Primer group for detecting pathogenic mutation of CCND2 gene and kit containing primer group
By designing primer sets for pathogenic mutations of CCND2 gene and combining with Sanger sequencing technology, the problem of long cycle and high cost of detection of pathogenic mutations of CCND2 gene in the prior art is solved, and a rapid and economical detection effect is achieved.
Patent Information
- Application Number
- CN202510071273.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-16
- Publication Date
- 2025-04-11
AI Technical Summary
The prior art is difficult to quickly and economically detect pathogenic mutations of CCND2 gene, especially functionally acquired pathogenic mutations, resulting in a long detection cycle and high cost.
A primer set for detecting pathogenic mutations of CCND2 genes, including upstream and downstream primers, was designed, and was detected by Sanger sequencing technology, which shortened the detection cycle and reduced the detection cost.
Through the design of this primer set, pathogenic mutations of the CCND2 gene, especially the c.842C>A mutation site, can be detected quickly and accurately, shortening the detection cycle and reducing the detection cost.
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Figure CN119799879A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of biotechnology, and in particular to a primer combination and a kit for detecting CCND2 gene pathogenic mutations. Background Art
[0002] The CCND2 (Cyclin D2) gene is located on human chromosome 12 and encodes cyclin D2, a protein that plays a key role in the cell cycle. It mainly affects cell growth and division by regulating the transition of cells from the growth phase (G1 phase) to the DNA replication phase (S phase).
[0003] Mutations in the CCND2 gene are associated with a variety of disorders, including a rare inherited brain development disorder called Megalencephaly-polymicrogyria-polydactyly-hydrocephalus (MPPH). This syndrome is characterized by an abnormally large brain and head (megalencephaly), excessive folding of the cerebral cortex (polymicrogyria), extra fingers or toes (polydactyly), and hydrocephalus.
[0004] The inheritance pattern of this syndrome is usually a de novo mutation, which means that there is usually no history of this disease in the patient's family. These new mutations occur during the formation of germ cells (such as sperm or eggs) or in the early embryonic development stage. Therefore, individuals with this disease are usually the first to have such mutations in the family. However, general genetic testing is performed through whole exome sequencing (WES) or whole genome sequencing (WES) technology. Both methods are second-generation sequencing technologies and are very expensive. Summary of the invention
[0005] The main purpose of the present invention is to propose a primer set for detecting pathogenic mutations in the CCND2 gene, aiming to determine whether the CCND2 gene has pathogenic mutations by Sanger sequencing, thereby shortening the detection cycle and reducing the detection cost.
[0006] To achieve the above purpose, the primer set for detecting pathogenic mutations of the CCND2 gene proposed in the present invention includes an upstream primer and a downstream primer, the nucleotide sequence of the upstream primer is shown in SEQ ID NO.7, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.8.
[0007] Optionally / in one embodiment, the first primer set is used to detect the c.842C>A mutation site of the CCND2 gene.
[0008] Optionally / in one embodiment, the primer set is used for Sanger sequencing.
[0009] Optionally / in one embodiment, the PCR reaction system for Sanger sequencing includes the following components:
[0010] 10×PCR Buffer;
[0011] dNTPs (2.5 mM);
[0012] Genomic DNA;
[0013] Upstream primer;
[0014] Downstream primer;
[0015] Taq DNA polymerase;
[0016] ddH2O.
[0017] Optionally / in one embodiment, the added concentration of the genomic DNA is 100 ng / μL.
[0018] Optionally / in one embodiment, the volumes of the components of the PCR reaction system for Sanger sequencing are as follows:
[0019] 10×PCR Buffer: 1.0 μL;
[0020] dNTP (2.5 mM): 0.8 μL;
[0021] Genomic DNA (100 ng / μL): 0.5 μL;
[0022] Upstream primer: 0.4 μL;
[0023] Downstream primer: 0.4 μL;
[0024] Taq DNA polymerase: 0.1 μL;
[0025] ddH2O: 6.8 μL;
[0026] Total volume: 10 μL.
[0027] The present invention also provides a kit for detecting CCND2 gene pathogenic mutations, comprising the above primer set.
[0028] The present invention also proposes the use of the above kit in detecting pathogenic mutations of the CCND2 gene.
[0029] The technical solution of the present invention adopts two sets of primers to detect two pathogenic mutations of the CCND2 gene, so that Sanger sequencing can be used to complete the detection of the pathogenic mutation of the CCND2 gene, that is, Sanger sequencing can be used to determine whether the CCND2 gene has a pathogenic mutation, thereby shortening the detection cycle and reducing the detection cost. BRIEF DESCRIPTION OF THE DRAWINGS
[0030] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the structures shown in these drawings without paying creative work.
[0031] Figure 1 The band result diagrams of primer set 1 to primer set 3 of the present invention;
[0032] Figure 2 The band result diagrams of primer set 4 to primer set 6 of the present invention;
[0033] Figure 3 The band result diagram of primer set 7 to primer set 9 of the present invention;
[0034] Figure 4 The band result diagram of primer set 10 to primer set 12 of the present invention;
[0035] Figure 5 Graph showing the band results of primer set 13 to primer set 15 of the present invention;
[0036] Figure 6 This is a fetal B-ultrasound image of Application Example 1 of the present invention;
[0037] Figure 7 This is a pedigree diagram of Application Example 1 of the present invention;
[0038] Figure 8 Schematic diagram of the c.842C>A mutation site in the present invention.
[0039] The realization of the purpose, functional features and advantages of the present invention will be further explained in conjunction with embodiments and with reference to the accompanying drawings. DETAILED DESCRIPTION
[0040] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.
[0041] In addition, if there are descriptions involving "first", "second", etc. in the embodiments of the present invention, the descriptions of "first", "second", etc. are only used for descriptive purposes and cannot be understood as indicating or implying their relative importance or implicitly indicating the number of technical features indicated. Therefore, the features defined as "first" and "second" may explicitly or implicitly include at least one of the features. In addition, if the meaning of "and / or" appearing in the full text is to include three parallel schemes, taking "A and / or B" as an example, it includes scheme A, or scheme B, or a scheme that satisfies both A and B. In addition, the technical solutions between the various embodiments can be combined with each other, but it must be based on the ability of ordinary technicians in this field to implement. When the combination of technical solutions is contradictory or cannot be implemented, it should be deemed that such a combination of technical solutions does not exist and is not within the scope of protection required by the present invention.
[0042] The CCND2 (Cyclin D2) gene is located on human chromosome 12 and encodes cyclin D2, a protein that plays a key role in the cell cycle. It mainly affects cell growth and division by regulating the transition of cells from the growth phase (G1 phase) to the DNA replication phase (S phase).
[0043] Mutations in the CCND2 gene are associated with a variety of disorders, including a rare inherited brain development disorder called Megalencephaly-polymicrogyria-polydactyly-hydrocephalus (MPPH). This syndrome is characterized by an abnormally large brain and head (megalencephaly), excessive folding of the cerebral cortex (polymicrogyria), extra fingers or toes (polydactyly), and hydrocephalus.
[0044] Although most human genetic diseases are caused by loss-of-function (LOF) mutations, that is, mutations that cause the gene to lose its normal function and cause disease. However, in the CCND2 gene, a specific type of mutation is called gain-of-function mutation, which leads to increased stability of cyclin D2. Usually, gain-of-function mutations increase the function of the protein, resulting in developmental abnormalities and are strongly associated with disease states. In the case of megalencephaly, microgyria, polydactyly, and hydrocephalus (MPPH), gain-of-function mutations lead to abnormal accumulation of cyclin D2 encoded by the CCND2 gene. This accumulation prevents the normal regulation of the cell cycle, leading to disordered proliferation of brain cells, and ultimately forming megalencephaly and other brain structural abnormalities.
[0045] Mutation sites in specific segments of the CCND2 gene have been identified as being associated with MPPH. For example, specific mutations in the coding region (such as missense mutations in the region encoding a certain amino acid codon) can lead to increased stability of cyclin D2. These mutation points include key regions in the protein structure, which are usually associated with protein degradation markers. When these regions mutate, they may block the degradation pathway, thereby enhancing the function of the protein and triggering cell proliferation.
[0046] The inheritance pattern of MPPH is usually a de novo mutation, which means that there is usually no history of the disease in the patient's family. These new mutations occur during the formation of reproductive cells (such as sperm or eggs) or in the early embryonic development stage, so individuals with this disease are usually the first to have such mutations in the family. Genetic etiology research on such patients, especially prenatal testing, will help improve the health of the newborn population.
[0047] However, there is currently no detection kit specifically for the gain-of-function pathogenic mutation of the CCND2 gene, and it can only be detected through whole exome sequencing (WES) or whole genome sequencing (WES) technology. Both methods are second-generation sequencing technologies, which have high requirements for detection technology and equipment, are expensive, and have a long cycle.
[0048] The inventors of the present invention identified a new gain-of-function pathogenic mutation of the CCND2 gene in clinical work, which caused a typical MPPH syndrome phenotype in a fetus. That is, NM_001759.3:c.842C>A (p.Pro281His). There is no report of this mutation site in the ESP database, the Thousand People database, the EXAC database, the GnomAD database and other databases. The wild-type CCND2 gene is coded as ENSG00000118971 in the Ensemble database (https: / / useast.ensembl.org / index.html). There is currently no detection method for the CCND2 gene c.842C>A mutation site.
[0049] To this end, the present invention aims to provide a primer set, a kit and its use for rapid detection of the CCND2 gene c.842C>A mutation site. The alignment information of the CCND2 gene c.842C>A mutation site and the human genome reference sequence GRCh37 is as follows:
[0050] The base C at position 4409147 on chromosome 12 mutated to A, i.e., c.842C>A mutation, located in exon 5 of CCND2 gene. This mutation will cause the proline (Pro) at position 281 to be converted to histidine (His), i.e., the amino acid change is (p.Pro281His).
[0051] Sanger sequencing, the full name of which is Sanger chain termination sequencing, is a DNA sequencing method invented by British scientist Frederick Sanger in 1977. The basic principle of Sanger sequencing is to use DNA polymerase and end-labeled ddNTPs (dideoxynucleotide triphosphates) to terminate the synthesis of the DNA chain. This method has long been the gold standard in genomics and molecular biology research due to its high accuracy and reliability. After years of development, Sanger sequencing has mature kits and automated equipment, which have the advantages of high accuracy, easy result reading and low cost.
[0052] In summary, the present invention firstly hopes to propose a primer set for detecting pathogenic mutations in the CCND2 gene, which can determine whether the CCND2 gene has pathogenic mutations through Sanger sequencing, thereby shortening the detection cycle and reducing the detection cost.
[0053] Accordingly, in the present invention, the normal human CCND2 gene coding sequence is shown as SEQ ID NO.31; the normal human CCND2 protein amino acid sequence is shown as SEQ ID NO.32; the CCND2 gene coding sequence of the c.842C>A pathogenic mutation is shown as SEQ ID NO.33; and the CCND2 protein amino acid sequence of the c.842C>A pathogenic mutation is shown as SEQ ID NO.34.
[0054] Specifically, the primer set for detecting the pathogenic mutation of the CCND2 gene proposed in the present invention comprises an upstream primer and a downstream primer, the nucleotide sequence of the upstream primer is shown in SEQ ID NO.7, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.8. The primer set proposed in the present invention is used to detect the c.842C>A mutation site of the CCND2 gene.
[0055] Among them, when performing Sanger sequencing, the volumes of the components of the PCR reaction system are as follows:
[0056] 10×PCR Buffer: 1.0 μL;
[0057] dNTP (2.5 mM): 0.8 μL;
[0058] Genomic DNA (100 ng / μL): 0.5 μL;
[0059] Upstream primer: 0.4 μL;
[0060] Downstream primer: 0.4 μL;
[0061] Taq DNA polymerase: 0.1 μL;
[0062] ddH2O: 6.8 μL;
[0063] Total volume: 10 μL.
[0064] The following is a description of the specific embodiments.
[0065] Example 1 Design of primer sets
[0066] The present invention designs 15 pairs of candidate primer sets for the c.842C>A mutation site of the CCND2 gene, and the designed primer sets follow the design principles of primers.
[0067] It should be noted that the primers in the following table have different binding sites. To show the difference, the bases of the binding sites on the exons of the primers are represented by capital letters, and the bases of the binding sites on the introns are represented by lowercase letters.
[0068] Table 1 Candidate primer set sequence list
[0069]
[0070]
[0071]
[0072]
[0073] For the above 15 primer sets, a 5-tube PCR reaction system was set up for each primer set to verify the feasibility of the primers.
[0074] Among them, the reaction settings of the 5-tube PCR reaction system are as follows
[0075] Table 2 5-tube reaction system
[0076] Components Reaction 1 Reaction 2 Reaction 3 Reaction 4 React 5 10×PCR Buffer 1.0 1.0 1.0 1.0 1.0 dNTP(2.5mM) 0.8 0.8 0.8 0.8 0.8 Genomic DNA (100 ng / μL) 0.5 0 0.5 0.5 0.5 Upstream primer 0 0.4 0.4 0 0.4 Downstream primer 0 0.4 0 0.4 0.4 Taq DNA polymerase 0.1 0.1 0.1 0.1 0.1 <![CDATA[ddH2O]]> 7.6 7.3 7.4 7.4 6.8 Total volume 10 10 10 10 10
[0077] Referring to the table above, the design idea of the 5-tube PCR reaction system is as follows: Reaction 1 is a negative control with only template DNA and no primers; Reaction 2 is a negative control with only upstream and downstream primers and no template DNA; Reaction 3 is to test the upstream primer; Reaction 4 is to test the downstream primer; Reaction 5 is a complete PCR system test. In this way, the feasibility of each primer set is verified.
[0078] Among them, a thermal cycler is used in PCR amplification, and the settings of the thermal cycler are shown in the following table:
[0079] Table 3 PCR amplification setting table
[0080] Temperature(℃) time Number of cycles 94 3 minutes 1 40 30 seconds 32 Tm 30 seconds 32 72 1 minute 32 72 5 minutes 1 10 ∞ 1
[0081] In the above table, T m The value is set according to the T in each primer set in Table 1 m It should be noted that, if only the upstream primer or the downstream primer is used in reaction 3 and reaction 4, the T value of each primer is used. m For example, reaction 2 and reaction 5 have both upstream primers and downstream primers, then the T values of the upstream primers and downstream primers in each primer set can be taken as m The mean of the values; it should be noted that the T m The values are sometimes quite different, so you can choose T according to the actual situation. m The specific value of the value.
[0082] Reference Figures 1 to 5 From the table below, it can be found that the primer set (primer set 4) has better reaction results than other primer sets.
[0083] Table 4 Reaction results of each primer set
[0084]
[0085]
[0086] The primer set will be used for subsequent testing.
[0087] Application Example 1
[0088] Reference Figures 6 to 8 , a non-consanguineous couple, denied family history of genetic diseases, pregnant woman 1, 0. Prenatal ultrasound at 11+6 weeks of pregnancy showed that the fetal NT was 3.2mm. Color photos at 24 weeks of pregnancy showed that the bilateral lateral cerebral fissures of the fetus showed platform-like changes, and the bilateral lateral ventricles were dilated, with the left side about 1.04cm wide and the right side about 1.13cm wide. Both hands had 6 fingers deformed, and both feet had 6 toes deformed. Further MRI verification showed that the sulci and fissures of the bilateral cerebral hemispheres were not obvious, the lateral fissures showed platform-like changes, the surface of the bilateral frontal lobes showed slightly fine serrated changes, and flake-like T2WI low-signal shadows were seen in the bilateral basal ganglia. The bilateral lateral ventricles were widened, with the left side about 12.4mm wide and the right side 10.3mm wide.
[0089] Fetal amniotic fluid cells were extracted for culture, and peripheral blood was drawn from the pregnant couple at the same time. After genomic DNA was extracted, PCR amplification was performed using primer set 4 of the present invention. The amplified product was analyzed by Sanger, and it was found that the fetal CCND2 gene carried a c.842C>A mutation, while neither the fetal parents carried this mutation, so this mutation was a new mutation.
[0090] According to the ACMG standard, the pathogenicity evidence of this mutation is PS2 (new mutation in the patient, and no family history verified by the parents) + PM2 (variation not found in the normal control population in the ESP database, the Thousand People database, and the EXAC database, or extremely low frequency sites in recessive genetic diseases) + PP3 (multiple statistical methods predict that the mutation will have a harmful effect on the gene or gene product, including conservative prediction, evolutionary prediction, splicing site effects, etc.). Due to its high pathogenicity, after detailed communication with the pregnant woman and her family, the pregnant woman chose to terminate the pregnancy. The fetus that was induced to abort was confirmed to have the characteristics of MPPH syndrome from the appearance. For example, six fingers on both hands, six toes on both feet, and a large head circumference.
[0091] The present invention also proposes a kit for detecting pathogenic mutations in the CCND2 gene, which includes the above-mentioned primer set. The specific sequence and usage of the primer set refer to the above description and embodiments. Since this kit adopts all the technical solutions of all the above-mentioned embodiments, it has at least all the beneficial effects brought by the technical solutions of the above-mentioned embodiments, which will not be repeated here one by one.
[0092] The present invention also proposes the use of the above-mentioned kit in detecting pathogenic mutations of the CCND2 gene. The above-mentioned kit can be used as one of the means for rapid screening of pathogenic mutations of the CCND2 gene; and because the present invention uses Sanger sequencing, the experimental conditions are very low and can be used in conventional biological laboratories, and the cost is very low and the detection cycle is short.
[0093] The above descriptions are only optional embodiments of the present invention, and are not intended to limit the patent scope of the present invention. All equivalent structural changes made using the contents of the present invention's specification and drawings, or directly / indirectly applied in other related technical fields, are included in the patent protection scope of the present invention.
Claims
1. A primer set for detecting pathogenic mutations in the CCND2 gene, characterized in that: It comprises an upstream primer and a downstream primer. The nucleotide sequence of the upstream primer is shown in SEQ ID NO.7, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.
8.
2. The primer set according to claim 1, characterized in that The first primer set is used to detect the c.842C>A mutation site of the CCND2 gene.
3. The primer set according to any one of claims 1 or 2, characterized in that The primer set was used for Sanger sequencing.
4. The primer set according to claim 3, characterized in that The PCR reaction system of the Sanger sequencing includes the following components: 10×PCR Buffer; dNTPs (2.5 mM); Genomic DNA; Upstream primer; Downstream primer; Taq DNA polymerase; ddH2O.
5. The primer set according to claim 4, characterized in that The added concentration of the genomic DNA was 100 ng / μL.
6. The primer set according to claim 5, characterized in that The volumes of the components of the PCR reaction system for Sanger sequencing are as follows: 10×PCR Buffer: 1.0 μL; dNTP (2.5 mM): 0.8 μL; Genomic DNA (100 ng / μL): 0.5 μL; Upstream primer: 0.4 μL; Downstream primer: 0.4 μL; Taq DNA polymerase: 0.1 μL; ddH2O: 6.8 μL; Total volume: 10 μL.
7. A kit for detecting pathogenic mutations in CCND2 gene, characterized in that: Comprising the primer set according to any one of claims 1 to 6.
8. Use of the kit as claimed in claim 7 in detecting pathogenic mutations in the CCND2 gene.