阅读说明：本技术 家族性高胆固醇血症相关基因及其检测试剂盒与应用 (Familial hypercholesterolemia related gene and detection kit and application thereof ) 是由 陈牧雷 高元丰 杨新春 王欣 董莹 杨晓艳 曹晔萱 付源 史琳影 张媛 梁庆渊 于 2021-06-23 设计创作，主要内容包括：本发明公开了“一种家族性高胆固醇血症相关基因及其检测试剂盒与应用”,属于诊断试剂开发技术,其特征在于,包含人类LDLR基因c.817+1G>C杂合突变,所述c.817+1G>C杂合突变位于SEQ ID NO:1所示核苷酸序列的第16个碱基位置处；该位点可以作为临床辅助诊断家族性高胆固醇血症的生物标志物；通过检测受试者是否携带上述变异,可以检测该变异的携带者,为受试者提供优生优育指导和遗传咨询,减少患儿出生；为人类攻克家族性高胆固醇血症提供可能的药物治疗靶点,促进创新药物研发。(The invention discloses a familial hypercholesterolemia related gene and a detection kit and application thereof, belonging to the development technology of diagnostic reagents, which is characterized by comprising human LDLR gene c.817+1G > C heterozygous mutation, wherein the c.817+1G > C heterozygous mutation is positioned at the 16 th base position of a nucleotide sequence shown in SEQ ID NO. 1; the site can be used as a biomarker for clinically auxiliary diagnosis of familial hypercholesterolemia; by detecting whether the subject carries the variation or not, the carrier of the variation can be detected, and the prenatal and postnatal care guidance and the genetic counseling are provided for the subject, so that the birth of the infant patient is reduced; provides possible drug treatment targets for human beings to overcome the familial hypercholesterolemia, and promotes the research and development of innovative drugs.)

1. An LDLR mutant gene, which is characterized by comprising a human LDLR gene c.817+1G > C hybrid mutation, wherein the c.817+1G > C hybrid mutation is positioned at the 16 th base position of a nucleotide sequence shown in SEQ ID NO. 1.

2. Use of a reagent for detecting the LDLR mutant gene of claim 1 in the preparation of a kit for detecting familial hypercholesterolemia.

3. Use according to claim 2, wherein the reagents comprise reagents required for PCR amplification.

4. The use according to claim 3, wherein the reagent comprises a forward primer of SEQ ID NO. 2 and a reverse primer of SEQ ID NO. 3.

5. The use according to claim 2, wherein the reagents comprise one or more of Sanger sequencing reagents, fluorescent quantitative PCR reagents, reagents for the restriction enzyme fragment length polymorphism method, reagents for single strand conformation polymorphism analysis, and reagents for allele-specific oligonucleotide hybridization.

6. A kit for detecting a familial hypercholesterolemia-associated gene, comprising a reagent for detecting the LDLR mutant gene according to claim 1.

7. The test kit of claim 6, wherein the reagents comprise reagents required for PCR amplification.

8. The detection kit of claim 6, wherein the reagent comprises a forward primer shown in SEQ ID NO. 2 and a reverse primer shown in SEQ ID NO. 3.

9. The test kit of claim 6, wherein the reagents comprise Sanger sequencing reagents.

Technical Field

The invention relates to the development of diagnostic reagents, in particular to a familial hypercholesterolemia related gene, a detection kit and application thereof.

Background

Familial Hypercholesterolaemia (FH), also known as familial hyperbetalipoproteinemia, is clinically characterized by hypercholesterolemia, characteristic yellow tumors, and a family history of early cardiovascular disease. FH is the most common hereditary hyperlipidemia in childhood and is also the most serious of the diseases of lipid metabolism, which can cause various complications of cardiovascular diseases threatening life, and is an important risk factor of coronary artery diseases. The most characteristic clinical manifestations of this disease are elevated blood LDL-C levels, yellow tumor, corneal arcus and early-onset coronary heart disease. The clinical manifestations of homozygotes are much more severe than those of heterozygotes. The clinical manifestations of FH patients depend on their genotype, which is also influenced by non-genetic factors. The relationship between the FH genotype and phenotype is complex, and even individuals belonging to the same family have large differences in clinical expression, even with the same mutation.

B-mode ultrasound examination of FH patients often reveals hardening of the aortic root, which is progressively aggravated, with calcification of the aortic valve and/or stenosis of the main left coronary artery. 15% of patients had coronary aneurysm-like dilatation (limited or diffuse dilatation of coronary arteries, with diameter 1.5-2 times larger than that of adjacent normal coronary arteries), while only 2.5% of age and gender matched controls (patients with non-FH coronary heart disease) had coronary aneurysm-like dilatation. And at the same time, found that coronary aneurysm-like expansion and plasma HDL-C level is negatively correlated, so FH people considered to be easy to develop coronary aneurysm-like disease. The imbalance of blood lipid metabolism of FH patients can accelerate the process of atherosclerosis and early coronary heart disease, so that FH can be discovered and diagnosed early and intervened early, the morbidity and mortality of coronary artery diseases can be reduced obviously, and the clinical prognosis is improved.

At present, gene detection is a novel means for determining FH. Any one or a group of FH related gene discovery and proposed will be the field of important technical contribution. Currently known FH-associated mutations exceed 2000, of which about 1000 have been supported by sufficient evidence to be classified into two classes of pathogenic and potentially pathogenic mutations, which are mainly distributed among three genes, LDLR (90% or more), APOB (5% to 10%), PCSK9 (1% or less). However, some patients with FH can not be explained by known pathogenic genes, suggesting the existence of undiscovered pathogenic genes, and further research and improvement of FH pathogenic gene data is necessary to improve the accuracy of early detection of FH.

Disclosure of Invention

Based on the needs in the field and the discovery of the inventor, the invention provides an application of a familial hypercholesterolemia related gene and a detection kit thereof, and the technical scheme is as follows:

1. an LDLR mutant gene, which is characterized by comprising a human LDLR gene c.817+1G > C hybrid mutation, wherein the c.817+1G > C hybrid mutation is positioned at the 16 th base position of a nucleotide sequence shown in SEQ ID NO. 1.

2. The application of the reagent for detecting the LDLR mutant gene in preparing a familial hypercholesterolemia detection kit.

3. According to the use, the reagent comprises a reagent required for PCR amplification.

4. According to the use, the reagent comprises a forward primer shown as SEQ ID NO. 2 and a reverse primer shown as SEQ ID NO. 3.

5. According to the use, the reagent comprises one or more of a Sanger sequencing reagent, a fluorescent quantitative PCR reagent, a reagent for a restriction enzyme fragment length polymorphism method, a reagent for single-strand conformation polymorphism analysis and a reagent for allele-specific oligonucleotide hybridization.

6. A kit for detecting a familial hypercholesterolemia-associated gene, which comprises a reagent for detecting the LDLR mutant gene.

7. The detection kit is characterized in that the reagent contains reagents required by PCR amplification.

8. The detection kit is characterized in that the reagent comprises a forward primer shown by SEQ ID NO. 2 and a reverse primer shown by SEQ ID NO. 3.

9. The detection kit is characterized in that the reagent contains a Sanger sequencing reagent.

The invention can distinguish the familial hypercholesterolemia variant gene carriers from normal people by detecting whether the LDLR gene c.817+1G > C is heterozygous for mutation, so the variant can be used as a biomarker for clinically and auxiliarily diagnosing the familial hypercholesterolemia;

by detecting whether the subject carries the variation or not, the carrier of the variation can be detected, and the prenatal and postnatal care guidance and the genetic counseling are provided for the subject, so that the birth of the infant patient is reduced; provides possible drug treatment targets for human beings to overcome the familial hypercholesterolemia, and promotes the research and development of innovative drugs.

The LDLR gene encodes a low density lipoprotein receptor, the family of which consists of cell surface proteins involved in receptor-mediated endocytosis of specific ligands. Low Density Lipoproteins (LDL) are usually bound to cell membranes, enter the cell and enter lysosomes where proteins are degraded and cholesterol is used to inhibit the microsomal enzyme 3-hydroxy-3-methylglutaryl coa (hmg coa) reductase, which is the rate-limiting step in cholesterol synthesis. Meanwhile, the synthesis of cholesterol ester is mutually stimulated, and plays an important role in maintaining the metabolic balance of plasma lipoprotein. LDLR gene mutations are associated with the development of familial hypercholesterolemia and elevated low density lipoprotein cholesterol levels with autosomal dominant inheritance.

Familial hypercholesterolemia (AD) is clinically manifested by elevated blood LDL-C levels, yellow tumors, corneal arcus and early-onset coronary heart disease. Clinical manifestations depend on their genotype, which is also influenced by non-genetic factors, and are more severe in homozygotes than in heterozygotes. The plasma cholesterol concentration of heterozygote is 2-3 times of that of normal people and is between 350-550 mg/L; the homozygote is 6-8 times higher than normal and is between 650-1000 mg/L. Heterozygote xanthomas mostly appear after the age of 20 years, and homozygotes appear before the age of 4 years. Heterozygotes mostly develop coronary artery disease after age 30, while homozygotes mostly develop in childhood. The incidence of diseases is as follows: heterozygote 1/500, homozygote 1/100 million.

The gene detection of a subject diagnosed as 'familial hypercholesterolemia' shows that the subject carries LDLR c.817+1G > C heterozygous variation; the mutation was found to be a rare mutation by querying the population frequency database (thousand genomes: none, ESP 6500: none, ExAC: none). The frequency of this mutation was 0.00000406 when the database of the hundred-kno local Chinese population was queried. The mutation is not found by querying ClinVar and HGMD databases, and the mutation sites c.814-817 del (p.Val271-Asn 272insTer), c.810C > A (p.Cys270Ter), c.817+2T > G, c.817+2T > C, c.818-2A > G and the like near the mutation site are reported to be pathogenic mutation of familial hypercholesterolemia, but the report that the mutation discovered by the invention is related to the disease is not seen in literature search. According to the existing evidence: the mutation is a rare mutation, the local database frequency is 0.00000406, nearby sites have been reported as pathogenic mutations, but family linkage and functional evidence support are lacked, so the mutation is presumed to be a highly suspicious pathogenic mutation of familial hypercholesterolemia; the research of the invention shows that: the examinee carries the heterozygous variation of highly suspicious pathogenic mutant LDLR gene c.817+1G > C of familial hypercholesterolemia, and the diagnosis of clinical familial hypercholesterolemia is supported; family verification that the variation is inherited from the subject's mother (FH-CF-48-1); the pathogenicity of the mutation is relatively clear, the mutation is transmitted in families in an autosomal dominant inheritance mode, and the inheritance probability is 50%.

The discovery of the heterozygous mutation enriches the target points of the familial hypercholesterolemia gene detection and improves the accuracy of the familial hypercholesterolemia gene detection.

Drawings

FIG. 1 is a graph showing the relationship between carriers of LDLR gene c.817+1G > C heterozygous mutant gene and familial hypercholesterolemia in the pedigree of the present invention;

FIG. 2 is a Sanger's profile of patients and other disease-causing members of the family in an example of the present invention.

Detailed Description

The following description of the embodiments of the present invention, which is made in connection with the accompanying drawings and the exemplary embodiments, should not be construed as limiting the scope of the present invention.

Example 1 LDLR Gene c.817+1G > C hybrid mutant Gene, kit for in vitro detection of LDLR Gene c.817+1G > C hybrid mutant Gene

This example provides a LDLR gene c.817+1G > C hybrid mutant gene, whose nucleotide sequence is shown in SEQ ID No. 1;

SEQ ID NO:1ttggctgcgt taatg g/c tgag cgctggccat。

this example also provides primers for detecting LDLR gene c.834delg heterozygous deletion mutant genes:

a forward primer: 5'-GCCTCTCAAGCAGTTGGAACCAC-3' (SEQ ID NO: 2);

reverse primer: 5'-TCACTTGCCCACAGACGCACA-3' (SEQ ID NO: 3).

The kit for in vitro detection of the LDLR gene c.817+1G > C heterozygous mutant gene provided by the embodiment of the invention comprises: 1) the primer for amplifying the LDLR gene c.817+1G > C hybrid mutant gene; 2) PCR amplification enzyme; 3) PCR buffer, divalent or monovalent cation, hybridization solution. Specifically, the components of the kit for in vitro detection of the LDLR gene c.817+1G > C hybrid mutant gene are shown in Table 1:

TABLE 1

Example 2 method for in vitro detection of FH related genes in a sample

The embodiment of the invention provides a method for in vitro detecting whether familial hypercholesterolemia related genes exist in a sample to be detected, which comprises the following steps:

1. extracting DNA of a sample to be detected, and carrying out PCR amplification aiming at the c.817+1G > C site of the LDLR gene; wherein the sample to be detected is blood, hair, saliva, hair or living tissue of an individual to be detected;

the DNA of the sample may be extracted using any known well-established technique. In the embodiment, the DNA extraction of the sample to be detected is carried out by using a whole blood genome DNA extraction kit (Baishinuo) based on a magnetic bead method, wherein 200 mu l of the sample (serum/whole blood) and 10 mu l of proteinase K are added into the 1 st and 7 th columns of a 96 deep-well plate, a pipette is used for blowing and beating the mixture evenly, the mixture is kept still at room temperature for 10-15 min, and then 150 mu l of binding solution is added. The 96 deep well plate is placed into a full-automatic nucleic acid extraction and purification instrument ZK-01, DNA extraction is started, and the program is set as shown in Table 2:

TABLE 2

The 96 deep-well plate is taken out of the instrument, and the 6 th column and the 12 th column are DNA of the extracted sample to be detected.

2. Analyzing the PCR amplification product, wherein in the step, the PCR amplification conditions are shown in Table 3:

TABLE 3

Taking 3 mul of PCR product, detecting the PCR product by using 1.5% agarose gel electrophoresis, and selecting 1000bp Marker as reference. After product purification, Sanger sequencing was performed and then the sequencing results were read.

3. Identifying whether c.817+1G > C site of LDLR gene is mutated

If the c.817+1G > C site mutation of the LDLR gene is to be determined, the carrier of the variant gene is considered as a highly suspected pathogenic mutation (B grade) of the familial hypercholesterolemia.

Therefore, the detection of c.817+1G > C site mutation of the LDLR gene can be used for clinical diagnosis of familial hypercholesterolemia, provides genetic block for families carrying early familial hypercholesterolemia mutation, and improves the quality of prenatal and postnatal care.

Experimental example 1 study on association between familial hypercholesterolemia and LDLR gene c.817+1G > C heterozygous mutation

1. Subject information, as shown in Table 4 below

TABLE 4

The LDLR gene of the subject was detected by the detection method using the detection kit of example 1 and the kit of example 2 of the present invention.

2. The detection result shows

And (3) detection results: the examinee carries the heterozygous variation of highly suspicious pathogenic mutant LDLR gene c.817+1G > C of familial hypercholesterolemia, and the diagnosis of clinical familial hypercholesterolemia is supported.

The detection reagent of the embodiment 1 and the detection method of the embodiment 2 are used for detecting the LDLR variant gene carried by the detected person, and the details are shown in the following table 6:

TABLE 6

Note: AD is autosomal dominant inheritance, AR is autosomal recessive inheritance, XLD is X-chromosomal dominant inheritance, XLR is X-chromosomal recessive inheritance, and OMIM database is a transient absence of inheritance.

A level: clear pathogenic mutations, family linkage or functional evidence support clear association with disease.

B stage: highly suspected pathogenic mutations, population data and bioinformatic analyses suggest a high probability of disease association.

Grade C1; suspected pathogenic variations, gene function, population data and bioinformatic analysis suggest possible association with disease, but lack evidence support.

Level C2: the clinical significance is unknown, and the relationship with the disease cannot be judged according to the current cognition on the disease and the genetic information. D stage: the possibility of benign mutation is low, and the possibility of causing diseases is judged according to the current cognition on the diseases and genetic information.

3. Family verification

The results of clinical diagnosis were shown in Table 7, in which the LDLR variant gene carried by the family of the subjects was examined using the detection reagent of example 1 and the detection method of example 2 of the present invention.

TABLE 7

Sample coding	In relation to the subject	LDLR:c.817+1G>C	Clinical diagnosis
				FH-CF-48-1	Mother of first person	Heterozygous variation	Familial hypercholesterolemia
FH-CF-48-2	First and second brother	Heterozygous variation	Familial hypercholesterolemia
				FH-CF-48-3	Syndrome of first-degree syndrome	No variation	Non-familial hypercholesterolemia

As shown in FIGS. 1 and 2, the pedigree verified that the mutation was inherited from the mother of the subject (FH-CF-48-1); the subject's brother (FH-CF-48-2) also carried the mutation, verifying that the mutation is autosomal dominant.