CN104131102B - A kind of judge that NSCLC patient is to the test kit of Gefitinib therapeutic response - Google Patents

A kind of judge that NSCLC patient is to the test kit of Gefitinib therapeutic response Download PDF

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CN104131102B
CN104131102B CN201410387024.7A CN201410387024A CN104131102B CN 104131102 B CN104131102 B CN 104131102B CN 201410387024 A CN201410387024 A CN 201410387024A CN 104131102 B CN104131102 B CN 104131102B
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gefitinib
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马飞
宁力
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Abstract

The present invention discloses a kind of for judging that patients with advanced NSCLC is to the test kit of Gefitinib therapeutic response, and this test kit comprises the reagent that can be used in detecting the genotype in rs9912773 and/or rs12949918 site in STAT3 gene. Contriver is by carrying out gene type to patient, analysis STAT3 gene SNP site and Gefitinib are to the reactive dependency of Advanced Non-Small Cell lung cancer therapy, find the genotype according to rs9912773 and/or rs12949918 site, it can be determined that patients with advanced NSCLC is to the therapeutic response of Gefitinib. The determination being developed as patients with advanced NSCLC treatment plan of the test kit of the present invention provides a kind of reliable, sensitive mode.

Description

A kind of judge that NSCLC patient is to the test kit of Gefitinib therapeutic response
Technical field
The invention belongs to biomedical sector, it relates to a kind of judge Advanced Non-Small Cell lung cancer (NSCLC) patient to the test kit of Gefitinib therapeutic response and SNP in the application prepared in this test kit.
Background technology
Lung cancer is one of the most common, mortality ratio is the highest malignant tumour, and its incidence and mortality is also increasing year by year. Wherein nonsmall-cell lung cancer (NSCLC) accounts for more than the 80% of whole lung cancer, and major part patient has been late period when diagnosing. Chemotherapy was once the topmost treatment means of Advanced Non-Small Cell lung cancer, even if but best chemotherapy regimen, at present treatment always has efficiency also less than 50%, 1 year survival rate only about 40%.
The appearance of target therapeutic agent changes the current treatment status of nonsmall-cell lung cancer, considerably improves the lifetime of patient. Wherein Gefitinib (Gefitinib, ZD1839) it is listing the earliest, at present clinical application nonsmall-cell lung cancer target therapeutic agent the most widely, belong to a kind of selectivity, reversibility EGFR (EGF-R ELISA) tyrosine kinase inhibitor, listing in the many countries comprising China at present, commodity are called Iressa (Iressa).
Gefitinib treatment the objective of nonsmall-cell lung cancer has efficiency widely different, and document report is from 8.9-69% not etc. Wherein in IDEAL1 clinical study, the efficiency that has of Japanese is 27%, and American is then only 12%. And in EAP studies, the efficiency that has of Chinese is about 30%. Below the clinical efficacy of Gefitinib treatment nonsmall-cell lung cancer is all pointed out to there is significant individual difference. Owing to patients with advanced NSCLC is very limited for lifetime, therefore select as early as possible may effective pharmacological agent survive particularly important to prolongation patient. In addition Gefitinib be representative target therapeutic agent costly, price is tens of times of common chemotherapy, also requires to select the patient that may benefit to accept targeted therapy from the angle of health economics. Therefore, find predicating curative effect of gefitinib prediction index, instruct individuation targeted therapy, be key clinical problem urgently to be resolved hurrily at present.
The biomarker being most commonly used to prediction predicating curative effect of gefitinib at present is the sudden change of EGFR gene, is mainly the base deletion (G2235-A2249 disappearance) of exons 19 and the point mutation (L858R) of exon 21.A biomarker retrospective analysis for the international multicenter III phase clinical study of ISEL finds, exist EGFR genetic mutation 16 example NSCLC patients accept Gefitinib treat after the efficiency that has be 37.5%, and the 116 of EGFR gene wild-type example patient's Gefitinib the efficiency that has be only 2.6%. But the biomarker that EGFR genetic mutation is predicted as predicating curative effect of gefitinib, there is certain deficiency: the detection 1. suddenlyd change needs tumor tissues, and major part lung cancer has been late period when initial diagnosis, lose the chance that operation obtains tumor specimen, even if small part patient is biopsy acquirement sample likely, also can limit with biopsy specimen amount because of recall rate is limited, be difficult to carry out abrupt climatic change, current clinical on can meet abrupt climatic change requirement patient's ratio less than 20% (ISEL is about 17% in studying); 2. transgenation is by the impact of Tumor Heterogeneity, and same patient is at the tumor specimen of different sites or the acquisition of disease different steps, and its abrupt climatic change the possibility of result exists difference, and the possibility of result therefore detected can not represent whole body and omnidistance tumour feature; 3. the flow process of abrupt climatic change is complicated, the requirement height of plant and instrument, and common laboratory is difficult to carry out, and testing cost is also high simultaneously, have impact on its clinical application widely.
Based on the weak point of above EGFR genetic mutation, the clinical demand developing more preferably outcome prediction biomarker and detection method thereof seems more urgent.
Summary of the invention
Contriver has found that rs9912773 and rs12949918 site and patients with advanced NSCLC are to the dependency of Gefitinib therapeutic response, and develop accordingly and can judge that patients with advanced NSCLC is to the test kit of Gefitinib therapeutic response, and patients with advanced NSCLC is being prepared to the application in the judgement test kit of Gefitinib therapeutic response in rs9912773 and/or rs12949918 site.
On the one hand, the present invention provides for judging that patients with advanced NSCLC is to the two of Gefitinib therapeutic response SNP site, described SNP site is rs9912773 and rs12949918 site, when rs9912773 loci gene type is GG or GC, patients with advanced NSCLC is poor to Gefitinib therapeutic response, and patient's curative ratio is low; When rs9912773 loci gene type is CC, patients with advanced NSCLC is good to Gefitinib therapeutic response, patient's curative ratio height. When rs12949918 loci gene type is CC or CT, patients with advanced NSCLC is poor to Gefitinib therapeutic response, and patient's curative ratio is low; When rs12949918 loci gene type is TT, patients with advanced NSCLC is good to Gefitinib therapeutic response, patient's curative ratio height.
On the other hand, the present invention provides the reagent for detecting SNP site genotype, described reagent can for adopting the reagent needed for any technology for detection SNP known in the art, as long as it can detect the genotype in rs9912773 and/or rs12949918 site in sample.
It is known to those with ordinary skill in the art that, it is possible to the pleomorphism site of multiple technologies vitro detection CETP gene order on DNA level. Can through with after the sense-rna of radio-labeling or DNA probe and amplification DNA sequence dna hybridize, to differentiate loci polymorphism. Can also based on the change of known nucleotide sequence, the normal PCR primer with having specific polymorphism of synthesis, the substrate of polymerase chain reaction (PCR reaction) adds the Nucleotide of fluorescent mark, occur according to reaction product has unstressed configuration, determine with or without base change in the primer that amplification is used, thus detect specific polymorphism.Directly checked order by DNA and can directly disclose crt gene and carry the sequence difference having between specific Genetic polymorphism. When being combined with PCR, the susceptibility of this kind of method improves greatly. The mensuration of the nucleotide sequence of various DNA and DNA fragmentation also can by ordinary method such as dideoxy chain termination. In addition, nucleotide sequencing also can with business sequencing kit or automatic sequencer etc. Conventional automatic sequencing method radio-labeling or fluorescent mark determine nucleotide sequence. Pvuii restriction fragment analysis (RFLP) can also be adopted to carry out the pleomorphism site of vitro detection CETP gene order.
Reagent for detecting SNP site genotype provided by the invention, comprises the primer of the nucleotide sequence for the SNP site that increases and/or MassARRAYSequenom can be used to detect the single-basic extension primer of SNP site genotype. For rs9912773, as follows at the primer sequence of interior nucleotide sequence for rs9912773 site of increasing: forward primer 5 '-ACGTTGGATGCAGTTGATTGTTAAAGCTGCC-3 '; Reverse primer 5 '-ACGTTGGATGGGTCTGTTTTGTGGGTTTTC-3 '; Described single-basic extension primer sequence is: 5 '-tttgTTGTAGAGCTCAGACCT-3 '. For rs12949918, as follows at the primer sequence of interior nucleotide sequence for rs12949918 site of increasing: forward primer 5 '-ACGTTGGATGATCATGGGCTATCCCCAACA-3 '; Reverse primer 5 '-ACGTTGGATGGAGAATACACGCTTAGAGAG-3 '; Described single-basic extension primer sequence is: 5 '-AGAGAGAATCTAGAAAGAAACA-3 '.
Again on the one hand, the present invention provides the test kit for detecting SNP site genotype, this test kit comprises the reagent adopted needed for any technology for detection SNP known in the art, as long as it can detect the genotype in rs9912773 and/or rs12949918 site in sample.
Test kit for detecting SNP site genotype provided by the invention comprises the primer of the nucleotide sequence for the SNP site that increases and/or MassARRAYSequenom can be used to detect the single-basic extension primer of SNP site genotype. For rs9912773, as follows at the primer sequence of interior nucleotide sequence for rs9912773 site of increasing: forward primer 5 '-ACGTTGGATGCAGTTGATTGTTAAAGCTGCC-3 '; Reverse primer 5 '-ACGTTGGATGGGTCTGTTTTGTGGGTTTTC-3 '; Described single-basic extension primer sequence is: 5 '-tttgTTGTAGAGCTCAGACCT-3 '. For rs12949918, as follows at the primer sequence of interior nucleotide sequence for rs12949918 site of increasing: forward primer 5 '-ACGTTGGATGATCATGGGCTATCCCCAACA-3 '; Reverse primer 5 '-ACGTTGGATGGAGAATACACGCTTAGAGAG-3 '; Described single-basic extension primer sequence is: 5 '-AGAGAGAATCTAGAAAGAAACA-3 '.
The present invention provides for judging that patients with advanced NSCLC is to the test kit of Gefitinib therapeutic response, the judgement principle of this test kit is the genotype in reagent detection rs9912773 and/or the rs12949918 site utilized in test kit, judges that patients with advanced NSCLC is to Gefitinib therapeutic response according to the genotype of the above-mentioned SNP site of surveyed patient. When rs9912773 loci gene type is GG or GC, patients with advanced NSCLC is poor to Gefitinib therapeutic response, and patient's curative ratio is low;When rs9912773 loci gene type is CC, patients with advanced NSCLC is good to Gefitinib therapeutic response, patient's curative ratio height. When rs12949918 loci gene type is CC or CT, patients with advanced NSCLC is poor to Gefitinib therapeutic response, and patient's curative ratio is low; When rs12949918 loci gene type is TT, patients with advanced NSCLC is good to Gefitinib therapeutic response, patient's curative ratio height.
This test kit comprises the reagent adopted needed for any technology for detection SNP genotype known in the art, as long as it can detect the genotype in rs9912773 and/or rs12949918 site in sample. Provided by the invention for judging that the test kit of Gefitinib therapeutic response is comprised the primer of the nucleotide sequence for the SNP site that increases and/or MassARRAYSequenom can be used to detect the single-basic extension primer of SNP site genotype by patients with advanced NSCLC. For rs9912773, as follows at the primer sequence of interior nucleotide sequence for rs9912773 site of increasing: forward primer 5 '-ACGTTGGATGCAGTTGATTGTTAAAGCTGCC-3 '; Reverse primer 5 '-ACGTTGGATGGGTCTGTTTTGTGGGTTTTC-3 '; Described single-basic extension primer sequence is: 5 '-tttgTTGTAGAGCTCAGACCT-3 '. For rs12949918, as follows at the primer sequence of interior nucleotide sequence for rs12949918 site of increasing: forward primer 5 '-ACGTTGGATGATCATGGGCTATCCCCAACA-3 '; Reverse primer 5 '-ACGTTGGATGGAGAATACACGCTTAGAGAG-3 '; Described single-basic extension primer sequence is: 5 '-AGAGAGAATCTAGAAAGAAACA-3 '.
Present invention also offers the application of rs9912773 and/or rs12949918 site in the reagent of preparation detection SNP site genotype. Described reagent can for adopting the reagent needed for any technology for detection SNP genotype known in the art, as long as it can detect the genotype in rs9912773 and/or rs12949918 site in sample.
Present invention also offers the application of rs9912773 and/or rs12949918 site in the test kit of preparation detection SNP site genotype. Described test kit comprises the reagent that can detect SNP site genotype. In a specific embodiment, described reagent comprises the primer of the nucleotide sequence for the SNP site that increases and/or MassARRAYSequenom can be used to detect the single-basic extension primer of SNP site genotype. For rs9912773, as follows at the primer sequence of interior nucleotide sequence for rs9912773 site of increasing: forward primer 5 '-ACGTTGGATGCAGTTGATTGTTAAAGCTGCC-3 '; Reverse primer 5 '-ACGTTGGATGGGTCTGTTTTGTGGGTTTTC-3 '; Described single-basic extension primer sequence is: 5 '-tttgTTGTAGAGCTCAGACCT-3 '. For rs12949918, as follows at the primer sequence of interior nucleotide sequence for rs12949918 site of increasing: forward primer 5 '-ACGTTGGATGATCATGGGCTATCCCCAACA-3 '; Reverse primer 5 '-ACGTTGGATGGAGAATACACGCTTAGAGAG-3 '; Described single-basic extension primer sequence is: 5 '-AGAGAGAATCTAGAAAGAAACA-3 '.
Present invention also offers rs9912773 and/or rs12949918 site for the preparation of judging that patients with advanced NSCLC is to the application in the test kit of Gefitinib therapeutic response.The judgement principle of described test kit is the genotype in reagent detection rs9912773 and/or the rs12949918 site utilized in test kit, judges that patients with advanced NSCLC is to Gefitinib therapeutic response according to the genotype of the above-mentioned SNP site of surveyed patient. When rs9912773 loci gene type is GG or GC, patients with advanced NSCLC is poor to Gefitinib therapeutic response, and patient's curative ratio is low; When rs9912773 loci gene type is CC, patients with advanced NSCLC is good to Gefitinib therapeutic response, patient's curative ratio height. When rs12949918 loci gene type is CC or CT, patients with advanced NSCLC is poor to Gefitinib therapeutic response, and patient's curative ratio is low; When rs12949918 loci gene type is TT, patients with advanced NSCLC is good to Gefitinib therapeutic response, patient's curative ratio height.
Present invention also offers the application of the reagent for detecting rs9912773 and/or rs12949918 loci gene type in the test kit for the preparation of detection rs9912773 and/or rs12949918 loci gene type.
Present invention also offers the reagent for detecting rs9912773 and/or rs12949918 loci gene type for the preparation of judging that patients with advanced NSCLC is to the application in the test kit of Gefitinib therapeutic response.
Advantage of the present invention and useful effect are as follows: contriver studies discovery, and STAT3 Polymorphism (rs9912773 and/or rs12949918) is the Biological indicators of a kind of measurable Gefitinib treatment nonsmall-cell lung cancer curative effect. Detect as predicating curative effect of gefitinib prediction index using STAT3rs9912773 and/or rs12949918 gene polymorphic, the part shortcoming of existing prediction index (EGFR genetic mutation) can be overcome to a certain extent: 1. the sample of rs9912773 and/or rs12949918 detection can be derived from the karyocyte of any normal or tumor tissues, the easiest method is 2ml periphery blood, and therefore nearly all patient all can obtain detection sample; 2. rs9912773 and/or rs12949918 belongs to heritable variation, and detected result, not by the impact of Tumor Heterogeneity, whenever patient obtains sample in throughout one's life, or takes from any position sample, and its detected result should be consistent; 3. rs9912773 and/or rs12949918 testing process relative ease, equipment requirements are low, with low cost, are conducive to clinical expansion.
Concrete enforcement mode
Below by specific embodiment, the present invention is described further, it should be noted that as without particularly pointing out, embodiments of the invention are only for explaining the present invention, and do not mean that restriction protection scope of the present invention.
1. study object: Advanced Non-Small Cell lung cancer, 86 examples. Selected patient (>=18 years old) is Advanced Non-Small Cell lung cancer through histology or cytology confirmation, and all patients all provide Informed Consent Form.
2. administrated method: Gefitinib 250mg, once a day, oral. The medication cycle is 1 year.
The selection of 3.STAT3 gene SNP site: retrieval STAT3 gene in the SNP public database (http://hapmap:ncbi.nlm.nih.gov/) that international human genome Haplotype map plan provides, first the SNP site of information in known Han nationality crowd is chosen, again using Tag SNP wherein as candidate's polymorphic site of further somatotype, then screen to draw best Tag SNP to candidate's Tag SNP in conjunction with statistical natures such as minimum gene frequency (MAF) and Ha Di-Weinberg equilibrium P value (HWPval).Choose requirement: MAF > 0.1 and HWPval > 0.1. Having carried out the comprehensive SNP site based on Tag SNP is main body for STAT3 gene to select, and replaced by the part function SNP of associated, the candidate SNP locus of STAT3 gene is rs9912773 and rs12949918.
4. experimental technique
4.1 extracting genome DNA
Gather case research object 2ml anticoagulation cirumferential blood sample, adopt phenol-chloroform method to extract genomic dna. DNA sample is separated from peripheral blood leucocyte, sketches as follows:
(1) from 80 DEG C of taking-ups, the anticoagulation sample of collection being placed on room temperature, blood proceeds to another clean centrifuge tube after dissolving, Gai Yanhou is in the centrifugal 15min of 5,000 × g;
(2) the blood upper strata after centrifugal is abandoned, stay hemocyte, add appropriate lysis buffer (containing 10mMpH be 8.0 Tris-HCl, 0.1MEDTA, 20 μ g/ml pancreas RNase, 0.5%SDS), mixed even, in 37 DEG C of temperature bath 1h, add the mixed even rear 37 DEG C of temperature baths of Proteinase K (100 μ g/ml) and spend the night;
(3) temperature bath adds equal-volume after the balance phenol (pH7.4) of the saturated mistake of Tris-HCl is fully mixed even in the centrifugal 15min of 8,000 × g after terminating;
(4) upper water phase shift to another centrifuge tube and is added equal-volume phenol: chloroform (1:1), the centrifugal 15min of 8,000 × g after fully mixed even;
(5) upper water is moved in another centrifuge tube mutually, add the ammonium acetate solution (10M) of 10% volume, after mixed even, add the dehydrated alcohol of 2 times of volumes, after mixed even, put into 20 DEG C. The DNA being settled out, after 75% washing with alcohol twice, is dissolved in appropriate TE damping fluid. Use spectrophotometric determination DNA concentration.
The DNA extracted measures its concentration by spectrophotometry, and Sequenom technical requirements DNA reaches following standard: concentration is at 10ng/ more than μ l; OD260/280=1.8～2.0. DNA sample is placed in 1.5mlEP pipe and is stored in-80 DEG C of Ultralow Temperature Freezers. When carrying out the detection of SNP somatotype, sample is sub-packed in 96 orifice plates after being encoded.
The genetic analysis that 4.2 candidates are polymorphic
4.2.1SNP classifying method
According to gene order relevant in NCBIGenBank database, design PCR primer and mononucleotide expansion primer, adopt high-throughout MassARRAY time-of-fight mass spectrometry biochip system (Sequenom) to analyze the genotype of candidate SNP. By winning, vast biotechnology (Beijing) company limited has assisted SNP somatotype. When the principle of work of MassARRAY system carries out extension after utilizing hybridization probe to combine from PCR primer, isoallele extension length is not different, differentiating that probe extends length and determines different genotype by mass spectrum flight time difference, its gene type rate of accuracy reached is to more than 97%. MassARRAY system is the equipment of unique employing mass spectroscopy direct-detection SNP at present, and its reaction system does not rely on hybridization, therefore by the interference of hybridization mispairing. This system is different from TaqMan somatotype platform, does not also need primer or probe are carried out fluorescent mark, avoids fluorescent substance to the interference of gene type process.
In order to quality control, all establishing parallel repeat samples and negative control (not containing the blank of DNA) during genotype tests, the repetition rate of parallel sample analytical results is 100%. In addition, somatotype operation and analysis personnel are kept blind state by the morbid state of each sample.
4.2.2Sequenom laboratory operating procedures
4.2.2.1 design of primers
According to SNP site sequence information, it may also be useful to the primer-design software Assaydesign3.1 of sequenom company, design and synthesize PCR reaction and single-basic extension primer (table 1).
The each SNP site PCR of table 1 reacts primer and extends primer
4.2.2.2PCR reaction
Reaction system (reagent loss of 384 hole PCR plate+38%)
Table 2 reaction system
Reagent Concentration Volume (μ l)
Water, HPLC level NA 927.5
Containing 15mM MgCl2PCR damping fluid 10x 331.25
MgCl2 25mM 172.25
DNTP mixture 25mM 53
Primer mixture 0.5μM 530
HotStar Taq 5U/μl 106
DNA profiling 10ng/μl 1/ hole
Amount to 5/ hole
Reaction conditions
Table 3 reaction conditions
4.2.2.3SAP digestion
Reaction system (reagent loss of 384 hole PCR plate+38%)
Table 4SAP digestion reaction system
Reagent Concentration Volume (μ l)
Water NA 810.9
SAP damping fluid 10x 90.1
SAP 1.7U/μl 159.0
Amount to 2/ hole
Reaction conditions
Table 5SAP digestion reaction condition
Temperature (DEG C) Time (min) Cycle number
37 40 1
85 5 1
4 ∞ 1
4.2.2.4 extension
Reaction system (reagent loss of 384 hole PCR plate+38%)
Table 6 reaction system
Reagent Concentration Volume (μ l)
Water NA 400.2
iPLEX buffer plus 10x 106
iPLEX terminator NA 106
Primer mixture 0.6-1.3μM 426.1
IPlex enzyme NA 21.7
Amount to 2/ hole
Reaction conditions
Table 7 reaction conditions
4.2.2.5 machine testing is gone up
Reaction product (totally 9 μ l) is diluted 3 times, it may also be useful to resin carries out desalination; By the sample spot after desalting treatment on sample target, spontaneous nucleation; Upper machine carries out mass spectrometric detection, and collects data.
5. statistical study
Statistical analysis SPSS16.0 software carries out. With χ2The difference of each allelotrope and genotype distribution between case group and control group is compared in inspection; The odds ratio (oddsratio, OR) and 95% credibility interval (confidenceinterval, CI) thereof that calculate with unconditional logistic regression model represent genotype and tumorigenic relative risk; All OR values all through Confounding Factor as age, smoking state etc. correction; All statistical tests are two sides probability inspection, using P < 0.05 as having statistical significant difference standard.
6. experimental result
6.1 gene type results
The genotype that Gefitinib effectively organizes each SNP site of invalid group of patient with Gefitinib distributes in table 8. STAT3 gene rs9912773 site detects CC, GC and GG tri-kinds of genotype, CC genotype is effectively organized the invalid group of proportion with Gefitinib in Gefitinib and is respectively 80.6% and 19.4%, the proportion of GG genotype between two groups is respectively 57.1% and 42.9%, GC genotype proportion between two groups is respectively 46.5% and 53.5%, and three groups of genotype proportions between two groups have remarkable significant difference (P=0.008). GG+GC genotype is effectively organized the invalid group of proportion with Gefitinib in Gefitinib and is respectively 48% and 52%, and the difference in distribution of CC between two groups has significance (P=0.002). STAT3 gene rs12949918 site detects TT, CC and CT tri-kinds of genotype, TT genotype is effectively organized the invalid group of proportion with Gefitinib in Gefitinib and is respectively 81.8% and 18.2%, the proportion of CC genotype between two groups is respectively 57.1% and 42.9%, CT genotype proportion between two groups is respectively 20% and 80%, and three groups of genotype proportions between two groups have remarkable significant difference (P=0.012). CC+CT genotype is effectively organized the invalid group of proportion with Gefitinib in Gefitinib and is respectively 41.7% and 58.3%, and the difference in distribution of TT between two groups has significance (P=0.009).
Each gene SNP site genotype distribution between table 8 liang group
Ji is not replaced the analysis of Buddhist nun's therapeutic response susceptibility by 6.2SNP site and Advanced Non-Small Cell lung cancer
Non-corditional regression analysis shows, and rs9912773, rs12949918 site and Advanced Non-Small Cell lung cancer are not closely related for Buddhist nun's therapeutic response susceptibility to Ji.
The explanation of above-described embodiment is method and the core concept thereof for understanding the present invention. , it is also possible to the present invention carries out some improvement and modification, it is noted that for the person of ordinary skill of the art, under the premise without departing from the principles of the invention these improve and modify also by the protection domain falling into the claims in the present invention.

Claims (4)

1. the reagent detecting rs12949918 loci gene type is for the preparation of judging that patients with advanced NSCLC is to the purposes in the test kit of Gefitinib therapeutic response.
2. purposes according to claim 1, it is characterized in that, described reagent comprises the primer of the nucleotide sequence for described rs12949918 site of increasing and/or MassARRAYSequenom can be used to detect the single-basic extension primer of rs12949918 loci gene type.
3. purposes according to claim 2, it is characterised in that, described as follows at the primer sequence of interior nucleotide sequence for rs12949918 site of increasing: forward primer 5 '-ACGTTGGATGATCATGGGCTATCCCCAACA-3 '; Reverse primer 5 '-ACGTTGGATGGAGAATACACGCTTAGAGAG-3 '.
4. purposes according to claim 2, it is characterised in that, described single-basic extension primer sequence is: 5 '-AGAGAGAATCTAGAAAGAAACA-3 '.
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