A primer composition, product, and application for assessing genetic susceptibility to sudden death from unknown causes.

Abstract

This invention relates to the fields of forensic pathology, forensic genetics, and molecular identification technology, and particularly to a primer composition, product, and application for assessing genetic susceptibility to sudden death from unknown causes. The primer composition provided by this invention comprises amplification primers with nucleotide sequences as shown in SEQ ID NO. 1~512, i.e., 256 pairs of primers involving 238 quantitative trait loci. This invention further provides products and methods for assessing the genetic susceptibility to sudden death from unknown causes in individuals. Experimental results show that the primer composition or kit provided by this invention can obtain highly consistent and reproducible genotyping results. Based on this, the genetic susceptibility to sudden death from unknown causes can be further assessed by calculating a polygenic risk score, which is of great significance for the genetic background research of sudden death from unknown causes and for the accurate identification of the cause of death in sudden death-related cases in forensic identification work.

Description

Technical Field

[0001] This invention relates to the fields of forensic pathology, forensic genetics, and molecular identification technology, and in particular to a primer composition, product, and application for assessing genetic susceptibility to sudden death of unknown cause. Background Technology

[0002] In forensic identification, the determination of sudden death has always been a challenge. Previous studies have shown that the diseases leading to sudden death vary greatly across different age groups. For middle-aged and elderly individuals, myocardial ischemia and fatal arrhythmias caused by coronary atherosclerotic heart disease are usually the main causes of sudden death. However, in infants and young adults, sudden death often occurs in seemingly healthy individuals with no history of heart disease. In this group of sudden deaths, about one-third of the deceased cannot be definitively identified as having a cause of death even after systematic autopsy and histopathological examination; these sudden deaths are therefore classified as sudden unexplained death (SUD). These characteristics of SUD make its cause of death determination particularly difficult and complex.

[0003] In recent years, some scholars have proposed that genetic analysis of post-mortem samples can provide genetic evidence for determining the cause of death; this technique is also known as molecular autopsy. Through molecular autopsy, forensic scientists can obtain disease evidence that is difficult to observe through traditional autopsies and histopathological examinations, thereby inferring potential hereditary fatal diseases in the deceased. In recent years, through large-scale cohort studies, pedigree analyses, and electrophysiological studies, increasing evidence has shown that primary arrhythmia syndromes (represented by ion channelopathies), primary cardiomyopathy, and some hereditary metabolic diseases are closely related to the occurrence of subarachnoid hemorrhage (SUD). The pathogenic genes of these diseases are therefore considered susceptibility genes for SUD. However, at present, the application of molecular autopsy in forensic identification is often limited to screening for rare pathogenic genetic variations in the coding regions of known SUD susceptibility genes. In individuals where rare pathogenic genetic variations in the coding regions are not detected, there is still a lack of methods to systematically assess the genetic susceptibility to SUD and provide statistical probabilities.

[0004] In recent years, studies have shown that non-coding region genetic variations play a crucial role in the development of human hereditary traits and complex diseases. Exploring non-coding region genetic variations and identifying loci closely related to the occurrence of SUD (substances susceptible to SUD) holds promise for addressing the inability to assess the genetic susceptibility to SUD in individuals with undetectable rare pathogenic genetic variations in coding regions. Non-coding regions contain a large number of quantitative trait loci (QTLs) that significantly regulate gene expression, mainly including three categories: aQTLs (alternative polyadenylation QTLs), eQTLs (expression QTLs), and sQTLs (splicing QTLs). The combined effects of some QTLs can lead to significant alterations in gene expression, producing gene dosage effects similar to those caused by loss-of-function (LOF) and gain-of-function (GOF) genetic variations. Against this backdrop, a systematic identification of key QTLs that can lead to abnormal expression of SUD susceptibility genes has been conducted. This invention aims to develop primer compositions, products, and methods based on key QTLs for assessing the genetic susceptibility to sudden death (SUD), thereby accurately identifying individuals at high genetic risk for SUD and providing scientific evidence for precise cause-of-death identification in sudden death-related cases. Summary of the Invention

[0005] The purpose of this invention is to provide a primer composition, product, and application for assessing genetic susceptibility to sudden death of unknown cause (SUD), thereby addressing the problems existing in the prior art. The primer composition or kit provided by this invention can detect highly consistent QTL genotyping results, which can be used for subsequent multigene risk scoring and Z-score calculation, and is of great significance for the genetic background study of SUD and the accurate identification of the cause of death in sudden death-related cases.

[0006] To achieve the above objectives, the present invention provides the following solution: The present invention provides a primer composition for assessing genetic susceptibility to sudden death from unknown causes, the primer composition comprising primer pairs as shown in the "Primer Sequence" column of Table 1.

[0007] This invention provides the use of the above-described primer composition in the preparation of products for assessing genetic susceptibility to sudden death from unknown causes.

[0008] Optionally, the product includes reagents, reagent kits, and chips.

[0009] The present invention provides a product for assessing genetic susceptibility to sudden death from unknown causes, characterized in that the product comprises the primer composition described above.

[0010] Optionally, the product includes reagents, reagent kits, and chips.

[0011] This invention provides the application of the above-described primer composition in constructing model products for assessing genetic susceptibility to sudden death from unknown causes.

[0012] This invention provides a model product for assessing genetic susceptibility to sudden death from unknown causes, wherein the model product uses a polygenic risk score obtained from the above primer pair as the output variable.

[0013] Optionally, when the polygenic risk score of the sample to be tested is higher than the average polygenic risk score of the general population, that is, when the Z score percentile of the sample to be tested is greater than 0.5, it can be identified as an individual with a high genetic risk of unexplained sudden death.

[0014] Optionally, the basic calculation formula for the polygenic risk score is as follows: ; Where, β i G represents the effect value of the amplification result of the i-th primer pair; i Σ represents the allele dose of the amplification result of the i-th primer pair; Σ represents the sum of the amplification results of all primer pairs.

[0015] This invention provides the application of the above-described model product in constructing a system or device for assessing genetic susceptibility to unexplained sudden death.

[0016] The present invention discloses the following technical effects: This invention discloses a primer composition for assessing SUD genetic susceptibility based on 238 QTLs. The primer composition provided by this invention is obtained using next-generation sequencing technology. Based on the obtained primer composition, a method for genotyping 238 QTLs (i.e., 256 primer pairs involving 238 QTLs) was developed and established, and a product for assessing an individual's SUD genetic susceptibility was developed. Using the primer composition or kit provided by this invention, highly consistent and reproducible genotyping results can be obtained, which can be used for subsequent multigene risk scoring and Z-score calculation. Based on this, the genetic susceptibility to SUD can be further assessed by calculating the multigene risk score, which is of great significance for the genetic background research of SUD and the accurate determination of the cause of death in sudden death-related cases in forensic identification work. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 This is the subject operating characteristic curve in Example 3. Detailed Implementation

[0019] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0020] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0021] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0022] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0023] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0024] Table 1 Information on primer compositions Example 1 A method for detecting samples based on QTL primer compositions is disclosed, wherein information on the specific primer compositions is detailed in Table 1. The detection steps are as follows: (1) Extract DNA from the sample; (2) Using MultipSeq ® The first round of multiplex PCR amplification was performed using the Library Prep Kit, and the reaction system is shown in Table 2.

[0025] Table 2 Library Construction First Round of Multiplex PCR Reaction System Note: When constructing the library, the Primer pool is a mixture of primers SEQ ID NO.1~512 in Table 1. The concentration and amount of each primer are shown in Table 1.

[0026] PCR reactions were amplified on a 9700 or other PCR instrument. The multiplex PCR reaction program was as follows: preheating at 105℃; pre-denaturation at 95℃ for 3 minutes and 30 seconds, denaturation at 98℃ for 20 seconds, annealing at 55℃ for 1 minute, slow extension at 60℃ for 1 minute, fast extension at 65℃ for 2 minutes, for a total of 18 cycles; final extension at 72℃ for 5 minutes.

[0027] (3) First round of magnetic bead purification products The first-round multiplex PCR product was purified using IGT™ Pure Beads to obtain the purified mixed PCR product. Before use, the magnetic beads need to be equilibrated at room temperature for 30 minutes and then resuspended.

[0028] (4) Second round of adapter sequence ligation PCR reaction, the reaction system is shown in Table 3.

[0029] Table 3 Joint Connection Reaction System Note: Connector primer sequence: 5'-AATGATACGGCGACCACCGAGATCTACAC-i5 Index'-ACACTCTTTCCCTACACGACGCTCTTCCGATCT-Insert-TGGAATTCTCTCGGGTGCCAAGGAACTCCAGTCAC-i7 Index'-ATCTCGTATGCCGTCTTCTGCTTG-3', SEQ ID NO.513.

[0030] PCR reactions were amplified on a 9700 or other PCR instrument. The reaction program for multiplex PCR was as follows: preheating at 105°C; pre-denaturation at 95°C for 3 minutes and 30 seconds, denaturation at 98°C for 20 seconds, annealing at 58°C for 1 minute, extension at 72°C for 30 seconds, for a total of 9 cycles; and final extension at 72°C for 5 minutes.

[0031] (5) Second round of magnetic bead purification products Purify the second-round PCR products using IGT™ Pure Beads. Before use, the beads should be equilibrated at room temperature for 30 minutes and then resuspended.

[0032] (6) Purification and quantification of libraries Library quantification and quality control were performed using a Qubit 2.0 fluorescence quantitative analyzer and a Qsep 400 fully automated nucleic acid and protein analysis system.

[0033] (7) Sequencing and data analysis Sequencing was performed on the Illumina NOVASEQ 6000 platform using the MiSeq Reagent Kit V2. After obtaining the data, sequencing adapters, low-quality bases, or N-base sequences were removed. The sequences were then compared and ordered with the human reference genome (GRCh38) using BWA software to obtain SAM result files. The SAM files were converted to BAM format using Samtools, and library quality was assessed. Genotyping was then performed using GATK-3.8.0 and Varscan-v2.4.3, and QTL genotyping results were summarized.

[0034] Example 2 Using the method provided in Example 1, SUD was detected in 204 unrelated East Asian individuals, and their QTL typing results were used as the training set case group data. Subsequently, based on the racial characteristics of the training set case group, the specific QTLs and their β values ​​for constructing the polygenic risk score were determined, and the steps are as follows: (1) Allele frequency information of 238 QTLs in 22,448 East Asian general populations was obtained from the gnomAD public database (https: / / gnomad.broadinstitute.org / ) and used as the training set control group data; (2) Based on the differences in allele frequencies of 238 QTLs in the East Asian SUD sample (i.e., the training set case group, 204 cases) and the general East Asian population (i.e., the training set control group), the odds ratio (OR) of each QTL was calculated using SPSS software. This OR can be used to assess the association strength between the genotype of each QTL and the risk of SUD. The formula for calculating the OR is shown below: ; Where, p case p represents the frequency of effect alleles in the case group; control This indicates the frequency of the effect isotope in the control group.

[0035] (3) To avoid accidental phenomena caused by random sampling errors, only retain p OR values ​​<0.05 were used to identify 25 QTLs suitable for constructing polygenic risk scores (see Table 4 for details). The β value for each QTL was then calculated using the following formula: .

[0036] Table 4. QTL information that can be used to calculate polygenic risk scores Example 3 Using the method provided in Example 1, 20 unrelated East Asian SUD test set samples, independent of the training set samples, were tested. The genotyping results of the 25 QTLs identified in Example 2 were used as the test set case group data. Subsequently, based on the racial characteristics of the test set case group, polygenic risk scores and Z scores were calculated to assess the genetic susceptibility of individuals with SUD, mainly including the following steps: (1) The typing results of 25 QTLs identified in Example 2 were obtained from 504 individuals from the East Asian general population in Phase 3 batch from the Thousand Human Genome Database (https: / / www.internationalgenome.org / ) and used as the control group data of the test set; (2) Based on the β values ​​of the 25 QTLs obtained in Example 2, polygenic risk scores were calculated for the East Asian SUD sample (i.e., the test set case group or case group) and the East Asian general population (i.e., the test set control group or control group) using PLINK 1.9 software: ; Where, β i G represents the effect size at the i-th site; i β represents the allele dose at the i-th locus; Σ represents the summation over all included loci; that is, β i G represents the effect value of the amplification result of the i-th primer pair; i Σ represents the allele dose of the amplification result of the i-th primer pair; Σ represents the sum of the amplification results of all primer pairs.

[0037] (3) Based on the polygenic risk scores of the case group and control group samples in the test set, a receiver operating characteristic (ROC) curve was generated, with an area under the curve (AUC) value of 0.7694. Figure 1 The 95% confidence interval of the model is 0.6873-0.8515, indicating that the prediction model based on polygenic risk scores has a certain discriminative ability. That is, when the polygenic risk score of the sample to be tested is higher than the average polygenic risk score of individuals in the general East Asian population (according to the control group data of the test set, the average value is -0.0947), the genetic risk of SUD is high.

[0038] Based on this, the test set control group can be used as a reference population. The Z score of the SUD individuals in the test set and their percentile in the reference population can be calculated according to the following formula. Table 5 shows the calculation results and interpretation of the significance of 20 SUD individuals. Among them, 16 SUD cases (80%) were accurately identified as individuals with high genetic risk of SUD (Z score percentile greater than 0.5), which confirms that the primer composition or kit provided by the present invention can provide scientific evidence for the accurate identification of the cause of death of SUD.

[0039] ; ; Where Φ(Z score) is the cumulative distribution function of the standard normal distribution.

[0040] Table 5. Calculation results and interpretation of significance for 20 individuals with SUD. As can be seen from the above embodiments, the primer composition or kit for assessing the genetic susceptibility of SUD based on 238 QTLs provided by the present invention can be applied to calculate the polygenic risk score of an individual with SUD and its Z-score in the general population of the same ethnicity. That is, the primer composition or kit provided by the present invention can be used to assist in assessing the genetic susceptibility of SUD, thereby providing a new molecular detection method for the study of the genetic background of SUD and for the accurate identification of the cause of death in sudden death-related cases in forensic identification work.

[0041] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A primer composition for assessing genetic susceptibility to sudden death from unknown causes, characterized in that, The primer composition comprises primer pairs as shown in the table below: ； The genome version number is GRCh38.

2. The use of the primer composition of claim 1 in the preparation of a product for assessing genetic susceptibility to sudden death from unknown causes.

3. The application according to claim 2, characterized in that, The products include reagents, reagent kits, and chips.

4. A product for assessing genetic susceptibility to sudden death from unknown causes, characterized in that, The product comprises the primer composition of claim 1.

5. The product according to claim 4, characterized in that, The products include reagents, reagent kits, and chips.

6. The use of the primer composition of claim 1 in constructing a model product for assessing genetic susceptibility to sudden death from unknown causes.

7. A model product for assessing genetic susceptibility to sudden death from unknown causes, characterized in that, The model product uses the polygenic risk score obtained from the primer pair described in claim 1 as the output variable.

8. The model product according to claim 7, characterized in that, The basic calculation formula for the polygenic risk score is as follows: ； Where, β i G represents the effect value of the amplification result of the i-th primer pair; i Σ represents the allele dose of the amplification result of the i-th primer pair; Σ represents the sum of the amplification results of all primer pairs.

9. The model product according to claim 8, characterized in that, When the polygenic risk score of the sample to be tested is higher than the average polygenic risk score of the general population, that is, when the Z score percentile of the sample to be tested is greater than 0.5, it can be identified as an individual with a high genetic risk of sudden death from unknown causes. The formula for calculating the Z score percentile is as follows: ； ； Where Φ(Z score) is the cumulative distribution function of the standard normal distribution.

10. The use of the model product according to any one of claims 7-9 in constructing a system or device for assessing genetic susceptibility to sudden death from unknown causes.

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