Gout-related mutated D-lactic dehydrogenase gene, kit and application of gout-related mutated D-lactic dehydrogenase gene

Abstract

The invention discloses a mutated D-lactic dehydrogenase gene related to gout, a kit and application of the mutated D-lactic dehydrogenase gene. A coding region (CDS) of the mutated D-lactic dehydrogenase gene contains c.1327Cgt; t, and c.1058Cgt; the D-lactic dehydrogenase gene is a mutation site A, and the Genbank login number of the D-lactic dehydrogenase gene before mutation is NM194436.3. According to the invention, high-throughput sequencing of all exon regions is carried out on a young gout patient of Chinese Han nationality and parents thereof by adopting a whole exon group sequencing technology, and biological information analysis is combined to find that c.1327Cgt simultaneously exists on an LDHD gene (reference sequence gene NM194436.3) of the gout patient; t (p.His443Tyr) and c.1058Cgt are selected from the group consisting of Cgt and Cgt; a (p.Ala353Glu) autosome recessive genetic mode mutation is carried out. The mutation is associated with causing an increase in D-lactic acid in serum and urine of a patient and with gout. According to the invention, the understanding of LDHD-related mutation genetic modes is expanded, the research direction of gout pathogenesis is expanded, and a technical means is provided for developing effective early pathogenic gene screening.

Description

[0001] Priority declaration

[0002] This application claims priority to application number 202410781435.8, filed on June 17, 2024, entitled “A mutated D-lactate dehydrogenase gene, kit and application thereof associated with gout”, the entire contents of which are incorporated herein by reference. Technical Field

[0003] The present invention relates to a mutated D-lactate dehydrogenase (LDHD) gene associated with gout, a kit for detecting the mutated gene and its application. The present invention belongs to the field of medical technology. Background Art

[0004] Lactic acid is an important metabolite that can be divided into two enantiomers, L-lactic acid and D-lactic acid, according to the chirality of its C2 atom. In humans, the accumulation of excess L- and D-lactic acid can lead to lactic acidosis. D-lactic acidosis is generally considered a complication of short bowel syndrome, which occurs after the removal of part of the small intestine due to malignancy, disease, or jejunoileal bypass surgery. Although both L- and D-lactic acid can pass through the blood-brain barrier, unlike L-lactic acid, the accumulation of D-lactic acid in the brain has neurotoxic effects, leading to a variety of neurological symptoms that are often confused with primary neurological diseases. Currently, the cause of D-lactic acidosis is unclear. Lactate dehydrogenase D (LDHD) exhibits dehydrogenase activity towards D-lactate.

[0005] Recently, researchers have discovered several missense mutations in LDHD in patients with neurological symptoms or autosomal recessive gout and hyperuricemia. Metabolic profile analysis showed that the concentration of D-lactic acid in the urine and plasma of these patients was significantly increased. Studies have shown that these disease-related mutations can cause loss of LDHD function, resulting in increased levels of D-lactic acid in urine and plasma.

[0006] The substrate binding pocket of LDHD consists of a positively charged subsite A and a hydrophobic subsite B. Subsite A binds Mn 2+ Subsite A binds to the glycolate group of the substrate or the glyoxylate group of the product, while subsite B binds to the hydrophobic group of the substrate or product. Activity analysis of disease-related LDHD mutants showed that these mutations all resulted in reduced activity of LDHD towards the substrate. Structural and biochemical data indicate that LDHD is a Mn 2+It is a broad-spectrum dehydrogenase that catalyzes the dehydrogenation of various D-2-hydroxy acids with hydrophobic groups on the C2 atom. Based on the structural and biochemical results, the substrate recognition and catalytic mechanism of LDHD were revealed, and the role of LDHD mutation in the occurrence of D-lactic acidosis was clarified. It was confirmed that LDHD mutation can cause D-lactic acidosis, and it laid a structural foundation and provided a theoretical basis for the treatment of this disease and the development of related drugs. Summary of the invention

[0007] The purpose of the present invention is to provide a mutated D-lactate dehydrogenase gene related to gout, a kit and an application thereof.

[0008] In order to achieve the above object, the present invention adopts the following technical means:

[0009] The present invention uses whole exome sequencing technology to perform high-throughput sequencing of all exon regions for a young gout patient and his parents, and combined with bioinformatics analysis, it is found that the LDHD gene (reference sequence gene NM_194436.3) of gout patients has both c.1327C>T (p.His443Tyr) and c.1058C>A (p.Ala353Glu) autosomal recessive mutations. This mutation causes an increase in D-lactic acid in the patient's serum and urine and is associated with gout.

[0010] Based on the above research, the present invention proposes a mutated D-lactate dehydrogenase (LDHD) gene associated with gout, wherein the coding region of the mutated D-lactate dehydrogenase gene contains both c.1327C>T and c.1058C>A mutation sites, and the Genbank accession number of the D-lactate dehydrogenase gene before mutation is: NM_194436.3, wherein the coding region (CDS) of the D-lactate dehydrogenase gene is located between nucleotides 61..1515, and the above mutation site is described based on the coding region (CDS) of the D-lactate dehydrogenase gene, that is, the starting nucleotide position "61" of the coding region (CDS) of the D-lactate dehydrogenase gene is recorded as "1".

[0011] Preferably, the coding sequence of the mutated D-lactate dehydrogenase gene is as shown in SEQ ID NO.1.

[0012] A mutated D-lactate dehydrogenase associated with gout is also within the protection scope of the present invention, wherein the mutated D-lactate dehydrogenase simultaneously contains the following mutation sites p.His443Tyr and p.Ala353Glu, and the D-lactate dehydrogenase before mutation is encoded by a D-lactate dehydrogenase gene with a Genbank accession number of NM_194436.3.

[0013] Preferably, the amino acid sequence of the mutated D-lactate dehydrogenase is as shown in SEQ ID NO.2.

[0014] Furthermore, the present invention also proposes a PCR primer set for detecting the mutated D-lactate dehydrogenase gene associated with gout, wherein the target fragment amplified by the PCR primer set includes the bases corresponding to the 1058th and 1327th positions of the D-lactate dehydrogenase gene coding region.

[0015] Preferably, the PCR primer set includes primer pair P1 and primer pair P2, the amplified fragment of primer pair P1 contains the base corresponding to position 1327, the amplified fragment of primer pair P2 contains the base corresponding to position 1058, and the sequence of primer pair P1 is:

[0016] LDHD-F1:5'-CTCCGTAGTCAGGAACTTG-3'

[0017] LDHD-R1:5'-CCAGTTCCTCGGGCTCATCA-3'

[0018] The sequence of the primer pair P2 is:

[0019] LDHD-F2:5'-GCCCTTGTTTCTAGCGCC-3'

[0020] LDHD-R2:5'-CTCCTCTGCAGTTGGGGA-3'.

[0021] Furthermore, the present invention also proposes the use of the PCR primer set in the following aspects:

[0022] (1) preparing a reagent for detecting a mutated D-lactate dehydrogenase gene associated with gout;

[0023] (2) Preparation of reagents for gout diagnosis or risk assessment.

[0024] Furthermore, the present invention also proposes a kit for gout diagnosis or risk assessment, wherein the kit comprises the PCR primer set.

[0025] Preferably, the kit also includes reagents for extracting template DNA required for PCR amplification from the individual to be tested.

[0026] Preferably, the kit is used for gout diagnosis or risk assessment according to the following steps:

[0027] 1) Extracting DNA from the blood collected from the individual to be tested;

[0028] 2) using the DNA extracted in step 1) as a template and the PCR primer set of claim 3 or 4 to perform a PCR reaction to obtain a PCR reaction product; separating the target fragment amplified by the PCR reaction from the PCR reaction product, and performing typing and identification on the bases corresponding to the 1058th and 1327th positions of the D-lactate dehydrogenase gene coding region contained in the target fragment;

[0029] 3) Determination: The genotype determined by comparison includes wild homozygous type, mutant heterozygous type or homozygous mutant type.

[0030] Among them, preferably, the typing identification adopts a method of directly sequencing the target fragment, and determines the genotype or allele type of the individual to be tested corresponding to positions 1058 and 1327 of the LDHD gene coding region by comparing the sequencing results with the reference sequence.

[0031] Among them, preferably, when the results of the two sites are both wild homozygous, it is considered that the individual to be tested does not suffer from gout and has a low risk of suffering from gout; when the results of the two sites are both mutant heterozygous, it is considered that the individual to be tested suffers from gout or has a high risk of suffering from gout; when the results of any one of the two sites is homozygous mutant, it is considered that the individual to be tested suffers from gout or has a high risk of suffering from gout.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] 1. The present invention proposes a new LDHD gene mutation site that can cause increased D-lactic acid, namely c.1327C>T (p.His443Tyr) and c.1058C>A (p.Ala353Glu). By detecting whether the LDHD gene c.1058C>A and c.1327C>T mutations exist in the DNA sample from the patient, the cause of gout caused by increased D-lactic acid in the patient can be determined, thereby providing a basis for the clinical diagnosis of gout.

[0034] 2. The present invention also relates to a kit for detecting the new mutation site. The kit is used to detect whether there are c.1058C>A and c.1327C>T mutations corresponding to positions 1058 and 1327 on the human LDHD gene in the sample to be tested (LDHD gene fragment from the patient), so as to determine the genetic cause of the patient's increased D-lactate. Among them, the c.1058G>T mutation of the LDHD gene causes the 353rd position to be converted from alanine to glutamic acid (p.Ala353Glu), and the c.1327C>T mutation causes the 443rd position to be converted from histidine to tyrosine (p.His443Tyr). The kit proposed by the present invention can be used to quickly detect specific mutation sites of the LDHD gene, which will help to quickly and efficiently detect mutation sites in a wider range of patients with increased D-lactate, and provide important guidance for early clinical intervention for increased D-lactate. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 The results of LDHD gene sequencing for gout patients;

[0036] Figure 2 The LDHD gene sequencing results for the father of a gout patient;

[0037] Figure 3 This is the LDHD gene sequencing result of the mother of a gout patient.

[0038] Figure 4 The results of the detection of enzyme activity loss caused by the new LDHD gene mutation site;

[0039] Figure 5 These are the test results of uric acid levels at different time points in hyperuricemia caused by D-lactic acid. DETAILED DESCRIPTION

[0040] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, it should be understood that the embodiments are exemplary only and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications or replacements all fall within the protection scope of the present invention.

[0041] Example 1 Discovery of new LDHD gene mutation sites associated with gout

[0042] The present invention uses whole exome sequencing technology to perform high-throughput sequencing of all exon regions for a young gout patient and his parents. Combined with bioinformatics analysis, it is found that the LDHD gene coding region (reference sequence gene NM_194436.3) of the gout patient has both c.1327C>T (p.His443Tyr) and c.1058C>A (p.Ala353Glu) autosomal recessive mutations. The characteristics of the gout patient are shown in Table 1. This mutation causes an increase in D-lactic acid in the patient's serum and urine and exhibits clinical manifestations related to gout. The sequencing results of the patient and his parents are shown in Table 1. Figure 1-3 As shown (the sequence shown in the figure is the reverse complementary sequence sequencing result of the reference sequence gene NM_194436.3, and only the region where the mutation exists is shown).

[0043] Table 1: Characteristics of patients with LDHD mutations and gout

[0044]

[0045] Example 2 Functional verification of new LDHD gene mutation sites associated with gout

[0046] Gout-related LDHD was confirmed by in vitro cell experiments and in vivo mouse experiments

[0047] After c.1327C>T (p.His443Tyr) and c.1058G>T (p.Ala353Glu) mutations, LDHD activity is lost. LDHD is a key catalytic enzyme in D-lactate metabolism. The loss of its activity will lead to increased serum D-lactate levels, followed by excessive secretion of D-lactate by the kidneys in exchange for uric acid reabsorption, ultimately leading to hyperuricemia and gout.

[0048] 1. In vitro experiments

[0049] In order to verify whether the activity of the enzyme is reduced after the mutation of LDHD c.1327C>T (p.His443Tyr) and c.1058G>T (p.Ala353Glu) sites, we constructed LDHD and LDHD mutant cells with the above mutations using the 293T cell line. The specific steps are as follows: two plasmids are transferred into 293T cells, one containing the original LDHD gene and the other containing the LDHD mutant (c.1327C>T (p.His443Tyr) and c.1058G>T (p.Ala353Glu)) genes. Green fluorescence can be seen under the microscope after 24 hours, and positive cell populations are sorted by flow cytometry after 48 hours. Collect the cells into a centrifuge tube, discard the supernatant after centrifugation, add the extract, and disrupt the cells by ultrasonic wave (ice bath, power 200W, ultrasonic wave 3s, interval 10s, repeat 30 times); centrifuge at 8000g 4℃ for 10min, take the supernatant, and put it on ice. Use D-Lactate DehydrogenaseActivityAssay Kit( 200PRO microplate reader) was used to measure the activity of LDHD in cells.

[0050] The LDHD enzyme activity of control cells (293T cells), cells overexpressing LDHD and mutant cells was detected. Figure 4 As shown in the figure, the LDHD activity of 293T normal control is close to 0 and almost undetectable. In cells overexpressing LDHD, the activity of LDHD is increased (0.06825U / 10 4 cell), but the LDHD enzyme activity in LDHD mutant cells was significantly decreased (0.018800.06825U / 10 4 cell).

[0051] 2. In vivo experiments

[0052] To verify whether hyperuricemia is caused by elevated plasma D-lactate levels, we measured plasma uric acid levels in mice after D-lactate injection. C57BL / 6J mice were intraperitoneally injected with 200 μL 3M D-lactate, and blood samples were collected before and at multiple time points after injection (before injection, 60 min, 120 min, 180 min, 300 min after injection) to monitor plasma uric acid levels. The specific experimental steps were as follows: C57BL / 6J mice were intraperitoneally injected with 200 μL 3M D-lactate (71716, Sigma-Aldrich) (n=3) and blood samples were collected before and at multiple time points after injection (60, 120, 180 and 300 minutes) to monitor plasma uric acid levels. Blood was collected from the medial angular vein, anticoagulated in EDTA tubes, and immediately centrifuged at 14,000 rpm for 10 minutes at 4°C to separate the plasma and freeze at -20°C. All plasma samples collected at different time points were thawed and analyzed simultaneously: using UricAcidAssay Kit ( 200PRO microplate reader) to measure the plasma uric acid level.

[0053] The results are as follows Figure 5 As shown, the plasma uric acid level of mice injected with D-lactic acid increased steadily, reaching a peak at about 180 minutes after injection, and the average increase in plasma uric acid level was 12.71 (ug / ml). Therefore, injection of D-lactic acid into C57BL / 6 mice can cause hyperuricemia, indicating that increased plasma D-lactic acid can lead to increased plasma uric acid.

[0054] The above experiments show that c.1327C>T (p.His443Tyr) and c.1058G>T (p.Ala353Glu) mutations lead to loss of LDHD activity, which in turn leads to increased serum D-lactic acid levels, and increased plasma D-lactic acid leads to increased plasma uric acid, ultimately leading to hyperuricemia and gout. Therefore, the mutation sites c.1327C>T (p.His443Tyr) and c.1058G>T (p.Ala353Glu) of the LDHD gene are associated with gout and can be used for the diagnosis or risk assessment of gout.

[0055] Example 3 Detection of c.1327C>T (p.His443Tyr) and c.1058G>T (p.Ala353Glu) mutation sites

[0056] 1) Extracting DNA from the blood collected from the individual to be tested;

[0057] 2) using the DNA extracted in step 1) as a template and a PCR primer set to perform a PCR reaction to obtain a PCR reaction product;

[0058] The PCR primer set includes primer pair P1 and primer pair P2. The amplified fragment of primer pair P1 contains the base corresponding to position 1327, and the amplified fragment of primer pair P2 contains the base corresponding to position 1058. The sequence of primer pair P1 is:

[0059] LDHD-F1:5'-CTCCGTAGTCAGGAACTTG-3'

[0060] LDHD-R1:5'-CCAGTTCCTCGGGCTCATCA-3'

[0061] The sequence of the primer pair P2 is:

[0062] LDHD-F2:5'-GCCCTTGTTTCTAGCGCC-3'

[0063] LDHD-R2:5'-CTCCTCTGCAGTTGGGGA-3'.

[0064] Amplification conditions:

[0065]

[0066] Store at 4℃

[0067] Separating the target fragment amplified by the PCR reaction from the PCR reaction product, and performing typing identification on the bases corresponding to the 1058th and 1327th bases of the D-lactate dehydrogenase gene coding region contained in the target fragment;

[0068] 3) Determination: The genotype determined by comparison includes wild homozygous type, mutant heterozygous type or homozygous mutant type.

[0069] Among them, the typing identification adopts a method of directly sequencing the target fragment, and determines the genotype or allele type of the individual to be tested corresponding to the 1058th and 1327th positions in the LDHD gene coding region by comparing the sequencing results with the reference sequence.

[0070] Among them, when the results of the two sites are both wild homozygous, it is considered that the individual to be tested does not suffer from gout and has a low risk of suffering from gout. When the results of the two sites are both mutant heterozygous, it is considered that the individual to be tested suffers from gout or has a high risk of suffering from gout. When the results of any one of the two sites are homozygous mutant, it is considered that the individual to be tested suffers from gout or has a high risk of suffering from gout.

[0071] Example 4 Kit for Gout Diagnosis or Risk Assessment

[0072] The kit comprises:

[0073] 1. Primer pair P1 and primer pair P2, the sequence of primer pair P1 is: LDHD-F1: 5'-CTCCGTAGTCAGGGAACTTG-3'

[0074] LDHD-R1:5'-CCAGTTCCTCGGGCTCATCA-3'

[0075] The sequence of the primer pair P2 is:

[0076] LDHD-F2:5'-GCCCTTGTTTCTAGCGCC-3'

[0077] LDHD-R2:5'-CTCCTCTGCAGTTGGGGA-3'.

[0078] 2. Reagents used to extract template DNA required for PCR amplification from the individual to be tested; 3. Reagents required for PCR amplification, including Taq enzyme, Buffer, and dNTP.

Claims

1. A mutated D-lactate dehydrogenase (LDHD) gene associated with gout, characterized in that: The coding region of the mutated D-lactate dehydrogenase gene contains both c.1327C>T and c.1058C>A mutation sites. The Genbank accession number of the D-lactate dehydrogenase gene before mutation is: NM_194436.3, wherein the coding region (CDS) of the D-lactate dehydrogenase gene is located between nucleotides 61..1515.

2. The mutated D-lactate dehydrogenase (LDHD) gene associated with gout according to claim 1, characterized in that: The coding sequence of the mutated D-lactate dehydrogenase gene is shown in SEQ ID NO.

1.

3. A mutated D-lactate dehydrogenase associated with gout, characterized in that: The mutated D-lactate dehydrogenase contains the following mutation sites p.His443Tyr and p.Ala353Glu at the same time. The D-lactate dehydrogenase before mutation is encoded by the D-lactate dehydrogenase gene with Genbank accession number NM_194436.

3.

4. The mutated D-lactate dehydrogenase associated with gout according to claim 3, characterized in that: The amino acid sequence of the mutated D-lactate dehydrogenase is shown in SEQ ID NO.

2.

5. A PCR primer set for detecting the mutated D-lactate dehydrogenase gene associated with gout according to claim 1, characterized in that: The target fragment amplified by the PCR primer set includes bases corresponding to the 1058th and 1327th positions of the D-lactate dehydrogenase gene coding region.

6. The PCR primer set according to claim 5, characterized in that: The PCR primer set includes primer pair P1 and primer pair P2. The amplified fragment of primer pair P1 contains the base corresponding to position 1327, and the amplified fragment of primer pair P2 contains the base corresponding to position 1058. The sequence of primer pair P1 is: LDHD-F1:5'-CTCCGTAGTCAGGAACTTG-3' LDHD-R1:5'-CCAGTTCCTCGGGCTCATCA-3' The sequence of the primer pair P2 is: LDHD-F2:5'-GCCCTTGTTTCTAGCGCC-3' LDHD-R2:5'-CTCCTCTGCAGTTGGGGA-3'.

7. Use of the PCR primer set according to claim 5 or 6 in the following aspects: (1) preparing a reagent for detecting a mutated D-lactate dehydrogenase gene associated with gout; (2) Preparation of reagents for gout diagnosis or risk assessment.

8. A kit for gout diagnosis or risk assessment, characterized in that: The kit comprises the PCR primer set according to claim 5 or 6.

9. The kit according to claim 8, characterized in that The kit also includes reagents for extracting template DNA required for PCR amplification from the individual to be tested.

10. The kit according to claim 8, characterized in that The kit is used for gout diagnosis or risk assessment according to the following steps: 1) Extracting DNA from the blood collected from the individual to be tested; 2) using the DNA extracted in step 1) as a template and the PCR primer set of claim 3 or 4 to perform a PCR reaction to obtain a PCR reaction product; separating the target fragment amplified by the PCR reaction from the PCR reaction product, and performing typing and identification on the bases corresponding to the 1058th and 1327th positions of the D-lactate dehydrogenase gene coding region contained in the target fragment; 3) Determination: The genotype determined by comparison includes wild homozygous type, mutant heterozygous type or homozygous mutant type; Preferably, the typing identification adopts a method of directly sequencing the target fragment, and determines the genotype or allele type of the tested individual corresponding to positions 1058 and 1327 of the LDHD gene coding region by comparing the sequencing results with the reference sequence; Among them, preferably, when the results of the two sites are both wild homozygous, it is considered that the individual to be tested does not suffer from gout and has a low risk of suffering from gout; when the results of the two sites are both mutant heterozygous, it is considered that the individual to be tested suffers from gout or has a high risk of suffering from gout; when the results of any one of the two sites is homozygous mutant, it is considered that the individual to be tested suffers from gout or has a high risk of suffering from gout.

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