CN111718989A - Axenfeld-Rieger syndrome causative gene deletion frameshift mutation detection kit and its application - Google Patents

Abstract

The invention discloses a detection kit for Axenfeld-Rieger syndrome pathogenic gene deletion frameshift mutation and its application. The gene mutation described is the first discovery of the PITX2 gene mutation c.525delC, which is closely related to Axenfeld-Rieger syndrome, that is, the deletion of the base C at the 525th position of the 3rd exon of the PITX2 gene. The genetic codon for aspartate at position 175 (GAC) becomes the genetic codon for glutamic acid (GAG), and thus frameshift mutations begin. The present invention also provides a kit for detecting the PITX2 gene mutation, comprising: 25mM dNTPs, 10× PCR reaction buffer, DNA polymerase, PCR amplification primer pair and ddH ₂ O, the PCR amplification primer pair It consists of primer 1 (shown in SEQ ID NO: 1) and primer 2 (SEQ ID NO: 2). The invention provides a detection method for ARS deletion frameshift mutation caused by PITX2 gene, thereby providing a new method for ARS gene diagnosis and laying a foundation for the research of molecular mechanism of the disease.

Description

Axenfeld-Rieger Syndrome Gene Deletion Frameshift Mutation Detection Kit and its application

technical field

The invention relates to a gene mutation detection kit and its application, in particular to a deletion frameshift mutation detection kit of the common pathogenic gene PITX2 of Axenfeld-Rieger syndrome and its application. The invention belongs to the technical field of gene diagnosis product preparation.

Background technique

Axenfeld-Rieger syndrome is a group of rare eye-related genetic diseases, which is autosomal dominant and has a penetrance of nearly 100%. Abnormal, deviated pupils or polypupillary, according to the different parts of the eye can be divided into Axenfeld's anomaly and Rieger's anomaly, can also be accompanied by syndromic systemic developmental defects such as jaw hypoplasia, missing or small teeth, hearing loss, umbilical Some patients have abnormal development of the heart and brain. About 50% of patients with glaucoma and more than adolescence onset. Therefore, the disease brings a heavy burden to the patients themselves and their families.

Axenfeld-Rieger syndrome involves multiple systems in the body, and has phenotypic heterogeneity, making it difficult to diagnose clinically. Therefore, a clear genetic diagnosis can guide clinical diagnosis. PITX2 gene and FOCX1 gene are the main pathogenic mutation genes. Therefore, pathogenic gene detection can not only provide a clear genetic diagnosis for the patient himself, but also help the family members of patients who have not been diagnosed to detect the disease early, early treatment, and available In prenatal diagnosis and genetic counseling.

At present, most of the PITX2 gene mutations associated with Axenfeld-Rieger syndrome are substitutions of a single base site, and there are few reports of base deletion/insertion frameshift mutations that have a greater impact on protein function. The c.5256delC pathogenic mutation involved in this case was discovered for the first time in a Chinese patient with Axenfeld-Rieger syndrome. The inventor of this case once proposed in the patent application with the patent number ZL201410057930.0 that the PITX2 gene base deletion carried by another Chinese patient’s family Mutation c.77delA mutation, but the detection kit described in this patent cannot detect the c.525delC mutation involved in the present invention. Therefore, new methods for c.525delC mutation screening are needed to improve the detection rate of PITX2 gene deletion frameshift mutations and enrich the detection methods of PITX2 gene deletion frameshift mutations.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a detection kit for Axenfeld-Rieger syndrome deletion frameshift mutation, which makes up for the deficiencies in the existing Axenfeld-Rieger syndrome pathogenic gene detection technology. Code mutation: c.525delC-based, providing a rapid detection method for Axenfeld-Rieger syndrome. The NCBI reference sequence number of the PITX2 gene gDNA is NC_000004, the NCBI reference sequence number of the mRNA is NM_000325.6, and the corresponding protein NCBI reference sequence number is NP_000316.2.

The purpose of this invention is to realize through the following technical solutions:

A kit for detecting frameshift mutation of Axenfeld-Rieger syndrome pathogenic gene deletion of the present invention,

25mM dNTPs, 10× PCR reaction buffer, DNA polymerase, PCR amplification primer pair and ddH ₂ O, characterized in that the PCR amplification primer pair consists of primer 1 and primer 2, wherein the nucleoside of primer 1 The acid sequence is shown in SEQ ID NO: 1, the nucleotide sequence of primer 2 is shown in SEQ ID NO: 2, the Axenfeld-Rieger syndrome pathogenic gene is the mutated PITX2 gene, and the NCBI reference sequence number of the mRNA For NM_000325.6, the first nucleotide of the cDNA is calculated as 1, and the mutation is the deletion of the 525th base C in the third exon of the PITX2 gene.

Wherein, the nucleotide sequence of the 3rd exon of the PITX2 gene before the mutation is shown in SEQ ID NO:3, and the nucleotide sequence of the 3rd exon of the PITX2 gene after the mutation is shown in SEQ ID NO:4; The amino acid sequence of the PITX2 protein before the mutation is shown in SEQ ID NO:5, and the amino acid sequence of the PITX2 protein after the mutation is shown in SEQ ID NO:6.

This mutation causes the genetic codon for aspartic acid at position 175 (GAC) to become the genetic codon for glutamic acid (GAG), and thus begins to undergo a frameshift mutation, resulting in premature termination of the protein, i.e. p.Asp175Glufs *33 Frameshift mutation.

Wherein, preferably, the kit utilizes PCR technology to carry out the amplification of the pathogenic gene of Axenfeld-Rieger syndrome, and the reaction program is:

Pre-denaturation at 98°C for 2 minutes, and then enter the first cycle: denaturation at 98°C for 10 seconds, annealing and renaturation at 60°C for 15 seconds, and extension at 72°C for 15 seconds, for a total of 40 cycles, 72°C for 1 min, and storage at 4°C.

Further, the present invention also proposes that the kit be used in the preparation of a detection reagent for the deletion of frameshift mutations in the pathogenic gene of Axenfeld-Rieger syndrome.

Compared with the prior art, the beneficial effects of the present invention are:

The invention utilizes the newly discovered PITX2 gene c.525delC deletion frameshift mutation in my country and the provided specific detection primers, which can quickly detect the pathogenic mutation of Axenfeld-Rieger syndrome and provide gene diagnosis. The invention has simple operation, low cost, direct and accurate results, is suitable for clinical application, and can lay a foundation for the study of molecular mechanism of diseases.

Description of drawings

Figure 1 is a forward sequencing diagram of a normal person;

Figure 2 is a forward sequencing diagram of a patient with Axenfeld-Rieger syndrome;

Figure 3 is a reverse sequencing map of a patient with Axenfeld-Rieger syndrome.

Detailed ways

The present invention will be further described below with reference to specific embodiments, and the advantages and characteristics of the present invention will become clearer with the description. However, these examples are only exemplary and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solutions of the present invention can be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the protection scope of the present invention.

Example 1 Preparation of Axenfeld-Rieger Syndrome Deletion Frameshift Mutation Detection Kit

1. Design and synthesis of PCR amplification primer pairs

Primer 1: 5'-CTGCACTGTGGCATCTGTTT-3' (shown in SEQ ID NO: 1)

Primer 2: 5'-GCCCACGACCTTCTAGCATA-3' (shown in SEQ ID NO: 2)

It was synthesized by an automatic DNA synthesizer and diluted to 10 pmol/L.

2. Assembly of the kit

Includes: 25mM dNTP, 10x PCR reaction buffer, DNA polymerase, PCR amplification primer pair and _ddH2O .

Example 2 Application of Axenfeld-Rieger Syndrome Deletion Frameshift Mutation Detection Kit

Application background: A Chinese male patient with dysplasia of both eyes was clinically found. After a detailed clinical examination, it was found that the patient had post-corneal embryonic rings, abnormal iris development, polypupillary, and maxillary hypoplasia, missing teeth, and umbilical cord. Axenfeld-Rieger syndrome was confirmed by detailed ophthalmological and general examination. At the same time, a detailed clinical examination was carried out on the immediate family members of the man, and it was found that the two daughters of the man had similar clinical manifestations. And the clinical diagnosis was Axenfeld-Rieger syndrome.

Purpose and method: The kit obtained in Example 1 was used to perform gene detection on the above-mentioned 3 patients clinically diagnosed with Axenfeld-Rieger syndrome to confirm the gene diagnosis. The genomic DNA of 3 subjects to be tested was extracted, and the Axenfeld-Rieger syndrome deletion frameshift mutation detection kit was used to amplify the PITX2 gene. After PCR, the products were subjected to Sanger sequencing, analysis, and the sequencing results were read.

1. The specific steps are as follows:

(1) Extract the genomic DNA of the person to be tested and dilute to 100-200ng/ul.

(2) Synthetic primers:

Primer 1: 5'-CTGCACTGTGGCATCTGTTT-3' (shown in SEQ ID NO: 1)

Primer 2: 5'-GCCCACGACCTTCTAGCATA-3' (shown in SEQ ID NO: 2)

(3) In vitro amplification (PCR) of sample DNA target fragments

The PCR reaction system is 50ul, and the specific components are shown in Table 1 below:

Table 1 PCR reaction system

10x PCR reaction buffer 5ul 25mM dNTPs 0.4ul DNA polymerase 0.5ul Primer 1 2ul Primer 2 2ul DNA template 1ul ddH₂O 39.1ul

After mixing the above mixed liquid, the target fragment was amplified on the PCR machine. The specific reaction procedure is shown in Table 2 below:

Table 2 Reaction program

(4) Amplify step (3) with primer 1: 5'-CTGCACTGTGGCATCTGTTT-3' (shown in SEQ ID NO: 1) and primer 2: 5'-GCCCACGACCTTCTAGCATA-3' (shown in SEQ ID NO: 2) The obtained target DNA fragments were subjected to Sanger sequencing, and the sequencing results were compared with SEQ ID NO: 3, SEQ ID NO: 4 and the sequencing peak map (Figures 1, 2, and 3).

2. The result is as follows:

The forward and reverse sequencing results of the three subjects to be tested are shown in Figures 2 and 3. Double peaks begin to appear at the arrow marks. After splicing the sequencing peaks in the forward and reverse directions with the normal people's sequencing peaks (Figure 1), it can be seen in Figure 1. A base C is missing from the arrow mark, that is, the 525th base of the 3rd exon of the PITX2 gene on one chromosome chain of the person to be tested is deleted, and the mutation results in the genetic codon encoding the 175th aspartic acid ( GAC) becomes the genetic codon (GAG) of glutamic acid, and thus begins to undergo frameshift mutation, resulting in premature termination of the protein, that is, the p.Asp175Glufs*33 frameshift mutation. The nucleotide sequence of the 3rd exon of the unmutated PITX2 gene is shown in SEQ ID NO:3, and the nucleotide sequence of the 3rd exon of the mutated PITX2 gene is shown in SEQ ID NO:4, The amino acid sequence of the PITX2 protein before the mutation is shown in SEQ ID NO:5, and the amino acid sequence of the PITX2 protein after the mutation is shown in SEQ ID NO:6. Therefore, the 3 patients to be tested all carry the PITX2 gene pathogenic mutation: c.525delC, and are heterozygous mutations. This gene mutation is the first PITX2 gene mutation found in my country, and is closely related to the occurrence of Axenfeld-Rieger syndrome.

sequence listing

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Claims (4)

1. A kit for detecting the deletion frame-shift mutation of pathogenic gene of Axenfeld-Rieger syndrome is composed of 25 mdNTPs, 10 × PCR buffer solution, DNA polymerase, PCR amplification primer pair and ddH₂O, characterized in that the PCR amplification primer pair consists of a primer 1 and a primer 2, wherein the nucleotide sequence of the primer 1 is shown as SEQ ID NO. 1, the nucleotide sequence of the primer 2 is shown as SEQ ID NO. 2, the pathogenic gene of the Axenfeld-Rieger syndrome is mutated PITX2 gene, the NCBI reference sequence number of mRNA is NM-000325.6, the mutation is the deletion of the 525 th base C of the 3 rd exon of the PITX2 gene by taking the first nucleotide of cDNA as 1.

2. The kit of claim 1, wherein the nucleotide sequence of exon 3 of the pre-mutation PITX2 gene is set forth in SEQ ID No. 3, and the nucleotide sequence of exon 3 of the post-mutation PITX2 gene is set forth in SEQ ID No. 4; the amino acid sequence of the PITX2 protein before mutation is shown as SEQ ID NO. 5, and the amino acid sequence of the PITX2 protein after mutation is shown as SEQ ID NO. 6.

3. The kit of claim 1, wherein the kit is used for amplifying the pathogenic gene of the Axenfeld-Rieger syndrome by using a PCR technology, and the reaction procedure is as follows:

pre-denaturation at 98 ℃ for 2 min, then entering the first cycle: denaturation at 98 deg.C for 10 seconds, annealing at 60 deg.C for 15 seconds, and extension at 72 deg.C for 15 seconds, for 40 cycles, at 72 deg.C for 1min, and storage at 4 deg.C.

4. The use of the kit of any one of claims 1 to 3 in the preparation of a reagent for detecting deletion frameshift mutation of a pathogenic gene of the Axenfeld-Rieger syndrome.

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