Application of reagents for detecting APOB gene mutation or protein mutation in samples in the preparation of products for screening patients with familial hypercholesterolemia

Abstract

The present disclosure describes the use of a reagent for detecting APOB gene mutation or protein mutation in a sample in the preparation of a product for screening patients with familial hypercholesterolemia, wherein the APOB gene mutation is APOB c.676G>A, and the APOB protein mutation is APOB p.A226T. The present disclosure also discloses the use of a reagent for detecting APOB gene mutation or protein mutation in a sample in the preparation of a product for assessing susceptibility to familial hypercholesterolemia. According to the present disclosure, new pathogenic gene sites for familial hypercholesterolemia or mutation sites with high risk of familial hypercholesterolemia can be provided, which is helpful for the diagnosis, treatment and prevention of familial hypercholesterolemia.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to the use of a reagent for detecting APOB gene mutation or protein mutation in a sample in the preparation of a product for screening patients with familial hypercholesterolemia. Background Art

[0002] Familial hypercholesterolemia (FH) is a common genetic disease characterized by elevated low-density lipoprotein cholesterol (LDL-C) levels, accompanied by skin / tendon xanthomas, lipocorticoids, and premature atherosclerotic cardiovascular disease (ASCVD). The incidence of FH is increasing year by year, but its medical consultation rate and social awareness are still low. Early identification and diagnosis of FH and intervention can effectively prevent the occurrence of cardiovascular events. So far, the APOB c.676G>A site mutation found in clinical practice has not been reported in domestic and foreign studies. This study combined the patient's clinical data and genetic test results, traced the blood lipid status of the patient's family members, performed functional verification and clinical characteristics analysis on the mutation, and explored the pathogenicity and possible pathogenic mechanism of the mutation site, thereby providing a genetic basis for finding new prevention and treatment measures for FH.

[0003] APOB protein is the main component of plasma lipoprotein and participates in lipid synthesis, secretion, metabolism and other aspects. APOB gene mainly encodes two APOB proteins, namely APOB100 and APOB48. APOB48 is synthesized in the small intestinal mucosa and mainly participates in the synthesis of CM and transports TG and cholesterol from food into the blood. CM is cleared quickly in the blood and has a short half-life of only 5-10 minutes. Therefore, the content of APOB48 in the blood is also relatively low, about 0.1% of APOB100. One molecule of CM contains only one molecule of APOB48, and it always exists in CM during blood circulation and metabolism, and is not transferred to other lipoprotein particles. Therefore, APOB48 can also be used as a marker of CM. Changes in the expression of APOB48 will have an important impact on the synthesis of lipoproteins. Studies have found that increased synthesis of APOB48 in the small intestine will cause more fat to be transported to the liver, thereby increasing the synthesis of VLDL in the liver. APOB100 is mainly involved in the formation of VLDL and LDL. Since LDL is absorbed slowly, in most individuals, LDL constitutes the majority of cholesterol transport particles. Polymorphism of the APOB gene may lead to a decrease in the ability of LDL to bind to LDLR on liver cells, slowing down the clearance of TG and cholesterol in plasma, and causing a large amount of cholesterol to accumulate in the arteries, thereby causing ASCVD.

[0004] At present, many studies have been conducted to explain the pathogenic mechanism of familial hypercholesterolemia, but there are still unknown pathogenic gene loci. Further research on the pathogenic mechanism of familial hypercholesterolemia and isolation of new pathogenic gene variants of familial hypercholesterolemia are of great significance for the diagnosis, treatment and prevention of familial hypercholesterolemia. Summary of the invention

[0005] 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 familial hypercholesterolemia or a mutation site with high risk of familial hypercholesterolemia, which is helpful for the diagnosis, treatment and prevention of familial hypercholesterolemia.

[0006] To this end, the first aspect of the present disclosure provides an application of a reagent for detecting APOB gene mutation or protein mutation in a sample in the preparation of a product for screening patients with familial hypercholesterolemia, characterized in that the APOB gene mutation is APOBc.676G>A, and the APOB protein mutation is. In the present disclosure, the APOB c.676G>A mutation (the 676th base guanine (G) in the DNA sequence coding region of the APOB gene is replaced by adenine (A), resulting in the 226th amino acid of the APOB protein being mutated from alanine (A) to threonine (T), i.e., APOB p.A226T)) is identified through family studies, and functional studies confirm that the APOB c.676G>A (APOB p.A226T) mutation affects APOB activity, thereby providing a new pathogenic gene site for familial hypercholesterolemia or a mutation site with a high risk of familial hypercholesterolemia, and by detecting whether the sample carries the APOB c.676G>A (APOB p.A226T) mutation, it can assist in screening patients with familial hypercholesterolemia.

[0007] In the application involved in the present disclosure, optionally, the reagent includes a primer pair for amplifying the APOB gene and / or a probe for detecting the APOB gene variation. Thus, the APOB gene variation c.676G>A can be captured and / or detected by the primer pair and / or the probe.

[0008] In the application involved in the present disclosure, optionally, the primer pair is designed based on the nucleotide sequence of the 676th base in the coding region of the APOB gene in the human genome, and the probe is designed based on the nucleotide sequence of the 676th base and its upstream and downstream in the coding region of the APOB gene in the human genome. Thus, the primer can bind to the sequence of the upstream and downstream region of the 676th base in the coding region of the APOB gene, and the probe can bind to the sequence of the 676th base and the upstream and downstream region of the coding region of the APOB gene, so as to detect the region.

[0009] In the application involved in the present disclosure, optionally, the reagent also includes dNTPs, DNA polymerase and PCR reaction buffer, thereby providing reaction substrate, catalytic enzyme and buffer to facilitate the detection of APOB c.676G>A.

[0010] In the application involved in the present disclosure, optionally, the reagent includes a reagent for detecting the APOB protein variation using at least one of the following methods: protein and peptide sequence analysis technology, mass spectrometry-related protein detection technology, antibody detection technology. Thus, a reagent can be provided to detect the APOB protein variation (also called amino acid variation) APOBp.A226T.

[0011] In the application of the present disclosure, optionally, the reagent includes an antibody that can recognize APOB protein with APOB p.A226T mutation. Thus, the antibody that can recognize APOB protein with APOB p.A226T mutation can be used to detect APOB protein with APOB p.A226T mutation.

[0012] In the application involved in the present disclosure, optionally, the product further comprises a nucleic acid extraction reagent and / or a protein extraction reagent, thereby facilitating the detection of APOB c.676G>A or APOB p.A226T.

[0013] In the application involved in the present disclosure, optionally, the sample is from at least one of the peripheral blood, saliva, and tissue samples of the subject, and the APOB gene variation refers to the germline variation of the APOB gene, and the APOB protein variation refers to the germline variation of the APOB protein. Thus, the germline mutation (germline mutation refers to a variation that is already carried during the embryonic development period of a person and is carried by every cell in the body) of the APOB gene of the subject can be detected by detecting the peripheral blood, saliva, and / or tissue samples of the subject.

[0014] In the applications involved in the present disclosure, optionally, the APOB gene variation is a heterozygous mutation or a homozygous mutation, and the APOB protein variation is a heterozygous mutation or a homozygous mutation.

[0015] The second aspect of the present disclosure provides an application of a reagent for detecting APOB gene variation or protein variation in a sample in the preparation of a product for assessing susceptibility to familial hypercholesterolemia, characterized in that the APOB gene variation is APOBc.676G>A, and the APOB protein variation is APOB p.A226T. In the present disclosure, the APOBc.676G>A mutation was identified through family studies (the 676th base guanine (G) in the DNA sequence coding region of the APOB gene was replaced by adenine (A), resulting in the 226th amino acid of the APOB protein changing from alanine (A) to threonine (T), i.e., APOB p.A226T), and functional studies confirmed that the APOB c.676G>A (APOB p.A226T) mutation would affect APOB activity, thereby providing a new pathogenic gene site for familial hypercholesterolemia or a mutation site with a high risk of familial hypercholesterolemia, and by detecting whether the sample carries the APOB c.676G>A (APOB p.A226T) mutation, it can assist in the assessment of susceptibility to familial hypercholesterolemia.

[0016] According to the present disclosure, a pathogenic mutation site of familial hypercholesterolemia or a mutation site with a high risk of familial hypercholesterolemia can be provided, which is helpful for the diagnosis, treatment and prevention of familial hypercholesterolemia. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The pedigree diagram of the proband involved in the embodiment of the present invention.

[0018] Figure 2 This is a diagram showing the APOB c.676G>A mutation site verification results of the proband and his relatives involved in the embodiments of the present invention.

[0019] Figure 3 This is a diagram showing the INSR c.4028G>A mutation site verification results of the proband and his relatives involved in the embodiments of the present invention.

[0020] Figure 4 The diagram is a protein structure diagram of APOB involved in the embodiments of the present invention and a diagram of the amino acid sequence in different species.

[0021] Figure 5 It is a predicted three-dimensional structure of the APOB protein involved in the embodiment of the present invention.

[0022] Figure 6 This is a diagram showing the transfection results of human embryonic kidney cells HEK 293T at different multiples involved in the embodiments of the present invention.

[0023] Figure 7 This is a diagram showing the transfection results of the human liver cancer cell HepG2 at different multiples involved in the embodiments of the present invention.

[0024] Figure 8 It is an immunofluorescence expression diagram of the wild-type APOB and the mutant A226T-APOB involved in the embodiments of the present invention.

[0025] Fig. 9 This is a diagram showing the functional study results of the Ala226Thr-APOB mutation involved in the embodiments of the present invention.

[0026] Fig.10 This is a diagram showing the effect of the Ala226Thr-APOB mutation on other lipogenic genes involved in the embodiments of the present invention. DETAILED DESCRIPTION

[0027] The embodiments of the present invention are described in detail below, and 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 with the same or similar functions. The embodiments described below by the accompanying drawings are exemplary and are only used to explain the present invention, and cannot be interpreted 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] In this embodiment, any of the following applications is involved:

[0030] Use of a reagent for detecting APOB gene variation in a sample in the preparation of a product for screening patients with familial hypercholesterolemia, acute pancreatitis and / or hypertriglyceridemia;

[0031] Use of a reagent for detecting APOB gene mutation in a sample in the preparation of a product for screening patients with familial hypercholesterolemia and acute pancreatitis;

[0032] Use of a reagent for detecting APOB amino acid mutations in a sample in the preparation of a product for screening patients with familial hypercholesterolemia, acute pancreatitis and / or hypertriglyceridemia;

[0033] Use of a reagent for detecting APOB amino acid mutations in a sample in the preparation of a product for screening patients with familial hypercholesterolemia and acute pancreatitis;

[0034] Use of a reagent for detecting a variation in the APOB gene in a sample in a product for evaluating susceptibility to familial hypercholesterolemia, acute pancreatitis, and / or hypertriglyceridemia;

[0035] Use of a reagent for detecting an APOB amino acid mutation in a sample in a product for evaluating susceptibility to familial hypercholesterolemia, acute pancreatitis and / or hypertriglyceridemia.

[0036] In the above-mentioned applications involved in this embodiment, APOB gene variation or APOB amino acid mutation is related to the risks of three diseases: familial hypercholesterolemia, acute pancreatitis and hypertriglyceridemia. Therefore, reagents for detecting APOB gene variation or APOB amino acid mutation can be used to screen patients with any one or any combination of familial hypercholesterolemia, acute pancreatitis and hypertriglyceridemia, and reagents for detecting APOB gene variation or APOB amino acid mutation can be used to assess the susceptibility to any one or any combination of diseases: familial hypercholesterolemia, acute pancreatitis and hypertriglyceridemia.

[0037] The new mutation sites provided by the present invention supplement the genetic mutation spectrum of familial hypercholesterolemia, which can be beneficial for diagnosing patients with familial hypercholesterolemia for treatment, and for genetic diagnosis of carriers in the family for health management. At the same time, it can guide fertility according to the genotypes of both parents, so as to avoid the inheritance of pathogenic genes and guide eugenics.

[0038] The above reagents involved in this embodiment may include reagents for detecting APOB gene mutation or APOB amino acid mutation. In some examples, the APOB gene mutation is APOB c.676G>A, and the APOB amino acid mutation (i.e., APOB protein mutation) is APOB p.A226T, and APOB p.A226T refers to the mutation of the 226th amino acid of the APOB protein from alanine (A) to threonine (T). In this embodiment, the APOB c.676G>A mutation was identified through family studies (the G base at position 676 of the DNA coding region sequence of the APOB gene was replaced by the A base, resulting in the mutation of the 226th amino acid of the APOB protein from alanine to threonine, i.e., APOB p.A226T). This embodiment also confirms through functional studies that the APOB c.676G>A (APOBp.A226T) mutation affects APOB activity. In some examples, the APOB c.676G>A (APOB p.A226T) mutation leads to decreased APOB activity. Therefore, a new pathogenic gene site for familial hypercholesterolemia or a high-risk mutation site for familial hypercholesterolemia is provided, which can assist in screening patients with familial hypercholesterolemia by detecting whether the sample carries the APOB c.676G>A (APOB p.A226T) mutation.

[0039] In some examples, the APOB gene variation can be detected using at least one of pyrophosphate sequencing technology, Sanger sequencing, NGS sequencing, polymerase chain reaction-single strand conformation polymorphism analysis, and TaqMan probe method. In some examples, the APOB gene variation can be APOB c.676G>A.

[0040] In some examples, the reagents involved in this embodiment may include primer pairs for amplifying the APOB gene and / or probes for detecting APOB gene mutations. Thus, APOB gene mutations can be captured and / or detected by primer pairs and / or probes. In some examples, the reagents may also include dNTPs, DNA polymerase, and PCR reaction buffer. Thus, APOB gene mutations can be easily detected.

[0041] In some examples, the primer pair can be designed based on the nucleotide sequence of the 676th base in the coding region of the APOB gene in the human genome, and the probe can be designed based on the nucleotide sequence of the 676th base and its upstream and downstream in the coding region of the APOB gene in the human genome. Thus, the primer can bind to the sequence of the upstream and downstream region of the 676th base in the coding region of the APOB gene, and the probe can bind to the sequence of the 676th base and the upstream and downstream region of the coding region of the APOB gene, so as to detect APOBc.676G>A.

[0042] In some examples, the above reagents involved in this embodiment may also include reagents for detecting other APOB mutations other than APOB c.676G>A (APOB p.A226T). In some examples, other APOB mutations may include all pathogenic mutations and suspected pathogenic mutations of the APOB gene currently known for familial hypercholesterolemia. In some examples, the above reagents involved in this embodiment may also include reagents for detecting other genes related to familial hypercholesterolemia other than the APOB gene. In some examples, genes related to familial hypercholesterolemia may include APOA2, GHR, GSBS, EPHX2, LDLR, PCSK9, and LDLRAP1. Thus, all related sites of familial hypercholesterolemia can be detected, which is conducive to a one-time and more comprehensive screening of familial hypercholesterolemia.

[0043] 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, reagents for detecting genes or proteins related to diseases that require differential diagnosis (such as familial combined hyperlipidemia type 3) may also be included. Thus, differential diagnosis can be performed on the subject at the same time. For example, reagents for detecting genes or proteins related to related inherited metabolic diseases (such as hyperlipidemia, diabetes) may also be included. Thus, the subject can be screened for multiple diseases at the same time.

[0044] In some examples, the above reagents involved in this embodiment may include reagents for detecting APOB 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. In this way, APOB protein variation can be detected. In some examples, APOB protein variation refers to APOB p.A226T. In some examples, the above reagents involved in this embodiment may also include reagents for detecting APOB proteins other than APOBp.A226T variant proteins. For example, reagents for detecting APOB protein variants carrying other known pathogenic / suspected pathogenic sites may also be included.

[0045] In some examples, sequence analysis techniques for proteins and peptides may include chemical methods for N-terminal sequence determination, the Edman method, C-terminal enzymatic methods, and C-terminal chemical degradation methods.

[0046] 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).

[0047] In some examples, antibody detection techniques can include preparing antibodies that can recognize different mutants, immunoblotting (such as western blot) and enzyme-linked immunosorbent assay (ELISA).

[0048] In some examples, the product involved in this embodiment may be in the form of a reagent, a reagent set or a reagent kit. In some examples, the product may also include a system composed of instruments.

[0049] In some examples, the above-mentioned products involved in this embodiment may also include a system composed of instruments for detecting APOB gene variation or APOB amino acid mutation. For example, the product may be a system composed of PCR reagents, DNA sequencing reagents and DNA sequencers, or a system composed of TaqMan probes, PCR primer pairs, quantitative PCR instruments and modules 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 ligase detection reaction (LDR), or a system composed of PCR primer pairs, single base extension primers, chips, PCR instruments, modules for genotyping and / or other reagents and instruments required for Sequenom MassArray technology. Thus, it is convenient to detect APOB genes or APOB proteins.

[0050] 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.

[0051] In some examples, the clinical manifestations of patients with familial hypercholesterolemia also include acute pancreatitis and hypertriglyceridemia. In some examples, patients with familial hypercholesterolemia are usually accompanied by acute pancreatitis. Therefore, the present disclosure provides new pathogenic gene sites for familial hypercholesterolemia (and / or acute pancreatitis, and / or hypertriglyceridemia) or mutation sites with high risk of familial hypercholesterolemia (and / or acute pancreatitis, and / or hypertriglyceridemia). By detecting the APOB c.676G>A mutation in the sample, it can assist in screening patients with familial hypercholesterolemia (and / or acute pancreatitis, and / or hypertriglyceridemia), and can assist in assessing the susceptibility of familial hypercholesterolemia, acute pancreatitis and / or hypertriglyceridemia. Susceptibility refers to the risk of an individual getting sick determined by genetic basis, and can also be understood as the risk of different individuals getting sick under the same environment.

[0052] In some examples, in the above applications involved in this embodiment, the APOB gene or APOB protein can be detected by detecting at least one of the peripheral blood, saliva, and tissue samples of the subject. In other words, the sample to be detected can come from at least one of the peripheral blood, saliva, and tissue samples of the subject.

[0053] In some examples, the test subjects may be the general population, individuals suspected of having familial hypercholesterolemia, or individuals at high risk of familial hypercholesterolemia. In some examples, individuals suspected of having familial hypercholesterolemia may be patients with acute pancreatitis, patients with recurrent pancreatitis, patients with familial hypercholesterolemia, and patients with hypertriglyceridemia. In some examples, individuals at high risk of familial hypercholesterolemia may be people with a family history of familial hypercholesterolemia, such as individuals with at least one immediate family member diagnosed with familial hypercholesterolemia.

[0054] In some examples, the germline mutation of the APOB gene in the sample can be detected. Germline mutation, also known as germ cell mutation, is a mutation carried by reproductive cells such as sperm or eggs. In some examples, the germline mutation result of the APOB gene can be obtained by extracting the gDNA (genomic DNA) of the subject and testing the genomic DNA.

[0055] In some examples, the 676th base of the coding region of the APOB gene can be detected. Further, the APOB c.676G>A mutation can be detected. In other words, it can be detected whether the subject carries the APOB c.676G>A mutation.

[0056] In some examples, as long as there is one APOB c.676G>A mutation in the APOB gene of the subject, the subject can be diagnosed as a patient with familial hypercholesterolemia. In other words, when the APOB c.676G>A of the subject is detected as a heterozygous mutation, the subject can be diagnosed as a patient with familial hypercholesterolemia. Of course, when the APOB c.676G>A of the subject is detected as a homozygous mutation, the subject can also be diagnosed as a patient with familial hypercholesterolemia.

[0057] In this embodiment, by using the gene mutation of APOB c.676G>A as a marker, patients with familial hypercholesterolemia can be screened, and then a reagent for detecting APOB gene mutations in a sample is provided for use in preparing a product for screening patients with familial hypercholesterolemia. Similarly, a reagent for detecting APOB gene mutations in a sample can be provided for use in preparing a product for screening acute pancreatitis, hypertriglyceridemia, and in preparing a product for assessing susceptibility to familial hypercholesterolemia, acute pancreatitis, or hypertriglyceridemia. Similarly, a reagent for detecting APOB amino acid mutations in a sample can be provided for use in preparing a product for screening familial hypercholesterolemia, acute pancreatitis, or hypertriglyceridemia, and in preparing a product for assessing susceptibility to familial hypercholesterolemia, acute pancreatitis, or hypertriglyceridemia.

[0058] 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 protection scope of the present invention.

[0059] [Example]

[0060] Clinical Cases

[0061] (1) Case information

[0062] The proband was a female with a history of type 2 diabetes for more than 4 years. She was admitted to the hospital for acute pancreatitis and hyperlipidemia. Routine biochemical test results showed that her total cholesterol (Cho 16.5mmol / L), low-density lipoprotein (LDL-C 10.66mmol / L) and triglyceride (TG 23.45mmol / L) levels were significantly elevated.

[0063] The mother of the proband was diagnosed with "type 2 diabetes" at the age of 40 and "acute pancreatitis and hyperlipidemia" at the age of 44, and received lipid-lowering treatment with "fenofibrate 200 mg qn". She had a history of "breast cancer" and underwent bilateral mastectomy.

[0064] The proband's uncle was diagnosed with "type 2 diabetes and hyperlipidemia" at the age of 51 and did not receive lipid-lowering treatment.

[0065] The proband's maternal grandmother was diagnosed with "hyperlipidemia and coronary heart disease" at the age of 58 and received lipid-lowering treatment with "atorvastatin calcium 10 mg qn".

[0066] Other family members in the proband's pedigree had no history of hyperlipidemia.

[0067] (2) Sample testing and results:

[0068] a) Peripheral blood was collected from the proband and related family members for lipid and blood sugar tests, and OGTT, insulin and C-peptide release tests were performed (the results are shown in Tables 1 and 2, respectively). The results showed that the proband, his mother, uncle, and grandmother all had hyperlipidemia and type 2 diabetes, and the proband's grandfather had type 2 diabetes. A pedigree chart was drawn based on the test results and the results of the proband's family history collection ( Figure 1 ).

[0069] Table 1 Blood lipid test results of the proband and family members

[0070]

[0071] Note: Reference value for total cholesterol (Cho): 2.80-6.00 mmol / L; reference value for high-density lipoprotein cholesterol (HDL-C): 0.80-2.00 mmol / L; reference value for low-density lipoprotein cholesterol (LDL-C): 1.00-3.37 mmol / L; reference value for triglycerides (TG): 0.30-1.70 mmol / L; reference value for free fatty acids (NEFA): 10.0-85.0 umol / dL.

[0072] Table 2 Results of OGTT, insulin release test and C-peptide release test in the proband and family members

[0073]

[0074] Note: Fasting reference value for blood glucose (GLU): 3.90-6.10 mmol / L; fasting reference value for C-peptide (CP): 0.81-3.85 ng / ml; fasting reference value for insulin (INS): 3-25 uIU / ml; reference value for glycosylated hemoglobin (HbA1c): 4-6%; reference value for average blood glucose concentration (eAG): 3.28-7.10 mmol / L.

[0075] b) Genomic DNA (gDNA) of the proband was extracted for whole exome sequencing (WES). Whole exome sequencing and sequence analysis were performed by Beijing Fujun Gene Biotechnology Co., Ltd.

[0076] Three gene variants highly associated with the patient's clinical phenotype were detected: APOB c.676G>A, INSR c.4028G>A, and AKT2 c.433G>C (see Table 3).

[0077] Table 3 Genetic testing results of the proband and his relatives

[0078]

[0079] In order to determine the genetic source of the proband's gene mutation, the corresponding mutation sites were verified in the proband's mother and father, and the results showed that the proband's gene mutation came from the mother. In order to further determine the gene mutation associated with hyperlipidemia in the proband's family, the proband's mother was sequenced for the whole exome gene again, and the results also found site mutations in the three genes APOB c.676G>A, INSR c.4028G>A, and AKT2 c.433G>C, which were highly correlated with the clinical phenotype.

[0080] The APOB c.676G>A mutation site and INSR c.4028G>A mutation site were verified in other maternal family members of the proband. The results showed that the proband and his mother, uncle, and grandmother all had APOB c.676G>A mutation site ( Figure 2 The proband, his mother, uncle, and grandfather all had INSR c.4028G>A mutation ( Figure 3 ).

[0081] Combined with the medical history of the proband's family, since the patient's maternal grandmother had a history of hyperlipidemia and early-onset coronary heart disease, while the maternal grandfather had no history of hyperlipidemia, the history of hyperlipidemia in the proband's family may not be related to the INSR c.4028G>A mutation. Therefore, it is speculated that the hyperlipidemia in this family may be associated with the APOB c.676G>A mutation. APOB (c.676G>A, p.A226T) is a missense heterozygous mutation, in which the nucleotide 676 in the coding region changes from guanine to adenine, resulting in the mutation of the amino acid at position 226 from alanine to threonine.

[0082] In this example, all data were obtained in compliance with legal regulations and based on user consent, and the data was used legally. The proband and his family members agreed and signed the informed consent form. In addition, unless otherwise specified, the reagents or instruments used in this example were commercially available.

[0083] Functional study of APOB c.676G>A

[0084] (1) Conservatism analysis

[0085] In order to further study the functional impact of APOB mutation on protein function, the effect of APOB mutation on protein structure was predicted. Figure 4 The diagram is a protein structure diagram of APOB involved in the embodiments of the present invention and a diagram of the amino acid sequence in different species.

[0086] The APOB gene is located at the end of the short arm of human chromosome 2. The gene is approximately 43kb in length and is responsible for encoding 4564 amino acids. It is the largest known protein in the human body. The APOB Ala226Thr mutation is located in the yolk saponin domain at the N-terminus of the APOB gene ( Figure 4 Part a of the figure), involved in the storage and transport of lipids.

[0087] According to conservation analysis, the position of the APOB mutation p.A226T is highly conserved, and multiple amino acid sequence alignments show that A226 is conserved among different species and is all alanine (A) ( Figure 4 Part b)

[0088] (2) Protein three-dimensional structure prediction and analysis

[0089] The three-dimensional (3D) structures of wild-type APOB48 (WT-APOB48) and its mutant APOB48 (A226T-APOB48) were predicted using the phyre2 server, and comprehensive analysis and modeling were performed by direct comparison with the final results. The 3D structural models of WT-APOB48 and A226T-APOB48 were established as reference models. In addition, the 3D structure was visualized using PyMOL software.

[0090] Figure 5 The three-dimensional structure prediction diagram of the APOB protein involved in the embodiment of the present invention. The results show that the mutation at this site leads to the addition of a hydrogen bond between the 226th amino acid and the 684th amino acid, affecting the spatial domain of the protein and causing a change in the biological function of the gene. In summary, the mutant APOB p.A226T may affect the tertiary structure of APOB.

[0091] (3) In vitro activity studies

[0092] 3-1) Cell culture

[0093] Human hepatocellular carcinoma cell (HepG2) and human embryonic kidney cell (HEK 293T) were purchased from the Stem Cell Bank of the Chinese Academy of Sciences.

[0094] Cell recovery

[0095] a.Ultraviolet disinfection of the clean bench for 30 minutes in advance, prepare cell culture bottles, centrifuge tubes, complete culture medium, etc. Preheat the water bath to 37°C in advance.

[0096] b. Carefully remove the cell cryopreservation tube from the liquid nitrogen tank and quickly thaw it in a 37°C water bath.

[0097] c. Transfer the cell suspension to a 15 ml centrifuge tube in a clean bench, add 4 ml of complete culture medium, and centrifuge at 1000 rpm for 3 minutes.

[0098] d. Discard the supernatant, resuspend the cell pellet in 1 ml of complete culture medium, and mix well by pipetting.

[0099] e. Transfer the cell suspension to a T25 culture flask, add 4 ml of complete culture medium, shake the culture flask slowly in an 8-shape until the cells are evenly distributed, and then place it in a 37°C constant temperature 5% CO2 incubator for culture.

[0100] Cell passaging

[0101] a. Observe the cells under a microscope until the confluence reaches more than 85% and then perform cell subculture. Aspirate the original culture medium.

[0102] b. Add 2 ml of PBS buffer and rinse twice.

[0103] c. Aspirate the PBS buffer and add 1 ml of trypsin for digestion.

[0104] d. After digestion is complete, add 2 ml of complete medium to terminate digestion.

[0105] e. Blow the adherent cells, collect the cell suspension and transfer it to a 15 ml centrifuge tube, and centrifuge it at 1000 rpm for 3 minutes.

[0106] f. Aspirate the supernatant and retain the cell pellet, then add 3 ml of complete culture medium to resuspend the cells.

[0107] g. Transfer the resuspended cells into new culture flasks or dishes in equal parts, add an appropriate amount of complete culture medium, shake slowly in an 8-shape until the cells are evenly distributed, and then place in a 37°C constant temperature 5% CO2 incubator for culture.

[0108] Cell cryopreservation

[0109] a. As in steps ae of “Cell passaging”.

[0110] b. Aspirate the supernatant and keep the cell pellet, add 1 ml of freezing solution, mix well by pipetting, transfer to a freezing tube, and mark the name, surname, and date.

[0111] c. Place the cryotube in a cryo box and place it in a -80℃ low-temperature refrigerator. After 24 hours, place it in a liquid nitrogen tank for long-term storage.

[0112] 3-2) Plasmid construction

[0113] a) Construction of pEnCMV-APOB-3×FLAG-SV40-Neo plasmid

[0114] Use high-fidelity DNA polymerase (Vazyme #P505) to amplify the fragments according to the following primers:

[0115] F1:5'-cttggtaccgagctcggatccATGGACCCGCCGAGGCCC-3' (sequence 1);

[0116] R1:5'-gtgatggaagagaaaCAGATTTGTAGAGTTGATAGTTCCGAG-3' (sequence 2);

[0117] F2:5'-atctgTTTCTCTTCCATCACTTGACCCA-3' (sequence 3);

[0118] R2:5'-gcggaccagttGTACAAGTTGCTGTAGACATTCGTGG-3' (sequence 4);

[0119] F3:5'-aacttgtacAACTGGTCCGCCTCCTACAGT-3' (sequence 5);

[0120] R3:5'-tgctggatatctgcagaattcTATCATATATGTCTGCAGTTGAGATAGTTTT-3' (sequence 6).

[0121] pEnCMV-MCS-3×FLAG-SV40-Ne was double-digested (BamHI / EcoRI, NEB) and then subjected to homologous recombination (Vazyme #C112).

[0122] b) Construction of pCMV-FLAG-APOB-Linker-EGFP plasmid

[0123] Use high-fidelity DNA polymerase (Vazyme #P505) to amplify the fragments according to the following primers:

[0124] F1:5'-aaggacgacgatgacaagcttATGGACCCGCCGAGGCCC-3' (sequence 7);

[0125] R1:5'-gtgatggaagagaaaCAGATTTGTAGAGTTGATAGTTCCGAG-3' (sequence 8);

[0126] F2:5'-atctgTTTCTCTTCCATCACTTGACCCA-3' (sequence 9);

[0127] R2:5'-gcggaccagttGTACAAGTTGCTGTAGACATTCGTGG-3' (sequence 10);

[0128] F3:5'-aacttgtacAACTGGTCCGCCTCCTACAGT-3' (sequence 11);

[0129] R3:5'-gcttccccctcctccggtaccTATCATATATGTCTGCAGTTGAGATAGTTTT-3' (sequence 12).

[0130] pCMV-FLAG-MCS-Linker-EGFP was double-digested (HindIII / KpnI, NEB) and then homologous recombination was performed (Vazyme #C112).

[0131] c) Construction of pEnCMV-APOB(A226T)-3×FLAG-SV40-Neo plasmid and pCMV-FLAGAPOB(A226T)-Linker-EGFP plasmid

[0132] Point mutagenesis was performed according to the instructions of the mutagenesis kit (Vazyme #C215) with the following primers:

[0133] F:5'-GCCCACTTaCTCTCATCAAAAGGCATGACCCGC-3' (sequence 13);

[0134] R: 5'-GATGAGAGtAAGTGGGCTGATGCCTGTGCGGA-3' (sequence 14).

[0135] d) All constructs were verified by sequencing on an ABI3730xl sequencer.

[0136] 3-3) Plasmid transfection

[0137] a) Adjust cell density 24 hours in advance: Count HepG2 cells and seed them in 6-well plates, and divide them into CON group (cells not transfected with plasmid), WT group (cells transfected with WT-APOB48-Flag plasmid), Mut group (cells transfected with A226T-APOB48-Flag plasmid), WT-EGFP group (cells transfected with plasmids expressing both EGFP and WT-APOB48-Flag), and Mut-EGFP group (cells transfected with plasmids expressing both EGFP and A226T-APOB48-Flag). Make sure that the cell confluence reaches 70-90% before transfection.

[0138] b) (Take a single well in a six-well plate as an example) Dissolve 5ul Lipofectamine™3000 in 125ul Opti-MEM, dissolve 5ul P3000™ and 2.5ug plasmid in 125ul Opti-MEM, mix the plasmid solution and Lipo solution and let stand at room temperature for 10min. At the same time, remove the original culture medium of the cells in the seed plate, add 1ml of fresh culture medium without double antibodies and plasmid-lipo mixture.

[0139] c) Observe the cell growth status and collect the cells for subsequent experiments when the transfection efficiency reaches about 70% under a fluorescence microscope.

[0140] d) The transfection steps of human embryonic kidney cells (HEK293T) were the same as above.

[0141] 3-3) DAPI staining

[0142] a) (Using 24-well plate as an example) Remove the original culture medium of HEK 293T cells and add 1 ml of paraformaldehyde fixative for 10 minutes.

[0143] b) Add 1 ml of PBS buffer and rinse twice, 3 minutes each time.

[0144] c) Aspirate the PBS buffer, add 100ul of DAPI reagent, and stain for 15 minutes.

[0145] d) Add 1 ml of PBS buffer and rinse three times, 5 minutes each time.

[0146] e) Take a glass slide, add 10ul of anti-fluorescence quenching sealing solution, take out the round cell slide from the 24-well plate and place it on the cover slip to seal the slide.

[0147] 3-4) Real-time PCR

[0148] a) Extraction of cellular RNA (column method)

[0149] Aspirate the medium in the 6-well plate and add 1 ml of Buffer RL for digestion and lysis for 10-15 minutes.

[0150] Transfer the lysed sample to FastPure gDNA-Filter Columns Ⅲ (FastPure gDNA-Filter Columns Ⅲ has been placed in the collection tube), centrifuge at 12000rpm for 30 seconds. Discard FastPure gDNA-Filter Columns Ⅲ and collect the filtrate.

[0151] Add 0.5 times the volume of anhydrous ethanol to the filtrate and mix thoroughly.

[0152] Transfer all the mixed solution to FastPure RNA Columns III (FastPure RNA Columns III has been placed in the collection tube), centrifuge at 12000rpm for 30 seconds, and discard the filtrate.

[0153] Add 700ul of BufferRW1 to FastPure RNA Columns Ⅲ, centrifuge at 12000rpm for 30 seconds, and discard the filtrate. Add 700ul of BufferRW2 (with anhydrous ethanol) to FastPure RNA Columns Ⅲ, centrifuge at 12000rpm for 30 seconds, and discard the filtrate. Add 500ul of BufferRW2 (with anhydrous ethanol) to FastPure RNA Columns Ⅲ, centrifuge at 12000rpm for 2 minutes, and carefully remove the adsorption column from the collection tube to avoid contact with the filtrate and cause contamination.

[0154] Carefully transfer the adsorption column to a new RNase-free Collection Tubes 1.5ml centrifuge tube, add 50ul of RNase-free ddH2O to the center of the adsorption column, let it stand at room temperature for 1 minute, centrifuge at 12000rpm for 1 minute to elute the RNA, and collect the filtrate.

[0155] The RNA concentration was tested, a portion was reverse transcribed, and the remaining portion was stored at -80°C for future use.

[0156] RNA concentration and purity determination: First, use DEPC water to zero the instrument, then test it again with DEPC water. If the error is not large, the measurement can be performed. Wipe the instrument with lens paper each time you add a sample. Pipette 1.5ul RNA extract and test it with the instrument. The OD260 / OD280 should be between 1.8-2.0 and the RNA concentration should be at least 50ng / ul. Test each sample at least twice, and take the average value as the RNA concentration of the sample.

[0157] b) Reverse transcription of mRNA into cDNA

[0158] Removal of sample DNA

[0159]

[0160] Add the above components to a 100ul PCR tube, gently pipette to mix, place on the RT-PCR instrument, and set the conditions to 42℃, 2 minutes → 4℃, ∞.

[0161] Reverse transcription reaction

[0162]

[0163] Add the above components to the PCR tube, mix them by gently pipetting, and place them on the RT-PCR instrument. The reaction conditions are 37°C, 15 minutes → 85°C, 5 minutes → 4°C, ∞.

[0164] The product can be used immediately for qPCR reaction, or stored at -20℃ and used within half a year; for long-term storage, it is recommended to store at -80℃ after aliquoting. Repeated freezing and thawing of cDNA should be avoided.

[0165] c) Real-time quantitative PCR (qPCR)

[0166] Computing system

[0167]

[0168] Take out the cNDA and put it in an ice box indoors away from light.

[0169] Prepare a 96-well PCR plate, add the corresponding volume of SYBR, ddH2O, cDNA and primers according to the number of replicate wells in the experimental design, centrifuge at 4°C to mix, and then insert into an ice box.

[0170] After adding samples, wear PE gloves, cover the plate with sealing film, wrap with tin foil to avoid light, and store at 4℃ for a short term.

[0171] Centrifuge the 96-well plate for 1 minute before loading it into the PCR instrument to start the amplification reaction.

[0172] The reaction time is as follows:

[0173]

[0174] Real-time PCR analysis was performed by LC480 LightCycler. Relative gene expression was determined using the comparative method (2-ΔΔCT). β-actin was used as an internal standard.

[0175] 3-5) Immunohistochemistry

[0176] a) RIPA method to extract cell proteins

[0177] Mix RIPA lysis buffer and PMSF protease inhibitor at a ratio of 100:1, discard the culture medium in the 6-well plate, wash the cells 3 times with pre-cooled PBS buffer, add 150uL of lysis buffer to each well, and lyse on ice for 10 minutes.

[0178] Label a 1.5 ml EP tube, collect the lysed cells into it with a cell scraper, vortex, and crush the cells using an ultrasonic cell crusher.

[0179] Place the EP tube in ice and continue lysis for 20 min, shaking every 10 min.

[0180] After 30 minutes of total lysis, the EP tube was placed in a 4°C centrifuge and centrifuged at 12,000 rpm for 15 minutes.

[0181] The supernatant after centrifugation was transferred into a new EP tube, a part of which was used to detect the protein concentration, and the remaining part was added with 5× SDS protein loading buffer, denatured in a 95°C metal bath for 15 minutes, and stored at -80°C for later use.

[0182] b) BCA detection of protein concentration

[0183] Prepare BCA working solution: the volume ratio of solution A to solution B is 50:1. Mix and place on ice for later use.

[0184] The standards were diluted to 0.5 mg / ml.

[0185] Prepare a standard curve (operate on ice):

[0186] Add different volumes of standard samples to the standard wells of the 96-well plate, and then fill up to 20ul with PBS buffer, as shown in the following table:

[0187]

[0188] Add 2ul of the sample to be tested and 18ul of PBS buffer into the sample well.

[0189] Add 200ul of pre-prepared BCA working solution to each well and incubate at 37°C for 20-30 minutes.

[0190] The absorbance of each well was detected at a wavelength of 562 nm using an enzyme reader, and a standard curve was prepared using the standard curve protein concentration and the corresponding OD value, so as to calculate the protein concentration of the sample well according to the OD value of the sample well to be tested.

[0191] c) Western blot

[0192] Preparation of SDS-PAGE gel: Select different gel concentrations according to the molecular weight of the target protein. Use low-concentration gel for high-molecular-weight proteins and high-concentration gel for low-molecular-weight proteins. Select 10% gel according to the molecular weight of the target protein APOB. First, fix the cleaned gel plate (1.5mm) on the gel rack and check for leakage with double distilled water. After the leak test, discard the double distilled water and prepare the lower separation gel according to the instructions of the gel preparation kit, that is, mix the separation gel solution and buffer solution 4ml / block, add 80ul / block ammonium persulfate solution to promote coagulation, shake and mix, quickly pour into the gel plate, and add an appropriate amount of anhydrous ethanol solution to seal the liquid surface to prevent gas from entering. After the separation gel solidifies, pour out the anhydrous ethanol, take 1ml / block of the upper concentrated gel solution and buffer solution, add 20ul / block ammonium persulfate solution, mix and quickly pour into the plate, insert a 1.5mm gel comb after filling, pay attention to prevent bubbles, and set aside after solidification.

[0193] Sample loading: Place the protein samples in the -80℃ refrigerator on ice to melt in advance. Fix the prepared gel in the electrophoresis tank, add 1× electrophoresis buffer, and slowly pull out the gel comb. Add 4ul protein marker on both sides first, and then slowly add the samples to be tested into the wells one by one, avoiding bubbles and sample overflow.

[0194] Electrophoresis: Cover the electrophoresis tank, connect the electrophoresis instrument, adjust to constant voltage, first set to 80V, the time is about 30 minutes, after the bromophenol blue band runs through the concentrated gel, adjust the voltage to 120V and continue electrophoresis, stop electrophoresis when the band runs to the bottom edge of the gel.

[0195] Transfer: Cut the PVDF membrane into a rectangle of appropriate size and activate it with methanol for 10-15 seconds in advance. Soak the transfer clip in 1× transfer solution, and install the transfer clip in order from positive to negative: sponge, filter paper, PVDF membrane, separation gel, filter paper, sponge, avoid bubbles between the gel and PVDF membrane, clamp the clamp and place it in the transfer tank, with the black side of the clip corresponding to the black side of the tank, and the white side of the clip corresponding to the red side of the tank. Fill it with transfer solution and put in an ice bag, and place the transfer tank in an ice box, connect the instrument, and transfer the membrane at a constant current of 300mA for 180 minutes.

[0196] Blocking: After the transfer is completed, take out the PVDF membrane and immerse it completely in 5% skim milk, with the front of the PVDF membrane facing up, and block it by slowly shaking at room temperature for 1 hour.

[0197] Primary antibody incubation: dilute the antibody at 1:1000 with primary antibody diluent (dilute the antibody according to the instructions), immerse the PVDF membrane completely in the antibody solution, and incubate on a shaker at 4°C overnight.

[0198] Washing membrane: On the next day, remove the PVDF membrane from the primary antibody solution and wash the membrane three times with 1× TBST, each time for 10 minutes.

[0199] Secondary antibody incubation: dilute the secondary antibody with 5% skim milk at a ratio of 1:20,000, immerse the membrane completely in it, and incubate on a shaker at room temperature for 1 hour.

[0200] Wash the membrane: Wash the membrane 3 times with 1× TBST, 10 min each time.

[0201] ECL chemiluminescence detection: according to the amount, take ECL luminescent liquid A and B and mix them in a ratio of 1:1. Keep the luminescent liquid away from light and use it immediately after preparation. Evenly cover the membrane and detect it with a fully automatic chemiluminescence analyzer. Use ImageJ software to analyze the grayscale value of the image.

[0202] 3-6) Statistical analysis

[0203] ImageJ software was used to analyze the grayscale of protein bands. The t-test was used for statistical comparison of PCR results and band grayscale analysis. All analysis data were statistically analyzed and plotted using Graph pad Prism 9. Each experiment was repeated three times or more. Representative experimental results are shown in the figure. P < 0.05 was considered statistically significant.

[0204] 3-7) Test results

[0205] a) Plasmid group transfection results

[0206] Human HEK 293T cells and HepG2 cells were selected and transfected with wild-type APOB48 (expressing both EGFP gene and WT-APOB48-Flag gene) plasmid and mutant APOB48 (expressing both EGFP gene and A226T APOB48-Flag gene) plasmid, respectively. Stably transfected cells expressed bright green fluorescence ( Figure 6 , Figure 7 ). In the control group, no intervention was performed, and human HEK 293T cells and HepG2 cells spontaneously emitted green fluorescence.

[0207] Note: Figure 6 In the figure, a, b, and c are the green fluorescence expressions of HEK 293T cells in the control group under the microscope of ×40, ×100, and ×400, respectively; d, e, and f are the green fluorescence expressions of HEK 293T cells transfected with wild-type plasmid under the microscope of ×40, ×100, and ×400, respectively; g, h, and i are the green fluorescence expressions of HEK 293T cells transfected with mutant plasmid under the microscope of ×40, ×100, and ×400, respectively. Figure 7In the figure, a, b, and c are the green fluorescence expressions of HepG2 cells in the control group under the microscope of ×40, ×100, and ×400, respectively; d, e, and f are the green fluorescence expressions of HepG2 cells transfected with the wild-type plasmid under the microscope of ×40, ×100, and ×400, respectively; g, h, and i are the green fluorescence expressions of HepG2 cells transfected with the mutant plasmid under the microscope of ×40, ×100, and ×400, respectively.

[0208] b) Immunofluorescence expression of APOB in cells

[0209] In order to verify the expression location of mutant A226T-APOB, human HEK 293T cells were selected and transfected with wild-type APOB48 (expressing EGFP gene and WT-APOB48-Flag gene) plasmid and mutant APOB48 (expressing EGFP gene and A226T-APOB48-Flag gene) plasmid, respectively. DAPI staining was performed 48 hours later, and the immunofluorescence expression location of mutant A226T-APOB was observed by fluorescence microscopy. The results showed that wild-type APOB and mutant A226T-APOB were mainly localized in the cytoplasm ( Figure 8 ).

[0210] c) Functional study of the Ala226Thr-APOB mutation

[0211] Compared with WT-APOB, the Ala226Thr-APOB mutation had no significant difference in transcriptional and protein expression levels ( Fig. 9 ). Fig. 9 In the figure, (a) is the histogram of RNA expression levels of WT-APOB and A226T-APOB in HEK 293T cells, (b) is the histogram of RNA expression levels of WT-APOB and A226T-APOB in HepG2 cells, (c) is the protein expression of WT-APOB and A226T-APOB in HEK 293T cells, (d) is the histogram of APOB protein levels in HEK 293T cells under incubation with Flag antibody, (e) is the histogram of APOB protein levels in HEK 293T cells under incubation with APOB antibody, (f) is the protein expression of WT-APOB and A226T-APOB in HepG2 cells, (g) is the histogram of APOB protein levels in HepG2 cells under incubation with APOB antibody, n represents the number of experiments, ns means that the expression of A226T-APOB is not statistically significant compared with WT-APOB (p>0.05).

[0212] Previous studies have shown that FASN, ACACA, SREBF1, NRIH3, PPARGC1B, LPIN1, PPARA, and CPT1A genes are involved in regulating lipid metabolism and are related to the function of the APOB gene. In order to further explore the pathogenicity of the A226TAPOB mutation, 14 lipid-regulating genes were selected, including genes involved in triacylglycerol (TAG) synthesis: LPIN1, LPIN2, LPIN3, DGAT1, DGAT2; genes involved in lipogenesis: FASN, ACACA; transcription factors involved in lipogenesis: SREBF1, NR1H3, PPARGC1A, PPARGC1B; genes involved in fatty acid oxidation: PPARA, CPT1A, and microsomal triglyceride transfer protein (MTTP). The effect of the A226T-APOB mutation on other lipid-regulating genes was further studied by primer design and q-PCR experiments.

[0213] q-PCR results showed that the A226T-APOB mutation led to increased mRNA expression levels of TAG synthesis genes (LPIN1, LPIN2, DGAT2), lipogenesis genes (FASN, ACACA, SREBF1, PPARGC1B), and fatty acid oxidation genes (CPT1A) ( Fig.10 ). Fig.10In the figure, (a) shows that the Ala226Thr-APOB mutation leads to increased expression of LPIN1, ** represents p < 0.01 for the A226T-APOB mutation compared with WT-APOB (p = 0.0021); the Ala226Thr-APOB mutation leads to increased expression of LPIN2, ** represents p < 0.01 for the A226T-APOB mutation compared with WT-APOB (p = 0.0015); the Ala226Thr-APOB mutation leads to increased expression of DAGT2, * represents p < 0.05 for the A226T-APOB mutation compared with WT-APOB (p = 0.0284); (b) shows that the Ala226Thr-APOB mutation leads to increased expression of FASN, * represents p < 0.05 for the A226T-APOB mutation compared with WT-APOB (p = 0.0 131); Ala226Thr-APOB mutation leads to increased expression of ACACA, * represents p < 0.05 for A226T-APOB mutation compared with WT-APOB (p = 0.0361); Ala226Thr-APOB mutation leads to increased expression of SREBF1, * represents p < 0.05 for A226T-APOB mutation compared with WT-APOB (p = 0.0129); Ala226Thr-APOB mutation leads to increased expression of PPARGC1B, * represents p < 0.05 for A226T-APOB mutation compared with WT-APOB (p = 0.0468); Ala226Thr-APOB mutation leads to increased expression of CPT1A, * represents p < 0.05 for A226T-APOB mutation compared with WT-APOB (p = 0.0361). ns indicates that the expression of A226T-APOB was not statistically significant compared with WT-APOB (p>0.05).

[0214] 3-8) Conclusion

[0215] In this study, the APOB mutation site (c.676G>A, p.A226T) was found through the collection of clinical history data and gene sequencing analysis of a FH family. This gene mutation is highly correlated with the clinical phenotype of FH. In vitro functional studies have confirmed that the APOB (c.676G>A, p.A226T) site mutation leads to increased expression levels of TAG synthesis genes (LPIN1, LPIN2, DGAT2) and lipogenesis genes (FASN, ACACA, SREBF1, PPARGC1B), causing increased cholesterol and triglycerides in the blood circulation, which is related to the proband's history of FH and repeated attacks of acute pancreatitis.

[0216] In summary, according to the above research and research results of the embodiment, APOB c.676G>A mutation or APOBp.A226T mutation will damage the function of APOB protein. APOB c.676G>A mutation or APOB p.A226T mutation is the pathogenic mutation of the familial hypercholesterolemia family in this embodiment. The APOB c.676G>A (p.A226T) mutation discovered in this embodiment is a new pathogenic gene for familial hypercholesterolemia and is autosomal dominant.

[0217] Although the present disclosure is specifically described above in conjunction with the accompanying drawings and embodiments, it is to 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 essential spirit and scope of the present disclosure, and these modifications and changes all fall within the scope of the present disclosure.

Claims

1. Use of a reagent for detecting APOB gene mutation or protein mutation in a sample in the preparation of a product for screening patients with familial hypercholesterolemia, characterized in that: The APOB gene variation or protein variation is used to diagnose patients with familial hypercholesterolemia. The APOB gene variation is APOB c.676G>A, the APOB protein variation is APOB p.A226T, and the transcript is NM_000384.

2. The use according to claim 1, characterized in that: The reagents include a primer pair for amplifying the APOB gene and / or a probe for detecting a variation of the APOB gene.

3. The use according to claim 2, characterized in that: The primer pair is designed based on the nucleotide sequence of the 676th base in the coding region of the APOB gene in the human genome, and the probe is designed based on the 676th base and the nucleotide sequence of the 676th base in the coding region of the APOB gene in the human genome.

4. The use according to claim 2 or 3, characterized in that: The reagents also include dNTPs, DNA polymerase and PCR reaction buffer.

5. The use according to claim 1, characterized in that: The reagents include reagents for detecting the APOB 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 5, characterized in that: The reagent includes an antibody that recognizes an APOB protein having an APOB p.A226T mutation.

7. The use according to claim 2 or 5, characterized in that: The product also includes a nucleic acid extraction reagent and / or a protein extraction reagent.

8. The use according to claim 1, characterized in that: The sample is from at least one of the peripheral blood, saliva, and tissue samples of the subject, and the APOB gene variation refers to the germline variation of the APOB gene, and the APOB protein variation refers to the germline variation of the APOB protein.

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

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