Nevus basal cell carcinoma syndrome gene mutation site and application thereof

Abstract

The invention provides a nevus basal cell carcinoma syndrome gene mutation site and application thereof, the gene mutation site is a frame-shift mutation c.724725delCA of a No. 5 exon of a PTCH1 gene, and the mutation causes the change of a subsequent codon reading frame and causes the frame-shift mutation p.Q242Vfs * 9 of PTCH1 protein. According to the c.724725delCA mutation site disclosed by the invention, the pathogenic gene spectrum of the NBCCS is expanded. The PTCH1 gene c.724725delCA mutation provided by the invention can be applied to NBCCS diagnosis, and can also be used for preparing a kit containing a mutation detection primer or probe, and a genetic counseling and prenatal diagnosis method based on the mutation.

Description

Technical Field

[0001] The present invention belongs to the technical field of nevoid basal cell carcinoma syndrome pathogenic genes, and particularly relates to an application of a primer for detecting gene mutation sites in the preparation of a nevoid basal cell carcinoma syndrome screening or auxiliary diagnosis kit. Background Art

[0002] Nevoid basal cell carcinoma syndrome (NBCCS) is a rare autosomal dominant genetic disorder characterized by multiple odontogenic keratocysts (OKCs), basal cell carcinomas (BCCs), and skeletal deformities. PTCH1 gene mutations are currently known to be the primary cause of NBCCS, but the reported mutation sites are limited, and the clinical phenotypes vary significantly across mutation sites. Currently, the diagnosis of NBCCS relies primarily on clinical criteria and genetic testing of known mutation sites. However, in some patients, the causative mutation remains unknown, limiting genetic counseling and prenatal diagnosis. Summary of the Invention

[0003] The present invention addresses the shortcomings of the prior art by providing primers for detecting gene mutations for use in preparing screening or auxiliary diagnostic kits for nevoid basal cell carcinoma syndrome. The c.724_725delCA mutation expands the spectrum of pathogenic genes for NBCCS. The present invention also provides for the use of the c.724_725delCA mutation in the PTCH1 gene in the diagnosis of NBCCS. 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.

[0004] To solve the above technical problems, the present invention adopts a technical solution: the use of a primer for detecting a gene mutation site in the preparation of a screening or auxiliary diagnosis kit for nevoid basal cell carcinoma syndrome. The gene mutation site is a frameshift mutation c.724_725delCA in exon 5 of the PTCH1 gene. This mutation results in the deletion of two bases (CA) at positions 724 and 725 in the coding region, altering the reading frame of subsequent codons. This mutation causes a frameshift mutation in the PTCH1 protein, p.Q242Vfs*9, in which the glutamine (Q) at amino acid position 242 of the PTCH1 protein mutates to valine (V). The frameshift causes a complete change in the subsequent amino acid sequence. Starting from position 242, the mutation causes the reading frame to shift, generating a new stop codon. After the frameshift, the new stop codon appears at the 9th amino acid position. The truncated protein after the mutation has a total of 1198 fewer amino acids (the protein molecular weight changes from 160KD to 20KD).

[0005] The nucleotide sequence of the wild-type PTCH1 cDNA is shown in SEQ ID No. 3; The nucleotide sequence of the cDNA of the PTCH1 c.724_725delCA mutant is shown in SEQ ID No. 4; 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 in the WIPO sequence.

[0006] The amino acid sequence of the PTCH1 mutant p.Q242Vfs*9 is shown in SEQ ID No. 6. Starting from amino acid position 242, a new stop codon appears at the 9th amino acid position. The stop codon is not included in SEQ ID No. 6 in the WIPO sequence.

[0007] Preferably, the primers for detecting gene mutation sites include a forward primer and a reverse primer, the nucleotide sequence of the forward primer is shown as SEQ ID No. 1, and the nucleotide sequence of the reverse primer is shown as SEQ ID No. 2.

[0008] Preferably, the method for detecting the gene mutation site is: Perform PCR on the genomic DNA to be tested using forward primers and reverse primers to obtain PCR products; The PCR amplification reaction system is as follows: 10× Taq Buffer 5 μL, 2.5 mM dNTPs 4 μL, 10 μM forward primer 2 μL, 10 μM forward primer 2 μL, 5 U / μL Taq DNA polymerase 0.5 μL, genomic DNA to be tested 1 μL, and ddH2O is added to 50 μL. The PCR amplification reaction program was as follows: pre-denaturation at 95°C for 5 min; 35 cycles of 95°C for 30 s, 58°C for 30 s, and 72°C for 30 s; final extension at 72°C for 5 min; When a 563 bp band of the target fragment appears in the electrophoresis result of the PCR product, a gene mutation site of nevoid basal cell carcinoma syndrome exists.

[0009] Preferably, the gene mutation site c.724_725delCA is used to prepare a kit comprising primers or probes for detecting the mutation; and is used for genetic counseling and prenatal diagnosis methods based on the mutation.

[0010] Compared with the prior art, the present invention has the following advantages: 1. This study discovered for the first time the association between the PTCH1 gene c.724_725delCA mutation and NBCCS; this mutation results in loss of PTCH1 protein function and may trigger abnormal activation of the Shh pathway; the mutation site is highly conserved across species and has clear pathogenicity.

[0011] 2. It is known in the prior art that most 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.

[0012] 3. The PTCH1 gene c.724_725delCA mutation of the present invention can be used in the diagnosis of NBCCS. Kits containing primers or probes for detecting the mutation can also be prepared, as well as genetic counseling and prenatal diagnosis methods based on the mutation.

[0013] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is the NBCCS pedigree chart of Example 1 of the present invention.

[0015] Figure 2 This is the clinical examination of the proband of Example 1 of the present invention.

[0016] Figure 3 These are the panoramic radiographs and chest radiographs of the proband in Example 1 of the present invention.

[0017] Figure 4 This is a pathological section of the proband in Example 1 of the present invention.

[0018] Figure 5 This is a gel electrophoresis diagram of the PCR amplification products of Example 1 of the present invention (proband: TQP; proband's brother: TQR; proband's father: TJ; proband's mother: W).

[0019] Figure 6 This is a diagram showing the quantitative PCR amplification efficiency of the primer specificity detection in Example 1 of the present invention.

[0020] Figure 7 This is a quantitative PCR melting curve diagram for primer specificity detection in Example 1 of the present invention.

[0021] Figure 8 This is the Sanger sequencing chart of the immediate family members of the proband in Example 1 of the present invention.

[0022] Figure 9 This is the prediction of the pathogenicity of the mutant protein in Example 1 of the present invention.

[0023] Figure 10This is the conservation analysis of the mutation sites in Example 1 of the present invention.

[0024] Figure 11 This is the secondary structure analysis of the mutant protein of Example 1 of the present invention.

[0025] Figure 12 This is the three-dimensional structure analysis of the mutant protein of Example 1 of the present invention.

[0026] Figure 13 This is the pcDNA3.1-3×Flag plasmid map of Example 1 of the present invention.

[0027] Figure 14 This is the change in molecular weight and expression level of the mutant protein of Example 1 of the present invention. DETAILED DESCRIPTION

[0028] Example 1 The primers for detecting gene mutation sites in this embodiment are used in the preparation of a screening or auxiliary diagnosis kit for nevoid 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 causes the deletion of two bases CA (cytosine and adenine) at positions 724 and 725 in the coding region, changing the reading frame of subsequent codons. This mutation causes the glutamine (Q) at amino acid position 242 of the PTCH1 protein to valine (V), resulting in the PTCH1 mutant type p.Q242Vfs*9. The frameshift causes a complete change in the subsequent amino acid sequence. Starting from position 242, the mutation causes the reading frame to shift, resulting in a new stop codon. After the frameshift, the new stop codon appears at the 9th amino acid position (p.Q242Vfs*9). The truncated protein after the mutation has a total of 1198 fewer amino acids (the protein molecular weight changes from 160KD to 20KD).

[0029] The nucleotide sequence of the wild-type PTCH1 cDNA is shown in SEQ ID No. 3; The nucleotide sequence of the cDNA of the PTCH1 c.724_725delCA mutant is shown in SEQ ID No. 4; 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 in the WIPO sequence.

[0030] The amino acid sequence of the PTCH1 mutant p.Q242Vfs*9 is: (SEQ ID No. 6) MASAGNAAEPQDRGGGGSGCIGAPGRPAGGGRRRRTGGLRRAAAPDRDYLHRPSYCDAAFALEQISKGKATGRKAPLWLRAKFQRLLFKLGCYIQKNCGKFLVVGLLIFGAFAVGLKAANLETNV EELWVEVGGRVSRELNYTRQKIGEEAMFNPQLMIQTPKEEGANVLTTEALLQHLDSALQASRVHVYMYNRQWKLEHLCYKSGELITETGYMDQIIEYLYPCLIITPLDCFWEGAKLVWDSIPPR*; 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 in the WIPO Sequence.

[0031] The new mutation site c.724_725delCA (p.Q242Vfs*9) in the PTCH1 gene solved the following technical problems: 1. Expanded the spectrum of pathogenic gene mutations in NBCCS and provided new molecular markers for clinical diagnosis; 2. It fills the gap in existing technologies where some NBCCS patients cannot be diagnosed through genetic testing; 3. Provides new targets for genetic counseling, prenatal diagnosis and development of disease detection kits.

[0032] 1. Technical solution: 2.1 Family investigation, clinical evaluation, and treatment This study recruited a three-generation Han Chinese family from Hunan Province, China. All family members underwent a full-body examination, dental specialist examination, and panoramic radiographs. A questionnaire was used to collect basic information and medical history of the participants, and a family tree was drawn 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 a paper informed consent form, and minors (under the age of 18) had their guardians agree and sign. The family has three generations, each with patients, for a total of four patients ( Figure 1 The black arrow represents the proband, the black box represents the male patient, the white box represents the normal male, and the white circle represents the normal female). The proband is an 11-year-old male. Gross physical examination showed left and right maxillofacial asymmetry, with obvious bulging of the right zygomatic area; the nasal floor was collapsed, and the nasal alar contour and position were asymmetrical ( Figure 2 Middle A); Multiple small pigmented moles appeared on both palms ( Figure 2There are two dark brown pigmented patches on the right side of the waist, with sizes of 5 mm × 10 mm and 7.5 mm × 10 mm, respectively ( Figure 2 Middle C); forehead protrusion, large occipital-forehead distance, and large head circumference ( Figure 2 In Figures A and D, the patient's head circumference is 57.8 mm, which is larger than that of normal people of the same age. The average head circumference of 11-year-old boys in China is about 52-55 cm. Oral imaging examination showed multiple cysts in the upper and lower jaws ( Figure 3 (As shown by the white arrows in middle A); Cystic lesions at the ascending ramus of the mandible on both sides; the chest anteroposterior radiograph shows forked rib deformity at the fourth rib on the left and the third and sixth ribs on the right ( Figure 3 Middle B, indicated by white arrows). Pathological examination of the lesion area showed that there were many cystic structures in the lesion area, surrounded by fibrous cystic tissue and lined with a thin layer of stratified squamous epithelium ( Figure 4 China A and Figure 4 Middle B), the epithelial surface is not completely keratinized, the basal cell nuclei are darkly stained and arranged in a palisade pattern, which is a typical OKC lining epithelium ( Figure 4 Middle C), a large number of inflammatory cells infiltrate below the fibrous cystic tissue ( Figure 4 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.

[0033] 2.2 Whole-exome sequencing and variant site screening Genomic DNA (gDNA) was extracted from peripheral blood samples from all participants using the phenol / chloroform method. Exome capture, high-throughput sequencing, and common variant filtering of the gDNA from the proband were performed by Beijing Berry Genomics Co., Ltd. The Agilent SureSelect Human All Exon V6 liquid phase capture system efficiently enriched the DNA for human exome regions, followed by high-throughput, deep sequencing on the Illumina sequencing platform.

[0034] After obtaining the original WES sequencing data, the data filtering strategy was as follows: (1) Based on the fact that the consanguineous family conforms to the autosomal dominant inheritance pattern, heterozygous mutations were prioritized; (2) non-synonymous mutations, splice site mutations, and mutations predicted to affect splicing were retained; (3) mutations with a minor allele frequency (MAF) greater than 0.1% in the 1000 Genomes Project database were excluded; (4) candidate pathogenic genes reported in relevant literature were screened in combination with bioinformatics prediction results.

[0035] Through the above filtering strategy, the candidate pathogenic gene PTCH1 was preliminarily screened in the proband. Subsequently, PrimerPremier 5.0 software was used to design specific primers targeting the region approximately 200 bp upstream and downstream of the PTCH1 gene mutation site: Forward primer: 5′-GCAAAAGCTCTGCTCGTT-3′ (SEQ ID No. 1); Reverse primer: 5′-TTCTGCTGAAATCCCCTC-3′ (SEQ ID No. 2).

[0036] The PTCH1 gene mutation sites of all family members were amplified by PCR and verified by Sanger sequencing.

[0037] PCR amplification: Reaction system: 10× Taq Buffer 5 μL, 2.5 mM dNTPs 4 μL, 10 μM forward primer 2 μL, 10 μM forward primer 2 μL, 5 U / μL Taq DNA polymerase 0.5 μL, genomic DNA to be tested 1 μL, ddH2O to 50 μL; Reaction conditions: initial denaturation at 95°C for 5 min; 35 cycles (95°C for 30 s, 58°C for 30 s, and 72°C for 30 s); final extension at 72°C for 5 min.

[0038] The DNA samples of the four family members (proband, proband's brother, father and mother) were amplified by PCR and the products were subjected to gel electrophoresis ( Figure 5Electrophoresis results showed that the target fragment (563 bp) was correctly positioned and clearly labeled, with no nonspecific bands or primer-dimer formation observed. This indicates that the primers designed in this protocol have good specificity. A negative control (DNA replaced with water) also did not amplify any bands, indicating that the primers do not complement each other to form hairpins or dimers, ensuring high primer efficiency during PCR amplification. In this study, DNA from the proband and the proband's mother was subjected to qPCR. The qPCR amplification reaction system (20 μL total) contained 10 μL of SYBR Green qPCR Master Mix (2X), 0.4 μL of primers (10 μM each), 2 μL of template DNA (50 ng), and 7.2 μL of ddH2O. Reaction conditions: 95°C pre-denaturation for 5 min; 40 cycles (95°C for 15 s, 60°C for 60 s); 95°C for 30 s, 60°C for 15 s. Primer specificity was tested using amplification and melting curve plots. The results showed an overall S-shaped amplification curve. In all curves, signals approached background (baseline) in the early cycles (<10 cycles) and gradually separated thereafter. Figure 6 ), while the melting curve showed a single main peak and the Tm value was in line with expectations, with no small peaks or shoulders, indicating that the primers had good specificity ( Figure 7 The PCR amplification product was sent to the company for Sanger sequencing. The results showed that the PTCH1 gene of the family patients had a heterozygous mutation. The heterozygous mutation was a frameshift mutation (c.724_725delCA). This mutation was not found in normal members. Figure 8 ).

[0039] 2.3 Bioinformatics Analysis Mutation Taster (http: / / www.mutationtaster.org / ) was used to predict the pathogenicity of the mutant protein and the conservation of the mutant amino acid. The results showed that the mutation was pathogenic (score: 1, Figure 9 ), the mutation site is highly conserved among different species ( Figure 10 The SOPMA online tool (https: / / npsa-prabi.ibcp.fr / cgi-bin / npsa_automat.pl?page=npsa_sopma.html) was used to analyze the secondary structure changes of the mutant protein. The software results showed that the mutant protein completely lost the C-terminal intracellular domain, and the number and proportion of α-helices, extended chains and random coils were significantly changed ( Figure 11and Table 1) (WT is PTCH1 wild type, MUT is PTCH1 mutant). SWISS-MODEL (https: / / swissmodel.expasy.org / ) software was used to compare the three-dimensional structural differences between the mutant and wild-type proteins. The results showed that the transmembrane domain (TM2-TM12) and the intracellular regulatory domain were completely lost after the protein mutation ( Figure 12 ).

[0040] Table 1 Specific values of secondary structure changes of wild-type protein and mutant protein 2.4 Effect of PTCH1 mutation on protein expression OBIO TECHNOLO (Shanghai) was commissioned to synthesize the full-length wild-type cDNA sequence and mutant cDNA sequence of PTCH1 and cloned into the pCDNA3.1-3×Flag vector ( Figure 13 ), PTCH1-WT and PTCH1-MUT vectors were obtained. HEK-293T was selected as the tool cell, and the experiment was divided into four groups, namely MOCK: untreated normal control group, Ctrl: empty group; WT: PTCH1 wild type group; MUT: PTCH1 mutant group. Each group of vectors was transfected into HEK-293T cells by lentivirus. The transfection efficiency was observed by fluorescence microscopy 48 hours after transfection. The transfection efficiency could reach more than 90%. The proteins in each group of cells were collected, and the expression level and molecular weight of PTCH1 protein in each group were detected by WB. The WB results showed that the MUT group (PTCH1 mutant group) produced a truncated protein (~20KD), and the expression level did not change significantly compared with the control group ( Figure 14 ).

[0041] 2. Diagnostic Applications: PTCH1 gene-specific primers or probes (SEQ ID No. 1-2) were designed to detect the c.724_725delCA mutation.

[0042] 3. Test kit development: Contains PCR primers, sequencing primers or gene chips targeting the mutation.

[0043] This study identified for the first time a frameshift mutation in exon 5 of the PTCH1 gene, c.724_725delCA. This mutation alters the reading frame of subsequent codons, resulting in a frameshift mutation in the PTCH1 protein, p.Q242Vfs*9, in which glutamine at position 242 of the PTCH1 protein is converted to valine. This mutation causes premature termination of PTCH1 protein at position 250, completely depleting the C-terminal intracellular domain (including the Sufu binding domain and Gli regulatory region). This mutation may prevent SMO binding and inhibit its activity, ultimately triggering abnormal activation of the Shh signaling pathway and leading to the development of NBCCS.

[0044] This study discovered for the first time the association between the PTCH1 gene c.724_725delCA mutation and NBCCS; this mutation leads to loss of PTCH1 protein function and may trigger abnormal activation of the Shh pathway; the mutation site is highly conserved across different species and has clear pathogenicity.

[0045] The difference between the present invention and the prior art is that: in the prior art, most 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.

[0046] The PTCH1 gene c.724_725delCA mutation of the present invention can be used in the diagnosis of NBCCS. A kit containing primers or probes for detecting the mutation can also be prepared, as well as genetic counseling and prenatal diagnosis methods based on the mutation.

[0047] The value of the PTCH1 gene c.724_725delCA mutation provided by the present invention: (1) Diagnostic value: Provide new molecular diagnostic markers for NBCCS patients and improve the diagnosis rate.

[0048] (2) Genetic counseling: Clarify the carrier status of family members and guide reproductive decisions.

[0049] (3) Kit development: Commercial kits can be developed to specifically detect this mutation and promote precision medicine.

[0050] (4) Scientific research value: Provide new clues for the study of Shh signaling pathway mechanism.

[0051] Specific application cases of the PTCH1 gene c.724_725delCA mutation of the present invention: Case 1: Family mutation detection and eugenics recommendations WES and Sanger sequencing were performed on the proband and his family members to confirm the cosegregation of the c.724_725delCA mutation. Pregnancy and childbearing advice was provided to the affected family members. Because the family's disease is inherited in an autosomal dominant pattern, with a 50% chance of inheriting the mutation, future prenatal diagnosis or assisted reproductive technology (preimplantation genetic testing) is recommended to prevent transmission of the mutation.

[0052] Case 2: Kit Validation This study randomly selected 20 human DNA samples from a biological sample bank, designed specific primers, and through PCR amplification and sequencing, all of them were able to effectively amplify the target DNA fragments with a sensitivity of 100%.

[0053] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent variation made to the above embodiment based on the essence of the invention technology shall still fall within the scope of protection of the technical solution of the present invention.

Claims

1. Use of a primer for detecting gene mutation sites in the preparation of a screening or auxiliary diagnosis kit for nevoid basal cell carcinoma syndrome, characterized in that: The gene mutation site is a frameshift mutation c.724_725delCA in exon 5 of the PTCH1 gene, which causes a frameshift mutation p.Q242Vfs*9 in the PTCH1 protein, and the glutamine at position 242 of the PTCH1 protein is mutated to valine.

2. Use of a primer for detecting gene mutation sites according to claim 1 in preparing a screening or auxiliary diagnosis 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. Use of a primer for detecting gene mutation sites according to claim 2 in preparing a screening or auxiliary diagnosis kit for nevus-like basal cell carcinoma syndrome, characterized in that: The method for detecting the gene mutation site is: Perform PCR on the genomic DNA to be tested using forward primers and reverse primers to obtain PCR products; The PCR amplification reaction system is as follows: 10× Taq Buffer 5 μL, 2.5 mM dNTPs 4 μL, 10 μM forward primer 2 μL, 10 μM forward primer 2 μL, 5 U / μL Taq DNA polymerase 0.5 μL, genomic DNA to be tested 1 μL, and ddH2O is added to 50 μL. The PCR amplification reaction program was as follows: initial denaturation at 95°C for 5 min; 35 cycles of 95°C for 30 s, 58°C for 30 s, and 72°C for 30 s; and final extension at 72°C for 5 min. When a 563 bp band of the target fragment appears in the electrophoresis result of the PCR product, a gene mutation site of nevoid basal cell carcinoma syndrome exists.

4. Use of a primer for detecting a gene mutation site according to claim 1 in preparing a screening or auxiliary diagnosis kit for nevus-like basal cell carcinoma syndrome, wherein the gene mutation site is used to prepare a kit comprising the mutation detection primer or probe; and for genetic counseling and prenatal diagnosis methods based on the mutation.

Images