Product for gene detection of congenital central pulmonary insufficiency syndrome and application thereof

Abstract

The invention provides a product for gene detection of congenital central pulmonary insufficiency syndrome and application of the product, namely, a method for screening patients with the congenital central pulmonary insufficiency syndrome is established on the basis of obtaining an SNP (Single Nucleotide Polymorphism) which can cause the congenital central pulmonary insufficiency syndrome through screening. Wherein the SNP site is located at the 194th site of a nucleotide fragment of which the nucleotide sequence is SEQ ID NO: 1, and is Tgt; c mutation. On the basis that the SNP which can cause the congenital central pulmonary insufficiency syndrome is obtained through screening, a method for screening the congenital central pulmonary insufficiency syndrome patient is established, so that an effective CCHS gene diagnosis approach is provided, and the CCHS can be quickly and accurately diagnosed. The method disclosed by the invention has the advantages of high sensitivity, strong specificity, simplicity and convenience in operation and the like, and is suitable for early diagnosis and genetic counseling of the CCHS.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular biomedicine and gene detection, and specifically relates to a product for gene detection of congenital central hypoventilation syndrome and its application. Background of the Invention

[0002] Congenital central hypoventilation syndrome Congenital Central Hypoventilation Syndrome PHOX2B hyperventilation (CCHS) is a rare autosomal dominant genetic disease characterized by severe respiratory depression or apnea during sleep, which can lead to hypoxemia and hypercapnia in severe cases. The pathogenesis of CCHS is closely related to mutations in the PHOX2B gene, especially the expansion mutation of the polyalanine repeat sequence (Polyalanine Repeat Expansion, PARM) in the exon 3 region of the gene. In addition, non-polyalanine repeat sequence mutations (Non-PARM) are also associated with CCHS.

[0003] Currently, the detection of PHOX2B gene mutations mainly relies on Sanger sequencing and next-generation sequencing (NGS). Although these methods have high accuracy, they also have disadvantages such as high cost, long time consumption, and complex operation, making them difficult to apply to newborn screening or large-scale population testing.

[0004] Therefore, developing a rapid, economical, highly sensitive and specific SNP detection method is of great significance for the early diagnosis and genetic counseling of CCHS. Real-time fluorescence quantitative PCR (qPCR) technology is an ideal choice for solving the above problems because of its rapidity, sensitivity, strong specificity and low cost. Summary of the invention

[0005] The purpose of the present invention is to provide a product for genetic testing of congenital central hypoventilation syndrome and its application, that is, to establish a method for screening patients with congenital central hypoventilation syndrome based on screening a SNP that causes congenital central hypoventilation syndrome (CCHS) The present invention first provides a SNP site that can cause congenital central pulmonary hypoventilation syndrome. The SNP site is located at the 945th position (ATG start) of the 3rd exon of the PHOX2B gene, that is, at the 194th position of the nucleotide fragment with the nucleotide sequence of SEQ ID NO: 1, and is a T>C mutation.

[0006] , Another aspect of the present invention also provides the use of the preparation for detecting the above-mentioned SNP site in the preparation of a product for detecting congenital central hypoventilation syndrome. The preparation, as a specific record of an embodiment, is a PCR amplification sequencing primer; Furthermore, the sequence information of the PCR amplification sequencing primers is as follows: F: 5'-GGGTAGGAGTGGGGTTGAAA-3' (SEQ ID NO: 2), R: 5'-GAGAGCAAAGAGGCCAAGAG-3' (SEQ ID NO: 3); In yet another aspect, the present invention provides a detection kit for screening patients with congenital central hypoventilation syndrome, wherein the kit comprises primers for detecting the above-mentioned SNP sites.

[0007] The present invention screens and obtains a SNP that causes congenital central hypoventilation syndrome (CCHS), and establishes a method for screening patients with congenital central hypoventilation syndrome, thereby providing an effective CCHS gene diagnosis approach that can quickly and accurately diagnose CCHS. The method of the present invention has the advantages of high sensitivity, strong specificity, and simple operation, and is suitable for early diagnosis and genetic counseling of CCHS. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Figure 1 : DNA sequencing of the proband; Figure 2 : DNA sequencing of the proband's family members, where a is the proband's mother, b is the proband's father, c is the proband's eldest sister, and d is the proband's second sister Figure 3 : DNA sequencing images of the proband and his family members, where a is the proband, b is the proband's mother, c is the proband's father, d is the proband's eldest sister, and e is the proband's second sister. DETAILED DESCRIPTION

[0009] The present invention is described in detail below in conjunction with embodiments and drawings.

[0010] Example 1: Screening for SNP markers associated with CCHS

[0011] 1. Extract DNA from patient's peripheral blood The patient was a 7-month-old boy with shallow breathing, drowsiness, and convulsions during the course of the disease. The patient needed continuous oxygen, and could not maintain normal breathing without oxygen. With the consent of the family and in compliance with medical ethics, 5 mL of peripheral blood sample was collected from the patient, collected in EDTA anticoagulant tubes, and the tubes were gently inverted several times to prevent blood coagulation and stored at 4°C.

[0012] DNA was extracted using the Qiagen QIAamp DNA Blood Mini Kit. The specific steps are as follows: Take 200 μL of peripheral blood and add it to a 1.5 mL centrifuge tube. Add 20 μL of proteinase K and 200 μL of lysis buffer (Buffer AL), mix thoroughly, and incubate at 56°C for 10 minutes to lyse the cells. Add 200 μL of anhydrous ethanol, mix well, transfer to a QIAamp spin column, centrifuge at 8000 rpm for 1 minute, and discard the filtrate. Add 500 μL of wash buffer (Buffer AW1), centrifuge at 8000 rpm for 1 minute, discard the filtrate, add 500 μL of wash buffer (Buffer AW2), centrifuge at 14000 rpm for 3 minutes, and discard the filtrate. Transfer the spin column to a new 1.5 mL centrifuge tube, add 50-100 μL of elution buffer (Buffer AE), let stand at room temperature for 1 minute, centrifuge at 8000 rpm for 1 minute, and collect the DNA solution. Use a spectrophotometer to detect the concentration and purity of DNA.

[0013] 2. Determine the gene mutation site The SNP was determined to be located in the third exon of the PHOX2B gene, which contains a polyalanine repeat sequence (GCN repeat) and is a hotspot for CCHS-related mutations.

[0014] The sequence information of the detection primers used is as follows: Upstream primer F: 5'-GGGTAGGAGTGGGGTTGAAA-3', Downstream primer R: 5′-GAGAGCAAAGAGGCCAAGAG-3′; The total volume of the PCR reaction was 25 μL, including: 2.0 μL of peripheral blood DNA (50 ng / μL), 1.0 μL of upstream and downstream primers (10 μM), 0.5 μL of dNTPs (10 mM), 2.5 μL of PCR buffer (10×), MgCl 2 1.5 μL (25 mM), DNA polymerase 0.2 μL (5 U / μL), sterile water to 25 μL. PCR reaction conditions: 95℃ 5min, 95℃ 30s, 60℃ 30s, primer 72℃ 30s, 35 cycles, 72℃ extension 5min, 4℃ storage.

[0015] Agarose gel electrophoresis was used to detect the PCR amplification products, and the NGS method was used to sequence the above PCR products. In the proband, a double peak was found at position 945 (ATG start) of exon 3 of the PHOX2B gene ( Figure 1 ), which was analyzed to be c.945A>G mutation.

[0016] The proband's family members were collected, and peripheral blood genomic DNA was extracted from their family members. PCR amplification was performed using upstream primer F and downstream primer R as primers, and the PCR products were directly sequenced. It was found that the SNP mutation found in the present invention did not exist in the PHOX2B gene of the other four family members of the proband who did not suffer from CCHS ( Figure 2 ).

[0017] Example 2: SNP site verification

[0018] Peripheral blood samples were collected from the proband and other family members, and the purified PCR products were used as templates, and SEQ ID NO: 1 and SEQ ID NO: 2 were used as templates for Sanger sequencing. The reaction system was 1.0 μL of peripheral blood DNA, 1.0 μL of primers (3.2 μM), 1.0 μL of BigDye Terminator v3.1, 2.0 μL of 5× sequencing buffer, and sterile water was added to 10 μL. Sequencing reaction conditions: 96℃ 1min, 96℃ 10s, 50℃ 5s, 60℃ extension 4min, 25 cycles, stored at 4℃, the PCR products were purified using a kit and transferred to a 96-well plate for sequencing.

[0019] The c.945A>G mutation (p.315Wext41) was detected in the proband in the exon 3 region of the PHOX2B gene, and the Sanger sequencing result was T>C ( Figure 3 ), which is consistent with the NGS result. The SNP site was also found in the detection of patients with congenital central hypoventilation syndrome using SEQ ID NO: 2 and SEQ ID NO: 3 primers, indicating that mutations at this site can cause congenital central hypoventilation syndrome.

[0020] In summary, the present invention provides a highly sensitive and specific SNP detection method, which can quickly diagnose congenital central hypoventilation syndrome (CCHS) by detecting PHOX2B gene mutations. It has the advantages of simple operation and non-invasive detection, and is suitable for clinical screening.

Claims

1. A SNP site that causes congenital central hypoventilation syndrome, characterized in that: The SNP site is located at the 194th position of the nucleotide fragment of SEQ ID NO: 1, and is a T>C mutation.

2. Use of the PCR amplification sequencing primer pair for detecting the SNP site described in claim 1 in the preparation of a product for detecting congenital central hypoventilation syndrome.

3. The use according to claim 2, characterized in that The upstream primer of the primer pair has a sequence of SEQ ID NO: 2, and the downstream primer has a sequence of SEQ ID NO:

3.

4. The use according to claim 2, characterized in that The product is a PCR amplification sequencing kit.

5. A detection kit for screening patients with congenital central hypoventilation syndrome, characterized in that: The kit comprises a primer pair for detecting the SNP site according to claim 1.

6. The kit according to claim 5, characterized in that The upstream primer of the primer pair has a sequence of SEQ ID NO: 2, and the downstream primer has a sequence of SEQ ID NO:

3.

7. A method for screening and detecting congenital central hypoventilation syndrome, characterized in that: The method is to detect the SNP site described in claim 1.

8. The method according to claim 7, characterized in that The method is to use the kit described in claim 5 for detection.

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