Mutated pabpcl gene fragment and detection method and application thereof

Abstract

The application relates to a mutant PABPC1L gene fragment and a detection method and application thereof, which has the following mutation: c.1121G>A compared with a wild-type PABPC1L gene, and the cDNA sequence of the wild-type PABPC1L gene is shown as SEQ ID NO:1. A pathogenic gene PABPC1L splicing site variation (c.1121G>A) of a female primary infertility patient is identified through a whole exon sequencing technology screening, which can assist in analyzing the genetic defects of the female primary infertility patient and providing a reference for selecting a suitable assisted reproduction method for the female primary infertility patient.

Description

Technical Field

[0001] This application relates to the field of molecular biology technology, and in particular to a mutated PABPC1L gene fragment and its detection method and application. Background Technology

[0002] Primary female infertility is a global health problem affecting most women of reproductive age. Many factors contribute to primary female infertility, including oocyte maturation disorders, ovulation disorders, fallopian tube factors, uterine factors, and cervical factors. Among these, oocyte immaturity is a common cause of primary female infertility. Oocyte maturation disorders manifest in various forms: germinal vesicle (GV) arrest, metaphase I (MI) arrest, metaphase II (M) arrest, and mixed arrest. Mutations or functional defects in any gene affecting meiosis can lead to oocyte maturation disorders. These patients, after repeated assisted reproductive technologies including in-vitro fertilization (IVF-ET), are unable to obtain mature oocytes, thus hindering further IVF procedures. Identifying the relevant pathogenic genes is crucial for the clinical diagnosis and classification of the disease. Current research has identified several genes that can lead to oocyte maturation disorders, such as PATL2, TUBB8, ZP1, ZP2, and ZP3.

[0003] However, these genes can only explain the causes in some patients, and the causes remain unknown in many others. Furthermore, in a significant number of women with primary infertility, genetic testing reveals gene variations lacking functional evidence, making it impossible to determine whether these variations are related to female infertility. This poses a significant challenge to the precise prevention and treatment of primary infertility in women. Summary of the Invention

[0004] This study found that a missense mutation c.1121G>A occurred in PABPC1L of women with primary infertility, affecting the expression of the PABPC1L protein (p.R374Q), thereby affecting the normal meiotic process and ultimately manifesting as primary infertility. This mutation site was not recorded in healthy population databases. Detecting the presence of this mutation site in PABPC1L carried by women with primary infertility helps to clarify the genetic causes of female primary infertility and can provide a reference for choosing appropriate assisted reproductive technologies. Based on this, this application provides a novel mutated PABPC1L gene fragment.

[0005] This application provides a mutated PABPC1L gene fragment, which has the following mutation compared with the wild-type PABPC1L gene: c.1121G>A, and the cDNA sequence of the wild-type PABPC1L gene is shown in SEQ ID NO: 1.

[0006] This application also provides a mutant PABPC1L protein, which has the following mutation compared to the wild-type PABPC1L protein: p.R374Q, the amino acid sequence of which is shown in SEQ ID NO: 2.

[0007] This application also provides the application of the above-described detection reagent for PABPC1L gene mutation in the preparation of diagnostic products for primary infertility, with reference to the cDNA sequence of the wild-type PABPC1L gene, wherein the PABPC1L gene mutation is c.1121G>A.

[0008] In some embodiments, the detection reagent performs detection at the nucleic acid level.

[0009] In some embodiments, the detection reagent includes reagents suitable for at least one of the following methods:

[0010] Restriction fragment length polymorphism (RFLP), single-strand conformation polymorphism (SCM), polymerase chain reaction (PCR), denaturing gradient gel electrophoresis (DLGGE), nucleic acid sequencing, nucleic acid typing chip detection, mass spectrometry of flight (MS / MS), denaturing high-performance liquid chromatography (HPLC), snap-shot method, in situ hybridization, biomolecular mass spectrometry (BMS), and HRM method.

[0011] In some embodiments, the detection reagent includes one or more of the following primer pairs:

[0012] Primer pairs with nucleotide sequences as shown in SEQ ID No. 3-4, primer pairs with nucleotide sequences as shown in SEQ ID No. 5-6, primer pairs with nucleotide sequences as shown in SEQ ID No. 7-8, primer pairs with nucleotide sequences as shown in SEQ ID No. 9-10, primer pairs with nucleotide sequences as shown in SEQ ID No. 11-12, primer pairs with nucleotide sequences as shown in SEQ ID No. 13-14, primer pairs with nucleotide sequences as shown in SEQ ID No. 15-16, primer pairs with nucleotide sequences as shown in SEQ ID No. 17-18, primer pairs with nucleotide sequences as shown in SEQ ID No. 19-20, primer pairs with nucleotide sequences as shown in SEQ ID No. 21-22, primer pairs with nucleotide sequences as shown in SEQ ID No. 23-24, primer pairs with nucleotide sequences as shown in SEQ ID No. 25-26, and primer pairs with nucleotide sequences as shown in SEQ ID No. 27-28.

[0013] In some embodiments, the diagnostic product is a reagent kit.

[0014] In some embodiments, the kit further includes at least one of a negative control, a positive control, a nucleic acid extraction reagent, a PCR amplification reagent, a sequencing reagent, and an electrophoresis detection reagent.

[0015] This application also provides a method for detecting PABPC1L gene mutations, which involves detecting whether the target individual's PABPC1L gene has the following mutation: c.1121G>A.

[0016] In some embodiments, the above-described method for detecting PABPC1L gene mutations includes the following steps:

[0017] Using the DNA of the sample to be tested as a template, PCR amplification was performed using the primer pairs in the above-mentioned detection reagent to obtain the amplification product; and the amplification product was analyzed to determine the mutation status of the PABPC1L gene. Attached Figure Description

[0018] Figure 1 This document presents the results of homozygous splicing site variants of the PABPC1L gene found in two female patients with primary infertility in this application. A represents a family pedigree with hereditary PABPC1L mutations, with black circles representing female patients with primary infertility. B shows the patient carrying a homozygous splicing site (c.1121G>A) mutation in the PABPC1L gene, confirmed by Sanger sequencing. C shows the sequence conservation of the mutant amino acid sequence compared to sequences from nine other species. D shows the domain locations of the PABPC1L protein. E, from left to right, shows the three-dimensional structures of the wild-type and mutant PABPC1L proteins. F shows fertilized eggs observed 20 hours after in vitro fertilization in the control group and patients during IVF. G shows fertilized eggs observed 3 hours and 20 hours after intracytoplasmic sperm injection in the control group and patients during ICSI.

[0019] Figure 2 This application describes the effects of PABPC1L mutation on the localization of PABPC1L protein and the meiotic process in mouse oocytes. A shows the effect of PABPC1L mutation on the localization of PABPC1L protein during the GOs and FGOs stages in mice; B shows the effect of PABPC1L mutation on the meiotic process of oocytes; C shows the effect of PABPC1L mutation on oocytes 6 hours after the GVBD stage; and D shows the effect of PABPC1L mutation on the chromosome morphology of oocytes. Detailed Implementation

[0020] To make the above-mentioned objectives, features, and advantages of this application more apparent and understandable, a detailed description of specific embodiments of this application is provided. Many specific details are set forth in the following description to provide a thorough understanding of this application. However, this application can be implemented in many other ways different from those described herein, and those skilled in the art can make similar modifications without departing from the spirit of this application. Therefore, this application is not limited to the specific embodiments disclosed below.

[0021] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. Unless otherwise specifically stated, all raw materials, reagents, instruments, and equipment used in this application are commercially available or can be prepared by existing methods.

[0022] The term "primer" as used herein refers to an oligonucleotide, whether naturally occurring in purified restriction digest or synthetically produced, which, when placed under conditions that induce the synthesis of primer extension products complementary to the nucleic acid strand (e.g., in the presence of nucleotides and inducers such as DNA polymerase and at suitable temperature and pH), functions as a site of synthesis initiation. "HIS" is a tag commonly used to detect protein expression. 。

[0023] A missense mutation occurs when a codon encoding a certain amino acid is replaced by a codon encoding a different amino acid, thus altering the amino acid composition and sequence of the polypeptide chain. Missense mutations typically result in the polypeptide chain losing its original function, causing protein abnormalities.

[0024] Primary female infertility is a disease caused by multiple factors, with high phenotypic and genetic heterogeneity, requiring continuous testing for new pathogenic genes.

[0025] The applicant, through extensive creative experimental research, discovered that mutations at the c.1121G>A site of the PABPC1L gene are associated with primary female infertility. Predictions using various bioinformatics software revealed that the missense mutations are pathogenic mutations, and the mutated amino acids are highly conserved across multiple species.

[0026] Therefore, one embodiment of this application provides a mutated PABPC1L gene fragment, which has the following mutation compared with the wild-type PABPC1L gene: c.1121G>A, and the cDNA sequence of the wild-type PABPC1L gene is shown in SEQ ID NO: 1.

[0027] This application also provides a mutant PABPC1L protein, which has the following mutation compared to the wild-type PABPC1L protein: p.R374Q. The amino acid sequence of the wild-type PABPC1L protein is shown in SEQ ID NO: 2, specifically:

[0028] .

[0029] Prediction using bioinformatics software showed that the above-mentioned gene mutation sites were missense mutations. The 3D structure of the protein showed that the gene mutation led to abnormal hydrogen bond connections of amino acids, making the protein spatial structure unstable and thus affecting the normal function of PABPC1L protein.

[0030] This application also provides the application of a PABPC1L gene mutation detection reagent in the preparation of diagnostic products for primary infertility, using the cDNA sequence of the wild-type PABPC1L gene as a reference, wherein the PABPC1L gene mutation is c.1121G>A.

[0031] In some of these embodiments, the detection reagents are used for detection at the nucleic acid level.

[0032] In one specific example, the above-mentioned detection reagents include reagents suitable for at least one of the following methods: restriction fragment length polymorphism, single-strand conformation polymorphism, polymerase chain reaction, denaturing gradient gel electrophoresis, nucleic acid sequencing, nucleic acid typing chip detection, mass spectrometry of flight, denaturing high performance liquid chromatography, snap-shot method, in situ hybridization, biomolecular mass spectrometry, and HRM method.

[0033] In some embodiments, the detection reagent includes one or more of the following primer pairs:

[0034] Primer pairs with nucleotide sequences as shown in SEQ ID No. 3-4, primer pairs with nucleotide sequences as shown in SEQ ID No. 5-6, primer pairs with nucleotide sequences as shown in SEQ ID No. 7-8, primer pairs with nucleotide sequences as shown in SEQ ID No. 9-10, primer pairs with nucleotide sequences as shown in SEQ ID No. 11-12, primer pairs with nucleotide sequences as shown in SEQ ID No. 13-14, primer pairs with nucleotide sequences as shown in SEQ ID No. 15-16, primer pairs with nucleotide sequences as shown in SEQ ID No. 17-18, primer pairs with nucleotide sequences as shown in SEQ ID No. 19-20, primer pairs with nucleotide sequences as shown in SEQ ID No. 21-22, primer pairs with nucleotide sequences as shown in SEQ ID No. 23-24, primer pairs with nucleotide sequences as shown in SEQ ID No. 25-26, and primer pairs with nucleotide sequences as shown in SEQ ID No. 27-28.

[0035] In some embodiments, the diagnostic product is a reagent kit. It is understood that the specific type is not limited to this, and various types of testing products are acceptable.

[0036] In some embodiments, the kit further includes at least one of a negative control, a positive control, a nucleic acid extraction reagent, a PCR amplification reagent, a sequencing reagent, and an electrophoresis detection reagent.

[0037] In one embodiment, the positive control is a DNA sample of PABPC1L that has undergone the above-mentioned mutation.

[0038] In one embodiment, the negative control is deionized water. It is understood that in other specific examples, the negative control may be other samples of PABPC1L that have not undergone mutation.

[0039] In one embodiment, the PCR amplification reagent includes a PCR premix. In an optional specific example, the PCR premix includes Mg. 2+ Reaction buffer, DNA polymerase, and dNTPs.

[0040] In one embodiment, the reaction buffer can be 1×, 2×, 5×, or 10×; the concentration of dNTPs is 8 mM to 12 mM; the concentration of MgCl2 is 20 mM to 30 mM; and the concentration of Taq DNA polymerase is 4 U / μL to 6 U / μL.

[0041] In a specific example, the concentration of the detection reagent was 10 μM; the PCR buffer was 5×; the concentration of dNTPs was 10 mM; the concentration of MgCl2 was 25 mM; and the concentration of Taq DNA polymerase was 5 U / μL.

[0042] It is understood that, in some other specific examples, the kit described above may not include any of the reagents among positive control, negative control, PCR amplification reagent, sequencing reagent, and electrophoresis detection reagent, and the excluded reagents may be reasonably obtained from external sources.

[0043] The above-mentioned kit for detecting PABPC1L gene mutations can be used to assist in the diagnosis of primary infertility in women, and is characterized by its simple and rapid operation.

[0044] This application also provides a method for detecting PABPC1L gene mutations, which involves detecting whether the target individual's PABPC1L gene contains the following mutation: c.1121G>A. In a specific example, the detection method includes the following steps S10, S20, S30, S40, and S50:

[0045] Step S10: Obtain the DNA of the sample to be tested.

[0046] In one embodiment, in step S10, the sample to be tested is one or more of peripheral blood and prenatal diagnosis-related samples.

[0047] Furthermore, in step S10, the sample to be tested is the patient's peripheral blood.

[0048] Step S20: Using the DNA of the sample to be tested as a template, perform PCR amplification using the primer pairs in the detection reagents of any of the above embodiments to obtain the amplification product.

[0049] In some embodiments, in step S20, the PCR amplification program is set as follows: pre-denaturation at 95°C for 5 min; denaturation at 94°C for 30-45 s, annealing at 56°C-60°C for 30-60 s, extension at 72°C for 45-60 s, for a total of 30-35 cycles; and finally extension at 72°C for 3-7 min, with the PCR amplification product stored at 4°C. It is understood that the PCR program can be reasonably adjusted in other specific examples.

[0050] In a specific example, the PCR amplification program was set as follows: 95℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 56.5℃ annealing for 30 s, 72℃ extension for 60 s, for a total of 34 cycles; and finally 72℃ extension for 5 min. The PCR amplification products were stored at 4℃.

[0051] Step S30: Perform agarose gel electrophoresis on the PCR amplification products.

[0052] In one embodiment, in step S30, 3 μL to 5 μL of PCR amplification product and DL2000 DNA Marker (TSJ011-100, Qingke Biotechnology) are respectively applied to the gel wells, electrophoresed at 220V for 9 min to 10 min, and then imaged.

[0053] In one embodiment, in step S30, the band size of the PCR amplification product is confirmed to be correct by agarose gel electrophoresis.

[0054] Step S40: Sequencing the amplification products.

[0055] In one embodiment, in step S40, the sequencing method is Sanger sequencing.

[0056] In one embodiment, in step S40, sequencing analysis is performed using the same primer pair as the PCR amplification primer pair.

[0057] Step S50: Analyze the mutation status of the PABPC1L gene.

[0058] In one embodiment, in step S50, the sequence obtained from sequencing in step S40 is compared with the cDNA sequence of the wild-type PABPC1L gene as shown in SEQ ID NO: 1 to analyze whether PABPC1L has undergone mutation.

[0059] The above-mentioned method for detecting PABPC1L gene mutations has at least the following advantages: (1) High detection efficiency: Only one pair of primers needs to be designed, and PABPC1L mutations can be detected by PCR amplification and sequencing analysis. The mutation detection can be completed within 1 to 2 days. (2) Simple operation and low cost: Compared with DNA library construction, whole exome sequencing combined with bioinformatics analysis, PCR amplification combined with Sanger sequencing technology is simpler and cheaper.

[0060] The present application will be further described below with reference to specific embodiments, but these should not be construed as limiting the scope of protection of the present application.

[0061] Example 1

[0062] In clinical research practice, peripheral blood samples were obtained from two female infertile patients from consanguineous families. They had no prior history of major illnesses, normal menstrual cycles, normal basal hormone levels (see Table 1 for details), and normal ovarian reserve. The male partners of these patients had normal semen analysis results, with sperm exhibiting normal morphology and motility. The proband (III-2) couple underwent IVF treatment in their first cycle using the long protocol, retrieving 11 oocytes. On day 1, 10 oocytes extruded polar bodies, and 1 oocyte was MI (miscarriage of conception). Nine oocytes were unfertilized, and 2 were 2PN (secondary nucleus implantation). The embryos subsequently did not cleave and continued to develop. In the second cycle, ICSI treatment was performed using the antagonist protocol. Oocyte retrieval was performed 35 hours after triggering, yielding 11 oocytes. One oocyte degenerated, and the remaining oocytes were all MI. After 10 hours of in vitro culture, 2 oocytes extruded polar bodies. These two oocytes did not form pronuclei during ICSI. Furthermore, the proband's sister (III-3) underwent one IVF cycle using the long protocol, retrieving 15 oocytes. On day 1, 10 oocytes extruded polar bodies, and 4 were MI. All 15 oocytes were unfertilized (e.g., ...). Figure 1 (as shown in F and G).

[0063] A novel pathogenic gene PABPC1L splice site variant (c.1121G>A) was identified using whole-exome sequencing technology, which causes oocyte maturation arrest in patients. Immature oocytes awaiting in vitro maturation from these patients were arrested during metaphase I of meiosis and failed to fertilize after IVF and ICSI, resulting in female infertility.

[0064] Table 1. Infertility-related tests in two patients with primary infertility.

[0065]

[0066]

[0067] To clarify the pathogenicity of this mutation site, we extracted peripheral blood DNA from the father (whose mother was deceased) and his fertile sister, and designed specific PCR primers (SEQ ID NO: 3-28). PCR technology was used to amplify the PABPC1L gene-related region in the DNA of family members. Further Sanger sequencing of the amplified products revealed that both patients indeed possessed a homozygous mutation at this site, and that the father and fertile sister were carriers of this mutation, consistent with familial co-segregation characteristics (e.g., ...). Figure 1 (As shown in A and B). Furthermore, we did not find this variant in 160 normally fertile Chinese women. Analysis of the conservation of this mutation site revealed that the amino acid sequence is highly conserved across species (e.g., ...). Figure 1 (As shown in C and D). The above results indicate that PCR amplification combined with Sanger sequencing using the above-mentioned specific primer sequences can be used to detect the pathogenicity of splice site variations in the peripheral blood oocyte maturation arrest pathogenic gene PABPC1L.

[0068] Experimental methods:

[0069] 1.1 Extraction and purification of peripheral blood gDNA

[0070] DNA was extracted from peripheral blood samples of patients and their families using a DNA extraction kit (DNA QIAamp DNA Blood Mini Kit; 51106) from QIAGEN (Germany). The specific extraction steps are as follows:

[0071] 1) Add 200 μL of whole blood and 20 μL of proteinase K to a 1.5 mL EP tube and mix well;

[0072] 2) Add 200 μL of AL, invert and mix well, shake for 30 seconds, centrifuge briefly for 3 seconds, and incubate in a 56°C water bath for 15 minutes;

[0073] 3) Add 200 μL of anhydrous ethanol, invert to mix, shake for 30 s, and centrifuge for 3 s.

[0074] 4) Pour the liquid into a filter column containing centrifuge tubes, centrifuge at 13000×g for 1 min, and discard the filtrate and centrifuge tubes;

[0075] 5) Place the filter column into a new centrifuge tube, add 500 μL AW1, centrifuge at 13000×g for 1 min, and discard the filtrate and centrifuge tube;

[0076] 6) Place the filter column into a new centrifuge tube, add 500 μL AW2, centrifuge at 13000×g for 1 min, and discard the filtrate; centrifuge again for 3 min, and discard the filtrate and centrifuge tube;

[0077] 7) Place the filter column in a new EP tube, add 80 μL of AE, let it stand at room temperature for 5 min, centrifuge at 13000×g for 1 min, and store at 4℃ for short-term storage or -70℃ for long-term storage.

[0078] 1.2 Whole-exome sequencing and data analysis

[0079] 30 μg of peripheral blood DNA was collected from the two patients with primary infertility and sent to BGI Genomics Co., Ltd. in Shenzhen for whole-exome sequencing and analysis (including DNA library construction, whole-exome sequencing, and data analysis). Mutation identification was performed using a genome analysis toolkit. The mutations found by sequencing were annotated using ANNOVAR software, and pathogenic genes were screened based on the annotation results.

[0080] The screening strategy for candidate pathogenic genes is as follows: (1) Filter mutations with a frequency greater than 1% in public genetic mutation databases (1000 Genomes, dbSNP, ExAC database); (2) Select mutations that are predicted to be pathogenic by bioinformatics software (Mutationtaster, SIFT, PloyPhen-2); (3) Give priority to homozygous mutations or compound heterozygous mutations; (4) Select mutations whose gene function is related to the occurrence of oocyte maturation arrest, or are specifically highly expressed in ovarian tissue, or exhibit female infertility phenotype in model animals.

[0081] Based on the above steps, the pathogenic gene PABPC1L associated with female primary infertility was identified; the mutation site associated with female primary infertility was c.1121G>A.

[0082] 1.3 PCR amplification

[0083] Based on the screened pathogenic genes and their pathogenic mutation sites, specific amplification primers were designed, and the primer information is shown in Table 2. The PCR reaction system for the test samples was prepared according to Table 3. The PCR premix solution was... Green PCR MasterMix (REF M7123, Promega, USA); DNA template is peripheral blood gDNA from female patients with primary infertility or their family members.

[0084] Table 2 Primer Information

[0085]

[0086]

[0087] Table 3 PCR reaction system

[0088] Element volume Green Master Mix, 2× 15μL RNase-free Water 13μL DNA template 1μL Primer F (10 μmol / L) 0.5μL Primer R (10 μmol / L) 0.5μL total 30μL

[0089] The prepared PCR reaction system was amplified according to the following procedure: 95℃ pre-denaturation for 90s; 95℃ denaturation for 30s, 60℃ annealing for 30s, 72℃ extension for 60s cycled 34 times; and finally 72℃ extension for 5min. The PCR amplification products were stored at 4℃.

[0090] 1.4 Agarose gel electrophoresis

[0091] (1) Preparation of 2% agarose gel: Weigh 2g agarose (TSJ001, Qingke Biotechnology) into an Erlenmeyer flask, add 100mL of 1×TAE buffer (TSG001, Qingke Biotechnology), microwave on high for 3min to dissolve, cool to about 35℃, add 4μL of nucleic acid dye (TSJ003, Qingke Biotechnology), mix well, pour into the assembled gel mold, and cool to room temperature to form.

[0092] (2) Take 3 μL of PCR amplification product and DL2000 DNA Marker (TSJ011-100, Qingke Biotechnology) and spot them into the gel wells respectively. Electrophoresis at 220V for 9 min and then take pictures.

[0093] 1.5 Sequencing Analysis

[0094] PCR amplification products that passed agarose gel electrophoresis and the corresponding PCR amplification primer pairs were sent to Qingke Biotechnology Co., Ltd. (Beijing, China) for Sanger sequencing analysis. The test results are as follows: Figure 1 As shown, female primary infertility patient 1 (F1II-2) and her sister (F1II-3) both have homozygous missense mutations in PABPC1L (c.1121G>A). Figure 1 In -A, the black circle represents a female patient with primary infertility.

[0095] 1.6 Protein Structure Analysis

[0096] To clarify the pathogenicity of the aforementioned homozygous missense mutations, a three-dimensional structural model of the wild-type PABPC1L protein was downloaded from the AlphaFold Protein Structure database, and the three-dimensional structure of the PABPC1L mutant protein was predicted using PyMOL visualization software. Figure 1 As shown in -E, the c.1121G>A mutation causes the corresponding amino acid to mutate (p.R374Q), and the three amino acids L-378, P-459 and A-1099 that form hydrogen bonds with the amino acid at position 374 are replaced by two amino acids K-371 and L-378, making the protein spatial structure unstable.

[0097] 1.7 Staining Analysis

[0098] Wild-type and mutant PABPC1L, both carrying the exogenous HIS marker, were overexpressed in mouse oocytes (GOs) during growth and fully developed oocytes (FGOs), and the oocytes were further stained using immunofluorescence staining. The results showed that the HIS marker signal was distributed in the cytoplasm of both oocyte stages, accumulating intranuclearly in GOs and around the chromosomes in FGOs. Compared to wild-type PABPC1L, mutant PABPC1L, while distributing the HIS signal throughout the oocyte cytoplasm, showed lower signal accumulation around the nucleus or chromosomes. Figure 2 -A).

[0099] To determine whether PABPC1L plays a role in oocyte meiosis, a point mutant plasmid of PABPC1L at c.1121G>A (p.R374Q) was constructed. After in vitro injection into oocytes at the germinal vesicle (GV) stage, a significant decrease in PABPC1L signal both intranuclearly and perinuclearly was observed in oocytes injected with the mutant plasmid. After 12 hours of inhibition in MEM medium containing 2.5 mM milrinone, oocytes overexpressing the PABPC1L gene were cultured again. The proportions of germinal vesicle breakdown (GVBD) were 88.6% and 75.4% after 200 minutes of culture for oocytes injected with the wild-type and mutant PABPC1L plasmids, respectively. Figure 2 B) That is, the GVBD rate of oocytes injected with the mutant PABPC1L gene was lower than that of wild-type PABPC1L oocytes. Secondly, 6 hours after entering GVBD, in most oocytes overexpressing wild-type PABPC1L, chromosomes were aligned on the equatorial plate; however, the chromosome condensation rate in the mutant group was higher than that in the wild-type group, at 16% and 52.4%, respectively. Figure 2 (C, D). These phenomena indicate that overexpression of the exogenous mutant PABPC1L leads to a corresponding delay in the time when oocytes resume meiosis.

[0100] This application found through research that PABPC1L in female patients with primary infertility has a missense mutation, mainly manifested as the c.1121G>A mutation, which causes abnormal hydrogen bond connections between adjacent amino acids, significantly reduces the expression level of PABPC1L protein, leads to delay in the meiotic process, affects the normal meiotic process, causes oocyte maturation arrest and fertilization failure, and thus infertility.

[0101] The proposed method, which uses the detection of human PABPC1L mutations to assist in the diagnosis or prediction of treatment outcomes for women with primary infertility, has advantages such as low cost and short processing time.

[0102] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0103] The embodiments described above are merely illustrative of several implementation methods of this application, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the protection scope of this application. Therefore, the protection scope of this patent application should be determined by the appended claims.

Claims

1. A kind PABPC1L The application of gene mutation detection reagents in the preparation of diagnostic products for primary infertility caused by oocyte maturation disorders is characterized by, With reference to the cDNA sequence of wild type PABPC1L gene, the cDNA sequence of wild type PABPC1L gene is shown as SEQ ID NO: 1, and the mutant gene is c.1121G>A, the detection reagent comprises a primer pair with nucleotide sequence as shown in SEQ ID NOs. 15-16. PABPC1L The cDNA sequence of wild type 2. Use according to claim 1, characterized in that, The mutant PABPC1L protein has the following mutation: p.R374Q, compared with the wild-type PABPC1L protein, the amino acid sequence of which is shown as SEQ ID NO:

2.

3. Use according to claim 1, characterized in that, The detection reagent is detected at the nucleic acid level.

4. Use according to claim 3, characterized in that, The detection reagent comprises reagents suitable for at least one of the following methods: Restriction fragment length polymorphism method, single-strand conformation polymorphism method, polymerase chain reaction, denaturing gradient gel electrophoresis, nucleic acid sequencing method, nucleic acid typing chip detection, denaturing high performance liquid chromatography, in situ hybridization, biological mass spectrometry and HRM method.

5. The use according to claim 4, wherein the compound is ###0002### The biological mass spectrometry comprises a flying mass spectrometer detection; And / or, the nucleic acid sequencing method comprises a Snapshot method.

6. The use according to any one of claims 1 to 5, characterized in that, The diagnostic product is a kit.

7. Use according to claim 6, characterized in that, The kit further comprises at least one of a negative control, a positive control, a nucleic acid extraction reagent, a PCR amplification reagent, a sequencing reagent, and an electrophoresis detection reagent.

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