Probe and chip for screening genes related to limb development defects and application of probe and chip

Abstract

The invention discloses a probe and a chip for screening genes related to limb development defects and application of the probe and the chip, belongs to the technical field of biomedicine, and provides the probe for screening the genes related to the limb development defects. The limb development defect related genes comprise a polydactylia related gene, a concomitant deformity related gene, a giant finger / toe disease related gene, a short finger / toe disease related gene, a polyarticular contracture disease related gene, a hand and foot deformity related gene and a congenital ulna and radius fusion related gene; the probes have nucleotide sequences as shown in SEQ ID NO. 1 to SEQ ID NO. 802. According to the invention, 140 genes corresponding to limb development defects are taken as target genes, probes are designed, and a gene sequencing chip is constructed, so that the detection rate is improved, the sequencing price is reduced, the cost performance is improved, and the method can be applied to clinical gene detection on a large scale and has a good effect.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology, and in particular relates to a probe, a chip and applications thereof for screening genes related to limb development defects. Background Art

[0002] Limb Developmental Defects (LDDs) are common birth defects. LDDs are numerous and include polydactyly, syndactyly, megadactyly, brachydactyly, arthrogryposis multiplex, split-hand / foot malformation, and congenital radioulnar synostosis. These conditions involve developmental abnormalities such as limb differentiation disorders and excessive growth, which can lead to malformations and disability. LDDs have both genetic and non-genetic causes (such as maternal illness, infection, and medication). Therefore, clarifying the cause through medical history analysis and molecular testing, and developing appropriate prevention and treatment plans, is the most effective approach for precise prevention, control, and treatment. However, precise prevention and treatment of LDDs still faces numerous challenges. The main reasons are: LDDs have high phenotypic heterogeneity, making typing difficult, and the cause of approximately one-third of LDD patients remains unknown. LDDs also have high genetic heterogeneity, with numerous pathogenic genes (over 200) identified, making genetic testing challenging. The use of candidate gene testing leads to a high false-negative rate. Chromosome microarrays, whole-exome sequencing, or whole-genome sequencing are expensive and unsuitable for large-scale screening, severely limiting their clinical application and resulting in a high birth rate of children with defects. Summary of the Invention

[0003] To solve the above technical problems, the present invention proposes a probe, chip and application thereof for screening genes related to limb development defects. The present invention takes 140 genes corresponding to limb development defects as target genes, designs probes, and constructs gene sequencing chips, which improves the detection rate while reducing the sequencing price, thereby improving the cost-effectiveness. It can be widely used in clinical genetic testing with good results.

[0004] To achieve the above-mentioned purpose, the present invention provides a probe for screening genes related to limb development defects, wherein the probe specifically captures genes related to limb development defects, wherein the genes related to limb development defects include genes related to polydactyly, genes related to syndactyly, genes related to macrodactyly, genes related to brachydactyly, genes related to multiple arthrogryposis, genes related to cleft limbs and genes related to congenital fusion of the radius and ulna; the probe is the nucleotide sequence shown in SEQ ID NO.1 to SEQ ID NO.802.

[0005] Preferably, the polydactyly-related genes include GLI3, LMBR1, HOXA13, HOXD13, ZNF141, IQCE, GLI1, KIAA0825, KIF7, GLI2, RAB23, ZSWIM6, BBS2, ARL6, BBS4, MKKS, BBS7, BBS9, BBS10, BBS12, SMO, EVC2, EVC, WNT7A, TBX5, HYLS1, FGFR2, MKS1, TMEM216, CEP290, TCTN3, DDX59, CPLAINE1, DHCR7, SALL1, KIAA0586, and DYNC2. LI1, IFT43, INTU, CCND2, MAP3K20, CC2D2A, TCTN2, IFT80, DYNC2H1, TTC21B, WDR19, NEK1, WDR35, IFT140, IFT172, CEP120, IFT52, IFT81, PITX 1. PIK3R2, AKT3, BBS1, BBS5, PTCH1, TFAP2A, CD96, GDF5, TMEM67, EBP, ALX3, PORCN, INPP5E, NAA10, PIK3CA, OFD1, FGFR1, GPC3, TBX3, BMP4, SD One or more of CCAG8, CREBBP, and TRIM32; the syndactyly-related genes include one or more of FBLN1, CPLAIN1, DCHS1, MMP1, CREBBP, LRP4, FGF16, CCNQ, and BHLHA9; the macrodactyly-related gene is PIK3CA; the brachydactyly-related genes include BMP2, GDF5, CHST11, EXT1, HDAC6, NBAS, RAD21, RUNX2, IHH, FGFR3, PDE3A, ROR2, BMPR1B, CHSY1, COL2A1, HOXD13, NOG, and PRMT 7. One or more of PTHLH, TRPS1, TRPV4, PDE4D, SMAD4, FGFR1, FGFR2, HOXA13, SHH, and TP63; the arthrogryposis-related genes include one or more of ERGIC1, LGI4, SCYL2, MMP1, TOR1A, VPS33B, TNNI2, PIEZO2, TPM2, GLE1, RYR1, SYNE1, NEB, COL4A5, NOTCH3, COL11A1, COL6A1, PIK3CA, COL1A1, COL1A2, COL25A1, COL2A1, and COL6A3;The genes associated with cleft limb deformity include one or more of FGF13, POLL, BTRC, DLX5, DLX6, FBXW4, GJA1, HOXD13, TP63, WNT10B, RUNX2, COL1A1, COL1A2, COL2A1, COL6A2, GDF5, and GLI3; the genes associated with congenital fusion of the radius and ulna include one or more of HOXA11, MECOM, and SMAD6.

[0006] The present invention also provides the use of the probe in preparing a product for screening genes related to limb development defects.

[0007] Preferably, the product is a chip or a kit.

[0008] The present invention also provides a chip for screening genes related to limb development defects, wherein the chip is fixed with the probe.

[0009] Preferably, the chip is a liquid phase chip.

[0010] The present invention also provides the use of the chip in preparing a screening kit for genes related to limb development defects.

[0011] The present invention also provides a kit for screening genes related to limb development defects, which includes the chip.

[0012] Compared with the prior art, the present invention has the following advantages and technical effects:

[0013] The present invention provides probes and chips for screening genes related to limb developmental defects. Based on the mutant genes of common acrodysplasia, more than 140 frequently mutated genes were selected and designed into a chip. This chip can add new target gene sequences and is not limited by existing genes. While reducing sequencing costs, it will greatly improve the efficiency and cost-effectiveness of genetic testing. It plays an important role in popularizing clinical applications. Compared with the clinical application of whole-exome sequencing, it can greatly reduce the cost of testing. The present invention uses 50 clinical cases confirmed by whole-exome sequencing for verification, demonstrating that the detection rate of the chip of the present invention can reach 100%, greatly promoting clinical application and achieving good social benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0015] Figure 1Flowchart for preparation of microarray for screening genes related to limb development defects;

[0016] Figure 2 Radar chart of chip performance for screening genes related to limb development defects. DETAILED DESCRIPTION

[0017] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0018] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each smaller range between any intermediate value within a stated value or stated range and any other stated value or intermediate value within the stated range is also encompassed by the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0019] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0020] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0021] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0022] Example 1

[0023] 1. Research Methods

[0024] 1. By searching published literature and the Human Online Mendelian Inheritance Database, we searched for known pathogenic genes for polydactyly, syndactyly, macrodactyly, brachydactyly, multiple arthrogryposis, cleft limbs, and congenital fusion of the radius and ulna. We selected relatively frequently mutated genes as target genes for inclusion in sequencing chips.

[0025] 2. Probe hybridization capture sequencing liquid phase chip is a targeted sequencing technology that combines probe hybridization technology with high-throughput sequencing technology. First, the probe is designed based on the sequence of the target region, and a biotin-labeled probe pool is synthesized and prepared. Then, the probe pool is used to perform probe hybridization with the whole genome library to specifically capture and enrich the library containing the target region. Second-generation sequencing and bioinformatics analysis are performed to obtain the base sequence information and mutation information of the target region.

[0026] 2. Preparation of a Screening Chip for Genes Related to Limb Development Defects

[0027] Step 1. Probe design:

[0028] We searched the HGMD database and local genome databases to identify 140 genes associated with limb developmental defects (see Table 1). Probe design was performed using these exons as target regions. The design results are as follows: the target region of gene exons is 810.92 kbp, the number of probes is 802, the probe density is 3.1x, and the average probe length is 100 nt. The nucleotide sequences of the probes are shown in SEQ ID NOs. 1 to 28754 (see Table 1).

[0029] Table 1 140 genes associated with limb development defects

[0030]

[0031]

[0032]

[0033]

[0034] Step 2: Synthesize and prepare RNA probe pool:

[0035] According to the base sequence of each probe, the probe sequence is synthesized by chemical synthesis and mixed to prepare a biotin-labeled RNA probe pool.

[0036] Step 3: Construction of whole genome library:

[0037] The extracted genomic DNA (gDNA) is enzymatically cut and sheared to obtain fragmented DNA with an average length of 200bp. The ends are then blunted, 5' phosphorylated, and A is added to the 3'. Finally, the second-generation sequencing adapters are connected to both sides of the fragments using ligase, and the Pre-PCR library is amplified to obtain a whole-genome library.

[0038] Step 4: Probe hybridization capture:

[0039] The whole genome library was mixed with a biotinylated probe pool, adapter blocking sequence, and hybridization reaction buffer. The mixture was incubated at 80°C for 5 minutes to denature the double-stranded DNA of the whole genome library into single strands. The temperature was then lowered to 50°C and incubated for 14 hours to allow the probe to fully hybridize and bind to the single-stranded DNA library containing the target region sequence.

[0040] After the hybridization reaction is completed, T1 magnetic beads are added, and the streptavidin on their surface binds to the biotin of the probe. The T1 magnetic beads are then recovered using a magnet to recover the probe and the single-stranded library of the target region, while the library to which the probe cannot bind is removed.

[0041] The recovered T1 magnetic beads and probes were rinsed with a buffer solution to further remove the single-stranded library non-specifically bound to the probe.

[0042] The PCR reaction was carried out using the linker sequence as a primer and the single-stranded library as a template. The product was then purified by magnetic beads to obtain a targeted library of the target region.

[0043] Step 4: Next-generation sequencing and data analysis:

[0044] The targeted library is sequenced using high-throughput sequencing technology, and then the base sequence of the target region is obtained through bioinformatics analysis. The type and frequency of base mutations in the target region are determined through sequence alignment. Figure 1 Shown is a flow chart for preparing a screening chip for genes related to limb development defects.

[0045] 3. Clinical testing using a gene screening chip related to limb developmental defects

[0046] Patients submitted for testing were diagnosed by Pediatric Surgery and Hand and Foot Surgery departments at the Ninth Affiliated Hospital of Shanghai Jiao Tong University School of Medicine, Qilu Hospital of Shandong University, West China Hospital of Sichuan, Xiangya Hospital of Central South University, and Xi'an Red Cross Hospital. Samples submitted for testing primarily consisted of, but were not limited to, pre-coagulated peripheral venous blood. Genomic DNA was extracted, and the test results for some samples are shown in Table 2.

[0047] Table 2 Test results of some samples

[0048]

[0049]

[0050]

[0051] This chip was used to sequence peripheral blood samples from congenital limb developmental defects such as brachydactyly, polydactyly, and syndactyly. A total of 308 samples were sequenced, and 281 samples were found to have a clear gene mutation, a detection rate exceeding 90%. The results of the 32 samples are attached in Table 2 above.

[0052] IV. Performance Determination of Chip Screening for Genes Related to Limb Development Defects

[0053] The average values ​​of the QC rate, alignment rate, coverage, capture rate, and uniformity indicators of the two samples were calculated and plotted as a radar chart.

[0054] Table 3 Comparison of chip expected performance and actual test performance

[0055] index QC rate (%) Comparison rate (%) Uniformity (%) Capture rate (%) Coverage (%) 20%X-coverage (%) expected 90 98 20 70 99 97 actual 95.43 99.76 17.66 87.38 99.82 98.76

[0056] like Figure 2 The chip performance radar chart is shown in Table 3 and Figure 2 It can be seen that the various indicators of the chip of the present invention are good and meet expectations.

[0057] Coverage indicates the coverage of the target region by sequencing data; uniformity indicates how much the actual sequencing depth at each site deviates from the average sequencing depth, generally measured as the proportion of regions reaching 20% ​​of the average sequencing depth. The probes achieve over 99% coverage of the target region, meeting the personalized needs of the target region and providing a high-quality, efficient chip. This ensures highly uniform probe coverage of the target region, resulting in a probe that balances data coverage, uniformity, and capture efficiency.

[0058] In liquid-phase probe hybridization capture sequencing, capture efficiency is used to describe the specificity of probe hybridization. It refers to the ratio of sequences falling in the target region to sequences on the genome, and has an inverse relationship with the off-target rate. During the probe sequence design process, the present invention fully considers the similarity between the probe sequence and the non-target region to avoid matching the probe sequence with more sequences in the non-target region. In addition, the probe sequence avoids high-copy sequences in the genome to avoid binding to high-copy sequences during the hybridization reaction.

[0059] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A probe for screening genes related to limb development defects, characterized in that: The probe specifically captures genes related to limb development defects, including genes related to polydactyly, syndactyly, macrodactyly, brachydactyly, multiple arthrogryposis, cleft limbs, and congenital fusion of the radius and ulna. The probe is a nucleotide sequence shown in SEQ ID NO.1 to SEQ ID NO.

802.

2. The probe according to claim 1, characterized in that The polydactyly-associated genes include GLI3, LMBR1, HOXA13, HOXD13, ZNF141, IQCE, GLI1, KIAA0825, KIF7, GLI2, RAB23, ZSWIM6, BBS2, ARL6, BBS4, MKKS, BBS7, BBS9, BBS10, BBS12, SMO, EVC2, EVC, WNT7A, TBX5, HYLS1, FGFR2, MKS1, TMEM216, CEP290, TCTN3, DDX59, CPLAINE1, DHCR7, SALL1, KIAA0586, and DYNC2LI1. , IFT43, INTU, CCND2, MAP3K20, CC2D2A, TCTN2, IFT80, DYNC2H1, TTC21B, WDR19, NEK1, WDR35, IFT140, IFT172, CEP120, IFT52, IFT81, PITX1, PIK3R2, AKT3, BBS1, BBS5, PTCH1, TFAP2A, CD96, GDF5, TMEM67, EBP, ALX3, PORCN, INPP5E, NAA10, PIK3CA, OFD1, FGFR1, GPC3, TBX3, BMP4, SDCC One or more of AG8, CREBBP, and TRIM32; the syndactyly-related genes include one or more of FBLN1, CPLAIN1, DCHS1, MMP1, CREBBP, LRP4, FGF16, CCNQ, and BHLHA9; the macrodactyly-related gene is PIK3CA; the brachydactyly-related genes include BMP2, GDF5, CHST11, EXT1, HDAC6, NBAS, RAD21, RUNX2, IHH, FGFR3, PDE3A, ROR2, BMPR1B, CHSY1, COL2A1, HOXD13, NOG, and PRMT7 , PTHLH, TRPS1, TRPV4, PDE4D, SMAD4, FGFR1, FGFR2, HOXA13, SHH, TP63; the arthrogryposis-related genes include one or more of ERGIC1, LGI4, SCYL2, MMP1, TOR1A, VPS33B, TNNI2, PIEZO2, TPM2, GLE1, RYR1, SYNE1, NEB, COL4A5, NOTCH3, COL11A1, COL6A1, PIK3CA, COL1A1, COL1A2, COL25A1, COL2A1, and COL6A3;The genes associated with cleft limb deformity include one or more of FGF13, POLL, BTRC, DLX5, DLX6, FBXW4, GJA1, HOXD13, TP63, WNT10B, RUNX2, COL1A1, COL1A2, COL2A1, COL6A2, GDF5, and GLI3; the genes associated with congenital fusion of the radius and ulna include one or more of HOXA11, MECOM, and SMAD6.

3. Use of the probe according to claim 1 or 2 in preparing a screening product for genes related to limb development defects.

4. The application according to claim 3, characterized in that The product is a chip or a kit.

5. A chip for screening genes related to limb development defects, characterized in that: The chip is fixed with the probe according to claim 1 or 2.

6. The chip according to claim 5, characterized in that: The chip is a liquid phase chip.

7. Use of the chip according to claim 5 or 6 in preparing a screening kit for genes related to limb development defects.

8. A kit for screening genes related to limb development defects, characterized in that: The kit comprises the chip according to claim 5 or 6.

Images