RNA methylation detection composition and its application in leukemia diagnosis

Abstract

The present invention provides an RNA methylation detection composition, comprising: 1) a first amplification primer pair, whose nucleotide sequences are shown in SEQ ID NO: 5 and 6 respectively; a first blocking primer, whose nucleotide sequence is shown in SEQ ID NO: 11; 2) a second amplification primer pair, whose nucleotide sequences are shown in SEQ ID NO: 8 and 9 respectively; a second blocking primer, whose nucleotide sequence is shown in SEQ ID NO: 12, wherein the 3'-end nucleotides of the first blocking primer and the second blocking primer are modified nucleotides to prevent DNA polymerase from performing primer extension using the first blocking primer or the second blocking primer as a primer. The present invention also provides the use of the above RNA methylation detection composition in the diagnosis and screening of acute myeloid leukemia (AML).

Description

Technical Field

[0001] The present invention relates to a composition for RNA methylation detection, in particular to a composition for detecting m5C methylation in RNA, and also to application of the composition in leukemia diagnosis. Background Art

[0002] Acute myeloid leukemia (AML) is a malignant disease originating from myeloid hematopoietic stem / progenitor cells in the bone marrow and is a common form of acute leukemia in adults. AML accounts for about 30% of pediatric leukemia. The clinical manifestations of AML include anemia, bleeding, infection and fever, organ infiltration, metabolic abnormalities, etc., which can often be life-threatening if not treated in time.

[0003] At present, the diagnosis of various leukemias, including AML, is mostly based on the examination of bone marrow cell morphology. For example, the doctor performs a bone marrow puncture on the patient to obtain the aspirate, stains it and observes it under an electron microscope, calculates the proportion of each type of cell, and then makes a diagnosis based on it. This AML diagnosis method is time-consuming and labor-intensive, and depends on the professional level of the inspector, and the test results are somewhat subjective. In addition, if the test requires a bone marrow puncture, it will also increase the patient's pain. The diagnosis of AML also includes the discovery of genetic characteristics such as gene mutations and gene rearrangements through molecular genetic testing, but this type of detection method often focuses on specific AML typing and is not suitable for early AML diagnosis. Some biomarkers for the diagnosis of AML have also been reported in this field, but so far they have not been widely used in clinical practice.

[0004] After the discovery of DNA cytosine methylation, technicians in this field also discovered the existence of RNA cytosine methylation (m5C) in cells, and have discovered a series of proteases related to RNA cytosine methylation and their mechanisms of action. Currently, differential RNA methylation has been found in some diseases, but there has been no report on the use of these methylation differences for diagnosing AML. Summary of the invention

[0005] In one aspect, the present invention provides an RNA methylation detection composition, comprising:

[0006] 1) a first amplification primer pair, whose nucleotide sequences are shown in SEQ ID NOs: 5 and 6, respectively; a first blocking primer, whose nucleotide sequence is shown in SEQ ID NO: 11;

[0007] 2) a second amplification primer pair, whose nucleotide sequences are shown in SEQ ID NOs: 8 and 9, respectively; a second blocking primer, whose nucleotide sequence is shown in SEQ ID NO: 12,

[0008] The 3'-end nucleotides of the first blocking primer and the second blocking primer are modified nucleotides, so as to prevent DNA polymerase from performing primer extension using the first blocking primer or the second blocking primer as a primer.

[0009] In some embodiments, the RNA methylation detection composition further includes a first fluorescent probe, whose nucleotide sequence is shown in SEQ ID NO:7; and a second fluorescent probe, whose nucleotide sequence is shown in SEQ ID NO:10.

[0010] In some embodiments, the modified nucleotide is a nucleotide in which the 3'-OH is modified by Spacer C3.

[0011] In some embodiments, the first fluorescent probe and the second fluorescent probe are both Taqman probes.

[0012] In some embodiments, different fluorescent groups are attached to the 5' ends of the first fluorescent probe and the second fluorescent probe.

[0013] In some embodiments, the first fluorescent probe is connected to a fluorescent group TEXAS RED at the 5' end and a fluorescent quencher group BHQ2 at the 3' end; the second fluorescent probe is connected to a fluorescent group FAM at the 5' end and a fluorescent quencher group BHQ2 at the 3' end.

[0014] On the other hand, the present invention also provides the use of the above RNA methylation detection composition in the preparation of a diagnostic kit for diagnosing acute myeloid leukemia. DETAILED DESCRIPTION

[0015] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.

[0016] The term "comprising" or "including" means including the elements, integers or steps mentioned, but does not exclude any other elements, integers or steps. In the text, when the term "comprising" or "including" is used, unless otherwise specified, it also covers the situation consisting of the elements, integers or steps mentioned. For example, when it is mentioned that a combination "comprising" certain components, it is also intended to cover that the combination consists of these components.

[0017] "RNA methylation" in this article refers to the phenomenon that cytosine residues in RNA (such as mRNA) are modified to 5-methylcytosine (5mC). "RNA methylation difference" refers to the overall different methylation levels in a part of the population (such as patients) relative to another part of the population (such as healthy people) at specific sites on the RNA molecule, such as methylation in one part of the population and no methylation in another part of the population; or the methylation level in one part of the population is higher and the methylation level in another part of the population is lower. The "methylation level" here refers to, for a specific RNA molecule, the ratio of the number of RNA molecules in the cell or human body that have the methylation phenomenon to the total amount of the RNA molecules.

[0018] "Blocking primer" herein refers to a small single-stranded DNA molecule that, after binding to a DNA molecule (such as ssDNA), prevents or weakens the amplification of other amplification primers using the DNA molecule as a template, and its 3' end is modified and has no extension ability in the presence of DNA polymerase.

[0019] "Taqman probe", also known as hydrolysis probe, dual-labeled probe, refers to an oligonucleotide labeled with a fluorescent group and a quencher group at an appropriate position in this article. When the Taqman probe remains intact, the quencher group inhibits the fluorescence emission of the fluorescent group through fluorescence resonance energy transfer. After the oligonucleotide is degraded by the 5'-3' nuclease activity of DNA polymerase, the fluorescent group and the quencher group are separated, and the fluorescence emission of the fluorescent group can be detected.

[0020] The present inventors discovered several RNA methylation differential sites by RNA bisulfite sequencing (RNA-BisSeq), and subsequently found that the combination of several of these sites could be used for AML diagnosis and early screening by simple multiplex RT-qPCR detection.

[0021] The present invention is described in detail below through specific examples.

[0022] Example 1 Screening for 5mC methylation differential sites

[0023] We used RNA-BisSeq to detect the 5mC methylation of mRNA in peripheral blood cells of patients with acute myelogenous leukemia (AML) and non-AML patients, and to find mRNA methylation differential sites that can be used to diagnose AML. Comparison of mRNA methylation in 56 AML patients and 62 non-AML patients (including healthy people and patients with other diseases, all from Zhanjiang Central People's Hospital) revealed 5 sites with different 5mC methylation levels. The methylation levels in AML patients were relatively high, and they were distributed on 4 different mRNA molecules. These mRNAs were transcribed from NPM1 (nucleophosmin1), KLF5 (KLF transcription factor5), RNF5 (ring finger protein5), and GATA2 (GATA binding protein2) genes, of which the mRNA from the GATA2 gene included two differential sites. Table 1 shows the 5mC methylation levels of specific sites on the mRNA fragments from these genes. The calculation method of the methylation level of a specific site is as follows: methylation level = number of reads determined to be C by sequencing / (number of reads determined to be C by sequencing + number of reads determined to be T by sequencing)*100%.

[0024] Table 1 5mC methylation levels on some mRNA fragments

[0025]

[0026]

[0027] We used the methylation levels of these sites for the diagnosis of AML in patients, but did not find any single site with diagnostic value, and the AUC values ​​were all less than 0.65. We then hoped to improve the diagnostic effect by selecting a combination of two sites to construct a diagnostic model, and only found that the AUC value of the diagnostic model constructed by combining the KLF5 methylation site and the downstream methylation site of the GATA2 gene mRNA exceeded 0.7, reaching 0.73, showing a certain diagnostic value.

[0028] Example 2 RT-PCR primer design and detection verification

[0029] Considering the relatively high cost of high-throughput sequencing of mRNA in peripheral blood cells using RNA-BisSeq to determine methylation levels in clinical AML diagnosis, we then attempted to determine the methylation levels of specific sites by reverse transcription-qPCR (RT-qPCR) after bisulfite treatment.

[0030] For the above-mentioned KLF5 differential methylation sites, the designed amplification primers are: KLF5-F: 5'-TGTGAAATGGAGAAGTATTTGATAC-3' (SEQ ID NO: 5); KLF5-R: 5'-TAATACTATAAAATAACCCTTCAATATCA-3' (SEQ ID NO: 6), and the fluorescent probe is 5'-TEXASRED-AAGTATAGATGAGATAGTGTTTTAGTTG-BHQ2-3' (SEQ ID NO: 7).

[0031] Since the GATA2 gene contains two differentially methylated sites, the upstream and downstream amplification primers designed used the differentially methylated sites as primer ends: GATA2-F: 5'-ATTTTGATTTATATTATTTGGGTTTGGC-3' (SEQ ID NO: 8), GATA2-R: 5'-ACAAACATACACAAAACTATAAAAAACG-3' (SEQ ID NO: 9), and the fluorescent probe was: 5'-FAM-CATCCACCTCATCTAAAAACAACAAC-BHQ2-3' (SEQ ID NO: 10).

[0032] When the above primers were used to detect mRNA methylation levels in peripheral blood cells, nonspecific amplification was found to be quite serious. Therefore, we designed blocking primers to minimize nonspecific amplification.

[0033] For the differential methylation sites of KLF5, the blocking primer used was: KLF5-blocker: 5'-TGAAATGGAGAAGTATTTGATAT-3' (SEQ ID NO: 11); for the differential methylation sites downstream of the GATA2 gene, the blocking primer used was 5'-TACACAAAACTATAAAAAACA-3' (SEQ ID NO: 12). The 3'-terminal hydroxyl groups of the blocking primers were all modified with 3'-Spacer C3, which can block the amplification of the template obtained by reverse transcription of unmethylated mRNA using the above amplification primers.

[0034] The brief experimental process of detecting mRNA methylation in peripheral blood cells using the above primers and probes is as follows. Peripheral blood was collected from patients or healthy subjects, and blood cells were separated by centrifugation. After lysis, total RNA was extracted using an RNA extraction kit, and mRNA was separated using Oligo (dT) magnetic beads. Unmethylated cytosine was deaminated by mRNA using a bisulfite conversion kit (Epigentek), and the mRNA after magnetic bead separation was quantified using a NanoDrop One / OneC spectrophotometer. SuperScriptTM III one-step RT-PCR kit for multiple RT-qPCR, and the amplification curves of KLF5 and GATA2 mRNA were obtained simultaneously. The concentrations of amplification primers and probes in the 50 μL reaction system were both 0.2 μM, the concentration of blocking primers was 0.5 μM, and the treated mRNA template was 0.5 μg. RT-PCR reaction conditions: 42℃5min; 95℃40s; 95℃15s, 50℃40s, 45 cycles, and the fluorescence signal was collected at 50℃.

[0035] Multiple batches of detection tests were conducted using the above method. In one batch, mRNA methylation levels were detected in 10 AML patients and 8 healthy subjects, and the amplified Ct results are listed in Table 2.

[0036] Table 2 RT-PCR test results (Ct value)

[0037]

[0038]

[0039] The test results of other batches were similar. As can be seen from the results in Table 2, for the KLF5 gene, the Ct value of most AML patients tested was <=32; for the GATA2 gene, the Ct value of most AML patients tested was <=35.

[0040] Example 3 AML Diagnosis

[0041] This embodiment uses the two critical values ​​(32 and 35) obtained in Embodiment 2 to perform patient diagnosis.

[0042] The subjects were selected from 85 patients who were determined to undergo AML examination by doctors at Zhanjiang Central People's Hospital based on their initial symptoms from May 2020 to October 2023. Of these 85 patients, 27 were eventually clinically diagnosed as AML patients.

[0043] Using the detection method described in Example 2, while clinically testing these 85 patients for AML, we diagnosed these 85 patients based on the method described in the example. Results: The sensitivity of diagnosis based only on the KLF5 gene detection results was 66.7% (18 / 27), and the specificity was 77.6% (45 / 58); the sensitivity of diagnosis based only on the GATA2 gene detection results was 77.7% (21 / 27), and the specificity was 75.9% (49 / 58). Based on the detection results of both the KLF5 gene and the GATA2 gene, that is, the Ct value of the KLF5 gene detection was <= 32 and the Ct value of the GATA2 gene detection was <= 35, the sensitivity of diagnosis was 63.0% (17 / 27), and the specificity was 96.6% (56 / 58). It can be seen that compared with the single gene methylation diagnosis results, the diagnostic specificity of the dual gene diagnosis has been greatly improved with a slightly reduced sensitivity. This result is even more effective than the methylation level detection by RNA-BisSeq. We analyzed that on the one hand, the multiplex RT-qPCR of the present invention actually utilizes three methylation difference sites. On the other hand, the library preparation and sequencing process in the RNA-BisSeq method is too complicated, resulting in serious distortion of the detection results. The RT-qPCR process is relatively simple and easy to operate, and it more accurately reflects the real methylation level differences.

[0044] Subsequently, we used the above two critical values ​​to screen inpatients over 75 years old who were not diagnosed with AML. Among 233 inpatients (133 males and 100 females), 3 patients were detected whose Ct values ​​met the above critical value requirements and should be diagnosed as AML patients. Two of these 3 patients were subsequently clinically diagnosed as AML patients. This shows that the diagnostic method of the present invention can also be used for early screening of AML patients.

Claims

1. A composition for detecting RNA methylation, comprising: 1) a first amplification primer pair, whose nucleotide sequences are shown in SEQ ID NOs: 5 and 6, respectively; a first blocking primer, whose nucleotide sequence is shown in SEQ ID NO: 11; 2) a second amplification primer pair, whose nucleotide sequences are shown in SEQ ID NOs: 8 and 9, respectively; a second blocking primer, whose nucleotide sequence is shown in SEQ ID NO: 12; as well as 3) a first fluorescent probe, whose nucleotide sequence is shown in SEQ ID NO: 7; a second fluorescent probe, whose nucleotide sequence is shown in SEQ ID NO: 10, The 3'-end nucleotides of the first blocking primer and the second blocking primer are modified nucleotides, so as to prevent DNA polymerase from performing primer extension using the first blocking primer or the second blocking primer as a primer.

2. The RNA methylation detection composition according to claim 1, wherein the modified nucleotide is a nucleotide whose 3'-OH is modified by Spacer C3.

3. The RNA methylation detection composition as claimed in claim 1, wherein the first fluorescent probe and the second fluorescent probe are both Taqman probes.

4. The RNA methylation detection composition as claimed in claim 3, wherein the 5' ends of the first fluorescent probe and the second fluorescent probe are connected with different fluorescent groups.

5. The RNA methylation detection composition as claimed in claim 4, wherein the first fluorescent probe is connected to a fluorescent group TEXAS RED at the 5' end and a fluorescent quencher group BHQ2 at the 3' end; the second fluorescent probe is connected to a fluorescent group FAM at the 5' end and a fluorescent quencher group BHQ2 at the 3' end. The RNA methylation detection composition according to claim 1 , wherein the RNA is mRNA.

7. Use of the RNA methylation detection composition according to any one of claims 1 to 6 in preparing a diagnostic kit for diagnosing acute myeloid leukemia.

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