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New gene mutation site causing congenital membranous cataract, detection method and application

A detection method and cataract technology, applied in biochemical equipment and methods, microbe measurement/testing, DNA/RNA fragments, etc., to achieve the effect of expanding understanding and expanding research directions

Active Publication Date: 2020-09-01
FOURTH MILITARY MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is still a lack of reports on such LIM2 gene mutation sites for East Asian populations

Method used

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  • New gene mutation site causing congenital membranous cataract, detection method and application
  • New gene mutation site causing congenital membranous cataract, detection method and application
  • New gene mutation site causing congenital membranous cataract, detection method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] In this example, a new generation of whole-exome sequencing technology was used to perform high-throughput sequencing of all exon regions in a family of autosomal dominant inherited congenital cataracts in the Han Chinese, and LIM2 was found in combination with biological information analysis. Genetically c.388C> T(p.R130C) mutation is related to congenital membranous cataract, and this mutation has been verified by co-separation experiments and other methods. details as follows:

[0067] 1. Sample collection

[0068] The congenital cataract family consists of 26 members, including 11 patients with congenital cataract ( figure 1 ). Eight congenital cataract patients and five normal controls were selected as exome sequencing samples in the family (Table 1).

[0069] Table 1. Basic situation of congenital cataract family samples

[0070] family member gender age Uncorrected vision right / left Lens opacity Ⅱ:1M56 20 / 20; 20 / 20 Transparent Ⅱ: 2F59 20 / 100; 20 / 63 Membranous Ca...

Embodiment 2

[0083] As a further verification of Example 1, the following example is provided. Sanger direct sequencing method was used to verify the mutation in the family, and it was confirmed that it was co-segregated with the phenotype and passed by autosomal dominant inheritance; at the same time, the mutation at this locus was carried out in 100 normal local population peripheral blood genomic DNA samples Screening did not find the mutation.

[0084] 1. Direct sequencing method to verify the mutation of LIM2 gene in patients in the family

[0085] 1) PCR amplification target fragment: reaction conditions and reaction system:

[0086] Table 1. PCR reaction system of LIM2 gene

[0087]

[0088] Among them, PCR amplification uses Tiangen Biochemical Technology Co., Ltd. PCR Mix (Taq enzyme, buffer, dNTP).

[0089] Reaction conditions: PCR reaction is carried out on BIORAD My Cycle thermal cycler, the reaction process (including temperature and time) is as Figure 4 Shown:

[0090] ①Pre-denaturat...

Embodiment 3

[0142] Detection of CC-related gene LIM2 mutation site (c.388C> T) Kit and its application

[0143] 1. The composition of the kit

[0144] 1) Kit 1: A kit for detecting mutant LIM2 gene, including one or more sets of primer pairs, wherein the mutation is a mutation of LIM2 gene c.388C> The mutation p.R130C of T or MP20 protein, wherein the primer pair is designed on the genomic sequence or cDNA coding region sequence based on a position selected from the following, so that the amplified product covers the position. The kit for detecting mutant LIM2 gene includes the following primers:

[0145] Primer p1 for amplification:

[0146] LIM2-F1:5’-CAGAGACAATGGCCAATTAC-3’

[0147] LIM2-R1:5’-GAGCCCAACACCCTACTCTC-3’

[0148] Primer p2 for amplification:

[0149] LIM2-F2:5’-AGACAGCATCGCATACTGGA-3’

[0150] LIM2-R2:5’-AATCCCTGCGAAGAACGTCA-3’

[0151] Kit 2: A kit for detecting mutant LIM2 gene, including one or more nucleic acid probes, the mutation is a mutation of LIM2 gene c.388C> T, the probe is...

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Abstract

The invention relates to the field of disease-related mutant genes and discloses a novel gene mutation site causing congenital membranous cataract, a detection method and application. The invention specifically relates to gene mutation c.388C > T (p.R130C) of LIM2 and a genetic mode of dominant inheritance of autosomes of the gene discovered for the first time, and discloses a congenital membranous cataract virulence gene LIM2 mutation detection kit at the same time. The kit disclosed by the invention is used for detecting whether a patient has LIM2 gene c.388C > T mutation or not; therefore,an effective way for congenital cataract gene diagnosis, prenatal gene screening and genetic counseling is provided. The kit is beneficial to clinical prenatal diagnosis screening and common screeningof newborn LIM2 gene mutation, early surgery and training rehabilitation intervention are carried out on diagnosis congenital membranous cataract children, and irreversible lifelong hypopsia is reduced; and a basis is provided for screening after preventive fertility.

Description

Technical field [0001] The present invention relates to the field of gene detection, in particular to a new gene mutation site that causes congenital membranous cataract, detection method and application, Background technique [0002] Congenital cataract (congenital cataract, CC) refers to the partial or complete opacity of the lens that exists before birth or gradually forms after birth and occurs within one year after birth. The global incidence of CC is 1-15 / 10,000, which is the second leading cause of blindness in children. CC due to abnormal lens metabolism in the embryonic stage causes its own transparency to decrease. Any genetic mutation that participates in or affects the development of the lens may lead to the occurrence of congenital cataracts. [0003] Among more than 100 genetic abnormalities that have been found to cause congenital cataracts, 39 are associated with only manifestations that cause simple cataracts. Among these 39 gene abnormalities, more than half of ...

Claims

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Application Information

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IPC IPC(8): C12Q1/6883C12N15/11C12Q1/6869
CPCC12Q1/6883C12Q1/6869C12Q2600/156C12Q2600/106C12Q2531/113
Inventor 周健裴瑞王芳叶巍李吉马济远何梦梅
Owner FOURTH MILITARY MEDICAL UNIVERSITY
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