Application of for ABCA3 genes to detecting congenital CCMC (cataract-microcornea syndrome)

A congenital cataract and gene technology, applied in the direction of DNA / RNA fragments, recombinant DNA technology, microbial measurement / inspection, etc., can solve the problem of limited understanding of disease-causing genes

Active Publication Date: 2016-01-27
SHANDONG EYE INST +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, the knowledge of the causative genes of CCMC is still limited, and the 7 known genes can only explain the pathogenesis of less than 30% of patients so far

Method used

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  • Application of for ABCA3 genes to detecting congenital CCMC (cataract-microcornea syndrome)
  • Application of for ABCA3 genes to detecting congenital CCMC (cataract-microcornea syndrome)
  • Application of for ABCA3 genes to detecting congenital CCMC (cataract-microcornea syndrome)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Embodiment 1: Screen the mutation site of ABCA3 gene from congenital CCMC family

[0029] 1. Extraction of peripheral blood genomic DNA:

[0030] In compliance with the relevant national policies and regulations, and on the basis of the consent of the sampling subject, draw 2-5ml of peripheral venous blood from the first family member, put it into an EDTA anticoagulant tube, and store it at -80°C for later use; the frozen EDTA anticoagulant blood After melting at room temperature, put 500 μL into a centrifuge tube, add an equal volume of TE (pH 8.0), mix well, centrifuge at 10,000 rpm for 10 minutes at 4°C, and discard the supernatant.

[0031] Add 180 μLTE, 20 μL SDS (10%), 8 μL proteinase K (10 mg / ml), mix well, and place in a 37° C. water bath overnight. Remove the sample from the water bath and briefly centrifuge to pellet the sample. Add an equal volume of Tris-saturated phenol (about 300 μL) to the reaction tube, mix thoroughly, centrifuge at 10,000 rpm for 10 m...

Embodiment 2

[0086] Example 2: Screening the mutation site of ABCA3 gene from another congenital CCMC family

[0087] 1. Extraction of genomic DNA from peripheral blood: the conventional phenol-chloroform method described in Example 1 was used to extract genomic DNA from the peripheral blood of each member of the family.

[0088] 2. PCR amplification target fragment: reaction conditions and reaction system:

[0089] (1) PCR reaction conditions: 94°C for 3min; 94°C for 40sec, 55±3°C for 40se, 72°C for 60sec, 30-35cycle; 72°C for 10min.

[0090] (2) Reaction system: (TAKARALATaqpolymerase)

[0091]

[0092] The reaction system was used to amplify each patient's genomic DNA template and 19 pairs of primers for the ABCA3 gene.

[0093] 3. Sequencing of PCR products: The genomic DNA templates of the patients in the family and the amplification products of 19 pairs of primers of the ABCA3 gene were sequenced using the conventional Sanger sequencing method. A heterozygous mutation occurred ...

Embodiment 3

[0094] Example 3: Screening the mutation site of ABCA3 gene from another 2 patients with congenital sporadic CCMC

[0095] Collection of sporadic patients with CCMC: A total of 2 sporadic patients with definite diagnosis of CCMC were collected in Shandong Eye Institute and Qingdao Eye Hospital.

[0096] Genomic DNA extraction from peripheral blood:

[0097] Take 2-5ml of peripheral venous blood from 2 patients with sporadic CCMC, put them into EDTA anticoagulant tubes, and freeze them at -80°C for later use; after the frozen EDTA anticoagulated blood is thawed at room temperature, put 500 μL into centrifuge tubes, and apply The conventional phenol-chloroform method described in Example 1 was used to extract genomic DNA from the peripheral blood of each member of the family.

[0098] PCR amplification target fragment: reaction conditions and reaction system:

[0099] (1) PCR reaction conditions: 94°C for 3min; 94°C for 40sec, 55±3°C for 40se, 72°C for 60sec, 30-35cycles; 72°C f...

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Abstract

The invention aims to provide application of ABCA3 genes to preparing diagnosis products for detecting congenital cataract-microcornea syndrome (CCMC). The application has the advantages that novel application of the ABCA3 genes is provided, and accordingly an effective way is provided for genetic diagnosis of congenital CCMC diseases, prenatal gene screening and genetic counseling; as shown by application effects, SNP (single nucleotide polymorphism) sites and detection primers of the genes can be effectively used for quickly detecting ABCA3 gene mutation sites for clinical patients and villus or amniotic fluid of fetuses.

Description

technical field [0001] The invention belongs to the related technical field of gene diagnosis and prenatal gene diagnosis, and specifically relates to the application of ATP junction box (ABC) transporter A3 (ABCA3) in the preparation and detection of congenital cataract-microcorneal syndrome (CCMC) gene diagnosis products. Background of the invention [0002] Congenital cataract is a common eye disease that causes low vision and blindness in children. The incidence rate is about 0.01% to 0.06%, and about 50% of them are related to genetics. The most common mode of inheritance is autosomal dominant inheritance. Congenital cataract can occur independently, or as a concomitant symptom of ocular or systemic syndromes, among which 12%-18% of congenital cataract patients often have microcorneal symptoms. Cataract-microcornea syndrome (Cataract-microcorneayndrome, CCMC, OMIM116200) is a congenital dysplasia eye disease, often involving both eyes, manifested as different phenotypes...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6883C12Q2600/156
Inventor 陈鹏代云海谢立信王晔肖晶晶张清岩管李萍王俊
Owner SHANDONG EYE INST
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