Application of C12orf40 gene

Abstract

The present invention relates to an application of C12orf40 gene. In clinical research practice, the inventor screened and identified a pathogenic gene C12orf40 splice site variation (NM_001031748.4, c.1286+1G>A) that causes non-obstructive azoospermia patients through whole exome sequencing technology. No haploid sperm was found in the testicular biopsy tissue of the patient, showing non-obstructive azoospermia. The gene is specifically expressed in testicular tissue and not expressed in other tissues. It can be used as a biomarker for non-obstructive azoospermia, which is helpful to further study the mechanism of non-obstructive azoospermia and explore the treatment of non-obstructive azoospermia.

Description

Technical Field

[0001] The present invention relates to the field of reproductive genetic technology, and in particular to an application of a C12orf40 gene associated with non-obstructive azoospermia. Background Art

[0002] Male infertility is a phenotypic heterogeneous disease caused by multiple factors, including oligospermia, asthenozoospermia, teratozoospermia, azoospermia and other unexplained male infertility. Among them, non-obstruction azoospermia (NOA) is the most serious phenotype of male infertility, mainly caused by spermatogenesis disorders caused by genetic defects including chromosomal abnormalities, Y chromosome AZF microdeletions, and gene mutations. There is currently a lack of clinical treatment methods to restore spermatogenesis in patients, and the main method of reproducing the next generation is through sperm donation. With the widespread application of new-generation sequencing technology represented by whole exome sequencing in the screening of pathogenic genes for genetic diseases, some pathogenic gene mutations that cause NOA have been discovered one after another, which provides important molecular basis for the diagnosis, prevention and fertility treatment of NOA patients.

[0003] However, after genetic testing for a considerable number of NOA patients, it is impossible to determine whether the detected gene variants are related to NOA due to the lack of functional research evidence. These variants are called "variants of unknown clinical significance", which poses a huge challenge to the precise prevention and treatment of NOA patients. Summary of the invention

[0004] Based on this, it is necessary to provide a new biomarker that can be used to detect or treat non-obstructive azoospermia.

[0005] The present invention provides an application of a C12orf40 gene as a biomarker in the preparation of a product for detecting or treating non-obstructive azoospermia.

[0006] The present invention also provides a mutant C12orf40 gene, wherein the nucleotide sequence of the mutant C12orf40 gene has a c.1286+1G>A mutation compared with the nucleotide sequence of the wild-type C12orf40 gene.

[0007] The present invention also provides a mutant C12orf40 protein, wherein the amino acid sequence of the mutant C12orf40 protein has a p.Lys405fs mutation compared with the amino acid sequence of the wild-type C12orf40 protein.

[0008] The present invention also provides a recombinant expression vector, which contains the mutant C12orf40 gene as described above.

[0009] The present invention also provides a host cell, wherein the genome of the host cell is incorporated with the mutant C12orf40 gene as described above.

[0010] The present invention also provides the use of the C12orf40 gene, mutant C12orf40 protein, recombinant expression vector or host cell described above in preparing a non-obstructive azoospermia animal model.

[0011] The present invention also provides use of the above-mentioned detection reagent of the C12orf40 gene or the C12orf40 protein encoded by it in the preparation of a product for detecting non-obstructive azoospermia.

[0012] In one embodiment, the detection reagent includes a reagent suitable for at least one of the following methods: fluorescent dye method, digital PCR, resonance light scattering method, real-time fluorescence quantitative PCR, sequencing or biological mass spectrometry.

[0013] The present invention also provides a detection reagent for non-obstructive azoospermia, comprising a PCR primer pair, wherein the PCR primer pair can amplify the full length or partial fragment of the C12orf40 gene as described above.

[0014] In one embodiment, the upstream primer of the PCR primer pair can complementarily pair with the exon 8 region of the C12orf40 gene, and the downstream primer of the PCR primer pair can complementarily pair with the exon 10 region of the C12orf40 gene.

[0015] In one embodiment, the nucleotide sequences of the upstream primer and the downstream primer are shown as SEQ ID NO: 2 and SEQ ID NO: 3, respectively.

[0016] The present invention also provides a kit for detecting non-obstructive azoospermia, which comprises the detection reagent as described above, and at least one of an RNA extraction reagent, a reverse transcriptase, a PCR reaction buffer, dNTPs and a DNA polymerase.

[0017] In clinical research practice, the inventor screened and identified a splice site variation (NM_001031748.4, c.1286+1G>A) of the pathogenic gene C12orf40 in patients with non-obstructive azoospermia by whole exome sequencing technology. No haploid sperm was found in the testicular biopsy tissue of the patient, showing non-obstructive azoospermia. The gene was specifically expressed in testicular tissue and not expressed in other tissues. In order to clarify the pathogenicity of the splice site variation, we extracted the patient's peripheral blood RNA and designed specific PCR primers (SEQ ID NO: 2 and SEQ ID NO: 3). RT-PCR technology (the first amplification used the patient's peripheral blood cDNA as a template, the second amplification used the first amplification product as a template, and both amplifications used the same pair of primers) was used to detect whether the splice site variation affected RNA splicing. The results showed that the target fragment could be amplified, and compared with the normal control, there was an abnormal splicing transcript, indicating that the splice site variation affected RNA splicing. Sanger sequencing was further performed on the above amplification products of normal controls and patients. The results showed that the homozygous splicing site mutation of C12orf40 (c.1286+1G>A) affected RNA splicing, resulting in the deletion of exon 9, causing the termination codon to be premature, and thus truncating the translated protein. It can be seen that the C12orf40 gene can be used as a biomarker for non-obstructive azoospermia, and the homozygous splicing site mutation of C12orf40 (c.1286+1G>A) can cause non-obstructive azoospermia. The above-mentioned specific primer sequence combined with RT-PCR technology can be used for the pathogenicity detection of splicing site mutations of NOA pathogenic gene C12orf40 that is not expressed or underexpressed in peripheral blood. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The results of homozygous splicing site variation of C12orf40 gene found in a NOA patient by the present invention; wherein A is the confirmation by Sanger sequencing that the patient carries homozygous splicing site (c.1286+1G>A) mutation of C12orf40 gene, B is the expression spectrum of C12orf40 in different tissues, and C is the testicular pathological staining result of a patient (P) carrying homozygous splicing site variation of C12orf40 gene. Compared with the control (NC), no haploid sperm was found in the testicular spermatogenic cells of the patient, showing meiotic arrest;

[0019] Figure 2The splicing pattern results of the homozygous splicing site variation of C12orf40 found in patients with non-obstructive azoospermia detected by the present invention; wherein A is the expression of C12orf40 in peripheral blood RNA of patients and normal controls detected by RT-PCR and the inferred possible splicing pattern, B is the PCR product sequence in patients and normal controls detected by Sanger sequencing, and the splicing pattern of the splicing site is clarified, and C is the bioinformatics analysis of the C12orf40 homozygous splicing site (c.1286+1G>A) affecting RNA splicing, resulting in the deletion of exon 9, causing the termination codon to be premature, thereby causing the translated protein to be truncated. DETAILED DESCRIPTION

[0020] In order to facilitate the understanding of the present invention, the present invention will be described more comprehensively below, and preferred embodiments of the present invention are provided. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0021] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more related listed items.

[0022] The present invention provides the use of C12orf40 gene as a biomarker in the preparation of a product for detecting or treating non-obstructive azoospermia. Optionally, the above product can be a reagent, a kit, a drug or a device.

[0023] The present invention also provides a mutant C12orf40 gene, wherein the nucleotide sequence of the mutant C12orf40 gene has a c.1286+1G>A mutation compared with the nucleotide sequence of the wild-type C12orf40 gene, and the nucleotide sequence of the wild-type C12orf40 gene is shown in NC_000012.12 (NCBI Reference Sequences).

[0024] Among the many unsignificant variants found clinically, about 20% are splice site variants, which may be related to RNA splicing. RNA splicing refers to the process of cutting out the intron part of the gene sequence (DNA) and splicing the exon fragments to form a new RNA sequence. The result of these abnormal splicing is the loss of a segment of amino acids in the protein or, more commonly, premature termination of translation. To determine whether splice site variants affect RNA splicing, it is first necessary to obtain biopsy tissue or peripheral blood RNA from the patient, and then design primers in the upstream and downstream exon regions of the splicing site of the pathogenic gene for RT-PCR and Sanger sequencing, so as to analyze the pathogenicity of the splice site variants.

[0025] In clinical research practice, the inventor screened and identified a splice site variation (NM_001031748.4, c.1286+1G>A) of the pathogenic gene C12orf40 in patients with non-obstructive azoospermia by whole exome sequencing technology. No haploid sperm was found in the testicular biopsy tissue of the patient, showing non-obstructive azoospermia. The gene was specifically expressed in testicular tissue and not expressed in other tissues. In order to clarify the pathogenicity of the splice site variation, we extracted the patient's peripheral blood RNA and designed specific PCR primers (SEQ ID NO: 2 and SEQ ID NO: 3). RT-PCR technology (the first amplification used the patient's peripheral blood cDNA as a template, the second amplification used the first amplification product as a template, and both amplifications used the same pair of primers) was used to detect whether the splice site variation affected RNA splicing. The results showed that the target fragment could be amplified, and compared with the normal control, there was an abnormal splicing transcript, indicating that the splice site variation affected RNA splicing. Sanger sequencing was further performed on the above amplified products of normal controls and patients. The results showed that the homozygous splicing site mutation of C12orf40 (c.1286+1G>A) affected RNA splicing, resulting in the deletion of exon 9, causing the termination codon to be premature, thereby truncating the translated protein (SEQ ID NO: 1). It can be seen that the C12orf40 gene can be used as a biomarker for non-obstructive azoospermia. The homozygous splicing site mutation of C12orf40 (c.1286+1G>A) can cause non-obstructive azoospermia, and the above-mentioned specific primer sequence combined with RT-PCR technology can be used for the pathogenicity detection of splicing site mutations of NOA pathogenic gene C12orf40 that is not expressed or underexpressed in peripheral blood. The discovery of this mutant gene provides a new experimental theoretical basis and new direction for further studying the mechanism of non-obstructive azoospermia and exploring the treatment of non-obstructive azoospermia, and enriches the diagnosis and detection methods of non-obstructive azoospermia.

[0026] The present invention provides a mutant C12orf40 protein. The amino acid sequence of the mutant C12orf40 protein has a p.Lys405fs mutation compared with the amino acid sequence of the wild-type C12orf40 protein. The amino acid sequence is specifically shown in SEQ ID NO: 1.

[0027] The present invention provides a recombinant expression vector, which contains the mutant C12orf40 gene as described above. It is understood that the vector types include, but are not limited to: plasmid; phagemid; cosmid; artificial chromosome, such as yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC) or P1-derived artificial chromosome (PAC); bacteriophage such as λ phage or M13 phage and animal virus, etc. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomas (such as SV40).

[0028] The present invention also provides a host cell, the genome of which is incorporated with the mutant C12orf40 gene as described above. The types of host cells include, but are not limited to, prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells. It is understood that the host cell type is not limited thereto and can be selected as needed.

[0029] The present invention also provides the use of the C12orf40 gene, mutant C12orf40 protein, recombinant expression vector or host cell as described above in preparing an animal model of non-obstructive azoospermia. By preparing an animal model of non-obstructive azoospermia, it is more convenient to study the pathogenic mechanism and explore the treatment of non-obstructive azoospermia. Optionally, the above-mentioned animal includes mammals, such as mice, rats, rabbits or monkeys.

[0030] The present invention also provides the use of the above-mentioned C12orf40 gene or the C12orf40 protein encoded by it in the preparation of a product for detecting non-obstructive azoospermia. It is understood that the detection reagent can be a detection reagent for a mutant gene or mutant protein, or a detection reagent for a wild-type gene or wild-type protein.

[0031] In a specific example, the above-mentioned product is a test kit, a test strip or a test chip, etc. It can be understood that the specific type is not limited thereto, and various detection products are acceptable.

[0032] In a specific example, the detection reagents include reagents suitable for at least one of the following methods: fluorescent dye method, digital PCR, resonance light scattering method, real-time fluorescence quantitative PCR, sequencing or biological mass spectrometry, etc.

[0033] A detection reagent for non-obstructive azoospermia according to an embodiment of the present invention comprises a PCR primer pair, which can amplify the full length or a partial fragment of the C12orf40 gene, thereby detecting whether the C12orf40 gene is mutated by PCR or RT-PCR.

[0034] In a specific example, the upstream primer of the PCR primer pair can be complementary to the exon 8 region of the C12orf40 gene, and the downstream primer of the PCR primer pair can be complementary to the exon 10 region of the C12orf40 gene. In this way, the cDNA product obtained by reverse transcription of the sample can be amplified, and then the length of the amplified product can be used to determine whether the sample has the c.1286+1G>A mutation without sequencing.

[0035] In a specific example, the nucleotide sequences of the upstream primer and the downstream primer are shown as SEQ ID NO: 2 and SEQ ID NO: 3, respectively.

[0036] A detection kit for non-obstructive azoospermia according to an embodiment of the present invention comprises the above detection reagent, and at least one of an RNA extraction reagent, a reverse transcriptase, a PCR reaction buffer, dNTPs and a DNA polymerase.

[0037] A method for detecting non-obstructive azoospermia according to an embodiment of the present invention comprises the following steps:

[0038] S1. Provide samples;

[0039] S2, extracting RNA from the above samples and reversely transcribing them to obtain cDNA products;

[0040] S3. Amplify the C12orf40 gene fragment of the above cDNA product using a PCR primer pair, and analyze the amplified product.

[0041] Optionally, the sample is selected from one or more of plasma, whole blood, lymph, urine, sweat, mucus, sputum and saliva, preferably a peripheral blood sample.

[0042] In a specific example, when amplifying the C12orf40 gene fragment of the cDNA product, the above-mentioned PCR primer pair is used for continuous amplification more than twice. Considering the difficulty of obtaining biopsy tissue in clinical practice, the extraction of peripheral blood RNA for pathogenic analysis of splicing site variation is a commonly used detection method in clinical practice. However, it has been found clinically that NOA pathogenic genes are specifically highly expressed in testicular tissue, not expressed or too low expressed in other tissues, making it difficult to verify the pathogenicity of splicing site variation by RT-PCR of peripheral blood RNA. At present, the Minigene technology of in vitro verification experiment of abnormal mRNA splicing is commonly used clinically to solve the above problems, but the Minigene experiment generally takes 1 to 2 months, resulting in an extension of the patient's diagnosis cycle. In addition, the Minigene experiment also has the characteristic of high cost, which makes it difficult to widely promote it in clinical practice. Therefore, the present invention can amplify DNA or RNA samples with extremely low abundance by using the same PCR primer pair for continuous amplification more than twice, which is more sensitive than ordinary PCR technology and overcomes the limitation of extremely low expression of sample target mRNA.

[0043] We constructed C12orf40 knockout mice and found that C12orf40 knockout male mice showed NOA phenotype. In addition, we found a NOA patient carrying a rare homozygous splice site mutation (c.1286+1G>A) in the clinic. Since we could not obtain the patient's testicular biopsy tissue, we extracted the patient's peripheral blood RNA and used the above PCR primer pair to perform two consecutive RT-PCR and single RT-PCR, respectively, to perform RNA splicing analysis on the newly discovered splice site mutation (c.1286+1G>A). The results showed that the single RT-PCR failed to amplify the target band, while the two consecutive RT-PCRs could amplify the corresponding target band. Therefore, the above detection method has the advantages of strong versatility, low cost and high efficiency.

[0044] The present invention will be further described in detail below mainly in conjunction with specific implementation methods and drawings.

[0045] Example 1

[0046] In clinical research practice, we identified a new pathogenic gene C12orf40 splice site variation (NM_001031748.4, c.1286+1G>A) that causes azoospermia through whole exome sequencing technology. No haploid sperm was found in the testicular biopsy tissue of the patient, showing non-obstructive azoospermia. This gene is specifically expressed in testicular tissue and not expressed in other tissues (such as Figure 1 shown).

[0047] In order to clarify the pathogenicity of the splicing site variation, we extracted the patient's peripheral blood RNA and designed specific PCR primers (SEQ ID NO: 2 and SEQ ID NO: 3). We used two consecutive RT-PCR techniques (i.e., the first amplification used the patient's peripheral blood cDNA as a template, and the second amplification used the first amplification product as a template, and both amplifications used the same pair of primers) to detect whether the splicing site variation affects RNA splicing. The results showed that the target fragment could be amplified, and compared with the normal control, there were abnormal splicing transcripts, indicating that the splicing site variation affected RNA splicing (such as Figure 2 As shown). Sanger sequencing of the above amplified products of normal controls and patients showed that the homozygous splicing site variation of C12orf40 (c.1286+1G>A) affected RNA splicing, resulting in the deletion of exon 9, causing the premature termination codon, and thus truncating the translated protein. The above results show that the above specific primer sequence combined with RT-PCR technology can be used for the pathogenicity detection of splicing site variation of NOA pathogenic gene C12orf40 that is not expressed or underexpressed in peripheral blood.

[0048] Experimental methods:

[0049] 1.1 Extraction and purification of gDNA from peripheral blood

[0050] A DNA extraction kit (DNA Qiamp DNA Blood Mini Kit; 51106) from QIAGEN, Germany, was used to extract DNA from peripheral blood. The steps are as follows:

[0051] (1) Add 200 μL of whole blood and 20 μL of proteinase K to a 1.5 mL EP tube and mix well;

[0052] (2) Add 200 μl AL, mix by inversion, shake for 30 seconds, centrifuge for 3 seconds, and place in a 56°C water bath for 15 minutes;

[0053] (3) Add 200 μL of anhydrous ethanol, mix by inversion, shake for 30 seconds, and centrifuge for 3 seconds;

[0054] (4) Pour the liquid into a column containing a centrifuge tube, centrifuge at 13,000 × g for 1 min, and discard the filtrate and centrifuge tube;

[0055] (5) Place the column in a new centrifuge tube, add 500 μL AW1, centrifuge at 13,000 × g for 1 min, and discard the filtrate and centrifuge tube;

[0056] (6) Place the column in a new centrifuge tube, add 500 μL AW2, centrifuge at 13,000 × g for 1 min, and discard the filtrate;

[0057] (7) Discard the filtrate, centrifuge for 3 min, and discard the filtrate and centrifuge tube;

[0058] (8) Discard the filtrate and centrifuge tube, place the column in a new EP tube, add 80 μL of AE, stand at room temperature for 5 min, centrifuge at 13,000 × g for 1 min, and store at 4°C or -70°C for long-term storage.

[0059] 1.2 Peripheral blood RNA extraction and purification

[0060] The RNA extraction kit (RNA Qiamp RNABlood Mini Kit, 52304) of QIAGEN, Germany, was used to extract RNA from peripheral blood. The steps are as follows:

[0061] (1) Take 1.5 mL of fresh whole blood and place it in a 15 mL centrifuge tube, add 7.5 mL of EL, and incubate on ice for 10 min;

[0062] (2) Centrifuge at 400 × g for 10 min at 4°C, remove the supernatant, add 3 mL of EL to the cell pellet, and mix well;

[0063] (3) Centrifuge at 400 × g for 10 min at 4°C, remove the supernatant, add 600 μL RLT + 6 μL β-ME mixture, and mix well;

[0064] (4) Pour the mixture into a QIAshredder spin column and spin at 13,000 × g for 2 min. Discard the column and add 600 μL of 70% anhydrous ethanol to the filtrate and mix well (do not centrifuge).

[0065] (5) Pour the filtrate into a new QIAmp spin column, 8000 × g, 15 s;

[0066] (6) Transfer the column to a new centrifuge tube, add 350 μL RW1, and centrifuge at 8000 × g for 15 s.

[0067] (7) Add 10 μL DNA digestion enzyme and 70 μL RDD, digest at room temperature for 25 min, 8000 × g, 15 s;

[0068] (8) Add 350 μL RW1, 8000 × g, 15 s;

[0069] (9) Add 500 μL RPE, centrifuge at 8000 × g for 15 s, transfer the column to a new centrifuge tube, and centrifuge at 13000 × g for 1 min;

[0070] (10) Add 40 μL of RNase-free water and centrifuge at 8000 × g for 1 min.

[0071] 1.3 Whole exome sequencing and data analysis

[0072] 30 μg of gDNA from the patient's peripheral blood was sent to BGI for whole exome sequencing. WES sequencing and analysis were performed by Shenzhen BGI Genomics Co., Ltd., including DNA library construction, WES machine and data analysis, and the genome analysis toolkit was used for variant identification. The variants found by sequencing (including single nucleotide variants, small insertions or deletions, etc.) were annotated using ANNOVAR software, and then the annotation results of whole exome sequencing were personalized for candidate pathogenic genes screening.

[0073] 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 predicted by bioinformatics software (Mutationtaster, SIFT, PloyPhen-2) as potentially pathogenic; (3) Give priority to homozygous mutations or compound heterozygous mutations; (4) Select mutations whose gene function is related to spermatogenesis, or is specifically highly expressed in testicular tissue, or whose model animals show a male sterility phenotype.

[0074] 1.4 PCR and Sanger sequencing to verify the splicing site mutation

[0075] The candidate pathogenic gene C12orf40 screened by full-length sequencing was used to design PCR amplification primers based on the mutation site. The primer sequences used were: F: 5′-ACCAGTGGTTTTCCTCTGTACG-3′, R: CACCCTCAAATGGCAATACGC-3′, and the PCR amplification system (30 μL) was as follows:

[0076]

[0077]

[0078] The PCR amplification reaction conditions were as follows: 95°C pre-denaturation for 5 min, 94°C denaturation for 45 s, 56-60°C annealing for 30-60 s, 72°C extension for 45 s, a total of 34 cycles, and finally 72°C extension for 5 min. The PCR amplification products were stored at 4°C. The PCR amplification products were subjected to agarose gel electrophoresis. After confirming that the amplified target band was correct, the remaining PCR amplification products were sent to Qingke Biotechnology Co., Ltd. (Beijing, China) for Sanger sequencing verification.

[0079] 1.5 RT-PCR and Sanger sequencing to verify splicing site mutations and RNA splicing changes

[0080] The RT-PCR amplification system (30 μL) is as follows (SEQ ID NO: 2 and SEQ ID NO: 3 are used as PCR primers for both rounds of PCR amplification):

[0081] Green Master Mix, 2× 15μL RNase-free water 13μL Patient peripheral blood cDNA 1μL (about 50ng) Upper and lower primers 0.5μL each (10μmol / L) total 30μL

[0082] The PCR amplification reaction conditions were as follows: pre-denaturation at 95°C for 5 min, denaturation at 94°C for 45 s, annealing at 56-60°C for 30-60 s, extension at 72°C for 45 s, for a total of 34 cycles, and finally extension at 72°C for 5 min. The PCR amplification product was stored at 4°C.

[0083] The second round of PCR amplification system (30 μL) is as follows:

[0084] Green Master Mix, 2× 15μL RNase-free water 13μL The first round of PCR amplification products 1μL (about 50ng) Upper and lower primers 0.5μL each (10μmol / L) total 30μL

[0085] The PCR amplification reaction conditions were as follows: 95°C pre-denaturation for 5 min, 94°C denaturation for 45 s, 56-60°C annealing for 30-60 s, 72°C extension for 45 s, a total of 34 cycles, and finally 72°C extension for 5 min. The PCR amplification products were stored at 4°C. The PCR amplification products were subjected to agarose gel electrophoresis. After confirming that the amplified target band was correct, the remaining PCR amplification products were sent to Qingke Biotechnology Co., Ltd. (Beijing, China) for Sanger sequencing verification.

[0086] 1.6 Agarose gel electrophoresis steps are as follows:

[0087] (1) Preparation of 2% agarose (TSJ001, Qingke Biotechnology) gel: weigh 2 g agarose into a 500 mL conical flask, add 100 mL 1×TAE buffer (TSG001, Qingke Biotechnology), heat in a microwave oven for 3 min, cool to about 35°C, add 4 μL of nucleic acid dye (TSJ003, Qingke Biotechnology), mix well, pour into a trough with a comb installed in advance, and cool at room temperature;

[0088] (2) Take 3 μL of PCR amplification product and DL2000 DNA Marker (TSJ011-100, Qingke Biotechnology) for electrophoresis at 220 V for 9 min and take images.

[0089] Experimental results:

[0090] 1. Homozygous splice site mutations in the C12orf40 gene were identified in NOA patients

[0091] Whole exome sequencing was used to identify a homozygous splice site mutation in the C12orf40 gene in a NOA patient (P) (NM_001031748.4, c.1286+1G>A). Sanger sequencing was used to confirm that the patient did have this homozygous splice site mutation (e.g. Figure 1 As the expression and function of this gene in tissues are unclear, we subsequently detected the expression profile of C12orf40 in different tissues. The results showed that C12orf40 was highly expressed in testis tissue and was not expressed or was too low expressed in other tissues (e.g. Figure 1 B). Further testicular biopsy tissue was obtained from the patient for pathological staining, and the results showed that compared with the normal control (NC), no haploid round spermatids were found in the patient's testicular seminiferous tubules, indicating meiotic arrest (as shown in Figure 1C).

[0092] 2. Homozygous splicing site mutations in the C12orf40 gene affect RNA splicing

[0093] To clarify the pathogenicity of the splicing site variation, we extracted the patient's peripheral blood RNA and designed specific PCR primers (F: 5′-CCAGAAAAATGTCAGCCAAACAAG-3′, R: 5′-TCCAAAGTCCCATGACTGCTTA-3′). We used RT-PCR to detect whether the splicing site variation affected RNA splicing. The results showed that the target fragment could be amplified, and compared with the normal control, there were abnormal splicing transcripts, indicating that the splicing site variation affected RNA splicing (such as Figure 2 A). Sanger sequencing of the above amplified products of normal controls and patients showed that the homozygous splicing site of C12orf40 (c.1286+1G>A) affected RNA splicing, resulting in the deletion of exon 9 and the premature termination codon, thus truncating the translated protein (as shown in Figure 1). Figure 2 B~2C). The above results show that the C12orf40 gene can be used as a biomarker for non-obstructive azoospermia, and the homozygous splice site mutation of C12orf40 (c.1286+1G>A) will lead to non-obstructive azoospermia. The above specific primer sequence combined with RT-PCR technology can be used for the pathogenicity detection of splice site variation of NOA pathogenic gene C12orf40 that is not expressed or underexpressed in peripheral blood.

[0094] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described 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.

[0095] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the invention patent. It should be pointed out that for ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be based on the attached claims. Sequence Listing <110> CITIC Xiangya Reproductive and Genetic Specialist Hospital Co., Ltd. central south university <120> Application of C12orf40 gene <160> 3 <170> SIPOSequenceListing 1.0 <210> 1 <211> 427 <212> PRT <213> Artificial Sequence <400> 1 Met Asn Trp Val Gly Gly Ser Arg Ser Arg Val Leu Ile Lys Gln Glu 1 5 10 15 Arg Arg Lys Gln Lys Glu Tyr Phe Glu Lys His Arg Leu Lys Ser Lys 20 25 30 Met Lys Ser Leu Gly Val Leu Ser Pro Val Lys Asn Ser Ala Val Ser 35 40 45 Leu Asp Ile Leu Asn Leu Tyr Met Val Asn Gln Ile Ser Cys Lys Lys 50 55 60 Lys Ile Pro Glu Thr Val Arg Lys Pro Thr His Val Asn Met Asn Arg 65 70 75 80 Asp Ile Lys Met Pro Leu Arg Lys His Asn Leu Glu Leu Thr Met Ser 85 90 95 Pro His Cys Val Pro Ser Lys Leu Cys Leu Asp Asp Thr Glu Thr Asn 100 105 110 Val Asn Cys Gln Arg Leu Ser Ser Lys Glu Asp Leu Gly Pro Val Gln 115 120 125 Ser Gln Gly Met Asp Ser Tyr Ser Met Leu His Pro Gln Phe Ser Lys 130 135 140 Ile Glu Asn Cys Ser Phe Thr Pro Ser Ser Phe Ser Val Glu Leu Pro 145 150 155 160 Ser Asn Arg His Ile Ser Lys Leu Asn Phe Thr Ser Gly Ile Ala Pro 165 170 175 Thr Pro Gln Lys Leu Ala Tyr Glu Lys Lys Gln Asn Asp Gln Arg Ser 180 185 190 Thr Val Asn Cys Ser Asp Ser Leu Leu Ser Lys Leu Asn Lys Ser Gln 195 200 205 Asp Val Phe Ser Pro Ser His Lys Thr Thr Arg Phe Gly Thr Leu Phe 210 215 220 Glu Arg Leu Asn Ser Leu Gly Asn Arg Asn Leu Leu Thr Lys Ser Pro 225 230 235 240 Ala Val Ile Met Asp Glu Asp Cys Arg Ser Thr Asp Glu Ile Arg Gln 245 250 255 Ser Asp Tyr Ile Thr Glu Lys His Ser Ile Gln His Ile Trp Gly Lys 260 265 270 Asn Gly Lys Glu Val Ser Asn Phe Leu Glu Asp Val Asn Gln Ser Thr 275 280 285 Pro Asn Leu Leu Ser Glu Asn Cys Asp Ser Phe Val Ser Gln Asn Met 290 295 300 Ile Asn Val Leu Asn Ile Asp Glu Gln Arg Ile Lys Lys Thr Phe Asn 305 310 315 320 Lys Cys Asp Tyr Asp Ser Met Gly Asp Thr Cys Val Val Thr Ser Ser 325 330 335 Asp Lys Asn His Val Thr Asp Arg Cys Ile Arg Asn Ile Phe Thr Val 340 345 350 Pro Glu Leu Thr Phe Ser Asn Ser Thr Leu Asn Lys Thr Ser Tyr Pro 355 360 365 Glu Lys Cys Gln Pro Asn Lys Lys Tyr Gln Arg Glu Tyr Asn Lys Asn 370 375 380 Glu Arg Asn Asp Leu Ser Thr Ser Phe Glu Asn Asp Tyr Tyr Pro Ser 385 390 395 400 Ser Ser Glu Arg Lys Gly Glu Ile Tyr Leu Arg Lys Asn Cys Thr Leu 405 410 415 Ser Ser His Gly Thr Leu Asp Leu Met Arg Phe 420 425 <210> 2 <211> 24 <212> DNA <213> Artificial Sequence <400> 2 ccagaaaaat gtcagccaaa caag 24 <210> 3 <211> 22 <212> DNA <213> Artificial Sequence <400> 3 tccaaagtcc catgactgct ta 22

Claims

1. Use of a detection reagent for C12orf40 gene splicing site variation in the preparation of a product for detecting non-obstructive azoospermia, characterized in that: The splicing site variation is NM_001031748.4, c.1286+1G>A.

2. Use of the detection reagent for C12orf40 gene splicing site variation according to claim 1 in the preparation of a product for detecting non-obstructive azoospermia, characterized in that: The detection reagents include a PCR primer pair.

3. Use of the detection reagent for C12orf40 gene splicing site variation according to claim 2 in the preparation of a product for detecting non-obstructive azoospermia, characterized in that: The nucleotide sequences of the upstream primer of the PCR primer pair and the downstream primer of the PCR primer pair are shown as SEQ ID NO: 2 and SEQ ID NO: 3, respectively.

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