Application of Smoc2 gene and SNP marker thereof in multiple epiphyseal dysplasia
阅读说明：本技术 Smoc2基因及其SNP标志物在多发性骨骺发育不良中的应用 (Application of Smoc2 gene and SNP marker thereof in multiple epiphyseal dysplasia ) 是由 刘奇迹 李江夏 李琳 龙逢 施宏彪 郭少嫱 马玉儿 李鹏宇 于 2020-12-21 设计创作，主要内容包括：本发明提供Smoc2基因及其SNP标志物在多发性骨骺发育不良中的应用,涉及生物医学技术领域。本发明在山东收集到一个骨骼发育异常家系,经最终筛选试验验证,SMOC2 c.1076T>G；p.L359R错义突变是该常染色体显性多发性骨骺发育不良家系的致病突变,SMOC2基因是新的多发性骨骺发育不良的致病基因。本发明为揭示多发性骨骺发育不良发生机制提供新的致病理论依据,也为开发适用于我国人群的疾病早期预警模型奠定基础,因此具有良好的实际应用之价值。(The invention provides an application of a Smoc2 gene and an SNP marker thereof in multiple epiphyseal dysplasia, and relates to the technical field of biomedicine. According to the invention, a bone dysplasia family is collected in Shandong, and is verified by a final screening test, so that SMOC2c.1076T is greater than G; the p.L359R missense mutation is a pathogenic mutation of the autosomal dominant multiple epiphyseal dysplastic family, and the SMOC2 gene is a novel pathogenic gene of multiple epiphyseal dysplasia. The invention provides a new pathogenic theoretical basis for disclosing the occurrence mechanism of multiple epiphyseal dysplasia and lays a foundation for developing early disease early warning models suitable for people in China, thereby having good practical application value.)
1. The application of the substance for detecting the polymorphism or genotype of SMOC2c.1076T > G in human genome in the preparation of products for screening multiple epiphyseal dysplasia patients.
2. The application of the substance for detecting the polymorphism or genotype of SMOC2c.1076T > G in human genome in the preparation of products for detecting susceptibility of multiple epiphyseal dysplasia.
3. The application of the substance for detecting the polymorphism or genotype of SMOC2c.1076T > G in human genome in preparing the product for detecting the single nucleotide polymorphism related to multiple epiphyseal dysplasia.
4. The application of the substance for detecting the polymorphism or genotype of SMOC2c.1076T > G in human genome in the preparation of products for identifying or assisting in identifying the single nucleotide polymorphism related to multiple epiphyseal dysplasia.
5. Any of the following applications:
B1) the application of the polymorphism or genotype of SMOC2c.1076T > G in human genome in the preparation of products for screening multiple epiphyseal dysplasia patients;
B2) the polymorphism or genotype of SMOC2c.1076T > G in the human genome is applied to the preparation of products for detecting multiple epiphyseal dysplasia susceptibility;
B3) the application of the substance for detecting the polymorphism or genotype of SMOC2c.1076T > G in human genome in screening multiple epiphyseal dysplasia patients;
B4) the application of the substance for detecting the polymorphism or genotype of SMOC2c.1076T > G in human genome in detecting susceptibility of multiple epiphyseal dysplasia;
B5) the application of the substance for detecting the polymorphism or genotype of SMOC2c.1076T > G in human genome in detecting the single nucleotide polymorphism related to multiple epiphyseal dysplasia;
B6) use of a substance that detects the polymorphism or genotype of SMOC2c.1076t > G in the human genome for identifying or aiding in the identification of single nucleotide polymorphisms associated with multiple epiphyseal dysplasia;
B7) use of a polymorphism or genotype of SMOC2c.1076t > G in the human genome for screening for multiple epiphyseal dysplastic patients;
B8) the application of the polymorphism or genotype of SMOC2c.1076T > G in human genome in detecting susceptibility to multiple epiphyseal dysplasia.
6. A product containing a substance for detecting a polymorphism or genotype of SMOC2c.1076t > G in the human genome, which is any one of a) to d):
a) products that detect single nucleotide polymorphisms or genotypes associated with multiple epiphyseal dysplasia;
b) identifying or aiding in identifying a product of a single nucleotide polymorphism or genotype associated with multiple epiphyseal dysplasia;
c) screening multiple epiphyseal dysplastic patient products;
d) and detecting multiple epiphyseal dysplasia susceptibility products.
7. The product of claim 6, wherein: the substance for detecting the polymorphism or genotype of the SMOC2c.1076T > G in the human genome is a PCR primer for amplifying a genomic DNA fragment including the SMOC2c.1076T > G.
8. The product of claim 7, wherein: PCR primers were SMOC2-F and SMOC 2-R.
9. The product of claim 8, wherein the SMOC2-F is any one of the following single stranded DNA of a1) to a 4):
a1) single-stranded DNA shown in a sequence 1 in a sequence table;
a2) single-stranded DNA obtained by adding one or several nucleotides to the 5 'end and/or 3' end of a 1);
a3) a single-stranded DNA having 85% or more identity to the single-stranded DNA defined in a1) or a 2);
a4) single-stranded DNA which hybridizes with the single-stranded DNA defined in a1) or a2) under stringent conditions;
the SMOC2-R is any one of the following single-stranded DNA from b1) to b 4):
b1) single-stranded DNA shown in a sequence 2 in a sequence table;
b2) single-stranded DNA obtained by adding one or several nucleotides to the 5 'end and/or 3' end of b 1);
b3) a single-stranded DNA having 85% or more identity to the single-stranded DNA defined in b1) or b 2);
b4) a single-stranded DNA which hybridizes with the single-stranded DNA defined in b1) or b2) under stringent conditions.
10. The following M1) or M2):
m1) a method of screening for multiple epiphyseal dysplastic patients comprising: detecting the genotype of the SMOC2c.1076T & gtG site in the genome of the object to be detected;
m2) a method for detecting susceptibility to multiple epiphyseal dysplasia comprising: and detecting the genotype of the SMOC2c.1076T > G site in the genome of the object to be detected.
Technical Field
The invention relates to the technical field of biomedicine, in particular to application of a Smoc2 gene and an SNP marker thereof in multiple epiphyseal dysplasia.
Background
The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.
Multiple Epiphyseal Dysplasia (MED) (OMIM132400) is a osteochondral Dysplasia disease that is clinically characterized by mild to moderate short stature and early-onset osteoarthritis in the joints of the knee, hip, etc. The prevalence rate of the disease is about 1/20000-1/10000, and the genetic mode and clinical expression of the disease are heterogeneous. The inheritance mode of the disease is mainly autosomal dominant inheritance, and some families of patients are autosomal recessive inheritance.
The pathogenic genes found by the research so far are 8, namely COMP gene, COL9A2 gene, COL9A3 gene, DTDST gene, MATN3 gene, COL9A1 gene, CANT1 gene and COL2A1 gene. However, there are still cases of multiple epiphyseal dysplasia with no identifiable genetic mutation, while other genetic causes of multiple epiphyseal dysplasia remain to be determined.
Disclosure of Invention
In view of the problems in the prior art, the invention aims to provide the application of the Smoc2 gene and the SNP marker thereof in multiple epiphyseal dysplasia. According to the invention, a bone dysplasia family is collected in Shandong, and is verified by a final screening test, so that SMOC2c.1076T is greater than G; the p.L359R missense mutation is a pathogenic mutation of the autosomal dominant multiple epiphyseal dysplastic family, and the SMOC2 gene is a novel pathogenic gene of multiple epiphyseal dysplasia. Based on the above results, the present invention has been completed.
Specifically, the invention firstly provides any one of the following applications:
a1, detecting the polymorphism or genotype of SMOC2c.1076T > G in human genome, and applying the substance in screening multiple epiphyseal dysplasia patient products;
a2, detecting the polymorphism or genotype of SMOC2c.1076T > G in human genome, and the application in preparing products for detecting multiple epiphyseal dysplasia susceptibility;
a3, the application of the substance for detecting the polymorphism or genotype of SMOC2c.1076T > G in human genome in preparing the product for detecting the single nucleotide polymorphism related to multiple epiphyseal dysplasia;
a4, detecting the polymorphism or genotype of SMOC2c.1076T > G in human genome, and the application in the preparation of products for identifying or assisting in identifying single nucleotide polymorphism related to multiple epiphyseal dysplasia;
B1) the application of the polymorphism or genotype of SMOC2c.1076T > G in human genome in the preparation of products for screening multiple epiphyseal dysplasia patients;
B2) the polymorphism or genotype of SMOC2c.1076T > G in the human genome is applied to the preparation of products for detecting multiple epiphyseal dysplasia susceptibility;
B3) the application of the substance for detecting the polymorphism or genotype of SMOC2c.1076T > G in human genome in screening multiple epiphyseal dysplasia patients;
B4) the application of the substance for detecting the polymorphism or genotype of SMOC2c.1076T > G in human genome in detecting susceptibility of multiple epiphyseal dysplasia;
B5) the application of the substance for detecting the polymorphism or genotype of SMOC2c.1076T > G in human genome in detecting the single nucleotide polymorphism related to multiple epiphyseal dysplasia;
B6) use of a substance that detects the polymorphism or genotype of SMOC2c.1076t > G in the human genome for identifying or aiding in the identification of single nucleotide polymorphisms associated with multiple epiphyseal dysplasia;
B7) use of a polymorphism or genotype of the human genome SMOC2c.1076t > G for screening for multiple epiphyseal dysplastic patients;
B8) the application of the polymorphism or genotype of SMOC2c.1076T > G in human genome in detecting susceptibility to multiple epiphyseal dysplasia.
The SMOC2c.1076t > G (NM — 001166412.2) variation is located in SMOC2 exon 11, and this missense mutation results in a change in the encoded amino acid 359 from lysine to arginine (p.l 359r).
In the above application, the substance for detecting the polymorphism or genotype of the SMOC2c.1076T > G in the human genome can be a PCR primer for amplifying a genomic DNA fragment including the SMOC2c.1076T > G and/or a probe for detecting the SMOC2c.1076T > G.
In the application, the multiple epiphyseal dysplasia can be multiple epiphyseal dysplasia of Chinese population.
In order to solve the above technical problems, the present invention also provides a product containing a substance for detecting a polymorphism or genotype of smoc2c.1076t > G in a human genome, which is any one of a) to d):
a) products that detect single nucleotide polymorphisms or genotypes associated with multiple epiphyseal dysplasia;
b) identifying or aiding in identifying a product of a single nucleotide polymorphism or genotype associated with multiple epiphyseal dysplasia;
c) screening multiple epiphyseal dysplastic patient products;
d) and detecting multiple epiphyseal dysplasia susceptibility products.
In the product, the substance for detecting the polymorphism or genotype of the SMOC2c.1076T > G in the human genome can be a PCR primer for amplifying a genomic DNA fragment including the SMOC2c.1076T > G and/or a probe for detecting the SMOC2c.1076T > G.
In the product, the multiple epiphyseal dysplasia can be multiple epiphyseal dysplasia of Chinese population.
The invention also provides the following methods of M1) or M2):
m1) a method of screening for multiple epiphyseal dysplastic patients comprising: detecting the genotype of the SMOC2c.1076T & gtG site in the genome of the object to be detected;
m2) a method for detecting susceptibility to multiple epiphyseal dysplasia comprising: and detecting the genotype of the SMOC2c.1076T > G site in the genome of the object to be detected.
In the method, the multiple epiphyseal dysplasia may be multiple epiphyseal dysplasia of Chinese population.
In the method, the detection of the genotype of the SMOC2c.1076T > G site in the genome of the object to be detected can be carried out by using the substance for detecting the polymorphism or the genotype of the SMOC2c.1076T > G.
In practical application, the substance for detecting the polymorphism (namely allele) or genotype of the SMOC2c.1076T > G and other substances (such as substances for detecting other single nucleotide polymorphism or genotype related to multiple epiphyseal dysplasia) can be combined together to prepare the product for screening the patients with multiple epiphyseal dysplasia.
The beneficial technical effects of one or more technical schemes are as follows:
according to the technical scheme, SMOC2c.1076T > G is found in a sample of a skeletal dysplastic family from Shandong to be a single nucleotide polymorphism related to multiple epiphyseal dysplasia, and the single nucleotide polymorphism can be used for diagnosing the multiple epiphyseal dysplasia. Meanwhile, the substance for detecting the polymorphism (namely allele) or genotype of the SMOC2c.1076T > G and other substances (such as the substance for detecting other single nucleotide polymorphism (namely allele) or genotype related to multiple epiphyseal dysplasia) can be combined together to prepare a product for screening patients with multiple epiphyseal dysplasia or prepare a product for detecting susceptibility of multiple epiphyseal dysplasia.
The technical scheme provides a new pathogenic theoretical basis for revealing the occurrence mechanism of multiple epiphyseal dysplasia and lays a foundation for developing early disease early warning models suitable for people in China, so that the method has good practical application value.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.
FIG. 1 is a diagram of multiple epiphyseal dysplasia pedigree in accordance with an embodiment of the present invention;
FIG. 2 is a graph showing clinical and imaging performance of 3 patients in a family according to an embodiment of the present invention;
FIG. 3 is STR linkage analysis according to an embodiment of the present invention;
FIG. 4 shows amino acid conservation analysis of the SMOC2 protein 359 th position according to the example of the present invention;
FIG. 5 is a graph showing the results of analysis by the bioinformatics analysis software Polyphen2 and mutation taster according to the embodiment of the present invention; wherein, A is bioinformatics analysis software Polyphen2, B is bioinformatics analysis software mutation task;
FIG. 6 shows the sequencing results of the missense mutation Sanger of the SMOC2 gene as a family member in the examples of the present invention.
Detailed Description
It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments according to the present application. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.
In an exemplary embodiment of the invention, any of the following applications is provided:
a1, detecting the polymorphism or genotype of SMOC2c.1076T > G in human genome, and applying the substance in screening multiple epiphyseal dysplasia patient products;
a2, detecting the polymorphism or genotype of SMOC2c.1076T > G in human genome, and the application in preparing products for detecting multiple epiphyseal dysplasia susceptibility;
a3, the application of the substance for detecting the polymorphism or genotype of SMOC2c.1076T > G in human genome in preparing the product for detecting the single nucleotide polymorphism related to multiple epiphyseal dysplasia;
a4, detecting the polymorphism or genotype of SMOC2c.1076T > G in human genome, and the application in the preparation of products for identifying or assisting in identifying single nucleotide polymorphism related to multiple epiphyseal dysplasia;
B1) the application of the polymorphism or genotype of SMOC2c.1076T > G in human genome in the preparation of products for screening multiple epiphyseal dysplasia patients;
B2) the polymorphism or genotype of SMOC2c.1076T > G in the human genome is applied to the preparation of products for detecting multiple epiphyseal dysplasia susceptibility;
B3) the application of the substance for detecting the polymorphism or genotype of SMOC2c.1076T > G in human genome in screening multiple epiphyseal dysplasia patients;
B4) the application of the substance for detecting the polymorphism or genotype of SMOC2c.1076T > G in human genome in detecting susceptibility of multiple epiphyseal dysplasia;
B5) use of a substance that detects a polymorphism or genotype of rs27044 and/or rs30187 in the human genome for detecting a single nucleotide polymorphism associated with multiple epiphyseal dysplasia;
B6) use of a substance that detects the polymorphism or genotype of SMOC2c.1076t > G in the human genome for identifying or aiding in the identification of single nucleotide polymorphisms associated with multiple epiphyseal dysplasia;
B7) use of a polymorphism or genotype of SMOC2c.1076t > G in the human genome for screening for multiple epiphyseal dysplastic patients;
B8) the application of the polymorphism or genotype of SMOC2c.1076T > G in human genome in detecting susceptibility to multiple epiphyseal dysplasia.
In still another embodiment of the present invention, in the above use, the substance for detecting a polymorphism or genotype of SMOC2c.1076t > G in the human genome may be a PCR primer for amplifying a genomic DNA fragment including SMOC2c.1076t > G and/or a probe for detecting SMOC2c.1076t > G.
In yet another embodiment of the invention, said product may comprise said PCR primers and/or said probe for detection of SMOC2c.1076T > G.
In another embodiment of the present invention, in the above application, the multiple epiphyseal dysplasia may be multiple epiphyseal dysplasia of chinese population.
In still another embodiment of the present invention, there is provided a product containing a substance for detecting a polymorphism or genotype of smoc2c.1076t > G in the human genome, wherein the product is any one of a) to d):
a) products that detect single nucleotide polymorphisms or genotypes associated with multiple epiphyseal dysplasia;
b) identifying or aiding in identifying a product of a single nucleotide polymorphism or genotype associated with multiple epiphyseal dysplasia;
c) screening multiple epiphyseal dysplastic patient products;
d) and detecting multiple epiphyseal dysplasia susceptibility products.
In still another embodiment of the present invention, in the above product, the substance for detecting a polymorphism or genotype of SMOC2c.1076t > G in the human genome may be a PCR primer for amplifying a genomic DNA fragment including SMOC2c.1076t > G.
In another embodiment of the present invention, in the above product, the multiple epiphyseal dysplasia may be multiple epiphyseal dysplasia of chinese population.
In still another embodiment of the present invention, the present invention further provides the following method of M1) or M2):
m1) a method of screening for multiple epiphyseal dysplastic patients comprising: detecting the genotype of the SMOC2c.1076T & gtG site in the genome of the object to be detected;
m2) a method for detecting susceptibility to multiple epiphyseal dysplasia comprising: and detecting the genotype of the SMOC2c.1076T > G site in the genome of the object to be detected.
In another embodiment of the present invention, in the above method, the multiple epiphyseal dysplasia may be multiple epiphyseal dysplasia of chinese population.
In still another embodiment of the present invention, in the above method, the detection of the genotype of the SMOC2c.1076t > G site in the genome of the test subject may be performed using the substance for detecting the polymorphism or genotype of SMOC2c.1076t > G.
In practical application, the substance for detecting the polymorphism (namely allele) or genotype of the SMOC2c.1076T > G and other substances (such as substances for detecting other single nucleotide polymorphism or genotype related to multiple epiphyseal dysplasia) can be combined together to prepare the product for screening the patients with multiple epiphyseal dysplasia.
Wherein the substance for detecting the polymorphism or genotype of SMOC2c.1076T > G in the human genome may be a reagent and/or an instrument required for determining the polymorphism or genotype of rs27044 and/or rs30187 by at least one of the following methods: DNA sequencing, restriction enzyme fragment length polymorphism, single-strand conformation polymorphism, denaturing high performance liquid chromatography, SNP chip, microfluidic chip technology, TaqMan probe technology and Sequenom MassArray technology. Wherein, the reagents and/or instruments required for determining the polymorphism or genotype of the SMOC2c.1076T > G by utilizing the Sequenom MassArray technology comprise PCR primer pairs, extension primers based on single-base extension reaction, phosphatase, resin, chips, MALDI-TOF (matrix-assisted laser desorption/ionization-time of flight mass spectrometry) and/or other reagents and instruments required by the Sequenom MassArray technology; reagents and/or instruments required for determining the polymorphism or genotype of rs27044 and/or rs30187 by using the TaqMan probe technology comprise a TaqMan probe, a PCR primer pair, a quantitative PCR instrument, a genotyping module and/or other reagents required by the TaqMan probe technology; the SNP chip includes a chip based on nucleic acid hybridization reaction, a chip based on single base extension reaction, a chip based on allele-specific primer extension reaction, a chip based on "one-step" reaction, a chip based on primer ligation reaction, a chip based on restriction enzyme reaction, a chip based on protein DNA binding reaction and/or a chip based on fluorescent molecule DNA binding reaction. The reagents and/or instruments required for determining the polymorphism or genotype of the SMOC2c.1076T > G by the microfluidic chip technology comprise a DNA extraction microfluidic module and reagents, a DNA amplification module and a PCR primer pair, a nucleic acid labeling module and related reagents, and an SNP chip and related hybridization, elution and scanning microfluidic modules and reagents.
The product can be a reagent or a kit, and can also be a system consisting of the reagent or the kit and an instrument, such as a system consisting of a primer and a DNA sequencer, a system consisting of a PCR reagent and a DNA sequencer, a system consisting of a TaqMan probe, a PCR primer pair, a quantitative PCR instrument and a module for genotyping and other reagents required by TaqMan probe technology, a system consisting of a probe, a PCR primer pair and other reagents and instruments required by Ligase Detection Reaction (LDR), and a system consisting of a PCR primer pair, a single base extension primer, a chip, a PCR instrument, a module for genotyping and/or other reagents and instruments required by Sequenom MassArray technology.
And (2) amplifying a genomic DNA fragment including the SMOC2c.1076T > G by using a PCR primer, taking the obtained PCR amplification product as a template, and detecting the sequence of the obtained extension product by using the probe for detecting the SMOC2c.1076T > G to determine the polymorphism (namely allele) and the genotype of the SMOC2c.1076T > G. The PCR primers have no special requirements on the sequence as long as the genomic DNA fragment including SMOC2c.1076T > G can be amplified. The probe for detecting the SMOC2c.1076T > G can be designed according to the upstream and downstream of the SMOC2c.1076T > G in the human genome, and the sequence of the probe covers the nucleotide of the SMOC2c.1076T > G in the human genome.
In yet another embodiment of the present invention, the PCR primers are SMOC2-F and SMOC 2-R;
the SMOC2-F is any one of the following single-stranded DNA from a1) to a 4):
a1) single-stranded DNA shown in a sequence 1 in a sequence table;
a2) single-stranded DNA obtained by adding one or several nucleotides to the 5 'end and/or 3' end of a 1);
a3) a single-stranded DNA having 85% or more identity to the single-stranded DNA defined in a1) or a 2);
a4) single-stranded DNA which hybridizes with the single-stranded DNA defined in a1) or a2) under stringent conditions;
the SMOC2-R is any one of the following single-stranded DNA from b1) to b 4):
b1) single-stranded DNA shown in a sequence 2 in a sequence table;
b2) single-stranded DNA obtained by adding one or several nucleotides to the 5 'end and/or 3' end of b 1);
b3) a single-stranded DNA having 85% or more identity to the single-stranded DNA defined in b1) or b 2);
b4) a single-stranded DNA which hybridizes with the single-stranded DNA defined in b1) or b2) under stringent conditions.
The term "identity" as used herein refers to sequence similarity to a native nucleic acid sequence. "identity" includes a nucleotide sequence having 85% or more, or 90% or more, or 95% or more identity to the nucleotide sequence shown in SEQ ID No.1 or SEQ ID No.2 of the present invention. Identity can be assessed visually or by computer software. Using computer software, the identity between two or more sequences can be expressed in percent (%), which can be used to assess the identity between related sequences.
The stringent conditions are hybridization and washing of the membrane 2 times, 5min each, at 68 ℃ in a solution of 2 XSSC, 0.1% SDS, and 2 times, 15min each, at 68 ℃ in a solution of 0.5 XSSC, 0.1% SDS; alternatively, hybridization was carried out at 65 ℃ in a solution of 0.1 XSSPE (or 0.1 XSSC), 0.1% SDS, and the membrane was washed.
In another embodiment of the present invention, the identity of 85% or more may be 85%, 90% or 95% or more.
In yet another embodiment of the invention, SMOC2c.1076T > G is typed using a Taqman (thermo Fisher) genotyping platform. The DNA template containing the SNP site region is amplified by PCR technology and then detected by using a probe.
The invention is further illustrated by the following examples, which are not to be construed as limiting the invention thereto. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.
Examples
A skeletal dysplastic family is collected in Shandong, and the four generations of 38 members are shared in the family, and the proband is IV-9. Through family map analysis, each generation of the family has patients and has diseases for both men and women, and the family accords with the genetic characteristics of autosomal dominant hereditary diseases (figure 1).
The ancestor of the pedigree is IV-9, and the patient is normal at birth and has no obvious clinical symptoms. After 2 years of age, parents find that the motor ability of the parents is slow to develop, and symptoms such as knee joint inversion, knee joint swelling and pain appear at 4 years of age. The imaging was characterized by knee swelling, uneven epiphyseal ossification of the distal femur and proximal tibia, in the form of "glacier fissures", with irregular jagged epiphyseal surfaces (fig. 2). Other patients of the family are adults, and the symptoms of mild short stature appear, and the height is between 1.4 and 1.6 meters. The large joints of knee joints, hip joints and the like of most patients are stiff and cannot be straightened; the fatigue is easy, the pain is obvious after fatigue, and the user can not rise by means of external force after squatting or squatting; symptoms worsen with seasonal changes. Some patients have osteoporosis symptoms, and joint swelling is obvious. II-7 swelling of the finger joints was evident. The X-ray of III-14 shows bilateral patellar superior apices and patellar position shift downward (FIG. 2). The final clinical diagnosis is multiple epiphyseal dysplasia.
In order to search for the pathogenic gene of the family, the peripheral blood of some patients and normal persons in the family is collected to extract genomic DNA. Firstly, STR loci closely linked with pathogenic genes of known multiple epiphyseal dysplasia genes are selected, family member DNA is taken as a template, PCR amplification and modified polyacrylamide gel electrophoresis are carried out, and then linkage analysis is carried out, and no linkage is found (figure 3), so that all known pathogenic genes are excluded.
Thus, we performed whole exome sequencing of proband IV-9 and found that there was a mutation site in each of the INSL5, MRPS5, ANXA5, SMOC2, KAT6B, MYO15A, DSCAM and PABPC3 genes. These mutations were retrieved in the ExAC (outer Aggregation Consortium, http:// ExAC. broadinfection. org) database, and all but those located in the SMOC2 and PABPC3 genes were found to be not retrieved in the ExAC database (Table 1). The ExAC database is a database of genetic variations that pooled the sequencing results of exons from 60,706 individuals of different ethnic groups. If a variation is retrieved from the database and occurs more frequently, it is suggested that the variation may not be a pathogenic variation. Thus, only SMOC2 and PABPC3 are likely to be the mutations that cause the disease in this family. However, further Sanger sequencing analysis revealed that the PABPC3 gene variation was not detected in other patients, and was not co-segregating with phenotype and was therefore excluded.
TABLE 1 candidate genes and their frequencies in the Normal population
The SPARC-related modular calcium binding 2(SMOC2) c.1076T > G (NM-001166412.2) variation is located in SMOC2 exon 11, and this missense mutation results in a change from lysine to arginine at amino acid 359 (p.L 359R). We first performed an analysis of this mutation site using online software. Analysis was performed by analyzing whether the 359 th amino acid (lysine) encoded by SMOC2 gene was evolutionarily conserved using Clustal Omega (https:// www.ebi.ac.uk/Tools/msa/clustalo), and it was revealed that the lysine at this site was conserved among species in many species (FIG. 4). The results of bioinformatics analysis software Polyphen2 (FIG. 5, A) and mutation taster (FIG. 5, B) both showed that the mutation at this site was pathogenic.
To verify whether the variation caused disease in patients in this family, we designed primers upstream and downstream of the variation site (SMOC2 forward 5 'TGAGACACCAGGACCATGAG 3', SEQ ID No. 1); SMOC2 reverse 5 'ACTCCCAGGCCATAAACACA 3', SEQ ID No.2), PCR amplification and Sanger sequencing were performed using the extracted genomic DNA of the family member as a template. Sequencing results showed that all patients in this family were heterozygous for the c.1076T > G variation, whereas there was no such variation in the normal members. Thus, this heterozygous variation was co-segregating with the phenotype of the patients in the family (FIG. 6).
The above results suggest that SMOC2c.1076t > G; the p.L359R missense mutation is a pathogenic mutation of the autosomal dominant multiple epiphyseal dysplastic family, and the SMOC2 gene is a novel pathogenic gene of multiple epiphyseal dysplasia.
It should be noted that the above examples are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to the examples given, those skilled in the art can modify the technical solution of the present invention as needed or equivalent substitutions without departing from the spirit and scope of the technical solution of the present invention.
SEQUENCE LISTING
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