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WISP3 gene mutation for PPD (Progressive Pseudorheumatoid Dysplasia)-assisted diagnosis and application thereof

An auxiliary diagnosis and gene technology, applied in the field of biomedicine, can solve the problem of "hot spots" where sufficient mutations have not been found

Active Publication Date: 2017-11-03
PEKING UNION MEDICAL COLLEGE HOSPITAL CHINESE ACAD OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, some mutation sites in the WISP3 gene related to PPD have been found in the process of molecular biology research, but a sufficient number of mutation "hotspots" have not been found. If PPD-related mutation sites can be screened out as biomarkers , and the development of corresponding diagnostic kits will be a powerful impetus to the screening and early diagnosis of PPD in my country

Method used

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  • WISP3 gene mutation for PPD (Progressive Pseudorheumatoid Dysplasia)-assisted diagnosis and application thereof
  • WISP3 gene mutation for PPD (Progressive Pseudorheumatoid Dysplasia)-assisted diagnosis and application thereof
  • WISP3 gene mutation for PPD (Progressive Pseudorheumatoid Dysplasia)-assisted diagnosis and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1 Whole Exome Sequencing

[0046] 1. Sample collection

[0047] In December 2016, a 17-year-old male patient with PPD who visited Peking Union Medical College Hospital had typical clinical manifestations and was diagnosed with X-ray examination; his parents were not sick after testing. Obtain the informed consent of the family subjects to collect the blood samples of the research subjects, and it has been approved by the ethics committee of the hospital. Stored in a -80°C low-temperature refrigerator after serial numbering.

[0048] 2. Extraction and purification of peripheral blood DNA

[0049] (1) Add hemolysis reagent (i.e. lysate, 40 parts) to the peripheral blood stored in the 2mL cryopreservation tube. Dilute the volume of TrisHcl solution to 2000mL, the same below), mix by inverting and transfer completely.

[0050] (2) Removal of red blood cells: Fill the 5mL centrifuge tube to 4mL with hemolysis reagent, mix by inversion, centrifuge at 4000rpm for 10 ...

Embodiment 2

[0070] Example 2 Sanger sequencing for verification of mutation sites

[0071] 1. Collect the blood samples of 3 samples in the family in Example 1, and extract the DNA samples with Example 1;

[0072] 2. PCR amplification

[0073] Design of PCR primers: The primers were designed and synthesized by Shanghai Sangon. A total of 2 pairs of primers were designed, as shown in Table 2.

[0074] Table 2 Primer Sequence

[0075]

[0076] The PCR reaction system is shown in Table 3, the preparation of 25 μL system:

[0077] Table 3 reaction system

[0078] components

Amount added

template

1μL

forward primer

0.5μL

reverse primer

0.5μL

dNTP 10mM

0.5μL

Taq Buffer

2.5μL

Taq enzyme 5U / μL

0.2 μL

water

19.8μL

[0079] The PCR amplification program is shown in Table 4.

[0080] Table 4 reaction system

[0081]

[0082] 3. Sequencing

[0083] After PCR amplification, take 5 μL of the ampl...

Embodiment 3

[0089] The making of embodiment 3 gene mutation detection kit

[0090] The production and operation process of the mutation site kit is based on Sanger sequencing scanning detection typing technology. The kit contains specific primers for amplifying the two mutation sites of the WISP3 gene in Example 2. The kit can also include reagents commonly used in PCR reactions, such as Taq enzyme, dNTP mixture, MgCl 2 solution, deionized water, etc.; these commonly used reagents are well known to those skilled in the art, and may also contain standards and / or references (such as standards for genotype determination and blank controls, etc.). The value of this kit is that only peripheral blood is needed and no other tissue samples are required. The most streamlined and specific primer pair is used to detect the mutation site, and then the mutation site spectrum is used to assist in the determination of PPD. It is not only stable, convenient, accurate, and greatly Improve the sensitivity...

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Abstract

The invention discloses WISP3 gene mutation for PPD (Progressive Pseudorheumatoid Dysplasia)-assisted diagnosis. The mutation comprises mutation of two loci of a WISP3 gene, namely, NM_198239.1:c.643+2T>C and NM_198239.1:c.1064_1065dupGT:p.Gln356ValfsTer33. The invention further discloses a PPD-assisted diagnostic kit. Diagnosis of the PPD is assisted by the mutation of the two loci of the WISP3 gene, so that a clinician can quickly and accurately grasp the condition of a patient, a basis is laid for the evaluation of the clinical treatment effect, and discovery of a novel small molecular medicament target with a potential therapeutic value is assisted.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to a WISP3 gene mutation for auxiliary diagnosis of PPD and its application. Background technique [0002] Progressive pseudorheumatoid dysplasia (PPD) is a rare, autosomal recessive disorder of chondrodysplasia due to Wnt1-induced mutations in the signal peptide pathway protein 3 gene, reported incidence in Commonwealth of England countries It is about 1 / 1 million, and there are very few domestic case reports. The age of onset is mostly 1 to 10 years old, and 77% of them are 3 to 5 years old. [0003] The clinical symptoms of children with PPD are mainly large and small joint pain, swelling, deformity, and limited movement. Clinically, they are often misdiagnosed as juvenile rheumatoid arthritis and mucopolysaccharidosis. Imaging examinations of the whole body skeleton (including the spine) are very important for the diagnosis, manifested as generalized flat vertebrae with ir...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/12C12N15/11
CPCC12Q1/6883C12Q2600/118C12Q2600/136C12Q2600/156
Inventor 肖刻翁习生鲁昕冯宾
Owner PEKING UNION MEDICAL COLLEGE HOSPITAL CHINESE ACAD OF MEDICAL SCI
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