Screening device and medium for candidate pathogenic genes and variations of hemophagocytic syndrome

Abstract

This application relates to a screening device and medium for candidate pathogenic genes and variants of hemophagocytic lymphohistiocytosis (HLH), belonging to the field of gene sequencing analysis technology. The device includes: an acquisition module for acquiring the gene sequencing variant file of the subject; a preliminary filtering module for initially filtering variant sites in the gene sequencing variant file; a variant annotation module for annotating the variant sites in the initially filtered gene sequencing variant file to generate a variant annotation file; a secondary filtering module for secondary filtering of variant sites in the variant annotation file; and a screening module for screening the variant sites in the secondary-filtered variant annotation file based on a local variant library to obtain candidate pathogenic genes and variants. The local variant library stores gene sequencing data of patients with similar HLH phenotypes and their non-phenotyped family members. This application can rapidly and accurately screen for harmful mutations related to HLH.

Description

Technical Field

[0001] This application relates to the field of gene sequencing analysis technology, and in particular to a device and medium for screening candidate pathogenic genes and variants of hemophagocytic lymphohistiocytosis syndrome. Background Technology

[0002] Hemophagocytic syndrome (HPS), also known as hemophagocytic lymphohistiocytosis (HLH), is a rare and life-threatening immune syndrome. It is characterized by excessive proliferation of lymphocytes and histiocytes, leading to cytokine-mediated tissue damage and multi-organ dysfunction. The disease has a rapid onset, progresses quickly, and has a high mortality rate.

[0003] Depending on the etiology, HLH can be divided into two forms: familial (i.e., primary) and acquired (i.e., secondary). Primary HLH is caused by gene mutations or inflammasome activation that affect cell lysis and lymphocyte survival, and mainly occurs in children at a young age. Secondary HLH is mainly triggered by acquired factors and is often associated with infections, malignancies, rheumatic diseases, and autoimmune diseases; it can occur in both children and adults. The most common infection-related HLH is EBV-HLH (Epstein-Barr virus-associated HLH). The most common HLH-related malignancy is lymphoma. When HLH occurs against a background of rheumatic diseases, such as systemic juvenile idiopathic arthritis (sJIA), adult-onset Still's disease (ASD), or systemic lupus erythematosus (SLE), it is often referred to as macrophage activation syndrome (MAS). It is currently believed that many cases of secondary HLH also have a certain genetic background, such as heterozygous alterations and polymorphisms in genes related to primary HLH, and manifest HLH after a "secondary blow" from external triggering factors (such as viral infections).

[0004] Due to the rapid progression of HLH, rapid and timely diagnosis is crucial. The currently accepted diagnostic criteria for HLH are the HLH-2004 criteria, which combine genetic testing, clinical manifestations, and laboratory test results. Commonly used genetic testing methods include first-generation (Sanger) sequencing, polymerase chain reaction (PCR), and next-generation sequencing (NGS). Next-generation sequencing mainly includes whole-exome sequencing (WES), whole-genome sequencing (WGS), and targeted sequencing. Whole-genome sequencing offers comprehensive detection, but it is relatively expensive, and the screening and interpretation of numerous non-coding region variants presents challenges. Targeted sequencing is relatively inexpensive, but its detection range is not comprehensive enough. With the continuous development of sequencing technology, whole-exome sequencing can detect not only protein-coding regions but also some non-coding regions at the boundaries of coding regions and known pathogenic mutations in deep intron regions. It is cost-effective, covers known pathogenic genes, and helps detect new pathogenic mutations, thus it is widely used. However, the number of original variants detected in each whole exome sequencing sample is over 100,000, especially in single-person test samples. Screening data from single-person samples is quite difficult, especially for diseases with inheritance patterns of AD, XLD, and XLR, where family co-segregation cannot be determined. This presents significant challenges in terms of both the accuracy of screening sites and the screening time.

[0005] Therefore, how to quickly and accurately screen for harmful mutations related to HLH is an urgent problem to be solved. Currently, there is no method for screening whole-exome sequencing data for HLH-related diseases. Summary of the Invention

[0006] To address the aforementioned technical problems, this application provides a device and medium for screening candidate pathogenic genes and variants of hemophagocytic lymphohistiocytosis (HLH).

[0007] In a first aspect, this application provides a screening device for candidate pathogenic genes and variants of hemophagocytic lymphohistiocytosis syndrome, employing the following technical solution:

[0008] A screening device for candidate pathogenic genes and variants of hemophagocytic lymphohistiocytosis syndrome, comprising:

[0009] The acquisition module is used to acquire the gene sequencing variant files of the test subject;

[0010] The preliminary filtering module is used to perform preliminary filtering of variant sites in the gene sequencing variant files;

[0011] The variant annotation module is used to annotate variant sites in the initially filtered gene sequencing variant files and generate variant annotation files.

[0012] A secondary filtering module is used to perform secondary filtering on the variant sites in the variant annotation file;

[0013] The screening module is used to screen the mutation sites in the secondary filtered mutation annotation file based on the local mutation library to obtain candidate pathogenic genes and mutations.

[0014] The local variant library stores gene sequencing data of patients with similar HLH phenotypes and their non-phenotyped family members.

[0015] Optionally, the acquisition module includes:

[0016] The quality control submodule is used to acquire the raw sequencing data of the subject, perform quality control on the raw sequencing data, and generate a fastq format file.

[0017] The alignment submodule is used to align the fastq format file with the base sequence in the reference genome to generate a sam file;

[0018] The conversion processing submodule is used to convert the SAM file into a BAM file and perform preset processing on the BAM file, wherein the preset processing includes deduplication and indexing;

[0019] The variant detection submodule is used to perform variant detection on the first-processed BAM file and generate the gene sequencing variant file.

[0020] Optionally, the preliminary filtering module is specifically used to filter out variant sites that meet the preliminary filtering conditions;

[0021] The preliminary filtering conditions include at least one of the following: low quality value, total sequencing depth less than a preset depth, mutation frequency less than a first preset frequency, and INDEL variants with a length not less than a preset length.

[0022] Optionally, the variant annotation file includes at least one of the following: basic variant information annotation, variant frequency database annotation, variant-related disease annotation, variant ACMG rating annotation, variant hazard prediction annotation, variant-related literature annotation, and HLH disease-related gene set annotation.

[0023] Optionally, the secondary filtering module includes:

[0024] The first filtering submodule is used to identify the variant sites belonging to the preset variant type as the first candidate filtering objects, retain the first candidate filtering objects that meet any one of the first retention condition, the second retention condition, the third retention condition, the fourth retention condition and the fifth retention condition, and filter out the remaining first candidate filtering objects.

[0025] The second filtering submodule is used to identify variant sites in the public population frequency database whose database frequency is greater than the second preset frequency as second candidate filtering objects, retain the second candidate filtering objects that meet any one of the first retention condition, the second retention condition, and the third retention condition, and filter out the remaining second candidate filtering objects; the third filtering submodule is used to identify benign or potentially benign variant sites belonging to the Clinvar database with 2 stars or more as third candidate filtering objects, retain the third candidate filtering objects that meet the second retention condition, and filter out the remaining third candidate filtering objects;

[0026] The preset mutation types include at least one of the following: unknown mutation, synonymous mutation, intergenic region mutation, non-coding RNA mutation, intron mutation, 5'UTR mutation, 3'UTR mutation, upstream mutation, and downstream mutation.

[0027] The first retention criterion is a variant site that belongs to the pathogenic or likely pathogenic variants in the Clinvar database;

[0028] The second retention condition is that the mutation tag in the Human Gene Mutation Database (HGMD) is any one of the following mutation sites: pathogenic mutation DM, suspected pathogenic mutation DM?, and disease-associated polymorphic mutation DFP.

[0029] The third retention condition is a variant site whose ACMG rating is pathogenic or likely pathogenic in the variant ACMG rating annotation.

[0030] The fourth retention criterion is the variant site with relevant literature annotations.

[0031] The fifth retention condition is that at least one variant site in the variant hazard prediction annotation is predicted as harmful by the software.

[0032] Optionally, the candidate pathogenic genes and variants include at least one of the following: candidate de novo mutation pathogenic genes and variants, candidate compound heterozygous mutation pathogenic genes and variants, candidate homozygous mutation pathogenic genes and variants, candidate sex chromosome mutation pathogenic genes and variants, and candidate AR single heterozygous mutation pathogenic genes and variants.

[0033] Optionally, the secondary filtering module includes:

[0034] The judgment module is used to determine whether the mutation sites in the mutation annotation file meet the sixth, seventh and eighth preservation conditions;

[0035] The first retention module is used to retain variant sites that simultaneously satisfy the sixth retention condition, the seventh retention condition, and the eighth retention condition, and to use unretained variant sites as first candidate retention objects;

[0036] The second retention module is used to retain the variant sites in the first candidate retention objects that meet the second retention conditions, and to use the unretained variant sites as the second candidate retention objects.

[0037] The third retention module is used to retain the variant sites in the second candidate retention objects that meet the ninth retention condition, and to use the unretained variant sites as the third candidate retention objects;

[0038] The fourth retention module is used to retain the variant sites that meet the tenth retention condition in the third candidate retention objects, and filter out the unretained variant sites.

[0039] The second retention condition is that the mutation tag in the Human Gene Mutation Database (HGMD) is any one of the following mutation sites: pathogenic mutation DM, suspected pathogenic mutation DM?, and disease-associated polymorphic mutation DFP.

[0040] The sixth retention condition is that the type of the mutation site does not belong to any of the following: unknown mutation, synonymous mutation, intergenic region mutation, non-coding RNA mutation, intron mutation, 5'UTR mutation, 3'UTR mutation, upstream mutation, and downstream mutation.

[0041] The seventh retention condition is that the frequency of the variant site in any public population frequency database is not greater than the second preset frequency or is not included in any public population frequency database.

[0042] The eighth retention criterion is a variant site belonging to the Clinvar database other than benign and likely benign variants with a rating of 2 stars or higher; the ninth retention criterion is a variant site belonging to the Clinvar database that is pathogenic or likely pathogenic, or a variant site whose ACMG rating is pathogenic or likely pathogenic in the ACMG rating annotation of the variant.

[0043] The tenth retention condition is a variant site with relevant literature annotations for the variant, or a variant site that is predicted as harmful by software in at least one of the variant hazard prediction annotations.

[0044] Optionally, the filtering module includes at least one of the following sub-modules:

[0045] The novel mutation screening submodule is used to screen for OMIM inheritance patterns of autosomal dominant AD, somatic mutation Smu, X-linked dominant XLD, X-linked recessive XLR, Y-linked YL, and blank; to screen for mutations in the local variant library where the number of heterozygous or hemizygous carriers is less than a first preset value or the population carrier frequency in the local variant library is less than a first preset frequency; and to select HLH-related genes from the HLH disease-related gene set annotation as candidate novel mutated pathogenic genes and variant sets that can explain the proband phenotype.

[0046] The compound heterozygous mutation screening submodule is used to screen for variants of OMIM with autosomal recessive AR and blank inheritance patterns; to screen for variants in the local variant library with a homozygous carrier count less than a second preset value or a population carrier frequency in the local variant library less than a second preset frequency; and to select HLH-related genes in the HLH disease-related gene set annotation as candidate compound heterozygous mutation pathogenic genes and variant sets that can explain the proband phenotype.

[0047] The homozygous mutation screening submodule is used to screen for variants with OMIM inheritance patterns of autosomal recessive AR and blank; to screen for variants with a homozygous carrier count less than a third preset value in all public population frequency databases and a homozygous carrier count less than a fourth preset value in the local variant library, or to screen for variants with a population carrier frequency less than a third preset frequency in the local variant library; and to select HLH-related genes from the HLH disease-related gene set annotation as candidate homozygous mutation pathogenic genes and variant sets that can explain the proband phenotype.

[0048] The sex chromosome mutation screening submodule is used to screen for variants of OMIM with X-linked dominant XLD, X-linked recessive XLR, and Y-linked YL inheritance patterns; to screen for variants with a number of hemizygous or homozygous carriers less than a fifth preset value in all public population frequency databases and a number of hemizygous or homozygous carriers less than a sixth preset value in the local variant library, or to screen for variants with a population carrier frequency less than a fourth preset frequency in the local variant library; and to select HLH-related genes from the HLH disease-related gene set annotation as candidate sex chromosome mutation pathogenic genes and variant sets that can explain the proband phenotype. The AR single heterozygous mutation screening submodule is used to screen for variants of OMIM with autosomal recessive AR inheritance; to screen for variants with a homozygous carrier count less than a seventh preset value in all public population frequency databases and a homozygous carrier count less than an eighth preset value in the local variant library, or to screen for variants with a population carrier frequency less than a fifth preset frequency in the local variant library; to screen for heterozygous variants carrying only one mutation on the same gene; and to select HLH-related genes from the HLH disease-related gene set annotation as candidate AR single heterozygous mutation pathogenic genes and variant sets that can explain the proband phenotype.

[0049] Secondly, this application provides an electronic device that adopts the following technical solution:

[0050] An electronic device includes a memory and a processor; the memory stores a computer program that can be loaded and executed by the processor, the computer program implementing the functions of the device according to any one of the first aspects.

[0051] Thirdly, this application provides a computer-readable storage medium, which adopts the following technical solution:

[0052] A computer-readable storage medium storing a computer program that can be loaded and executed by a processor, the computer program implementing the functions of the apparatus according to any one of the first aspects.

[0053] By adopting the above technical solution and adding the screening of the local variant library, it is possible to retain variants carried by patients with certain similar phenotypes but insufficient pathogenic evidence, which greatly improves the positive detection rate of individual samples, while filtering out too many variants carried by normal people in the local variant library, which greatly improves the accuracy of screening and reduces the false positive rate.

[0054] Since the local variant library is a database that specifically stores gene sequencing data of patients with similar HLH phenotypes and their non-phenotyped relatives, this screening device can achieve screening of HLH candidate pathogenic genes and variants more quickly.

[0055] Furthermore, this screening device can not only screen for HLH-related disease gene variants associated with the subject's phenotype, but also screen for suspected pathogenic mutations in disease phenotypes that the subject has not yet shown (possibly due to the subject not being old enough to develop the disease or the disease phenotype being mild and not easily detected), prompting clinicians and the subject to pay attention, so as to achieve comprehensive screening of harmful mutations in the subject.

[0056] In addition, the large accumulation of local variant libraries can help identify the same variants detected in HLH patients with similar phenotypes, facilitating the discovery of new genes in this disease area. Attached Figure Description

[0057] Figure 1 This is a structural block diagram of a screening device for candidate pathogenic genes and variants of hemophagocytic lymphohistiocytosis (HLH) according to an embodiment of this application.

[0058] Figure 2 This is a structural block diagram of the sub-modules included in the acquisition module of this application embodiment.

[0059] Figure 3 This is a structural block diagram of the sub-modules included in the secondary filtering module of this application embodiment.

[0060] Figure 4This is a structural block diagram of the sub-modules included in the filtering module of this application embodiment.

[0061] Figure 5 This is a structural block diagram of an electronic device according to an embodiment of this application. Detailed Implementation

[0062] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings.

[0063] Figure 1 This is a structural block diagram of a screening device 100 for candidate pathogenic genes and variants of hemophagocytic lymphohistiocytosis (HLH) provided in an embodiment of this application. Figure 1 As shown, the device 100 mainly includes:

[0064] Module 101 is used to acquire the gene sequencing variant files of the examinee;

[0065] The preliminary filtering module 102 is used to perform preliminary filtering of variant sites in gene sequencing variant files;

[0066] The variant annotation module 103 is used to annotate the variant sites in the preliminarily filtered gene sequencing variant files and generate variant annotation files.

[0067] The secondary filtering module 104 is used to perform secondary filtering on the variant sites in the variant annotation file.

[0068] The screening module 105 is used to screen the variant sites in the secondary filtered variant annotation file based on the local variant library to obtain candidate pathogenic genes and variants.

[0069] In this embodiment, the original gene sequencing data in the gene sequencing variant file can be any one of whole exome sequencing data, targeted sequencing data, and whole genome sequencing data, and this embodiment does not make specific limitations on this.

[0070] In this embodiment, the local variant database stores gene sequencing data of patients with similar HLH phenotypes and their family members without the phenotype. Here, phenotype refers to the sum of traits exhibited by an individual with a specific genotype under certain environmental conditions. Trait refers to the morphological, structural, physiological, and biochemical characteristics of an organism.

[0071] The local variant database will statistically analyze the sequencing data of nearly a thousand HLH patients and their families (without phenotype) collected from clinical information, and record the number of times each variant was detected, whether each variant corresponds to an HLH patient or a family member without phenotype, and whether the person being tested carries a homozygous or heterozygous variant.

[0072] The specific construction process of the local variant library is as follows: Sequencing data from a large number of samples are processed through a series of bioinformatics analyses to obtain annotation files. The annotation files of all sample data are then extracted and processed to obtain the following three tables:

[0073] (1) Sample feature table:

[0074] Sample characteristics are derived from clinical information collection. The sample characteristic table is used to record sample name, sample order, and other information about the sample (such as age, gender, presence of HLH phenotype, etc.). The sample order is a custom order, generally set according to the order of sample testing time.

[0075] (2) First mutation attribute table:

[0076] It includes the mutation ID and the first mutation attribute. The first mutation attribute refers to the attribute of the mutation itself, which can be understood as database annotation information. It is independent of the sample and does not vary with different samples. It consists of four columns: the gene where the mutation is located, the amino acid and exon changes caused by the mutation, the rs number corresponding to the mutation (the ID of the mutation in dbSNP), and the frequency of occurrence of the mutation in different databases.

[0077] (3) Second mutation attribute table:

[0078] It includes mutation ID and second mutation attributes. The second mutation attributes include information such as mutation carrying method, mutation quality and mutation depth. They are sample-related and can be understood as unannotated mutation information, which may manifest differently in different samples.

[0079] In this embodiment, the mutation ID is used to identify the mutation location and mutation type. It can be composed of four columns connected by hyphens "-", namely Chr (chromosome number), Pos (absolute position of the mutation start site on the chromosome, such as 764634 representing the 764634th base on the corresponding chromosome where the mutation begins), ALT (base information after the mutation), and REF (base information of the reference genome).

[0080] The local mutation library includes a sample feature library, a first mutation attribute library, and a second mutation attribute library. The sample feature library stores data from the sample feature table, the first mutation attribute library stores data from the first mutation attribute table, and the second mutation attribute library stores data from the second mutation attribute table.

[0081] For any mutation ID, all identical common mutation attributes corresponding to that mutation ID can be queried from the first mutation attribute table. These identical common mutation attributes are then merged and deduplicated. The data in the merged and deduplicated first mutation attribute table is then merged and deduplicated with the data in the existing first mutation attribute library to update the first mutation attribute library.

[0082] In addition, the local mutation library can be updated periodically by periodically searching for the mutation ID against the latest annotation database.

[0083] It should be noted that the difference between the first mutation attribute table and the second mutation attribute table is that the former specifies the mutation attributes at a specific site, which does not change with different samples, while the latter may vary in different samples.

[0084] In some embodiments, such as Figure 2 As shown, the acquisition module 101 includes the following sub-modules:

[0085] The quality control submodule 1011 is used to acquire the raw sequencing data of the subject, perform quality control on the raw sequencing data, and generate fastq format files.

[0086] The alignment submodule 1012 is used to align the fastq format file with the base sequence in the reference genome to generate a sam file;

[0087] The conversion processing submodule 1013 is used to convert the sam file into a bam file and perform preset processing on the bam file, including deduplication and indexing.

[0088] The variant detection submodule 1014 is used to perform variant detection on the first-processed BAM file and generate a gene sequencing variant file.

[0089] In this embodiment, the quality control submodule 1011 removes low-quality reads, reads that are too long, or reads containing too many N bases. For example, reads with more than 50% of bases having a quality value less than 5, reads shorter than 150 bp, and reads containing more than 15 N bases are filtered out. Here, "reads" refers to the base sequence obtained from a single sequencing run, i.e., a series of ATCGGGTA sequences, and is not part of the genome.

[0090] The variant detection submodule 1014 converts BAM file analysis into VCF format gene sequencing variant files. Variants typically include point mutations (SNVs) and short insertions / deletions (INDELs). SNVs and INDELs can be detected using software such as GATK and samtools.

[0091] It should be noted that FASTQ format is a text-based format primarily used to store nucleic acid sequences and sequencing quality. Both the sequence and quality are represented using ASCII encoding, and it is currently the standard for high-throughput sequencing results. SAM files are generally the standard output files of sequence alignment programs; SAM format is a plain text format using tabs as delimiters. BAM files are binary encoded versions of SAM files, significantly compressing their size.

[0092] In this embodiment, after generating the gene sequencing variant file, the variant sites need to be preliminarily filtered by the preliminary filtering module 102 to remove variants with low reliability. Optionally, the preliminary filtering conditions include at least one of the following: low quality value (variables with the LowQual marker in the original VCF file), total sequencing depth less than a preset depth (e.g., 10), mutation frequency less than a first preset frequency (e.g., 10%), and INDEL variants with a length not less than a preset length (e.g., 20). The mutation frequency is the ratio of mutation depth to total sequencing depth.

[0093] Since the variant sites in gene sequencing variant files only indicate that a change has occurred at a certain location in the genome, it is unclear whether this change will affect biological function. Therefore, it is necessary to annotate the detected variant sites through various variant information databases to transform the genome sequence variant data into information on changes in biological function, so as to further screen and interpret the variants.

[0094] In some implementations, variant annotation files include at least one of the following: basic variant information annotation, variant frequency database annotation, variant-related disease annotation, variant ACMG rating annotation, variant hazard prediction annotation, variant-related literature annotation, and HLH disease-related gene set annotation.

[0095] The following is a detailed explanation of the above annotations.

[0096] (1) Basic information notes on the mutation:

[0097] This includes the gene name associated with the variant, the nucleic acid changes, amino acid changes, and transcript numbers that conform to the Human Genome Variation Society (HGVS) nomenclature, the variant type, whether it is a LOF variant (including nonsense mutations, frameshift mutations, start codon deletions, and splice site mutations), the variant carrying pattern of the proband and parents (heterozygous, homozygous, hemizygous, wild-type), the mutation frequency (mutation depth / total sequencing depth), the rs number (the variant's ID in dbSNP) recorded in the dbSNP database (a database in the National Center for Biotechnology Information (NCBI) specifically used to store species SNP locus information), whether it is a de novo mutation (applicable to 3 samples from a family, where both parents are wild-type and the proband is heterozygous or hemizygous), whether it is a compound heterozygous mutation (preliminary judgment is that the proband carries 2 or more mutations on the same gene), and whether the variant is located in a repeating region and the number of repeating bases.

[0098] (2) Annotations of variations in population frequency databases:

[0099] This includes public population frequency databases (e.g., gnomAD_Exomes_All, gnomAD_Exomes_EastAsian, gnomAD_Genomes_All, gnomAD_Genomes_EastAsian, 1000g2015aug_all, ExAC_ALL), as well as accumulated local variant databases of HLH probands and their families.

[0100] (3) Notes on variant-related diseases:

[0101] This includes Clinvar, HGMD, OMIM, and Orphanet databases.

[0102] The Clinvar database, hosted by NCBI, is a database of disease-related human genome variations that stores the relationships between human variant sites and phenotypes. Clinvar database annotations include variant pathogenicity ratings: pathogenic, likely pathogenic, uncertain significance, likely benign, benign, etc., as well as review status (0 stars, 1 star, 2 stars, 3 stars, 4 stars).

[0103] The results annotated using the Human Gene Mutation Database (HGMD) include the relevant disease name, confidence labels {DM - pathogenic mutation, DM? - suspected pathogenic mutation, DP - disease-associated polymorphic mutation, DFP - disease-associated polymorphic mutation (supported by functional studies), FP - functional polymorphism (supported by in vivo / in vitro experiments but unclear disease relationship)}, and the associated literature PMID number.

[0104] The results of the OMIM database annotation include the disease phenotype OMIM number, disease name, mode of inheritance, disease overview, and disease phenotype.

[0105] The results of the Orphanet database annotations include the Orphanet ID and the disease name.

[0106] (4) ACMG rating notes for variants:

[0107] Using the ACMG rating from the InterVar database (which is part of the site pathogenicity assessment database) as a reference, the rating results are displayed as Pathogenic, Likely Pathogenic, Uncertain Significance, Likely Benign, and Benign.

[0108] (5) Notes on the prediction of the hazards of mutations:

[0109] This includes prediction of protein hazard and prediction of splicing hazard. Specifically, software such as Polyphen2, SIFT, MutationTaster, and REVEL can be used to predict the protein hazard of variants, while software such as SPLICE-AI, MaxEntScan, and dbscSNV can be used to predict the splicing hazard of variants.

[0110] (6) Reference notes related to the variation:

[0111] Retrieve PMID numbers of literature associated with variants from databases such as Clinvar and HGMD.

[0112] (7) Annotation of HLH disease-related gene sets:

[0113] Variants in the HLH_EBV_MAS_lymphoma column of the annotation results table are displayed in the gene set related to HLH (including EB virus, macrophage activation syndrome, lymphoma, etc.), while variants outside this gene set are displayed in the others column.

[0114] In this embodiment, before using the screening module 105 to screen candidate pathogenic genes and variants, it is necessary to use the secondary filtering module 104 to filter out variants with low pathogenicity.

[0115] In some embodiments, such as Figure 3 As shown, the secondary filtering module 104 includes the following sub-modules:

[0116] The first filtering submodule 1041 is used to determine the variant sites belonging to the preset variant type as the first candidate filtering objects, retain the first candidate filtering objects that meet any one of the first retention condition, the second retention condition, the third retention condition, the fourth retention condition and the fifth retention condition, and filter out the remaining first candidate filtering objects.

[0117] The second filtering submodule 1042 is used to identify variant sites in the public population frequency database whose database frequencies are all greater than the second preset frequency (e.g., 1%) as second candidate filtering objects, retain the second candidate filtering objects that meet any one of the first retention condition, the second retention condition, and the third retention condition, and filter out the remaining second candidate filtering objects.

[0118] The third filtering submodule 1043 is used to identify benign or potentially benign variant sites with 2 or more stars in the Clinvar database as third candidate filtering objects, retain the third candidate filtering objects that meet the second retention conditions, and filter out the remaining third candidate filtering objects.

[0119] The preset mutation types include at least one of the following: unknown mutation, synonymous mutation, intergenic region mutation, non-coding RNA mutation, intron mutation, 5'UTR mutation, 3'UTR mutation, upstream mutation, and downstream mutation.

[0120] The first retention criterion is that the variant site belongs to the pathogenic or likely pathogenic variant in the Clinvar database;

[0121] The second retention condition is that the mutation site in the Human Genetic Mutation Database (HGMD) is any of the following: pathogenic mutation DM, suspected pathogenic mutation DM?, or disease-associated polymorphic mutation DFP.

[0122] The third retention condition is for variant sites that have an ACMG rating of pathogenic or likely pathogenic in the variant ACMG rating annotation.

[0123] The fourth retention criterion is for variant sites with relevant literature annotations related to the aforementioned variants;

[0124] The fifth retention condition is that at least one variant site in the variant hazard prediction annotation is predicted to be harmful by the software.

[0125] It should be noted that the variants described in 2-star entries in the Clinvar database represent a consensus reached by multiple submitters without conflict. 3-star and 4-star entries are submitted by an expert panel using the ClinGen certification method. 1-star entries or entries without 1 star should be treated with caution. Therefore, the third filtering submodule identifies benign or potentially benign variant sites with 2 or more stars in the Clinvar database as third candidate filtering objects.

[0126] In addition, unknown mutations refer to undetected known gene mutations; synonymous mutations refer to changes in a base that do not alter the codon of the product's amino acid sequence; intergenic mutations refer to variations occurring within segments that do not have a genetic effect; non-coding RNA mutations refer to variations occurring in regions that do not encode proteins; intron mutations refer to variations occurring within introns; 5'UTR mutations refer to variations in the region of mature mRNA upstream of the coding region (CDS) and downstream of the 5' cap that does not encode proteins; 3'UTR mutations refer to variations in the region of mature mRNA at the 3' end that does not encode proteins; upstream mutations refer to variations in the first predetermined region (e.g., 1Kb, 2kb, 5kb, etc.) upstream of the transcription start site; and downstream mutations refer to variations in the second predetermined region (e.g., 1Kb, 2kb, 5kb, etc.) downstream of the transcription termination site.

[0127] It should be noted that the execution order of the first filtering submodule 1041, the second filtering submodule 1042, and the third filtering submodule 1043 can be set arbitrarily. However, after the previous filtering submodule performs filtering, the next filtering submodule filters the remaining (unfiltered) mutation sites, and so on.

[0128] Of course, the secondary filtering module 104 can also be of other structures. Figure 4 Another structural schematic diagram of the secondary filtering module 104 is shown. The secondary filtering module 104 includes the following sub-modules:

[0129] The judgment module 1044 is used to determine whether the variant sites in the variant annotation file meet the sixth, seventh and eighth retention conditions.

[0130] The first retention module 1045 is used to retain variant sites that simultaneously meet the sixth, seventh, and eighth retention conditions, and to use unretained variant sites as first candidate retention objects.

[0131] The second retention module 1046 is used to retain the variant sites that meet the second retention conditions in the first candidate retention objects, and to use the unretained variant sites as the second candidate retention objects.

[0132] The third retention module 1047 is used to retain the variant sites that meet the ninth retention condition in the second candidate retention objects, and to use the unretained variant sites as the third candidate retention objects;

[0133] The fourth retention module 1048 is used to retain the variant sites that meet the tenth retention condition in the third candidate retention objects and filter out the unretained variant sites.

[0134] The second retention condition is that the mutation tag in the Human Gene Mutation Database (HGMD) is any of the following mutation sites: pathogenic mutation DM, suspected pathogenic mutation DM?, and disease-associated polymorphic mutation DFP.

[0135] The sixth retention condition is that the type of mutation site does not belong to any of the following: unknown mutation, synonymous mutation, intergenic region mutation, non-coding RNA mutation, intron mutation, 5'UTR mutation, 3'UTR mutation, upstream mutation, and downstream mutation.

[0136] The seventh retention condition is that the frequency of the variant site in any public population frequency database is not greater than the second preset frequency (e.g., 1%) or is not included in any public population frequency database.

[0137] The eighth retention criterion is that the variant sites belong to the Clinvar database, excluding benign and possibly benign variants with 2 or more stars.

[0138] The ninth retention criterion is a variant site that is pathogenic or likely pathogenic in the Clinvar database, or a variant site that is rated as pathogenic or likely pathogenic in the ACMG rating annotation.

[0139] The tenth retention criterion is that the variant site has a variant-related literature annotation, or at least one variant site predicted as harmful by software in the variant hazard prediction annotation.

[0140] In some embodiments, candidate pathogenic genes and variants include at least one of candidate de novo mutation pathogenic genes and variants, candidate compound heterozygous mutation pathogenic genes and variants, candidate homozygous mutation pathogenic genes and variants, candidate sex chromosome mutation pathogenic genes and variants, and candidate AR single heterozygous mutation pathogenic genes and variants.

[0141] It's important to note that de novo mutations are mutations that are not carried by the parents' somatic cells but are carried by the child; they can include various forms of variation such as large (chromosomal disorders), medium (exon deletions / duplications), and small (point mutations). Generally, heterozygous mutations conforming to autosomal dominant, X-linked dominant, Y-linked inheritance, or hemizygous mutations in somatic cells or X-linked recessive inheritance can lead to the disease phenotype. Compound heterozygous mutations occur when heterozygous mutations occur at different locations on two chromosomes of a patient's pair of chromosomes. Homozygous mutations refer to mutations at the same location in both alleles of a pair of alleles.

[0142] like Figure 4 As shown, for the above-mentioned different types of candidate pathogenic genes and variants, the screening module 105 can be composed of at least one of the following sub-modules.

[0143] (1) Novel Mutation Screening Submodule 1051:

[0144] The variants used to screen for OMIM with the following inheritance patterns: autosomal dominant AD, somatic mutation Smu, X-linked dominant XLD, X-linked recessive XLR, Y-linked YL, and blank.

[0145] Filter for variants in the local variant library where the number of heterozygous or hemizygous carriers is less than a first preset value (e.g., 5) or the population carrier frequency in the local variant library is less than a first preset frequency (e.g., 1%).

[0146] If it is a standard family of three samples (proband, father and mother), then the father and mother are selected to be wild-type carriers of the variant, and the proband is a heterozygous variant.

[0147] If it is a non-standard family 2-sample (proband and father or mother), then the father or mother is selected as wild type and the proband is selected as heterozygous variant;

[0148] If it is a single-person sample (the first witness), then the first witness is selected as a heterozygous variant;

[0149] Genes in the HLH_EBV_MAS_lymphoma column were selected as candidate novel pathogenic mutant genes and variant sets that could explain the proband phenotype.

[0150] Furthermore, LOF variants, or variants predicted as harmful mutations by any two or more protein hazard prediction software or splicing prediction software, or variants reported as pathogenic or likely pathogenic in the Clinvar database, or variants included in the HGMD database as DM or DM?, or variants in the local variant library where probands already carry the same variant and have similar phenotypes, or variants rated as pathogenic or likely pathogenic by ACMG, or variants with further artificial ratings as pathogenic or likely pathogenic reported in the literature, are selected as candidate pathogenic genes and variants that can explain the proband phenotype.

[0151] Genes in the "others" column were further screened, with priority given to LOF variants, or variants predicted as harmful by all protein hazard prediction software or splicing prediction software, or variants reported as pathogenic or likely pathogenic by the Clinvar database, or variants rated as pathogenic or likely pathogenic by ACMG, or variants further artificially rated as pathogenic or likely pathogenic by literature reports, as candidate novel pathogenic genes and variants that are not related to the proband phenotype but may cause disease.

[0152] (2) Composite heterozygous mutation screening submodule 1052:

[0153] The OMIM inheritance pattern was screened for variants with autosomal recessive AR and blank.

[0154] Filter for variants in the local variant library where the number of homozygous carriers is less than a second preset value (e.g., 10) or where the frequency of carriers in the local variant library is less than a second preset frequency (e.g., 1%).

[0155] If it is a standard family three-sample, the proband is selected to carry two or more heterozygous variants on the same gene, and one of the variants is carried by a heterozygous father and a wild-type mother, and one or more variants are carried by a heterozygous mother and a wild-type father; or one of the variants is carried by a heterozygous mother and a wild-type father, and one or more variants are carried by a heterozygous father and a wild-type mother; or one of the variants is carried by both parents and a wild-type mother, and one or more variants are carried by one parent and a wild-type mother.

[0156] If it is a non-standard family 2 sample, the proband is selected to carry two or more heterozygous variants on the same gene, and one of the variants is carried by a heterozygous father or mother, while the other one or more variants are carried by wild-type father or mother, or one of the variants is carried by a wild-type father or mother, while the other one or more variants are carried by heterozygous father or mother.

[0157] If it is a single-person sample, the proband is selected if he carries two or more heterozygous variants on the same gene.

[0158] Genes in the HLH_EBV_MAS_lymphoma column were selected as candidate complex heterozygous mutation pathogenic genes and variant sets that could explain the proband phenotype.

[0159] Furthermore, priority is given to selecting LOF variants, or variants predicted as harmful mutations by any two or more protein hazard prediction software or splicing prediction software, or variants reported as pathogenic or likely pathogenic in the Clinvar database, or variants included in the HGMD database as DM or DM?, or variants in the local variant library where probands already carry the same variant and have similar phenotypes, or variants rated as pathogenic or likely pathogenic by ACMG, or variants with further artificial ratings as pathogenic or likely pathogenic reported in the literature as candidate complex heterozygous pathogenic genes and variants that can explain the proband phenotype.

[0160] Genes in the "others" column were further screened, with priority given to LOF variants, or variants predicted as harmful by all protein hazard prediction software or splicing prediction software, or variants reported as pathogenic or likely pathogenic by the Clinvar database, or variants rated as pathogenic or likely pathogenic by ACMG, or variants artificially rated as pathogenic or likely pathogenic by literature reports, as candidate compound heterozygous pathogenic genes and variants that are not related to the proband phenotype but may cause disease.

[0161] (3) Homozygous mutation screening submodule 1053:

[0162] The OMIM inheritance pattern was screened for variants with autosomal recessive AR and blank.

[0163] Filter variants with a homozygous carrier count less than a third preset value (e.g., 10) in all public population frequency databases and a homozygous carrier count less than a fourth preset value (e.g., 10) in the local variant database; or filter variants with a population carrier frequency less than a third preset frequency (e.g., 1%) in the local variant database.

[0164] If it is a standard family of three samples, select the proband as homozygous and the father is heterozygous and the mother is heterozygous, or the father is heterozygous and the mother is wild-type, or the mother is heterozygous and the father is wild-type.

[0165] If it is a non-standard family 2-sample, select the variant where the proband is homozygous and the father or mother is heterozygous, or the variant where the proband is homozygous and the father or mother is wild-type.

[0166] If it is a single-person sample, the proband is selected as the homozygous variant.

[0167] Genes in the HLH_EBV_MAS_lymphoma column were selected as candidate homozygous pathogenic genes and variant sets that could explain the proband phenotype.

[0168] Furthermore, priority is given to selecting LOF variants, or variants predicted as harmful mutations by any two or more protein hazard prediction software or splicing prediction software, or variants reported as pathogenic or likely pathogenic in the Clinvar database, or variants included in the HGMD database as DM or DM?, or variants in the local variant library where probands already carry the same variant and have similar phenotypes, or variants rated as pathogenic or likely pathogenic by ACMG, or variants artificially rated as pathogenic or likely pathogenic in the literature as candidate homozygous pathogenic genes and variants that can explain the proband phenotype.

[0169] Genes in the "others" column were further screened, with priority given to LOF variants, or variants predicted as harmful by all protein hazard prediction software or splicing prediction software, or variants reported as pathogenic or likely pathogenic by the Clinvar database, or variants rated as pathogenic or likely pathogenic by ACMG, or variants artificially rated as pathogenic or likely pathogenic by literature reports, as candidate homozygous pathogenic genes and variants that are not related to the proband phenotype but may be pathogenic.

[0170] (4) Sex chromosome mutation screening submodule 1054:

[0171] Screening for OMIM variants with X-linked dominant (XLD), X-linked recessive (XLR), and Y-linked YL inheritance patterns.

[0172] Filter variants in the public population frequency database where the number of hemizygous or homozygous carriers is less than the fifth preset value (e.g., 5), and in the local variant database where the number of hemizygous or homozygous carriers is less than the sixth preset value (e.g., 5); or filter variants in the local variant database where the population carrier frequency is less than the fourth preset frequency (e.g., 1%).

[0173] If it is a standard family of three samples, the proband is selected as a homozygous or hemizygous variant, with the father being wild-type and the mother being heterozygous.

[0174] If it is a non-standard family 2-sample, select a proband who is homozygous or hemizygous and whose father is wild-type variant, or a proband who is homozygous or hemizygous and whose mother is heterozygous variant.

[0175] If it is a single-person sample, the proband is selected as a homozygous or hemizygous variant.

[0176] Genes related to the HLH_EBV_MAS_lymphoma column were selected as candidate pathogenic genes and variant sets of sex chromosome mutations that could explain the proband phenotype.

[0177] Furthermore, priority is given to selecting LOF variants, or variants predicted as harmful mutations by any two or more protein hazard prediction software or splicing prediction software, or variants reported as pathogenic or likely pathogenic in the Clinvar database, or variants included in the HGMD database as DM or DM?, or variants in the local variant library where probands already carry the same variant and have similar phenotypes, or variants rated as pathogenic or likely pathogenic by ACMG, or variants artificially rated as pathogenic or likely pathogenic in the literature as candidate sex chromosome mutations and pathogenic genes and variants that can explain the proband phenotype.

[0178] Genes in the "others" column were further screened, with priority given to LOF variants, or variants predicted as harmful by all protein hazard prediction software or splicing prediction software, or variants reported as pathogenic or likely pathogenic by the Clinvar database, or variants rated as pathogenic or likely pathogenic by ACMG, or variants artificially rated as pathogenic or likely pathogenic by literature reports, as candidate sex chromosome mutations and pathogenic genes and variants that are not related to the proband phenotype but may cause disease.

[0179] (5) AR single heterozygous mutation screening submodule 1055:

[0180] Screening for OMIM variants with an autosomal recessive AR inheritance pattern.

[0181] Filter variants with a homozygous carrier count less than the seventh preset value (e.g., 10) in all public population frequency databases and a homozygous carrier count less than the eighth preset value (e.g., 10) in the local variant database; or filter variants with a population carrier frequency less than the fifth preset frequency (e.g., 1%) in the local variant database.

[0182] Screening for heterozygous variants that carry only one mutation on the same gene.

[0183] If it is a standard family of three samples, select the proband as heterozygous, and the father is heterozygous and the mother is wild-type, or the mother is heterozygous and the father is wild-type.

[0184] If it is a non-standard family 2-sample, select the variant where the proband is heterozygous and the father or mother is heterozygous, or the variant where the proband is heterozygous and the father or mother is wild-type.

[0185] If it is a single-person sample, the proband is selected as a heterozygous variant.

[0186] Genes related to the HLH_EBV_MAS_lymphoma column were selected as candidate AR single heterozygous mutation pathogenic genes and variant sets that can explain the proband phenotype.

[0187] Furthermore, priority is given to selecting LOF variants, or variants predicted as harmful mutations by any two or more protein hazard prediction software or splicing prediction software, or variants reported as pathogenic or likely pathogenic in the Clinvar database, or variants included in the HGMD database as DM or DM?, or variants in the local variant database where probands already carry the same variant and have similar phenotypes (this is the focus), or variants rated as pathogenic or likely pathogenic by ACMG, or variants with artificially rated as pathogenic or likely pathogenic reported in the literature as candidate AR single heterozygous pathogenic genes and variants that can explain the proband phenotype.

[0188] The following is an example of screening the whole exome sequencing data of a single sample from the laboratory. The candidate pathogenic genes and variants of HLH were screened using screening methods with and without the local variant library. The comparison results are shown in Table 1.

[0189] Table 1

[0190]

[0191] As shown in Table 1, after screening with the local variant library, the total number of candidate pathogenic variants related to the proband phenotype was 68, compared to the total number of 122 variants without screening with the local library. The number of retained candidate pathogenic sites decreased by (122-68) / 122 = 44.26%, of which the number of newly discovered candidate pathogenic sites decreased by (66-25) / 66 = 62.12%.

[0192] The functional modules in the embodiments of this application can be integrated together to form an independent unit, such as integrated into a processing unit, or each module can exist physically separately, or two or more modules can be integrated to form an independent unit. The integrated unit can be implemented in hardware or as a software functional unit. If the function is implemented as a software functional module and sold or used as an independent product, it can be stored in a computer-readable storage medium. Based on this understanding, the technical solution of this application, in essence, or the part that contributes to the prior art, or part of the technical solution, can be embodied in the form of a software product. This computer software product is stored in a storage medium and includes several instructions to cause an electronic device (which may be a personal computer, server, or network device, etc.) or processor to execute all or part of the steps of the methods of the various embodiments of this application. The aforementioned storage medium includes various media that can store program code, such as USB flash drives, mobile hard drives, read-only memory, random access memory, magnetic disks, or optical disks.

[0193] Figure 5 This is a structural block diagram of an electronic device 500 provided in an embodiment of this application. For example... Figure 5 As shown, the electronic device 500 includes a memory 501, a processor 502, and a communication bus 503; the memory 501 and the processor 502 are connected through the communication bus 503.

[0194] The memory 501 can be used to store instructions, programs, code, code sets, or instruction sets. The memory 501 may include a program storage area and a data storage area. The program storage area may store instructions for implementing an operating system, instructions for at least one function, and instructions for implementing the function of the screening device for candidate pathogenic genes and variants of hemophagocytic lymphohistiocytosis (HLH) provided in the above embodiments. The data storage area may store data involved in the screening device for candidate pathogenic genes and variants of HLH provided in the above embodiments.

[0195] Processor 502 may include one or more processing cores. Processor 502 executes instructions, programs, code sets, or instruction sets stored in memory 501, and calls data stored in memory 501 to perform various functions and process data as described in this application. Processor 502 may be at least one of the following: Application Specific Integrated Circuit (ASIC), Digital Signal Processor (DSP), Digital Signal Processing Device (DSPD), Programmable Logic Device (PLD), Field Programmable Gate Array (FPGA), Central Processing Unit (CPU), Controller, Microcontroller, and Microprocessor. It is understood that, for different devices, the electronic devices used to implement the functions of processor 502 may also be other types, and this application embodiment does not specifically limit the specific devices used.

[0196] The communication bus 503 may include a path for transmitting information between the aforementioned components. The communication bus 503 may be a PCI (Peripheral Component Interconnect) bus or an EISA (Extended Industry Standard Architecture) bus, etc. The communication bus 503 can be divided into an address bus, a data bus, a control bus, etc. For ease of representation, Figure 5 The symbol is represented by only one double arrow, but this does not indicate that there is only one bus or one type of bus. Figure 5 The electronic device shown is merely an example and should not impose any limitation on the functionality and scope of use of the embodiments of this application.

[0197] This application provides a computer-readable storage medium storing a computer program that can be loaded by a processor and executed to implement the screening device for candidate pathogenic genes and variants of hemophagocytic lymphohistiocytosis provided in the above embodiments.

[0198] In this embodiment, the computer-readable storage medium can be a tangible device that holds and stores instructions used by an instruction execution device. The computer-readable storage medium can be, but is not limited to, an electrical storage device, a magnetic storage device, an optical storage device, an electromagnetic storage device, a semiconductor storage device, or any combination thereof. Specifically, the computer-readable storage medium can be a portable computer disk, a hard disk, a USB flash drive, random access memory (RAM), read-only memory (ROM), erasable programmable read-only memory (EPROM or flash memory), spoofing random access memory (SRAM), portable compact disc read-only memory (CD-ROM), digital multifunction disc (DVD), memory stick, floppy disk, optical disk, magnetic disk, mechanical encoding device, or any combination thereof.

[0199] In the embodiments provided in this application, it should be understood that the disclosed systems and devices can be implemented in other ways. For example, the device embodiments described above are merely illustrative. For instance, the division of modules or units is only a logical functional division, and in actual implementation, there may be other division methods. For example, multiple units or components may be combined or integrated into another system, or some features may be ignored or not executed. Furthermore, the coupling or direct coupling or communication connection shown or discussed may be through some interfaces; the indirect coupling or communication connection between devices or units may be electrical, mechanical, or other forms.

[0200] Additionally, it should be understood that relational terms such as "first" and "second" are used merely to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. The terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0201] The above are merely preferred embodiments of this application and are not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A screening device for candidate pathogenic genes and variations of hemophagocytic syndrome, characterized by, The method comprises the following steps: an acquisition module is configured to acquire a gene sequencing variant file of a subject; a preliminary filtering module is configured to preliminarily filter variant sites in the gene sequencing variant file; a variant annotation module is configured to annotate the variant sites in the preliminarily filtered gene sequencing variant file to generate a variant annotation file; a secondary filtering module is configured to secondarily filter the variant sites in the variant annotation file; a screening module is configured to screen the variant sites in the secondarily filtered variant annotation file based on a local variant library to obtain candidate pathogenic genes and variants; wherein the local variant library stores gene sequencing data of patients with similar HLH phenotypes and family members without phenotypes; the variant annotation file comprises at least one of variant basic information annotation, annotation of the variant in a population frequency database, annotation of a variant-related disease, annotation of a variant ACMG rating, annotation of a variant harmfulness prediction, annotation of a variant-related literature, and HLH disease-related gene set annotation; wherein the annotation of the variant in the population frequency database comprises annotation of the local variant library; the candidate pathogenic genes and variants comprise at least one of candidate novel mutation pathogenic genes and variants, candidate complex heterozygous mutation pathogenic genes and variants, candidate homozygous mutation pathogenic genes and variants, candidate sex chromosome mutation pathogenic genes and variants, and candidate AR single heterozygous mutation pathogenic genes and variants; the screening module comprises at least one of the following sub-modules: a novel mutation screening sub-module is configured to screen variants with OMIM genetic modes of autosomal dominant AD, somatic mutation Smu, X-linked dominant XLD, X-linked recessive XLR, Y-linked YL, and blank; screen variants with a number of heterozygous or hemizygous carriers in the local variant library less than a first preset value or a population carrying frequency in the local variant library less than a first preset frequency; and select genes related to HLH in the HLH disease-related gene set annotation as a candidate set of novel mutation pathogenic genes and variants that can explain the phenotype of the proband; a complex heterozygous mutation screening sub-module is configured to screen variants with OMIM genetic modes of autosomal recessive AR and blank; screen variants with a number of homozygous carriers in the local variant library less than a second preset value or a population carrying frequency in the local variant library less than a second preset frequency; and select genes related to HLH in the HLH disease-related gene set annotation as a candidate set of complex heterozygous mutation pathogenic genes and variants that can explain the phenotype of the proband; a homozygous mutation screening sub-module is configured to screen variants with OMIM genetic modes of autosomal recessive AR and blank; screen variants with a number of homozygous carriers in all public population frequency databases less than a third preset value, and a number of homozygous carriers in the local variant library less than a fourth preset value, or screen variants with a population carrying frequency in the local variant library less than a third preset frequency; and select genes related to HLH in the HLH disease-related gene set annotation as a candidate set of homozygous mutation pathogenic genes and variants that can explain the phenotype of the proband. The sex chromosome mutation screening submodule is configured to screen mutations with OMIM genetic modes of X-linked dominant XLD, X-linked recessive XLR, and Y-linked YL; screen mutations with half-mutant or homozygous carrying numbers less than a fifth preset value in all public population frequency databases, and with half-mutant or homozygous carrying numbers less than a sixth preset value in a local mutation library, or screen mutations with population carrying frequencies less than a fourth preset frequency in the local mutation library; and select genes related to HLH in the HLH disease-related gene set annotation as candidate sex chromosome mutation pathogenic genes and mutation sets that can explain the phenotype of the proband. The AR single heterozygous mutation screening submodule is configured to screen mutations with OMIM genetic modes of autosomal recessive AR; screen mutations with homozygous carrying numbers less than a seventh preset value in all public population frequency databases, and with homozygous carrying numbers less than an eighth preset value in a local mutation library, or screen mutations with population carrying frequencies less than a fifth preset frequency in the local mutation library; screen heterozygous mutations carrying only one mutation on the same gene; and select genes related to HLH in the HLH disease-related gene set annotation as candidate AR single heterozygous mutation pathogenic genes and mutation sets that can explain the phenotype of the proband.

2. The apparatus of claim 1, wherein, The acquisition module comprises: The quality control submodule is configured to acquire raw sequencing data of the subject, perform quality control on the raw sequencing data, and generate a fastq format file; The alignment submodule is configured to align the fastq format file with base sequences in a reference genome, and generate a sam file; The conversion processing submodule is configured to convert the sam file into a bam file, and perform preset processing on the bam file, wherein the preset processing comprises deduplication and index establishment; The variant detection submodule is configured to perform variant detection on the first processed bam file, and generate the gene sequencing variant file.

3. The apparatus of claim 1, wherein, The preliminary filtering module is specifically configured to filter out variant sites that meet preliminary filtering conditions. The preliminary filtering conditions comprise at least one of a low quality value, a total sequencing depth less than a preset depth, a mutation frequency less than a first preset frequency, and an INDEL mutation with a length not less than a preset length.

4. The apparatus of claim 1, wherein, The secondary filtering module comprises: The first filtering submodule is configured to determine variant sites belonging to preset mutation types as first candidate filtering objects, retain first candidate filtering objects that meet any one of first retention conditions, second retention conditions, third retention conditions, fourth retention conditions, and fifth retention conditions, and filter out the remaining first candidate filtering objects; The second filtering submodule is configured to determine variant sites with all database frequencies greater than a second preset frequency in a public population frequency database as second candidate filtering objects, retain second candidate filtering objects that meet any one of the first retention conditions, the second retention conditions, and the third retention conditions, and filter out the remaining second candidate filtering objects; and The third filtering submodule is configured to determine variant sites with all database frequencies less than a third preset frequency in the public population frequency database as third candidate filtering objects, retain third candidate filtering objects that meet any one of the first retention conditions, the second retention conditions, the third retention conditions, the fourth retention conditions, and the fifth retention conditions, and filter out the remaining third candidate filtering objects. The third filtering submodule is configured to determine a variation site belonging to a benign or possibly benign variation site with a star of 2 or more in the Clinvar database as a third candidate filtering object, retain the third candidate filtering object satisfying the second retention condition, and filter out the remaining third candidate filtering objects; The preset variation type includes at least one of unknown mutation, synonymous mutation, intergenic mutation, non-coding RNA mutation, intron mutation, 5'UTR mutation, 3'UTR mutation, upstream mutation, and downstream mutation. The first retention condition is a variation site belonging to a pathogenic Pathogenic or possibly pathogenic Likely Pathogenic variation site in the Clinvar database. The second retention condition is a variation site with a mutation label being any one of a pathogenic mutation DM, a suspected pathogenic mutation DM?, and a disease-related polymorphic mutation DFP in the human gene mutation database HGMD. The third retention condition is a variation site with an ACMG rating of pathogenic Pathogenic or possibly pathogenic Likely Pathogenic in the variation ACMG rating annotation. The fourth retention condition is a variation site with the variation-related literature annotation. The fifth retention condition is a variation site with at least one software prediction being harmful in the variation harmfulness prediction annotation.

5. The apparatus of claim 1, wherein, The secondary filtering module includes: The judgment module is configured to judge whether a variation site in the variation annotation file satisfies a sixth retention condition, a seventh retention condition, and an eighth retention condition. The first retention module is configured to retain a variation site satisfying the sixth retention condition, the seventh retention condition, and the eighth retention condition, and take a variation site not retained as a first candidate retention object. The second retention module is configured to retain a variation site satisfying a second retention condition in the first candidate retention object, and take a variation site not retained as a second candidate retention object. The third retention module is configured to retain a variation site satisfying a ninth retention condition in the second candidate retention object, and take a variation site not retained as a third candidate retention object. The fourth retention module is configured to retain a variation site satisfying a tenth retention condition in the third candidate retention object, and filter out a variation site not retained. The second retention condition is a variation site with a mutation label being any one of a pathogenic mutation DM, a suspected pathogenic mutation DM?, and a disease-related polymorphic mutation DFP in the human gene mutation database HGMD. The sixth retention condition is that the type of the variation site does not belong to any one of unknown mutation, synonymous mutation, intergenic mutation, non-coding RNA mutation, intron mutation, 5'UTR mutation, 3'UTR mutation, upstream mutation, and downstream mutation. The seventh retention condition is that the frequency of the variation site in any public population frequency database is not greater than a second preset frequency or is not included in any public population frequency database. The eighth retention condition is a variation site belonging to the Clinvar database except a benign and possibly benign variation site with a star of 2 or more. The ninth reserved condition is a variant site belonging to Pathogenic or Likely Pathogenic in Clinvar database, or a variant site with an ACMG rating of Pathogenic or Likely Pathogenic in the variant ACMG rating annotation; The tenth reserved condition is a variant site with the variant related literature annotation, or a variant site predicted to be deleterious by at least one software in the variant deleteriousness prediction annotation.

6. An electronic device, comprising: A computer program product comprising a memory and a processor; the memory stores a computer program capable of being loaded and executed by the processor, and the computer program implements the functions of the device according to any one of claims 1 to 5.

7. A computer-readable storage medium, characterized in that, A computer program product comprising a memory and a processor; the memory stores a computer program capable of being loaded and executed by the processor, and the computer program implements the functions of the device according to any one of claims 1 to 5.

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