.

Further, the mutational site is FBN1 gene c.7785 position, c.7785delC the mutation is.

The present invention also provides a kind of for diagnosing the reagent of marfan's syndrome, and the reagent is the mentioned-above base of detection Because of the reagent of mutation.

In the prior art to the detection of gene mutation site genotype, various conventional methods can be used, as DNA sequencing, Restrictive fragment length polymorphism (RFLP), single-strand conformation polymorphism (SSCP), allele specific oligonucleotide Hybridize (ASO).Therefore the mentioned-above reagent of the present invention includes the reagent used in the above method.

Preferably, the specificity of nucleic acid sequence of the reagent including containing mentioned-above gene mutation site expands Increase primer.

Further, the reagent can also include other conventional reagents and/or DNA extraction process in pcr amplification reaction Used in reagent used in reagent and/or DNA sequencing process.

Further, other conventional reagents in the pcr amplification reaction include but is not limited to dNTP, PCR buffer, magnesium from Son, Tap polymerase etc..

The present invention provides mentioned-above gene mutations to prepare the application in mentioned-above reagent.Art technology Personnel are according to gene mutation site upstream and downstream sequence design specificity amplification primer or specificity detection probe.Primer and probe Design method be the ordinary skill in the art.

The present invention also provides mentioned-above gene mutations to prepare the application in marfan's syndrome diagnostic kit.

The present invention also provides mentioned-above reagents to prepare the application in marfan's syndrome diagnostic kit.

Further, whether the diagnostic kit diagnoses individual with horse by mentioned-above mutation in detection sample All syndrome.

In specific embodiments of the present invention, the samples sources are blood.

The present invention also provides a kind of marfan's syndrome diagnostic kit, the diagnostic kit includes that detection is noted earlier Gene mutation reagent.

The reagent can be capable of detecting when any reagent of mentioned-above gene mutation site genotype.Including but not It is limited to DNA sequencing, Restrictive fragment length polymorphism (RFLP), single-strand conformation polymorphism (SSCP), allele specific Oligonucleotide hybridization (ASO) used in reagent.

Specific embodiment according to the present invention, the reagent of the invention include containing mentioned-above gene mutation position The specificity amplification primer of point.

Further, the reagent can also include other conventional reagents and/or DNA extraction process in pcr amplification reaction Used in reagent used in reagent and/or DNA sequencing process.

Further, other conventional reagents in the pcr amplification reaction include but is not limited to dNTP, PCR buffer, magnesium from Son, Tap polymerase etc..

The present invention also provides detection FBN1 genes with the presence or absence of the method for mutation, and described method includes following steps:

(1) sample genomic dna is extracted；

(2) FBN1 gene order is expanded；

(3) DNA sequencing；

(4) sample DNA sequencing result to be detected is compared with normal person's genomic dna sequence, if it find that in the presence of C.7785delC, then judge that FBN1 gene has mutation.

The present invention also provides a kind of methods for diagnosing marfan's syndrome, and described method includes following steps: detection FBN1 The genotype in gene c.7785 site, if existed c.7785delC on FBN1 gene, diagnosing individual test subjects is all synthesis of horse Levy patient.

Advantages of the present invention: the present invention has found that c.7785delC FBN1 gene is mutated by two generation sequencing technologies for the first time can Lead to marfan's syndrome.It on the one hand, can be with early screening marfan's syndrome by detecting whether subject carries above-mentioned mutation Disease-causing gene carriers of mutation provides prenatal and postnatal care guidance；Molecular diagnosis foundation can also be provided for marfan's syndrome patient.Especially Ground, diagnostic kit provided by the present invention can be used for quickly and efficiently predicting or diagnosing marfan's syndrome.On the other hand, originally Invention be marfan's syndrome study of incident mechanism established important foundation, for marfan's syndrome patient treatment provide it is completely new Theoretical foundation.The third aspect, the present invention can provide possible drug target for treatment marfan's syndrome.

" Tyr2596Thrfs*86 ", " p.Tyr2596Thrfs*86 ", synonymous with " Y2596Tfs*86 " herein.

" Gly2003Arg ", " G2003R " are synonymous with " p.G2003R " herein.

Detailed description of the invention

Fig. 1 shows the clinical manifestation of subject XH253, wherein A: hand bilateral dolichostenomelia；B: sufficient bilateral spider foot sample Refer to；C: the front elevation of entire backbone X-ray imaging display scoliosis；D: entire backbone X-ray imaging display scoliosis Side view；

Fig. 2 shows subject XH253 genomic DNA Sanger sequence results；

Fig. 3 shows the protein electrophoresis figure of SDD-AGE；

Fig. 4 shows Western blot result figure, wherein A: albumen development figure；B: gray-scale statistical figure.

Specific embodiment

In the present invention, unless otherwise stated, Science and Technology noun used herein has art technology The normally understood meaning of personnel institute.Also, molecular genetics used herein, nucleic acid chemistry and molecular biology relational language With widely used term and the conventional steps that laboratory operation step is in corresponding field.Meanwhile in order to better understand The definition and explanation of relational language is provided below in the present invention.

Diagnosis whether term " diagnosis " includes the prediction of disease risks, disease incidence in text further includes to disease prognosis Assessment.

Term " mutation " refers to that the polynucleotide sequence of wild type changes in text, becomes variant, and variant can be with Be naturally occur or non-natural occur.

In the present invention, " primer " refers to the polynucleotide passage for expanding target nucleic acids in PCR reacts, it typically is Oligonucleotides, such as containing at least five base, for example, 5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20, 21, the polynucleotide passage of 22,23,24,25,30,35,40,45,50 or more bases.Primer need not with it is to be amplified Target gene or its complementary strand complete complementary, as long as it being capable of specific amplification target gene.As used herein,

Term " specific amplification " refers to that primer can react amplifying target genes by PCR, without expanding other genes. For example, specific amplification FBN1 gene refers to, primer only expands FBN1 gene in PCR reaction, without expanding other genes, this The design of class primer is well known to those skilled in the art.

Present invention will be further explained below with reference to specific examples.It should be understood that these embodiments are merely to illustrate the present invention Rather than it limits the scope of the invention.In the following examples, the experimental methods for specific conditions are not specified, usually according to conventional strip Part such as Sambrook et al., molecular cloning: laboratory manual (New York:ColdSpring Harbor Laboratory Press, 1989) condition described in, or according to the normal condition proposed by manufacturer.

The gene mutation screening relevant to marfan's syndrome of embodiment 1

1, participant recruits

Barrier study is interpreted by the scoliosis & complication (DISCO) of BJ Union Hospital (PUMCH) (www.discostudy.org) 1 patient MFS has been recruited.At least two clinical experts independently confirm that the clinic of MFS is examined It is disconnected.The age is 18 years old or more in registration patient or its parent (participants of under-18s when entering group), which provide, written to know Letter of consent.Germline DNA is collected from impacted propositus and corresponding unaffected parent's (if any).

2, sample preparation

Peripheral blood sample is collected from participant.According to the explanation of manufacturer QIAamp DNA Blood MiniKit (QIAGEN, Germany) extracts genomic DNA.By the visual inspection after ethidium bromide staining and use 1% Ago-Gel The size fractionation of electrophoresis characterizes the quality and purity of the DNA of extraction.By QubitBR measurement (Invitrogen, USA) to base Because a group DNA is quantified, for the preparation of the next-generation sequencing library (NGS).

3, targeting two generations sequencing

In order to establish one to greatest extent covering be related to vertebra abnormality phenotype the Mendel disease cause of disease known NGS (next-generation sequencing) panel, this research carry out congenital scoliosis (CS) related disease The document and database retrieval of system.After manual examination and verification, 344 genes are found altogether, are equivalent to the relevant single-gene table of 457 CS Type.Also include 220 genes of another set, be related to approach relevant to vertebra development or induce CS table in animal model Type, but it is not yet associated with the human diseases with CS phenotype.Targeting panel includes 564 genes, 6458 exons and side The region wing 30bp, total Genome Size 2.97Mb.Designed for target capture DNA probe and online purchased from NimbleGen (Roche, Switzerland) (https: //design.nimblegen.com/nimbledesign/app).It is expected that final The target area of one group of DNA probe covering 99.7%.In addition to the TBX6 gene with compound heterozygosis genetic model of previous publications Except, FBN1 gene is also the target in panel.Targeting sequence enrichment library is prepared according to conventional technical means.For each Sample uses 1 μ g genomic DNA as starting material.By the size of genomic DNA fragmentization to~200-250bp.By segment The DNA of change carries out end reparation, and the connection of adapter is covered and be indexed with A tail.After 5 PCR amplifications recycle, merge every A index product simultaneously hybridizes with the DNA probe of the targeted capture in a solution capture reaction.The product of enrichment DNA is targeted into one Step carries out 14 PCR amplification circulations, is then quantitatively carried out finally by Bioanalyzer analysis (Agilent, USA) and qPCR The verifying of library yield.It is sequenced on Illumina Hiseq2500 sequenator using PE100 mode.

4, variant is handled, filtering and annotation

After generating original sequence data, low quality is deleted using perl script and is adapted the reads of device pollution.It uses Remaining reads is navigated to ginseng and examines genome hg19 (GRCh37, http://genome.ucsc.edu/) by BWA mapper On.In addition, calling mononucleotide variant (SNV) and small insertion and missing (indel) using GATK bioinformatics packet.Using interior The annotation pipeline of portion's exploitation annotates variant.In public database (1000Genomes Project, Exome Sequencing Project 6500 (it can be obtained from following website: http://evs.gs.washington.edu/EVS/), ExAC (can obtain from http://exac.broadinstitute.org) or inside WES control data set) in filter out and have Common and high-frequency (minorAllele frequency > 1%) variant.This research is also using based on human mutation database (HGMD) and the customized databank of mankind's online Mendelian inheritance in man (OMIM) (can be obtained from following network address: https: // Omim.org/ the variant detected) is annotated.

5, Sanger is sequenced

Sanger sequencing is carried out to the genomic DNA from all individuals, correctly to confirm the variant of all identifications.It uses 5 software Design primers of Primer Premier are with the specific variants comprising FBN1 gene.It is analyzed using SNAPGENE software Sanger reading.

6, result

Clinical signs and new allelic variant with MFS diseased individuals

Subject XH253.Propositus is 10 years old Chinese girl (phenotypic characteristic is shown in Figure 1A-D), passes through radiophotography Confirm that scoliokyphosis is presented in she.The time of assessment is 4 years old for the first time.Patient moves in local hospital and is diagnosed as the upper respiratory tract Infection.Full X-ray of spine shows slight scoliosis, the non-surgical treatment guarded.After 2 years, the state of an illness of patient is disliked Change: the degree of scoliosis becomes more serious.It is performed for more than 22 hours conservative therapys daily with bracket.She by referral to I Orthopedic spinal expert further assessed and managed.Full X-ray of spine shows there is three curve scoliosis. The angle Cobb of primary curvatures is 43 degree, back it is flat with Thoracolumbar disk after it is convex.Transthoracic echocardiography assessment display moderate bicuspid valve The aortic root of incompetence and 3.6cm measure (when being standardized as age and body surface area, score=4.5 Z).Formerly card Skeletal system exception, such as long and thin limbs and spider web spasm are observed in person.Visual impairment occur she 5 years old when.Root Classify according to Ghent, propositus meets the clinical criteria of classical MFS, and score is total up to 7 points in the Gent standard of revision.Pass through Targeting NGS identifies heterozygosis frameshift deletion c.7785delC (p.Tyr2596Thrfs*86) in the FBN1 of propositus, then passes through It is further verified using the orthogonal experimental method that Sanger is sequenced to confirm, as a result as shown in Figure 2.

2 Candidate Mutant functional verification of embodiment

1, plasmid construction and mutagenesis

Contain the DNA piece of the overall length FBN1cDNA of derived from human muscle cDNA and suitable restriction site by PCR amplification Section, and be cloned into the carrier pEGFP of NheI and SacII digestion.PCR product is digested with restriction enzyme KpnI and EcoRI, and is connected To the identical restriction site of pEGFP, recombinant plasmid pEGFP-FBN1 is generated.Mutation EGFP-FBN1 is generated by direct mutagenesis Plasmid.The G2003R previously reported in adolescent idiopathic scoliosis (AIS) case is used as positive control.According to manufacture The explanation of quotient utilizes QuikChange Lightning site directed mutagenesis kit (Agilent Technologies, Santa Clara, CA, USA) will c.7785delC (p.Tyr2596Thrfs*86) and G2003R mutation introduce wild type (WT) pEGFP-FBN1.It is sequenced by Sanger dideoxy to 2 kinds of obtained mutant plasmid pEGFP-FBN1-Tyr2596Thrfs*86, PEGFP-FBN1-Gly2003Arg is sequenced, to confirm that base needed for incorporation changes.

2, cell culture and transfection

HEK293T cell culture medium (Dulbecco's Modified Eagle Medium (Gibco, Waltham, MA, USA)+10% fetal calf serum (Biological Industries, Cromwell, USA)+1% penicillin/streptomycin (Gibco-Life Technologies, USA)) in cultivated in 37 DEG C, the cell incubator containing 5% carbon dioxide.According to system The explanation for making quotient, using Lipofectamine3000 (Invitrogen, CA, USA), with mutant or/and WT pEGFP- FBN1 transfection/cotransfection cells.6 hours after transfection, culture medium is replaced with fresh complete DMEM culture medium, and by cell into one Step culture 48 hours.

3、SDD-AGE

In order to carry out half denaturing detergent-agarose gel electrophoresis (SDD-AGE), by HEK293T cell and wild type (pEGFP-FBN1) and mutant (pEGFP-FBN1-Tyr2596Thrfs*86) cotransfection 6 hours it, then incubates 48 hours.? 5%CO is used at 37 DEG C₂After incubating 48 hours in hot tank, by being centrifuged 2 minutes harvest cells in 3000rcf, it is resuspended in 200 μ L In water, then it is centrifuged.Then about 100 μ l pickling cells are added into each hole, are subsequently added into 120 μ L lysis buffer (100mM Tris pH 8,1%Triton X-100,50mM beta -mercaptoethanol, 3%HALT protease inhibitor cocktail, 30mM N- second Base maleimide and 12.5U/mL nuclease).Then rubber pad (Nunc 276002) sealing block is used, in Qiagen On Tissuelyzer 2 twice with maximum speed oscillation, continue 3 minutes.Then 35 μ L 4X Sample Buffers are added into each hole Liquid (2X TAE, 20% glycerol, 8%SDS, 0.01% bromophenol blue).Then by block it is of short duration vortex and so that it is incubated at room temperature 3 points Then clock is centrifuged 2 minutes at 3000rcf to remove cell fragment.Then carry out Hybond ECL nitrocellulose electrophoresis and Capillary trace.By on Protein transfer to PVDF membrane, and it is anti-with the monoclonal of the antifibrin 1 sufficiently characterized Body (Abcamab124334, Cambridge, UKAbcam) is detected.

4, Western blotting

With RIPA (50mM Tris-HCL, 1%NP40,0.25%Na- deoxycholic acid, 150mMNaCl and the 1mM of improvement EDTA；CompleteTM protease inhibitor cocktail) lytic cell, and protein concentration is measured with BCA-.It is poly- in 8%SDS The protein that total amount is 5mg is separated on acrylamide gel, and electrophoresis is carried out to protein and is transferred on nitrocellulose filter.It will Film is closed 30 minutes in milk powder at room temperature (RT, 25 degrees Celsius), and primary antibody (Phospho-Smad2 (Ser245/250/ is added 255) antibody, CellSignaling Technology；Anti- GAPDH, Millipore) it is incubated overnight at 4 DEG C.After washing, by phase The goat antirabbit secondary antibody (KPL) for the horseradish peroxidase answered incubates 1 hour at (25 degrees Celsius) of room temperature.With WesternBright ECL chemiluminescence system (Advansta, CA, USA) visualizes band.With different cell lysates Carry out the experiment three times.Use ImageJ quantitative chemical luminous signal.

5, it statisticallys analyze

SPSS software, version 17.0 (SPSS) are examined for carrying out double tail t, compare the value of test sample and check sample. P value is considered to have statistical significance less than 0.05.

6, result

Altogether by the plasmid (pEGFP-FBN1-Tyr2596Thrfs*86) and WT plasmid (pEGFP-FBN1) of expressing mutant It is transfected into HEK293T cell.After expression 48 hours, splitting for the cell of expression EGFP-FBN1 fusion is analyzed by SDD-AGE Solve object.With in SDD-AGE and the HEK293T cell lysate of western blot research transient expression EGFP-FBN1 fusion Detection of the SDD-AGE to SDS resistance aggregation.Inducible protein matter is expressed 48 hours and is examined with monoclonal FBN1 specific antibody It surveys.It is worth noting that, WT plasmid (EGFP-FBN1 fusion) has the SDS resistance of amyloid protein structure.Due to starch The formation of sample albumen is time-and concentration-dependent, and the time of transient expression is set as 48 hours.After expression in 48 hours, WT plasmid (EGFP-FBN1 fusion) aggregation, and the cotransfection of pEGFP-FBN1-Tyr2596Thrfs*86 and WT plasmid is presented Sub-fraction collectin out.Since the concentrating portions are less than the concentrating portions of WT plasmid, show that Tyr2596Thrfs*86 may NMD mechanism is undergone, therefore leads to FBN1 haploinsufficiency (Fig. 3).

Previous studies show that causing harmful FBN1 of marfan's syndrome to be mutated leads to endogenous transforming growth factor β The up-regulation of (TGF-β) receptor signal.This signal transduction can measure or indirect by the abnormal activation of downstream targets in blood plasma Measurement, including SMAD2 (pSMAD2), the Hyperphosphorylationof of ERK1/2 and JNK1.In order to determine that newly truncating FBN1 in MFS patient becomes The functional outcome of body Tyr2596Thrfs*86, the plasmid transfection for expressing mutant EGFP-FBN1cDNA is thin to HEK293T In born of the same parents.With truncated FBN1 variant Tyr2596Thrfs*86 (P=0.007), the transfection of Gly2003Arg (P=0.006) is thin Born of the same parents show the significant raising (Fig. 4 A, B) of phosphorylation of SMAD2 (pSMAD2) in western blot.

3 marfan's syndrome diagnostic kit of embodiment

1, kit forms: primer/Taq enzyme/buffer/dNTP

It is extracted and purified genes material (DNA sample) from organism sample to be detected.

2, application method:

(1) extracting genome DNA: peripheral blood sample genomic dna is extracted using genome DNA extracting reagent kit.

(2) pcr amplification reaction is first carried out using PCR primer, Taq enzyme, sample genomic dna, buffer, dNTP etc.；

(3) pcr amplification product is purified；

(4) BigDye reaction is carried out to the PCR product of purifying；

(5) BiyDye reaction product is purified；

(6) BiyDye reaction product is sequenced, sequencing sequence is compared with normal sequence.

In addition, it should also be understood that, after reading the above teachings of the present invention, those skilled in the art can be to this hair Bright to make various changes or modifications, these equivalent forms also fall within the scope of the appended claims of the present application.

Claims (10)

1. a kind of relevant gene mutation of marfan's syndrome, which is characterized in that the gene mutation is the mutation on FBN1 gene.

2. gene mutation according to claim 1, which is characterized in that the mutational site is FBN1 gene c.7785 position, C.7785delC the mutation is.

3. a kind of for diagnosing the reagent of marfan's syndrome, which is characterized in that the reagent is described in detection as claimed in claim 1 or 22 Gene mutation reagent.

4. reagent according to claim 3, which is characterized in that the reagent includes being directed to base of any of claims 1 or 2 Because of the specificity amplification primer in mutational site.

5. reagent according to claim 4, which is characterized in that the reagent includes the conventional reagent in pcr amplification reaction, And/or reagent used in reagent used in DNA extraction process and/or DNA sequencing process.

6. application of the mutation of any of claims 1 or 2 in the reagent described in any one of preparation claim 3-5.

7. reagent described in mutation of any of claims 1 or 2 or any one of claim 3-5 is examined preparing marfan's syndrome Application in disconnected kit.

8. application according to claim 7, which is characterized in that the diagnostic kit passes through claim in detection sample Whether mutation described in 1 or 2 is individual with marfan's syndrome to diagnose.

9. application according to claim 8, which is characterized in that the samples sources are blood.

10. a kind of marfan's syndrome diagnostic kit, which is characterized in that the diagnostic kit includes appointing in claim 3-5 Reagent described in one.