CN112813156A - DNA library for detecting and diagnosing bone development disorder pathogenic gene and application thereof - Google Patents
DNA library for detecting and diagnosing bone development disorder pathogenic gene and application thereof Download PDFInfo
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Abstract
The invention relates to a DNA library for detecting and diagnosing skeletal developmental disorder pathogenic genes by a targeted high-throughput sequencing technology and application thereof, wherein the library comprises 507 skeletal developmental disorder pathogenic genes. 507 skeletal developmental disorder pathogenic genes are optimized, a probe pool is designed, a target region library aiming at the 507 skeletal developmental disorder pathogenic genes is established, sequencing is carried out on the library by utilizing a high-throughput sequencing technology, pathogenic mutation is searched, and genetics and molecular biology bases are provided for clinical diagnosis. The 507 genes related to the invention comprise pathogenic genes of genetic diseases which are almost all clinically expressed by skeletal developmental disorders, such as collagen dysplasia, metaphyseal dysplasia, osteogenesis imperfecta, bone density reduction, mucopolysaccharide storage disease, cartilage dysplasia and the like, and have important significance and clinical value for diagnosis, differential diagnosis and accurate treatment of the skeletal developmental disorders.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to a DNA library for detecting and diagnosing skeletal developmental disorder pathogenic genes by a targeted high-throughput sequencing technology and application thereof.
Background
Skeletal Dysplasia (SD) is a genetic disease affecting the composition and structure of bone and cartilage tissues, and is clinically manifested by abnormal growth and development of various skeletal tissues, such as short and small body, joint dislocation, skull and limb deformity, abnormal curvature of spine, change in bone density, and the like. The incidence of the disease is about 3/10000, with a conservative estimate of millions of patients nationwide. Patients often have skeletal deformities of different parts of the body, and clinically have different phenotypes, with chondrodysplasia and osteogenesis imperfecta being the most common types. The general symptoms are short stature, joint dislocation, deformity of head and limbs, abnormal curvature of spine, change of bone density, etc. The disease is sometimes mild, and can also be severe until spontaneous abortion before birth. However, most of them develop rapidly in the developmental stage, and the disease condition is severe, and even combine with other organ dysfunction, so once the manifestation of the skeletal developmental disorder is found, the cause of disease should be determined in time, and targeted treatment and consultation should be adopted.
Currently, skeletal developmental disorders include dozens of major types, such as collagen dysplasia, metaphyseal dysplasia, osteogenesis imperfecta, bone density reduction, mucopolysaccharidosis, chondrodysplasia, etc., depending on their molecular genetic basis, etiology and phenotype, and only by using genetic testing, a definitive diagnosis of skeletal developmental disorders can be made. At present, the traditional Sanger sequencing method is mostly adopted in clinical laboratories to detect gene mutation, and if a plurality of pathogenic genes of skeletal developmental disorders are simultaneously detected, the workload is huge, the detection efficiency is low, more importantly, precious DNA samples are wasted, the detection cost of gene diagnosis is obviously increased, and the large-scale application of the gene diagnosis in clinical molecular diagnosis is severely restricted. Therefore, there is a need to find a new method for detecting the mutation of the pathogenic gene of the skeletal developmental disorder, which can improve the diagnosis accuracy, reduce the cost and labor intensity, and improve the timeliness.
Disclosure of Invention
The invention aims to provide a DNA library for detecting and diagnosing skeletal developmental disorder pathogenic genes by a targeted high-throughput sequencing technology, which can improve the diagnosis accuracy and reduce the cost and labor intensity.
The second object of the present invention is to provide the use of said DNA library.
The purpose of the invention is realized by the following technical scheme: a DNA library for diagnosing skeletal developmental disorders based on high throughput sequencing technology, the library comprising 507 skeletal developmental disorder-causing genes, wherein the 507 skeletal developmental disorder-causing genes are as follows:
ABCC6、ABCC9、ACAN、ACP5、ACTB、ACTG1、ACVR1、ADAMTS10、 ADAMTS17、ADAMTSL2、AGA、AGPS、AKT1、ALDH3A2、ALG9、ALPL、 ALX1、ALX3、ALX4、AMER1、ANKH、ANKRD55、ANO5、ANOS1、ANTXR2、 AP2S1、APC、ARHGAP31、ARID1B、ARSB、ARSE、ARVCF、ASXL1、ATP6V0A2、 ATP6V0A4、ATP7A、B3GALT6、B3GAT3、B4GALT7、BAAT、BAZ1B、BGN、 BHLHA9、BMP1、BMP2、BMP4、BMPER、BMPR1B、BRAF、BRF1、BUB1、 BUB1B、BUB3、C21orf2、CA2、CANT1、CASR、CAV1、CC2D2A、CCBE1、 CCDC141、CCDC8、CCNQ、CCR6、CD247、CD96、CDC45、CDC6、CDH3、 CDKN1C、CDT1、CEP120、CEP290、CEP57、CHD7、CHEK2、CHRNA1、 CHRND、CHRNG、CHST14、CHST3、CHSY1、CKAP2L、CLCN5、CLCN7、 CLIP2、COG1、COL10A1、COL11A1、COL11A2、COL1A1、COL1A2、COL2A1、 COL3A1、COL5A1、COL5A2、COL9A1、COL9A2、COL9A3、COLEC11、COMP、 COMT、CORO7-PAM16、CREB3L1、CREBBP、CRTAP、CTGF、CTNNB1、 CTNS、CTSA、CTSC、CTSK、CUL7、CYP26B1、CYP27A1、CYP27B1、CYP2R1、 DCHS1、DDR2、DHCR24、DHODH、DLL3、DLX3、DLX5、DLX6、DMP1、 DNASE1L3、DOCK6、DOK7、DUSP6、DVL1、DYM、DYNC2H1、DYNC2LI1、 EBP、EFNB1、EFTUD2、EIF2AK3、ELN、ENPP1、EOGT、EP300、EPHX1、 ERCC1、ERF、ESCO2、EVC、EVC2、EXT1、EXT2、EXTL3、EZH2、FAH、 FAM111A、FAM20C、FAS、FAT4、FBLN1、FBN1、FBN2、FBXW4、FERMT3、 FEZF1、FGF10、FGF16、FGF17、FGF23、FGF3、FGF8、FGF9、FGFR1、FGFR2、 FGFR3、FIG4、FKBP10、FLNA、FLNB、FLRT3、FMN1、FN1、FREM1、FUCA1、 FZD2、GALNS、GALNT3、GBA、GCDH、GDF3、GDF5、GDF6、GJA1、GJB2、 GJB6、GLB1、GLE1、GLI3、GMNN、GNA11、GNAS、GNE、GNPAT、GNPTAB、 GNPTG、GNS、GORAB、GP1BB、GPC3、GPC6、GPX4、GREM1、GTF2IRD1、 GUSB、HBB、HDAC4、HDAC8、HES7、HESX1、HFE、HGD、HGSNAT、 HIRA、HNF4A、HNRNPA1、HNRNPA2B1、HOXA11、HOXA13、HOXD13、 HPGD、HS6ST1、HSPG2、IARS2、ICK、IDH1、IDH2、IDS、IDUA、IFITM5、IFT122、IFT140、IFT172、IFT43、IFT80、IGF1R、IHH、IKBKG、IL11RA、IL17RD、 IL1RN、IL2RA、IL2RB、IMPAD1、INPPL1、IRF5、IRX5、JAG1、JMJD1C、 KAT6A、KAT6B、KCNJ2、KCNJ8、KIF22、KIF7、KISS1R、KL、KRAS、LBR、 LEMD3、LFNG、LIFR、LIMK1、LMBR1、LMNA、LMX1B、LONP1、LPIN2、 LRP4、LRP5、LTBP2、MAFB、MAN2B1、MAN2C1、MASP1、MATN3、MEGF8、 MEOX1、MESP2、MGAT2、MGP、MITF、MKS1、MMP13、MMP2、MMP9、 MNX1、MSX2、MUSK、MYCN、NAGLU、NANS、NDUFS8、NEK1、NEU1、 NF1、NFIX、NIPBL、NKX3-2、NLRC4、NLRP3、NOG、NOTCH2、NOTCH3、 NPPC、NPR2、NSD1、NSDHL、NSMCE2、NSMF、OBSL1、OCRL、OFD1、 ORC1、ORC4、ORC6、OSTM1、P3H1、P4HB、PAM16、PANK2、PAPSS2、 PCNT、PCYT1A、PDE3A、PDE4D、PDGFRB、PEX10、PEX11B、PEX12、PEX13、 PEX14、PEX16、PEX19、PEX2、PEX26、PEX3、PEX5、PEX6、PEX7、PHEX、 PHYH、PIGV、PIK3CA、PITX1、PLEKHM1、PLOD1、PLOD2、PLS3、POLR1C、 POLR1D、POP1、POR、PPIB、PPP3CA、PRKAR1A、PROK2、PROKR2、PTDSS1、 PTEN、PTH1R、PTHLH、PTPN11、PTPN2、PTPN22、PYCR1、RAB23、RAB33B、 RAD21、RAPSN、RASGRP2、RB1、RBM8A、RBPJ、RECQL4、RFC2、RMRP、 RNU4ATAC、ROR2、RPGRIP1L、RREB1、RUNX2、SALL1、SALL4、SBDS、 SCARB2、SCARF2、SEC23A、SEC24C、SEC24D、SEMA3A、SERPINF1、 SERPINH1、SETD2、SF3B4、SFRP4、SFTPC、SGSH、SH3BP2、SH3PXD2B、 SHH、SHOX、SKI、SLC17A5、SLC25A12、SLC26A2、SLC29A3、SLC34A1、 SLC34A3、SLC35D1、SLC39A13、SLC40A1、SLC4A1、SLC9A3R1、SLCO2A1、 SLCO5A1、SMAD3、SMAD4、SMARCA2、SMARCAL1、SMC1A、SMC3、 SNRPB、SNX10、SNX22、SOST、SOX10、SOX6、SOX9、SP7、SPAG8、SPARC、 SPECC1L、SPRY4、SQSTM1、SSR4、STAT3、STAT4、SULF1、SUMF1、TACR3、 TBCE、TBL2、TBX1、TBX15、TBX3、TBX4、TBX5、TBX6、TBXAS1、TCF12、 TCIRG1、TCOF1、TCTN3、TGDS、TGFB1、TGFB2、TGFBR1、TGFBR2、 THPO、TJP2、TMCO1、TMEM165、TMEM216、TMEM38B、TMEM67、 TNFRSF11A、TNFRSF11B、TNFSF11、TP53、TP63、TRAPPC2、TREM2、TRIP11、 TRIP13、TRPS1、TRPV4、TTC21B、TWIST1、TYK2、TYROBP、UROS、VCP、 VDR、VPS33A、WDR11、WDR19、WDR34、WDR35、WDR60、WISP3、WNT1、WNT10B、WNT3、WNT5A、WNT6、WNT7A、XYLT1、XYLT2、ZMPSTE24。
the DNA library provided by the invention covers almost all genetic diseases which are clinically manifested by skeletal developmental disorders, such as collagen protein generation failure, metaphyseal dysplasia, osteogenesis imperfecta, bone density reduction, mucopolysaccharidosis, cartilage dysplasia and the like, and comprises 507 skeletal developmental disorder pathogenic genes. These 507 gene selections are based on the internationally reported and recognized clinical pathogenic gene database (OMIM database, HGMD database, ClinVar database). The 507 gene pathogenic gene variation can cause diseases alone or in combination with the disease to cause more serious and complex clinical phenotypes, and the invention can comprehensively sequence the 507 gene pathogenic gene variation at one time for the first time.
The invention designs a probe pool which can cover the exon of the gene and the adjacent +/-20 bp intron region according to 507 skeletal developmental disorder pathogenic genes, utilizes the probe target capture to establish a target region library containing the 507 skeletal developmental disorder pathogenic genes, utilizes a high-throughput sequencing technology to sequence the library, searches pathogenic mutation, determines the genetic cause of skeletal developmental disorder, and provides a theoretical basis of genetics and molecular biology for clinical diagnosis.
The invention also provides an application of the DNA library in preparing a diagnostic kit, wherein the kit is used for searching pathogenic genes of skeletal developmental disorders and further diagnosing the skeletal developmental disorders.
Further, the application comprises the following steps:
1) collecting clinical data and clinical biological samples of the patient with the skeletal developmental disorder, preferably, the clinical biological samples refer to various samples from human body, including but not limited to peripheral blood, body fluid, tissue and organ samples from the subject, such as saliva, hair or oral mucosa of the subject.
2) Extracting the genome DNA of the sample, preferably, the extraction method comprises a DNA extraction kit or various manual extraction methods;
3) quantifying the extracted genomic DNA and constructing a library, preferably, the quantification methods include, but are not limited to, fluorescence quantification methods and electrophoresis; wherein, the library construction comprises the following steps:
a) fragmenting genomic DNA, preferably by methods including but not limited to ultrasonication, transposase cleavage, and restriction endonuclease cleavage;
b) carrying out end repair on the fragmented genomic DNA and simultaneously carrying out 3' end addition of A;
c) ligating the product of adding A at the 3' end to a linker for amplification of the ligation-effective product, preferably from a high throughput sequencing library kit;
d) performing PCR amplification on the ligation product by using a universal primer, and adding a complete joint, wherein the universal primer is preferably from a high-throughput sequencing and library building kit;
e) the 507 skeletal development disorder pathogenic genes are targeted and captured in a target area by using probes aiming at the 507 skeletal development disorder pathogenic genes, and preferably, the capture method comprises but is not limited to liquid phase probe capture and solid phase chip hybridization capture;
f) washing off the uncaptured library, and only reserving the target area library;
j) obtaining a target region capture library.
4) Performing quantitative operation on the library, preferably, the quantitative method includes but is not limited to fluorescence quantitative method and electrophoresis;
5) performing high throughput sequencing of the library using a sequencing device, preferably, the sequencing device includes, but is not limited to, a Novaseq series, a Hiseq series, a Nexeseq series, a BGIseq series second generation nucleic acid sequencer;
6) and comparing the obtained sequencing data by bioinformatics, and obtaining the related information of the pathogenic site by mutation interpretation.
A diagnostic kit comprising said DNA library.
Compared with the prior art, the invention has the advantages that:
1. the skeletal developmental disorder is related to various hereditary diseases, has different degrees of severity and various clinical manifestations, is from death before birth to lateral bending of the spine, even has no obvious symptom, and is difficult to make accurate etiological diagnosis clinically. The invention aims to establish a new method for quickly, accurately and high-flux detecting the pathogenic genes of the skeletal developmental disorder, thereby helping to understand pathogenesis and laying a foundation for assisting clinical diagnosis, prognosis judgment, prenatal diagnosis and accurate treatment.
2. The gene diagnosis is helpful for clinical genetic counseling, prenatal diagnosis and the like. Through clinical genetic consultation, family members can know the possibility of suffering from diseases; parents or the patients can guide the breeding through gene detection, and the health of future breeding offspring is ensured. Skeletal developmental disorders are often one of the clinical phenotypes of genetic diseases, and accurate health and reproductive guidance can be provided only by performing genetic testing. Finding carriers of asymptomatic young virulence genes in families also helps to develop home medical and rehabilitation programs earlier.
3. The DNA library of the application refers to a large number of documents by the inventor on the basis of developing high-throughput gene detection service for many years and accumulating a large number of clinical cases of the bone dysplasia in China, and finally selects 507 skeletal dysplasia pathogenic genes by adopting a disease-gene correlation screening method provided by ClinGen and other power databases, thereby giving consideration to comprehensiveness, accuracy and scientificity. 507 skeletal developmental disorder pathogenic genes are optimized, a probe pool is designed, a target region library aiming at the 507 skeletal developmental disorder pathogenic genes is established, sequencing is carried out on the library by utilizing a high-throughput sequencing technology, pathogenic mutation is searched, and genetics and molecular biology bases are provided for clinical diagnosis. The 507 gene detection areas related by the invention can detect almost all genetic diseases which take skeletal developmental disorders as clinical manifestations, such as collagen dysplasia, metaphyseal dysplasia, osteogenesis imperfecta, bone density reduction, mucopolysaccharide storage disease, cartilage dysplasia and the like, and have important significance and clinical value for diagnosis and differential diagnosis of skeletal developmental disorders.
4. The invention adopts a high-throughput sequencing technology to sequence the target region capture library, and can simultaneously detect all exons and adjacent regions of 507 skeletal developmental disorder pathogenic genes involved in the invention in one sequencing reaction. Compared with the traditional sequencing technology, the method has the advantages of obviously improved detection efficiency and obviously reduced cost, has great advantages in aspects of bone development disorder gene excavation and pathogenic gene screening, and is an efficient, reliable and economic bone development disorder pathogenic gene detection technology.
5. In conclusion, the DNA library and the application thereof have the characteristics of accuracy, flexibility, rapidness and low cost; through clinical evaluation, the invention has good auxiliary diagnosis value on the skeletal developmental disorder.
Drawings
FIG. 1 is the quality control information for the 507 skeletal developmental disorder-causing genes of proband samples to establish a target region-targeting DNA library in example 1;
FIG. 2 is data coverage information for sequencing the target region of 507 bone dysplasia causative genes of the proband sample in example 1;
FIG. 3 is data amount information for sequencing the target regions of 507 skeletal developmental disorder-causing genes of proband samples in example 1;
FIG. 4 is a pedigree map of the pedigree of skeletal developmental disorders described in example 1, in which arrows indicate probands, solid icons indicate patients, and open icons indicate healthy individuals.
Detailed Description
The invention provides a DNA library for detecting and diagnosing a pathogenic gene of skeletal developmental disorder and application thereof, which are further described in the following with reference to specific embodiments.
Unless otherwise indicated, the techniques used in the examples are conventional and well known to those skilled in the art, and may be performed according to the fourth edition of the molecular cloning, laboratory Manual, or related information, and the reagents and products used are also commercially available. Various procedures and methods not described in detail are conventional methods well known in the art, and the sources, trade names, and components of the reagents used are indicated at the time of first appearance, and the same reagents used thereafter are the same as those indicated at the first appearance, unless otherwise specified.
Example 1:
in this embodiment, a Hiseq sequencing platform of Illumina corporation is used to detect genomic DNA of peripheral blood of a human subject, and the specific implementation steps are as follows:
1. sample source
A congenital dysostosis patient from Shandong province in China, the proband is a 12-year-old male, the proband is the same as a normal child before the age of 8, and multiple fractures occur repeatedly after the age of 8: fracture occurs after light physical activity of unilateral clavicle fracture, right lower limb tibia fracture and fibula fracture. Physical examination: normal physique and intelligence development, no abnormal hair, blue sclera and normal hearing. Laboratory examination: normal blood, normal autoantibody, normal blood sedimentation, and no abnormality of liver and kidney function. The family members were healthy, denying family history with skeletal developmental disorders. The family tree of this family is shown in fig. 4, with arrows pointing to probands, solid icons representing patients, and open icons representing healthy individuals. A total of 3 family members were involved in the test, including proband (diseased), proband mother (healthy), proband father (healthy), informed consent of the proband parents was obtained, and 10mL of venous blood (EDTA anticoagulated) was collected from the elbow veins of the family members of this example for testing.
2. Extraction of specimen genomic DNA:
a genomic DNA extraction kit (HiPure Blood) from magenta was used according to the instructions provided by the trade company&Tissue DNA Kit) genomic DNA was extracted from peripheral blood samples, the purity of the DNA was measured using Nanodrop one, and OD of the genomic DNA obtained260nm/OD280nmAll are located between 1.7 and 2.0, and the concentration of the DNA is measured by using Nanodrop one, and the concentration of the obtained genomic DNA is 50 to 100 ng/. mu.L, and the total amount is 5 to 10. mu.g.
3. Establishing a genome amplification library:
according to the instructions provided by the trade company, the Kit of KAPA company (KAPA Hyperplus Library Preparation Kit) is used for carrying out enzyme digestion and fragmentation, end repair, 3' end A addition, linker ligation and PCR amplification on genomic DNA, and finally, the obtained Library is quantified and quality checked, and the specific implementation steps are as follows:
1) and (3) carrying out genome DNA fragmentation reaction, wherein the reaction system is as follows:
reaction conditions are as follows: the reaction was carried out at 37 ℃ for 12 min.
2) End repairing and 3' end adding A reaction, wherein the reaction system is as follows:
reaction conditions are as follows: reacting at 20 ℃ for 1 min; reacting at 65 ℃ for 30 min; constant temperature of 20 DEG C
3) Linker linking reaction, the reaction system is as follows:
| name of reagent | Dosage (mu L) |
| DNA obtained in step 2) | 30 |
| NF H2O | 2.5 |
| KAPA Ligation Buffer | 15 |
| Adaptor(15μM) | 2.5 |
| KAPA DNA Ligase | 5 |
| Total volume | 55 |
Reaction conditions are as follows: the reaction was carried out at 20 ℃ for 20 min.
4) The first PCR amplification reaction comprises the following reaction systems:
| name of reagent | Dosage (mu L) |
| DNA obtained in step 3) | 16 |
| KAPA HiFi HotStart Ready Mix(2×) | 20 |
| T5*Primer(10μM) | 1.5 |
| T8*Primer(10μM) | 1.5 |
| Total volume | 39 |
Reaction conditions are as follows: 45s at 98 ℃; (98 ℃ for 15s,60 ℃ for 30s,72 ℃ for 30 s). times.6 cycles; 1min at 72 ℃; storing at 12 deg.C.
5) DNA quantification and quality control:
measuring the concentration and the strip distribution condition of the PCR amplification product by using Nanodrop one; preparing 2% agarose gel, mixing the obtained PCR amplification product with the sample loading buffer solution, and observing the position, brightness and uniformity of the band by electrophoresis, wherein the size of the band is qualified between 200 and 800 bp.
4. Constructing a target region targeting DNA library based on probe hybridization capture:
the library is constructed by adopting a library construction kit of IGT company according to the operation of the instruction provided by a merchant, a probe is designed according to 507 candidate skeletal developmental disorder pathogenic gene sequences, synthesized and labeled by biotin, and the specific implementation steps are as follows:
1) and (3) probe hybridization:
mixing 5 mu g of PCR product obtained in the step 3 with 5 mu L of Cot-1human DNA, and carrying out vortex oscillation; adding 2.5 × Ampure XP beads according to the total volume, uniformly mixing and centrifuging, and standing for 5min at room temperature; the magnetic beads are centrifugally washed for 2 times by 80% ethanol, and are eluted by a hybridization solution for hybridization reaction, wherein the system of the hybridization solution is as follows:
| name of reagent | Dosage (mu L) |
| IDT 2×Hybridization Buffer | 8.5 |
| Hybridization Enhancer | 2.75 |
| NF H2O | 1.75 |
| Universal Blockers-TS Mix | 2 |
| Biotin-labeled Probe (507 genes) | 4 |
| Total volume | 19 |
Hybridization conditions: reacting at 95 ℃ for 10 min; at 65 ℃ overnight
2) The probes hybridized with the sample target sequences were captured on magnetic beads by binding biotin to Streptavidin using 80. mu.L of Streptavidin-labeled magnetic beads (M-270Streptavidin beads). Washing with 200 μ L of 1 × Bead Wash Buffer at 65 deg.C and room temperature for three times, each for 3min, and washing the magnetic beads with 20 μ L of NF H2And (4) resuspending the solution.
3) And carrying out a second PCR amplification reaction on the captured target sequence, wherein the reaction system is as follows:
| name of reagent | Dosage (mu L) |
| KAPA HiFi Hot start Readymix(2×) | 25 |
| X Gen Library Amplification primer-Ts Mix | 5 |
| DNA with magnetic beads obtained in step 2) | 20 |
| |
50 |
Reaction conditions are as follows: 45s at 98 ℃; (98 ℃ for 15s,60 ℃ for 30s,72 ℃ for 30 s). times.7 cycles; 1min at 72 ℃; storing at 4 ℃.
5) And (3) PCR product quantification and quality inspection:
measuring the concentration and the strip distribution condition of the PCR amplification product by using Nanodrop one; preparing 2% agarose gel, mixing the obtained PCR amplification product with the sample loading buffer solution, and observing the position, brightness and uniformity of the band by electrophoresis, wherein the size of the band is qualified between 200 and 800 bp.
5. Generating information analysis and variant interpretation of sequencing data:
NGS sequencing results were aligned to the human reference genome UCSC NCBI37/hg19 using Novocraft Novoalign to obtain a unique aligned sequence aligned to the genome. The variation of 507 skeletal developmental disorder-causing gene regions is determined by VarScan mpileup2snp and VarScan mpileup2indel detection. Common variations in dbSNP and ExAC databases were removed using Remove Run Common Variants and Remove Global Common Variants software. The variants were then annotated using Interactive Biosoftware Alamut Batch. The database used for annotation includes: dbSNP, ExAC, 1000g, ClinVar, OMIM, etc., and utilizes the software FATHMM, FATHMMMKL, METALR, METASVM, MUTATIONASSESSOR, MUTATIONTASTERAGGGD, AGVGD, LRT, PROVEAN, SIFT to predict the variant function. According to the ACMG genetic variation classification standard and guideline, mutation sites which are meaningful for diagnosing skeletal developmental disorders are obtained by analysis.
6. Sequencing results interpretation and analysis
507 skeletal developmental disorder pathogenic genes of 3 examinees are sequenced at one time by the DNA library for detecting and diagnosing skeletal developmental disorder pathogenic genes provided by the invention. Taking prover as an example, through quality control analysis, the size of the obtained DNA library is mainly distributed in 200-800bp, the average length is 496bp, the concentration and the fragment size meet the sequencing requirement, and the quality of library construction is prompted to be qualified (as shown in FIG. 1). The DNA library comprises 507 genes, wherein the length of a coding region is 3942109bp, the length of a non-coding region is 766722bp, and the total length is 4708831 bp. Wherein the 10 × coverage of the coding region is 99.47%, the 20 × coverage is 99.34%, and the 50 × coverage is 95.78% (see fig. 2). A Total of 7065041 fragment read length data (Total Reads) were obtained with a match rate (Aligned) of 86.87% (see FIG. 3). The sequencing result meets the data requirement of variation interpretation. Through analysis of the sequencing data, the pathogenic gene variation of skeletal developmental disorder (osteogenesis imperfecta) related to the clinical phenotype of the tested person is found, and is shown in the following table:
a heterozygous variation was found in the subject COL1A1 gene, NM-000088.3: c.159del (p.Trp53). COL1A1 autosomal dominant variants are associated with the development of Ehlers-Danlos syndrome types I and VII (Ehlers-Danlos syndrome, types I and VII), osteogenesis imperfecta types I-IV (types I-IV), and Caffey's disease (PubMed: 20301472, 22855962; OMIM: 120150).
Osteogenesis Imperfecta (OI), also called brittle bone disease and porcelain doll, is a group of rare hereditary diseases, the sick children are easy to fracture, even if the sick children collide slightly, serious fracture can be caused, and partial subtypes have the typical expression of blue sclera. Osteogenesis imperfecta type I is mainly due to decreased collagen I leading to bone fragility and appearance of blue sclera; the symptoms of patients with osteogenesis imperfecta type II are the most serious, which are marked by serious bending and fracture of long bones and are usually dead in perinatal period; osteogenesis imperfecta type III is characterized by repeated fracture and skeletal deformity, progressive aggravation of symptoms, short and small final stature, and blue sclera of part of patients; osteogenesis imperfecta has large variation of IV type phenotype, and the severity is different, the type can only show osteoporosis and bone fragility without fracture, and the common sclera is normal.
The c.159del (p.trp53) mutation results in a premature stop codon, which may cause protein truncation or activate nonsense-mediated mRNA degradation, thereby affecting the function of the protein product encoded by COL1a1 gene. This variation has not been reported in the literature, and another nonsense variation at the same amino acid position (c.158G > A, p.Trp53) has been reported in patients with osteogenesis imperfecta (PubMed: 30886339). The mutation was not detected in the DNA sample of the parent of the proband, suggesting that the mutation is a newly developed pathogenic mutation. The pathogenic variation suggests that the proband is the osteogenesis imperfecta patient, and the clinical accurate diagnosis is successfully realized. The patient belongs to the delayed type of osteogenesis imperfecta, the clinical manifestation is relatively light, and the prevention of fracture mainly avoids unnecessary movement injury. Delayed type tends to be gradually improved, and no special treatment is required, such as recurrence of fracture, shortening of fixation time, and prevention of extensive bone atrophy can be considered. For genetic counseling, patients have a lower risk of recurrent development in their offspring with osteogenesis imperfecta, usually less than 1%, due to new onset of the mutation. Since the disease is an autosomal dominant disease, the patient has a chance of developing a diseased child at 1/2. (see fig. 4)
Reference throughout this specification to the description of "one embodiment," "this embodiment," "an embodiment," or the like, means that a particular feature, structure, material, or characteristic described in connection with the embodiment is included in at least one embodiment of the invention and is not specifically referred to.
It should be noted that, although the above embodiments have been described herein, the invention is not limited thereto. Therefore, based on the innovative concepts of the present invention, the technical solutions of the present invention can be directly or indirectly applied to other related technical fields by making changes and modifications to the embodiments described herein, or by using equivalent structures or equivalent processes performed in the content of the present specification and the attached drawings, which are included in the scope of the present invention.
Claims (8)
1. A DNA library for diagnosing skeletal developmental disorders based on high throughput sequencing technology, wherein the library comprises 507 causative genes of skeletal developmental disorders, wherein the 507 causative genes of skeletal developmental disorders are as follows:
ABCC6、ABCC9、ACAN、ACP5、ACTB、ACTG1、ACVR1、ADAMTS10、ADAMTS17、ADAMTSL2、AGA、AGPS、AKT1、ALDH3A2、ALG9、ALPL、ALX1、ALX3、ALX4、AMER1、ANKH、ANKRD55、ANO5、ANOS1、ANTXR2、AP2S1、APC、ARHGAP31、ARID1B、ARSB、ARSE、ARVCF、ASXL1、ATP6V0A2、ATP6V0A4、ATP7A、B3GALT6、B3GAT3、B4GALT7、BAAT、BAZ1B、BGN、BHLHA9、BMP1、BMP2、BMP4、BMPER、BMPR1B、BRAF、BRF1、BUB1、BUB1B、BUB3、C21orf2、CA2、CANT1、CASR、CAV1、CC2D2A、CCBE1、CCDC141、CCDC8、CCNQ、CCR6、CD247、CD96、CDC45、CDC6、CDH3、CDKN1C、CDT1、CEP120、CEP290、CEP57、CHD7、CHEK2、CHRNA1、CHRND、CHRNG、CHST14、CHST3、CHSY1、CKAP2L、CLCN5、CLCN7、CLIP2、COG1、COL10A1、COL11A1、COL11A2、COL1A1、COL1A2、COL2A1、COL3A1、COL5A1、COL5A2、COL9A1、COL9A2、COL9A3、COLEC11、COMP、COMT、CORO7-PAM16、CREB3L1、CREBBP、CRTAP、CTGF、CTNNB1、CTNS、CTSA、CTSC、CTSK、CUL7、CYP26B1、CYP27A1、CYP27B1、CYP2R1、DCHS1、DDR2、DHCR24、DHODH、DLL3、DLX3、DLX5、DLX6、DMP1、DNASE1L3、DOCK6、DOK7、DUSP6、DVL1、DYM、DYNC2H1、DYNC2LI1、EBP、EFNB1、EFTUD2、EIF2AK3、ELN、ENPP1、EOGT、EP300、EPHX1、ERCC1、ERF、ESCO2、EVC、EVC2、EXT1、EXT2、EXTL3、EZH2、FAH、FAM111A、FAM20C、FAS、FAT4、FBLN1、FBN1、FBN2、FBXW4、FERMT3、FEZF1、FGF10、FGF16、FGF17、FGF23、FGF3、FGF8、FGF9、FGFR1、FGFR2、FGFR3、FIG4、FKBP10、FLNA、FLNB、FLRT3、FMN1、FN1、FREM1、FUCA1、FZD2、GALNS、GALNT3、GBA、GCDH、GDF3、GDF5、GDF6、GJA1、GJB2、GJB6、GLB1、GLE1、GLI3、GMNN、GNA11、GNAS、GNE、GNPAT、GNPTAB、GNPTG、GNS、GORAB、GP1BB、GPC3、GPC6、GPX4、GREM1、GTF2IRD1、GUSB、HBB、HDAC4、HDAC8、HES7、HESX1、HFE、HGD、HGSNAT、HIRA、HNF4A、HNRNPA1、HNRNPA2B1、HOXA11、HOXA13、HOXD13、HPGD、HS6ST1、HSPG2、IARS2、ICK、IDH1、IDH2、IDS、IDUA、IFITM5、IFT122、IFT140、IFT172、IFT43、IFT80、IGF1R、IHH、IKBKG、IL11RA、IL17RD、IL1RN、IL2RA、IL2RB、IMPAD1、INPPL1、IRF5、IRX5、JAG1、JMJD1C、KAT6A、KAT6B、KCNJ2、KCNJ8、KIF22、KIF7、KISS1R、KL、KRAS、LBR、LEMD3、LFNG、LIFR、LIMK1、LMBR1、LMNA、LMX1B、LONP1、LPIN2、LRP4、LRP5、LTBP2、MAFB、MAN2B1、MAN2C1、MASP1、MATN3、MEGF8、MEOX1、MESP2、MGAT2、MGP、MITF、MKS1、MMP13、MMP2、MMP9、MNX1、MSX2、MUSK、MYCN、NAGLU、NANS、NDUFS8、NEK1、NEU1、NF1、NFIX、NIPBL、NKX3-2、NLRC4、NLRP3、NOG、NOTCH2、NOTCH3、NPPC、NPR2、NSD1、NSDHL、NSMCE2、NSMF、OBSL1、OCRL、OFD1、ORC1、ORC4、ORC6、OSTM1、P3H1、P4HB、PAM16、PANK2、PAPSS2、PCNT、PCYT1A、PDE3A、PDE4D、PDGFRB、PEX10、PEX11B、PEX12、PEX13、PEX14、PEX16、PEX19、PEX2、PEX26、PEX3、PEX5、PEX6、PEX7、PHEX、PHYH、PIGV、PIK3CA、PITX1、PLEKHM1、PLOD1、PLOD2、PLS3、POLR1C、POLR1D、POP1、POR、PPIB、PPP3CA、PRKAR1A、PROK2、PROKR2、PTDSS1、PTEN、PTH1R、PTHLH、PTPN11、PTPN2、PTPN22、PYCR1、RAB23、RAB33B、RAD21、RAPSN、RASGRP2、RB1、RBM8A、RBPJ、RECQL4、RFC2、RMRP、RNU4ATAC、ROR2、RPGRIP1L、RREB1、RUNX2、SALL1、SALL4、SBDS、SCARB2、SCARF2、SEC23A、SEC24C、SEC24D、SEMA3A、SERPINF1、SERPINH1、SETD2、SF3B4、SFRP4、SFTPC、SGSH、SH3BP2、SH3PXD2B、SHH、SHOX、SKI、SLC17A5、SLC25A12、SLC26A2、SLC29A3、SLC34A1、SLC34A3、SLC35D1、SLC39A13、SLC40A1、SLC4A1、SLC9A3R1、SLCO2A1、SLCO5A1、SMAD3、SMAD4、SMARCA2、SMARCAL1、SMC1A、SMC3、SNRPB、SNX10、SNX22、SOST、SOX10、SOX6、SOX9、SP7、SPAG8、SPARC、SPECC1L、SPRY4、SQSTM1、SSR4、STAT3、STAT4、SULF1、SUMF1、TACR3、TBCE、TBL2、TBX1、TBX15、TBX3、TBX4、TBX5、TBX6、TBXAS1、TCF12、TCIRG1、TCOF1、TCTN3、TGDS、TGFB1、TGFB2、TGFBR1、TGFBR2、THPO、TJP2、TMCO1、TMEM165、TMEM216、TMEM38B、TMEM67、TNFRSF11A、TNFRSF11B、TNFSF11、TP53、TP63、TRAPPC2、TREM2、TRIP11、TRIP13、TRPS1、TRPV4、TTC21B、TWIST1、TYK2、TYROBP、UROS、VCP、VDR、VPS33A、WDR11、WDR19、WDR34、WDR35、WDR60、WISP3、WNT1、WNT10B、WNT3、WNT5A、WNT6、WNT7A、XYLT1、XYLT2、ZMPSTE24。
2. use of a DNA library according to claim 1 for the preparation of a diagnostic kit, characterized in that: the kit is used for diagnosing skeletal development disorder.
3. The application according to claim 2, characterized in that it comprises the following steps:
1) extracting genomic DNA of a sample of a subject;
2) quantifying the extracted genomic DNA and constructing a library according to the following steps:
a) fragmenting the genomic DNA;
b) adding a base A to the 3' end of the fragmented genomic DNA while performing end repair;
c) connecting a product with a base A added at the 3' end with a joint;
d) performing PCR amplification on the ligation product, and adding a complete linker;
e) targeting capture of a target region using probes for the 507 skeletal developmental disorder-causing genes of claim 1;
f) washing off the uncaptured library, and only reserving the target area library;
j) obtaining a target region capture library;
3) performing quantitative operation on the library;
4) high-throughput sequencing;
5) and (5) analyzing the data to obtain the related information of the pathogenic site.
4. Use according to claim 3, characterized in that: the sample in the step 1) is from peripheral blood, body fluid and a tissue organ sample of a subject.
5. Use according to claim 3, characterized in that: the quantitative method in the step 2) comprises a fluorescence quantitative method and electrophoresis.
6. Use according to claim 3, characterized in that: the fragmentation method in the step a) comprises ultrasonic crushing, transposase enzyme digestion and restriction enzyme digestion.
7. Use according to claim 3, characterized in that: the quantitative method in the step 3) comprises a fluorescence quantitative method and electrophoresis.
8. A diagnostic kit comprising the DNA library of claim 1.
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