CN117821584A - Androgen alopecia risk gene variation detection kit and application thereof - Google Patents
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Abstract
The invention discloses an androgenetic alopecia risk gene variation detection site, a detection kit and application of the kit, and belongs to the technical field of biological detection. The detection primers are respectively designed based on polymorphic ectopic sites, and a method for detecting androgenetic alopecia with simple, convenient, rapid, accurate and high flux is established by using a multiplex PCR-sanger sequencing method, and a reference basis is provided for detecting androgenetic alopecia and assisting doctors in treatment. Fills up the clinical blank; establishing a method for carrying out integrated analysis on AGA related gene locus detection results, and making great sense for assisting doctors in early diagnosis and treatment of AGA, thus filling the blank in the field; the PCR reaction of 7 sites is carried out by only using one reaction hole, and the operation is simple; the universal primer is arranged to ensure the consistent amplification efficiency of the detection system; and introducing all gene detection results into a comprehensive scoring system formed by autonomous modeling, and comprehensively scoring the results of 7 genotype loci.
Description
Technical Field
The invention belongs to the technical field of biological detection, and particularly relates to an androgenetic alopecia risk gene variation detection site, a detection kit and application of the kit.
Background
Androgenetic alopecia (androgenetic alopecia, AGA) is one of the most common types of alopecia, a progressive hair follicle miniaturization of alopecia disease that begins at puberty or post puberty. Can be suffered from both men and women. The prevalence rate of men in China is about 21.3%, and the prevalence rate of women is about 6.0%. AGA does not affect physical health, but severely affects mental health and quality of life of patients, and if diagnosis and treatment are performed early, the progress of alopecia can be obviously delayed, and the quality of life and mental health of patients can be obviously improved. The normal hair growth cycle includes four phases: growing period or period; a retirement period or degeneration; rest period or rest; and exogenous, or abscission. About 80-90% of hair follicles are in anagen phase at any time, with about 100 hair follicles in anagen phase per day. This physiological process results in the development of large end hair fibers. In AGA, the hair follicle stays less long in the growth phase and is "miniaturized", resulting in an abnormally short and thin hair shaft. Androgens cause gradual conversion of terminal hair to intermediate hair and villi, resulting in gradual thinning and loss of hair in AGA patients.
Epidemiological investigation shows that the ratio of patients with family genetic history in AGA patients is 53.3% -63.9%, and the father line is obviously higher than the mother line. Several susceptibility genes are found in current whole genome sequencing and localization studies. The family history positive male AGA patient rate was 76.6% significantly higher than female 56.0%. Of the family history positive male AGA patients, the male patient was 44.9% larger, well above the female (17.2%) and the parent (14.4%). From a severity point of view, the average baldness score was higher for male AGA family history positive patients than for family history negative patients. It was suggested that male AGA family history positives are more severe than those without family history and that hair loss is faster during the same time of onset. In terms of age of onset, patients with a family history of AGA had an average age of onset earlier than those without a family history. Patients with a history of maternal AGA positivity developed earlier than father positivity. The onset of parental double family history positives was earliest in male patients. The influence of maternal inheritance on the age of onset of children is more than that of the father, and if both parents have history of AGA, the onset of children is earlier.
The above study shows that AGA is a polygenic recessive genetic disease with genetic predisposition. Since AGA patients are mostly in the process of progressive exacerbation until alopecia ends, the importance of early diagnosis and treatment and long-term treatment should be emphasized for patients with the susceptibility to alopecia gene AGA. Therefore, the clear AGA patient alopecia susceptibility gene mutation has important predictive value for the progress and severity of the AGA disease, and also has important reference significance for the selection of subsequent treatment schemes and the establishment of treatment periods.
However, there are two significant problems in the current AGA diagnosis and treatment: problem 1: at present, no method or kit for detecting AGA related gene polymorphism exists in the market of medical appliances. Therefore, it is highly desirable to establish a detection method capable of comprehensively detecting the polymorphism of the AGA-related gene, and to provide a more effective detection means for AGA from the gene level. Problem 2: AGA is controlled by multiple genes, and even if polymorphism of related genes can be detected, it cannot provide clinical detection and treatment with a definite directivity due to the complexity of the results.
Therefore, a method for detecting the polymorphism of AGA related genes and comprehensively evaluating the polymorphism of a plurality of sites is needed, and more reference guidance is provided for clinic.
Disclosure of Invention
The invention aims to provide an androgenetic alopecia risk gene mutation detection site, and based on the polymorphic mutation sites (rs 6493497, rs7176005, rs913063, rs1160312, rs2180439, rs11010734 and rs2420640 sites), detection primers are respectively designed, and a method for detecting androgenetic alopecia by using a multiplex PCR-sanger sequencing method is established, so that the method is simple, rapid, accurate and high-throughput, and a reference basis is provided for detecting androgenetic alopecia and assisting doctors in treatment.
The invention is realized by the following technical scheme:
the androgen alopecia risk gene mutation detection site comprises 7 sites, specifically: rs6493497, rs7176005, rs913063, rs1160312, rs2180439, rs11010734 and rs2420640 sites.
Preferably, the amplification primers of the sites rs6493497, rs7176005, rs913063, rs1160312, rs2180439, rs11010734 and rs2420640 have nucleotide sequences specifically shown in SEQ ID NO. 1-SEQ ID NO. 14.
The primer concentration is 50-400nM.
A kit for detecting androgenetic alopecia risk gene variation, which comprises an amplification primer in the androgenetic alopecia risk gene variation detection site.
Preferably, the kit further comprises a sequencing primer and a PCR premix of the androgenetic alopecia risk gene mutation detection site.
An application of an androgenetic alopecia risk gene variation detection kit is provided, wherein the kit is used for detecting 7-site polymorphism of an androgenetic alopecia risk gene for non-diagnosis purpose.
The detection method is a multiplex PCR-sanger sequencing method.
Multiplex PCR-sanger sequencing, i.e., chemical synthesis, synthesizes nucleotides with no hydroxyl group at the 3' position, i.e., dideoxynucleotides (ddNTPs). It has one less hydroxyl group at the 3 'position than the monodeoxynucleotide, and only remains at the 5' position, and it can be incorporated into the DNA strand by the polymerase during the polymerization reaction. Because it lacks the hydroxyl group at the 3' position, there is no way to bind to the next dNTP and the polymerization reaction of the DNA strand is stopped and does not extend down. Thus, during the polymerization of the DNA strand, a series of DNA fragments of different lengths are obtained by polymerization of dNTPs with ddNTPs, each of which has a dideoxy nucleotide residue at its 3' -end that is complementary to the base at the corresponding position on the template.
Compared with the prior art, the invention has at least the following technical effects:
the invention provides an androgenetic alopecia risk gene mutation detection site (rs 6493497, rs7176005, rs913063, rs1160312, rs2180439, rs11010734 and rs2420640 sites), detection primers are respectively designed based on the polymorphic mutation sites, and a method for detecting androgenetic alopecia by using a multiplex PCR-sanger sequencing method is established, so that the method is simple, convenient, rapid, accurate and high-throughput, and provides reference basis for detecting androgenetic alopecia and assisting doctors in treatment.
Secondly, the method comprises the steps of extracting nucleic acid from an acquired blood sample by a specific method, using multiplex PCR amplification and sanger sequencing technology, using one reaction hole of multiplex PCR to realize amplification of all target gene loci under the action of primers, then using sanger sequencing technology to sequence amplified sequences, further obtaining specific bases of relevant loci, and then introducing gene detection results of all the loci into AGA sequencing scoring analysis software.
The AGA sequencing scoring analysis software is AGA scoring software obtained by comprehensively analyzing and modeling a plurality of indexes such as related gene loci, attack time, family history, alopecia type, severity, treatment period, recurrent index and the like of hundreds of AGA patients and control groups by adopting a machine learning algorithm. The software can comprehensively predict and evaluate the genetic susceptibility, severity, treatment cycle and recurrence of AGA according to genotype detection results.
The method provided by the invention has the following advantages:
(1) fills the clinical blank: currently, no product for detecting AGA related gene polymorphism exists in the medical appliance market; establishing a method for integrating AGA related gene locus detection results, which has great significance for assisting doctors in early diagnosis and AGA treatment and fills the blank in the field;
(2) the operation is simple: according to the invention, only one reaction hole is utilized, PCR reaction is carried out aiming at the positions of rs6493497, rs7176005, rs913063, rs1160312, rs2180439, rs11010734 and rs2420640 7, and the operation is simple;
(3) the detection result is accurate: the primer design of the invention comprises a specific primer section and a universal primer section, the universal primer ensures the consistent amplification efficiency of the detection system, the specific primer ensures the specificity of the detection result, and the accuracy of the detection result is greatly provided;
(4) and (3) comprehensively analyzing the results: another innovation point of the invention is that: and (3) aiming at the detection results, which are not simple single analysis, introducing all the gene detection results into a comprehensive scoring system formed by autonomous modeling, and comprehensively scoring the results of 7 genotype loci. The trouble of inconsistent single results on clinical interpretation is avoided, and the reliability and referenceability of the results are more significant due to comprehensive interpretation, so that the method has great significance on the detection and interpretation of related genes of multiple gene factors and is more convenient for clinical popularization and application.
Drawings
FIG. 1 shows the correlation of each gene locus with disease occurrence and type;
FIG. 2 is a graph of 7 point scoring weights and total alignment;
FIG. 3 is a ROC curve showing the prediction ability of the present detection method and scoring system for AGA occurrence.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to the following examples, which are to be construed as merely illustrative and not limitative of the scope of the invention, but are not intended to limit the scope of the invention to the specific conditions set forth in the examples, either as conventional or manufacturer-suggested, nor are reagents or apparatus employed to identify manufacturers as conventional products available for commercial purchase.
Example 1:
1. rs6493497, rs7176005, rs913063, rs1160312, rs2180439, rs11010734 and rs2420640 locus polymorphism detection methods.
The invention establishes a method for detecting the locus polymorphism of the rs6493497, rs7176005, rs913063, rs1160312, rs2180439, rs11010734 and rs2420640 of suspected AGA patients by using a PCR-sanger sequencing technology simply, conveniently, rapidly and accurately with high flux, thereby providing a reference basis for diagnosis of AGA, and storing the kit at-20 ℃. The specification of the kit is as follows: 50 person/box. The following components of the kit are as follows:
。
the invention provides an amplification and sequencing primer, PCR and sequencing method for detecting rs6493497, rs7176005, rs913063, rs1160312, rs2180439, rs11010734 and rs2420640 locus polymorphism and a required reagent, which comprises the following steps:
(1) Amplification reaction solution: purchased from eastern spinning (Shanghai) biotechnology limited (cat No.:) The reaction system comprises taq DNA polymerase, 4 dNTPs and ions required by various PCR amplification reaction solutions.
(2) Primer working solution for each site: amplification primer pair, universal amplification primer and sequencing primer pair for sites rs6493497, rs7176005, rs913063, rs1160312, rs2180439, rs11010734 and rs2420640 and D/Rnase Free ddH 2 O。
Site primer sequence: the primer concentration is 50 to 400nM, preferably 200 nM.
The amplification primers of the sites rs6493497, rs7176005, rs913063, rs1160312, rs2180439, rs11010734 and rs2420640 are specifically shown in SEQ ID NO. 1-SEQ ID NO. 14; the general amplification primer is shown as SEQ ID NO. 15-SEQ ID NO. 16.
2. Kit performance verification
2.1 sample processing
The method is used for performing performance verification on the enterprise references for detecting the site polymorphisms rs6493497, rs7176005, rs913063, rs1160312, rs2180439, rs11010734 and rs 2420640. Extracting nucleic acid from all samples according to a universal DNA extraction kit (product number: IVD 3018) of the American-based bio-column method, diluting the obtained DNA sample to 20 ng/mu L, and storing at 2-8 ℃; if the sample is not suitable for long time storage at-20 ℃.
2.2 PCR amplification
And adding 20 mu L of the prepared PCR amplification reaction solution into 5 mu L of the extracted sample DNA (20 ng/mu L), and carrying out PCR amplification on the machine.
2.3 amplification procedure:
2.4 Sequencing:
amplification PCR product purification: after the PCR amplification is finished, taking 5 mu L of PCR products to carry out 1-2% agarose gel electrophoresis, observing whether target bands are amplified (the size of the target bands is 500 bp+), if the target bands are observed, purifying the PCR products in time, and specifically, carrying out detailed operation see a PCR product purification kit instruction book. The PCR products purified by the kit steps are subjected to electrophoresis identification and fixed value (the concentration range of the PCR products is 10-50 ng), and sequencing PCR can be immediately carried out or the PCR products can be stored at-20+/-2 ℃ for standby (the storage time is not longer than 2 days).
Sequencing PCR: the PCR products with the defined values were identified by electrophoresis, and the sequencing PCR system was prepared as follows.
Qualitative PCR reaction conditions are shown in the following table:
sequencing preparation:
(1) And taking PCR reaction tubes after the sequencing PCR reaction is finished, and adding 2 mu L of 125 mmol/L EDTA and 2 mu L of 3 mol/L sodium acetate (pH 5.2) into the bottoms of the PCR reaction tubes.
(2) And adding 50 mu L of 100% absolute ethyl alcohol, covering a tube cover, oscillating for a short time, and standing at room temperature in a dark place for 15 minutes.
(3) Centrifugation was performed at 12000rpm for 30 minutes at 4℃and the supernatant was immediately removed with care (if no immediate handling was possible, please re-centrifuge for 3 minutes before handling).
(4) 150 μl of pre-chilled 70% ethanol was added to each tube, centrifuged at 12000rpm for 10 minutes at 4deg.C, the supernatant was immediately removed carefully (if no immediate operation was possible, the tube bottom was centrifuged again for 3 minutes before operation), and this step was repeated once.
(5) The product of the step can be sealed and kept at minus 20 plus or minus 2 ℃ for 5 days in dark place for 15 to 30 minutes at room temperature (70 percent ethanol is volatilized cleanly when the liquid at the bottom of the tube is observed).
(6) Adding 10 mu L Hi-Di Formamide, dissolving DNA by short-time shaking, and centrifuging for short time to completely centrifuge the liquid on the pipe wall to the bottom of the pipe.
(7) The dissolved sample was denatured on a qualitative PCR instrument at 95℃for 5 minutes, rapidly cooled in ice for 4 minutes, and then prepared for electrophoresis.
Sequencing by a gene analyzer:
(1) Sample adding: adding the denatured sequencing product into a 96-well plate matched with a gene analyzer, covering, and editing a sample list according to the sample adding sequence. Sequencing by selecting the seq_std_BDTV3.1_ASSYXL_POP7 or the seq_std_BDTV3.1_ASSY_POP7 with IVD mark according to the model of the sequencer
(2) When the ABI gene analyzer was used, data collection and analysis were performed using the ABI company Data collection and Sequencing Analysis software. For further information on Data Collection and Sequencing Analysis analysis software, please refer to Data Collection and Analysis Software user manual. And automatically storing the sequencing result in a preset position, and opening the sequencing result for analysis after the reaction is finished to obtain files in the format of ab1 and phd 1.
2.5 Sequencing result analysis:
(1) The SeqScanner program was run and sequencing results were imported.
(2) Sequencing results: and comparing the wild type sequence with each other base by application software, searching mutation points, and recording the base types of the mutation.
(3) And (3) introducing the gene detection results of each site into AGA sequencing scoring analysis software, and then comprehensively predicting and evaluating the genetic susceptibility, severity, treatment period and recurrence of the AGA according to the genotype detection results by the software.
2.6 results of detection of the performance of the kit:
3. AGA sequencing scoring analysis model and software
The invention establishes a alopecia disease scoring model by calculating the classification probability based on a multiple logistic regression algorithm and fitting a decision boundary.
Based on the SNP detection result, the genotype result is converted into a numerical result, the homozygous site corresponding to REF is scored as 2, the heterozygous mutation score is 1, the homozygous mutation score is 0, and the genotypes of the SNP sites are graded.
Firstly, constructing a first edition model of preprocessed data, primarily screening the original data, deleting data with a difference between a predicted value and a true value of more than 0.55 in control as outliers, further constructing a multiple logistic regression model, calculating data distribution conditions and model performance conditions, and checking an AUC value of the model, namely the area under an ROC curve, wherein the AUC value is between 0.5 and 1, so that the performance of the predicted model is better than that of random guessing; the closer the AUC is to 1, the better, indicating that the model has at least one threshold that can accurately separate the data above and below the threshold.
Furthermore, the scoring model disclosed by the invention is used for calculating the alopecia score (total points) of a patient based on a logistic algorithm and combining with a nomogram, and can calculate the alopecia trend of a certain stage according to the prediction score of each time period so as to predict the recurrence, wherein the scoring system performs normalization processing according to the weight of each index in the model, so that the classification prediction probability is associated with the scoring result to obtain an evaluation result.
Multiple logistic regression model formula:
glm (group to rs6493497+rs7176005+rs913063+rs1160312+rs2180439+rs11010734+rs2420640, data=data, family=binominal); wherein, the numerical values of rs6493497, rs7176005, rs913063, rs1160312, rs2180439, rs11010734 and rs2420640 are all respective fractional values.
As shown in FIG. 1, the correlation between each gene locus and the occurrence and type of disease is shown.
As shown in fig. 2, 7 points are scored for weight and total alignment.
Test example: detection of 58 clinical samples
1. Sample processing
Blood samples of 30 AGA patients and 28 normal volunteers, diagnosed in the Peking water pool hospital hair medicine research center affiliated to the university of Country medical science, were collected using EDTA anticoagulant tubes for 2022 years.
200 mu L of each sample is taken, and DNA is extracted from nucleic acid according to a universal DNA extraction kit (product number: IVD 3018) of the American-based bio-column method for later use.
The PCR was performed in the same manner as in example 1 at 2.2 to 2.6.
2. Clinical sample detection results:
/>
。
3. analysis of results
By analyzing the detection results of 58 samples (30 AGA patients and 28 normal volunteers), the AGA risk gene detection method and the scoring method can effectively (AUC value of 0.8214) prompt the type and the risk of the variation of the AGA risk gene of the AGA patient by taking 0.78 as an optimal threshold (specificity of 0.929 and sensitivity of 0.638), and prompt the selection of a further treatment scheme of the AGA patient.
As shown in fig. 3, the prediction ability ROC curve of the detection method and the scoring system for the occurrence of AGA is shown.
In 58 samples, the coverage of the detection result of the kit compared with the second generation sequencing result is 100%.
The detection duration of the method is 24 hours (3 working days of the detection period), the detection duration of the second generation sequencing is 96 hours (15 working days of the detection period), and the kit has obvious advantages in the aspect of saving time and cost. The detection cost of the kit is 100 yuan, the detection cost of second generation sequencing is 2000 yuan, and the kit has obvious advantages in the aspect of saving economic cost.
The detection method of the invention is compared with other methods:
in conclusion, the invention provides an economic, convenient, quick and effective means for AGA risk gene detection and occurrence risk assessment.
Finally, it should be noted that: the foregoing description is only of the preferred embodiments of the invention and is not intended to limit the scope of the invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (4)
1. An androgenetic alopecia risk gene variation detection site, which is characterized by comprising 7 sites, specifically: rs6493497, rs7176005, rs913063, rs1160312, rs2180439, rs11010734 and rs2420640 sites.
2. The androgenic alopecia risk gene mutation detection site of claim 1, wherein the amplification primers of the sites rs6493497, rs7176005, rs913063, rs1160312, rs2180439, rs11010734 and rs2420640 have nucleotide sequences specifically shown in SEQ ID NO. 1-SEQ ID NO. 14.
3. The kit for detecting androgenetic alopecia risk gene variation, which is characterized by further comprising a sequencing primer and a PCR premix of the androgenetic alopecia risk gene variation detection site according to claim 2.
4. The use of the kit for detecting an androgenic alopecia risk gene variation according to claim 3, wherein the kit is used for detecting 7-locus polymorphism of the androgenic alopecia risk gene for non-diagnostic purposes.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20110104691A1 (en) * | 2008-04-07 | 2011-05-05 | Life & Brain Gmbh | Androgenetic alopecia |
| KR20180125058A (en) * | 2017-04-25 | 2018-11-22 | 연세대학교 산학협력단 | Specific primers for predicting hair loss and uses thereof |
| KR102061317B1 (en) * | 2018-11-22 | 2020-01-02 | 주식회사 다우진유전자연구소 | Kits for detecting hair loss disorders with Single Nucleotide Polymorphism and method for detecting hair loss disorders thereby |
-
2024
- 2024-03-06 CN CN202410251251.0A patent/CN117821584A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20110104691A1 (en) * | 2008-04-07 | 2011-05-05 | Life & Brain Gmbh | Androgenetic alopecia |
| KR20180125058A (en) * | 2017-04-25 | 2018-11-22 | 연세대학교 산학협력단 | Specific primers for predicting hair loss and uses thereof |
| KR102061317B1 (en) * | 2018-11-22 | 2020-01-02 | 주식회사 다우진유전자연구소 | Kits for detecting hair loss disorders with Single Nucleotide Polymorphism and method for detecting hair loss disorders thereby |
Non-Patent Citations (3)
| Title |
|---|
| IN-YOUNG KIM BS1等: "The first broad replication study of SNPs and a pilot genome-wide association study for androgenetic alopecia in Asian populations", 《J COSMET DERMATOL》, vol. 21, no. 11, 19 July 2022 (2022-07-19), pages 6174 - 6183 * |
| WENLONG RUI等: "Association of Single Nucleotide Polymorphisms in the CYP19A1 Gene with Female Pattern Hair Loss in a Chinese Population", 《DERMATOLOGY》, vol. 231, no. 3, 24 July 2015 (2015-07-24), pages 239 - 244, XP055909817, DOI: 10.1159/000433597 * |
| 叶萌等: "20号染色体与X染色体上的8个SNP位点与云南汉族雄激素性脱发的相关性", 《中华医学遗传学杂》, vol. 33, no. 3, 30 June 2016 (2016-06-30), pages 383 - 387 * |
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