SNP (Single Nucleotide Polymorphism) combination for predicting hepatic fibrosis of female metabolism-related fatty liver disease patient, kit and application of SNP combination and kit

Abstract

The invention provides an SNP (Single Nucleotide Polymorphism) site combination for predicting the hepatic fibrosis risk of a patient with a female metabolism-related fatty liver disease (MAFLD), a kit and application of the SNP site combination and the kit. The SNP locus combination comprises two SNP loci, namely, PNPLA3rs738409C > G and MBOAT7rs641738C > T, and the SNP locus combination comprises two SNP loci, namely, PNPLA3rs738409C > G and The kit comprises a detection system aiming at the two SNP sites. The SNP site combination and the kit for predicting the hepatic fibrosis risk of the female MAFLD patient can rapidly, accurately, simply and conveniently detect the genotype of the patient with high throughput, and the conformity of the detection result and the sequencing result is 100%; and the accuracy and the sensitivity of predicting the hepatic fibrosis risk of the female MAFLD patient based on the detection result are relatively high, and the method has important clinical application significance and value for prognosis and risk assessment of the female MAFLD patient.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a SNP combination, a kit and an application thereof for predicting the risk of liver fibrosis in female patients with metabolic-related fatty liver disease. Background Art

[0002] With the improvement of the quality of life, fatty liver has become a disease that has received great attention internationally in recent years. Because its serious consequences have gradually become apparent, it has become the second leading cause of liver transplantation in the United States and is showing a trend of continued rise. The prevalence of fatty liver in my country is as high as 30%, and the number of patients with cirrhosis and liver cancer caused by it is gradually increasing. It is expected that by 2030, China will surpass the United States to become the country with the highest prevalence of fatty liver.

[0003] The occurrence and development of metabolic associated fatty liver disease (MAFLD) is the result of genetic-environmental interactions, and about 50% of MAFLD is genetically driven. Therefore, some experts suggest that MAFLD be divided into genetic and non-genetic (metabolic) types, which have significant differences in liver outcomes and complications. There are hundreds of millions of MAFLD patients, but only a small number of them have adverse outcomes. Therefore, it is necessary to risk stratify patients and predict their prognosis in order to carry out individualized management.

[0004] Currently, the only predictor of liver cirrhosis is liver fibrosis. Recently, several studies have shown that the additive effect analysis of single nucleotide polymorphisms (SNPs) of pathogenic genes is significantly associated with increased risk of liver steatosis, liver fibrosis, cirrhosis and liver cancer, which is significantly better than single gene SNPs.

[0005] Currently, there are no SNP test kits available on the market at home or abroad for prediction, diagnosis, auxiliary diagnosis or risk stratification of MAFLD patients.

[0006] In addition, the current commercial method for SNP detection is mainly sequencing. It can be used for whole genome sequencing or for sequencing of a gene fragment. Single gene sequencing only detects the SNP of one gene at a time. If multi-gene testing is required, multiple sequencing is required. The cost is relatively high and the operation is complicated. Summary of the invention

[0007] Purpose of the Invention

[0008] In response to the needs in the above-mentioned prior art, the object of the present invention is to provide a SNP combination, a kit and its application that can predict the risk of liver fibrosis in female metabolic-associated fatty liver disease (MAFLD) patients with high sensitivity and high accuracy.

[0009] The inventors found that the combined detection of SNP site polymorphisms of the two genes, MBOAT7 and PNPLA3, can predict liver fibrosis in female MAFLD patients with high accuracy and sensitivity, and thus developed a SNP kit that can predict the risk of liver fibrosis in female MAFLD patients, solving the current problems of low sensitivity of single SNP detection in predicting liver fibrosis and expensive sequencing costs.

[0010] Solution

[0011] To achieve the purpose of the present invention, the technical solution adopted by the present invention is as follows:

[0012] In a first aspect, the present invention provides a SNP site combination for predicting the risk of liver fibrosis in female patients with metabolic-related fatty liver disease, wherein the SNP site combination comprises the following two SNP sites:

[0013] PNPLA3 rs738409C>G and MBOAT7 rs641738C>T.

[0014] In a second aspect, the present invention provides a kit for predicting the risk of liver fibrosis in female patients with metabolic-related fatty liver disease, characterized in that the kit comprises primers and probes for detecting polymorphisms of the following two SNP sites:

[0015] PNPLA3 rs738409C>G and MBOAT7 rs641738C>T.

[0016] In a feasible embodiment, the primers used to detect PNPLA3 rs738409C>G are the following primer pair: an upstream primer having a nucleotide sequence as shown in SEQ ID NO: 1 and a downstream primer having a nucleotide sequence as shown in SEQ ID NO: 2; preferably, the probe used to detect PNPLA3 rs738409C>G is the following probe pair: probe 1 having a nucleotide sequence as shown in SEQ ID NO: 3 and one base thereon, preferably the 7th base, modified with a locked nucleic acid, and probe 2 having a nucleotide sequence as shown in SEQ ID NO: 4.

[0017] In a feasible embodiment, the primers used to detect MBOAT7rs641738C>T are the following primer pair: an upstream primer having a nucleotide sequence as shown in SEQ ID NO:5 and a downstream primer having a nucleotide sequence as shown in SEQ ID NO:6; preferably, the probe used to detect MBOAT7rs641738C>T is the following probe pair: probe 1' having a nucleotide sequence as shown in SEQ ID NO:7, and probe 2' having a nucleotide sequence as shown in SEQ ID NO:8 and one base thereon, preferably the 7th base, has been modified with locked nucleic acid.

[0018] For each of the above probe pairs, the 5' end of each probe is modified with a fluorescent group, the 3' end is modified with a quenching group, and the 5' end fluorescent groups of the two probes in the probe pair are different;

[0019] Preferably, the fluorescent group is selected from FAM, VIC, TET, JOE, HEX, CY3, CY5, ROX and TAMRA, and the quenching group is MGB.

[0020] In a preferred embodiment:

[0021] The 5' end of the probe 1 has a HEX modification, and the 3' end has a MGB modification;

[0022] The probe 2 has a ROX modification at the 5' end and a MGB modification at the 3' end;

[0023] The probe 1' has a VIC modification at the 5' end and a MGB modification at the 3' end;

[0024] The 5' end of the probe 2' has 6-FAM modification, and the 3' end has MGB modification.

[0025] Preferably, the kit further comprises a PCR reaction solution, a positive quality control product and a negative quality control product;

[0026] Preferably, the PCR reaction solution includes Taq enzyme, UDG enzyme, dNTP, Mg 2+ , TrisHCl, KCl;

[0027] Preferably, the positive quality control product is a mixture of PNPLA3 rs738409C>G site wild-type plasmid, PNPLA3rs738409C>G site mutant plasmid, MBOAT7rs641738C>T site wild-type plasmid, and MBOAT7rs641738C>T site mutant plasmid; further preferably, the requirements for the positive quality control product are: the detection result of PNPLA3 rs738409C>G site is CG heterozygous, and the detection result of MBOAT7rs641738C>T site is CT heterozygous;

[0028] Preferably, the negative quality control product is enzyme-free water; further preferably, the requirements for the negative quality control product are: the PNPLA3 rs738409C>G site detection result is negative, and the MBOAT7rs641738C>T site detection result is negative.

[0029] In a third aspect, the present invention provides the use of primers and probes for detecting polymorphisms of the following two SNP sites in the preparation of a product for predicting the risk of liver fibrosis in female patients with metabolic-related fatty liver disease:

[0030] PNPLA3 rs738409C>G and MBOAT7 rs641738C>T.

[0031] In a feasible embodiment, the primers used to detect PNPLA3 rs738409C>G are the following primer pair: an upstream primer having a nucleotide sequence as shown in SEQ ID NO: 1 and a downstream primer having a nucleotide sequence as shown in SEQ ID NO: 2; preferably, the probe used to detect PNPLA3 rs738409C>G is the following probe pair: probe 1 having a nucleotide sequence as shown in SEQ ID NO: 3 and one base thereon, preferably the 7th base, modified with a locked nucleic acid, and probe 2 having a nucleotide sequence as shown in SEQ ID NO: 4.

[0032] In a feasible embodiment, the primers used to detect MBOAT7rs641738C>T are the following primer pair: an upstream primer having a nucleotide sequence as shown in SEQ ID NO:5 and a downstream primer having a nucleotide sequence as shown in SEQ ID NO:6; preferably, the probe used to detect MBOAT7rs641738C>T is the following probe pair: probe 1' having a nucleotide sequence as shown in SEQ ID NO:7, and probe 2' having a nucleotide sequence as shown in SEQ ID NO:8 and one base thereon, preferably the 7th base, has been modified with locked nucleic acid.

[0033] For each of the above probe pairs, the 5' end of each probe is modified with a fluorescent group, the 3' end is modified with a quenching group, and the 5' end fluorescent groups of the two probes in the probe pair are different;

[0034] Preferably, the fluorescent group is selected from FAM, VIC, TET, JOE, HEX, CY3, CY5, ROX and TAMRA, and the quenching group is MGB.

[0035] In a preferred embodiment:

[0036] The 5' end of the probe 1 has a HEX modification, and the 3' end has a MGB modification;

[0037] The probe 2 has a ROX modification at the 5' end and a MGB modification at the 3' end;

[0038] The probe 1' has a VIC modification at the 5' end and a MGB modification at the 3' end;

[0039] The 5' end of the probe 2' has 6-FAM modification, and the 3' end has MGB modification.

[0040] Preferably, the product includes a diagnostic reagent, a diagnostic device and a diagnostic system.

[0041] In a fourth aspect, the present invention provides an application of PNPLA3 rs738409C>G and MBOAT7 rs641738C>T in constructing a computational model for predicting the risk of liver fibrosis in female patients with metabolic-related fatty liver disease, wherein the computational model uses the genotypes of PNPLA3 rs738409C>G and MBOAT7 rs641738C>T as input variables.

[0042] Beneficial Effects

[0043] The present invention first discovered the correlation between the combination of the two SNP sites, PNPLA3 rs738409C>G and MBOAT7 rs641738C>T, and liver fibrosis in female MAFLD patients. Based on this, the present invention proposes a SNP site combination for predicting the risk of liver fibrosis in female MAFLD patients, a kit for predicting the risk of liver fibrosis in female MAFLD patients developed based on the combined detection of the SNP site combination, and the use of the detection primers and probes of the SNP site combination in the preparation of a product for predicting the risk of liver fibrosis in female MAFLD patients.

[0044] Clinical experimental studies have confirmed that the kit of the present invention has high accuracy and sensitivity for predicting the risk of liver fibrosis in female MAFLD patients, and the consistency between the detection results and the sequencing results is 100%. The homozygous wild type, homozygous mutant type and heterozygous type of two sites of the sample can be detected in two tubes at the same time; and the kit adopts the PCR method for detection, without the need for expensive equipment, and can be carried out in medical institutions or testing institutions with PCR detection qualifications, and the detection cost is low, the operation is simple, and the cycle is short. Therefore, it has important clinical application significance and value for the prognosis and risk assessment of female MAFLD patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] One or more embodiments are exemplarily described by the pictures in the accompanying drawings, and these exemplary descriptions do not constitute limitations on the embodiments. The special word "exemplary" here means "used as an example, embodiment or illustrative". Any embodiment described as "exemplary" here is not necessarily interpreted as being superior or better than other embodiments.

[0046] Figure 1 The CC-type amplification curve of the rs738409 site of the PNPLA3 gene is shown.

[0047] Figure 2 The GG-type amplification curve of the rs738409 site of the PNPLA3 gene is shown.

[0048] Figure 3 The CG-type amplification curve of the rs738409 site of the PNPLA3 gene is shown.

[0049] Figure 4 Shows the CC-type amplification curve of the rs641738 site of the MBOAT7 gene.

[0050] Figure 5 Shows the CT-type amplification curve of the rs641738 site of the MBOAT7 gene.

[0051] Figure 6 Shows the TT-type amplification curve of the rs641738 site of the MBOAT7 gene.

[0052] Figure 7 The CC-type amplification curve of the rs738409 site of the PNPLA3 gene is shown.

[0053] Figure 8 Shows the CC sequencing results of the PNPLA3 gene rs738409 site.

[0054] Fig. 9 The GG-type amplification curve of the rs738409 site of the PNPLA3 gene is shown.

[0055] Fig.10 Showing the sequencing results of the GG type at the rs738409 site of the PNPLA3 gene.

[0056] Fig.11 The CG-type amplification curve of the rs738409 site of the PNPLA3 gene is shown.

[0057] Fig.12 Shows the CG sequencing results of the PNPLA3 gene rs738409 site.

[0058] Fig.13 Shows the CC-type amplification curve of the rs641738 site of the MBOAT7 gene.

[0059] Fig.14 Shows the CC sequencing results of the rs641738 site of the MBOAT7 gene.

[0060] Fig.15 Shows the CT-type amplification curve of the rs641738 site of the MBOAT7 gene.

[0061] Fig.16 Shows the CT sequencing results of the MBOAT7 gene rs641738 site.

[0062] Fig.17 Shows the TT-type amplification curve of the rs641738 site of the MBOAT7 gene.

[0063] Fig.18 Shows the TT sequencing results of the MBOAT7 gene rs641738 site.

[0064] Fig.19 PRS of MALFD patients in F0, F1, F2, F3, and F4 groups classified according to the METAVIR score of liver fibrosis MBOAT7 Score statistics chart.

[0065] Fig. 20 PRS of MALFD patients divided into fibrosis group and non-fibrosis group according to whether liver fibrosis occurs MBOAT7 Score statistics chart.

[0066] Fig.21 The ROC curve of the logistic regression model established based on the genotype score of the rs641738 locus of the MBOAT7 gene is shown.

[0067] Fig. 22 PRS of MALFD patients in F0, F1, F2, F3, and F4 groups classified according to the METAVIR score of liver fibrosis PNPLA3 Score statistics chart.

[0068] Fig.23 PRS of MALFD patients divided into fibrosis group and non-fibrosis group according to whether liver fibrosis occurs PNPLA3 Score statistics chart.

[0069] Fig.24 The ROC curve of the logistic regression model established based on the genotype score of the rs738409 locus of the PNPLA3 gene is shown.

[0070] Fig.25 The statistical graph shows the double-locus weighted PRS scores of MALFD patients divided into fibrosis group and non-fibrosis group according to whether liver fibrosis occurs.

[0071] Fig.26 The statistical graph shows the double-locus weighted PRS scores of MALFD patients in groups F0, F1, F2, F3, and F4 classified according to the METAVIR score of liver fibrosis.

[0072] Fig. 27 The ROC curves of the logistic regression model established based on the genotype score of the rs641738 site of the MBOAT7 gene and the genotype score of the rs738409 site of the PNPLA3 gene are shown. DETAILED DESCRIPTION

[0073] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described in conjunction with the embodiments below. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0074] In addition, in order to better illustrate the present invention, numerous specific details are provided in the specific embodiments below. It should be understood by those skilled in the art that the present invention can also be implemented without certain specific details. In some embodiments, raw materials, elements, methods, means, etc. well known to those skilled in the art are not described in detail, so as to highlight the main purpose of the present invention.

[0075] Unless explicitly stated otherwise, throughout the specification and claims, the term “comprise” or variations such as “include” or “comprising”, etc., will be understood to include the stated elements or components but not to exclude other elements or components.

[0076] The present invention is further described in detail below by way of examples.

[0077] About the kit of the present invention

[0078] 1. Detection principle

[0079] Using PCR-fluorescent probe technology, two SNP sites can be detected. The SNP site of a gene can be detected in one reaction system, which contains a pair of amplification primers specific to the site and two differently labeled fluorescent MGB probes (the two probes are used to distinguish wild type and mutant type, respectively). After adding template DNA to the reaction system, the PCR amplification reaction begins. During the reaction, the instrument collects and records the fluorescence signal intensity released by the fluorescent probe after enzyme cleavage in real time. After the reaction is completed, the genotype is determined by analyzing the threshold cycle number (Ct value) of each detection channel.

[0080] Compared with ordinary TaqMan probes, the detection method uses MGB probes with short design sequences, which can reduce nonspecific hybridization. The MGB structure increases the Tm value and enhances the target binding stability. Some bases are locked in nucleic acid modification to improve target recognition and stability.

[0081] 2. Kit Composition

[0082] The kit of the present invention is essentially a PCR detection kit for SNP polymorphism of two key genes related to MAFLD (PCR-fluorescent probe method), which comprises primer-probe mixture A, primer-probe mixture B, positive quality control product and negative quality control product.

[0083] In a feasible embodiment, the specific composition of the kit of the present invention is shown in Table 1 below.

[0084] Table 1. Kit composition

[0085]

[0086]

[0087] Remark:

[0088] Requirements for positive quality control products: PNPLA3 rs738409C>G site detection result is CG heterozygous; MBOAT7rs641738C>T site detection result is CT heterozygous.

[0089] Requirements for negative quality control products: PNPLA3 rs738409C>G site test result is negative; MBOAT7rs641738C>T site test result is negative.

[0090] The primer and probe sequence information involved in Table 1 is shown in Table 2 below.

[0091] Table 2. Primers and probes used

[0092]

[0093] Example 1: SNP site genotype detection and result determination of the kit of the present invention

[0094] The above kit of the present invention was used, and the ABI7500 fluorescence quantitative PCR instrument was used to perform genotype detection of two SNP sites on 33 clinical blood samples from Beijing You'an Hospital affiliated to Capital Medical University (all patients' informed consent had been obtained). The specific detection process is as follows:

[0095] 1) The magnetic bead blood genomic DNA extraction kit produced by Tiangen Biochemical Technology (Beijing) Co., Ltd. was used to extract nucleic acid from 33 clinical samples. The specific procedures are as follows:

[0096] A. Add 200uL of blood, 20uL of proteinase K and 300uL of lysis buffer GHL to a 2mL centrifuge tube, shake and mix, incubate at 65℃ for 15min, invert and mix several times, 3-5 times each time;

[0097] B. Place at room temperature for 5 minutes, add 350uL isopropanol, and shake to mix for 10 seconds;

[0098] C. Add 20uL of magnetic bead suspension G, oscillate and mix for 1min, let stand for 9min, oscillate and mix for 1min every 3min;

[0099] D. Place the centrifuge tube on the magnetic stand and let it stand for 30 seconds. After the magnetic beads are completely adsorbed, carefully aspirate the liquid;

[0100] E. Remove the centrifuge tube from the magnetic rack, add 700uL of buffer GDA, and shake to mix for 5 minutes;

[0101] F. Place the centrifuge tube on the magnetic rack and let it stand for 30 seconds. After the magnetic beads are completely adsorbed, carefully aspirate the liquid;

[0102] G. Remove the centrifuge tube from the magnetic rack, add 700uL of rinse solution PWD, and shake and mix for 2 minutes;

[0103] H. Place the centrifuge tube on the magnetic rack and let it stand for 30 seconds. After the magnetic beads are completely adsorbed, carefully aspirate the liquid;

[0104] I. Repeat steps GH once, attach the centrifuge tube to the magnet, and dry at room temperature for 10-15 minutes.

[0105] J. Remove the centrifuge tube from the magnetic field, add 50-100uL of elution buffer TB, vortex to mix, place at 56℃, and incubate for 10 minutes. During this time, invert and mix 3 times, 3-5 times each time.

[0106] K. Place the centrifuge tube on the magnetic rack and let it stand for 2 minutes. After the magnetic beads are completely adsorbed, carefully transfer the DNA solution to a new centrifuge tube and store it under appropriate conditions.

[0107] 2) PCR detection (system preparation and sample addition)

[0108] A. Take out the PCR reaction solution, primer-probe mixture A and primer-probe mixture B in the kit (PCR-fluorescent probe method), thaw them at room temperature, and shake and mix before use;

[0109] B. Preparation of amplification system: According to Table 3 below, the amplification reaction solutions of PNPLA3 rs738409 site and MBOAT7 rs641738 site were prepared respectively;

[0110] Table 3

[0111]

[0112] C. Dispense the prepared reaction solution into PCR reaction tubes at 23uL / tube.

[0113] D. Take out the positive control and negative control from the kit, thaw at room temperature, and shake to mix before use.

[0114] E. Take 2uL of the extracted nucleic acid, positive quality control and negative quality control respectively and add them into the corresponding PCR reaction tubes, cover the tubes tightly, centrifuge them instantly, and move them to the amplification area for PCR detection.

[0115] F. Place the prepared system into the qPCR instrument and perform amplification according to the program shown in Table 4 below;

[0116] Table 4

[0117]

[0118] 3) PCR result analysis

[0119] Calculate the ΔCt value of the two SNP sites: According to the difference in ΔCt values, determine the genotype of each detection target; the determination criteria of each genotype of the two SNP sites are shown in Table 5, and the schematic diagram of the amplification curve of each genotype of the two SNP sites is shown in Figure 1-Figure 6 The results showed that there were differences in the ΔCt values ​​of different genotypes, and the genotypes could be determined by different difference ranges, proving that the kit was effective in detecting genotypes (in the case of no Ct value, the value was substituted into 40 for calculation).

[0120] Table 5. Genotype determination criteria for two SNP loci

[0121]

[0122] The test results and genotype determination results of 33 clinical blood samples are shown in Table 6.

[0123] Table 6. Results of testing and genotyping of 33 clinical blood samples

[0124]

[0125]

[0126] Example 2: Consistency evaluation of the SNP site genotype detection results of the kit of the present invention and the first generation sequencing results

[0127] The method described in Example 1 was used to extract nucleic acids from 292 clinical blood samples (all with informed consent from the patients), and PCR amplification was performed. The ΔCt values ​​of the two SNP sites were calculated, and the genotype of each test sample was determined based on the ΔCt value difference and the genotype determination criteria described in Example 1. At the same time, the PCR products of each test sample were sent to Shanghai Bioengineering for first-generation sequencing.

[0128] For these 292 clinical samples, the genotype detection results detected by the kit of the present invention and the genotype detection results by first-generation sequencing are shown in the following Table 7.

[0129] The amplification curves and sequencing results of each genotype of the two SNP sites are shown in Figure 7-18 .

[0130] Table 7. Comparison of SNP genotype detection results of the kit of the present invention with those of first generation sequencing

[0131]

[0132]

[0133]

[0134]

[0135]

[0136]

[0137]

[0138]

[0139]

[0140] The results in Table 7 show that the genotypes of PNPLA3 rs738409 and MBOAT7 rs641738 detected by the kit of the present invention are completely consistent with the first generation sequencing results, that is, the consistency between the detection results of the kit of the present invention and the sequencing results is as high as 100%.

[0141] Example 3: Determination of PRS weights of MBOAT7 gene rs641738 site and PNPLA3 gene rs738409 site for liver fibrosis

[0142] This example aims to evaluate the effect of the rs641738 site of the MBOAT7 gene and the rs738409 site of the PNPLA3 gene on the risk of liver fibrosis in female patients with metabolic fatty liver disease (MAFLD) and to determine the weights of these sites in the polygenic risk score (PRS).

[0143] Specifically, we collected clinical data from 680 female MAFLD patients, including 600 patients with fibrosis and 80 patients without fibrosis. All of these patients were confirmed to have MAFLD through strict clinical diagnosis and the degree of liver fibrosis was evaluated.

[0144] The degree of liver fibrosis was evaluated using the METAVIR scoring system, which divided the degree of fibrosis into five levels: F0, F1, F2, F3 and F4, where F0 means no fibrosis, and F1 to F4 respectively indicate mild fibrosis in the portal area, a small amount of fibrous septa but no change in the liver structure, a large number of fibrous septa and the beginning of deformation of the liver structure, and cirrhosis.

[0145] Using the kit of the present invention, the genotype of the patient's MBOAT7 and PNPLA3 gene mutation sites was detected. Specifically, we detected the rs641738 site of the MBOAT7 gene and the rs738409 site of the PNPLA3 gene. According to the genotype, the wild type, heterozygote and homozygote were assigned 0, 1, and 2 points respectively to quantify the contribution of the genotype to the PRS. In order to evaluate the effect of the MBOAT7 and PNPLA3 gene mutation sites on the risk of liver fibrosis, we used the logistic regression analysis method. The logistic regression model is used to determine the effect of the genotype score on the probability of fibrosis, and the formula is as follows:

[0146]

[0147] Among them, p is the probability that the patient has fibrosis, α is the intercept term of the model, β is the regression coefficient of the genotype score, that is, the effect size of the mutation site, and Gi is the genotype score in which wild type, heterozygous and homozygous are assigned 0, 1, and 2, respectively.

[0148] By logistic regression analysis, we calculated the effect size (β value) of the rs641738 site of the MBOAT7 gene and the rs738409 site of the PNPLA3 gene. These β values ​​represent the relative contribution of each mutation site to the risk of liver fibrosis, and the results are summarized in Table 8.

[0149] Table 8

[0150]

[0151] This example evaluates the effect of MBOAT7 and PNPLA3 gene mutation sites on the risk of fibrosis in female MAFLD patients by logistic regression analysis. The results show that both mutation sites significantly affect the risk of fibrosis, and the effect of MBOAT7 gene mutation is greater; among them, the MBOAT7 rs641738 site significantly increases the risk of liver fibrosis, with an OR value of 13.369; the PANPLA3 gene rs738409 site also significantly increases the risk of liver fibrosis, with an OR value of 2.073. The PRS weights of the MBOAT7 gene rs641738 site and the PNPLA3 gene rs738409 site for liver fibrosis are 2.5929 and 0.7289, respectively. According to the parameter values ​​shown in Formula 1 and Table 8, the liver fibrosis risk assessment based on the MBOAT7 gene rs641738 site and the PNPLA3 gene rs738409 site are derived as Formula 2 and Formula 3, respectively.

[0152]

[0153] Among them G i The wild type, heterozygous type and homozygous type are assigned 0, 1 and 2 respectively. Example 4: PRS score of MBOAT7 locus rs641738 and construction of liver fibrosis prediction model based on it

[0154] The PRS score calculation formula of MBOAT7 gene rs641738 is as follows:

[0155] PRS MBOAT7 =β MBOAT7 ×G i (Formula 4)

[0156] where β MBOAT7 That is, the weight 2.5929 calculated in Example 3, G i The wild type, heterozygous, and homozygous of the rs641738 locus have scores of 0, 1, and 2, respectively.

[0157] According to the METAVIR score of liver fibrosis, 680 MALFD patients were divided into 5 groups: F0, F1, F2, F3, and F4. The PRS of each group was MBOAT7 Score Fig.19 ; According to whether the liver fibrosis occurs, the patients were divided into fibrotic and non-fibrotic groups. The PRS of each group MBOAT7 Score Fig. 20 ;

[0158] In a cohort of 600 patients with fibrosis and 80 patients without fibrosis, PRS MBOAT7 The logistic regression algorithm was used to build the model for the features. The model was constructed and tested 1000 times using bootstrap sampling. The number of fibrosis and non-fibrosis samples in the training set was equal to ensure the balance of the model. Finally, the predicted type of each sample was calculated by voting. The ROC curve of the model is shown in Fig.21 ,according to Fig.21 From the ROC curve, we can see that its AUC = 0.72, the sensitivity is 0.51 and the specificity is 0.94 under the optimal cutoff.

[0159] The above results show that: based on the genotype score PRS of the MBOAT7 gene rs641738 locus MBOAT7 The established logistic regression model has good diagnostic performance for liver fibrosis in female MAFLD patients, especially the model has high specificity.

[0160] Example 5: PRS score of PNPLA3 locus rs738409 and construction of liver fibrosis prediction model based on it

[0161] The PRS score calculation formula for PNPLA3 gene rs738409 is as follows:

[0162] PRS PNPLA3 =β PNAPLA3 ×G i (Formula 5)

[0163] where β PNPLA3 That is, the weight 0.7289 calculated in Example 3, G i The wild type, heterozygote and homozygote of rs738409 locus were assigned 0, 1 and 2 points respectively. According to the METAVIR score of liver fibrosis, 680 MALFD patients were divided into 5 groups: F0, F1, F2, F3 and F4. The PRS PNPLA3 Score Fig. 22 ; According to whether the liver fibrosis occurs, the patients were divided into fibrotic and non-fibrotic groups. The PRS of each group PNPLA3 Score Fig.23 ;

[0164] In a cohort of 600 patients with fibrosis and 80 patients without fibrosis, PRS PNPLA3 The logistic regression algorithm was used to build the model for the features. The model was constructed and tested 1000 times using bootstrap sampling. The number of fibrosis and non-fibrosis samples in the training set was equal to ensure the balance of the model. Finally, the predicted type of each sample was calculated by voting. The ROC curve of the model is shown in Fig.24 ,according to Fig.24 From the ROC curve, we can see that its AUC = 0.64, the sensitivity is 0.53 and the specificity is 0.75 under the optimal cutoff.

[0165] The above results show that: PRS based on the genotype score of the PNPLA3 gene rs738409 locus PNPLA3 The established logistic regression model has a certain diagnostic performance for liver fibrosis in female MAFLD patients, with high specificity and sensitivity.

[0166] Example 6: Calculation and modeling of weighted PRS at the rs641738 site of the MBOAT7 gene and the rs738409 site of the PNPLA3 gene

[0167] The weighted PRS calculation formula for the mutation sites of the MBOAT7 gene rs641738 site and the PNPLA3 gene rs738409 site is: PRSj = β j ×G j ; Wherein β is the weight of the mutation site calculated in Example 3; G j is the number of effect alleles of the mutation site detected for the individual, and its possible values ​​are 0, 1, and 2.

[0168] The polygenic risk score is the sum of the weighted PRS of individual genes, that is, PRS = ΣPRS j The PRS scores were divided into 2 groups according to the presence or absence of liver fibrosis. The distribution of PRS is shown in Fig.25 According to the liver fibrosis stage, it is divided into 5 groups: F0, F1, F2, F3, and F4. The distribution of PRS is shown in Fig.26 .

[0169] In a cohort of 600 patients with fibrosis and 80 patients without fibrosis, a logistic regression algorithm was used to build a model with the PRS calculated in this example as the feature. The model was constructed and tested 1000 times using bootstrap sampling. The number of fibrosis and non-fibrosis samples in the training set was equal to ensure the balance of the model. Finally, the predicted type of each sample was calculated by voting. The ROC curve of the model is shown in Fig. 27 ,according to Fig. 27From the ROC curve, we can see that its AUC = 0.79, the sensitivity is 0.66 and the specificity is 0.81 under the optimal cutoff.

[0170] The above results show that the logistic regression model established based on the PRS of the PNPLA3 gene rs738409 site and the MBOAT7 gene rs641783 site has good diagnostic performance for liver fibrosis in female MAFLD patients, and its overall performance is better than that of the single sites of the PNPLA3 gene rs738409 site and the MBOAT7 gene rs641783 site.

[0171] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A SNP site combination for predicting the risk of liver fibrosis in female patients with metabolic-related fatty liver disease, characterized in that: The SNP locus combination includes the following two SNP loci: PNPLA3 rs738409C>G and MBOAT7 rs641738C>T.

2. A kit for predicting the risk of liver fibrosis in female patients with metabolic-related fatty liver disease, characterized in that: The kit includes primers and probes for detecting polymorphisms of the following two SNP sites: PNPLA3 rs738409C>G and MBOAT7 rs641738C>T.

3. The kit according to claim 2, characterized in that The primers used to detect PNPLA3 rs738409C>G are the following primer pair: an upstream primer having a nucleotide sequence as shown in SEQ ID NO: 1 and a downstream primer having a nucleotide sequence as shown in SEQ ID NO: 2; And / or, the primers used to detect MBOAT7rs641738C>T are the following primer pair: an upstream primer having a nucleotide sequence as shown in SEQ ID NO:5 and a downstream primer having a nucleotide sequence as shown in SEQ ID NO:

6.

4. The kit according to claim 3, characterized in that The probes used to detect PNPLA3 rs738409C>G are the following probe pair: probe 1 having a nucleotide sequence as shown in SEQ ID NO:3 and one base, preferably the 7th base, thereof modified with a locked nucleic acid, and probe 2 having a nucleotide sequence as shown in SEQ ID NO:4; And / or, the probe used to detect MBOAT7rs641738C>T is the following probe pair: probe 1' having a nucleotide sequence as shown in SEQ ID NO:7, and probe 2' having a nucleotide sequence as shown in SEQ ID NO:8 and one base, preferably the 7th base, thereof is modified with locked nucleic acid.

5. The kit according to claim 4, characterized in that For each probe pair, the 5' end of each probe is modified with a fluorescent group, the 3' end is modified with a quenching group, and the 5' end fluorescent groups of the two probes in the probe pair are different; Preferably, the fluorescent group is selected from FAM, VIC, TET, JOE, HEX, CY3, CY5, ROX and TAMRA, and the quenching group is MGB.

6. The kit according to claim 5, characterized in that The 5' end of the probe 1 has a HEX modification, and the 3' end has a MGB modification; The probe 2 has a ROX modification at the 5' end and a MGB modification at the 3' end; The probe 1' has a VIC modification at the 5' end and a MGB modification at the 3' end; The 5' end of the probe 2' has 6-FAM modification, and the 3' end has MGB modification.

7. The kit according to any one of claims 2 to 6, characterized in that The kit also includes a PCR reaction solution, a positive quality control product and a negative quality control product; Preferably, the PCR reaction solution includes Taq enzyme, UDG enzyme, dNTP, Mg 2+ , TrisHCl, KCl; Preferably, the positive quality control product is a mixture of PNPLA3 rs738409C>G site wild-type plasmid, PNPLA3rs738409C>G site mutant plasmid, MBOAT7rs641738C>T site wild-type plasmid, and MBOAT7rs641738C>T site mutant plasmid; further preferably, the requirements for the positive quality control product are: the detection result of PNPLA3 rs738409C>G site is CG heterozygous, and the detection result of MBOAT7rs641738C>T site is CT heterozygous; Preferably, the negative quality control product is enzyme-free water; further preferably, the requirements for the negative quality control product are: the PNPLA3rs738409C>G site detection result is negative, and the MBOAT7rs641738C>T site detection result is negative.

8. Use of primers and probes for detecting polymorphisms of the following two SNP sites in the preparation of a product for predicting the risk of liver fibrosis in female patients with metabolic-related fatty liver disease: PNPLA3 rs738409C>G and MBOAT7 rs641738C>T.

9. The use according to claim 8, characterized in that: The primers used to detect PNPLA3 rs738409C>G are the following primer pair: an upstream primer having a nucleotide sequence as shown in SEQ ID NO:1 and a downstream primer having a nucleotide sequence as shown in SEQ ID NO:2; preferably, the probe used to detect PNPLA3 rs738409C>G is the following probe pair: probe 1 having a nucleotide sequence as shown in SEQ ID NO:3 and one base, preferably the 7th base, thereof being modified with a locked nucleic acid, and probe 2 having a nucleotide sequence as shown in SEQ ID NO:4; And / or, the primers used to detect MBOAT7rs641738C>T are the following primer pair: an upstream primer having a nucleotide sequence as shown in SEQ ID NO:5 and a downstream primer having a nucleotide sequence as shown in SEQ ID NO:6; preferably, the probe used to detect MBOAT7rs641738C>T is the following probe pair: probe 1' having a nucleotide sequence as shown in SEQ ID NO:7, and probe 2' having a nucleotide sequence as shown in SEQ ID NO:8 and one base thereon, preferably the 7th base, has been modified with locked nucleic acid. Wherein, for each probe pair, the 5' end of each probe is modified with a fluorescent group, the 3' end is modified with a quenching group, and the 5' end fluorescent groups of the two probes in the probe pair are different; Preferably, the fluorescent group is selected from FAM, VIC, TET, JOE, HEX, CY3, CY5, ROX and TAMRA, and the quenching group is MGB; Further preferably, the 5' end of the probe 1 is modified with HEX, and the 3' end is modified with MGB; the 5' end of the probe 2 is modified with ROX, and the 3' end is modified with MGB; the 5' end of the probe 1' is modified with VIC, and the 3' end is modified with MGB; the 5' end of the probe 2' is modified with 6-FAM, and the 3' end is modified with MGB; Preferably, the product includes a diagnostic reagent, a diagnostic device and a diagnostic system.

10. Application of PNPLA3 rs738409C>G and MBOAT7 rs641738C>T in constructing a computational model for predicting the risk of liver fibrosis in female patients with metabolic-related fatty liver disease, characterized in that: The computational model used the genotypes of PNPLA3rs738409C>G and MBOAT7rs641738C>T as input variables.

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