WO2022134165A1

WO2022134165A1 - Pathogenic gene col1a2 mutation of bone dysplasia disease and detection reagent thereof

Info

Publication number: WO2022134165A1
Application number: PCT/CN2020/141426
Authority: WO
Inventors: 黄欢
Original assignee: 黄欢
Priority date: 2020-12-24
Filing date: 2020-12-30
Publication date: 2022-06-30
Also published as: CN112608925B; CN112608925A

Abstract

A pathogenic mutation of osteogenesis imperfecta and a detection reagent thereof. A new type mutant COL2A1 gene, the mutated COL2A1 gene being a single point mutation c.3944G>A(chr12:48368588), and a heterozygous mutation being pathogenic, which is a dominant inheritance pattern. The amino acid change p.Cys1315Tyr and the site mutation results in the structure of the Type II collagen in the articular cartilage being changed, thereby causing the Type II collagen disease. A kit for detecting osteogenesis imperfecta, which comprises: a reagent for detecting site 3944bp of COL2A1 gene CDS, or a reagent for detecting the amino acid site at position 1315 of the COL2A1 protein. Provided is a pathogenic mutation (c.3944G>A on the COL2A1 gene) for osteogenesis imperfecta, and the diagnosis of osteogenesis imperfecta can be made by detecting the mutation.

Description

A causative gene COL1A2 mutation of a bone dysplasia disease and its detection reagent

technical field

The invention belongs to the fields of biomedicine and genetics, and relates to a COL1A2 mutation of a pathogenic gene of abnormal bone development and a detection reagent thereof.

Background technique

Skeletal dysplasia, also known as dwarfism, is a general term for a group of diseases that cause short stature due to abnormal bones or cartilage. Although most of these patients have normal intelligence, these diseases will affect the normal development of bone and cartilage tissue, and sometimes also It can cause bone deformation and even affect other systems of the body. At present, more than 200 kinds of skeletal dysplasia diseases have been reported. Many congenital skeletal dysplasias are caused by genetic mutations. Generally, skeletal dysplasias are diagnosed by X-ray films and MRI examinations. and evaluation, generally appear after symptoms, because the genetic disease cannot be treated once it occurs, which brings a heavy burden to the society and the family, the only measure is early diagnosis, informed choice, so search and verify the pathogenic location of the genetic disease It is of great significance to establish a genetic diagnosis method for skeletal dysplasia, conduct prenatal genetic diagnosis, and avoid the birth of such children.

More than ten years ago, for the diagnosis of genetic diseases, it was generally through physical examination, biochemical and mass spectrometry examinations, and then auxiliary examinations such as nuclear magnetic resonance and CT, and finally targeted genetic testing. Prenatal diagnosis cannot be achieved. There are many types of genetic diseases and various clinical symptoms. Therefore, the diagnosis efficiency of genetic diseases is not high. Many diseases have not been clearly diagnosed, or even misdiagnosed. These have always been problems that plagued clinical practice. The emergence of high-throughput sequencing technology (Next generation sequencing, NGS) provides a good solution to the above problems. NGS has fast speed, high throughput, and high degree of automation. From DNA sample preparation, on-board sequencing, to data analysis, it only takes 8-9 hours to detect all disease-related genes at one time, which greatly improves the performance of genetic diseases. The efficiency of diagnosis, especially the emergence of whole exome sequencing, has greatly increased the detection rate of pathogenic mutations in genetic diseases. This technology can detect about more than 20,000 gene exon regions in the human genome at one time. The number of genes that reached more than 95% was 18,105, including more than 4,000 genes with clear pathogenic relationships in the OMIM database, more than 5,000 diseases, and 68 microdeletion-microduplication syndromes were detected. It is especially suitable for ultrasound during pregnancy to indicate that there are problems in the skeletal system, but there is no pedigree (new mutation), and the acquisition of clinical features in the fetal period is limited. The whole exome sequencing of fetal parents and fetal trio can be fast, accurate, and large-scale (all exons) to search for pathogenic mutation sites.

Based on the emergence of NGS, the clinical diagnosis strategy for single-gene genetic diseases has changed greatly, and different detection schemes have emerged for three clinical detection needs: (1) For a single disease, Sanger sequencing method and qPCR method are used. Or Panel single disease detection, the detection platform is next-generation sequencing, MLPA or NGS, its advantages and limitations lie in amplifying a trait or a disease gene, detecting point mutations and deletions, and discovering new mutations, but not new genes; ( 2) For subjects with a clear single clinical phenotype, family history or other obvious genetic factors, and high requirements for diagnosis cycle and efficiency, panel disease combination or whole panel detection is used, and the detection platform is NGS. Its advantages and limitations lie in Detecting related gene combinations, the results have clear clinical significance, easy to interpret, relatively short cycle, cost-effective, new mutations can be found, but no new genes; (3) For difficult cases with complex clinical manifestations or unclear disease direction, or panel The test results are negative, or based on clinical research of multiple disease families, NGS-based whole exome sequencing is used to capture and sequence the entire exome, and point mutations and deletion duplications are found, which can detect 75-85% of the disease. Mutations, new genes are found, but mutations of unknown clinical significance may be found, which are difficult to interpret; NGS-based whole genome sequencing can also be used to detect the entire human genome, sequence the entire genome, and find point mutations and deletions. Duplication, structural variation, Dynamic mutations can simultaneously detect mitochondrial mutations and discover new genes, but mutations of unknown clinical significance may be found, which are difficult to interpret.

The COL2A1 gene is located at the position 12q13.11-q13.2 of the long arm of chromosome 12. The gene is 31.538kb long, has 54 exons and 53 introns, and encodes 1487 amino acids. The COL2A1 gene encodes the pre-α1(II) chain of collagen type II. Type II collagen adds structure and strength to the connective tissues that support the muscles, joints, organs, and skin of the body. Type II collagen is primarily found in cartilage and most Cartilage is then transformed into bone and participates in the regulation of intraperiosteal osteogenesis and endochondral osteogenesis. Type II collagen has a homotrimeric structure consisting of three triple helix α polypeptides folded into rods. The collagen region is composed of multiple amino acid sequences arranged repeatedly in a Gly-X-Y pattern (Gly is glycine, X usually represents proline, and Y usually represents hydroxyproline). There are many COL2A1 gene exons. So far, more than 100 mutations have been reported. COL2A1 gene-related diseases include Torrance type vertebral body flattening lethal skeletal dysplasia, chondrogenesis hypoplasia type 2, congenital vertebral epiphyseal dysplasia, Kniest dysplasia, Strudwick-type spondylometaphyseal dysplasia, etc. (https://mirror.omim.org/entry/120140), the COL2A1 gene-related diseases shown by OMIM are all autosomal dominant inheritance, but the COL2A1 gene c.3944G >A (p.Cys1315Tyr) mutation (based on the above strategy, detected by NGS technology) causes osteogenesis imperfecta (OI) has never been reported and confirmed.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a COL1A2 mutation in the pathogenic gene of bone dysplasia and a detection reagent thereof in view of the above-mentioned defects.

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

The purpose of the present invention can be realized by following technical scheme:

A mutated COL2A1 gene for detecting osteogenesis imperfecta disease, the mutated COL2A1 is a heterozygous mutation or a homozygous mutation c.3944G>A, the gene number of the wild-type COL2A1 gene in the NCBI database is: NM_001844.4, The base at 3944bp of CDS of this gene was mutated from G to A, and other parts were the same as wild type. The CDS sequence of the wild-type COL2A1 gene is shown in SEQ ID NO.1.

A mutated COL2A1 protein, the gene transcript number of the wild-type COL2A1 protein in the NCBI database is: NP_001835.3, the mutated COL2A1 protein is mutated from cysteine to tyrosine at the 1315th amino acid of the wild-type protein , and other parts are the same as wild type. The amino acid sequence of wild-type COL2A1 protein is shown in SEQ ID NO.2,

Application of the reagent for detecting the mutated COL2A1 gene or the mutated COL2A1 protein of the present invention in the preparation of a detection reagent or a detection device for osteogenesis imperfecta.

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

Said primer pair preferably consists of SEQ ID NO.3 and SEQ ID NO.4.

The detection equipment preferably includes a gene chip containing the mutated COL2A1 gene, a high-throughput sequencing, and a Sanger sequencing detection platform.

A kit for detecting osteogenesis imperfecta, the kit includes:

(1) A reagent for detecting the nucleotide at 3944bp of CDS of COL2A1 gene; or a reagent for detecting the 1315th amino acid site of COL1A1 protein;

(2) The product instruction manual, which clearly records that the nucleotide at 3944bp of CDS of COL2A1 gene is mutated from G to A, or the 1315th amino acid position of COL2A1 protein is changed from C to Y, which is a pathogenic mutation of osteogenic dysplasia.

Wherein, the reagents are preferably selected from primers or primer pairs, probes, antibodies, or nucleic acid chips.

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

Described reagent is further preferably the primer pair that detects COL21A1 gene CDS 3944bp place nucleotide; More preferably by 5'-TGGACTTAGCTCATGCAGAT-3' (SEQ ID NO.3) and 5'-TGGATTGGGGTAGACGC-3' (SEQ ID NO.3) NO.4) primer pair composed of.

The gene chip hybridization probe sequence for detecting the nucleotide at the 3944 bp of COL2A1 gene CDS in the kit is preferably as shown in SEQ ID NO.5.

A method for screening new COL2A1 gene mutations in OI patients using deep sequencing as a platform, and zebrafish mutation model combined with SIFT and Polyphen protein function prediction to verify that the gene mutation is a pathogenic gene mutation: including the following steps:

(1) For families with abnormal skeletal development (+/- with other abnormalities) shown by fetal ultrasound, or families with a genetic history of OI, collect clinical data and DNA-containing specimens such as blood and tissues, and extract genomic DNA;

(2) Detect 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, TGFB1, TNFRS, F11A, TRAPPC2, TREM2, TYROBP, WNT5A, WNT7A, ZMPSTE24, COL1A1, COL1A2, CRTAP, P3H1, SERPINF1, IFITM5, FKBP10, PPIB, SP7, BMP1, SERPINH1, TMEM38B, WNT1B, WNT1B, WNT1; Sub-detection, detection of mutations including point mutations of about 20,000 genes and small fragment deletion and insertion mutations within 20bp.

(3) The DNA is interrupted and the library is prepared, and then the coding region or the whole exon region of the target gene and the adjacent sheared DNA are captured and enriched by the chip, and finally the mutation detection is carried out using a high-throughput sequencing platform.

(4) The optimized bioinformatics analysis of the sequencing results revealed a new OI pathogenic mutation as COL2A1.Cys1315Tyr. The mutation was located on chromosome 12, and the base at physical position 48368588 (NCBI database) was mutated from G to A; protein level: The 1315th amino acid of the COL2A1 gene encoded protein was mutated from cysteine to tyrosine.

(5) The high-throughput sequencing described in step (3), the length of the target region based on whole exome sequencing is 58682415bp, the coverage of the target region reaches at least 99.91%, the average depth of the target region is at least 83.48X, and the average depth of the target region> The proportion of 30X sites is at least 98.70%.

(6) For the new mutation site COL2A1.Cys1315Tyr, use SIFT and Polyphen to predict its protein function.

(7) The zebrafish model was used to verify that the c.3944G>A point mutation on the COL2A1 gene affects skeletal development. The gene col2a1a (ENSDARG00000069093), which is highly similar to the human COL2A1 gene, was found in the ENSEMBL database. By expressing col2a1a in wild-type zebrafish The same point mutation gene was used to simulate the dominant expression in humans, and the skeletal development of zebrafish embryos was observed to verify that the new mutation site COL2A1.c.3944G>A resulted in abnormal skeletal development OI.

(8) Based on the test results of the whole exome test of the Trio family, identify whether the source of the mutation is a de novo mutation or a hereditary mutation. If it is a hereditary mutation, it should be determined whether it is a gonad mosaic according to the mutation ratio of one parent. combined mutation.

(9) If it is a new mutation, the next pregnancy can choose natural conception + prenatal diagnosis, and the probability of having the same mutation again is very low; if it is a hereditary mutation, you can choose IVF + preimplantation genetic diagnosis + prenatal diagnosis Diagnosis, excluding implanted embryos inherited the same mutation COL2A1.c.3944G>A.

beneficial effect

1. The present invention reports for the first time a new mutation site in the OI pathogenic gene COL2A1.c.3944G>A, which is an autosomal dominant genetic disease, regardless of heterozygous mutation or homozygous mutation. The diagnosis of the disease provides new pathogenic loci.

2. Provide new ideas for finding and verifying new clinical mutations, by performing Trio whole-exome sequencing for difficult cases with complex clinical characterization or unclear disease direction, and bioinformatics analysis of the harmfulness of new mutations in zebrafish, etc. Validated on animal models, and with informed consent, IVF combined with preimplantation diagnosis was performed, and embryos that excluded the mutation COL2A1.c.3944G>A were transplanted to obtain normal children without clinical signs of abnormal bone development.

3. The method of the present invention can be extended to the diagnosis and analysis of difficult cases with complex clinical manifestations or unclear disease direction in other fields other than bone dysplasia, and provides an effective method for finding new mutation sites or even new mutation genes for clinical practice. platform.

Description of drawings

Figure 1 Fetal ultrasound results

Figure 2 Sanger sequencing results of the family

Figure 3 Conservation of mutation positions between human COL2A1 gene and homologous genes in zebrafish, the mutation site (G3944A) of the present invention and its corresponding mutation site in zebrafish are represented by boxes

Figure 4 Nucleotide and amino acid correspondence map of zebrafish mutation sequences

Figure 5 Construction of zebrafish transcription and microinjection plasmids

Figure 6 SIFT prediction results of new sites in each database.

Figure 7. Polyphen prediction results of new sites in each database.

Figure 8 Wild-type zebrafish and COL1A2 gene c.3944G>A mutant zebrafish

Figure 9 Fetal Sanger sequencing results

Figure 10 PGT-M fetal ultrasound results

Detailed ways

After extensive and in-depth research, the present inventors discovered a new mutation site of the OI-related gene COL2A1, which can be used for diagnosing the above-mentioned diseases and for developing effective gene therapy drugs for the above-mentioned diseases.

When detecting variants at relevant loci, the detection can be on genomic DNA, cDNA or mRNA, or protein. Mutations can be detected using established techniques such as Western blotting, Southern blotting, DNA sequence analysis, PCR and in situ hybridization, and the like.

Various techniques can be employed to detect the presence of a G to A mutation at position 3944 of the wild-type COL2A1 gene (SEQ ID NO. 1), and these techniques are encompassed by the present invention. For example, gene chips and high-throughput sequencing capture probes are prepared based on relevant loci. In addition, the identification can be carried out by PCR with primers specific to the relevant site; or the identification can be carried out by designing a probe that can specifically bind according to the relevant site; or the identification can be carried out using specific restriction enzymes.

As an optional method, the single-base extension technology based on PCR technology can also be used to detect the variant site. One base at the variant site. Different fluorescently labeled ddNTPs are added for the reaction, or dNTPs and related reaction enzymes are added by pyrosequencing, and the primers can be extended only when the added ddNTPs or dNTPs are complementary to the base of the variation site. The type of mutation can be determined by detecting the fluorescence emitted by the extended bases or the visible light emitted by a series of enzymatic reactions in pyrosequencing.

The present invention also includes a reagent for detecting whether the analyte contains the variant site (whether there is a G to A mutation at position 3944 of CDS of the COL2A1 gene). The reagents are, for example: primers specific to the relevant mutation sites, and the amplified product contains a base corresponding to the 3944th position of the COL1A1 gene; probes specific to the relevant mutation sites can be combined with the mutated region. Specific binding occurs without specific binding to the unmutated region, and the probe carries a detectable signal; or a restriction endonuclease specific for the relevant mutated site.

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

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

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

Example 1

Genetic testing of a fetus whose ultrasound suggests skeletal abnormalities.

experimental method:

1. Collection of serial ultrasound results for pregnant women undergoing IVF-ET (PGT-A) pregnancy single embryo transfer due to her husband's chromosome 46,XY,inv(1)(p11q12), and collection of case data: collection of umbilical cord tissue of the induced fetus and the parents of the fetus to collect peripheral blood for genetic diagnosis. Genomic DNA from blood and tissue samples of each member of the family was extracted with a blood genomic DNA extraction kit (Tiangen Biochemical Technology Co., Ltd.).

2. High-throughput sequencing technology was used to mine the pathogenic mutations in this family: CNV-seq was used to detect deletions and duplication syndromes of more than 100kb in the whole genome and mutation detection with an accuracy of 1bp in the whole exon. Clinical whole exome test-Trio detects the exons of about 20,000 genes in the human genome, and the detection strategy is to target all 3,583 definite pathogenic relationships included in the OMIM database involved in the patient's clinical symptom complaint. Gene-by-gene analysis.

3. Trio whole exon information analysis includes filtering out the junctions, low-quality bases and undetected bases from the original off-board data, comparing them to the reference genome, performing SNP detection and InDel or CNV analysis, and then passing the database Note, the results of variant detection are screened for disease-related deleterious loci or genes through three analysis strategies based on variant deleteriousness, sample family status, and gene functional phenotype.

4. The pathogenic gene is identified by Sanger sequencing verification: PCR method is used to amplify the screened mutation sites and adjacent DNA sequences in the corresponding families, and the primer sequences used are designed by Primer 5 primer design software to detect the present invention. The sequences of primer pairs for pathogenic mutations are shown in SEQ ID NO.3 and SEQ ID NO.4. The PCR reaction system (50 μl system) used was: 10*buffer 5 μl, 25 mM MgCl ₂ 3 μl, Taq DNA polymerase 5 U, 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°C, 3min; (94°C, 25s; 55°C, 25s; 72°C, 20s) 35 cycles; 72°C 7min; 4°C preservation. After 2% agarose electrophoresis, the gel imager was used for detection. Marker was added to determine the size of the fragments. Sanger sequencing was performed on samples with a single band and the size of the fragments matched to determine whether the site was mutated or whether there was chimerism according to the height of the mutation peak.

Experimental results:

1. The director of the ultrasound department conducted an ultrasound examination of the fetus in the abdomen of the pregnant woman, and found that the fetus was congenitally abnormal, the fetal neck lymphatic sac tumor, fetal edema, short limb development (that is, the long bones of the limbs were short strip echoes), fetal head and torso It was wrapped in a cocoon-like shape, with a narrow thorax and a bulging abdomen (Figure 1). It was preliminarily judged that the fatal skeletal dysplasia was possible, and no members of the family had similar symptoms.

2. After CNV-seq detection and whole-exon detection of fetal induced labor tissue sample DNA, it was found that the fetus has c.3944G>A mutation in COL2A1 gene, which is a VOUS mutation, that is, a mutation of unknown clinical significance, and no other mutations were found. For the suspected pathogenic gene mutation site, the results of whole exome next-generation sequencing showed that the father had a low proportion of mosaicism at the same mutation site, and the same mutation was not found in the peripheral blood DNA of the fetal mother. Sanger sequencing confirmed that the mutation of this gene locus was a heterozygous mutation in the fetal sample, and there was a low proportion of chimeric mutation in the peripheral blood of the father, and no mutation was found in the peripheral blood of the mother, which was consistent with the second-generation sequencing results (Fig. 2). If the father is suspected of having gonadal mosaicism, preimplantation diagnosis in IVF can prevent the birth of a child with the same mutation.

3. According to the design scheme of the present invention, it is successfully confirmed that the detected COL2A1 gene c.3944G>A mutation is a new pathogenic locus of OI.

Example 2:

A functional study and a gene knockout animal model study were performed on the pathogenic gene detected in Example 1. Here, the detected new mutation c.3944G>A of the COL2A1 gene is taken as an example.

experimental method:

1. Conservation analysis: The frequency of occurrence of this locus in each database is evaluated.

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

3. The gene knockout animal model confirmed that the mutation site is the pathogenic mutation site.

(1) Analyze the homologous gene and point mutation position of COL2A1 in zebrafish, and select the correct homologous gene in zebrafish to prepare point mutation; find a gene highly similar to the human COL2A1 gene on the ENSEMBL website, which is ENSDARG00000069093, and analyze The conservation of mutation positions is shown in Figure 3. The above alignment results show that this locus is conserved in zebrafish genes, implying the importance of its function. In order to verify that the mutation at this site in zebrafish leads to a similar phenotype, the gene col2a1a (ENSDARG00000069093) in zebrafish with high similarity to human COL2A1 was selected for experiments.

(2) Method to verify the function of COL2A1(G3944A) point mutation: In humans, COL2A1(G3944A) exhibits a dominant skeletal dysplasia phenotype during embryonic stage, so it can be expressed by expressing the same point mutation gene of col2a1a in wild-type zebrafish To simulate the dominant expression in the human body, it was verified by observing the skeletal development of zebrafish embryos. Taking the sequence of the transcript ENSDARG00000069093 of the col2a1a gene as a reference, primers were designed to clone the full length of the gene and construct a point mutation of col2a1a (G3944A).

(3) Construct a plasmid expressing col2a1a (G3944A) in zebrafish: mutation point: G3944A, mutation primer: the original plasmid is used as a template, amplify the promoter region, and select the primer col2a1a-F1 (5'-CCT CTG ACA CCT GAT GCC AAT TGC-3') and col2a1a-R1 (5'-ATG CAG GTC CTA AGG GGT GAA AGT CG-3'), zebrafish genomic DNA was used as the template, and three pairs of primers were used to amplify the col2a1a mutant fragment, respectively col2a1a-M1F1 (5'-ATG TTC AGA TTG CTG GAT TCA CG-3') and col2a1a-CDSR4(5'-GCC AAT TGG ACC AGT CAA ACC T-3'), col2a1a-F4(5'-AAG AGG TTT GAC TGG TCC AAT-3') and col2a1a-mutR(5'-CCA TGT TGT AGA AAA CTT TGA TGG CAT CAG CAG-3'), col2a1a-mut(5'-GCC ATC AAA GTT TTC TAC AAC ATG GAG ACC GGA GAG ACC T -3') and col2a1a-CDSR5 (5'-CAA GAA GCA GAC TGC GCC AAT GTC-3'). The plasmid was synthesized by the method of homologous recombination. After the plasmid was constructed, it was sent to a sequencing company for sequencing. After analysis of the sequencing results, the plasmid was constructed correctly; the strain was preserved, and the plasmid was extracted and purified. The concentration after plasmid purification was 400 ng/μL. The method of expressing col2a1a (G3944A) selects DNA microinjection (using tol2 transposase to mediate high-efficiency transgene), using the overexpression plasmid constructed by microinjection (Figure 5) and transposase mRNA overexpression, confirmed by phenotype whether there is an impact.

(4) Phenotypic observation after expressing col2a1a (G3944A): After microinjection, the overall morphological development was continuously observed, especially the development of trunk bones (with or without bending).

Experimental results:

1. COL2A1; NM_001844.4; c.3944G>A; p.Cys1315Tyr|p.C1315Y; EX52; CDS52: missense mutation, there is no relevant literature report on the pathogenicity of this locus. Using SIFT and Polyphen to predict its protein function, the results are both harmful, and the probability of this site occurring in normal people is extremely low (Figure 6-7). COL2A1-related Torrance-type vertebral body flattening lethal skeletal dysplasia, chondrogenesis hypoplasia type 2, congenital spondylometaphyseal dysplasia, Kniest dysplasia, Strudwick-type spondylometaphyseal dysplasia are all autosomal dominant inheritance, i.e., etc. The presence of a deleterious mutation in the allele may cause the disease.

2. Through spine curvature phenotype analysis, it was found that injection of col2a1a plasmid resulted in curvature of the zebrafish spine (Figure 8).

Example 3:

Formulate an assisted reproduction strategy, and both spouses have informed consent to choose assisted reproduction for implantation diagnosis PGT-M and prenatal diagnosis

experimental method:

The remaining embryos after transplantation in Example 1 were subjected to PGT-M detection, and embryos that did not contain the mutation (with normal chromosomal CNV) were selected for transplantation, at 18 weeks of pregnancy, and amniocentesis karyotype analysis was performed, and the COL2A1 gene c.3944G>A was performed. (p.Cys1315Tyr) mutation detection to further confirm that there is no mutation at this site, genomic DNA is directly extracted from the subject's peripheral blood, tissue or amniotic fluid, and PCR amplification of COL2A1 gene exon 52 c.3944G>A (p. .Cys1315Tyr) was located in the fragment and Sanger sequencing, and the sequencing results were compared with the COL2A1 gene reference sequence (NM_001844). Ultrasound follow-up, and postnatal follow-up.

Experimental results:

1. It is not suggested that the fetus (M3944, II-2) carries the COL2A1 gene c.3944G>A (p.Cys1315Tyr) mutation from the gene level (Fig. 9).

2. Ultrasound showed no abnormality, and the neonatal development after birth was normal, and there was no skeletal deformity (Figure 10).

Claims

A mutated COL2A1 gene for detecting osteogenesis imperfecta diseases, characterized in that the mutated COL2A1 is a heterozygous mutation or a homozygous mutation c.3944G>A, and the gene number of the wild-type COL2A1 gene in the NCBI database is: NM_001844 .4, the base at 3944bp of CDS of this gene is mutated from G to A, and other parts are the same as wild type.
A mutated COL2A1 protein, characterized in that the number of the wild-type COL2A1 protein in the NCBI database is: NP_001835.3, and the mutated COL2A1 protein is mutated from cysteine to tyrosine at the 1315th amino acid of the wild-type protein , and other parts are the same as wild type.
Application of the reagent for detecting the mutated COL2A1 gene of claim 1 or the mutated COL2A1 protein of claim 2 in the preparation of a detection reagent or detection device for osteogenic dysplasia.
The application according to claim 3, wherein the detection reagent is selected from one or more of probes, primers and antibodies.
The application according to claim 3, wherein the detection equipment comprises a gene chip for detecting the mutated COL2A1 gene of claim 1, a detection platform for high-throughput sequencing and Sanger sequencing.
A test kit for detecting osteogenesis imperfecta, characterized in that the test kit comprises:

(1) A reagent for detecting the nucleotide at 3944bp of CDS of COL2A1 gene; or a reagent for detecting the 1315th amino acid site of COL2A1 protein;

(2) The product instruction manual, which clearly records that the nucleotide at 3944bp of CDS of COL2A1 gene is mutated from G to A, or the 1315th amino acid position of COL2A1 protein is changed from C to Y, which is a pathogenic mutation of osteogenic dysplasia.
The kit according to claim 6, wherein the reagent is 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 probes Needle.
The test kit according to claim 7, wherein the reagent is a primer pair for detecting the nucleotide at the 3944 bp of COL2A1 gene CDS; preferably a primer pair consisting of SEQ ID NO.3 and SEQ ID NO.4.
kit according to claim 7, it is characterized in that the gene chip hybridization probe sequence that detects COL2A1 gene CDS nucleotide 3944bp place is as shown in SEQ ID NO.5.
A method for screening COL2A1 gene new mutations in OI patients with deep sequencing as a platform, characterized in that it comprises the following steps:

(1) For families with abnormal skeletal development shown by fetal ultrasound, or with a history of OI genetic disease, collect clinical data and DNA-containing specimens such as blood and tissue, and extract genomic DNA;

(2) Detect 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, TGFB1, TNFRS, F11A, TRAPPC2, TREM2, TYROBP, WNT5A, WNT7A, ZMPSTE24, COL1A1, COL1A2, CRTAP, P3H1, SERPINF1, IFITM5, FKBP10, PPIB, SP7, BMP1, SERPINH1, TMEM38 B, WNT1 B, WNT1B, WNT1; Exon detection, detection of mutations including point mutations of about 20,000 genes and small fragment deletion and insertion mutations within 20bp;

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

(4) The optimized bioinformatics analysis of the sequencing results showed that a new OI pathogenic mutation was COL2A1.Cys1315Tyr, the mutation was located on chromosome 12, and the base at physical position 48368588 was mutated from G to A; protein level: The amino acid 1315 of the COL2A1 gene encoded protein was mutated from cysteine to tyrosine;

(5) The high-throughput sequencing described in step (3), the length of the target region based on whole exome sequencing is 58682415bp, the coverage of the target region reaches at least 99.91%, the average depth of the target region is at least 83.48X, and the average depth of the target region> The proportion of 30X sites is at least 98.70%;

(6) For the new mutation site COL2A1.Cys1315Tyr, use SIFT and Polyphen to predict its protein function;

(7) The zebrafish model was used to verify that the c.3944G>A point mutation on the COL2A1 gene affects skeletal development. The gene col2a1a, which is highly similar to the human COL2A1 gene, was found in the ENSEMBL database. The gene number is ENSDARG00000069093. The same point mutation gene of col2a1a was expressed to simulate the dominant expression in humans, and the skeletal development of zebrafish embryos was observed to verify that the new mutation site COL2A1.c.3944G>A resulted in abnormal skeletal development OI.