CN106834509B - Application of primer and reagent in preparation of reagent group for screening growth hormone deficiency - Google Patents
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Abstract
The invention relates to a forward primer with a sequence shown as SEQ ID NO.1, a reverse primer with a sequence shown as SEQ ID NO.2, a dual gene primer with a sequence shown as SEQ ID NO.3 and application of a reagent for amplifying a nucleic acid fragment in preparation of a reagent group for screening growth hormone deficiency, and then the primer is used for detecting the amplified nucleic acid fragment of Single Nucleotide Polymorphism (SNP) rs9368758(A/G) to screen growth hormone deficiency and susceptibility thereof, and the method is used for evaluating and predicting the curative effect of a growth hormone deficiency patient after treatment by using recombinant human growth hormone.
Description
Technical Field
The invention relates to a single gene mononucleotide polymorphism site related to growth hormone deficiency disease in the field of biomedicine and application thereof
Background
Dwarfism is a disease which is relatively common in pediatric endocrine, and the most common cause of the disease is growth hormone deficiency. At present, the diagnosis of the dwarf disease in clinical work is based on the clinical guidelines of the dwarf disease, laboratory indexes, genetic markers and the like, wherein the main is growth hormone stimulation test. The specific diagnostic criteria may be (1) the definition of compliance with dwarfism: the height is lower than the average value of the heights of normal children of the same age, sex and race minus 2 standard deviations or is below the 3 rd percentile, wherein part of the heights belong to physiological variation; (2) the results of the two drugs (insulin and levodopa) sequential growth hormone stimulation test show that the peak value is less than 10 ng/ml; (3) no obvious abnormality is found in the blood routine, liver and kidney function, blood fat and thyroid gland functional test; (4) growth rate (GV) <5 cm/year (3 years to prepubertal); <6 cm/year (in puberty); (5) bone Age (BA) lags behind the actual age (CA). On the premise of meeting the clinical characteristics of the short stature on the basis of excluding other diseases, a growth hormone stimulation test is carried out, and the growth hormone deficiency patients can be divided into complete growth hormone deficiency and partial growth hormone deficiency according to the growth hormone deficiency level according to the peak value.
However, this can only be seen in a proportion of patients who have already appeared to be short, thereby further defining the cause of the short stature. Yet another group of potential patients may have difficulty reaching the intended end height without prior intervention, developing dwarfism. There is therefore an urgent need for genetic markers that can be screened for dwarfism or to assess the efficacy of treatment after intervention.
rs9368758 is a SNP site of a biallelic polymorphism on human chromosome 6q21.3, altered to be a switch (G/A, C/T on its complementary strand). The rs9368758 genotype is GG, AA or GA. Wherein AA is homozygous at locus A of rs9368758, GA is heterozygous between G and A, and GG is homozygous at G. Wherein the polymorphism (allele) or genotype for detecting rs9368758 in human genome can be specifically the nucleotide type for detecting rs 9368758.
Disclosure of Invention
The technical problem to be solved by the invention is how to screen growth hormone deficiency patients in Chinese continental population and how to evaluate and predict the curative effect of the growth hormone deficiency patients after the treatment by the recombinant human growth hormone.
In order to solve the above technical problems, the present invention provides a reagent set containing a substance for detecting a polymorphism (allele) or genotype of rs9368758 in a human genome, which is any one of the following:
a product for detecting or identifying a single nucleotide polymorphism (allele) or genotype associated with growth hormone deficiency.
A product for screening patients with growth hormone deficiency and susceptibility thereof is provided.
A product for predicting the curative effect of growth hormone deficiency patient after recombinant human growth hormone therapy.
The product comprises a forward primer with a sequence shown as SEQ ID NO.1, a reverse primer with a sequence shown as SEQ ID NO.2, a dual gene primer with a sequence shown as SEQ ID NO.3 and a reagent for amplifying nucleic acid fragments, wherein the sequence of the forward primer is 5'-GAGCAGCCCAAGGTTAC-3'; the sequence of the reverse primer is: 5'-TCCCTTGAGACCAATGTGTCT-3' are provided.
Preferably, the product comprises dNTPs, TaqDNA polymerase and Mg2+And PCR reaction buffer.
More preferably, the product comprises a standard positive template.
In practical application, the substance for detecting the polymorphism (allele) or genotype of the rs9368758 and other substances can be combined together to prepare a product for screening patients with growth hormone deficiency or predicting the curative effect after the treatment of the recombinant human growth hormone.
The product can be a reagent or a kit, and can also be a system consisting of the reagent or the kit and an instrument, such as a system consisting of a primer and a DNA sequencer, a system consisting of a PCR kit DNA sequencing reagent and a DNA sequencer, a system consisting of a Taqman probe and the like.
The technical scheme provided by the invention can be used for detecting the rs9368758 polymorphism or genotype substance in the human genome to prepare a product for screening growth hormone deficiency patients or predicting the curative effect of the growth hormone deficiency patients after the recombinant human growth hormone is applied to treatment.
The invention also provides any one of the following uses:
a method of using the aforementioned product, comprising the steps of:
(1) extracting the genome DNA of a receptor to be detected; (2) taking genome DNA of a receptor to be detected as a template, and amplifying fragments through PCR reaction by using a forward primer with a sequence shown as SEQ ID NO.1 and a reverse primer with a sequence shown as SEQ ID NO. 2; (3) the base at the 74 th base (bp) in the PCR amplification product sequence is detected to be A or G by utilizing the dual gene primer of the sequence shown as SEQ ID NO. 3.
Preferably, the genotype of the individual to be detected can be determined by at least one of matrix-assisted laser desorption ionization time-of-flight mass spectrometry, gene chip detection, DNA sequencing, restriction enzyme fragment length polymorphism, Taqman probe, SnapShot and the like.
Use of the aforementioned product for assessing the risk of an individual for deficiency of growth hormone, comprising the steps of:
(1) screening fragments amplified by a forward primer with a sequence shown as SEQ ID NO.1 and a reverse primer with a sequence shown as SEQ ID NO.2 by adopting a polymerase chain reaction and sequencing technology; (2) adopting a dual gene primer with a sequence shown as SEQ ID NO.3 to detect the genotype; (3) assessing the lack of growth hormone in the subject based on genotype, wherein the presence of the allele G or genotype GG of the single nucleotide polymorphism rs9368758(A/G) detected in vitro indicates that the subject is at increased risk for a lack of growth hormone.
Preferably, the use increases the relative risk of the individual for the lack of growth hormone by 1.10-fold to 2.65-fold when the presence of the allele G or genotype GG of the single nucleotide polymorphism rs9368758(A/G) is detected, compared to the detection of the allele A of the single nucleotide polymorphism rs9368758 (A/G).
Preferably, the use increases the risk of deficiency of growth hormone in the individual by a relative risk of 1.71-fold when the presence of the allele G or genotype GG of the single nucleotide polymorphism rs9368758(A/G) is detected, compared to the detection of the allele A of the single nucleotide polymorphism rs9368758 (A/G).
Preferably, for said use, the biological sample comprises blood or saliva.
Preferably, in the application, the single nucleotide polymorphism rs9368758(A/G) is obtained by amplifying a nucleic acid fragment through a forward primer with a sequence shown as SEQ ID NO.1 and a reverse primer with a sequence shown as SEQ ID NO.2, and then using a dual gene primer with a sequence shown as SEQ ID NO.3 to detect the dual gene A or G of the single nucleotide polymorphism rs9368758 (A/G).
Use of a product as described above for assessing the effect of an individual on the prediction of the lack of growth hormone on the treatment with growth hormone, characterized in that it comprises the following steps:
(1) screening fragments amplified by a forward primer with a sequence shown as SEQ ID NO.1 and a reverse primer with a sequence shown as SEQ ID NO.2 by adopting a polymerase chain reaction and a sequencing technology; (2) adopting a dual gene primer with a sequence shown as SEQ ID NO.3 to detect the genotype; (3) predicting the effect after administration of growth hormone treatment based on the genotype, wherein the nucleotide at the 74bp site of the amplified fragment is G or A, and wherein the presence of G or GG, which is the allele of the single nucleotide polymorphism rs9368758(A/G), is detected in vitro, the therapeutic effect is poor compared to an individual in which A, which is the allele of the single nucleotide polymorphism rs9368758(A/G), is detected in vitro.
Preferably, the use is carried out in a case where the allele G or genotype GG of the SNP rs9368758(A/G) is detected to be present, in the case of a non-pubertal subject, in an amount of 0.42-fold to 1.02-fold of the therapeutic dose of growth hormone, as compared to the amount of the allele A of the SNP rs9368758(A/G) detected.
Preferably, the use is carried out in a case where the allele G or genotype GG of the SNP rs9368758(A/G) is detected to be present, the treatment dose of growth hormone for the non-pubertal individual is 1.02 times that for the single nucleotide polymorphism rs9368758(A/G) detected as allele A.
Preferably, the use is for the treatment of a pubertal puberty in a subject 1.38 times more active in growth hormone than the treatment of pubertal puberty in a subject in which the allele G or genotype GG of the SNP rs9368758(A/G) is detected.
In the application, the growth hormone deficiency disease is specifically growth hormone deficiency disease of Chinese mainland population.
In the experiment of the present invention, it was confirmed that in the case group consisting of 229 patients with growth hormone deficiency and the control group consisting of 314 normal healthy people in mainland China, the risk allele of rs9368758 is G, and the proportion of the allele in the case growth hormone deficiency patient population is increased by about 30% compared with the proportion in the normal population. The relative risk of developing homozygote A carrying allele A at locus rs9368758 (AA genotype) and heterozygote A at locus rs9368758(A genotype) compared to homozygote G carrying allele at risk (G genotype) for growth hormone deficiency was 0.4645 and 0.7013. The P value is 0.01, which indicates that rs9368758 is a single nucleotide polymorphism related to growth hormone deficiency.
When the polymorphic site of the growth hormone deficiency rs9368758 is homozygous wild type (i.e. the genotype is GG), the effect of predicting the medicament to improve the height is relatively weaker after the recombinant human growth hormone is subcutaneously injected for treatment;
after the recombinant human growth hormone is injected subcutaneously to treat a growth hormone deficiency patient in the adolescence period without starting, when the polymorphic site of rs9368758 is subjected to homozygous mutation, the first dosage of the recombinant human growth hormone medicine is smaller when the ideal height improving effect is predicted to be achieved, 0.1U/kg.d can be recommended according to the conventional dwarf clinical guideline, and the dosage is adjusted according to the relevant laboratory examination indexes to achieve the optimal height improving effect; when the rs9368758 polymorphic locus wild type is used for a growth hormone deficiency patient, the first dose of the recombinant human growth hormone medicine is relatively large for predicting to achieve an ideal height improving effect, and the first recombinant human growth hormone dose can be properly increased within a safe dose range suggested by a microsomia clinical guideline.
After the adolescent growth hormone deficiency patient is treated by subcutaneous injection of the recombinant human growth hormone, no matter the rs9368758 polymorphic site is homozygous mutation or wild type, under the guidance of the microsomia clinical guideline, even if the first dose is relatively large, the height improving effect of the recombinant human growth hormone medicament after the dose is adjusted for many times is poor.
The invention seeks to find out the relation between the mutation of the rs9368758 polymorphic site and the treatment opportunity of the recombinant human growth hormone, and the opportunity is easily ignored by doctors and parents. Therefore, the growth hormone deficiency infant who is screened for homozygous mutation of rs9368758 polymorphic sites aiming at genes can find potential patients through a screening product or a predicted curative effect product which is prepared from substances of rs9368758 polymorphism or genotype in human genome, and guide the growth hormone treatment in the early clinical stage, and the optimal treatment time is in the puberty non-starting period (9.0 +/-2.5 y).
Drawings
FIG. 1 is a diagram of a genotype sequencing peak.
FIG. 2 is a diagram of a genotype sequencing peak.
FIG. 3 is a diagram of peaks from genotype sequencing.
FIG. 4 is a diagram of SNP typing cluster analysis of three genotype time-of-flight mass spectra, in which the horizontal axis region (i.e., regular triangle) represents the individual genotype GG; the vertical axis region (i.e., inverted triangle) represents an AA genotype individual; the middle region (i.e., square) represents a GA genotype individual; no call indicates that individual idiotype is missing; low Mass Height represents a Low molecular weight site; high Mass Height indicates a High molecular weight site.
Detailed Description
The technical means adopted by the invention to achieve the predetermined object of the invention are further described below with reference to the drawings and the preferred embodiments of the invention.
Example 1 collecting blood sample of rs9368758 Single nucleotide polymorphism site to be detected
TABLE 1 basic characteristics of case group and control group of patients with growth hormone deficiency
Table 1 shows a total of 229 Growth Hormone Deficiency (GHD) and 314 normal controls. In the study subjects, the proportion of males to females accounted for 70.7% and 29.3% in the cases, respectively, and the proportion of males to females accounted for 58.3% and 41.7% in the control group, respectively. The mean age of the cases was 10.2. + -. 3.0 years, which approximately coincided with the mean age of the control group of 7.4. + -. 3.0 years.
Table 2.rs9368758 basic information of single nucleotide polymorphism sites
| SNP | Chromosome zone | Position of | Alleles |
| Rs9368758 | 6p21.32 | 33170244 | G/A |
Table 2 shows that the locus rs9368758 is on chromosome band 6p21.32, at position 33170244, and the allele is G/A.
A sample of venous blood (5 ml) of 229 patients with GHD, shown in Table 1, was collected, dispensed through an EDTA anticoagulant tube, and stored in a freezer at-80 ℃. The experiment uses a blood sample to prepare DNA, and can leave a saliva sample to extract DNA if the blood sample is inconvenient to leave in clinic, so that the DNA can be extracted.
Example 2 Collection of genomic DNA of blood sample to be tested
1. The blood sample obtained in example 1 was extracted, and DNA (obtained from TIANGEN, model DP318) was extracted by a blood genome DNA extraction kit, while ensuring that the DNA concentration was 20ng/ul or more and the purity A260/A280 was 1.7-2.0.
2. Amplification of nucleotide fragment containing rs9368758 site
Primers were designed based on the SNP site sequence information provided by NCBI (see table 2), including the forward primer: 5'-GAGCAGCCCAAGGTTAC-3' (shown as SEQ ID NO. 1) and a reverse primer: 5'-TCCCTTGAGACCAATGTGTCT-3' (shown in SEQ ID NO. 2), the nucleotide fragment in which the SNP to be detected is amplified is shown as follows:
GAGCAGCCCAAGGTTACAGCAGTGAGGCAGTGGAGGCCTCCCGGGAGTAAGGGCTTCTCTTGGCCCCTGAGAC [ G/A ] ATACTAGAGTTTATGGTCTGGGAAAGGGAGGCAGAAGACCAGACACATTGGTCTCAAGGGA (shown as SEQID NO. 4)
The SNP site is located at the 74bp position of the PCR amplified fragment, and the base can be G or A.
Wherein the PCR reaction content is 1. mu.l template DNA, 0.05 pmol/. mu.l forward primer F and reverse primer R each 0.5. mu.l, 0.1. mu.l, 25nM dNTPmix, 0.1. mu.l Hotstar
(5U/ul), 1.0. mu.l Primer mix (500nM each), 0.5. mu.l 10 XPCR reaction buffer, the remainder being double distilled water (HPLC grade).
The PCR reaction conditions were 94 ℃ pre-denaturation for 15 minutes (min), 94 ℃ denaturation for 20 seconds(s), 56 ℃ annealing for 30 seconds, 72 ℃ extension for 1 minute, and 45 cycles followed by 72 ℃ extension for 3 minutes. Then Shrimp alkaline phosphatase (Shrimp Alkalinephosphophosphatase, SAP) was added to digest the PCR product; alkaline phosphatase, exonuclease I (SAP enzyme, concentration of 1.7U/. mu.l) and SAP reaction buffer are added into PCR amplification products in the SAP reaction system to digest 5' -terminal phosphate groups, redundant products and primers in the PCR products.
And then carrying out extension reaction on the PCR product after SAP reaction: taking an amplification product obtained after SAP reaction purification as a template, adding a raw material ddNTP including a dual gene primer with a sequence shown in SEQ ID NO.3 to detect the dual gene of the single nucleotide polymorphism rs9368758(A/G) as A or G to carry out PCR reaction, and terminating the primer extension by one base (a kit for the extension reaction is purchased from iPLEX company and has the model of Agena).
Adding water and resin into the product after single base amplification for desalting, and finally performing mass spectrum detection on a sample machine.
Example 3 rs9368758 site genotyping
The rs9368758 locus genotyping was performed by Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF MS) on the sample obtained in example 2, and the specific results are as follows:
as shown in FIG. 1, the typing results are GA; as shown in FIG. 2, the typing results were AA; as shown in FIG. 3, the typing result was GG.
Example 4 correlation analysis of site rs9368758 and growth hormone deficiency
The results of the statistical analysis of the specific data obtained in example 3 and the raw typing by applying the statistical method of the taber-side test of R × C are shown in table 3.
TABLE 3 Association analysis of rs9368758 locus and growth hormone deficiency
As shown in table 3, after mass spectrometry, the distribution of each genotype in the case group and the height normal group was found to be different in the case group and the control group by chi-square test, i.e., the P value was 0.012, and it was considered that the site has a certain correlation with the occurrence of the disease. Therefore, the gene distribution frequency of the locus rs9368758 in the case group and the control group is compared, and the genotype distribution frequency of the two groups has statistical significance (P ═ 0.012).
Genotype case/control analysis under dominant model at site G of rs9368758 in Table 4
The results of the G dominant mode of genotypes by chi-square assay with 4 tables show that in the G dominant mode, G containing alleles (GA, GG) are at 1.71 times higher risk of GHD than the AA genotype, with statistical differences (P0.02, OR 1.71, 95% CI 1.11-2.65). Indicating that allele G is a risk factor for the onset of GHD.
Example 5 locus rs9368758 Association with efficacy of recombinant human growth hormone treatment
A total of 106 growth hormone deficiency patients of the subject clinically meet the diagnosis standard of the growth hormone deficiency and meet the indication of applying growth hormone treatment, and are subcutaneously injected every night according to the dwarf disease guide established by Chinese medical society, pediatrics, genetics, endocrine and metabolism in 2008, by 0.1-0.15 u/(kg.d).
During the experiment: the general treatment period is 1 month, then the clinic visits height, weight and biochemical index (such as IGF-1), if no obvious abnormality is found in biochemical index and the height rises well (about 1 cm/month), then the continuous medication is recommended. If no obvious abnormality is detected after 3 months at the time of the consultation and the growth rate is still acceptable (>1.5cm/3 months), the application of growth hormone is recommended, and therefore data analysis of repeated measurements is adopted.
Table 5, rs9368758 locus and the therapeutic effect correlation analysis after the recombinant human growth hormone treatment
P value <0.05, # P value, genotype and treatment time interaction.
"Times × GA" means that for each 30 day increase in treatment, the height of patients with genotype GA is 0.11cm less than that of patients with genotype AA for Tanner stage I, and this difference is statistically significant (P0.025) taking into account the effect of time factors; similarly, the height of a patient with genotype GG is increased by 0.13cm less than that of a patient with genotype AA, and the difference is statistically significant (P ═ 0.01) considering the influence of time factors.
The height increased gradually with the treatment time, and the height increased differently between the three groups of genotypes. The AA genotype increased most rapidly, followed by the GA genotype and the GG genotype.
After the first growth hormone dosage of the 106 GHD patients receiving recombinant human growth hormone treatment and the medical advice at each consultation are adjusted to perfect the database, the statistical method of multiple regression analysis is adopted, when the genotypes are GG and GA, the growth hormone treatment dosage of the individuals without the initiation/initiation of adolescence is several times of that of the genotype AA, and the specific results and steps are as follows:
(1) aiming at GHD patients without adolescence starting
And importing the database into statistical software, and putting the patient's applied growth hormone dosage at the dependent variable position and putting the patient's genotype at the exposed variable position by adopting a statistical method of multiple regression analysis, wherein the genotypes of the patients are AA and GG + GA genotypes. Under the condition of not adjusting other factors, when the genotype is analyzed to be GG and GA, the dose of the growth hormone treatment applied to the individuals without promoting puberty is 0.42 times compared with the genotype AA; however, considering that the dose is related to weight, IGF-1, height level, it is adjusted by three variables, Weight (WT), IGF1 and Height (HT). When the genotypes are analyzed to be GG and GA, the dose of growth hormone treatment applied to the individuals without promoting puberty is 1.02 times compared with the genotype AA.
(2) Aiming at GHD patients after the onset of puberty
As described above, in the case of analyzing genotypes GG and GA under the influence of body weight, IGF-1 and body level, the dose of growth hormone treatment administered to a non-pubertal subject was 1.38 times as much as that administered to genotype AA.
The results show that the rs9368758 not only has relevance to the occurrence of growth hormone deficiency, but also has relevance to the difference of curative effects of the recombinant human growth hormone after treatment.
Although the present invention has been described with reference to a preferred embodiment, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.
Sequence listing
<110> hospital affiliated with Jining medical college
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Claims (4)
1. The application of a forward primer with a sequence shown as SEQ ID NO.1, a reverse primer with a sequence shown as SEQ ID NO.2, a dual gene primer with a sequence shown as SEQ ID NO.3 and a reagent for amplifying a nucleic acid fragment in the preparation of a reagent group for screening growth hormone deficiency or predicting the effect of a growth hormone deficiency patient after recombinant human growth hormone treatment is applied.
2. The use of claim 1, wherein the reagent set comprises a probe set consisting of Taqman probe G and Taqman probe A.
3. The use of claim 1, wherein said set of reagents comprises dNTPs, Taq DNA polymerase, Mg2+And PCR reaction buffer.
4. The use of claim 1, wherein said set of reagents further comprises a standard positive template.
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| ISS患儿NPR3、COL11A2基因单核苷酸多态性的研究;李正兰;《中国优秀硕士学位论文全文数据库（电子期刊）医药卫生科技辑》;20120615(第6期);中文摘要2-5段，第1页第3段，第2页第2段，第3页第2段，第26页表8.1-8.2，第27页第1段，第40页第4段 * |
| rhGH治疗儿童矮小症促身高增长影响因素的研究;王颖;《中国优秀硕士学位论文全文数据库（电子期刊）医药卫生科技辑》;20130215(第2期);全文 * |
| 生长激素受体基因多态性与广西瑶族儿童特发性矮小症的相关性研究;曾婷等;《中国儿童保健杂志》;20150731;第23卷(第7期);全文 * |
| 矮小症生长激素受体基因外显子3多态性与重组人生长激素疗效相关性的Meta分析;李鑫;《中国优生与遗传杂志》;20170131;第25卷(第1期);摘要，第1.5节 * |
| 矮小症的病因与遗传学现状;云素芳等;《中国优生与遗传杂志》;20120331;第20卷(第3期);全文 * |
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