CN112824537A - Kit for specifically detecting sarcopenia through rs524533 - Google Patents

Abstract

The invention discloses a kit for specifically detecting sarcopenia by detecting rs 524533. The kit comprises a specific primer pair and a specific fluorescent probe pair for detecting the rs524533SNP locus, a conventional component for fluorescent quantitative PCR detection and the like. The kit of the invention estimates the individual sarcopenia genetic susceptibility by simultaneously detecting the single nucleotide polymorphism locus genotype of rs524533 closely related to the sarcopenia genetic susceptibility.

Description

Kit for specifically detecting sarcopenia through rs524533

Technical Field

The invention relates to the field of SNP typing detection in the technical field of genetic engineering, in particular to a kit for specifically detecting sarcopenia by detecting rs 524533.

Background

Sarcopenia, also called Sarcopenia (Sarcopenia), is a chronic metabolic disease of the elderly, which is mainly characterized by the decrease of the quality of skeletal muscle fibers, the decrease of muscle strength, the decrease of muscle endurance and metabolic capability, the increase of connective tissues and fat, and the like with the increase of age. Sarcopenia often causes the mobility of the old to be reduced, daily actions such as walking, sitting, climbing, lifting heavy objects and the like are influenced, and the hand is gradually free from binding the force of chicken. The muscle function is reduced, and the probability of accidental falling injury of the old is increased by 40 percent. Statistically, 50% of the elderly die from accidents due to falls. The risk of inconvenient actions of the sarcopenia old people is 2-5 times that of the old people of the same age, and the life quality and the self-care ability of the old people are seriously influenced.

Sarcopenia not only increases the risk of disability and loss of self-care ability of life, but also promotes the development of diseases such as osteoporosis and arthritis, and is also an important reason for inducing senile diseases such as hypertension, diabetes and hyperlipidemia. Sarcopenia has become one of the leading causes of disability and death worldwide. Sarcopenia occurs in 13-24% of the elderly under age 70, with a prevalence of up to 50% and even higher in those over age 80. Annual economic losses associated with sarcopenia exceed $ 1000 billion.

Numerous studies of candidate genes in the past have shown that many genes are associated with changes in human muscle mass. More than 40% of the genetic variations associated with the human lean body mass index (LMI) can produce differences in traits. With the progress of molecular genetics, it is found that sarcopenia is a polygenic genetic disease, and genetic association research of sarcopenia is carried out in many countries. At least 20 human genes or chromosomal regions have been detected by molecular genetics and reported to be associated with sarcopenia to date. In addition, both genetic and molecular epidemiological evidence suggests that genetic factors are involved in determining susceptibility to increased or lost muscle mass in specific dietary or drug regimes, as well as a high risk of developing other related diseases in sarcopenia patients. At present, many candidate genes exist, but most of the candidate genes are required to be further improved in detection accuracy.

As for the method for detecting sarcopenia genes, the conventional detection method at present includes Restriction Fragment Length Polymorphism (RFLP), which is a method for simply treating the PCR product of a patient by enzyme digestion and then detecting whether there is variation when the variation affects the enzyme cutting site of a certain Restriction enzyme. However, the method has the defects of long time consumption, complex operation, low accuracy and the like. The detection of gene polymorphism sites by PCR combined with DNA sequencing is also useful, but the application of this method in large-scale population screening or detection of multiple sites of multiple genes is limited. Therefore, it is necessary to establish a high-throughput, high-efficiency and low-cost SNP (single nucleotide polymorphism) typing method for sarcopenia susceptibility genes to realize clinical rapid detection or large-scale population screening.

Disclosure of Invention

The invention provides a kit for specifically detecting sarcopenia by detecting rs 524533.

One of the purposes of the invention is to provide a screening method of SNP molecular markers of sarcopenia susceptibility genes, which is to find a molecular marker locus rs524533 which is positioned on a No. 6 chromosome 44240559 by analyzing genome sequences of three samples of Fremingham's heart disease research (FHS), Kansasicheng osteoporosis research sample (KCOS) and woman health primary program (WHI) and the muscle content of limbs measured by a dual-energy X-ray analyzer, wherein the mutation into C/T is directly related to the muscle content of human.

The invention provides an rs524533 molecular marker amplification primer pair on a 6p21.1 chromosome according to an rs524533 molecular marker sequence disclosed on NCBI, and an upstream primer of the primer pair: ACATCTTCTGTTTCATTGCC, respectively; a downstream primer: TTCTCTCCATATCCCTGTT, respectively; amplified fragment size: 260 bp. Simultaneously, a pair of detection primer pairs is provided, and genotype-fluorescent probe sequences are as follows: 5 '-FAM-CTCTGCACTcTCCTGTACT-TAMRA-3'; genotype two fluorescent probe sequence: 5 '-VIC-CTCTGCACTtTCCTGTACT-TAMRA-3'.

The rs524533SNP detection chip on the chromosome 6p21.1 can be used for detecting the mutation of the C/T of the chromosome 6p21.1 segment individually or in parallel. The detection chip is prepared by adopting a conventional construction method in the field.

The invention provides a kit for detecting sarcopenia genetic susceptibility. The kit comprises:

the specific primer pair for detecting the rs524533SNP polymorphism genotype has the sequence shown in SEQ ID NO: 2 and 3; PCR reaction components (including Taq enzyme, dNTP mixed solution, MgCl2 solution, reaction buffer solution, deionized water and the like).

The invention has the main advantages that:

the rs524533SNP marker obtained by identification from the chromosome 6p21.1 can be used for identifying the symptoms of sarcopenia of human beings, and the identification has the advantages of good accuracy and high specificity;

the detection method of the invention has simple steps, the SNP locus detection can be completed by one-step PCR, the amplification of the target sequence containing the SNP locus avoids a plurality of uncertain factors existing in the complex operation processes of repeated PCR and the like, thereby greatly improving the detection accuracy and embodying the accurate and simultaneously qualitative and quantitative analysis characteristics.

Drawings

FIG. 1 is a graph of the results of an rs524533 gel mobility (EMSA) experiment.

FIG. 2 is a graph of the results of dual luciferase reporter gene experiments in rs 524533.

Detailed Description

EXAMPLE 1 obtaining molecular markers for SNPs

The present invention uses three samples in total, including the framingham heart disease study (FHS), the women health initiative study african american (WHI-AA) subsample, and the Kansas City Osteoporosis Study (KCOS). The first two samples were obtained from the U.S. genotype and phenotype database (dbGAP), and the third sample was a sample that was coordinated with the Dunhong teaching lesson, an sarcopenia research expert. In which FHS was used as the finding sample and WHI-AA and KCOS were used as the verifying samples.

1, finding sample (FHS)

FHS is a vertical prospective queue comprising 16000 participants across three generations of european ancestry. The first generation consisted of 5209 participants residing in the town of foming hamsters, massachusetts, usa. The second generation was an adult child of the members of the first generation and his spouse, with a total of 5124 participants. The third generation consists of 4000 participants, who are grandchildren of the first generation.

The first generation participants received a scan of a dual energy X-ray apparatus (DXA) during the 22 th or 24 th examination. The instrument is capable of not only measuring bone density, but also accurately measuring the content of body components including fat and muscle. The second generation participant accepted the DXA scan during the 6 th or 7 th exam, and the third generation participant accepted the DXA scan during the 2 nd exam.

2, verifying the sample

The first validation sample was a WHI-AA sample, which was a subsample of the WHI observation study accessed by dbGaP and comprised a total of 845 African American participants. Genotyping was performed by Affymetrix SNP 6.0 microarray and muscle content was determined by the Hologic QDR DXA instrument.

The second validation sample was a KCOS sample containing 2286 participants. Genotyping was also performed by Affymetrix SNP 6.0 microarray, and muscle content was determined by the Hologic QDR DXA instrument.

3, phenotypic treatment

In all samples, covariates such as gender, age squared, height squared, and the first 5 principal components calculated from the genomic data (to measure population stratification effects) were screened for significance using stepwise regression. To correct for the effects of fat content, fat content was also used for covariate correction. The residuals of the original muscle content after covariate correction were normalized using the quantile of the normal distribution. The normalized residuals are used for downstream correlation analysis.

4, genotyping and quality control

All samples were typed using a high throughput genotyping chip. Quality control is realized at a sample level and an SNP level. At the sample level, sex was inferred by analyzing the X chromosome using the plink software and compared to sex in a questionnaire. Individuals of non-conforming gender were deleted from the data. At the SNP level, SNPs that do not meet the Ha-Weathering equilibrium are deleted from the data. Since FHS is a pedigree sample, SNPs that do not comply with mendelian rules are also deleted.

Genotype complementation

All samples were genotypically complemented using sequencing data for the thousand human genome (2013 version 5 months). Haplotypic data of 240 sequenced individuals are first downloaded from a thousand human genome website, and allele of a reference genome (i.e. thousand human genome data) and a target genome (i.e. a GWAS sample) are checked for consistency, and the individuals with inconsistency are deleted from the target genome. The genotype complementation is carried out by using software FISH, and the algorithm has the advantages of high operation speed, small occupied memory, no need of phase splitting (phasing) in advance on a target genome and the like. And setting software parameters by default.

6, correlation analysis

In finding sample FHS, a mixed linear model was used to examine the genetic association between corrected muscle content and SNPs. In both validation samples, the correlation signal of the discovery sample is validated using a linear model.

Electrophoretic mobility assay (EMSA)

The cells used for EMSA experiments were mouse C2C12 myoblasts. The culture conditions were 37 ℃ and 5% CO2 humidified incubator. Growth was performed in medium supplemented with 10% fetal bovine serum and 100U/mL penicillin and 100. mu.g/mL streptomycin.

EMSA was used to investigate the ability of rs524533 to bind transcription factors. Firstly, a single-stranded DNA probe sequence which takes rs524533 as a center and is 93bp in length is synthesized, biotin is used for marking at the 3' end, and then the sequence is synthesized into a double-stranded DNA probe sequence through a PCR reaction. Nucleoprotein extracted from C2C12 cells was incubated with DNA probe sequences for 20 min at room temperature using a chemiluminescent EMSA kit. The incubation products were electrophoresed in 6% native polyacrylamide gels for 1 hour. After UV cross-linking, the image was developed after incubation for 15 min in a blocking solution containing streptavidin-hrp conjugate.

The experiment set up a comparison group and a blank control group, each containing two alleles.

8, Dual luciferase reporter Gene assay

The dual luciferase reporter gene assay examines the effect of rs524533 on downstream gene expression. First, a double-stranded DNA sequence with rs524533 as the center and 213bp in length is synthesized. Then inserted upstream of the pGL 3-promoter reporter plasmid. The plasmid was then transfected into C2C12 cells using jetPRIME transfection reagent, and PRL-TK vector transfected to express the renin luciferase gene was used as an internal control for transfection efficiency. Luciferase activity was measured 24 hours later with a dual luciferase kit, and normalized and analyzed for renin luciferase activity.

Similarly, the experiment set up a comparative group and a blank control group containing two alleles, respectively.

9, conclusion

The sample size of sample FHS was found to be 6587, of which 55% were female. Significant in the whole genome (GWS, 5.0X 10)^-8) Horizontally, a total of 15 SNPs were associated with muscle content. Of these 12 were located at 6p21.1, one at 5q22.3, one at 9q21.13 and the last at 10q 24.33.

In the validation sample WHI-AA (N-847), 4 SNPs at 6p21.1 were significant (p <0.05) and had the same direction as the effect found in the sample. SNPs at the remaining sites were not significant.

In the validation sample KCOS (N ═ 2219), all of the 4 SNPs validated above were also significant (p < 0.05). At the same time, their effect direction is the same as that of the found sample.

Combining the analysis of the discovery sample and the validation sample, 4 SNPs located at the 6p21.1 site had a very significant and reliable genetic association with muscle content. One of the major SNPs was rs524533, the results are shown in Table 1 below, and the genetic association results for rs524533 are shown in Table 1 below

Note: c is the functional allele in allele C/T.

The three genotypes TT, CT and CC of rs524533 correspond to leg muscle content of on average 15.49 kg, 15.09 kg and 14.56 kg, respectively, corresponding to a reduction of about 0.47 kg per allele C.

To further determine whether rs524533 is able to bind to intracellular transcription factors, we performed EMSA experiments, the results of which are shown in figure 1. rs524533 shows a migration band at the same position in both alleles, indicating that rs524533 does bind to a protein transcription factor or factors. In addition, the binding capacity of the two alleles to transcription factors is different, and is expressed by the difference in gray scale of the corresponding binding bands.

Based on the EMSA results, we further investigated the regulation pattern of rs524533 on gene expression in cells by dual luciferase assay, and the results are shown in fig. 2. Compared with pGL 3-promoter plasmid, the plasmid containing rs524533 reduces the expression of luciferase by more than 30 times, and shows that the SNP has a regulation mode of inhibiting gene expression. Furthermore, there was a statistical difference between luciferase expression activities of the two alleles T and C of rs524533 (p ═ 8.41 × 10^-4) It is shown that this regulatory pattern is allele specific, consistent with the aforementioned EMSA results, better substantiatingrs524533 is a genetic risk factor for muscle content.

Taken together, the two allelic loci T and C of rs524533 are able to discriminate well between high and low muscle groups. When the allele is C, it shows sarcopenia property, and when it is T, it does not show sarcopenia property. Allele type C results in a decrease in muscle content that ultimately leads to the development of sarcopenia, which is statistically extremely significant, and thus can be detected by detecting the site genotype.

The above description of the embodiments is only intended to illustrate the method of the invention and its core idea. It should be noted that, for those skilled in the art, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications will also fall into the protection scope of the claims of the present invention.

Sequence listing

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Claims (7)

1. A kit for detecting sarcopenia, characterized in that: the kit comprises a probe and a primer which can specifically detect SNP sites related to sarcopenia.

2. The kit of claim 1, comprising a probe and primers capable of specifically detecting rs 524533.

3. The kit of claim 2, wherein: the kit comprises the nucleotide sequence shown in SEQ ID NO: 2-5.

SEQ ID NO: 2-5 in the preparation of a kit for sarcopenia detection.

Use of rs524533 as a target for detecting sarcopenia.

Use of rs524533 in the preparation of a kit for the detection of sarcopenia.

7. A method of detecting sarcopenia comprising using a kit of any one of claims 1 to 3.

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