New applications of the BCAS2 gene

Abstract

This invention provides a novel application of the BCAS2 gene, belonging to the fields of biomedical technology and diagnostics. By detecting the expression status of the BCAS2 gene or identifying T→G mutations occurring in its 3'UTR region, this invention reveals the association between BCAS2 and the pathogenesis of HIGM1 patients, potentially greatly expanding the clinical diagnostic scope of HIGM1 and significantly improving diagnostic accuracy. Furthermore, this discovery also holds promise for opening up new target directions for the diagnosis and treatment of other immunodeficiency diseases.

Description

Technical Field

[0001] This invention belongs to the fields of biomedical technology and diagnostics, and specifically relates to a new application of the BCAS2 gene. Background Technology

[0002] Hyper IgM syndrome (HIGM) falls under the category of primary immunodeficiency diseases. Its pathogenesis involves a defect in the B-cell immunoglobulin class switching process, preventing the normal conversion of IgM into high-titer antibodies such as IgG, IgA, and IgE. This results in serum IgM levels being normal or elevated, while IgG, IgA, and IgE levels are decreased. Currently, three main types of HIGM are known: Type I Hyper IgM syndrome (HIGM1), Type II Hyper IgM syndrome (HIGM2), and Type III Hyper IgM syndrome (HIGM3).

[0003] Among them, type I hyper-IgM syndrome (HIGM1) is caused by mutations in the DNA sequence encoding CD40L on the surface of T cells, which leads to the loss of CD40L. Clinically, HIGM1 patients usually experience recurrent infection symptoms in childhood. Previous studies have shown that defects in the interaction between TB cells or dendritic cells can cause abnormal T cell function and eventually induce HIGM1. HIGM1 has now been classified as a combined immunodeficiency disease. Recent studies have further revealed that most of the mutation sites of CD40L are concentrated in integrin sites such as αvβ3, α5β1 and αIIbβ3. Mutations at these sites affect the transport process of CD40L on platelets and have become one of the key factors in the pathogenesis of HIGM1[7].

[0004] Under different exogenous stimuli, B cells can produce different transcripts (Germline transcripts, GLTs; Post-switch transcripts, PLTs), which then undergo class-switching recombination (CSR). GLTs and PLTs belong to a class of long non-coding RNAs containing an S region. Targeting and regulating GLTs or PLTs is one of the effective means of regulating CSR, with alternative splicing (AS) being a commonly used method. Alternative splicing refers to the splicing of exons in the same gene in different combinations, allowing the gene to generate different transcripts under different spatiotemporal conditions. RNA-binding proteins (RBPs) are a collective term for proteins that are persistent throughout RNA regulation and can bind to RNA. Alternative splicing events involving RNA-binding proteins play a crucial role in various physiological processes of the cell. RNA-binding proteins such as HuR, HNRNP F, and PTBP1 can affect B cell activation and antibody CSR processes by regulating alternative splicing.

[0005] Breast carcinoma amplified sequence 2 (BCAS2), also known as splicing factor 27 (SPF27), is highly expressed in human breast cancer cells

[16] . As an RNA-binding protein, BCAS2 is a highly conserved component of the CDC5L / PRP19 splicing complex. It is widely involved in the alternative splicing regulation of various cancer cells, including sperm cells, oocytes, pancreatic β cells, pancreatic cancer cells, and esophageal cancer cells. Studies have found that BCAS2 can form a splicing complex with SRSF7 and bind to the mRNA sequence of CD72 to perform splicing function, ultimately changing the expression level of CD72 protein, thereby interfering with the antibody class switching of B cells and causing an imbalance in the ability of B cells to produce antibodies in HIGM1 patients. Based on the above research conclusions, it is hoped that a diagnostic method for HIGM1 based on BCAS2 can be constructed to help with the accurate subtyping and effective treatment of HIGM. Summary of the Invention

[0006] The purpose of this invention is to further broaden the clinical diagnostic scope of HIGM1 disease, significantly improve the accuracy of diagnosis, and open up new target directions for the diagnosis and treatment of other immunodeficiency diseases, thereby providing new applications for the BCAS2 gene.

[0007] To achieve the above objectives, the technical solution of the present invention is as follows:

[0008] In a first aspect, the present invention provides the application of the BCAS2 gene as a biomarker in the preparation of kits for predicting and / or diagnosing high IgM syndrome.

[0009] Secondly, the present invention provides reagents for detecting the expression level of the BCAS2 gene, said reagents being reagents for genome, transcriptome and / or proteome sequencing.

[0010] Preferably, the expression level is the protein expression level and / or the mRNA transcription level.

[0011] Preferably, the reagent is a biomolecular reagent that specifically binds to the BCAS2 gene, or specifically binds to the target gene of the BCAS2 gene, or specifically hybridizes with the nucleic acid encoding the BCAS2 gene, or forms an immunoprecipitation product with the BCAS2 gene.

[0012] Preferably, the target genes of the BCAS2 gene are CD72, Rps6ka5, Las1l, Nfat5, Rpn1, and Top1.

[0013] Preferably, the biomolecular reagent is selected from primers, probes, or antibodies.

[0014] Thirdly, the present invention provides reagents for detecting mutations in the 3'UTR region of the BCAS2 gene.

[0015] Preferably, the 3'UTR region mutation is a T→G mutation.

[0016] Preferably, the reagent is a reagent used for genome sequencing.

[0017] Fourthly, the present invention provides the use of reagents for detecting the BCAS2 gene in the preparation of kits for predicting and / or diagnosing high IgM syndrome.

[0018] Preferably, the reagent is the reagent described in the second or third aspect.

[0019] Fifthly, the present invention provides a kit for predicting and / or diagnosing high IgM syndrome, the kit comprising the reagents described in the second aspect and / or the reagents described in the third aspect.

[0020] In a sixth aspect, the present invention provides a method for detecting high IgM syndrome for non-diagnostic purposes, the method comprising detecting the expression level of the BCAS2 gene in a sample to be tested, and / or detecting mutations in the 3'UTR region of the BCAS2 gene in the sample to be tested.

[0021] In a seventh aspect, the present invention provides a prediction system for high IgM syndrome, the prediction system comprising a detection module and an analysis and judgment module; the detection module detects the expression level of the BCAS2 gene in the sample to be tested or detects mutations in the 3'UTR region of the BCAS2 gene in the sample to be tested, and transmits the expression level data or mutation data to the analysis and judgment module, the analysis and judgment module predicting whether the sample to be tested suffers from high IgM syndrome based on the expression level data or the mutation data.

[0022] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0023] Firstly, this invention is the first to construct a BCAS2 knockout mouse B cell line and the first to study the changes in CSR in this B cell line; it demonstrates that BCAS2 knockout directly leads to a significant decrease in the CSR frequency of the B cell line, and that restoring BCAS2 expression will restore the original CSR frequency.

[0024] Secondly, this invention demonstrates that BCAS2 knockout mice exhibit a phenotype similar to high IgM syndrome, with unchanged or increased IgM antibody expression detected in serum, while the expression of high-titer antibodies such as IgA, IgG, and IgE is reduced; and the ability of B cells to produce IgG1, IgG2b, IgG3, and IgE in vivo is significantly reduced.

[0025] Third, this invention is the first to use RNA-seq analysis to confirm that after BCAS2 specific knockout, the expression of CD72 changed significantly, and the alternative splicing of its exon 3 also changed significantly.

[0026] Fourth, this invention is the first to confirm that BCAS2 regulates its target site recognition through alternative splicing: UV crosslinking immunoprecipitation and CLIP-seq analysis confirmed that CD72 is a target gene for significant BCAS2 binding. Furthermore, this invention also discovered that the motif for significant BCAS2 binding to the CD72 gene is GAAGAA. And for the first time, this motif was mutated in the CD72 gene of B cells, revealing a decrease in exon skipping frequency and a significant reduction in antibody class conversion ability after mutation.

[0027] Fifth, this invention is the first to perform immunoprecipitation on BCAS2 in activated B cells: proteins interacting with BCAS2 are captured using a BCAS2 antibody and analyzed by mass spectrometry, with the immunoprecipitation products used to verify the mass spectrometry results. This invention also co-knocks out the interacting protein SRSF7 with BCAS2, finding that knocking out BCAS2 has a more significant impact on CD72 alternative splicing and B cell antibody class conversion compared to knocking out BCAS2 and SRSF7 alone. Furthermore, this invention analyzes the binding of the conserved sequence of BCAS2 to SRSF7, finding that SRSF7 binds significantly to both coil 1 and coil 2 of BCAS2.

[0028] Sixth, this invention is the first to utilize Sanger sequencing of BCAS2 in the blood of HIGM1 patients. The sequencing results show that a T→G mutation exists in BCAS2 in the blood of HIGM1 patients, and this mutation leads to downregulation of BCAS2 expression and changes in alternative splicing of related genes. This invention also uses RNA-seq analysis to examine the expression changes of different genes in the blood of HIGM1 patients. Many gene expression and alternative splicing changes observed in BCAS2-deficient mice are also present in the blood of HIGM1 patients.

[0029] Seventh, this invention discloses for the first time a method for studying the effects of multiple factor interactions on biological functions. First, interactions are identified and characterized using immunoprecipitation and protein mass spectrometry. Then, key binding sites are determined through high-throughput sequencing or staining-crosslinked immunoprecipitation sequencing, followed by further investigation of their biological functions. Ultimately, the association of BCAS2 in the pathogenesis of HIGM1 patients is revealed, and a diagnostic method based on BCAS2 gene downregulation or 3'UTR mutations is proposed. Attached Figure Description

[0030] The accompanying drawings, which are included to provide a further understanding of the invention and form part of this invention, illustrate exemplary embodiments of the invention and are used to explain the invention, but do not constitute an undue limitation of the invention. In the drawings:

[0031] Figure 1The effect of siRNA knockdown of Bcas2 on antibody production in the CH12 cell line was investigated. Figure A illustrates the locations of the Bcas2 transcript variants and the two Bcas2 siRNA sequences. Figure B shows the quantitative PCR analysis of B cell mRNA from the siCtrl and two Bcas2 siRNA groups. In the quantitative PCR analysis, Bcas2 expression levels were normalized to the Gapdh transcript and the siCtrl group (n=3, mean ± SD). Figure C shows the Western blots of B cell mRNA from the siCtrl group and the two Bcas2 siRNA groups. Figure D shows the CSR flow cytometry analysis of the CH12 cell lines after CIT stimulation in the NC and two Bcas2 siRNA groups. Figure E shows the CSR quantification shown in Figure D, with each symbol representing a CH12 cell culture (n≥3, mean ± SD). Figure F shows the concentrations of IgM and IgA in the CH12 cell culture supernatant from the siCtrl and two Bcas2 siRNA groups. Each symbol represents a technical repetition, and the column represents the mean; G represents the quantitative PCR analysis of mRNA 24 hours after CIT stimulation, with the expression levels of μGLT and aGLT normalized to β-actin transcript and the CIT-stimulated siCtrl group (n=3, mean ± SD); H represents the expression of Bcas2 protein in the Bcas2 knockdown and rescue groups. (I) Flow cytometry analysis of CSRs in CH12 cell lines used for Bcas2 knockdown and rescue; J is the quantitative display of CSRs in Figure I, with each symbol representing a CH12 cell culture (n=3, mean ± SD); K represents alternative splicing events in CH12 cell lines with Bcas2 knockdown and rescue.

[0032] Figure 2 Knockdown of Bcas2 with AID-Cre reduces the ability of B cells to produce antibodies. A represents a schematic diagram of conditional Bcas2 (targeted allele, obtained by gene editing of the Bcas2 WT ​​allele using a targeting vector) and the Cre-deficient Bcas2 allele (Bcas2 KO allele). Indicator PCR primers are shown; B represents Bcas2. fl / flGenotypic results of AID Cre / + (Bcas2-cKO) mice; C represents the serum IgM and IgG3 concentrations of 7-week-old WT and Bcas2-cKO mice, with each symbol representing an individual mouse and the horizontal line representing the mean; D represents the serum IgA, IgE, IgG1, IgG2b, and IgG2c concentrations of 7-week-old WT and Bcas2-cKO mice, with each symbol representing an individual mouse and the horizontal line representing the mean; E represents the proportion of GC B cells in the Peyer's node of WT and Bcas2-cKO mice analyzed by flow cytometry; F represents the quantitative statistical results of GC B in Figure E, with each symbol representing a B cell culture from an individual mouse (n≥4, mean ± SD); G represents genomic DNA PCR analysis and LPS and LPS+IL4 stimulated WT and Bcas2-cKO mice. fl / fl Western blot analysis of AID Cre / + (Bcas2-cKO) mice (day 3); H shows quantitative PCR analysis of mRNA in WT and Bcas2-cKO B cells 72 hours after LPS and LPS+IL4 stimulation. Bcas2 expression levels were normalized to β-actin transcripts under LPS and LPS+IL4 stimulation (n=3, mean ± SD); I shows quantitative PCR analysis of mRNA 72 hours after LPS and LPS+IL4 stimulation. Expression levels of variable and constant regions of different Ig molecules were normalized to β-actin transcripts and LPS and LPS+IL4 stimulation (n=3, mean ± SD); J shows flow cytometry analysis of surface IgG1 expression after LPS+IL4 stimulation; K shows flow cytometry analysis of surface IgG3 expression after LPS stimulation; L shows the quantitative statistical results of IgG1 and IgG3 in LPS and LPS+IL4 cultures in Figures J and K (n≥4, mean ± SD).

[0033] Figure 3To illustrate how BCAS2 affects antibody production in B cells by regulating alternative splicing of Cd72, A shows a bar chart of Cd72 RNA-seq expression values; B shows co-immunostained Ctrl and cKO cells using antibodies against the target proteins Cd72 and B220 from the CH12 cell line, with DNA stained using DAPI (scale bar, 1 μm); C shows the analysis of LPS+IL4-stimulated WT and Bcas2-cKO cells by RT-PCR. Alternative splicing of Cd72 in B-cell spleen (n≥3) is shown, correspondingly displaying the inclusion (Incl) to exclusion (Excl) ratio. Analysis of CSR-related, DNA recombination and repair-related, and Bcas2-binding gene expression and exon-exon linkages shows Bcas2 binding peaks in meiosis-related gene transcripts; purple arrows indicate transcription direction. F represents the forward primer, R the reverse primer; D shows abundant Bcas2-binding motifs, displaying the first five abundant motifs; E is a schematic diagram of the construction of Cd72-targeting mutations (locations of motifs significantly binding to Bcas2); F represents OE, EV, and KD. Quantitative PCR analysis of B cells: In the quantitative PCR analysis, Bcas2 expression levels were normalized to Gapdh transcripts and EV groups (n=3, mean ± SD); G represents Western blot analysis of Bcas2 expression levels in EV and KDB cells; H represents alternative splicing of Cd72 in WT and OE, EV and KD mutant CH12 cell lines by RT-PCR analysis (n=3), showing the inclusion (Incl) to exclusion (Excl) ratio accordingly; I represents CSR flow cytometry analysis of WT and OE, EV and KD mutant CH12 cell lines after CIT stimulation; J represents CSR quantification as shown in Figure I, where each symbol represents CH12 cell culture (n=3, mean ± SD), where OE indicates overexpression; EV indicates empty vector; and KD indicates knockout.

[0034] Figure 4Bcas2 recruits AS-related proteins to regulate AS in B cells. A shows a silver-stained gel of Bcas2 and a control immunoprecipitate from spleen B cells; B is a scatter plot of protein scores indicating significant differences in protein between the two parallel pooled samples; C shows an IP assay performed on spleen B cell extracts after LPS stimulation; D shows a network displaying GO enrichment analysis of Bcas2-binding proteins; E shows co-immunostaining and intensity scans of CH12 cells using antibodies against Bcas2 and Bcas2-binding proteins (SRSF7 and DHX15), with DNA stained with DAPI; scale bar, 1 μm; F shows quantitative PCR analysis of B cell mRNAs of NC, siBcas2, siSRSF7, and siS+B. In quantitative PCR analysis, the expression levels of Bcas2 and SRSF7 were normalized to gapdh transcripts and the NC group (n=3, mean ± SD); G shows the CSR of CH12 cell lines with NC, siBcas2, siSRSF7, and siS+B after CIT stimulation, analyzed by flow cytometry; H shows the CSR quantification shown in Figure G; I shows the alternative splicing of Bcas3 and Cd72 in CH12 cell lines with NC, siBcas2, siSRSF7, and siS+B by RT-PCR analysis (n=3), correspondingly showing the inclusion (Incl) to exclusion (Excl) ratio; J is a model diagram of the sequence positions of CC1 and CC2 in Bcas2 protein; K is Bcas2 Immunoprecipitation of flag and SRSF7-HA; L represents the conserved sequence of Bcas2 protein across different species; M represents the CSR of CH12 cell lines with NC, CC1CC2 deletion, siSRSF7, and CC1CC2 deletion + siSRSF7 after CIT stimulation, analyzed by flow cytometry; N represents the CSR quantification shown in Figure M; O represents the alternative splicing of Bcas3 and Nfat5 in CH12 cell lines with NC, CC1CC2 deletion, siSRSF7, and CC1CC2 deletion + siSRSF7 (n=3) analyzed by RT-PCR, correspondingly showing the ratio of inclusion (Incl) to exclusion (Excl).

[0035] Figure 5Bcas2 may be involved in the expression and alternative splicing of CSR-related genes in hyper-IgM. A represents the concentrations of IgA, IgG, and IgM in the blood of hyper-IgM patients and their normal reference values; B represents the concentrations of IgG1, IgG2, IgG3, IgG4, IgA1, and IgE in the serum of hyper-IgM patients, with each symbol representing a technical repeat and columns representing averages; C represents the expression of Bcas2 mRNA in normal individuals and HIGM1 patients; D represents the quantitative PCR analysis of B cell mRNA in normal individuals and hyper-IgM patients. In the quantitative PCR analysis, the expression levels of genes altered by Bcas2 knockout (Cd72, Rps6ka5, Las1l, Rexo5, Nfat5) were normalized to Gapdh transcripts and the normal control group (n=3, mean ± SD); E represents the alternative splicing of genes in the Bcas2 knockout model in the serum of normal individuals and Hyper IgM patients analyzed by RT-PCR; F is a volcano plot showing the distribution of differentially expressed genes in the RNA-seq data. The horizontal axis in the figure represents the fold change in gene ploidy in the blood of normal individuals and Hyper IgM patients. |FoldChange|≥1.5.Padj≤0.05. Upregulated genes are shown as red dots, and downregulated genes are shown as green dots; G is a cluster heatmap of differentially expressed genes. The horizontal axis is genotype, and the vertical axis is the normalized FPKM value of differentially expressed genes. Blue indicates higher expression levels, while yellow indicates lower expression levels; H is a network showing GO enrichment analysis of differentially expressed genes in Hyper IgM patients; I is a cluster heatmap of several genes related to Bcas2-cKO differentiated in RNA-seq and gene expression columns reflecting FPKM values. Blue indicates higher expression levels, while yellow indicates lower expression levels; JK shows the mRNA expression of upregulated and downregulated genes as shown in Figure I.

[0036] Figure 6 To illustrate the presence of BCAS2 mutation-induced expression loss in HIGM1 patients, leading to antibody production loss via the same mechanism, A represents RNA-seq data showing Bcas2 and Cd72 expression between normal individuals and HIGM1 patients; B represents Sanger sequencing results of the target gene region, with each peak representing a nucleotide and peak height reflecting the relative abundance of nucleotides; C and D represent mRNA expression in wild-type cells and cells with the Bcas2 mutant plasmid, as well as alternative splicing events of Bcas3 and Nfat5; E represents flow cytometry analysis of CSR in the CH12 cell line after CIT stimulation; F represents CSR quantification as shown in Figure E, with each symbol representing a CH12 cell culture (n≥3, mean ± SD). Detailed Implementation

[0037] This invention innovatively reveals a novel mechanism that triggers antibody rearrangement diversity, and further exploration based on this mechanism has uncovered a series of application scenarios. For example, by accurately detecting low expression of the BCAS2 gene, or by identifying T→G mutations occurring in its 3'UTR region, it is expected to greatly broaden the clinical diagnostic scope of HIGM1 disease and significantly improve diagnostic accuracy. Simultaneously, this discovery also holds promise for opening up new target directions for the diagnosis and treatment of other immunodeficiency diseases. It is worth emphasizing that any new mechanism revealed by this invention, and all related applications, are covered within the scope of protection of this invention.

[0038] The novel mechanism involved in this invention involves the following detailed steps: When the body encounters antigens such as pathogenic microorganisms, numerous genes within B cells initiate transcription processes, thereby generating mRNA. During this process, BCAS2, along with related proteins such as SRSF7, co-constructs a cleavage complex. This cleavage complex, leveraging the specific recognition and binding capabilities of BCAS2, precisely anchors to the CD72 mRNA sequence, thereby exercising alternative splicing function. This series of complex molecular biological processes ultimately leads to a decrease in the ratio of short to long CD72 transcripts, thereby causing changes in CD72 expression and strongly driving antibody diversity generation within B cells. Disruption of this signaling pathway can trigger immunodeficiency diseases. Specifically, if a mutation occurs in the 3'UTR region of BCAS2 (such as a typical T→G mutation), the expression stability of the BCAS2 gene will decrease, leading to abnormal fluctuations in CD72 expression, preventing normal antibody class switching and ultimately inducing HIGM1 syndrome.

[0039] In the aforementioned new mechanism, "when the body encounters antigen stimulation such as pathogenic microorganisms, many genes inside B cells will initiate the transcription process and generate mRNA" refers to the addition of bacterial lipopolysaccharide LPS or LPS and IL-4 as stimulating factors to B cells during the experimental operation, followed by detection using transcriptome sequencing technology, which revealed many differentially expressed mRNAs.

[0040] In the aforementioned new mechanism, "BCAS2 will synergistically construct a cleavage complex with related proteins such as SRSF7 during this process" refers to the high proportion of binding proteins (SRSF7, DHX15, and HNRNPK, etc.) obtained by mass spectrometry analysis of BCAS2 immunoprecipitation products, and experimental evidence showing that the absence of these proteins leads to the alternative cleavage of CD72 and the conversion of B cell antibody classes.

[0041] In the aforementioned new mechanism, "the scission complex, with the specific recognition and binding ability of BCAS2, precisely anchors to the mRNA sequence of CD72, thereby exerting alternative scission function" refers to CLIP-seq of BCAS2 showing significant binding to the CD72 gene sequence; and RNA-seq analysis of activated B cells after BCAS2 knockout revealed changes in alternative scission of CD72 in the BCAS2 binding region.

[0042] In the aforementioned new mechanism, "this series of complex molecular biological processes ultimately leads to a decreasing ratio of short to long CD72 transcripts, thereby triggering changes in CD72 expression and strongly driving antibody diversity within B cells" refers to alternative splicing causing exon skipping in exon 3 of CD72, and a decrease in the frequency of exon skipping, resulting in a reduction in the proportion of short transcripts. This change leads to the upregulation of CD72 expression. CD72 is currently considered to affect antibody class conversion in B cells, and its expression changes ultimately influence antibody diversity.

[0043] In the aforementioned new mechanism, "if a mutation occurs in the 3'UTR region of BCAS2 (such as a typical T→G mutation), it will lead to a decrease in the expression stability of the BCAS2 gene" can refer to mutations in the 3'UTR region, which is widely considered to be related to transcriptional stability. If a mutation occurs, it will lead to a decrease in expression.

[0044] In this embodiment, it should be noted that unless specific conditions are specified, they are performed under conventional conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0045] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0046] Example 1

[0047] The following research was conducted in this embodiment:

[0048] First, a BCAS2 knockout mouse B cell line was constructed, and the changes in CSR in this B cell line were studied. It was demonstrated that BCAS2 knockout directly led to a significant decrease in the CSR frequency of the B cell line, and that BCAS2 expression restoration would restore the original CSR frequency.

[0049] Secondly, BCAS2 knockout transgenic mice were constructed, and the changes in B cell CSR in these mice were studied. This demonstrated that BCAS2 knockout had no effect on B cell development, proliferation, and differentiation, further proving that BCAS2 knockout directly led to a significant decrease in CSR frequency. BCAS2 knockout mice exhibited a phenotype similar to high IgM syndrome: IgM antibody expression remained unchanged or increased in serum, while the expression of high-titer antibodies such as IgA, IgG, and IgE decreased; and the production capacity of B cells for IgG1, IgG2b, IgG3, and IgE was significantly reduced.

[0050] Third, RNA-seq analysis confirmed that after BCAS2-specific knockout, CD72 expression changed significantly, and alternative splicing of its exon 3 also changed significantly.

[0051] Fourth, it was confirmed that BCAS2 regulates its target site recognition through alternative splicing: UV crosslinking immunoprecipitation and CLIP-seq analysis confirmed that CD72 is a significant target gene for BCAS2 binding. The motif that significantly binds to the CD72 gene was found to be GAAGAA. Furthermore, this motif was mutated in the CD72 gene of B cells for the first time, revealing a decrease in exon skipping frequency and a significant reduction in antibody class conversion ability in B cells after mutation.

[0052] Fifth, immunoprecipitation was performed on BCAS2 in activated B cells: proteins interacting with BCAS2 were captured using a BCAS2 antibody and analyzed by mass spectrometry. The mass spectrometry results were then validated using the immunoprecipitation products. Co-knockout of the interacting protein SRSF7 with BCAS2 revealed that, compared to knocking out BCAS2 and SRSF7 alone, knocking out BCAS2 had a more significant impact on CD72 alternative splicing and B cell antibody class conversion. Analysis of the binding of the conserved sequence of BCAS2 to SRSF7 showed that SRSF7 binds significantly to both coil 1 and coil 2 of BCAS2.

[0053] Sixth, Sanger sequencing was performed on BCAS2 in the blood of HIGM1 patients. The sequencing results showed that BCAS2 in the blood of HIGM1 patients had a T→G mutation, and this type of mutation led to downregulation of BCAS2 expression and changes in alternative splicing of related genes.

[0054] Seventh, RNA-seq analysis was used to examine the expression changes of different genes in the blood of HIGM1 patients. Many gene expression and alternative splicing changes that were observed in BCAS2 defective mice were also found in the blood of HIGM1 patients.

[0055] Eighthly, a method for studying the interaction of multiple factors affecting biological functions is revealed. First, interactions are identified and characterized using immunoprecipitation and protein mass spectrometry. Then, high-throughput sequencing or staining-crosslinked immunoprecipitation sequencing is used to determine key binding sites, followed by further investigation of their biological functions. Ultimately, the association of BCAS2 in the pathogenesis of HIGM1 patients is revealed, and a diagnostic method based on BCAS2 gene downregulation or 3'UTR mutation is proposed.

[0056] The specific experimental method is as follows:

[0057] 1) Animal breeding and genotyping

[0058] C57BL / 6J and ICR mice were purchased from Beijing Huafukang Biotechnology Co., Ltd. Bcas2fl / + mice were generated, bred, and kindly provided by Professor Li Lei of the Chinese Academy of Sciences. The preparation method of these mice is disclosed in the following literature: Wenbo Liu, et al. BCAS2 is involved in alternative mRNA splicing in spermatogonia and the transition to meiosis[J]. Nature Communications volume 8, Article number:14182(2017).

[0059] AID-Cre mice were purchased from the Jackson laboratory. The AID-Cre mice were bred by crossing them with their parent Bcas2 mice. fl / + Bcas2 derived from mice fl / fl Bcas2-cKO mice were generated by backcrossing mice (obtained through crossbreeding parental Bcas2fl / + mice). Primers were designed and PCR was used to target Bcas2... fl / + AID-Cre and Bcas2 fl / fl Mice were genotyped. Primers used for genotyping are summarized in Table 1. Mice were grouped and housed in a temperature-controlled environment (22℃±2℃) under specific pathogen-free (SPF) conditions with a 12-hour:12-hour light-dark cycle. Male SPF C57BL / 6J mice, approximately 7 weeks old, were purchased from Beijing Huafukang Biotechnology Co., Ltd., located in Beijing, China. Throughout the experiment, mice had unrestricted access to sterile water and standard rodent food from China Agricultural University. All animal-related procedures were performed in accordance with the Animal Care and Use Guidelines. All animal experiments in this invention were approved by the Laboratory Animal Welfare and Animal Experimentation Ethics Review Committee of China Agricultural University and conducted in accordance with the guidelines (Approval No.: AW02504202-5-1).

[0060] 2) Cell Culture

[0061] Eight-week-old Bcas2-cKO mice and their control littermates (Bcas2) fl / fl Primary B cells were freshly isolated from mice. CH12F3 cells (commercially available) and mouse spleen B cells were cultured in RPMI 1640 medium (Catalog#sh30809.01, Hyclone) supplemented with 5% fetal bovine serum (Catalog#P30-3302, PAN), L-glutamine (Catalog#25030081, Thermo), sodium pyruvate (Catalog#11360070, Thermo), and penicillin-streptomycin antibiotics (Catalog#15070063, Thermo). 293T cells were cultured in DMEM medium (Catalog#12430054, Gibco) supplemented with 10% fetal bovine serum (Catalog#P30-3302, PAN) and penicillin-streptomycin antibiotics (Catalog#15070063, Thermo). The cells were stored in a humidified incubator at 37°C and 5% CO2.

[0062] 3) Cell transfection detection

[0063] Transient knockdown of the Bcas2 gene in the CH12F3 cell line was achieved via its siRNA. In short, the cells were knocked down at a rate of 1 × 10⁻⁶. 6 Cells were seeded at a density of 100 cells / mL. 1 nM siRNA from the electroporation mixture (catalog #MPK10025, Thermo) was added to the cells, followed by electroporation transfection using a Neon transfection device (Invitrogen). Subsequently, cells were seeded at a density of 1 × 10⁻⁶ cells / mL in the presence of 1 μg / mL purified anti-mouse / rat CD40 (catalog #16-0401-82, Invitrogen), 10 ng / mL recombinant mouse IL-4 (catalog #214-14, Peprotech), and 2 ng / mL recombinant human TGF-β1 (catalog #315-15, Peprotech). 5Cells were cultured at a density of [number] cells / mL for 3 days. Transient knockdown efficiency was verified by qRT-PCR. Bcas2 siRNA was synthesized by Guangzhou Ribobio Biotechnology Co., Ltd. according to the designed sequence CAGCTTACAGCTGGATCTA (SEQ ID NO.1). For transfection of 293T cells, GeneTwin™ gene transfection reagent (Biomed, China) was used. A total of 6 μg of plasmid DNA was dissolved in 100 μL of PBS. Then, 18 μL of transfection reagent was mixed with PBS to obtain a final volume of 100 μL. This mixture was then mixed with the plasmid solution to a final volume of 200 μL and incubated for 20 minutes. The transfection complex was then added to the 293T cell culture at ~70% confluence, gently mixed, and incubated for 48 hours.

[0064] 4) B cell activation

[0065] Mouse spleen B cells were isolated using a B cell isolation kit according to the manufacturer's instructions (Catalog #19854, Stem cell). Cells were washed three times with 1×PBS and then... 5 Cells were cultured at a density of 10 cells / mL. Then, the cells were exposed to 25 μg / mL LPS (Catalog#L3024-10MG, Sigma-Aldrich) or LPS plus 10 ng / mL recombinant mouse IL-4 (Catalog#214-14, Peprotech) for 3 days.

[0066] 5) qRT-PCR detection

[0067] Total RNA was extracted using RNAiso Plus (Catalog #9109, Takara) and quantified using a NanoDrop One spectrophotometer (Thermo Scientific). cDNA synthesis was performed according to the instructions provided with the HiScript kit (Catalog #Q711, Vazyme, China). Gene expression was measured using a QuantStudio™ 5 real-time PCR instrument (384-well module) (Applied Biosystems). Assay analysis was performed using a PCR amplification instrument (Eppendorf), and the PCR products were then visualized on agarose gels using a gel imaging system (Tanon 1600, China). The relative expression of the target genes was normalized using Gapdh or Actb as reference genes. Primers for qRT-PCR and Assay analysis were synthesized by Sangon Biotech, and their sequences are listed in Table 1.

[0068] Table 1 Primer sequences

[0069]

[0070]

[0071] 6) RNA sequencing analysis

[0072] First, B cells from the spleen of control and Bcas2-cKO mice were isolated using a B cell sorting kit. The control mice were Bcas2-cKO mice. fl / fl Mice were separated and cultured in 1640 medium. Control and Bcas2-cKO cells were co-treated with LPS and IL4. Samples were collected after 3 days for further processing.

[0073] Total RNA was extracted from mouse spleen B cells treated with LPS+IL4, and mRNA was enriched using poly-T oligonucleotide-linked magnetic beads. The purified mRNA was fragmented to construct transcriptome sequencing libraries. The indexed samples were clustered according to the manufacturer's guidelines using the TruSeq PE Cluster kit v3-cBot-HS (Illumina) on the cBot ClusterGeneration System. Library preparation was then sequenced on the Illumina NovaSeq platform, which generated 150 bp paired-end reads. Strict quality control measures were implemented after sequencing. A reference genome index was constructed, and clean paired-end reads were aligned to this reference genome using HISAT2 software (version 2.0.5). FeatureCounts (version 1.5.0) was then used to enumerate reads mapped to each gene. Following this, the FPKM value for each gene was calculated, taking into account both gene length and the read count mapped to it. Differential gene expression between Bcas2-cKO and control mice (including biological replicates for each condition) was performed using the DESeq2 R software package (version 1.20.0) 72. Genes identified by DESeq2 with a padj value ≤0.05 were described as differentially expressed.

[0074] 7) Plasmid construction

[0075] The pcDNA3.0-Bcas2 plasmid containing the ampicillin resistance gene was designed to include a CMV promoter upstream of the Bcas2 coding sequence, followed by a bovine growth hormone polyadenylation signal (polyA). Restriction endonuclease digestion with two enzymes (EcoR I and Kpn I) linearized the pcDNA3.0-Bcas2 vector backbone to generate compatible ends for ligation with the PCR-amplified Bcas2 fragment. After generating the target plasmid with T4 DNA ligase, the ligated plasmid was transformed into competent *E. coli* cells, which were cultured on LB agar plates containing ampicillin for selection of positive transformations. Plasmid extraction was performed using a plasmid extraction kit (Catalog #AG21032, Accurate Biology, China) according to the manufacturer's instructions.

[0076] 8) Flow cytometry

[0077] Using BD LSRFortessa TM Flow cytometry analysis was performed using a Becton, Dickinson and Company, US. Prior to analysis, cells were resuspended in phosphate-buffered saline (PBS) supplemented with 1% bovine serum albumin (BSA) to a final concentration of 1 × 10⁻⁶. 6 Cells / mL. For immunophenotyping assays, cells were treated with a combination of fluorescently labeled antibodies targeting cell surface markers (B220, Catalog #103224, Biolegend; CD19, Catalog #152418, Biolegend; IgD, Catalog #405711, Biolegend; IgM, Catalog #406511, Biolegend; CD43, Catalog #143203, Biolegend; CD25, Catalog #113703, Biolegend; CD5, Catalog #100641, Biolegend; CD23, Catalog #101607, Biolegend; CD93, Catalog #136505, Biolegend). Initially, 1 × 10⁻⁶ cells / mL were used. 6Cells were allocated to 5 mL polystyrene tubes and exposed to an appropriate antibody mixture at 4°C in the dark for 30 minutes. After this incubation period, the cells were washed twice with PBS and then resuspended in 300 μL of PBS-BSA solution. In the RNA silencing assay, B220 (Catalog#103224, Biolegend), CD19 (Catalog#152418, Biolegend), IgM (Catalog#406511, Biolegend), and IgA (Catalog#407003, Biolegend) were introduced into the cell mixture at a concentration ratio of 1:400. For cytokine induction, cells co-cultured with LPS were labeled with B220, CD19, IgG2b (Catalog#406705, Biolegend), IgG3 (Catalog#406803, Biolegend), and secondary antibody (Catalog#405203, Biolegend) at a uniform ratio of 1:400. Conversely, cells co-cultured with a combination of LPS and IL-4 were labeled with B220, CD19, IgG1 (Catalog#406605, Biolegend), and IgE (Catalog#406907, Biolegend) at a ratio of 1:400. For in vivo immunization, spleen B cells were collected and then labeled with CD95 (Catalog#152606, Biolegend), GL7 (Catalog#144614, Biolegend), CD138 (Catalog#142509, Biolegend), CD38 (Catalog#102741, Biolegend), and IgD (Catalog#405727, Biolegend). The ratio of each antibody was 1:400. Before labeling the cells, each cell mixture was incubated with a uniform CD16 / CD32 antibody at a 1:400 ratio (Catalog#BE0307-1mg, BioXcell). BD FACSDiva was used. TM Data acquisition was performed using software version 8.0.1, while FlowJo was used. TM Data analysis was performed using software version 10.7.1. A gating strategy was employed to exclude debris, aggregates, and non-viable cells based on forward scattering (FSC), side scattering (SSC), and PI staining. Fluorescence compensation was performed using single-stain controls for each fluorescent dye.

[0078] 9) Western imprint

[0079] Total protein was extracted using cell lysis buffer (P0013, Beyotime) supplemented with PMSF (1:100, Catalog#ST506, Beyotime) and a mixture of protease inhibitors (1:100, Catalog#P1005, Beyotime). Pierce was used. TM Protein concentration was determined using a BCA protein assay kit (Catalog #23227, ThermoFisher). Protein lysates were then separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride membrane (Catalog #IPVH00010, Millipore). Afterwards, the membrane was blocked with 5% BSA for 2 hours and incubated at 4°C with primary antibodies (β-actin, Catalog#66009-1-Ig, Proteintech; BCAS2, Catalog#PA2466, Abmart; Tubulin, Catalog#11224-1-AP, Proteintech; Dhx15, Catalog#82137-1-RR, Proteintech; Srsf7, Catalog#PA3317S, Abmart; HNRNP K, Catalog#67708-1-IG, Proteintech; HNRNP D, Catalog#ab259895, Abcam; AID, Catalog#4975S, CST). The membrane was then incubated with secondary antibodies (goat anti-rabbit IgG (H+L), Catalog #31460, Invitrogen; goat anti-mouse IgG (H+L), Catalog #31430, Invitrogen) at room temperature for 2 hours. After incubation with ECL Plus (Catalog #PE0010, Solarbio), protein visualization was performed using a Tanon 5200 chemiluminescence imaging system.

[0080] 10) Immunofluorescence staining

[0081] Immunofluorescence staining was performed on the following proteins in CH12 cells: Bcas2 (Catalog#AB108330, Abcam), Srsf7 (Catalog#sc-390126, Santa), HNRNP K (Catalog#67708-1-Ig, Proteintech), Dhx15 (Catalog#sc-271686, Santa), Msi2 (Catalog#T55629S, Abmart), CD72 (Catalog#PH4955S, Abmart), and RPS6KA5 (Catalog#PA6507S, Abmart). Cells were washed with PBS and then fixed with 4% paraformaldehyde (PFA) in PBS at room temperature for 15 minutes. Cells were incubated at RT with 0.1% Triton X-100 in PBS for 5–10 minutes. Cells were washed three times with PBS to remove Triton X-100. Non-specific binding sites were blocked by incubating cells with blocking solution (Catalog#P0260, Beyotime) for 1 hour at room temperature. The sample was incubated overnight with primary antibody at 4°C. Cells were washed three times with PBS and then incubated for 1 hour at room temperature in the dark at a uniform ratio of 1:400 (anti-rabbit antibody, Catalog#P0183, Beyotime; anti-mouse antibody, Catalog#P0196, Beyotime). Coverslips containing cells were mounted onto slides using a mounting medium (Catalog#P0265, Beyotime). Images were taken using a confocal microscope (Zeiss LSM 900, Germany).

[0082] 11) Co-immunoprecipitation (co-IP) and silver staining

[0083] Total protein was extracted using cell lysis buffer (P0013, Beyotime) supplemented with PMSF (1:100, Catalog#ST506, Beyotime) and a protease inhibitor mixture (1:100, Catalog#P1005, Beyotime). After incubation on ice for 20 minutes, the lysate was pre-clarified at 4°C for 1 hour with 10 μl of protein A / G beads (Catalog#80104G, Invitrogen). Subsequently, 5 μg of Bcas2 antibody (Catalog#PA2466, Abmart) and normal mouse IgG (Catalog#2729S, CST) were added to the lysate. The mixture was incubated overnight at 4°C. The next day, the lysate was incubated with 50 μl of protein A / G beads on a vortex mixer at 4°C for 4 hours. The agarose complex containing the antibody and target protein was washed, eluted, and Western blotted. In the protein mass spectrometry facility, the complex structures of proteins were examined using immunoprecipitation-mass spectrometry (IP-MS) on a Thermo Q-Exactive high-resolution mass spectrometer (Thermo Scientific, Waltham, MA, Tsinghua University). Subsequently, the raw data acquired from the mass spectrometer were preprocessed using a Mascot Distiller 2.4 for peak identification. The peak list was then retrieved from the UniProt mouse database using the Mascot 2.5 search engine. For silver staining, Pierce was used according to the instructions. TM Thermo Scientific Silver Staining Kit TM

[0084] 12) GO enrichment analysis

[0085] GO enrichment analysis was performed on differentially expressed genes and AS genes using the clusterProfiler R package (version 3.4.4). To correct for gene length bias, genes presenting a total TPM sum in all samples greater than 1 were included in the background list. Analysis was performed using the mouse reference genome GRCm38 / mm10. Multiple testing was adjusted using the Benjamini-Hochberg method. Enrichment analyses with a corrected p-value less than 0.05 were considered to significantly enrich differentially expressed genes and AS genes.

[0086] 13) Sanger sequencing

[0087] The PCR reaction mixture consisted of 10-50 ng genomic DNA, 0.2 μM of each primer, 200 μM dNTP, 1.5 mM MgCl2, 1× PCR buffer, and 1 unit of Taq DNA polymerase (Thermo Fisher Scientific), for a total volume of 50 μL. PCR conditions were as follows: initial denaturation at 95°C for 2 minutes; denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, extension at 72°C for 1 minute; and a final extension at 72°C for 10 minutes. A NanoDrop spectrophotometer (Thermo Fisher Scientific) was used to ensure sufficient sequencing concentration. A sequencing reaction was constructed in a 10 μL volume containing 1-2 μL of purified PCR product, 1 μL of BigDye Terminator v3.1 ready-to-use reaction mixture, 1.5 μL of 5× sequencing buffer, 1 μL of 3.2 pmol / μL primers, and nuclease-free water to bring the volume to 10 μL. The cyclic sequencing conditions were: initial denaturation at 96°C for 1 min; followed by 25 cycles of 96°C for 10 s, 50°C for 5 s, and 60°C for 4 min; finally, maintenance at 4°C. The sequencing reaction was purified using an ethanol / EDTA precipitation method. Briefly, 1 μL of 125 mM EDTA and 1 μL of 3 M sodium acetate (pH 4.6) were added to the sequencing reaction, followed by 30 μL of 100% ethanol. The mixture was incubated at room temperature for 15 min, then centrifuged at 12000 rpm for 15 min at 4°C. The supernatant was then carefully discarded, and the precipitate was washed with 70 μL of 70% ethanol. After centrifugation at 12000 rpm for 5 min, the supernatant was removed, and the precipitate was air-dried. The dried precipitate was resuspended in 10 μL of Hi-Di formamide (Applied Biosystems). The resuspended sample was then loaded into an Applied Biosystems 3500 gene analyzer. Sequencing runs were performed according to the manufacturer's instructions. 3.2.1 The software visualizes the final sequencing data to identify mutations.

[0088] The experimental content and results are as follows:

[0089] (1) The antibody production capacity of B cells with specific knockout of BCAS2 is reduced.

[0090] B cells are the sole site of antibody production, and they can respond to different antigens by producing multiple types of immunoglobulins (Ig). Bcas2 (breast carcinoma amplified sequence 2), as an RNA-binding protein, can perform alternative splicing (AS) on RNA. This example first investigated the effect of B cell-specific Bcas2 knockout on class switch recombination (CSR). The CH12 cell line was used, which has been shown to effectively perform CSR and produce IgA under cytokine stimulation. In this example, Bcas2-deficient CH12 cells were generated by knocking down Bcas2 with siRNA. Figure 1 (AC). Compared with the control group, Bcas2-deficient cells showed increased IgM levels upon stimulation with CD40, IL-4, and TGF-β. - IgA + The proportion of positive cells decreased significantly. Figure 1 (D and E). Consistent with this, in the supernatant of Bcas2 knockdown cell culture, the level of antibody IgA after CSR was significantly reduced, while the level of the original IgM remained unchanged. Figure 1 In addition, Bcas2 deficiency led to a significant reduction in IgA class-switching recombinant transcription following cytokine stimulation, while the initial transcription level remained unchanged. Figure 1 (G). To further verify the role of Bcas2, a Bcas2 recovery experiment was performed in this embodiment, and the results showed that the protein expression level returned to normal. In addition, compared with the Bcas2 knockdown group, IgM - IgA + The proportion of cells increased significantly, indicating that Bcas2 has a significant impact on antibody production. Figure 1 (HJ). Bcas2 recovery experiments showed that AS events in CH12 cells were restored in the Bcas2 recovery group compared to the Bcas2 knockdown group. Figure 1 (Middle K).

[0091] After validating the cell line, this embodiment also constructed a mouse model to further investigate the effect of Bcas2 on antibody CSR. A conditional knockout (cKO) mouse model (genotype Bcas2fl / flAID-Cre) was established by mating mice with the Bcas2 locus containing two loxP sites (Bcas2fl / fl) with AID-Cre mice. In this model, the Bcas2 gene is knocked out in activated B cells (…). Figure 2(A). The principle is that the deletion of exons 3 and 4 of Bcas2 leads to the premature introduction of the stop codon during transcription, triggering mRNA degradation through a nonsense-mediated RNA degradation pathway. Genotypic analysis confirmed that the loxP site was successfully inserted into the intron region of Bcas2, and the expression of AID-Cre was validated in Bcas2-cKO mice. Figure 2 (B) Next, this embodiment measures the concentration of different types of antibodies in mice. Compared with the control group (Bcas2fl / fl) mice, the serum levels of high-affinity antibodies (including IgA, IgE, IgG1, IgG2b, and IgG2c) in Bcas2-cKO mice were significantly reduced, while the levels of IgM and IgG3 remained unchanged after conditional knockout of Bcas2 in B cells. Figure 2 (C and D). Consistent with changes in immunoglobulins in the blood, the proportions of GC B cells and IgA+GC B cells in the Painley lymph nodes of Bcas2-cKO mice were significantly lower than those in the control group (C and D). Figure 2 (E and F). These data indicate that Bcas2 is crucial for the generation of high-affinity, class-switching recombinant antibodies.

[0092] Since cell-mediated antibody regeneration (CSR) is an indispensable process in B cell antibody production, this embodiment further determined whether Bcas2 affects in vitro antibody CSR. Primary B cells were isolated from the spleens of Bcas2-cKO mice and control mice, and these cells were stimulated with LPS or LPS+IL-4 to induce class switching of different antibodies. Under stimulation with LPS and LPS+IL-4, exons of the Bcas2 gene were excised from the genome in primary B cells of Bcas2-cKO mice. Figure 2 (G). A significant decrease in Bcas2 mRNA and protein levels was also observed, while AID expression remained unchanged. Figure 2 (G and H). In Bcas2-cKO B cells, upon stimulation with LPS or LPS+IL-4, the expression of all post-CSR transcripts was significantly reduced, while no significant changes were observed in pre-CSR transcripts. Figure 2 This indicates that B cells exhibit a specific CSR deficiency in the absence of Bcas2. Furthermore, compared to the control group, the absence of Bcas2 in activated B cells significantly reduced the proportion of IgG1+ and IgG3+ B cells (I). Figure 2 (JL). In summary, these results reveal that Bcas2 has a broad regulatory role in B cell antibody CSR.

[0093] (2) BCAS2 affects antibody production in B cells by regulating alternative splicing of Cd72.

[0094] First, in this embodiment, we found that the expression of the Cd72 gene, which is involved in the CSR process, was significantly increased in the Bcas2 knockout group in mouse samples. Figure 3 (A). Immunofluorescence experiments showed that the expression level of Cd72 protein was increased in the Bcas2 knockout group. Figure 3 (B). DNA gel electrophoresis results revealed alterations in the AS event of Cd72. Figure 3 (C). Analysis using HOMER software showed that the RNA motif bound by Bcas2 is mainly the GAAGAA motif (C). Figure 3 (D). Therefore, in order to investigate whether Bcas2 regulates Cd72 and whether it directly affects the CSR of B cells, the GAAGAA motif in exon 3 of the Cd72 gene was deleted. Figure 3 The gene was mutated (E). Subsequently, the AS status of wild-type and mutant Cd72 cells was detected in CH12 cells transfected with empty vector, Bcas2 overexpression plasmid, and Bcas2 silencing RNA. This example confirmed the increase in Bcas2 gene expression during overexpression, and that Bcas2 expression was significantly decreased at both the transcriptional and protein levels during Bcas2 knockdown. Figure 3 (F and G). Following an endogenous splicing pattern, Bcas2 overexpression, compared to the empty vector, promoted the proportion of exon skipping in the CD72 vector gene; while transient knockdown of Bcas2 reduced the proportion of related exon skipping; deletion of the GAAGAA motif in CD72 significantly reduced Bcas2-dependent exon skipping (F and G). Figure 3 (H). Meanwhile, in the presence of normal Bcas2 expression, the Cd72 mutation significantly reduced the proportion of IgM-IgA+ cells, while this change was not observed in the absence of Bcas2. Figure 3 (I and J). These results indicate that Bcas2 further influences antibody CSRs in activated B cells by regulating Cd72 splicing in a manner dependent on the GAAGAA motif in the Cd72 transcript.

[0095] This embodiment then uses interaction proteomics analysis to identify proteins that interact with Bcas2. Through co-immunoprecipitation, silver staining, and co-localization staining analysis, several AS-related proteins were identified, including SRSF7, HNRNP K, HNRNP D, and DHX15. Protein validation was also performed in cells before and after LPS treatment. Figure 4 (AC). The interaction between Bcas2 and DHX15 was not detected in spleen B cells not stimulated by LPS. Figure 4(C). These results reveal the dynamic changes in Bcas2-interacting proteins during B cell activation. GO enrichment analysis showed that Bcas2-interacting proteins are mainly concentrated in the RNA splicing and processing processes (C). Figure 4 (D). This embodiment uses immunofluorescence to detect the interaction between Bcas2 and SRSF7. Figure 4 (E). Further investigation was conducted into the role of SRSF7 in Bcas2-mediated antibody CSR. First, decreased expression of both Bcas2 and SRSF7 was detected in both the Bcas2 knockdown and SRSF7 knockdown groups. Figure 4 In CH12 cells lacking Bcas2 and / or SRSF7, the proportion of IgA+ cells was significantly reduced (F). Figure 4 (G and H). Similarly, suppressing Bcas2 and SRSF7 expression alone or in combination leads to a decrease in the exclusion / inclusion ratio of Bcas3 and CD72 genes (G and H). Figure 4 (I). To investigate the structural basis of the interaction between Bcas2 and SRSF7, this embodiment generated three plasmids containing the deletion of the Bcas2 helix-coil (CC) domain—ΔCC1, ΔCC2, or both—in the pcDNA3.0 vector. Figure 4 (These three plasmids have been published in the following literature: BCAS2 and hnRNPH1 orchestrate alternative splicing for DNA double-strand break repair and synapsis in meiotic prophase I. Longjie Sun, etc. Cell Mol Life Sci. 2024 Nov 9; 81(1):449. doi:10.1007 / s00018-024-05479-7.). The sequences of the binding sites are conserved among different species. Figure 4 (L). Immunoprecipitation analysis was performed on 293T cells after FLAG-tagged transfection. Results showed that SRSF7-HA (purchased from BGI Genomics) bound strongly to the full-length Bcas2, but bound weakly to ΔCC1 and ΔCC2, with significantly reduced binding to ΔCC1CC2, indicating that both CC1 and CC2 sequences of Bcas2 are involved in the binding to SRSF7. Figure 4 In this embodiment, siSRSF7 and ΔCC1CC2 were co-transfected into CH12 cells, and the proportion of IgA+ cells was significantly reduced compared to the control group. Figure 4 The presence of MN in the middle of the body leads to a decrease in the exclusion / inclusion ratio of the Bcas3 and Nfat5 genes. Figure 4(O). The amino acid sequences of ΔCC1, ΔCC2, and ΔCC1CC2 are shown in Table 2.

[0096] Table 2. Amino acid sequences of ΔCC1, ΔCC2, and ΔCC1CC2

[0097] name amino acid sequence ΔCC1 NEKHIQDLNWQRKNMQLT(SEQ ID NO.26) ΔCC2 YEIERTIVQLENEIYQIKQQHGEANKEN(SEQ ID NO.27) ΔCC1CC2 NEKHIQDLNWQRKNMQLTYEIERTIVQLENEIYQIKQQHGEANKEN(SEQ ID NO.28)

[0098] (3) In HIGM1 patients, there is a loss of expression due to BCAS2 mutation, which leads to a lack of antibody production through the same mechanism.

[0099] Hyper-IgM syndrome (HIGM1) is a rare primary immunodeficiency disease, often characterized by recurrent infections in early life. Studies have shown that HIGM1 patients have significantly reduced levels of IgA and IgG in their blood, while IgM levels are normal or elevated. The genomics, proteomics lineage, and pathogenesis of HIGM1 remain unclear. In this study, we report a clinical case of HIGM1 in a child. The sample was obtained from the Department of Pediatrics, General Hospital of the Chinese People's Liberation Army. The patient was 7 months old and male. Clinical test results: SMZ(11-29)2024-1-17. Completed genetic testing revealed a CD40LG gene mutation, inherited from the mother, suggesting X-linked immunodeficiency with hyper-IgM. This case presented with low circulating levels of IgG, IgA, and IgE, but IgM levels were within the normal range. Figure 5 (A). Further analysis of IgA and IgG subclasses showed that serum levels of IgA1, IgG1, IgG3, and IgG4 in HIGM1 patients were significantly lower than in age-matched healthy individuals, while IgG2 levels remained unchanged. Figure 5 (B)

[0100] Next, this example investigated whether the pathological changes observed in HIGM1 were related to changes in the anti-atherosclerotic response (AS) of antibody CSR. This example found that Bcas2 expression was significantly reduced in the aforementioned HIGM1 patients (…). Figure 5 (C). Target genes of Bcas2, including Cd72, Rps6ka5, Las1l, and Nfat5, are transcribed upregulated in HIGM1 B cells. Figure 5 The expression pattern of D in mouse activated Bcas2-cKO B cells is consistent with that of D. Figure 5 In addition, the AS values ​​of Rps6ka5, Slc25a19, and Las1l changed significantly in HIGM1 (D). Figure 5(E). To gain a broader understanding of the molecular changes in HIGM1, peripheral blood mononuclear cells (PBMCs) from this patient were obtained and transcriptomic and proteomic studies were performed. Transcriptomic analysis revealed that the transcriptional levels of 4775 genes were significantly downregulated in the PBMCs of HIGM1 patients. Figure 5 (F and G). GO enrichment analysis showed that genes with altered transcriptional levels were mainly enriched in pathways related to double-strand break repair, mRNA splicing, immune responses, and B cell activation. Figure 5 (H). Genes targeted by Bcas2, including Rpn1 and Top1, are downregulated in HIGM1 patients, while Cd72 and Nfat5 are upregulated. Figure 5 (I, J, and K). Compared with healthy individuals, the expression of exon Cd72 was significantly enhanced ( Figure 6 (A). Sanger sequencing of genomic DNA from the blood of HIGM1 patients revealed a T-to-G mutation in the 3'UTR region of Bcas2 DNA. Figure 6 The mutation (B in the 3'UTR) suggests that this mutation may lead to instability of the Bcas2 transcript. Subsequently, in this example, the wild-type Bcas2 gene and the Bcas2 mutant gene (Bcas2-Mut) were overexpressed in the CH12 cell line. The mutation in the 3'UTR significantly altered the stability of Bcas2 mRNA. Figure 6 (C). In this embodiment, it was observed that the splicing changes of Bcas3 and Nfat5 in the Bcas2-mutant CH12 cell line were similar to those in the Bcas2 knockout CH12 cells. Figure 6 (D). Furthermore, the antibody production capacity of CH12 cell lines with the Bcas2 mutant gene was significantly reduced, similar to the effect observed when Bcas2 was knocked down in CH12 cell lines. Figure 6 (EF). In summary, the data from this embodiment reveal that Bcas2 may regulate AS in the pathogenesis of HIGM1.

[0101] Although embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents. The embodiments described above merely illustrate several implementations of the invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make several modifications and improvements without departing from the concept of the invention, and these all fall within the protection scope of the invention. Therefore, the protection scope of this invention should be determined by the appended claims.

Claims

1. The application of reagents for detecting the BCAS2 gene in the preparation of kits for predicting and / or diagnosing type I hyper-IgM syndrome, characterized in that, The reagent is used to detect the expression level of the BCAS2 gene.

2. The application according to claim 1, characterized in that, The reagent used to detect the expression level of the BCAS2 gene is a reagent used for genome, transcriptome and / or proteome sequencing.

3. The application according to claim 2, characterized in that, The expression level refers to the protein expression level and / or mRNA transcription level.

4. The application according to claim 2, characterized in that, The reagent used to detect the expression level of the BCAS2 gene is a biomolecular reagent that specifically binds to the BCAS2 gene or specifically hybridizes with the nucleic acid encoding the BCAS2 gene.

5. The application according to claim 4, characterized in that, The biomolecular reagents are selected from primers, probes, or antibodies.

6. The application of reagents for detecting mutations in the 3'UTR region of the BCAS2 gene in the preparation of kits for predicting and / or diagnosing type I hyper-IgM syndrome, characterized in that, The 3'UTR region mutation is a nucleotide sequence as shown in CACAAAAGGCATCTGAACTTTTAATGAACTTTGAAGGACAACAGCATCTTCCCAAAACCA, with a T→G mutation at position 31.

7. The application according to claim 6, characterized in that, The reagent used to detect mutations in the 3'UTR region of the BCAS2 gene is a reagent used for genome sequencing.

8. A predictive system for type I hyper-IgM syndrome, characterized in that, The prediction system includes a detection module and an analysis and judgment module. The detection module detects the expression level of the BCAS2 gene in the sample to be tested or detects mutations in the 3'UTR region of the BCAS2 gene in the sample to be tested. The 3'UTR region mutation is a nucleotide sequence as shown in CACAAAAGGCATCTGAACTTTTAATGAACTTTGAAGGACAACAGCATCTTCCCAAAACCA, with a T→G mutation at position 31. The expression level data or mutation data is transmitted to the analysis and judgment module, which predicts whether the sample to be tested suffers from type I high IgM syndrome based on the expression level data or the mutation data.

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