Use of a reagent in the preparation of a product for screening hyperlipidemia or assessing the susceptibility to hyperlipidemia

Abstract

The present disclosure describes the use of a reagent in the preparation of a product for screening patients with hyperlipidemia and / or acute pancreatitis, or assessing the susceptibility to hyperlipidemia and / or acute pancreatitis. The reagent is used to detect LPL gene mutations or protein mutations in a sample. The LPL gene mutation is that the A base at the 1015th position of the DNA sequence of the LPL gene is replaced by a C base, and the LPL amino acid mutation is that the amino acid at the 339th position of the LPL protein is mutated from lysine (K) to glutamine (Q). According to the present disclosure, it is possible to provide new pathogenic gene loci for hyperlipidemia and / or acute pancreatitis or mutation loci with high risk of hyperlipidemia and / or acute pancreatitis, which is helpful for the diagnosis, treatment and prevention of hyperlipidemia and / or acute pancreatitis.

Description

[0001] This application is a divisional application of a patent application with an application date of May 16, 2023, an application number of 202310561811.8, and an invention title of "Application of a reagent for detecting LPL gene variation or amino acid mutation in a sample in the preparation of a product for screening hyperlipidemia patients". Technical Field

[0002] The present invention relates to the field of biomedical technologies, and particularly relates to the application of a reagent in the preparation of a product for screening hyperlipidemia and / or acute pancreatitis patients, or for evaluating the susceptibility to hyperlipidemia and / or acute pancreatitis. Background Art

[0003] With the continuous improvement of people's living standards, there have been great changes in the daily dietary structure, resulting in an increasing number of people with imbalances in the content of nutritional components in the blood. For example, hyperlipidemia (HLP) caused by excessive blood lipid levels. The risk of atherosclerotic cardiovascular disease (ASCVD) and acute pancreatitis (AP) in hyperlipidemia patients will be significantly increased. Lipids specifically cover lipoids and fats. Fats refer to triglycerides, that is, triacylglycerol (TG). If there are factors that cause a decrease in triglyceride decomposition or an increase in synthesis, then hypertriglyceridemia (HTG) may occur, leading to hyperlipidemia.

[0004] There are many factors causing hyperlipidemia, mainly including genetic factors, unhealthy lifestyles, type 2 diabetes, etc. Currently, numerous studies have found that genes that can cause severe hypertriglyceridemia after mutation include: apolipoprotein C-II (APOC2), lipoprotein lipase (LPL), lipase maturation factor 1 (LMF1), apolipoprotein V (APOA5), glycosylphosphatidylinositol-anchored high-density lipoprotein-binding protein 1 (GPIHBP1), etc. Among them, the defect of lipoprotein lipase (LPL) is an important cause of hypertriglyceridemia. The human lipoprotein lipase (LPL) gene is mainly located on chromosome 8p22. It is an esterifying enzyme with the activity of triglyceride hydrolase and plays an extremely important role in lipid metabolism. Some studies have shown that LPL gene variation leads to a decrease in lipoprotein lipase activity, which can affect the catabolism of triglycerides, resulting in the obstruction of chylomicron metabolism and an increasing accumulation of chylomicrons in the body, thus leading to the occurrence of hyperlipidemia.

[0005] At present, many studies have elaborated on the pathogenic mechanism of hyperlipidemia, but there are still unknown pathogenic gene loci. Further studying the pathogenic mechanism of hyperlipidemia and isolating new pathogenic gene variations of familial hyperlipidemia is of great significance for the diagnosis, treatment, and prevention of hyperlipidemia. Summary of the Invention

[0006] The present disclosure is completed in view of the above-mentioned state of the prior art, and its purpose is to provide a pathogenic mutation site of hyperlipidemia or a mutation site with a high risk of hyperlipidemia, which is helpful for the diagnosis, treatment and prevention of hyperlipidemia.

[0007] To this end, the first aspect of the present disclosure provides an application of a reagent for detecting LPL gene variation or protein variation in a sample in the preparation of a product for screening hyperlipidemia patients, wherein the LPL gene variation is LPL c.1015A>C, and the LPL protein variation is. In the present disclosure, the LPL c.1015A>C mutation is identified through pedigree research (the A base at the 1015th position of the DNA sequence of the LPL gene is replaced by the C base, resulting in the amino acid at the 339th position of the LPL protein being mutated from lysine (K) to glutamine (Q), that is, LPL p.K339Q), and through functional research, it is confirmed that the LPL c.1015A>C (LPL p.K339Q) mutation will affect the activity of lipoprotein lipase (LPL). Therefore, a new pathogenic gene site of hyperlipidemia or a mutation site with a high risk of hyperlipidemia is provided. By detecting whether the sample carries the LPL c.1015A>C (LPL p.K339Q) mutation, it is possible to assist in screening hyperlipidemia patients.

[0008] In the application involved in the present disclosure, optionally, the reagent includes a primer pair for amplifying the LPL gene and / or a probe for detecting the LPL gene variation. Thus, it is possible to capture and / or detect the LPL gene variation c.1015A>C by the primer pair and / or the probe.

[0009] In the application involved in the present disclosure, optionally, the primer pair is designed according to the nucleotide sequences upstream and downstream of the 1015th base in the coding region of the LPL gene in the human genome, and the probe is designed according to the nucleotide sequences of the 1015th base and its upstream and downstream in the coding region of the LPL gene in the human genome. Thus, the primer can bind to the sequences in the upstream and downstream regions of the 1015th position in the coding region of the LPL gene, and the probe can bind to the sequences of the 1015th position and its upstream and downstream in the coding region of the LPL gene to detect this region.

[0010] In the application involved in the present disclosure, optionally, the reagent further includes dNTPs, DNA polymerase and PCR reaction buffer. Thus, it is possible to provide reaction substrates, catalytic enzymes and buffers to facilitate the detection of LPL c.1015A>C.

[0011] In the applications related to the present disclosure, optionally, the reagent includes a reagent for detecting LPL protein variants using at least one of the following methods: sequence analysis techniques of proteins and peptides, mass spectrometry-related protein detection techniques, and antibody detection techniques. Thus, a reagent can be provided to detect the LPL protein variant (also known as amino acid variant) LPL p.K339Q.

[0012] In the applications related to the present disclosure, optionally, the reagent includes an antibody that can recognize the LPL protein with the LPL p.K339Q mutation. Thus, the antibody that can recognize the LPL protein with the LPL p.K339Q mutation can be used to detect the LPL protein with the LPL p.K339Q mutation.

[0013] In the applications related to the present disclosure, optionally, the product further includes a nucleic acid extraction reagent and / or a protein extraction reagent. Thus, it is convenient to detect LPL c.1015A>C or LPL p.K339Q.

[0014] In the applications related to the present disclosure, optionally, the sample is from at least one of the peripheral blood, saliva, and tissue sample of the subject to be tested, and the LPL gene variant refers to the germline variant of the LPL gene, and the LPL protein variant refers to the germline variant of the LPL protein. Thus, by detecting the peripheral blood, saliva, and / or tissue sample of the subject to be tested, the germline mutation of the LPL gene of the subject (germline mutation refers to the variant already carried during the embryonic development period of a human, and every cell in the body carries it) can be detected.

[0015] In the applications related to the present disclosure, optionally, the LPL gene variant is a heterozygous mutation or a homozygous mutation, and the LPL protein variant is a heterozygous mutation or a homozygous mutation.

[0016] The second aspect of the present disclosure provides an application of a reagent for detecting LPL gene mutations or protein mutations in a sample in the preparation of a product for evaluating hyperlipidemia susceptibility, characterized in that the LPL gene mutation is LPL c.1015A>C, and the LPL protein mutation is LPL p.K339Q. In the present disclosure, through pedigree research, the LPL c.1015A>C mutation (the A base at the 1015th position of the DNA sequence of the LPL gene is replaced by the C base, resulting in the amino acid at the 339th position of the LPL protein being mutated from lysine (K) to glutamine (Q), i.e., LPL p.K339Q) is identified, and through functional research, it is confirmed that the LPL c.1015A>C (LPL p.K339Q) mutation affects the activity of lipoprotein lipase (LPL). Thus, a new pathogenic gene locus for hyperlipidemia or a mutation locus with high risk of hyperlipidemia is provided. By detecting whether the sample carries the LPL c.1015A>C (LPL p.K339Q) mutation, the susceptibility to hyperlipidemia can be assisted in evaluation.

[0017] According to the present disclosure, a pathogenic mutation locus for hyperlipidemia or a mutation locus with high risk of hyperlipidemia can be provided, which is helpful for the diagnosis, treatment and prevention of hyperlipidemia. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 It is a graph showing the change in triglyceride level of the proband involved in the embodiment of the present invention.

[0019] Figure 2 It is a pedigree chart of the proband involved in the embodiment of the present invention.

[0020] Figure 3 It is a sequencing result graph of the proband involved in the embodiment of the present invention.

[0021] Figure 4 It is a protein structure diagram of LPL and an amino acid sequence diagram among different species involved in the embodiment of the present invention.

[0022] Figure 5 It is a three-dimensional structure prediction graph of the LPL protein involved in the embodiment of the present invention.

[0023] Figure 6 It is a schematic diagram of the expression level results of LPL WT and LPL p.K339Q.

[0024] Figure 7 It is a histogram of the expression levels of LPL WT and LPL p.K339Q.

[0025] Figure 8 It is a graph of the LPL activity results of LPL WT and LPL p.K339Q in cells.

[0026] Figure 9 Results of LPL activity in the cell culture supernatant of LPL WT and LPL p.K339Q Detailed implementation mode

[0027] The following details the implementation modes of the present invention. Examples of the implementation modes are shown in the accompanying drawings, where the same or similar reference numerals represent the same or similar elements or elements with the same or similar functions throughout. The implementation modes described through the accompanying drawings are exemplary and are only used to explain the present invention, and should not be construed as a limitation to the present invention.

[0028] Those skilled in the art can understand that, unless otherwise defined, all terms (including technical terms and scientific terms) used herein have the same meaning as the general understanding of those of ordinary skill in the technical field to which the present invention belongs.

[0029] It should also be understood that terms such as those defined in a general dictionary should be understood to have a meaning consistent with the meaning in the context of the prior art, and will not be interpreted with an idealized or overly formal meaning unless defined as here.

[0030] Those skilled in the art can understand that, unless specifically stated, the singular forms "a", "an", "the", and "said" used herein may also include the plural forms. It should be further understood that the term "comprising" used in the description of the present invention means the presence of the described features, integers, steps, operations, elements, and / or components, but does not exclude the presence or addition of one or more other features, integers, steps, operations, elements, and / or their groups.

[0031] For ease of understanding the present invention, the following further explains the present invention with specific examples in conjunction with the accompanying drawings, and the specific examples do not constitute a limitation to the embodiments of the present invention. Those skilled in the art should understand that the drawings are only schematic diagrams of the embodiments, and the components in the drawings are not necessarily essential for implementing the present invention.

[0032] In this implementation mode, it relates to any of the following applications:

[0033] Use of a reagent for detecting LPL gene mutations in a sample in the preparation of a product for screening patients with hyperlipidemia, acute pancreatitis, and / or hypertriglyceridemia;

[0034] Use of a reagent for detecting LPL gene mutations in a sample in the preparation of a product for screening patients with hyperlipidemia accompanied by acute pancreatitis;

[0035] Use of a reagent for detecting LPL amino acid mutations in a sample in the preparation of a product for screening patients with hyperlipidemia, acute pancreatitis, and / or hypertriglyceridemia;

[0036] Use of a reagent for detecting LPL amino acid mutations in a sample in the preparation of a product for screening patients with hyperlipidemia accompanied by acute pancreatitis;

[0037] Use of a reagent for detecting LPL gene variations in a sample in the preparation of a product for evaluating the susceptibility to hyperlipidemia, acute pancreatitis, and / or hypertriglyceridemia;

[0038] Use of a reagent for detecting LPL amino acid mutations in a sample in the preparation of a product for evaluating the susceptibility to hyperlipidemia, acute pancreatitis, and / or hypertriglyceridemia.

[0039] In the above applications involved in this embodiment, both LPL gene variations or LPL amino acid mutations are related to the risks of the three diseases of hyperlipidemia, acute pancreatitis, and hypertriglyceridemia. Therefore, a reagent for detecting LPL gene variations or LPL amino acid mutations can be used to screen patients with any one or more of the diseases of hyperlipidemia, acute pancreatitis, and hypertriglyceridemia, and a reagent for detecting LPL gene variations or LPL amino acid mutations can be used to evaluate the susceptibility to any one or more of the diseases of hyperlipidemia, acute pancreatitis, and hypertriglyceridemia.

[0040] The new mutation sites provided by the present invention supplement the genetic mutation spectrum of familial hyperlipidemia, which is beneficial for diagnosing hyperlipidemia patients for treatment, and for genetic diagnosis of carriers in the family for health management. At the same time, it can guide childbirth according to the genotypes of both parents to avoid the inheritance of pathogenic genes and guide eugenics and good child-rearing.

[0041] Among the above-mentioned reagents involved in this embodiment, reagents for detecting LPL gene mutations or LPL amino acid mutations may be included. In some examples, the LPL gene mutation is LPL c.1015A>C. LPL c.1015A>C means that the 1015th base in the coding region of the wild-type LPL gene is mutated from A (adenine) to C (cytosine); the LPL amino acid mutation (i.e., LPL protein variation) is LPL p.K339Q. LPL p.K339Q means that the 339th amino acid of the LPL protein is mutated from lysine (K) to glutamine (Q). In this embodiment, the LPL c.1015A>C mutation (the 1015th A base in the DNA sequence of the LPL gene is replaced by a C base, resulting in the 339th amino acid of the LPL protein being mutated from lysine to glutamine, i.e., LPL p.K339Q) was identified through family studies. This embodiment also confirmed through functional studies that the LPL c.1015A>C (LPL p.K339Q) mutation affects lipoprotein lipase (LPL) activity. In some examples, the LPL c.1015A>C (LPL p.K339Q) mutation causes a decrease in LPL activity. Thus, a new pathogenic gene locus for hyperlipidemia or a mutation locus with a high risk of hyperlipidemia is provided. By detecting whether the sample carries the LPL c.1015A>C (LPL p.K339Q) mutation, it is possible to assist in screening patients with hyperlipidemia.

[0042] In some examples, at least one of pyrosequencing technology, Sanger sequencing, NGS sequencing, polymerase chain reaction - single-strand conformation polymorphism analysis, and TaqMan probe method can be used to detect LPL gene mutations. In some examples, the LPL gene mutation can be LPL c.1015A>C.

[0043] In some examples, the above-mentioned reagents involved in this embodiment may include primer pairs for amplifying the LPL gene and / or probes for detecting LPL gene mutations. Thus, it is possible to capture and / or detect LPL gene mutations through the primer pairs and / or probes. In some examples, the reagent may further include dNTPs, DNA polymerase, and PCR reaction buffer. Thus, it is convenient to detect LPL gene mutations.

[0044] In some examples, the primer pair can be designed based on the nucleotide sequences upstream and downstream of the 1015th base in the coding region of the LPL gene in the human genome, and the probe can be designed based on the nucleotide sequences of the 1015th base in the coding region of the LPL gene in the human genome and its upstream and downstream regions. Thus, the primer can bind to the sequences in the upstream and downstream regions of the 1015th base in the coding region of the LPL gene, and the probe can bind to the sequences of the 1015th base in the coding region of the LPL gene and its upstream and downstream regions to detect LPL c.1015A>C.

[0045] In some examples, the above-mentioned reagents involved in this embodiment may also include reagents for detecting other LPL mutations other than LPL c.1015A>C (LPLp.K339Q). In some examples, other LPL mutations may include all pathogenic mutations and suspected pathogenic mutations of the LPL gene currently known for hyperlipidemia. In some examples, the above-mentioned reagents involved in this embodiment may also include reagents for detecting other genes related to hyperlipidemia other than the LPL gene. In some examples, genes related to hyperlipidemia may include APOC2, LMF1, APOA5, GPIHBP1. Thus, it is possible to detect all relevant sites of hyperlipidemia, which is beneficial for a one-time and more comprehensive screening of hyperlipidemia.

[0046] In some examples, the above-mentioned reagents involved in this embodiment may also include reagents for detecting genes or proteins related to other diseases. For example, it may also include reagents for detecting genes or proteins related to inherited metabolic diseases. Thus, it is possible to screen multiple diseases in the subject being tested simultaneously.

[0047] In some examples, the above-mentioned reagents involved in this embodiment may include reagents for detecting LPL protein variations using at least one of the following methods: sequence analysis techniques for proteins and peptides, mass spectrometry-related protein detection techniques, and antibody detection techniques. Thus, it is possible to detect LPL protein variations. In some examples, the LPL protein variation refers to LPL p.K339Q. In some examples, the above-mentioned reagents involved in this embodiment may also include reagents for detecting LPL proteins other than the protein with the LPL p.K339Q variation. For example, it may also include reagents for detecting LPL protein variants carrying other known pathogenic / suspected pathogenic sites.

[0048] In some examples, the sequence analysis techniques for proteins and peptides may include chemical methods for N-terminal sequence determination, the Edman method, C-terminal enzymatic cleavage methods, and C-terminal chemical degradation methods.

[0049] In some examples, mass spectrometry-related protein detection techniques may include matrix-assisted laser desorption ionization, time-of-flight mass spectrometry (MALDI-TOF MS), and electrospray ionization mass spectrometry (ESI-MS).

[0050] In some examples, antibody detection techniques may include methods for preparing antibodies that can recognize different mutants, immunoblotting (such as western blot) methods, and enzyme-linked immunosorbent assay (ELISA) methods.

[0051] In some examples, the form of the above-mentioned products involved in this embodiment may be in the form of reagents, reagent sets, or kits. In some examples, the product may also include a system composed of instruments.

[0052] In some examples, the above-mentioned products involved in this embodiment may also include a system composed of instruments for detecting LPL gene variations or LPL amino acid mutations. For example, the product may be a system composed of PCR reagents, DNA sequencing reagents, and a DNA sequencer, or a system composed of TaqMan probes, PCR primer pairs, a quantitative PCR instrument, a module for genotyping, and other reagents required for TaqMan probe technology, or a system composed of probes, PCR primer pairs, and other reagents and instruments required for ligation detection reaction (LDR), or a system composed of PCR primer pairs, single-base extension primers, chips, PCR instruments, a module for genotyping, and / or other reagents and instruments required for Sequenom MassArray technology. Thus, it is possible to facilitate the detection of the LPL gene or LPL protein.

[0053] In some examples, the above-mentioned products involved in this embodiment may also include nucleic acid extraction reagents and / or protein extraction reagents. Thus, it is possible to facilitate gene detection or protein detection.

[0054] In some examples, the clinical manifestations of patients with hyperlipidemia also include acute pancreatitis and hypertriglyceridemia. In some examples, patients with hyperlipidemia are usually accompanied by acute pancreatitis. Therefore, the present disclosure provides new pathogenic gene loci for hyperlipidemia (and / or acute pancreatitis, and / or hypertriglyceridemia) or mutation sites with high risk of hyperlipidemia (and / or acute pancreatitis, and / or hypertriglyceridemia). By detecting the LPL c.1015A>C mutation in a sample, it is possible to assist in screening patients with hyperlipidemia (and / or acute pancreatitis, and / or hypertriglyceridemia) and to assist in evaluating the susceptibility to hyperlipidemia, acute pancreatitis, and / or hypertriglyceridemia. Susceptibility refers to the risk of an individual getting sick determined by the genetic basis, and can also be understood as the risk of different individuals getting sick under the same environment.

[0055] In some examples, in the above applications involved in the present embodiment, the LPL gene or LPL protein can be detected by detecting at least one of the peripheral blood, saliva, and tissue samples of the subject to be detected. In other words, the sample to be detected can be from at least one of the peripheral blood, saliva, and tissue samples of the subject to be detected.

[0056] In some examples, the subject to be detected can be the general population, an individual suspected of having hyperlipidemia, or a population at high risk of hyperlipidemia. In some examples, an individual suspected of having hyperlipidemia can be a patient with acute pancreatitis, a patient with recurrent pancreatitis, a patient with hyperlipidemia, or a patient with hypertriglyceridemia. In some examples, a population at high risk of hyperlipidemia can be a population with a family history of hyperlipidemia, such as an individual with at least one immediate family member diagnosed with hyperlipidemia.

[0057] In some examples, the germline mutation of the LPL gene in the sample can be detected. Germline mutation, also known as germ cell mutation, is a mutation carried by germ cells such as sperm or eggs. In some examples, the germline mutation result of the LPL gene can be obtained by extracting the gDNA (genomic DNA) of the subject to be detected and detecting the genomic DNA.

[0058] In some examples, the 1015th base in the coding region of the LPL gene can be detected. Further, the LPL c.1015A>C mutation can be detected. In other words, it can be detected whether the subject to be detected carries the LPL c.1015A>C mutation.

[0059] In some examples, as long as there is one LPL c.1015A>C mutation in the LPL gene of the subject to be detected, it can assist in diagnosing that the subject to be detected is a patient with hyperlipidemia. In other words, when it is detected that the LPL c.1015A>C of the subject to be detected is a heterozygous mutation, it can assist in diagnosing that the subject to be detected is a patient with hyperlipidemia. Of course, when it is detected that the LPL c.1015A>C of the subject to be detected is a homozygous mutation, it can also assist in diagnosing that the subject to be detected is a patient with hyperlipidemia.

[0060] In this embodiment, by using the gene variation of LPL c.1015A>C as a biomarker, patients with hyperlipidemia can be screened, and further, an application of a reagent for detecting gene variation of LPL in the gene in the preparation of a product for screening patients with hyperlipidemia is provided. Similarly, an application of a reagent for detecting gene variation of LPL in the gene in the preparation of a product for screening acute pancreatitis and hypertriglyceridemia, and in the preparation of a product for evaluating the susceptibility to hyperlipidemia, acute pancreatitis or hypertriglyceridemia can also be provided. Similarly, an application of a reagent for detecting LPL amino acid mutation in the sample in the preparation of a product for screening hyperlipidemia, acute pancreatitis or hypertriglyceridemia, and in the preparation of a product for evaluating the susceptibility to hyperlipidemia, acute pancreatitis or hypertriglyceridemia can also be provided.

[0061] Next, the above applications involved in the present invention will be further explained in detail in combination with examples, but they should not be construed as limiting the protection scope of the present invention.

[0062] [Examples]

[0063] In this example, the English abbreviations or symbols of the professional terms that may be involved are explained as shown in Table 1.

[0064] Table 1 Explanation of Professional Terms

[0065]

[0066] Clinical Case:

[0067] (1) Case Information

[0068] The proband is a 32-year-old woman who has given birth to a son and a daughter. At 26 weeks of gestation, she complained of abdominal pain and vomiting for one week. Laboratory tests showed triglyceride (TG) level of 15.21 mmol / L and total cholesterol level of 11.08 mmol / L. Abdominal MR showed manifestations of acute pancreatitis and a slightly enlarged spleen. She was admitted to the hospital with acute pancreatitis. After one week of treatment, the triglyceride level decreased to 9.28 mmol / L and she was discharged. At 29 weeks of gestation, the triglyceride level increased to 33.98 mmol / L. At 33 weeks of gestation, the triglyceride level continued to increase to 46.39 mmol / L. She was admitted to the endocrinology department and the nutrition department for consultation, given low molecular weight heparin treatment, controlled diet, and supplemented with short peptide enteral preparations. After one week of review, the triglyceride level was 12.01 mmol / L and she was discharged for delivery. At 35 weeks of gestation, the triglyceride level was 58.23 mmol / L and she was admitted to the obstetrics department due to placental abruption. At 35 weeks + 3 days of gestation, the triglyceride level was 32.45 mmol / L. She gave birth to a female infant weighing 2420 g through vaginal delivery. The triglyceride test result of the neonate was 0.33 mmol / L. After delivery, the triglyceride level decreased. It was 28.94 mmol / L on the first day after delivery, 18.68 mmol / L on the third day after delivery, 22.72 mmol / L on the fifth day after delivery, and 8.3 mmol / L on the twelfth day after delivery. She resumed breastfeeding. The triglyceride level was 20.25 mmol / L three weeks after delivery. She stopped breastfeeding and took Lipanthyl (fenofibrate capsules). For details, please refer to Figure 1 , Figure 1 which is the graph of the change in the triglyceride level of the proband involved in the embodiment of the present invention.

[0069] (2) Sample testing:

[0070] a) Collect peripheral blood from the proband, the proband's father, the proband's mother, the proband's younger brother, and the proband's son, completely anticoagulate and centrifuge, and then collect plasma. Measure plasma CHO (total cholesterol), HDL-C (high-density lipoprotein cholesterol), LDL-C (low-density lipoprotein cholesterol), TG (triglyceride), Hcy (homocysteine), NEFA (non-esterified fatty acid), lipoprotein a, apolipoprotein A1, and apolipoprotein B.

[0071] b) Extract genomic DNA (gDNA) from the proband, the proband's father, the proband's mother, the proband's older brother, the proband's son, and the proband's daughter for whole exome sequencing (WES). The whole exome sequencing and sequence analysis were both carried out by Jinan Aixinzuoer Medical Testing Co., Ltd., a cooperative unit of Qilu Hospital of Shandong University.

[0072] In this embodiment, the acquisition of all data is based on compliance with legal norms and user consent, and is a legal application of the data. The proband and her family members have all agreed and signed the informed consent form. In addition, unless otherwise specified, the reagents or instruments used in this embodiment are all commercially available.

[0073] (3) Detection results:

[0074] The detection results of plasma CHO (total cholesterol), HDL-C (high-density lipoprotein cholesterol), LDL-C (low-density lipoprotein cholesterol), TG (triglyceride), Hcy (homocysteine), NEFA (non-esterified fatty acid), lipoprotein a, apolipoprotein A1, and apolipoprotein B are shown in Table 2 below:

[0075] Table 2 Metabolite detection results

[0076]

[0077] Gene detection results: As Figure 2 , Figure 3 shown, Figure 2 is the pedigree chart of the proband involved in the embodiment of the present invention, Figure 3 is the sequencing result chart of the proband involved in the embodiment of the present invention.

[0078] Figure 2 Among them, the circle represents a female, the square represents a male, and the arrow indicates the proband. According to Figure 2 and Figure 3 , it can be seen that I-1 (proband's father), II-2 (proband), and III-1 (proband's son) all carry the common mutation (LPL c.1015A>C (p.K339Q), transcript NM_000237.2). The 1015th A base of the DNA sequence of the LPL gene is replaced by a C base, resulting in the 339th amino acid of the LPL protein changing from lysine (K) to glutamine (Q). And it is a heterozygote. The proband's mother and the proband's daughter (III-2) did not carry this gene mutation after DNA sequencing. Among them, for the LPL c.1015A>C mutation, according to the "ACMG Genetic Variant Classification Standards and Guidelines", this mutation is not included in the normal control population in the GmomAD database (PM2 evidence), and the bioinformatics analysis software Mutation_Taster, SIFT, and PolyPhen2 all predict it as a harmful mutation (PP3). In summary, this mutation is tentatively determined to have an unclear clinical significance.

[0079] In this example, the carrier status of the heterozygous mutation (LPL c.1015A>C) is consistent with the clinical phenotype of the carrier. Therefore, it is speculated that this mutation may weaken the activity of LPL, and it may be the pathogenic mutation of HTG (hypertriglyceridemia). In order to further study the pathogenicity of this mutation, functional studies were carried out subsequently.

[0080] Functional studies:

[0081] (1) Conservation analysis

[0082] To further study the functional impact of LPL mutations on protein function, we predicted the effects of LPL mutations on protein structure. Figure 4 For the protein structure diagram of LPL and the amino acid sequence diagram among different species involved in the embodiments of the present invention, it can be seen that the LPL gene encodes a protein of 472 amino acid residues, and the mutation site c.1015A>C / p.K339Q is located on the conserved domain of LPL. The LPL conserved domain consists of a lipoprotein lipase superfamily region ( Figure 4 part A in), and LPL, as a homodimer, has dual functions of triglyceride hydrolase and ligand / bridging factor for receptor-mediated lipoprotein uptake. According to conservative analysis, the position where the mutant LPL p.K339Q is located is highly conserved. Multiple amino acid sequence alignments show that K339 is conserved among different species and is lysine (K) in all four plants ( Figure 4 part B in).

[0083] (2) Prediction and analysis of protein three-dimensional structure

[0084] Figure 5 For the three-dimensional structure prediction diagram of the LPL protein involved in the embodiments of the present invention.

[0085] Using I-TASSER modeling and PyMOL drawing analysis, the three-dimensional (3D) structure of LPL-WT and its variant (LPLp.K339Q) were predicted by threading method, and comprehensive analysis was carried out through head-to-head comparison of the final models. And the surface charges of LPL-WT and its variant (LPL p.K339Q) were analyzed using the Adaptive Poisson-Boltzmann Solver (APBS) of PyMOL.

[0086] The results showed that by using the mutagenesis module of pyMol to mutate the protein residue 339 lysine to glutamine, changes in the electrostatic potential energy around the residue and the surrounding hydrogen bonds were observed, and the electrostatic potential energy after mutation shifted to the left. According to I-TASSER modeling and PyMOL drawing analysis, after Lys339 was mutated to Gln, a pair of hydrogen bonds were formed between Gln339 and Val340 at the distance indicating that the LPL p.K339Q mutation may affect the main chain structure of LPL. In summary, the mutant LPLp.K339Q may affect the tertiary structure of LPL.

[0087] (3) In vitro activity study

[0088] Construct LPL-WT (wide type, wild type) plasmid and LPL c.1015A>C plasmid, transfect cells to detect protein expression, and collect supernatant and cell microspheres to measure the expression and activity of LPL. Specifically:

[0089] The Homo-Lpl (NM_000237.3) plasmid was purchased from BioSun Biotechnology (Shanghai) Co., Ltd. The Lpl mutant plasmid LPLc.1015A>C was constructed using the QuikChange site-directed mutagenesis kit (Agilent Technologies, Santa Clara, CA) according to the manufacturer's instructions. All constructs were verified by sequencing using an ABI 3730xl sequencer, and their structures were fully sequenced and used as templates in other cloning designs.

[0090] LO2 cells (human normal hepatocytes) and HEK-293 cells (human embryonic kidney cells 293) (both purchased from ATCC) were seeded in 10 cm 2 culture dishes for 24 h. On the next day, transfection was performed. 3 μg of each plasmid, pCDNA3.1, pCDNA3.1-LPL-WT, and pCDNA3.1-LPL-p.K339Q, was added to the culture dishes, and 500 μL of opti-MEM (Gbico, cat: 31985-070) was added and incubated for 3-5 min. 6 μL of liposome TMAdd 2000 transfection reagent (Invitrogen, cat: 11668-027) to opti-MEM and incubate for 3-5 min. Add the DNA mixture to the liposome mixture and incubate for 15-20 min. Add the above mixture to the cell culture medium and use fresh medium after 4-6 h. The control group is not transfected. Culture at 37 °C with 5% carbon dioxide for 48 h. Then, collect the cells and perform sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Immunoblotting was performed using anti-lipoprotein lipase (ab172953, Abcam, 1:1000), anti-GAPDH antibody (ab9485, Abcam, 1:1000), and goat anti-rabbit IgG H&L secondary antibody (ab7064, Abcam 1:5000). The signal was detected using a chemiluminescence kit (Millipore Corporation, CA, USA, WBKLS0050), and imaging was performed using a chemiluminescence imaging system (Shanghai Qinxiang Scientific Instruments Co., Ltd.). Obtain the cell supernatant and measure the total esterase activity using a total esterase detection kit (cat: A067-1-2, Nanjing Jiancheng Bioengineering Institute). The calculation formula is shown in the figure below. LPL activity (μ / mL) = (LPL OD value - blank tube) / (standard tube OD value - blank tube) × concentration standard (500 μmol / L) × dilution ratio, before sample testing × 60 min / 20 min ÷ 1000 (OD: optical density).

[0091] Figure 6 Schematic diagram of the expression level results of LPL WT and LPL p.K339Q, Figure 7 Histogram of the expression levels of LPL WT and LPL p.K339Q, Figure 8 Result graph of LPL activity of LPL WT and LPL p.K339Q in cells, Figure 9 Result graph of LPL activity of LPL WT and LPL p.K339Q in cell culture supernatant. From Figure 6 and Figure 7 it can be seen that the LPL c.1015A>C / p.K339Q mutation does not affect the protein expression level of LPL. From Figure 8 and Figure 9 it can be seen that in transfected cells and cell culture supernatant, the protein activity of the LPL c.1015A>C / p.K339Q variant is weaker than that of the LPL wild type, and the difference is statistically significant (P < 0.01). Therefore, it is speculated that LPL c.1015A>C / p.K339Q will reduce the activity of LPL, affect the hydrolysis of TG, and thus cause familial combined hyperlipidemia. It is speculated that it is a pathogenic mutation.

[0092] In this example, unless otherwise specified, the reagents or instruments used are all obtained from ordinary commercial sources. In this example, the research data are expressed as mean ± standard deviation (standard deviation). A two-tailed, paired Student's t-test was used for statistical comparison of the data sets between the two groups of GraphPad Prism 8. Each experiment was independently repeated at least three times. Representative experimental results are shown in the figure. p < 0.05 was considered significant.

[0093] In summary, according to the above research and research results of the examples, the LPL c.1015A>C mutation or the LPL p.K339Q mutation impairs the function of the LPL protein. The LPL c.1015A>C mutation or the LPL p.K339Q mutation is the pathogenic mutation of the hyperlipidemia pedigree in this example. The LPL c.1015A>C (p.K339Q) mutation discovered in this example is a new pathogenic gene for familial hyperlipidemia and shows autosomal dominant inheritance.

[0094] Although the present disclosure has been specifically described above in conjunction with the accompanying drawings and embodiments, it is understood that the above description does not limit the present disclosure in any way. Those skilled in the art can make modifications and variations to the present disclosure as needed without departing from the essential spirit and scope of the present disclosure, and these modifications and variations all fall within the scope of the present disclosure.

Claims

1. Use of a reagent in the preparation of a product for screening hyperlipidemia or assessing the susceptibility to hyperlipidemia, Characterized in that, The reagent is used to detect LPL amino acid mutations in a sample, the LPL amino acid mutation is that the 339th amino acid of the LPL protein is mutated from lysine (K) to glutamine (Q), the transcript of the LPL gene is NM_000237.2, and the reagent for detecting LPL amino acid mutations includes at least one of the reagent for detecting LPL amino acid mutations using protein and peptide sequence analysis techniques, mass spectrometry-related protein detection techniques, and antibody detection techniques.

2. The use according to claim 1, Characterized in that, The protein and peptide sequence analysis technique includes the Edman method; the antibody detection technique includes the method of preparing antibodies that can recognize different mutants.

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