CN106282349B - A kind of full-length genome copy number detection chip of X chromosome high-density probe customization - Google Patents
A kind of full-length genome copy number detection chip of X chromosome high-density probe customization Download PDFInfo
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Abstract
The present invention provides a kind of full-length genome copy number detection chip of X chromosome high-density probe customization for Mental development impaired patients, being capable of gene relevant to Mental development obstacle on high density detection X chromosome.Genetic chip high sensitivity of the invention, specificity and high reliablity, can science be used for screening Mental development obstacle infant inherited pathogenic factor, for clinical unknown cause Mental development disorder with children, especially boy provides gene diagnosis, to improve the diagnosis of the disease, authentic communication is provided for the prognosis and genetic counselling of the disease.
Description
Technical field
The present invention relates to a kind of full-length genome copy number detection chip fields, more particularly to one kind for detecting spirit
The full-length genome chip of developmental disorder patient copy number variation.
Background technique
Mental development obstacle (or neurodevelopmental disorder, neuro-developmental disorders, NDD) is
One group of common illness of section, including mental retardation, autism-spectrum obstacle, difficulty of learning, Gilles de la Tourette's syndrome, attention deficit,
A variety of Mental development obstacles such as mostly dynamic obstacle.NDD is coefficient by h and E factor as a result, wherein about 25-
50% NDD is caused by having inherent cause.In these inherent causes, there is 25-30% mutation to be likely to occur in X chromosome
On.The report of clinical research simultaneously support NDD male patient more than women 30% viewpoint, reason is that male only has an X
Chromosome, gene are in hemizygosity (hemizygote), therefore male patient is easier that exposure recessive gene variation occurs,
Cause the dyskinetic phenotype of Mental development.
In full genome in 25000 functional genes, gene about 1700 of psychiatric developmental regulation are participated in, in the mankind
6.8% is accounted in genome, wherein X chromosome just there are 107, accounts for the 13.1% of X chromosome gene number (818).Biology letter
Expression quantity of the breath credit analysis discovery mankind's X chromosome gene in central nervous system is 2.8 times in other tissues, further
Show that the related gene of X chromosome plays an important role in brain normal development and function.Bioinformatic analysis shows, from complete
Consider in genome, Mental development related gene is significantly more than other non-X chromosome (1.146/Mb comparisons on X chromosome
0.69/Mb), simultaneously in Mental development disorder with children, genome mutation is significantly more than other non-X dyeing on X chromosome
Body.These Preliminary Results prompt: for Mental development impaired patients, especially male's Mental development impaired patients, it should
It first or must genetic mutation on screening X chromosome.
Genome copies number variation (copy number variants, CNVs) is to be occurred to reset and cause by genome
, it is widely present in human genome, from 1bp (base-pair) to the missing (deletion) within the scope of millions of bp, insertion
(insertion), the variation of the complicated multidigit point such as (duplication) is repeated.The mutation rate of human genome CNVs is higher,
CNVs covers the 35% of human genome complete nucleotide, and single nucleotide polymorphism (SNP) proportion is less than 1%.
CNVs can be family heredity, is also possible to newly send out, not only has facilitation to genome adaptive evolution, and to Meng De
Your heredity is distributed and is played an important role with complex disease.Previously the study found that CNVs with direct interference gene or can change
Becoming gene dosage influences gene expression, so as to cause some special genetic diseases, such as micro- repetition and micro-deleted comprehensive
Simulator sickness.As the genome-wide screening research of various diseases is spread out in succession, it is each that discovery CNVs is related to series of human disease
The hereditary basis that class human diseases, especially Mental development class illness occur.In Patients with Mental Retardation, 15-20%'s
NDD patient carries rare/newborn pathogenic gene group CNVs, wherein the chain mental retardation of X caused by X chromosome CNVs
Syndrome (XLMR) patient accounts for the 10%-15% for distributing amentia patient.It therefore, can very great Cheng by detection X chromosome CNVs
The diagnosis of the raising Mental development obstacle disease of degree.
At present there are many detection that method can be used for CNVs, traditional CNVs detection method has FISH, MLPA, qPCR.
Though these technologies can complete the detection of CNVs to a certain extent, also differ greatly away from quick, efficient, automation target.
DNA chip technology is a kind of gene detection tool newly developed in recent years.Its principle is that a large amount of oligonucleotide probe is had rule
It is arranged on a slide to rule, these probes can show the complementary of the extension increasing sequence of the sample DNA of mass signatures with signal
Sequence is combined, and by detecting to semiochemicals, software processing analysis is carried out to results of hybridization, to obtain
Obtain the intensity and its distribution pattern figure of hybridization signal.The method of full-length genome chip based on oligonucleotides provides for analysis CNVs
A kind of technology of high throughput, thus it is external at present clearly propose, for NDD patient, especially NDD child patient, Ying Shou
The detection of full-length genome chip is first completed, excludes genome C NV to the pathogenic of disease.Domestic and international various big hospital is all carried out within nearly 5 years
The inspection of genome chip.
But there are some drawbacks for commercial chip currently on the market: due to the limitation of number of probes on chip, existing market
The 60K chip (number of probes is about 60,000 on chip) of upper mainstream could not be preferentially to disease on disease related gene, X chromosome
Sick correlation important gene carries out probe design or probe density, and too low (the X chromosome probe on existing conventional die only accounts for
The 4.79% of full-length genome), therefore the CNV screening resolution ratio on X chromosome is extremely low.Lead to a major issue in this way: if
The pathogenic gene group CNV that patient carries is too small, when using common genome C NV chip screening, cannot be detected, and reports
Patient's false negative result is given, thus delay diagnosis and diagnosis.Although some present companies also develop 180K chip and (visit on chip
Needle quantity is about 180,000), resolution ratio increases, but the inspection for Mental development obstacle related gene on X chromosome
It surveys still without specific aim, and 180K chip price is expensive, full-length genome chip belongs to project at one's own expense at present, leads to many patients
Family can not undertake this testing cost, and final choice is abandoned.On the whole, the current chip for detecting CNVs is not due to having
There is the importance for considering X chromosome in Mental development, therefore do not carry out probe design in X chromosome, causes some heredity
Patient fails to complete genetic diagnosis in time.
The present invention in view of the above technical problems, develops a kind of 60K chip, related for X chromosome Mental development obstacle
Gene carry out probe high density designs, can high density detection X chromosome on gene relevant to Mental development obstacle (differentiate
Rate is up to 1kb)；Also, on non-X chromosome carry out low-density design, can detect simultaneously on non-X chromosome with essence
The relevant gene of refreshing developmental disorder (resolution ratio is up to 500kb).Compared with conventional 180K chip, number of probes on chip of the present invention
Reduce 120K, price is greatly reduced, and clinically has higher operability, and price also makes it easy to by patient home institute
Receive, there is very high economic and practical.
Summary of the invention
The object of the present invention is to provide a kind of X chromosome high-density probe customization full-length genome copy number detection chip,
Can high density detection X chromosome on gene relevant to Mental development obstacle.Genetic chip high sensitivity of the invention, it is special
Anisotropic and high reliablity, can science be used for the inherited pathogenic factor of screening Mental development obstacle infant, for clinical unknown cause spirit hair
It educates disorder with children, especially boy and gene diagnosis is provided, so that the diagnosis of the disease is improved, for the prognosis and genetic counselling of the disease
Authentic communication is provided.
In order to achieve the above objectives, the present invention provides a kind of full-length genome copy number inspection of X chromosome high-density probe customization
Chip is surveyed, in the genetic chip of the present invention, probe design both was carried out to disease related gene on non-X chromosome, simultaneously
Detailed, professional optimization is carried out to the probe of X chromosome, high-density probe is carried out to wherein 90 important diseases related genes and is set
Meter so that common commercial chip can not screening go out pathogenic gene group variation, through the invention chip can screening come out, mention
The gene diagnosis ability of Gao Liao hospital or doctor to the disease.
Specifically, the present invention provides a kind of full-length genome copy number detection chip of X chromosome high-density probe customization, institute
The number of probes for stating the detection X chromosome that chip includes accounts for the 30%-40% of whole number of probes.
Further, the present invention provides a kind of full-length genome copy number detection chip of X chromosome high-density probe customization,
The chip includes the probe for detecting following 90 genes:
ABCD1、ACSL4、AFF2、AP1S2、ARHGEF9、ARX、ATP7A、ATRX、AVPR2、BCOR、BRWD3、CASK、
CDKL5、CUL4B、DCX、DKC1、DLG3、DMD、EIF2S3、FANCB、FGD1、FLNA、FMR1、FTSJ1、GDI1、GK、GPC3、
GRIA3、HCCS、HDAC6、HDAC8、HPRT1、HSD17B10、HUWE1、IDS、IGBP1、IKBKG、IL1RAPL1、KDM5C、
KIAA2022、L1CAM、LAMP2、LAS1L、MAOA、MBTPS2、MECP2、MED12、MID1、MTM1、NAA10、NDP、
NDUFA1、NHS、NLGN3、NLGN4X、NSDHL、OCRL、OFD1、OPHN1、OTC、PAK3、PCDH19、PDHA1、PGK1、
PHF6、PHF8、PLP1、PORCN、PQBP1、PRPS1、RAB39B、RAB40AL、RBM10、RPL10、RPS6KA3、SHROOM4、
SLC16A2、SLC6A8、SLC9A6、SMC1A、SMS、SOX3、SYN1、SYP、TIMM8A、TSPAN7、UBE2A、UPF3B、
ZDHHC9 and ZNF711；
Above-mentioned 90 genes are important gene relevant to Mental development obstacle on X chromosome.
Preferably, the number of probes for above-mentioned 90 genes of detection that chip of the present invention includes accounts for whole number of probes
25%-40%, preferably from about 30%.
Further, the present invention provides a kind of full-length genome copy number detection core of X chromosome high-density probe customization
Piece, the chip include probe (wherein chrX expression X chromosome, the number of 1502 exons described in detection embodiment table 1
Genome accurate location is indicated between block).
Above-mentioned 1502 exons are the exon of 90 important genes relevant to Mental development obstacle on X chromosome.
Preferably, the number of probes for above-mentioned 1502 exons of detection that chip of the present invention includes accounts for whole number of probes
10% or more of amount, preferably 10%-20%.
It is furthermore preferred that in the probe for above-mentioned 1502 exons of detection that chip of the present invention includes, greater than 98%
Exon has at least two probe, and the exon greater than 88% has at least three probe, and the exon greater than 69% has at least four spy
Needle, the exon greater than 44% have at least five probe.
Further, genetic chip of the present invention also includes solid phase carrier, and the solid phase carrier is selected from slide, metal
One of piece, silicon wafer, potsherd, plastic sheet, nitrocellulose membrane, nylon membrane are a variety of.
On the other hand, the present invention also provides a kind of genetic chips in the copy number for detecting Mental development impaired patients
Purposes in variation.
The beneficial effects of the present invention are: the probe designed in genetic chip of the present invention is not only distributed in full-length genome, but also can be high
Density is distributed on X chromosome, and probe is relatively concentrated in relevant 90 genes of Mental development obstacle on X chromosome again
On, the chip both can on the non-X chromosome of screening be greater than 500kb disease related gene group CNVs, can also screening X chromosome list
Gene, even single exon CNVs, can more more effectively screening goes out the Disease-causing gene of patient extensively so that common commercial chip
Can not screening go out pathogenic gene group variation, by the chip can screening come out, improve hospital or doctor to the disease
The gene diagnosis ability of disease.And the genetic chip high degree of automation, it is easy to promote and utilize, it is adapted to different levels hospital
It needs.
Detailed description of the invention
Fig. 1: the hybridization signal figure of genetic chip of the present invention.
Fig. 2: the micro- comparison diagram for repeating syndrome of existing 180K chip and genechip detection MECP2 of the present invention is utilized.
Fig. 3: the comparison diagram of existing 180K chip and genechip detection 16p11.2 microdeletion syndrome of the present invention is utilized
Specific embodiment
The present invention is described in more detail for following example, artisan will appreciate that not departing from this hair
Made change, all belongs to the scope of the present invention in the case where bright scope and spirit.
Embodiment 1: the design principle and preparation step of chip of the present invention
1, chip probe designs:
Tiled probe library (https: the //earray.chem.agilent.com/suredesign/) first in full genome
In 30000 probes, at this time on X chromosome only have 2509 probes.Later, for 90 important diseases on X chromosome
Related gene adds 18837 probes, is designed specifically to: first first tiling 10000 probes to 90 genes of X chromosome, so
1502 exon regions and its beside 200bp (each 100bp in left and right) for picking out 90 genes afterwards design 9712 spies
Needle, wherein the exon design greater than 98% has at least two probe, and the exon design greater than 88% has at least three probe,
Exon design greater than 69% has at least four probe, has at least five probe (specific probe greater than the design of 44% exon
1) design section see the table below, finally pick out repetition probe, totally 18837 probes；Repetition probe 5000 are designed simultaneously, and control is visited
5580, needle, totally 59417 probes.Finally, according to the above-mentioned gene order picked out, by well known to those skilled in the art
, general method determines probe sequence and synthesizes.
1502 exon regions and number of probes of 90 genes corresponding to the present embodiment chip middle probe see the table below
1:
For the probe design section of 1502 exons of 90 important diseases related genes in 1 the present embodiment chip of table
And number
As can be seen that the probe design of the present embodiment chip is dramatically different with conventional die.It is with conventional 60K chip
Example, in about 60000 probes of full-length genome, number of probes is generally 2874 on X chromosome, accounts for 4.79%；Above-mentioned 90
The number of probes of important diseases related gene is generally 301, accounts for 0.5%；And 90 important diseases related genes is outer
The number of probes for showing son is then less, can not count ratio.And in 60K chip of the invention, number of probes is on X chromosome
21346,35.9% is accounted for, and the number of probes of 90 important diseases related genes is 18837,31.7% is accounted for, wherein 90
The number of probes of 1502 exons of important diseases related gene is 9712, accounts for 16.3%.It can be seen that of the present invention
Full-length genome chip, since important gene devises high-density probe on X chromosome, X chromosome pathogenic for screening
There is absolute predominance in the work of genome C NV.
2, chip preparation step:
(1) processing of chip carrier: glass slide is washed with chromic acid lotion, soaked overnight, and washing, 24wt% ammonium hydroxide impregnated
Night, washing, be then immersed in 95% (v/v) ethanol solution of aminopropyl trimethoxysilane, with glacial acetic acid adjust pH value to
4.0, the cleaning of 95% (v/v) EtOH Sonicate, 150 DEG C dry 4 hours, the processing of 9% (v/v) glutaraldehyde aldehyde radicalization.
(2) preparation and post-processing of genetic chip: the probe sequence designed according to step 1 passes through those skilled in the art
Every probe is prepared in well known method, and every probe is then diluted to final concentration of 50 μM/L, institute with sampling liquid respectively
The sampling liquid stated is 750mmol/L sodium chloride, 75mmol/L sodium acetate, 5% (v/v) glycerol, 1wt% ficoll and 0.1wt%
Dodecyl yellow acid sodium solution, and be the bodies such as the PBS solution (containing 0.05wt%SDS) of 200mmol/L with concentration in 96 orifice plates
After product mixing, by contact point sample or ink jet type point sample, chip carrier is downloaded to according to gene chip probes arrangement schematic diagram point
On, every point sample volume is about 0.5nL, and dessert spacing is 500 μm, and spot diameter is about 200 μm, and every probe repeats point sample 2 times,
After point sample, immobilization is placed at room temperature for 24 hours up to genetic chip.
Embodiment 2: the detection embodiment of chip of the present invention
(1) it the complete genome DNA extracting of sample to be examined: can be extracted according to conventional method in that art, sample can be people
Vein anticoagulated blood, the present invention use business DNA extraction kit (Qiagen, QIAamp DNA Blood Mini Kit, Cat
No.51104 complete genome DNA extraction) is carried out；DNA mass is carried out using Nanodrop 2000, Qubit 2.0 and concentration is examined
It surveys.
(2) digestion (Agilent, SureTag DNA the Labeling Kit, Cat of check sample and sample to be examined
No.5190-3399): sample to be examined and check sample respectively take 0.2-0.5ug genomic DNA, place in different Ep pipes, add water
Be diluted to 10.1ul, check sample it is consistent with sample to be examined gender (check sample Agilent, Cat No.5190-4370,
5190-4371), following Double digestion digestive juice (including restriction enzyme A luI and RsaI etc.) is added according to 2.9ul system,
37 DEG C digest 2 hours, 65 DEG C be incubated for 20 minutes, 4 DEG C 10 minutes, it is ensured that restriction endonuclease inactivation.
| Component | Each system (ul) | 16.2 reaction systems (ul) |
| Water | 1 | 17.82 |
| 10 times of buffers | 1.3 | 21.06 |
| BSA | 0.1 | 1.62 |
| Alu | 0.25 | 4.05 |
| Rsa | 0.25 | 4.05 |
| It amounts to | 2.9 | 48.6 |
(3) fluorescein marks: taking out postdigestive sample from 4 DEG C, 2.5ul random primer is added in each Ep pipe, rapidly
95 DEG C are denaturalized 3 minutes, place 5 minutes on ice, and following mixed mark liquid (including fluorescence then is added according to 9.5ul system rapidly
Element, marker enzyme etc.), 37 DEG C digest 2 hours, later 65 DEG C be incubated for 10 minutes, 4 DEG C 10 minutes, it is ensured that restriction endonuclease inactivation.
(4) purify and hybridize: the sample after 4 DEG C of taking-up fluorescent markers is separately added into the mixing of 430ul TE solution, mixes
Business purification kit (SureTag DNA Labeling Kit Purification Columns, Cat No.5190- is used afterwards
3391) purifying is completed, 14000g is centrifuged 10 minutes, abandons liquid, and 480ul TE is added again, and 17000g is centrifuged 12 minutes, abandons liquid
Collecting pipe is inverted in new Ep pipe by body, and 1000g is centrifuged 1 minute, is saved collection liquid and is redeterminated absorbance value, respectively
8-11ug DNA is taken, check sample and sample to be examined are mixed, following bulk crossing liquid, 95 DEG C of changes are added according to 9.5ul system
Property 3 minutes, 37 DEG C be incubated for 30 minutes, 4 DEG C 10 minutes, it is ensured that restriction endonuclease inactivation.
| Component | Each system (ul) | 10 reaction system rxn (ul) |
| 10 times of confining liquids | 4.5 | 45 |
| 2 times of HIRPM buffers | 22.5 | 225 |
| It amounts to | 27 | 270 |
(5) chip loading and incubation: every 8 sample to be examined are loaded on a chip.Be put into hybrid heater (SHELDON,
Model No.G2545A), 67 DEG C incubation 24-40 hours.
(6) it washes chip: 2 box of washing lotion being placed in 37 DEG C of water-baths preheat overnight in advance, core is taken out out of hybrid heater
Piece is removed remaining hybridization solution, is then placed in 1 box of washing lotion with 1 previous cleaning of washing lotion, sufficiently cleaning 5 minutes, is then put rapidly
2 box of washing lotion of preheating is set, is cleaned 1 minute.Taking out rapidly ensures that chip does not speckle with liquid.
(7) chip data scans: bootrom scanner (Agilent, DNA Microarray Scanner), by chip
It places built in scanner in box, starts scanner program in computer, start to scan.
(8) initial cross signal is obtained from scanner, extracts hybridization signal using Feature Extract software, and
It is changed into genomic locations and hybridization ratio, into data analysis program.From picture 1 as it can be seen that the figure in the upper left corner is array quadrangle
Dot chart respectively has 2,4,3,5 control probe signals in picture up and down, and control probe can normally be shown, illustrates this time real
Test success.The red green hybridization signal column diagram (upper figure is red, and the following figure is green) in the lower left corner, this two figures indicate red green hybridization letter
Number equilibrium.Upper right corner figure shows that red and green signals numerical value is close, all reach " high-quality " (the 1st, 2,3,7,11 rows) or " good " (the 4th, 6,
8,10,12,13 row), illustrate red green fluorescence hybridization zero deflection, fluorescence power energy actual response genome copies number.The lower right corner
It shows that red green signal has obvious positive correlation, illustrates that whole genome structure is normal.
Embodiment 3: two contrasting detection embodiments of chip of the present invention and existing chip
1.MECP2 is micro- to repeat syndrome
Utilize the chip of existing SurePrint G3 180K chip (Agilent, Cat.G4884A) and the embodiment of the present invention
The micro- DNA sample for repeating syndrome patient of MECP2 is detected respectively according to the method for embodiment 2.As it is clear from fig. 2 that in core of the present invention
In the testing result of piece, an abnormal peak (at dotted line), the position on the exception peak can clearly be seen that in the region Xq28
It is almost the same with the position 180K, but its peak value is apparently higher than 180K chip.Experimental result confirms that 60K chip of the invention can be quasi-
It really detects that MECP2 is micro- and repeats syndrome.Since the price of 60K chip will be lower than business 180K chip, if using the customization
60 chips, the economic pressures for patient home will be reduced being born, but to some pathogenic CNV, the recall rate and indifference of the two.
2.16p11.2 microdeletion syndrome
Utilize the chip of existing SurePrint G3 180K chip (Agilent, Cat.G4884A) and the embodiment of the present invention
Detect the DNA sample of 16p11.2 microdeletion syndrome patient respectively according to the method for embodiment 2.It can be seen from figure 3 that in the present invention
In the testing result of chip, an abnormal ebb (recessed gray bars), the region can clearly be seen that in the region 16p11.2
Abnormal ebb is almost the same in size and 180K chip.Experimental result confirms that 60K chip of the invention can not only not only may be used
Minigene group to detect X chromosome is unbalance, and the genome that can also detect non-X chromosome is micro- unbalance.Therefore it avoids suffering from
Person is to complete to diagnose and multiple repairing weld, repeated detection, while decreasing testing cost.
In conclusion the present invention provides it is a kind of be suitable for detect Mental development disorder Disease-causing gene variation full genome
Group 60K chip 60K chip identical with specification is compared, which goes out more microcosmic, more tiny cause Mental development
The genetic mutation of obstacle, therefore there is stronger practical application value.
Claims (3)
1. a kind of full-length genome copy number detection chip, wherein the number of probes for the detection X chromosome that the chip includes is
21346, the number of probes for the 90 important diseases related genes on detection X chromosome for including is 18837, the inspection for including
The number of probes for surveying 1502 exons of 90 important diseases related genes on X chromosome is 9712；Wherein 90 bases
The probe of cause are as follows: ABCD1, ACSL4, AFF2, AP1S2, ARHGEF9, ARX, ATP7A, ATRX, AVPR2, BCOR, BRWD3,
CASK、CDKL5、CUL4B、DCX、DKC1、DLG3、DMD、EIF2S3、FANCB、FGD1、FLNA、FMR1、FTSJ1、GDI1、GK、
GPC3、GRIA3、HCCS、HDAC6、HDAC8、HPRT1、HSD17B10、HUWE1、IDS、IGBP1、IKBKG、IL1RAPL1、
KDM5C、KIAA2022、L1CAM、LAMP2、LAS1L、MAOA、MBTPS2、MECP2、MED12、MID1、MTM1、NAA10、NDP、
NDUFA1、NHS、NLGN3、NLGN4X、NSDHL、OCRL、OFD1、OPHN1、OTC、PAK3、PCDH19、PDHA1、PGK1、
PHF6、PHF8、PLP1、PORCN、PQBP1、PRPS1、RAB39B、RAB40AL、RBM10、RPL10、RPS6KA3、SHROOM4、
SLC16A2、SLC6A8、SLC9A6、SMC1A、SMS、SOX3、SYN1、SYP、TIMM8A、TSPAN7、UBE2A、UPF3B、
ZDHHC9 and ZNF711；Wherein detect the design area of 9712 probes of 1502 exons of 90 important diseases related genes
Domain and each Area Probe number are as follows:
In 9712 probes of 1502 exons of detection, the exon greater than 98% has at least two probe, greater than 88%
Exon has at least three probe, and the exon greater than 69% has at least four probe, and the exon greater than 44% has at least five spy
Needle.
2. chip according to claim 1 is used to detect the copy number variation of Mental development impaired patients.
3. chip according to claim 2 also includes solid phase carrier, the solid phase carrier is selected from slide, sheet metal, silicon
One of piece, potsherd, plastic sheet, nitrocellulose membrane, nylon membrane are a variety of.
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| CN108642174B (en) * | 2018-07-17 | 2022-04-05 | 大连医科大学附属第一医院 | Neuropsychiatric development retardation and advanced cognitive dysfunction (ADF) pathogenic gene set, and detection primer and kit thereof |
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