A molecular marker, kit and application for diagnosing sperm head malformation

Abstract

The present invention relates to a molecular marker, a kit and an application for diagnosing sperm head malformation, belonging to the technical field of gene diagnosis. By collecting genomic DNA samples from patients with abnormal sperm and healthy controls, whole-exome sequencing was performed, and a gene mutation site significantly associated with sperm head malformation was screened out, that is, the first base of the intron downstream of exon 5 of the polypeptide N-acetylgalactosaminyltransferase-like protein 5 encoding gene GALNTL5. Specific amplification primers were designed for this gene mutation site, which can be used to prepare a diagnostic kit for sperm head malformation, and applied to screen patients with sperm head malformation, providing more accurate diagnosis and treatment guidance for infertility caused by teratospermia, and also providing a new theoretical basis for the treatment of patients with sperm head malformation.

Description

Technical Field

[0001] The present invention relates to the technical field of gene diagnosis, and in particular to a molecular marker, a kit and an application for diagnosing sperm head deformities. Background Art

[0002] Infertility is a global public health problem of human reproductive health, affecting multiple aspects such as physiological, psychological and social factors.

[0003] Teratozoospermia can cause a decrease in sperm motility and forward motility, or be accompanied by varying degrees of asthenozoospermia, which is one of the common causes of male infertility. Teratozoospermia has various manifestations, including abnormal development of any one or more parts of the head (giant head, amorphous, double head, etc.), body (thick, cracked, incomplete, etc.), and tail (curled tail, double tail, absent tail, etc.), which poses an obstacle to accurate diagnosis of different parts.

[0004] Currently, patients with teratozoospermia usually receive assisted reproductive technology - intracytoplasmic sperm injection (ICSI) treatment to obtain biological offspring, but the cause remains unknown in some cases. At the same time, genetic defects are one of the important factors leading to teratozoospermia, which significantly increases the risk of passing the pathogenic gene to the offspring through assisted reproductive technology. Therefore, carrying out systematic genetic screening before ICSI has important clinical value. If it is diagnosed as an irreparable genetic factor, it can avoid patients from receiving ineffective or harmful treatment, and guide them to consider assisted reproductive technology and undergo genetic examinations. Through accurate genetic diagnosis, patients can be prevented from receiving ineffective drug treatment (such as antioxidants being ineffective for round-headed spermatozoa), and at the same time, the risk of birth defects in offspring can be reduced.

[0005] Therefore, accurate diagnosis of teratozoospermia is the core link connecting etiological analysis, treatment plan formulation and optimization of fertility outcomes. By integrating morphology, genetics and clinical evaluation, precision medicine can be maximally achieved, the risk of offspring can be reduced, the fertility success rate can be improved, and waste of medical resources can be reduced. Although many genetic screening methods have emerged in the prior art, teratozoospermia often involves abnormal development of multiple parts, and there is often a lack of gene screening sites for a single part, especially for sperm head deformities, in the prior art. Moreover, the existing genetic screening methods usually require collecting peripheral blood samples of the subjects to be tested for sequencing, which not only increases the cost of screening, but also causes damage and pain to the subjects to be tested. Summary of the Invention

[0006] To solve the above technical problems, according to the diagnostic criteria for abnormal spermatozoa in the "Laboratory Manual for the Examination and Processing of Human Semen (Sixth Edition)" issued by the World Health Organization, genomic DNA samples of 421 patients with abnormal spermatozoa were collected. At the same time, genomic DNA samples of 2,664 healthy men with offspring were collected as controls. Through whole-exome sequencing, Sanger sequencing, and sperm sample staining, a mutation site significantly associated with sperm head abnormalities was screened out. This site is located in the gene GALNTL5 encoding polypeptide N-acetylgalactosaminyltransferase-like protein 5 on human chromosome 7, at the first base of the intron downstream of exon 5, where guanine mutates to adenine.

[0007] The first object of the present invention is to provide a molecular marker for diagnosing sperm head abnormalities. The molecular marker includes a mutant of polypeptide N-acetylgalactosaminyltransferase-like protein 5, and the mutation occurs at the first base of the intron downstream of exon 5 of the gene GALNTL5 encoding polypeptide N-acetylgalactosaminyltransferase-like protein 5, and the mutation is from guanine to adenine.

[0008] The GALNTL5 gene is located on human chromosome 7, with a gene length of 63,484 base pairs, containing 9 exons, and is highly specifically expressed in the testis. The above mutation site is at the first base of the intron downstream of exon 5 of the GALNTL5 gene, where guanine mutates to adenine, corresponding to position 658 of the coding sequence (cDNA sequence), and is named GALNTL5-c.658+1G>A according to the Human Genome Variation Society (HGVS) nomenclature. This alternative splicing mutation results in the occurrence of abnormal exon 5 skipping events.

[0009] The second object of the present invention is to provide the application of the gene GALNTL5 encoding polypeptide N-acetylgalactosaminyltransferase-like protein 5 in the preparation of a diagnostic kit for sperm head abnormalities. The diagnostic kit for sperm head abnormalities detects the mutation of the first base in the intron downstream of exon 5 of the gene GALNTL5 encoding polypeptide N-acetylgalactosaminyltransferase-like protein 5, and the mutation is from guanine to adenine.

[0010] Furthermore, the detection method of the diagnostic kit for sperm head abnormalities includes extracting genomic DNA of the test sample, amplifying the DNA, sequencing the amplification product, and judging whether the base has mutated according to the sequencing result. When the base mutates from guanine to adenine, it is judged that the test sample is from a patient with sperm head abnormalities.

[0011] Furthermore, the test sample is semen.

[0012] Furthermore, the sequencing uses the Sanger sequencing method.

[0013] In one embodiment of the present invention, genomic DNA of a subject to be tested is extracted from semen and subsequent amplification and sequencing are performed. The steps of DNA extraction are as follows:

[0014] (1) Aspirate the sperm sample to be tested and transfer it into a centrifuge tube containing the first buffer, and vortex.

[0015] (2) Centrifuge to remove most of the supernatant, leaving part of the first buffer and the precipitate.

[0016] (3) After vortexing, add the first buffer again and vortex at full speed to collect the sample adhering to the tube wall.

[0017] (4) Centrifuge to remove the supernatant, taking care not to aspirate the sperm precipitate during this process.

[0018] (5) Add the second buffer and resuspend.

[0019] (6) Add proteinase K, incubate, then add proteinase K and dithiothreitol again and continue incubating.

[0020] (7) Extract DNA according to the steps of the DNA extraction kit.

[0021] Among them, the addition of dithiothreitol cuts the disulfide bond, opens the protein structure, facilitates subsequent DNA extraction, and increases the amount of genomic DNA extracted.

[0022] The third object of the present invention is to provide a sperm head malformation diagnosis kit, which detects a mutation in the first base in the intron downstream of exon 5 of the gene GALNTL5 encoding polypeptide N-acetylgalactosaminyltransferase-like protein 5, and the mutation is a change from guanine to adenine.

[0023] Furthermore, the kit contains primers for gene amplification.

[0024] Furthermore, the primers include a forward primer and a reverse primer. The forward primer is as shown in SEQ ID NO.1, and the reverse primer is as shown in SEQ ID NO.2.

[0025] Specifically, the sequence of the forward primer is 5’-AGAACTTCTGTGGAATAGCATGGA-3’ (SEQ ID NO.1).

[0026] Specifically, the sequence of the reverse primer is 5’-GTGTCGGGTCAGAAGCATCA-3’ (SEQ ID NO.2).

[0027] Specifically, the expected product size is 716 base pairs.

[0028] Furthermore, the kit includes reagents for gene amplification and gene sequencing.

[0029] Furthermore, the kit includes reagents for extracting DNA from a sample to be tested.

[0030] In one embodiment of the present invention, Sanger sequencing is used to sequence the amplification product.

[0031] The fourth object of the present invention is to provide the use of a primer composition in the preparation of a sperm head malformation diagnostic kit, wherein the primer composition is used to detect the first base mutation in the intron downstream of exon 5 of the polypeptide N-acetylgalactosaminyltransferase-like protein 5 encoding gene GALNTL5, and the mutation is a change from guanine to adenine.

[0032] Furthermore, the primer composition includes a forward primer and a reverse primer, the forward primer is as shown in SEQ ID NO.1, and the reverse primer is as shown in SEQ ID NO.2.

[0033] The fifth object of the present invention is to provide a kit for detecting the deletion of exon 5 of the polypeptide N-acetylgalactosaminyltransferase-like protein 5 encoding gene GALNTL5, wherein the kit detects the first base mutation in the intron downstream of exon 5 of GALNTL5, and the mutation is a change from guanine to adenine.

[0034] Advantages of the present invention:

[0035] The present invention first reveals the direct association between the mutation of the first base from adenine to guanine in the intron downstream of exon 5 of the GALNTL5 gene and sperm head malformation, providing a key molecular marker for the etiological diagnosis of male infertility. By designing specific primers and combining sequencing techniques, the mutation site can be quickly and highly sensitively identified, and the detection process is standardized and the results are reliable. The kit integrates DNA extraction, amplification and sequencing modules, simplifies the operation steps, is suitable for batch detection of clinical samples, improves the diagnostic efficiency, and provides a scientific basis for gene screening, genetic counseling and personalized treatment of infertile patients. The method provided by the present invention extracts and sequences from semen DNA. Compared with peripheral blood commonly used in the prior art, semen DNA can reflect the status and functional information of the male reproductive system, is more beneficial for accurately identifying the GALNTL5 genotype, and also avoids causing damage and pain to the person to be tested. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] In order to make the content of the present invention easier to be clearly understood, the following further details the present invention according to specific embodiments of the present invention in conjunction with the drawings, wherein:

[0037] Figure 1The figure shows the results of Sanger sequencing of peripheral blood DNA samples from 10 sperm head malformation cases in Example 1 of the present invention;

[0038] Figure 2 The figure shows partial results of periodic acid Schiff (PAS) staining of semen samples from sperm head malformation cases in Example 1 of the present invention;

[0039] Figure 3 The figure shows the results of Sanger sequencing of semen DNA samples from 8 sperm head malformation cases and 2 fertile controls in Example 2 of the present invention;

[0040] Figure 4 The figure shows the process of in vitro verification experiment of abnormal mRNA splicing for sperm head malformation cases and control groups in Example 3 of the present invention;

[0041] Figure 5 The figure shows the results of in vitro verification experiment of abnormal mRNA splicing for sperm head malformation cases and control groups in Example 3 of the present invention;

[0042] Figure 6 The figure shows the results of digital PCR for quantifying abnormal transcripts in sperm head malformation cases and control groups in Example 3 of the present invention. Detailed implementation manners

[0043] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, so that those skilled in the art can better understand the present invention and be able to implement it, but the specific embodiments cited are not intended to limit the present invention.

[0044] Example 1: Screening of mutant genes for sperm head malformation

[0045] 421 cases of teratozoospermia were collected, and at the same time, 2664 fertile men with at least one healthy offspring were collected as the control group. 5 mL of peripheral blood samples were collected from 421 cases and 2664 control groups respectively, and the DNA in the samples was extracted for whole exome sequencing.

[0046] Quality control, alignment, annotation and other processes were performed on the obtained whole-exome sequencing results to obtain the variant information carried by the case-control. Then, according to the evaluation criteria of the American College of Medical Genetics and Genomics (ACMG), pathogenic / likely pathogenic loss-of-function variants were included for association analysis. The analysis results are shown in Table 1, where OR is the risk score (odds ratio, OR), CI is the confidence interval, and Inf represents infinity. From the results in Table 1, it can be seen that the mutation site GALNTL5-c.658+1G>A is the most significant variant, carried by 10 cases of teratospermia and 0 fertile control samples.

[0047] The GALNTL5 gene is located on human chromosome 7 and encodes the polypeptide N-acetylgalactosaminyltransferase 5, which is highly specifically expressed in the testis. The mutation site is located in exon 5 of the GALNTL5 gene, where the first base of the downstream intron is mutated from guanine to adenine, corresponding to position 658 of the coding sequence (cDNA sequence), and is named GALNTL5-c.658+1G>A according to the Human Genome Variation Society (HGVS) nomenclature.

[0048] Table 1 Results of mutation association analysis

[0049]

[0050] Peripheral blood samples of the 10 involved cases were collected, and genomic DNA was extracted from the samples. Amplification primers were designed for the mutant GALNTL5-c.658+1G>A with reference to the human genome sequence database hg19 (Genome Reference Consortium human genome build 37, GRCh37). The forward primer sequence is 5’-AGAACTTCTGTGGAATAGCATGGA-3’ (shown in SEQ ID NO.1), and the reverse primer sequence is 5’-GTGTCGGGTCAGAAGCATCA-3’ (shown in SEQ ID NO.2), which were synthesized by Sangon Biotech (Shanghai) Co., Ltd. The expected product size is 716 base pairs.

[0051] The extracted peripheral blood DNA was loaded into the reaction system as shown in Table 2, gently mixed, centrifuged briefly, and then placed in a PCR instrument to perform the reaction according to the amplification reaction program shown in Table 3.

[0052] Table 2 PCR reaction system (25 μL)

[0053]

[0054] Table 3 PCR Amplification Reaction Program

[0055]

[0056] After obtaining the amplification primers, Sanger sequencing was performed, and the sequencing results are as follows Figure 1 shown. Mutations were found in the GALNTL5-c.658+1G locus of the genomic DNA of 10 cases of teratospermia, proving the authenticity of this variant site.

[0057] Semen samples from 10 cases were collected for PAS staining (Periodic Acid Schiff reaction). The staining results of semen samples from some cases are as follows Figure 2 shown, indicating that the case samples were macrocephalic sperm with acrosome abnormalities, which further suggested that this variant might have potential application value in the classification of teratospermia.

[0058] Example 2: Verification of the Detection Effect of Actual Samples

[0059] To further verify the authenticity of this variant, 360 cases of teratospermia and 1400 fertile control samples were further included using the same criteria as in Example 1, and Sanger sequencing of the GALNTL5-c.658+1G>A variant site was performed on them. Considering that the process of collecting peripheral blood from the subjects to be tested will cause damage to the subjects' bodies, and semen DNA can reflect the status and functional information of the male reproductive system, an attempt was made to collect semen samples from the subjects to be tested and extract genomic DNA from them for sequencing. The specific process includes the following steps

[0060] I. Somatic Cell Lysis

[0061] (1) Pipette 500 μL of the semen sample to be tested into a centrifuge tube containing 9.5 mL of the first buffer (composition shown in Table 4), and vortex at full speed for 10 seconds;

[0062] (2) Centrifuge at 4000 r / min for 10 minutes;

[0063] (3) Carefully remove most of the supernatant, leaving about 1 mL of the first buffer and the precipitate;

[0064] (4) Vortex at full speed for 10 seconds, and then transfer to a 2 mL centrifuge tube;

[0065] (5) Add 0.5 mL of the first buffer to a 15 mL centrifuge tube, and vortex at full speed for 10 seconds to collect the sample adhering to the tube wall and then incorporate it into the 2 mL EP tube;

[0066] (6) Centrifuge the centrifuge at full speed (20,000 x g) for 2 minutes;

[0067] (7) Carefully remove the supernatant, taking care not to aspirate the sperm pellet during this process.

[0068] Table 4 Composition of the First Buffer

[0069]

[0070] II. DNA Extraction (using QIAamp DNA Micro Kit(50) kit for extraction)

[0071] (1) Add 300 µL of the second buffer (composition shown in Table 5) in a fume hood and resuspend.

[0072] (2) Add 100 µL of proteinase K and incubate at 55°C with a rotation speed of 250 r / min for 2 hours.

[0073] (3) Add 20 µL of proteinase K and 34 µL of 2M dithiothreitol (DTT), and incubate at 55°C and 250 r / min for another 2 hours.

[0074] (4) Add 400 µL of lysis buffer (Buffer AL) and 400 µL of absolute ethanol to the sample, and vortex briefly and centrifuge briefly.

[0075] (5) Transfer all of the mixture to a silica membrane column (QiAamp Mini) (placed in a 2 mL collection tube) using a pipette, centrifuge at 6000 x g for 1 minute, and discard the filtrate.

[0076] (6) Add 500 µL of deproteinization solution (Buffer AW1). Centrifuge at 6000 x g for 1 minute and discard the filtrate.

[0077] (7) Add 500 µL of desalting ionic solution (Buffer AW2). Centrifuge at full speed (20,000 x g) for 3 minutes and discard the filtrate.

[0078] (8) Centrifuge at full speed (20,000 x g) for 1 minute with no sample to completely remove Buffer AW2.

[0079] (9) Place the QIAamp Mini column in a new 1.5 mL EP tube, add 200 µL of sterile and enzyme-free water, incubate at room temperature for 1 minute. Centrifuge at 6000 x g for 1 minute to elute the DNA.

[0080] (10) The elution step can be repeated once to increase the DNA yield.

[0081] Table 5 Composition of the Second Buffer

[0082]

[0083] III. Measuring DNA Concentration and Quality

[0084] Use a Thermo Scientific NanoDrop spectrophotometer to measure the DNA concentration and quality, and make the absorbance ratio OD 260 / OD 280 between 1.8 - 2.0, with a concentration greater than 30 ng / μL and a volume of 200 μL for further detection.

[0085] IV. Polymerase Chain Reaction (PCR)

[0086] Refer to the steps and procedures described in Example 1.

[0087] V. Sanger Sequencing and Result Analysis

[0088] Entrust Sangon Biotech (Shanghai) Co., Ltd. to perform Sanger sequencing on the PCR products to obtain base peak maps. Use biological software such as SnapGene to read and analyze the maps. The detection results are shown in Table 6. Among 1760 samples to be tested, a total of 8 samples carried the GALNTL5-c.658+1G>A mutation. All 8 samples were from the teratozoospermia group. The sequencing results of the semen samples are as Figure 3 shown. PAS staining was performed on these 8 samples, and the results were all macrocephalic sperm with abnormal acrosomes. The remaining 352 teratozoospermia samples did not carry the GALNTL5-c.658+1G>A mutation after Sanger sequencing. PAS staining was performed on these 352 semen samples, and the results were not macrocephalic sperm with abnormal acrosomes. No mutation carriers were found in the 1400 fertile control group. The true negative number was 1400, and the false positive number was 0. The detection specificity was 100%, verifying the great potential of this mutation site for diagnosing teratozoospermia. At the same time, it also showed that DNA extracted from semen samples could also be used to detect the GALNTL5-c.658+1G>A mutation, thereby further judging whether the tested person has sperm head deformity.

[0089] Table 6 Detection Results of 1760 Samples to be Tested

[0090]

[0091] Example 3: In Vitro Verification of Mutant Genes for Sperm Head Deformity

[0092] The in vitro verification experiment of abnormal mRNA splicing was used to verify the cases of sperm head deformity and the control group respectively. The in vitro verification of abnormal mRNA splicing was carried out by cloning the target genomic fragment with the mutation site GALNTL5-c.658+1G>A, constructing a recombinant expression vector, transfecting cell lines, extracting RNA and reverse transcribing cDNA, and then using electrophoresis and sequencing techniques to verify the effect of this mutation on mRNA splicing. The verification process is as Figure 4 shown, and the verification results are as Figure 5 shown, which proves that the GALNTL5-c.658+1G>A mutation can lead to the occurrence of abnormal exon 5 skipping events.

[0093] After that, semen samples of patients with sperm head deformity and control group subjects to be tested were collected respectively. Somatic cell lysis was carried out according to the method provided in Example 2, RNA was extracted therefrom, and digital PCR (dPCR) technology was used to analyze the transcripts (mature mRNAs that can be encoded into proteins through transcription) of patients with sperm head deformity and the control group. The results are as Figure 6 shown. The number of abnormal transcripts with exon 5 skipping in the transcripts of patients with sperm head deformity is significantly more than that of the control group subjects to be tested.

[0094] Obviously, the above examples are only for illustration purposes and are not intended to limit the implementation methods. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all implementation methods here. And the obvious changes or modifications derived therefrom are still within the protection scope of the present invention.

Claims

1. Use of a primer composition in the preparation of a sperm head malformation diagnostic kit, characterized in that: The primer composition is used to amplify a target sequence including the first base in the intron downstream of exon 5 of the polypeptide N-acetylgalactosaminyltransferase-like protein 5 encoding gene GALNTL5, where the first base is mutated from guanine to adenine, corresponding to position 658 of the coding sequence, and named GALNTL5-c.658+1G>A according to the Human Genome Variation Society nomenclature. The primer composition includes a forward primer and a reverse primer, and the gene GALNTL5 refers to the human genome sequence database hg19.

2. The application according to claim 1, wherein: The forward primer is as shown in SEQ ID NO.1, and the reverse primer is as shown in SEQ ID NO.

2.

3. The application according to claim 1, characterized in that: The detection method of the application includes extracting genomic DNA of the sample to be tested, amplifying the DNA, sequencing the amplification product, and judging whether the base is mutated according to the sequencing result. When the base is mutated from guanine to adenine, it is judged that the sample to be tested is from a patient with sperm head deformity.

4. The application according to claim 3, wherein: The sample to be tested is semen.

5. The application according to claim 3, characterized in that: Sanger sequencing is used for the sequencing.

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