Systems for screening patients for hyperlipidemia and / or acute pancreatitis or assessing susceptibility to hyperlipidemia and / or acute pancreatitis

Abstract

This disclosure describes a system for screening patients for hyperlipidemia and / or acute pancreatitis or assessing susceptibility to hyperlipidemia and / or acute pancreatitis. The system includes reagents, reagent sets, kits, or instruments for detecting LPL gene or protein mutations in samples. LPL gene mutations involve a substitution of an A base at position 1015 of the LPL gene's DNA sequence with a C base, and LPL protein mutations involve a mutation of an amino acid at position 339 from lysine (K) to glutamine (Q). This disclosure can provide novel pathogenic gene loci for hyperlipidemia and / or acute pancreatitis or mutation sites associated with a high risk of hyperlipidemia and / or acute pancreatitis, facilitating 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, application number 202310561811.8, and invention name being Application of Reagents for Detecting LPL Gene Mutations or Protein Mutations in Samples in the Preparation of Products for Screening Hyperlipidemia Patients. Technical Field

[0002] The present invention relates to the field of biomedicine technology, and in particular to a system for screening patients with hyperlipidemia and / or acute pancreatitis or evaluating susceptibility to hyperlipidemia and / or acute pancreatitis. Background Art

[0003] As living standards continue to improve, daily dietary patterns have undergone significant changes, leading to an increasing number of people experiencing nutrient imbalances in their blood. For example, elevated blood lipid levels can lead to hyperlipidemia (HLP). Patients with HLP are at significantly increased risk for atherosclerotic cardiovascular disease (ASCVD) and acute pancreatitis (AP). Lipids specifically encompass lipids and fats, with fats referring to triglycerides (TG). Factors that reduce triglyceride breakdown or increase triglyceride synthesis can lead to hypertriglyceridemia (HTG), which in turn contributes to hyperlipidemia.

[0004] Hyperlipidemia is caused by a variety of factors, including genetics, unhealthy lifestyles, and type 2 diabetes. Numerous studies have identified genes whose mutations can lead to severe hypertriglyceridemia, including apolipoprotein C-II (APOC2), lipoprotein lipase (LPL), lipase maturation factor 1 (LMF1), apolipoprotein V (APOA5), and glycosylphosphatidylinositol-anchored high-density lipoprotein binding protein 1 (GPIHBP1). Among these, defects in lipoprotein lipase (LPL) are a key cause of hypertriglyceridemia. The human lipoprotein lipase (LPL) gene, primarily located on chromosome 8p22, is an esterifying enzyme with triglyceride hydrolase activity, playing a crucial role in lipid metabolism. Studies have shown that LPL gene mutations lead to reduced lipoprotein lipase activity, which can affect triglyceride catabolism, hindering chylomicron metabolism and increasing chylomicron accumulation in the body, leading to hyperlipidemia.

[0005] While numerous studies have explored the pathogenic mechanisms of hyperlipidemia, unknown pathogenic gene loci remain. Further research into the pathogenic mechanisms of hyperlipidemia and the isolation of novel pathogenic gene variants in familial hyperlipidemia will be crucial for the diagnosis, treatment, and prevention of hyperlipidemia. Summary of the Invention

[0006] The present disclosure is made 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 high risk of hyperlipidemia, which is helpful for the diagnosis, treatment and prevention of hyperlipidemia.

[0007] To this end, a first aspect of the present disclosure provides the use of a reagent for detecting LPL gene or protein mutations in a sample in the preparation of a product for screening patients for hyperlipidemia. The LPL gene mutation is LPL c.1015A>C, and the LPL protein mutation is LPL p.K339Q. In this disclosure, the LPL c.1015A>C mutation (the A at position 1015 in the DNA sequence of the LPL gene is replaced by a C, resulting in a mutation of amino acid 339 of the LPL protein from lysine (K) to glutamine (Q), i.e., LPL p.K339Q)) was identified through family studies. Functional studies confirmed that the LPL c.1015A>C (LPL p.K339Q) mutation affects lipoprotein lipase (LPL) activity. This provides a new pathogenic gene locus for hyperlipidemia or a mutation site with a high risk for hyperlipidemia. Detecting whether a sample carries the LPL c.1015A>C (LPL p.K339Q) mutation can assist in screening patients for hyperlipidemia.

[0008] In the applications of the present disclosure, the reagents may optionally include a primer pair for amplifying the LPL gene and / or a probe for detecting the LPL gene variation. Thus, the LPL gene variation c.1015A>C can be captured and / or detected using the primer pair and / or probe.

[0009] In the applications involved in the present disclosure, optionally, the primer pair is designed based on the nucleotide sequence upstream and downstream of base 1015 of the LPL gene coding region in the human genome, and the probe is designed based on the nucleotide sequence upstream and downstream of base 1015 of the LPL gene coding region in the human genome. Thus, the primers can bind to sequences upstream and downstream of base 1015 of the LPL gene coding region, and the probe can bind to sequences upstream and downstream of base 1015 of the LPL gene coding region, to detect the region.

[0010] In the applications of the present disclosure, the reagents may optionally further include dNTPs, DNA polymerase, and PCR reaction buffer, thereby providing reaction substrates, catalytic enzymes, and buffers to facilitate the detection of LPL c.1015A>C.

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

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

[0013] In the applications of the present disclosure, the product may optionally further include a nucleic acid extraction reagent and / or a protein extraction reagent, thereby facilitating the detection of LPL c.1015A>C or LPL p.K339Q.

[0014] In the applications of the present disclosure, the sample may optionally be from at least one of a subject's peripheral blood, saliva, or tissue sample, and the LPL gene variation refers to a germline variation of the LPL gene, and the LPL protein variation refers to a germline variation of the LPL protein. Thus, by testing the subject's peripheral blood, saliva, and / or tissue sample, it is possible to detect a germline mutation in the subject's LPL gene (a germline mutation refers to a mutation carried during human embryonic development and present in every cell in the body).

[0015] In the applications involved in the present disclosure, optionally, the LPL gene variation is a heterozygous mutation or a homozygous mutation, and the LPL protein variation is a heterozygous mutation or a homozygous mutation.

[0016] A second 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 assessing susceptibility to hyperlipidemia, characterized in that the LPL gene variation is LPL c.1015A>C, and the LPL protein variation is LPL p.K339Q. In the present disclosure, the LPL c.1015A>C mutation (the A base at position 1015 in the DNA sequence of the LPL gene is replaced by the C base, resulting in the mutation of the amino acid at position 339 of the LPL protein from lysine (K) to glutamine (Q), i.e., LPL p.K339Q)) was identified through family studies, and functional studies confirmed that the LPL c.1015A>C (LPL p.K339Q) mutation affects lipoprotein lipase (LPL) activity. This provides a new pathogenic gene site for hyperlipidemia or a mutation site with a high risk of hyperlipidemia. By detecting whether a sample carries the LPL c.1015A>C (LPL p.K339Q) mutation, it can assist in the assessment of susceptibility to hyperlipidemia.

[0017] According to the present disclosure, a pathogenic mutation site of hyperlipidemia or a mutation site 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 This is a graph showing changes in triglyceride levels of the proband involved in the examples of the present invention.

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

[0020] Figure 3 This is a diagram showing the sequencing results of the proband involved in the embodiments of the present invention.

[0021] Figure 4 The diagram shows the protein structure of LPL involved in the embodiments of the present invention and its amino acid sequence in different species.

[0022] Figure 5 This is a predicted three-dimensional structure of the LPL protein involved in the embodiments of the present invention.

[0023] Figure 6 Schematic diagram of the expression level results of LPL WT and LPL p.K339Q.

[0024] Figure 7 The histogram shows the expression levels of LPL WT and LPL p.K339Q.

[0025] Figure 8 The graph shows the LPL activity results of LPL WT and LPL p.K339Q in cells.

[0026] Figure 9 The graph shows the LPL activity results of LPL WT and LPL p.K339Q in the cell culture supernatant. DETAILED DESCRIPTION

[0027] The embodiments of the present invention are described in detail below. Examples of the embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain the present invention and are not to be construed as limiting the present invention.

[0028] It should be understood by those skilled in the art that unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

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

[0030] Those skilled in the art will appreciate that, unless otherwise stated, the singular forms "a," "an," "said," and "the" used herein may also include plural forms. It should be further understood that the term "comprising" used in the specification of the present invention refers to the presence of the stated features, integers, steps, operations, elements, and / or components, but does not preclude the presence or addition of one or more other features, integers, steps, operations, elements, and / or groups thereof.

[0031] To facilitate understanding of the present invention, the present invention is further explained below with reference to specific embodiments in conjunction with the accompanying drawings. However, the specific embodiments do not constitute a limitation of the embodiments of the present invention. Those skilled in the art should understand that the accompanying drawings are only schematic diagrams of the embodiments, and the components in the accompanying drawings are not necessarily required to implement the present invention.

[0032] In this embodiment, any of the following applications is involved:

[0033] Use of a reagent for detecting LPL gene mutation 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 and 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] The use of a reagent for detecting LPL amino acid mutations in a sample in the preparation of a product for screening patients with hyperlipidemia and acute pancreatitis;

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

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

[0039] In the above-mentioned applications involved in this embodiment, LPL gene variation or LPL amino acid mutation is associated with the risk of three diseases: hyperlipidemia, acute pancreatitis, and hypertriglyceridemia. Therefore, the reagent for detecting LPL gene variation or LPL amino acid mutation can be used to screen patients for any one or any combination of hyperlipidemia, acute pancreatitis, and hypertriglyceridemia. The reagent for detecting LPL gene variation or LPL amino acid mutation can be used to assess susceptibility to any one or any combination of diseases: hyperlipidemia, acute pancreatitis, and hypertriglyceridemia.

[0040] The new mutation sites provided by the present invention supplement the genetic mutation spectrum of familial hyperlipidemia, which can facilitate the diagnosis of hyperlipidemia patients for treatment, as well as genetic diagnosis of carriers in the family for health management. At the same time, it can guide fertility based on the genotypes of both parents, so as to avoid the inheritance of pathogenic genes and guide eugenics.

[0041] The above-mentioned reagents involved in this embodiment may include reagents for detecting LPL gene mutations or LPL amino acid mutations. In some examples, the LPL gene mutation is LPL c.1015A>C, which refers to a mutation from A (adenine) to C (cytosine) at position 1015 in the coding region of the wild-type LPL gene. The LPL amino acid mutation (also known as the LPL protein mutation) is LPL p.K339Q, which refers to a mutation from lysine (K) to glutamine (Q) at amino acid position 339 in the LPL protein. In this embodiment, the LPL c.1015A>C mutation (the substitution of A at position 1015 in the DNA sequence of the LPL gene with C, resulting in a mutation from lysine to glutamine at amino acid position 339 in the LPL protein, i.e., LPL p.K339Q) was identified through family studies. This embodiment also confirms through functional studies that the LPL c.1015A>C (LPL p.K339Q) mutation affects lipoprotein lipase (LPL) activity. In some cases, the LPL c.1015A>C (LPL p.K339Q) mutation leads to decreased LPL activity. This provides a new pathogenic gene locus for hyperlipidemia or a mutation site that indicates a high risk of hyperlipidemia. By detecting whether a sample carries the LPL c.1015A>C (LPL p.K339Q) mutation, it can assist in screening patients for hyperlipidemia.

[0042] In some examples, the LPL gene variant can be detected using at least one of pyrosequencing, Sanger sequencing, NGS sequencing, polymerase chain reaction-single-strand conformation polymorphism analysis, and TaqMan probe analysis. In some examples, the LPL gene variant can be LPL c.1015A>C.

[0043] In some examples, the reagents involved in this embodiment may include primer pairs for amplifying the LPL gene and / or probes for detecting LPL gene variants. Thus, LPL gene variants can be captured and / or detected using the primer pairs and / or probes. In some examples, the reagents may also include dNTPs, DNA polymerase, and PCR reaction buffer. This facilitates detection of LPL gene variants.

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

[0045] In some examples, the reagents described in this embodiment may also include reagents for detecting LPL mutations other than LPL c.1015A>C (LPLp.K339Q). In some examples, these other LPL mutations may include all currently known and suspected pathogenic mutations in the LPL gene for hyperlipidemia. In some examples, the reagents described in this embodiment may also include reagents for detecting genes other than the LPL gene that are associated with hyperlipidemia. In some examples, genes associated with hyperlipidemia may include APOC2, LMF1, APOA5, and GPIHBP1. This allows for detection of all relevant loci for hyperlipidemia, facilitating a more comprehensive, one-time screening for hyperlipidemia.

[0046] In some examples, the reagents described in this embodiment may also include reagents for detecting genes or proteins associated with other diseases. For example, they may also include reagents for detecting genes or proteins associated with inherited metabolic diseases. This allows for simultaneous screening of multiple diseases in the subject.

[0047] In some examples, the reagents described in this embodiment may include reagents for detecting LPL protein variants using at least one of the following methods: protein and peptide sequence analysis techniques, mass spectrometry-related protein detection techniques, and antibody detection techniques. This allows for the detection of LPL protein variants. In some examples, the LPL protein variant is LPL p.K339Q. In some examples, the reagents described in this embodiment may also include reagents for detecting LPL proteins other than the LPL p.K339Q variant. For example, they may also include reagents for detecting LPL protein variants carrying other known or suspected pathogenic loci.

[0048] In some examples, protein and peptide sequence analysis techniques can include chemical methods for N-terminal sequence determination, the Edman method, C-terminal enzymatic methods, and C-terminal chemical degradation methods.

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

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

[0051] In some examples, the product involved in this embodiment may be in the form of a reagent, a reagent set, or a test kit. In some examples, the product may also include a system composed of instruments.

[0052] In some examples, the aforementioned products involved in this embodiment may also include systems comprised of instruments for detecting LPL gene variants or LPL amino acid mutations. For example, the product may be a system comprised of PCR reagents, DNA sequencing reagents, and a DNA sequencer; or a system comprised of TaqMan probes, PCR primer pairs, a quantitative PCR instrument, a genotyping module, and other reagents required for TaqMan probe technology; or a system comprised of probes, PCR primer pairs, and other reagents and instruments required for a ligase detection reaction (LDR); or a system comprised of PCR primer pairs, single-base extension primers, a chip, a PCR instrument, a genotyping module, and / or other reagents and instruments required for Sequenom MassArray technology. This facilitates the detection of the LPL gene or LPL protein.

[0053] In some examples, the above-mentioned product involved in this embodiment may also include a nucleic acid extraction reagent and / or a protein extraction reagent, thereby facilitating 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 often accompanied by acute pancreatitis. Therefore, the present disclosure provides new pathogenic gene sites 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 the sample, it can assist in screening patients with hyperlipidemia (and / or acute pancreatitis, and / or hypertriglyceridemia) and can assist in assessing the susceptibility to hyperlipidemia, acute pancreatitis and / or hypertriglyceridemia. Susceptibility refers to the risk of an individual developing a disease determined by genetic basis, and can also be understood as the risk of different individuals developing the disease under the same environment.

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

[0056] In some examples, the subjects may be the general population, individuals suspected of having hyperlipidemia, or individuals at high risk of hyperlipidemia. In some examples, individuals suspected of having hyperlipidemia may be patients with acute pancreatitis, recurrent pancreatitis, hyperlipidemia, or hypertriglyceridemia. In some examples, individuals at high risk of hyperlipidemia may be those with a family history of hyperlipidemia, such as individuals with at least one immediate family member diagnosed with hyperlipidemia.

[0057] In some cases, a sample can be tested for germline mutations in the LPL gene. Germline mutations, also known as germline mutations, are mutations that originate in reproductive cells such as sperm or eggs. In some cases, LPL gene germline mutation results can be obtained by extracting and testing gDNA (genomic DNA) from the individual being tested.

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

[0059] In some examples, the presence of just one LPL c.1015A>C mutation in the subject's LPL gene can assist in diagnosing the subject as a patient with hyperlipidemia. In other words, if the subject's LPL c.1015A>C mutation is detected as a heterozygous mutation, the subject can be diagnosed as a patient with hyperlipidemia. Of course, if the subject's LPL c.1015A>C mutation is detected as a homozygous mutation, the subject can also be diagnosed as a patient with hyperlipidemia.

[0060] In this embodiment, by using the LPL c.1015A>C gene mutation as a marker, patients with hyperlipidemia can be screened, thereby providing a reagent for detecting LPL gene mutations in a sample for use in preparing a product for screening patients with hyperlipidemia. Similarly, a reagent for detecting LPL gene mutations in a sample can be provided for use in preparing a product for screening acute pancreatitis, hypertriglyceridemia, or in preparing a product for assessing susceptibility to hyperlipidemia, acute pancreatitis, or hypertriglyceridemia. Similarly, a reagent for detecting LPL amino acid mutations in a sample can be provided for use in preparing a product for screening hyperlipidemia, acute pancreatitis, or hypertriglyceridemia, or in preparing a product for assessing susceptibility to hyperlipidemia, acute pancreatitis, or hypertriglyceridemia.

[0061] The above applications involved in the present invention are further explained in detail below in conjunction with the embodiments, but they should not be understood as limiting the scope of protection of the present invention.

[0062] [Example]

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

[0064] Table 1 Description of professional terms

[0065]

[0066] Clinical Cases:

[0067] (1) Case information

[0068] The proband was a 32-year-old woman with a son and a daughter. At 26 weeks of gestation, she complained of abdominal pain and vomiting for a week. Laboratory tests revealed triglycerides (TG) of 15.21 mmol / L and total cholesterol of 11.08 mmol / L. Abdominal MRI showed features of acute pancreatitis and a slightly enlarged spleen, leading to hospital admission for acute pancreatitis. After one week of treatment, the TG level decreased to 9.28 mmol / L, and she was discharged. At 29 weeks of gestation, the TG level increased to 33.98 mmol / L. At 33 weeks of gestation, the TG level continued to rise to 46.39 mmol / L, leading to consultation with the endocrinology and nutrition departments. Low-molecular-weight heparin was administered, along with a controlled diet and a short peptide enteral preparation. A week later, the TG level was 12.01 mmol / L, and she was discharged pending delivery. At 35 weeks of gestation, the TG level was 58.23 mmol / L, leading to a placental abruption and subsequent admission to the obstetrics department. At 35 weeks + 3 days of pregnancy, the triglyceride level was 32.45mmol / L. A baby girl weighing 2420g was delivered vaginally. The newborn's triglyceride level was 0.33mmol / L. Triglyceride levels decreased after delivery, with the level at 28.94mmol / L on the first day after delivery, 18.68mmol / L on the third day after delivery, 22.72mmol / L on the fifth day after delivery, 8.3mmol / L on the twelfth day after delivery, and breastfeeding was resumed. At three weeks after delivery, the triglyceride level was 20.25mmol / L, and breastfeeding was stopped. Lipingzhi (fenofibrate capsules) was used for treatment. Figure 1 , Figure 1 This is a graph showing changes in triglyceride levels of the proband involved in the examples of the present invention.

[0069] (2) Sample testing:

[0070] a) Peripheral blood was collected from the proband, the proband's father, the proband's mother, the proband's brother, and the proband's son. The blood was then fully anticoagulated and centrifuged, and plasma was collected. Plasma levels of total cholesterol (CHO), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), triglycerides (TG), homocysteine ​​(Hcy), non-esterified fatty acids (NEFA), lipoprotein a, apolipoprotein A1, and apolipoprotein B were measured.

[0071] b) Genomic DNA (gDNA) was extracted from the proband, the proband's father, mother, brother, son, and daughter for whole-exome sequencing (WES). Whole-exome sequencing and sequence analysis were performed by Jinan Aixinzuoer Medical Testing Co., Ltd., a partner of Qilu Hospital of Shandong University.

[0072] In this example, all data were obtained in compliance with legal regulations and based on user consent, ensuring the legal use of the data. The proband and family members agreed and signed the informed consent form. Furthermore, unless otherwise specified, all reagents and instruments used in this example were commercially available.

[0073] (3) Test results:

[0074] The test 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 acids), lipoprotein a, apolipoprotein A1 and apolipoprotein B are shown in Table 2 below:

[0075] Table 2 Metabolite detection results

[0076]

[0077] Genetic test results: Figure 2 、 Figure 3 As shown, Figure 2 is a pedigree chart of the proband involved in the embodiments of the present invention, Figure 3 This is a diagram showing the sequencing results of the proband involved in the embodiments of the present invention. Figure 2 In the figure, the circle represents female, the square represents male, and the arrow points to the proband. Figure 2 and Figure 3 I-1 (the proband's father), II-2 (the proband), and III-1 (the proband's son) all carry a common mutation (LPL c.1015A>C (p.K339Q), transcript NM_000237.2). This mutation involves a C substitution at position 1015 of the LPL gene, resulting in a lysine (K) to glutamine (Q) mutation at position 339 of the LPL protein. These individuals are heterozygous. DNA sequencing of the proband's mother and the proband's daughter (III-2) revealed no carriers of this gene mutation. The LPL c.1015A>C mutation, according to the ACMG Criteria and Guidelines for the Classification of Genetic Variation, is not included in the GmomAD database of healthy controls (PM2 evidence). Bioinformatics analysis software Mutation_Taster, SIFT, and PolyPhen2 all predict it to be deleterious (PP3). Therefore, this mutation is tentatively classified as of unknown clinical significance.

[0078] In this example, the presence of a heterozygous mutation (LPL c.1015A>C) was consistent with the carrier's clinical phenotype, leading to speculation that this mutation may impair LPL activity and be a pathogenic mutation for hypertriglyceridemia (HTG). To further investigate the pathogenicity of this mutation, functional studies were subsequently performed.

[0079] Functional studies:

[0080] (1) Conservative analysis

[0081] To further investigate the functional impact of LPL mutations on protein function, we predicted the effects of LPL mutations on protein structure. Figure 4 The protein structure diagram of LPL involved in the embodiment of the present invention and the amino acid sequence diagram of different species show that the LPL gene encodes a protein of 472 amino acid residues, and the mutation site c.1015A>C / p.K339Q is located in the conserved domain of LPL. The conserved domain of LPL consists of a lipoprotein lipase superfamily region ( Figure 4 Part A of the ), LPL, as a homodimer, has the dual functions of triglyceride hydrolase and ligand / bridging factor for receptor-mediated lipoprotein uptake. According to conservation analysis, the position of the LPL mutation p.K339Q is highly conserved. Multiple amino acid sequence alignments show that K339 is conserved among different species. In all four plants, it is lysine (K) ( Figure 4 B in the .

[0082] (2) Protein three-dimensional structure prediction and analysis

[0083] Figure 5 This is a predicted three-dimensional structure of the LPL protein involved in the embodiments of the present invention.

[0084] The three-dimensional (3D) structures of LPL-WT and its variant (LPL p.K339Q) were predicted using I-TASSER modeling and PyMOL graphics analysis. A comprehensive analysis was conducted through head-to-head comparison of the final models. The surface charges of LPL-WT and its variant (LPL p.K339Q) were analyzed using the Adaptive Poisson-Boltzmann Solver (APBS) in PyMOL.

[0085] Results showed that using the pyMol mutagenesis module to mutate protein residue 339 lysine to glutamine revealed changes in the electrostatic potential energy and surrounding hydrogen bonds around the residue, with the electrostatic potential energy shifting to the left after the mutation. I-TASSER modeling and PyMOL mapping analysis revealed that the mutation of Lys339 to Gln formed a pair of hydrogen bonds between Gln339 and Val340 at a distance of 2.9 Å, suggesting that the LPL p.K339Q mutation may affect the main chain structure of LPL. In summary, the LPL p.K339Q mutation may affect the tertiary structure of LPL.

[0086] (3) In vitro activity studies

[0087] LPL-WT (wide type, wild type) plasmid and LPL c.1015A>C plasmid were constructed, transfected into cells to detect protein expression, and supernatant and cell microspheres were collected to measure LPL expression and activity. Specifically:

[0088] 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, their structures were fully sequenced, and they were used as templates in other cloning designs.

[0089] LO2 cells (human normal liver cells) and HEK-293 cells (human embryonic kidney cells 293) (both purchased from ATCC) were seeded in 10 cm 2 The cells were cultured in a dish for 24 hours. The next day, transfection was performed by adding 3 μg of each plasmid, pCDNA3.1, pCDNA3.1-LPL-WT, and pCDNA3.1-LPL-p.K339Q, to the dish, along with 500 μL of Opti-MEM (Gbico, cat: 31985-070) and incubating for 3-5 minutes. Six μL of Lipofectamine™ 2000 transfection reagent (Invitrogen, cat: 11668-027) was added to Opti-MEM and incubated for 3-5 minutes. The DNA mixture was added to the lipofectamine mixture and incubated for 15-20 minutes. This mixture was then added to the cell culture medium, and fresh culture medium was replaced 4-6 hours later. A control group was not transfected. The cells were cultured at 37°C with 5% CO2 for 48 hours. The cells were then harvested and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Immunoblotting was performed using anti-lipoprotein lipase (ab172953, Abcam, 1:1000), anti-GAPDH (ab9485, Abcam, 1:1000), and goat anti-rabbit IgG H&L secondary antibody (ab7064, Abcam, 1:5000). Signals were detected using a chemiluminescence kit (Millipore, CA, USA, WBKLS0050), and imaging was performed using a chemiluminescence imaging system (Shanghai Qinxiang Scientific Instrument Co., Ltd.). Cell supernatants were obtained and total esterase activity was measured using a total esterase assay 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) × standard concentration (500 μmol / L) × dilution ratio, sample pre-assay × 60 min / 20 min ÷ 1000 (OD: optical density).

[0090] Figure 6 Schematic diagram of the expression levels of LPL WT and LPL p.K339Q. Figure 7 is the expression level histogram of LPL WT and LPL p.K339Q, Figure 8 This is the result of LPL activity in cells of LPL WT and LPL p.K339Q. Figure 9 The figure shows the results of LPL activity of LPL WT and LPL p.K339Q in cell culture supernatant. 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. Figure 8 and Figure 9 The protein activity of the LPL c.1015A>C / p.K339Q variant was weaker than that of the wild-type LPL in both transfected cells and culture supernatants, with the difference being statistically significant (P < 0.01). Therefore, it is speculated that LPL c.1015A>C / p.K339Q reduces LPL activity and affects triglyceride hydrolysis, thereby causing familial combined hyperlipidemia, and is speculated to be a pathogenic mutation.

[0091] Unless otherwise specified, all reagents and instruments used in this example were commercially available. Data are presented as mean ± standard deviation (SD). Statistical comparisons between two GraphPad Prism 8 datasets were performed using a two-tailed, paired Student's t-test. Each experiment was repeated at least three times. Representative experimental results are shown in the figure. A p value < 0.05 was considered significant.

[0092] In summary, the above studies and results in this example demonstrate that the LPL c.1015A>C mutation or the LPL p.K339Q mutation impairs LPL protein function. These mutations are the causative mutations in the hyperlipidemia families described in this example. The LPL c.1015A>C (p.K339Q) mutation discovered in this example represents a novel causative gene for familial hyperlipidemia, and is inherited in an autosomal dominant manner.

[0093] Although the present disclosure has been described in detail above with reference to the accompanying drawings and embodiments, it will be understood that the above description does not limit the present disclosure in any form. Those skilled in the art may modify and change the present disclosure as needed without departing from the spirit and scope of the present disclosure, and such modifications and variations all fall within the scope of the present disclosure.

Claims

1. Use of a reagent for detecting LPL gene mutation or protein mutation in a sample in the preparation of a system for screening patients with familial hyperlipidemia, characterized in that: The LPL gene mutation is that the A base at position 1015 of the DNA sequence of the LPL gene is replaced by the C base, and the LPL protein mutation is that the amino acid at position 339 of the LPL protein is mutated from lysine (K) to glutamine (Q). As long as there is one LPL c.1015A>C mutation in the LPL gene of the person being tested, the person being tested is considered to be a patient with familial hyperlipidemia, and the transcript of the LPL gene is NM_000237.

2.

2. The use according to claim 1, characterized in that The system uses at least one of Sanger sequencing, NGS sequencing, polymerase chain reaction-single-strand conformation polymorphism analysis, and TaqMan probe method to detect LPL gene variation.

3. The use according to claim 2, characterized in that When the system uses the Sanger sequencing method or the NGS sequencing method, the system includes PCR reagents, DNA sequencing reagents, and a DNA sequencer.

4. The use according to claim 2, characterized in that When the system uses the TaqMan probe method, the system includes a TaqMan probe, a PCR primer pair, a quantitative PCR instrument, a module for performing genotyping, and other reagents required for the TaqMan probe technology.

5. The use according to claim 1, characterized in that Reagents for LPL protein variation include reagents for detecting LPL protein variation using at least one of the following methods: protein and peptide sequence analysis technology, mass spectrometry-related protein detection technology, and antibody detection technology.

6. The use according to claim 1 or 5, characterized in that The reagent includes an antibody that can recognize LPL protein having the LPLp.K339Q mutation.

7. The use according to claim 1, characterized in that The sample comes from at least one of the peripheral blood, saliva, and tissue sample of the subject.

8. The use according to claim 1, characterized in that The LPL gene variation refers to the germline variation of the LPL gene, and the LPL protein variation refers to the germline variation of the LPL protein.

9. The use according to claim 1 or 8, characterized in that The LPL gene mutation is a heterozygous mutation or a homozygous mutation, and the LPL protein mutation is a heterozygous mutation or a homozygous mutation.

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