A nevus-like basal cell carcinoma syndrome gene mutation site and application thereof

Abstract

The application provides a nevus basal cell carcinoma syndrome gene mutation site and application thereof, the gene mutation site is a frame shift mutation c.724_725delCA of a 5th exon of a PTCH1 gene, the mutation causes a subsequent codon reading frame change, and causes a PTCH1 protein frame shift mutation p.Q242Vfs*9.The c.724_725delCA mutation site of the application expands the pathogenic gene spectrum of NBCCS.The PTCH1 gene c.724_725delCA mutation in the application can be applied in NBCCS diagnosis, and a kit containing a mutation detection primer or probe and a genetic counseling and prenatal diagnosis method based on the mutation can be prepared.

Description

Technical Field

[0001] This invention belongs to the field of pathogenic gene technology for nevus-like basal cell carcinoma syndrome, specifically relating to the application of a primer for detecting gene mutation sites in the preparation of a screening or auxiliary diagnostic kit for nevus-like basal cell carcinoma syndrome. Background Technology

[0002] Nevus-like basal cell carcinoma syndrome (NBCCS) is a rare autosomal dominant genetic disorder, clinically manifested as multiple odontogenic keratocysts (OKC), basal cell carcinoma (BCC), and skeletal deformities. Currently, PTCH1 gene mutations are known to be the main cause of NBCCS, but the reported mutation sites are limited, and significant heterogeneity in clinical phenotypes exists among different mutation sites. In current technology, the diagnosis of NBCCS mainly relies on clinical criteria and gene testing for known mutation sites, but some patients still lack a clearly identifiable pathogenic mutation, leading to limitations in genetic counseling and prenatal diagnosis. Summary of the Invention

[0003] The technical problem this invention aims to solve is to address the shortcomings of the prior art by providing a primer for detecting gene mutation sites in the preparation of screening or auxiliary diagnostic kits for nevus-like basal cell carcinoma syndrome (NBCCS). This c.724_725delCA mutation site expands the pathogenic gene spectrum of NBCCS. The PTCH1 gene c.724_725delCA mutation in this invention can be used in the diagnosis of NBCCS, and kits containing primers or probes for detecting this mutation can also be prepared, as well as methods for genetic counseling and prenatal diagnosis based on this mutation.

[0004] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is: the application of a primer for detecting gene mutation sites in the preparation of a screening or auxiliary diagnostic kit for nevus-like basal cell carcinoma syndrome, wherein the gene mutation site is a frameshift mutation c.724_725delCA in exon 5 of the PTCH1 gene, which causes the deletion of two CA bases (cytosine and adenine) at positions 724 and 725 in the coding region, thereby changing the subsequent codon reading frame;

[0005] This mutation causes a frameshift mutation p.Q242Vfs*9 in the PTCH1 protein, meaning that glutamine (Q) at position 242 of the PTCH1 protein is replaced by valine (V). The frameshift completely changes the subsequent amino acid sequence. Starting from position 242, the mutation causes a reading frame shift, resulting in a new stop codon. After the frameshift, the new stop codon appears at the 9th amino acid position. The truncated protein after the mutation is 1198 amino acids shorter (the protein molecular weight changes from 160KD to 20KD).

[0006] The nucleotide sequence of the wild-type cDNA of PTCH1 is shown in SEQ ID No. 3;

[0007] The nucleotide sequence of the cDNA of the PTCH1 c.724_725delCA mutant is shown in SEQ ID No. 4;

[0008] The amino acid sequence of the wild-type PTCH1 protein is shown in SEQ ID No. 5. The stop codon appears at the last position of the amino acid sequence of the wild-type PTCH1 protein and is not included in SEQ ID No. 5 of the WIPO Sequence.

[0009] The amino acid sequence of the PTCH1 mutant p.Q242Vfs*9 is shown in SEQ ID No. 6. Starting from the 242nd amino acid, a new stop codon appears at the 9th amino acid position. The stop codon is not included in SEQ ID No. 6 of the WIPO Sequence.

[0010] Preferably, the primers for detecting gene mutation sites include a forward primer and a reverse primer, wherein the nucleotide sequence of the forward primer is shown in SEQ ID No. 1 and the nucleotide sequence of the reverse primer is shown in SEQ ID No. 2.

[0011] Preferably, the method for detecting the gene mutation site is as follows:

[0012] PCR was performed on the genomic DNA to be tested using forward and reverse primers to obtain PCR products;

[0013] The PCR amplification reaction system consisted of: 5 μL of 10×Taq Buffer, 4 μL of 2.5 mM dNTPs, 2 μL of 10 μM forward primer, 2 μL of 10 μM forward primer, 0.5 μL of 5 U / μL Taq DNA polymerase, 1 μL of genomic DNA to be tested, and ddH2O to a final volume of 50 μL.

[0014] The PCR amplification reaction program was as follows: 95℃ pre-denaturation for 5 min; 95℃ for 30 s, 58℃ for 30 s, 72℃ for 30 s, 35 cycles; 72℃ final extension for 5 min.

[0015] When the electrophoresis results of the PCR product show a 563bp band of the target fragment, a gene mutation site for nevus-like basal cell carcinoma syndrome is present.

[0016] Preferably, the gene mutation site c.724_725delCA is used to prepare a kit containing primers or probes for detecting the mutation; and is used in genetic counseling and prenatal diagnostic methods based on the mutation.

[0017] Compared with the prior art, the present invention has the following advantages:

[0018] 1. This invention is the first to discover the association between the c.724_725delCA mutation in the PTCH1 gene and NBCCS; this mutation leads to the loss of function of the PTCH1 protein and may trigger abnormal activation of the Shh pathway; the mutation site is highly conserved in different species and has clear pathogenicity.

[0019] 2. In the existing technology, most known PTCH1 gene mutations are nonsense or frameshift mutations, but c.724_725delCA is a newly discovered mutation site, which expands the pathogenic gene spectrum of NBCCS.

[0020] 3. The PTCH1 gene c.724_725delCA mutation in this invention can be used in the diagnosis of NBCCS, and kits containing primers or probes for detecting this mutation can also be prepared, as well as genetic counseling and prenatal diagnosis methods based on this mutation.

[0021] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments. Attached Figure Description

[0022] Figure 1 This is the NBCCS pedigree chart of Embodiment 1 of the present invention.

[0023] Figure 2 This is the clinical examination of the proband in Embodiment 1 of the present invention.

[0024] Figure 3 These are panoramic and chest X-rays of the proband in Embodiment 1 of the present invention.

[0025] Figure 4 This is a pathological section of the proband from Embodiment 1 of the present invention.

[0026] Figure 5 This is a gel electrophoresis image of the PCR amplification product of Example 1 of the present invention (proband: TQP; proband brother: TQR; proband father: TJ; proband mother: W).

[0027] Figure 6 This is a graph showing the quantitative PCR amplification efficiency of primer specificity detection in Example 1 of the present invention.

[0028] Figure 7 This is a quantitative PCR melting curve of primer-specific detection in Example 1 of the present invention.

[0029] Figure 8 This is the Sanger sequencing data of the first-line relatives of the proband in Embodiment 1 of the present invention.

[0030] Figure 9This is the pathogenicity prediction of the mutant protein in Example 1 of the present invention.

[0031] Figure 10 This is the conservation analysis of mutation sites in Example 1 of the present invention.

[0032] Figure 11 This is the secondary structure analysis of the mutant protein in Example 1 of the present invention.

[0033] Figure 12 This is a three-dimensional structural analysis of the mutant protein in Example 1 of the present invention.

[0034] Figure 13 This is the pcDNA3.1-3×Flag plasmid map of Example 1 of the present invention.

[0035] Figure 14 This refers to the changes in the molecular weight and expression level of the mutant protein in Example 1 of the present invention. Detailed Implementation

[0036] Example 1

[0037] This embodiment describes the application of primers for detecting gene mutation sites in the preparation of screening or auxiliary diagnostic kits for nevus-like basal cell carcinoma syndrome. The gene mutation site is a frameshift mutation in exon 5 of the PTCH1 gene (NM_000264.5:exon5:c.724_725delCA). This mutation results in the deletion of two CA bases (cytosine and adenine) at positions 724 and 725 in the coding region, altering the subsequent codon reading frame. This mutation causes glutamine (Q) to be replaced by valine (V) at amino acid position 242 of the PTCH1 protein, resulting in the PTCH1 mutant p.Q242Vfs*9.

[0038] The frameshift caused a complete change in the subsequent amino acid sequence. Starting from position 242, the reading frame shifted due to the mutation, resulting in a new stop codon. After the frameshift, the new stop codon appeared at the 9th amino acid position (p.Q242Vfs*9). The truncated protein after the mutation was 1198 amino acids shorter (the protein molecular weight changed from 160KD to 20KD).

[0039] The nucleotide sequence of the wild-type cDNA of PTCH1 is shown in SEQ ID No. 3;

[0040] The nucleotide sequence of the cDNA of the PTCH1 c.724_725delCA mutant is shown in SEQ ID No. 4;

[0041] The amino acid sequence of the wild-type PTCH1 protein is shown in SEQ ID No. 5. The stop codon appears at the last position of the amino acid sequence of the wild-type PTCH1 protein and is not included in SEQ ID No. 5 of the WIPO Sequence.

[0042] The amino acid sequence of the PTCH1 mutant p.Q242Vfs*9 is as follows: (SEQ ID No. 6)

[0043] MASAGNAAEPQDRGGGGSGCIGAPGRPAGGGRRRRTGGLRRAAAPDRDYLHRPSYCDAAFALEQISKGKATGRKAPLWLRAKFQRLLFKLGCYIQKNCGKFLVVGLLIFGAFAVGLKAANLETNV EELWVEVGGRVSRELNYTRQKIGEEAMFNPQLMIQTPKEEGANVLTTEALLQHLDSALQASRVHVYMYNRQWKLEHLCYKSGELITETGYMDQIIEYLYPCLIITPLDCFWEGAKLVWDSIPPR*;

[0044] Note: * indicates that the new stop codon appears at the 9th amino acid position, starting from the 242nd amino acid (V); this * is not included in SEQ ID No. 6 of the WIPO Sequence.

[0045] The novel mutation site c.724_725delCA (p.Q242Vfs*9) in the PTCH1 gene solves the following technical problems:

[0046] 1. It broadened the spectrum of pathogenic gene mutations in NBCCS, providing new molecular markers for clinical diagnosis;

[0047] 2. It fills the gap in existing technologies where some NBCCS patients cannot be diagnosed through genetic testing;

[0048] 3. It provides new targets for the development of genetic counseling, prenatal diagnosis and disease detection kits.

[0049] I. Technical Solution:

[0050] 2.1 Family history investigation, clinical assessment, and treatment

[0051] This study recruited a three-generation Han Chinese family from Hunan Province, China. All family members underwent physical examinations, oral examinations, and panoramic radiographs. Basic information and medical history were collected through questionnaires, and a family pedigree was created to analyze the genetic characteristics of the disease. This study was approved by the Ethics Committee of Xiangya Stomatological Hospital, Central South University, Hunan Province. All participants signed written informed consent forms; minors (under 18 years of age) received consent and signatures from their guardians. The family consisted of three generations, with patients in each generation, totaling four patients. Figure 1 (The black arrows represent the proband, the black squares represent male patients, the white squares represent normal males, and the white circles represent normal females.) The proband is an 11-year-old male. Physical examination revealed asymmetry of the maxillofacial region, with significant bulging of the right zygomatic region; a sunken nasal base and asymmetrical nasal alar contours and positions. Figure 2 (A) Multiple small pigmented moles appeared on both palms ( Figure 2 (See white box in section B); Two dark brown pigmented patches, approximately 5mm x 10mm and 7.5mm x 10mm in size, are present on the skin of the right lower back. Figure 2 (C) Prominent forehead, excessively large occiput-frontal distance, and large head circumference. Figure 2 In cases A and D, the patient's head circumference was 57.8 mm, larger than that of normal individuals of the same age. The average head circumference of an 11-year-old boy in China is approximately 52-55 cm. Oral imaging revealed multiple cysts within the maxilla and mandible. Figure 3 (Indicated by the white arrow in center A); cystic lesions in the bilateral mandibular ramus; chest X-ray showing a forked rib deformity on the left fourth rib and right third and sixth ribs (indicated by white arrow in center A). Figure 3 (Indicated by white arrow B). Pathological examination of the lesion area revealed numerous cystic structures surrounded by fibrous cystic wall tissue, with a thin stratified squamous epithelial lining above them. Figure 4 China A and Figure 4 (Medium B), the epithelial surface is incompletely keratinized, the basal cell nuclei are deeply stained and arranged in a palisade pattern, which is typical of OKC lining epithelium ( Figure 4 In the middle C), a large number of inflammatory cells infiltrated beneath the fibrous capsule wall tissue. Figure 4 (D). The patient was ultimately diagnosed with NBCCS syndrome. The proband's brother and father had similar clinical presentations and were also diagnosed with NBCCS syndrome.

[0052] 2.2 Whole exome sequencing and variant site screening

[0053] Genomic DNA (gDNA) was extracted from peripheral blood samples of all participants using the phenol / chloroform method. GDNA exon capture, high-throughput sequencing, and common variant filtering for the proband of the family lineage were performed by Berry Genomics Co., Ltd. The Agilent SureSelect Human All Exon V6 liquid phase capture system efficiently enriched human whole-exon regions in the DNA, followed by high-throughput, high-depth sequencing on the Illumina sequencing platform.

[0054] After obtaining the raw WES sequencing data, the data filtering strategy is as follows: (1) Based on the fact that the close relatives' lineage conforms to the autosomal dominant inheritance pattern, heterozygous mutations are prioritized for screening; (2) Non-synonymous mutations, splice site mutations, and mutations that are predicted to affect splicing are retained; (3) Mutations with a minor allele frequency (MAF) greater than 0.1% in the 1000 Genomes Project database are excluded; (4) Screening is carried out by combining the bioinformatics prediction results and referring to the candidate pathogenic genes reported in relevant literature.

[0055] Using the above filtering strategy, the candidate pathogenic gene PTCH1 was initially screened out from the probands. Subsequently, specific primers were designed using PrimerPremier 5.0 software targeting a region approximately 200 bp upstream and downstream of the PTCH1 gene mutation site.

[0056] Forward primer: 5'-GCAAAAGCTCTGCTCGTT-3' (SEQ ID No. 1);

[0057] Reverse primer: 5'-TTCTGCTGAAATCCCCTC-3' (SEQ ID No. 2).

[0058] The PTCH1 gene mutation sites of all family members were amplified by PCR and verified by Sanger sequencing.

[0059] PCR amplification: Reaction system: 10×Taq Buffer 5μL, 2.5 mM dNTPs 4μL, 10 μM forward primer 2μL, 10 μM forward primer 2μL, 5 U / μL Taq DNA polymerase 0.5μL, genomic DNA to be tested 1μL, ddH2O to make up to 50μL;

[0060] Reaction conditions: 95℃ pre-denaturation for 5 min; 35 cycles (95℃ for 30 s, 58℃ for 30 s, 72℃ for 30 s); 72℃ final extension for 5 min.

[0061] DNA samples from four members of the family (the proband, the proband's brother, the father, and the mother) were amplified by PCR, and the products were then subjected to gel electrophoresis. Figure 5 The proband's DNA was: TQP; proband's brother: TQR; proband's father: TJ; proband's mother: W. Electrophoresis results showed that the target fragment (563bp) band was correctly positioned and clear, with no non-specific bands or primer dimer structures observed. This indicates that the primers designed in this protocol have good specificity. The negative control group (DNA replaced with water) also did not amplify any non-specific bands, indicating that the primers themselves do not form complementary hairpin or dimer structures, ensuring the high efficiency of the primers in the PCR amplification process. In this study, the DNA of the proband and the proband's mother was used for qPCR. The qPCR amplification reaction system (total 20μL) contained: 10μL of SYBR Green qPCR Master Mix (2X), 0.4μL of primers (10μM each), 2μL of template DNA (50 ng), and 7.2μL of ddH2O. Reaction conditions: 95℃ pre-denaturation for 5 min; 40 cycles (95℃ for 15 s, 60℃ for 60 s); 95℃ for 30 s, 60℃ for 15 s. Primer specificity was detected by amplification and melting curves. Results showed that the amplification curves were generally S-shaped, with all curves showing signals close to the background (baseline) in the early cycles (<10 cycles), then gradually separating. Figure 6 The melting curve showed a single main peak and the Tm value met expectations, with no small peaks or shoulder peaks, all indicating good primer specificity. Figure 7 The PCR amplification products were sent to the company for Sanger sequencing, and the results showed that the PTCH1 gene in this family patient group had a heterozygous mutation. This heterozygous mutation was a frameshift mutation (c.724_725delCA), which was not found in normal members. Figure 8 ).

[0062] 2.3 Bioinformatics Analysis

[0063] Mutation Taster (http: / / www.mutationtaster.org / ) was used to predict the pathogenicity of the mutant protein and the conservation of the mutant amino acids. The results showed that this mutation was pathogenic (score: 1, Figure 9 ), mutation sites are highly conserved across different species ( Figure 10 The secondary structure changes of the mutant protein were analyzed using the SOPMA online tool (https: / / npsa-prabi.ibcp.fr / cgi-bin / npsa_automat.pl?page=npsa_sopma.html). The software results showed that the mutant protein completely lost its C-terminal intracellular domain, and the number and proportion of α-helices, extended strands, and random coils were significantly altered. Figure 11 (See Table 1) (WT represents PTCH1 wild-type, MUT represents PTCH1 mutant). The three-dimensional structural differences between the mutant and wild-type proteins were compared using SWISS-MODEL software (https: / / swissmodel.expasy.org / ). The results showed that the transmembrane domains (TM2-TM12) and intracellular regulatory domains were completely lost after the protein mutation. Figure 12 ).

[0064] Table 1. Specific numerical values ​​of secondary structure changes in wild-type and mutant proteins.

[0065]

[0066] 2.4 Effect of PTCH1 mutation on protein expression levels

[0067] The full-length wild-type and mutant cDNA sequences of PTCH1 were synthesized by OBIO TECHNOLO (Shanghai) Co., Ltd., and cloned into the pCDNA3.1-3×Flag vector. Figure 13 PTCH1-WT and PTCH1-MUT vectors were obtained. HEK-293T cells were selected as the experimental group, and the experiment was divided into four groups: MOCK (untreated normal control group), Ctrl (empty vector group), WT (PTCH1 wild-type group), and MUT (PTCH1 mutant group). The vectors of each group were transfected into HEK-293T cells using lentivirus. Transfection efficiency was observed under a fluorescence microscope 48 hours after transfection, and the transfection efficiency reached over 90%. Proteins were collected from each group of cells, and Western blotting (WB) was used to detect the expression level and molecular weight of PTCH1 protein in each group. WB results showed that the MUT group (PTCH1 mutant group) produced truncated protein (~20KD), and the expression level was not significantly different from the control group. Figure 14 ).

[0068] II. Diagnostic Applications:

[0069] Design PTCH1 gene-specific primers or probes (SEQ ID No. 1-2) to detect the c.724_725delCA mutation.

[0070] III. Reagent Kit Development:

[0071] Includes PCR primers, sequencing primers, or gene chips targeting this mutation.

[0072] This invention identifies for the first time a frameshift mutation c.724_725delCA in exon 5 of the PTCH1 gene. This mutation alters the subsequent codon reading frame, causing a frameshift mutation p.Q242Vfs*9 in the PTCH1 protein, replacing glutamine with valine at position 242. This mutation leads to premature termination of the PTCH1 protein at position 250, resulting in the complete loss of its C-terminal intracellular domain (including the Sufu binding domain and Gli regulatory region). This prevents the binding of SMO to inhibit its activity, ultimately leading to abnormal activation of the Shh signaling pathway and the occurrence of NBCCS.

[0073] This invention is the first to discover the association between the c.724_725delCA mutation in the PTCH1 gene and NBCCS; this mutation leads to loss of function of the PTCH1 protein and may trigger abnormal activation of the Shh pathway; the mutation site is highly conserved in different species and has clear pathogenicity.

[0074] The difference between this invention and the prior art is that most known PTCH1 gene mutations in the prior art are nonsense or frameshift mutations, but c.724_725delCA is a newly discovered mutation site, which expands the pathogenic gene spectrum of NBCCS.

[0075] The PTCH1 gene c.724_725delCA mutation in this invention can be used in the diagnosis of NBCCS, and kits containing primers or probes for detecting this mutation can also be prepared, as well as genetic counseling and prenatal diagnosis methods based on this mutation.

[0076] The value of the c.724_725delCA mutation in the PTCH1 gene provided by this invention:

[0077] (1) Diagnostic value:

[0078] To provide new molecular diagnostic biomarkers for NBCCS patients and improve diagnostic rates.

[0079] (2) Genetic counseling:

[0080] Clarifying the carrier status of family members can guide reproductive decisions.

[0081] (3) Reagent kit development:

[0082] Commercial kits that specifically detect this mutation can be developed, advancing precision medicine.

[0083] (4) Scientific research value:

[0084] This provides new clues for the study of the Shh signaling pathway mechanism.

[0085] Specific application examples of the PTCH1 gene c.724_725delCA mutation of this invention:

[0086] Case 1: Family mutation testing and recommendations for optimal birth outcomes

[0087] WES and Sanger sequencing were performed on the proband and his family members to verify the co-segregation of the c.724_725delCA mutation. Eugenics recommendations were provided to affected family members. Since the disease follows an autosomal dominant inheritance pattern, patients have a 50% probability of inheriting the mutation. Prenatal diagnosis or assisted reproductive technology (preimplantation genetic testing) is recommended to avoid passing on the mutation.

[0088] Case 2: Reagent Kit Validation

[0089] This study randomly selected 20 human DNA samples from the biobank, designed specific primers, and successfully amplified the target DNA fragments through PCR amplification and sequencing, with a sensitivity of 100%.

[0090] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any way. Any simple modifications, alterations, and equivalent changes made to the above embodiments based on the inventive essence shall still fall within the protection scope of the present invention.

Claims

1. The application of a primer for detecting gene mutation sites in the preparation of a screening or auxiliary diagnostic kit for nevus-like basal cell carcinoma syndrome, characterized in that, The gene mutation site is PTCH1 A frameshift mutation c.724_725delCA in exon 5 of the gene causes a frameshift mutation p.Q242Vfs*9 in the PTCH1 protein, in which glutamine at position 242 of the PTCH1 protein is mutated to valine; PTCH1 The nucleotide sequence of the wild-type cDNA of the gene is shown in SEQ ID No.

3.

2. The application of the primer for detecting gene mutation sites according to claim 1 in the preparation of a screening or auxiliary diagnostic kit for nevus-like basal cell carcinoma syndrome, characterized in that, The primers for detecting gene mutation sites include a forward primer and a reverse primer. The nucleotide sequence of the forward primer is shown in SEQ ID No. 1, and the nucleotide sequence of the reverse primer is shown in SEQ ID No.

2.

3. The application of the primer for detecting gene mutation sites according to claim 2 in the preparation of a screening or auxiliary diagnostic kit for nevus-like basal cell carcinoma syndrome, characterized in that, The method for detecting the gene mutation site is as follows: PCR was performed on the genomic DNA to be tested using forward and reverse primers to obtain PCR products; The PCR amplification reaction system consisted of: 5 μL of 10×Taq Buffer, 4 μL of 2.5 mM dNTPs, 2 μL of 10 μM forward primer, 2 μL of 10 μM forward primer, 0.5 μL of 5 U / μL Taq DNA polymerase, 1 μL of genomic DNA to be tested, and ddH2O to a final volume of 50 μL. The PCR amplification reaction program was as follows: 95℃ pre-denaturation for 5 min; 95℃ for 30 s, 58℃ for 30 s, 72℃ for 30 s, 35 cycles; 72℃ final extension for 5 min. When the electrophoresis results of the PCR product show a 563bp band of the target fragment, a gene mutation site for nevus-like basal cell carcinoma syndrome is present.

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