Gene panel for detecting retinal pigmentosa and use thereof

Abstract

The application discloses a gene panel for detecting retinal pigment degeneration and application thereof, the gene panel comprising PRPF31, TFPT, NDUFA3, OSCAR, USH2A, EYS, RPGR, RHO, RP1, ABCA4, RDH12, CRB1, CNGA1, SNRNP200, CERKL, PDE6B, PROM1, CEP290, RP2, CYP4V2, RPE65, PRPF6 and CNGB1. The gene panel comprises PRPF31 and its upstream and downstream genes, and covers high-frequency mutation genes in an RP population. Through high-density probe design on target regions of the genes, not only the detection cost is reduced, but also precise diagnosis of clinical typing of RP and comprehensive genetic evaluation of PRPF31-RP patients can be realized.

Description

Technical Field

[0001] This application belongs to the field of biological detection technology, specifically relating to a gene panel for detecting retinitis pigmentosa and its application. Background Technology

[0002] Retinitis Pigmentosa (RP) is a group of inherited retinal degenerative diseases characterized by progressive photoreceptor cell dysfunction and apoptosis, exhibiting high clinical and genetic heterogeneity. Typical clinical manifestations include night blindness, progressive narrowing of the visual field (tunnel vision), and eventual loss of central vision, making it a leading cause of blindness in young and middle-aged adults. Genetically, RP can be inherited in autosomal dominant, autosomal recessive, and X-linked modes. The PRPF31 gene (pre-mRNA processing factor 31) has been identified as one of the main pathogenic genes causing autosomal dominant retinitis pigmentosa (adRP). The PRPF31 protein encoded by this gene is a core component of the mRNA spliceosome complex and is widely expressed in various tissues and cells; however, heterozygous mutations in this protein only lead to dominant retinal lesions. Currently, over 350 pathogenic mutations in the PRPF31 gene have been identified. These mutations are widely distributed across various exons and non-coding regions (introns and expression regulatory regions) of the PRPF31 gene, with non-coding mutations accounting for approximately 20%. Besides SNVs and InDels, mutation types include many large deletions / duplications (copy number variations, CNVs), large insertions, and structural rearrangements, ranging in size from several kb to tens of kb to over a hundred kb. The affected areas may involve multiple upstream and downstream genes (especially simultaneous deletions of parts of several adjacent upstream genes). CNV variations account for approximately 15% of all PRPF31 gene mutations. Therefore, accurate detection of gene mutations in PR patients is crucial for clarifying the molecular etiology of RP, differentiating clinical phenotypes caused by different genotypes, guiding family genetic counseling, and assessing prognosis.

[0003] Currently, traditional molecular biology methods such as Sanger sequencing, real-time quantitative PCR (qPCR), and MLPA can only detect or verify known sites in gene mutation detection technologies. They cannot be used to screen patients with unknown mutation sites, nor can they provide mutation information for other RP-related genes, nor can they indicate or rule out the potential contribution of other RP-related gene mutations to the patient's clinical phenotype. On the other hand, high-throughput sequencing technologies, represented by whole-genome sequencing (WGS) and whole-exome sequencing (WES), can theoretically capture genetic variations across gene coding regions and even the entire genome. However, the large data output of WGS and WES leads to high sequencing and bioinformatics analysis costs, complex data analysis processes, and high requirements for data storage and computing resources. Furthermore, WES still has significant limitations in detecting variations in non-coding regions, restricting its routine application in large-scale clinical screening.

[0004] Next-generation sequencing (NGS) gene panels based on target sequence capture have become the mainstream method for genetic disease diagnosis, offering significant advantages in cost control and throughput. However, conventional gene panel design has technical limitations for PRPF31 gene detection: firstly, due to limitations in the design principles and algorithms of capture probes, they can only detect SNV and InDel type mutations, failing to cover the detection of variants such as large deletions / duplications (CNVs), which account for a higher proportion; secondly, standard capture probes typically focus on exon regions, making it difficult for existing panels to effectively cover and detect pathogenic variants in non-coding regions located deep within introns or upstream / downstream regulatory regions of the gene (such as deep intron mutations affecting splicing or promoter region mutations). This technical limitation may lead to missed detection of RP patients caused by variants in the non-coding region or CNVs of the PRPF31 gene, thus affecting the accuracy of genetic testing and analysis results. Summary of the Invention

[0005] In view of this, the primary objective of this application is to provide a gene panel for detecting retinitis pigmentosa. The gene panel is based on the PRPF31 gene, and also incorporates upstream and downstream genes of the PRPF31 gene region, covering high-frequency mutated genes in the RP population. This allows for high-density probe design targeting the target regions of the aforementioned genes. The target regions include most of the non-coding regions and all exons of the PRPF31 gene and its upstream and downstream genes; as well as all exons of high-frequency mutated RP-related genes and their adjacent intron regions within 50-100 bp. This reduces detection costs while improving the clinical subtyping and diagnostic capabilities for PRPF31-RP, enabling comprehensive genetic assessment of PRPF31-RP patients.

[0006] To achieve the above objectives, this application adopts the following technical solution: One aspect of this application discloses a gene panel for detecting retinitis pigmentosa, which includes PRPF31 and its upstream and downstream related genes and frequently mutated RP-related pathogenic genes. The upstream and downstream related genes of PRPF31 are TFPT, NDUFA3, and OSCAR, and the frequently mutated RP-related pathogenic genes are USH2A, EYS, RPGR, RHO, RP1, ABCA4, RDH12, CRB1, CNGA1, SNRNP200, CERKL, PDE6B, PROM1, CEP290, RP2, CYP4V2, RPE65, PRPF6, and CNGB1.

[0007] Another aspect of this application discloses the use of the described gene panel in the preparation of products for diagnosing the clinical phenotype of retinitis pigmentosa or for genetic assessment of retinitis pigmentosa.

[0008] Another aspect of this application discloses a product for detecting gene panels, which includes a capture probe targeting the gene panel.

[0009] Another aspect of this application discloses the use of the gene panel, or the product described herein, in the preparation of a device for diagnosing the clinical phenotype of retinitis pigmentosa or for genetic assessment of retinitis pigmentosa.

[0010] Another aspect of this application discloses an apparatus for diagnosing the clinical phenotype of retinitis pigmentosa or for genetic assessment of retinitis pigmentosa, comprising: The detection module, including the reagents described in this application, is configured to perform panel detection on the genomic DNA genes of the sample to be tested to obtain raw data; The data filtering module is configured to filter raw data to obtain high-quality clean reads; The data analysis module is configured to perform bioinformatics analysis on clean reads. This application has at least the following beneficial effects: The gene panel provided in this application primarily detects the PRPF31 gene, while also covering upstream and downstream genes of PRPF31, as well as other frequently mutated RP-related pathogenic genes. High-density capture probes are designed using most of the non-coding regions (including intronic regions and upstream and downstream regulatory regions) and all exons of the PRPF31 gene and multiple upstream and downstream (especially upstream) genes, as well as all exons and adjacent 50-100 bp introns of frequently mutated RP-related pathogenic genes as target regions (Table 1). This not only reduces detection costs and improves clinical accessibility but also enhances the detection capability of genetic variations in the PRPF31 intronic region and expression regulatory region. Furthermore, in addition to the target regions, the detection capability for PRPF31 gene copy number variations (CNVs) is added. Therefore, it can indicate or rule out the potential contribution of other RP-related gene variations to the patient's clinical phenotype, thereby achieving a comprehensive genetic assessment of RP11 patients (PRPF31-RP), which is of great significance for accurate clinical diagnosis and treatment. Attached Figure Description

[0011] Figure 1 This is a schematic diagram of the device for detecting gene panels in this application.

[0012] Figure 2 To visualize probe coverage on a gene panel using IGV graphics, among other things, Figure 2 In Figure A, the probe coverage of the PRPF31 gene is shown. Figure 2 In Figure B, the probe coverage of the upstream and downstream genes of PRPF31 is shown.

[0013] Figure 3 This is a schematic diagram of the gene panel detection process in this application. Detailed Implementation

[0014] The embodiments of this application will be clearly and completely described below. The technical solutions in the embodiments described below are exemplary and only possible technical implementations of this application, not all possible implementations. Those skilled in the art can combine the embodiments of this application to obtain other embodiments without creative effort, and these embodiments are also within the protection scope of this application.

[0015] The first aspect of this application discloses a gene panel for detecting retinitis pigmentosa, which includes PRPF31 and its upstream and downstream related genes and frequently mutated RP-related pathogenic genes. The upstream and downstream related genes of PRPF31 are TFPT, NDUFA3 ​​and OSCAR, and the frequently mutated RP-related pathogenic genes are USH2A, EYS, RPGR, RHO, RP1, ABCA4, RDH12, CRB1, CNGA1, SNRNP200, CERKL, PDE6B, PROM1, CEP290, RP2, CYP4V2, RPE65, PRPF6 and CNGB1.

[0016] This application provides PRPF31 as the main gene, covering its upstream and downstream genes (TFPT, NDUFA3 ​​and OSCAR), as well as other frequently mutated RP-related pathogenic genes (USH2A, EYS, RPGR, RHO, RP1, ABCA4, RDH12, CRB1, CNGA1, SNRNP200, CERKL, PDE6B, PROM1, CEP290, RP2, CYP4V2, RPE65, PRPF6 and CNGB1). By designing comprehensive and high-density probes targeting the non-coding regions (including intronic regions and upstream and downstream regulatory regions) and all exons of PRPF31 and its upstream and downstream genes, as well as all exons and 50-100bp regions of adjacent introns of frequently mutated RP-related pathogenic genes, we can not only reduce detection costs, but also efficiently and accurately obtain genetic information on the PRPF31 gene and other RP-related genes in a single parallel detection. At the same time, we can indicate or rule out the potential contribution of other RP-related gene variants to the patient's clinical phenotype, thus achieving a comprehensive genetic assessment of RP11 patients (PRPF31-RP).

[0017] The second aspect of this application discloses the use of the gene panel described in this application in products for diagnosing the clinical phenotype of retinitis pigmentosa or for genetic assessment of retinitis pigmentosa.

[0018] Furthermore, this application discloses products for detecting gene panels. Specifically, these products can be any of reagents, kits, or chips. The term "reagent" refers to a collective term for various biological reagents capable of specifically identifying, capturing, or amplifying the target region (RP) within the aforementioned gene panel. Its core function is to enrich RP-related gene fragments from complex genomic DNA for subsequent high-throughput sequencing analysis.

[0019] In some examples, the reagent includes a capture probe targeting the gene panel described in this application, the sequence of which is designed to be complementary to the target region in the gene panel. As a specific example, the target regions of the capture probe sequence are designed as shown in Table 1. These target regions cover most of the non-coding regions (including intron regions and upstream and downstream regulatory regions) and all exons of PRPF31 and its upstream and downstream genes in the gene panel, as well as all exons and 50-100 bp regions of adjacent introns of frequently mutated RP-related pathogenic genes. This ensures that splice site variations can be detected as much as possible, including pathogenic deep intron mutation regions, and improves the detection capability of genetic variations in expression regulatory regions and CNV variations, thereby further achieving comprehensive genetic assessment.

[0020] It should be understood that, given a specific target area, the specific probe design and synthesis can be carried out with reference to existing technologies, and therefore there are no special limitations or instructions. In this application, the synthesis can be directly entrusted to a third party.

[0021] This application discloses, in a third aspect, the use of the gene panel described herein, or the product described herein, in a device for diagnosing the clinical phenotype and etiology of retinitis pigmentosa or for genetic assessment of retinitis pigmentosa. It further discloses related devices.

[0022] In this application, the "device" is a functional system integrating hardware and software for automating the entire process of analysis, from sample processing to report output. Through the synergistic action of its various modules, the device transforms raw sequencing data into clinically relevant diagnostic or genetic assessment information.

[0023] In some specific examples, the device includes a detection module, a data filtering module, a data analysis module, and a result output module.

[0024] The "detection module" is a functional unit that enables target sequence capture and high-throughput sequencing. It contains the reagents described in this application to detect the preset target region of the gene panel of the sample to be tested and obtain raw data.

[0025] The term "sample to be tested" refers to biological material derived from a subject and containing their genomic DNA. The selection of the sample should ensure that sufficient quality and quantity of DNA can be extracted for subsequent analysis. In this application, suitable subjects are those who have been definitively diagnosed with RP or asymptomatic individuals within a patient's family.

[0026] Specific examples of the test sample include: any one of the following: anticoagulated blood (such as EDTA-anticoagulated blood), serum, plasma, tissue (such as skin tissue), saliva, dried swabs, dried blood smears, and cultured cells (such as oral mucosal cells). Preferably, for ease of collection and transportation, the sample can be a dried blood smear or saliva. However, it is not limited to these; any other body fluid or tissue containing nucleated cells of the subject can be used as the test sample in this application.

[0027] When the detection module runs, it performs the following steps: Step S101: Extract genomic DNA from the sample to be tested.

[0028] This is the initial step in the detection process, aimed at isolating intact, purified genomic DNA from the sample to be tested. This is typically performed using methods known in the art, such as commercial DNA extraction kits, following the manufacturer's instructions.

[0029] It should be noted that the extracted DNA needs to undergo sample quality control, including testing for DNA purity, concentration, degree of DNA degradation, and the presence of contamination such as RNA or protein. For specific examples, the total DNA volume should be no less than 50 ng, with A260 / A280 = 1.8-2.0 and A260 / A230 > 2.0.

[0030] Step S102: Library construction.

[0031] In this step, the extracted genomic DNA is fragmented (e.g., using an ultrasonic disruptor or enzyme digestion) to form fragments of approximately 150-250 bp. The fragmented DNA is then end-repaired, an A-tail is added to the 3' end, and a sequencing adapter with a specific tag sequence (Index) is ligated to form a DNA library ready for sequencing.

[0032] Step S103: Probe hybridization capture.

[0033] The constructed DNA library was hybridized in liquid phase with capture probes designed for the target region of the gene panel described in this application (Table 1) to enable the probes to specifically bind to the DNA fragments in the target region. Subsequently, after eluting unbound probes and library fragments with unstable non-specific binding, post-PCR library amplification was performed to complete the specific capture of the target region library and achieve efficient enrichment of the target region.

[0034] Step S104: High-throughput sequencing.

[0035] The enriched and qualified target gene library is amplified, quantified, and then sequenced on a high-throughput sequencing platform, such as Illumina NovaSeq, NextSeq, MGIseq, etc., to obtain raw sequencing image data, which is then converted into raw nucleic acid sequence data (FASTQ format), i.e., raw data.

[0036] The "Data Filtering Module" is a functional unit that preprocesses the raw data generated by the detection module, ensuring the quality of subsequent analysis through strict quality control. When this module runs, it performs the following steps: Step S201: Remove the connector.

[0037] Because sequencing reads may exceed the length of the target DNA fragment, the sequence of the sequencing adapter will be read at the end of the read. This step uses specialized bioinformatics software commonly used in the field (such as Cutadapt, Trimmomatic, etc.) to identify and remove the adapter sequence from the reads.

[0038] Step S202: Remove low-quality reads.

[0039] The raw data undergoes base quality assessment, and reads with low sequencing quality are removed. Specific filtering criteria can be adjusted based on the specific experiment. For example, in a particular case, a sliding window method can be used to remove bases with a quality value below Q20 from the ends of reads, ultimately discarding reads shorter than 36 bp. After adapter removal and removal of low-quality reads, the resulting high-quality sequences are termed "clean reads."

[0040] The "Data Analysis Module" is the core unit for performing in-depth bioinformatics analysis on the filtered clean reads. When this module runs, it performs the following steps: Step S301: Sequencing quality assessment.

[0041] Statistical analysis was performed on indicators such as the amount of clean reads, alignment rate, Q20 / Q30 ratio, sequencing depth, and coverage uniformity to evaluate the overall quality of this sequencing experiment and ensure that the data can be used for subsequent variant detection.

[0042] Step S302: Sequence alignment.

[0043] Using well-known biological sequence alignment tools (such as BWA, Bowtie2, etc.), clean reads were aligned with human reference genomes (such as GRCh37 / hg19 or GRCh38 / hg38) to generate SAM / BAM files containing location information. Key capture metrics for the target region were statistically analyzed, including the capture chip coverage of the target region, sequencing depth, capture efficiency, coverage uniformity, and the number of successfully aligned sequences and bases, to verify the effectiveness of the capture experiment.

[0044] Step S303: Mutation detection.

[0045] The compared BAM files are processed and sorted, and variant detection software (such as GATK, VarScan, FreeBayes, CNVkit, Lumpy, etc.) is used to identify and detect variant sites. Depending on the analysis purpose and the software algorithm, the types of variants detected can cover a variety of forms. As a preferred example, the variant detection includes: SNVs (single nucleotide variants), InDels (insertions and deletions), and CNVs (copy number variants).

[0046] It is worth mentioning that this application introduces the detection of CNVs (copy number variations), which are identified by analyzing a negative baseline database established based on normal human samples using CNVkit software.

[0047] Step S304: Mutation annotation.

[0048] The detected variants are filtered through a pre-established mutation background library (a database of benign variants commonly found in the normal population) and mutation feature models to remove interference signals such as benign variants and sequencing errors from the original variant results identified in the previous analysis (including single nucleotide variants, insertion / deletion variants, copy number variants, etc.), thus obtaining reliable mutation results of the target gene and providing core data for subsequent pathogenicity analysis.

[0049] The "Results Output Module" automatically generates structured and standardized clinical test reports based on the annotation results from the data analysis module. Specifically, it clearly defines the association between detected variants (SNV, InDel, CNV, etc.) and RP, strictly classifying variants into five categories—"pathogenic, suspected pathogenic, undetermined significance, suspected benign, and benign"—according to the ACMG / AMP guidelines, and outputs standardized test reports.

[0050] The present application will be further illustrated below with reference to specific embodiments. It should be noted that the specific embodiments below are for illustrative purposes only and do not limit the scope of the present application in any way.

[0051] 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 this application belongs. The terminology used herein in the specification of this application is for the purpose of describing particular embodiments only and is not intended to be limiting of this application.

[0052] In addition, unless otherwise specified, methods without detailed conditions or steps are conventional methods, and the reagents and materials used are commercially available.

[0053] Example 1: Design and Synthesis of Capture Probes 1.1 Selection of target regions for gene panels Through extensive review of literature related to retinitis pigmentosa (RP) and genetic testing, and systematic searching of disease databases (such as OMIM, ClinVar, DISEASE, GeneReviews, RetNet, etc.): (1) Based on the reported variation types and location distribution characteristics of the PRPF31 gene, probes were designed for the PRPF31 gene and its upstream and downstream regions of approximately 50 kb (specifically, focusing on covering the adjacent upstream genes such as TFPT, NDUFA3, and OSCAR). The probes covered most of the sequence positions within this region, including all exons of the genes involved, most intron regions, and other non-coding regions such as upstream and downstream expression regulatory regions. For details, please refer to [link to relevant documentation]. Figure 2 .

[0054] (2) Select other RP-related genes with sufficient evidence to cause retinitis pigmentosa and relatively high mutation frequency, and target 19 core genes including USH2A, EYS, RPGR, RHO, RP1, ABCA4, RDH12, CRB1, CNGA1, SNRNP200, CERKL, PDE6B, PROM1, CEP290, RP2, CYP4V2, RPE65, PRPF6, and CNGB1 (based on RP patient cohort data reported in the literature, this group of genes can cover the genetic mutations of about 80% of the RP patient population). Probes were designed for all exons and 50-100bp regions of adjacent introns of each of these 19 genes.

[0055] 1.2 Probe Design and Synthesis In this embodiment, the gene panel corresponds to a target genomic region totaling 250.54 kb. A specific probe set of 275.67 kb (number of probes: 8390) was designed, achieving 100% coverage of the target region with a probe density of 3.2209×, ensuring targeted and accurate capture. The chemical synthesis of the panel probes was commissioned to a third-party organization, Aijitaikang. For details on the target genomic region corresponding to the gene panel and its probe coverage, please refer to Table 1 (reference genome is hg19 / GRCh37).

[0056] Table 1. Target genomic regions corresponding to gene panels and their probe coverage.

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[0066] Example 2: Sample processing 2.1 Sample Extraction Extraction was performed using a commercially available magnetic bead-based universal nucleic acid extraction kit, following the instructions.

[0067] 2.2 DNA Sample Quality Requirements DNA concentration was determined using a quantitative PCR instrument (Qubit 4.0 Fluorometer), with a total DNA volume of at least 50 ng. Agarose gel electrophoresis was used to assess DNA integrity and protein residue. Purity was determined using Nanodrop, with A260 / A280 = 1.8-2.0 and A260 / A230 > 2.0.

[0068] Example 3: Library Construction 3.1 DNA fragmentation, end repair, addition of an "A" to the 3' end (1) Take Fragment&ERA Buffer v3 and Fragment&ERA Enzyme Mix v3 out of the -20℃ freezer in advance, place them on an ice box to melt, vortex mix and centrifuge briefly, and place them on an ice box for later use.

[0069] (2) Prepare the reaction system on the ice box according to the table below, as shown in Table 2: Table 2 Reaction System

[0070] (3) Mix the contents using a pipette and place it in the PCR instrument. The temperature of the hot lid is 85℃. The reaction program is as follows: 4℃, 1 min; 30℃, 40 min; 65℃, 20 min; 4℃, Hold. (Control the enzyme digestion time to randomly break the genomic DNA into fragments of 150~250 bp.) 3.2 Connector Connection (1) Take the Adapter Ligation Buffer v3, Adapter (15 μM) and Adapter Ligasev3 out of the -20℃ freezer in advance, place them on an ice box to melt, vortex / or invert to mix and centrifuge briefly, and place them on an ice box for later use.

[0071] (2) Based on the amount of DNA used for library construction, dilute the Adapter (15 μM) to an appropriate concentration in advance.

[0072] (3) Prepare the reaction system on the ice box according to Table 3: Table 3 Reaction System

[0073] (4) Pipette the mixture and place it in the PCR instrument. Close the hot cap or leave it uncovered. The reaction procedure is as follows: 20℃, 15min; 4℃, Hold.

[0074] 3.3 Purification of Ligation Products (1) Take out the purification magnetic beads (IGT® Pure Beads) from the 4℃ refrigerator, mix well and equilibrate at room temperature for 30 min; after equilibration, vortex mix again for later use. At the same time, prepare 80% ethanol fresh with anhydrous ethanol and Nuclease-Free Water and store at room temperature for later use (preferably use freshly prepared 80% ethanol to ensure purification effect).

[0075] (2) Add 70 μL (for Illumina adaper) or 55 μL (for MGI adapter) of purified magnetic beads to the 100 μL reaction system after step 3.2. Vortex to mix, let stand at room temperature for 5 min; centrifuge briefly, place the PCR tube on a magnetic rack for 3 min, and wait for the solution to become clear.

[0076] (3) Keep the PCR tube on the magnetic rack, carefully remove the supernatant, add 200 μL of 80% ethanol to the tube, let stand for 30 seconds, and remove the supernatant; repeat this washing step once.

[0077] (4) After aspirating the supernatant from the last wash, centrifuge briefly and carefully aspirate the residual ethanol at the bottom of the tube with a 10 μL pipette (be careful not to aspirate the magnetic beads). Let stand at room temperature for 3-5 minutes to dry the magnetic beads and allow the residual ethanol to evaporate completely.

[0078] (5) Add 22 μL of Nuclease-Free Water to the PCR tube, remove the magnetic rack, vortex to mix, and let stand at room temperature for 2 min. Centrifuge briefly, place back on the magnetic rack and let stand for 2 min until the solution is clear. Take 20 μL of the supernatant, transfer it to a new PCR tube and label it for later use.

[0079] 3.4 PCR Amplification (1) Take out the PCR Master Mix and UDI Primer from the kit from the -20℃ refrigerator, thaw them on the ice box, invert them or vortex briefly to mix, centrifuge briefly, and store them on the ice box for later use.

[0080] (2) Prepare the PCR reaction system as shown in Table 4 on an ice box: Table 4 PCR reaction system

[0081] (3) Pipette the tubes to mix thoroughly and centrifuge briefly. Place the PCR tubes into the PCR instrument, set the hot cap temperature to 105°C, and run the reaction program shown in Table 5: Table 5 PCR reaction procedure

[0082] 3.5 Purification of PCR Amplification Products (1) Take out the purification magnetic beads (IGT® Pure Beads) from the 4℃ refrigerator, mix well and equilibrate at room temperature for 30 min; after equilibration, vortex mix again for later use. At the same time, prepare 80% ethanol fresh with anhydrous ethanol and Nuclease-Free Water and store at room temperature for later use (preferably use freshly prepared 80% ethanol to ensure purification effect).

[0083] (2) Add one volume (50 μL) of magnetic beads to the 50 μL reaction system after step 3.4, mix by pipetting or vortexing, and let stand at room temperature for 5 min. Centrifuge briefly, place the PCR tube on a magnetic rack for 3 min, and wait for the solution to become clear.

[0084] (3) Keep the PCR tube on the magnetic rack, carefully remove the supernatant, add 200 μL of 80% ethanol solution to the tube, let stand for 30 seconds, and remove the supernatant; repeat this washing step once.

[0085] (4) After aspirating the supernatant from the last wash, centrifuge briefly, place on a magnetic rack, and carefully aspirate the residual ethanol at the bottom of the tube using a 10 μL pipette (be careful not to aspirate the magnetic beads). Let stand at room temperature for 3-5 minutes to dry the magnetic beads and allow the residual ethanol to evaporate completely.

[0086] (5) Add 30 μL of Nuclease-Free Water to the PCR tube, remove the magnetic rack, vortex to mix, and let stand at room temperature for 2 min. Centrifuge briefly, place back on the magnetic rack and let stand for 2 min until the solution is clear. Take 28 μL of the supernatant, transfer it to a new PCR tube, label it, and set it aside for later use.

[0087] (6) Take 1 μL of the library, use the Qubit dsDNA HS Assay Kit reagent to determine the concentration on a Qubit 4.0 Fluorometer and record it; take another 1 μL of the library and use a fragment analyzer to perform fragment quality control to ensure that the library meets the requirements of subsequent experiments.

[0088] Example 4: Probe Hybridization and Capture 4.1 Library and probe hybridization (1) Add an appropriate amount of the library to be hybridized to the PCR tube (750 ng for hybridization of a single library, 500 ng / library for hybridization of multiple libraries) and label it; open the cap and place it in a vacuum concentrator to concentrate until completely dry.

[0089] (2) After the library is concentrated, prepare the hybridization reaction solution according to Table 6: Table 6 Hybridization reaction solution system

[0090] (3) Add 30 μL of the prepared hybridization reaction solution to the dried library, vortex for 30 s to fully dissolve the dried DNA at the bottom of the tube, and then briefly centrifuge.

[0091] (4) Place the PCR tube into the PCR instrument, set the hot cap temperature to 85℃, and run the reaction program: 80℃, 5min; 4℃, hold. Continue hybridization for 12~18h.

[0092] 4.2 Target Region DNA Capture, Cleaning, and Amplification (1) Cap Beads were removed in advance and equilibrated at room temperature for 30 minutes; Wash Buffer 1 was placed in a water bath and preheated to 37°C, and Wash Buffer 2 was placed in a water bath and preheated to 50°C.

[0093] (2) Add 50 μL of capture magnetic beads to the PCR tube, place it on a magnetic rack to adsorb for 1 min, wait for the solution to become clear, and then discard the supernatant.

[0094] (3) Add 180 μL Binding Buffer and mix well. After a brief centrifugation, place the tube on a magnetic rack for 1 min to absorb the solution. Once the solution is clear, discard the supernatant. Repeat this washing step 3 times. Remove the PCR tube from the magnetic rack, add 180 μL Binding Buffer, and mix by pipetting or vortexing.

[0095] (4) Quickly add the capture magnetic beads containing 180 μL Binding Buffer from step (3) to the PCR hybridization product in section 4.1 (keeping the hybridization product on the PCR instrument), and mix by pipetting. Remove the PCR tube from the PCR instrument and place it in a vertical rotary mixer (speed ≤10 rpm) for 30 min at room temperature. After incubation, centrifuge briefly, place on a magnetic rack for 2 min to adsorb, and discard the supernatant after the solution becomes clear.

[0096] (5) Add 150 μL of Wash buffer 1, gently pipette to mix, replace with a new tube cap, place in a vertical rotary mixer, wash at room temperature for 15 min (rotation speed not exceeding 10 rpm), centrifuge briefly, place on a magnetic rack for adsorption for 2 min, wait for the solution to become clear, and discard the supernatant. Repeat this step twice.

[0097] (6) Add 150µL of TargetSeq One®Wash Buffer 2 v2 preheated at 50℃, gently aspirate and mix, centrifuge briefly, and place in a constant temperature shaker or metal bath for 10 min at 50℃; centrifuge again briefly, gently aspirate and mix, and transfer all liquid (including magnetic beads) to a new PCR tube. Place the new PCR tube on a magnetic rack for 2 min to adsorb the liquid. After the solution becomes clear, discard the supernatant.

[0098] (7) Add 200µL of 80% ethanol, let stand for 30s, then completely remove the ethanol solution. Air dry the magnetic beads at room temperature to allow the residual ethanol to evaporate completely. Add 24µL of Nuclease-Free Water, remove the PCR tube, briefly vortex to resuspend the magnetic beads, and set aside. Prepare the PCR reaction solution according to Table 7: Table 7 PCR reaction solution system

[0099] After mixing with a pipette, place the mixture into the PCR instrument and run the reaction program in Table 8. The hot cap temperature is 105℃. Table 8 PCR reaction procedure

[0100] 4.3 Purification after PCR reaction (1) Take out the purified magnetic beads, mix well, and equilibrate at room temperature for 30 min. Add 55 μL (1.1 times the volume) of purified magnetic beads to the PCR product in section 4.2, mix well by pipetting, and let stand at room temperature for 5 min. Collect the liquid on the tube wall by instant centrifugation, place the PCR tube on a magnetic rack for adsorption for 3 min, and wait for the solution to become clear.

[0101] (2) Discard the supernatant, add 200 μL of 80% ethanol solution to the tube, and let stand for 30 seconds. Discard the supernatant, and repeat the 80% ethanol washing operation once to completely discard the supernatant. Let stand at room temperature for 3-5 minutes to air dry the magnetic beads until the residual ethanol has completely evaporated.

[0102] (3) Add 25 μL of Nuclease-Free Water, remove the PCR tube, pipette and mix well, and let stand at room temperature for 2 min. After a brief centrifugation, put it back on the magnetic rack and let it adsorb for 2 min until the solution is clear.

[0103] (4) Take 23 μL of supernatant, transfer it to a new PCR tube and label it; store the captured library in a -20℃ freezer for later use.

[0104] (5) Take 1 μL of the purified library and use the Qubit dsDNA HS Assay Kit reagent to determine and record the concentration on a Qubit 4.0 Fluorometer; take another 1 μL of the library and use a fragment analyzer to perform fragment quality control to ensure that the library meets the requirements of subsequent experiments.

[0105] Example 5: Sequencing Based on the results of Examples 3 and 4, sequencing was performed on either an Illumina or MGI high-throughput gene sequencer.

[0106] Example 6: Data Analysis After generating the raw sequencing data in Example 5, bioinformatics analyses, including "data filtering and quality control → reference sequence alignment analysis → result filtering and annotation," were used to accurately detect mutation sites in the target region. The specific steps are as follows: (1) Data filtering and quality control: For each sample's sequencing library and the FASTQ format raw data generated by the corresponding sequencing channel, adapter sequence removal and low-quality reads (sequence fragments with unqualified sequencing quality values) filtering operations are first performed to obtain high-quality clean reads. At the same time, a detailed quality control report is generated, covering core indicators such as adapter residue, Q20 and Q30 values ​​before and after data filtering, GC base content, quality control pass rate (QCrate), and base quality distribution, to ensure the reliability of subsequent analysis data.

[0107] (2) Sequence alignment: Using BWA bioinformatics software, the clean data obtained in the previous step was aligned with the human reference gene hg19 / GRCh38. Simultaneously, repetitive sequence labeling (removing repetitive sequence interference generated by PCR amplification), sequence sorting and other subsequent processing were completed to finally generate a BAM file. Further, key capture indicators of the target region were statistically analyzed, including the coverage of the capture chip to the target region, sequencing depth, capture efficiency, coverage uniformity, and the number of sequences and bases that could be successfully aligned, to verify the effectiveness of the capture experiment.

[0108] (3) Results filtering and annotation: SNVs, InDels, SVs and CNVs calling were performed using GATK software, CNVkit software and Lumpy software (the CNVs identification was obtained by CNVkit software analysis based on the negative baseline database established by normal human samples). Then, the original mutation results (including single nucleotide variants SNVs, insertion and deletion variants InDels, copy number variants CNVs, structural variants SVs, etc.) identified in the previous analysis were filtered through the pre-established mutation background library (database of common benign variants in normal populations) and mutation feature model, etc., to remove interference signals such as benign variants and sequencing errors, and finally obtain reliable mutation results of the target gene, providing core data for subsequent pathogenicity analysis.

[0109] Example 7: Interpretation of Results The core of result interpretation is to clearly define the association between detected variants (SNV, InDel, CNV, SV, etc.) and RP. Variants are strictly categorized into five classes according to the ACMG / AMP guidelines: "pathogenic, suspected pathogenic, of unknown significance, suspected benign, and benign." Accurate interpretation is achieved through a combination of initial software assessment and manual correction. The specific steps are as follows: (1) Loading databases: Load authoritative disease databases (such as OMIM, ClinVar, GeneReviews, etc., which store known RP pathogenic variant information) and custom databases (containing internally accumulated RP variant data of the Chinese population, benign background variant library, etc.) to provide data support for subsequent variant analysis.

[0110] (2) Collect variation information: Read the basic variation information (such as mutation site, variation type), population variation frequency data (the probability of the occurrence of the variation in the normal population), disease association data (whether the variation has been reported to be related to RP), variation function prediction results (predicting whether the variation will affect gene / protein function), and variation conservation (the degree of evolutionary conservation of the gene region in different species) for each input variation information to ensure comprehensive information coverage.

[0111] (3) Processing and interpreting evidence: (a) Evidence assessment: Using the collected variant information, each variant is assessed to determine whether it meets the criteria for pathogenicity or benignity—pathogenicity evidence is divided into four levels: extremely pathogenic (PVS), highly pathogenic (PS), moderately pathogenic (PM), and weakly pathogenic (PP); benignity evidence is divided into three levels: independently benign (BA), strongly benign (BS), and weakly benign (BP). (b) Evidence combination and classification: In accordance with the evidence combination rules of the ACMG / AMP guidelines, the evidence assessed above is integrated, and each variant is assigned a final classification (pathogenic, suspected pathogenic, undetermined significance, suspected benign, benign). (c) Manual correction: Combining the examinee's clinical phenotype (such as RP symptoms and course), family history (whether relatives have the disease), and the latest research literature, the initial judgment results of the software are adjusted and optimized to ensure that the interpretation results are highly consistent with clinical reality.

[0112] Example 8: Clinical Sample Validation Using the gene detection panel designed in Example 1 and the experimental and analytical methods described in Examples 2-7, panel gene detection was performed on samples from 38 RP patients with PRPF31 gene mutations from 10 families. In each of the 10 families, at least one RP patient had been found to have a pathogenic PRPF31 gene variant of P or LP by other detection methods (such as WES, QPCR, or Sanger). The types of these PRPF31 gene variants included SNV, InDel, CNV, etc., and the variant sites were distributed in intron regions, exon regions, and upstream expression regulatory regions (also covering multiple genes adjacent to PRPF31 upstream).

[0113] The test results show that the gene panel designed in this application can not only detect the above-mentioned PRPF31 gene mutation sites with 100% accuracy, but also effectively detect mutation sites on other RP genes in these patients that may affect the RP phenotype, as detailed in Table 9: Table 9 Clinical Sample Validation Results

[0114] Note: For mutations in other RP-related genes, Table 9 only shows P / LP / VUS sites that are consistent with the disease phenotype inheritance pattern, as well as P / LP sites that are inconsistent with the inheritance pattern.

[0115] It should be noted that this application is not limited to the above-described embodiments. The above embodiments are merely examples, and any embodiments with the same structure and effect as the technical concept within the scope of this application are included in the technical scope of this application. Furthermore, various modifications that can be conceived by those skilled in the art to the embodiments, and other ways of constructing by combining some of the constituent elements of the embodiments, without departing from the spirit of this application, are also included in the scope of this application.

Claims

1. A gene panel for detecting retinitis pigmentosa, characterized in that, It includes PRPF31 and its upstream and downstream related genes, as well as frequently mutated RP-related pathogenic genes. Among them, the upstream and downstream related genes of PRPF31 are TFPT, NDUFA3 ​​and OSCAR, and the frequently mutated RP-related pathogenic genes are USH2A, EYS, RPGR, RHO, RP1, ABCA4, RDH12, CRB1, CNGA1, SNRNP200, CERKL, PDE6B, PROM1, CEP290, RP2, CYP4V2, RPE65, PRPF6 and CNGB1.

2. The use of the gene panel as described in claim 1 in the preparation of products for diagnosing the clinical phenotype and etiology of retinitis pigmentosa or for genetic assessment of retinitis pigmentosa; Preferably, the product is one of a reagent, a reagent kit, or a chip.

3. A product for detecting gene panels, characterized in that, It includes a capture probe targeting the gene panel of claim 1.

4. The product as described in claim 3, characterized in that, The target regions of the capture probe sequence are most of the non-coding regions and all exons of PRPF31 and its upstream and downstream related genes. The non-coding regions include intron regions and upstream and downstream regulatory regions; as well as all exons and 50-100 bp regions of adjacent introns of RP-related pathogenic genes with high frequency mutations. The specific target regions are shown in Table 1.

5. The use of the gene panel as described in claim 1, or the product as described in claim 3 or 4, in the preparation of a device for diagnosing the clinical phenotype of retinitis pigmentosa or for genetic assessment of retinitis pigmentosa.

6. A device for diagnosing the clinical phenotype and etiology of retinitis pigmentosa or for genetic assessment of retinitis pigmentosa, characterized in that, include: The detection module includes a capture probe as defined in claim 3 or 4, which is configured to perform gene panel detection on the genomic DNA of the sample to be tested to obtain raw data; The data filtering module is configured to filter raw data to obtain high-quality clean reads; The data analysis module is configured to perform bioinformatics analysis on clean reads. And a results output module, which is configured to output a standardized test report based on the results of bioinformatics analysis.

7. The apparatus as claimed in claim 6, characterized in that, The sample to be tested can be any one of the subject's anticoagulated blood, serum, plasma, tissue, saliva, dried swab, dried blood smear, or cultured cells.

8. The apparatus as claimed in claim 6, characterized in that, When the detection module is running, it performs the following steps: extracting genomic DNA from the sample to be tested; library construction; probe hybridization capture; and high-throughput sequencing.

9. The apparatus as claimed in claim 6, characterized in that, When the data filtering module is running, the steps include: removing connectors from the raw data and filtering out low-quality reads.

10. The apparatus as claimed in claim 6, characterized in that, When the data analysis module is running, it performs the following steps: sequencing quality assessment, sequence alignment, variant detection, and variant annotation. Preferably, the mutation detection includes SNVs, InDels, and CNVs, wherein CNV mutation detection is performed based on a negative baseline database established from normal human samples.

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