WO2020133233A1

WO2020133233A1 - Pathogenic mutation of osteogenesis imperfecta disease and detection reagent therefor

Info

Publication number: WO2020133233A1
Application number: PCT/CN2018/124930
Authority: WO
Inventors: 黄欢
Original assignee: 黄欢
Priority date: 2018-12-27
Filing date: 2018-12-28
Publication date: 2020-07-02
Also published as: CN109897894A; CN109897894B

Abstract

Provided are a pathogenic mutation of osteogenesis imperfecta and a detection reagent therefor. For a mutant COL1A1 gene, single site mutation c.1822G>A (chr17:48270211) is related in the mutated COL1A1 gene, whereof the heterozygous mutation is pathogenic, the genetic mode is dominant inheritance, and for the amino acid change at p.Gly608Ser, the site mutation causes dyssynthesis of I-type collagen in connective tissue, so that a lesion is formed. Provided is a kit for detecting osteogenesis imperfecta, comprising a reagent for detecting the 1822bp-th site of a COL1A1 gene CDS or a reagent for detecting the 608-th amino acid site of a COL1A1 protein. The osteogenesis imperfecta disease can be diagnosed by detecting the pathogenic mutation (c.1822G>A on the COL1A1 gene).

Description

Pathogenic mutation of osteogenesis imperfecta disease and its detection reagent

Technical field

The invention belongs to the field of biomedicine, and relates to a pathogenic mutation of osteogenesis imperfecta and its detection reagent.

Background technique

Osteogenesis imperfecta (osteogenesis imperfecta, OI) is a rare congenital skeletal developmental disorder, also known as brittle bone disease, porcelain doll, or brittle bone-blue sclera-deafness syndrome. It is characterized by fragile bone, blue sclera, deafness, and loose joints. It is a congenital hereditary pain caused by the development of mesenchymal tissues and the formation of collagen. Children are prone to fractures, and minor collisions can also cause severe fractures. At present, OI has no special treatment methods, mainly to prevent fractures, and the efficacy of drug treatment is uncertain. Stem cell therapy and gene therapy methods need to be further studied and identified, and they cannot be applied to clinics in a short time. OI is hereditary and familial, but there are a few cases of single disease. OI has a high incidence in my country and the world, about 3 in 100,000, and the proportion of men and women with the disease is about the same. my country is a large country of OI genetic resources, but most of the genetic information related to OI comes from Western countries. Therefore, it is particularly important to carry out in-depth genetic research on OI patients in my country to explore potential new pathogenic genes and mutations related to OI.

OI is a single-gene inherited disease, and its common genetic patterns include autosomal dominant inheritance and autosomal recessive inheritance. OI occurs mainly due to mutations in the genes of the alpha 1 or alpha 2 procollagen (Pro-α1 or Pro-α2) chains that make up type I collagen (ie, COL1A1 and COL1A2), leading to type I collagen synthesis disorders, especially the amount of collagen in connective tissue. The content of type I collagen is reduced. Collagen is the main collagen component of bones, skin, sclera, and dentin, so the disease changes in these parts are obvious. According to gene mutations, it can be divided into 4-11 types, but it is controversial, and the severity and prognosis of different types of osteogenesis imperfecta are different. . At present, there are still many OI patients whose disease-causing genes have not been found, especially in the fetal period, many clinical manifestations cannot be revealed in the womb by ultrasound and other technical methods, so it may be confused with various genetic diseases of abnormal bone development, suggesting that there are a large number of New disease-causing genes such as OI and abnormal bone development and new disease-causing mutations need to be discovered.

The molecular genetics research for OI must be based on certain molecular biology techniques. An important purpose of studying OI disease-causing genes is to conduct molecular diagnosis of OI. How to detect mutations of many disease-causing genes is one of the current problems. The location-based cloning strategy of gene linkage analysis is a classic method for identifying single-gene genetic disease-causing genes, but it also faces some difficulties: (1) Multi-generation families are usually required, and it is difficult to analyze small families and sporadic cases. (2) Sometimes multigenerational families cannot locate the site of disease. (3) It is difficult to select the correct disease-causing genes in the linked region. Prenatal diagnosis relies on ultrasound to examine the fetal skeletal system. Only a few bone development disorders can be found, and it is difficult to distinguish different types of bone development abnormalities. Therefore, in view of the indistinguishable nature of fetal OI diseases and the limitations of traditional analysis techniques, it is particularly urgent to seek a new research method for OI pathogenic genes.

COL1A1 is located at the position of 17q21-22 on the long arm of chromosome 17. The gene is 18kb long and has 51 exons. Each type III collagen triple gel helix region contains 338 consecutive repeating three amino acids GXY, where G is glycine, X and Y are usually proline and hydroxyproline. The presence of glycine is necessary for the formation of the triple gel helix. In this region, glycine residues caused by point mutations are replaced, and exon loss caused by mutations at the cleavage site is the main form of type Ⅰ collagen gene mutations. . Other mutations such as insertions, deletions, and duplications are relatively few. There are many exons of COL1A1 gene. So far, more than one hundred mutations have been reported. However, c.1822G>A gene mutation caused OI has never been reported or confirmed.

Summary of the invention

The object of the present invention is to provide a new pathogenic mutation for osteogenesis imperfecta in view of the above defects.

Another object of the present invention is to provide the application of the pathogenic mutation.

The object of the present invention can be achieved by the following technical solutions:

A mutant COL1A1 gene for detecting osteogenesis imperfecta disease, the mutated COL1A1 is a heterozygous mutation or a homozygous mutation c.1822G>A, the gene number of the wild-type COL1A1 gene in the NCBI database is: NM_000088.3, The base at the 1822 bp of the gene CDS was mutated from G to A, and the other parts were the same as the wild type. The CDS sequence of the wild-type COL1A1 gene is shown in SEQ ID NO.1.

A mutated COL1A1 protein. The gene transcript number of the wild-type COL1A1 protein in the NCBI database is: NP_000079. The mutated COL1A1 protein is mutated from glycine to serine at the 608th amino acid of the wild-type protein. Other parts are the same as the wild-type COL1A1 protein. . The amino acid sequence of the wild-type COL1A1 protein is shown in SEQ ID NO. 2,

The application of the reagent for detecting the mutated COL1A1 gene or the mutated COL1A1 protein according to the present invention in the preparation of an osteogenesis imperfecta disease detection reagent or detection equipment.

The detection reagents described herein are preferably selected from one or more of primers or primer pairs, probes, antibodies, or nucleic acid chips, high-throughput sequencing, and Sanger sequencing.

The detection device preferably includes a gene chip containing a mutant COL1A1 gene, a high-throughput sequencing, and Sanger sequencing detection platform.

A kit for detecting osteogenesis imperfecta disease, the kit includes:

(1) A reagent for detecting the nucleotide at 1822 bp of CDS of COL1A1 gene; or a reagent for detecting the 608th amino acid position of COL1A1 protein;

(2) The product manual clearly states that the nucleotide at the 1822 bp CDS of the COL1A1 gene is mutated from G to A, or that the 608th amino acid position of the COL1A1 protein is changed from G to S as a pathogenic mutation for osteogenesis imperfecta.

Wherein, the reagent is preferably from a primer or a primer pair, a probe, an antibody, or a nucleic acid chip.

As a preferred aspect of the present invention, the reagent is a gene chip hybridization probe based on deep sequencing as a platform.

The reagent is further preferably a primer pair for detecting the nucleotide at the 1822 bp of the CDS of the COL1A1 gene; further preferably 5'- TGGCGCTGTCGTAAGTAT-3' ( SEQ ID NO. 3) and 5'- CCTGTAGGTGGGAAATGG-3' ( SEQ ID NO.4 ) Primer pair consisting of.

The sequence of the gene chip hybridization probe for detecting the nucleotide at the 1822 bp of the CDS of the COL1A1 gene in the kit is preferably shown in SEQ ID NO. 5.

A method for screening new mutations of the COL1A1 gene in patients with OI using deep sequencing. The zebrafish mutation model combines SIFT and Polyphen protein function prediction to verify that the gene mutation is a pathogenic gene mutation: it includes the following steps:

(1) For families with fetal ultrasound showing abnormal bone development or a history of OI genetic disease, collect clinical data and blood, tissue and other DNA-containing specimens to extract genomic DNA;

(2) Detection of a series of genes related to abnormal bone development, including genes ADAMTSL2, AGPS, ANKH, ARSE, CCDC8, CHST3, COL10A1, COL2A1, COL9A1, COL9A2, COL9A3, COMP, CTSK, CUL7, DLL3, EBP, EVC, EVC2, FBN1, FGFR1, FGFR2, FGFR3, FLNB, GNAS, GNPAT, HES7, LFNG, LMNA, MATN3, MESP2, OBSL1, PEX7, PTH1R, ROR2, RUNX2, SLC26A2, SLC35D1, SMARCAL1, SOST, SOX9, TGFB F11A, TRAPPC2, TREM2, TYROBP, WNT5A, WNT7A, ZMPSTE24, COL1A1, COL1A2, CRTAP, P3H1, SERPINF1, IFITM5, FKBP10, PPIB, SP7, BMP1, SERPINH1, TMEM38B, WNT1B, WNT1B, WNT.

(3) Break the DNA and prepare the library, then capture and enrich the target gene coding region and the adjacent cut DNA through the chip, and finally use a high-throughput sequencing platform for mutation detection.

(4) The bioinformatics analysis of the sequencing results was optimized, and a new OI pathogenic mutation was selected as COL1A1.Gly608Ser. The mutation is located on chromosome 17, and the physical position is 48270211 (NCBI database). The base is mutated from G to A; protein level: the amino acid at position 608 of the protein encoded by the COL1A1 gene is mutated from glycine to serine.

(5) The high-throughput sequencing described in step (3), the length of the sequencing target area is 131271bp, the coverage of the target area reaches at least 99.95%, the average depth of the target area is at least 171.42X, and the proportion of the average depth of the target area is >30X sites At least 96.99%.

(6) For the new mutation site COL1A1.Gly608Ser, SIFT and Polyphen were used to predict protein function.

(7) Use the zebrafish model to verify that the c.1822G>A point mutation on the COL1A1 gene affects bone development. Find the gene col1a1a (ENSDARG00000012405) highly similar to the human COL1A1 gene in the ENSEMBL database, by expressing col1a1a in wild-type zebrafish The same point mutation gene was used to simulate the dominant expression in the human body, and the bone development of the zebrafish embryo was observed to verify that the new mutation site COL1A1.c.1822G>A resulted in abnormal bone development OI.

Beneficial effect

1. OI is a serious congenital skeletal dysplasia disease, which has a high incidence of genetic diseases in my country, which is harmful to national health. At present, there is no effective treatment for OI. The prevention is mainly to carry out prenatal diagnosis and prevent the birth of children. OI has obvious genetic heterogeneity, which is divided into autosomal dominant inheritance and autosomal recessive inheritance. There are currently hundreds of known mutations, but there are still a large number of unknown pathogenic genes. The discovery of OI's new disease-causing mutation genes is conducive to further exploring the molecular genetic causes of OI, thereby helping to understand the pathogenesis, assisting clinical diagnosis, prenatal diagnosis and transgenic therapy.

2. For the first time, the new mutation site c.1822G>A in the OI pathogenic gene COL1A1 was reported, which is an autosomal dominant genetic disease. Both heterozygous and homozygous mutations are pathogenic.

3. Provide a solution for discovering new mutations, that is, by designing a series of genetic test panels for a certain human system, high-throughput sequencing detection of DNA extracted from biological samples, and bioinformatics analysis of the harmfulness of new mutations. Gene knockout verification has been performed on animal models such as zebrafish. The present invention provides a new pathogenic site of OI, and provides a new molecular biological basis for the diagnosis of the disease.

BRIEF DESCRIPTION

Figure 1 Fetal ultrasound results

Figure 2 Sanger sequencing results of pedigree

Figure 3 Conservation of the mutation positions of human COL1A1 gene and homologous genes in zebrafish, the mutation site (G1822A) described in the present invention and its corresponding mutation site in zebrafish are underlined

Figure 4 The nucleotide and amino acid correspondence of the sequence of the zebrafish mutation

Figure 5 Construction of zebrafish transcription and microinjection plasmids

Figure 6 Mutation frequency of new loci in each database

Figure 7 SIFT prediction results of new sites in various databases.

Figure 8. The prediction results of the new site polyphen in various databases.

Figure 9 Wild type zebrafish and COL1A1 gene c.1822G>A mutant zebrafish

detailed description

After extensive and in-depth research, the present inventors have discovered a new mutation site of OI-related gene COL1A1, which can be used to diagnose the above diseases and to develop effective gene therapy drugs for the above diseases.

When detecting mutations at related sites, the detection can be directed to genomic DNA, cDNA or mRNA, or protein. Existing techniques such as Western blotting, Southern blotting, DNA sequence analysis, PCR and in situ hybridization can be used to detect mutations.

Various techniques can be used to detect the presence of a G to A mutation at position 1822 of the wild-type COL1A1 gene (SEQ ID NO. 1). These techniques are included in the present invention. For example, gene chip and high-throughput sequencing capture probes are prepared based on relevant sites. In addition, primers specific to the relevant sites can be used for PCR identification; or probes that can specifically bind can be designed according to the relevant sites for identification; or specific restriction enzymes can be used for identification.

As an alternative method, single-base extension technology based on PCR technology can also be used to detect the mutation site. The principle is to design a primer located upstream of the mutation site to be tested, and the 3'end distance of the primer The mutation site is one base. Add different fluorescently labeled ddNTPs for reaction, or add dNTPs and related enzymes for reaction by pyrosequencing. Only when the added ddNTPs or dNTPs are complementary to the base of the mutation site, the primers can be extended. The type of mutation can be determined by detecting the fluorescence emitted by the extended base or the visible light emitted by a series of enzyme reactions in pyrosequencing.

The present invention also includes a reagent for detecting whether the mutation site (the G1 to A mutation at position 1822 of the CDS of the COL1A1 gene) is contained in the analyte. The reagent is, for example, a primer specific to the relevant mutation site, and the amplified product contains the base corresponding to the 1822th position of the COL1A1 gene; a probe specific to the relevant mutation site can be used in conjunction with the mutation region The specific binding occurs but cannot specifically bind to the region where the mutation has not occurred, and the probe carries a detectable signal; or a restriction enzyme specific for the relevant mutation site.

The kit can also include various reagents required for DNA, RNA, hybridization, color development, etc., including but not limited to: extraction solution, amplification solution, hybridization solution, enzyme, control solution, display solution Color liquid, lotion, etc.

In addition, the kit can also include instructions for use and nucleic acid sequence analysis software.

The present invention will be further described below in conjunction with specific embodiments.

Example 1

Genetic testing of a fetus whose ultrasound suggests abnormal bone development.

experimental method:

1. Collection of ultrasound results of pregnant women and collection of case data such as family genetic history: collection of clinical data and blood samples of members of the family, the parents of the fetus collect peripheral blood, and umbilical cord blood puncture for pregnant women Make a genetic diagnosis. Using blood genomic DNA extraction kit (Tiangen Biochemical Technology Co., Ltd.) to extract blood genomic DNA of each member of the family.

2. Use high-throughput sequencing technology to mine the family's pathogenic mutations: detect 61 genes related to bone development abnormalities, first fragment the genomic DNA, and carry out end labeling, perform liquid phase hybridization with genomic DNA, and then target the genome The DNA fragments in the region are enriched and then sequenced using second-generation sequencing technology. The specific solution is based on the 120mer RNA oligonucleotide probe or "baits". The biotin attached to Baits can be adsorbed by magnetic beads labeled with streptavidin. The interrupted genome fragment is hybridized with baits to capture the target fragment. After the magnetic beads are used to adsorb the DNA fragments with baits, the magnetic beads are eluted and the RNA probe is degraded to finally obtain the DNA fragments in the target region.

3. The process of standard information analysis includes: removal of joint contamination and low-quality data. The data is compared with BWA and UCSC hg19 databases. Statistical analysis of data output, depth analysis of sequencing, uniformity analysis of coverage, and SNP variation information detection (SAMtools, SOAPsnp, GATK), SNP RefGene annotation, SNP database analysis (database annotation analysis with dbSNP, Thousand Genome data, ESP exome database, and Yanhuang genome (Asia Pacific only) data), SNP conservative prediction, pathogenicity Sexual analysis (only for human samples, software: SIFT, Polyphen-2, Phylop, GERP scores, Mutation assets, Condel, FATHMM), SNP distribution statistics on each gene functional element, InDel mutation information detection (SAMtools, GATK), InDel's RefGene annotation, InDel database analysis (database annotation analysis with dbSNP, Thousand Human Genome Data, ESP Exome Database, Yanhuang Genome (Asia Pacific only)), InDel distribution statistics on each gene functional element.

4. Verification by Sanger sequencing to identify disease-causing genes: PCR method was used to amplify the selected mutation sites and adjacent DNA sequences in the corresponding families. The primer sequences used were designed using Primer 5 primer design software to detect the invention. The sequence of the primer pair for pathogenic mutation is shown in SEQ ID NO.3 and SEQ ID NO.4. The reaction system (50 μl system) of the PCR used was: 10*buffer 5 μl, 25 mM MgCl ₂ 3 μl, Taq DNA polymerase 5U, dNTP mixture 2 mM, forward and reverse primers 1.2 μM each, and sterile distilled water was added to 50 μl. Put into the PCR machine, 94 ℃, 3min; (94 ℃, 25s; 55 ℃, 25s; 72 ℃ 15s) 35 cycles; 72 ℃ 7min; 4 ℃ stored. After 2% agarose electrophoresis, the gel imager was used to detect the size of the fragments. Marker was added to determine the size of the fragments. Samples with a single band and the same size were subjected to Sanger sequencing to determine whether the site was mutated.

Experimental results:

1. Ultrasound examination of the fetus in the abdomen of the pregnant woman found that the fetal skull had abnormal morphology, which was a "strawberry" shape. Both femurs were bent at an angle, both tibias were bent, the limbs were dysplastic, and the fetal cardiothoracic ratio increased 1. The ratio of the diameter of the pulmonary artery is abnormal, and the tricuspid regurgitation is mild (Figure 1). The initial judgment may be osteogenesis imperfecta, and members of the family who have not experienced similar symptoms.

2. Through the target area capture sequencing and bioinformatics analysis of the fetal cord blood sample DNA, it is found that the fetus has a c.1822G>A mutation on the COL1A1 gene, which is a VOUS mutation, that is, a mutation of unknown clinical significance, no other suspicious was found. Of mutations in pathogenic genes. Sanger sequencing confirmed that the mutation of this gene locus has never been found in OI patients, nor in the peripheral blood DNA of fetal parents (Figure 2).

3. According to the design scheme of the present invention, it is successfully confirmed that the detected COL1A1 gene c.1822G>A mutation is a new pathogenic site of OI.

Example 2:

Functional studies and gene knockout animal model studies were conducted on the pathogenic genes detected in Example 1. Here, the detected new mutation of COL1A1 gene c.1822G>A is taken as an example.

experimental method:

1. Conservative analysis: evaluate the frequency of occurrence of the locus in each database.

2. Predict the pathogenic ability of mutations based on SIFT and polyphen values.

3. The animal model of gene knockout confirms that the mutation site is a pathogenic mutation site.

(1) Analyze the homologous genes and point mutation positions of COL1A1 in zebrafish, select the correct homologous genes in zebrafish for the preparation of point mutations; find the highly similar gene to human COL1A1 gene on the ENSEMBL website, which is ENDARG00000012405, analysis The conservative position of the mutation is shown in Figure 3. The above comparison results indicate that this locus is conserved in the zebrafish gene, suggesting the importance of its locus function. In order to verify that the zebrafish mutation at this site caused a similar phenotype, the gene col1a1a (ENSDARG00000012405) with high similarity to human COL1A1 in zebrafish was selected for experiment.

(2) Method of verifying the point mutation function of COL1A1 (G1822A): In humans, COL1A1 (G1822A) exhibits a dominant skeletal dysplasia phenotype during the embryonic stage, so the same point mutation gene can be expressed by expressing col1a1a in wild-type zebrafish To simulate the dominant expression in the human body, and then verify it by observing the bone development of the zebrafish embryo. Using the sequence of the col1a1a gene transcript ENSDART00000009393.8 as a reference, design primers to clone the full length of the gene and construct a col1a1a (G1774A) point mutation. The nucleotide and amino acid correspondences of the sequence of the mutation are shown in FIG. 4.

(3) Construction of a zebrafish-expressing col1a1a (G1774A) plasmid: mutation point: G1774A, mutation primer: the original plasmid has been used as a template to amplify the vector backbone, select the primer col1a1a-VF (5'-GCC, CAG, TCT, GCT, TCT, TGT, AAG) GAT, CCA, CCG, GAT, CTA, GAT, A-3') and col1a1a-VR (5'-GCT, TAA, ACA, AGA, ATC, TCT, AG-3'); the original plasmid is used as a template, the promoter region is amplified, and primer CMV-T7-F is selected (5'-CTA, GAG, ATT, CTT, GTT, TAA, GCG, ACA, TTG, ATT, ATT, GAC, TAG-3') and CMV-T7-R (5'-AAT, ATC, CAC, AAA, GCT, GAA, CAT, GGT, GGC, AAG, CTT, AAC, TAG, CCA, GCT, TG-3) ); using zebrafish genomic DNA as a template, amplify col1a1aM fragment 1, select primers col1a1a-F1 (5'-ATG TTC AGC TTT GTG GAT AT-3') and col1a1a-R1 (5'-TAG TTG CAC CAA TAG CAC CC-3'); In order to overcome non-specific amplification, a primer is extended at both ends of col1a1aM fragment 2 to design primers. The primer sequence is as follows: 5'-GAG GAA GGC AAG AGA GGA CC-3' and 5'-CCA GGG GGA TTT TAC ACG CT-3', using zebrafish genomic DNA as a template, PCR amplification, using PCR recovered product as a template, using amplified col1a1aM fragment 2 primer col1a1a-F2 (5'-GGG TGC TTA TTG TGC TGC CAC TAG CGC TCC TGG, TAA, GGA, TGG, T-3') and col1a1a-R2 (5'-TTA, CAA, GAA, GCA, GAC, TGG, GC-3') were amplified to obtain col1a1aM fragment 2. Use homologous recombination to synthesize plasmids. After the construction of the plasmid is completed, send it to the sequencing company for sequencing. After analysis of the sequencing results, the plasmid is constructed correctly. The successfully constructed plasmid sequence is shown in SEQ ID NO.5; strain preservation, plasmid extraction And purification. The concentration of plasmid after purification was 400ng/μL. The method of expressing col1a1a (G1774A) uses DNA microinjection (using tol2 transposase to mediate efficient transgenes), using overexpression plasmids constructed by microinjection (Figure 5) and transposase mRNA to express, confirmed by phenotype Does it have an impact.

(4) Phenotype observation after expression of col1a1a (G1774A): After microinjection, continue to observe its overall morphological development, paying special attention to the development of trunk bones (with or without bending).

Experimental results:

1.COL1A1; NM_000088.3; c.1822G>A; p.Gly608Ser|p.G608S; EX27; CDS27: missense mutation, there is no relevant literature report on the pathogenicity of this site. Using SIFT and Polyphen to predict protein function, the results are harmful, the probability of this site occurring in normal people is extremely low (Figure 6-8). Osteogenesis imperfecta 1/2/3/4 types associated with the COL1A1 gene are autosomal dominant inheritance.

2. Through the analysis of spine curvature phenotype, it was found that injection of col1a1a plasmid caused zebrafish spine curvature (Figure 9).

Claims

A mutant COL1A1 gene for detecting osteogenesis imperfecta disease, characterized in that the mutated COL1A1 is a heterozygous mutation or a homozygous mutation c. 1822G>A, and the gene number of the wild-type COL1A1 gene in the NCBI database is: NM_000088 .3, the base at the 1822 bp of the gene CDS is mutated from G to A, and the other parts are the same as the wild type.
A mutant COL1A1 protein, characterized in that the wild-type COL1A1 protein is numbered in the NCBI database: NP_000079, the mutant COL1A1 protein is mutated from glycine to serine at the 608th amino acid of the wild-type protein, and other parts are the same as the wild-type .
The reagent for detecting the mutant COL1A1 gene according to claim 1 or the mutant COL1A1 protein according to claim 2 in the preparation of an osteogenesis imperfecta disease detection reagent or a detection device.
The use according to claim 3, characterized in that the detection reagent is selected from one or more of probes, primers and antibodies.
The application according to claim 3, characterized in that the detection device comprises a detection platform containing a gene chip for detecting the COL1A1 gene of claim 1, high-throughput sequencing, and Sanger sequencing.
A kit for detecting osteogenic hypoplasia, characterized in that the kit includes:

(1) A reagent for detecting the nucleotide at 1822 bp of CDS of COL1A1 gene; or a reagent for detecting the 608th amino acid position of COL1A1 protein;

(2) The product manual clearly states that the nucleotide at the 1822 bp CDS of the COL1A1 gene is mutated from G to A, or that the 608th amino acid position of the COL1A1 protein is changed from G to S as a pathogenic mutation for osteogenesis imperfecta.
The kit according to claim 6, characterized in that the reagents are selected from one or more of primers or primer pairs, probes, antibodies, or nucleic acid chips; preferably, deep sequencing is a platform for gene chip hybridization needle.
The kit according to claim 7, characterized in that the reagent is a primer pair for detecting the nucleotide at 1822 bp of the CDS of the COL1A1 gene; preferably a primer pair consisting of SEQ ID NO. 3 and SEQ ID NO. 4.
The kit according to claim 7, wherein the sequence of the gene chip hybridization probe for detecting the nucleotide at the 1822 bp of the CDS of the COL1A1 gene is shown in SEQ ID NO. 5.
A method for screening new mutations of the COL1A1 gene in OI patients using deep sequencing as a platform, which is characterized by the following steps:

(1) For families whose fetal ultrasound shows abnormal bone development, or a history of OI genetic disease, collect clinical data and blood, tissue and other DNA-containing specimens to extract genomic DNA;

(2) Detection of a series of genes related to abnormal bone development, including genes ADAMTSL2, AGPS, ANKH, ARSE, CCDC8, CHST3, COL10A1, COL2A1, COL9A1, COL9A2, COL9A3, COMP, CTSK, CUL7, DLL3, EBP, EVC, EVC2, FBN1, FGFR1, FGFR2, FGFR3, FLNB, GNAS, GNPAT, HES7, LFNG, LMNA, MATN3, MESP2, OBSL1, PEX7, PTH1R, ROR2, RUNX2, SLC26A2, SLC35D1, SMARCAL1, SOST, SOX9, TGFB F11A, TRAPPC2, TREM2, TYROBP, WNT5A, WNT7A, ZMPSTE24, COL1A1, COL1A2, CRTAP, P3H1, SERPINF1, IFITM5, FKBP10, PPIB, SP7, BMP1, SERPINH1, TMEM38B, WNT1B, WNT1B, WNT1;

(3) Break the DNA and prepare the library, then capture and enrich the target gene coding region and the adjacent cut DNA through the chip, and finally use a high-throughput sequencing platform for mutation detection;

(4) The bioinformatics analysis of the sequencing results was optimized, and a new OI pathogenic mutation was selected as COL1A1.Gly608Ser. The mutation was located on chromosome 17 and the base at physical position 48270211 was mutated from G to A; protein level: COL1A1 gene encodes protein 608 amino acid mutation from glycine to serine;

(5) The high-throughput sequencing described in step (3), the length of the sequencing target area is 131271bp, the coverage of the target area reaches at least 99.95%, the average depth of the target area is at least 171.42X, and the proportion of the average depth of the target area is >30X sites At least 96.99%;

(6) For the new mutation site COL1A1.Gly608Ser, use SIFT and Polyphen to predict protein function;

(7) Use the zebrafish model to verify that c.1822G>A point mutation on the COL1A1 gene affects skeletal development. A gene col1a1a highly similar to the human COL1A1 gene was found in the ENSEMBL database. The gene number is ENDARG00000012405. The expression of col1a1a is the same point mutation gene to simulate the dominant expression in humans. Observing the bone development of zebrafish embryos verifies that the new mutation site COL1A1.c.1822G>A leads to abnormal bone development OI.