Probe set, kit and library construction method for motor neuron disease related genes

Abstract

The invention provides a probe group, a kit and a library construction method for motor neuron disease related genes. 241 related genes which are queried through a database and literature and newly identified by a team are comprehensively incorporated. The invention provides a probe group, a kit and a library construction method for related genes of motor neuron disease (ALS). The probe group comprises coding regions and adjacent shearing regions of 241 genes, and can capture specific coding regions and adjacent shearing regions and perform high sequencing depth, so that the sequencing target gene is accurate, and the sequencing cost is reduced. The gene detection panel provided by the invention can be used for carrying out economical and efficient genetic detection on patients with motor neuron disease (ALS), helps to find genetic causes and guides clinical practice.

Description

Technical Field

[0001] This invention relates to the field of gene detection technology, and in particular to a probe set, kit, and library construction method for genes related to motor neuron disease. Background Technology

[0002] Motor neuron disease mainly refers to amyotrophic lateral sclerosis (ALS), commonly known as "Lou Gehrig's disease." It is a chronic, progressive neurodegenerative disease that selectively affects the upper and lower motor neurons. Clinical manifestations include limb movement disorders, dysphagia, dysarthria, and dyspnea, which progress and seriously affect the patient's quality of life.

[0003] Diagnostic methods for motor neuron disease (ALS) involve many aspects, including a detailed clinical history, neurological physical examination, laboratory tests, electrophysiological examinations, and genetic testing. Since genetic factors play a crucial role in the pathogenesis of ALS, genetic testing can determine the type of gene mutation in a patient. Positive gene testing can accelerate the diagnostic process for ALS, allowing patients to begin drug treatment earlier. Some pathogenic gene variants are associated with disease-specific phenotypes, which can also be used for prognostic evaluation and genetic counseling. With the widespread implementation of gene testing and the potential for targeted gene therapies, abandoning the dichotomy of familial and sporadic ALS and supporting both genetically confirmed and non-genetically confirmed ALS—that is, the presence and absence of ALS gene mutations supporting a molecular subclassification of the disease—may be more helpful for early diagnosis, early treatment, and prognosis.

[0004] Motor neuron disease (ALS) progresses rapidly, and there is currently no specific treatment. Clinical supportive care, such as non-invasive ventilation for patients with respiratory muscle involvement, can improve symptoms and quality of life. Gastrostomy is also an effective treatment for providing nutritional support. Drugs already approved for clinical use, such as riluzole and eradavone, can slow disease progression and improve patients' daily lives, but their effects are limited. In 2024, the first antisense nucleotide drug targeting mutations in the superoxide dismutase 1 (SOD1) gene, "tofersen," was launched for the treatment of ALS patients with SOD1 mutations. This is the first gene-targeted therapy for ALS. SOD1-ALS is a rare inherited form of ALS, accounting for only about 2% of all ALS cases. However, in Chinese patients with fALS, SOD1 mutations are the most common, with most patients experiencing rapid disease progression and a survival period of no more than 3 years after onset. Clinical trials have shown that the use of this drug slows the progression of nerve damage and disease, and reduces the levels of neurofilament light chain protein (NfL) in neurons or their axons that are damaged. In addition, several clinical trials targeting other genes (such as FUS, ATXN2, and C9ORF72) are underway, including anti-transcriptional drugs, antibodies, small molecules, and cell and gene therapies. These trials aim to evaluate the efficacy and safety of new drugs, gene therapies, immunotherapies, stem cell therapies, and other emerging treatments for motor neuron disease (ALS), with the hope of achieving personalized precision treatment.

[0005] Given the crucial role of genetic factors in the diagnosis and treatment of motor neuron disease (ALS), identifying known pathogenic mutations through gene testing not only aids in diagnosis but also offers potential therapeutic guidance. This necessitates highly efficient and accurate gene testing methods. Traditional Sanger sequencing can only detect one DNA fragment at a time, limiting its high-throughput and high-coverage capabilities. Previous gene chip technologies, based on existing sequence designs, can only detect some known gene mutations, failing to effectively identify low-frequency, rare, or novel mutations. In recent years, next-generation sequencing (NGS) has seen continuous development, with whole-genome sequencing (WGS) and whole-exome sequencing (WES) capturing, enriching, and analyzing exons of the entire human genome and known genes, respectively. These methods have been widely used in recent years, but they also have drawbacks, such as high testing costs, high expenses, and the large volume of results obtained, leading to complex data processing and difficulties in variant interpretation.

[0006] Furthermore, with the continuous progress in genetic research on motor neuron disease (ALS) in recent years, an increasing number of pathogenic or risk genes for ALS have been discovered. It is essential to develop a comprehensive (including new, undisclosed pathogenic genes) and cost-effective genetic testing method for ALS to help clinicians and patients identify the causes and guide subsequent clinical practice. Summary of the Invention

[0007] This invention provides a multi-gene detection panel for motor neuron disease, wherein the gene detection panel includes the following motor neuron disease-related genes:

[0008] The present invention also proposes a probe set for motor neuron disease-related genes, wherein the probe set consists of detection probes generated for the aforementioned 241 motor neuron disease-related genes; The probe group was designed according to the following rules: (a) The probe length is 90-150 bp; (b) Probe design merges adjacent regions; (c) The probe design range is 20 bp extended to both sides of the target range; (d) The probe reference is the positive strand of the genome; (e) The common sequences on both wings of the probe are shown in SEQ ID NO.1 and SEQ ID NO.2; (f) Probe specificity is ensured by comparing whole-genome information in probe design; (g) The standard for probe GC content is 40%-60%. For every 10% increase in GC content deviation from the standard, the number of probes in that region increases by 50%. (h) Probes with approximate sequences in non-capture regions of the genome, with the number of probes in that region increasing by 100% for each approximate sequence; the approximate sequence is a sequence with at least 95% homology to the probe sequence; (i) Capture and detect the base sequences of oligonucleotide probes for the above 241 genes according to the above design rules.

[0009] Furthermore, the probes in the probe group are labeled with a marker, preferably biotinylated on the U and T bases.

[0010] The present invention also proposes a kit for detecting genes related to motor neuron disease, the kit comprising the above-mentioned probe set, PCR amplification primers for enriching and capturing target sequences, adapters for multi-sample hybridization capture, Hyb Blocks for blocking before library hybridization, and magnetic beads that are compatible with the markers on the probes.

[0011] This invention also proposes a method for constructing a library for detecting genes related to motor neuron disease, the library construction comprising the following steps: a) Sample collection and extraction: Collect the samples to be tested and extract genomic DNA; b) DNA fragmentation: breaking DNA into 150-200bp fragments; c) End repair: The fragmented DNA sample is repaired at the end and A-Tailing is added, and then the product is purified. d) Add adapters: Purify after adding adapters; e) Adding sequencing tags: Adding sequencing tags to the DNA library that has already been fitted with adapters by PCR amplification, wherein the DNA library contains adapters and is then purified; f) Target gene hybridization and capture: The DNA library is blocked before hybridization using Hyb Block, and then the capture probe set is used to hybridize and capture the library, and the captured target sequences are enriched.

[0012] The probe set, kit, and library construction method for ALS-related genes proposed in this invention involve 241 genes, including both reported and newly identified ALS-related genes, providing broad gene coverage. The probe set contains the coding regions and adjacent splice regions of these 241 ALS genes, enabling targeted capture of specific coding and adjacent splice regions and high sequencing depth, resulting in precise sequencing of target genes and reduced sequencing costs. Liquid-phase hybridization-based sequence capture technology enriches the target genes effectively, reducing sample size requirements. Attached Figure Description

[0013] Figure 1 This is a pedigree chart of consanguineous marriage in Embodiment 1 of the present invention and KIF3C Sequencing map of gene mutation sites; Figure 2 To knock down KIF3C The following figures show the apoptosis status of cells and the quantitative results of cleaved caspase-3 fluorescence intensity. Figure 3 For patients with motor neuron disease (ALS) in Embodiment 1 of the present invention MAIP1 Distribution map of rare and harmful gene variant sites; Figure 4 This refers to the overexpression in HEK293T cells in Example 1 of the present invention. MAIP1 WT&mut Effect on apoptosis (Figure); Figure 5In the ALS patient of Embodiment 1 of the present invention PARP16 Distribution of gene mutation sites; Figure 6 This refers to the knockdown of U2OS cells in Embodiment 1 of the present invention. PARP16 Diagram showing post-stress particle formation; Figure 7 Sample 1 in Embodiment 3 of the present invention SOD1 PCR sequencing verification diagram of gene mutation. Detailed Implementation

[0014] To facilitate understanding of the present invention, a more complete description will be given below with reference to the accompanying drawings. Several embodiments of the invention are illustrated in the drawings. However, the invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

[0015] 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 invention pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0016] Example 1: Preparation of capture probes for genes related to motor neuron disease (ALS) This embodiment provides a set of capture probes for capturing genes related to motor neuron disease (ALS), and the design of the capture probe set conforms to the following rules: (a) The probe length is 121 bp; (b) Probe design merges adjacent regions; (c) The probe design range is 20 bp extended to both sides of the target range; (d) The probe reference is the positive strand of the genome; (e) The common sequences on both wings of the probe are shown in SEQ ID NO.1 and SEQ ID NO.2;

[0017] (f) Probe specificity is ensured by comparing whole-genome information in probe design; (g) The standard for probe GC content is 40%-60%. For every 10% increase in GC content deviation from the standard content, the number of probes in that region increases by 50%. (h) Probes with approximate sequences in non-capture regions of the genome, with the number of probes in that region increasing by 100% for each approximate sequence; the approximate sequence is a sequence with at least 95% homology to the probe sequence.

[0018] The base sequences of oligonucleotide probes used to capture and detect 241 ALS-related genes according to the above design rules are shown in the table below:

[0019] The genes covered in this embodiment were selected by searching databases including the Online Mendelian Inheritance in Man (OMIM) database, the NCBI ClinVar database, the Neuromuscular Disease Center database, and research reports on motor neuron disease (ALS) related genes in various literature databases, as well as newly identified unpublished pathogenic and susceptibility genes, ultimately resulting in 241 genes.

[0020] Among them, this gene panel contains 3 new motor neuron disease (ALS)-related genes: KIF3C , MAIP1 , PARP16 .in, KIF3C Genes were identified through homozygous mapping studies based on WES data in families of motor neuron disease (ALS) excluding known disease-causing genes from consanguineous marriages or other recessive inheritance patterns. KIF3C The gene sequence variation c.C2272T:p.R758X was identified as the pathogenic variant in this family. The pedigree and PCR sequencing data of the mutation site are shown below. Figure 1 As shown.

[0021] Primary neurons from the cerebral cortex at E16.5 (gestational age 17.5 days) were cultured and infected with AAV-tdTomato + AAV-Ctrl and AAV-tdTomato + AAV-shKIF3C on day 5. Cells were fixed and collected on day 10, and apoptosis was detected using activated Case-3 antibody staining. Images were acquired using a laser confocal microscope. The results showed... KIF3C Knockdown led to a significant increase in activated casepase-3, suggesting early apoptosis. Cellular experiments showed... KIF3C The knockdown of this substance led to the activation of apoptosis, suggesting that the mutation caused... KIF3C Loss of gene function has potential pathogenic effects. Please refer to [link / reference]. Figure 2 The figure shows the apoptosis status after KIF3C knockdown. Primary cultured neurons were infected with AAV-tdTomato to label cells, and then cleaved caspase-3 was detected to assess the level of apoptosis. The bar chart on the right shows the quantitative results of cleaved caspase-3 fluorescence intensity. Compared with the pSuper control, the cleaved caspase-3 level in the KIF3C-shRNA treatment group was significantly increased, suggesting that KIF3C knockdown induced apoptosis.

[0022] MAIP1 Genes were identified through screening for mitochondrial-related gene variants, which revealed that rare and harmful variants were enriched in patients with motor neuron disease (ALS). MAIP1 Rare harmful variant sites and PCR sequencing images are shown below. Figure 3 As shown. PARP16 Genes were identified through genome-wide association analysis (GWAS) by finding that rare nonsynonymous variants were significantly enriched in patients with motor neuron disease (ALS). The Manhattan plot of the GWAS rare variant association analysis is shown below. Figure 3 As shown.

[0023] The probe sequence was synthesized using methods known in the art and uniformly mixed in a 1 ml TE buffer. 1 μl of this buffer was then used for PCR amplification using universal PCR primers (SEQ ID NO. 3 and SEQ ID NO. 4). The PCR amplification system consisted of: 1 μl of the probe solution; 1 μl of the forward primer (20 μM); 1 μl of the reverse primer (20 μM); and 47 μL of Platinum PCR Supermix (Thermo Fisher Scientific), for a total volume of 50 μL. Amplification conditions were: 95 °C for 2 min; 20 cycles of (94 °C, 30 s, 55 °C, 30 s, 72 °C, 30 s); and 72 °C for 5 min.

[0024]

[0025] The PCR product was purified using the MinElute PCR Purification Kit (Qiagen) and stored at -20°C. 500 ng of the purified PCR product was used for in vitro transcription using the Ambion SP6 megascript kit (Thermo Fisher Scientific). The transcribed RNA was purified using the Qiagen RNeasy minikit (Qiagen), and the purified product yielded the biotin-labeled probe of this embodiment.

[0026] In overexpression MAIP1 WT&mut In the HEK293T stable cell line containing the protein, after 72 hours of DOX-induced expression, cells were collected, and late apoptosis was detected in each group using PI single staining combined with flow cytometry. HEK293T cells were used as a negative control. Blank tubes, EGFP single-stained tubes, and PI single-stained tubes were set up for voltage regulation and compensation regulation, respectively. The control group underwent PI single staining, while each experimental group underwent EGFP and PI double staining. Please refer to [link to relevant documentation]. Figure 4 The figure shows the overexpression of [a specific substance] in HEK293T cells. MAIP1 WT&mut Effects on apoptosis. AE represents the control group and the overexpressing group, respectively. MAIP1 WT , MAIP1 G114V , MAIP1 L176P , MAIP1 R283W The cell apoptosis status at the time, Q2 and Q3 represent successful expression MAIP1 WT&mut -EGFP cells (EGFP) + Q1 and Q2 represent late-stage apoptotic cells (PI). + Successfully expressed MAIP1 WT&mut The proportion of late apoptosis in the -EGFP cell population (Q2 / (Q2+Q3) %) was statistically compared with that in the control group (Q1 / (Q1+Q2+Q3+Q4) %) (F). This was compared with the control and overexpressing cells. MAIP1 WT In comparison, overexpression MAIP1 L176P The number of cells leading to late apoptosis was significantly increased; similarly, compared with wild-type, only overexpression of MAIP1 L176P The above results collectively indicate that wild-type MAIP1 protein has no significant effect on apoptosis, while overexpression of mutant MAIP1 leads to increased late-stage apoptosis. L176P A significant pro-apoptotic effect was observed, suggesting that MAIP1...L176P Potential pathogenicity.

[0027] PARP16 Genes were identified through genome-wide association analysis that showed significant enrichment of rare nonsynonymous variants in ALS patients. PARP16 Distribution of variation Figure 5 As shown. In U2OS cells, after PARP16 was knocked down using siRNA, we systematically evaluated the temporal dynamics of stress granule (SG) formation induced by sodium arsenite (NaAsO2). Figure 6 Immunofluorescence staining was used to label PARP16, the stress granule marker protein G3BP1, and the cell nucleus (Hoechst), and cells were collected at 0, 15, 30, 60, and 120 minutes after treatment for observation. In contrast, the PARP16 knockdown group (si-PARP16) showed a significantly delayed stress granule formation kinetics under the same treatment conditions. At 15 minutes, significant G3BP1 aggregation was barely observable in the knockdown group; at 30 minutes, only a few scattered granular structures were observed; at 60 minutes, the number of stress granules was still significantly lower than that of the control group; and it was not until 120 minutes that the number of stress granules in the knockdown group gradually approached the level of the control group at the earlier time points. Overall, PARP16 knockdown significantly delayed the NaAsO2-induced stress granule formation process and reduced the number of stress granules at the same time points, suggesting that PARP16 may play a key role in the early stages of the stress response, and that the weakened PARP16 function caused by mutation may affect the stress response and play a role in the pathogenesis of ALS.

[0028] This embodiment also proposes a kit for detecting genes related to motor neuron disease (ALS), which contains the above-described probe set.

[0029] Example 2: Specific Capture and Sequencing This embodiment uses the capture probe set of motor neuron disease (ALS) related genes provided in Embodiment 1, and enriches the target sequence based on liquid-phase hybridization sequence capture technology. The liquid-phase hybridization technology route is as follows: 1) prepare a hybridization probe library, 2) enrich the target gene using the probe, and 3) sequence the enriched DNA sequence using a high-throughput sequencer. Using liquid-phase hybridization technology can effectively enrich the target gene, reduce the sample size requirement, and better control the sequencing cost.

[0030] The method for building this library includes the following steps: 1. DNA extraction and fragmentation DNA was extracted from four peripheral blood samples using the QIAamp DNA Blood Mini Kit (Qiagen).

[0031] Using the Bioruptor Pico DNA fragmentation instrument (Bioruptor Corporation), after the temperature of the cold cycler drops to 4°C, set the parameters ON 30 s and OFF 30 s as one cycle, and perform 10 cycles as one round, for a total of 3 rounds. After each round, place the sample on a shaker to mix thoroughly, centrifuge briefly, and then proceed to the next round of fragmentation.

[0032] Take 1µl of sample and use QSEP 100 biological fragment analyzer (Guangding Biotechnology Co., Ltd.) to detect fragments. After normal fragmentation, the main peak of the sample is about 150bp-200bp.

[0033] 2. End-of-pipe repair Take 10×T4 PNK Buffer and Natural dNTP Mix (Yisheng Biotechnology) from the kit stored at -20℃, thaw them on ice and mix thoroughly with a Vortex until there are no solid insolubles in the buffer. Remove the enzyme from the -20℃ freezer and place it on an ice box at -20℃.

[0034] The end-repair reaction system was prepared in 1.5 ml centrifuge tubes as shown below: incubate at 20°C for 30 min. The product was purified using Ampure XP magnetic beads and dissolved in 20 µl TE.

[0035]

[0036] 3. Add an "A" at the end (A-Tailing) Prepare reaction systems with "A" added to the end in 1.5 ml centrifuge tubes as shown below: incubate at 37°C for 30 min. The reaction product was purified using Ampure XP magnetic beads and dissolved in 25 µl TE buffer.

[0037]

[0038] 4. Adapter Connection Prepare the adapters for connecting the reaction mixture in 1.5 ml centrifuge tubes. Incubate at 20°C for 15 min. Purify the product using Ampure XP magnetic beads, dissolving it in 21 µl TE buffer.

[0039] 5. PCR amplification increases index The PCR reaction system and reaction conditions are as follows:

[0040] Run the following program on the PCR instrument:

[0041] The reaction product was purified using Ampure XP magnetic beads and dissolved in 50 µl TE buffer. The PCR product concentration was detected using a NanoDrop 1000 (NanoDrop Corporation). Library quantification was performed using Qubit 3.0; a library concentration >25 ng / µl was considered acceptable. QSEP 100 was used for detection; the main peak of the library should be around 220-320 bp, with no extraneous peaks before or after it.

[0042] 6. Hybridization Remove the Hyb Block and Hyb Buffer from the refrigerator and thaw them on ice. After thawing, place the Hyb Buffer in a metal bath and preheat it to 65°C.

[0043] Mix the sample library with the Hyb block according to the following system and label it as B.

[0044]

[0045] Melt the Hyb Buffer at room temperature. A precipitate will appear before heating. Mix well and preheat in a 65°C water bath. Once completely dissolved (without precipitate or turbidity), take 20 μl of Hyb Buffer and place it in a new 200 μl PCR tube. Cap the tube and label it A. Continue to incubate in a 65°C water bath until ready for use.

[0046] Take 5 μl of RNase Block and 2 μl of Probe and place them in a 200 μl PCR tube. Gently pipette and mix well. After a short centrifugation, place on ice and set aside. Label as C.

[0047] Set the PCR instrument parameters as follows: heat lid 100℃, 95℃, 5 min; 65℃, hold. Place PCR tube B on the PCR instrument and run the above procedure.

[0048] When the PCR instrument temperature drops to 65℃, place PCR tube A on the PCR instrument for incubation and cover with the PCR instrument's hot cap. After 5 minutes, place PCR tube C on the PCR instrument for incubation and cover with the PCR instrument's hot cap. After placing PCR tube C in the PCR instrument for 2 minutes, adjust the pipette to 13µl, transfer 13µl of Hyb Buffer from PCR tube A to PCR tube C, and transfer all the sample from PCR tube B to PCR tube C. Gently pipette 10 times to mix thoroughly, avoiding the generation of a large number of air bubbles. Seal the tube cap, cover with the PCR instrument's hot cap, and incubate at 65℃ overnight (8-16h).

[0049] 7. Capture Pre-parsed Wash Buffer 2 (1.8 ml per capture) and preheated on a ThermoMixer at 65°C.

[0050] Keep the hybridization product in PCR tube C on the PCR instrument. Add the hybridized product from PCR tube C to 200µL of Biotin-bed. Mix by pipetting 6 times. Place the tube on a centrifuge (10rpm / min) and incubate at room temperature for 30 minutes. Then place the centrifuge tube on a magnetic rack for 2 minutes and remove the supernatant.

[0051] Add 500µL of Wash Buffer 1 to the centrifuge tube, gently aspirate and shake 6 times to mix, resuspend the magnetic bead, and vortex mix the sample for 5 seconds. Incubate the sample at room temperature for 15 minutes.

[0052] Add 500µl of preheated Wash Buffer 2 at 65°C, vortex mix for 5 seconds, incubate on a ThermoMixer at 65°C for 10 minutes, and wash at 800 rpm.

[0053] Briefly centrifuge the tubes on a magnetic rack for 2 minutes, then remove the supernatant. Repeat the washing process twice, for a total of three times. For the final wash, thoroughly remove all Wash Buffer 2 (residue can be removed using a 10µl pipette).

[0054] Add 25µL of Nuclease-free water to the centrifuge tube, remove the centrifuge tube from the magnetic rack, and gently aspirate 6 times to resuspend the magnetic beads for later use.

[0055] 8. Enrichment After obtaining the DNA, it needs to be enriched into a DNA library. Prepare the Mix according to the table below.

[0056]

[0057] After purifying the PCR products, the library was quantified using the Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific), and the library fragment length was determined using QSEP 100. The library length was approximately 220-320 bp.

[0058] Example 3: Screening for genes related to motor neuron disease (ALS) This embodiment provides a method for screening genes related to motor neuron disease (ALS). This method involves sequencing the library constructed in Example 2 to screen for ALS-related genes. The library prepared in Example 2 was subjected to paired-end sequencing on an Illumina sequencing platform. Simultaneously, the tag sequences on the samples were also sequenced. The source of the sequencing data was analyzed, and the data was compared with the BWA and UCSC hg19 databases. The results are shown in the table below:

[0059] Sample 1 was detected SOD1 c.335G>A heterozygous mutation. The results were validated by Sanger sequencing and are reliable. Figure 7 ).

[0060] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0061] The embodiments described above are merely illustrative of several implementation methods of this application, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the protection scope of this application. Therefore, the protection scope of this patent application should be determined by the appended claims.

Claims

1. A motor neuron disease multi-gene detection panel comprising, The gene detection panel comprises the following motor neuron disease related genes: 。 2. A probe set for capturing genes related to motor neuron disease, characterized in that, The probe set is a detection probe generated for the 241 motor neuron disease related genes of claim 1; The probe set is designed according to the following rules: (a) The probe length is 90-150bp; (b) The probe design combines adjacent regions; (c) The probe design interval is 20bp extended to both wings of the target interval; (d) The probe reference is the genomic sense strand; (e) The common sequences of the probe wings are shown in SEQ ID NO. 1 and SEQ ID NO. 2; (f) The probe design ensures probe specificity by comparing whole genome information; (g) The GC content of the probe is standardized at 40%-60%, and for every 10% deviation from the standard content, the number of probes in this region is increased by 50%; (h) In the non-capture region of the genome, there are probes with similar sequences, and for every similar sequence, the number of probes in this region is increased by 100%; the similar sequence is a sequence with at least 95% homology to the probe sequence; (i) The base sequence of the oligonucleotide probe for capturing and detecting the above 241 genes according to the above design rules.

3. A kit for detecting a motor neuron disease-associated gene, characterized by, The kit comprises the probe set of claim 1 or 2.

4. A library construction method for detecting a motor neuron disease-related gene, characterized by, The method comprises the probe set of claim 1 or 2.

5. The library construction method of the motor neuron disease-related gene according to claim 4, wherein The library construction comprises the following steps: a) Sample collection and extraction: collect the sample to be tested and extract the genomic DNA; b) DNA fragmentation: break the DNA into 150-200bp DNA fragments; c) End repair, end repair and A-Tailing are performed on the broken DNA sample, and then the product is purified; d) Add adapters: add adapters and purify; e) Add sequencing tags: add sequencing tags to the DNA library with adapters through PCR amplification, and the DNA library includes adapters containing adapters, and then purify; f) Target gene hybridization and capture: use Hyb Block to block the DNA library before hybridization, then use the capture probe set to hybridize and capture the library, and enrich the captured target sequence.

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