Application of N-acetylserotonin in early diagnosis or prevention of non-syndromic cleft lip and palate during pregnancy

Abstract

The invention discloses application of N-acetylserotonin in early diagnosis or prevention of non-syndromic cleft lip and palate during pregnancy, and the application screens, identifies and finally confirms that the N-acetylserotonin in serum can be used as a biomarker for early diagnosis of the non-syndromic cleft lip and palate through metabonomics. A diagnostic product for early diagnosis of the non-syndromic cleft lip and palate is developed based on the biomarker, and the diagnostic product has high sensitivity and high specificity, and can solve the problems of difficult early diagnosis and easy misdiagnosis of the non-syndromic cleft lip and palate. Meanwhile, the method also has the characteristics of low detection cost and good repeatability, the stability and reliability of the N-acetylserotonin in the serum for diagnosing the non-syndromic cleft lip and palate are verified by means of discovery and verification, and the method has important clinical development and application values.

Description

Technical Field

[0001] The present invention relates to the technical field of medical biological detection, and particularly to the use of N-acetylserotonin as a biomarker or drug target in the preparation of products for diagnosing or treating non-syndromic cleft lip and palate. Background Art

[0002] Orofacial clefts are one of the most common congenital developmental defects in humans, bringing heavy economic and psychological pressures to patients and their families. According to statistics, the incidence of orofacial clefts varies by ethnicity and region. Among them, the incidence in Asians and American Indians is as high as 1 / 500, in Europeans is 1 / 1000, and in Africans is the lowest, about 1 / 2500.

[0003] Orofacial clefts are divided into two categories: 30% are syndromic orofacial clefts (SOCs) accompanied by other systemic tissue and organ malformations, which are caused by chromosomal abnormalities, single gene mutations, and certain specific teratogens; 70% are non-syndromic cleft lip with or without cleft palate (NSCL / P) without other system or organ malformations. NSCL / P belongs to a complex disease and is the result of the combined action of genetic factors (family linkage studies and twin analyses have confirmed that the heritability of NSCL / P reaches 70%) and environmental factors (such as active or passive smoking, alcohol consumption, vitamin deficiency, viral infection, and exposure to radiation in the early pregnancy of pregnant women). Establishing sensitive, specific, and reproducible detection methods for analyzing the serum metabolomic characteristics and potential biomarkers of NSCL / P patients will contribute to timely diagnosis in prenatal examinations. Summary of the Invention

[0004] Based on the above background, to solve the problems of no recognized biomarkers, small sample sizes in most studies, and poor diagnostic efficacy, the present invention provides a blood metabolite biomarker and its application in the early screening of non-syndromic cleft lip and palate.

[0005] To achieve the above object, the present invention is realized through the following technical solutions:

[0006] The present invention provides the application of a blood metabolite as a biomarker in the preparation of products for the early diagnosis or auxiliary diagnosis of non-syndromic cleft lip and palate, and the blood metabolite is selected from N-acetylserotonin.

[0007] The present invention also provides the use of a substance for detecting N-acetylserotonin in the preparation of a product for the early diagnosis or auxiliary diagnosis of non-syndromic cleft lip and palate. The product can be in the form of a product commonly used in the art for diagnosis or auxiliary diagnosis, such as a reagent, test strip, kit or probe, etc. The product diagnoses non-syndromic cleft lip and palate by detecting the content level of N-acetylserotonin.

[0008] In a specific example, the present invention provides the use of a substance for detecting N-acetylserotonin in the preparation of a kit for the early diagnosis or auxiliary diagnosis of non-syndromic cleft lip and palate. The diagnostic kit contains a detection reagent for specifically detecting N-acetylserotonin in a biological sample.

[0009] The present invention also provides a kit for the auxiliary diagnosis of non-syndromic cleft lip and palate, which contains a detection reagent for specifically detecting N-acetylserotonin in a biological sample.

[0010] For the use or detection kit of the present invention, the sample to be detected is the serum of a subject.

[0011] The method for detecting N-acetylserotonin can be carried out according to the conventional methods in the art, for example, analyzed by the method of liquid chromatography-mass spectrometry, or detected by designing chemical reagents.

[0012] The method of the present invention is based on a judgment model of mass spectrometry detection data: a disease judgment probability p is established based on the data of the non-syndromic cleft lip and palate case group and the healthy control group, and the regression equation is as follows:

[0013] logit(p) = β0 + β1 × X

[0014] Where: β0 = 77.61, β1 = -2.221×10 -3

[0015] Obtained: p = e 77.61-0.002221×X / (1 + e 77.61-0.002221×X )

[0016] Wherein, X is the detected value of N-acetylserotonin in the serum sample taken from the subject, and the cutoff value = 0.5, that is, when the P value is greater than 0.5, it is diagnosed that the subject has non-syndromic cleft lip and palate.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0018] The present invention studies non-syndromic cleft lip and palate through blood combined with metabolomics. The screened metabolic markers can distinguish non-syndromic cleft lip and palate from the control, and its AUC value can reach 1.0, with good sensitivity and specificity, and has been well verified in the verification group, and can be used as potential markers for non-syndromic cleft lip and palate.

[0019] In addition, the detection sample of the biomarker of the present invention is serum, which is different from traditional biomarkers. It is stable, minimally invasive, and easy to detect, which will greatly improve the sensitivity and specificity of disease diagnosis, making the diagnosis of non-syndromic cleft lip and palate more convenient and feasible, and laying a foundation for the evaluation of clinical prevention effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 For the screening results of Example 1; among them Figure 1 A is the PCA score plot of the samples in the disease group and the control group, observing the degree of variation between the samples in the disease group and the control group; Figure 1 B is the volcano plot of the samples in the disease group and the control group, mainly used to show the relative content differences of metabolites in the two (groups) of samples and the significance of the differences statistically; Figure 1 C is to screen the key differential metabolites in the samples of the disease group and the control group according to the VIP value; Figure 1 D is the expression quantification of N-acetylserotonin, mainly used to show the data distribution and its probability density; Figure 1 E is the ROC curve of the external test samples of the model.

[0021] Figure 2 For the ROC curve predicted by N-acetylserotonin in Example 2. DETAILED DESCRIPTION OF THE INVENTION

[0022] The following examples are used to illustrate the present invention, but are not used to limit the scope of the present invention. Without departing from the spirit and essence of the present invention, any modification or replacement of the methods, steps or conditions of the present invention belongs to the scope of the present invention.

[0023] For the convenience of understanding the present invention, the present invention will be described more comprehensively below. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, these embodiments are provided to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0024] Example 1 Screening of Differential Metabolites

[0025] 1. Instruments and Reagents: As shown in Table 1 and Table 2

[0026] Table 1 Instrument Information List

[0027]

[0028] Table 2 Information of Full-spectrum Standards and Reagents

[0029]

[0030]

[0031] 2. Sample collection

[0032] 2.1 A total of 46 sporadic non - syndromic cleft lip and palate patients with clear diagnosis, age - and sex - matched, and without family history were collected from Xuzhou First Hospital, Nanjing Children's Hospital, and Nanjing Stomatological Hospital Affiliated to Nanjing Medical University, and 23 healthy controls without a family history of cleft lip and palate were collected. All research subjects signed informed consent forms, which complied with the regulations of medical ethics.

[0033] 2.2 Blood collection: Fasting venous blood was drawn, centrifuged at 3000 r / min for 5 min at 4 °C, and the supernatant was taken.

[0034] 3. Sample metabolite extraction

[0035] Blood metabolite extraction: Take 50 μL of serum sample, add 150 μL of mass spectrometry water and 400 μL of acetonitrile, vortex, and let it stand at 20 °C for 1 hour. Centrifuge at 14000 g for 10 min, take the supernatant for centrifugal concentration, re - dissolve it with 50 μL of 2% acetonitrile - water, centrifuge at 14000 g for 10 min, and take 10 μL for injection after passing through a 10 kD filter membrane.

[0036] 4. Liquid - phase analysis

[0037] The data acquisition instrument system mainly includes ultra - performance liquid chromatography (Ultra Performance Liquid Chromatography, UPLC) (ExionLC AD, https: / / sciex.com.cn / ) and tandem mass spectrometry (Tandem mass spectrometry, MS / MS) ( https: / / sciex.com.cn / ).

[0038] 5. Principles of metabolite qualitative and quantitative analysis

[0039] Based on the targeted standard substance database MWDB (metware database), qualitative analysis was carried out according to the retention time RT (Retention time) of the detected substances, the information of parent - daughter ion pairs, and the secondary spectrum data. Metabolite quantification was completed by using the multiple reaction monitoring mode (multiple reaction monitoring, MRM) analysis of triple quadrupole mass spectrometry.

[0040] 6. Quality control:

[0041] The preparation method of quality control samples is to mix small portions of serum samples into mixed serum, and then analyze them using the same method as the analysis samples. During the whole analysis process, QCs were injected at regular time intervals (every ten samples) to provide a set of data that can be used to evaluate repeatability.

[0042] 7. Screening Results

[0043] 7.1 The unsupervised PCA plot shows a certain degree of discrimination between the non-syndromic cleft lip and palate group and the normal control group (see Figure 1 A); further, a supervised OPLS-DA model was constructed, and the discrimination between the two groups became more obvious (see Figure 1 B). 237 differential metabolites were screened between the non-syndromic cleft lip and palate group and the normal control group.

[0044] 8. Screening Criteria:

[0045] 1) Metabolites with fold change ≥ 2 and fold change ≤ 0.5 were selected. If the difference in metabolites between the control group and the experimental group is more than 2-fold or less than 0.5, the difference is considered significant.

[0046] 2) If there are biological replicates in the sample grouping, on the basis of the above, metabolites with VIP ≥ 1 were selected. The VIP value represents the influence intensity of the between-group difference of the corresponding metabolite in the classification and discrimination of each group of samples in the model. Generally, metabolites with VIP ≥ 1 are considered to have significant differences.

[0047] For the differential metabolites identified based on the screening criteria in each group comparison, the top 10 metabolites with the largest VIP value in the OPLS-DA model were selected for display (see Figure 1 C). A violin plot was used to show the comparison of the peak area expression of N-acetylserotonin in the control group and the experimental group (see Figure 1 D). The ROC curve was used to evaluate the predictive effect of N-acetylserotonin on non-syndromic cleft lip and palate. The results showed that the predicted AUC value was 1.0 (see Figure 1 E).

[0048] According to the judgment model of the mass spectrometry detection data in Example 1, the inventor found that based on the data of the non-syndromic cleft lip and palate case group and the healthy control group, the disease judgment probability p was established, and the regression equation was as follows:

[0049] logit(p) = β0 + β1 × X

[0050] Where: β0 = 77.61, β1 = -2.221×10 -3

[0051] Obtained: p = e 77.61-0.002221×X / (1 + e 77.61-0.002221×X )

[0052] Among them, X is the detected value of N-acetylserotonin in the serum sample taken from the subject, and the cutoff value = 0.5. That is, when the P value is greater than 0.5, it is diagnosed that the subject has non-syndromic cleft lip and palate.

[0053] Example 2 verifies the differentially expressed metabolites obtained by screening

[0054] The screened N-acetylserotonin was used as a biomarker for the early diagnosis of non-syndromic cleft lip and palate for verification. Another 20 patients with non-syndromic cleft lip and palate and 11 healthy controls without a family history of cleft lip and palate were selected. All the study subjects signed the informed consent form, which complied with the regulations of medical ethics.

[0055] The blood metabolites were detected by UPLC MS in the full scan mode. Based on the quantitative analysis of the first-order peak area of the biomarker, the expression levels of N-acetylserotonin metabolites between the non-syndromic cleft lip and palate group and the healthy control group were obtained, and the P value was calculated according to the formula in Example 1. The result showed that the value of the healthy control group was 1, while the value of the patient group was 0. The ROC curve was used to evaluate the predictive effect of the three metabolites on non-syndromic cleft lip and palate. The results showed that the AUC value predicted by the metabolite was 1.0 (see Figure 2 ), with a good predictive effect. N-acetylserotonin can be used as a potential biomarker for non-syndromic cleft lip and palate.

[0056] The technical features of the above embodiments can be combined arbitrarily. For the sake of brevity of description, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, it should be considered as the scope recorded in this specification.

Claims

1. Use of N-acetylserotonin as a biomarker in the preparation of a product for early diagnosis or auxiliary diagnosis of non-syndromic cleft lip and palate.

2. Use of a substance for detecting N-acetylserotonin in the preparation of a product for early diagnosis or auxiliary diagnosis of non-syndromic cleft lip and palate.

3. The use according to claim 2, wherein, the product is a reagent, test strip, kit or probe.

4. Use of a substance for detecting N-acetylserotonin in the preparation of a kit for early diagnosis or auxiliary diagnosis of non-syndromic cleft lip and palate.

5. The use according to claim 4, wherein, the diagnostic kit contains a detection reagent for specifically detecting N-acetylserotonin in a biological sample.

6. For the use according to any one of claims 1 to 5, establishing a disease judgment probability p based on the data of the non-syndromic cleft lip and palate case group and the healthy control group, and its regression equation is as follows: logit(p) = β 0 + β 1 × X Wherein: β0＝77.61,β1＝-2.221×10 -3 Obtained: p = e 77.61-0.002221×X / (1 + e 77.61-0.002221×X ) wherein, p represents the probability that the subject has non-syndromic cleft lip and palate, X is the detected value of N-acetylserotonin in the serum sample taken from the subject, and the cut-off value (Cutoff) = 0.5, that is, when the P value is greater than 0.5, it is diagnosed that the subject has non-syndromic cleft lip and palate.

7. For the use according to any one of claims 1 to 5, wherein, the detected sample is serum.

8. A kit for auxiliary diagnosis of non-syndromic cleft lip and palate, wherein, it contains a detection reagent for specifically detecting N-acetylserotonin in a biological sample.

9. The diagnostic kit according to claim 7, wherein, the detected sample is serum.

10. For the kit according to claim 8 or 9, wherein, establishing a disease judgment probability p based on the data of the non-syndromic cleft lip and palate case group and the healthy control group, and its regression equation is as follows: logit(p) = β 0 + β 1 × X where: β0 = 77.61, β1 = -2.221×10 -3 Obtained: p = e 77.61-0.002221×X / (1 + e 77.61-0.002221×X ) wherein, p represents the probability that the subject has non-syndromic cleft lip and palate, X is the detected value of N-acetylserotonin in the serum sample taken from the subject, and the cut-off value (Cutoff) = 0.5, that is, when the P value is greater than 0.5, it is diagnosed that the subject has non-syndromic cleft lip and palate.

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