CN106650310A - Method for identifying embryos carrying chromosomal balanced translocation and normal embryos - Google Patents
Method for identifying embryos carrying chromosomal balanced translocation and normal embryos Download PDFInfo
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- CN106650310A CN106650310A CN201710013009.XA CN201710013009A CN106650310A CN 106650310 A CN106650310 A CN 106650310A CN 201710013009 A CN201710013009 A CN 201710013009A CN 106650310 A CN106650310 A CN 106650310A
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- G16B20/00—ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations
Abstract
The invention relates to the field of genetic diagnosis and human assisted reproduction, and more particularly relates to an embryo pre-implantation genetic diagnosis technique (PGD). According to the method provided by the invention, for patients suffering from chromosomal balanced translocation (including reciprocal translocation and Robertson translocation) and mates thereof, family haplotype linkage analysis is performed on the chromosomes of the embryos obtained by in vitro fertilization and the relatives of the translocation carrier, the embryos with chromosomal balanced translocation and the embryos with normal chromosomes can be rapidly, simply and accurately distinguished, and at the same time, the chromosome copy number variation of the embryo is screened, so that the clinical pregnancy rate is improved, the genetic transmission of the chromosomal balanced translocation to the next generation is timely blocked before embryo implantation, and thus the development and progress of the human assisted reproductive technology are promoted to a certain extent.
Description
Technical field
The invention belongs to gene diagnosis and human ancillary reproductive field, in particular to diagnostic techniques before Embryonic limb bud cell
(PGD) it is, a kind of can to differentiate the haplotype linkage analysts side whether embryo carries parent's balanced translocation chromosomal inheritance information
Method.
Background technology
Translocation is a kind of common chromosomal structural variation, is because the fracture of coloured differently body is distributed
The chromosome abnormality that raw mistake is spliced and caused.Probably there is 0.19% incidence in normal population, in recurrent abortion or anti-
The probability occurred in the patient of multiple IVF kinds plant failure is about 5%.Although balance translocation carrier ordinary circumstance phenotype without exception,
But there is the gamete that up to 19%-77% is unbalanced type in the gamete that its archaeocyte meiosis is produced, this and dye
The breakaway poing of colour solid transposition is relevant with the concrete chromosome that transposition occurs.The quadrivalent structure of chromosome, generally occurs in cell
The meiosis I phase, produced by two chromosomes and corresponding two normal homologue pairings of transposition.This four
The common form of valency structure has 2:2,3:1 or 4:0 three kinds of clastotypes.Only 2:Under 2 pattern, clastotype energy is aligned
Normal or balance gamete is produced, other clastotypes produce the gamete of non-equilibrium type.Nonequilibrium gamete can lead
Cause infertile, recurrent abortion, or the offspring of fertility dysnoesia or other congenital anomalies.
PGD is that " embryo transfer enters diagnostic techniques " is successfully applied to first clinic by Handyside AH earliest, initial
PGD technologies detect sex using PCR or FISH methods, help the X such as risky fertility hemophilia A, progressive muscular dystrophy to connect
Mr. and Mrs' pregnancy and delivery of lock hereditary disease offspring goes out normal girl baby；Over 2006, there is researcher to pass through to select with Disease-causing gene in dye
The STR marker sites of close linkage carry out single-gene disorder diagnosing embryo's whether heredity Disease-causing gene on the position of colour solid
Embryonic limb bud cell before diagnose, the method is otherwise known as science of heredity haplotype analysis (preimplantation genetic before implantation
Haplotyping, PGH).PGD technologies in the patient of translocation due to examination haunting and have chromosome large stretch of
The healthy embryo of Duan Yichang, improves Clinical Pregnancy Rate in, is widely used in supplementary reproduction field.For with known dye
The Mr. and Mrs of colour solid balanced translocation, the PGD based on FISH (FISH) is used for filtering out unbalanced embryo earliest, but
Due to technology restriction of the FISH technology in terms of cell is fixed with signal-obtaining, the accuracy that result can be affected to understand.In recent years,
Biochip technology and sequencing technologies can be widely used in each due to it to the advantage that 23 pairs of chromosomes carry out examination comprehensively
Reproductive center, improves Clinical Pregnancy Rate in.But despite of that, these traditional PGD technologies can only become to the copy number of embryo
It is different to be detected.
Up till now, some article reports that embryo and complete fetal tissues are carried with regard to differential staining body balanced translocation are had
Road.Initially, researcher designs the special specific FISH probe across breakaway poing for chromosome translocation breakaway poing, can be used for difference dye
Normal and balanced translocation the embryo of colour solid, although the method has certain feasibility, but method design is complicated, time-consuming it is difficult to face
It is applied in bed work.In the recent period, the research of Treff et al. is then employed based on SNP chip technology, with translocation carrier Mr. and Mrs
Both sides and uneven embryo are reference, are differentiated by building the haplotype within breakaway poing 5Mb, can identify dyeing body phase
The chromosomal condition of the carrier embryo of mutual transposition.A kind of technology of MicroSeq-PGD has been invented by Ling You research groups, passes through
With reference to chromosome translocation breakaway poing microdissection technology and NGS, the sequence of the DNA near transposition breakaway poing is detected, so as to area
Not normal embryo and the embryo of balanced translocation.But, all there are following defects in said method：1) method and technology flow process is all very
Complexity, takes longer；2) the homologous recombination situation in breakaway poing region cannot be judged；3) it is not suitable for the carrying of Robertsonian translocation
Person.It can be seen that, still lack in prior art can accurately, be quickly applied to clinical differential staining body balanced translocation (phase reciprocity
Position and Robertsonian translocation) embryo auxiliary procreation technology.
The content of the invention
For defect present in above-mentioned prior art, the present invention establishes a kind of based on the extensive SNP bases of full-length genome
Because of the method that genotyping result carries out haplotyping (PGH) before Embryonic limb bud cell.It is flat for chromosome according to the method for the invention
Embryo and translocation carrier parent after the patient of weighing apparatus transposition (including mutual transposition and Robertsonian translocation) and its spouse, inseminatio externalis
The chromosome of category carries out family haplotype linkage analysts, can embryo that is quick, easy, distinguishing translocation exactly
With the normal embryo of chromosome, and while carry out examination to the chromosome copies number variation of embryo, Clinical Pregnancy Rate in is improved, can
Distinguish the embryo for carrying the embryo and normal chromosomal of balanced translocation chromosome before the Embryonic limb bud cell being fertilized in vitro in time, so as to
Preferential to transplant the completely normal embryo of chromosome, in time blocking translocation entails the next generation.Push away to a certain extent
The progress of Issues of Human Assisted Reproductive Technologies is moved.
The concrete technical scheme of the present invention is as follows：
The invention provides a kind of carry embryo and the complete fetal tissues of chromosome for differential staining body balanced translocation
Family haplotype reconstruction method, comprises the steps of：
1) sample Genotyping：By translocation carrier Mr. and Mrs both sides, at least one carrier relatives and dye
Colour solid balance translocation carrier Mr. and Mrs IVF Embryos carry out extensive SNP genotype detections；Carrier relatives can be and take
There are the relatives of identical balanced translocation, the alternatively normal relatives of chromosome with person；
2) information SNPs site is determined：The selection standard of information SNPs is：It is miscellaneous in translocation carrier
Mould assembly, is homozygous in its spouse, and is also homozygous SNP site in carrier relatives；Select covering coloring body
Information SNPs on transposition breakaway poing, translocation chromosome and normal homologue corresponding thereto, chromosome is per Mb models
1 information SNP is at least selected in enclosing；Breakaway poing region is being covered, information SNPs is selected from the range of breakaway poing upstream and downstream 1-5Mb
Select；
3) family haplotype is built：Set step 2) determine information SNPs site covered by family linkage analysis
The haplotype of the whole chromosome of the region of fracture, translocation chromosome whole chromosome and its homologue, coloured differently at two
The collection of body haplotype is collectively referred to as family haplotype.
The invention provides a kind of differential staining body balanced translocation carries the PGH side of embryo and the complete fetal tissues of chromosome
Method, comprises the steps of：
1) sample Genotyping
Translocation carrier Mr. and Mrs both sides, at least one carrier relatives and carrier Mr. and Mrs are in vitro fertilization
Embryo carries out extensive SNP genotype detections；Carrier relatives can be the relatives for having identical balanced translocation with carrier, also may be used
For the normal relatives of chromosome；Translocation carrier Mr. and Mrs both sides, carrier relatives are referred to as into sample for reference, are carried
Person's Mr. and Mrs' IVF Embryos are referred to as sample undetermined；
2) information SNPs site is determined
The selection standard of information SNPs is：It is heterozygous in translocation carrier, is pure in its spouse
Mould assembly, and be also homozygous SNP site in carrier relatives；Select covering coloring body transposition breakaway poing, transposition dyeing
Information SNPs on body and normal homologue corresponding thereto, in the range of the every Mb of chromosome 1 information is at least selected
SNP；In covering coloring body transposition breakaway poing region, information SNPs is selected from the range of chromosome translocation breakaway poing upstream and downstream 1-5Mb
Select；
3) family haplotype is built
Set step 2) determine information SNPs site by family linkage analysis obtain covering two at the region of fracture, transposition
The haplotype of the whole chromosome of chromosome whole chromosome and its homologue, the collection of coloured differently body haplotype is collectively referred to as
Family haplotype；
4) data collection and analysis
By the chromosome monosomy type of the haplotype information in chromosome translocation breakaway poing region and sample for reference in sample undetermined
Information is compared, and judges whether chromosome translocation breakaway poing region there occurs by the haplotype of whole chromosome homologous heavy
Group：
I) when being reference with the relatives with carrier with identical balanced translocation：
If a. sample chromosomes transposition breakaway poing region undetermined is not recombinated, when sample breakaway poing region monomer undetermined
In type information and sample for reference translocation carrier and have with carrier identical balanced translocation relatives list
When body-shape information is consistent, then embryo is carried for balanced translocation；When sample breakaway poing region haplotype information undetermined and sample for reference
Middle translocation carrier haplotype information is consistent, but has the haplotype of the relatives of identical balanced translocation with carrier
Then it is the completely normal embryo of chromosome when information is inconsistent；
If b. sample chromosomes transposition breakaway poing region undetermined there occurs homologous recombination, balanced translocation is carried or chromosome
The criterion of fetal tissues completely is contrary with a；
Ii) when being reference with the normal relatives of chromosome：
If a. sample chromosomes transposition breakaway poing region undetermined is not recombinated, when sample breakaway poing region monomer undetermined
Translocation carrier and the haplotype with the normal relatives of the chromosome of carrier in type information and sample for reference
Then it is the completely normal embryo of chromosome when information is consistent；When sample breakaway poing region haplotype information undetermined and sample for reference
Middle translocation carrier haplotype information is consistent, but with the haplotype information of the normal relatives of the chromosome of carrier
When inconsistent, then embryo is carried for balanced translocation；
If b. sample chromosomes transposition breakaway poing region undetermined there occurs homologous recombination, balanced translocation is carried or chromosome
The criterion of fetal tissues completely is contrary with a.
Extensive SNP genotype detections of the present invention cover 23 pairs of chromosomes；Extensive SNP genotype detection methods are excellent
Select genetic chip and gene sequencing.
When the present invention selects embryonic development to the 3-7 days, biopsy obtains 1-10 cell as step 1) described in vitro
The detection sample of fertilized embryo；It is preferably derived from the cell of embryonic blastomeres biopsy or blastaea trophectoderm biopsy.
At covering coloring body transposition breakaway poing region, described information SNPs is from breakaway poing in information SNPs site of the present invention
Select in the range of the 2-4Mb of downstream, select preferably from the range of breakaway poing upstream and downstream 2Mb.
When the present invention builds family haplotype, every chromosome per Mb in the range of at least select 2,3,4,5,6,7,8,9 or
10 information SNPs.
The cell lysis that the present invention obtains biopsy, carries out whole genome amplification；Whole genome amplification method may be selected from MDA
Method, MALBAC methods or other whole genome amplification methods.
Translocation carrier Mr. and Mrs both sides of the present invention are that body is thin with the source of the detection sample of carrier relatives
Born of the same parents, preferred peripheral blood.
The invention provides a kind of system for carrying embryo for differential staining body balanced translocation, the system is included can
Calculate the software and the hardware for carrying above-mentioned software for processing sample data, it is characterised in that
1) system is also included and stores translocation carrier Mr. and Mrs both sides, at least one carrier relatives
With the genotype data that translocation carrier Mr. and Mrs IVF Embryos carry out extensive SNP genotype detections
Hardware；Carrier relatives can be the relatives for having identical balanced translocation with carrier, alternatively the normal relatives of chromosome；Will dye
Colour solid balance translocation carrier Mr. and Mrs both sides, carrier relatives are referred to as sample for reference, and carrier's Mr. and Mrs' IVF Embryos are referred to as
Sample undetermined；
2) software is according to following regular determination information SNPs sites：The selection standard of information SNPs is：In chromosome
It is heterozygous in balance translocation carrier, is homozygous in its spouse, and is also homozygous SNP in carrier relatives
Site；Select the letter on covering coloring body transposition breakaway poing, translocation chromosome and normal homologue corresponding thereto
Breath SNPs, in the range of the every Mb of chromosome 1 information SNP is at least selected；Breakaway poing region is being covered, information SNPs is from breakaway poing
Select in the range of the 1-5Mb of downstream；
3) software is according to following principle construction family haplotypes：The letter chosen according to above-mentioned standard on every chromosome
Breath SNPs sites, obtain covering the region of fracture at two, translocation chromosome whole chromosome and its homologous by family linkage analysis
The haplotype of the whole chromosome of chromosome, the collection of coloured differently body haplotype is collectively referred to as family haplotype；
4) software is by the dye of the haplotype information in chromosome translocation breakaway poing region and sample for reference in sample undetermined
Colour solid haplotype information is compared, and judges whether chromosome translocation breakaway poing region occurs by the haplotype of whole chromosome
Homologous recombination：
I) when being reference with the relatives with carrier with identical balanced translocation：
If a. sample chromosomes transposition breakaway poing region undetermined is not recombinated, when sample breakaway poing region monomer undetermined
In type information and sample for reference translocation carrier and have with carrier identical balanced translocation relatives list
When body-shape information is consistent, then embryo is carried for balanced translocation；When sample breakaway poing region haplotype information undetermined and sample for reference
Middle translocation carrier haplotype information is consistent, but the haplotype of the carrier relatives of balanced translocation identical with carrying
Then it is the completely normal embryo of chromosome when information is inconsistent；
If b. sample chromosomes transposition breakaway poing region undetermined there occurs homologous recombination, balanced translocation is carried or chromosome
The criterion of fetal tissues completely is contrary with a；
Ii) when being reference with the normal relatives of chromosome：
If a. sample chromosomes transposition breakaway poing region undetermined is not recombinated, when sample breakaway poing region monomer undetermined
Translocation carrier and the haplotype with the normal relatives of the chromosome of carrier in type information and sample for reference
Then it is the completely normal embryo of chromosome when information is consistent；When sample breakaway poing region haplotype information undetermined and sample for reference
Middle translocation carrier haplotype information is consistent, but with the haplotype information of the normal relatives of the chromosome of carrier
When inconsistent, then embryo is carried for balanced translocation；
If b. sample chromosomes transposition breakaway poing region undetermined there occurs homologous recombination, balanced translocation is carried or chromosome
The criterion of fetal tissues completely is contrary with a.
Differential staining body balanced translocation of the present invention carries embryo and the PGH methods of the complete fetal tissues of chromosome have
Following advantage：1) while being predicted to chromatin state in embryo, examination is carried out to the 23 pairs of chromosome；2) except
The haplotype in breakaway poing region, can simultaneously set up the whole of the whole chromosome that is related to transposition and corresponding normal homologue
The haplotype of bar chromosome, this can show the restructuring situation of two homologues in breakaway poing region of patient, and PGH's is pre-
Surveying can be comprising breakpoint region to whole chromosome；3) suitable for all genetic translocation familys (including phase
Mutually transposition and Robertsonian translocation)；4) method is relatively easy, takes shorter, and whole discrimination process can be completed in 48h, is suitable for
In routine clinical work.
Description of the drawings
Fig. 1. the family collection of illustrative plates for carrying translocation is illustrated
Fig. 2. No. 3 family peripheral blood caryograms
Fig. 3. No. 3 family Haplotype maps
Specific embodiment
Embodiment 1：The sample for reference of patient and patient's parental generation is collected
9 are raised and the translocation for receiving supplementary reproduction have been carried into family, those selected has been all from Shanghai collection love
Heredity and supplementary reproduction research institute.Each family is required for signing Written informed consent, and research approach is produced by Fudan University woman
Human experimenter Ethics Committee of hospital of section ratifies.
From in June, 2014 in March, 2016, there are recurrent spontaneous history or chromosome abnormality in all 9 families
History, the side of Mr. and Mrs one that translocation is carried is referred to as below " patient ", the opposing party's referred to as " spouse of patient ",
The family collection of illustrative plates for carrying translocation is illustrated to ask for an interview Fig. 1.Each pair patient Mr. and Mrs and patient parent are extracted while raising
The peripheral blood 10ml of category (patient father and mother pay the utmost attention to, alternatively other relatives).A part of peripheral blood is used for lymphocyte culture,
Carry out chromosome karyotype analysis；Another part peripheral blood extracts DNA according to this area usual manner, makes for follow-up SNPs partings
With.
Peripheral blood chromosome preparation method is as follows：
1st, cell culture
1). blood sampling：Alcohol disinfecting skin, ulnar vein blood sampling, by injection needle directly through blake bottle rubber stopper, to 10ml
30-40 drop whole bloods, the even rearmounted 37 DEG C of insulating box cultures of jog are injected in culture medium.
2). culture：Time is 68 hours.During culture, regular jog is even, makes cell be fully contacted culture medium.
3). colchicine process：Terminate 2-4 hours before culture, add colchicin (to use 1ml syringes 5 in nutrient solution
Number needle point is added dropwise 2 and drips, and makes final concentration of 0.07 μ g/ml).
Above step is both needed to sterile working
2nd, prepared by chromosome
1). collect cell：Culture is all proceeded in clean centrifuge tube, 8-10 minutes are centrifuged with 1000rpm, abandon supernatant
Liquid.
2). Hypotonic treatment：The hypotonic medium 8ml of 37 DEG C of pre-temperature is added in graduated centrifuge tube, is mixed with dropper, put 37 DEG C of perseverances
Hypotonic 15-25 minutes in tepidarium.
3). pre-fix：0.5ml fixers are added after hypotonic, 1000rpm centrifugation 8-10 minutes after gently mixing.
4). one fixes：Supernatant is abandoned, 5ml fixers are added, is gently mixed, stand 20 minutes.1000rpm is centrifuged, and abandons
Clear liquid.
5). two fix, three fix：Same fixation.
6). suspension processed：After abandoning supernatant, visual cell's quantity adds appropriate fixer to make cell suspension.
7). drop piece：Cell suspension is drawn from the slide that 10-20cm height drops in a dried and clean, featheriness dissipates, gas
It is dry.
8). dyeing：1:10Giemsa dyes 5-10 minutes, and thin washing goes unnecessary dye liquor, gas to do.
9). microscopy：Split coil method well dispersed, that dyeing is moderate is found under low power lens, oily Microscopic observation chromosome morphology is simultaneously
Count.
If the PBC caryogram of patient is identical with its mother, the balanced translocation heredity of patient is from maternal；With father
Parent is identical, then the balanced translocation heredity of patient is from father.When patient father and mother cannot take blood (such as dying) or disagree take blood when, suffer from
The siblings of person or other relatives also can use peripheral blood and do karyotyping, it is also possible to used as reference during structure family haplotype
Sample.
The translocation chromosome caryogram of 1-9 familys asks for an interview table 1.Fig. 2 illustrates the peripheral blood caryogram of No. 3 familys.
The 1-9 family translocation chromosome caryogram tables of table 1.
| Family is numbered | Chromosome number | Carrier | Translocation chromosome caryogram |
| 1 | 46 | XY | t(5；22)(q33；q12)mat |
| 2 | 46 | XX | t(16；18)(q22；q21.1)pat |
| 3 | 46 | XX | t(12；22)(p12；q13)pat |
| 4 | 46 | XX | t(11；16)(p11.2；p12.3)pat |
| 5 | 46 | XY | t(1；19)(q12；p13)mat |
| 6 | 45 | XX | rob(14；21)(q10；q10)mat |
| 7 | 45 | XY | rob(14；21)(q10；q10)pat |
| 8 | 45 | XX | rob(14；15)(q10；q10)pat |
| 9 | 46 | XY | t(6；9)(q27；q22)mat/pat |
Embodiment 2：Blastaea biopsy and whole genome amplification (WGA)
1st, it is in vitro fertilization
9 families to raising carry out (IVF) in vitro fertilization, and external fertilization method follows this area conventional method and carries out；
Female parent/the paternal age of these families, phenotype, ovulation result, embryonated egg quantity and the blastaea quantity eventually for biopsy are listed in
Table 2.By above-mentioned in vitro fertilization, 9 families obtain altogether 44 blastaeas carries out follow-up biopsy and haplotyping research.
The 1-9 familys basic condition of table 2. and situation in vitro fertilization
2nd, blastaea biopsy and whole genome amplification
The above-mentioned embryo in blastocyst stage is taken, the 5th or 6 day in embryonic development removes 3 to 10 from trophectoderm
Individual cell.Biopsy cells are placed in the PCR pipe equipped with alkaline denaturation buffer (KOH) carries out cell lysis.Again by multiple
Displacement amplification (MDA) method carries out whole genome amplification (WGA).According to method described in kit specification, phi 29DNA are used
Polymerase carry out isothermal DNA amplification (the unicellular whole genome amplification kits of Repli-g, QIAGEN GmbH, Hilden,
Germany), concrete operations flow process is as follows：
1) pre-process：Buffer DLB are resuspended
Buffer DLB add 500 μ l H2O sc to mix centrifugation and are stored in -20 DEG C, 6 months；
2) buffer D2 are prepared
The μ l of DTT, 1M 3
Buffer DLB(reconstituted) 33μl
Total volume 36μl
Buffer D2 are frozen to be less than 3 months in -20 DEG C；
3) take 4 μ l biopsy cells lysate samples to mix with 3 μ l buffer D2,65 DEG C of reaction 10min；
4) in step 3) 3 μ l terminate liquid terminating reactions are added in reactant liquor；
5) Master mix are prepared
6) in step 4) 40 μ l Master mix are added in each reaction for obtaining, overall reaction system totally 50 μ l；
7) by step 6) reaction system be placed in 30 DEG C reaction 8h, 65 DEG C, 3min terminating reactions；
8) product enters row agarose gel electrophoresis detection, in -20 DEG C of preservations.
Embodiment 3：SNP genotypings and haplotype (haplotypes) are analyzed
1st, SNP genotype detections
SNP genotype detections are carried out using Illumina human Karyomap-12V1.0 microarrays.Each
Karyomap-12 chips include nearly 300,000 SNPs, can comprehensively cover people 23 to chromosome.Blastaea will be carried out in embodiment 2
44 samples that biopsy and whole genome amplification are obtained are grouped according to family, respectively with family patient Mr. and Mrs, patients' relatives
It is one group that 3 parts of whole genome amplification sample is compiled, and carries out microarray SNP genotype detections and analysis, and packet situation is as shown in table 3.
Specific experiment method is carried out with reference to specification, be will not be described here.
The 1-9 familys SNP array experiment of table 3. is grouped
Note：F represents women, and M represents the male sex
2nd, haplotyping
After obtaining whole SNPs information of chip detection, embryo's Molecu- lar karyotyping and family haplotype are built, be respectively used to mirror
Determine embryo chromosome copy number situation and differentiate embryo chromosome balanced translocation carrier state.Concrete operation method is as follows：
A. family haplotype reconstruction：
1) sample Genotyping：Patient Mr. and Mrs both sides, patients' relatives and embryo's (blastaea) are carried out into SNP Genotypings；
2) information SNPs site is determined：The selection standard of information SNPs is：It is miscellaneous in translocation carrier
Mould assembly, is homozygous in its spouse, and is also homozygous SNP site in patients' relatives；Select covering coloring body easy
Information SNPs on position breakaway poing, translocation chromosome and normal homologue corresponding thereto, chromosome is per Mb scopes
Inside at least select 1 information SNP；Breakaway poing region is being covered, information SNPs is selected from the range of breakaway poing upstream and downstream 2Mb；
3) family haplotype is built：Set step 2) determine information SNPs site covered by family linkage analysis
The haplotype of the whole chromosome of the region of fracture, translocation chromosome whole chromosome and its homologue, coloured differently at two
The collection of body haplotype is collectively referred to as family haplotype.
As shown in table 4, Fig. 3 illustrates the family list of No. 3 familys to 1-9 family partial embryonic family haplotype reconstructions information
Build figure：
The 1-9 family partial embryonic family haplotype reconstruction information of table 4.
1Breakaway poing is determined by the Molecu- lar karyotyping of uneven embryo
2The elder brother of patient is used as reference
3The elder sister of patient is used as reference
4Because lacking information SNPs at this, the region can not build haplotype
B. the method for distinguishing the embryo for carrying balanced translocation or normal chromosomal：
By the haplotype information in breakaway poing region in embryo's (blastaea) cell and the chromosome monosomy type information of sample for reference
Compare, judge whether chromosome translocation breakaway poing region there occurs by the haplotype information of whole chromosome homologous heavy
Group：
I) when being reference with the relatives with carrier with identical balanced translocation：
If a. sample chromosomes transposition breakaway poing region undetermined is not recombinated, when sample breakaway poing region monomer undetermined
In type information and sample for reference translocation carrier and have with carrier identical balanced translocation relatives list
When body-shape information is consistent, then embryo is carried for balanced translocation；When sample breakaway poing region haplotype information undetermined and sample for reference
Middle translocation carrier haplotype information is consistent, but the haplotype of the carrier relatives of balanced translocation identical with carrying
Then it is the completely normal embryo of chromosome when information is inconsistent；
If b. sample chromosomes transposition breakaway poing region undetermined there occurs homologous recombination, balanced translocation is carried or chromosome
The criterion of fetal tissues completely is contrary with a；
Ii) when being reference with the normal relatives of chromosome：
If a. sample chromosomes transposition breakaway poing region undetermined is not recombinated, when sample breakaway poing region monomer undetermined
The haplotype letter of the normal relatives of the chromosome of translocation carrier and carrier in type information and sample for reference
Then it is the completely normal embryo of chromosome, when in sample breakaway poing region haplotype information undetermined and sample for reference when ceasing consistent
Translocation carrier haplotype information is consistent, but with the haplotype information of the normal relatives of the chromosome of carrier not
When consistent, then embryo is carried for balanced translocation；
If b. sample chromosomes transposition breakaway poing region undetermined there occurs homologous recombination, balanced translocation is carried or chromosome
The criterion of fetal tissues completely is contrary with a.
3rd, 1-9 familys embryo chromosome balanced translocation screening results
Through family haplotype linkage analysts, treat that transplanting embryo is diagnosed to 44,20 is balanced translocation or just
The euploid of normal chromosome, 11 is the related variation of transposition, and 13 show and the unrelated variation of transposition；Use respectively and take
It is reference to have the normal relatives of the chromosome of relatives or carrier of identical balanced translocation with person, analyzes above-mentioned 20 balances easy
Position or fetal tissues, using above-mentioned Bi) or Bii) described in differentiating method each embryo is predicted respectively, predict the outcome
Unanimously：9 is chromosome translocation embryo, and remaining 12 is complete fetal tissues.And each embryo's difference transposition breakaway poing
There is uniformity between the predicting the outcome of region.
Embodiment 4：Embryo transfer and embryo chromosome balanced translocation haplotype screening method compliance test result
1st, embryo transfer
The embryo being related in the table 4 of above-described embodiment 3 is used for into embryo transfer according to this area conventional method.9 freezing capsules
Embryo is survived after melting, and 9 transplanting cycles, the successful pregnancies after first set grafting are have passed through altogether.
2nd, second trimester of pregnancy row amniocentesis carries out CYTOGENETIC ANALYSIS OF ONE checking
After embryo transfer success, by relatively of the present invention differentiating dye to verify with second trimester of pregnancy routine amniotic fluid caryogram
Colour solid balanced translocation carries the forecasting accuracy of the PGH methods of embryo and the complete fetal tissues of chromosome.
Amniocyte method of chromosome preparation (in-situ method)
A. cell culture
1). during amniotic fluid (about 20ml) is transferred into aseptic centrifuge tube, 1000rpm is centrifuged 10 minutes；
2). supernatant is removed, for other analyses, retains cell suspension about 0.5～1ml, mixed to 2 with culture medium～
2.5ml or so；
3). cell suspension is divided equally in 2～4 Chromslide culture dishes；
4). after cultivating 24/48 hour, in every Chromslide culture dish about 2.5ml amniotic fluid culture mediums are added；
5). after cultivating the 5th～6 day, cell growth situation is observed, the culture medium for more renewing；
6) behind .1～2 day observation of cell upgrowth situation, if number of cell clones enough, in culture dish colchicum is added
Element, harvesting, process time determines according to autumn waters -- limid eyes solution concentration.
B. prepared by chromosome
1). Chromslide Tissue Culture Dish is inclined, culture medium is removed completely；
2). add 3～4ml hypotonic mediums in each culture dish, room temperature treatment 10 minutes；
3). 0.5～0.7ml fixers, room temperature treatment 5 minutes are directly added in hypotonic medium；
4). supernatant is removed, the fixer for adding 3～4ml fresh, room temperature treatment；
5). repeat the 4th step 1～2 time；
6). fixer is removed, in Maxchrome Chromosome spread instrument (the appropriate parameter of setting) Chromosome spread is carried out
Process；
7). it is aging after slide is dried, show band.
Table 5 show used in the embryo of successful implantation differential staining body balanced translocation of the present invention carry embryo or
The diagnostic result that chromosome fetal tissues method is obtained carries out the tool of the result of CYTOGENETIC ANALYSIS OF ONE checking with amniocentesis
Body information.
Table 5.PGH predicts the outcome and the contrast of amniocentesis cytogenetics the result
As shown in Table 5, through checking, the genetic analysis predicted the outcome with cell in amniocentesis of family haplotype
As a result it is completely the same, it was demonstrated that differential staining body balanced translocation of the present invention carries the spirit of the PGH methods of embryo or fetal tissues
Sensitivity and specificity are 100%.
Claims (9)
1. a kind of family haplotype reconstruction that embryo and the complete fetal tissues of chromosome are carried for differential staining body balanced translocation
Method, it is characterised in that comprise the steps of：
1) sample Genotyping：By translocation carrier Mr. and Mrs both sides, at least one carrier relatives and chromosome
Balance translocation carrier Mr. and Mrs IVF Embryos carry out extensive SNP genotype detections；Carrier relatives can be and carrier
The normal relatives of relatives with identical balanced translocation, alternatively chromosome；
2) information SNPs site is determined：The selection standard of information SNPs is：It is heterozygous in translocation carrier,
It is homozygous in its spouse, and is also homozygous SNP site in carrier relatives；The transposition of covering coloring body is selected to break
Information SNPs on knick point, translocation chromosome and normal homologue corresponding thereto, in the range of the every Mb of chromosome extremely
1 information SNP is selected less；Breakaway poing region is being covered, information SNPs is selected from the range of breakaway poing upstream and downstream 1-5Mb；
3) family haplotype is built：Set step 2) determine information SNPs site by family linkage analysis obtain covering two at
The haplotype of the whole chromosome of the region of fracture, translocation chromosome whole chromosome and its homologue, coloured differently body list
The collection of build is collectively referred to as family haplotype.
2. a kind of differential staining body balanced translocation carries the PGH methods of embryo and the complete fetal tissues of chromosome, it is characterised in that
Comprise the steps of：
1) sample Genotyping
By translocation carrier Mr. and Mrs both sides, at least one carrier relatives and carrier's Mr. and Mrs' IVF Embryos
Carry out extensive SNP genotype detections；Carrier relatives can be the relatives for having identical balanced translocation with carrier, alternatively contaminate
The normal relatives of colour solid；Translocation carrier Mr. and Mrs both sides, carrier relatives are referred to as into sample for reference, carrier husband
Woman's IVF Embryos are referred to as sample undetermined；
2) information SNPs site is determined
The selection standard of information SNPs is：Be heterozygous in translocation carrier, in its spouse be it is homozygous,
And it is also homozygous SNP site in carrier relatives；Select covering coloring body transposition breakaway poing, translocation chromosome and with
Information SNPs on its corresponding normal homologue, in the range of the every Mb of chromosome 1 information SNP is at least selected；
Covering coloring body transposition breakaway poing region, information SNPs is selected from the range of chromosome translocation breakaway poing upstream and downstream 1-5Mb；
3) family haplotype is built
Set step 2) determine information SNPs site by family linkage analysis obtain cover two at the region of fracture, transposition dye
The haplotype of the whole chromosome of body whole chromosome and its homologue, the collection of coloured differently body haplotype is collectively referred to as family
Haplotype；
4) data collection and analysis
The haplotype information in chromosome translocation breakaway poing region in sample undetermined and the haplotype information of sample for reference are compared
It is right, judge whether chromosome translocation breakaway poing region there occurs homologous recombination by the haplotype of whole chromosome：
I) when being reference with the relatives with carrier with identical balanced translocation：
If a. sample chromosomes transposition breakaway poing region undetermined is not recombinated, when sample breakaway poing region haplotype undetermined letter
Breath and sample for reference in translocation carrier and have with carrier identical balanced translocation relatives haplotype
When information is consistent, then embryo is carried for balanced translocation；When dye in sample breakaway poing region haplotype information undetermined and sample for reference
Colour solid balance translocation carrier haplotype information is consistent, but the haplotype information of the carrier relatives of balanced translocation identical with carrying
Then it is the completely normal embryo of chromosome when inconsistent；
If b. sample chromosomes transposition breakaway poing region undetermined there occurs homologous recombination, balanced translocation is carried or chromosome is complete
The criterion of fetal tissues is contrary with a；
Ii) when being reference with the normal relatives of chromosome：
If a. sample chromosomes transposition breakaway poing region undetermined is not recombinated, when sample breakaway poing region haplotype undetermined letter
Translocation carrier and the haplotype information with the normal relatives of the chromosome of carrier in breath and sample for reference
Then it is the completely normal embryo of chromosome when consistent；When dye in sample breakaway poing region haplotype information undetermined and sample for reference
Colour solid balance translocation carrier haplotype information is consistent, but differs with the haplotype information of the normal relatives of the chromosome of carrier
During cause, then embryo is carried for balanced translocation；
If b. sample chromosomes transposition breakaway poing region undetermined there occurs homologous recombination, balanced translocation is carried or chromosome is complete
The criterion of fetal tissues is contrary with a.
3. method as claimed in claim 1 or 2, it is characterised in that step 1) the extensive SNP genotype detections cover 23 pairs
Chromosome；Extensive SNP genotype detection method preferred gene chips and gene sequencing.
4. method as claimed in claim 1 or 2, it is characterised in that when embryonic development was to the 3-7 days, biopsy obtain 1-10 it is thin
Born of the same parents are used as step 1) described in IVF Embryos detection sample；It is preferably derived from embryonic blastomeres biopsy or blastaea nourishes outer
The cell of germinal layer biopsy.
5. method as claimed in claim 1 or 2, it is characterised in that step 2) in covering coloring body transposition breakaway poing region, it is described
Information SNPs is selected from the range of chromosome translocation breakaway poing upstream and downstream 2-4Mb, preferably from chromosome translocation breakaway poing upstream and downstream
Select in the range of 2Mb.
6. method as claimed in claim 1 or 2, it is characterised in that step 2) in chromosome per Mb in the range of at least select 2,3,
4th, 5,6,7,8,9 or 10 information SNPs.
7. method as claimed in claim 4, it is characterised in that the cell lysis for obtaining biopsy, carries out whole genome amplification；Entirely
Genome amplification method may be selected from MDA methods, MALBAC methods or other whole genome amplification methods.
8. method as claimed in claim 1 or 2, it is characterised in that step 1) in translocation carrier Mr. and Mrs both sides
It is body cell with the detection samples sources of carrier relatives, preferred peripheral blood.
9. a kind of system for carrying embryo and the complete fetal tissues of chromosome for differential staining body balanced translocation, the system bag
Hardware containing the software that can calculate process sample data and for carrying above-mentioned software, it is characterised in that
1) system is also included and stores translocation carrier Mr. and Mrs both sides, at least one carrier relatives and dye
Colour solid balance translocation carrier Mr. and Mrs IVF Embryos carry out the hard of the genotype data of extensive SNP genotype detections
Part；Carrier relatives can be the relatives for having identical balanced translocation with carrier, alternatively the normal relatives of chromosome；Will dyeing
Body balance translocation carrier Mr. and Mrs both sides, carrier relatives are referred to as sample for reference, and carrier's Mr. and Mrs' IVF Embryos are referred to as treating
Random sample sheet；
2) software is according to following regular determination information SNPs sites：The selection standard of information SNPs is：In balance of chromosome
It is heterozygous in translocation carrier, is homozygous in its spouse, and is also homozygous SNP positions in carrier relatives
Point；Select the information on covering coloring body transposition breakaway poing, translocation chromosome and normal homologue corresponding thereto
SNPs, in the range of the every Mb of chromosome 1 information SNP is at least selected；Breakaway poing region is being covered, information SNPs is upper and lower from breakaway poing
Select in the range of trip 1-5Mb；
3) software is according to following principle construction family haplotypes：The information chosen according to above-mentioned standard on every chromosome
SNPs sites, obtain covering the region of fracture, translocation chromosome whole chromosome and its homologous dye at two by family linkage analysis
The haplotype of the whole chromosome of colour solid, the collection of coloured differently body haplotype is collectively referred to as family haplotype；
4) software is by the chromosome of the haplotype information in chromosome translocation breakaway poing region and sample for reference in sample undetermined
Haplotype information is compared, and judges whether chromosome translocation breakaway poing region there occurs together by the haplotype of whole chromosome
Recombinate in source：
I) when being reference with the relatives with carrier with identical balanced translocation：
If a. sample chromosomes transposition breakaway poing region undetermined is not recombinated, when sample breakaway poing region haplotype undetermined letter
Breath and sample for reference in translocation carrier and have with carrier identical balanced translocation relatives haplotype
When information is consistent, then embryo is carried for balanced translocation；When dye in sample breakaway poing region haplotype information undetermined and sample for reference
Colour solid balance translocation carrier haplotype information is consistent, but the haplotype information of the carrier relatives of balanced translocation identical with carrying
Then it is the completely normal embryo of chromosome when inconsistent；
If b. sample chromosomes transposition breakaway poing region undetermined there occurs homologous recombination, balanced translocation is carried or chromosome is complete
The criterion of fetal tissues is contrary with a；
Ii) when being reference with the normal relatives of chromosome：
If a. sample chromosomes transposition breakaway poing region undetermined is not recombinated, when sample breakaway poing region haplotype undetermined letter
Translocation carrier and the haplotype information with the normal relatives of the chromosome of carrier in breath and sample for reference
Then it is the completely normal embryo of chromosome when consistent；When dye in sample breakaway poing region haplotype information undetermined and sample for reference
Colour solid balance translocation carrier haplotype information is consistent, but differs with the haplotype information of the normal relatives of the chromosome of carrier
During cause, then embryo is carried for balanced translocation；
If b. sample chromosomes transposition breakaway poing region undetermined there occurs homologous recombination, balanced translocation is carried or chromosome is complete
The criterion of fetal tissues is contrary with a.
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| CN113436680B (en) * | 2020-05-22 | 2022-03-25 | 复旦大学附属妇产科医院 | Method for simultaneously identifying chromosome structural abnormality and carrier state of pathogenic gene of embryo |
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