CYP21A2 gene variation detection method and application

Abstract

The invention provides a CYP21A2 gene variation detection method and application, and relates to the technical field of molecular diagnosis. The amplification primer group comprises a target gene amplification primer pair and a fusion gene amplification primer pair; the target gene amplification primer pair can specifically amplify the full-length sequence of the CYP21A2 gene; a forward primer in the fusion gene amplification primer pair can be combined in an upstream specific sequence of the CYP21A1P, and a reverse primer in the fusion gene amplification primer pair can be combined in a downstream specific sequence of the CYP21A2 gene. The amplification and sequencing primer group can be used for simply, quickly and accurately detecting various mutations of the CYP21A2 gene and the fusion gene and the fusion type of the CYP21A1P-CYP21A2.

Description

Technical Field

[0001] The present invention relates to the field of molecular diagnostic technologies, and more particularly, to a method for detecting CYP21A2 gene mutations and its applications. Background Art

[0002] Congenital adrenal hyperplasia (CAH) is an autosomal recessive genetic disorder caused by defects in adrenal steroid synthesis enzymes. Among them, 21-hydroxylase deficiency (21-OHD) is the most common type, accounting for 90% - 95% of CAH cases. The pathogenic gene CYP21A2 of 21-OHD is located on the short arm of chromosome 6 (6p21.3) and is responsible for encoding 21-hydroxylase, which is involved in the synthesis of glucocorticoids and mineralocorticoids. There are various types of mutations in the CYP21A2 gene, including small mutations, large fragment deletions caused by fusion with pseudogenes (forming fusion genes), etc. Mutations in the CYP21A2 gene lead to the absence or impaired function of 21-hydroxylase, causing a series of clinical symptoms, including androgen excess, disorders of sex development (DSD), and electrolyte disorders. Globally, the incidence of CAH is approximately 1 / 14000 to 1 / 18000, while the incidence in China is between 1 / 16466 and 1 / 12200. Since CAH can lead to severe metabolic disorders, early and accurate molecular diagnosis is crucial for early intervention and management of the disease.

[0003] Currently, the clinical diagnosis of 21-OHD mainly relies on hormone level detection and gene detection. Hormone detection methods (such as serum 17-OHP concentration measurement) are affected by individual differences, diurnal fluctuations, age, and drug interference, and are prone to false positives or false negatives. Therefore, gene detection is considered the gold standard for diagnosing 21-OHD. The CYP21A2 gene is tandemly arranged in the genome with the highly homologous pseudogene CYP21A1P, and the homology between the two is 98.4%. The high similarity of the true and false gene sequences and the easy fusion of the two to produce the CYP21A1P-CYP21A2 fusion gene both pose challenges to gene diagnosis.

[0004] Currently, common gene detection methods include Sanger sequencing + multiplex ligation-dependent probe amplification (MLPA) after PCR amplification (referred to as Sanger sequencing + MLPA) and third-generation single-molecule sequencing. Sanger sequencing is mainly used for the detection of point mutations, and MLPA is used for exon copy number analysis and the speculation of fusion genes; while the recently emerged third-generation single-molecule sequencing can detect point mutations and fusion genes at one time, which is an ideal detection method.

[0005] There are still multiple problems in the existing technology for CYP21A2 gene detection. First, the MLPA method has limitations in detecting copy number changes. Its quantitative detection mode based on probe hybridization determines a limited detection range, which can only cover some exons (such as 1, 3, 4, 7) where there are differences between true and false gene sequences. The probe length is short, so the detection range is limited; moreover, if there are variations in the probe binding region, it may lead to inaccurate copy number detection. More importantly, MLPA cannot directly detect the fusion breakpoints of true and false genes, and its recognition of fusion genes can only be speculated through copy number changes, with a certain possibility of misjudgment. Second, Sanger sequencing has limitations in the length of sequencing fragments. Generally, the single-read length does not exceed 1000bp, resulting in the need for multiple primer segmental amplifications for CYP21A2 gene detection. Due to the high homology between true and false genes, the amplification primers need to be strictly designed at specific positions, otherwise non-specific amplification of pseudogenes may occur, affecting the detection accuracy. In addition, Sanger sequencing usually does not cover intron regions and untranslated regions (UTRs), which may miss mutations affecting splicing and regulation, reducing the comprehensiveness of detection. Third, although the third-generation sequencing method can detect point mutations and fusion genes at one time, its detection cost is high, the instrument and equipment are expensive, and the error rate of single-molecule sequencing is relatively high. It still needs to be verified by Sanger sequencing or MLPA, which limits its practical application. At the same time, for a small number of samples, the experimental cost of third-generation sequencing is relatively high, which is not suitable for routine detection in hospitals or primary laboratories.

[0006] In summary, there are certain limitations in the current molecular diagnostic methods for detecting CYP21A2 gene mutations. The MLPA has a limited detection range and cannot accurately detect the specific breakpoints of fusion genes. The segmented amplification method of Sanger sequencing increases the experimental complexity and may have the risk of missed mutation detection. Although the third-generation sequencing method has advantages in detection integrity, it still faces challenges in terms of cost, operation difficulty, and data interpretation. Therefore, there is an urgent need to develop a detection method with low cost, simple operation, strong specificity, and comprehensive coverage of various CYP21A2 gene mutations (including point mutations, microdeletions, fusion genes, and fusion position types) to improve the gene diagnosis efficiency and accuracy of 21-OHD.

[0007] In view of this, the present invention is specifically proposed. Summary of the Invention

[0008] The object of the present invention is to provide an amplification primer set, a sequencing primer set, a detection product, and a detection method. The amplification primer set can simply, quickly, and accurately detect various mutations of the CYP21A2 gene, as well as the CYP21A1P-CYP21A2 fusion gene and its fusion type.

[0009] In order to achieve the above object of the present invention, the following technical solutions are specifically adopted: In the first aspect, the present invention provides an amplification primer set, including a target gene amplification primer pair and a fusion gene amplification primer pair; The target gene amplification primer pair can specifically amplify the full-length sequence of the CYP21A2 gene; The forward primer in the fusion gene amplification primer pair can bind to the upstream specific sequence of CYP21A1P, and the reverse primer can bind to the downstream specific sequence of the CYP21A2 gene.

[0010] In an optional embodiment, the nucleotide sequence of the forward primer of the target gene amplification primer pair is as shown in SEQ ID NO.1; The nucleotide sequence of the forward primer of the fusion gene amplification primer pair is as shown in SEQ ID NO.2; The nucleotide sequences of the reverse primer of the target gene amplification primer pair and the reverse primer of the fusion gene amplification primer pair are both as shown in SEQ ID NO.3.

[0011] In the second aspect, the present invention provides a sequencing primer set. The sequencing primer set can specifically bind to the template sequence with the amplification product of the amplification primer set described in any one of the foregoing embodiments as the template, and can cover the full-length sequence of the CYP21A2 gene.

[0012] In an alternative embodiment, the sequencing primer set includes a forward sequencing primer and / or a reverse sequencing primer; The nucleotide sequence of the forward sequencing primer is shown as at least one of SEQ ID NO.4 to SEQ ID NO.10; The nucleotide sequence of the reverse sequencing primer is shown as at least one of SEQ ID NO.11 to SEQ ID NO.16.

[0013] In a third aspect, the present invention provides a detection product, including the amplification primer set as described in any one of the foregoing embodiments, and the sequencing primer set as described in any one of the foregoing embodiments 3-4.

[0014] In a fourth aspect, the present invention provides a detection method, including: Collecting a target DNA sample; Using the target gene amplification primer pair in the amplification primer set as described in any one of the foregoing embodiments to perform a first PCR amplification on the target DNA sample to obtain a first amplification result; and using the fusion gene amplification primer pair in the amplification primer set as described in any one of the foregoing embodiments to perform a second PCR amplification on the target DNA sample to obtain a second amplification result; Determining the fusion gene detection result of the target DNA sample according to the first amplification result and the second amplification result.

[0015] In an alternative embodiment, the amplification reaction systems of the first PCR amplification and the second PCR amplification include: 2×GC Buffer I, 15 μL; dNTP Mixture, with the concentration of each dNTP being 2.5 mM, 4.8 μL; For the target gene amplification primer pair or the fusion gene amplification primer pair, the concentrations of the forward primer and the reverse primer of the primer pair used are both 10 uM, and the amounts used are both 1 μL; Amplification enzyme, 0.3 μL; Target DNA sample, 30 ng; And controlling the total volume to be 30 μL.

[0016] In an alternative embodiment, the determining the fusion gene detection result of the target DNA sample according to the first amplification result and the second amplification result includes: If there is an amplification product in the first amplification result and there is no amplification product in the second amplification result, then there is no fusion gene in the target DNA sample; If amplification products are present in both the first amplification result and the second amplification result, then there is a fusion gene in the target DNA sample, and the fusion gene is in a heterozygous state; If no amplification product is present in the first amplification result and an amplification product is present in the second amplification result, then there is a fusion gene in the target DNA sample, and the fusion gene is in a homozygous state or a compound heterozygous state.

[0017] In an alternative embodiment, after determining the fusion detection result of the target DNA sample based on the first amplification result and the second amplification result, the method further includes: Performing Sanger sequencing on the amplification products in the first amplification result and / or the second amplification result using the sequencing primer set described in any of the foregoing embodiments to obtain a sequencing result; Analyzing the sequencing result to obtain an analysis result; Wherein, the analysis result includes at least one of the following results: A. Determining whether there is a fusion gene and confirming the specific break and rejoining positions of the fusion gene; C. Confirming the specific mutation types and positions within the CYP21A2 gene.

[0018] In a fifth aspect, the present invention provides an application of the amplification primer set described in any of the foregoing embodiments and the sequencing primer set described in any of the foregoing embodiments in the preparation of a detection product for detecting full-length mutations and / or fusion genes of the CYP21A2 gene.

[0019] The amplification primer set provided by the present invention can amplify the full length of the CYP21A2 gene at one time, covering all exons, introns and UTR regions, ensuring that all mutation types can be detected, improving the integrity and comprehensiveness of detection, and avoiding missed detection of mutations caused by segmented amplification.

[0020] This primer set has high specificity. The primer binding sites are selected in regions where there are significant differences between the true and false genes. When specifically amplifying the "true gene", it can effectively avoid the interference of the false gene CYP21A1P, ensuring that the amplification product is only from the CYP21A2 gene and improving the accuracy of detection.

[0021] This primer set can be used to detect the CYP21A1P-CYP21A2 fusion gene, can accurately identify the presence of the fusion gene, and can determine the heterozygous, homozygous or compound heterozygous state of the fusion gene based on the presence or absence of the products of the first and second amplification products. At the same time, combined with sequencing, the break and rejoining positions can be further determined, improving the detection ability for fusion variations.

[0022] This primer set has strong compatibility and is suitable for the PCR + Sanger sequencing scheme. At the same time, it can be combined with detection methods such as real-time fluorescence PCR or next-generation sequencing, meeting the needs of different laboratories, and having strong flexibility and applicability.

[0023] This amplification primer set has a low cost and a simple detection process, and is suitable for routine gene detection in hospitals and primary-level laboratories. Compared with the expensive third-generation sequencing scheme, it is more clinically feasible, providing an efficient and economical detection strategy for the accurate gene diagnosis of 21-OHD. Brief Description of the Drawings

[0024] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for use in the description of the specific embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0025] Figure 1 It shows the positional relationship of the CYP21A2 gene on the chromosome and the generation of fusion genes; Figure 2 It is a schematic diagram of the principle of specific amplification of the CYP21A2 gene and fusion gene amplification; Figure 3 It is the alignment result of 4 specific internal sequencing primers on UCSC; Figure 4 It is a schematic flow chart of the detection method in the embodiment of the present application; Figure 5 It is a schematic flow chart of the refinement of step S3 in the detection method in the embodiment of the present application; Figure 6 It is an electrophoresis diagram of specific amplification of the CYP21A2 gene in Example 1 of the present application; Figure 7 It is a Sanger sequencing diagram (selecting 2 typical positions at the beginning and end) after specific amplification of the CYP21A2 gene in Example 1 of the present application; Figure 8 It is a diagram of the specific amplification results of CYP21A2 and fusion genes using 2 reaction systems (target gene amplification primer pair and fusion gene amplification primer pair) in Example 2 of the present application; Figure 9 It is a sequencing result diagram of Case 1 in Example 3 of the present application; Figure 10 It is a sequencing result diagram of Case 2 in Example 3 of the present application; Figure 11 It is a sequencing result diagram of Case 3 in Example 3 of the present application; Figure 12This is the alignment result diagram of the E5R sequencing sequence of Fusion Gene Case 1 in Example 4 of this application; Figure 13 This is the sequencing result diagram (partial, after reverse) of CYP21A2-E5R after amplification in Fusion Gene Case 1 of Example 4 of this application; Figure 14 This is the alignment result of the E5R sequencing sequence and its specific position on the gene in Fusion Gene Case 2 of Example 4 of this application; Figure 15 This is the sequencing result diagram (partial, after reverse) of CYP21A2-E7R after amplification in Fusion Gene Case 2 of Example 4 of this application. Detailed implementation manners

[0026] The following will describe the implementation schemes of the present invention in detail in conjunction with examples. However, those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention. For those not specified in the examples, they are carried out according to conventional conditions or conditions recommended by the manufacturer. Those reagents or instruments not specified by the manufacturer can be obtained as conventional products through commercial purchase.

[0027] In the examples of this application, an amplification primer set is provided, including a target gene amplification primer pair and a fusion gene amplification primer pair; the target gene amplification primer pair can specifically amplify the full-length sequence of the CYP21A2 gene; the forward primer in the fusion gene amplification primer pair can bind to the upstream specific sequence of CYP21A1P, and the reverse primer can bind to the downstream specific sequence of the CYP21A2 gene.

[0028] It should be noted that referring to Figure 1 , the CYP21A2 gene is located on the short arm of chromosome 6 (6p21.3), and there is a highly homologous pseudogene CYP21A1P (with a homology of up to 98.4%). The two are arranged in tandem, with a distance of about 30 kb, and are jointly located in the class III region of the human leukocyte antigen (HLA) locus in the major histocompatibility complex (MHC). Together with the RP genes (RP1 and RP2) encoding tryptophan / threonine kinase, the genes encoding complement C4 (C4A and C4B), and the TNX genes (TNXA and TNXB) encoding the extracellular matrix protein - tenascin X, they jointly form a 2-copy repeated RP C4 CYP21 TNX structure, called the RCCX unit.

[0029] In the embodiments of the present application, an amplification primer set is provided to achieve specific amplification of CYP21A2. Among them, for the target gene amplification primer pair, the forward primer (CYP21A2-F) binds to the upstream specific sequence of the CYP21A2 gene. The reverse primer (CYP21A2-R) binds to the downstream specific sequence of the CYP21A2 gene. Through PCR amplification, the full-length sequence (5700bp) of the CYP21A2 gene can be specifically amplified. For the fusion gene amplification primer pair, its forward primer (CYP21A1P-F) binds to the upstream specific sequence of the CYP21A1P gene. The reverse primer (CYP21A2-R) binds to the downstream specific sequence of the CYP21A2 gene. In the presence of the fusion gene, the sequence of the fusion gene (5700bp) can be specifically amplified.

[0030] When designing the primers in this embodiment, special attention is paid to the specific matching of the 3'-end of the primers with the target gene sequence to ensure that the primers can specifically amplify the target gene and avoid non-specific amplification. The specificity of the primers is verified through tools such as BLAST to ensure that the primers only bind to the CYP21A2 gene or the fusion gene.

[0031] In this embodiment, through the design of specific primers, the "true gene" CYP21A2 gene and the "pseudo gene" CYP21A1P can be effectively distinguished, avoiding the interference of the pseudo gene and improving the accuracy of detection. The target gene amplification primer pair can amplify the full-length sequence of the CYP21A2 gene, including all exons, introns, and untranslated regions (UTRs), ensuring the comprehensiveness and integrity of the detection. The fusion gene amplification primer pair can specifically detect the presence of the CYP21A1P-CYP21A2 fusion gene. Combined with Sanger sequencing, it solves the problems that traditional methods are not easy to detect fusion genes and cannot accurately locate the fusion position. By amplifying the full-length gene sequence at one time, the experimental steps are simplified, and the experimental time and cost are reduced.

[0032] Furthermore, the nucleotide sequence of the forward primer of the target gene amplification primer pair is as shown in SEQ ID NO.1; the nucleotide sequence of the forward primer of the fusion gene amplification primer pair is as shown in SEQ ID NO.2; the nucleotide sequences of the reverse primer of the target gene amplification primer pair and the reverse primer of the fusion gene amplification primer pair are both as shown in SEQ ID NO.3.

[0033] In this embodiment, when analyzing CYP21A2, it is required that the amplification can meet the specific amplification of the full-length of the true gene to detect all variations within the range of the true gene. At the same time, it is necessary to avoid the interference of the pseudo gene and make the operation as simple as possible.

[0034] Given that the full length of the CYP21A2 gene is only 3230 bp, which is relatively short, in this example, the strategy of "amplifying the full-length gene sequence at one time" is adopted to simplify the amplification conditions and ensure the fidelity of amplification. This strategy covers all regions of the gene, including the 5' and 3' untranslated regions (UTRs), exons, and introns. The advantage of amplifying the full-length gene at one time is that it can completely cover all sequences of the gene, which is beneficial for detecting various types of gene mutations. Compared with segmental amplification, this method not only ensures the specificity of amplification but also significantly simplifies the operation process.

[0035] Specifically amplifying the CYP21A2 gene and excluding the interference of the pseudogene CYP21A1P is the key to ensuring the accurate detection of CYP21A2 gene mutations. For this purpose, primer design is crucial, especially the 3' end of the primer, which must ensure its highly specific binding to the target gene sequence. To achieve the specific amplification of the full-length gene, a pair of primers need to be designed, which are located in the outer regions upstream and downstream of the gene respectively. However, due to the extremely high homology of the upstream and downstream sequences between the CYP21A2 gene and the CYP21A1P gene, finding the sequence differences between the two and designing specific primers become the key.

[0036] Approximately 300 bp upstream of the coding region of the CYP21A2 gene, there is a 20-bp sequence, in which there are 3-bp base differences between the true and false genes. Based on this, in this example, the forward primer of the target gene amplification primer pair is designed in this region, and it is ensured that the 3' end of the primer contains 2 consecutive different bases to enhance specificity. In addition, using a specific sequence after 2 kb downstream of the CYP21A2 gene, the reverse primer of the target gene amplification primer pair is designed to further ensure the specificity of primer binding. Through this design, the specificity of the full-length amplification product of the CYP21A2 gene can be effectively guaranteed, providing a reliable basis for subsequent mutation detection.

[0037] Table 1. Sequences and related information of the target gene amplification primer pair (reaction system 1)

[0038] As can be seen from Table 1, the forward primer of the target gene amplification primer pair has 3 specific bases of the CYP21A2 gene (the 6th, 18th, and 19th bases from the 5' end), and the full length of the reverse primer is the specific base of the CYP21A2 gene. The specific bases are shown in bold and italic font.

[0039] CYP21A1P (defined as a pseudogene) is arranged in tandem with CYP21A2 (defined as a true gene), and the distance between the two is approximately 30 kb. Due to their high homology, gene fusion is likely to occur, resulting in the fusion of the original two gene copies into one fusion gene, with a deletion of approximately 30 kb of the intervening sequence. This fusion phenomenon can occur at different exon positions of the gene, and at least 9 different fusion forms have been reported in the literature. The breakpoints mainly lie between the 2nd intron and the 8th exon of the CYP21A2 gene. Although there are diverse fusion forms, each form leads to the deletion of the 30 kb intervening sequence, only with different specific starting and ending positions of the deletion.

[0040] Generally speaking, before fusion, the total length from the start of the CYP21A1P gene to the end of the CYP21A2 gene is approximately 33 kb; after fusion, the length of the formed fusion gene is approximately 3.2 kb.

[0041] Normally, a sequence length of 33 kb exceeds the amplification range of ordinary PCR enzymes, but the two ends of the fusion gene are only about 3.2 kb apart, which provides a condition for detecting the fusion gene in this example. Because generally, sequences within 10 kb can be successfully amplified using specific enzymes.

[0042] Based on the change in sequence length before and after fusion and the amplification ability of the enzyme, in the fusion gene amplification primer pair of this example, a forward primer CYP21A1P-F was designed at the position corresponding to CYP21A2-F and paired with the reverse primer CYP21A2-R that specifically amplifies the CYP21A2 gene. In the absence of the fusion gene, the distance between these two primers is 35.7 kb, exceeding the amplification ability of the enzyme, so no amplification product will be generated; while in the presence of the fusion gene, the distance between the primers is shortened, and an amplification product can be generated, with the same length of 5700 bp. The amplification conditions are the same as those for specifically amplifying the CYP21A2 gene, and the primer positions are referenced Figure 2 。

[0043] Table 2. Sequences and related information of the fusion gene amplification primer pair (reaction system 2)

[0044] In the above table, in the case of the fusion of the true and false genes, a 5700 bp amplification product will appear.

[0045] In this embodiment, the design of the target gene amplification primer pair and the fusion gene amplification primer pair in the amplification primer set is not simply based on the local sequence of the gene to be detected, but is based on in-depth research on the unique structures and sequence characteristics of the CYP21A2 gene and its pseudogene CYP21A1P, as well as a profound understanding of PCR amplification and detection techniques. By combining the two primer pairs for detection, unexpected technical effects can be obtained, significantly improving the sensitivity and accuracy of the scope and discrimination of the object to be detected.

[0046] First, the CYP21A2 gene and the pseudogene CYP21A1P have a high degree of homology (up to 98.4%), which makes it difficult for conventional primer design methods to effectively distinguish between the two, easily leading to non-specific amplification and unable to accurately detect the target gene. Therefore, the primer pair design in this application is not simply based on the local gene sequence design, but through careful screening and optimization to ensure that the primers can specifically bind to the specific sequences of the target gene, thereby achieving accurate detection of the CYP21A2 gene and its fusion gene.

[0047] Second, in this application, the combined use of the target gene amplification primer pair and the fusion gene amplification primer pair can achieve specific amplification of the full-length sequence of the CYP21A2 gene and detection of the CYP21A1P-CYP21A2 fusion gene. This combined detection method has the following unexpected technical effects: by specifically amplifying the full-length sequence of the CYP21A2 gene, all regions of the gene can be covered, including exons, introns, and untranslated regions, so as to detect various types of variations, such as point mutations, small insertions and deletions, etc.; and, the design of the fusion gene amplification primer pair utilizes the change in sequence length before and after fusion, enabling a specific product with a length of 5700bp to be amplified in the presence of the fusion gene. This design can not only effectively detect the presence of the fusion gene, but also visually judge the presence and status of the fusion gene through methods such as electrophoresis, significantly improving the sensitivity and accuracy of detection.

[0048] Third, the primer pair design in this application has the following innovative points: (1) Selection of specific binding sites: At 300bp upstream of the coding region of the CYP21A2 gene, there is a 20bp sequence with a 3bp base difference between the true and false genes. Based on this, the forward primer is designed in this region and it is ensured that the 3' end of the primer contains 2 consecutive different bases. At the same time, using the specific sequence after 2kb downstream of the CYP21A2 gene, the reverse primer is designed to further ensure the specificity of primer binding.

[0049] (2)Ingenious Design of Fusion Gene Detection: By pairing the forward primer CYP21A1P-F with the reverse primer CYP21A2-R and utilizing the change in sequence length before and after fusion, specific detection of the fusion gene was achieved. In the absence of the fusion gene, the distance between the primers exceeded the amplification ability of the enzyme, and no amplification product was generated; while in the presence of the fusion gene, the distance between the primers was shortened, enabling the generation of specific amplification products.

[0050] Finally, Application Examples The primer pair design in this application demonstrated significant advantages in practical applications. For example, when detecting point mutations in the CYP21A2 gene, by specifically amplifying the full-length gene sequence and combining internal sequencing primers for Sanger sequencing, various mutations within the gene could be accurately detected. When detecting the CYP21A1P-CYP21A2 fusion gene, by electrophoretically detecting the amplification products, the presence and status of the fusion gene could be visually judged. This combined detection method not only improved the accuracy and sensitivity of detection but also simplified the experimental operation process and reduced the experimental cost.

[0051] In summary, the primer pair design provided in the embodiments of this application is not simply based on local gene sequences but through careful screening and optimization, achieved specific amplification and detection of the CYP21A2 gene and its fusion gene. This combined detection method has unexpected technical effects of expanding the detection range, increasing sensitivity, and improving accuracy, providing strong technical support for the diagnosis and research of CYP21A2 gene-related diseases.

[0052] The embodiments of this application provide a sequencing primer set that can specifically bind to a template sequence with the amplification product of the amplification primer set described in any of the previous embodiments as a template and can cover the full-length sequence of the CYP21A2 gene.

[0053] Based on the target gene amplification primer pair and the fusion gene amplification primer pair in the amplification primer set provided in the embodiments of this application, a 5700bp amplification product was obtained by pairing CYP21A2-F and CYP21A2-R for amplification, which specifically contains the complete exon, intron, and untranslated region sequences of the CYP21A2 gene.

[0054] Therefore, based on this amplification product, this embodiment provides a sequencing primer set whose core function is to specifically bind to the amplification product of the amplification primer set and cover the full-length sequence of the CYP21A2 gene, adopting a strategy of "single amplification product, multi-directional sequencing" to detect all possible mutation types in the full-length region within the gene. The key to this design lies in ensuring that the sequencing primers can comprehensively cover the target gene region, thereby achieving comprehensive detection of gene mutations.

[0055] Compared with the traditional technology of "segmented amplification and sequencing", this strategy has the following advantages: true gene-specific amplification is more guaranteed, the experimental conditions are simpler and more economical, and there is no need to set different amplification conditions.

[0056] The sequencing primer set must be able to specifically bind to the amplification product, which means that the primer sequences need to be highly complementary to specific regions of the CYP21A2 gene to avoid sequencing errors caused by non-specific binding. The design goal of the sequencing primer set is to cover the full length of the CYP21A2 gene, including exons, introns, and untranslated regions (UTRs). This comprehensive coverage design can detect various types of mutations within the gene, such as point mutations, small insertions, and deletions.

[0057] Furthermore, the sequencing primer set includes a sequencing forward primer and / or a sequencing reverse primer; wherein, the nucleotide sequence of the sequencing forward primer is shown as at least one of SEQ ID NO.4 to SEQ ID NO.10; the nucleotide sequence of the sequencing reverse primer is shown as at least one of SEQ ID NO.11 to SEQ ID NO.16.

[0058] In this embodiment, to detect the mutation types within the CYP21A2 gene, fully considering the length limitation of Sanger sequencing, multiple internal sequencing primers are designed. Using the 5700bp full-length amplification product as the sequencing template, different internal sequencing primers are used alone or in any combination to detect mutations within the full length of the gene, including the 5' untranslated region (5' UTR), 3' untranslated region (3' UTR), each exon, and each intron. There are a total of 13 internal sequencing primers, including 7 forward primers (F) and 6 reverse primers (R). Specifically shown in Table 3 and Table 4.

[0059] The sequencing primer set provided in this embodiment includes a sequencing forward primer and a sequencing reverse primer. These primers can be used as individual forward sequencing primers or reverse sequencing primers, or a primer pair composed of upstream and downstream forward sequencing primers and reverse sequencing primers can be selected. This design allows for flexible selection and sequencing according to different situations of the samples to be detected, thereby improving the accuracy and reliability of sequencing.

[0060] For example, when sequencing an unknown sample (i.e., a suspected CAH patient sample without genetic diagnosis), in order to sequence the full length, considering that the Sanger sequencing read length usually does not exceed 1000bp, internal sequencing primers are required for multi-directional sequencing. At this time, all internal sequencing primers of the sequencing primer set in this embodiment can be selected.

[0061] For patient samples that have been confirmed by test results (such as those diagnosed in other institutions or tested by other methods), or parental samples of patients, when it is known that they have the target variation and reconfirmation or prenatal diagnosis is required, internal sequencing primers in the sequencing primer set can be selected according to the position of the mutation, and usually 1 or 1 pair can be selected at this time.

[0062] The amplification and sequencing forward primers have nucleotide sequences shown in at least one of SEQ ID NO.1, SEQ ID NO.4 to SEQ ID NO.10. These primers are used for sequencing from the 5' end to the 3' end of the gene, and the obtained sequence is the sense strand sequence of the gene. When used in combination, the full length of CYP21A2 can be sequenced, and the upstream region of the gene can be covered. The amplification and sequencing reverse primers have nucleotide sequences shown in at least one of SEQ ID NO.3, SEQ ID NO.11 to SEQ ID NO.16. These primers are used for sequencing from the 3' end to the 5' end of the gene, and the obtained sequence is the antisense strand sequence of the gene. When used in combination, the full length of the CYP21A2 gene can also be sequenced, and the downstream region of the gene can be covered, and it can be mutually verified with the sequencing results of the forward primers. This combined design of forward and reverse primers not only ensures the comprehensiveness and integrity of sequence detection, but also improves the accuracy of variant detection through bidirectional sequencing, thereby reducing the occurrence of false positives or false negatives.

[0063] Table 3. Nucleotide sequences of sequencing forward primers

[0064] In the above table, NO. represents the sequence number, that is, SEQ ID NO.; the primer name is the naming of this primer in this embodiment; the position represents the position on the genome (GRCh37 / hg19), and the same applies hereinafter.

[0065] Table 4. Nucleotide sequences of sequencing reverse primers

[0066] The sequencing primer set designed in the examples that can cover the full length of the CYP21A2 gene significantly improves the detection range coverage. This means that not only common point mutations can be detected, but also potential pathogenic variants located in intron regions, as well as the specific breakpoints of gene structure variations (such as fusion genes) can be detected. This comprehensive detection ability is crucial for deeply understanding the genetic basis of CYP21A2 gene-related diseases.

[0067] The combined use of forward sequencing primers and reverse sequencing primers can be flexibly selected according to different samples. This sequencing strategy not only improves the sensitivity of sequencing, enabling the detection of rare variations, but also enhances the accuracy of detection through the mutual verification of results. For example, when detecting fusion genes, the breakpoints of fusion genes can be accurately determined through the sequencing results of reverse primers and non-specific forward primers, thereby providing more accurate information for clinical diagnosis. This application uses an innovative primer design strategy. The primer design in this application is not simply based on local gene sequences, but through careful screening and optimization to ensure that the primers can specifically bind to the specific sequences of target genes. This design strategy not only considers the specificity of primers but also the efficiency of amplification and sequencing. For example, when selecting sequencing primers, special attention is paid to the complementarity of the 3' end of the primers with the template and the distribution of the primers in the entire gene sequence to ensure comprehensive coverage and efficient amplification.

[0068] In summary, the sequencing primer set, through the design of specific binding and comprehensive coverage, significantly improves the detection range, sensitivity, and accuracy of CYP21A2 gene variations. This innovative primer design strategy can not only effectively distinguish the CYP21A2 gene from its highly homologous pseudogene CYP21A1P but also improve the reliability of variation detection through bidirectional sequencing. This design provides strong technical support for the diagnosis and research of CYP21A2 gene-related diseases.

[0069] In some embodiments, among the primers in Table 3 and Table 4, 4 primers are specific to the CYP21A2 gene, including 2 forward primers (CYP21A2-E4F, CYP21A2-E7F) and 2 reverse primers (CYP21A2-E5R, CYP21A2-E9R). When necessary, these specific primers can also be directly used to amplify partial sequences of the CYP21A2 gene.

[0070] Table 5. Specific Primers

[0071] The blast alignment results for the above specific primers are as follows: Reference Figure 3 , the blast results of each specific primer in the sequencing primer set all show that the primer is completely consistent with the CYP21A gene sequence (shown in the figure above each group), while the 3' end of the primer is different from the CYP21A2P pseudogene (shown in the figure below each group).

[0072] The embodiments of this application provide a detection product, including the amplification primer set as described in the foregoing embodiments and the sequencing primer set as described in the foregoing embodiments.

[0073] The above-mentioned detection products may include, but are not limited to, the following products: (1) kits, kits containing amplification primer sets and sequencing primer sets, for gene detection in laboratories. (2) Diagnostic tools: tools for clinical diagnosis, capable of quickly and accurately detecting variations in the CYP21A2 gene, such as diagnostic reagents, gene detection reagent combinations, and multi-method combined detection systems, mainly used for clinical diagnosis, with the characteristics of high specificity and high sensitivity. (3) Research tools: tools for the scientific research field, helping researchers deeply study the variations of the CYP21A2 gene and its relationship with diseases, such as high-throughput sequencing kits, gene editing tools, and bioinformatics analysis software, mainly used for the scientific research field, with the characteristics of high throughput, high sensitivity, and high specificity.

[0074] Reference Figure 4 , in the embodiments of the present application, a detection method is provided, including: Step S1, obtaining a target DNA sample by collection.

[0075] In this step, DNA is extracted from a biological sample as the target sample for subsequent detection. The processing process may include: selecting a suitable biological sample (such as peripheral blood, saliva, tissue sample, etc.), and using a standard DNA extraction method (such as a commercial kit) to extract high-quality DNA, so as to obtain a pure DNA sample with a concentration and purity meeting the requirements of subsequent PCR amplification.

[0076] It should be noted that the target DNA sample may include, but is not limited to, being extracted from the peripheral blood samples of patients, parents, or relatives, or fetal DNA extracted from amniotic fluid, chorionic villi, or umbilical cord blood in prenatal diagnosis. In this step, the quality of the DNA sample should be ensured to improve the accuracy and reliability of the detection. Specifically, a commercial DNA extraction kit can be used, and DNA can be extracted and quantitatively analyzed according to the instructions.

[0077] Step S2, using the target gene amplification primer pair in the amplification primer set as described in any one of the foregoing embodiments to perform the first PCR amplification on the target DNA sample to obtain a first amplification result; and using the fusion gene amplification primer pair in the amplification primer set as described in any one of the foregoing embodiments to perform the second PCR amplification on the target DNA sample to obtain a second amplification result.

[0078] In this step, the target DNA sample is subjected to PCR amplification using the target gene amplification primer pair (CYP21A2-F and CYP21A2-R) to specifically amplify the full-length sequence of the CYP21A2 gene. Additionally, the target DNA sample is subjected to PCR amplification using the fusion gene amplification primer pair (CYP21A1P-F and CYP21A2-R) to detect the presence of the CYP21A1P-CYP21A2 fusion gene. The reaction systems and amplification conditions for the two amplification reactions are the same.

[0079] Step S3: Determine the fusion gene detection result of the target DNA sample based on the first amplification result and the second amplification result.

[0080] Based on the results of the first PCR amplification and the second PCR amplification, determine whether the CYP21A1P-CYP21A2 fusion gene is present in the target DNA sample. Specifically, it can be carried out by electrophoresis detection. The amplified products are subjected to agarose gel electrophoresis to observe the bands.

[0081] Furthermore, the amplification reaction systems for the first PCR amplification and the second PCR amplification are specifically shown in Table 6 as follows: Table 6: Amplification reaction system

[0082] In the table, the forward primer and the reverse primer respectively correspond to the forward and reverse primers in the target gene amplification primer pair or the fusion gene amplification primer pair. The concentrations of the forward primer and the reverse primer of the primer pair used are both 10 uM, and the amounts used are both 1 μL.

[0083] In addition, the amplification conditions for the first PCR amplification and the second PCR amplification can be shown in Table 7 as follows: Table 7: Amplification conditions

[0084] Furthermore, referring to Figure 5 , step S3, determining the fusion gene detection result of the target DNA sample based on the first amplification result and the second amplification result, may include the following three situations (specifically, refer to Table 8): (1) Result 1 (step S31): If there is an amplified product in the first amplification result and no amplified product in the second amplification result, then there is no fusion gene in the target DNA sample; that is, if there is an amplified product in reaction system 1 (CYP21A2-F and CYP21A2-R) and no amplified product in reaction system 2 (CYP21A1P-F and CYP21A2-R): there is no fusion gene.

[0085] In this case, PCR amplification using the target gene amplification primer pair (CYP21A2-F and CYP21A2-R) produced a specific amplification product, indicating that the CYP21A2 gene itself was present and could be amplified normally. At the same time, PCR amplification using the fusion gene amplification primer pair (CYP21A1P-F and CYP21A2-R) did not produce an amplification product, which meant that the CYP21A1P-CYP21A2 fusion gene was not present in this sample. The results showed that only the normal CYP21A2 gene was present in the sample, and the presence of the fusion gene was not detected.

[0086] (2) Result 2 (step S32): If amplification products are present in both the first amplification result and the second amplification result, then a fusion gene is present in the target DNA sample, and the fusion gene is in a heterozygous state; if there is an amplification product in reaction system 1 and an amplification product in reaction system 2: a fusion gene is present and is in a heterozygous state.

[0087] When amplification products are produced in both reaction systems, this indicates that both the normal CYP21A2 gene and the CYP21A1P-CYP21A2 fusion gene are present in the sample. In this case, the fusion gene coexists with the normal gene, that is, it presents a heterozygous state. The results showed that both the normal CYP21A2 gene and the CYP21A1P-CYP21A2 fusion gene were present in the sample. By this method, the presence of the fusion gene can be accurately judged, and its heterozygous state can be determined, which is of great significance for genetic counseling and clinical diagnosis.

[0088] (3) Result 3 (step S33): If no amplification product is present in the first amplification result and an amplification product is present in the second amplification result, then a fusion gene is present in the target DNA sample, and the fusion gene is in a homozygous state or a compound heterozygous state. That is, if there is no amplification product in reaction system 1 and an amplification product in reaction system 2: a fusion gene is present and is in a homozygous or compound heterozygous state.

[0089] If there is no amplification product in reaction system 1 but there is an amplification product in reaction system 2, this indicates that there is no normal CYP21A2 gene in the sample, but there is a CYP21A1P-CYP21A2 fusion gene. In this case, the fusion gene may be in a homozygous state (i.e., both alleles are the same type of fusion gene), or it may be in a compound heterozygous state (i.e., one allele is one type of fusion gene, and the other allele is another type of fusion gene or there may be a larger fragment deletion and other variations affecting amplification). The results show that there is no normal CYP21A2 gene in the sample, but there is a fusion gene, and the fusion gene may be in a homozygous or compound heterozygous state. This method can detect the presence of the fusion gene and can indicate the possible genetic status, which is of great value for in-depth understanding of the genetic background of the disease.

[0090] Table 8. Corresponding relationship between amplification results and determination of fusion genes

[0091] In the above table, "√" represents the presence of an amplification product, and "×" represents the absence of an amplification product.

[0092] In the above steps, according to the results of the first PCR amplification and the second PCR amplification, it is determined whether there is a CYP21A1P-CYP21A2 fusion gene in the target DNA sample, and it is clarified whether there is a CYP21A1P-CYP21A2 fusion gene and its status in the target DNA sample. This method has the advantages of high specificity, high sensitivity and simple operation, and is suitable for clinical diagnosis and scientific research applications. This method realizes the accurate detection of the CYP21A2 gene and its fusion gene through the design of specific primers and PCR amplification, providing strong technical support for clinical diagnosis and genetic research.

[0093] Furthermore, after step S3, after determining the fusion detection result of the target DNA sample according to the first amplification result and the second amplification result, it further includes: Step S4, performing Sanger sequencing on the amplification products in the first amplification result and / or the second amplification result using the sequencing primer set described in one of the foregoing embodiments to obtain a sequencing result.

[0094] It should be noted that after performing two amplifications (the first PCR amplification and the second PCR amplification) on the target DNA sample, in the obtained first amplification result and second amplification result, there may be an amplification product in one of the amplification results (such as results 1 and 3 in Table 8), or there may be amplification products in both amplification results (such as result 2 in Table 8).

[0095] How to perform further sequencing on the obtained amplification products can be differentiated according to the specific clinical situation at this time: (1) If the person to be tested corresponding to the target DNA sample is suspected of being a CAH patient, then for the first PCR amplification and the second PCR amplification, regardless of whether 1 or 2 amplification products are obtained, sequencing is required to identify the 2 possible mutations. (2) If the person to be tested corresponding to the target DNA sample is an asymptomatic parent or asymptomatic family member of the proband, then only one of the products needs to be sequenced. For example, for suspected CAH patient A, a target DNA sample is collected.

[0096] First, in the first step, after performing the first PCR amplification (reaction system 1) using the target gene amplification primer pair and the second PCR amplification (reaction system 2) using the fusion gene amplification primer pair respectively, amplification products are obtained after amplification of the 2 reaction systems.

[0097] a. Perform reverse sequencing on the amplification product obtained by the second PCR amplification to confirm a deletion of exons 1-3 of the CYP21A2 gene caused by the fusion gene; b. Perform multi-directional sequencing on the amplification product obtained by the first PCR amplification to confirm a heterozygous mutation of c.1069C>T in the CYP21A2 gene. The conclusion is that the patient has a compound heterozygous mutation of CYP21A2 exon1-3del and c.1069C>T mutation.

[0098] In the second step, the parents can be tested. Similarly, perform the first PCR amplification (reaction system 1) and the second PCR amplification (reaction system 2) respectively, that is, amplification of the 2 reaction systems. Then, in the amplification results, it is very likely that one person (such as the father) among the parents has an amplification product for the fusion gene amplification, which proves that the fusion gene comes from this parent (father). At this time, the source can actually be judged, and it is possible to choose to sequence only the amplification product (fusion gene product) of the second PCR amplification, or not to perform sequencing; while the other party (such as the mother) only has a specific amplification product of the CYP21A2 gene. According to the position of the 1069 mutation in the patient, appropriate primers can be selected for internal sequencing to detect whether the 1069 mutation in the patient is indeed derived from this parent (mother).

[0099] If it is clear that the patient does not have the fusion gene, that is, there is no amplification product in the second amplification result after the second PCR amplification using the fusion gene amplification primer pair, while there is an amplification product in the first amplification result after amplification using the target gene amplification primer pair, then the amplification product in the first amplification result after amplification can be subjected to multi-directional sequencing, and it may be clear that the patient has 2 point mutations, such as the compound heterozygous mutations of c.293-13C>G and c.1069C>T. Then, specific amplification of the CYP21A2 gene (the first PCR amplification) is performed on the parents, and appropriate primers are selected for sequencing of the amplification product, so as to clarify the origin of these two mutations, which usually come from the father and the mother respectively.

[0100] After completing the PCR amplification, Sanger sequencing is performed on the first amplification result (the full-length sequence of the CYP21A2 gene) and / or the second amplification result (the amplification product of the fusion gene). Specifically, it may include: (1)Template preparation: Purify the PCR amplification product to remove the unbound primers and dNTPs to obtain pure template DNA.

[0101] (2)Sequencing reaction: Perform Sanger sequencing reaction using the sequencing primer set. The sequencing primer set includes multiple pairs of forward and reverse primers that can cover the full-length sequence of the CYP21A2 gene.

[0102] (3)Sequencing conditions: Perform the reaction according to the standard conditions of Sanger sequencing, which usually include denaturation, annealing, and extension steps.

[0103] Through the above steps, the sequencing results of the CYP21A2 gene and its fusion gene can be obtained, and the specific DNA sequence can be determined.

[0104] Sanger sequencing has high accuracy and can accurately detect variation types such as point mutations, small insertions, and deletions inside the gene. Specifically, a commercial Sanger sequencing kit can be used, and the sequencing product is subjected to electrophoresis analysis according to the instructions, and the DNA sequence is read through the fluorescence signal.

[0105] That is, after amplification of the "true gene" and the fusion gene, the PCR product is subjected to gel cutting and purification and used as a template, and then Sanger sequencing is performed using the amplification primer or the internal sequencing primer. The instrument used can be the ABI 3500 sequencer, and the sequencing reagents and methods are the same as those of other Sanger sequencing, so they will not be elaborated here.

[0106] Step S5, analyze the sequencing results to obtain an analysis result.

[0107] Among them, the analysis result includes at least one of the following results: A. Determine whether there is a fusion gene and confirm the specific breakage and rejoining positions of the fusion gene (i.e., the fusion type); C. Confirm the specific mutation types and positions within the CYP21A2 gene.

[0108] In this step, analyze the Sanger sequencing results to determine whether there is a fusion gene, confirm the fusion gene type, and the specific mutation types and positions within the CYP21A2 gene. Specifically, for the obtained sequencing results, use software or manual interpretation and align them using the UCSC website (http: / / genome.ucsc.edu / ) to analyze the mutation sites and the breakage and rejoining positions of the fusion gene.

[0109] Based on the above detection method, further confirm the specific mutation types and positions in the amplification products, as well as the existence and fusion positions of the fusion gene through Sanger sequencing. This method has high accuracy and can accurately detect the mutation types and positions within the gene, as well as the existence and fusion positions of the fusion gene, providing strong technical support for clinical diagnosis and genetic research.

[0110] The principle is that a 5700bp fusion gene can be amplified using the upstream primer of the "pseudogene" and the downstream primer of the "true gene". In the embodiments of the present application, the entire gene can be sequenced using the internal sequencing primers of the CYP21A2 gene (sequencing primer set). With the above conditions, the amplification products of the fusion gene can be fully utilized, and multiple reverse primers of CYP21A2 and the common forward primer of the true and false genes, such as CYP21A2-E1F, CYP21A2-E5R, etc., can be used as internal sequencing primers to detect the breakage and rejoining sites of the fusion of the true and false genes.

[0111] The embodiments of the present application provide the use of an amplification primer set as described in the foregoing embodiments and a sequencing primer set as described in the foregoing embodiments in the preparation of a detection product for detecting full-length mutations and / or fusion genes of the CYP21A2 gene.

[0112] For example, it may include but is not limited to: (1) detecting 21-hydroxylase deficiency; (2) determining the breakage and rejoining positions of the CYP21A1P-CYP21A2 fusion gene; (3) detecting CYP21A2 gene mutations and clinical diagnosis in suspected CAH patients, which can detect point mutations within the full length of CYP21A2 and exon deletion mutations of the CYP21A2 gene caused by CYP21A1-CYP21A2 fusion; (4) prenatal genetic diagnosis when the carrier couple of CYP21A2 gene mutations (including point mutations and fusion genes) have a second child.

[0113] The present invention will be further illustrated by specific implementation cases below. However, it should be understood that these implementation cases are only for more detailed illustration and should not be construed as limiting the present invention in any way.

[0114] Implementation Case 1: Specific Amplification of CYP21A2 In this implementation case, for the family with the number 150, the patient is a point mutation patient verified by other methods. For the patient and his / her parents in the family, the CYP21A2 gene was specifically amplified using the CYP21A2-F and CYP21A2-R primers (target gene amplification primer pair). The electrophoresis results are referred to Figure 6 , where the maker is the Shengong Biology B500352 reagent. Figure 6 It shows that the amplified fragment conforms to the 5700bp product and the product specificity is good.

[0115] Taking the purified 5700bp product as a template, PCR product head-to-tail sequencing was performed using the upstream primer CYP21A2-F and the inner primer CYP21A2-10R. By aligning in the UCSC database, it can be seen that the products are all CYP21A2 gene-specific sequences, which are different from the pseudogene CYP21A1P, indicating that the CYP21A2 gene can be specifically amplified (refer to Figure 7 ).

[0116] Implementation Case 2: Amplification of CYP21A1P-CYP21A2 Fusion Gene Experimental Method: In this implementation case, for 4 cases (numbered C1 - C4) that have been clearly identified as carrying the fusion gene by the MLPA method (the fusion genes are all in heterozygous state), reaction system 1 (i.e., the first amplification) (target gene amplification primer pair, CYP21A2-F and CYP21A2-R paired) and reaction system 2 (i.e., the second amplification) (fusion gene amplification primer pair, CYP21A1P-F and CYP21A2-R) were used for amplification to detect the presence and status of the fusion gene.

[0117] At the same time, 2 normal samples (N1, N2) detected by MLPA were used as normal controls. Amplification and electrophoresis were carried out according to the aforementioned method.

[0118] Experimental Results: Refer to Figure 8 , the electrophoresis results show that for the normal control samples N1 and N2, there are amplification products in reaction system 1 (the first amplification result) but no amplification products in reaction system 2 (the second amplification result), indicating that they do not carry the fusion gene; for the C1 - C4 samples, there are products in both reaction system 1 (the first amplification result) and reaction system 2 (the second amplification result), indicating the presence of the fusion gene and being carried in a heterozygous state.

[0119] Implementation case 3: Detection of different mutations of CYP21A2 gene Experimental methods: In this implementation case, three samples of mutation cases at different locations and of different types were selected, and the full-length CYP21A2 gene was amplified according to the aforementioned method, and the presence of mutations was detected using full-length amplification primers or internal sequencing primers.

[0120] (1) Case 1: splicing mutation, missense mutation The boy had external genital malformation and was clinically diagnosed with CAH. Sanger sequencing + MLPA testing confirmed that he had compound heterozygous mutations of c.293-13C>G and c.1069C>T in the CYP21A2 gene, which were located in intron 2 and exon 8, respectively.

[0121] The CYP21A2 whole gene amplification method in part 1 was used to amplify the full-length sequence of the child's true gene, and then Sanger forward and reverse sequencing were performed using internal sequencing primers E2F and E3R to detect the c.293-13C>G splicing mutation; Sanger sequencing was performed using internal sequencing primers CYP21A2-E7F and CYP21A2-E9R to detect the c.1069C>T missense mutation.

[0122] refer to Figure 9 As shown, the upper figure shows the sequencing results of CYP21A2-E2F as the internal sequencing primer; the lower figure shows the sequencing results of CYP21A2-E9R as the internal sequencing primer (after reverse complementation). It can be seen that the splicing mutation c.293-13C>G (upper figure) and c.1069C>T (lower figure) are compound heterozygous mutations. The arrow indicates the location of the mutation. The results show that the above two mutations can be clearly detected, and the same detection method has been used to confirm that the mutations come from the parents (the results of the parents are not shown).

[0123] (2) Case 2: frameshift mutation and missense mutation caused by base insertion The patient was a 3-year-old boy who was clinically diagnosed with CAH. PCR-NGS+MLPA method diagnosed him with compound heterozygous mutations of c.707A>C (p.H236P) and c.923dupT (p.L308Ffs*6) in the CYP21A2 gene, which were located in exons 6 and 7, respectively.

[0124] The above method was also used to amplify the CYP21A2 gene, and CYP21A2-E6F and E7R were used as internal sequencing primers to detect the c.707A>C mutation, and CYP21A2-E7F and CYP21A2-E9R were used as internal sequencing primers to detect the c.923dupT mutation.

[0125] refer to Figure 10As shown in the figure above, the sequencing result using CYP21A2-E6R as the internal sequencing primer (after reverse complementation); the figure below shows the sequencing result of the CYP21A2-E7F internal sequencing primer. It can be seen that there are compound heterozygous mutations of c.707A>C (p.H236P) and c.923dupT (p.L308Ffs*6). The arrows indicate the mutation positions. The sequencing results show that the method used in this example can detect the above two mutations.

[0126] (3)Case 3: 5’UTR variation, missense mutation A female patient was clinically diagnosed with CAH. Two variations (c.-126C>T, -113G>A) in the 5’UTR region of the CYP21A2 gene were detected by Sanger sequencing + MLPA. At the same time, she also carried a nonsense mutation c.955C>T (p.Gln319*) located in exon8, which can be rated as a pathogenic variation according to ACMG.

[0127] The CYP21A2 gene was amplified using the above method. CYP21A2-E1F and E1R were used as internal sequencing primers to detect 5’UTR variations. E7F and E9R were used as internal sequencing primers to detect the c.955C>T variation.

[0128] Reference Figure 11 to the sequencing result figure, where the upper figure shows the sequencing result using CYP21A2-E1F as the internal sequencing primer; the lower figure shows the sequencing result using CYP21A2-E7F as the internal sequencing primer. The left arrow in the upper figure is the c.-126C>T variation, and the right arrow is the -113G>A variation; the arrow in the lower figure is the c.955C>T heterozygous mutation.

[0129] Example 4: Detection of the specific fusion position of the CYP21A1P-CYP21A2 fusion gene The fusion of the CYP21A1P “pseudogene” and the CYP21A2 “true gene” will lead to the deletion of the upstream exon of the functional CYP21A2 gene. As previously mentioned, multiple types of fusion genes have been found. However, since the clinical methods for detecting fusion genes are mainly based on MLPA, and MLPA, as a method based on probe amplification and quantification for detection, cannot clarify the specific breakage and rejoining (i.e., fusion) position. Therefore, the result usually reports “deletion of exons 1 - a certain exon of the CYP21A2 gene”, such as “deletion of exons 1 - 3 of the CYP21A2 gene”.

[0130] In this example, using the method of the present invention, by combining fusion gene amplification + CYP21A2 gene internal sequencing primer Sanger sequencing, the breakage and rejoining (i.e., fusion) position of the fusion gene can be clarified.

[0131] (1)Case 1, a case of deletion of exons 1-3 of the CYP21A2 gene Clinical MLPA detection suggested "heterozygous deletion of exons 1-3 of the CYP21A2 gene". Amplification was carried out using reaction system 2 (fusion gene amplification primer pair) (primers: CYP21A1P-F, CYP 21A2-R). After amplification, gel cutting and purification were performed, and then sequencing was carried out with the sequencing primer sets CYP21A2-E5R and CYP21A2-E7R.

[0132] Reference Figure 12 , after reverse complementation (reverse) of the sequencing results, the sequences were respectively aligned and analyzed with the CYP21A1P and CYP21A2 gene sequences. Because the homology between the true and false genes is too high, there are large segments of common sequences. Figure 12 In [reference], the sequences within the red square are the specific sequences of the pseudogene, the sequences within the blue frame are the specific sequences of the true gene, and the middle part is the common sequence. There are 2 possible breakage and rejoining positions. Position 1 (the upper arrow) is ch6:31974184-ch6:32006921; Position 2 (the lower arrow) is ch6:31974466-32007203.

[0133] According to Figure 12 , the possible specific breakage and rejoining position 1 is chr6:31,974,184--chr6:32,006,921, that is, the deletion range is chr6:31,974,185-32,006,920, totaling 32738bp; the possible specific breakage point 2 is ch6:31974466-32007203, and the deleted bases are the middle ch6:31974467-32007202, and the deletion length still totals 32738bp.

[0134] Analyzing the sequence of the breakage and rejoining region in this implementation case, it can be found that the fusion region is indeed near CYP21A2 exon3. Reference Figure 13 , before 574bp (pink and blue part), it is the specific sequence of the CYP21A1P pseudogene; in the middle 575-856 (pink part), it is the common sequence of the true and false genes; after 857 (light green part), it is the specific sequence of the CYP21A2 true gene; the yellow dotted line is the corresponding region of CYP21A2 gene intron2; the brown dotted line is the corresponding region of CYP21A2 gene exon3, and it can be seen that the front 50bp sequence has been completely replaced by the pseudogene sequence.

[0135] The exon3 of the CYP21A2 gene is 155bp in total (c.293 - c.447). Whether the breakpoint is at position 1 at the front end or position 2 at the back end, the effect is that the first 50bp (c.293 - c.342) of the normal authentic gene exon3 is replaced by the CYP21A1P gene sequence, forming an incomplete authentic gene exon3. The deletion of the fusion gene, including the first 50bp of exon3 and the previous sequence, results in the incompleteness of the authentic gene and the loss of its function.

[0136] Therefore, by applying the detection method in this embodiment, it can be clarified that the deletion of exons 1 - 3 detected by MLPA is actually a deletion of a total of 32738bp of the partial sequence of exon3 of the CYP21A2 gene and the previous sequence caused by the fusion gene. This is different from the generally understood deletion of exons 1 - 3, which should include the deletion of all sequences of exons 1 - 3.

[0137] (2) Case 2, a case of deletion of the 5’UTR - exon6 of the CYP21A2 gene For the DNA sample of a case clinically detected by MLPA as "heterozygous deletion of the 5’UTR - exon6 of the CYP21A2 gene", amplification was carried out using reaction system 2 (fusion gene amplification primer pair) (primers: CYP21A1P - F, CYP21A2 - R). After amplification, gel cutting and purification were performed, and then sequencing was carried out with the forward sequencing primer CYP21A2 - E7R and the reverse sequencing primer CYP21A2 - E9R in the sequencing primer set as a primer pair.

[0138] After reverse complement (reverse) of the sequencing results, the sequences were respectively compared and analyzed with the CYP21A1P and CYP21A2 gene sequences, referring to Figure 14 , where the sequence in the pink area is the corresponding position of exon6 of the CYP21A2 gene, the light green area is the corresponding position of exon7 of the CYP21A2 gene, and the middle is the corresponding position of intron6. It can be seen that all sequences of exon6 and intron6 of the CYP21A2 gene are replaced by the pseudogene and extend to the 9th base of exon7.

[0139] Through Figure 14 it can be found that there may still be 2 possible specific breakpoint positions. The possible position 1 is ch6:31975053 - 32007790, that is, the deletion range is ch6:31975054 - 32007789, totaling 32736bp; the possible position 2 is ch6:31975150 - ch6:32007887, and the deleted bases are ch6:31975151 - 32007886 in the middle, and the deletion length still totals 32736bp.

[0140] Reference Figure 15 By analyzing the gene fusion position, it can be found that near intron6 of the CYP21A2 gene, which results in the entire exon6 and intron6 sequences being replaced by pseudogenes and extending to the 9th base of exon7, generating a c.749C>T substitution, which is a synonymous mutation and a benign variation.

[0141] In summary, from the experimental results in the above implementation cases, the detection method provided by the present invention, based on PCR and Sanger sequencing technologies, can simply, quickly, and accurately detect various mutations of the CYP21A2 gene, as well as the CYP21A1P-CYP21A2 fusion gene and its fusion types.

[0142] This method is applicable to all clinical cases suspected of CYP21A2 gene mutations. By combining PCR amplification and Sanger sequencing with different internal primers, it is possible to determine at one time whether there are fusion genes, fusion position types, and all mutation types within the entire CYP21A2 gene range. Compared with the currently commonly used methods of detecting CYP21A2 gene mutations, such as MLPA+Sanger or third-generation sequencing, it has the advantages of low cost, simple protocol, rapid process, comprehensive detection of mutations, and accurate detection sites, and has great value for clinical application and promotion.

[0143] Finally, it should be noted that the above implementation cases are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing implementation cases, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing implementation cases, or perform equivalent replacements for some or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. An amplification primer set, characterized in that: It includes a target gene amplification primer pair and a fusion gene amplification primer pair; The target gene amplification primer pair can specifically amplify the full-length sequence of the CYP21A2 gene; The forward primer in the fusion gene amplification primer pair can bind to the upstream specific sequence of CYP21A1P, and the reverse primer can bind to the downstream specific sequence of CYP21A2 gene.

2. The amplification primer set according to claim 1, characterized in that: The nucleotide sequence of the forward primer of the target gene amplification primer pair is shown in SEQ ID NO.1; The nucleotide sequence of the forward primer of the fusion gene amplification primer pair is shown in SEQ ID NO.2; The nucleotide sequences of the reverse primer of the target gene amplification primer pair and the reverse primer of the fusion gene amplification primer pair are both shown as SEQ ID NO.

3.

3. A sequencing primer set, characterized in that: The sequencing primer set can specifically bind to a template sequence using the amplification product of the amplification primer set according to any one of claims 1 to 2 as a template, and can cover the full-length sequence of the CYP21A2 gene.

4. The sequencing primer set according to claim 3, characterized in that: The sequencing primer set includes a sequencing forward primer and / or a sequencing reverse primer; The nucleotide sequence of the sequencing forward primer is shown in at least one of SEQ ID NO.4 to SEQ ID NO.10; The nucleotide sequence of the sequencing reverse primer is shown as at least one of SEQ ID NO.11 to SEQ ID NO.

16.

5. A detection product, characterized in that: It comprises the amplification primer set as described in any one of claims 1-2, and the sequencing primer set as described in any one of claims 3-4.

6. A detection method, characterized in that: include: Collecting and obtaining target DNA samples; Using the target gene amplification primer pair in the amplification primer set according to any one of claims 1 to 2, performing a first PCR amplification on the target DNA sample to obtain a first amplification result; and using the fusion gene amplification primer pair in the amplification primer set according to any one of claims 1 to 2, performing a second PCR amplification on the target DNA sample to obtain a second amplification result; The fusion gene detection result of the target DNA sample is determined according to the first amplification result and the second amplification result.

7. The detection method according to claim 6, characterized in that: The amplification reaction system of the first PCR amplification and the second PCR amplification includes: 2×GC Buffer I, 15 μL; dNTP Mixture, where each dNTP concentration is 2.5 mM, 4.8 μL; The target gene amplification primer pair or the fusion gene amplification primer pair uses a forward primer and a reverse primer with a concentration of 10uM and a dosage of 1μL; amplification enzyme, 0.3 μL; Target DNA sample, 30 ng; And control the total volume to 30μL.

8. The detection method according to claim 6, characterized in that: Determining the fusion gene detection result of the target DNA sample according to the first amplification result and the second amplification result includes: If the amplification product exists in the first amplification result and the amplification product does not exist in the second amplification result, then the fusion gene does not exist in the target DNA sample; If amplification products exist in both the first amplification result and the second amplification result, then the target DNA sample contains a fusion gene, and the fusion gene is in a heterozygous state; If no amplification product exists in the first amplification result, and an amplification product exists in the second amplification result, then the target DNA sample contains a fusion gene, and the fusion gene is in a homozygous state or a compound heterozygous state.

9. The detection method according to claim 6, characterized in that: After determining the fusion gene detection result of the target DNA sample according to the first amplification result and the second amplification result, the method further includes: Performing Sanger sequencing on the amplification products in the first amplification result and / or the second amplification result using the sequencing primer set according to any one of claims 3 to 4 to obtain a sequencing result; Analyze the sequencing results to obtain analysis results; wherein the analysis results include at least one of the following results: A. Determine whether there is a fusion gene and confirm the specific breakage and reconnection position of the fusion gene; C. Confirm the specific mutation type and location within the CYP21A2 gene.

10. Use of the amplification primer set according to any one of claims 1 to 2 and the sequencing primer set according to any one of claims 3 to 4 in preparing a detection product for detecting full-length mutations and / or fusion genes of the CYP21A2 gene.

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