CN113684215A

CN113684215A - Novel mutated gene associated with hypercholesterolemia and use thereof

Info

Publication number: CN113684215A
Application number: CN202111041174.9A
Authority: CN
Inventors: 程翔; 查灵凤; 王景林; 刘美琳; 杨芬; 郭爽
Original assignee: Union Hospital Tongji Medical College Huazhong University of Science and Technology
Current assignee: Union Hospital Tongji Medical College Huazhong University of Science and Technology
Priority date: 2021-09-07
Filing date: 2021-09-07
Publication date: 2021-11-23

Abstract

The invention belongs to the field of LDLR gene research, and particularly relates to a new mutant gene related to hypercholesterolemia and its application. Nucleic acids, including the following target fragments, compared with wild-type LDLR gene fragments, the target fragment has a c.1879G>A mutation. A polypeptide having the p.A627T mutation compared to wild-type LDLR. The application of a gene fragment in the preparation of a drug for preventing and treating hypercholesterolemia, the gene fragment is a gene fragment whose single nucleotide at the c.1879 site of LDLR is G. A drug for preventing and treating hypercholesterolemia, containing at least one of the following a. c.1879G>A mutation inhibitor of LDLR gene, b. p.A627T mutation inhibitor of LDLR gene, c. gene fragment, said gene fragment is LDLR A gene fragment in which the single nucleotide at the c.1879 position of the gene is G. The invention discovers a new mutation site c.1879G>A of the LDLR gene, and the detection of the mutation site can be used for auxiliary diagnosis of hypercholesterolemia patients; it is of great significance for the diagnosis and treatment of hypercholesterolemia .

Description

Novel mutated gene associated with hypercholesterolemia and use thereof

Technical Field

The invention belongs to the field of LDLR gene research, and particularly relates to a novel mutant gene related to hypercholesterolemia and application thereof.

Background

Familial Hypercholesterolemia (FH) is one of the most common metabolic inherited diseases characterized by extremely high levels of Total Cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C), with excess cholesterol precipitating in the tissues, resulting in xanthoma, acceleration of the progression of atherosclerosis and increased risk of early coronary artery disease. Dyslipidemia, characterized by increased LDL-C or TC, is an important risk factor for atherosclerotic cardiovascular disease. Therefore, the reduction of LDL-C level is the primary intervention target for preventing and controlling atherosclerotic cardiovascular diseases, and the discovery and accurate regulation of novel medicaments for LDL-C level have great significance for maintaining human health. Without proper preventive work, coronary artery disease occurs before 65 years of age in approximately 85% of male FH patients and 50% of female FH patients, while homozygous familial hypercholesterolemia usually dies before 30 years of age. Screening of myocardial infarction patients < 50 years of age showed that about 2% of patients have familial hypercholesterolemia. Thus, early CVD (especially early myocardial infarction), stroke and increased risk of total mortality are the main features of untreated familial hypercholesterolemia. Increased age, increased body mass index, type 2 diabetes, hypertension and a history of smoking can increase the risk of cardiovascular disease in heterozygous familial hypercholesterolemia patients.

At present, three gene mutations are considered as main pathogenic mutations, (1) mutation of low-density lipoprotein receptor gene (LDLR) causes reduction of the number of LDLR molecules or reduction of LDLR activity, thereby reducing clearance of LDL-C; (2) mutation of apolipoprotein B gene (APOB), deficient APOB100 on LDL unable to bind LDLR; (3) mutation of proprotein convertase subtilisin kexin 9 gene (PCSK9) results in rapid internalization and degradation of LDLR. In rare cases, mutations in other genes involved in lipoprotein metabolism APOE (encoding apolipoprotein E) and STAP1 (encoding signal transduction adaptor 1) may also lead to familial hypercholesterolemia. At present, FH related pathogenic sites still remain to be further studied. Thus, the discovery and proposal of any one or a group of genes associated with familial hypercholesterolemia would be an important technical contribution to the art.

Disclosure of Invention

Aiming at the problems, the invention provides the LDLR gene mutant and the application thereof, mainly discovers a new LDLR gene mutant, fills up the missing in the gene aspect of familial hypercholesterolemia, discovers some new mutant genes and provides a new scheme for the treatment and screening of subsequent diseases.

In order to solve the problems, the invention adopts the following technical scheme:

a nucleic acid comprising a nucleic acid sequence comprising a target fragment,

the target fragment has a c.1879G > A mutation compared to the wild-type LDLR gene fragment.

A polypeptide having a p.a627t mutation compared to wild type LDLR.

A gene mutation having a c.1879G > A mutation compared to the wild-type LDLR gene.

Use of a biological model for the preparation of a hypercholesterolemia screening formulation, said biological model carrying at least one of:

a. the aforementioned nucleic acids;

b. the aforementioned polypeptides;

c. mutation of the aforementioned gene.

A kit for screening a biological sample for hypercholesterolemia, comprising

A reagent capable of detecting an LDLR gene mutant, wherein the LDLR gene mutant is at least one of the following compared with a wild-type LDLR gene

a. The aforementioned nucleic acids;

b. the aforementioned polypeptides;

c. mutation of the aforementioned gene.

In some embodiments, the reagent in the kit is a nucleic acid probe or primer;

preferably, the nucleotide sequence of the primer includes:

a forward primer of sequence tcatcccagtgtttaacgg,

the reverse primer of sequence gtcagggcaggaacgaga.

A construct comprising a nucleic acid or a genetic mutation thereof.

Use of an inhibitor for the manufacture of a medicament for the prevention or treatment of hypercholesterolemia, said inhibitor having an inhibitory effect on at least any one of:

a.1879G > A mutation of LDLR gene,

p.a627t mutation of ldlr gene.

The application of the gene segment in preparing the medicine for preventing and treating hypercholesterolemia is the gene segment with G as c.1879 site mononucleotide of LDLR.

A medicine for preventing and treating hypercholesterolemia, at least one of the following medicines

a c.1879G > A mutation inhibitor of LDLR gene,

a p.A627T mutation inhibitor of LDLR gene,

c. a gene segment, wherein the gene segment is a gene segment with C.1879 site mononucleotide of LDLR gene as G,

d. a gene carrier, which contains a gene segment capable of replacing mononucleotide A at the c.1879 site of the LDLR gene with mononucleotide G and expressing.

The invention has the beneficial effects that:

(1) a new mutation site c.1879G > A of the LDLR gene is found, and the detection of the mutation site can be used for auxiliary diagnosis of hypercholesteremia patients; has important significance for diagnosing and treating hypercholesterolemia;

(2) the LDLR mutation site c.1879G > A is rare, and the mutation site in the invention is included in a diagnostic kit, so that the diagnostic site of the hypercholesterolemia diagnostic kit is further enriched;

(3) under the premise of not violating relevant regulations such as law and the like, the mutant site is repaired by means of gene engineering, and related diseases can be prevented and treated;

(4) the genotype of c.1879G > A locus in a human genome can be detected as a means for screening and identifying hypercholesterolaemia; or the substance for detecting the c.1879G > A mutation or the genotype in the human genome is used for preparing products for screening patients with hypercholesterolemia, detecting products with hypercholesterolemia, and detecting, identifying or assisting in identifying single nucleotide polymorphism related to the hypercholesterolemia. The above-mentioned substances for detecting c.1879G > A mutation or genotype in human genome include, but are not limited to, PCR primer pairs and probe sets for amplifying genomic DNA fragments containing c.1879G > A sites.

Drawings

FIG. 1 is a family map of a hypercholesterolemic patient;

FIG. 2 is a comparison of the Sanger sequencing verification peaks of the LDLR gene c.1879G > A mutation in proband normal human patients with hypercholesterolemia;

FIG. 3 is a screen of c.1879G > A mutation in the normal population.

Detailed Description

The invention is further illustrated below:

the first aspect of this section discloses the following technical contents:

a nucleic acid comprising a nucleic acid sequence comprising a target fragment,

the target fragment has a c.1879G > A mutation compared to the wild-type LDLR gene (one reference sequence is SEQ NO: 1). Other existing or subsequently discovered LDLR genes, provided that there is a c.1879G > A mutation, and mutants having the same, similar effects as those of the present invention, should fall within the scope of the present invention. It is not limited that other sequences than the c.1879g > a mutation must be identical to SEQ NO: 1 are identical. The nucleic acid is DNA.

A polypeptide having a p.a627t mutation compared to wild type LDLR (one is SEQ NO: 2). As before, it is not limited that other sequences than the p.a627t mutation must be identical to SEQ NO: 2 are identical.

Use of a biological model in the preparation of a screening formulation, said biological model carrying at least one of the following:

a. the aforementioned nucleic acid;

b. the aforementioned polypeptides;

c. mutation of the aforementioned gene.

The screening agent is an agent for screening familial hypercholesterolemia.

A kit for screening a biological sample for familial hypercholesterolemia, comprising

An agent capable of detecting a LDLR gene mutant, said LDLR gene mutant having a c.1879G > A mutation compared to a wild-type LDLR gene.

In a parenchymal mode, the LDLR gene mutant is at least one of the following:

a. the aforementioned nucleic acid;

b. the aforementioned polypeptides;

c. mutation of the aforementioned gene.

The reagent is a nucleic acid probe or a primer; in one embodiment, the method comprises the step of,

the nucleotide sequence of the primer comprises:

forward primer with sequence tcatcccagtgtttaacgg

The reverse primer of sequence gtcagggcaggaacgaga.

A construct comprising the aforementioned nucleic acid or the aforementioned gene mutation.

Recombinant cells obtained by transforming a recipient cell with the aforementioned constructs or expressing the aforementioned polypeptides.

The recombinant protein for preparing the medicine for preventing and treating hypercholesterolemia is expressed by the recombinant cell.

a.1879G > A mutation of LDLR gene,

p.a627t mutation of a ldlr gene polypeptide.

a c.1879G > A mutation inhibitor of LDLR gene,

a p.a627t mutation inhibitor of a ldlr gene polypeptide.

a c.1879G > A mutation inhibitor of LDLR gene,

a p.A627T mutation inhibitor of LDLR gene,

c. a gene segment, wherein the gene segment is a gene segment with the C.1879 site mononucleotide of No. 13 exon of LDLR as G,

d. a gene vector, which comprises a gene segment capable of replacing a single nucleotide A at a position c.1879 of an exon13 of an LDLR gene with a single nucleotide G and expressing the gene segment.

The second aspect of this section introduces a method for preparing a portion of the plasmid and pharmaceutical intermediate:

the preparation method of the plasmid for preparing the medicine for preventing and treating hypercholesterolemia comprises the following steps:

(A) taking a wild type LDLR gene as a template, and amplifying to obtain a mutant LDLR cDNA fragment;

(B) carrying out enzyme digestion on the GATC sequence methylated at the adenine N6 position in the DNA double strand, and purifying a mutant LDLR cDNA fragment;

(C) carrying out double enzyme digestion on the mutant LDLR cDNA fragment and an expression vector by using HindIII enzyme and Kpn1 enzyme, and carrying out T4 DNA ligase enzyme linkage to obtain an expression plasmid;

(D) adding the system obtained by the enzyme-linked reaction into an escherichia coli competent cell to obtain plasmid bacterial liquid;

(E) extracting plasmid from the plasmid bacterial liquid.

More specific description of the above steps:

in the step (A), a wild LDLR gene is used as a template, an upstream primer F-mut: 5'-tcatcccagtgtttaacgg-3' is subjected to site-directed mutagenesis, and a downstream primer R-mut: 5'-gtcagggcaggaacgaga-3' and DNA polymerase Primer Star are amplified by polymerase chain reaction to obtain mutant LDLR cDNA segment; and/or

In the step (B), a DpnI enzyme is used for enzyme digestion of a methylated GATC sequence at the adenine N6 position in a DNA double strand, a template plasmid is removed, a PCR mutation product is left, and a mutation LDLR cDNA fragment is subjected to preliminary purification; and/or

In the step (C), the expression vectors are pCMV-3XFLAg and pEGFP-C1, and the expression plasmids are pCMV-3XFLAg-LDLR-Mut and pEGFP-C1-LDLR-Mut; and/or

In the step (E), the correct plasmid bacterial liquid is determined through monoclonal antibody identification and/or sequencing; and/or

In step (E), Plasmid extraction was performed by using the Endo-free Plasmid DNA Mini Kit II.

The use of an inhibitor in a medicament for the prevention and treatment of familial hypercholesterolemia, the inhibitor having an inhibitory effect on at least any one of:

a.1879G > A mutation of LDLR gene,

p.a627t mutation of a ldlr gene polypeptide.

a c.1879G > A mutation inhibitor of LDLR gene,

a p.A627T mutation inhibitor of LDLR gene,

c. a gene segment, wherein the gene segment is a gene segment with C.1879 site mononucleotide of LDLR as G,

d. a gene vector which contains a gene segment capable of replacing mononucleotide A at the c.1879 site of the TTN gene with mononucleotide G and expressing

A specific construction step of the plasmid for preparing the medicament for preventing and treating the hypercholesterolemia comprises the following steps:

cDNA (CCDS12254, NM-000527.4) expression plasmids of the wild-type LDLR gene were purchased from Weizhen Biotech, Inc.

(1) Taking wild type LDLR cDNA as a template, and an upstream primer LDLR-HindIII-F with a HindIII enzyme cutting site: tcatcccagtgtttaacgg and a downstream primer gtcagggcaggaacgaga with a Kpn1 restriction site;

and amplifying by Polymerase Chain Reaction (PCR) with high fidelity DNA polymerase Primer Star to obtain a wild type LDLR cDNA fragment with the enzyme cutting site.

(2) The wild-type LDLR cDNA in the above step is used as a template, and a mutant LDLR cDNA fragment is obtained by Polymerase Chain Reaction (PCR) amplification of a site-specific mutagenesis upstream Primer tcatcccagtgtttaacgg, a site-specific mutagenesis downstream Primer gtcagggcaggaacgaga and a high fidelity DNA polymerase Primer Star. The specific reaction system and the amplification steps are as follows:

reaction system:

and (3) amplification procedure:

(3) DpnI enzyme digestion of mutant PCR products

DpnI recognizes and cleaves the GATC sequence methylated at adenine N6 (N6-methyaddine) in the DNA duplex. The plasmid extracted from E.coli is methylated and the PCR product is not methylated, so that the DpnI enzyme can specifically cleave the template plasmid without affecting the PCR product, thereby removing the template plasmid to leave the PCR mutation product.

DpnI enzyme digestion system and steps:

buffer and water are mixed fully and evenly, then DpnI enzyme is added, after the DpnI enzyme is added, the mixture can be blown by a gun or mixed evenly by gentle vortex, and the mixture is incubated for 1 hour or more at 37 ℃.

(4) Enzyme digestion of the target fragment and the vector: the fragment and the expression vector were digested simultaneously with HindIII enzyme and Kpn1 enzyme.

Cleavage system of target fragment (wild type LDLR cDNA and mutant LDLR cDNA):

expression vector (pCMV-3XFlag and pEGFP-C1) digestion system:

and carrying out enzyme digestion reaction at 37 ℃ for 6-8 hours, and respectively purifying and recovering fragments and the vector.

(5) The target fragment is enzymatically linked to a vector

And (3) purifying and recovering the double-enzyme-digested fragments and the vector, and measuring the concentration of the double-enzyme-digested fragments and the vector, wherein the concentration of the double-enzyme-digested fragments and the vector is determined according to a target fragment (mol): vector (mol) ═ 3:1, enzyme using T4 DNA ligase at 4 ℃ overnight.

The wild LDLR cDNA segment and the mutant LDLR cDNA segment are respectively connected with an expression vector pCMV-3XFlag enzyme to obtain expression plasmids pCMV-3XFlag-LDLR-Wt and pCMV-3 XFlag-LDLR-Mut.

The wild type LDLR cDNA fragment and the mutant LDLR cDNA fragment are respectively connected with an expression vector pEGFP-C1 enzyme to obtain expression plasmids pEGFP-C1-LDLR-Wt and pEGFP-C1-LDLR-Mut.

The concrete system is as follows:

note the negative control with only vector and no target fragment in the set-up system.

(6) And (3) transformation: respectively adding 10L of system and negative control system after enzyme-linked reaction into 100L of DH5 alpha escherichia coli competent cells, placing on ice for 30 minutes, thermally shocking at 42 ℃ for 90 seconds, immediately placing on ice, standing for 3 minutes, then adding 500L of LB culture medium without antibiotics, placing on a shaking table at 37 ℃ for 100 revolutions per minute, recovering and activating for 2 hours, 3000 Xrpm, centrifuging for 5 minutes, reserving 100uL of supernatant, slightly suspending the thallus, coating the thallus on an LB solid culture plate containing corresponding resistance, and culturing in a 37 ℃ culture box for 12-16 hours.

(7) Identification of the single clone: plates with enzyme linked transformation and negative control transformation were observed and, under normal conditions, the negative plates showed little or no long clones. Selecting a plurality of monoclonals in 3mL of LB liquid culture medium with corresponding resistance, oscillating the shaking table at 37 ℃ for 200 r/min, culturing for 12 hours, firstly taking 200uL of bacterial liquid and preserving the seeds with 20% of glycerol, and extracting plasmids from the residual bacterial liquid by using a plasmid miniprep kit to perform double enzyme digestion detection.

(8) Sequencing: and (3) if the plasmid detected by double enzyme digestion in the last step is subjected to agarose gel detection to detect two bands of a target fragment and a vector fragment, selecting 1-2 tubes of plasmids for sequencing.

The bacterial liquid of the Plasmid with correct sequencing is inoculated into 15mL LB liquid culture medium with corresponding resistance, the Plasmid is extracted by using an Endo-free Plasmid DNA Mini Kit II, the agarose gel is detected, the concentration is measured, and the bacterial liquid is stored at-20 ℃.

(9) High purity plasmid extraction

Taking the plasmid bacterial liquid with correct sequencing to a 50ml centrifugal tube containing LB culture medium with 20ml corresponding antibiotics, and culturing for 12-16 hours at 37 ℃ by vibrating a shaking table at 200 rpm. And (3) rotating a low-speed centrifuge 3600, centrifuging for 20 minutes, removing supernatant, reversely buckling a tube on clean paper for 5 minutes, and depositing thalli at the bottom of the tube for later use. Using the Endo-free Plasmid DNA Mini Kit II Kit of OMEGA, high quality endotoxin-free Plasmid was extracted by the following procedure

1. Adding 500 mul of equilibrium buffer solution into an adsorption column, standing for 5 minutes, centrifuging at 12000g for 2 minutes, and discarding the liquid in a collection tube;

2. adding 500 μ l Solution of Solution I (RNase A is added before use) into the thallus precipitate, blowing or swirling by using a pipette, and completely blowing the precipitate until the sterile body is agglomerated;

3. transferring the resuspended bacteria liquid into a 2ml EP tube, adding 500 mul solution II, immediately and slightly reversing and uniformly mixing for 6-8 times, completely cracking the thallus, clarifying the bacteria liquid, and standing for 5 minutes;

4. 250 μ l of ice bath N3 buffer was immediately added and the mixture was gently inverted 6-8 times until complete mixing and a white flocculent precipitate formed. 12000g and rotating and centrifuging for 15 minutes;

5. carefully pipette the supernatant into a new 1.5ml EP tube, add 0.1 volume of an ice bath ETR solution, mix by turning upside down and turn the solution cloudy. Standing on ice for 10 min, reversing and mixing for several times, and making the solution clear;

incubation with a metal thermostat at 6.42 ℃ for 5 minutes, the solution became cloudy, centrifuged at 12000Xg for 5 minutes, and blue ETR at the bottom of the tube;

7. carefully sucking the supernatant, transferring the supernatant into a 2ml EP tube, adding 0.5-time volume of absolute ethyl alcohol, turning the mixture up and down, uniformly mixing, and standing at room temperature for 5 minutes;

8. sucking 700ul (step 7) of the solution by a liquid transfer machine into the adsorption column after balanced treatment, standing for 5 minutes, centrifuging at 12000Xg for 2 minutes, and discarding the liquid in the collection tube;

9. repeating the steps 8 to 7, wherein all the liquid passes through the adsorption column;

10. adding 500 μ l HBCbuffer into adsorption column, standing at room temperature for 2 min, centrifuging at 12000Xg for 1 min, and discarding the liquid in the collection tube;

11. add 600. mu.l DNA wash buffer to the adsorption column, let stand at room temperature for 2 minutes, centrifuge at 12000Xg for 1 minute, discard the waste. Washing is repeated once;

12. the adsorption column was idle at 12000Xg and centrifuged for 2 min to completely remove the washing buffer;

13. placing the adsorption column in a new collecting pipe, standing at room temperature until the absolute ethyl alcohol is volatilized;

14. carefully adding 80 mul of elution buffer solution or ultrapure water on an adsorption column membrane, standing for 10 minutes at room temperature, and centrifuging for 2 minutes at 12000Xg to obtain a plasmid solution;

15. the plasmid concentration was determined by using a NanoDrop2000 c ultramicro UV-visible spectrophotometer and stored at-20 ℃ for future use.

Because LDLR is crucial to cholesterol metabolism, any mutation in any part of the gene can cause diseases. Mutations based on LDLR protein synthesis and function can be classified as type 5: the l-type mutation is a non-expression allele and comprises promoter sequence mutation, nonsense mutation, frame shift mutation and splicing mutation, and the mutation causes the cell not to express LDLR; the 2-type mutation is a transport-defective allele, mainly occurs in a ligand binding domain and an epidermal growth factor precursor domain, can synthesize a receptor, but is blocked in the transport from the endoplasmic reticulum to the Golgi apparatus, and is finally degraded after being accumulated in the endoplasmic reticulum; the 3-type mutation is combined with a defective allele, and the type is common, and is characterized in that an abnormal receptor protein coded by the mutant gene can reach the cell surface, but loses the function of combining LDL, and the mutation also occurs in a ligand binding domain and an epidermal growth factor precursor domain; the 4-type mutation is an inward shift defective allele, the type is rare, the inward shift defective allele occurs in a cytoplasmic region or a transmembrane domain, and the inward shift defective allele is caused by the mutation of a codon encoding an NPVY sequence and is characterized in that a receptor can be combined with LDL but can not transport the LDL to the cell surface and gather in a covered pit; the 5-type mutation is a recycling defect allele, a receptor can be combined with LDL and can also move into a human cell, but can not be separated from the LDL in a lysosome, the receptor and a ligand are simultaneously degraded, so that LDLR can not be recycled to the cell surface, and the mutation is mostly generated in an epidermal growth factor precursor structural domain.

It should be noted that the mutation sites and sequences given above are all referred to by the contents of the proteon sequencing platform, and it should be understood by those skilled in the art that the mutation sites and sequences shown may be slightly different or changed due to the update of the database or the difference of the database, and these differences or changes can be found by the contents given in the database as the standard, and these differences or changes are also included in the protection scope of the present invention.

Thus, the recombinant cells obtained by transforming the receptor cells with the constructs according to the embodiments of the present invention can be effectively used as a model for research related to familial hypercholesterolemia, particularly heterozygous familial hypercholesterolemia. Wherein the kind of the recipient cell is not particularly limited.

The term "construct" as used in the present invention refers to a genetic vector comprising a specific nucleic acid sequence and capable of transferring the nucleic acid sequence of interest into a host cell to obtain a recombinant cell. According to an embodiment of the present invention, the form of the construct is not particularly limited. According to an embodiment of the present invention, it may be at least one of a plasmid, a phage, an artificial chromosome, a Cosmid (Cosmid), a virus, preferably a plasmid. The plasmid is used as a genetic vector, has the characteristics of simple operation and capability of carrying a larger fragment, and is convenient to operate and process. The form of the plasmid is not particularly limited, and may be a circular plasmid or a linear plasmid, and may be either single-stranded or double-stranded. The skilled person can select as desired. The term "nucleic acid" used in the present invention may be any polymer comprising deoxyribonucleotides or ribonucleotides, including but not limited to modified or unmodified DNA, RNA, the length of which is not subject to any particular limitation. For constructs used to construct recombinant cells, it is preferred that the nucleic acid be DNA, as DNA is more stable and easier to manipulate than RNA.

The third aspect of this section is illustrated in connection with some research examples:

sample collection

The inventor collected 1 family of hypercholesterolemia in wuhan, as shown in figure 1,

the family members were collected for 3 total, with 1 patient and 2 normal persons.

□ indicates normal males, ■ indicates diseased males, o indicates normal females, ● indicates diseased females, and ↗ indicates probands. All family members participating in the present invention signed informed consent for a total of 3 persons participating in the present invention. The inventors collected peripheral blood samples of patients in the family of the aforementioned hypercholesterolemic patients and normal persons in the family.

DNA extraction: extracting DNA of human peripheral blood leukocytes by using a TIANAmp blood genome DNA extraction kit, and detecting the concentration and purity of the extracted DNA sample by using a NanoDrop2000 spectrophotometer and agarose gel electrophoresis detection. The obtained genomic DNA OD260/OD280 of each specimen is between 1.8 and 2.0, and the concentration is not less than 80 ng/ml.

Whole exon sequencing

The inventor carries out whole exoscope group sequencing on probands in a hypercholesterolemia family by using a proteon sequencing platform.

Mutation detection, annotation, and database comparison

The results of variation detection and annotation provided by a sequencing company are researched on two types of mutations which are most likely to be related to pathology, namely nonsynonymous mutation, coding region insertion and deletion. Results in these samples, the proband was found to have 82570 SNV mutations, 15209 indel mutations, and 79 CNVs. And (3) removing all known variants with allele frequency more than 0.01 in the database according to the filtration of public databases of dbSNP database, HapMap database and thousand human genome database. Screening for mutations in the common gene LDLR, PCSK9, APOB leading to familial hypercholesterolemia. Thus, the inventors found that proband had a heterozygous mutation shown in FIG. 2, missense mutation c.1879G > A, p.A627T: GCC > ACC (p.Ala627Thr), in exon13 of the LDLR gene.

The mutation type is heterozygous mutation, missense mutation, the mutation frequency is 0.000004, the mutation belongs to rare mutation, and the mutation type is not reported in Chinese population.

Sequencing verification by Sanger method

1. Primer design

Designing a mutation site amplification primer by using Genetool software:

a forward primer: 5'-tcatcccagtgtttaacgg-3'

Reverse primer: 5'-gtcagggcaggaacgaga-3' are provided.

DNA amplification (PCR)

The PCR reaction system amounted to 25 ul: 1ul of sample DNA, forward and reverse primers 0.5ul each, 12.5ul of 2 XTSINGKE Master Mix and 10.5ul of ddH 2O.

PCR amplification procedure: the sample is loaded on a machine, preheated at 94 ℃ for 3 minutes, circularly denatured at 94 ℃ for 30s, annealed at 59 ℃ for 30s with gradient temperature, extended at 72 ℃ for 35 times, kept at 72 ℃ for 10 minutes, and then the end temperature is 4 ℃.

PCR product detection

And detecting the PCR product by agarose gel electrophoresis.

3. Sequencing

After successful PCR, Sanger sequencing was performed using an ABI3730XL type sequencer for forward and reverse sequencing.

Analysis of sequencing results

Finch TV Version 1.4.0 was used to read Sanger sequencing results.

The proband carries a mutation in the LDLR gene, which is located in the No. 13 exon, c.1879G > A, p.A627T: GCC > ACC (p.Ala627Thr), resulting in the change of alanine at position 627 to threonine.

The mutation type is heterozygous mutation, missense mutation, the mutation frequency is 0.000004, the mutation belongs to rare mutation, and the mutation type is not reported in Chinese population. Predicted to be harmful is: pathogenicity (SIFT, Polyphen2, LRT, MutationTaster, FATHMM), changing the mutated region to conservative. The literature search did not find the relationship between the mutations found in the present invention and familial hypercholesterolemia.

Bioinformatics prediction

(1) Analysis of hazard of mutation

According to analysis of harmfulness prediction software SIFT, Polyphen2_ HVAR _ pred and MutationTaster _ pred, the site mutation is found to be the harmfulness of the mutation, and diseases can be caused after the mutation. Predicted to be harmful is: the mutagenic region was conserved by the change in pathogenicity (SIFT, Polyphen2, LRT, MutationTaster, FATHMM).

(2) Conservation assay

The site is located at the 627th amino acid position of the LDLR protein, and the site is conservative and can cause the protein to be abnormally functional if the site is mutated.

(3) Function prediction

The mutation exon13: c.1879G > A, p.A627T: GCC > ACC (p.Ala627Thr) results in that the 627 alanine is changed into threonine, and the normal termination code can not appear, thus influencing the normal translation of LDLR protein and further influencing blood fat.

The region of the site is the epidermal growth factor precursor domain of the LDLR protein, the region is responsible for regulating and controlling the transport and the combination of the LDLR, if the region is changed, the endocytosis and intracellular transport of the LDLR are abnormal, and the blood fat is increased.

Sixth, clinical analysis

(1) ACMG Standard rating

This locus can be scored as pathogenic according to ACMG genetic variation classification criteria and guidelines.

(2) The condition of medication

Proband maximal LDL-C: 8.05mmol/L, the LDL-C of the proband is reduced from the maximum 8.05 to 5.36mmol/L after the atorvastatin is taken at the daily dose, the statins are continuously added with ezetimibe, and the level of the LDL-C is kept at 1.2mmol/L after the PCSK9 inhibitor is injected for two weeks, so that the treatment effect is better. A PCSK9 inhibitor that binds to PCSK9 and inhibits the binding of circulating PCSK9 to Low Density Lipoprotein Receptor (LDLR) thereby preventing PCSK 9-mediated degradation of low density lipoprotein receptor and controlling blood lipids, the medicament being for the treatment of adult heterozygous familial hypercholesterolemia and not for homozygous familial hypercholesterolemia. The family applied PCSK9 inhibitor can reduce blood fat obviously, which shows that the proband also keeps part of LDLR function, and proves that the proband is heterozygosis mutation.

(3) Screening for c.1879G > A mutation in healthy population

Healthy populations were screened for c.1879G > A mutations using the Taqman probe method.

The specific operation of the Taqman probe method for genotyping experiments is as follows:

2ml of genome DNA sample, 4ml of 2 XTaqMan GT master mix, 0.2ul of 40 XTaqMan SNP probe and 1.8ul of deionized water were mixed uniformly (10ul reaction system). And adding a 96-well PCR plate into each well of one-person reaction system, and carrying out reaction in an ABI 7300 real-time fluorescent quantitative PCR instrument. Denaturation at 95 ℃ for 30 seconds, annealing at 60 ℃ for 1 minute, and repeating 35 cycles. After the reaction was completed, genotyping was performed using 7500System SDS software, and it was determined whether the above samples carried mutations.

The nucleic acid probe is a Taqman probe, and the nucleic acid sequence of the Taqman probe is as follows:

forward probe sequence: tcatcccagtgtttaacgg the flow of the air in the air conditioner,

reverse probe sequence: gtcagggcaggaacgaga are provided.

The results showed that the genotypes in 200 populations were all CC, carrying no mutations.

Based on the evidence, we find that a new site mutation c.1879G > A in the LDLR gene can cause familial hypercholesterolemia. The locus can be used for gene screening treatment of familial hypercholesterolemia.

It will be apparent to those skilled in the art that various modifications may be made to the above embodiments without departing from the general spirit and concept of the invention. All falling within the scope of the present invention. The protection scheme of the invention is subject to the appended claims.

Claims

1. nucleic acid, is characterized in that, comprises and contains following target fragment,

Compared with the wild-type LDLR gene fragment, the target fragment has the c.1879G>A mutation.

2. A polypeptide, characterized in that,

Compared to wild-type LDLR, the polypeptide has the p.A627T mutation.

3. A gene mutation, characterized in that, compared with the wild-type LDLR gene, the gene mutation has a c.1879G>A mutation.

4. The application of biological models in the preparation of hypercholesterolemia screening preparations, wherein the biological models carry at least one of the following

a. the nucleic acid of claim 1,

b. the polypeptide of claim 2,

c. The gene mutation of claim 3.

5. A test kit for screening a hypercholesterolemic biological sample, characterized in that it comprises

A reagent capable of detecting a LDLR gene mutant, which is at least one of the following as compared with the wild-type LDLR gene

a. the nucleic acid of claim 1,

b. the polypeptide of claim 2,

c. The gene mutation of claim 3.

6. kit according to claim 5, is characterized in that, described reagent is nucleic acid probe or primer;

Preferably, the nucleotide sequence of the primer includes:

The sequence is the forward primer of tcatcccagtgtttaacgg,

The sequence is the reverse primer for gtcagggcaggaacgaga.

7 . A construct comprising the nucleic acid of claim 1 or the genetic mutation of claim 3 . 8 .

8. The use of an inhibitor in the preparation of a drug for preventing and treating hypercholesterolemia, wherein the inhibitor has an inhibitory effect on at least any of the following

a. c.1879G>A mutation of the LDLR gene,

b. p.A627T mutation of the LDLR gene.

9. The application of a gene fragment in the preparation of a drug for preventing and treating hypercholesterolemia, wherein the gene fragment is a gene fragment whose single nucleotide at the c.1879 position of LDLR is G.

10. A drug for preventing and treating hypercholesterolemia, characterized in that it contains at least one of the following

a. c.1879G>A mutation inhibitor of LDLR gene,

b. p.A627T mutation inhibitor of LDLR gene,

c. a gene fragment, the gene fragment is a fragment whose single nucleotide at site c.1879 in the LDLR gene is G,

d. A gene vector containing a gene fragment capable of replacing the single nucleotide A at the c.1879 site of the LDLR gene with a single nucleotide G and expressing it.