CN106701924A - Application of single-nucleotide-polymorphism rs55882956 in screening of Hansen's disease sufferers - Google Patents
Application of single-nucleotide-polymorphism rs55882956 in screening of Hansen's disease sufferers Download PDFInfo
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Abstract
The invention discloses application of single-nucleotide-polymorphism rs55882956 in screening of Hansen's disease sufferers. The technical scheme protected by the invention is as follows: application of substances for detecting polymorphism or genotype of rs55882956 in a human genome in preparation of products for screening the Hansen's disease sufferers and application of the substances for detecting the polymorphism or genotype of the rs55882956 in the human genome in the preparation of products for detecting single nucleotide polymorphism related to Hansen's disease. The substances for detecting the polymorphism or genotype of the rs55882956 and other substances (such as substances for detecting other single nucleotide polymorphisms or genotypes related to the Hansen's disease) can be combined together so as to prepare products for screening the Hansen's disease sufferers.
Description
Technical field
The answering in examination leper the present invention relates to SNP rs55882956 in biological technical field
With.
Background technology
Leprosy, also known as Hansen ' s diseases, are a kind of communicable diseases caused by Mycobacterium leprae (M.Leprae), main
Invade skin and peripheral nerve.2014, there were 213,889 new cases in the whole world.Although presently, there are effective treatment side
Method, but in some countries, particularly developing country, leprosy is still teratogenesis and causes the main cause of some social concerns.For
Understand the genetic base of vulnerability to leprosy, the whole-genome association (GWAS) of leprosy located 17 common change dystopys
Point, discloses effect of the inherent immunity with adaptive immunity in leprosy morbidity.However, these common risk variant sites are big
Part is located at noncoding region, is only capable of the onset risk of partial interpretation leprosy.At present, also not to the change of protein encoding regions
Different, particularly low frequency variation and rare variation carries out system research.And these variations have proven to the inheritance susceptible with some diseases
Property it is related.
The content of the invention
The technical problems to be solved by the invention be how examination leper with detection vulnerability to leprosy.
In order to solve the above technical problems, present invention firstly provides following any purposes：
A1) polymorphism (i.e. allele) of rs55882956 or the material of genotype are preparing sieve in detection human genome
The application looked into leper's product；
A2) polymorphism (i.e. allele) of rs55882956 or the material of genotype are preparing inspection in detection human genome
The application surveyed in vulnerability to leprosy product；
A3) polymorphism (i.e. allele) of rs55882956 or the material of genotype are preparing inspection in detection human genome
The application surveyed in the product of the SNP related to leprosy；
A4) polymorphism (i.e. allele) of rs55882956 or the material of genotype are preparing mirror in detection human genome
Application in fixed or the auxiliary identification SNP related to leprosy product；
B1) polymorphism (i.e. allele) or genotype of rs55882956 are preparing examination leper in human genome
Application in product；
B2) polymorphism (i.e. allele) or genotype of rs55882956 are preparing detection vulnerability to leprosy in human genome
Application in property product；
B3) polymorphism (i.e. allele) of rs55882956 or the material of genotype are numb in examination in detection human genome
Application in wind patient；
B4) polymorphism (i.e. allele) of rs55882956 or the material of genotype are numb in detection in detection human genome
Application in wind neurological susceptibility；
B5) in detection human genome the polymorphism (i.e. allele) of rs55882956 or the material of genotype detection with
Application in the related SNP of leprosy；
B6) in detection human genome the polymorphism (i.e. allele) of rs55882956 or the material of genotype in identification or
Application in the auxiliary identification SNP related to leprosy；
B7) in human genome the polymorphism (i.e. allele) or genotype of rs55882956 in examination leper
Using；
B8) in human genome rs55882956 polymorphism (i.e. allele) or genotype detect vulnerability to leprosy in
Application.
Rs55882956 is a SNP site for two equipotential polymorphisms on human chromosome 19p13.2, belongs to low frequency variation
(MAF=3.63%), in the extron of TYK2 genes, the variation is conversion (A/G is then T/C on its complementary strand).Institute
It is AA, AG or GG to state rs55882956 genotype.The AA is that rs55882956 sites are the homozygous of A, and the GG is
Rs55882956 sites are homozygous for G's, and the AG is the heterozygous that rs55882956 sites are A and G.The detection people's gene
The polymorphism (i.e. allele) or genotype of rs55882956 concretely detect the nucleotides species of rs55882956 in group.
In such use, the polymorphism of rs55882956 or the material of genotype can be amplification in the detection human genome
PCR primer and/or Single base extension primer including the genomic DNA fragment including rs55882956.
In such use, the individual ratio in leper colony of the AA and the AG genotype is respectively higher than right
Ratio of the genotype answered in normal person colony.The product may include that the PCR primer and/or the Single base extension draw
Thing.
In such use, the leprosy concretely Chinese population leprosy.
In order to solve the above technical problems, present invention also offers the polymorphism containing rs55882956 in detection human genome
Or the product of the material of genotype.
The product of the material of the polymorphism or genotype of rs55882956 in the human genome containing detection provided by the present invention
Product, are a)-d) in any one product：
A) product of the detection SNP (i.e. allele) related to leprosy or genotype；
B) product of identification or the auxiliary identification SNP (i.e. allele) related to leprosy or genotype；
C) examination leper product；
D) vulnerability to leprosy product is detected.
In the said goods, the polymorphism of rs55882956 or the material of genotype can be amplification in the detection human genome
PCR primer and/or Single base extension primer including the genomic DNA fragment including rs55882956.
In the said goods, the leprosy concretely Chinese population leprosy.
In order to solve the above technical problems, present invention also offers following M1) or method M2)：
M1) the method for examination leper, including：Detect the gene in rs55882956 sites in subject gene group to be measured
Type, such as genotype in rs55882956 sites are AA genotype, and the object to be measured is or candidate is leper；Such as
The genotype in rs55882956 sites is AG genotype, and the object to be measured is or candidate is leper；Such as rs55882956
The genotype in site is GG genotype, and the object to be measured is or candidate is non-leper；
M2 the method for) detecting vulnerability to leprosy, including：Detect the gene in rs55882956 sites in subject gene group to be measured
Type, such as genotype in rs55882956 sites are AA genotype, and the object to be measured is susceptible or the susceptible leprosy of candidate；Such as
The genotype in rs55882956 sites is AG genotype, and the object to be measured is susceptible or the susceptible leprosy of candidate；Such as rs55882956
The genotype in site is GG genotype, and the object to be measured is not susceptible or the not susceptible leprosy of candidate.
In the above method, the leprosy concretely Chinese population leprosy.
In the above method, detecting the genotype in rs55882956 sites in subject gene group to be measured can use the detection
The polymorphism of rs55882956 or the material of genotype are carried out.
It is demonstrated experimentally that the risk allele of rs55882956 is A, ratio of the allele in leper colony
It is higher by 45.73% than ratio of the allele in normal health crowd.The P values of rs55882956 are 1.04 × 10-6, and
The relative risk of rs55882956 is 1.30, illustrates that rs55882956 is the SNP related to leprosy.
In three genotype of rs55882956, the individual ratio in leper colony of the individual and AG genotype of AA genotype
The individual ratio in normal person colony of respectively higher than corresponding genotype, the individuality of GG genotype is in leper colony
Individual ratio in normal person colony of the ratio less than the genotype.
The present invention in actual applications, can be by the polymorphism (i.e. allele) or the thing of genotype that detect rs55882956
Matter is united to other materials (such as detecting the material of other SNPs related with leprosy or genotype)
Prepare the product of examination leper.
Wherein, the polymorphism of rs55882956 or the material of genotype can be by following at least one in detection human genome
Kind of method determine the polymorphism or genotype of rs55882956 needed for reagent and/or instrument：DNA sequencing, Restriction Enzyme section
Segment length polymorphism, single-strand conformation polymorphism, denaturing high-performance chromatography, SNP chip, TaqMan probe technology and Sequenom
MassArray technologies.Wherein, polymorphism or the genotype institute of rs55882956 are determined using Sequenom MassArray technologies
Need reagent and/or instrument include PCR primer to, the extension primer based on single base extension, phosphatase, resin, chip,
MALDI-TOF (matrix-assisted laser desorption/ionization-time of fligh, Matrix-assisted
Laser desorption ionization flight time mass spectrum) and/or Sequenom MassArray technologies required for other reagents and instrument；
Reagent and/or instrument needed for the polymorphism or genotype of rs55882956 are determined using TaqMan probe technology include TaqMan
Probe, PCR primer to, quantitative PCR apparatus, the module for carrying out Genotyping and/or other examinations required for TaqMan probe technology
Agent；SNP chip is including the chip based on nucleic acid hybridization reaction, the chip based on single base extension, based on allele spy
The chip of specific primer extension, based on " one-step method " reaction chip, the chip based on primer coupled reaction, based on limitation
The chip of property inscribe enzyme reaction, the chip based on protein D NA association reactions and/or the core based on fluorescence molecule DNA association reactions
Piece.In one embodiment of the invention, that utilize is the Infinium Human Exome BeadChip of Illumina companies
Chip.
The product can be reagent or kit, can also be the system being made up of reagent or kit and instrument, such as by drawing
The system of thing and DNA sequencer composition, the system being made up of PCR reagent and DNA sequencing reagent and DNA sequencer, by TaqMan
Other examinations of probe, PCR primer to, quantitative PCR apparatus and required for carrying out the module and TaqMan probe technology of Genotyping
The system of agent composition, other reagents and instrument group as required for probe, PCR primer pair and Ligase detection reaction (LDR)
Into system, by PCR primer to, Single base extension primer, chip, PCR instrument, the module for carrying out Genotyping and/or
The system of other reagents and the instrument composition required for Sequenom MassArray technologies.
Using PCR primer amplification including the genomic DNA fragment including rs55882956, with the pcr amplification product for obtaining
It is template, single base extension is carried out using Single base extension primer, the sequence of the extension products to obtaining is detected, really
Determine the polymorphism (i.e. allele) and genotype of rs55882956.The PCR primer does not have particular/special requirement in sequence, as long as
Can amplify including the genomic DNA fragment including rs55882956.The extension primer is according in human genome
Rs55882956 upstreams (not including the SNP site) are designed, and last 1 nucleotides of the extension primer corresponds to human genome
Preceding 1 nucleotides of middle rs55882956.The extension primer (does not include also dependent on rs55882956 downstreams in human genome
The SNP site) design, the 1st nucleotides of the extension primer corresponds to rear 1 nucleosides of rs55882956 in human genome
Acid.
In the present invention, the PCR primer can be made up of rs55882956-F and rs55882956-R；
The rs55882956-F is following a1) to a4) in any one single stranded DNA：
A1) the single stranded DNA in sequence table shown in sequence 10；
A2) in a1) the single stranded DNAs that obtain of 5 ' ends and/or 3 ' end one or several nucleotides of addition；
A3) and a1) or a2) single stranded DNA that limits have more than 85% homogeneity single stranded DNA；
A4) under strict conditions with a1) or a2) limit single stranded DNA hybridize single stranded DNA；
The rs55882956-R is following b1) to b4) in any one single stranded DNA：
B1) the single stranded DNA in sequence table shown in sequence 11；
B2) in b1) the single stranded DNAs that obtain of 5 ' ends and/or 3 ' end one or several nucleotides of addition；
B3) and b1) or b2) single stranded DNA that limits have more than 85% homogeneity single stranded DNA；
B4) under strict conditions with b1) or b2) limit single stranded DNA hybridize single stranded DNA.
The Single base extension primer (rs55882956-E) can be following c1) to c4) in any one single stranded DNA：
C1) the single stranded DNA in sequence table shown in sequence 12；
C2) in c1) the single stranded DNAs that obtain of 5 ' ends and/or 3 ' end one or several nucleotides of addition；
C3) and c1) or c2) single stranded DNA that limits have more than 85% homogeneity single stranded DNA；
C4) under strict conditions with c1) or c2) limit single stranded DNA hybridize single stranded DNA.
A2 it is) described in a1) 5 ' ends and/or the single stranded DNA that obtains of 3 ' end one or several nucleotides of addition be in sequence
The single stranded DNA that 5 ' ends of the single stranded DNA shown in 10 and/or 3 ' end one to ten nucleotides of addition are obtained.B2 it is) described in b1)
The single stranded DNA that 5 ' ends and/or 3 ' end one or several nucleotides of addition are obtained is at 5 ' ends of the single stranded DNA shown in sequence 11
And/or 3 ' end addition single stranded DNAs for obtaining of one to ten nucleotides.C2 it is) described in c1) 5 ' ends and/or 3 ' end additions one
Or the single stranded DNA that several nucleotides are obtained is in 5 ' ends of the single stranded DNA shown in sequence 12 and/or 3 ' one to ten core of end addition
The single stranded DNA that thuja acid is obtained.
Term " homogeneity " used herein refers to the sequence similarity with native sequence nucleic acid." homogeneity " includes and this hair
Nucleotide sequence shown in bright sequence 10, sequence 11 or sequence 12 have 85% or higher, or 90% or higher, or 95% or
The nucleotide sequence of homogeneity higher.Homogeneity can with the naked eye or computer software is evaluated.Using computer software, two
Homogeneity between individual or multiple sequences can represent with percentage (%), and it can be used to evaluate same between correlated series
Property.
The stringent condition is, in 2 × SSC, in the solution of 0.1%SDS, to hybridize at 68 DEG C and wash film 2 times, every time
5min, and in 0.5 × SSC, the solution of 0.1%SDS, hybridize at 68 DEG C and wash film 2 times, each 15min；Or, 0.1 ×
Hybridize and wash film in SSPE (or 0.1 × SSC), the solution of 0.1%SDS, under the conditions of 65 DEG C.
Above-mentioned more than 85% homogeneity, can be 85%, 90% or more than 95% homogeneity.
In an embodiment of the present invention, rs55882956 applications SEQUENOM Genotyping platforms arePoint
Son amount array technique, SequenomSNP detection process combinations multiple PCR technique, MassARRAY iPLEX are mono-
Base elongation technology, and matrix solid-dispersion flying time mass spectrum analysis mass-spectrometric technique (matrix-assisted
Laser desorption/ionization-time of flight, MALDI-TOF) carry out parting detection.Will be comprising SNP
The DNA profiling in point region is expanded by round pcr, and reusing special extension primer, to carry out Single base extension with PCR primer anti-
Should.Because pleomorphism site base is different, the different terminal bases of extension products will cause the difference of the molecular weight of product after extending
It is different, therefore embodied by the difference of molecular weight by the base difference that SNP polymorphisms cause, by matrix assisted laser desorption ionization
Ionization time of flight mass spectrometry analyzes mass-spectrometric technique, detects the size of extension products molecular weight, and the analysis software of application specific passes through
Judge the difference of molecular weight and carry out SNP parting detections.
The present invention has found in a sample (7048 lepers and 14398 healthy persons) from Chinese population
Rs55882956 is the SNP related to leprosy.Can will detect the polymorphism (i.e. allele) of rs55882956
Or the material of genotype and other materials (such as detect other SNPs (i.e. allele) related with leprosy or
The material of genotype) it is united the product for preparing examination leper.
Brief description of the drawings
Fig. 1 is that all samples (7,048 cases and 14,398 normal healthy controls) are carried out using fixed effect model
The result of meta analyses.
Specific embodiment
The present invention is further described in detail with reference to specific embodiment, the embodiment for being given is only for explaining
The bright present invention, rather than in order to limit the scope of the present invention.
Experimental technique in following embodiments, unless otherwise specified, is conventional method.
Material used, reagent etc. in following embodiments, unless otherwise specified, commercially obtain.
Embodiment 1, rs145562243, rs149308743, rs76418789, rs146466242, rs55882956,
Rs780668 and rs181206 are the SNPs related to leprosy
Method：Inventor by three phases to Chinese population in 7,048 lepers and 14,398 it is normal right
Shine into the whole-genome association research of row protein encoding regions.First, by Exon chip to 1,648 lepers
With 2,318 SNP site (MAF of the 40,491 of normal control coding regions>0.1%) it is analyzed (first stage).So
34 candidate SNP locus are verified in north of China crowd (3,169 lepers and 9,814 normal controls) afterwards
(second stage).It is last in the other southern china crowd (2,231 lepers and 2,266 normal controls) for choosing and
8 candidate locus are verified (phase III) in all samples in first and second stage.
Research object
The research object of Exon chip discovery phase (first stage) is 1,670 lepers of Northern Part of China
With 2,321 normal controls.Qualify Phase (second stage) by 39 variant sites Northern Part of China other 3,169
Verified in example leper and 9,814 normal controls.The repeated authentication stage of final 8 variant sites to filtering out
The research object of (phase III) selection is three independent samples and all samples in first and second stage of southern area of China
This：(1) 906, Sichuan Province leper and 878 normal controls；(2) 829, Yunnan Province leper and 589 are normal right
According to；(3) 496, Guizhou Province leper and 799 normal controls.Leper and normal control are Chinese han population,
And geographically match.The clinical information of research object is shown in Table 1.
Table 1, research object information
Wherein, the diagnostic criteria of leper is to meet 2 or more than 2 in following 4, or to meet the 3rd article of person true
Vertical diagnosis：1st, skin damaged is with sensory disturbance and closes sweat, or has numb area；2nd, peripheral nerve involvement, shows as the thick companion of nerve cord
Corresponding function obstacle；3rd, skin damaged histotomy or tissue fluid smear find Mycobacterium leprae；4th, pathology visible features are sexually revised.
The diagnostic criteria of normal control (normal healthy people)：Family history (including leper without History of Leprosy and without leprosy
One-level, two grades and third degree relative)；Without animal infectious diease medical history；Without other autoimmune disease medical histories, systemic disease disease
History and family history.
This research has passed through Shandong Academy of Medical Sciences, Shandong Dermatopathy Cypridopathy Prevention and Cure Institute's Institutional Review Board
Approval.
Parting and Quality Control
From Illumina companies Infinium Human Exome BeadChip chips to 1,670 lepers
And 2,321 normal controls carry out parting, are had to 1 on chip, and 998 sample of Chinese carry out full sequencing of extron group identification
Go out 27,089 (>Exist in 1 sample) non-synonymous coding region variation.Exclude noncoding region variation and MAF<0.1%
Variation, to 40, the variation of 491 coding regions is associated analysis.In Qualify Phase and repeated authentication stage, select
Sequenom MassARRAY system (Agena Bioscience), QuantStudioTM 12K Flex Real-Time
PCR (Applied Biosystems) and 7900HT Fasr Real-Time PCR System (Applied Biosystems)
Carry out parting research.Quality Control filter criteria is as follows：Exclude variation, the recall rate of uncertain colony<90% and Hardy- is not met
Weinberg equilibrium laws (P<1×10-3), sample recall rate<95% situation.
Statistical analysis
In the first stage, the correlation analysis between phenotype and single mutant gene type is carried out by GMMAT-v0.7.
Second and third stage, by the linear regression model (LRM) in PLINK to MAF>1% being associated property of SNPs analysis, using PLINK
Middle Fisher's exact methods method is to MAF<1% SNPs is associated analysis.Using fixed effect model (by META-
V7.0 softwares are to MAF>1% SNPs uses inverse variance method, to MAF<1% SNPs is divided using z- statistic combined methods
Analysis).Meta analyses are carried out to the data that discovery phase, Qualify Phase, the part of repeated authentication stage three combine.Meet simultaneously
p<1.23×10-6(0.05/40,491) variation is the variation of statistically significant extron group.For second and the 3rd rank
The association analysis of section, p<0.05 and to have the variation of same effect with discovery phase be the site with statistical significance.Profit
Checked with Cochran ' sQ and the heterogeneity of independent sample is analyzed, p<0.05 thinks with statistical significance.
Extron group discovery phase is analyzed
Parting is carried out to 1,670 lepers and 2,321 the 273,028 of normal control variant sites.By master
It is of Chinese origin that constituent analysis determines that all samples are, and it was found that has preferable gene in leper and normal control
The matching of type.By a series of filtering of samples and variant sites, 40,491 MAF are found altogether>0.1% code area becomes dystopy
Point (38,068 nonsynonymous mutations and 2,423 same sense mutations), after by it 1,648 lepers and 2,318 health
It is analyzed in control.
Full exon analysis find there is stronger relevance in MHC regions.All variant sites in removal MHC regions
Afterwards, the Q-Q icons of remaining SNP site close zero cloth, show extron association analysis result caused by crowd's Confounding Factor
Possibility is preferably minimized (lambda value=0.99).Meanwhile, to different MAF threshold values (MAF>0.5%, MAF>1% and MAF>
5%) SNP site carries out the analysis of Q-Q figures, consistent with the above results.
Found by studying, five are pointed out relevant property (P<1.0×10-3) coding region variant sites (four often
See variation and a low frequency variation) it is also included within the site that the GWAS for reporting before has found.Four common variant sites are therewith
The SNP site of preceding report has stronger linkage disequilibrium, and only low frequency variation (IL-23R gene rs76418789) is independent.
In addition, Q-Q figures (SNP site in removal MHC regions) tail end distribution has deviation with the Q-Q figures of standard zero distribution, show exist
New association site, has the variant sites in 38 new coding regions to be found to have relevance (P<1.0×10-3)。
It is the effect of the further variation of research low frequency and rare variation, by the method pair of SKAT-O and berden-test
Exclude the variant sites and 38 new coding regions of the variant sites in MHC regions and 5 coding regions in known site
Interior variant sites (P<1.0×10-3) after SNP site be analyzed.Two methods prove that gene association is not accidental.
Qualify Phase is analyzed
To 38 newfound volumes in 3,169 lepers and 9,814 normal healthy controls in north of China region
The variant sites in code region and the variant sites for low frequency coding region that GWAS has found before carry out parting.There are 34 variations
Site can succeed parting, wherein 15 sites are consistent (in Qualify Phase, P in discovery phase and Qualify Phase<0.05, OR
The effect of value is consistent with discovery phase), 6 sites are respectively provided with the statistics of extron group analysis in discovery phase and Qualify Phase
Meaning (P<1.23×10-6) and without genetic heterogeneity, this 6 sites are respectively：NCKIPSD gene rs145562243 (P=
1.44×10-8, OR=4.35), CARD9 gene rs149308743 (P=4.99 × 10-10, OR=4.75), IL23R genes
Rs76418789 (P=6.28 × 10-9, OR=1.37), FLG gene rs146466242 (P=1.44 × 10-10, OR=1.45),
SLC29A3 gene rs780668 (P=2.89 × 10-7, OR=1.14) and IL27 gene rs181206 (P=5.92 × 10-7,OR
=0.83) (table 3).
In addition, two variant sites of coding region：TYK2 gene rs55882956 (P=2.75 × 10-5, OR=1.29)
With USP49 gene rs75746803 (P=3.45 × 10-6, OR=1.28) it is respectively provided with leprosy in discovery phase and Qualify Phase
Uniformity and relevance, but only just reached the statistical significance (P of extron group analysis<5.0×10-5) (table 3).
Wherein, to rs145562243, rs149308743, rs76418789, rs146466242, rs55882956,
Rs780668 and rs181206 partings use Sequenom MassARRAY system (Agena Bioscience),
QuantStudioTM12K Flex Real-Time PCR (Applied Biosystems) and 7900HT Fasr Real-
Time PCR System (Applied Biosystems) are carried out, and specific method is as follows：
1.rs181206, rs780668, rs76418789, rs149308743, rs145562243 and rs55882956's
Parting
Rs181206, rs780668, rs76418789, rs149308743, rs145562243 and rs55882956 this 6
SNP application SEQUENOM Genotyping platforms areMolecular weight array technique, Sequenom
SNP detection process combinations multiple PCR technique, MassARRAY iPLEX Single base extension technologies, and matrix assisted laser desorption ionization
Ionization time of flight mass spectrometry analysis mass-spectrometric technique (matrix-assisted laser desorption/ionization-time
Of flight, MALDI-TOF) carry out parting detection.DNA profiling comprising SNP site region is expanded by round pcr, then
Single base extension is carried out using special extension primer and PCR primer (table 2).Because pleomorphism site base is different, prolong
Stretching the different terminal bases of product will cause the difference of the molecular weight of product after extending, therefore the base caused by SNP polymorphisms
Difference is embodied by the difference of molecular weight, by matrix solid-dispersion flying time mass spectrum analysis mass spectrum skill
Art, detects the size of extension products molecular weight, and the analysis software of application specific carries out SNP by judging the difference of molecular weight
Parting is detected.
2,6 SNP serotype specific primers of table
Operating procedure is as follows：
6 SNP use Sequenom MassArray (San Diego, USA) platform validation, and each sample is about used
15ngDNA.The genomic DNA of peripheral blood is extracted first, and after standardization, sample DNA includes SNP through multi-PRC reaction amplification
In interior genomic DNA fragment, amplified production carries out the extension of the SNP site single chain of specificity to point, and extension products desalination simultaneously turns
Move on on the chip in 384 holes.Mass spectrograph (MALDI-TOF MS) carries out the detection of allele, using Sequenom
MassARRAY parting softwares are analyzed to testing result.
1.1st, whole blood sample collection
In informed consent, and collection research object peripheric venous blood 5ml in the case of written consent book is signed, be positioned over
EDTANa2In anticoagulant tube, put -80 DEG C of refrigerator-freezers and store for future use.
1.2nd, DNA concentration standardization comprises the following steps：
1) using NanoDrop-1000 concentration testers Accurate Determinings per it is a need standardized sample DNA concentration and
OD ratios (A260/A280, A260/A230).
2) electrical form is set up, each sample aperture that is ranked needs the DNA numberings for adding.
The sample of Sequenom MassArray partings is carried out, blank and repeated sample pair are left on every 96 orifice plate
According to.
3) according to the order of electrical form, addition has determined the DNA of concentration.
For carrying out the sample requirement experimental concentration of Sequenom MassArray partings for 12-30ng/ μ l, typically with
18ng/ μ l are preferred.And A260/A280 ratios are between 1.5-2.0, A260/230 between 1.5-2.3, such as DNA concentration is high
Appropriate FG3 is then added in 18ng/ μ l, by concentration mark to 18ng/ μ l；As DNA concentration is less than 12ng/ μ l, then again from blood
Extract qualified DNA.Concentration is directly added between 12-18ng/ μ l.
Sticky masking foil is sticked after centrifugation, and the information such as sample plate mark, sample type, source place are put on marker pen.
4) on plate centrifuge, 3000g be centrifuged 3 minutes, deposit in -20 DEG C it is standby.
1.3rd, multiplex PCR is carried out using the forward primer and reverse primer of each SNP
1.4th, the extension of the single chain of SNP site specificity
Wherein, extension primer is designed according to SNP site upstream in human genome (not including the SNP site), the extension
Last 1 nucleotides of primer corresponds to preceding 1 nucleotides of the SNP site in human genome.
1.5th, data quality control
1) SNP to parting carries out call rate calculating, removal call rate<95% SNP or gene frequency<
0.01 SNPs；
2) genetic equilibrium inspection is carried out to SNP, removal deviates the SNP (Hardy- in check sample of the law of genetic equilibrium
The P of Weinberg balance checks≤0.001).
3) the parting dendrogram of SNP is checked in Sequenom MassArray systems, removal dendrogram point heap is unclear
SNP。
4) sample Quality Control：The sample that directly removal parting fails.
Statistical analysis will be carried out by the sample and SNP of Quality Control.
1.6th, data statistic analysis
SNP using the softwares of Plink 1.07 to parting success and by Quality Control does gene phenotype in case group and control group
Correlation analysis, the genotype of each sample and the relevance of phenotype, Ran Houyong are checked with Cochran-Armitage trend
The genotype of all samples of Cochran-Mantel-Haenszel comprehensive analysis and the correlation of phenotype.Evaluate individual with Q inspections
Heterogeneity between body, in this experiment, with p<0.05 used as inspection level.Multiple logistic regression analyses are used for detection zone
The independence of interior signal.Inspection level α is inspection level divided by the SNP numbers by quality control with 0.05.Q is checked to be used for
The conspicuousness of genetic heterogeneity is assessed, P values after SNP correction detections are considered as less than 0.05 notable genetic heterogeneity.
The parting of 2.rs146466242
Rs146466242 application 7900HT/Taqman genotyping systems carry out SNP partings.
Primer and probe sequence are：
Forward primer rs146466242-F:CCACATAAACCTGGGTCCTTATTAA (sequence 19)
Reverse primer rs146466242-R:GGAAAGATCTGATATCTGTAAAGCAAGTG (sequence 20)
Probe 1rs146466242-P1:Marked by reporter fluorescence dyestuff FAM at CTTGGATGATCTTTAC (sequence 21), 5 ' ends
Note, 3 ' ends are marked by quencher fluorescent dye NFQ
Probe 2rs146466242-P2:Marked by reporter fluorescence dyestuff VIC at CTTGGATGATCTTAAC (sequence 22), 5 ' ends
Note, 3 ' ends are marked by quencher fluorescent dye NFQ
Operating procedure is as follows：
The peripheric venous blood 5ml of each object is extracted respectively, genomic DNA is extracted, and (DNA concentration is equal for genomic DNA respectively
Between 50-100ng/ microlitres).
Real-time fluorescence quantitative PCR reaction reaction system be：The μ L of genomic DNA 1,2 × TaqMan GT master mix
(Life technology companies) 2.5 μ L, the μ L of 20 × TaqMan probe mixture 0.65, the μ L of deionized water 0.85.Will be above-mentioned anti-
Answer in system 96 hole PCR plates of addition, entered using the real-time fluorescence quantitative PCR instrument of ABI 7900 (Applied Biosystems companies)
Row reaction.Reaction condition is：95 DEG C are denatured 30 seconds, and 60 DEG C are annealed 1 minute, 40 circulations.
In above-mentioned reaction system, 20 × TaqMan probe mixture includes the gene including rs146466242 including amplification
The PCR primer pair and complete probe of group DNA fragmentation, wherein PCR primer are complete to being made up of above-mentioned forward primer and reverse primer
Probe is made up of probe 1 and probe 2.
After reaction terminates, genotyping is carried out using 7900System SDS software, determine each research object
The genotype in rs146466242 sites.
Repeated authentication is analyzed
In order to further verify 8 non-synonymous variant sites (6 extron group analysis have statistical significance site and
2 promptings may be related sites) whether there is relevance, and choose three independent samples totally 2,231 in southern china region
Example leper and 2,266 normal controls.Then all samples (7,048 cases and 14,398 normal healthy controls) are carried out
Parting, wherein, to rs145562243, rs149308743, rs76418789, rs146466242, rs55882956,
The same Qualify Phase of specific method that rs780668 and rs181206 partings are used.
Then meta is carried out to all samples (7,048 cases and 14,398 normal healthy controls) using fixed effect model
Analysis.7 encoding mutants show that the uniformity in three phases is associated, and without heterogeneous evidence, and have in all samples
Significant difference (the P of extron<0.05/40,491=1.23 × 10-6)：Two rare mutation, positioned at 3p21.31's
NCKIPSD genes rs145562243 (MAF=0.4%, P=1.71 × 10-9, OR=4.35) and positioned at the CARD9 bases of 9q34.3
Because of rs149308743 (MAF=0.5%, P=2.09 × 10-8, OR=4.75)；Three low frequency variations, positioned at 1p31.3's
IL23R genes rs76418789 (MAF=4.32%, P=1.03 × 10-10, OR=1.36), positioned at the FLG genes of 1q21.3
Rs146466242 (MAF=3.54%, P=3.39 × 10-12, OR=1.45) and positioned at the TYK2 genes of 19p13.2
Rs55882956 (MAF=3.63%, P=1.04 × 10-6, OR=1.30)；Two common variations, positioned at 10q22.1's
SLC29A3 genes rs780668 (MAF=42%, P=2.17 × 10-9, OR=1.14) and positioned at the IL27 genes of 16p11.2
Rs181206 (MAF=15%, P=1.08 × 10-7, OR=0.83).Although in all independence sample analyses, being located at
The USP49 genes rs75746803 of 6p21.1 shows uniformity, but the site is less than extron significance (MAF=
4.8%, P=3.57 × 10-6, OR=1.25) and (table 3 and Fig. 1).
Genotype frequencies of 7 SNP in 7048 lepers and 14398 normal controls is as shown in table 4 and table 5.
Result shows：In three genotype of rs145562243, the individual ratio in leper colony of TC genotype is higher than right
The individual ratio in normal person colony of the genotype answered, the individual ratio in leper colony of CC genotype is less than
The individual ratio in normal person colony of the genotype；
In three genotype of rs149308743, the individual ratio in leper colony of TC genotype is higher than right
The individual ratio in normal person colony of the genotype answered, the individual ratio in leper colony of CC genotype is less than
The individual ratio in normal person colony of the genotype；
In three genotype of rs76418789, the individuality of the individual and AG genotype of AA genotype is in leper colony
In ratio be respectively higher than the individual ratio in normal person colony of corresponding genotype, the individual of GG genotype is suffered from leprosy
Individual ratio in normal person colony of the ratio less than the genotype in person colony；
In three genotype of rs146466242, the individuality of the individual and AT genotype of AA genotype is in leper group
Ratio in body is respectively higher than the individual ratio in normal person colony of corresponding genotype, and the individuality of TT genotype is in leprosy
Individual ratio in normal person colony of the ratio less than the genotype in PATIENT POPULATION；
In three genotype of rs55882956, the individuality of the individual and AG genotype of AA genotype is in leper colony
In ratio be respectively higher than the individual ratio in normal person colony of corresponding genotype, the individual of GG genotype is suffered from leprosy
Individual ratio in normal person colony of the ratio less than the genotype in person colony；
In three genotype of rs780668, the individuality of the individual and TC genotype of TT genotype is in leper colony
Ratio be respectively higher than the individual ratio in normal person colony of corresponding genotype, the individuality of CC genotype is in leper
Individual ratio in normal person colony of the ratio less than the genotype in colony；
In three genotype of rs181206, the individual ratio in leper colony of AA genotype is higher than corresponding
The individual ratio in leper colony of the individual ratio in normal person colony of genotype, GG genotype and AG genotype
The individual ratio in normal person colony of example respectively lower than correspondence genotype.
Genotype individuals number of the table 4, SNP in leper and normal population
Genotype frequency of the table 5, SNP in leper and normal population
Gene frequency (%) and variable quantity compared with the control of the table 6, SNP in leper colony
In note table 4 and table 5, in the genotype of rs145562243, A1*A1 represents that CC, A1*A2 represent that TC, A2*A2 are represented
TT；
In the genotype of rs149308743, A1*A1 represents that CC, A1*A2 represent that TC, A2*A2 represent TT；
In the genotype of rs76418789, A1*A1 represents that AA, A1*A2 represent that AG, A2*A2 represent GG；
In the genotype of rs146466242, A1*A1 represents that AA, A1*A2 represent that AT, A2*A2 represent TT；
In the genotype of rs55882956, A1*A1 represents that AA, A1*A2 represent that AG, A2*A2 represent GG；
In the genotype of rs780668, A1*A1 represents that TT, A1*A2 represent that TC, A2*A2 represent CC；
In the genotype of rs181206, A1*A1 represents that GG, A1*A2 represent that AG, A2*A2 represent AA.
Using two logistic regression models (log-additive models) the calculating leper colonies classified and normally
Gene frequency difference P values in people colony, determine SNP whether there is significant, and wherein genotype is united using additive models
Meter.Result find, rs145562243, rs149308743, rs76418789, rs146466242, rs55882956,
The gene frequency of each allele of rs780668 and rs181206 has significantly in normal person colony and leper colony
Sex differernce.
As shown in Table 6, the risk allele of rs145562243 is T, and gene of the allele in leper is frequently
Rate is 0.0039, and the gene frequency in normal control is 0.0004, and compared with normal control, the allele is in leper
In increased 875.00%；The risk allele of rs149308743 is T, and gene of the allele in leper is frequently
Rate is 0.0055, and the gene frequency in normal control is 0.0006, and compared with normal control, the allele is in leper
In increased 816.67%；The risk allele of rs76418789 is A, gene frequency of the allele in leper
It is 0.0610, the gene frequency in normal control is 0.0432, and compared with normal control, the allele is in leper
Increased 41.20%；The risk allele of rs146466242 is A, and gene frequency of the allele in leper is
0.0558, the gene frequency in normal control is 0.0354, and compared with normal control, the allele increases in leper
Add 57.63%；The risk allele of rs55882956 is A, and gene frequency of the allele in leper is
0.0529, the gene frequency in normal control is 0.0363, and compared with normal control, the allele increases in leper
Add 45.73%；The risk allele of rs780668 is T, and gene frequency of the allele in leper is
0.4648, the gene frequency in normal control is 0.4213, and compared with normal control, the allele increases in leper
Add 10.33%；The risk allele of rs181206 is A, and gene frequency of the allele in leper is
0.8829, the gene frequency in normal control is 0.8503, and compared with normal control, the allele increases in leper
Add 3.83%.
Test result indicate that, rs145562243, rs149308743, rs76418789, rs146466242,
The polymorphism or genotype or gene frequency of rs55882956, rs780668 and rs181206 can be used for the sieve of leper
Look into.
In addition, carrying out joint survey by age in sample and gender information, the shadow of age and sex ratio is analyzed
Ring.Closely similar (the tables of ORs that (age and sex) in two common mutations and three low frequency mutation SNPs is not adjusted and adjusted
7), disclose in the correlation analysis of these SNPs, there are less effect at sex and age.
Table 7, sex and age are on two common mutations and the analysis result of three low frequency mutation SNP influences
Secondary allele/main allele；
OR, OR are according to inferior for gene is calculated；
F () refers to fixed-effect model.
<110>Shandong Dermatopathy Cypridopathy Prevention and Cure Institute
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Claims (10)
1. in detection human genome the polymorphism of rs55882956 or the material of genotype in examination leper's product is prepared
Application.
2. the polymorphism of rs55882956 or the material of genotype are preparing detection vulnerability to leprosy product in detection human genome
In application.
3. the polymorphism of rs55882956 or the material of genotype are preparing the detection list related to leprosy in detection human genome
Application in the product of nucleotide polymorphisms.
4. the polymorphism of rs55882956 or the material of genotype are preparing identification or auxiliary identification and fiber crops in detection human genome
Application in the product of the SNP that wind facies is closed.
5. following any applications：
B1) the application of the polymorphism or genotype of rs55882956 in examination leper's product is prepared in human genome；
B2) the application of the polymorphism or genotype of rs55882956 in detection vulnerability to leprosy product is prepared in human genome；
B3) the application of the polymorphism of rs55882956 or the material of genotype in examination leper in detection human genome；
B4) the polymorphism of rs55882956 or the material of genotype answering in vulnerability to leprosy is detected in detection human genome
With；
B5) in detection human genome the polymorphism of rs55882956 or the material of genotype in the detection monokaryon glycosides related to leprosy
Application in sour polymorphism；
B6) polymorphism of rs55882956 or the material of genotype are being identified or are aiding in identification and leprosy phase in detection human genome
Application in the SNP of pass；
B7) the application of the polymorphism or genotype of rs55882956 in examination leper in human genome；
B8) the application of the polymorphism or genotype of rs55882956 in vulnerability to leprosy is detected in human genome.
6., containing the product of the material of the polymorphism or genotype of rs55882956 in detection human genome, be a)-d) in appoint
A kind of product：
A) product of the detection SNP related to leprosy or genotype；
B) product of identification or the auxiliary identification SNP related to leprosy or genotype；
C) examination leper product；
D) vulnerability to leprosy product is detected.
7. product according to claim 6, it is characterised in that：The polymorphism of rs55882956 in the detection human genome
Or the material of genotype is PCR primer and/or Single base extension of the amplification including the genomic DNA fragment including rs55882956
Primer.
8. product according to claim 7, it is characterised in that：The PCR primer by rs55882956-F and
Rs55882956-R is constituted；
The rs55882956-F is following a1) to a4) in any one single stranded DNA：
A1) the single stranded DNA in sequence table shown in sequence 10；
A2) in a1) the single stranded DNAs that obtain of 5 ' ends and/or 3 ' end one or several nucleotides of addition；
A3) and a1) or a2) single stranded DNA that limits have more than 85% homogeneity single stranded DNA；
A4) under strict conditions with a1) or a2) limit single stranded DNA hybridize single stranded DNA；
The rs55882956-R is following b1) to b4) in any one single stranded DNA：
B1) the single stranded DNA in sequence table shown in sequence 11；
B2) in b1) the single stranded DNAs that obtain of 5 ' ends and/or 3 ' end one or several nucleotides of addition；
B3) and b1) or b2) single stranded DNA that limits have more than 85% homogeneity single stranded DNA；
B4) under strict conditions with b1) or b2) limit single stranded DNA hybridize single stranded DNA.
9. the product according to claim 7 or 8, it is characterised in that：The Single base extension primer is following c1) to c4)
In any one single stranded DNA：
C1) the single stranded DNA in sequence table shown in sequence 12；
C2) in c1) the single stranded DNAs that obtain of 5 ' ends and/or 3 ' end one or several nucleotides of addition；
C3) and c1) or c2) single stranded DNA that limits have more than 85% homogeneity single stranded DNA；
C4) under strict conditions with c1) or c2) limit single stranded DNA hybridize single stranded DNA.
10. following M1) or method M2)：
M1) the method for examination leper, including：The genotype in rs55882956 sites in subject gene group to be measured is detected, such as
The genotype in rs55882956 sites is AA genotype, and the object to be measured is or candidate is leper；Such as rs55882956
The genotype in site is AG genotype, and the object to be measured is or candidate is leper；Such as the gene in rs55882956 sites
Type is GG genotype, and the object to be measured is or candidate is non-leper；
M2 the method for) detecting vulnerability to leprosy, including：The genotype in rs55882956 sites in subject gene group to be measured is detected,
Genotype such as rs55882956 sites is AA genotype, and the object to be measured is susceptible or the susceptible leprosy of candidate；Such as
The genotype in rs55882956 sites is AG genotype, and the object to be measured is susceptible or the susceptible leprosy of candidate；Such as rs55882956
The genotype in site is GG genotype, and the object to be measured is not susceptible or the not susceptible leprosy of candidate.
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Cited By (2)
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| CN110029162A (en) * | 2019-05-22 | 2019-07-19 | 中国科学院生物物理研究所 | A kind of SNP marker and its application being located at Noncoding gene area for detection system lupus erythematosus neurological susceptibility |
| CN113151452A (en) * | 2021-05-31 | 2021-07-23 | 山东第一医科大学附属皮肤病医院(山东省皮肤病性病防治研究所、山东省皮肤病医院) | Application of single nucleotide polymorphism rs13259978 in screening of leprosy patients |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110029162A (en) * | 2019-05-22 | 2019-07-19 | 中国科学院生物物理研究所 | A kind of SNP marker and its application being located at Noncoding gene area for detection system lupus erythematosus neurological susceptibility |
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| CN113151452A (en) * | 2021-05-31 | 2021-07-23 | 山东第一医科大学附属皮肤病医院(山东省皮肤病性病防治研究所、山东省皮肤病医院) | Application of single nucleotide polymorphism rs13259978 in screening of leprosy patients |
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