Pou3f4 gene mutant and application thereof

Abstract

This application belongs to the field of molecular biology technology, specifically relating to a POU3F4 gene mutant and its applications. The POU3F4 gene mutant has a c.703T>A mutation relative to the wild-type POU3F4 gene. The POU3F4 mutant gene provided by this invention is a novel pathogenic variant of type 2 X-linked hereditary deafness. This pathogenic variant can be used to screen asymptomatic or mildly phenotypically presenting female carriers of DFNX2, indicating the risk to their offspring, and to perform genetic diagnosis on male patients to provide scientific surgical plans for hearing intervention.

Description

Technical Field

[0001] This application belongs to the field of molecular biology technology, specifically relating to a POU3F4 gene mutant and its applications. Background Technology

[0002] Hereditary deafness is a significant disease affecting human health, with approximately 50% of congenital hearing loss caused by genetic factors. Its inheritance patterns are diverse, including autosomal dominant, autosomal recessive, X-linked, and mitochondrial inheritance. X-linked deafness (DFNX) accounts for about 1% to 2% of hereditary hearing loss. Although the proportion is low, its unique inheritance pattern—the disease-causing gene is located on the X chromosome—means that male patients typically exhibit a fully penetrating phenotype, while female carriers may only show mild symptoms or be asymptomatic. However, they can pass the disease-causing gene to their offspring, causing male offspring to be affected. Therefore, it holds special and important significance in genetic counseling and prenatal diagnosis.

[0003] In DFNX, X-linked deafness type 2 (DFNX2) is the most common type. DFNX2 has typical anatomical features, namely incomplete partition type III (IP-III) inner ear malformation. Its pathological features mainly include: spherical enlargement of the base of the internal auditory canal, lack of bony septum between the cochlea and the internal auditory canal, and absence or dysplasia of the cochlear axis. Clinically, patients usually present with prelingual, progressive, sensorineural hearing loss. Computed tomography (CT) is the key imaging tool for diagnosing IP-III malformation. In terms of treatment, due to the frequent presence of stapes footplate fixation or abnormalities, these patients face special risks such as cerebrospinal fluid "gushing" during cochlear implantation. Therefore, preoperative genetic diagnosis and accurate imaging evaluation are crucial for developing a safe and effective surgical plan.

[0004] Currently, over 100 pathogenic mutations in the POU3F4 gene have been reported globally, including point mutations, small insertions / deletions, and large deletions of the entire gene or regulatory regions. However, the mutation spectrum of the POU3F4 gene exhibits significant racial and regional differences. In the Chinese population, the number of identified mutation sites remains limited, and the molecular causes of many families carrying suspected hereditary deafness phenotypes remain unclear. This means that a large number of new pathogenic mutations still await discovery and identification.

[0005] The discovery and functional validation of the new POU3F4 mutant has multiple important implications: First, it can further expand and improve the gene mutation spectrum of hereditary deafness in the Chinese population, providing new targets for precise molecular diagnosis; second, it is crucial for clarifying the pathogenic mechanism and understanding the functional domains of the POU3F4 protein; finally, it can provide accurate genetic counseling, risk assessment, and fertility guidance for families carrying this mutation, especially effectively screening out phenotypically normal or mildly carrier females and warning them of the risk of having male children with the mutation, thereby achieving primary prevention.

[0006] Therefore, discovering and identifying new pathogenic mutants of the POU3F4 gene and developing targeted detection methods and kits are of urgent practical need and important clinical application value for filling existing technological gaps and promoting the development of precision medicine for DFNX2. Summary of the Invention

[0007] The purpose of this invention is to overcome the shortcomings of the prior art and provide a POU3F4 gene mutant and its application, specifically by adopting the following technical solution:

[0008] In a first aspect, the present invention provides a POU3F4 gene mutant, wherein the nucleotides of the POU3F4 gene mutant have a c.703T>A mutation relative to the wild-type POU3F4 gene.

[0009] As a further preferred embodiment, the nucleotide sequence of the wild-type POU3F4 gene is shown in SEQ ID NO.1.

[0010] SEQ ID NO. 1:

[0011]

[0012] This invention is based on a self-collected family with X-linked recessive hearing loss. Through whole-exome sequencing, whole-genome linkage analysis, and Sanger sequencing verification, it was successfully discovered that only the c.703T>A (p.Phe235Ile) mutation of the POU3F4 gene co-segregated with the disease phenotype in the linkage region, identifying it as the pathogenic variant of this family. This was ultimately verified in the family and in normal controls. The POU3F4 mutant gene provided by this invention is a novel pathogenic variant of X-linked deafness type 2 (DFNX2). This pathogenic variant can be used to screen asymptomatic or mildly phenotypically identifiable female carriers of DFNX2, indicating the risk to their offspring, and to perform genetic diagnosis on male patients to provide scientific surgical plans for hearing intervention.

[0013] Secondly, the present invention provides a protein encoded by the above-mentioned POU3F4 gene mutant, wherein the amino acid sequence of the protein has a p.Phe235IIe mutation relative to the amino acid sequence of the protein expressed by the wild-type POU3F4 gene.

[0014] As a further preferred embodiment, the amino acid sequence of the protein expressed by the wild-type POU3F4 gene is shown in SEQ ID NO.2.

[0015] SEQ ID NO.2:

[0016] MATAASNPYSILSSTSLVHADSAGMQQGSPFRNPQKLLQSDYLQGVPSNGHPLGHHWVTLSDGGPWSSTLATSPLDQQDVKPGREDLQLGAIIHHRSPHVAHHSPHTNHPNAWGASPAPNPSITSSGQPLNVYSQPGFTVSGMLEHGGLTPPPAAASAQSLHPVLREPPDHGELGSHHCQ DHSDEETPTSDELEQFAKQFKQRRIKLGFTQADVGLALGTLYGNVFSQTTICRFEALQLSFKNMCKLKPLLNKWLEEADSSTGSPTSIDKIAAQGRKRKKRTSIEVSVKGVLETHFLKCPKPAAQEISSLADSLQLEKEVVRVWFCNRRQKEKRMTPPGDQQPHEVYSHTVKTDTSCHDL.

[0017] Thirdly, the present invention provides the application of reagents for detecting POU3F4 gene mutants in the preparation of DFNX2 pathogenic gene diagnostic reagents or DFNX2 pathogenic gene diagnostic kits.

[0018] As a further preferred embodiment, the reagent for detecting the POU3F4 gene mutant includes a primer pair, wherein the sequence of the forward primer is shown in SEQ ID NO. 3 and the sequence of the reverse primer is shown in SEQ ID NO. 4.

[0019] SEQ ID NO.3: CATTGCCAGGATCACTCCGA;

[0020] SEQ ID NO.4: GTCTGTTTTCACGGTGTGCG.

[0021] Fourthly, the present invention provides a detection kit for screening DFNX2 pathogenic genes, the detection kit comprising reagents for detecting POU3F4 gene mutants having a c.703T>A mutation compared to SEQ ID NO. 1;

[0022] Alternatively, reagents for detecting POU3F4 gene mutant proteins with the p.Phe235IIe mutation compared to SEQ ID NO.2.

[0023] As a further preferred embodiment, the reagent is an antibody or a primer.

[0024] As a further preferred embodiment, for the POU3F4 gene mutant having the c.703T>A mutation compared to SEQ ID NO. 1, the nucleotide sequences of the primers are shown in SEQ ID NO. 3-SEQ ID NO. 4.

[0025] As a further preferred embodiment, the POU3F4 gene mutant protein having the p.Phe235IIe mutation compared to SEQ ID NO.2, and the antibody includes GAPDH, HRP-conjugated Affinipure Goat Anti-Mouse IgG (H+L), and HRP-conjugated Affinipure Goat Anti-Rabbit IgG (H+L).

[0026] The beneficial effects of this invention are as follows: Currently reported harmful variants of POU3F4 are mainly nonsense variants, which cause protein truncation and provide strong evidence for pathogenicity scoring. However, missense variants have not been verified by functional experiments, making it difficult to determine pathogenicity. The detection method used in this invention can confirm that the c.703T>A missense mutation affects transcriptional activity and reduces protein stability, thus confirming that c.703T>A is a harmful variant affecting protein function. Attached Figure Description

[0027] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0028] Figure 1 The image shows a pedigree of a family with type 2 X-linked inherited hearing loss.

[0029] Figure 2 The image shown is a pure-tone audiometry result diagram of the proband.

[0030] Figure 3 The image shown is a CT scan of the temporal bone of the proband.

[0031] Figure 4 The image shows the Sanger sequencing peaks at the c.703 site of the POU3F4 gene, representing a member of the family.

[0032] Figure 5 The results show the analysis of luciferase activity data between wild-type and POU3F4c.703T>A mutant.

[0033] Figure 6 The results show the stability test results of wild-type and POU3F4c.703T>A mutant proteins. A represents the relative expression level (WB) of POU3F4 mutant (c.703T>A p.Phe235IIe) and wild-type protein; B is a grayscale graph of the relative expression level (WB) of POU3F4 mutant (c.703T>A p.Phe235IIe) and wild-type protein. Detailed Implementation

[0034] The technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0035] Example 1

[0036] Identification of POU3F4 gene mutants

[0037] Sample collection:

[0038] A new X-linked recessive hereditary deafness family was discovered during large-scale eugenic genetic screening and molecular diagnosis of deafness genes, named DFNA-2023-17 (see pedigree chart). Figure 1 (Where, ○ represents a normal female, □ represents a normal male, and ■ represents a male patient.) The arrow indicates a female carrier, and the proband is indicated by the arrow. Patients in this family present with non-syndromic prelingual sensorineural hearing loss. Figure 2 (The left and right images represent the left and right ears, respectively). Whole exome sequencing was performed on this family, and the results showed that the only X-linked recessive genetic variant was the c.703T>A variant in the POU3F4 gene. It was preliminarily determined that the POU3F4 c.703T>A variant was the cause of the disease in this family.

[0039] Whole exome sequencing:

[0040] After obtaining informed consent, 2-3 mL of peripheral venous blood was drawn from each of the DFNA-2023-17 proband and their family members, anticoagulated with EDTA, and genomic DNA was extracted according to the instructions of the Qiagen Universal DNA Extraction Kit (catalog number: 51304). After obtaining the genomic DNA, the DNA was quality controlled using an Agilent 5200 fragment analyzer, and whole exome capture sequencing was performed using an Illumina HiSeq sequencer. The specific procedures are as follows:

[0041] After passing quality control, the DNA was randomly fragmented into 200-500 bp segments using a Covaris ultrasonic disruptor. T4 DNA polymerase and Klenow fragment repair were then used to create blunt ends. Amplification was performed using a few cycles (4-8 rounds) with a high-fidelity DNA polymerase (such as KAPA HiFi) to enrich the adapter-containing DNA fragments for exon capture sequencing. Sequencing results showed an average sequencing depth of 138X for 99% of the targeted regions. A total of 138,176 single nucleotide variants (SNVs) and 21,808 insertion / deletion variants (Indels) were detected in all samples. These variants were filtered using multidimensional databases, including population databases (dbSNP, 1000 Genome, ExAC), disease databases (OMIM, HGMD, Clinvar), and bioinformatics prediction tools (SIFT, Polyphen2, and Mutation Taster). Based on the clinical phenotypic correlation, the pathogenicity of the mutation was rated according to the ACMG genetic mutation classification guidelines and the ClinGen hearing loss expert group's recommendations on ACMG mutation interpretation. Combined with the suspected X-linked recessive inheritance pattern (XR) in this family, POU3F4c.703T>A was finally identified as a potential candidate variant site.

[0042] The nucleotide sequence of the POU3F4 gene mutant is shown in SEQ ID NO. 5:

[0043]

[0044] Example 2

[0045] Family co-segregation and Sanger sequencing verification

[0046] Candidate pathogenic variants screened using polymerase chain reaction (PCR) and Sanger sequencing were validated in probands and their families using whole-exome sequencing. Primers for the POU3F4c.703T>A site were designed using NCBI Primer-BLAST software. The primer sequences are as follows: forward primer (CATTGCCAGGATCACTCCGA) and reverse primer (GTCTGTTTTCACGGTGTGCG). The primers were synthesized and provided by Beijing Qingke Biotechnology Co., Ltd. After primer synthesis, the selected candidate variant POU3F4c.703T>A was subjected to PCR using a Tiangen KT201-02 2xTag_PCR Mastermix (Tiangen Biotech (Beijing) Co., Ltd.). The PCR reaction conditions were: 94℃ pre-denaturation for 5 minutes; 94℃ denaturation for 30 seconds, 58℃ annealing for 30 seconds, 72℃ extension for 1 minute, repeated 35 times; and a final extension at 72℃ for 10 minutes. The PCR reaction volume was 20 μL, as shown in Table 1 below. The fragment size of the amplified products was identified by 2.2% agarose gel electrophoresis. The purified PCR products were then sequenced using a Sanger sequencer from Applied Biosystems.

[0047] Table 1 PCR reaction system

[0048]

[0049] A family study revealed that all affected males carried the POU3F4c.703T>A hemizygous mutation, while female carriers showed no hearing abnormalities. All males with normal hearing were wild-type at this locus. Figure 4 Therefore, it is believed that POU3F4c.703T>A is co-separated in this family lineage, which is the pathogenic variant of this family lineage.

[0050] Example 3

[0051] Investigating the luciferase activity of wild-type POU3F4 and mutant POU3F4

[0052] 1. Carrier Construction

[0053] 1.1 Carrier Construction

[0054] (1) Construction of pCMV-3Xflag-Neo(EGFP)-POU3F4-wt vector

[0055] (2) Fragment amplification:

[0056] Using pCMV-3XFlag-Neo(EGFP)-POU3F4-HindⅢ-F and pCMV-3XFlag-Neo(EGFP)-POU3F4-KpnI-R as primers, PCR amplification was performed using the synthesized POU3F4 DNA template to obtain the HindⅢ-wt-KpnI fragment.

[0057] Enzyme digestion: The vector pCMV-3Xflag-Neo (EGFP) and the fragment HindⅢ-wt-KpnI were double-digested with HindⅢ and KpnI.

[0058] Ligation: After recovering the enzyme-digested vector and fragment, ligation is performed at a fragment:vector ratio of 3:1.

[0059] Transformation: The ligation product is transformed into DH5α competent cells.

[0060] Pick bacteria and perform colony PCR identification;

[0061] Positive clone sequencing.

[0062] (2) Construction of pCMV-3Xflag-Neo(EGFP)-POU3F4-mut vector

[0063] Fragment amplification: Using the synthesized DNA of POU3F4 as a template, and using POU3F4-mut-F and pCMV-3XFlag-Neo(EGFP)-POU3F4-KpnI-R as primers, the mut fragment was amplified to obtain the fragment.

[0064] Enzyme digestion: The vector pCMV-3Xflag-Neo (EGFP) and the fragment HindⅢ-mut-KpnI were double-digested with HindⅢ and KpnI.

[0065] Ligation: After recovering the enzyme-digested vector and fragment, ligate them at a ratio of fragment:vector = 3:1.

[0066] Transformation: The ligation product was transformed into DH5α competent cells, and colony PCR was performed to identify the bacteria. Positive clones were sequenced.

[0067] (3) Construction of pProDuoLuci-POU3F4 (c.-472_+25) vector

[0068] Fragment amplification:

[0069] Using normal human gDNA as a template, the target fragment of 519bp was amplified using pProDuoLuci-POU3F4-NotI-F and pProDuoLuci-POU3F4-XhoI-R as primers.

[0070] Enzyme digestion: The vector pProDuoLuci and the fragment were digested with NotI and XhoI.

[0071] Ligation: After recovering the enzyme-digested vector and fragment, ligation is performed at a fragment:vector ratio of 3:1.

[0072] Transformation: The ligation product was transformed into DH5α competent cells, and colony PCR was performed to identify the bacteria. Positive clones were sequenced.

[0073] 2. Cell culture and transfection

[0074] 293T and HeLa cells were cultured in DMEM medium containing 10% fetal bovine serum. The constructed empty vector pCMV-3Xflag-Neo(EGFP) and the recombinant vector pCMV-3Xflag-Neo(EGFP)-POU3F4-wt / mut were transiently co-transfected with the luciferase vector pProDuoLuci-POU3F4(c.-472_+25) into 293T and HeLa cells according to the Lipo2000 liposome instructions. Luciferase activity was measured 48 hours after transfection.

[0075] 3. Luciferase activity detection

[0076] Forty-eight hours after transfection, the culture medium was removed from the culture plate, and the cells were washed with 1×PBS. Following the instructions of the Dual Luciferase Reporter Gene Assay Kit, 250 μL of freshly prepared 1×PLB lysis buffer was added to each well of a 12-well plate. The plate was gently agitated at room temperature for 15 min, and the lysis buffer was collected. Detection was performed using a full-function microplate reader (PerkinElmer). 20 μL of cell lysis buffer was added to each well, followed by 100 μL of LARII to detect firefly luciferase activity. Then, 100 μL of freshly prepared 1×Stop&Glo® reagent was added to detect Renilla luciferase activity. The expression activity of luciferase was calculated using Renilla luciferase activity as a control.

[0077] 4. Experimental Results

[0078] 4.1 Carrier Construction

[0079] The constructed vector was transformed and subjected to bacterial culture PCR followed by sequencing. Sequence alignment was performed, and wild-type and mutant positive clones were selected and sequenced.

[0080] 4.2 Luciferase activity detection

[0081] Luciferase activity was measured 48 hours after transfection, according to the Dual Luciferase Reporter Gene Assay Kit instructions. The results are shown in Tables 2-3 and... Figure 5 As shown in the figure. The results indicate that the transcriptional level of the detected reporter gene was significantly downregulated compared to the wild type, and slightly lower than that of the control group.

[0082] Table 2

[0083]

[0084] Table 3

[0085]

[0086] Example 4

[0087] Stability assay of biotype and POU3F4c.703T>A mutant protein

[0088] 1. Cell culture and transfection

[0089] HEK293T cells were cultured in DMEM medium containing 10% fetal bovine serum. The constructed wild-type and mutant eukaryotic recombinant expression vectors pCMV-3Xflag-Neo(EGFP)-POU3F4 were transiently transfected into 293T cells according to the Lipo2000 liposome instructions. After 48 hours of transfection, the cells were treated with inhibitors, and Western blotting was performed after sample collection.

[0090] 2. Protein stability assay

[0091] After transfection with wild-type and mutant eukaryotic recombinant expression vectors for 48 hours, cells were treated with 10 μg / mL CHX for 0, 4, 8, and 24 hours, respectively, and cell pellets were collected. Total protein was extracted from the cell pellets using RIPA lysis buffer. Protein concentration was determined using a BSA kit, followed by protein denaturation treatment. The difference in degradation rate between wild-type and mutant target proteins was detected by Western blotting.

[0092] 3. Experimental Results

[0093] Stability assay of POU3F4-mut(c.703T>A p.Phe235Ile) protein: After transfection for 48 hours, cells were treated with 10ug / mL CHX for 0, 4, 8 and 24 hours respectively, and samples were collected for Western blot analysis.

[0094] CHX is a protein synthesis inhibitor. After treatment with the same concentration (10ug / mL) for different times (0, 4, 8, 24hr), the expression level of flag-POU3F4-wt began to decrease from the 8th hour of treatment; while the expression level of flag-POU3F4-mut2 decreased significantly from the 4th hour of treatment, and the expression level was extremely low at the 24th hour.

[0095] The results showed that CHX treatment resulted in a faster degradation rate of the mutated protein, suggesting that the mutation altered the protein's degradation rate. Figure 6 A, Figure 6 B), protein stability is reduced.

[0096] The embodiments of this application have been described above with reference to the accompanying drawings. Specific examples have been used to illustrate the principles and implementation methods of this application. The description of the above embodiments is only for the purpose of helping to understand the core ideas of this application. However, this application is not limited to the specific embodiments described above. The specific embodiments described above are merely illustrative and not restrictive. Those skilled in the art can make many other forms under the guidance of this application without departing from the spirit and scope of the claims, and all of these forms are within the protection scope of this application.

Claims

1. A POU3F4 gene mutant, characterized in that, The nucleotide sequence of the POU3F4 gene mutant is shown in SEQ ID NO.

5.

2. A protein encoded by the POU3F4 gene mutant according to claim 1, characterized in that, The amino acid sequence of the protein is a p.Phe235IIe mutation relative to the amino acid sequence of the protein expressed by the wild-type POU3F4 gene; The amino acid sequence of the protein expressed by the wild-type POU3F4 gene is shown in SEQ ID NO.2.

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