PMFBP1 mutant gene related to azoosperm syndrome and application of PMFBP1 mutant gene

Abstract

The invention provides a PMFBP1 mutant gene related to azoospermia syndrome and an application of the PMFBP1 mutant gene. The mutant gene is a PMFBP1 gene c.2641Cgt; the protein p.Arg881Ter is subjected to non-sense mutation, so that the encoded protein p.Arg881Ter is truncated, and the function of a sperm head-tail connection device is damaged. The invention also provides a specific Sanger sequencing detection method and a kit of the mutation, which can be used for rapid molecular diagnosis, genetic counseling and assisted reproduction guidance of the disease, and provides a basis for precise medical treatment.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biomedical detection, in particular to a PMFBP1 mutant gene related to acaphalic spermatozoa syndrome and application thereof. BACKGROUND

[0002] Acephalic spermatozoa syndrome (ASS) is a severe teratozoospermia, in which most of the sperm in the semen are headless, leading to male infertility. Even if intracytoplasmic sperm injection is used, the clinical outcome is usually poor. The disease has obvious genetic heterogeneity, and current studies have shown that it is associated with double allelic mutations of multiple genes. Among them, PMFBP1 gene has been confirmed to be one of the important pathogenic genes leading to acaphalic spermatozoa syndrome. The protein encoded by this gene plays a key role in the assembly and maintenance of the head-tail junction device of sperm. The mutation types of PMFBP1 gene associated with acaphalic spermatozoa syndrome reported in existing literature mainly include missense mutations, splice site variations, etc. These mutations may lead to protein function defects, but still retain a certain protein length.

[0003] In the aspect of clinical diagnosis, there are certain limitations in the genetic detection of acaphalic spermatozoa syndrome. The conventional targeted detection method (such as PCR-RFLP or qPCR probe method for known hot spot mutations) has limited detection range, and it is easy to miss detection for rare mutations or new mutations with extremely low frequency in the database. Although high-throughput sequencing technologies such as whole exome sequencing can comprehensively detect, they are high in cost, complex in data analysis and long in cycle, and are difficult to popularize and apply as a first-line screening method in clinical practice.

[0004] Therefore, there is still a need in the art to explore new pathogenic mutations related to acaphalic spermatozoa syndrome, especially mutation types that may lead to more severe loss of function. At the same time, it is urgent to develop targeted, accurate, rapid and low-cost detection methods to perfect the pathogenic gene mutation spectrum of the disease and provide effective tools for clinical precision diagnosis and genetic counseling. SUMMARY

[0005] The present application aims to provide a PMFBP1 mutant gene related to acaphalic spermatozoa syndrome and application thereof, which enriches the gene library for diagnosis and treatment of male infertility, provides a new molecular marker for the etiological study of acaphalic spermatozoa syndrome (ASS), provides more powerful detection tools for the early discovery of acaphalic spermatozoa syndrome through biological gene screening methods in clinical practice, and enables timely treatment intervention.

[0006] To achieve the above object, the present application adopts the following technical solutions: In the first aspect of the present application, a PMFBP1 mutant gene associated with headless sperm syndrome is provided, and the nucleotide sequence of the mutant pathogenic gene is shown as SEQ ID NO: 2. Compared with the nucleotide sequence of the PMFBP1 gene shown as SEQ ID NO: 1, the nucleotide changes are as follows: the nucleotide sequence contains a c.2641C>T mutation, and the encoded PMFBP1 protein has an arginine to stop codon mutation at amino acid position 881. In the second aspect of the present application, a nucleic acid fragment is provided, which comprises the PMFBP1 mutant gene or a gene sequence fragment comprising the c.2641C>T mutation site.

[0007] In the third aspect of the present application, a primer pair for detecting the PMFBP1 mutant gene is provided, which can specifically amplify the DNA region containing the c.2641 site of the PMFBP1 gene.

[0008] Further, the sequences of the primer pair are shown as SEQ ID NO: 3-4: Forward primer: 5'-GAATGTCAGTTCCTAATG-3', Reverse primer: 5'-TCAAGCTTCTGCTGCTGCTG-3'.

[0009] In the fourth aspect of the present application, a kit for detecting headless sperm syndrome is provided, which comprises a component for detecting the PMFBP1 mutant gene.

[0010] Further, the component comprises the primer pair.

[0011] Further, the component further comprises one or more of PCR reaction reagents and sequencing reagents.

[0012] Further, the PCR reaction reagents comprise: dNTPs, Taq enzyme, Mg 2+ , and PCR reaction buffer.

[0013] In the fifth aspect of the present application, the PMFBP1 mutant gene is used in the preparation of a reagent or kit for diagnosing or headless sperm syndrome.

[0014] In a sixth aspect of the present application, a method for detecting the PMFBP1 mutant gene for non-diagnostic purposes is provided, comprising the following steps: a) obtaining genomic DNA of the sample to be tested; b) using specific primers to perform PCR amplification on the PMFBP1 gene region containing the c.2641 site; c) performing sequencing analysis on the amplification product to determine the nucleotide type of the c.2641 site.

[0015] Further, the sequencing analysis is Sanger sequencing method.

[0016] The one or more technical solutions in the embodiments of the present application have at least the following technical effects or advantages: 1. Filling the blank of pathogenic mutation spectrum, with important scientific value: The present application first identifies and reports a homozygous nonsense mutation c.2641C>T (p.Arg881Ter) located at the end of exon 17 of the PMFBP1 gene. This mutation type (nonsense mutation leading to protein truncation) is fundamentally different from the missense mutation commonly seen in the past, which theoretically will lead to more complete loss of protein function. This discovery perfects the pathogenic gene mutation spectrum of ASS, and provides a new molecular target and theoretical basis for studying the function of PMFBP1 gene and the pathogenesis of ASS.

[0017] 2. Sufficient evidence of mutation pathogenicity, providing a reliable target for precise diagnosis: Through family cosegregation analysis (proband homozygous, parents heterozygous) and population frequency verification (extremely low frequency <8x10 -6 ), combined with bioinformatics software prediction that the mutation has high harmfulness, multiple evidence chains are provided for the pathogenicity of the mutation, making it a high-confidence genetic marker in the molecular diagnosis of ASS.

[0018] 3. The detection method is highly targeted, accurate, fast and low-cost, and easy to promote in clinical practice: The present application develops a Sanger sequencing verification method based on specific primers for the specific mutation site c.2641C>T. Compared with whole-exome sequencing, this method does not require complex data analysis, has a short cycle and low cost; compared with traditional targeted detection methods, it has higher accuracy and will not miss this new mutation. The method is stable, easy to operate, and is very suitable for promotion as a first-line screening or verification means in clinical laboratories, solving the clinical pain points of high-throughput sequencing high cost and incomplete coverage of traditional methods.

[0019] 4. Clear clinical significance and direct social and economic benefits: Based on the clear pathogenicity of the mutation, clear genetic counseling and assisted reproductive strategy guidance can be provided directly to positive patients detected. For example, patients can be advised to avoid using their own sperm for potentially ineffective ICSI cycles, and instead consider sperm donation or adoption. This can effectively avoid the economic burden and psychological trauma of repeated treatment failures for patient families, while saving valuable medical resources and achieving true precision medicine. BRIEF DESCRIPTION OF DRAWINGS

[0020] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings needed in the embodiment description will be briefly introduced. Obviously, the drawings in the following description are some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor.

[0021] Figure 1 Family tree for the embodiment; Figure 2 Sanger sequencing peak chart of the nonsense mutation c.2641C>T(p.Arg881Ter) site of the PMFBP1 gene in the proband family of the test example; Figure 3 Amino acid sequence alignment chart of the wild type (WT) and mutant (Mutant) of the PMFBP1 protein corresponding to the gene site.

[0022] Figure 4 Results of sperm morphology analysis, wherein, Figure 4 A: Diff-Quik staining image, showing the morphologies of normal sperm and patient's headless sperm. Figure 4 B: PNA (red) and DAPI (blue) fluorescence staining image, showing abnormal acrosome structure of patient's sperm. Figure 4 C: Transmission electron microscope image, revealing the ultrastructure disintegration of the head-tail junction of the patient's sperm and the disorder of the mitochondrial sheath.

[0023] Figure 5 Results of, wherein, Figure 5 A: Western Blot result chart, showing the complete absence of PMFBP1 protein in patient's sperm. Figure 5 B: Schematic diagram of construction of wild type and mutant PMFBP1 expression plasmids. Figure 5 C: Stability analysis results chart of mutant and wild type PMFBP1 protein in HEK-293T cells under CHX and MG132 treatment. DETAILED DESCRIPTION

[0024] The advantages and various effects of the present application will be more clearly presented hereinafter in conjunction with specific embodiments and examples. Those skilled in the art should understand that these embodiments and examples are used to illustrate the present application, rather than limit the present application.

[0025] Throughout the specification, unless otherwise specifically indicated, the terms used herein are to be understood as having the meanings commonly used in the art. Therefore, 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 the present application belongs. If there is a contradiction, the present specification takes precedence.

[0026] Unless otherwise specifically indicated, various raw materials, reagents, instruments and equipment used in the present application can be purchased on the market or obtained by existing methods.

[0027] The prior art has a blind spot in the genetics of azoospermia factor syndrome (ASS), especially the lack of research on the mutation type on the PMFBP1 gene that can cause complete loss of protein function. At the same time, there is a lack of detection means that can balance precision and low cost and is suitable for specific new mutations in clinical practice.

[0028] Based on this, the general idea of the present application is: First, a new pathogenic mutation (c.2641C>T) that can directly cause severe truncation of the key functional domain of PMFBP1 protein is identified through genetic analysis; Then, a highly specific and simple Sanger sequencing detection scheme is designed for this specific mutation site. The core of this general idea is to combine "new target discovery" with "special detection method development" to form a complete solution.

[0029] Specifically, first, starting from clinical cases, the root genetic cause of the disease is excavated in depth using gene sequencing technology, filling the blank of known pathogenic mutation spectrum; second, after discovering a new target, instead of relying on expensive and complex general detection platforms, the most suitable precision diagnostic tool is developed for it, so as to overcome the defects of existing detection methods in terms of pertinence, cost and efficiency, and finally realize the connection from scientific discovery to clinical application.

[0030] This scheme skillfully avoids the high cost and complexity of whole exome sequencing, and overcomes the limitation of traditional targeted detection methods that cannot cover new mutations, providing a new effective path for the precision diagnosis and genetic counseling of ASS.

[0031] The present application will be described in detail below in conjunction with examples and experimental data.

[0032] Example 1: Discovery and identification of a novel mutation site in PMFBP1 gene 1. Sample source A 34-year-old male patient (proband) with infertility was selected. He visited the Reproductive Center of Renmin Hospital, Wuhan University for infertility after 5 years of marriage. The patient's semen analysis showed that the semen volume and sperm concentration were normal, but >90% of the sperm were headless or only with a "pinpoint" head, which met the diagnostic criteria for headless sperm syndrome. The patient's parents were phenotypically normal, and there was no history of other known genetic diseases in the family. All subjects signed an informed consent form, and the study was approved by the Ethics Committee of Wuhan University.

[0033] 2. Whole exome sequencing (WES) Peripheral venous blood (5 mL) was collected from the proband and his parents, and genomic DNA was extracted using QIAamp Blood DNA Kit (QIAGEN). The DNA concentration was determined by Qubit 3.0, and all were diluted to 10 ng / μL.

[0034] The library construction used Agilent SureSelect Human All Exon V6 capture kit, and the end repair, A addition, linker ligation and PCR enrichment were completed according to the instructions. After hybrid capture, the library was purified by AMPure XP magnetic beads, quantified by Qubit, and detected by Agilent 2100.

[0035] The sequencing platform was Illumina NovaSeq 6000, PE150 mode, and the average sequencing depth of the target region was >100x. After the data was quality controlled by FastQC, it was aligned to the GRCh37 / hg19 reference genome by BWA v0.7.17; GATK v4.0 completed base quality correction, variant detection (HaplotypeCaller) and VQSR filtering; ANNOVAR was used for variant annotation. The variant screening criteria were: located in exon or splice site, minor allele frequency (MAF) <1% in gnomAD, ExAC, 1000 Genomes database, and non-synonymous or nonsense mutation, predicted as deleterious.

[0036] 3. Results The results showed that the proband carried a homozygous nonsense mutation PMFBP1 (NM_031293.3) c.2641C>T (p.Arg881Ter), and his parents were heterozygous carriers of this site (see Figure 1 ). The mutation was not included in the above public databases or had a very low frequency (MAF <8x10 -6 ), and the CADD score was 36.0, indicating that it was a deleterious variation.

[0037] Example 2: Sanger sequencing verification of PMFBP1 c.2641C>T mutation site 1. Primer design and PCR amplification Forward primer: 5'-GAATGTCAGTTCCTAATG-3' (SEQ ID NO. 3); Reverse primer: 5'-TCAAGCTTCTGCTGCTGCTG-3' (SEQ ID NO. 4); The PCR reaction system (50 μL) is as follows Table 1

[0038] The PCR reaction program is as follows: Pre-denaturation: 95℃, 5 min Cycle amplification (35 cycles): 95℃ 30s → 55℃ 30s → 72℃ 30s Final extension: 72℃, 10 min Storage: 4℃ 2. Sequencing and result analysis After the PCR product is verified by agarose gel electrophoresis, it is sent to a sequencing company for bidirectional Sanger sequencing. Chromas or SnapGene software is used to analyze the sequencing peak chart.

[0039] 3. Results The Sanger sequencing results (see Figure 2 ) are completely consistent with the WES results ( Figure 3 ): the proband is homozygous T / T genotype at the c.2641 site, and his parents are C / T heterozygous genotype at the site, successfully verifying the authenticity of the c.2641C>T mutation site.

[0040] Example 3: Detection kit based on c.2641C>T mutation site The present embodiment provides a kit for detecting PMFBP1 gene c.2641C>T mutation, which comprises the following components: (1) DNA extraction component: QIAamp Blood DNA Kit or other equivalent genomic DNA extraction reagent.

[0041] (2) PCR amplification component: Specific primer dry powder or storage solution: containing the forward primer and reverse primer described in Example 2.

[0042] 2x Taq PCR Master Mix: containing a thermostable DNA polymerase, dNTPs, Mg2+ and reaction buffer. (Other Mixes can also be used in other embodiments) Sterile deionized water.

[0043] (3) Positive control: Plasmid DNA or genomic DNA sample containing PMFBP1 gene c.2641C>T mutation (homozygous or heterozygous).

[0044] (4) Negative control: Genomic DNA sample of wild-type PMFBP1 gene.

[0045] (5) Instructions: Detailed operation steps, result interpretation criteria (such as wild-type C / C, heterozygous C / T, and mutant homozygous T / T peak chart examples), and precautions.

[0046] Method of use: Follow the instructions to perform DNA extraction, PCR amplification (system and procedure same as Example 2), then send the PCR product to a sequencing service, and analyze the sequencing results according to the provided interpretation criteria. This can genotype the subject's sample.

[0047] Example 4, Functional and morphological analysis The semen sample of the proband was subjected to a number of morphological analyses.

[0048] I. Sperm morphological analysis 1. Diff-Quik staining analysis Fresh semen samples from the patient were taken, and smears were prepared according to the World Health Organization (WHO) 5th edition standard. Diff-Quik staining kit was used for staining.

[0049] Observed under an optical microscope (see Figure 4 A) shows: the head and tail of the control group are connected normally, and the morphology is complete; while the vast majority of sperm in the patient's semen exhibit a "needle-like" tail structure with missing heads (i.e. headless sperm), and only a few sperm retain abnormal small heads, consistent with the typical characteristics of headless sperm syndrome.

[0050] 2. Peanut agglutinin (PNA) staining To evaluate the structure of the sperm acrosome, the patient's sperm smear was taken, fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and stained with TRITC-labeled PNA, and the nucleus was counterstained with DAPI.

[0051] Observed under a fluorescence microscope (see Figure 4 B) shows: the patient's sperm acrosome structure is abnormal, with disordered or missing PNA signals, suggesting defects in acrosome development or assembly.

[0052] 3. Transmission electron microscopy (TEM) analysis The patient and normal control semen samples were taken, double-fixed with 2.5% glutaraldehyde and 1% osmium acid, dehydrated with gradient ethanol, embedded with epoxy resin EPON 812, ultra-thin sectioned (60-80 nm), and then lead-uranium double-stained.

[0053] Transmission electron microscopy observation (see Figure 4 C) shows that the structure of the patient's sperm head-neck junction is severely damaged, the connection between the basal plate and the nuclear membrane is interrupted, and the mitochondrial sheath arrangement is disordered, further confirming that the PMFBP1 mutation leads to the disintegration of the sperm head-tail connection apparatus (HTCA).

[0054] II. Protein expression and stability analysis 1. Protein expression analysis The total protein of the patient and normal control sperm was extracted, and the protein was extracted using RIPA lysis buffer (Servicebio), and quantified by BCA method. Equal amounts of protein were subjected to 10% SDS-PAGE electrophoresis, transferred to PVDF membrane, blocked with 5% skim milk, and then incubated with PMFBP1 primary antibody (rabbit) and HRP-labeled secondary antibody, respectively, and developed by ECL.

[0055] Results (see Figure 5 A) shows that no PMFBP1 protein band is detected in the patient's sperm, while clear PMFBP1 protein expression is observed in the control group, indicating that the c.2641C>T nonsense mutation leads to complete loss of PMFBP1 protein.

[0056] 2. Protein stability analysis Wild-type (WT) and mutant (c.2641C>T) PMFBP1 cDNA were cloned into pcDNA3.1+C-HA vector (Sangon Biotech) and transfected into HEK-293T cells. After 24 hours of transfection, the cells were treated with protein synthesis inhibitor cycloheximide (CHX, 100 μmol / L) and proteasome inhibitor MG132 (10 μmol / L) for 0, 4, 8, and 12 hours, respectively. The total protein was extracted and Western blot was performed to detect the expression of HA-tagged protein.

[0057] Results (see Figure 5 B) shows that under CHX treatment, the degradation rate of mutant PMFBP1 protein is significantly slower than that of wild-type, indicating that the truncated protein has abnormal stability in the in vitro system; while in the presence of MG132, the stability of the mutant protein is further enhanced (see Figure 5 C).

[0058] Example 5: Bioinformatics and population genetics analysis of PMFBP1 c.2641C>T mutation 1. Objective To assess the frequency of PMFBP1 gene c.2641C>T (p.Arg881Ter) mutation in the general population and its potential pathogenicity based on international public databases and bioinformatics algorithms, and to provide population statistical and computational biology evidence for the mutation as a pathogenic marker for the absence of head syndrome.

[0059] 2. Materials and methods Data sources: The following public genomic databases were queried to obtain the allele frequency of the c.2641C>T site: 1000 Genomes Project (1000G); Genome Aggregation Database (gnomAD); Exome Aggregation Consortium (ExAC); Bioinformatics prediction: The pathogenicity of the mutation was predicted using the following authoritative algorithms: CADD (Combined Annotation Dependent Depletion): A score of ≥20 usually indicates harm.

[0060] MutationTaster: Directly gives classification results such as "pathogenic" or "possibly pathogenic".

[0061] Analysis criteria: According to the ACMG / AMP guidelines, very low population frequency (e.g. <0.0001) and high CADD score (e.g. ≥20) are considered as evidence of pathogenicity.

[0062] 3. Results The results are summarized in the following table: Table 2

[0063] From Table 2, we can see that: Population frequency: The mutation is not included in the main public databases (1000G, ExAC), and the frequency in gnomAD is also very low (8.25 x 10 -6 ), which meets the rareness characteristics of pathogenic mutations.

[0064] Harm prediction: The CADD Phred score is as high as 36.0, strongly suggesting that the mutation has high harm; MutationTaster software directly predicts it as "pathogenic".

[0065] 4. Conclusion This example demonstrates that the PMFBP1 c.2641C>T mutation is extremely rare in the normal population and is consistently predicted to be a deleterious variant by multiple algorithms. These data provide strong supportive evidence for the pathogenicity of this mutation, and in combination with the clinical fact that it was discovered in the proband of the headless sperm syndrome, further consolidates its reliability as a genetic marker of the disease.

[0066] Finally, it is also necessary to note that the terms "comprising", "including", or any other variant thereof, are intended to cover non-exclusive inclusions, so that processes, methods, articles or devices that include a series of elements not only include those elements, but also other elements not expressly listed, or inherent to such processes, methods, articles or devices.

[0067] Although embodiments of the present application have been described, those skilled in the art will recognize that the application can be practiced with additional modifications and variations without departing from the spirit and scope of the application. Accordingly, the appended claims are intended to embrace all such modifications and variations as fall within the scope of the present application.

[0068] Obviously, numerous modifications and variations of the present application are possible in light of the above teachings. It is therefore to be understood that within the scope of the appended claims and their equivalents, the application can be practiced otherwise than as specifically described.

Claims

1. A PMFBP1 mutant gene associated with headless sperm syndrome, characterized in that, The nucleotide sequence of the mutant pathogenic gene is shown as SEQ ID NO: 2, and the nucleotide sequence of the PMFBP1 gene is shown as SEQ ID NO: 1, wherein the nucleotide sequence contains a c.2641C>T mutation, and the encoded PMFBP1 protein has a mutation from arginine to stop codon at the 881st amino acid.

2. A nucleic acid fragment, characterized in that, The nucleic acid fragment comprises the PMFBP1 mutant gene according to claim 1, or a gene sequence fragment containing the c.2641C>T mutation site.

3. A primer pair for detecting the PMFBP1 mutant gene of claim 1, characterized in that, The primer pair can specifically amplify the DNA region containing the c.2641 site of the PMFBP1 gene.

4. The primer pair according to claim 3, characterized in that, The sequence of the primer pair is shown as SEQ ID NO: 3-4.

5. A kit for detecting headless sperm syndrome, characterized by, The kit comprises components for detecting the PMFBP1 mutant gene according to claim 1.

6. The kit of claim 5, wherein The components comprise the primer pair according to claim 3 or 4.

7. The kit of claim 6, wherein The components further comprise one or more of PCR reaction reagents and sequencing reagents.

8. Use of the PMFBP1 mutant gene according to claim 1 in the preparation of a reagent or kit for diagnosing or headless sperm syndrome.

9. A method for detecting the PMFBP1 mutant gene of claim 1 for non-diagnostic purposes, characterized by, The method comprises the following steps: a) obtaining genomic DNA of a sample to be tested; b) using specific primers to perform PCR amplification on the PMFBP1 gene region containing the c.2641 site; c) performing sequencing analysis on the amplification product to determine the nucleotide type of the c.2641 site.

10. The method of claim 9, wherein, The sequencing analysis is Sanger sequencing method.

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