Diagnostic marker for a neural crest disease and its applications

Abstract

The present invention belongs to the field of biotechnology, and particularly relates to a diagnostic marker for neural crest diseases. The diagnostic marker includes a mutant Dph1 gene, which is a Dph1 gene carrying gene mutation sites c.724G>C, p.E242Q and c.136C>T, p.Q46X. The present application first discovers that neural crest diseases are directly related to the Dph1 gene mutation sites c.724G>C, p.E242Q and c.136C>T, p.Q46X, providing a theoretical basis for the gene diagnosis, prenatal diagnosis and gene therapy of neural crest diseases.

Description

Technical Field

[0001] This application belongs to the field of biotechnology, and particularly relates to a diagnostic marker for neural crest diseases and its application. Background Art

[0002] The neural crest refers to a pair of cell cords that are parallel to the neural tube and formed by some cells on the neural folds detaching from the neural tube when the neural groove closes into the neural tube. The neural crest is located dorsolaterally to the neural tube. Neural crest cells can differentiate into all nerve cells and glial cells in the peripheral nervous system, chromaffin cells in the adrenal medulla, melanocytes, parafollicular cells, and other types of cells. The formation, migration, and differentiation of neural crest cells are strictly regulated, and any factor that disrupts the development of neural crest cells may lead to embryonic developmental malformations.

[0003] Neural crest diseases are a collective term for a series of diseases caused by abnormal development of neural crest cells. Neural crest diseases are diverse in type and complex in phenotype, involving multiple parts such as the craniofacial region, heart, gastrointestinal tract, and skin, seriously endangering the physical function and mental health of patients. Neural crest diseases account for about 1 / 3 of children with birth defects. Genetic factors are the main risk factors for neural crest diseases, but environmental risk factors and abnormal gene-environment interactions may also cause neural crest diseases. The clinical phenotypes of neural crest diseases are complex and show significant genetic heterogeneity. There are many types of neural crest diseases, mostly sporadic rare cases, and it is difficult to collect samples, resulting in difficulties in systematic research; the clinical characteristics of different neural crest diseases are chaotic and overlapping, the case symptoms of neural crest diseases are diverse, and the phenotypes are redundant and overlapping, and there is currently no systematic and effective classification system; the causes of neural crest diseases lack systematic revelation. Neural crest diseases are basically studied independently, such as the typical representatives - cleft lip and palate and Hirschsprung's disease, but systematic research on various neural crest diseases at the level of neural crest diseases has not been carried out. Summary of the Invention

[0004] In view of this, this application provides a diagnostic marker for neural crest diseases and its application, thereby providing a theoretical basis for the gene diagnosis, prenatal diagnosis, and gene therapy of neural crest diseases.

[0005] In the first aspect, this application provides a diagnostic marker for neural crest diseases, and the diagnostic marker includes:

[0006] The mutant Dph1 gene, which is the Dph1 gene carrying the gene mutation sites c.724G>C, p.E242Q and c.136C>T, p.Q46X.

[0007] In a second aspect, the present application provides the use of a reagent for detecting a Dph1 gene mutant in the preparation of a product for diagnosing a neural crest disease, wherein the mutation sites of the Dph1 gene mutant are the mutation of the 724th nucleotide of the Dph1 gene from G to C and the mutation of the 136th nucleotide from C to T.

[0008] Further, the reagent includes primers for detecting the mutation site of the 724th nucleotide of the Dph1 gene from G to C and primers for detecting the mutation site of the 136th nucleotide of the Dph1 gene from C to T.

[0009] Further, the primers for detecting the mutation site of the 724th nucleotide of the Dph1 gene from G to C include an upstream amplification primer for c.724G>C and a downstream amplification primer for c.724G>C. The nucleotide sequence of the upstream amplification primer for c.724G>C is as shown in SEQ ID NO:1, and the nucleotide sequence of the downstream amplification primer for c.724G>C is as shown in SEQ ID NO:2.

[0010] Further, the primers for detecting the mutation site of the 136th nucleotide of the Dph1 gene from C to T include an upstream amplification primer for c.136C>T and a downstream amplification primer for c.136C>T. The nucleotide sequence of the upstream amplification primer for c.136C>T is as shown in SEQ ID NO:3, and the nucleotide sequence of the downstream amplification primer for c.136C>T is as shown in SEQ ID NO:4.

[0011] In a third aspect, the present application provides the use of the above-mentioned diagnostic marker in the preparation of a kit for detecting a neural crest disease.

[0012] In a fourth aspect, the present application provides the use of the primers as shown in SEQ ID NO:1 - SEQ ID NO:4 in the preparation of a kit for detecting a neural crest disease.

[0013] In a fifth aspect, the present application provides the use of the above-mentioned diagnostic marker in the preparation of an animal model of a neural crest disease.

[0014] In another aspect, the present application provides a method for constructing an animal model of a neural crest disease, including: changing the Dph1 gene of an animal to mutate the 709th nucleotide of the Dph1 gene from G to C and changing the Dph1 gene of the animal to mutate the 121st nucleotide from C to T.

[0015] In yet another aspect, the present application provides a method for screening a mouse model of a neural crest disease with a Dph1 mutant gene for non-diagnostic purposes, including:

[0016] (1) Extracting nucleic acid DNA from a biological sample to be tested;

[0017] (2) determining the sequence of the nucleic acid DNA;

[0018] (3) The nucleic acid sequence or its complementary sequence described in step (2) has mutation sites c.709G>C, p.E237Q (corresponding to the mutation site c.724G>C, p.E242Q of the human Dph1 gene) and c.121C>T, p.Q41X (corresponding to the mutation site c.136C>T, p.Q46X of the human Dph1 gene) compared to the wild-type Dph1 gene, and the mutation is an indicator marker of neural crest disease.

[0019] Furthermore, the biological sample to be tested is a blood sample or a tissue sample.

[0020] As described above, the diagnostic markers for neural crest diseases of the present application and their applications have the following beneficial effects:

[0021] (1) This application discovered for the first time that neural crest diseases are directly associated with the Dph1 gene mutation sites c.724G>C, p.E242Q and c.136C>T, p.Q46X, providing a theoretical basis for the genetic diagnosis, prenatal diagnosis and gene therapy of neural crest diseases.

[0022] (2) The primers of the present application can directly perform exon sequencing on the Dph1 gene and can correctly detect exon mutations, eliminating the tedious procedure of high-throughput screening. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 These are the c.724G>C, p.E242Q mutation site and c.136C>T, p.Q46X mutation site of the Dph1 gene; WT is the normal gene sequence.

[0024] Figure 2 The Dph1 gene mutation status of the patient and his family members, where Proband is the patient, Father is the father, Mother is the mother, and Sister is the sister.

[0025] Figure 3 Results of mouse embryos and pathological sections, where a is 10.5 days Dph1 + / + Wild-type embryos and Dph1 E237Q / Q41X Double mutant embryo, b is 10.5 days Dph1 + / + Wild-type embryos and Dph1 + / Q41X Mutant embryo, c is 10.5 days Dph1 + / + Wild-type embryos and Dph1 + / E237Q Mutant embryo, d is 14.5 days Dph1+ / + Wild-type embryonic anterior palatofacial region, e is Dph1 at 14.5 days + / + Wild-type embryonic posterior palatofacial region, f is Dph1 at 14.5 days E237Q / Q41X Mutant embryonic anterior palatofacial region, g is Dph1 at 14.5 days E237Q / Q41X Mutant embryonic posterior palatofacial region, h is Dph1 at 15.5 days + / + Wild-type embryonic anterior palatofacial region, i is Dph1 at 15.5 days + / + Wild-type embryonic posterior palatofacial region, j is Dph1 at 15.5 days E237Q / Q41X Mutant embryonic anterior palatofacial region, k is Dph1 at 15.5 days E237Q / Q41X Mutant embryonic posterior palatofacial region, t is the embryonic tongue, ps is the embryonic palatal process, Anterior is the anterior palatofacial region, Posterior is the posterior palatofacial region, E10.5 is the embryo at 10.5 days, E14.5 is the embryo at 14.5 days, E15.5 is the embryo at 15.5 days. Detailed implementation mode

[0026] The present invention is further described below through specific specific examples. However, it should be noted that the specific material ratios, process conditions, results, etc. described in the embodiments of the present invention are only used to illustrate the present invention and cannot be used to limit the protection scope of the present invention. Any equivalent changes or modifications made according to the spirit and essence of the present invention should be covered by the protection scope of the present invention.

[0027] The present invention is described in detail below through specific exemplified embodiments. Similarly, it should be understood that the following embodiments are only used to specifically illustrate the present invention and cannot be understood as limiting the protection scope of the present invention. Some non-essential improvements and adjustments made by those skilled in the art based on the above content of the present invention all fall within the protection scope of the present invention. The specific process parameters and the like in the following examples are also only an example within a suitable range, that is, those skilled in the art can make selections within a suitable range according to the description herein, rather than being limited to the specific values in the following examples.

[0028] I. Obtaining samples

[0029] Collect 1 case of a child, female, 4 years and 9 months old, with clinical manifestations of growth retardation and language development retardation; mental retardation, sparse eyebrows, eyelashes and hair, and facial deformity features including scaphocephaly, frontal prominence, micrognathia, low-set ears, downward displacement of the palpebral fissure, epicanthus and nasal bridge depression.

[0030] The father, mother and sister of the child did not show symptoms of neural crest diseases.

[0031] Collect the peripheral whole blood of the child and family members (including the child's father, mother and sister). The specific steps are as follows:

[0032] 1. Lymphocyte Separation

[0033] (1) Take 5 ml of anticoagulated peripheral whole blood from each of the patient and family members (including the patient's father, mother, and sister) (add EDTA to the peripheral whole blood to obtain anticoagulated peripheral whole blood) and add it to a centrifuge tube. Add an equal volume of PBS buffer (commercially available, can be purchased) to the centrifuge tube and mix well to obtain a mixed blood sample;

[0034] (2) Add lymphocyte separation solution (commercially available, can be purchased) with an equal volume to the anticoagulated peripheral whole blood to another centrifuge tube. Gently add the mixed blood sample to the separation solution, and then centrifuge horizontally at 500 g / min for 20 min.

[0035] (3) After centrifugation, there is stratification: After centrifugation, aspirate the lymphocyte layer, add 10 ml of PBS buffer (commercially available, can be purchased), mix well, then centrifuge at 1500 r / min for 10 min. After centrifugation, aspirate the lymphocyte layer, add 10 ml of PBS buffer (commercially available, can be purchased), mix well, and then centrifuge at 1500 r / min for 10 min to obtain the sample to be tested.

[0036] 2. Total DNA Extraction

[0037] (1) Add 20 - 30 μl of DNA extraction reagent (Lucigen catalog number: QE09050) to each sample to be tested, and centrifuge at 1500 r / min at room temperature for 2 min to precipitate the sample and the added reagent to the bottom of the test tube.

[0038] (2) Heat at 65 °C for 10 - 15 min. After heating, centrifuge at 1500 r / min at room temperature for 2 min again to make the liquid on the tube wall sink.

[0039] (3) Heat at 98 °C for 2 min to inactivate the enzyme. After heating, centrifuge at 1500 r / min at room temperature for 2 min again to make the liquid on the tube wall sink to obtain total DNA.

[0040] (4) Take 1 μL of the sample and measure the DNA concentration of the sample on a nucleic acid quantifier NanoDrop 2000 to ensure that the DNA concentration in the extracted sample is 50 - 100 ng / μL to prepare for subsequent PCR amplification.

[0041] II. Primer Design

[0042] Design upstream and downstream primers for amplifying two mutation sites of the Dph1 gene on primer 5.0, and send the primer sequences to Beijing Tsingke Biotechnology Co., Ltd. for synthesis.

[0043] Among them, the nucleotide sequences of the upstream amplification primer and the downstream amplification primer for the c.724G>C, p.E242Q mutation site of the Dph1 gene are shown in SEQ ID NO:1 and SEQ ID NO:2 respectively;

[0044] The nucleotide sequences of the upstream amplification primer and the downstream amplification primer for the c.136C>T, p.Q46X mutation site of the Dph1 gene are shown in SEQ ID NO:3 and SEQ ID NO:4 respectively.

[0045] Table 1 Primers for Mutation Sites

[0046]

[0047]

[0048] III. PCR Amplification

[0049] Using the T3 super PCR mix high-fidelity enzyme (purchased from Beijing Tsingke Biotechnology Co., Ltd.), PCR amplification was performed with the total DNA obtained in Step 2 as the template. The PCR reaction system was: 18.6 μl of TAQ enzyme, 0.2 μl of upstream amplification primer, 0.2 μl of downstream amplification primer, and 2 μl of DNA template. PCR reaction conditions: denaturation at 98°C for 2 min, 35 amplification cycles: denaturation at 98°C for 15 s, annealing at 60°C for 15 s, extension at 72°C for 20 s; then extension at 72°C for 5 min, and finally stored at 12°C.

[0050] Among them, the amplification product of the 724th nucleotide of the Dph1 gene is 329 bp, and the amplification product of the 136th nucleotide of the Dph1 gene is 423 bp.

[0051] IV. Sequencing

[0052] Beijing Tsingke Biotechnology Co., Ltd. was entrusted to sequence the PCR products amplified in Step 3 to obtain the sequencing result ab1 file.

[0053] V. Result Judgment

[0054] The sequencing result ab1 file feedback by the sequencing company was opened with Chromas software. The Dph1 gene mutation site was judged by the direct sequencing method of PCR amplification products. The basis for judgment was the "peak pattern" of the sequencing result. The "peak pattern" of the sequencing result was: at the mutated base site, the peak pattern was a double peak; the wild-type PCR product was a single DNA molecule, and at the mutation site, it was a normal single peak. Therefore, at the Dph1 gene mutation sites c.724G>C, p.E242Q and c.136C>T, p.Q46X, double peak graphs appeared (as Figure 1 shown).

[0055] It can be seen from Figure 1 that compared with the wild type, the 724th nucleotide of the Dph1 gene has a mutation from G to C, and the 136th nucleotide has a mutation from C to T.

[0056] The sequencing results show that (as Figure 2 shown), there are heterozygous mutations at two loci in the Dph1 gene of the child, c.724G>C, p.E242Q (located in exon 2) and c.136C>T, p.Q46X (located in exon 7). The mother's Dph1 gene carries the heterozygous mutation c.136C>T, p.Q46X, the father's Dph1 gene carries c.724G>C, p.E242Q, and the Dph1 gene of the child's sister is wild type. This result indicates that the heterozygous mutation sites c.724G>C, p.E242Q and c.136C>T, p.Q46X of the Dph1 gene have caused neurocristopathy, and the heterozygous mutation sites c.724G>C, p.E242Q and c.136C>T, p.Q46X of the Dph1 gene are directly related to neurocristopathy.

[0057] VI. Construction of gene-modified mice

[0058] Entrust Shanghai Model Organisms Center, Inc. to obtain Cas9 mRNA and guideRNA by in vitro transcription using CRISPR / Cas9 technology, and obtain oligo donor DNA by synthesis. Microinject Cas9 mRNA, gRNA and donor DNA into the fertilized eggs of C57BL / 6J mice to introduce point mutations in the Dph1 gene, thereby obtaining gene-modified mice with heterozygous mutations c.709G>C, p.E237Q (corresponding to the mutation site c.724G>C, p.E242Q of the human Dph1 gene) and c.121C>T, p.Q41X (corresponding to the mutation site c.136C>T, p.Q46X of the human Dph1 gene), and then cross-breed the Dph1 + / E237Q mice with Dph1 + / Q41X to obtain Dph1 E237Q / Q41X gene-modified mice with the same genotype as the child (i.e., the heterozygous mutant Dph1 gene containing the mutation sites c.724G>C, p.E242Q and c.136C>T, p.Q46X).

[0059] Take 10.5-day-old mouse embryos and observe the pathological characteristics of the mouse embryos.

[0060] It was observed that Dph1 gene - modified mice carrying heterozygous mutations c.709G>C, p.E237Q (corresponding to the mutation site c.724G>C, p.E242Q in the human Dph1 gene) and c.121C>T, p.Q41X (corresponding to the mutation site c.136C>T, p.Q46X in the human Dph1 gene) had abnormal physical development, were smaller in size, and had growth retardation. E237Q / Q41X

[0061] Mouse embryos at 14.5 days and 15.5 days were taken respectively for pathological section analysis. The specific steps were as follows:

[0062] (1) Pregnant mice at 14.5 days and 15.5 days of pregnancy were anesthetized and sacrificed. The mouse embryos were dissected out, and 1 - 2 mm of the embryo tails were cut and used as genotype identification samples. Then the embryos were fixed in 4% (mass concentration) paraformaldehyde for 48 h.

[0063] (2) Tissue dehydration: The heads of the fixed embryos were cut off, and the head tissues were dehydrated step - by - step. The dehydration process was as follows: first, treated in 75% (volume concentration) ethanol for 1 h 40 min, then in 85% (volume concentration) ethanol for 1 h 20 min, then in 95% (volume concentration) ethanol for 1 h 20 min, then in 100% ethanol for 1 h, and then in 100% ethanol for another 1 h.

[0064] (3) Tissue clearing: The tissues were placed in xylene for three consecutive 30 - min periods.

[0065] (4) Tissue infiltration with paraffin wax: The tissues were placed in 65°C liquid paraffin for 20 min, 30 min, and 40 min respectively.

[0066] (5) Tissue embedding and sectioning: The mold was filled with paraffin wax, and then the embryos were placed with their nose - lip parts facing down and allowed to solidify naturally to make tissue paraffin blocks; the blocks were trimmed and sectioned, with a section thickness of 4 μm. The sections were spread and attached to adhesive - free glass slides for HE staining.

[0067] ​(6) Tissue staining: The sections were dewaxed with xylene for 5 min, twice consecutively, then with 100% ethanol for 2 min, 95% ethanol for 2 min, 80% ethanol for 2 min, 75% ethanol for 3 min, washed with tap water for 5 min, stained with hematoxylin for 5 min, rinsed with running water for 10 min, differentiated with 1% hydrochloric acid alcohol for 5 seconds, washed with water for 5 min, blued with saturated lithium carbonate for 10 seconds, rinsed with tap water for 5 min, stained with eosin staining solution for 1 min. The stained sections were dehydrated with 75% ethanol for 2 min, 95% ethanol for 2 min, 95% ethanol for another 2 min, 100% ethanol for 2 min, 100% ethanol for another 2 min, and then made transparent with xylene for 2 min twice. Neutral gum was dropped on the glass slide and covered with a coverslip for sealing. After the gum was slightly dry, the results were observed under a microscope, and the results were as Figure 3 shown.

[0068] As Figure 3 can be seen, the Dph1 gene of the heterozygous mutant Dph1 gene containing the c.724G>C, p.E242Q mutation site and the c.136C>T, p.Q46X mutation site E237Q / Q41X gene-modified mice have pathological characteristics related to neural crest diseases, that is, Dph1 E237Q / Q41X gene-modified mice have facial developmental defects such as cleft lip and palate (see the palate part of the embryos indicated by the arrows in Figure 3 j, k, and there is a large-distance cleft lip and palate between the two palatal processes).

[0069] In summary, the present application has first discovered two heterozygous mutation sites c.724G>C, p.E242Q and c.136C>T, p.Q46X of the Dph1 gene and the abnormal transcription and translation generated by these heterozygous mutation sites, clarified the pathogenicity of the mutation, revealed that the heterozygous mutation sites c.724G>C, p.E242Q and c.136C>T, p.Q46X can cause neural crest diseases, clarified the relationship between genotype and phenotype, and can be used for the auxiliary detection and molecular diagnosis of Dph1 gene-related neural crest diseases.

[0070] The above embodiments are only illustrative of the principles and effects of the present invention, and are not used to limit the present invention. Any person familiar with this technology can modify or change the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or changes made by those with ordinary knowledge in the technical field without departing from the spirit and technical ideas disclosed by the present invention should still be covered by the claims of the present invention.

Claims

1. Use of a reagent for detecting Dph1 gene mutants in the preparation of a product for diagnosing neurocristopathy, characterized in that, the Dph1 gene mutant is the Dph1 gene carrying gene mutation sites c.724G>C, p.E242Q and c.136C>T, p.Q46X, and the neurocristopathy is cleft lip and palate.

2. The use according to claim 1, characterized in that, the reagent includes primers for detecting the mutations of c.724G>C, p.E242Q of the Dph1 gene and primers for detecting the mutations of c.136C>T, p.Q46X of the Dph1 gene.

3. The use according to claim 2, characterized in that, the primers for detecting the mutations of c.724G>C, p.E242Q of the Dph1 gene include a c.724G>C upstream amplification primer and a c.724G>C downstream amplification primer. The nucleotide sequence of the c.724G>C upstream amplification primer is shown in SEQ ID NO:1, and the nucleotide sequence of the c.724G>C downstream amplification primer is shown in SEQ ID NO:

2.

4. The use according to claim 2, characterized in that, the primers for detecting the mutations of c.136C>T, p.Q46X of the Dph1 gene include a c.136C>T upstream amplification primer and a c.136C>T downstream amplification primer. The nucleotide sequence of the c.136C>T upstream amplification primer is shown in SEQ ID NO:3, and the nucleotide sequence of the c.136C>T downstream amplification primer is shown in SEQ ID NO:

4.

5. Use of the primers shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4 in the preparation of a kit for detecting neurocristopathy, characterized in that, the neurocristopathy is cleft lip and palate.

6. A method for constructing a mouse model of neurocristopathy, characterized in that, comprising: using the CRISPR / Cas9 technology to modify the Dph1 gene of a mouse, so that the Dph1 gene has mutation sites c.709G>C, p.E237Q and c.121C>T, p.Q41X compared with the wild-type Dph1 gene, and the neurocristopathy is cleft lip and palate.

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