CN106650310B - A kind of method that differential staining body balanced translocation carries embryo and normal fetus - Google Patents
A kind of method that differential staining body balanced translocation carries embryo and normal fetus Download PDFInfo
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- CN106650310B CN106650310B CN201710013009.XA CN201710013009A CN106650310B CN 106650310 B CN106650310 B CN 106650310B CN 201710013009 A CN201710013009 A CN 201710013009A CN 106650310 B CN106650310 B CN 106650310B
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Abstract
The invention belongs to genetic diagnosis and human ancillary reproductive field, more specifically to diagnostic techniques before embryo implantation (PGD).The method according to the present invention, the chromosome of the embryo and translocation carrier relatives after patient and its spouse, inseminatio externalis for translocation (including mutual transposition and Robertsonian translocation) carries out family haplotype linkage analysts, can quickly, embryo and the normal embryo of chromosome easy, that accurately distinguish translocation, and screening is carried out to the chromosome copies number variation of embryo simultaneously, Clinical Pregnancy Rate in is improved, realization blocks translocation to follow-on heredity transmitting in time before embryo transfer.The progress of Issues of Human Assisted Reproductive Technologies has been pushed to a certain extent.
Description
Technical field
The invention belongs to gene diagnosises and human ancillary reproductive field, in particular to diagnostic techniques before embryo implantation
It (PGD), is that one kind can identify the haplotype linkage analysts side whether embryo carries parent's balanced translocation chromosomal inheritance information
Method.
Background technique
Translocation is a kind of common chromosomal structural variation, is since the fracture of different chromosomes is distributed
Raw mistake splicing and caused by chromosome abnormality.Probably there is 0.19% incidence in normal population, in recurrent abortion or instead
It is about 5% that multiple IVF kind, which plants the probability occurred in the patient to fail,.Although balance translocation carrier ordinary circumstance phenotype without exception,
But having up to 19%-77% in the gamete that generates of its archaeocyte meiosis is the gamete of unbalanced type, this and dye
The breaking point of colour solid transposition is related with the specific chromosome of transposition occurs.The quadrivalent structure of chromosome usually occurs in cell
The meiosis I phase is generated by two chromosomes of transposition and corresponding two normal homologue pairings.This four
The common form of valence structure has tri- kinds of clastotypes of 2:2,3:1 or 4:0.Only under the mode of 2:2, clastotype energy is aligned
Normal or balance gamete is generated, other clastotypes generate the gamete of non-equilibrium type.Nonequilibrium gamete can be led
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 clinic by Handyside AH earliest first, initial
PGD technology detects gender using PCR or FISH method, and the X such as risky fertility hemophilia A, progressive muscular dystrophy is helped to connect
Mr. and Mrs' pregnancy and delivery of lock hereditary disease offspring goes out normal girl baby；Over 2006, there is researcher contaminating by selection and Disease-causing gene
On the position of colour solid the STR marker site of close linkage come diagnose embryo whether heredity Disease-causing gene, carry out single-gene disorder
Embryo implantation before diagnose, this method is otherwise known as is implanted into before science of heredity haplotype analysis (preimplantation genetic
Haplotyping, PGH).PGD technology is due to that screening can haunt in the patient of translocation 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 fluorescence in situ hybridization (FISH) are used to filter out unbalanced embryo earliest, still
Due to technical restriction of the FISH technology in terms of cell is fixed with signal-obtaining, the accuracy of result interpretation will affect.In recent years,
Biochip technology and sequencing technologies due to the advantages of it can carry out comprehensively screening to 23 pairs of chromosomes be widely used in it is each
Reproductive center improves Clinical Pregnancy Rate in.But despite of that these traditional PGD technologies can only become the copy number of embryo
It is different to be detected.
To currently, having some article reports for carrying embryo and complete normal fetus about differential staining body balanced translocation
Road.Initially, researcher is broken specific FISH probe of the point design spy across breaking point for chromosome translocation, can be used for distinguishing dye
Normally and the embryo of balanced translocation, although this method has certain feasibility, method design complexity, time-consuming are difficult facing colour solid
It is applied in bed work.In the recent period, the research of Treff et al. is then used based on SNP chip technology, with translocation carrier Mr. and Mrs
Both sides and uneven embryo are reference, are identified by the haplotype within building breaking point 5Mb, can identify chromosome phase
The chromosomal condition of the carrier embryo of mutual transposition.The technology of MicroSeq-PGD a kind of has been invented by Ling You research group, passes through
In conjunction with chromosome translocation breaking point microdissection technology and NGS, the sequence of the DNA near transposition breaking point is detected, thus area
The embryo of not normal embryo and balanced translocation.But the above method all has following defects: 1) method and technology process is all very
Complexity takes a long time；2) it can not judge the homologous recombination situation in breaking point region；3) it is not suitable for the carrying of Robertsonian translocation
Person.It being capable of differential staining body balanced translocation (phase reciprocity that is accurate, being quickly applied to clinic as it can be seen that still lacking in the prior art
Position and Robertsonian translocation) embryo assisted reproductive technology.
Summary of the invention
For above-mentioned defect existing in the prior art, the present invention establishes a kind of based on the extensive SNP base of full-length genome
The method for carrying out haplotyping (PGH) before embryo implantation because of genotyping result.The method according to the present invention, it is flat for chromosome
Weigh the patient of transposition (including mutual transposition and Robertsonian translocation) and its embryo after spouse, inseminatio externalis and translocation carrier parent
The chromosome of category carries out family haplotype linkage analysts, can embryo that is quick, easy, accurately distinguishing translocation
With the normal embryo of chromosome, and screening is carried out to the chromosome copies number variation of embryo simultaneously, improves Clinical Pregnancy Rate in, it can
The embryo and the embryo of normal chromosomal for carrying balanced translocation chromosome are distinguished before the embryo implantation being fertilized in vitro in time, thus
The preferential transplanting completely normal embryo of chromosome, blocks translocation to be hereditary to the next generation in time.It pushes away to a certain extent
The progress of Issues of Human Assisted Reproductive Technologies is moved.
The specific technical solution of the present invention is as follows:
Embryo and the complete normal fetus of chromosome are carried for differential staining body balanced translocation the present invention provides a kind of
Family haplotype reconstruction method comprising the steps of:
1) sample Genotyping: by translocation carrier Mr. and Mrs both sides, at least a carrier relatives and dye
Colour solid balance translocation carrier Mr. and Mrs' IVF Embryos carry out extensive SNP genotype detection；Carrier relatives can be and take
There are the relatives of identical balanced translocation with person, can also be the normal relatives of chromosome；
2) site information SNPs: the selection criteria of information SNPs is determined are as follows: in translocation carrier be miscellaneous
Mould assembly is homozygous in its spouse, and is also homozygous SNP site in carrier relatives；Select covering coloring body
Information SNPs on transposition breaking point, translocation chromosome and normal homologue corresponding thereto, the every Mb model of chromosome
1 information SNP is at least selected in enclosing；In covering breaking point region, information SNPs is selected out of breaking point upstream and downstream 1-5Mb
It selects；
3) construct family haplotype: the site information SNPs that set step 2) determines is covered by family linkage analysis
The haplotype of the whole chromosome of the region of fracture, translocation chromosome whole chromosome and its homologue at two, difference dyeing
The collection of body haplotype is collectively referred to as family haplotype.
The present invention provides the sides PGH that a kind of differential staining body balanced translocation carries embryo and the complete normal fetus of chromosome
Method comprising the steps of:
1) sample Genotyping
By translocation carrier Mr. and Mrs both sides, at least a carrier relatives and carrier Mr. and Mrs are in vitro fertilization
Embryo carries out extensive SNP genotype detection；Carrier relatives can be to have the relatives of identical balanced translocation with carrier, can also
For the normal relatives of chromosome；Translocation carrier Mr. and Mrs both sides, carrier relatives are known as sample for reference, are carried
Person's Mr. and Mrs' IVF Embryos are known as sample undetermined；
2) site information SNPs is determined
The selection criteria of information SNPs are as follows: in translocation carrier be heterozygous, be pure in its spouse
Mould assembly, and be also homozygous SNP site in carrier relatives；Select covering coloring body transposition breaking point, transposition dyeing
1 information is at least selected within the scope of information SNPs on body and normal homologue corresponding thereto, the every Mb of chromosome
SNP；In covering coloring body transposition breaking point region, information SNPs is selected out of chromosome translocation breaking point upstream and downstream 1-5Mb
It selects；
3) family haplotype is constructed
The site information SNPs that set step 2) determines obtains the region of fracture, transposition at covering two by family linkage analysis
The haplotype of the whole chromosome of chromosome whole chromosome and its homologue, the collection of different chromosomes haplotype are collectively referred to as
Family haplotype；
4) data collection and analysis
By the chromosome monosomy type of the haplotype information and sample for reference in chromosome translocation breaking point region in sample undetermined
Information is compared, and it is homologous heavy to judge whether chromosome translocation breaking point region has occurred by the haplotype of whole chromosome
Group:
I) when there are the relatives of identical balanced translocation as reference with carrier:
If a. there is no recombinations in sample chromosomes transposition breaking point region undetermined, when sample breaking point undetermined region monomer
The list of translocation carrier and the relatives with carrier with identical balanced translocation in type information and sample for reference
When body-shape information is consistent, then embryo is carried for balanced translocation；When sample breaking point undetermined region haplotype information and sample for reference
Middle translocation carrier haplotype information is consistent, but the haplotype of the relatives with carrier with identical balanced translocation
It is then the completely normal embryo of chromosome when information is inconsistent；
If b. homologous recombination has occurred in sample chromosomes transposition breaking point region undetermined, balanced translocation carries or chromosome
The judgment criteria of complete normal fetus is opposite with a；
Ii) when using the normal relatives of chromosome as reference:
If a. there is no recombinations in sample chromosomes transposition breaking point region undetermined, when sample breaking point undetermined region monomer
Translocation carrier and the haplotype with the normal relatives of chromosome of carrier in type information and sample for reference
It is then the completely normal embryo of chromosome when information is consistent；When sample breaking point undetermined region haplotype information and sample for reference
Middle translocation carrier haplotype information is consistent, but the haplotype information with the normal relatives of the chromosome of carrier
When inconsistent, then embryo is carried for balanced translocation；
If b. homologous recombination has occurred in sample chromosomes transposition breaking point region undetermined, balanced translocation carries or chromosome
The judgment criteria of complete normal fetus is opposite with a.
Extensive SNP genotype detection of the present invention covers 23 pairs of chromosomes；Extensive SNP genotype detection method is excellent
Select genetic chip and gene sequencing.
When the present invention selects embryonic development to the 3-7 days, biopsy obtain 1-10 cell as described in step 1) 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 breaking point region, the information SNPs is from breaking point in the site information SNPs of the present invention
It selects within the scope of the 2-4Mb of downstream, is preferably selected out of breaking point upstream and downstream 2Mb.
At least selected when the present invention constructs family haplotype, within the scope of the every Mb of every chromosome 2,3,4,5,6,7,8,9 or
10 information SNPs.
The cell cracking that the present invention obtains biopsy carries out whole genome amplification；Whole genome amplification method can be selected from MDA
Method, MALBAC method or other whole genome amplification methods.
Translocation carrier Mr. and Mrs both sides of the present invention and the source of the detection sample of carrier relatives are that body is thin
Born of the same parents, preferably peripheral blood.
The present invention provides a kind of systems for carrying embryo for differential staining body balanced translocation, and the system includes can
The software of calculation processing sample data and hardware for carrying above-mentioned software, which is characterized in that
1) system also includes to store translocation carrier Mr. and Mrs both sides, at least a carrier relatives
The genotype data of extensive SNP genotype detection is carried out with translocation carrier Mr. and Mrs IVF Embryos
Hardware；Carrier relatives can also be able to be the normal relatives of chromosome to have the relatives of identical balanced translocation with carrier；It will dye
Colour solid balance translocation carrier Mr. and Mrs both sides, carrier relatives are known as sample for reference, and carrier's Mr. and Mrs' IVF Embryos are known as
Sample undetermined；
2) software determines the site information SNPs: the selection criteria of information SNPs are as follows: in chromosome according to following rules
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 breaking point, translocation chromosome and normal homologue corresponding thereto
SNPs is ceased, at least selects 1 information SNP within the scope of the every Mb of chromosome；In covering breaking point region, information SNPs is from breaking point
It is selected within the scope of the 1-5Mb of downstream；
3) software is according to following principle construction family haplotypes: the letter chosen on every chromosome according to above-mentioned standard
The site SNPs is ceased, the region of fracture, translocation chromosome whole chromosome and its homologous at covering two are obtained by family linkage analysis
The haplotype of the whole chromosome of chromosome, the collection of different chromosomes haplotype are collectively referred to as family haplotype；
4) software is by the dye of the haplotype information and sample for reference in chromosome translocation breaking point region in sample undetermined
Colour solid haplotype information is compared, and judges whether chromosome translocation breaking point region occurs by the haplotype of whole chromosome
Homologous recombination:
I) when there are the relatives of identical balanced translocation as reference with carrier:
If a. there is no recombinations in sample chromosomes transposition breaking point region undetermined, when sample breaking point undetermined region monomer
The list of translocation carrier and the relatives with carrier with identical balanced translocation in type information and sample for reference
When body-shape information is consistent, then embryo is carried for balanced translocation；When sample breaking point undetermined region haplotype information and sample for reference
Middle translocation carrier haplotype information is consistent, but the haplotype of the carrier relatives of balanced translocation identical as carrying
It is then the completely normal embryo of chromosome when information is inconsistent；
If b. homologous recombination has occurred in sample chromosomes transposition breaking point region undetermined, balanced translocation carries or chromosome
The judgment criteria of complete normal fetus is opposite with a；
Ii) when using the normal relatives of chromosome as reference:
If a. there is no recombinations in sample chromosomes transposition breaking point region undetermined, when sample breaking point undetermined region monomer
Translocation carrier and the haplotype with the normal relatives of chromosome of carrier in type information and sample for reference
It is then the completely normal embryo of chromosome when information is consistent；When sample breaking point undetermined region haplotype information and sample for reference
Middle translocation carrier haplotype information is consistent, but the haplotype information with the normal relatives of the chromosome of carrier
When inconsistent, then embryo is carried for balanced translocation；
If b. homologous recombination has occurred in sample chromosomes transposition breaking point region undetermined, balanced translocation carries or chromosome
The judgment criteria of complete normal fetus is opposite with a.
Differential staining body balanced translocation of the present invention, which carries embryo and the PGH method of the complete normal fetus of chromosome, to be had
Following advantage: 1) carry out screening while can predicting chromatin state in embryo, to 23 pairs of chromosome；2) in addition to
The haplotype in breaking point region can establish the whole of the whole chromosome for being related to transposition and corresponding normal homologue simultaneously
The haplotype of chromosome, this can show recombination situation of two homologues in breaking point region of patient, and PGH's is pre-
Survey may include breakpoint region to whole chromosome；3) it is suitable for all genetic translocation familys (including phase
Mutual transposition and Robertsonian translocation)；4) method is relatively easy, takes shorter, and entire discrimination process can be completed in 48h, is applicable in
It works in routine clinical.
Detailed description of the invention
Fig. 1 carries the family map signal of translocation
No. 3 family peripheral blood caryograms of Fig. 2
No. 3 family Haplotype maps of Fig. 3
Specific embodiment
Embodiment 1: the sample for reference of patient and patient's parental generation is collected
It has raised 9 and the translocation for receiving supplementary reproduction is carried into family, those selected is all from Shanghai collection love
Heredity and supplementary reproduction research institute.Each family requires signature Written informed consent, and research approach is produced by Fudan University woman
The approval of human experimenter Ethics Committee of hospital of section.
From in June, 2014 in March, 2016, there are recurrent spontaneous history or chromosome abnormality in all 9 families
History, translocation carry one side of Mr. and Mrs be referred to as below " patient ", another party referred to as " spouse of patient ",
The family map for carrying translocation is illustrated see Fig. 1.Each pair of patient Mr. and Mrs and patient parent are extracted while raising
Belong to the peripheral blood 10ml of (patient parent pays the utmost attention to, and can also be 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 field usual manner, makes for subsequent SNPs parting
With.
Peripheral blood chromosome the preparation method is as follows:
1, cell culture
1) takes a blood sample: alcohol disinfecting skin, ulnar vein blood sampling, by injection needle directly through the rubber stopper of culture bottle, to 10ml
30-40 drop whole blood, jog 37 DEG C of insulating box cultures of even postposition are injected in culture medium.
2) is cultivated: the time is 68 hours.During culture, regular jog is even, and cell is made to come into full contact with culture medium.
3) colchicine is handled: terminating before culture 2-4 hours, colchicin is added in culture solution and (uses 1ml syringe 5
2 drops are added dropwise in number needle point, make final concentration of 0.07 μ g/ml).
Above step is both needed to sterile working
2, prepared by chromosome
1) collects cell: culture being all transferred in clean centrifuge tube, with 1000rpm centrifugation 8-10 minutes, abandons supernatant
Liquid.
2) Hypotonic treatment: 37 DEG C of pre-temperature of hypotonic medium 8ml is added into graduated centrifuge tube, is mixed with dropper, sets 37 DEG C of perseverances
It is 15-25 minutes hypotonic in tepidarium.
3) is pre-fixed: 0.5ml fixer being added after hypotonic, mixes gently rear 1000rpm and is centrifuged 8-10 minutes.
4) mono- is fixed: abandoning supernatant, 5ml fixer is added, mixes gently, stand 20 minutes.1000rpm is centrifuged, in abandoning
Clear liquid.
5) bis- is fixed, three fix: same fixation.
6) suspension: after abandoning supernatant, visual cell's quantity is added appropriate fixer and cell suspension is made.
7) it drop piece: draws cell suspension and is dripped on the glass slide of a dried and clean from 10-20cm high, featheriness dissipates, gas
It is dry.
8) is dyed: 1:10Giemsa is dyed 5-10 minutes, and extra dye liquor is removed in thin washing, and gas is dry.
9) microscopy: split coil method well dispersed, that dyeing is moderate is found under low power lens, oily microscopic observation chromosome morphology is simultaneously
It counts.
If the peripheral blood cells caryogram of patient is identical as 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 parent can not 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, can also be used as reference when building family haplotype
Sample.
The translocation chromosome caryogram of 1-9 family is see table 1.Fig. 2 illustrates the peripheral blood caryogram of No. 3 familys.
1. 1-9 family translocation chromosome caryogram table of table
| Family number | 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)
1, in vitro fertilization
(IVF) in vitro fertilization is carried out to 9 families of recruitment, external fertilization method follows conventional method in that art progress；
Female parent/paternal age of these families, phenotype, ovulation result, fertilized eggs quantity and the blastaea quantity eventually for biopsy are listed in
Table 2.By above-mentioned in vitro fertilization, 9 families obtain 44 blastaeas altogether and carry out subsequent biopsy and haplotyping research.
2. 1-9 family basic condition of table and situation in vitro fertilization
2, 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
A cell.Biopsy cells are placed in the PCR pipe equipped with alkaline denaturation buffer (KOH) and carry out cell cracking.Again by multiple
Displacement amplification (MDA) method carries out whole genome amplification (WGA).According to method described in kit specification, with phi 29DNA
Polymerase progress isothermal DNA amplification (Repli-g single cell whole genome amplification kit, QIAGEN GmbH, Hilden,
Germany), concrete operations process is as follows:
1) pre-process: Buffer DLB is resuspended
Buffer DLB is added 500 μ l H2O sc mixing centrifugation and is stored in -20 DEG C, 6 months；
2) buffer D2 is prepared
3 μ l of DTT, 1M
Buffer DLB(reconstituted) 33μl
Total volume 36μl
Buffer D2 was frozen in -20 DEG C of no more than 3 moons；
3) 4 μ l biopsy cells lysate samples and 3 μ l buffer D2 are taken to mix, 65 DEG C of reaction 10min；
4) 3 μ l terminate liquids are added in step 3) reaction solution and terminate reaction；
5) Master mix is prepared
6) 40 μ l Master mix are added in each reaction that step 4) obtains, overall reaction system totally 50 μ l；
7) the step 6) reaction system is placed in 30 DEG C of reaction 8h, 65 DEG C, 3min termination reaction；
8) reaction product carries out agarose gel electrophoresis detection, saves in -20 DEG C.
Embodiment 3:SNP genotyping and haplotype (haplotypes) analysis
1, SNP genotype detection
SNP genotype detection is carried out using Illumina human Karyomap-12V1.0 microarray.Each
Karyomap-12 chip includes nearly 300,000 SNPs, can cover people 23 comprehensively to chromosome.Blastaea will be carried out in embodiment 2
44 samples that biopsy and whole genome amplification obtain are grouped according to family, respectively with family patient Mr. and Mrs, patients' relatives
Compiling for 3 parts of whole genome amplification sample is one group, carries out microarray SNP genotype detection and analysis, and grouping situation is as shown in table 3.
Specific experiment method is carried out referring to specification, and details are not described herein.
The grouping of 3. 1-9 family SNP array experiment of table
Note: F represents women, and M represents male
2, haplotyping
After the whole SNPs information for obtaining chip detection, embryo's Molecu- lar karyotyping and family haplotype are constructed, is respectively used to reflect
Determine embryo chromosome copy number situation and identifies embryo chromosome balanced translocation carrier state.The specific operation method is as follows:
A. family haplotype reconstruction:
1) patient Mr. and Mrs both sides, patients' relatives and embryo's (blastaea) sample Genotyping: are subjected to SNP Genotyping；
2) site information SNPs: the selection criteria of information SNPs is determined are as follows: in translocation carrier be miscellaneous
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 breaking point, translocation chromosome and normal homologue corresponding thereto, the every Mb range of chromosome
Inside at least select 1 information SNP；In covering breaking point region, information SNPs is selected out of breaking point upstream and downstream 2Mb；
3) construct family haplotype: the site information SNPs that set step 2) determines is covered by family linkage analysis
The haplotype of the whole chromosome of the region of fracture, translocation chromosome whole chromosome and its homologue at two, difference dyeing
The collection of body haplotype is collectively referred to as family haplotype.
1-9 family partial embryonic family haplotype reconstruction information is as shown in table 4, and Fig. 3 illustrates the family list of No. 3 familys
Figure figure:
4. 1-9 family partial embryonic family haplotype reconstruction information of table
1The Molecu- lar karyotyping that breaking point passes through uneven embryo determines
2The elder brother of patient is as reference
3The elder sister of patient is as reference
4Because lacking information SNPs at this, which cannot construct haplotype
B. the method for carrying the embryo of balanced translocation or normal chromosomal is distinguished:
By the chromosome monosomy type information of the haplotype information and sample for reference in breaking point region in embryo (blastaea) cell
It is compared, it is homologous heavy to judge whether chromosome translocation breaking point region has occurred by the haplotype information of whole chromosome
Group:
I) when there are the relatives of identical balanced translocation as reference with carrier:
If a. there is no recombinations in sample chromosomes transposition breaking point region undetermined, when sample breaking point undetermined region monomer
The list of translocation carrier and the relatives with carrier with identical balanced translocation in type information and sample for reference
When body-shape information is consistent, then embryo is carried for balanced translocation；When sample breaking point undetermined region haplotype information and sample for reference
Middle translocation carrier haplotype information is consistent, but the haplotype of the carrier relatives of balanced translocation identical as carrying
It is then the completely normal embryo of chromosome when information is inconsistent；
If b. homologous recombination has occurred in sample chromosomes transposition breaking point region undetermined, balanced translocation carries or chromosome
The judgment criteria of complete normal fetus is opposite with a；
Ii) when using the normal relatives of chromosome as reference:
If a. there is no recombinations in sample chromosomes transposition breaking point region undetermined, when sample breaking point undetermined region monomer
The haplotype letter of the normal relatives of the chromosome of translocation carrier and carrier in type information and sample for reference
It is then the completely normal embryo of chromosome when ceasing consistent, when in sample breaking point undetermined region haplotype information and sample for reference
Translocation carrier's haplotype information is consistent, but not with the haplotype information of the normal relatives of the chromosome of carrier
When consistent, then embryo is carried for balanced translocation；
If b. homologous recombination has occurred in sample chromosomes transposition breaking point region undetermined, balanced translocation carries or chromosome
The judgment criteria of complete normal fetus is opposite with a.
3,1-9 family embryo chromosome balanced translocation screening results
By family haplotype linkage analysts, 44 are diagnosed to transplanting embryo, 20 are balanced translocations or just
The euploid of normal chromosome, 11 are the relevant variation of transposition, 13 displays variation unrelated with transposition；It uses and takes respectively
Having the normal relatives of chromosome of the relatives or carrier of identical balanced translocation with person is reference, and it is easy to analyze above-mentioned 20 balances
Position or normal fetus, using above-mentioned Bi) or Bii) described in differentiating method each embryo is predicted respectively, prediction result
Consistent: 9 are chromosome translocation embryos, remaining 12 are complete normal fetus.And each embryo's difference transposition breaking point
It is with uniformity between the prediction result in region.
Embodiment 4: embryo transfer and embryo chromosome balanced translocation haplotype screening method compliance test result
1, embryo transfer
Embryo involved in 3 table 4 of above-described embodiment is used for embryo transfer according to conventional method in that art.9 freezing capsules
Embryo survives after melting, and have passed through 9 transplanting periods, the successful pregnancies after first set grafting altogether.
2, second trimester of pregnancy row amniocentesis carries out CYTOGENETIC ANALYSIS OF ONE verifying
After embryo transfer success, contaminated by verifying identification of the present invention compared with second trimester of pregnancy routine amniotic fluid caryogram
Colour solid balanced translocation carries the forecasting accuracy of the PGH method of embryo and the complete normal fetus of chromosome.
Amniocyte method of chromosome preparation (in-situ method)
A. cell culture
1) amniotic fluid (about 20ml) is transferred in sterile centrifuge tube by, and 1000rpm is centrifuged 10 minutes；
2) removes supernatant, is used for other analyses, retains cell suspension about 0.5~1ml, mixed to 2 with culture medium~
2.5ml left and right；
3) divides cell suspension in 2~4 Chromslide culture dishes equally；
4) after is cultivated 24/48 hour, about 2.5ml amniotic fluid culture medium is added into every Chromslide culture dish；
5) after is cultivated the 5th~6 day, cell growth condition is observed, the culture medium more renewed；
6) colchicum is added into culture dish if number of cell clones is enough for the upgrowth situation that cell is observed behind .1~2 day
Element harvests cell, and the processing time determines according to autumn waters -- limid eyes solution concentration.
B. prepared by chromosome
1) tilts Chromslide Tissue Culture Dish, completely removes culture medium；
2) 3~4ml hypotonic medium is added into each culture dish in, and room temperature is handled 10 minutes；
3) 0.5~0.7ml fixer is added in directly into hypotonic medium, and room temperature is handled 5 minutes；
4) removes supernatant, and the fresh fixer of 3~4ml, room temperature processing is added；
5) is repeated the 4th step 1~2 time；
6) removes fixer, and Chromosome spread is carried out in Maxchrome Chromosome spread instrument (setting parameter appropriate)
Process；
7) after slide is dry, aging shows band.
Table 5 show in the embryo of successful implantation using differential staining body balanced translocation of the present invention carry embryo or
Chromosome normal fetus method diagnostic result obtained and amniocentesis carry out the tool of the result of CYTOGENETIC ANALYSIS OF ONE verifying
Body information.
Table 5.PGH prediction result and amniocentesis cytogenetics verification result compare
As shown in Table 5, by verifying, the genetic analysis of cell in the prediction result and amniocentesis of family haplotype
As a result completely the same, it was demonstrated that differential staining body balanced translocation of the present invention carries the spirit of the PGH method of embryo or normal fetus
Sensitivity and specificity are 100%.
Claims (14)
1. joining in a kind of family haplotype for carrying embryo and the complete normal fetus of chromosome for differential staining body balanced translocation
The construction method of this haplotype in the same old way, which is characterized in that comprise the steps of:
1) sample Genotyping: by translocation carrier Mr. and Mrs both sides, at least a carrier relatives are advised greatly
Mould SNP genotype detection；Carrier relatives can be to have the relatives of identical balanced translocation with carrier, can also be normal for chromosome
Relatives；Translocation carrier Mr. and Mrs both sides, carrier relatives are known as sample for reference；
2) site information SNPs: the selection criteria of information SNPs is determined are as follows: it is heterozygous in translocation carrier,
It is homozygous in its spouse, and is also homozygous SNP site in carrier relatives；Select the transposition of covering coloring body disconnected
Within the scope of information SNPs on knick point, translocation chromosome and normal homologue corresponding thereto, the every Mb of chromosome extremely
1 information SNP is selected less；In covering breaking point region, information SNPs is selected out of breaking point upstream and downstream 1-5Mb；
3) construct sample for reference haplotype in family haplotype: the site information SNPs that set step 2) determines is chain by family
Analysis obtains the monomer of the whole chromosome of the region of fracture, translocation chromosome whole chromosome and its homologue at covering two
Type obtains sample for reference haplotype in family haplotype.
2. method as described in claim 1, which is characterized in that step 1) the extensive SNP genotype detection covers 23 pairs of dyes
Colour solid.
3. method as claimed in claim 1 or 2, which is characterized in that the extensive SNP genotype detection using genetic chip or
Gene order surveying method.
4. method as claimed in claim 1 or 2, which is characterized in that covering coloring body transposition breaking point region in step 2), it is described
Information SNPs is selected out of chromosome translocation breaking point upstream and downstream 2-4Mb.
5. method as claimed in claim 1 or 2, which is characterized in that covering coloring body transposition breaking point region in step 2), it is described
Information SNPs is selected out of chromosome translocation breaking point upstream and downstream 2Mb.
6. method as claimed in claim 1 or 2, which is characterized in that at least select 2 in step 2) within the scope of the every Mb of chromosome, 3,
4,5,6,7,8,9 or 10 information SNPs.
7. method as claimed in claim 1 or 2, which is characterized in that translocation carrier Mr. and Mrs both sides in step 1)
Detection samples sources with carrier relatives are body cell.
8. method as claimed in claim 1 or 2, which is characterized in that translocation carrier Mr. and Mrs both sides in step 1)
Detection samples sources with carrier relatives are peripheral blood.
9. joining in a kind of family haplotype for carrying embryo and the complete normal fetus of chromosome for differential staining body balanced translocation
The building system of this haplotype in the same old way, the system include to be capable of the software of calculation processing sample data and above-mentioned for carrying
The hardware of software, which is characterized in that
1) system also includes to store translocation carrier Mr. and Mrs both sides, at least a carrier relatives progress
The hardware of the genotype data of extensive SNP genotype detection；Carrier relatives can be easy with phase homostasis with carrier
The relatives of position, can also be the normal relatives of chromosome；Translocation carrier Mr. and Mrs both sides, carrier relatives are known as
Sample for reference；
2) software determines the site information SNPs: the selection criteria of information SNPs are as follows: in balance of chromosome according to following rules
It is heterozygous in translocation carrier, is homozygous in its spouse, and is also the homozygous position SNP in carrier relatives
Point；Select the information on covering coloring body transposition breaking point, translocation chromosome and normal homologue corresponding thereto
1 information SNP is at least selected within the scope of SNPs, the every Mb of chromosome；In covering breaking point region, information SNPs is above and below breaking point
It swims within the scope of 1-5Mb and selects；
3) software is according to sample for reference haplotype in following principle construction family haplotypes: the information that set step 2) determines
The site SNPs obtains the region of fracture, translocation chromosome whole chromosome and its homologous dyeing at covering two by family linkage analysis
The haplotype of the whole chromosome of body obtains sample for reference haplotype in family haplotype.
10. a kind of structure for the family haplotype for carrying embryo and the complete normal fetus of chromosome for differential staining body balanced translocation
System is built, the system includes the software for capableing of calculation processing sample data and the hardware for carrying above-mentioned software, feature
It is,
1) system also includes to store translocation carrier Mr. and Mrs both sides, at least a 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 detection
Part；Carrier relatives can also be able to be the normal relatives of chromosome to have the relatives of identical balanced translocation with carrier；It will dyeing
Body balance translocation carrier Mr. and Mrs both sides, carrier relatives are known as sample for reference, carrier's Mr. and Mrs' IVF Embryos be known as to
Random sample sheet；
2) software determines the site information SNPs: the selection criteria of information SNPs are as follows: in balance of chromosome according to following rules
It is heterozygous in translocation carrier, is homozygous in its spouse, and is also the homozygous position SNP in carrier relatives
Point；Select the information on covering coloring body transposition breaking point, translocation chromosome and normal homologue corresponding thereto
1 information SNP is at least selected within the scope of SNPs, the every Mb of chromosome；In covering breaking point region, information SNPs is above and below breaking point
It swims within the scope of 1-5Mb and selects；
3) software is according to following principle construction family haplotypes: the information chosen on every chromosome according to above-mentioned standard
The site SNPs obtains the region of fracture, translocation chromosome whole chromosome and its homologous dye at covering two by family linkage analysis
The haplotype of the whole chromosome of colour solid, the collection of different chromosomes haplotype are collectively referred to as family haplotype.
11. a kind of system for carrying embryo and the complete normal fetus of chromosome for differential staining body balanced translocation, the system
Hardware comprising capableing of the software of calculation processing sample data and for carrying above-mentioned software, which is characterized in that
1) system also includes to store translocation carrier Mr. and Mrs both sides, at least a 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 detection
Part；Carrier relatives can also be able to be the normal relatives of chromosome to have the relatives of identical balanced translocation with carrier；It will dyeing
Body balance translocation carrier Mr. and Mrs both sides, carrier relatives are known as sample for reference, carrier's Mr. and Mrs' IVF Embryos be known as to
Random sample sheet；
2) software determines the site information SNPs: the selection criteria of information SNPs are as follows: in balance of chromosome according to following rules
It is heterozygous in translocation carrier, is homozygous in its spouse, and is also the homozygous position SNP in carrier relatives
Point；Select the information on covering coloring body transposition breaking point, translocation chromosome and normal homologue corresponding thereto
1 information SNP is at least selected within the scope of SNPs, the every Mb of chromosome；In covering breaking point region, information SNPs is above and below breaking point
It swims within the scope of 1-5Mb and selects；
3) software is according to following principle construction family haplotypes: the information chosen on every chromosome according to above-mentioned standard
The site SNPs obtains the region of fracture, translocation chromosome whole chromosome and its homologous dye at covering two by family linkage analysis
The haplotype of the whole chromosome of colour solid, the collection of different chromosomes haplotype are collectively referred to as family haplotype；
4) software is by the chromosome of the haplotype information and sample for reference in chromosome translocation breaking point region in sample undetermined
Haplotype information is compared, and judges whether chromosome translocation breaking point region has occurred together by the haplotype of whole chromosome
Source recombination:
I) when there are the relatives of identical balanced translocation as reference with carrier:
If a. there is no recombinations in sample chromosomes transposition breaking point region undetermined, when sample breaking point undetermined region haplotype is believed
The haplotype of translocation carrier and the relatives with carrier with identical balanced translocation in breath and sample for reference
When information is consistent, then embryo is carried for balanced translocation；It is contaminated when in sample breaking point undetermined region haplotype information 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 as carrying
It is then the completely normal embryo of chromosome when inconsistent；
If b. homologous recombination has occurred in sample chromosomes transposition breaking point region undetermined, balanced translocation is carried or chromosome is complete
The judgment criteria of normal fetus is opposite with a；
Ii) when using the normal relatives of chromosome as reference:
If a. there is no recombinations in sample chromosomes transposition breaking point region undetermined, when sample breaking point undetermined region haplotype is believed
Translocation carrier and the haplotype information with the normal relatives of chromosome of carrier in breath and sample for reference
It is then the completely normal embryo of chromosome when consistent；It is contaminated when in sample breaking point undetermined region haplotype information and sample for reference
Colour solid balance translocation carrier haplotype information is consistent, but different with the haplotype information of the normal relatives of the chromosome of carrier
When cause, then embryo is carried for balanced translocation；
If b. homologous recombination has occurred in sample chromosomes transposition breaking point region undetermined, balanced translocation is carried or chromosome is complete
The judgment criteria of normal fetus is opposite with a.
12. the system as described in claim 10 or 11, which is characterized in that when embryonic development was to the 3-7 days, biopsy obtains 1-10
Detection sample of the cell as IVF Embryos described in step 1).
13. the system as described in claim 10 or 11, which is characterized in that the detection sample of IVF Embryos described in step 1)
From the cell of embryonic blastomeres biopsy or blastaea trophectoderm biopsy.
14. the system as described in claim 10 or 11, which is characterized in that the cell cracking for obtaining biopsy carries out full-length genome
Amplification；Whole genome amplification method can be selected from MDA method, MALBAC method or other whole genome amplification methods.
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| CN107267628A (en) * | 2017-07-13 | 2017-10-20 | 苏州贝康医疗器械有限公司 | Embryonic limb bud cell prochromosome abnormality detection kit |
| CN112639129A (en) * | 2018-09-03 | 2021-04-09 | 深圳华大智造科技有限公司 | Method and apparatus for determining the genetic status of a new mutation in an embryo |
| CN109920479B (en) * | 2019-03-13 | 2023-08-15 | 复旦大学附属妇产科医院 | Method for identifying chromosome inversion carrying state of embryo |
| CN111276189B (en) * | 2020-02-26 | 2020-12-29 | 广州市金域转化医学研究院有限公司 | Chromosome balance translocation detection and analysis system based on NGS and application thereof |
| 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 |
| CN114480667A (en) * | 2022-01-21 | 2022-05-13 | 复旦大学附属妇产科医院 | Method for detecting fetal chromosome balance structural variation through pregnant woman peripheral blood free DNA |
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