WO2022199682A1 - 先天性巨结肠的诊断或早期诊断标志物及其应用 - Google Patents
先天性巨结肠的诊断或早期诊断标志物及其应用 Download PDFInfo
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
Definitions
- the invention belongs to the field of biomedicine, and specifically relates to a marker for diagnosis or early diagnosis of Hirschsprung's disease and its application.
- Hirschsprung disease is a birth defect disease with abnormal enteric nerve development in children.
- the pathological mechanism is the migration and differentiation of enteric neural crest cells into enteric neurons. It is one of the common congenital intestinal diseases in children.
- the early manifestations of Hirschsprung's disease are vomiting, abdominal distension, diarrhea, etc., which can clinically cause neonatal death, recurrent enteritis after surgery, refractory constipation and other complications, which seriously affect the growth and development and quality of life of children.
- Timely diagnosis and treatment of Hirschsprung's disease can reduce the risk of Hirschsprung's colitis and obtain a good prognosis.
- the diagnosis of the disease requires postoperative pathological sections of the diseased tissue.
- the main methods of preoperative diagnosis are barium enema, rectal biopsy, and rectal manometry to determine whether to implement "radical resection of megacolon".
- barium enema is the most important diagnostic method. The principle is that there is stenosis and proximal dilatation of the intestine in children with Hirschsprung's congenital. However, this method can only diagnose children with typical intestinal morphological changes, the sensitivity needs to be improved, and the diagnostic accuracy is about 80%.
- Rectal biopsy is to directly take rectal tissue to detect the absence of ganglion cells, with high accuracy, but the sampling site has an impact on the results.
- This method is invasive and very expensive. Generally, this method is used in children whose barium enema is not obvious or suitable. When the child has the possibility of necrotizing enterocolitis (NEC), barium enema can lead to intestinal perforation. At this time, barium enema is not suitable for diagnosis, and rectal biopsy is considered. It can be seen that the barium enema and rectal biopsy methods commonly used in Hirschsprung's disease are invasive and bring certain physical and mental pain to neonatal patients and their parents who wish to obtain a diagnosis of the disease.
- the purpose of the present invention is to provide a diagnostic marker for Hirschsprung's disease and its application, and to provide a new accurate and sensitive method for the diagnosis of Hirschsprung's disease (especially early diagnosis). detection way.
- the present invention adopts the following technical solutions to realize:
- a first aspect of the present invention provides a combination of diagnostic markers for Hirschsprung's disease, comprising PRDM9 transposon fusion and cytomegalovirus (Cytomegalovirus, CMV).
- the PRDM9 transposon fusion comprises at least one, at least two, or three selected from PRDM9-type I, PRDM9-type II-1, PRDM9-type II-2.
- the second aspect of the present invention provides a combination of diagnostic markers for Hirschsprung's disease, comprising at least two or three selected from the following: PRDM9-type I; PRDM9-type II-1; PRDM9-type II- 2.
- the diagnostic marker combination comprises: PRDM9-type II-1, and PRDM9-type II-2. In some embodiments, the diagnostic marker combination further comprises cytomegalovirus.
- the diagnostic marker combination comprises: PRDM9-type I, and PRDM9-type II-1. In some embodiments, the diagnostic marker combination further comprises cytomegalovirus.
- the diagnostic marker combination comprises: PRDM9-type I, and PRDM9-type II-2. In some embodiments, the diagnostic marker combination further comprises cytomegalovirus.
- the diagnostic marker combination comprises: PRDM9-type I, PRDM9-type II-1, and PRDM9-type II-2. In some embodiments, the diagnostic marker combination further comprises cytomegalovirus.
- a third aspect of the present invention provides a diagnostic product for Hirschsprung's disease, the product comprising a PRDM9 transposon fusion detection agent and/or a cytomegalovirus detection agent.
- the product is in the form of test strips or strips, test chips, reagents, kits, or other conventional products in the art.
- the PRDM9 transposon fusion comprises at least one, at least two, or three selected from PRDM9-type I, PRDM9-type II-1, PRDM9-type II-2.
- a fourth aspect of the present invention provides a diagnostic product for Hirschsprung's disease, the product comprising: at least one, at least two of PRDM9-type I, PRDM9-type II-1, and PRDM9-type II-2 or detection agents for three diagnostic markers.
- the product is in the form of test strips or strips, test chips, reagents, kits, or other conventional products in the art.
- the diagnostic product comprises: a detection agent of PRDM9-type II-1 and/or a detection agent of PRDM9-type II-2. In some embodiments, the diagnostic product further comprises a detection agent for cytomegalovirus.
- the diagnostic product comprises: a detection agent of PRDM9-type I and/or a detection agent of PRDM9-type II-1. In some embodiments, the diagnostic product further comprises a detection agent for cytomegalovirus.
- the diagnostic product comprises: a detection agent of PRDM9-type I and/or a detection agent of PRDM9-type II-2. In some embodiments, the diagnostic product further comprises a detection agent for cytomegalovirus.
- the diagnostic product comprises: PRDM9-type I, PRDM9-type II-1, and PRDM9-type II-2. In some embodiments, the diagnostic product further comprises a detection agent for cytomegalovirus.
- the fifth aspect of the present invention provides the use of the diagnostic product according to the third aspect or the fourth aspect of the present invention in the preparation of a reagent or a kit for the diagnosis or auxiliary diagnosis of Hirschsprung's disease.
- the fusion site of the PRDM9 transposon fusion comprises at least one, at least two, or three selected from the group consisting of: PRDM9-type I; PRDM9-type II -1; and PRDM9-type II-2.
- the fusion site of the PRDM9 transposon fusion comprises: PRDM9-type I.
- the fusion site of the PRDM9 transposon fusion comprises: PRDM9-type II-1.
- the fusion site of the PRDM9 transposon fusion comprises: PRDM9-type II-2.
- the fusion site of the PRDM9 transposon fusion is selected from the group consisting of: PRDM9-type I and PRDM9-type II-1. In some embodiments, the fusion site of the PRDM9 transposon fusion is selected from the group consisting of: PRDM9-type I and PRDM9-type II-2. In some embodiments, the fusion site of the PRDM9 transposon fusion is selected from the group consisting of: PRDM9-type II-1 and PRDM9-type II-2. In some embodiments, the PRDM9 transposon fusion comprises PRDM9-type I, PRDM9-type II-1, PRDM9-type II-2.
- the PRDM9-type I is located at chr5:23299411.
- the unfused sequence of PRDM9-type I comprises the sequence shown in SEQ ID NO: 13, and the fused sequence comprises the sequence shown in SEQ ID NO: 16.
- the PRDM9-type II-1 is located at chr5:23262356.
- the unfused sequence of PRDM9-type II-1 comprises the sequence shown in SEQ ID NO: 14, and the fused sequence comprises the sequence shown in SEQ ID NO: 17.
- the PRDM9-type II-2 is located at chr5:23245181.
- the unfused sequence of PRDM9-type II-2 comprises the sequence shown in SEQ ID NO:15, and the fused sequence comprises the sequence shown in SEQ ID NO:18.
- the cytomegalovirus is human cytomegalovirus.
- detecting the cytomegalovirus is detecting infection with cytomegalovirus; preferably, detecting an antibody to cytomegalovirus; more preferably, detecting lgM antibody to cytomegalovirus; or more preferably, Detection of cytomegalovirus IgG antibody and IgG antibody.
- the diagnosis is an early diagnosis.
- the subject of the early diagnosis is a newborn who is less than 3 months old. In some embodiments, the subject of the early diagnosis is a newborn who is less than 1 month old.
- the detection is a quantitative detection.
- the PRDM9 transposon fusion detection agent or quantitative detection agent is used to perform any one of the following methods: polymerase chain reaction, denaturing gradient gel electrophoresis, nucleic acid typing chip detection, denaturing high performance liquid phase Chromatography, in situ hybridization, biological mass spectrometry, and HRM.
- the detection or quantitative detection of cytomegalovirus is used to perform any one of the following methods: radioimmunoassay, indirect immunofluorescence, dot immunogold diafiltration, biological mass spectrometry, immunoblotting And enzyme-linked immunosorbent assay, polymerase chain reaction, denaturing gradient gel electrophoresis, nucleic acid typing chip detection, denaturing high performance liquid chromatography, in situ hybridization and HRM method.
- the detection agent or quantitative detection agent comprises a primer.
- the primers include at least one of a-c:
- the primers further include a wild-type corresponding to at least one of PRDM9-type I, PRDM9-type II-1 and PRDM9-type II-2.
- the internal reference primers include at least one of d ⁇ f:
- the detection agent or quantitative detection agent further contains a DNA extraction reagent, a double-strand-specific fluorescent dye, dNTPs, DNA polymerase, a double-strand-specific fluorescent dye, and a one or more.
- the test sample of the reagent or kit is selected from the group consisting of blood, plasma, serum, feces, urine, saliva, amniotic fluid, villi, tissue, cells, At least one of tissue or cell lysate samples; in some preferred embodiments of the present invention, the test sample of the reagent or kit is blood, plasma or serum, more preferably blood, plasma or blood from peripheral blood. serum. In some preferred embodiments of the present invention, the test sample is blood; in some more preferred embodiments of the present invention, the test sample is from peripheral blood.
- primers as defined above are provided.
- the seventh aspect of the present invention provides a reagent, kit or detection chip comprising the primers described in the sixth aspect of the present invention.
- the eighth aspect of the present invention provides the use of the primers described in the sixth aspect of the present invention in the preparation of Hirschsprung's disease diagnosis or auxiliary diagnosis products.
- the product includes a reagent, kit or detection chip.
- the diagnosis is an early diagnosis.
- the subject of the early diagnosis is a newborn who is less than 3 months old. In some embodiments, the subject of the early diagnosis is a newborn who is less than 1 month old.
- the present invention utilizes DNA diagnostic technology, takes the lead in adopting the highly specific PRDM9 transposon fusion site to carry out the diagnosis of children's Hirschsprung's disease, and the product and method adopted can not only make the diagnosis of Hirschsprung's disease conform to specificity It also has many comprehensive advantages such as simple and rapid operation, no intervention, high throughput, and low cost, effectively making up for the deficiencies in the above-mentioned existing technologies, and is an effective diagnosis or treatment for Hirschsprung's disease.
- Alternative or auxiliary detection methods for screening filling the gap in the diagnosis of Hirschsprung's disease.
- the present invention through screening and verification of Hirschsprung's transposon fusion genetic markers, found that when PRDM9 transposon fusion was used to diagnose children with Hirschsprung's disease, the PRDM9-type I AUC value was 0.7885, and the PRDM9-type II- 1 is 0.8503 and PRDM9-type II-2 is 0.7791.
- the AUC of the three combined detection was 0.9666, the best limit corresponding to the specificity was 95.35%, and the sensitivity was 84.38%. The accuracy is even better than existing diagnostic methods.
- the specificity is 76.74%, which is suitable for disease screening.
- the present invention finds the virus CMV closely related to Hirschsprung's disease by screening to Hirschsprung's disease factor, and confirms that quantitative detection CMV is to the diagnosis of children's Hirschsprung's disease, especially early diagnosis, It has a good diagnostic effect, and the AUC reached the level of 0.7173 and 0.8571 in two independent detection cohorts respectively; it fills the gap in the early diagnosis of Hirschsprung's disease.
- CMV combined with PRDM9 transposon fusion level detection further improved the diagnosis, especially the effect of early diagnosis of Hirschsprung's disease.
- the combined detection results were as follows: AUC 0.9267 (95%CI, 0.8403to 1.000), the best limit corresponds to a specificity of 91.48% and a sensitivity of 84.62%; AUC 0.8899 (95%CI, 0.7842to 0.9956), the best limit corresponds to a specificity of 82.25% and a sensitivity Sexuality was 83.33%; it filled the gap of early diagnosis of Hirschsprung's disease.
- FIG. 1 is a schematic diagram of the screening process of Hirschsprung's congenital transposon fusion genetic marker in the embodiment of the present invention
- Fig. 2 is the transcriptome sequencing and differentially expressed transposon analysis results of Hirschsprung's disease in the embodiment of the present invention
- Fig. 3 is machine learning in the embodiment of the present invention that it is clear that PRDM9 transposon fusion is closely related to Hirschsprung's disease segmental type;
- Fig. 4 is the dual fluorescent reporter gene in the embodiment of the present invention to detect that PRDM9 transposon fusion occurs in the transcriptional regulatory region;
- Fig. 5 is the quantitative PCR detection of PRDM9 transposon fusion in the embodiment of the present invention and ROC curve to evaluate the diagnostic effect of PRDM9 transposon fusion on Hirschsprung's disease;
- Fig. 6 is the ROC curve in the embodiment of the present invention to evaluate the early diagnosis effect of PRDM9 transposon fusion on Hirschsprung's disease
- FIG. 7 is a virus closely related to Hirschsprung's colon obtained by screening in the embodiment of the present invention, wherein, the data marked in red is closely related to the severity of CMV infection and Hirschsprung's colon;
- Figure 8 is a comparison of the serum levels of CMV antibodies in children with Hirschsprung's disease of different months and other types of abdominal distention in the embodiment of the present invention; wherein, A is an IgG antibody, and B is an IgG antibody;
- Fig. 9 is the diagnostic effect of CMV lgM antibody and PRDM9 transposon fusion combined with early diagnosis of Hirschsprung's disease in children with Hirschsprung's disease and children without Hirschsprung's disease in the embodiment of the present invention; wherein, A is PRDM9-type I content, B is PRDM9-type II-1 content, C is PRDM9-type II-2 content, D is PRDM9-TE ratio, E is CMV lgM content, F is ROC curve;
- Figure 10 is another abdominal distension cohort in the embodiment of the present invention to verify the diagnostic effect of CMV lgM antibody and PRDM9 transposon fusion combined with early diagnosis of Hirschsprung's disease; wherein, A is PRDM9-type I content, B is PRDM9-type II -1 content, C is PRDM9-type II-2 content, D is PRDM9-TE ratio, E is CMV lgM content, and F is ROC curve.
- marker refers to a molecule to be used as a target for analysis of a patient's experimental sample.
- CMV cytomegalovirus
- Herpes B virus a genus of the subfamily Herpes B virus, the congenital infection of which is closely related to birth defect disorders.
- the "transposon” in the present invention refers to a DNA sequence that can be independently replicated or fragmented from in situ, inserted into another site after circularization, and has a regulatory effect on subsequent genes.
- PRDM9 in the present invention refers to PR domain containing 9, which is a DNA-binding protein, a methyltransferase that catalyzes the trimethylation of histones by H3K4, and an important transcriptional regulator and recombination hotspot of meiosis. the main control factor.
- PRDM9 transposon fusion refers to the transposon fusion that occurs on the PRDM9 gene.
- PRDM9 -type I the following positions in the upstream regulatory region of PRDM9 can be selected as transposon fusions: PRDM9 -type I, PRDM9-type II-1, PRDM9-type II-2.
- PRDM9-type I is an aneurysmal segment down-regulated to a type I transposon located at position 23299411 (hg19) of human chromosome 5.
- PRDM9-type II-1, PRDM9-type II-2 are named for the transposons that are upregulated as type II in aneurysmal segments, and type II has two loci named 1 and 2, which are located on chromosome 5, respectively. Bits 23262356 and 23245181 (hg19).
- the "Hirschsprung's disease” of the present invention is also called Hirschsprung's disease, which is caused by the lack of ganglion cells in the colon leading to continuous spasm of the intestinal tract, stagnation of feces in the proximal colon, and hypertrophy and dilation of the proximal colon. It is common in children.
- Short-segment lesions are located in the proximal and middle rectum, no more than 6.5cm away from the anal canal; common lesions are located in the proximal rectum or distal rectosigmoid colon, about 9cm away from the anal canal; long-segment lesions extend to the sigmoid colon or descending colon; full-colonic type The lesions spread to the entire colon and terminal ileum, within 30cm from the ileocecal valve.
- the "early diagnosis” in the present invention refers to the early diagnosis of a specific disease.
- the current diagnosis time for Hirschsprung's disease is 3 months to 3 years old.
- the early diagnosis of Hirschsprung's disease refers to the diagnosis of neonates within 3 months, preferably within 1 month of age.
- Early diagnosis of neonates with symptoms such as abdominal distension, delayed meconium, neonatal intestinal obstruction, neonatal constipation.
- the "ROC curve” in the present invention is a curve of 1-specificity (false positive rate) and sensitivity (true positive rate), which reflects the diagnostic ability of the binary classifier. For a good classifier, the ratio of true positive rate to false positive rate change is greater than 1, away from the 45 degree straight line.
- the "AUC" in the present invention refers to the area under the ROC curve, which is between 0.1 and 1, and is used to evaluate the quality of the classifier, and the closer the classifier is to 1, the better. AUC greater than 0.5 can be considered as a diagnostic marker of the disease.
- chr5:23299411 is the positions in the hg19 version.
- the present invention analyzes differentially expressed transposon fusions through whole transcriptome sequencing of neural segments and non-neural segments of tissues from patients with Hirschsprung's disease, and uses machine learning to screen translocations associated with Hirschsprung's subtypes
- the transposon fusion was further verified by quantitative PCR technology and ROC curve analysis in the DNA of Hirschsprung's cohort.
- the present invention finds for the first time that PRDM9 transposon fusion can be used for the diagnosis and early diagnosis of Hirschsprung's disease.
- the detection method provided by the present invention can not only make the diagnosis of Hirschsprung's disease consistent with It also has many comprehensive advantages such as strong specificity and high sensitivity, and has many comprehensive advantages such as earlier diagnosis time, simple and fast operation, no intervention, high throughput, and low cost, which effectively makes up for the above-mentioned deficiencies in the existing technology, and is an effective method.
- the diagnosis of Hirschsprung's disease especially the alternative or auxiliary detection method for early diagnosis.
- the PRDM9-type I, PRDM9-type II-1, and PRDM9-type II-2 can be used for Hirschsprung's disease.
- the level of fusion at the PRDM9-type I site is shown to be low in Hirschsprung's disease patient DNA relative to healthy controls and other intestinal disease controls.
- the PRDM9-type II-1, PRDM9-type II-2 locus fusion levels are expressed at high levels in Hirschsprung's disease patient DNA relative to healthy controls and other intestinal disease controls.
- the research results of the present invention also find for the first time that the quantitative detection of CMV independent or combined with PRDM9 transposon fusion can be used as the diagnosis and early diagnosis of Hirschsprung's disease.
- the detection method provided by the present invention It can not only make the diagnosis of Hirschsprung's disease meet the requirements of strong specificity and high sensitivity, but also has many comprehensive advantages such as earlier diagnosis time, simple and rapid operation, no intervention, high throughput, and low cost, which effectively makes up for the above-mentioned current situation. It is an effective alternative or auxiliary detection method for the early diagnosis of Hirschsprung's disease.
- the present invention finds viral CMV closely related to Hirschsprung's colon by screening Hirschsprung's disease factors, and confirms that quantitative detection of CMV has good diagnostic effect on diagnosis of children's Hirschsprung's disease, especially early diagnosis.
- CMV combined with PRDM9 transposon fusion level detection further improves the diagnosis or early diagnosis of Hirschsprung's disease.
- the subject of the early diagnosis is a newborn who is less than 3 months old.
- the subject of the early diagnosis is a newborn who is less than 1 month old.
- the present invention also relates to a method for the diagnosis or early diagnosis of Hirschsprung's disease, which comprises the detection of said PRDM9 transposon fusion fusion site and/or cytomegalovirus in a sample using a diagnostic product as defined above.
- the method for diagnosing Hirschsprung's disease by the product is: using the detection agent (quantitative detection agent) as defined above to detect (or quantitatively detect) the fusion site of the PRDM9 transposon fusion in the sample and CMV antibodies.
- the diagnosis or auxiliary diagnosis is Hirschsprung's disease.
- the expression level of the PRDM9 transposon fusion is defined as PRDM9-typeII-1+PRDM9-typeII-2.
- the expression level of the PRDM9 transposon fusion used is PRDM9-typeI, PRDM9-typeII-1, PRDM9-typeII-2, (PRDM9-typeII) -1+PRDM9-typeII-2) and one or more of PRDM9-TE.
- cytomegalovirus infection when cytomegalovirus infection is detected (preferably, CMV antibodies are detected, more preferably, the presence of CMV IgM antibodies is detected, further preferably, within 1-2 months of infection with CMV virus, CMV IgG will be significantly decreased and CMV IgG will be significantly increased), the diagnosis or auxiliary diagnosis of Hirschsprung's disease.
- the detection agent or quantitative detection agent is used to detect expression levels of PRDM9 fusions and/or to detect antibody levels to cytomegalovirus.
- the abnormal fusion level of CMV combined with PRDM9-TE is fitted as 0.1 ⁇ CMV lgM+PRDM9-TE ratio, and the fitting parameters are also obtained by those skilled in the art with reference to the parameters of logistic regression according to the actual detection situation of the sample.
- the combination of the CMV antibody level of the present invention and the expression level of the PRDM9 fusion plays a synergistic diagnostic role.
- fusion site detection of PRDM9 transposon fusions is concluded by comparison with a control group (eg, healthy individuals or other enteropathy groups that are not Hirschsprung's disease).
- a control group eg, healthy individuals or other enteropathy groups that are not Hirschsprung's disease.
- An increase or decrease is usually significant, and determining whether the subject is significantly different from the baseline in the healthy population can be performed using statistical methods known in the art, using confidence intervals and/or p value to confirm.
- the confidence interval can be 90%, 95%, 97.5%, 98%, 99%, 99.5%, 99.9% or 99.99% and the p-value can be 0.1, 0.05, 0.025, 0.02, 0.01, 0.005, 0.001 or 0.0001.
- one or more of the above-mentioned PRDM9-typeI, PRDM9-typeII-1, PRDM9-typeII-2, PRDM9-TE ratio and CMV antibody levels can be used to diagnose Hirschsprung's disease threshold according to those skilled in the art The actual situation is detected.
- the determination of the threshold value is a conventional technical means in the art.
- the threshold can be selected according to the expression level of each marker in a control group (healthy people or other enteropathy group without Hirschsprung's disease, etc.).
- a threshold can select a threshold, and test the detection accuracy of the selected threshold (such as detection rate, accuracy rate, false positive rate, false negative rate, sensitivity, specificity, etc.), and adjust it according to the detection effect Or lower the threshold until a better or optimal detection effect is achieved.
- detection accuracy such as detection rate, accuracy rate, false positive rate, false negative rate, sensitivity, specificity, etc.
- the detection or quantitative detection of CMV is used to perform any one of the following methods: radioimmunoassay, indirect immunofluorescence, dot immunogold diafiltration, biological mass spectrometry, immunoblotting, and enzymatic methods Linked immunosorbent assay, polymerase chain reaction, denaturing gradient gel electrophoresis, nucleic acid typing chip detection, denaturing high performance liquid chromatography, in situ hybridization and HRM method.
- the PRDM9 transposon fusion detection agent or quantitative detection agent is used to perform any one of the following methods: polymerase chain reaction, denaturing gradient gel electrophoresis, nucleic acid typing chip detection, denaturing high performance liquid phase Chromatography, in situ hybridization, biological mass spectrometry, and HRM.
- the polymerase chain reaction is selected from the group consisting of restriction fragment length polymorphism, single-strand conformation polymorphism, Taqman probe, qPCR, competitive allele-specific PCR, and allele specific PCR.
- the biological mass spectrometry is selected from mass spectrometry-in-flight detection.
- the detection agent or quantitative detection agent comprises a primer.
- the primers are detectably labeled.
- label refers to any atom or molecule that can be used to provide a detectable (preferably quantifiable) effect and that can be attached to a nucleic acid or protein. Labels include, but are not limited to, dyes; radiolabels, such as 32 P; binding moieties such as biotin; haptens such as digoxin; luminescent, phosphorescent, or fluorescent moieties; FRET) Fluorescent dyes that inhibit or shift parts of the emission spectrum combined.
- Labels can provide a signal detectable by fluorescence, radioactivity, colorimetry, gravimetry, X-ray diffraction or absorption, magnetism, enzymatic activity, and the like. Labels can be charged moieties (positive or negative) or, alternatively, can be charge neutral. Labels can include nucleic acid or protein sequences or a combination thereof, so long as the sequences comprising the label are detectable. In some embodiments, the nucleic acid is detected directly without labeling (eg, by reading the sequence directly).
- the labels are fluorophores, colorimetric labels, quantum dots, biotin, and other labeling molecules that can be used for detection (eg, alkyne groups for Raman imaging, rings for click reactions)
- Olefins, which are used for polymer labeling initiating groups can also be selected from polypeptide/protein molecules, LNA/PNA, unnatural amino acids and their analogs (such as peptidomimetics), unnatural nucleic acids and their analogs (nucleotide mimetics) ) and nanostructures (including inorganic nanoparticles, NV-centers, aggregation/assembly-induced luminescent molecules, rare earth ion ligand molecules, polyoxometalates, etc.).
- nucleotide sequences of the primers for detecting the PRDM9-type I are shown in SEQ ID NOs: 1-2.
- nucleotide sequences of the primers for detecting the PRDM9-type II-1 are shown in SEQ ID NOs: 3-4.
- nucleotide sequences of the primers for detecting the PRDM9-type II-2 are shown in SEQ ID NOs: 5-6.
- the primers include at least one of a-c:
- the detection agent or quantitative detection agent is used to detect or quantitatively detect at least one fragment set forth in SEQ ID NOs: 16-18.
- nucleotide sequence of the internal reference primer of the wild-type sequence corresponding to the PRDM9-type I is shown in SEQ ID NOs: 7-8.
- nucleotide sequence of the internal reference primer of the wild-type sequence corresponding to the PRDM9-type II-1 is shown in SEQ ID NOs: 9-10.
- nucleotide sequence of the internal reference primer of the wild-type sequence corresponding to the PRDM9-type II-2 is shown in SEQ ID NOs: 11-12.
- the primers further comprise an internal reference primer for detecting the wild-type sequence corresponding to at least one of PRDM9-type I, PRDM9-type II-1 and PRDM9-type II-2.
- the internal reference primers include at least one of d ⁇ f:
- the internal reference primer is used to detect or quantitatively detect at least one fragment shown in SEQ ID NOs: 13-15.
- the detection agent or quantitative detection agent further contains one or more of DNA extraction reagents, duplex-specific fluorescent dyes, dNTPs, DNA polymerase, duplex-specific fluorescent dyes, and water.
- the double-strand-specific fluorescent dye quantification is selected from any one of Ethidium Bromide, SYBR Green, PicoGreen, RiboGreen.
- the water is typically nucleic acid and/or nuclease-free water.
- the water can be distilled water (Distilled Water), deionized water (Deionized Water) or reverse osmosis water (Reverse osmosis Water).
- the DNA polymerase is selected from Taq, Bst, Vent, Phi29, Pfu, Tru, Tth, Tl1, Tac, Tne, Tma, Tih, Tf1, Pwo, Kod, Sac, Sso, Poc, Pab Any of , Mth, Pho, ES4 DNA polymerase, Klenow fragment.
- the kit further includes a sample processing reagent; further, the sample processing reagent includes at least one of a sample lysis reagent, a sample purification reagent, and a sample nucleic acid extraction reagent.
- the test sample suitable for the detection agent or quantitative detection agent or diagnostic product is blood, plasma, serum, feces, urine, saliva, amniotic fluid, villi, tissue, cells, tissue or cell lysates At least one of the samples is preferably blood, plasma or serum, more preferably blood, plasma or serum from peripheral blood.
- the present invention also relates to primers as defined above.
- the present invention also relates to a detection agent or a kit for quantifying a detection agent as defined above.
- the fusion site of described PRDM9 transposon fusion is selected from:
- application according to 4 is characterized in that, described primer also comprises the internal reference that is used to detect at least one corresponding wild-type sequence in PRDM9-type I, PRDM9-type II-1 and PRDM9-type II-2 primers.
- the quantitative detection agent further contains DNA extraction reagent, double-strand-specific fluorescent dye, dNTP, DNA polymerase, double-strand-specific fluorescent dye and water one or more of.
- test sample of the reagent or kit is selected from at least one of blood, tissue, and cell samples.
- a kit comprising the quantitative detection agent as defined in any one of claims 4 to 8.
- the fusion site of described PRDM9 transposon fusion is selected from:
- the diagnostic object of the early diagnosis is a newborn whose age is less than 3 months.
- diagnosis object of the early diagnosis is a newborn whose age is less than 1 month.
- application according to 5 is characterized in that, the nucleotide sequence of the primer that detects described PRDM9-type II-1 is shown in SEQ ID NO:1 ⁇ 2, detects the nucleotide sequence of described PRDM9-type II-2.
- the nucleotide sequences of the primers are shown in SEQ ID NOs: 3-4.
- nucleotide sequence of the internal reference primer of the wild-type sequence corresponding to the PRDM9-type II-1 is shown in SEQ ID NOs: 5 ⁇ 6, and the internal reference primer of the wild-type sequence corresponding to the PRDM9-type II-2
- the nucleotide sequence is shown in SEQ ID NO: 7-8.
- the quantitative detection agent also contains a kind of DNA extraction reagent, double-strand-specific fluorescent dye, dNTP, DNA polymerase, double-strand-specific fluorescent dye and water or more.
- test sample of the reagent or kit is selected from at least one of blood, tissue, and cell samples.
- Primer characterized in that it is as defined in claim 7 or 8.
- a kit comprising the quantitative detection agent as defined in any one of claims 6 to 9.
- the detection reagent of CMV combined with PRDM9 transposon fusion in the preparation of Hirschsprung's disease diagnosis product; the product includes detection test paper or test strip, detection chip or kit.
- the CMV detection reagent is used to perform any one of the following methods: radioimmunoassay, indirect immunofluorescence, spot immunogold filtration, biological Mass spectrometry, western blotting and enzyme-linked immunosorbent assay, polymerase chain reaction, denaturing gradient gel electrophoresis, nucleic acid typing chip detection, denaturing high performance liquid chromatography, in situ hybridization and HRM method;
- the detection agent for PRDM9 transposon fusion is used to perform any one of the following methods: polymerase chain reaction, denaturing gradient gel electrophoresis, nucleic acid typing chip detection, denaturing high performance liquid chromatography, in situ hybridization, biological mass spectrometry and HRM method.
- the primers preferably include at least one of a to c:
- the CMV detection reagent comprises a CMV antibody
- the CMV antibody is preferably a CMV 1gM antibody.
- a product for diagnosing Hirschsprung's disease characterized in that the product comprises a detection reagent of CMV and a detection reagent of PRDM9 transposon fusion.
- the primers preferably include at least one of a to c:
- sample to be tested for which the product is applicable is blood, plasma, serum, feces, urine, saliva, amniotic fluid, villi, tissue, cell, tissue or At least one of the cell lysate samples is preferably blood, plasma or serum, more preferably blood, plasma or serum from peripheral blood.
- diagnostic marker combination according to 1 is characterized in that: described PRDM9 transposon fusion comprises and is selected from PRDM9-type I at chr5:23299411, PRDM9-type II-1 at chr5:23262356, PRDM9-type II-1 at chr5 : at least one, at least two or three of PRDM9-type II-2 of 23245181.
- the diagnostic marker combination according to 1 or 2 characterized in that: comprising cytomegalovirus and PRDM9-type I.
- the diagnostic marker combination according to 1 or 2 characterized in that: comprising cytomegalovirus and PRDM9-type II-1.
- the diagnostic marker combination according to 1 or 2 characterized in that: comprising cytomegalovirus and PRDM9-type II-2.
- the diagnostic marker combination according to 1 or 2 characterized in that: comprising cytomegalovirus, PRDM9-type II-1 and PRDM9-type II-2.
- the diagnostic marker combination according to 1 or 2 characterized in that: comprising cytomegalovirus, PRDM9-type I and PRDM9-type II-1.
- the diagnostic marker combination according to 1 or 2 characterized in that: comprising cytomegalovirus, PRDM9-type I and PRDM9-type II-2.
- the diagnostic marker combination according to 1 or 2 characterized in that: comprising cytomegalovirus, PRDM9-type I, PRDM9-type II-1 and PRDM9-type II-2.
- a diagnostic marker combination for Hirschsprung's disease comprising at least two, at least three or four selected from the following: comprising PRDM9-type I selected from the group at chr5:23299411, PRDM9-type at chr5:23262356 type II-1, PRDM9-type II-2 at chr5:23245181, and cytomegalovirus.
- the diagnostic marker combination according to any one of 12-15, characterized in that it further comprises cytomegalovirus.
- a diagnostic product for Hirschsprung's disease comprising a detection agent for PRDM9 transposon fusion, and/or a detection agent for cytomegalovirus.
- the PRDM9 transposon fusion comprises and is selected from the PRDM9-type I at chr5:23299411, the PRDM9-type II-1 at chr5:23262356, and the PRDM9-type II-1 at chr5:23262356. : at least one, at least two or three of PRDM9-type II-2 of 23245181.
- a diagnostic product for Hirschsprung's disease comprises: PRDM9-type I located at chr5:23299411, PRDM9-type II-1 located at chr5:23262356, PRDM9-type II-2 located at chr5:23245181 , and a detection agent for at least one, at least two, at least three or four diagnostic markers of cytomegalovirus.
- a detection agent in the preparation of a Hirschsprung's disease diagnostic product; the product includes a detection test paper or strip, a detection chip, a reagent or a kit; the detection agent comprises: a detection agent for PRDM9 transposon fusion, and/ or cytomegalovirus detection agent.
- the detection agent comprises a detection agent for cytomegalovirus.
- the detection agent comprises a PRDM9 transposon fusion detection agent.
- PRDM9 transposon fusion comprises and is selected from PRDM9-type I that is positioned at chr5:23299411, PRDM9-type II-1 that is positioned at chr5:23262356, is positioned at chr5: At least one, at least two or three of PRDM9-type II-2 of 23245181.
- the detection agent comprises a PRDM9-type I detection agent.
- the detection agent comprises a detection agent of PRDM9-typeII-1.
- the detection agent comprises a detection agent of PRDM9-typeII-2.
- the detection agent comprises a PRDM9-type I detection agent and a PRDM9-typeII-1 detection agent.
- the detection agent comprises a PRDM9-type I detection agent and a PRDM9-typeII-2 detection agent.
- the detection agent comprises a PRDM9-typeII-1 detection agent and a PRDM9-typeII-2 detection agent.
- the detection agent comprises detection agents of PRDM9-type I, PRDM9-typeII-1 and PRDM9-typeII-2.
- the detection agent further comprises a detection agent for cytomegalovirus.
- a detection agent in the preparation of a Hirschsprung's disease diagnostic product; the product includes a detection test paper or test strip, a detection chip, a reagent or a kit; the detection agent comprises: PRDM9-type I located at chr5:23299411, located at A detection agent for at least one, at least two, at least three or four diagnostic markers of PRDM9-type II-1 at chr5:23262356, PRDM9-type II-2 at chr5:23245181, and cytomegalovirus.
- the detection agent comprises a detection agent for four diagnostic markers of PRDM9-type I, PRDM9-type II-1, PRDM9-type II-2, and cytomegalovirus.
- the diagnostic marker combination, diagnostic product or use according to any one of the preceding items characterized in that: the diagnostic object of the early diagnosis is a newborn who is less than 1 month old.
- the diagnostic marker combination, diagnostic product or use according to any one of the preceding items, characterized in that: the cytomegalovirus detection agent or quantitative detection agent is used to perform any one of the following methods: radioimmunoassay, indirect Immunofluorescence, dot immunogold filtration, biological mass spectrometry, western blotting and enzyme-linked immunosorbent assay, polymerase chain reaction, denaturing gradient gel electrophoresis, nucleic acid typing chip detection, denaturing high performance liquid chromatography, original Bit hybridization and HRM method.
- the cytomegalovirus detection agent or quantitative detection agent is used to perform any one of the following methods: radioimmunoassay, indirect Immunofluorescence, dot immunogold filtration, biological mass spectrometry, western blotting and enzyme-linked immunosorbent assay, polymerase chain reaction, denaturing gradient gel electrophoresis, nucleic acid typing chip detection, denaturing high performance liquid chromatography, original Bit hybridization and HRM method.
- cytomegalovirus detection of the cytomegalovirus is to detect the lgM antibody of cytomegalovirus; or to detect cytomegalovirus lgM antibody and lgG antibody .
- the primers also include at least one that is used to detect PRDM9-type I, PRDM9-type II-1 and PRDM9-type II-2.
- An internal reference primer corresponding to the wild-type sequence.
- test sample to which the detection agent or quantitative detection agent is applicable is selected from the group consisting of blood, plasma, serum, feces, urine at least one of fluid, saliva, amniotic fluid, villi, tissue, cells, tissue or cell lysate samples.
- test sample to which the detection agent or quantitative detection agent is applicable is selected from at least one of blood, plasma or serum. kind.
- test sample for which the detection agent or quantitative detection agent is applicable is selected from blood, plasma or serum from peripheral blood at least one of.
- a primer characterized in that it comprises at least one selected from the following a ⁇ c:
- the primer according to 67 characterized in that: comprising SEQ ID NOs: 1 ⁇ 2 for detecting PRDM9-type I.
- the primer according to 67 characterized in that it comprises SEQ ID NOs: 3-4 for detecting PRDM9-type II-1.
- the primer according to 67 characterized in that it comprises SEQ ID NOs: 5-6 for detecting PRDM9-type II-2.
- the primer according to 67 characterized in that: it comprises SEQ ID NOs: 1-2 for detecting PRDM9-type I; and SEQ ID NOs: 3-4 for detecting PRDM9-type II-1.
- the primer according to 67 characterized in that: it comprises SEQ ID NOs: 1-2 for detecting PRDM9-type I; and SEQ ID NOs: 5-6 for detecting PRDM9-type II-2.
- the primer according to 67 characterized in that it comprises: SEQ ID NOs: 3-4 for detecting PRDM9-type II-1; and SEQ ID NOs: 5-6 for detecting PRDM9-type II-2.
- primers according to 67 are characterized in that: comprise the SEQ ID NOs: 1 ⁇ 2 for detecting PRDM9-type I; SEQ ID NOs: 3 ⁇ 4 for detecting PRDM9-type II-1; Detection of SEQ ID NOs: 5-6 of PRDM9-type II-2.
- a reagent, kit or detection chip comprising the primer of any one of 66-76.
- the diagnosis object of the early diagnosis is a newborn whose age is less than 3 months.
- the diagnostic object of the early diagnosis is a newborn who is less than 1 month old.
- test results of the present invention are all statistically analyzed, t test is used to evaluate the difference between the two groups, and machine learning evaluates the difference in clinical parameters such as gender, age, disease phenotype, etc. of transposon fusion, and the statistical method is altmann. p ⁇ 0.05 was used to indicate statistical significance and all p-values were tested using two-sided. Statistical analysis was performed using R and Graphpad 8.0 software.
- Example 1 Screening process of Hirschsprung's transposon fusion genetic markers
- the present invention proves that the PRDM9 transposon fusion can be used for early diagnosis and screening of Hirschsprung's disease, and fills the gap in the field of early diagnosis of Hirschsprung's disease.
- the following are specific embodiments of the present invention.
- the screening process of Hirschsprung's congenital transposon fusion genetic marker used in this example is shown in FIG. 1 .
- the specific steps include: taking tissue samples from Hirschsprung's disease surgery, selecting paired tissue samples without nerve segments and with nerve segments for whole transcriptome sequencing, detecting transposons and genome chimeric sequences, and searching for chimeric paired Half of the reads of -end are reads of transposon sequences, determine the positions where these transposons are inserted, and statistically analyze the total number of reads in the ganglioned and non-neural segments of the chimeric transcription of each insertion site, using the total number of reads.
- the number of sequencing sequences was normalized, and limma R was used for differential expression analysis to analyze significantly changed transposon fusion sites.
- the found transposon fusion sites are divided into two types: down-regulated (type I) and up-regulated (type II) in non-neural segments.
- type I down-regulated
- type II up-regulated
- the genes in the sample are divided into three types. , including carrying I and II transposons (gene I, II), and not carrying transposons (gene N).
- the phenotypes of the disease were collected, these phenotypes were closely related to the disease, including the subtype of the disease (increasing in severity: short-segment, normal, long-segment, pancolonic), whether there was enteritis, whether there was virus infection , gender and age.
- machine learning methods analyze the relationship between disease phenotypes and gene transposon types, and predict possible driver genes.
- the screened transposon fusion genes were validated by qPCR in Hirschsprung's cohort and neonatal bloating cohort.
- Hirschsprung's cohort Blood DNA samples were divided into Hirschsprung's disease children group (32 cases), other enteropathy control group (16 cases), healthy children group (27 cases), age ranged from 3 months to 3 years, gender Three-quarters of males were male, and disease and controls were matched for age and sex. All samples were obtained from Guangzhou Women's and Children's Medical Center, and the healthy children group was the remaining blood samples after physical examination of healthy children.
- the blood collection method is anticoagulation blood collection, centrifugation to separate leukocytes to extract DNA.
- Colon tissue samples were surgically resected diseased tissues from 52 children with Hirschsprung's disease, including nerve segments and non-nervous segments.
- Neonatal bloating cohort 1 Whole blood was collected from neonates diagnosed with neonatal bloating, within 1 month of age, and followed up for one year. Finally, it was determined that the cohort included the group of children with Hirschsprung's disease (13 cases) and the group of children without Hirschsprung's disease (28 cases). 15 cases of other enteropathy (including intestinal atresia, intestinal stricture, neonatal necrotizing enterocolitis) and 13 cases of physiological abdominal distension in non Hirschsprung's disease, DNA was extracted from blood.
- enteropathy including intestinal atresia, intestinal stricture, neonatal necrotizing enterocolitis
- Neonatal abdominal distension cohort 2 a total of 44 cases, this cohort is derived from children admitted to the NICU due to unexplained abdominal distension within 1 month. A total of 13 neonates in this cohort were confirmed to be Hirschsprung patients, and 16 were controls for other intestinal diseases (including anal atresia, intestinal stricture, etc.). Another 15 patients with gastrointestinal disorders served as controls for nonorganic lesions.
- Neonatal abdominal distension cohort 3 a total of 40 cases, this cohort was derived from neonates within 1 month, including the group of children with Hirschsprung's disease (12 cases); the group of children without Hirschsprung's disease (28 cases), non-congenital Hirschsprung's children included 15 cases of other enteropathy (including intestinal atresia, intestinal stricture, neonatal necrotizing enterocolitis), and 13 cases of physiological abdominal distension.
- sequenced fastq files were de-linked with Trimmomatic-0.36, short and low-quality sequences were removed, and STAR was used to align with the human genome (hg19), HTSeq-count was used to calculate gene expression, and edgeR was used to calculate log2 (cpm).
- Gene expression was denormalized with voom, differentially expressed genes were calculated with limma R package, and FDR was used for multiple comparisons. Genes with significant differential expression were defined as P-value ⁇ 0.05 and log2FC>1. Gene expression values were converted into Z-scores, and heatmaps of differentially expressed genes were drawn using pheatmap. DAVID (version 6.8) was used for signal pathway enrichment analysis of differentially expressed genes, and the parameter was set to EASE value ⁇ 0.1 as cut-off.
- transposon fusion sites using the same method to detect chimeric sequences, find half of the chimeric paired-end reads are reads of transposon sequences, determine the positions where these transposons are inserted, and statistically analyze each insertion
- the chimeric transcription of the site was in the total number of reads with and without ganglion segments, normalized by the total sequencing number, and differential expression analysis was performed with limma R to obtain differential transposon fusion sites.
- differential transposon fusion sites fall into two categories, one that is down-regulated (type I) in ganglionic segments and one that is up-regulated (type II).
- FIG 2 shows differentially expressed transposon fusion sites in Hirschsprung's disease. There are significant changes in the expression of transposon fusion sites in Hirschsprung's disease, and these differential transposon fusion sites are divided into two categories, one is downregulated in aneurysmal segments (ganglionic segments) (type I) and a type of up-regulation (type II).
- type I aneurysmal segments
- type II a type of up-regulation
- the found non-neural segment down-regulated (type I) and up-regulated (type II) transposon fusion sites are associated with their most adjacent genes, and according to the type of transposon fusion sites carried by the most adjacent genes in the sample.
- the genes in the samples were divided into three types, including type I and II transposon fusion sites (gene I, II), and those without transposon fusion sites (gene N).
- type I and II transposon fusion sites gene I, II
- those without transposon fusion sites gene N
- the phenotypes of the disease were collected, these phenotypes were closely related to the disease, including the subtype of the disease (increasing in severity: short-segment, normal, long-segment, pancolonic), whether there was enteritis, whether there was virus infection , gender and age.
- the random forest method was used for phenotypic feature selection, and the phenotype-related transposon fusion genes of the disease were analyzed to predict possible driver genes. Using machine learning methods, the transposon fusion genes most significantly associated with Hirschsprung's subtypes were selected.
- the three transposon fusion positions of PRDM9 are chr5 23299411, 23262356, 23245181 (the version number of position resolution is hg19).
- the PRDM9 fusion transposon is located in the upstream non-coding region of PRDM9, and it is speculated that it may affect the transcription of PRDM9.
- the sequences (E1+TE, E2+TE, E3+TE) fused with PRDM9 and three transposons were combined with (E1+TE+C1, E2+TE+C2, E3+TE+C3), two control sequences ( E1+C1, E2+C2, E3+C3) and (C1, C2, C3) were put into the nano-luciferase reporter gene PNL3.1, respectively, with firefly luciferase as a control, and transiently transformed into 293T and SKNSH cell lines , two-channel detection of the intensity of the reporter gene to determine the effect of transposon insertion on PRDM9 expression.
- TE refers to transposon
- E(E1, E2, E3) refers to the gene sequence of the fusion site with the transposon for the three sites
- C(C1, C2, C3) refers to the three sites For each site, when there is no transposon fusion, the sequence downstream of the fusion site.
- PRDM9-type I fusion would further activate PRDM9 enhancer expression
- PRDM9-type II-1, PRDM9-type II-2 fusion would inhibit PRDM9 enhancer expression (see Figure 4).
- the three transposon fusion positions of PRDM9 are chr5 23299411, 23262356, 23245181, and the sequences of the three and the control sequences are shown below.
- PRDM9-type I is located at position 23299411 of human chromosome 5 (chr5 23299411), and its nucleotide sequence is:
- PRDM9-type II-1 is located at position 23262356 of human chromosome 5 (chr5 23262356), and its nucleotide sequence is:
- PRDM9-type II-2 is located at position 23245181 of human chromosome 5 (chr5 23245181), and its nucleotide sequence is:
- Two pairs of primers were designed for each fusion site to detect the DNA levels of wild-type (wt) and fusion-type respectively.
- the wild-type was used as an internal reference, and the primer sequences were shown below.
- Upstream primer F 5'-GGGTGACAGAGTGAGACTCCA-3' (SEQ ID NO.1);
- Downstream primer R 5'-CTGAACCCACCTCTGTCCTG-3' (SEQ ID NO. 2).
- Upstream primer F 5'-GGGTGACAGAGTGAGACTCCA-3' (SEQ ID NO.7);
- Downstream primer R 5'-TTGAAGAGGCTTGTCCAGAGA-3' (SEQ ID NO. 8).
- Upstream primer F 5'-GCTCTAGGAAGCAAATAAAAAGGA-3' (SEQ ID NO.3);
- Downstream primer R 5'-CTGTCTCAAAAAAAAAAAAAAAA-3' (SEQ ID NO. 4).
- Upstream primer F 5'-ATTGGGTTTTATCCCGGTTT-3' (SEQ ID NO.9);
- Downstream primer R 5'-CTGTCTCAAAAAAAAAAAAAAAA-3' (SEQ ID NO. 10).
- Upstream primer F 5'-TATTCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT
- Downstream primer R 5'-AAAATTAACTGGGCGTGGTG-3' (SEQ ID NO. 6).
- Upstream primer F 5'-TATTCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT
- Downstream primer R 5'-TGCATGTATATACTTTGAGACAAAACC-3' (SEQ ID NO. 12).
- the ROC curve was used to evaluate the diagnostic effect of PRDM9 transposon fusion on Hirschsprung's disease (Figure 5).
- the AUC values for PRDM9-type I were 0.7885, 0.8503 for PRDM9-type II-1, and 0.7791 for PRDM9-type II-2.
- PRDM9-type I was not effective in distinguishing megacolon from non-megacolon in neonatal abdominal distension, with an AUC value of 0.6032.
- PRDM9-type II was able to better distinguish megacolon from non-megacolon, among which the AUC of PRDM9-type II-1 was 0.7414, and the AUC of PRDM9-type II-2 was 0.7098.
- the AUC of the two PRDM9-type II combined detection (PRDM9-typeII-1+PRDM9-typeII-2) reached 0.7586, and the best limit corresponds to a specificity of 72.41% and a sensitivity of 83.33% (see Figure 6). It can be seen that PRDM9 transposon fusion, especially PRDM9-type II transposon fusion, can be used for the early diagnosis of Hirschsprung's disease.
- Example 9 CMV infection is positively correlated with Hirschsprung's severity
- the inventors In order to study the correlation between CMV infection and the severity of Hirschsprung's colon, the inventors analyzed the virus infection and HSCR subtypes (short segment type, common type, long segment type, whole colon) in the tissue samples extracted in Example 2. Correlation analysis was carried out.
- Example 10 CMV lgM antibodies in neonates are higher than other types of abdominal distension in children with abdominal distension finally diagnosed with Hirschsprung's disease
- the inventors compared the cryopreserved serum of neonates with abdominal distension of Hirschsprung's disease and the cryopreserved serum of neonates with abdominal distension without Hirschsprung's disease to explore whether CMV infection in the neonatal period is related to the occurrence of HSCR.
- CMV lgM and IgG in serum By detecting the content of CMV lgM and IgG in serum (detection method adopts the laboratory detection method routine in the field, in the following examples, all use ELISA method to detect).
- the inventor found that in the neonatal period, the CMV lgM antibody in the body of the neonatal abdominal distension patient who was finally diagnosed as megacolon was significantly higher than that of the non-megacolon neonatal abdominal distention patient. Moreover, within 1 to 2 months of diagnosis and treatment, it was also found that the CMV lgM of these children diagnosed with megacolon would be significantly decreased, but the IgG would be significantly increased. This opposite change often predicts acute CMV infection. It can be seen that CMV infection in the neonatal period is related to Hirschsprung's disease. Detection of CMV lgM antibodies in neonates can provide strong evidence for the early diagnosis of Hirschsprung's disease. .
- Example 11 Diagnostic effect of CMV combined with PRDM9 transposon fusion for early diagnosis of Hirschsprung's disease
- the invention People have been followed up by telephone for at least half a year (2019-2020), and finally confirmed that a total of 13 neonates in this cohort were Hirschsprung patients and 16 were controls with other intestinal diseases (including anal atresia, intestinal stricture, etc.). Another 15 patients with gastrointestinal disorders served as controls for nonorganic lesions.
- qPCR was performed on its sample DNA to analyze the level of PRDM9-TE fusion, and ELISA was used to detect the level of CMV lgM in its serum.
- PRDM9-TE ratio (PRDM9-type II-1)+(PRDM9-type II-2)-(PRDM9-type I); corresponding to the same person
- the serum of CMV was tested by ELISA to detect the level of lgM antibody to CMV.
- the abnormal fusion level of CMV combined with PRDM9-TE was fitted as 0.1 ⁇ CMV+PRDM9-TE ratio, and the fitting parameters were obtained by referring to the parameters of logistic regression.
- the AUC of PRDM9-TE to identify megacolon and non-megacolon can reach 0.8425 (95% CI, 0.7122 to 0.9728).
- the content of CMV lgM antibody in megacolon was also higher than that in non-megacolon, with an AUC of 0.8571 (95% CI, 0.7290 to 0.9853).
- the AUC of the combined detection for distinguishing Hirschsprung from Hirschsprung's colon was significantly improved compared with the single detection, which could reach 0.9267 (95%CI, 0.8403to 1.000). When the two reached the best limit, the corresponding specificity was 91.48% and the sensitivity was 84.62%. This result indicates that PRDM9-TE fusion and CMV infection can synergistically and effectively predict the occurrence of megacolon, and the combination of the two can be used as an effective marker combination for the diagnosis or early diagnosis of megacolon.
- Example 12 The effect of CMV combined with PRDM9 transposon fusion on early diagnosis of Hirschsprung's disease was verified by the abdominal distension cohort of cryopreserved samples
- CMV lgM antibody and PRDM9 transposon fusion In order to further evaluate the stability of the diagnostic effect of the above combined diagnostic markers (CMV lgM antibody and PRDM9 transposon fusion), the inventors used a retrospective cohort of neonatal abdominal distension with cryopreserved DNA and serum as the test object for verification. sex test.
- the cryopreserved DNA was used to perform qPCR based on the primers described in Example 6 to detect the level of the PRDM9-TE fusion site, and then the serum corresponding to the same person was used to perform ELISA to detect the level of lgM antibody to CMV.
- the serum of CMV was tested by ELISA to detect the level of lgM antibody to CMV.
- the abnormal fusion level of CMV combined with PRDM9-TE was fitted as 0.1 ⁇ CMV+PRDM9-TE ratio, and the fitting parameters were obtained by referring to the parameters of logistic regression.
- the present invention through the screening and verification of Hirschsprung's congenital transposon fusion genetic markers, found that when PRDM9 transposon fusion was used to diagnose children with Hirschsprung's disease, the PRDM9-type I AUC value was 0.7885, and the PRDM9- Type II-1 was 0.8503 and PRDM9-type II-2 was 0.7791.
- the AUC of the three combined detection was 0.9666, the best limit corresponding to the specificity was 95.35%, and the sensitivity was 84.38%.
- the blood DNA of neonatal infants with abdominal distension within one month was collected, and the PRDM9 transposon fusion was detected. It was found that PRDM9-type II could better distinguish megacolon from non-megacolon, and the PRDM9-type II-1AUC was 0.7414, and 0.7098 for PRDM9-type II-2. The AUC of the combined detection of the two was 0.7586, the best limit corresponds to a specificity of 72.41% and a sensitivity of 83.33%. It can be seen that the PRDM9 transposon fusion can be used for the early diagnosis and screening of Hirschsprung's disease, filling the gap in the early diagnosis of Hirschsprung's disease.
- the present invention finds the viral CMV closely related to Hirschsprung's disease through the screening of Hirschsprung's disease factors, and confirms that the quantitative detection of CMV for the diagnosis of children's Hirschsprung's disease, especially the early diagnosis, has good results.
- the diagnostic effect of , the AUC reached levels of 0.7173 and 0.8571 in two independent testing cohorts, respectively.
- CMV combined with PRDM9 transposon fusion level detection can further improve the diagnosis or early diagnosis of Hirschsprung's disease.
- the best cutoff corresponds to a specificity of 91.48% and a sensitivity of 84.62%; AUC 0.8899 (95%CI, 0.7842 to 0.9956), the best cutoff corresponds to a specificity of 82.25% and a sensitivity of 83.33%.
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Abstract
本发明公开了先天性巨结肠诊断或早期诊断标志物及其应用，其利用PRDM9转座子融合和/或CMV中的一种或两种检测作为先天性巨结肠的标记物，以及所述标志物的检测剂在制备试剂或试剂盒等先天性巨结肠诊断产品中的应用，达到了相对于传统诊断方法(钡灌肠等)，具有确诊时间更早、操作简单快速、无介入、通量高、成本低等众多综合优势，有效地弥补了现有技术中的不足，是一种有效的先天性巨结肠诊断(尤其是早期诊断)的替代或辅助检测手段。而且，本方法稳定性好，在多个独立队列中都得到较好的诊断效果，填补了在先天性巨结肠诊断或早期诊断领域中的空白。
Description
本发明属于生物医学领域，具体涉及先天性巨结肠的诊断或早期诊断标志物及其应用。
先天性巨结肠(Hirschsprung disease，HSCR)是一种儿童肠神经发育异常的出生缺陷疾病，病理机制为肠神经嵴细胞迁移和分化成肠神经元发生障碍，导致肠神经缺乏而发生持续性痉挛，是小儿常见的先天性肠道疾病之一。先天性巨结肠的早期表现为呕吐、腹胀、腹泻等，临床上会造成新生儿死亡或手术后反复肠炎、难治性便秘等并发症，严重影响患儿的生长发育和生活质量。
先天性巨结肠的及时诊治可以减少先天性巨结肠肠炎发生的危险，获得良好的预后。该疾病确诊需要术后病变组织的病理切片。术前诊断方法主要为钡剂灌肠，直肠活检、直肠测压，以判断是否要实施“巨结肠根治术”。目前，钡灌肠为最重要的诊断方法，原理为先天性巨结肠患儿的肠道存在无神经节段的狭窄、近端的扩张，钡灌肠后可见扩张和狭窄段而诊断为巨结肠。但该方法只能诊断出有典型肠道形态改变的患儿，灵敏度需要提高，诊断的准确度80％左右。直肠活检是直接取直肠组织，检测是否有神经节细胞的缺失，准确率高，但取样部位对结果有影响。该方法为介入有创型，且价格非常高，一般情况下该方法应用于钡灌肠不明显，或不适用的患儿。当患儿有坏死性小肠结肠炎(NEC)这种可能的时候，钡灌肠会导致肠穿孔，这种时候不适合用钡灌肠去诊断，才考虑用直肠活检。可见，先天性巨结肠所常用的钡灌肠检测、直肠活检方法具有有创性，对希望获得疾病确诊的新生儿患者及其父母均带来一定的身心痛苦。先天性巨结肠患儿表现为呕吐、腹胀、便秘、肠炎等，此类症状的患儿群体很大，其中为先天性巨结肠的儿童占比又很少，上述方法对仪器设备的要求高，价格也昂贵，不适合用于疾病的筛选。除了钡灌肠检测、直肠活检外，还可采用无创的直肠测压方法，直肠测压是通过检测肛门内括约肌的松弛缺乏判定肠神经的支配异常，虽然无创，但其假阳性、假阴性率均较高，并不能有效和准确地对先天性巨结肠疾病进行确诊。因而直肠测压方法仅为辅助诊断方法，不能用于单独检测。
此外，先天性巨结肠患儿大部分早在刚出生的新生儿期(一个月内)就会表现出新生儿腹胀、呕吐、肠梗阻等症状，但大部分从钡灌肠中不会看到明显的扩张和狭窄段。大多数患儿都是在3个月后经过反复的腹胀后，在钡灌肠下可见明显的扩张、狭窄段才诊断为巨结肠，确定行巨结肠根治术。由于引起早期新生儿腹胀可能的疾病还可能有肠紊乱、蛋白过敏、肠闭锁、肠狭窄、新生儿坏死性小肠结肠炎等，只有很少一部分是先天性巨结肠，钡灌肠又没有那么灵敏，这就给先天性巨结肠患儿的诊断或早期诊断带来了很大挑战。
因此，急需一种灵敏度高的诊断方法，特别是早期诊断方法，将这些先天性巨结肠患儿筛查出来，以便早干预，减少疾病的死亡率和改善预后。
发明内容
为克服上述现有技术的缺点，本发明的目的在于提供先天性巨结肠的诊断标志物及其应用，为先天性巨结肠的诊断(特别是早期诊断)提供了一种新的准确、灵敏的检测途径。
为了实现上述目的，本发明采用以下技术方案予以实现：
本发明的第一方面，提供一种先天性巨结肠的诊断标志物组合，包含PRDM9转座子融合和巨细胞病毒(Cytomegalovirus,CMV)。
在一些实施方式中，所述PRDM9转座子融合包含选自PRDM9-type I、PRDM9-type II-1、PRDM9-type II-2中的至少一种、至少两种或三种。
本发明的第二方面，提供一种先天性巨结肠的诊断标志物组合，包含选自以下的至少两种、或三种：PRDM9-type I；PRDM9-type II-1；PRDM9-type II-2。
在一些实施方式中，所述诊断标志物组合包含：PRDM9-type II-1、和PRDM9-type II-2。在一些实施方式中，所述诊断标志物组合还包含巨细胞病毒。
在一些实施方式中，所述诊断标志物组合包含：PRDM9-type I、和PRDM9-type II-1。在一些实施方式中，所述诊断标志物组合还包含巨细胞病毒。
在一些实施方式中，所述诊断标志物组合包含：PRDM9-type I、和PRDM9-type II-2。在一些实施方式中，所述诊断标志物组合还包含巨细胞病毒。
在一些实施方式中，所述诊断标志物组合包含：PRDM9-type I、PRDM9-type II-1、和PRDM9-type II-2。在一些实施方式中，所述诊断标志物组合还包含巨细胞病毒。
本发明的第三个方面，提供一种先天性巨结肠的诊断产品，所述产品包含PRDM9转座子融合的检测剂，和/或巨细胞病毒的检测剂。
在一些实施方式中，所述产品是检测试纸或试纸条、检测芯片、试剂、试剂盒、或本领域其他常规的产品形式。
在一些实施方式中，所述PRDM9转座子融合包含选自PRDM9-type I、PRDM9-type II-1、PRDM9-type II-2中的至少一种、至少两种或三种。
本发明的第四个方面，提供一种先天性巨结肠的诊断产品，所述产品包含：PRDM9-type I、PRDM9-type II-1、PRDM9-type II-2中至少一种、至少两种或三种诊断标志物的检测剂。
在一些实施方式中，所述产品是检测试纸或试纸条、检测芯片、试剂、试剂盒、或本领域其他常规的产品形式。
在一些实施方式中，所述诊断产品包含：PRDM9-type II-1的检测剂和/或PRDM9-type II-2的检测剂。在一些实施方式中，所述诊断产品还包含巨细胞病毒的检测剂。
在一些实施方式中，所述诊断产品包含：PRDM9-type I的检测剂和/或PRDM9-type II-1的检测剂。在一些实施方式中，所述诊断产品还包含巨细胞病毒的检测剂。
在一些实施方式中，所述诊断产品包含：PRDM9-type I的检测剂和/或PRDM9-type II-2的检测剂。在一些实施方式中，所述诊断产品还包含巨细胞病毒的检测剂。
在一些实施方式中，所述诊断产品包含：PRDM9-type I、PRDM9-type II-1、和PRDM9-type II-2。在一些实施方式中，所述诊断产品还包含巨细胞病毒的检测剂。
本发明的第五个方面，提供本发明第三方面或第四方面所述诊断产品在制备用于先天性巨结肠诊断或辅助诊断 的试剂或试剂盒中的用途。
根据本发明的前述方面，在一些实施方式中，所述PRDM9转座子融合的融合位点包含选自以下的至少一种、至少两种、或三种：PRDM9-type I；PRDM9-type II-1；和PRDM9-type II-2。在一些实施方式中，所述PRDM9转座子融合的融合位点包含：PRDM9-type I。在一些实施方式中，所述PRDM9转座子融合的融合位点包含：PRDM9-type II-1。在一些实施方式中，所述PRDM9转座子融合的融合位点包含：PRDM9-type II-2。在一些实施方式中，所述PRDM9转座子融合的融合位点选自：PRDM9-type I和PRDM9-type II-1。在一些实施方式中，所述PRDM9转座子融合的融合位点选自：PRDM9-type I和PRDM9-type II-2。在一些实施方式中，所述PRDM9转座子融合的融合位点选自：PRDM9-type II-1和PRDM9-type II-2。在一些实施方式中，所述PRDM9转座子融合包含PRDM9-type I、PRDM9-type II-1、PRDM9-type II-2。
根据本发明的前述方面，在一些实施方式中，所述PRDM9-type I位于chr5:23299411。在一些实施方式中，PRDM9-type I未发生融合的序列包含如SEQ ID NO:13所示序列，发生融合后的序列包含如SEQ ID NO:16所示序列。在一些实施方式中，所述PRDM9-type II-1位于chr5:23262356。在一些实施方式中，PRDM9-type II-1未发生融合的序列包含如SEQ ID NO:14所示序列，发生融合后的序列包含如SEQ ID NO:17所示序列。在一些实施方式中，所述PRDM9-type II-2位于chr5:23245181。在一些实施方式中，PRDM9-type II-2未发生融合的序列包含如SEQ ID NO:15所示序列，发生融合的序列包含如SEQ ID NO:18所示序列。
根据本发明的前述方面，在一些实施方式中，所述巨细胞病毒是人巨细胞病毒。在一些实施方式中，检测所述巨细胞病毒是检测巨细胞病毒的感染；优选的，是检测巨细胞病毒的抗体；更优选的，是检测巨细胞病毒的lgM抗体；或更优选的，是检测巨细胞病毒lgM抗体和lgG抗体。
根据本发明的前述方面，在一些实施方式中，所述诊断是早期诊断。在一些实施方式中，所述早期诊断的诊断对象为年龄小于3个月的新生儿。在一些实施方式中，所述早期诊断的诊断对象为年龄小于1个月的新生儿。
根据本发明的前述方面，在一些实施方式中，所述检测为定量检测。在一些实施方式中，所述PRDM9转座子融合的检测剂或定量检测剂用于执行以下任一种方法：聚合酶链反应、变性梯度凝胶电泳、核酸分型芯片检测、变性高效液相色谱法、原位杂交、生物质谱法以及HRM法。在一些实施方式中，所述巨细胞病毒的检测剂或定量检测剂用于执行以下任一种方法：放射免疫法、间接免疫荧光法、斑点免疫金渗滤法、生物质谱法、免疫印记法和酶联免疫吸附法，聚合酶链反应、变性梯度凝胶电泳、核酸分型芯片检测、变性高效液相色谱法、原位杂交以及HRM法。
根据本发明的前述方面，在一些实施方式中，所述检测剂或定量检测剂包括引物。在一些实施方式中，所述引物包括a～c中的至少一种：
a.用于检测PRDM9-type I的SEQ ID NO：1～2；
b.用于检测PRDM9-type II-1的SEQ ID NO：3～4；和
c.用于检测PRDM9-type II-2的SEQ ID NO：5～6。
根据本发明的前述方面，可选的，如上所述的应用，所述引物还包括用于检测PRDM9-type I、PRDM9-type II-1 和PRDM9-type II-2中至少一个所对应的野生型序列的内参引物。在一些实施方式中，所述内参引物包括d～f中的至少一种：
d.用于检测PRDM9-type I所对应的野生型序列的SEQ ID NO：7～8；
e.用于检测PRDM9-type II-1所对应的野生型序列的SEQ ID NO：9～10；以及
f.用于检测PRDM9-type II-2所对应的野生型序列的SEQ ID NO：11～12。
根据本发明的前述方面，在一些实施方式中，所述检测剂或定量检测剂还含有DNA提取试剂、双链特异性荧光染料、dNTP、DNA聚合酶、双链特异性荧光染料以及水中的一种或多种。
根据本发明的前述方面，在本发明的一些实施方式中，所述试剂或试剂盒的受试样品选自血液、血浆、血清、粪便、尿液、唾液、羊水、绒毛、组织、细胞、组织或细胞裂解物样品中至少一种；在本发明的一些优选实施方式中，所述试剂或试剂盒的受试样品为血液、血浆或血清，更优选为来自外周血的血液、血浆或血清。在本发明的一些优选实施方式中，检测样品为血液；在本发明的一些更优选实施方式中，检测样品来自外周血。
本发明的第六方面，提供如上文所定义的引物。
本发明的第七方面，提供包含本发明第六方面所述引物的试剂、试剂盒或检测芯片。
本发明的第八方面，提供本发明第六方面所述引物在制备先天性巨结肠诊断或辅助诊断产品中的用途。
在一些实施方式中，所述产品包括试剂、试剂盒或检测芯片。
在一些实施方式中，所述诊断是早期诊断。在一些实施方式中，所述早期诊断的诊断对象为年龄小于3个月的新生儿。在一些实施方式中，所述早期诊断的诊断对象为年龄小于1个月的新生儿。
本发明的有益效果为：
1)本发明利用DNA诊断技术、率先采用高特异性的PRDM9转座子融合位点进行儿童先天性巨结肠的诊断，所采用的产品和方法，不仅能够使得先天性巨结肠的诊断符合特异性强、灵敏度高的要求，还具有操作简单快速、无介入、通量高、成本低等众多综合优势，有效地弥补了上述现有技术中的不足，是一种有效的先天性巨结肠诊断或筛选的替代或辅助检测手段，填补了在先天性巨结肠诊断领域的空白。
2)本发明通过先天性巨结肠转座子融合遗传标记物的筛选和验证，发现PRDM9转座子融合诊断先天性巨结肠患儿时，PRDM9-type I AUC值为0.7885，PRDM9-type II-1为0.8503，PRDM9-type II-2为0.7791。三者联合检测AUC为0.9666，最佳界限对应特异性为95.35％，灵敏性为84.38％。准确性甚至优于现有诊断方法。而且本方法灵敏性为100％时，特异性为76.74％，适合用于疾病筛选。
3)进一步地，收集一个月内的新生儿腹胀患儿的血液DNA，检测PRDM9转座子融合，结果发现PRDM9-type II则能比较好的区分巨结肠和非巨结肠，PRDM9-type II-1AUC为0.7414，PRDM9-type II-2为0.7098。两者联合检测AUC为0.7586，最佳界限对应特异性为72.41％，灵敏性为83.33％。由此可见，PRDM9转座子融合可以用于先天性巨结肠的早期诊断和筛查，填补了先天性巨结肠早期诊断的空白。
4)再进一步地，本发明通过对先天性巨结肠病毒因素的筛选，找到与先天性巨结肠密切相关的病毒CMV，并证实定量检测CMV对儿童先天性巨结肠的诊断，尤其是早期诊断，具有良好的诊断效果，在两个独立的检测队列 中AUC分别达到了0.7173和0.8571的水平；填补了先天性巨结肠早期诊断的空白。
5)再进一步地，CMV联合PRDM9转座子融合水平检测，进一步提高了诊断，特别是早期诊断先天性巨结肠的效果，在两个独立的检测队列中，联合检测的诊断效果分别为：AUC 0.9267(95％CI,0.8403to 1.000)，最佳界限对应特异性为91.48％，灵敏性为84.62％；AUC 0.8899(95％CI，0.7842to 0.9956)，最佳界限对应特异性为82.25％，灵敏性为83.33％；填补了先天性巨结肠早期诊断的空白。
为了更清楚地说明本发明具体实施方式或现有技术中的技术方案，下面将对具体实施方式或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图是本发明的一些实施方式，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1为本发明实施例中的先天性巨结肠转座子融合遗传标记物的筛选流程示意图；
图2为本发明实施例中的先天性巨结肠的转录组测序及差异表达转座子分析结果；
图3为本发明实施例中的机器学习明确PRDM9转座子融合与先天性巨结肠段型密切相关；
图4为本发明实施例中的双荧光报告基因检测PRDM9转座子融合发生在转录调控区域；
图5为本发明实施例中的PRDM9转座子融合的定量PCR检测及ROC曲线评价PRDM9转座子融合对先天性巨结肠的诊断效果；
图6为本发明实施例中的ROC曲线评价PRDM9转座子融合对先天性巨结肠的早期诊断效果；
图7为本发明实施例中的筛查得到的先天性巨结肠密切有关的病毒，其中，标红的为CMV感染与巨结肠严重程度密切相关的数据；
图8为本发明实施例中的CMV抗体在不同月龄的先天性巨结肠患儿中与其它类型腹胀患儿血清中的含量对比；其中，A为lgG抗体，B为lgM抗体；
图9为本发明实施例中先天性巨结肠患儿和非先天性巨结肠患儿的CMV lgM抗体和PRDM9转座子融合联合早期诊断先天性巨结肠的诊断效果；其中，A为PRDM9-type I含量，B为PRDM9-type II-1含量，C为PRDM9-type II-2含量，D为PRDM9-TE ratio，E为CMV lgM含量，F为ROC曲线；
图10为本发明实施例中的另外一个腹胀队列验证CMV lgM抗体和PRDM9转座子融合联合早期诊断先天性巨结肠的诊断效果；其中，A为PRDM9-type I含量，B为PRDM9-type II-1含量，C为PRDM9-type II-2含量，D为PRDM9-TE ratio，E为CMV lgM含量，F为ROC曲线。
现将详细地提供本发明实施方式的参考，其一个或多个实例描述于下文。提供每一实例作为解释而非限制本发明。实际上，对本领域技术人员而言，显而易见的是，可以对本发明进行多种修改和变化而不背离本发明的范围或精神。例如，作为一个实施方式的部分而说明或描述的特征可以用于另一实施方式中，来产生更进一步的实施方式。
除非另有定义，本文所使用的所有的技术和科学术语与属于本发明的技术领域的技术人员通常理解的含义相同。本文中在本发明的说明书中所使用的术语只是为了描述具体的实施例的目的，不是旨在于限制本发明。本文所使用 的术语“和/或”包括一个或多个相关的所列项目的任意的和所有的组合。
一.术语
本文使用的术语“标志物”或“生化标志物”指要用作分析患者实验样品的靶标的分子。
本文使用的术语“CMV”为巨细胞病毒，是乙型疱疹病毒亚科的一个属，其先天感染与出生缺陷性疾病有密切关系。
本发明所述“转座子”是指一段可以从原位上单独复制或断裂下来，环化后插入另一位点，并对其后的基因起调控作用的DNA序列。
本发明所述“PRDM9”是指PR domain containing 9，是一种DNA结合蛋白，为催化组蛋白发生H3K4三甲基化的甲基转移酶，是减数分裂的重要转录调控因子和重组热点定位的主控因子。
本发明所述“PRDM9转座子融合”是指发生在PRDM9基因上的转座子融合，如本实施例所示，可选为位于PRDM9上游调控区域的如下位点有转座子融合：PRDM9-type I，PRDM9-type II-1，PRDM9-type II-2。PRDM9-type I的命名是无神经节段下调为type I的转座子，位于人第5号染色体的第23299411位(hg19)。PRDM9-type II-1，PRDM9-type II-2的命名是无神经节段上调为type II的转座子，type II有两个位点分别命名为1和2，分别位于第5号染色体的第23262356位和第23245181位(hg19)。
本发明所述“先天性巨结肠疾病”又称希尔施普龙病，是由于结肠缺乏神经节细胞导致肠管持续痉挛，粪便淤滞于近端结肠，近端结肠肥厚、扩张，是小儿常见的先天性肠道疾病之一，其中对于先天性巨结肠疾病而言，按照严重程度递增，临床上可分为短段型，普通型，长段型，全结肠型。短段型病变位于直肠近、中段，距肛管不超过6.5cm；常见型病变位于直肠近端或直肠乙状结肠远端，距肛管约9cm；长段型病变延至乙状结肠或降结肠；全结肠型病变波及全部结肠及回肠末端，距回盲瓣30cm以内。
本发明所述“早期诊断”是指对具体疾病的早期诊断。先天性巨结肠目前的诊断时间是3个月-3岁，先天性巨结肠疾病的早期诊断，是指对3个月内的新生儿进行诊断，优选在年龄为1个月内的有新生儿腹胀、胎粪延迟、新生儿肠梗阻、新生儿便秘等症状的新生儿进行的早期诊断。
本发明所述“ROC曲线”是1-特异性(假阳性率)和敏感性(真阳性率)变化的曲线，反应了二分类器的诊断能力。一个好的分类器真阳性率与假阳性率的变化比值是大于1的，远离45度直线。
本发明所述“AUC”是指ROC曲线下面积，介于0.1和1之间，用于评价分类器的好坏，越接近1分类器越好。AUC大于0.5可考虑用作疾病的诊断标志物。
本发明所述“chr5:23299411”、“chr5:23262356”、“chr5:23245181”为hg19版本中的位置。
二.诊断先天性巨结肠
早期诊断新生儿是否患有先天性巨结肠，可以早干预，减少疾病的死亡率和改善预后。先天性巨结肠患儿大部分早期诊断为新生儿腹胀，但是新生儿腹胀中只有很少一部分是先天性巨结肠。目前先天性巨结肠诊断的年龄绝大部分为3个月及以后，更早的检测因为钡灌肠中缺乏明显的扩张和狭窄段，使诊断不准确；而且，钡灌肠检测、直肠活检方法具有有创性，对希望获得疾病确诊的新生儿患者及其父母均带来一定的身心痛苦；再次，直肠测压方法 虽然无创，但其假阳性、假阴性率均较高，并不能有效和准确地对先天性巨结肠疾病进行确诊。
本发明通过来自于先天性巨结肠患者的组织的有神经节段和无神经节段的全转录组测序，分析差异表达的转座子融合，并用机器学习筛选先天性巨结肠亚型相关的转座子融合，并在先天性巨结肠队列的DNA中用定量PCR技术、ROC曲线分析，对其进行了进一步效果验证。
本发明首次发现PRDM9转座子融合可用作先天性巨结肠的诊断和早期诊断，相对于传统诊断方法(钡灌肠等)，本发明所提供的检测方法不仅能够使得先天性巨结肠的诊断符合特异性强、灵敏度高的要求，还具有确诊时间更早、操作简单快速、无介入、通量高、成本低等众多综合优势，有效地弥补了上述现有技术中的不足，是一种有效的先天性巨结肠诊断，尤其是早期诊断的替代或辅助检测手段。所述PRDM9-type I、PRDM9-type II-1、PRDM9-type II-2可以用于先天性巨结肠。在一些实施方式中，相对于健康对照组和其他肠疾病对照，所述PRDM9-type I位点融合水平在先天性巨结肠患者DNA中表现为低水平。在一些实施方式中，相对于健康对照组和其他肠疾病对照，所述PRDM9-type II-1、PRDM9-type II-2位点融合水平在先天性巨结肠患者DNA中表现为高水平。
本发明的研究结果还首次发现，CMV独立或联合PRDM9转座子融合定量检测可作为先天性巨结肠的诊断和早期诊断，相对于传统诊断方法(钡灌肠等)，本发明所提供的检测方法不仅能够使得先天性巨结肠的诊断符合特异性强、灵敏度高的要求，还具有确诊时间更早、操作简单快速、无介入、通量高、成本低等众多综合优势，有效地弥补了上述现有技术中的不足，是一种有效的先天性巨结肠早期诊断的替代或辅助检测手段。本发明通过对先天性巨结肠病毒因素的筛选，找到与先天性巨结肠密切相关的病毒CMV，并证实定量检测CMV对儿童先天性巨结肠的诊断，尤其是早期诊断，具有良好的诊断效果。同时CMV联合PRDM9转座子融合水平检测，进一步提高了诊断或早期诊断先天性巨结肠的效果。
在一些实施方式中，所述早期诊断的诊断对象为年龄小于3个月的新生儿。
在一些实施方式中，所述早期诊断的诊断对象为年龄小于1个月的新生儿。
本发明还涉及一种先天性巨结肠的诊断或早期诊断方法，其包括使用如上所定义的诊断产品检测样品中所述PRDM9转座子融合的融合位点和/或巨细胞病毒。在一些实施方式中，该产品对于先天性巨结肠的诊断方法为：使用如上所定义的检测剂(定量检测剂)检测(或定量检测)样品中所述PRDM9转座子融合的融合位点和CMV抗体。
在一些实施方式中，当检测到发生PRDM9发生转座子融合水平发生变化(例如，检测到PRDM9-typeI水平降低，PRDM9-type II-1水平升高、PRDM9-typeII-2水平升高中任意一个或多个)，则诊断或辅助诊断为先天性巨结肠。在一些实施方式中，所述PRDM9转座子融合的表达水平定义为PRDM9-typeII-1+PRDM9-typeII-2。在一些实施方式中，所述PRDM9转座子融合的表达水平定义为PRDM9-TE异常融合水平，用PRDM9-TE ratio来表示，PRDM9-TE ratio＝PRDM9-typeII-1+PRDM9-typeII-2-PRDM9-typeI。在一些实施方式中，在先天性巨结肠的诊断或者早期诊断中，所采用的PRDM9转座子融合的表达水平采用PRDM9-typeI、PRDM9-typeII-1、PRDM9-typeII-2、(PRDM9-typeII-1+PRDM9-typeII-2)和PRDM9-TE中的一个或多个来定义。
在一些实施方式中，当检测到巨细胞病毒感染(优选为检测到CMV抗体，更优选为检测到CMV IgM抗体的 存在，进一步优选地，检测到在感染CMV病毒的1～2个月中，CMV lgM会出现显著降低而CMV lgG会显著升高)，则诊断或辅助诊断为先天性巨结肠。在一些实施方式中，所述检测剂或定量检测剂用于检测PRDM9融合的表达水平和/或检测巨细胞病毒的抗体水平。在一些实施方式中，CMV联合PRDM9-TE异常融合水平拟合为0.1×CMV lgM+PRDM9-TE ratio，该拟合参数亦由本领域技术人员根据样本的实际检测情况参考逻辑回归的参数得到。本发明的CMV抗体水平与PRDM9融合的表达水平组合起到了协同诊断的作用。
在一些实施方式中，PRDM9转座子融合的融合位点检测是与对照组(例如健康人或非先天性巨结肠的其他肠病组)比较而得出结论。升高或者降低通常是显著性的，确定受试者和健康群体的初始状态(baseline)相比，是否具有显著性的差异可用本领域公知的统计学方法进行，并使用置信区间和/或p值进行确认。在一些实施方案中，置信区间可以为90％、95％、97.5％、98％、99％、99.5％、99.9％或99.99％并且p值可以为0.1、0.05、0.025、0.02、0.01、0.005、0.001或0.0001。
在一些实施方式中，上述PRDM9-typeI、PRDM9-typeII-1、PRDM9-typeII-2、PRDM9-TE ratio和CMV抗体水平中的一个或多个用于诊断巨结肠的阈值可由本领域技术人员根据实际情况检测得出。所述阈值的确定是本领域的常规技术手段。在一些实施方式中，可根据对照组(健康人或非先天性巨结肠的其他肠病组等)的各标志物表达水平选定阈值。本领域技术人员可以选定一个阈值，并测试选定所述阈值检测的准确率(如检出率、检准率、假阳性率、假阴性率、灵敏度、特异性等)，根据检测效果上调或者下调所述阈值，直至达到较佳或最佳的检测效果。
三.标志物的检测
在一些实施方式中，所述CMV的检测剂或定量检测剂用于执行以下任一种方法：放射免疫法、间接免疫荧光法、斑点免疫金渗滤法、生物质谱法、免疫印记法和酶联免疫吸附法，聚合酶链反应、变性梯度凝胶电泳、核酸分型芯片检测、变性高效液相色谱法、原位杂交以及HRM法。
在一些实施方式中，所述PRDM9转座子融合的检测剂或定量检测剂用于执行以下任一种方法：聚合酶链反应、变性梯度凝胶电泳、核酸分型芯片检测、变性高效液相色谱法、原位杂交、生物质谱法以及HRM法。
在一些实施方式中，所述聚合酶链反应选自限制性片段长度多态性法、单链构象多态性法、Taqman探针法、qPCR、竞争性等位基因特异性PCR和等位基因特异性PCR。
在一些实施方式中，所述生物质谱法选自飞行质谱仪检测。
在一些实施方式中，所述检测剂或定量检测剂包括引物。
在一些实施方式中，所述引物带有可检测的标记。如本文所用的术语“标记”指可用于提供可检测的(优选可定量的)效果且可以连接至核酸或蛋白的任何原子或分子。标记包括但不限于染料；放射性标记，诸如
32P；结合部分诸如生物素；半抗原诸如地高辛；发光、发磷光或发荧光部分；和单独的荧光染料或与可以通过荧光共振能量转移(FRET)抑制或移动发射光谱的部分组合的荧光染料。标记可以提供可通过荧光、放射性、比色、重量测定、X射线衍射或吸收、磁性、酶活性等检测的信号。标记可以是带电荷的部分(正电荷或负电荷)或可选地，可以是电荷中性的。标记可以包括核酸或蛋白序列或由其组合，只要包含标记的序列是可检测的。在一些实施方案中，核酸在没有标记的情况下直接检测(例如，直接读取序列)。
在一些实施方式中，所述标记是荧光团、比色标记、量子点、生物素以及其他可以用于探测的标签分子(如用于拉曼衍射成像的炔烃基团，用于click反应的环烯烃，用于聚合物标记的引发集团)，也可以选自多肽/蛋白分子，LNA/PNA，非天然氨基酸及其类似物(比如拟肽)，非天然核酸及其类似物(拟核苷酸)和纳米结构(包括无机纳米颗粒，NV-center，聚集/组装诱导发光分子，稀土离子配体分子，多金属氧簇等)。
在一些实施方式中，检测所述PRDM9-type I的引物的核苷酸序列为SEQ ID NO：1～2所示。
在一些实施方式中，检测所述PRDM9-type II-1的引物的核苷酸序列为SEQ ID NO：3～4所示。
在一些实施方式中，检测所述PRDM9-type II-2的引物的核苷酸序列为SEQ ID NO：5～6所示。
在一些实施方式中，所述引物包括a～c中的至少一种：
a.用于检测PRDM9-type I的SEQ ID NO：1～2；
b.用于检测PRDM9-type II-1的SEQ ID NO：3～4；以及
c.用于检测PRDM9-type II-2的SEQ ID NO：5～6。
在一些实施方式中，所述检测剂或定量检测剂用于检测或定量检测SEQ ID NO：16～18所示的至少一个片段。
在一些实施方式中，所述PRDM9-type I所对应的野生型序列的内参引物的核苷酸序列为SEQ ID NO：7～8所示。
在一些实施方式中，所述PRDM9-type II-1所对应的野生型序列的内参引物的核苷酸序列为SEQ ID NO：9～10所示。
在一些实施方式中，所述PRDM9-type II-2所对应的野生型序列的内参引物的核苷酸序列为SEQ ID NO：11～12所示。
在一些实施方式中，所述引物还包括用于检测PRDM9-type I、PRDM9-type II-1和PRDM9-type II-2中至少一个所对应的野生型序列的内参引物。
在一些实施方式中，所述内参引物包括d～f中的至少一种：
d.用于检测PRDM9-type I所对应的野生型序列的SEQ ID NO：7～8；
e.用于检测PRDM9-type II-1所对应的野生型序列的SEQ ID NO：9～10；以及
f.用于检测PRDM9-type II-2所对应的野生型序列的SEQ ID NO：11～12。
在一些实施方式中，所述内参引物用于检测或定量检测SEQ ID NO：13～15所示的至少一个片段。
在一些实施方式中，所述检测剂或定量检测剂还含有DNA提取试剂、双链特异性荧光染料、dNTP、DNA聚合酶、双链特异性荧光染料以及水中的一种或多种。
在一些实施方式中，所述双链特异性荧光染料定量选自溴化乙锭、SYBR Green、PicoGreen、RiboGreen中的任一种。
在一些实施方式中，所述水通常为核酸和/或无核酸酶的水。水可以为蒸馏水(Distilled Water)、去离子水(Deionized Water)或反渗水(Reverse osmosis Water)。
在一些实施方式中，所述DNA聚合酶选自Taq、Bst、Vent、Phi29、Pfu、Tru、Tth、Tl1、Tac、Tne、Tma、 Tih、Tf1、Pwo、Kod、Sac、Sso、Poc、Pab、Mth、Pho、ES4DNA聚合酶、Klenow片段中的任一种。
在一些实施方式中，所述试剂盒还包括样品的处理试剂；进一步地，所述样品的处理试剂包括样品裂解试剂、样品纯化试剂以及样品核酸提取试剂中的至少一种。
在一些实施方式中，所述检测剂或定量检测剂或诊断产品适用的待检样本为为血液、血浆、血清、粪便、尿液、唾液、羊水、绒毛、组织、细胞、组织或细胞裂解物样品中至少一种，优选为血液、血浆或血清，更优选为来自外周血的血液、血浆或血清。
本发明还涉及如上所定义的引物。
本发明还涉及如上所定义的检测剂或定量检测剂的试剂盒。
四.关于PRDM9转座子融合作为先天性巨结肠疾病标志物的应用
1.PRDM9转座子融合的定量检测剂在制备先天性巨结肠诊断试剂或试剂盒中的应用；
所述PRDM9转座子融合的融合位点选自：
位于chr5:23299411的PRDM9-type I；
位于chr5:23262356的PRDM9-type II-1；以及
位于chr5:23245181的PRDM9-type II-2。
2.根据1所述的应用，其特征在于，所述定量检测剂用于执行以下任一种方法：
聚合酶链反应、变性梯度凝胶电泳、核酸分型芯片检测、变性高效液相色谱法、原位杂交、生物质谱法以及HRM法。
3.根据1所述的应用，其特征在于，所述定量检测剂为引物。
4.根据3所述的应用，其特征在于，所述引物包括a～c中的至少一种：
a.用于检测PRDM9-type I的SEQ ID NO：1～2；
b.用于检测PRDM9-type II-1的SEQ ID NO：3～4；以及
c.用于检测PRDM9-type II-2的SEQ ID NO：5～6。
5.根据4所述的应用，其特征在于，所述引物还包括用于检测PRDM9-type I、PRDM9-type II-1和PRDM9-type II-2中至少一个所对应的野生型序列的内参引物。
6.根据5所述的应用，其特征在于，所述内参引物包括d～f中的至少一种：
d.用于检测PRDM9-type I所对应的野生型序列的SEQ ID NO：7～8；
e.用于检测PRDM9-type II-1所对应的野生型序列的SEQ ID NO：9～10；以及
f.用于检测PRDM9-type II-2所对应的野生型序列的SEQ ID NO：11～12。
7.根据4～6任一项所述的应用，其特征在于，所述定量检测剂还含有DNA提取试剂、双链特异性荧光染料、dNTP、DNA聚合酶、双链特异性荧光染料以及水中的一种或多种。
8.根据1～6任一项所述的应用，其特征在于，所述试剂或试剂盒的受试样品选自血液、组织、细胞样品中至少一种。
9.引物，其特征在于，其如权利要求4～6任一项中所定义。
10.试剂盒，其特征在于，含有权利要求4～8任一项中所定义的定量检测剂。
五.先天性巨结肠早期诊断标志物及其应用
1.PRDM9转座子融合的定量检测剂在制备先天性巨结肠早期诊断试剂或试剂盒中的应用；
所述PRDM9转座子融合的融合位点选自：
位于chr5:23262356的PRDM9-type II-1；和/或
位于chr5:23245181的PRDM9-type II-2。
2.根据1所述的应用，其特征在于，所述早期诊断的诊断对象为年龄小于3个月的新生儿。
3.根据1所述的应用，其特征在于，所述早期诊断的诊断对象为年龄小于1个月的新生儿。
4.根据1所述的应用，其特征在于，所述定量检测剂用于执行以下任一种方法：
聚合酶链反应、变性梯度凝胶电泳、核酸分型芯片检测、变性高效液相色谱法、原位杂交、生物质谱法以及HRM法。
5.根据1所述的应用，其特征在于，所述定量检测剂为引物。
6.根据5所述的应用，其特征在于，检测所述PRDM9-type II-1的引物的核苷酸序列为SEQ ID NO：1～2所示，检测所述PRDM9-type II-2的引物的核苷酸序列为SEQ ID NO：3～4所示。
7.根据6所述的应用，其特征在于，所述引物还包括内参引物；
所述PRDM9-type II-1所对应的野生型序列的内参引物的核苷酸序列为SEQ ID NO：5～6所示，所述PRDM9-type II-2所对应的野生型序列的内参引物的核苷酸序列为SEQ ID NO：7～8所示。
8.根据6或7所述的应用，其特征在于，所述定量检测剂还含有DNA提取试剂、双链特异性荧光染料、dNTP、DNA聚合酶、双链特异性荧光染料以及水中的一种或多种。
9.根据1～7任一项所述的应用，所述试剂或试剂盒的受试样品选自血液、组织、细胞样品中至少一种。
10.引物，其特征在于，其如权利要求7或8中所定义。
11.试剂盒，其特征在于，含有权利要求6～9任一项中所定义的定量检测剂。
六.CMV与PRDM9转座子融合作为先天性巨结肠早期诊断标志物及其应用
1.人巨细胞病毒检测试剂在制备先天性巨结肠诊断产品中的应用；所述产品包括检测试纸或试纸条、检测芯片或试剂盒。
2.CMV联合PRDM9转座子融合的检测试剂在制备先天性巨结肠诊断产品中的应用；所述产品包括检测试纸或试纸条、检测芯片或试剂盒。
3.根据2所述的应用，其特征在于，所述PRDM9转座子融合的融合位点选自：
位于chr5:23299411的PRDM9-type I；和/或
位于chr5:23262356的PRDM9-type II-1；和/或
位于chr5:23245181的PRDM9-type II-2。
4.根据1～3任一项所述的应用，其特征在于，所述早期诊断的诊断对象为年龄小于3个月的新生儿，优选为年龄小于1个月的新生儿。
5.根据1～3任一项所述的应用，其特征在于，所述CMV的检测试剂用于执行以下任一种方法：放射免疫法、间接免疫荧光法、斑点免疫金渗滤法、生物质谱法、免疫印记法和酶联免疫吸附法，聚合酶链反应、变性梯度凝胶电泳、核酸分型芯片检测、变性高效液相色谱法、原位杂交以及HRM法；
所述PRDM9转座子融合的检测剂用于执行以下任一种方法：聚合酶链反应、变性梯度凝胶电泳、核酸分型芯片检测、变性高效液相色谱法、原位杂交、生物质谱法以及HRM法。
6.根据3所述的应用，其特征在于，所述PRDM9转座子融合检测试剂包括引物；
所述引物优选包括a～c中的至少一种：
a.用于检测PRDM9-type I的SEQ ID NO：7～8；
b.用于检测PRDM9-type II-1的SEQ ID NO：11～12；以及
c.用于检测PRDM9-type II-2的SEQ ID NO：15～16。
7.根据3-6任一所述的应用，其特征在于，所述CMV检测试剂包括CMV抗体，所述CMV抗体优选为CMV lgM抗体。
8.一种先天性巨结肠诊断产品，其特征在于，所述产品中包括CMV的检测试剂和PRDM9转座子融合的检测试剂。
9.根据8所述的先天性巨结肠诊断产品，其特征在于，所述PRDM9转座子融合检测试剂包括引物；
所述引物优选包括a～c中的至少一种：
a.用于检测PRDM9-type I的SEQ ID NO：7～8；
b.用于检测PRDM9-type II-1的SEQ ID NO：11～12；以及
c.用于检测PRDM9-type II-2的SEQ ID NO：15～16。
10.根据8所述的先天性巨结肠诊断产品，其特征在于，所述CMV检测试剂包括CMV抗体，所述CMV抗体优选为CMV lgM抗体。
11.根据9-10任一项所述的产品，其特征在于，所述产品适用的待检样本为血液、血浆、血清、粪便、尿液、唾液、羊水、绒毛、组织、细胞、组织或细胞裂解物样品中至少一种，优选为血液、血浆或血清，更优选为来自外周血的血液、血浆或血清。
七.关于先天性巨结肠的诊断或早期诊断标志物及其应用
1.一种先天性巨结肠的诊断标志物组合，包含PRDM9转座子融合和巨细胞病毒。
2.根据1所述的诊断标志物组合，其特征在于：所述PRDM9转座子融合包含选自位于chr5:23299411的PRDM9-type I、位于chr5:23262356的PRDM9-type II-1、位于chr5:23245181的PRDM9-type II-2中的至少一种、至少两种或三种。
3.根据1或2所述的诊断标志物组合，其特征在于：包含巨细胞病毒和PRDM9-type I。
4.根据1或2所述的诊断标志物组合，其特征在于：包含巨细胞病毒和PRDM9-type II-1。
5.根据1或2所述的诊断标志物组合，其特征在于：包含巨细胞病毒和PRDM9-type II-2。
6.根据1或2所述的诊断标志物组合，其特征在于：包含巨细胞病毒、PRDM9-type II-1和PRDM9-type II-2。
7.根据1或2所述的诊断标志物组合，其特征在于：包含巨细胞病毒、PRDM9-type I和PRDM9-type II-1。
8.根据1或2所述的诊断标志物组合，其特征在于：包含巨细胞病毒、PRDM9-type I和PRDM9-type II-2。
9.根据1或2所述的诊断标志物组合，其特征在于：包含巨细胞病毒、PRDM9-type I、PRDM9-type II-1和PRDM9-type II-2。
10.根据前述1-9中任一所述的诊断标志物组合，其特征在于：所述诊断标志物是早期诊断标志物组合。
11.一种先天性巨结肠的诊断标志物组合，包含选自以下的至少两种、至少三种或四种：包含选自位于chr5:23299411的PRDM9-type I、位于chr5:23262356的PRDM9-type II-1、位于chr5:23245181的PRDM9-type II-2、和巨细胞病毒。
12.根据11所述的诊断标志物组合，其特征在于：包含PRDM9-type II-1和PRDM9-type II-2。
13.根据11所述的诊断标志物组合，其特征在于：包含PRDM9-type I和PRDM9-type II-1。
14.根据11所述的诊断标志物组合，其特征在于：包含PRDM9-type I和PRDM9-type II-2。
15.根据11所述的诊断标志物组合，其特征在于：包含PRDM9-type I、PRDM9-type II-1和PRDM9-type II-2。
16.根据12-15中任一项所述的诊断标志物组合，其特征在于：还包含巨细胞病毒。
17.根据11-16中任一项所述的诊断标志物组合，所述诊断标志物是早期诊断标志物组合。
18.一种先天性巨结肠的诊断产品，所述产品包含PRDM9转座子融合的检测剂，和/或巨细胞病毒的检测剂。
19.根据18所述的诊断产品，其特征在于：所述产品包含巨细胞病毒的检测剂。
20.根据18所述的诊断产品，其特征在于：所述产品包含PRDM9转座子融合的检测剂。
21.根据18或20所述的诊断产品，其特征在于：所述PRDM9转座子融合包含选自位于chr5:23299411的PRDM9-type I、位于chr5:23262356的PRDM9-type II-1、位于chr5:23245181的PRDM9-type II-2中的至少一种、至少两种或三种。
22.根据21所述的诊断产品，其特征在于：所述产品包含PRDM9-type I的检测剂。
23.根据21所述的诊断产品，其特征在于：所述产品包含PRDM9-typeII-1的检测剂。
24.根据21所述的诊断产品，其特征在于：所述产品包含PRDM9-typeII-2的检测剂。
25.根据21所述的诊断产品，其特征在于：所述产品包含PRDM9-type I的检测剂和PRDM9-typeII-1的检测剂。
26.根据21所述的诊断产品，其特征在于：所述产品包含PRDM9-type I的检测剂和PRDM9-typeII-2的检测剂。
27.根据21所述的诊断产品，其特征在于：所述产品包含PRDM9-typeII-1的检测剂和PRDM9-typeII-2的检测剂。
28.根据21所述的诊断产品，其特征在于：所述产品包含PRDM9-type I、PRDM9-typeII-1和PRDM9-typeII-2三者的检测剂。
29.根据22-28中任一项所述的诊断产品，其特征在于：所述产品还包含巨细胞病毒的检测剂。
30.一种先天性巨结肠的诊断产品，所述产品包含：位于chr5:23299411的PRDM9-type I、位于chr5:23262356的PRDM9-type II-1、位于chr5:23245181的PRDM9-type II-2、和巨细胞病毒中至少一种、至少两种、至少三种或四种诊断标志物的检测剂。
31.根据30所述的诊断产品，其特征在于：所述产品包含PRDM9-type I、PRDM9-type II-1、PRDM9-type II-2、和巨细胞病毒四种诊断标志物的检测剂。
32.根据18-21和23-31任一项所述的诊断产品，所述产品用于先天性巨结肠的早期诊断。
33. 18-32任一项所述的诊断产品在制备用于先天性巨结肠诊断或辅助诊断的试剂或试剂盒中的用途。
34.检测剂在制备先天性巨结肠诊断产品中的用途；所述产品包括检测试纸或试纸条、检测芯片、试剂或试剂盒；检测剂包含：PRDM9转座子融合的检测剂，和/或巨细胞病毒的检测剂。
35.根据34所述的用途，其特征在于：所述检测剂包含巨细胞病毒的检测剂。
36.根据34所述的用途，其特征在于：所述检测剂包含PRDM9转座子融合的检测剂。
37.根据34或36所述的用途，其特征在于：所述PRDM9转座子融合包含选自位于chr5:23299411的PRDM9-type I、位于chr5:23262356的PRDM9-type II-1、位于chr5:23245181的PRDM9-type II-2中的至少一种、至少两种或三种。
38.根据34所述的用途，其特征在于：所述检测剂包含PRDM9-type I的检测剂。
39.根据34所述的用途，其特征在于：所述检测剂包含PRDM9-typeII-1的检测剂。
40.根据34所述的用途，其特征在于：所述检测剂包含PRDM9-typeII-2的检测剂。
41.根据34所述的用途，其特征在于：所述检测剂包含PRDM9-type I的检测剂和PRDM9-typeII-1的检测剂。
42.根据34所述的用途，其特征在于：所述检测剂包含PRDM9-type I的检测剂和PRDM9-typeII-2的检测剂。
43.根据34所述的用途，其特征在于：所述检测剂包含PRDM9-typeII-1的检测剂和PRDM9-typeII-2的检测剂。
44.根据34所述的用途，其特征在于：所述检测剂包PRDM9-type I、PRDM9-typeII-1和PRDM9-typeII-2三者的检测剂。
45.根据36-44中任一项所述的用途，其特征在于：所述检测剂还包含巨细胞病毒的检测剂。
46.检测剂在制备先天性巨结肠诊断产品中的用途；所述产品包括检测试纸或试纸条、检测芯片、试剂或试剂盒；检测剂包含：位于chr5:23299411的PRDM9-type I、位于chr5:23262356的PRDM9-type II-1、位于chr5:23245181的PRDM9-type II-2、和巨细胞病毒中至少一种、至少两种、至少三种或四种诊断标志物的检测剂。
47.根据46所述的用途，其特征在于：所述检测剂包含PRDM9-type I、PRDM9-type II-1、PRDM9-type II-2、和巨细胞病毒四种诊断标志物的检测剂。
48.根据34-37和39-47任一项所述的用途，所述产品用于先天性巨结肠的早期诊断。
49.根据前述任一项所述的诊断标志物组合、诊断产品或用途，其特征在于：所述诊断是早期诊断。
50.根据前述任一项所述的诊断标志物组合、诊断产品或用途，其特征在于：所述早期诊断的诊断对象为年龄小 于3个月的新生儿。
51.根据前述任一项所述的诊断标志物组合、诊断产品或用途，其特征在于：所述早期诊断的诊断对象为年龄小于1个月的新生儿。
52.根据前述任一项所述的诊断标志物组合、诊断产品或用途，其特征在于：所述检测是定量检测，所述检测剂是定量检测剂。
53.根据前述任一项所述的诊断标志物组合、诊断产品或用途，其特征在于：所述PRDM9转座子融合的检测剂或定量检测剂用于执行以下任一种方法：聚合酶链反应、变性梯度凝胶电泳、核酸分型芯片检测、变性高效液相色谱法、原位杂交、生物质谱法以及HRM法。
54.根据前述任一项所述的诊断标志物组合、诊断产品或用途，其特征在于：所述巨细胞病毒的检测剂或定量检测剂用于执行以下任一种方法：放射免疫法、间接免疫荧光法、斑点免疫金渗滤法、生物质谱法、免疫印记法和酶联免疫吸附法，聚合酶链反应、变性梯度凝胶电泳、核酸分型芯片检测、变性高效液相色谱法、原位杂交以及HRM法。
55.根据前述任一项所述的诊断标志物组合、诊断产品或用途，其特征在于：检测所述巨细胞病毒是检测巨细胞病毒的感染。
56.根据前述任一项所述的诊断标志物组合、诊断产品或用途，其特征在于：检测所述巨细胞病毒是检测巨细胞病毒的抗体。
57.根据前述任一项所述的诊断标志物组合、诊断产品或用途，其特征在于：检测所述巨细胞病毒是检测巨细胞病毒的lgM抗体；或者是检测巨细胞病毒lgM抗体和lgG抗体。
58.根据前述任一项所述的诊断标志物组合、诊断产品或用途，其特征在于：所述检测剂或定量检测剂包括引物。
59.根据58所述的诊断标志物组合、诊断产品或用途，其特征在于：所述引物包括a～c中的至少一种：
a.用于检测PRDM9-type I的SEQ ID NO：1～2；
b.用于检测PRDM9-type II-1的SEQ ID NO：3～4；和
c.用于检测PRDM9-type II-2的SEQ ID NO：5～6。
60.根据前述59所述的诊断标志物组合、诊断产品或用途，其特征在于：所述引物还包括用于检测PRDM9-type I、PRDM9-type II-1和PRDM9-type II-2中至少一个所对应的野生型序列的内参引物。
61.根据权利要求60所述的诊断标志物组合、诊断产品或用途，其特征在于：所述内参引物包括d～f中的至少一种：
d.用于检测PRDM9-type I所对应的野生型序列的SEQ ID NO：7～8；
e.用于检测PRDM9-type II-1所对应的野生型序列的SEQ ID NO：9～10；和
f.用于检测PRDM9-type II-2所对应的野生型序列的SEQ ID NO：11～12。
62.根据前述任一项所述的诊断标志物组合、诊断产品或用途，其特征在于：所述检测剂或定量检测剂还含有DNA提取试剂、双链特异性荧光染料、dNTP、DNA聚合酶、双链特异性荧光染料以及水中的一种或多种。
63.根据前述任一项所述的诊断标志物组合、诊断产品或用途，其特征在于：所述检测剂或定量检测剂所适用的受试样品选自血液、血浆、血清、粪便、尿液、唾液、羊水、绒毛、组织、细胞、组织或细胞裂解物样品中至少一种。
64.根据前述任一项所述的诊断标志物组合、诊断产品或用途，其特征在于：所述检测剂或定量检测剂所适用的受试样品选自血液、血浆或血清中的至少一种。
65.根据前述任一项所述的诊断标志物组合、诊断产品或用途，其特征在于：所述检测剂或定量检测剂所适用的受试样品选自来自外周血的血液、血浆或血清的至少一种。
66.引物，其特征在于：如58-61任一项所定义。
67.引物，其特征在于：包括选自以下a～c中的至少一种：
a.用于检测PRDM9-type I的SEQ ID NO：1～2；
b.用于检测PRDM9-type II-1的SEQ ID NO：3～4；以及
c.用于检测PRDM9-type II-2的SEQ ID NO：5～6。
68.根据67所述引物，其特征在于：包括用于检测PRDM9-type I的SEQ ID NO：1～2。
69.根据67所述引物，其特征在于：包括用于检测PRDM9-type II-1的SEQ ID NO：3～4。
70.根据67所述引物，其特征在于：包括用于检测PRDM9-type II-2的SEQ ID NO：5～6。
71.根据67所述引物，其特征在于：包括用于检测PRDM9-type I的SEQ ID NO：1～2；和用于检测PRDM9-type II-1的SEQ ID NO：3～4。
72.根据67所述引物，其特征在于：包括用于检测PRDM9-type I的SEQ ID NO：1～2；和用于检测PRDM9-type II-2的SEQ ID NO：5～6。
73.根据67所述引物，其特征在于：包括用于检测PRDM9-type II-1的SEQ ID NO：3～4；和用于检测PRDM9-type II-2的SEQ ID NO：5～6。
74.根据67所述引物，其特征在于：包括用于检测PRDM9-type I的SEQ ID NO：1～2；用于检测PRDM9-type II-1的SEQ ID NO：3～4；和用于检测PRDM9-type II-2的SEQ ID NO：5～6。
75.根据67-74任一项所述引物，其特征在于：还包括用于检测PRDM9-type I、PRDM9-type II-1和PRDM9-type II-2中至少一个所对应的野生型序列的内参引物。
76.根据67-75任一项所述引物，其特征在于：所述内参引物包括d～f中的至少一种：
d.用于检测PRDM9-type I所对应的野生型序列的SEQ ID NO：7～8；
e.用于检测PRDM9-type II-1所对应的野生型序列的SEQ ID NO：9～10；和
f.用于检测PRDM9-type II-2所对应的野生型序列的SEQ ID NO：11～12。
77.包含66-76任一项所述引物的试剂、试剂盒或检测芯片。
78. 66-76任一项所述引物在制备先天性巨结肠诊断或辅助诊断产品中的用途。
79.根据78所述的用途，所述产品包括试剂、试剂盒或检测芯片。
80.根据78-79任一项所述的用途，其特征在于：所述诊断是早期诊断。
81.根据78-80任一项所述的用途，其特征在于：所述早期诊断的诊断对象为年龄小于3个月的新生儿。
82.根据78-81任一项所述的用途，其特征在于：所述早期诊断的诊断对象为年龄小于1个月的新生儿。
下面将结合实施例对本发明的实施方案进行详细描述。
本发明试验结果均采用统计学分析，t检验用来评估两组间的差异，机器学习评估转座子融合在性别、年龄、疾病表型等临床参数方面的差异，统计方法为altmann。p<0.05用于表示统计学显著性，所有p值均使用双侧检验。统计分析采用R和Graphpad 8.0软件进行。
实施例1.先天性巨结肠转座子融合遗传标记物的筛选流程
本发明证明了PRDM9转座子融合可以用于先天性巨结肠的早期诊断和筛查，填补了在先天性巨结肠早期诊断领域的空白，以下是本发明的具体实施例。
本实施例所采用的先天性巨结肠转座子融合遗传标记物的筛选流程如图1所示。具体步骤包括，取先天性巨结肠手术的组织样品，其中选取无神经节段、有神经节段的配对组织样品进行全转录组测序，检测转座子和基因组嵌合序列，查找嵌合的paired-end的reads中有一半是转座子序列的reads，确定这些转座子插入的位置，统计分析各插入位点的嵌合转录在有神经节和无神经节段的总reads数，用总的测序数目去做归一化，用limma R去做差异表达分析，从而分析显著变化的转座子融合位点。将发现的转座子融合位点分成无神经节段下调(type I)和上调(type II)两种，按照样品中基因携带转座子融合位点的类型，将样品中基因分为三型，包括携带I和II型转座子(gene I，II)，及不携带转座子(gene N)。同时收集疾病的表型，这些表型与疾病密切相关，包括疾病的亚型(按照严重程度递增：短段型，普通型，长段型，全结肠型)，是否有肠炎，是否有病毒感染，性别和年龄。用机器学习的方法，分析疾病的表型与基因转座子类型的关系，预测可能的驱动基因。筛选得到的转座子融合基因在巨结肠队列和新生儿腹胀队列中用qPCR验证。
实施例2.血液DNA和组织样本采集和分组
巨结肠队列：血液DNA样品分为先天性巨结肠患儿组(32例)，其它肠病对照组(16例)，健康儿童组(27例)，年龄介乎3个月到3岁，性别男性3/4为男性，疾病和对照组年龄和性别匹配。所有样品来源于广州市妇女儿童医疗中心，健康儿童组为健康儿童体检后剩余血样。采血方式为抗凝采血，离心分离白细胞提取DNA。结肠组织样品为52个先天性巨结肠患儿手术切除的病变组织，包括有神经节段和无神经节段。
新生儿腹胀队列1：收集诊断为新生儿腹胀的新生儿，年龄在1个月内的全血，并进行后续随访，随访时间为一年。最后确定该队列中含有先天性巨结肠患儿组(13例)；非先天性巨结肠患儿组(28例)。非先天性巨结肠中其它肠病15例(包括肠闭锁、肠狭窄、新生儿坏死性小肠结肠炎)，生理性腹胀13例，提取血中DNA。
新生儿腹胀队列2：共44例，这个队列来源于1个月内因为不明原因腹胀而入住NICU的儿童，对这些患儿，发明人进行了至少半年的电话随访(2019-2020)，并最终确认这个队列中共13个新生儿为巨结肠病人，16个为其他肠疾病对照(包括肛门闭锁、肠狭窄等)。另外15个胃肠功能紊乱患者作为非器质性病变的对照。
新生儿腹胀队列3：共40例，这个队列来源于1个月内的新生儿，包括先天性巨结肠患儿组(12例)；非先 天性巨结肠患儿组(28例)，非先天性巨结肠患儿包括其它肠病15例(包括肠闭锁、肠狭窄、新生儿坏死性小肠结肠炎)，生理性腹胀13例。
实施例3.先天性巨结肠全转录组测序及差异表达转座子分析
在先天性巨结肠病人52例，手术的组织样品，取有神经节段、无神经节段的配对组织样品，用RNAeasy kit(Qiagen)提取总RNA，用illumina TruSeq RNA Library Prep Kit v2建库，进行转录组测序，得到每个样品6GB paired-end的测序reads。
测序的fastq文件用Trimmomatic-0.36去接头，去短和低质量序列，用STAR与人的基因组(hg19)比对，用HTSeq-count计算基因表达量，edgeR计算log2(cpm)。基因表达量用voom去归一化，用limma R package计算差异表达基因，用FDR做多重比较。差异表达显著的基因定义为P-value<0.05且log2FC>1。基因表达值转化成Z-score，用pheatmap画出差异表达基因的热图。差异表达基因的信号通路富集分析用DAVID(version 6.8)，参数设置为EASE值<0.1作为cut-off。
转座子融合位点分析，用同上的方法检测嵌合序列，查找嵌合的paired-end的reads中有一半是转座子序列的reads，确定这些转座子插入的位置，统计分析各插入位点的嵌合转录在有神经节和无神经节段的总reads数，用总的测序数目去做归一化，用limma R去做差异表达分析，得到差异的转座子融合位点。这些差异的转座子融合位点分为两类，一类在无神经节段(aganglionic segments)下调(type I)和一类上调(type II)。
图2示出了先天性巨结肠中差异表达的转座子融合位点。在先天性巨结肠中存在转座子融合位点表达的显著变化，这些差异的转座子融合位点分为两类，一类在无神经节段(无神经节段，aganglionic segments)下调(type I)和一类上调(type II)。
实施例4.机器学习明确PRDM9转座子融合与先天性巨结肠段型密切相关
将发现的无神经节段下调(type I)和上调(type II)的转座子融合位点，关联其最相邻基因，按照样品中最相邻基因携带转座子融合位点的类型，将样品中基因分为三型，包括携带I和II型转座子融合位点(gene I，II)，及不携带转座子融合位点(gene N)。同时收集疾病的表型，这些表型与疾病密切相关，包括疾病的亚型(按照严重程度递增：短段型，普通型，长段型，全结肠型)，是否有肠炎，是否有病毒感染，性别和年龄。用随即森林方法进行表型的特征选择(feature selection)，分析疾病的表型相关的转座子融合基因，预测可能的驱动基因。用机器学习的方法，选择与先天性巨结肠亚型最显著相关的转座子融合基因。
结果见图3，其显示PRDM9转座子融合，与先天性巨结肠段型密切相关(p＝0.001)，在所有转座子融合的基因中p值排名靠前，重要性(importance)的排名也靠前。具体而言，PRDM9-type I融合在短段型中更多；而PRDM9-type II融合与长段型有关。这些结果提示，PRDM9的转座子融合是一个与先天性巨结肠严重程度密切相关的标志物。
实施例5.双荧光报告基因检测PRDM9转座子融合发生在转录调控区域
PRDM9的三个转座子融合位置为chr5 23299411，23262356，23245181(位置解析的版本号为hg19)。PRDM9融合转座子位于PRDM9上游非编码区，推测可能影响PRDM9的转录。将PRDM9和三个转座子融合的序列(E1+TE， E2+TE，E3+TE)与(E1+TE+C1，E2+TE+C2，E3+TE+C3)，两种对照序列(E1+C1，E2+C2，E3+C3)和(C1，C2，C3)分别放入纳米荧光素酶报告基因PNL3.1，用萤火虫荧光素酶做对照，用瞬时转化293T和SKNSH细胞系中，双通道检测报告基因的强度，以此来确定转座子插入对PRDM9对表达的影响。其中，TE是指转座子；E(E1，E2，E3)指针对三个位点而言，与转座子发生融合的位点的基因序列；C(C1，C2，C3)指针对三个位点而言，没有转座子融合时，融合位点的下游序列。结果显示PRDM9-type I融合会进一步激活PRDM9的增强子表达，PRDM9-type II-1，PRDM9-type II-2融合会抑制PRDM9增强子的表达(见图4)。
实施例6.PRDM9转座子融合的定量PCR
为了验证PRDM9转座子融合对先天性巨结肠的诊断效果，我们收集来源于巨结肠病人(32例，即HSCR)、其它肠疾病对照(包括肛门狭窄和肠狭窄造瘘儿童的血液DNA样品共16例，即HD)、健康儿童对照(27例，即Control)的血细胞DNA，用定量PCR检测血液DNA中PRDM9转座子融合的水平。每个融合位点设计2对引物，分别检测野生型(wt)和融合型的DNA水平，其中野生型用来做内参，引物序列如下所示。结果显示巨结肠病人的血液DNA中PRDM9-type I转座子融合显著低于健康对照组和肠疾病对照；PRDM9-type II-1在巨结肠中显著高于健康对照和肠疾病对照；PRDM9-type II-2在巨结肠中显著高于健康对照，但和其它肠疾病对照差异不显著(图5)。
PRDM9的三个转座子融合位置为chr5 23299411，23262356，23245181，三者序列及对照序列如下所示。
PRDM9-type I位于人第5号染色体的第23299411位(chr5 23299411)，其核苷酸序列为：
5’-GGGTGACAGAGTGAGACTCCATCTCAAAAATTTAAAAAAAAAAAAAAAAGCCTGTGGTTATTTTGATGGGATAGCAAGGATAACATGAACCATGTAAGGTTTGGCATAAATAGTTCATAGTTTACCTGGTCTGTCTGACTGTCTGAGATGTCACTGAGTCATTTGATGACCTTGAAATGCTAAAGCATTGGAATGAATGTCTGTAGATCAGGACAGAGGTGGGTTCAGAGAACTATTTAGGAGACATCTGGGTGGGAGTTGTGGCCAAC-3’(SEQ ID NO.16)；
其未发生融合的野生型序列(wt1)为：
5’-GGGTGACAGAGTGAGACTCCATCTCAAAAATTTTATTTCCTTAACAAGTACTTATTTTTTTATTTTTATTTTTTTAAGACTAGTCAAGTGCAATAATGAGAAGGAGGAAAAGAGTAGAACAGAAGTTCGATCTGTGACTGTGAACAATCAATTGAGATAATGCGTTATCTCTGGACAAGCCTCTTCAAGTATTTAATGAACACATTATAATCATATTGAACTAATTTTAAAATTATGTAATTTATATAAACTTATATGTAAATTGCACAT-3’(SEQ ID NO.13)。
PRDM9-type II-1位于人第5号染色体的第23262356位(chr5 23262356)，其核苷酸序列为：
5’-AACCTTCTCACTGAATAGAGCTCTAGGAAGCAAATAAAAAGGAAAAAATGTTTTTACAACAAAGGTAACCAAAAATCACATAATTTTCGCATGAAAAATCTTTTTTAGTCCAGGTACAGTGGCTCATGCCTATAATTCCAGCACCTTGAGACTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGAGACAG-3’(SEQ ID NO.17)；
其未发生融合的野生型序列(wt2)为：
5’-TTCAAAAACACAAAATATTCTGGGGGGATTGGGTTTTATCCCGGTTTAAAGCTGAAACCACTTTAAGGCATGTTGTGGCAATACAGTGATGGCAGGGTGGTTCTGGAAACTGTTAAATCTACAGTGTTCAGCTAACAAAAAGAATGTTTTACTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGAGACAG-3’(SEQ ID NO.14)。
PRDM9-type II-2位于人第5号染色体的第23245181位(chr5 23245181)，其核苷酸序列为：
5’-TATTCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTACAGTCTCACTCTGTCGTCCAGGCTGGAGTGCAGTGGAGTGATCTTGGCTCACTACATTTCTCTCTTAGGTTCAAGCGATTCTTCTGCCTCAGCCTCCTGAGTAGCTAGAATTACAGGCATGCACCACCACGCCCAGTTAATTTTTGTATTTT-3’(SEQ ID NO.18)；
其未发生融合的野生型序列(wt3)为：
5’-TATTCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTACAAATACAGTGTTTCCTCTTCCAATTTTGAGAAGATTTCTTATTTTTAAAGGTTTGTCTCAAAGTATATACATGCATATTTTTCATTTAAATCATTTTTCCTTTTCTTAGCTTTGAAATATGTCTTTCATTATATATTTTCCTGAAATGCCT-3’(SEQ ID NO.15)。
每个融合位点设计2对引物，分别检测野生型(wt)和融合型的DNA水平，其中野生型用来做内参，引物序列如下所示。
PRDM9-type I的引物对：
上游引物F：5’-GGGTGACAGAGTGAGACTCCA-3’(SEQ ID NO.1)；
下游引物R：5’-CTGAACCCACCTCTGTCCTG-3’(SEQ ID NO.2)。
PRDM9-type I的内参wt1的引物对：
上游引物F：5’-GGGTGACAGAGTGAGACTCCA-3’(SEQ ID NO.7)；
下游引物R：5’-TTGAAGAGGCTTGTCCAGAGA-3’(SEQ ID NO.8)。
PRDM9-type II-1的引物对：
上游引物F：5’-GCTCTAGGAAGCAAATAAAAAGGA-3’(SEQ ID NO.3)；
下游引物R：5’-CTGTCTCAAAAAAAAAAAAAAAA-3’(SEQ ID NO.4)。
PRDM9-type II-1的wt2的引物对：
上游引物F：5’-ATTGGGTTTTATCCCGGTTT-3’(SEQ ID NO.9)；
下游引物R：5’-CTGTCTCAAAAAAAAAAAAAAAA-3’(SEQ ID NO.10)。
PRDM9-type II-2的引物对：
上游引物F：5’-TATTCTTTTTTTTTTTTTTTTTTTTT-3’(SEQ ID NO.5)；
下游引物R：5’-AAAATTAACTGGGCGTGGTG-3’(SEQ ID NO.6)。
PRDM9-type II-2的wt3的引物对：
上游引物F：5’-TATTCTTTTTTTTTTTTTTTTTTTTT-3’(SEQ ID NO.11)；
下游引物R：5’-TGCATGTATATACTTTGAGACAAACC-3’(SEQ ID NO.12)。
采用PCR方法检测PRDM9转座子融合。
实施例7.ROC曲线评价PRDM9转座子融合对先天性巨结肠的诊断效果
ROC曲线用于评价PRDM9转座子融合对先天性巨结肠的诊断效果(图5)。PRDM9-type I的AUC值为0.7885，PRDM9-type II-1为0.8503，PRDM9-type II-2为0.7791。三者联合(PRDM9-TE ratio＝PRDM9-typeII-1+ PRDM9-typeII-2-PRDM9-typeI)检测AUC为0.9666，最佳界限对应特异性为95.35％，灵敏性为84.38％。用于疾病筛选时，灵敏性为100％时，特异性为76.74％。由此可见，PRDM9转座子融合可以有效诊断和筛选先天性巨结肠，表现出很好的效果。
实施例8.ROC曲线评价PRDM9转座子融合对先天性巨结肠早期诊断效果
为了评价PRDM9转座子是否可以用于早期新生儿腹胀样品中筛选先天性巨结肠患儿，本实验的实验对象选择新生儿腹胀队列1(腹胀队列1，n＝41)，包括先天性巨结肠患儿组(13例)；非先天性巨结肠患儿组(28例)，非先天性巨结肠患儿包括其它肠病15例(包括肠闭锁、肠狭窄、新生儿坏死性小肠结肠炎)，生理性腹胀13例，提取实验对象血中DNA，来确定PRDM9转座子融合检测技术是否能用于年龄在1个月内的腹胀新生儿样品。结果发现，PRDM9-type I区分新生儿腹胀中巨结肠和非巨结肠的效果欠佳，AUC值为0.6032。而PRDM9-type II则能够较好地区分巨结肠和非巨结肠，其中，PRDM9-type II-1AUC为0.7414，PRDM9-type II-2为0.7098。两种PRDM9-type II联合检测(PRDM9-typeII-1+PRDM9-typeII-2)AUC达到0.7586，最佳界限对应特异性为72.41％，灵敏性为83.33％(见图6)。由此可见，PRDM9转座子融合尤其是PRDM9-type II的PRDM9转座子融合可以用于先天性巨结肠的早期诊断。
实施例9.CMV感染与巨结肠严重程度正相关
为了研究CMV感染与巨结肠的严重程度之间的关联性，发明人对实施例2中提取的组织样品中病毒感染和HSCR亚型(短段型，常见型，长段型，全结肠)的相关性进行比对分析。
通过比对149种人类病毒在组织样品中的基因组上的整合情况，来判断组织样品中病毒的感染情况。其中，在排除掉感染人数低于8％的病毒后，发明人发现巨结肠组织样品中共发现28种病毒在肠道中有整合，其中只有CMV与巨结肠亚型显著相关，而且，在严重的巨结肠病人(全结肠和长段型)中，CMV感染比率远远高于轻微的巨结肠病人(短段型和常见型)(见图7)。这些结果提示CMV感染跟巨结肠相关。
实施例10.新生儿中CMV lgM抗体在最终诊断为先天性巨结肠的腹胀患儿中高于其它类型腹胀
基于实施例9的结果，发明人进一步对此进行试验验证。
发明人分别以先天性巨结肠新生儿腹胀新生儿冻存血清和非先天性巨结肠新生儿腹胀新生儿冻存血清进行比对，以此探究新生儿期的CMV感染是否与HSCR发生有关。
其中，所选新生儿腹胀病人共计159例，100例小于1个月，59例为1～2个月的新生儿。
结果如图8所示(图片黑白处理后，无法对应组别，故解释如下：不同检测时间的两个检测结果，左边为HSCR，右边为non-HSCR)。
通过检测血清中CMV lgM和lgG的含量(检测方法采用本领域常规的实验室检测方法，在下述实施例中，均使用ELISA方法进行检测)。发明人发现，在新生儿时期，最终被确诊为巨结肠的新生儿腹胀患者体内的CMV lgM抗体显著高于非巨结肠新生儿腹胀患者。而且，在诊治的1～2个月中，还发现这些被确诊巨结肠的患儿的CMV lgM会出现显著降低，但lgG会显著升高。这种相反的变化常常预示了急性CMV感染，由此可知，新生儿期的CMV感染与先天性巨结肠有关，检测新生儿中CMV lgM抗体，可以为先天性巨结肠的早期诊断提供有力的证据。
实施例11.CMV联合PRDM9转座子融合用于早期诊断先天性巨结肠的诊断效果
为了进一步验证联合CMV lgM抗体和PRDM9转座子融合水平(PRDM9-typeI，PRDM9-typeII-1和PRDM9-typeII-2)是否能在先天性巨结肠的诊断中，尤其选择早期诊断中，达到更好的甚至协同的效果，发明人收集了一个新生儿腹胀队列(腹胀队列2，n＝44)，这个队列来源于1个月内因为不明原因腹胀而入住NICU的儿童，对这些患儿，发明人进行了至少半年的电话随访(2019-2020)，并最终确认这个队列中共13个新生儿为巨结肠病人，16个为其他肠疾病对照(包括肛门闭锁、肠狭窄等)。另外15个胃肠功能紊乱患者作为非器质性病变的对照。使用实施例6所述引物对其样品DNA进行qPCR，分析PRDM9-TE融合水平，对其血清使用ELISA来检测CMV lgM的水平。PRDM9-TE异常融合水平用PRDM9-TE ratio来表示，其中，PRDM9-TE ratio＝(PRDM9-type II-1)+(PRDM9-type II-2)-(PRDM9-type I)；用同一个人对应的血清做ELISA检测CMV的lgM抗体的水平，CMV联合PRDM9-TE异常融合水平拟合为0.1×CMV+PRDM9-TE ratio，该拟合参数参考逻辑回归的参数得到。
结果如图9所示。
可以发现，PRDM9-TE识别巨结肠和非巨结肠的AUC可以到0.8425(95％CI，0.7122to 0.9728)。CMV lgM抗体在巨结肠中的含量也高于非巨结肠，AUC为0.8571(95％CI，0.7290to 0.9853)。将两者联合检测用于区分巨结肠和非巨结肠的AUC相比单一检测有显著提高，可以达到0.9267(95％CI，0.8403to 1.000)。两者达到最佳界限时对应的特异性为91.48％，灵敏性为84.62％。此结果表明PRDM9-TE融合和CMV感染可以协同有效的预测巨结肠的发生，两者联合应用可以作为巨结肠的诊断或早期诊断的有效标志物组合。
实施例12.冻存样品的腹胀队列验证CMV联合PRDM9转座子融合早期诊断先天性巨结肠的效果
为了进一步评估以上联合诊断标志物(CMV lgM抗体和PRDM9转座子融合)的诊断效果的稳定性，发明人用同时冻存有DNA和血清的一个回顾性的新生儿腹胀队列作为试验对象进行验证性试验。
该新生儿腹胀队列的患儿共40例(腹胀队列3)，均为一个月以内新生儿，包括先天性巨结肠患儿组(12例)；非先天性巨结肠患儿组(28例)，非先天性巨结肠患儿包括其它肠病15例(包括肠闭锁、肠狭窄、新生儿坏死性小肠结肠炎)，生理性腹胀13例。
用冻存的DNA基于实施例6所述引物进行qPCR，检测PRDM9-TE融合的位点的水平，然后用同一个人对应的血清做ELISA检测CMV的lgM抗体的水平。PRDM9-TE异常融合水平用PRDM9-TE ratio来表示，其中，PRDM9-TE ratio＝(PRDM9-type II-1)+(PRDM9-type II-2)-(PRDM9-type I)；用同一个人对应的血清做ELISA检测CMV的lgM抗体的水平，CMV联合PRDM9-TE异常融合水平拟合为0.1×CMV+PRDM9-TE ratio，该拟合参数参考逻辑回归的参数得到。
结果如图10所示。
可以发现，CMV lgM单独区分新生儿腹胀中巨结肠和非巨结肠时，AUC值仅为0.7173(95％CI，0.5477to0.8868)；PRDM9-TE融合位点单独区分新生儿腹胀中巨结肠和非巨结肠时，AUC值为0.8363(95％CI，0.6894to0.9892)。而当两者联合诊断时，AUC可以达到更优秀的协同诊断效果，AUC提升至0.8899(95％CI，0.7842to 0.9956)；最佳界限对应特异性为82.25％，灵敏性为83.33％。由此可见，CMV的lgM抗体和PRDM9转座子融合的联合可以 用于先天性巨结肠的诊断，尤其是早期诊断，且具有相对单一性检测更加优异的特异性和灵敏度，达到了协同作用，与实施例11的结果吻合。
综上所述，本发明通过先天性巨结肠转座子融合遗传标记物的筛选和验证，发现PRDM9转座子融合诊断先天性巨结肠患儿时，PRDM9-type I AUC值为0.7885，PRDM9-type II-1为0.8503，PRDM9-type II-2为0.7791。三者联合检测AUC为0.9666，最佳界限对应特异性为95.35％，灵敏性为84.38％。
进一步地，收集一个月内的新生儿腹胀患儿的血液DNA，检测PRDM9转座子融合，结果发现PRDM9-type II则能比较好的区分巨结肠和非巨结肠，PRDM9-type II-1AUC为0.7414，PRDM9-type II-2为0.7098。两者联合检测AUC为0.7586，最佳界限对应特异性为72.41％，灵敏性为83.33％。由此可见，PRDM9转座子融合可以用于先天性巨结肠的早期诊断和筛查，填补了先天性巨结肠早期诊断的空白。
再进一步地，本发明通过对先天性巨结肠病毒因素的筛选，找到与先天性巨结肠密切相关的病毒CMV，并证实定量检测CMV对儿童先天性巨结肠的诊断，尤其是早期诊断，具有良好的诊断效果，在两个独立的检测队列中AUC分别达到了0.7173和0.8571的水平。同时CMV联合PRDM9转座子融合水平检测，进一步提高了诊断或早期诊断先天性巨结肠的效果，在两个独立的检测队列中，联合检测的诊断效果分别为：AUC 0.9267(95％CI,0.8403 to 1.000)，最佳界限对应特异性为91.48％，灵敏性为84.62％；AUC 0.8899(95％CI，0.7842 to 0.9956)，最佳界限对应特异性为82.25％，灵敏性为83.33％。
以上所述实施例的各技术特征可以进行任意的组合，为使描述简洁，未对上述实施例中的各个技术特征所有可能的组合都进行描述，然而，只要这些技术特征的组合不存在矛盾，都应当认为是本说明书记载的范围。
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准。
Claims (14)
- 一种先天性巨结肠的诊断标志物组合，包含PRDM9转座子融合和巨细胞病毒。
- 根据权利要求1所述的诊断标志物组合，其特征在于：所述PRDM9转座子融合包含选自位于chr5:23299411(hg19)的PRDM9-type I、位于chr5:23262356(hg19)的PRDM9-type II-1、位于chr5:23245181(hg19)的PRDM9-type II-2中的至少一种、至少两种或三种。
- 一种先天性巨结肠的诊断标志物组合，包含选自以下的两种、或三种：位于chr5:23299411(hg19)的PRDM9-type I、位于chr5:23262356(hg19)的PRDM9-type II-1、位于chr5:23245181(hg19)的PRDM9-type II-2。
- 一种先天性巨结肠的诊断产品，所述产品包含PRDM9转座子融合的检测剂，和/或巨细胞病毒的检测剂。
- 根据权利要求4所述的诊断产品，所述PRDM9转座子融合包含选自位于chr5:23299411(hg19)的PRDM9-type I、位于chr5:23262356(hg19)的PRDM9-type II-1、位于chr5:23245181(hg19)的PRDM9-type II-2中的至少一种、至少两种或三种。
- 一种先天性巨结肠的诊断产品，所述产品包含：位于chr5:23299411(hg19)的PRDM9-type I、位于chr5:23262356(hg19)的PRDM9-type II-1、位于chr5:23245181(hg19)的PRDM9-type II-2中至少一种、至少两种或三种诊断标志物的检测剂。
- 根据权利要求4-6任一项所述的诊断产品，其特征在于：所述诊断是早期诊断；优选的，所述早期诊断的诊断对象为年龄小于3个月的新生儿；进一步优选的，所述早期诊断对象为年龄小于1个月的新生儿。
- 根据权利要求4-7任一项所述的诊断产品，其特征在于：所述PRDM9转座子融合的检测剂用于执行以下任一种方法：聚合酶链反应、变性梯度凝胶电泳、核酸分型芯片检测、变性高效液相色谱法、原位杂交、生物质谱法以及HRM法；所述巨细胞病毒的检测剂用于执行以下任一种方法：放射免疫法、间接免疫荧光法、斑点免疫金渗滤法、生物质谱法、免疫印记法和酶联免疫吸附法，聚合酶链反应、变性梯度凝胶电泳、核酸分型芯片检测、变性高效液相色谱法、原位杂交以及HRM法；优选的，所述巨细胞病毒的检测剂是巨细胞病毒抗体的检测剂，更优选的，是巨细胞病毒lgM抗体的检测剂，或更优选的，是巨细胞病毒lgM抗体和lgG抗体的检测剂；优选的，所述检测剂为定量检测剂。
- 根据权利要求8所述的诊断产品，其特征在于：所述检测剂或定量检测剂包括引物；优选的，所述引物包括a～c中的至少一种：a.用于检测PRDM9-type I的SEQ ID NO：1～2；b.用于检测PRDM9-type II-1的SEQ ID NO：3～4；c.用于检测PRDM9-type II-2的SEQ ID NO：5～6。
- 根据权利要求9所述的诊断产品，其特征在于；所述引物还包括用于检测PRDM9-type I、PRDM9-type II-1和PRDM9-type II-2中至少一个所对应的野生型序列的内参引物；优选的，所述内参引物包括d～f中的至少一种：d.用于检测PRDM9-type I所对应的野生型序列的SEQ ID NO：7～8；e.用于检测PRDM9-type II-1所对应的野生型序列的SEQ ID NO：9～10；f.用于检测PRDM9-type II-2所对应的野生型序列的SEQ ID NO：11～12。
- 根据权利要求8-10任一项所述的诊断产品，其特征在于：所述检测剂或定量检测剂还含有DNA提取试剂、双链特异性荧光染料、dNTP、DNA聚合酶、双链特异性荧光染料以及水中的一种或多种。
- 根据权利要求8-10任一项所述的诊断产品，其特征在于，所述检测剂或定量检测剂所适用的受试样品选自血液、血浆、血清、粪便、尿液、唾液、羊水、绒毛、组织、细胞、组织或细胞裂解物样品中至少一种，优选为血液、血浆或血清，更优选为来自外周血的血液、血浆或血清。
- 权利要求4-12任一项所述的诊断产品在制备用于先天性巨结肠诊断或辅助诊断的试剂或试剂盒中的用途。
- 引物，其特征在于：如权利要求9-10任一项所定义。
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102358900A (zh) * | 2011-11-09 | 2012-02-22 | 南京医科大学 | 与人类先天性巨结肠发生相关的血浆微小核糖核酸标志物及其应用 |
| US20150323546A1 (en) * | 2012-12-21 | 2015-11-12 | University of Tromsø | Biomarkers in inflammatory bowel disease |
| CN108699528A (zh) * | 2015-12-23 | 2018-10-23 | 纪念斯隆-凯特琳癌症中心 | 在先天性巨结肠症中通过衍生自多能干细胞的人肠神经嵴谱系可行的基于细胞的治疗和药物开发 |
| CN112708673A (zh) * | 2021-03-26 | 2021-04-27 | 广州市妇女儿童医疗中心 | Prdm9转座子融合作为先天性巨结肠疾病标志物的应用 |
| CN112852957A (zh) * | 2021-03-26 | 2021-05-28 | 广州市妇女儿童医疗中心 | 先天性巨结肠早期诊断标志物及其应用 |
-
2022
- 2022-03-25 WO PCT/CN2022/082967 patent/WO2022199682A1/zh active Application Filing
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102358900A (zh) * | 2011-11-09 | 2012-02-22 | 南京医科大学 | 与人类先天性巨结肠发生相关的血浆微小核糖核酸标志物及其应用 |
| US20150323546A1 (en) * | 2012-12-21 | 2015-11-12 | University of Tromsø | Biomarkers in inflammatory bowel disease |
| CN108699528A (zh) * | 2015-12-23 | 2018-10-23 | 纪念斯隆-凯特琳癌症中心 | 在先天性巨结肠症中通过衍生自多能干细胞的人肠神经嵴谱系可行的基于细胞的治疗和药物开发 |
| CN112708673A (zh) * | 2021-03-26 | 2021-04-27 | 广州市妇女儿童医疗中心 | Prdm9转座子融合作为先天性巨结肠疾病标志物的应用 |
| CN112852957A (zh) * | 2021-03-26 | 2021-05-28 | 广州市妇女儿童医疗中心 | 先天性巨结肠早期诊断标志物及其应用 |
Non-Patent Citations (2)
| Title |
|---|
| AMBARTSUMYAN LUSINE, SMITH CAITLIN, KAPUR RAJ P: "Diagnosis of Hirschsprung Disease", PEDIATRIC AND DEVELOPMENTAL PATHOLOGY, vol. 23, no. 1, 1 January 2020 (2020-01-01), US , pages 8 - 22, XP055969013, ISSN: 1093-5266, DOI: 10.1177/1093526619892351 * |
| YAMADA SHINTARO, KIM SEOYOUNG, TISCHFIELD SAM E., JASIN MARIA, LANGE JULIAN, KEENEY SCOTT: "Genomic and chromatin features shaping meiotic double-strand break formation and repair in mice", CELL CYCLE, vol. 16, no. 20, 18 October 2017 (2017-10-18), US , pages 1870 - 1884, XP055969007, ISSN: 1538-4101, DOI: 10.1080/15384101.2017.1361065 * |
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