CN106834448B - With the associated interleukins 16 of primary biliary cholangitis and its application - Google Patents
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Abstract
本发明公开白细胞介素16（IL‑16）在制备检测或辅助检测、筛查或预测原发性胆汁性胆管炎(Primary Biliary Cholangitis,PBC)的产品中的应用。本发明还公开与原发性胆汁性胆管炎易感性显著相关的IL‑16的单核苷酸多态位点为：rs17875509、rs11857713、rs4778636、rs11073001、rs11630277、rs56859031、rs57763246、rs17875523、rs3898677、rs139879640、rs4577037、rs3899547、rs17875532、rs17875533、rs11556218、rs1803275、rs3926277、rs3926278、rs3926279、rs17875542、rs17875543、rs7166271、rs11325、rs4778640。本发明还公开IL‑16在制备PBC动物模型中的应用以及在制备PBC治疗药物方面的应用。本发明首次发现IL‑16的多个基因多态性位点与PBC的发生显著相关，可用于PBC发病风险预测的指标之一。本发明首次发现IL‑16在PBC病人血液表达异常升高。
The invention discloses the application of interleukin-16 (IL-16) in preparing a product for detection or auxiliary detection, screening or prediction of primary biliary cholangitis (PBC). The present invention also discloses that the single nucleotide polymorphism sites of IL-16 significantly related to the susceptibility to primary biliary cholangitis are: rs17875509, rs11857713, rs4778636, rs11073001, rs11630277, rs56859031, rs57763246, rs17875523, rs3898677, rs1398777, rs1398 , rs4577037, rs3899547, rs17875532, rs17875533, rs11556218, rs1803275, rs3926277, rs3926278, rs3926279, rs17875542, rs17875543, rs7166271, rs11325, rs477864. The invention also discloses the application of IL-16 in the preparation of PBC animal model and the application in the preparation of PBC therapeutic medicine. The present invention finds for the first time that multiple gene polymorphism sites of IL-16 are significantly correlated with the occurrence of PBC, and can be used as one of the indicators for predicting the risk of PBC onset. The present invention finds for the first time that the expression of IL-16 in the blood of PBC patients is abnormally elevated.
Description
Technical field
The invention belongs to field of immunology, and in particular to the associated interleukins 16 of primary biliary cholangitis
(IL-16) it and its applies.
Background technique
Primary biliary cholangitis (primary biliary cholangitis, PBC), is once called as primary biliary
Cirrhosis (primary biliary cirrhosis) is a kind of Chronic Progressive autoimmunity for involving stones in intrahepatic bile duct system
Property disease, be mainly shown as in liver that small bile duct progressive is destroyed sexually revises with Portal inflammation, eventually leads to fibrosis and cirrhosis.
This disease takes place mostly in women above middle age, and male's case only accounts for 10%.With the continuous improvement and diagnosis to PBC disease cognitive
The foundation of method, the disease incidence and illness rate of PBC are in rising trend in the whole world.2003, in District of Shanghai to 5011 health
(26-85 years old) PBC specificity anti-mitochondrial antibody (AMA) (PDC-E2) of examinee carries out screening, and discovery AMA positive rate is higher than
0.16%, 0.3% [5] are higher than in female middle-aged.2006, in In Guangdong Province to the general of 8126 adults (18-83 years old)
The positive rate for looking into middle discovery AMA (PDC-E2) is higher than 0.05%, and 0.15% is higher than in female middle-aged.Recently in District of Shanghai pair
The AMA screening results of 19012 residents find that 0.40% male and 0.94% women AMA are positive, wherein 25 people
(0.13%) PBC is suffered from.With the aging of China's population, PBC will be rapidly developed in China into a kind of more typical disease
Disease was likely to be breached ten thousand patient of 20-30 in 2025, may be up to ten thousand patient of 35-50 to the year two thousand fifty.
Early diagnosis and therapy is the most effective means for controlling the development of PBC disease.The treatment of PBC is lacked effectively at present
Method, ursodesoxycholic acid are the medicines uniquely controling effectively to early stage PBC patient (especially jaundice do not occur patient)
Object, but acted on without healing, and the only resource for saving advanced stage PBC patient is to implement liver transplant.China is virus hepatitis height
The clinical symptoms of the country of hair, PBC are similar to virus hepatitis, and PBC patient is often misdiagnosed as virus hepatitis or drug early stage
Property hepatitis, has seriously affected the diagnosis and treatment of PBC patient.Therefore, early diagnosis and therapy is carried out to PBC patient, there is important society
It can meaning.
PBC has very strong genetic predisposition, and the PBC disease incidence of the first generation relatives of patient is 100 times higher than general population.
The report of comprehensive North America, Europe and Japan, the familial incidence of PBC are 3.8-9%.2005, one in the extensive of North America
Investigation result shows that the relative risk (odds ratio) of patient PBC lineal relative morbidity is up to 10.7.Pass through PBC patient
Homozygotic twin analyzed, find its co-morbid rate up to 63%.Our team are at the past 4 years to domestic PBC patient's
Having found the PBC patient of 3 pairs of enzygotic twins in investigation, the sister's disease symptom and disease time of three couples of patients is all very close,
In conjunction with international data with existing, the homozygotic twin co-morbid rate of PBC patient is up to 72.7%.
Since two thousand nine, PBC tool is shown to the series of results of white man PBC patient whole-genome association (GWAS)
There is very strong genetic predisposition.To apply the North America PBC cooperation group of artificial core member using GWAS and specific site high density
The method of Polymorphism Analysis carries out cohort analysis to North America PBC patient and control crowd, it was confirmed that the inheritance susceptible of PBC
Property and the close phase in the sites such as HLA-II class antigen, IL12A, IL12RB2, STAT4, IRF5, ch17q12-21, MMEL1 and SPIB
It closes, and disclose IL12 signal transduction pathway and Toll-like receptor (TLR)/TNF signal transduction pathway exception to lead
Cause the generation of PBC.It is closely related with PBC that the small-scale GWAS research of Japanese scholars has also discovered the site TNFSF15.China pair
The genetics research of PBC has certain basis, and domestic scholar loses with regard to the gene locis such as HLA and CTLA4 and Han nationality PBC respectively
It passes neurological susceptibility and has carried out Primary Study.
We start and complete the Illumina China chip scanning work to 1122 Han nationality's PBC samples, in conjunction with
4046 contrasting datas, have carried out large-scale cohort analysis, find for the first time in the world and confirm the gene loci of IL-16
Reach GWAS conspicuousness, and finds IL-16 overexpression in PBC blood samples of patients for the first time.
Patent document there is no to refer to the relationship of IL-16 gene loci and IL-16 and PBC in the world at present.
Summary of the invention
Goal of the invention: there is provided IL-16 in preparation detection or auxiliary for first technical problem to be solved by this invention
Application in the product of detection, screening or prediction primary biliary cholangitis.
That there is provided a series of is significant with primary biliary cholangitis for second technical problem to be solved by this invention
Relevant mononucleotide polymorphic site (SNP), and find out wherein be associated with primary biliary cholangitis neurological susceptibility it is most significant
Representative site, to provide the mononucleotide polymorphic site (SNP) is easy for assessing primary biliary cholangitis
The purposes of perception.
There is provided IL-16 to prepare the application in PBC animal model for third technical problem to be solved by this invention.
4th technical problem to be solved by this invention answering in terms of preparing PBC therapeutic agent there is provided IL-16
With.
Technical solution: in order to solve the above-mentioned technical problem, the technical scheme adopted by the invention is as follows: in a first aspect, providing
IL-16 preparation detection or auxiliary detection, screening or predict primary biliary cholangitis product in application.We open
The Illumina China chip scanning work to 1122 Han nationality's PBC samples is moved and completes, in conjunction with 4036 contrasting datas, into
It has gone large-scale cohort analysis, has found for the first time in the world and confirm that the gene loci of IL-16 has reached GWAS conspicuousness,
And IL-16 overexpression in PBC blood samples of patients is found for the first time；Especially by whole-genome association, find IL-16's
Gene pleiomorphism and PBC morbidity have substantial connection；By the serological analysis to PBC patient and normal person, it is found that IL-16 exists
Overexpression in PBC patient.
Second aspect provides a series of to the monokaryon glycosides of the significant relevant IL-16 of primary biliary cholangitis neurological susceptibility
Sour polymorphic site is used by being research object to 1122 Chinese Han nationality PBC Patient Sample As and 4036 normal human samples
HumanOmniZhongHua-8 (v1.1) scanning carries out the analysis of full-length genome associated data, carries out point of gene pleiomorphism (SNP)
Type, the data obtained using chip, the comparative analysis using PLINK software to all polymorphic sites find the more of IL-16 gene
There were significant differences in PBC and normal population for the frequency of a SNP: significant relevant to primary biliary cholangitis neurological susceptibility
The mononucleotide polymorphic site of IL-16 are as follows: rs17875509, allele G/C；Rs11857713, allele C/T；
Rs4778636, allele G/A；Rs11073001, allele A/G；Rs11630277, allele T/C；
Rs56859031, allele A/AGT；Rs57763246, allele C/T；Rs17875523, allele C/T；
Rs3898677, allele T/C；Rs139879640, allele TCTCA/T；Rs4577037, allele T/
G；Rs3899547, allele A/G；Rs17875532, allele C/T；Rs17875533, allele C/A；
Rs11556218, allele T/G；Rs1803275, allele G/A；Rs3926277, allele C/A；
Rs3926278, allele C/G；Rs3926279, allele A/G；Rs17875542, allele G/C；
Rs17875543, allele C/T；Rs7166271, allele C/T；Rs11325, allele G/T；
Rs4778640, allele A/G.
The third aspect, the present invention carry out linkage disequilibrium value by SNP to the site IL16, find multiple SNP with
The linkage relationship of rs11556218.It is more for the rs11556218 of wherein most significant difference using the iPlex method of Sequenom
State property site carries out verification test；And Impute2 and PLINK software is used, logistic regression principle carries out, using gender as co-variation
Amount, by synergistic effect model analysis；It is again seen that there were significant differences；All SNP in table 1 be have between PBC and control it is aobvious
The gene polymorphic site for writing difference, can be used for the risk profile to PBC.
Fourth aspect, the present invention is by having found the analysis for having gene expression analysis data library in the world, PBC associated bit
The expression of point polymorphism and IL-16 in lymphocyte, monocyte has significant association；Using PLINK software, logistic regression is former
Reason carries out the relationship that each SNP site and primary biliary cholangitis are assessed in analysis, calculates the opposite danger of dangerous allele
Dangerous degree (Odd ratio, OR) and 95% credibility interval, the dangerous allele for obtaining a series of this SNP site is respectively:
Rs17875509 polymorphic site is C；Rs11857713 polymorphic site is T；Rs4778636 polymorphic site is A；
Rs11073001 polymorphic site is G；Rs11630277 polymorphic site is A；Rs56859031 polymorphic site is AGT；
Rs57763246 polymorphic site is T；Rs17875523 polymorphic site is T；Rs3898677 polymorphic site is C；
Rs139879640 polymorphic site is T；Rs4577037 polymorphic site is G；Rs3899547 polymorphic site is G；
Rs17875532 polymorphic site is T；Rs17875533 polymorphic site is A；Rs11556218 polymorphic site is G；
Rs1803275 polymorphic site is A；Rs3926277 polymorphic site is A；Rs3926278 polymorphic site is G；
Rs3926279 polymorphic site is G；Rs17875542 polymorphic site is C；Rs17875543 polymorphic site is T；
Rs7166271 polymorphic site is T；Rs11325 polymorphic site is T；Rs4778640 polymorphic site is G.
5th aspect, the present invention suffer from 120 PBC by the quantitative Enzyme-Linked Immunospot (ELISA) special to IL-16
The concentration of the serum IL -16 of person and 120 normal persons is measured, find IL-16 in PBC patient significantly increase (P <
0.0001)。
In terms of 6th, chronic IL-16 expression preparation PBC animal model is introduced in the animal model that the present invention establishes, and will
This model is used for the screening of PBC therapeutic agent.
The utility model has the advantages that the present invention have the advantages that following characteristic and:
1) present invention firstly discovers that multiple gene polymorphism sites of IL-16 are significant related to the generation of PBC, can be used for
One of the index of PBC onset risk prediction.
It 2) is the target that can be used for PBC targeted therapy present invention discover that IL-16 abnormal expression in PBC blood samples of patients increases
Point.
3) discovery invented prepares PBC animal model possibly through chronic IL-16 expression is introduced in animal model, and
This model is used for the screening of PBC therapeutic agent.
Detailed description of the invention
Fig. 1 is the associated diagram of identified IL16 gene region mononucleotide polymorphism site and PBC in embodiment 1；Fig. 1
In: abscissa " Position on chr " is the position (unit Mb) on chromosome；Ordinate is mononucleotide polymorphism site
With being associated with conspicuousness P value (- log10 (p-value)) for PBC, right side Recombination rate is recombination fraction, unit (CM/
Mb)；Represent site rs17875509 with ◆ indicate.
Fig. 2 is that ELISA detects differential expression of the IL-16 in PBC and normal control population (HC) serum in embodiment 3
(* * * * is P < 0.0001)；In Fig. 2: abscissa, which is shown, is divided into two groups of PBC and normal control population (HC), and ordinate is to detect
Serum IL -16 concentration, unit (pg/ml, pg/ml).
Specific embodiment
Below by specific embodiment and attached drawing, the present invention is further described.
Experimental material used, main agents and formula are as follows in the embodiment of the present invention:
Main experimental materials and main agents:
1, the DNA sample and serum of primary biliary cholangitis patient and normal control population；
2, it whole-genome association chip analysis kit: is produced by Illumina company
HumanOmniZhongHua-8 (v1.1) assay kit includes chip and reagent.
3, IL-16 quantifies enzyme-linked immunologic detecting kit: being purchased from R&D Systems company, Human IL-
16Quantikine ELISA Kit, production number D1600.
Main solution is prepared:
1, phosphate buffer (PBS, 1L): 8g NaCl, 0.2g KCl, 0.24g KH2PO4, 3.628g Na2HPO4·
12H2O, distilled water are settled to 1L；
2, saturated sodium chloride solution (5M NaCl, 1L): 292.5g NaCl, distilled water dissolution is settled to 1L；
3,1M Tris-HCl buffer (pH 8.0): 30.3g Tris base is dissolved in 200ml distilled water, and hydrochloric acid is adjusted
PH value is settled to 250ml to 8.0；
4,0.5M EDTA (PH 8.0): 36.5g EDTA is dissolved in NaOH solution, is adjusted pH to 8.0, is settled to 250ml；
5, nuclei lysis buffer (Nuclei Lysis Buffer, 500ml): 40ml 5M NaCl, 2ml 0.5M EDTA
(PH 8.0), 5ml 1M Tris-HCl (pH 8.0), 453ml distilled water；
6, proteinase K buffer (Proteinase K buffer, 100ml): 50ml glycerol, 100ul 1M
Tris-HCl (pH 7.5), 200 μ l 1M CaCl2, 47ml distilled water；
7, Proteinase K (Proteinase K Solution, 10mg/ml): 100mg Proteinase K powder is dissolved in 10ml albumen
In enzyme K buffer；
8, digestive juice (Nuclei-SDS-Proteinase K-Master Mix Lysis buffer, 504ml): 420ml
Nuclei Lysis Buffer, 3.5ml Proteinase K (10mg/ml), 17.5ml 10%SDS, 0.35ml 0.5M
EDTA (PH 8.0), 62.65ml distilled water；
9, TE buffer (PH 8.0): 1ml 1M Tris-HCl buffer (pH 8.0), 0.2ml 0.5M EDTA (PH
8.0), distilled water is settled to 100ml；
10,6 × DNA spotting buffer (10ml): 88mg EDTA, 5mg bromophenol blue, 5mg dimethylbenzene is green, is dissolved in the bis- steamings of 4ml
In water, adds after 3.6ml glycerol and be adjusted to PH7.0 using NaOH, be settled to 10ml；
11,50 × TAE buffer: 242g Tris-base, 57.1ml glacial acetic acid, 200ml 0.5M EDTA (PH 8.0),
Distilled water is settled to 1L.
1 whole-genome association of embodiment
The present invention has found being associated with for IL-16 gene loci and primary biliary cholangitis by following methods and step.
One, method
1122 Chinese Han nationality PBC Patient Sample As and 4036 normal human samples are had chosen in Chinese han population first
For research object, HumanOmniZhongHua-8 (v1.1) scanning is carried out, the parting of gene pleiomorphism (SNP) is carried out, is used
Comparative analysis of the PLINK software to all polymorphic sites finds the frequency of multiple SNP of IL-16 gene in PBC and normal population
In there were significant differences.
Two, case and check sample inclusion criteria
All PBC patients for being included in research meet following standard: 1), all patients be Han nationality；2), AMA-M2 serum
Learn tests positive；3), biochemical indicator shows cholestasis evidence, is mainly shown as that ALP, GGT are increased；5), all kinds of hepatitis
Negative (hepatitis B virus surface antigen is negative, hepatitis B virus DNA in serum for poison infection and HIV infection index
Less than Hepatitis C virus RNA in 200 copy numbers/milliliter, serum less than 500 copy numbers/milliliter, viral hepatitis type E antigen negative, human immunity
Defective virus negative antibody)；6), without infection by Schistosoma history；7) patient main suit's drinking amount is less than 100ml/ days.
Genome-wide screening compares population sample and comes from Anhui, and Jiangsu, the normal Check-up crowd in Shandong, biochemical indicator is just
Often, no clinical disease characterization, readme body are strong.Case was screened between 22~85 years old, and average age is 54.7 years old；Control is 15 years old
It is screened between by 96 years old, average age is 34.8 years old.
Three, the essential characteristic of crowd is tested
Test crowd is that chip detection its essential characteristic of sample is as follows:
Table 1
Four, the preparation of the genome DNA sample of primary biliary cholangitis (PBC) patient and normal control population
1, the non-anticoagulation of patient and normal person are acquired in non-anticoagulation collecting pipe by each hospital.Collecting pipe is centrifuged,
Separate serum and blood clot.The serum on the blood sample upper layer of defrosting is mixed gently with pipettor in superclean bench, is dispensed
Enter 4 1.5ml blind nuts and save pipe, is stored in -80 DEG C.
2, separation gel is taken out with ladle, clot is poured into weighing boat, is sufficiently shredded clot with scissors, then inhaled with plastics
Broken clot is transferred in 50ml centrifuge tube by pipe.
3, weighing boat is rinsed with PBS buffer solution and heparin tube, recovery and rinsing liquid are mixed to 50ml centrifuge tube with plastic suction pipe
Liquid, centrifugation, 2500rpm, 15min, abandon supernatant by 4 DEG C.
4, plus 5ml digestive juice, mixing are put into water-bath constant temperature oscillator, 50 DEG C, 200rpm is digested overnight.
5, add 1.5ml 5M NaCl after digestion completely, mix；It is stored at room temperature 10min, is during which mixed by inversion repeatedly centrifugation,
3800rpm, 30min, room temperature.
6, transfer supernatant adds 2 times of volume dehydrated alcohols, mixes well in 50ml centrifuge tube.
7, the cotton-shaped DNA precipitating that will be seen that with pipettor is transferred to the 2ml EP pipe containing 500 μ l, 70% ethyl alcohol and carries out clearly
It washes.
8, it is centrifuged, 13000rpm, 5min, room temperature, abandons supernatant；It is centrifuged again, 13000rpm, 1min, room temperature is abandoned with pipettor
Raffinate to the greatest extent, dries, is dissolved with distilled water.
Five, HumanOmniZhongHua-8 (v1.1) chip scanning parting and the analysis of full-length genome associated data
HumanOmniZhongHua-8 (v1.1) chip be purchased from ILLUMINA Inc., parting according to method for product require into
Row.Concrete operations severe steps are carried out by the experimental procedure of Illumina company HumanOmniZhongHua-8 (v1.1) chip,
Associated description can equally be shown in document [Adler, A.J., Wiley, G.B., Gaffney, the P.M.Infinium Assay delivered
for Large-scale SNP Genotyping Applications.J.Vis.Exp.(81),e50683,doi:
10.3791/50683(2013).].Each DNA sample uses 200 nanograms (ng), in strict accordance with Illumina company
HumanOmniZhongHua-8 (v1.1) chip illustrates that experimental procedure is handled.
HumanOmniZhongHua-8 (v1.1) chip typing data by ILLUMINA Inc. BeadStudio software
It manages and exports, associated data analysis is equally carried out [Zuo, X.et al.Whole-exome SNP array by the document delivered
Identifies 15new susceptibility loci for psoriasis.Nat Commun 6,6793,2015].
The typing data of BeadStudio output is analyzed using PLINK, according to the same pairing mechanism of state (pairwise identity-
By-state), repeat samples and relationship sample are excluded.Using smartpca software, according to principal component analysis principle
(principal component analysis) excludes group's heterogeneous samples, shares 1,122PBC patient and 4, and 036 is normal right
According to as a result, for statisticalling analyze.The SNP that parting success rate is lower than 98% is excluded, the frequency (minor in analysis sample is excluded
Allele frequency, MAF) it is lower than 0.01 SNP, exclude the Hardy-Weinberg in normal reference sample
Equilibrium value P < 1 × 10-4SNP.By excluding, 776,516 autosomal SNP are shared for comparing.Using
PLINK software, SNP correlation analysis are imitated using gender as covariate (covariate) using additivity using the principle of linear regression
Principle (additive allelic effect) is answered, each SNP site and the associated conspicuousness of PBC are assessed, calculates dangerous equipotential
The relative risk (Odds ratio, OR) of gene and 95% credibility interval, directly obtain in analysis OR value, credibility interval and
P value, to obtain the dangerous allele in the site.(GWAS) analytical calculation, which is associated with, by full-length genome obtains SNP chip
In each site significance (P value) is associated with PBC, found out between PBC according to significance (P value is less than 0.0001)
There are potential associated chromosomal regions, and obtain the significantly associated mononucleotide polymorphism site of PBC neurological susceptibility and its equipotential
Gene.The reckoning (Imputation) of SNP be using the Hans' result in thousand Human Genome Programs as reference value (Reference),
Using IMPUTE2 software, in conjunction with 1,122PBC patient and 4, the result of 036 normal control is calculated.
Result is analyzed referring to table 2 and Fig. 1, as the result is shown in the gene regions IL16, there are 7 mononucleotide polymorphism sites
The disease of (rs11857713, rs4778636, rs11073001, rs11556218, rs1803275, rs11325, rs4778640)
Sick associated haplotype frequency is significantly higher than control crowd in PBC, and p value is 3.33-9.67 × 10-6.Have 7 SNP with
Rs11857713, rs4778636, rs11073001, rs11556218, rs1803275, rs11325, rs4778640's is chain
Imbalance (r2=0.9-1) corresponding p value is extrapolated, control crowd (p=9.91-3.15 × 10 are significantly higher than in PBC-6).Linkage disequilibrium (Linkage disequilibrium, LD) situation between chromosomal region mononucleotide polymorphism site,
Refer between different mononucleotide polymorphism sites there is Non random association, use r2It indicates (0-1).
A series of this mononucleotide polymorphism site and its allele are: rs17875509, allele G/C；
Rs11857713, allele C/T；Rs4778636, allele G/A；Rs11073001, allele A/G；
Rs11630277, allele C/A；Rs56859031, allele A/AGT；Rs57763246, allele C/T；
Rs17875523, allele C/T；Rs3898677, allele T/C；Rs139879640, allele TCTCA/
T；Rs4577037, allele T/G；Rs3899547, allele A/G；.Rs17875532, allele C/T；
Rs17875533, allele C/A；Rs11556218, allele T/G；Rs1803275, allele G/A；
Rs3926277, allele C/A；Rs3926278, allele C/G；Rs3926279, allele A/G；
Rs17875542, allele G/C；Rs17875543, allele C/T；Rs7166271, allele C/T；
Rs11325, allele G/T；Rs4778640, allele A/G.Wherein dangerous allele is respectively:
Rs17875509 polymorphic site is C；Rs11857713 polymorphic site is T；Rs4778636 polymorphic site is A；
Rs11073001 polymorphic site is G；Rs11630277 polymorphic site is A；Rs56859031 polymorphic site is AGT；
Rs57763246 polymorphic site is T；Rs17875523 polymorphic site is T；Rs3898677 polymorphic site is C；
Rs139879640 polymorphic site is T；Rs4577037 polymorphic site is G；Rs3899547 polymorphic site is G；
Rs17875532 polymorphic site is T；Rs17875533 polymorphic site is A；Rs11556218 polymorphic site is G；
Rs1803275 polymorphic site is A；Rs3926277 polymorphic site is A；Rs3926278 polymorphic site is G；
Rs3926279 polymorphic site is G；Rs17875542 polymorphic site is C；Rs17875543 polymorphic site is T；
Rs7166271 polymorphic site is T；Rs11325 polymorphic site is T；Rs4778640 polymorphic site is G (being shown in Table 2).It takes
The relative risk of individual generation PBC with these dangerous allele is 1.27-1.31 times of not carrier.
IL-16 gene region mononucleotide polymorphism site sequence:
As shown in the SEQ ID NO.1 in sequence table:
rs17875509CCTTACCCATGCAGATATGATGCTG[C/G]TTCAATGCTGGCTTCTGAGAAAGAC
As shown in the SEQ ID NO.2 in sequence table:
rs11857713GTCAAAGCACAAACAGCTGCCAACT[C/T]ATGACCTTTGTCTTAAAAGTTTAAA
As shown in the SEQ ID NO.3 in sequence table:
rs4778636CTCCTCCTCTTGAATCCTTCTTGCT[A/G]TTCAGCTTGGAAACTAGAATTTAGG
As shown in the SEQ ID NO.4 in sequence table:
rs11073001CTGCTGAAACATCTGCCTTGGACAC[A/G]GGGTTCTCGCTCAAGTGAGTTTCTA
As shown in the SEQ ID NO.5 in sequence table:
rs11630277TCTCATCTTTATTTTTAAAAATAAT[C/T]CTATATATAATTTAAAAAATTCCCA
As shown in the SEQ ID NO.6 in sequence table:
rs56859031TTTCCATGTGTGTGCAGATGTCTGA[AGT/A]GTGTGTGTGTGTCTGTCTGTAGGTA
As shown in the SEQ ID NO.7 in sequence table
rs57763246ATAACAAATCAGTCTGATGTCAGTC[C/T]GATGTTAAATTGTTCATCCTCTTGC
As shown in the SEQ ID NO.8 in sequence table
rs17875523TGTCAGTGGTGACTTCCTTGATTTC[C/T]TGATAAGTTTTCTATCACATAAAAA
As shown in the SEQ ID NO.9 in sequence table
rs3898677
TTTTAAGTGTTTTTTATGTGATAGA[A/G]AACTTATCAGGAAATCAAGGAAGTC
As shown in the SEQ ID NO.10 in sequence table
rs139879640TTAAAACCTGTAAGTCTCTATTTC[T/TCTCA]CTGAGTGCAGCTGAGTATTACAA
As shown in the SEQ ID NO.11 in sequence table
rs4577037AAGATTCCTGACCGTGTAGTTTACT[G/T]TCTACTTGAAGAGGAGGAAAGAGAG
As shown in the SEQ ID NO.12 in sequence table
rs3899547GGTAAAGAGTCAGAATTTCTAGGGG[A/G]GGAGCTATAAAAATGTCTAGACTGC
As shown in the SEQ ID NO.13 in sequence table
rs17875532ATGCTGGCCTCTGTGCCAGCAGCTC[C/T]AATCTAGGACACAATTATCTTTAAT
As shown in the SEQ ID NO.14 in sequence table
rs17875533GGACTGGACTTGTGTGATTTCTGGT[A/C]CTGACTTCCTTTGGTTTGCTCAGGT
As shown in the SEQ ID NO.15 in sequence table
rs11556218
TGGTTTGCTCAGGTTCACAGAGTGTTTCCAAA[G/T]GGGCTGGCCTCCCAGGAAGGGACTATTCAGA
AGGGCAATGAGGTTCT
As shown in the SEQ ID NO.16 in sequence table
rs1803275CCAGGCAAGCTGTGATTGTCACAAG[A/G]AAGCTGACTCCAGAGGCCATGCCCG
As shown in the SEQ ID NO.17 in sequence table
rs3926277AAAAAAACATGTTGGACAAAATATC[A/C]AAGTTTAAATCAAGACAGAGTCTGA
As shown in the SEQ ID NO.18 in sequence table
rs3926278CACACTTGGCCTCATTTGCCTTACC[C/G]TAGTCCTGGACACGTCAGCTCCTGC
As shown in the SEQ ID NO.19 in sequence table
rs3926279GGATTGGAGTACAAACCAGTCTGAT[A/G]TGGGGGTCACTTGGATTTCCCTGTG
As shown in the SEQ ID NO.20 in sequence table；
rs17875542CTCCTGCTGCTGACTGGTTTCGTTA[C/G]AGGAAGTTCTGCTGCGGCTGCAGAA
As shown in the SEQ ID NO.21 in sequence table；
rs17875543GGTTTCGTTAGAGGAAGTTCTGCTG[C/T]GGCTGCAGAAACCCAGAAGGTAGAG
As shown in the SEQ ID NO.22 in sequence table
rs7166271ACAAGTCACTTCACCACCATGGGCC[C/T]ATTTGCTTAAATGTTTAGGATGAGA
As shown in the SEQ ID NO.23 in sequence table
rs11325TCCTAAAATAAGGGCAGAGTCACAC[G/T]GGGGCAGCTGATACAAATTGCAGAC
As shown in the SEQ ID NO.24 in sequence table
rs4778640AATGAATGAATTCTAAGTCAATCCA[A/G]GAGTCTGATGATTTCTTGAAAAGGG
Table 2 and the significantly associated IL-16 gene region mononucleotide polymorphism site of primary biliary cholangitis
Wherein " OR (95%CI) " indicates to carry disease associated haplotype compared with carrying normal haploid individual
Individual occur PBC relative risk.Chromosome location is referring to Human Genome GRCh37/hg19..
Embodiment 2rs11556218 confirmatory experiment
By being directed to rs11556218 therein in the verifying of 907 PBC patients and 2027 normal controls, it is again seen that
There were significant differences.PBC patient's verification sample is as the standard in embodiment 1；IL-16 serum analysis normal reference sample comes from
Southeast China University's teacher's physical examination, biochemical indicator is normal, and ultrasound diagnosis is good for without liver and spleen exception, no clinical disease characterization, readme body.Verifying
Material and time in experiment implementation are provided such as without explanation by Sequenom company.
Experimental procedure:
1.rs11556218 sequence
TGGTTTGCTCAGGTTCACAGAGTGTTTCCAAA[G/T]GGGCTGGCCTCCCAG
GAAGGGACTATTCAGAAGGGCAATGAGGTTCT
2. design of primers
It is to be measured using Sequenom company Genotyping Tools and MassARRAY Assay Design software design
The PCR amplification primer and Single base extension primer of SNP site, and Integrated DNATechnologies is transferred to synthesize.
Design is as follows using DNA primer and probe:
rs11556218:
1 sequence of primer, ACGTTGGATGAGAACCTCATTGCCCTTCTG；
Primer 2 sequence, ACGTTGGATGTGGTTTGCTCAGGTTCACAG；
Probe sequence, GGTTCACAGAGTGTTTCCAAA
3.PCR amplification
PCR amplification uses multiple PCR technique, carries out in 384 orifice plates, and each reaction system total volume is 5 μ l.At one
PCR master mix solution is prepared in new 1.5ml EP pipe.
Prepare PCR master mix liquid:
PCR Mix is to each reaction, μ L
10×PCR Buffer 0.5μL
MgCl2(25mM)0.4μL
dNTP mix(25mM)0.1μL
HotStar Taq(5U/μL)0.1μL
Water 1.9μL
PCR primer mix (each 0.5pmol of primer 1 and 2) 1 μ L
Total Volume 4μL
Using 24 channel sample injectors, adjusting injection volume is 4 μ L, and PCR is added in each well of 384 orifice plates
Master mix liquid.384 orifice plate is PCR reaction plate.384 orifice plate of DNA sample prepared is taken out, 24 channels are used
Sample injector, adjusting injection volume are 1 μ L, include template DNA 20-50ng, Hotstar Taq in each 5 μ lPCR reaction system
0.5U, every amplimer 0.5pmol, the 25mM dNTPs of 0.1 μ l.The setting PCR reaction in the PCR instrument of compatible 384 orifice plates
Condition are as follows: 94 DEG C 4 minutes；94 DEG C 20 seconds, 56 DEG C 30 seconds, 72 DEG C 1 minute, 45 circulation；72 DEG C 3 minutes；4 DEG C of holdings.By 384
Hole PCR reaction plate is placed in PCR instrument, starting PCR reaction.
4.PCR product alkaline phosphatase treatment
PCR after reaction, by PCR product SAP (shrimp alkaline phosphatase, shrimp alkaline phosphatase
Enzyme) processing, with remove system middle reaches from dNTPs.
(1) alkaline phosphatase treatment reaction solution, SAP Mix are prepared.
SAP Mix is to each reaction, μ L
H2O1.53
SAP Buffer(10x)0.17
SAP Enzyme(1.7U/μL)0.3
Total Volume 2
(2) 24 channel sample injectors are used, adjusting injection volume is 2 μ L, and 384 hole PCR reaction plates are added in SAP Mix.It is right
In each alkaline phosphatase treatment reacting hole, reaction system total volume is 7 μ l, wherein 5 μ l, SAP Mix of PCR product, 2 μ l.
(3) 384 orifice plates are placed in the PCR instrument of compatible 384 orifice plates, set PCR reaction condition: 37 DEG C 40 minutes；85
DEG C 5 minutes；4 DEG C of maintenances, starting PCR instrument carry out alkaline phosphatase treatment.
5. Single base extension
(1) after alkaline phosphatase treatment, single base extension, 9 μ l of reaction system total volume are carried out.
(2) single base extension liquid, EXTEND Mix are prepared.
(3) 24 channel sample injectors are used, adjusting injection volume is 2 μ L, and corresponding 384 holes that are added EXTEND Mix are reacted
Plate.For each reacting hole, single base extension system includes 7 μ l and EXTEND Mix liquid of PCR product, 2 μ l after SAP processing
(wherein each 0.94 μ l, iPLEX enzyme of extension primer mixture, 0.041 μ l extends 0.2 μ l of mixture).
(4) 384 orifice plates are placed in the PCR instrument of compatible 384 orifice plates, set PCR reaction condition:
I.94℃for 30seconds；
II.94℃for 5seconds；
III.52℃for 5seconds；
IV.80℃for 5seconds；
V.GOTO III, 4more times；
VI.GOTO II, 39more times；
VII.72℃for 3minutes；
VIII.4℃forever；
Start PCR instrument and carries out single base extension.
6. purifying resin
(1) by the tiling of Clean Resin resin into the resin plate of 6mg；
(2) plus in 16 μ l water to the corresponding aperture of extension products；
(3) resin after drying is poured into extension products plate, sealer, slow speed vertical rotates 30 minutes, makes resin and anti-
Object is answered to come into full contact with；
(4) centrifugation makes resin sink to hole bottom.
7. chip point sample
Start MassARRAY Nanodispenser RS1000 point sample instrument, the extension products after purifying resin are moved to
On 384 hole SpectroCHIP chips.
8. Mass Spectrometer Method
SpectroCHIP chip after point sample is used into MALDI-TOF (matrix-assisted laser
Desorption/ionization-time of fligh, matrix solid-dispersion flight time mass spectrum) analysis,
Testing result is using 4.0 software of TYPER (sequenom) parting and exports result.Totally 907 PBC samples and 2127 controls
Sample has obtained effective result.
Using PLINK software, logistic regression principle is carried out, using gender as covariate, by synergistic effect model to 907
The result of PBC sample and 2127 control samples is statisticallyd analyze.Disease associated haplotype (G) frequency of rs11556218
(0.2326) is significantly higher than control crowd (0.1885) in PBC, and p value is 7.0 × 10-4, by rs11556218 1,122PBC
Patient and 4, the GWAS of 036 normal control as a result, and rs11556218 907 PBC samples and 2127 control samples knot
Fruit is overlapped analysis, and using the principle of fixed-effect model (I2 < 25%), statistical analysis includes 2029 PBC altogether
Sample and 6163 control samples.The result shows that disease associated haplotype (G) frequency of rs11556218 is significantly higher than in PBC
Control crowd, p value are 8.99 × 10-9, odds ratio value (96%CI) is 1.29 (1.18-1.41) (being shown in Table 3).
Result of the table 3:IL-16 polymorphic site rs11556218 in whole-genome association and verifying discovery
- 16 quantitative analysis of serum IL of 3 primary biliary cholangitis patient of embodiment and normal control population
It is to be purchased from R&D Systems company, Human IL- using mature kit that IL-16, which quantifies enzyme linked immunosorbent detection,
16Quantikine ELISA Kit, production number D1600.The step of according to kit, using 2 microlitres of serum.
Standard curve is prepared according to the standard items that kit provides, it is quantitative to test sample.
1. 100 microlitres of dilutions (providing in kit) are added in elisa plate detection hole；
2. 100 microlitres of standard samples, negative control (providing in kit) and 1:5 are added in elisa plate detection hole
The serum of diluted test sample (using the diluted provided in kit)；
3. sealing plate, room temperature is kept for 2 hours；
4. sucking liquid is washed 4 times with 200 microlitres of washing lotions (providing in kit), clear dry；
5. 100 microlitres of conjugates (providing in kit) are added, sealing plate, room temperature is kept for 2 hours；
6. sucking liquid is washed 4 times with 200 microlitres of washing lotions (providing in kit), clear dry；
7. 200 microlitres of substrate solutions (providing in kit) are added, room temperature is protected from light holding 30 minutes；
8. 50 microlitres of terminate liquids (providing in kit) are added, in 30 minutes, using ELISA plate reading machine
(iMarkMicroplate Absorbance Reader, Bio-Rad company), wavelength are set as 450nm, read result.
The experiment has carried out quantitative analysis to 120 PBC patients and 120 normal control serum, using GraphPad
The mapping of Prism6 software is simultaneously for statistical analysis, and all data are indicated with mean ± standard deviation, to patient's group and two groups of control group
Data are examined using Mann-Whiteny U, use Kruskal- to IL-16 expression in rs11556218 different genotype
Wallis is examined, and is compared two-by-two, and two-tailed test P thinks with statistical significance value < 0.05.Statistics discovery IL-16 serum is dense
Degree compares normal control in patient PBC and significantly increases (P < 0.0001).
It above are only the preferred embodiment of the invention, for those of ordinary skill in the art, do not departing from this hair
It under the premise of bright principle, can also make other variations or changes in different ways, these also should belong to protection model of the invention
It encloses.
SEQUENCE LISTING
<110>Southeast China University
<120>with the associated interleukins 16 of primary biliary cholangitis and its application
<130> SG20161114001
<160> 27
<170> PatentIn version 3.3
<210> 1
<211> 51
<212> DNA
<213> rs17875509
<221> misc_feature
<223> n is c or g
<400> 1
ccttacccat gcagatatga tgctgnttca atgctggctt ctgagaaaga c 51
<210> 2
<211> 51
<212> DNA
<213> rs11857713
<220>
<221> misc_feature
<223> n is c or t
<400> 2
gtcaaagcac aaacagctgc caactnatga cctttgtctt aaaagtttaa a 51
<210> 3
<211> 51
<212> DNA
<213> rs4778636
<221> misc_feature
<223> n is a or g
<400> 3
ctcctcctct tgaatccttc ttgctnttca gcttggaaac tagaatttag g 51
<210> 4
<211> 51
<212> DNA
<213> rs11073001
<221> misc_feature
<223> n is a or g
<400> 4
ctgctgaaac atctgccttg gacacngggt tctcgctcaa gtgagtttct a 51
<210> 5
<211> 51
<212> DNA
<213> rs11630277
<221> misc_feature
<223> n is c or t
<400> 5
tctcatcttt atttttaaaa ataatnctat atataattta aaaaattccc a 51
<210> 6
<211> 51
<212> DNA
<213> rs56859031
<221> misc_feature
<223> n is g or t
<400> 6
tttccatgtg tgtgcagatg tctgangtgt gtgtgtgtct gtctgtaggt a 51
<210> 7
<211> 51
<212> DNA
<213> rs57763246
<221> misc_feature
<223> n is c or t
<400> 7
ataacaaatc agtctgatgt cagtcngatg ttaaattgtt catcctcttg c 51
<210> 8
<211> 51
<212> DNA
<213> rs17875523
<221> misc_feature
<223> n is c or t
<400> 8
tgtcagtggt gacttccttg atttcntgat aagttttcta tcacataaaa a 51
<210> 9
<211> 51
<212> DNA
<213> rs3898677
<221> misc_feature
<223> n is a or g
<400> 9
ttttaagtgt tttttatgtg ataganaact tatcaggaaa tcaaggaagt c 51
<210> 10
<211> 48
<212> DNA
<213> rs139879640
<221> misc_feature
<223> n is t or tctca
<400> 10
taaaacctg taagtctcta tttcnctgag tgcagctgag tattacaaaa 50
<210> 11
<211> 51
<212> DNA
<213> rs4577037
<221> misc_feature
<223> n is g, or t
<400> 11
aagattcctg accgtgtagt ttactntcta cttgaagagg aggaaagaga g 51
<210> 12
<211> 51
<212> DNA
<213> rs3899547
<221> misc_feature
<223> n is a or g
<400> 12
ggtaaagagt cagaatttct aggggnggag ctataaaaat gtctagactg c 51
<210> 13
<211> 51
<212> DNA
<213> rs17875532
<221> misc_feature
<223> n is c or t
<400> 13
atgctggcct ctgtgccagc agctcnaatc taggacacaa ttatctttaa t 51
<210> 14
<211> 51
<212> DNA
<213> rs17875533
<221> misc_feature
<223> n is a or c
<400> 14
ggactggact tgtgtgattt ctggtnctga cttcctttgg tttgctcagg t 51
<210> 15
<211> 51
<212> DNA
<213> rs11556218
<221> misc_feature
<223> n is g or t
<400> 15
ctcaggttca cagagtgttt ccaaangggc tggcctccca ggaagggact a 51
<210> 16
<211> 51
<212> DNA
<213> rs1803275
<221> misc_feature
<222> (26)..(26)
<223> n is a or g
<400> 16
ccaggcaagc tgtgattgtc acaagnaagc tgactccaga ggccatgccc g 51
<210> 17
<211> 51
<212> DNA
<213> rs3926277
<221> misc_feature
<223> n is a or c
<400> 17
aaaaaaacat gttggacaaa atatcnaagt ttaaatcaag acagagtctg a 51
<210> 18
<211> 51
<212> DNA
<213> rs3926278
<221> misc_feature
<223> n is c or g
<400> 18
cacacttggc ctcatttgcc ttaccntagt cctggacacg tcagctcctg c 51
<210> 19
<211> 51
<212> DNA
<213> rs3926279
<221> misc_feature
<223> n is a or g
<400> 19
ggattggagt acaaaccagt ctgatntggg ggtcacttgg atttccctgt g 51
<210> 20
<211> 51
<212> DNA
<213> rs17875542
<221> misc_feature
<223> n is c or g
<400> 20
ctcctgctgc tgactggttt cgttanagga agttctgctg cggctgcaga a 51
<210> 21
<211> 51
<212> DNA
<213> rs17875543
<221> misc_feature
<223> n is c or t
<400> 21
ggtttcgtta gaggaagttc tgctgnggct gcagaaaccc agaaggtaga g 51
<210> 22
<211> 51
<212> DNA
<213> rs7166271
<221> misc_feature
<223> n is c or t
<400> 22
acaagtcact tcaccaccat gggccnattt gcttaaatgt ttaggatgag a 51
<210> 23
<211> 51
<212> DNA
<213> rs11325
<221> misc_feature
<223> n is g or t
<400> 23
tcctaaaata agggcagagt cacacngggg cagctgatac aaattgcaga c 51
<210> 24
<211> 51
<212> DNA
<213> rs4778640
<221> misc_feature
<223> n is a or g
<400> 24
aatgaatgaa ttctaagtca atccangagt ctgatgattt cttgaaaagg g 51
<210> 25
<211> 30
<212> DNA
<213>1 sequence of primer
<400> 25
acgttggatg agaacctcat tgcccttctg 30
<210> 26
<211> 30
<212> DNA
<213>primer 2 sequence
<400> 26
acgttggatg tggtttgctc aggttcacag 30
<210> 27
<211> 21
<212> DNA
<213>probe sequence
<400> 27
ggttcacaga gtgtttccaa a 21
Claims (1)
1. the reagent for detecting IL-16 gene polymorphic site rs11556218 is former in preparation detection or auxiliary detection, screening or prediction
Application in the product of the biliar cholangitis of hair property, rs11556218 sequence as shown in the SEQ ID NO.15 in sequence table,
In, mononucleotide n is that the risk of carrier's primary biliary cholangitis of G is higher than allele T.
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| CN201710015508.2A CN106834448B (en) | 2017-01-10 | 2017-01-10 | With the associated interleukins 16 of primary biliary cholangitis and its application |
| PCT/CN2017/092502 WO2018129886A1 (en) | 2017-01-10 | 2017-07-11 | Primary biliary cholangitis-associated interleukin 16 and application thereof |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201710015508.2A CN106834448B (en) | 2017-01-10 | 2017-01-10 | With the associated interleukins 16 of primary biliary cholangitis and its application |
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| WO2010102387A1 (en) * | 2009-03-09 | 2010-09-16 | University Health Network (Uhn) | Interleukin-12 polymorphisms for identifying risk for primary biliary cirrhosis |
| JP6245796B2 (en) * | 2012-09-03 | 2017-12-13 | 公益財団法人ヒューマンサイエンス振興財団 | Markers, probes, primers and kits for predicting the risk of developing primary biliary cirrhosis and methods for predicting the risk of developing primary biliary cirrhosis |
| CN103602671A (en) * | 2013-10-16 | 2014-02-26 | 中国人民解放军第四军医大学 | SNP in KRT8 gene exon area and determination method thereof |
| CN106834448B (en) * | 2017-01-10 | 2019-05-31 | 东南大学 | With the associated interleukins 16 of primary biliary cholangitis and its application |
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| CN106834448A (en) | 2017-06-13 |
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