A method of screening for congenital apergy- microphthalmia- cataract syndrome, a screening kit

Abstract

The present application relates to the field of molecular diagnosis, in particular to a congenital aperular-microphthalmos-cataract syndrome screening method, a screening kit and application thereof. GJA8 The patient with congenital aperular-microphthalmos-cataract syndrome has a hybrid mutation site c.137G>A on the gene, while the normal person does not have the mutation at the site. The present application develops a group of identification primers according to the mutation site, the nucleotide sequence of the identification primers is shown as SEQ ID NO. 1 and SEQ ID NO. 2, and the screening kit prepared by the identification primers can be used for early diagnosis and screening of congenital aperular-microphthalmos-cataract syndrome, such as early screening of pregnancy, etc., which can make early warning for eugenics and child rearing, and has good application prospect.

Description

Technical Field

[0001] This invention relates to the field of in vitro molecular diagnostics, specifically to a screening method, screening kit, and application of congenital apuprana-microphthalmia-cataract syndrome. Background Technology

[0002] Congenital apupil-microphthalmia-cataract syndrome is a rare ocular disease clinically characterized by the absence of a pupil in the eye, accompanied by the combined symptoms of cataracts and microphthalmia. It is inherited in an autosomal dominant pattern. To date, this syndrome was first reported by Kondo, with only one family and two isolated cases reported. Genetic studies have mapped candidate genes to chromosomes 1, 5, 8, 11, and 17, but a specific gene has not yet been identified. The causative gene associated with congenital apupil-microphthalmia-cataract syndrome remains undetermined.

[0003] GJA8 (Gap Junction Protein Alpha 8, GJA8 The gene is located on chromosome 1q21.1 and consists of two exons. This gene encodes a transmembrane connective protein, GJA8, or CX50, which is a member of the gap junction protein family and plays a key role in the formation of gap junction channels. GJA8 This gene is related to the development of the lens of the eye, and its mutations may be associated with congenital cataracts, cataracts, congenital cataract-microkeratosis syndrome, glaucoma, and microphthalmia. Currently, screening for congenital microphthalmia-microkeratosis syndrome is mainly based on ophthalmological examination, and can only diagnose patients who have already been born. Therefore, developing a rapid screening method and kit for patients with congenital microphthalmia-microkeratosis syndrome is of great significance for early diagnosis of this disease and for providing early warning for healthy births. Summary of the Invention

[0004] The purpose of this invention is to provide a screening method and kit for congenital microphthalmia-congenital cataract syndrome, which can be used for early diagnosis and screening of congenital microphthalmia-congenital cataract syndrome, including screening in early pregnancy, to provide early warning for eugenics and has good application prospects.

[0005] To achieve the above objectives, the present invention provides a screening method for congenital apuprana-microphthalmia-cataract syndrome, the screening method comprising, according to GJA8 Gene mutation sites are used to determine whether the subject of the test has congenital microphthalmia-congenital cataract syndrome.

[0006] Furthermore, the above GJA8 The gene mutation site is c.137G>A, and its mutation information is as follows: GJA8 When the 137th base in the gene coding region changes from G to A, the target being tested... GJA8 The mutation of the base from G to A at this gene locus indicates that the person being tested has congenital microphthalmia-cataract syndrome.

[0007] This invention also provides a set of identification primers for detecting congenital apuprana-microphthalmia-cataract syndrome, which are derived from... GJA8 The primers for identification were developed based on the gene mutation site c.137G>A. The nucleotide sequences of these primers are shown in SEQ ID NO.1 and SEQ ID NO.2.

[0008] The identification primers provided by this invention can be used for screening of congenital microphthalmia-congenital cataract syndrome.

[0009] The present invention also provides a screening kit for congenital apuprosis-microphthalmia-cataract syndrome, which contains identification primers with nucleotide sequences as shown in SEQ ID NO.1 and SEQ ID NO.2.

[0010] The screening kit provided by this invention can be used for screening congenital microphthalmia-congenital cataract syndrome.

[0011] Furthermore, the screening kits described above can be used for the early diagnosis of congenital microphthalmia-congenital cataract syndrome and / or for early warning of prenatal and postnatal care.

[0012] The screening method and kit for congenital microphthalmia-congenital cataract syndrome provided by this invention have the following advantages for the diagnosis and screening of this syndrome:

[0013] (1) This invention provides gene sequence polymorphism sites for screening congenital apupil-microphthalmia-cataract syndrome, which can be used as biological targets for screening and early intervention of congenital apupil-microphthalmia-cataract syndrome, and has broad application prospects.

[0014] (2) This invention directly uses Sanger first-generation sequencing, which can simply, directly and effectively screen patients with congenital apupa-microphthalmia-cataract syndrome, providing guidance for timely detection and treatment of patients.

[0015] (3) This invention can be used for prenatal diagnosis, provide early warning for eugenics, and is of great significance for preventing congenital apuccal-microphthalmia-cataract syndrome in offspring, thus reducing the economic burden on society. Attached Figure Description

[0016] Figure 1 This is a pedigree chart of the congenital apupa-microphthalmia-cataract syndrome in this invention.

[0017] Figure 2 This is a sequencing diagram of the c.137 mutation status in the genomes of normal individuals and patients in this invention. Detailed Implementation

[0018] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0019] 1) Collection of clinical samples

[0020] A family with congenital microphthalmia-apatia cataract syndrome was recruited at the First Affiliated Hospital of Zhengzhou. The family consists of 3 patients and 4 healthy family members. The family pedigree is shown below. Figure 1 As shown, solid circles represent patients with congenital apuprana-microphthalmia-cataract syndrome; hollow circles represent normal individuals; and arrows represent probands.

[0021] In addition, peripheral blood samples were collected from 400 healthy individuals, and genomic DNA was extracted from each sample.

[0022] Note: All family members involved in this invention research signed informed consent forms, and peripheral blood samples were obtained from patients and related family members.

[0023] 2) Sequencing and acquisition of mutation sites

[0024] Peripheral venous whole blood samples (2 mL, EDTA anticoagulated) were collected from all family members and labeled. Genomic DNA was extracted from the blood samples using a blood genomic DNA extraction kit from Tiangen Biotech Co., Ltd. The concentration and purity of the DNA were measured using a micro-spectrophotometer. The OD260 / OD280 value of the genomic DNA from each sample was between 1.7 and 1.9, with a concentration of not less than 50 ng / μL and a total amount of not less than 20 μg. Whole-exome sequencing was performed on the extracted DNA to screen for... GJA8 A novel heterozygous mutation site in the gene was found to be present in patients compared to normal individuals. GJA8 A change from G to A at position 137 of the coding region of a gene results in a change from G to E at position 46 of the encoded protein. This mutation site is denoted as c.137G>A. A set of identification primers can be developed based on this mutation site for screening for congenital microphthalmia-congenital cataract syndrome.

[0025] Based on the mutation sites screened in Experimental Example 1, this invention developed a set of identification primers (F and R), wherein primer F is Primer-F and primer R is Primer-R. The specific primer sequences are shown below:

[0026] The primer sequences are as follows (5'→3'):

[0027] The forward primer F is Primer-F (SEQ ID NO.1):

[0028] CGCGTTAGCAAAAACAGA;

[0029] The forward primer R is Primer-R (SEQ ID NO.2):

[0030] TCGTAGCAGACGTTCTCG.

[0031] A screening kit was prepared based on the developed identification primers, including amplification reagents and sequencing reagents, as shown in Tables 1 and 2 below:

[0032] Table 1 PCR Amplification Reagents

[0033] Components concentration volume PCR mixture 2× 1.5 mL Forward primer F 10 μM 150 μL Forward primer R 10 μM 150 μL <![CDATA[ddH2O]]> 2 mL

[0034] The PCR mixture in Table 1 includes components required for conventional PCR, such as Taq enzyme and dNTPs.

[0035] Table 2 GJA8 Gene mutation typing detection reagents (including purification reagents)

[0036] Components volume alkaline phosphatase 300 μL Restriction endonucleases 20 μL Bigdye 40 μL 5×buffer 200 μL F primer or R primer 100 μL <![CDATA[ddH2O]]> 1 mL

[0037] The kit also includes DNA standards: normal and those with DNA staining. GJA8 150 μL each of homozygous and heterozygous DNA from mutants of the c.137G>A site.

[0038] PCR amplification

[0039] Using the mutation site c.137G>A and genomic DNA samples screened in Experiment 1, as well as the identification primers and screening kit developed in Experiment 2, DNA samples from the subjects (family members extracted in Experiment 1 and 400 normal individuals) were amplified by PCR using the screening kit. The PCR reaction system for each genomic DNA sample was prepared according to the following ratios, and the PCR amplification reaction was carried out to obtain PCR amplification products. The PCR amplification products were detected by 2% agarose gel electrophoresis to preliminarily determine whether the size of the PCR amplification products was correct for subsequent experiments.

[0040] The specific conditions for PCR amplification are as follows:

[0041] The PCR amplification system was 20 μL: 1 μL DNA sample, 10 μL 2× Mix, 1 μL each of 10 μM identification primers (F and R), and ddH2O added to 20 μL.

[0042] PCR amplification conditions were as follows: 95 ℃ for 3 min; 95 ℃ for 15 s, 58 ℃ for 15 s, 72 ℃ for 10 s, 35 cycles; 72 ℃ for 5 min; stored at 4 ℃.

[0043] 2) Purification of PCR amplification products

[0044] Prepare a PCR amplification product purification system (10 μL) as shown in Table 3 below, and then perform PCR reaction according to the reaction conditions in Table 4 below.

[0045] Table 3 PCR product purification system

[0046] Components volume PCR amplification products 7.3 μL alkaline phosphatase 2.5 μL Restriction endonucleases 0.2 μL

[0047] Table 4 PCR product purification procedures

[0048] reaction temperature reaction time 37 ℃ 30 min 80 ℃ 15 min 12 ℃ Hold

[0049] 3) Sanger sequencing detection

[0050] The purified PCR products obtained above were subjected to Sanger sequencing. The PCR sequencing system was prepared according to Table 5, with a purification volume of 10 μL. The prepared PCR sequencing system was then subjected to the PCR sequencing reaction according to the reaction conditions in Table 6. If the sequencing result shows a mutation from base G to A at position 137 of the coding region, it indicates that the patient has congenital microphthalmia-congenital cataract syndrome.

[0051] Table 5 Sequencing system for PCR purification products

[0052] Components volume PCR purified products 2 μL 5×buffer 1.75 μL F primer or R primer (10 μM) 0.64 μL Bigdye 0.32 μL <![CDATA[ddH2O]]> Up to 10 μL

[0053] Table 6 Sequencing Procedures for PCR Purified Products

[0054] reaction temperature reaction time 96 ℃ 15 s 50 ℃ 10 s 60 ℃ 4 min(go to1 for 29 cycle) 12 ℃ Hold

[0055] Sequencing results showed that all three patients in the family diagnosed with congenital microphthalmia-congenital cataract syndrome were... GJA8 The patient had a heterozygous c.137G>A mutation. Other family members and 400 healthy controls did not have this heterozygous mutation; all were wild-type homozygous. Sequencing diagrams of the c.137 mutation in the genomes of healthy individuals and patients are shown below. Figure 2 As shown in the figure, II-2, II-1, and III-1 refer to... Figure 1 Three members in the family tree.

[0056] In summary, this invention provides a mutation site associated with congenital microphthalmia-congenital cataract syndrome and a screening method for congenital microphthalmia-congenital cataract syndrome. The identification primers and screening kits developed using this mutation site can be used for the early diagnosis and screening of congenital microphthalmia-congenital cataract syndrome, including screening in early pregnancy, providing early warning for eugenics and having significant implications for preventing congenital microphthalmia-congenital cataract syndrome in offspring.

[0057] Although the present invention has been described in detail through the preferred embodiments above, it should be understood that the above description should not be considered as a limitation of the present invention. Various modifications and substitutions to the present invention will be apparent to those skilled in the art after reading the above description. Therefore, the scope of protection of the present invention should be defined by the appended claims.

Claims

1. The application of a set of identification primers for in vitro detection of the c.137G>A mutation site in the GJA8 gene in the preparation of a screening kit for the auxiliary diagnosis of congenital microphthalmia-congenital cataract syndrome, characterized in that, The nucleotide sequences of the identification primers are shown in SEQ ID NO.1 and SEQ ID NO.2.

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