IGH rearrangement detection reverse transcription primer group, quantitative screening detection kit and application
By designing a reverse transcription primer set and specific PCR primer probes for IGH rearrangement detection, and combining them with digital PCR technology, the sensitivity and cost issues of IGH rearrangement detection were solved, and efficient quantitative detection of IGH-DUX4, IGH-EPOR, and IGH-CRLF2 was achieved.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- WUHAN XINO MEDICAL LABORATORY CO LTD
- Filing Date
- 2026-02-05
- Publication Date
- 2026-05-12
AI Technical Summary
Existing technologies are difficult to detect IGH rearrangements efficiently and at low cost, especially common fusion genes such as IGH-DUX4, IGH-EPOR, and IGH-CRLF2. Traditional methods such as karyotype analysis and FISH probes have low sensitivity, are difficult to design, and are costly.
A reverse transcription primer set for IGH rearrangement detection was designed, and specific PCR primers and probes were combined to perform quantitative screening using digital PCR technology. Specific reverse transcription primers and UNG enzyme digestion were used to avoid non-specific amplification, thus achieving efficient detection of IGH rearrangements.
It achieves highly sensitive, low-cost, and rapid quantitative detection of IGH rearrangements, accurately identifying three common fusion types and their proportions, reducing detection difficulty and equipment requirements.
Smart Images
Figure CN122012710A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of detection kits, specifically to IGH rearrangement detection reverse transcription primer sets, quantitative screening detection kits, and their applications. Background Technology
[0002] B-cell acute lymphoblastic leukemia (B-ALL) is characterized by a variety of genomic alterations, among which gene fusions involving immunoglobulin heavy chain (IGH) sites are one of the most common. However, due to the large size and high polymorphism of the IGH region, IGH-related fusion genes are difficult to detect using conventional detection methods. More than a dozen IGH fusion partners have been reported, including DUX4, EPOR, CRLF2, CEBPD, SPIDR, CEBPB, MYC, BCL2, BCL3, BCL6, CCND1, and MUM1, among which DUX4, EPOR, and CRLF2 are relatively common.
[0003] DUX4 is a dual-homeobox embryonic transcription factor (TF) that is normally expressed in germline cells but silenced in somatic cells. However, under pathological conditions, such as malnutrition, leukemia, and other types of cancer, DUX4 reexpression in somatic cells has been found to be a pathogenic factor. DUX4 is a gene located in the D4Z4 megasatellite repeat sequence on chromosome 4, and has a homologous polymorphic repeat sequence on chromosome 10. DUX4 rearrangements are found in 4%–7% of B-ALL cases, primarily affecting children and adolescents. Compared to other types of B-ALL, these patients often have lower white blood cell counts and are prone to lineage conversion during treatment, especially monocytic conversion. Current research has identified IGH as the most common DUX4 partner gene. The DUX4 gene can undergo insertional translocation with IGH in different forms, rather than balanced translocation, ultimately resulting in DUX4 overexpression.
[0004] The EPOR::IGH fusion gene is formed by the t(14;19)(q32;p13) translocation. The EPOR gene is the erythropoietin receptor gene. Located at 19p13 in the human genome, the EPOR gene encodes a membrane-bound receptor protein called the epidermal growth factor receptor (EPOR). EPOR is a cell membrane receptor protein that binds to erythropoietin (EPO) and mediates EPO signaling. EPO is a hormone secreted by the kidneys that plays a crucial role in erythrocyte production and maturation. The IGH gene refers to the immunoglobulin heavy chain gene. Located at 14q32, the IGH gene is responsible for the breakage and rearrangement of immunoglobulin genes, including the immunoglobulin heavy chain gene (IGH), the light chain (κ) chain gene (IGK), and the light chain (λ) chain gene (IGL). These genes are frequently broken and rearranged in B-cell tumors. These rearrangements result in the IG enhancer merging (fusion) with oncogenes such as MYC and BCL2, leading to their overexpression and activation. According to literature reports, the EPOR::IGH gene is common and highly aggressive in young B-ALL patients. Traditional diagnostic methods such as clinical examination, morphology, immunology, and cytogenetic studies often fail to detect the presence of EPOR::IGH.
[0005] Philadelphia chromosome-like (Ph-like) ALL is a newly established subtype of acute lymphoblastic leukemia. Although Ph-like ALL does not express the BCR-ABL fusion gene, it behaves similarly to true BCR / ABL1 positive cases. This subtype carries various molecular variants, the most common being CRLF2 rearrangements. CRLF2 gene rearrangements are found in 50% of Ph-like ALL cases, with CRLF2::IGH fusions and P2RY8::CRLF2 being the most common. CRLF2::IGH fusions are more common in adults and are consistently associated with Ph-like characteristics.
[0006] Currently, common methods for detecting gene rearrangements include G-banded chromosome karyotype analysis, FISH probes, RNA-Seq, and whole-genome sequencing. However, for the detection of IGH rearrangements, methods based on next-generation sequencing, such as RNA-Seq and whole-genome sequencing, are typically expensive and have low sensitivity; conventional detection methods like G-banded chromosome karyotype analysis and FISH probes cannot detect it well, for the following reasons: 1. For karyotype analysis, because the repetitive sequence inserted into the IGH site is small, or in the case of reverse translocation, IGH and fusion partners such as DUX4 are located in the subtelomere region, IGH rearrangement is cryptic in cytogenetics and is difficult to detect; 2. The main difficulty in FISH detection lies in the significant changes in the sequence and position of the IGH rearrangement breakpoints, which makes the design of the separating fluorescent probes difficult; in addition, fusion partners such as DUX4 have significant homology interference, causing cross-reactions. Summary of the Invention
[0007] This invention proposes a reverse transcription primer set for IGH rearrangement detection and a quantitative screening kit for quantitative screening of common fusion types of the IGH gene, so as to achieve auxiliary diagnosis in clinical practice.
[0008] The technical solution of this invention is implemented as follows: A first aspect of the present invention is to provide a reverse transcription primer set for detecting IGH rearrangements, comprising a reverse transcription primer set for detecting IGH rearrangements, wherein the rearrangement type includes at least one of IGH-DUX4, IGH-EPOR, and IGH-CRLF2; wherein: The reverse transcription primer set of the IGH-DUX4 includes the nucleotide sequence shown in SEQ ID NO: 1-34; And / or, the reverse transcription primer set of the IGH-EPOR includes nucleotide sequences as shown in SEQ ID NO: 35-44; And / or, the reverse transcription primer set of the IGH-CRLF2 includes nucleotide sequences as shown in SEQ ID NO: 45-55.
[0009] A second aspect of the invention is to provide applications of the reverse transcription primer set described in the first aspect, the applications including: a. Preparation of IGH rearrangement type detection products; b. Prepare products for quantitative detection of IGH fusion ratio.
[0010] In the above applications, the IGH rearrangement type or IGH fusion includes any one of IGH-DUX4, IGH-EPOR and IGH-CRLF2, including the detection of any reported rearrangement break site and fusion type.
[0011] A third aspect of the present invention is to provide an IGH quantitative screening kit, comprising the reverse transcription primer set described in the first aspect, and a PCR primer and probe set for detecting at least one rearrangement of IGH-DUX4, IGH-EPOR, and IGH-CRLF2.
[0012] Furthermore, in the above-mentioned reagent kit: The PCR primer and probe set for detecting IGH-DUX4 rearrangement includes probes with nucleotide sequences as described in SEQ ID NO: 57, and primers as shown in SEQ ID NO: 58-59; And / or, the PCR primer and probe set for detecting IGH-EPOR rearrangement includes probes with nucleotide sequences as described in SEQ ID NO: 60, and primers as shown in SEQ ID NO: 61-62; And / or, the PCR primer and probe set for detecting IGH-EPORIGH-CRLF2 rearrangement includes probes with nucleotide sequences as described in SEQ ID NO: 63, and primers as shown in SEQ ID NO: 64-65.
[0013] Furthermore, the kit also includes internal reference gene reverse transcription primers, and primer sets and probes for PCR amplification.
[0014] Preferably, in the above kit: The nucleotide sequence of the reverse transcription primer for the internal reference gene is shown in SEQ ID NO: 56; And / or, the primer set and probe for PCR amplification of the internal reference gene include a probe with a nucleotide sequence as shown in SEQ ID NO: 66, and a primer set with a nucleotide sequence as shown in SEQ ID NO: 67-68.
[0015] Preferably, any of the probes in the kit are modified with different fluorescent labels, and different modifications are used to collect signals of different colors to achieve efficient detection.
[0016] Further, the concentration of any of the reverse transcription primers in the reverse transcription primer set is 0.1-1 μM, for example 0.1 μM, 0.2 μM, 0.3 μM, 0.4 μM, 0.5 μM, 0.6 μM, 0.7 μM, 0.8 μM, 0.9 μM, 1 μM; preferably 0.4 μM or 0.5 μM.
[0017] Furthermore, the kit also includes at least one of the following: reverse transcription reaction reagent, PCR reaction reagent, RNA digesting enzyme, and UNG enzyme.
[0018] A fourth aspect of the present invention is to provide a method for quantitative screening of IGH rearrangements for non-diagnostic purposes, comprising using the kit described in the third aspect and performing the following steps: 1) Extract RNA from the sample; 2) RNA is reverse transcribed using reverse transcription primers to generate cDNA; 3) Using cDNA as a template, add PCR primers, probes, and UNG enzyme, and perform PCR amplification; 4) Determine the detection type and fusion ratio based on the probe detection signal.
[0019] Furthermore, the above method preferably uses digital PCR technology, by pre-preparing PCR reaction droplets, and then obtaining detection signals through amplification and fluorescence capture to obtain detection results, including fusion gene type and / or fusion ratio.
[0020] Specifically, the formula for calculating the fusion ratio is as follows: Fusion ratio = IGH fusion transcript copy number / internal reference ABL1 transcript copy number 100%.
[0021] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention designs specific reverse transcription primers targeting common IGH breakpoints. All reverse transcription primers underwent specificity evaluation and verification to ensure they do not interfere with each other. Specifically, the T bases near the 3' end of the reverse transcription primers are unmodified to ensure they do not affect the normal function of reverse transcriptase; the T base at the 5' end of the reverse transcription primers is replaced with a U base, allowing them to be digested by uracil-N-glycosylation enzyme (UNG); the remaining reverse transcription primers after reverse transcription are partially digested by UDG, enabling further detection of any break / fusion mode of at least three IGH rearrangement types using commonly used PCR amplification methods.
[0022] The IGH quantitative screening kit of this invention uses a reverse transcription primer set to obtain IGH gene cDNA and different types of fusion gene cDNA, which are then quantitatively detected using specific primers and probes designed upstream of the DUX4, EPOR, CRLF2, and ABL1 genes. Specifically, it includes the use of UNG and RNase A to rapidly digest residual RNA and reverse transcription primers in the system, avoiding non-specific amplification in subsequent PCR processes. This enables quantitative detection of any of the three rearrangement types, and has significant application value for clinical auxiliary diagnosis. Attached Figure Description
[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.
[0024] Figure 1 This is a schematic diagram of the quantitative detection process for IGH rearrangement described in this invention.
[0025] Figure 2 This is the Sanger sequencing result of the DUX4-IGH positive sample in Example 2 of the present invention.
[0026] Figure 3 This is the Sanger sequencing result of the EPOR-IGH positive sample in Example 2 of the present invention.
[0027] Figure 4 This is the Sanger sequencing result of the CRLF2-IGH positive sample in Example 2 of the present invention.
[0028] Figure 5 This is the digital PCR detection result of the DUX4-IGH positive sample in Example 2 of the present invention.
[0029] Figure 6 This is the digital PCR detection result of the EPOR-IGH positive sample in Example 2 of the present invention.
[0030] Figure 7 This is the digital PCR detection result of the CRLF2-IGH positive sample in Example 2 of the present invention.
[0031] Figure 8 This is the digital PCR detection result of the IGH rearrangement negative sample in Example 2 of the present invention. Detailed Implementation
[0032] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.
[0033] This invention uses publicly available data from stjude and RNA-Seq data from our laboratory to identify common IGH fusion partners DUX4 / EPOR / CRLF2 and their breakpoints, as shown in Table 1.
[0034] Table 1:
[0035] To target the breakpoints of IGH, specific reverse transcription primers were designed in conjunction with the internal reference gene ABL1, as shown in Table 2. All reverse transcription primers were evaluated for specificity using the BLAST tool on the NCBI website, and experimental verification was conducted to ensure that they did not interfere with each other.
[0036] Table 2:
[0037] If the sequence list provided by this invention is inconsistent with Table 2, the sequence shown in Table 2 shall prevail.
[0038] Specifically, the T bases near the 3' end of the above reverse transcription primers are unmodified, ensuring they do not affect the normal function of reverse transcriptase and guaranteeing high efficiency in the reverse transcription process; the T bases at the 5' end of the reverse transcription primers are replaced by U bases, allowing them to be digested by uracil-N-glycosylation enzyme (UNG); the remaining reverse transcription primers after reverse transcription are partially digested by UDG. A schematic diagram of the quantitative detection method provided by this invention is shown below. Figure 1 As shown, the steps include: 1) After digesting the trace DNA remaining in the total RNA extracted with DNase I, reverse transcription was performed using the aforementioned IGH (SEQ ID NO: 1-55) and ABL1 (SEQ ID NO: 56) specific primers to obtain ABL1 gene cDNA, IGH gene cDNA and DUX4 / EPOR / CRLF2::IGH fusion gene cDNA; 2) Use UNG and RNase A to rapidly digest residual RNA and reverse transcription primers in the system to avoid non-specific amplification in subsequent PCR processes; 3) Specific primers and probes were designed upstream of the DUX4 (primer SEQ ID NO: 58-59), EPOR (primer SEQ ID NO: 61-62), CRLF2 (primer SEQ ID NO: 64-65) and ABL1 (primer SEQ ID NO: 67-68) genes, respectively. The DUX4 probe was labeled with 5'FAM-3'MGB (SEQ ID NO: 57), the EPOR probe was labeled with 5'VIC-3'MGB (SEQ ID NO: 60), the CRLF2 probe was labeled with 5'ROX-3'MGB (SEQ ID NO: 63), and the ABL1 probe was labeled with 5'CY5-3'MGB (SEQ ID NO: 66).
[0039] 4) Perform dPCR reaction based on the specific primers and probes designed in step 3). IGH fusion partners and ABL1 internal reference genes are identified according to different fluorescent labels, and the detection results are obtained, including any fusion gene type, break site and fusion ratio shown in Table 1.
[0040] The specific primers and probes used in step 3) above are shown in Table 3.
[0041] Table 3:
[0042] It features simple and fast operation, high accuracy and low cost, and a sensitivity of 0.01%. It can detect common IGH rearrangements (IGH-DUX4, IGH-EPOR, IGH-CRLF2) quantitatively, and can quantitatively detect the fusion ratio. It has low analytical difficulty and low requirements for personnel skills and facilities and equipment, which has significant advantages.
[0043] In comparison, the DUX4-IGH rearrangement detection method described in this invention has significant advantages over methods such as karyotype analysis, FISH, whole-genome sequencing, and transcriptome sequencing, as detailed below:
[0044] Example 1: Quantitative Detection Method for IGH Rearrangements
[0045] 1. RNA extraction
[0046] RNA samples were extracted from bone marrow or peripheral blood using Trizol or other methods.
[0047] 2. Reverse transcription (Novazia, HiScript IV 1st Strand cDNA Synthesis Kit (+gDNAwiper), R412)
[0048] 1) RNA thermal denaturation
[0049] The extracted total RNA was heated at 65°C for 5 minutes and then rapidly cooled on ice for 2 minutes. This step aims to open up the secondary structure of the RNA as much as possible, keeping the RNA linear and improving reverse transcription efficiency.
[0050] 2) DNA digestion
[0051] Configure the DNA digestion system according to the table below, and incubate the system at 42°C for 2 minutes.
[0052] 3) Reverse transcription
[0053] Based on the DUX4 / EPOR / CRLF2-IGH fusion break site and the ABL1 internal reference gene transcript, specific reverse transcription primers were designed for IGH and ABL1. T bases near the 5' end of the primers were replaced with U bases to facilitate UNG digestion of all reverse transcription primers during subsequent detection, thus avoiding interference with PCR. The reverse transcription primer set is shown in Table 1.
[0054] In the above reverse transcription primers, the working solution concentration of any primer is 0.4 μM.
[0055] 4) Reverse transcription reaction system
[0056] 3. Digital PCR Detection Process
[0057] 1) Design the PCR primer and probe set as shown in Table 2. The DUX4 probe is labeled with 5'FAM-3'MGB, the EPOR probe with 5'VIC-3'MGB, the CRLF2 probe with 5'ROX-3'MGB, and the ABL1 probe with 5'CY5-3'MGB.
[0058] 2) Configuration of digital PCR instrument and detection system
[0059] Equipment: Sinaf Medical DQ24 Digital PCR System and related consumables
[0060] Testing system:
[0061] Because Taq enzyme has low reverse transcription activity, the purpose of using RNase A to digest RNA is to avoid Taq enzyme reverse transcription and amplification of the mRNA of the DUX4 / EPOR / CRLF2 gene during subsequent amplification, which would result in false positive results with low abundance.
[0062] 3) PCR amplification program
[0063] A higher annealing extension temperature (63℃) is beneficial to improving the specificity of the reaction system, resulting in better separation of positive and negative droplets in digital PCR.
[0064] During the amplification process, Taq enzyme cleaves the fluorescently labeled specific probe, which emits fluorescence. This fluorescence is ultimately recognized and recorded by a digital PCR droplet reader, and the fusion ratio is calculated according to the following formula: Fusion ratio = Copy number of any IGH fusion transcript / Copy number of internal reference ABL1 transcript 100%.
[0065] Example 2
[0066] 1) Collect one positive RNA sample each of DUX4 / EPOR / CRLF2-IGH fusion and one negative RNA sample of IGH rearrangement using RNA-Seq method, and verify and confirm the break position using PCR and Sanger sequencing methods.
[0067] Sample 1: DUX4-IGH chr4:191006703 / chr14:106815724, Sanger sequencing results are as follows Figure 2 As shown.
[0068] Sample 2: EPOR-IGH chr19:11488972 / chr14:106715347, Sanger sequencing results are as follows Figure 3 As shown.
[0069] Sample 3: CRLF2-IGH chrX:1345107 / chr14:106329470, Sanger sequencing results are as follows Figure 4 As shown.
[0070] 2) The aforementioned digital PCR method was used to quantitatively detect the above IGH fusion positive and IGH fusion negative samples.
[0071] The digital PCR test results of sample 1 are as follows Figure 5 As shown.
[0072] The digital PCR test results of sample 2 are as follows Figure 6 As shown.
[0073] The digital PCR test results of sample 3 are as follows: Figure 7 As shown.
[0074] In addition, digital PCR was performed on IGH rearranged negative RNA samples, and the results are as follows: Figure 8 As shown.
[0075] As can be seen, the results of digital PCR detection of the above different types of samples are consistent with those of Sanger sequencing.
[0076] Example 3
[0077] Fifty-five RNA samples that were positive for DUX4-IGH / EPOR-IGH / CRLF2-IGH by NGS and one negative RNA sample were selected and ddPCR was performed using the PCR system described in Example 1. The results are shown in the table below.
[0078]
[0079] The above detection results demonstrate that the IGH rearrangement screening kit provided by this invention can effectively detect the fusion types caused by the known breakpoints in Table 1, and can quantitatively detect the fusion ratio, indicating that the design of the reverse transcription primer set, PCR primers and probes can meet the requirements for specific detection.
[0080] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.
Claims
1. A reverse transcription primer set for IGH rearrangement detection, characterized in that, This includes a set of reverse transcription primers for detecting IGH rearrangements, with rearrangement types including at least one of IGH-DUX4, IGH-EPOR, and IGH-CRLF2; wherein: The reverse transcription primer set of the IGH-DUX4 includes the nucleotide sequence shown in SEQ ID NO: 1-34; And / or, the reverse transcription primer set of the IGH-EPOR includes nucleotide sequences as shown in SEQ ID NO: 35-44; And / or, the reverse transcription primer set of the IGH-CRLF2 includes nucleotide sequences as shown in SEQ ID NO: 45-55.
2. The application of the reverse transcription primer set according to claim 1, characterized in that, The applications include: a. Preparation of IGH rearrangement type detection products; b. Prepare products for quantitative detection of IGH fusion ratio.
3. An IGH quantitative screening kit, characterized in that, It includes the reverse transcription primer set as described in claim 1, and the PCR primer and probe set for detecting at least one rearrangement of IGH-DUX4, IGH-EPOR and IGH-CRLF2.
4. The kit according to claim 3, characterized in that, The PCR primer and probe set for detecting IGH-DUX4 rearrangement includes probes with nucleotide sequences as described in SEQ ID NO: 57, and primers as shown in SEQ ID NO: 58-59; And / or, the PCR primer and probe set for detecting IGH-EPOR rearrangement includes probes with nucleotide sequences as described in SEQ ID NO: 60, and primers as shown in SEQ ID NO: 61-62; And / or, the PCR primer and probe set for detecting IGH-EPORIGH-CRLF2 rearrangement includes probes with nucleotide sequences as described in SEQ ID NO: 63, and primers as shown in SEQ ID NO: 64-65.
5. The reagent kit as described in claim 3, characterized in that, The kit also includes internal reference gene reverse transcription primers, and primer sets and probes for PCR amplification.
6. The reagent kit as described in claim 5, characterized in that, The nucleotide sequence of the reverse transcription primer for the internal reference gene is shown in SEQ ID NO: 56; And / or, the primer set and probe for PCR amplification of the internal reference gene include a probe with nucleotide sequences as shown in SEQ ID NO: 66, and a primer set with nucleotide sequences as shown in SEQ ID NO: 67-68.
7. The kit as described in claim 5 or 6, characterized in that, Each probe in the kit is modified with a different fluorescent label.
8. The reagent kit as described in claim 3, characterized in that, The concentration of any of the reverse transcription primers in the reverse transcription primer set is 0.1-1 μM.
9. The reagent kit as described in claim 3, characterized in that, The kit also includes at least one of the following: reverse transcription reaction reagent, PCR reaction reagent, RNA digesting enzyme, and UNG enzyme.
10. A method for quantitative screening of IGH rearrangements, for non-diagnostic purposes, characterized in that, This includes using the kit described in claim 3 and performing the following steps: 1) Extract RNA from the sample; 2) RNA is reverse transcribed using reverse transcription primers to generate cDNA; 3) Using cDNA as a template, add PCR primers, probes, and UNG enzyme, and perform PCR amplification; 4) Determine the detection type and fusion ratio based on the probe detection signal.