Method and device for judging dysfunction type pathogenic evidence

Abstract

The invention relates to a method and a device for judging function loss type pathogenicity evidence, and the method comprises the following steps: obtaining the category of target variation according to the influence of the target variation on transcription; according to the category of the target variation, judging the influence of the target variation on the function of a target gene; and according to the obtained influence result of the target variation on the function of the target gene, judging whether the target variation information is the function loss type pathogenicity evidence of the target variation or not. Through the category of the target variation, the influence of the target variation on the function of the target gene can be quickly clarified. Furthermore, according to the influence result of the target variation on the function of the target gene, whether the target variation information is the function loss type pathogenicity evidence of the target variation can be judged, and the function loss type pathogenicity evidence is determined.

Description

A method and device for determining evidence of loss-of-function pathogenicity Technical Field

[0001] The present invention relates to the field of bioinformatics, and in particular to a method and device for determining evidence of loss-of-function pathogenicity. Background Art

[0002] With the development and popularization of genetic technology, laboratories are detecting an increasing number of new sequence variants. Deciphering the clinical significance of a given sequence variant and clarifying the relationship between the variant sequence and the disease are key to achieving precision medicine.

[0003] Currently, manual interpretation is usually based on the guidelines issued by the American College of Medical Genetics and Genomics (ACMG) in 2015 (Richards S, Aziz N, Bale S, et al. Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology [J]. Genetics in medicine, 2015, 17 (5): 405-423.). The interpretation process requires the use of a variety of professional databases, prediction software, and the review of a large amount of medical literature for evidence-based analysis. At the same time, the interpreter is required to have a multidisciplinary background in molecular biology, genetics, medicine, etc.

[0004] A study showed that when the pathogenicity classification results of 99 variant sequences from nine top laboratories in the United States were evaluated using the aforementioned ACMG guidelines, the consistency of interpretation results was only 34%. After multi-party consultation and detailed review of ACMG standards, the consistency of interpretation results increased to 71%. (Amendola LM, Jarvik GP, Leo MC, et al. Performance of ACMG-AMP variant-interpretation guidelines among nine laboratories in the Clinical Sequencing Exploratory Research Consortium[J]. The American Journal of Human Genetics, 2016, 98(6):1067-1076.) It can be seen that the consistency of interpretation results cannot be guaranteed based on the personal understanding and experience of the interpreters.

[0005] Of the 28 variant grading criteria specified in the ACMG guidelines, only one, very strong evidence of pathogenicity, is crucial for determining the pathogenicity of variants that cause loss of gene function. Specifically, it defines "non-functional variants (nonsense mutations, frameshift mutations, canonical ±1 or 2 splicing mutations, start codon mutations, and single or multiple exon deletions) when the pathogenic mechanism of a disease is loss of function (LOF)," i.e., loss-of-function pathogenicity. According to the guidelines' combined standard rules for classifying genetic variants, one very strong evidence of pathogenicity only needs to be combined with one or two other levels of pathogenicity evidence to designate a variant as likely pathogenic or pathogenic. Statistics show that the average usage rate of loss-of-function pathogenicity evidence is approximately 20%, ranking fourth among all levels of evidence. In actual use, there is still the practice of manually adjusting the strength of evidence. However, the detailed rules for the use of this evidence are not perfect.

[0006] Determining evidence for loss-of-function pathogenicity is typically done manually, a complex process involving pathogenic mechanisms, biologically relevant transcripts, prediction of nonsense-mediated mRNA decay (NMD), and gene function. Furthermore, different types of non-functional variants exhibit distinct biological characteristics, leading to differing approaches for their identification. Among the various types of non-functional variants, the impact of nonsense mutations, frameshift mutations, canonical ±1 or 2 splicing mutations, and start codon mutations on gene function is relatively easy to predict. In particular, for non-functional variants involving single or multiple exons, the situation is more complex, as factors such as the specific location and length of the variant are considered. Conventional variant detection or annotation tools lack the ability to predict the effect of the variant on the encoded protein. For these reasons, in practice, identification is often not performed according to guidelines, but rather based on personal experience or simply omitted. Existing interpretation often relies on manual effort and individual understanding of interpretation guidelines. However, these guidelines only provide general guidance, making them difficult to implement. Consequently, standards are inconsistent, consistency is low, accuracy is low, and efficiency is low.

[0007] Summary of the Invention

[0008] In one embodiment, a method for determining evidence of loss-of-function pathogenicity is provided, comprising the following steps:

[0009] S101: Obtain the category of the target variant based on its effect on transcription;

[0010] S102: Determine the effect of the target mutation on the function of the target gene based on the type of the target mutation; the target gene refers to the gene where the target mutation occurs;

[0011] S103: Based on the results of the target variant's effect on the target gene function obtained in S102, determine whether the target variant information is evidence of loss-of-function pathogenicity of the target variant;

[0012] The target variation information is the category and / or impact result of the target variation in the target variation information.

[0013] The method provided herein for determining loss-of-function pathogenicity evidence allows rapid identification of the target variant's impact on target gene function based on its classification. Furthermore, based on the target variant's impact on target gene function, it is possible to determine whether the target variant meets the requirements for loss-of-function pathogenicity, thereby confirming the presence of loss-of-function pathogenicity. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] FIG1 is a flow chart of a method for determining evidence of loss-of-function pathogenicity provided by an embodiment of the present invention;

[0015] FIG2 is a flow chart of a method for determining the strength of evidence for loss-of-function pathogenicity provided by an embodiment of the present invention;

[0016] FIG3 is a flow chart of a method for correcting the strength of evidence for loss-of-function pathogenicity provided by an embodiment of the present invention. DETAILED DESCRIPTION

[0017] The present invention is described in further detail below by specific embodiments. In the following embodiments, many details are described in order to make the present application better understood. However, those skilled in the art can easily recognize that some of the features can be omitted in different situations, or can be replaced by other materials or methods. In some cases, some operations related to the present application are not shown or described in the specification. This is to avoid the core part of the present application being overwhelmed by too much description, and for those skilled in the art, it is not necessary to describe these related operations in detail. They can fully understand the related operations based on the description in the specification and the general technical knowledge in this area.

[0018] In addition, the features, operations, or characteristics described in this specification may be combined in any appropriate manner to form various embodiments. Furthermore, the steps or actions in the method description may be reordered or adjusted in a manner readily apparent to those skilled in the art. Therefore, the various sequences in this specification are provided solely for the purpose of clearly describing an embodiment and are not intended to be mandatory, unless otherwise specified.

[0019] The serial numbers assigned to the components in this document, such as "first", "second", etc., are only used to distinguish the objects described and do not have any order or technical meaning.

[0020] As used herein, "CDS" (Coding sequence) refers to the coding sequence region in mature mRNA that can be translated into amino acids, starting from the start codon and ending at the stop codon.

[0021] As used herein, the "5' untranslated region" (5' UTR) is a short sequence between the cap and the start codon of the coding region, which includes sequences that mark translation initiation. The 5' UTR is a highly sensitive region for translation initiation, and its length, secondary structure, and number of AUGs all affect the efficiency of translation initiation.

[0022] As used herein, an "open reading frame" (ORF) is the normal nucleotide sequence of a structural gene, from the start codon to the stop codon, which encodes a complete polypeptide chain without the presence of a stop codon that would interrupt translation. In the mRNA sequence, every three consecutive bases (i.e., a triplet "codon") encodes the corresponding amino acid.

[0023] In this document, “judge,” “determine,” and “determine” are used interchangeably.

[0024] In this article, "target variant" can include one or more variant categories (two or more variant categories). There are no restrictions on the detection methods of target variants, such as first-generation sequencing, second-generation sequencing, third-generation sequencing, PCR, etc. There are no restrictions on the diseases associated with target variants, such as various single-gene genetic diseases and various hereditary tumors.

[0025] In this article, "affect" and "damage" are synonymous. When a target variant causes a change in target gene function, the target variant is judged to have an impact on the target gene's function and constitutes damage. Determining the impact of a target variant on target gene function specifically refers to determining whether the target variant disrupts the target gene's function. A target variant's impact on target gene function specifically refers to the situation where the target variant disrupts the target gene's function. The impact of a target variant on target gene function is unclear, specifically when it cannot be determined to have disrupted the target gene's function.

[0026] In one embodiment, the method provided herein for determining the effect of a variant on gene function is applicable to non-functional variants, such as nonsense mutations, frameshift mutations, canonical ±1 or ±2 splicing mutations, and single or multiple exon deletion variants. This method is particularly applicable to single or multiple exon deletion variants. Compared to existing technologies, the present invention provides an executable technical solution with more prominent technical effects.

[0027] In one embodiment, the target variation may be of one or more categories. In addition to the first and second categories, the target variation may also include other categories, such as variations that result in open reading frame changes or induce nonsense-mediated mRNA degradation.

[0028] In one embodiment, the source of information regarding the last pathogenic variant at the 3' end of the target gene and the key functional domain of the target gene is not limited and can be obtained from literature reports or public database records. For example, the former can be obtained from https: / / www.ncbi.nlm.nih.gov / clinvar / , and the latter can be obtained from https: / / asia.ensembl.org / index.html. Furthermore, the collected information regarding the last pathogenic variant at the 3' end of the target gene and the key functional domain of the target gene can be generated into a data table or a custom configuration library for direct access.

[0029] In this article, "target variant-affected regions" refer to all regions that differ from the reference sequence due to the target variant.

[0030] In this article, “the proportion of the target variant-affected region” refers to the ratio of the length of the target variant-affected region to the length of the reference sequence.

[0031] In this article, "target gene start codon pathogenicity" means that other variants (non-target variants) at the target gene start codon are determined to be pathogenic based on industry standards and / or expert consensus. For example, the pathogenic (Pathogenic / P) or suspected pathogenic (Likely pathogenic / LP) results given according to the guidelines issued by ACMG (American College of Medical Genetics and Genomics). There is no restriction on the source of information on target gene start codon pathogenicity reports, which can come from literature reports or public database records (for example, https: / / www.ncbi.nlm.nih.gov / clinvar / ). In addition, the collected target gene start codon pathogenicity report information can be generated into a data table or a custom configuration library for direct access.

[0032] According to the first aspect, in one embodiment, a method for determining evidence of loss-of-function pathogenicity is provided, comprising the following steps:

[0033] S101: Obtain the category of the target variant based on its effect on transcription;

[0034] S102: Determine the effect of the target mutation on the function of the target gene based on the type of the target mutation; the target gene refers to the gene where the target mutation occurs;

[0035] S103: Based on the results of the target variant's effect on the target gene function obtained in S102, determine whether the target variant information is evidence of loss-of-function pathogenicity of the target variant;

[0036] The target variation information is the category and / or impact result of the target variation in the target variation information.

[0037] Determining the effect of the target variant on the target gene's function is a key step in determining loss-of-function pathogenicity. Obtaining the classification of the target variant allows for rapid identification of its effect on the target gene's function. This impact can then be used to determine whether the target variant meets the requirements for loss-of-function pathogenicity and confirm that the target variant information constitutes loss-of-function pathogenicity.

[0038] In one embodiment, the target variant is a single or multiple exon deletion variant.

[0039] In one embodiment, the effect of the target variant on transcription includes whether the target variant results in a change in the open reading frame and / or whether the target variant triggers nonsense-mediated mRNA degradation.

[0040] In one embodiment, the category of the target variation includes one or more of a first category, a second category, a third category, and a fourth category;

[0041] The first category includes mRNAs that result in changes in the open reading frame but do not trigger nonsense-mediated degradation;

[0042] The second category included those that did not result in a change in the open reading frame;

[0043] The third category includes deletions covering the first CDS but not the whole gene;

[0044] The fourth category includes deletions covering the last CDS but not whole-gene deletions.

[0045] In one embodiment, if the target variant affects the function of the target gene, the target variant information is determined to be evidence of loss-of-function pathogenicity of the target variant;

[0046] If the target variant has no effect on the function of the target gene, the target variant information is determined to be non-target variant loss-of-function pathogenicity evidence.

[0047] In this embodiment, taking the target variant as the first category as an example, when the target variant is the first category, it can be directly determined that the target variant affects the function of the target gene, thereby determining that the first category is loss-of-function pathogenicity evidence of the target variant. In this case, the target variant information is the first category;

[0048] If the category of the target variant cannot directly determine whether it affects the function of the target gene, and further judgment of the impact of the target variant on gene function is required, the category of the target variant and the impact results obtained by further judgment are evidence of the loss-of-function pathogenicity of the target variant.

[0049] In one embodiment, when the category of the target variant includes: category 1, category 2, category 3, or category 4, it is determined that the target variant affects the function of the target gene, and the category of the target variant is evidence of loss-of-function pathogenicity of the target variant;

[0050] The first category includes mRNAs that result in changes in the open reading frame but do not trigger nonsense-mediated degradation;

[0051] The second category included those that did not result in a change in the open reading frame;

[0052] The third category includes deletions covering the first CDS but not the whole gene;

[0053] The fourth category includes deletions covering the last CDS but not whole-gene deletions.

[0054] It can be understood that when the target variant is of the first category, the target variant information is of the first category, and the first category is determined to be evidence of loss-of-function pathogenicity of the target variant;

[0055] When the target variant is in the second category, the target variant information is in the second category, and the second category is determined to be evidence of loss-of-function pathogenicity of the target variant;

[0056] When the target variant is in the third category, the target variant information is in the third category, and the third category is determined as evidence of loss-of-function pathogenicity of the target variant;

[0057] When the target variant is of the fourth category, the target variant information is of the fourth category, and the fourth category is determined as evidence of loss-of-function pathogenicity of the target variant.

[0058] In one embodiment, when the category of the target variant includes: category 5 and / or category 6, the target variant has no effect on the function of the target gene, and the category of the target variant is evidence of non-loss-of-function pathogenicity of the target variant;

[0059] The fifth category includes: variants that cannot be annotated to coding transcripts;

[0060] The sixth category includes: variants can be annotated to coding transcripts, but the region affected by the target variant does not contain coding sequences.

[0061] It is understood that when the target variant is classified as Category 5 and / or Category 6, Category 5 and / or Category 6 do not constitute evidence of loss-of-function pathogenicity for the target variant. In determining evidence of loss-of-function pathogenicity, Category 5 and / or Category 6 can be directly excluded.

[0062] According to the second aspect, in one embodiment, a method for determining the strength of evidence for loss-of-function pathogenicity comprises the following steps:

[0063] S104: Determine the strength of the loss-of-function pathogenicity evidence obtained by the method of the first aspect based on the degree of impact of the target variant on the function of the target gene.

[0064] In this example, the extent to which the target variant affects the function of the target gene is positively correlated with the strength of the evidence for loss-of-function pathogenicity.

[0065] The present invention adopts different processing methods for different categories of target variants to determine the degree of impact of the target variant on the function of the target gene, thereby determining the strength of the evidence of loss-of-function pathogenicity.

[0066] In one embodiment, determining the degree of impact of a target variant on a target gene function includes the following processing methods: a first processing and / or a second processing;

[0067] The first process includes: determining the degree of impact of the target variant on the target gene function by comparing the positional relationship between the target variant and the last pathogenic variant at the 3' end of the target gene;

[0068] The second process includes: determining the extent of the impact of the target variant on the function of the target gene by comparing the positional relationship between the target variant-affected region and the important functional domains of the target gene.

[0069] In one embodiment, in the first process, if the target variant is located before the last pathogenic variant at the 3' end of the target gene, the target variant information is determined to be strong loss-of-function pathogenicity evidence of the target variant; the target variant information includes the category of the target variant and the target variant is located before the last pathogenic variant at the 3' end of the target gene;

[0070] In the second process, if the target variant affected region overlaps with the important functional domain of the target gene, the target variant information is determined to be strong loss-of-function pathogenicity evidence of the target variant; the target variant information is the category of the target variant and the target variant affected region overlaps with the important functional domain of the target gene.

[0071] It can be understood that in the first processing, if the target variant is located before the last pathogenic variant at the 3' end of the target gene, it can be judged that the target variant has a large impact on the function of the target gene, and therefore the target variant information is determined to be strong loss-of-function pathogenicity evidence of the target variant.

[0072] The target variant information includes the target variant category and the location of the target variant before the last pathogenic variant at the 3' end of the target gene. Specifically, the target variant information includes the target variant category and the effect of the target variant on the target gene function. For example, if the target variant category is Category 1, the target variant information indicates that it causes an open reading frame change, does not trigger nonsense-mediated mRNA degradation, and is located before the last pathogenic variant at the 3' end of the target gene, then the target variant information is determined to be strong loss-of-function pathogenicity evidence for the target variant (or the strength of the evidence for loss-of-function pathogenicity of the target variant is strong).

[0073] It can be understood that in the second processing, if the target variant affected region overlaps with the important functional domain of the target gene, it can be judged that the target variant has a large impact on the function of the target gene, and therefore the target variant information is determined to be strong functional pathogenicity evidence of the target variant.

[0074] The target variant information includes the target variant category and whether the target variant-affected region overlaps with the target gene's important functional domains. Specifically, the target variant information includes the target variant category and the effect of the target variant on the target gene's function. For example, if the target variant category is the second category, the target variant information indicates that the target variant does not result in an open reading frame change, and the target variant-affected region overlaps with the target gene's important functional domains, then the target variant information is determined to be strong loss-of-function pathogenicity evidence for the target variant (or the strength of the loss-of-function pathogenicity evidence for the target variant is strong).

[0075] In one embodiment, in the first process, if the target variant is located after the last pathogenic variant at the 3' end of the target gene, the degree of influence of the target variant on the function of the target gene is determined based on the proportion of the affected region of the target variant;

[0076] In the second process, if the affected region of the target variant does not overlap with the important functional domain of the target gene, the degree of influence of the target variant on the function of the target gene is determined based on the proportion of the affected region of the target variant.

[0077] It is understandable that in the first process, if the target variant is located after the last pathogenic variant at the 3' end of the target gene, the extent of the target variant's effect on the target gene is unclear. It is necessary to further determine the extent of the target variant's effect on the target gene function based on the proportion of the target variant's affected region to determine the strength of the evidence for the loss-of-function pathogenicity of the target variant.

[0078] In the second process, if the affected region of the target variant does not overlap with the important functional domain of the target gene, the extent of the impact of the target variant on the target gene is unclear. It is necessary to further determine the extent of the impact of the target variant on the function of the target gene based on the proportion of the affected region of the target variant to determine the strength of the evidence for the loss-of-function pathogenicity of the target variant.

[0079] In one embodiment, determining the degree of influence of the target variation on the target gene function based on the impact region ratio of the target variation specifically includes:

[0080] When the proportion of the target variant-affected region is ≥ the preset value, the target variant information is strong loss-of-function pathogenicity evidence of the target variant;

[0081] In the first process, the target variant information includes the category of the target variant, the target variant is located after the last pathogenic variant at the 3' end of the target gene, and the proportion of the target variant-affected region is ≥ the preset value;

[0082] In the second processing, the target variation information includes the category of the target variation, the target variation-affected region has no overlap with the important functional domain of the target gene, and the proportion of the target variation-affected region is ≥ a preset value.

[0083] In this embodiment, in the first process, if the target variant is classified as the first category, the target variant information is that it causes an open reading frame change, does not trigger nonsense-mediated mRNA degradation, is located after the last pathogenic variant at the 3' end of the target gene, and the target variant-affected region ratio is ≥ a preset value, the target variant information is determined to be strong loss-of-function pathogenicity evidence for the target variant (or the strength of the loss-of-function pathogenicity evidence for the target variant is: strong);

[0084] In the second processing, if the category of the target variant is the second category, the target variant information is that it does not cause a change in the open reading frame, the target variant affected region does not overlap with the important functional domain of the target gene, and the proportion of the target variant affected region is ≥ the preset value, the target variant information is determined to be strong loss-of-function pathogenicity evidence of the target variant (or the strength of the loss-of-function pathogenicity evidence of the target variant is: strong).

[0085] Preferably, determining the degree of influence of the target variant on the target gene function according to the affected region ratio of the target variant further includes: when the affected region ratio of the target variant is less than a preset value, the target variant information is evidence of moderate loss-of-function pathogenicity of the target variant;

[0086] In the first process, the target variant information includes the category of the target variant, the target variant is located after the last pathogenic variant at the 3' end of the target gene, and the proportion of the target variant-affected region is less than the preset value;

[0087] In the second processing, the target variation information includes the category of the target variation, the target variation-affected region does not overlap with the important functional domain of the target gene, and the proportion of the target variation-affected region is less than a preset value.

[0088] In this embodiment, in the first process, if the target variant is classified as the first category, the target variant information is that it causes an open reading frame change, does not trigger nonsense-mediated mRNA degradation, is located after the last pathogenic variant at the 3' end of the target gene, and the target variant-affected region ratio is less than a preset value, the target variant information is determined to be evidence of moderate loss-of-function pathogenicity of the target variant;

[0089] In the second process, if the category of the target variant is the second category, the target variant information is that it does not cause a change in the open reading frame, the target variant-affected region does not overlap with the important functional domain of the target gene, and the proportion of the target variant-affected region is less than the preset value, the target variant information is determined to be evidence of moderate loss-of-function pathogenicity of the target variant.

[0090] In one embodiment, in the first process, the category of the target variation is the first category;

[0091] In the second treatment, the category of the target variant is the second category;

[0092] The first category includes variants that result in changes in the open reading frame and do not trigger nonsense-mediated mRNA degradation;

[0093] The second category included those that did not result in a change in the open reading frame.

[0094] It can be understood that, in this embodiment, when the category of the target variation is the first category, the first processing method is used to determine the degree of influence of the target variation on the target gene;

[0095] When the category of the target variation is the second category, the second processing method is used to determine the degree of influence of the target variation on the target gene.

[0096] In one embodiment, determining the extent to which a target variant affects the function of a target gene includes the following processing methods:

[0097] Third treatment;

[0098] The third process includes: determining the extent of the impact of the target mutation on the function of the target gene by querying the pathogenic reports of the target gene start codon where the target mutation occurs.

[0099] In one embodiment, in the third process, if the target gene in which the target variant occurs has a start codon pathogenicity report, the target variant information is determined to be strong loss-of-function pathogenicity evidence for the target variant;

[0100] The target mutation information includes the category of the target mutation and the target gene where the target mutation occurs and whether the start codon has been reported to be pathogenic.

[0101] It is understandable that in the third process, if the target gene in which the target mutation occurs has a report of pathogenicity of the start codon, it can be judged that the target mutation has a large impact on the function of the target gene, and therefore the target mutation information is determined to be strong loss-of-function pathogenicity evidence of the target mutation.

[0102] The target variant information includes the target variant category and reports of pathogenic start codons in the target gene where the target variant occurs. Specifically, the target variant information includes the target variant category and the effect of the target variant on the target gene function. For example, if the target variant category is Category 3, the target variant information covers the first CDS and is not a whole-gene deletion, and the target gene where the target variant occurs has reports of pathogenic start codons, then the target variant information is determined to be strong loss-of-function pathogenicity evidence for the target variant (or the strength of the evidence for loss-of-function pathogenicity is strong).

[0103] In one embodiment, in the third process, if there is no report of a target gene with a target mutation having a pathogenic start codon, the degree of impact of the target mutation on the function of the target gene is determined based on the proportion of regions affected by the target mutation.

[0104] It is understandable that in the third process, if there is no report of pathogenicity of the start codon of the target gene where the target mutation occurs, the extent of the impact of the target mutation on the target gene is unclear, and it is necessary to further determine the extent of the impact of the target mutation on the function of the target gene based on the proportion of the affected area of ​​the target mutation to determine the evidence of loss-of-function pathogenicity of the target mutation.

[0105] In one embodiment, in the third process, determining the degree of influence of the target variation on the target gene function based on the impact region ratio of the target variation specifically includes:

[0106] When the proportion of the target variant-affected region is ≥ the preset value, the target variant information is strong loss-of-function pathogenicity evidence of the target variant;

[0107] The target variation information includes the category of the target variation, the target gene of the target variation has no reports of pathogenicity caused by the start codon, and the proportion of the target variation-affected region is ≥ the preset value.

[0108] In this embodiment, if the category of the target variant is the third category, the target variant information covers the first CDS and is not a whole-gene deletion, there is no report of pathogenicity caused by the start codon of the target gene where the target variant occurs, and the proportion of the target variant-affected region is ≥ a preset value, the target variant information is determined to be strong loss-of-function pathogenicity evidence of the target variant (or the strength of the loss-of-function pathogenicity evidence of the target variant is: strong).

[0109] In one embodiment, in the third process, determining the degree of impact of the target variant on the target gene function based on the affected region ratio of the target variant further includes: when the affected region ratio of the target variant is less than a preset value, the target variant information is evidence of moderate loss-of-function pathogenicity of the target variant;

[0110] The target variation information includes the category of the target variation, the target gene of the target variation has no reports of pathogenicity caused by the start codon, and the proportion of the target variation-affected region is less than the preset value.

[0111] In this embodiment, if the target variant is of the third category, the target variant information covers the first CDS and is not a whole-gene deletion, there is no report of pathogenicity of the start codon of the target gene where the target variant occurs, and the proportion of the target variant-affected region is less than a preset value, the target variant information is determined to be moderate loss-of-function pathogenicity evidence of the target variant (or the strength of the loss-of-function pathogenicity evidence of the target variant is: moderate).

[0112] In one embodiment, in the third process, the category of the target variation is the third category;

[0113] The third category includes deletions that cover the first CDS but are not whole-gene deletions.

[0114] It can be understood that, in this embodiment, when the category of the target variation is the third category, the third processing method is used to determine the degree of influence of the target variation on the target gene.

[0115] In one embodiment, determining the extent to which a target variant affects the function of a target gene includes the following processing methods:

[0116] Fourth treatment;

[0117] The fourth process includes: determining the extent of the impact of the target mutation on the function of the target gene by predicting whether the sequence of the target gene in which the target mutation occurs has a stop codon.

[0118] In one embodiment, in the fourth process, if the sequence of the target gene in which the target mutation is predicted to occur does not contain a stop codon, the target mutation information is determined to be strong evidence of loss-of-function pathogenicity;

[0119] The target mutation information includes the category of the target mutation and the absence of a stop codon in the sequence of the target gene where the target mutation occurs.

[0120] It is understandable that if the sequence of the target gene where the target mutation occurs does not contain a stop codon, the target mutation has a great impact on the target gene, and therefore the target mutation information is determined to be strong loss-of-function pathogenicity evidence of the target mutation.

[0121] The target variant information includes the target variant category and the absence of a stop codon in the target gene sequence where the target variant occurs. For example, if the target variant category is Category 4, the target variant information includes the absence of a stop codon in the target gene sequence where the target variant occurs, covering the last CDS and not a whole-gene deletion, and the target variant is determined to be strongly pathogenic.

[0122] In one embodiment, if, in the fourth process, the sequence of the target gene in which the target mutation is predicted to occur contains a stop codon, the fifth process is then used to determine the extent to which the target mutation affects the function of the target gene;

[0123] The fifth treatment involves determining whether the target variant triggers nonsense-mediated mRNA degradation.

[0124] It is understandable that if the sequence of the target gene in which the target mutation occurs contains a stop codon, the extent of the impact of the target mutation on the target gene is unknown, and the extent of the impact of the target mutation on the function of the target gene needs to be determined according to the fifth process.

[0125] In one embodiment, in the fifth process, if the target variant triggers nonsense-mediated mRNA degradation, the target variant information is determined to be strong loss-of-function pathogenicity evidence of the target variant;

[0126] The target variation information includes the category of the target variation, the presence of a stop codon in the sequence of the target gene where the target variation occurs, and whether the target variation triggers nonsense-mediated mRNA degradation.

[0127] It can be understood that in the fifth treatment, if the target variant triggers nonsense-mediated mRNA degradation and the target variant has a great impact on the target gene, the target variant information is determined to be the strength of the evidence of extremely strong loss-of-function pathogenicity of the target variant.

[0128] In this embodiment, if the category of the target variation is the fourth category, the target variation information covers the last CDS and is not a whole-gene deletion, the sequence of the target gene where the target variation occurs contains a stop codon, and the target variation triggers nonsense-mediated mRNA degradation, the target variation information is determined to be extremely strong loss-of-function pathogenicity evidence of the target variation.

[0129] In one embodiment, if the target variant does not induce nonsense-mediated mRNA degradation, the extent of the effect of the target variant on the target gene function is determined according to the first treatment;

[0130] The first step involves comparing the positional relationship between the target variant and the last pathogenic variant at the 3' end of the target gene to determine the effect of the target variant on the function of the target gene.

[0131] It is understandable that if the target variant does not trigger nonsense-mediated mRNA degradation, the extent of the impact of the target variant on the target gene is unknown, and the extent of the impact of the target variant on the function of the target gene needs to be determined based on the first treatment.

[0132] In one embodiment, in the first process, if the target variant is located before the last pathogenic variant at the 3' end of the target gene, the target variant information is determined to be strong loss-of-function pathogenicity evidence; the target variant information includes the category of the target variant, the presence of a stop codon in the sequence of the target gene where the target variant occurs, the target variant does not trigger nonsense-mediated mRNA degradation, and the target variant is located before the last pathogenic variant at the 3' end of the target gene.

[0133] In this embodiment, if the category of the target variant is the fourth category, the target variant information covers the last CDS and is not a whole-gene deletion, the sequence of the target gene where the target variant occurs contains a stop codon, the target variant does not trigger nonsense-mediated mRNA degradation, and the target variant is located before the last pathogenic variant at the 3' end of the target gene, the target variant information is determined to be strong loss-of-function pathogenicity evidence of the target variant.

[0134] In one embodiment, in the first process, if the target variant is located after the last pathogenic variant at the 3' end of the target gene, the degree of influence of the target variant on the function of the target gene is determined based on the proportion of the affected region of the target variant.

[0135] In one embodiment, in the first process, determining the degree of influence of the target variation on the target gene function based on the impact region ratio of the target variation specifically includes:

[0136] When the proportion of the target variant affected region is ≥ the preset value, the target variant information is evidence of strong loss-of-function pathogenicity of the target variant; the target variant information includes the category of the target variant, the presence of a stop codon in the sequence of the target gene where the target variant occurs, the target variant does not trigger nonsense-mediated mRNA degradation, the target variant is located after the last pathogenic variant at the 3' end of the target gene, and the proportion of the target variant affected region is ≥ the preset value.

[0137] In this embodiment, if the category of the target variant is the fourth category, the target variant information covers the last CDS and is not a whole-gene deletion, the sequence of the target gene where the target variant occurs contains a stop codon, the target variant does not trigger nonsense-mediated mRNA degradation, the target variant is located after the last pathogenic variant at the 3' end of the target gene, and the proportion of the target variant-affected region is ≥ a preset value, the target variant information is determined to be strong loss-of-function pathogenicity evidence.

[0138] In one embodiment, determining the degree of influence of the target variation on the function of the target gene based on the impact region ratio of the target variation specifically includes: when the impact region ratio of the target variation is less than a preset value, the target variation information is evidence of moderate loss-of-function pathogenicity of the target variation; the target variation information includes the category of the target variation, the presence of a stop codon in the sequence of the target gene where the target variation occurs, the target variation does not trigger nonsense-mediated mRNA degradation, the target variation is located after the last pathogenic variation at the 3' end of the target gene, and the impact region ratio of the target variation is less than a preset value.

[0139] In this embodiment, if the category of the target variant is the fourth category, the target variant information covers the last CDS and is not a whole-gene deletion, the sequence of the target gene where the target variant occurs contains a stop codon, the target variant does not trigger nonsense-mediated mRNA degradation, the target variant is located after the last pathogenic variant at the 3' end of the target gene, and the proportion of the target variant-affected region is less than a preset value, the target variant information is determined to be evidence of moderate loss-of-function pathogenicity.

[0140] In one embodiment, in the fourth process, the category of the target variation is the fourth category;

[0141] The fourth category includes deletions covering the last CDS but not whole-gene deletions.

[0142] It is understood that in this embodiment, when the target variant is classified as the fourth category, the fourth process is used to determine the impact of the target variant on the target gene. If the fourth process fails to determine the impact of the target variant on the target gene, the fifth process is used. If the fifth process still fails to determine the impact of the target variant on the target gene, the first process is used to determine the impact of the target variant on the target gene.

[0143] According to the third aspect, in one embodiment, a method for correcting the strength of evidence for loss-of-function pathogenicity is provided, comprising:

[0144] Correction is made for the strength of evidence for loss-of-function pathogenicity determined by any of the methods of the second aspect.

[0145] In one embodiment, the correction includes correcting based on the strength of association between the target gene and the disease.

[0146] In one embodiment, performing correction based on the strength of association between the target gene and the disease includes:

[0147] If the target gene is strongly associated with the disease, no correction is required;

[0148] If the target gene has a moderate association with the disease, the evidence for loss-of-function pathogenicity is downgraded by one level;

[0149] If the target gene is weakly associated with the disease, the evidence for loss-of-function pathogenicity is downgraded to the second level.

[0150] The method of the present invention determines the strength of evidence for loss-of-function pathogenicity of the target variant, and the loss-of-function pathogenicity evidence can be further corrected based on the strength of the association between the target gene and the disease to obtain more accurate results.

[0151] According to the fourth aspect, in one embodiment, a device for determining evidence of loss-of-function pathogenicity is provided, comprising:

[0152] The category acquisition module is used to obtain the category of the target variant based on its impact on transcription;

[0153] The impact judgment module is used to judge the impact of the target variant on the target gene function based on the category of the target variant;

[0154] The evidence judgment module is used to determine whether the target variant is evidence of loss-of-function pathogenicity based on the impact of the target variant on the target gene function.

[0155] According to the fifth aspect, in one embodiment, a device for determining the strength of evidence for loss-of-function pathogenicity is provided, comprising:

[0156] A strength determination module is used to determine the strength of the loss-of-function pathogenicity evidence obtained by the device of the fourth aspect based on the degree of impact of the target variant on the function of the target gene.

[0157] According to the sixth aspect, in one embodiment, a device for correcting the strength of evidence for loss-of-function pathogenicity is provided, comprising:

[0158] A correction module is used to correct the strength of the evidence of loss-of-function pathogenicity determined by the device of the fifth aspect.

[0159] According to the seventh aspect, in one embodiment, a device for determining evidence of loss-of-function pathogenicity is provided, comprising:

[0160] Memory, used to store programs;

[0161] A processor, configured to implement any one of the methods of the first aspect by executing a program stored in a memory.

[0162] According to the eighth aspect, in one embodiment, a device for determining the strength of evidence for loss-of-function pathogenicity is provided, comprising:

[0163] Memory, used to store programs;

[0164] A processor, configured to implement any one of the methods of the second aspect by executing a program stored in a memory.

[0165] According to the ninth aspect, in one embodiment, a device for correcting the strength of evidence for loss-of-function pathogenicity is provided, comprising:

[0166] Memory, used to store programs;

[0167] A processor, configured to implement any one of the methods of the third aspect by executing a program stored in a memory.

[0168] According to the tenth aspect, in one embodiment, a computer-readable storage medium is provided, on which a program is stored, and the program can be executed by a processor to implement the method of any one of the first aspect, the second aspect or the third aspect.

[0169] Example 1

[0170] In this embodiment, the target mutation is an exon deletion mutation.

[0171] 1. Evidence of loss-of-function pathogenicity

[0172] Based on the effect of the target variant on transcription, the categories of target variants obtained include:

[0173] Category 5: variants cannot be annotated to coding transcripts;

[0174] Category 6: The variant can be annotated to the coding transcript, but the missing part does not contain the coding sequence (CDS).

[0175] 1.2 Determine the impact of target variants on target gene function

[0176] Target variants in the fifth category or the sixth category do not affect the function of the target gene.

[0177] 1.3 Determine whether the target variant information is evidence of loss-of-function pathogenicity

[0178] Category 5 or 6 target variants do not affect the function of the target gene, so category 5 or 6 is not evidence of loss-of-function pathogenicity for exon deletion variants.

[0179] This example simplifies the determination of loss-of-function pathogenicity for exon deletion variants. Conventional methods require multiple steps to determine whether the target variant information is evidence of loss-of-function pathogenicity, such as whether it causes an open reading frame change, whether it triggers nonsense-mediated mRNA degradation, the function of the corresponding amino acid region, and whether it is derived from the predominantly expressed transcript. This step greatly simplifies the multi-pathway and multi-step judgment process, making the judgment logic clearer.

[0180] It is understandable that embodiments of the present invention can directly exclude target variants that meet the fifth and sixth categories before determining the loss-of-function pathogenicity evidence of the target variant.

[0181] Example 2

[0182] In this example, the target mutation is an exon deletion mutation.

[0183] 1. Evidence of loss-of-function pathogenicity

[0184] 1.1 Based on the effect of target variants on transcription, the categories of target variants obtained include:

[0185] Category 1: results in open reading frame changes without triggering nonsense-mediated mRNA degradation;

[0186] Category 2: does not result in changes in the open reading frame.

[0187] The method for determining whether nonsense-mediated mRNA degradation is triggered is as follows: the amino acid position value 2 corresponding to the post-mutation stop codon and the theoretical critical amino acid position value 3 for triggering nonsense-mediated mRNA degradation are calculated; if the amino acid position value 2 is less than the amino acid position value 3, the variant is determined to trigger nonsense-mediated mRNA degradation; if the amino acid position value 2 is not less than the amino acid position value 3, the variant is determined not to trigger nonsense-mediated mRNA degradation. The amino acid position value 3 can be obtained from a public database (e.g., https: / / www.mutationtaster.org / ). In a preferred embodiment, the collected amino acid position values ​​3 can be preprocessed to generate a custom configuration library, which can be directly retrieved during the judgment process, further improving interpretation efficiency and reducing waste of computing resources. It should be noted that the amino acid position value 2 is predicted based on the post-mutation CDS sequence. Therefore, the post-mutation CDS sequence must first be obtained through splicing, and then the amino acid position value 2 is determined based on the amino acid length of the post-mutation CDS sequence. In this case, the mutated CDS sequence is based on the reference CDS sequence. According to the annotation results, the CDS deletion sequence corresponding to the mutation is directly deleted, and the remaining sequence is spliced ​​in the original order to obtain it.

[0188] Open reading frame alterations include situations where the variant sequence in the CDS region is not a multiple of three nucleotides, or where the variant sequence is a multiple of three nucleotides but the amino acid position value 2 differs from the theoretical stop codon position. For exon deletions, the theoretical stop codon position is calculated as the difference between the amino acid lengths of the reference and deleted sequences plus 1. In other words, the only situation where the open reading frame remains unchanged is when the variant sequence is a multiple of three nucleotides and the amino acid position value 2 is the same as the theoretical stop codon position. It is important to note that classifying a variant sequence that is a multiple of three nucleotides but the amino acid position value 2 differs from the theoretical stop codon position as an open reading frame alteration, rather than simply judging based on whether the variant is a multiple of three nucleotides as is done in conventional methods. This approach addresses the source of evidence for loss-of-function pathogenicity and encompasses special cases, such as deletions that are not multiples of three nucleotides but result in a new stop codon leading to premature termination. Furthermore, evidence for loss-of-function pathogenicity can be determined by assessing the impact of premature termination on gene function.

[0189] This example provides more comprehensive and reasonable coverage of evidence for loss-of-function pathogenicity. Compared with conventional interpretation methods, it can avoid omissions of special cases and improve the accuracy of detection.

[0190] 1.2 Determine the impact of target variants on target gene function

[0191] When the target variant is of the first category: causing a change in the open reading frame and not triggering nonsense-mediated mRNA degradation, it can be directly determined that the target variant affects the function of the target gene.

[0192] When the target variant falls into the second category: does not cause a change in the open reading frame, it can be directly determined that the target variant affects the function of the target gene.

[0193] 1.3 Determine whether the target variant information is evidence of loss-of-function pathogenicity

[0194] If the target variant in the first category affects the function of the target gene, the first category (open reading frame change without nonsense-mediated mRNA degradation) is determined to be evidence of loss-of-function pathogenicity for exon deletion variants.

[0195] If the target variant in the second category affects the function of the target gene, the second category (not resulting in an open reading frame change) is determined to be exon deletion variant with evidence of loss-of-function pathogenicity.

[0196] 2. Determination of the strength of evidence for loss-of-function pathogenicity

[0197] 2.1 Specific method for determining the impact of target variants on target gene function

[0198] a. Category 1 target variants: exon deletion variants that result in open reading frame changes but do not trigger nonsense-mediated mRNA degradation

[0199] The first treatment is used to determine the extent of the first-category target variant's effect on the target gene's function: the amino acid position value 1 corresponding to the first-category target variant's start position is compared with the amino acid position value 4 corresponding to the last pathogenic variant at the 3' end of the target gene. If the amino acid position value 1 is not greater than the amino acid position value 4, the first-category target variant is considered to have a high degree of effect on the target gene's function. If the variant causes an open reading frame change, does not trigger nonsense-mediated mRNA degradation, and the amino acid position value 1 is not greater than the amino acid position value 4, it is considered to have strong evidence of loss-of-function pathogenicity for an exon deletion variant. If the amino acid position value 1 is greater than the amino acid position value 4, the extent of the first-category target variant's effect on the gene's function is unknown.

[0200] Among them, the calculation method of amino acid position value 1 is: after left-aligning the reference CDS sequence and the mutated CDS sequence, the sequence consistency is compared, and the length value 3 from the A base in the start codon ATG of the reference CDS sequence to the first inconsistent base is calculated, and the length value 3 is divided by 3; if the result is an integer, it is the amino acid position value 1; if the result is not an integer, the amino acid position value 1 is the integer part plus 1. Among them, the splicing method of the mutated CDS sequence is the same as 1.1. The amino acid position value 4 represents the amino acid position corresponding to the last mutation at the 3' end among the credible pathogenic variants. Credible pathogenic variants can be recorded in public databases (for example, https: / / www.ncbi.nlm.nih.gov / gene / ) or from experimental verification. In a preferred embodiment, the collected position values ​​4 can be preprocessed to generate a custom configuration library, which can be directly called during the judgment process, which can further improve the interpretation efficiency and reduce the waste of computing resources.

[0201] For target variants classified as category 1, the extent to which their effect on the target gene function is unclear, further evidence of loss-of-function pathogenicity must be determined based on the proportion of regions affected by the variant.

[0202] b. Second category of target variants: exon deletion variants that do not result in an open reading frame change

[0203] The second category of target variants uses the second processing method to determine the impact of the second category of target variants on the target gene function:

[0204] Compare the coverage between the affected regions of the second-category target variant and the key functional domains of the target gene. If the affected regions of the target variant overlap with the key functional domains of the target gene, the second-category target variant has a significant impact on the function of the target gene. Overlap between the affected regions of the second-category target variant and the key functional domains of the target gene is considered strong evidence of loss-of-function pathogenicity for exon deletion variants. If the affected regions of the second-category target variant do not overlap with the key functional domains of the target gene, the impact of the second-category target variant on the function of the target gene is considered unclear.

[0205] The starting position of the target variant-affected region is position 1, and the ending position is position 1 + length 2 - (position 2 - 1). The calculation of amino acid position 1 is the same as in step 2.1a, and length 2 is the amino acid length encoded by the reference CDS sequence. The calculation of amino acid position 2 is the same as in step 1.1.

[0206] This information on important gene functional domains can be obtained from public databases (e.g., https: / / www.ebi.ac.uk / interpro / ) or scientific literature. In a preferred approach, the collected gene functional domain information can be preprocessed to generate a custom configuration library, which can be directly accessed during the judgment process, further improving interpretation efficiency and reducing the waste of computing resources.

[0207] For target variants determined to be in the second category and whose effects on the function of the target gene are unclear, further evidence of loss-of-function pathogenicity needs to be determined based on the proportion of regions affected by the target variant.

[0208] Evaluating the extent of the impact of the target variant on the function of the target gene is a key step in determining the strength of evidence for loss-of-function pathogenicity, especially for variants that only result in the deletion of part of the coding sequence. Such variants are usually unable to directly determine whether they will lead to complete loss of gene function. However, the prior art only generally summarizes whether the region to be determined as variant affects gene function, which obviously lacks operability. This step was designed for operability based on expert consensus and guidelines, and automated design was performed on this basis. It should be noted that this embodiment fully considers the biological characteristics of different subdivisions of exon deletion-type variants for different subdivisions, and uses differentiated judgment methods, which is more comprehensive and reasonable. For example, exon deletion-type variants that meet the first category result in changes in the open reading frame, so the functional domain information based on the reference genome is not fully applicable, that is, the second processing may not accurately determine the extent of the impact of the target variant on the function of the target gene.

[0209] 2.2 Specific method for determining the proportion of target variation-affected areas

[0210] The target variant's impact ratio specifically refers to the ratio of the length of the variant's impact region to the length of the reference sequence. If this ratio is ≥ a preset value (e.g., 10%), the target variant has a high degree of impact on the target gene's function. A first category (where amino acid position value 1 is greater than amino acid position value 4 and the ratio of the length of the variant's impact region to the length of the reference sequence is ≥10%) is determined as strong loss-of-function pathogenicity for an exon deletion variant (or a second category (where the target variant's impact region does not overlap with a key functional domain of the target gene and the ratio of the length of the variant's impact region to the length of the reference sequence is ≥10%). If this ratio is less than a preset value (e.g., 10%), the target variant is judged to have a low degree of impact on the target gene's function. A first category (where amino acid position value 1 is greater than amino acid position value 4 and the ratio of the length of the variant's impact region to the length of the reference sequence is <10%) is determined as moderate loss-of-function pathogenicity for an exon deletion variant (or a second category (where the target variant's impact region does not overlap with a key functional domain of the target gene and the ratio of the length of the variant's impact region to the length of the reference sequence is <10%). It is understood that the target variant's impact region and the reference sequence must be compared at the same level.

[0211] Using the amino acid level as an example, we calculate the ratio of the amino acid length of the target variant-affected region (length value 1) to the length of the reference amino acid sequence (length value 2). Specifically, length value 1 = length value 2 - (amino acid position value 2 - 1). Length value 2 is the amino acid length encoded by the reference CDS sequence. The calculation of amino acid position value 2 is the same as in step 1.1.

[0212] Example 3

[0213] In this embodiment, the target mutation is an exon deletion mutation.

[0214] 1. Evidence of loss-of-function pathogenicity

[0215] 1.1 Based on the effect of target variants on transcription, the categories of target variants obtained also include:

[0216] Category 3: covers the first CDS and is not a whole-gene deletion.

[0217] The third category of target variation is a special case and can be treated specially. The order of treating the third category of target variation is not limited to that of Example 2.

[0218] 1.2 Determine the impact of target variants on target gene function

[0219] When the target variant is of the third category: covering the first CDS and not a whole-gene deletion, it can be directly determined that the target variant of the third category affects the function of the target gene.

[0220] 1.3 Determine whether the target variant information is evidence of loss-of-function pathogenicity

[0221] If the target variant in the third category affects the function of the target gene, the third category is determined to be loss-of-function pathogenic evidence of the target variant, that is, loss-of-function pathogenic evidence of the variant covering the first CDS and not the whole gene deletion being an exon deletion.

[0222] 2. Determination of the strength of evidence for loss-of-function pathogenicity

[0223] 2.1 Specific judgment on the impact of the target variant on the target gene:

[0224] The third category of target variants uses the third processing method to determine the degree of impact of the third category of target variants on the target gene function: if the target gene where the third category of target variant occurs has a reported start codon pathogenic variant, then the third category of target variant is judged to have a large impact on the target gene function, and strong loss-of-function pathogenicity evidence is determined for the target gene where the third category of target variants and the target gene where the third category of target variant occurs have a reported start codon pathogenic variant as an exon deletion variant; if the target gene where the third category of target variant occurs has no reported start codon pathogenic variant, then the degree of impact of the third category of target variant on the target gene function is judged to be unclear.

[0225] Reports of start codon pathogenic variants can be obtained from publicly available databases (e.g., https: / / www.ncbi.nlm.nih.gov / gene / ) or from literature reports. In a preferred approach, the collected start codon pathogenic variant information can be preprocessed to generate a custom configuration library, which can be directly accessed during the judgment process, further improving interpretation efficiency and reducing the waste of computing resources.

[0226] For target variants that are determined to have unclear effects on target gene function, further determination of the proportion of regions affected by the target variant is required to determine evidence of loss-of-function pathogenicity.

[0227] 2.2 Specific method for determining the proportion of target variation-affected areas

[0228] The proportion of the target variant affected region specifically refers to the ratio of the length of the target variant affected region to the length of the reference sequence. If the ratio is ≥ the preset value (for example, 10%), the target variant has a large impact on the function of the target gene, and the target gene of the third category and the target variant occurring in the third category has no start codon pathogenicity report and the ratio of the length of the variant affected region to the length of the reference sequence is ≥10%, which is strong loss-of-function pathogenicity evidence for exon deletion variants. If the ratio is < the preset value (for example, 10%), the target variant of the third category has a small impact on the function of the target gene, and the target gene of the third category and the target variant occurring in the third category has no start codon pathogenicity report and the ratio of the length of the variant affected region to the length of the reference sequence is <10%, which is moderate loss-of-function pathogenicity evidence for exon deletion variants. It is understandable that the target variant affected region and the reference sequence need to be compared at the same level.

[0229] The following uses the amino acid level as an example to calculate the ratio of the amino acid length of the variant-affected region (length 1) to the length of the reference amino acid sequence (length 2). Length 1 is calculated by dividing the length of the missing CDS nucleotide sequence by 3. If the value is an integer, length 1 is calculated. If the value is not an integer, length 1 is calculated by adding 1 to the integer portion.

[0230] It is important to note that the target variant impact region is calculated differently than in 2.2 of Example 2. This example clarifies and refines the impact region based on the specific variant types and the unique biological functional impact of each type, resulting in a more scientific and reasonable differentiated design.

[0231] Exon deletion variants that cover the first CDS and are not full-gene deletions usually involve multiple functional elements and have diverse effects on gene function, which may involve changes in the start codon or fragment deletions. This embodiment creatively designs a hierarchical judgment of exon deletion variants containing the first CDS, and comprehensively covers various situations. This embodiment fully mines and utilizes public data, and can quickly determine the pathogenicity evidence of some variants through the pathogenicity judgment of the start codon, and quickly determine the strength of the pathogenicity evidence. For those that cannot be quickly determined, the target variant is further creatively combined with the determination of the proportion of the affected area to determine the evidence and the strength of the evidence from the fundamental perspective of whether it causes the loss of gene function.

[0232] Example 4

[0233] In this embodiment, the target mutation is an exon deletion mutation.

[0234] 1. Evidence of loss-of-function pathogenicity

[0235] 1.1 Based on the effect of target variants on transcription, the categories of target variants obtained also include:

[0236] Category 4: covers the last CDS but not the whole gene deletion.

[0237] The fourth category of exon deletion variants is a special case and can be treated with special treatment. The treatment of the fourth category of exon deletion variants is not limited to the order of Example 2 and Example 3.

[0238] 1.2 Determine the impact of target variants on target gene function

[0239] When the exon deletion variant is of the fourth category: covering the last CDS and not a whole-gene deletion, it can be directly determined that the fourth category exon deletion variant affects the function of the target gene.

[0240] 1.3 Determine whether the target variant information is evidence of loss-of-function pathogenicity

[0241] If the target variant in the fourth category affects the function of the target gene, the fourth category is determined to be loss-of-function pathogenic evidence for exon deletion variants, that is, loss-of-function pathogenic evidence for exon deletion variants that cover the last CDS and are not whole-gene deletions.

[0242] 2. Determination of the strength of evidence for loss-of-function pathogenicity

[0243] 2.1 Specific method for determining the impact of target variants on target gene function

[0244] The fourth category of target variants uses the fourth processing method to determine the degree of impact of the fourth category of target variants on the target gene function:

[0245] If the sequence of the target gene predicted to have the fourth category of target variation does not contain a stop codon, then the fourth category of target variation is judged to have a great degree of impact on the function of the target gene. The fourth category of target gene predicted to have the fourth category of target variation does not contain a stop codon, and the fourth category of target gene sequence is determined to have strong loss-of-function pathogenicity evidence for exon deletion-type variation. If the sequence of the target gene predicted to have the fourth category of target variation contains a stop codon, then the fourth category of target variation is judged to have an unknown degree of impact on the function of the target gene.

[0246] In the conventional annotation process, the specific situation of the stop codon is not considered. However, the lack of a stop codon will cause NSD (Non-stop decay), which will further affect gene function. This embodiment comprehensively considers the special type of mutation without a stop codon, and starting from the source of the impact of the mutation on gene function, clarifies its loss-of-function pathogenicity evidence. This special type of mutation corresponds to extremely strong loss-of-function pathogenicity evidence. According to existing expert consensus and guideline rules, only a few other evidences are needed to determine the association between the mutation and the disease. It can be seen that compared with the conventional interpretation method, omissions can be avoided and the accuracy of detection can be improved.

[0247] 2.2 For exon deletion variants in Category 4 whose impact on target gene function is unclear, the fifth step will be used to determine the impact of the target variant in Category 4 on target gene function. The fifth step includes determining whether the target variant in Category 4 induces nonsense-mediated mRNA degradation.

[0248] If the target variant in the fourth category triggers nonsense-mediated mRNA degradation, the target variant in the fourth category has a great impact on the function of the target variant. In this case, the sequence of the target gene in the fourth category, where the target variant in the fourth category is predicted to occur, contains a stop codon, and the target variant in the fourth category triggers nonsense-mediated mRNA degradation, which is strong evidence of loss-of-function pathogenicity, is determined to be an exon deletion variant.

[0249] If the target variant in the fourth category does not induce nonsense-mediated mRNA degradation, the target variant in the fourth category is subjected to the first treatment to determine the effect of the target variant in the fourth category on the function of the target gene. The aforementioned determination method is the same as the determination method of the first treatment for the target variant in the first category in 2.1 of Example 2.

[0250] Compare the amino acid position value 1 corresponding to the start position of the fourth-category target variant with the amino acid position value 4 corresponding to the last pathogenic variant at the 3' end of the target gene. If the amino acid position value 1 is not greater than the amino acid position value 4, the fourth-category target variant is considered to have a high degree of effect on the target gene function. This provides strong evidence of loss-of-function pathogenicity for the fourth-category target gene, including the presence of a stop codon in the sequence of the target gene predicted to harbor the fourth-category target variant, the absence of nonsense-mediated mRNA degradation by the fourth-category target variant, and the presence of amino acid position value 1 not greater than amino acid position value 4, as an exon deletion variant. If the amino acid position value 1 is greater than the amino acid position value 4, the first-category target variant is considered to have an unknown effect on gene function.

[0251] 2.3 The method for determining the proportion of the target variation-affected region in the fourth category is the same as 2.2 in Example 2 and will not be repeated here.

[0252] The fourth category of target variant affected region ratio specifically refers to the ratio of the length of the variant affected region to the length of the reference sequence. If this ratio is ≥ a preset value (e.g., 10%), the fourth category target variant has a high degree of effect on the function of the target gene. The fourth category is determined to be strong loss-of-function pathogenicity for exon deletion variants if the sequence of the target gene predicted to harbor the fourth category target variant contains a stop codon, the fourth category target variant does not trigger nonsense-mediated mRNA degradation, the amino acid position value 1 is greater than the amino acid position value 4, and the ratio of the length of the variant affected region to the length of the reference sequence is ≥10%. If this ratio is less than a preset value (e.g., 10%), the fourth category target variant is determined to have a low degree of effect on the function of the target gene. The fourth category is determined to be moderate loss-of-function pathogenicity for exon deletion variants if the sequence of the target gene predicted to harbor the fourth category target variant contains a stop codon, the fourth category target variant does not trigger nonsense-mediated mRNA degradation, the amino acid position value 1 is greater than the amino acid position value 4, and the ratio of the length of the variant affected region to the length of the reference sequence is <10%.

[0253] Example 5

[0254] In this embodiment, the target mutation is an exon deletion mutation.

[0255] 1. Evidence of loss-of-function pathogenicity

[0256] 1.1 Based on the effect of target variants on transcription, the categories of target variants obtained also include:

[0257] Category 7: Exon deletion variants that result in the loss of all coding sequences.

[0258] 1.2 Determine the impact of target variants on target gene function

[0259] For the seventh category of target variants: exon deletion variants that cause the loss of all coding sequences, it can be directly determined that the seventh category of target variants has an impact on the function of the target gene.

[0260] 1.3 Determine whether the target variant information is evidence of loss-of-function pathogenicity

[0261] The target variant in the seventh category has an impact on the function of the target gene, which can directly determine that the seventh category is evidence of loss-of-function pathogenicity of the target variant.

[0262] 2. Determination of the strength of evidence for loss-of-function pathogenicity

[0263] When the target variant is in category 7, it can be directly determined that category 7 is extremely strong evidence of loss-of-function pathogenicity of the target variant.

[0264] In this embodiment, for the target variant of the seventh category, it can be directly determined that the target variant of the seventh category has an impact on the function of the target gene, and through this impact, it can be directly determined that the seventh category is extremely strong loss-of-function pathogenic evidence of the target variant.

[0265] Conventional methods determine whether a variant results in a whole-gene deletion. However, a complete gene includes both coding and non-coding regions. For genes with loss-of-function pathogenicity, the absence of coding sequences alone can indicate an inability to synthesize biologically functional transcripts, which in turn leads to loss of function of the corresponding protein. This example more comprehensively encompasses exon deletion variants that meet the criteria for extremely strong loss-of-function pathogenicity.

[0266] Example 6

[0267] 1. Evidence of loss-of-function pathogenicity

[0268] In this embodiment, the target mutation is an exon deletion mutation.

[0269] 1.1 Based on the effect of target variants on transcription, the categories of target variants obtained also include:

[0270] Category 8: Causes changes in the open reading frame and triggers nonsense-mediated mRNA degradation.

[0271] 1.2 Determine the impact of target variants on target gene function

[0272] For target variants in the eighth category, it can be directly determined that the target variants in the eighth category have an impact on the function of the target gene.

[0273] 1.3 Determine whether the target variant information is evidence of loss-of-function pathogenicity

[0274] The target variants in the eighth category have an impact on the function of the target gene, and the target variants in the eighth category can be directly determined to be evidence of loss-of-function pathogenicity.

[0275] 2. Determination of the strength of evidence for loss-of-function pathogenicity

[0276] When the target variant is in category eight, it can be directly determined that category eight is extremely strong evidence of loss-of-function pathogenicity of the target variant.

[0277] In this embodiment, for the target variant of the eighth category, it can be directly determined that the target variant of the eighth category has an impact on the function of the target gene, and through this impact, it can be directly determined that the eighth category is extremely strong loss-of-function pathogenic evidence of the target variant.

[0278] The principle for determining whether the open reading frame has changed is the same as that in 2.1 of Example 2.

[0279] The embodiment of the present invention does not particularly limit the order of classifying the target variants of the first to eighth categories and the corresponding order of the method for determining the impact of the target variant on the target gene function.

[0280] Correction of the evidence for loss-of-function pathogenicity is performed based on the strength of the association between the target gene and the disease. The strength of the association between the gene and the disease can be obtained from public databases (e.g., https: / / www.clinicalgenome.org / , https: / / www.omim.org / ) or from literature reports. Specific correction methods include:

[0281] When the association strength between the target gene and the disease is strong, no correction is required;

[0282] When the target gene is moderately associated with the disease, the loss-of-function pathogenicity evidence is downgraded by one level. For example, very strong loss-of-function pathogenicity evidence is adjusted to strong loss-of-function pathogenicity evidence, strong loss-of-function pathogenicity evidence is adjusted to moderate loss-of-function pathogenicity evidence, and so on.

[0283] When the target gene is weakly associated with the disease, the loss-of-function pathogenicity evidence is downgraded by two levels. For example, very strong loss-of-function pathogenicity evidence is adjusted to moderate loss-of-function pathogenicity evidence, strong loss-of-function pathogenicity evidence is adjusted to supporting loss-of-function pathogenicity evidence, and so on.

[0284] The beneficial effects achieved by the embodiments of the present invention include:

[0285] Clarifying the applicable circumstances and strength of evidence can not only improve the standardization and accuracy of the use of such evidence, but also greatly reduce the investment in manual query of other types of evidence, improve the efficiency and consistency of interpretation, and further improve batch and automated interpretation.

[0286] Those skilled in the art will appreciate that all or part of the functions of the various methods in the above embodiments can be implemented by hardware or by computer program. When all or part of the functions in the above embodiments are implemented by computer program, the program can be stored in a computer-readable storage medium, and the storage medium can include: read-only memory, random access memory, disk, optical disk, hard disk, etc., and the program is executed by a computer to implement the above functions. For example, the program is stored in the memory of the device, and when the program in the memory is executed by the processor, all or part of the above functions can be implemented. In addition, when all or part of the functions in the above embodiments are implemented by computer program, the program can also be stored in a storage medium such as a server, another computer, disk, optical disk, flash disk or mobile hard disk, and saved in the memory of the local device by downloading or copying, or the system of the local device is updated. When the program in the memory is executed by the processor, all or part of the functions in the above embodiments can be implemented.

[0287] The above examples are used to illustrate the present invention, which are only used to help understand the present invention and are not intended to limit the present invention. Those skilled in the art can make several simple deductions, modifications or substitutions based on the concept of the present invention.

Claims

1. A method for determining evidence of loss-of-function pathogenicity, characterized in that: The following steps are involved: S101: Obtaining a category of the target variation according to the effect of the target variation on transcription; S102: Determine, based on the category of the target variation, the effect of the target variation on the function of the target gene; the target gene refers to the gene in which the target variation occurs; S103: Based on the results of the effect of the target variant on the target gene function obtained in S102, determining whether the target variant information is evidence of loss-of-function pathogenicity of the target variant; The target variation information is the category of the target variation and / or the impact result in the target variation information.

2. The method according to claim 1, wherein The target variation is a single or multiple exon deletion variation.

3. The method according to claim 1 or 2, wherein: The effects of the target variant on transcription include: Whether the target variant results in a change in the open reading frame; and / or Whether the target variant triggers nonsense-mediated mRNA degradation.

4. The method according to any one of claims 1 to 3, wherein: The category of the target variation includes one or more of the first category, the second category, the third category, and the fourth category; The first category includes mRNAs that result in changes in the open reading frame but do not induce nonsense-mediated degradation; The second category includes not resulting in a change in the open reading frame; The third category includes deletions that cover the first CDS but are not whole-gene deletions. The fourth category includes deletions that cover the last CDS and are not whole-gene deletions.

5. The method according to any one of claims 1 to 4, wherein: If the target variant affects the function of the target gene, determining the target variant information as loss-of-function pathogenicity evidence of the target variant; If the target variation has no effect on the function of the target gene, it is determined that the target variation information is not loss-of-function pathogenicity evidence of the target variation.

6. The method according to claim 5, wherein When the category of the target variant includes one or more of the first category, the second category, the third category, and the fourth category, determining that the target variant affects the function of the target gene, the category of the target variant is loss-of-function pathogenicity evidence of the target variant; The first category includes mRNAs that result in changes in the open reading frame but do not induce nonsense-mediated degradation; The second category includes not resulting in a change in the open reading frame; The third category includes deletions covering the first CDS but not the entire gene; The fourth category includes deletions that cover the last CDS and are not whole-gene deletions.

7. The method according to claim 5, wherein When the category of the target variant includes: category 5 and / or category 6, the target variant has no effect on the function of the target gene, and the category of the target variant is evidence of non-loss-of-function pathogenicity of the target variant; The fifth category includes: variants that cannot be annotated to coding transcripts; The sixth category includes: the variant can be annotated to a coding transcript, but the region affected by the target variant does not contain a coding sequence.

8. A method for determining the strength of evidence for loss-of-function pathogenicity, characterized in that The following steps are involved: S104: Determine the strength of the loss-of-function pathogenicity evidence obtained by the method described in any one of claims 1 to 7 based on the degree of impact of the target variant on the function of the target gene.

9. The method according to claim 8, characterized in that The determination of the degree of impact of the target variant on the target gene function includes the following processing methods: First treatment or second treatment; The first processing includes: determining the degree of influence of the target variant on the function of the target gene by comparing the positional relationship between the target variant and the last pathogenic variant at the 3' end of the target gene; The second processing includes: determining the degree of influence of the target variation on the function of the target gene by comparing the positional relationship between the target variation-affected region and the important functional domain of the target gene.

10. The method according to claim 9, characterized in that In the first process, if the target variant is located before the last pathogenic variant at the 3' end of the target gene, the target variant information is determined to be strong loss-of-function pathogenicity evidence of the target variant; the target variant information includes the category of the target variant and the target variant is located before the last pathogenic variant at the 3' end of the target gene; In the second processing, if the target variant affected region overlaps with the important functional domain of the target gene, the target variant information is determined to be strong loss-of-function pathogenicity evidence of the target variant; the target variant information is the category of the target variant and the overlap between the target variant affected region and the important functional domain of the target gene.

11. The method according to claim 9 or 10, characterized in that In the first process, if the target variant is located after the last pathogenic variant at the 3' end of the target gene, the degree of influence of the target variant on the function of the target gene is determined based on the proportion of the affected region of the target variant; In the second process, if the target variant affected region does not overlap with the important functional domain of the target gene, the degree of influence of the target variant on the target gene function is determined based on the proportion of the target variant affected region; Preferably, determining the degree of influence of the target variation on the target gene function according to the proportion of the affected region of the target variation specifically includes: When the proportion of the target variant-affected region is greater than or equal to a preset value, the target variant information is strong loss-of-function pathogenicity evidence of the target variant; In the first process, the target variation information includes the category of the target variation, the target variation is located after the last pathogenic variation at the 3' end of the target gene, and the proportion of the region affected by the target variation is greater than or equal to a preset value; In the second process, the target variation information includes the category of the target variation, the target variation-affected region does not overlap with the important functional domain of the target gene, and the target variation-affected region ratio is greater than or equal to a preset value; Preferably, determining the degree of influence of the target variant on the target gene function based on the affected region ratio of the target variant further comprises: when the affected region ratio of the target variant is less than a preset value, the target variant information is evidence of moderate loss-of-function pathogenicity of the target variant; In the first process, the target variation information includes the category of the target variation, the target variation is located after the last pathogenic variation at the 3' end of the target gene, and the proportion of the target variation-affected region is less than a preset value; In the second process, the target variation information includes the category of the target variation, the target variation-affected region does not overlap with the important functional domain of the target gene, and the target variation-affected region ratio is less than a preset value.

12. The method according to any one of claims 9 to 11, characterized in that In the first process, the category of the target variation is the first category; In the second process, the category of the target variation is the second category; The first category includes variants that result in changes in the open reading frame and do not trigger nonsense-mediated mRNA degradation; The second category includes those that do not result in a change in the open reading frame.

13. The method according to claim 8, characterized in that The determination of the degree of impact of the target variant on the target gene function includes the following processing methods: Third treatment; The third process includes: determining the extent of the impact of the target variation on the function of the target gene by querying reports on the pathogenicity of the start codon of the target gene where the target variation occurs.

14. The method according to claim 13, characterized in that In the third process, if the target gene in which the target variant occurs has a start codon pathogenicity report, the target variant information is determined to be strong loss-of-function pathogenicity evidence of the target variant; The target variation information includes the category of the target variation and reports that the target gene where the target variation occurs has a pathogenic start codon.

15. The method according to claim 13 or 14, wherein in the third step, if the target gene in which the target variant occurs has no reported pathogenic start codon, the degree of impact of the target variant on the function of the target gene is determined based on the proportion of regions affected by the target variant; Preferably, determining the degree of influence of the target variation on the target gene function according to the proportion of the affected region of the target variation specifically includes: When the proportion of the target variant-affected region is greater than or equal to a preset value, the target variant information is strong loss-of-function pathogenicity evidence of the target variant; The target variation information includes the category of the target variation, the target gene of the target variation has no reports of pathogenicity caused by the start codon, and the proportion of the region affected by the target variation is ≥ a preset value; Preferably, determining the degree of influence of the target variant on the target gene function based on the affected region ratio of the target variant further comprises: when the affected region ratio of the target variant is less than a preset value, the target variant information is evidence of moderate loss-of-function pathogenicity of the target variant; The target variation information includes the category of the target variation, the target gene of the target variation has no reports of pathogenicity caused by the start codon, and the proportion of the target variation-affected region is less than a preset value.

16. The method according to any one of claims 13 to 15, characterized in that: The category of the target variation is the third category; The third category includes deletions that cover the first CDS but are not whole-gene deletions.

17. The method according to claim 8, characterized in that The determination of the degree of impact of the target variant on the target gene function includes the following processing methods: Fourth treatment; The fourth process includes: determining the extent of the impact of the target variation on the function of the target gene by predicting whether the sequence of the target gene in which the target variation occurs contains a stop codon.

18. The method according to claim 17, characterized in that In the fourth process, if the sequence of the target gene in which the target mutation occurs does not contain a stop codon, the target mutation information is determined to be strong evidence of loss-of-function pathogenicity; The target variation information includes the category of the target variation and the absence of a stop codon in the sequence of the target gene where the target variation occurs.

19. The method according to claim 17 or 18, characterized in that If the sequence of the target gene in which the target mutation occurs contains a stop codon, determining the extent of the effect of the target mutation on the function of the target gene according to the fifth process; The fifth process includes determining whether the target variant triggers nonsense-mediated mRNA degradation.

20. The method according to claim 19, wherein In the fifth process, if the target variant triggers nonsense-mediated mRNA degradation, determining the target variant information as strong loss-of-function pathogenicity evidence of the target variant; The target variation information includes the category of the target variation, the presence of a stop codon in the sequence of the target gene where the target variation occurs, and the target variation triggers nonsense-mediated mRNA degradation.

21. The method according to claim 19 or 20, characterized in that If the target variant does not induce nonsense-mediated mRNA degradation, determining the extent of the effect of the target variant on the target gene function according to the first treatment; The first processing includes: determining the degree of influence of the target variant on the function of the target gene by comparing the positional relationship between the target variant and the last pathogenic variant at the 3' end of the target gene; Preferably, in the first processing, if the target variant is located before the last pathogenic variant at the 3' end of the target gene, then the target variant information is determined to be strong loss-of-function pathogenicity evidence; the target variant information includes the type of the target variant, the presence of a stop codon in the sequence of the target gene where the target variant occurs, the target variant does not trigger nonsense-mediated mRNA degradation, and the target variant is located before the last pathogenic variant at the 3' end of the target gene; Preferably, in the first process, if the target variant is located after the last pathogenic variant at the 3' end of the target gene, the degree of influence of the target variant on the function of the target gene is determined based on the proportion of the affected region of the target variant; Preferably, determining the degree of influence of the target variation on the target gene function according to the proportion of the affected region of the target variation specifically includes: When the target variant affects a region with a ratio greater than or equal to a preset value, the target variant information is considered strong loss-of-function pathogenicity evidence for the target variant; the target variant information includes the type of target variant, the presence of a stop codon in the sequence of the target gene where the target variant occurs, the target variant does not trigger nonsense-mediated mRNA degradation, the target variant is located after the last pathogenic variant at the 3' end of the target gene, and the target variant affects a region with a ratio greater than or equal to a preset value. Preferably, the determining the degree of influence of the target variation on the function of the target gene based on the proportion of the affected region of the target variation specifically further includes: when the proportion of the affected region of the target variation is less than a preset value, the target variation information is evidence of moderate loss-of-function pathogenicity of the target variation; the target variation information includes the category of the target variation, the presence of a stop codon in the sequence of the target gene where the target variation occurs, the target variation does not trigger nonsense-mediated mRNA degradation, the target variation is located after the last pathogenic variation at the 3' end of the target gene, and the proportion of the affected region of the target variation is less than a preset value.

22. The method according to any one of claims 17 to 21, characterized in that The category of the target variation is the fourth category; The fourth category includes deletions that cover the last CDS and are not whole-gene deletions.

23. A method for correcting the strength of evidence for loss-of-function pathogenicity, characterized in that The following steps are involved: S105: Correct the strength of the loss-of-function evidence determined by the method described in any one of claims 8 to 22.

24. The method according to claim 23, wherein The correction includes correction based on the strength of association between the target gene and the disease.

25. The method according to claim 22, wherein The correction according to the strength of association between the target gene and the disease includes: If the target gene is strongly associated with the disease, no correction is required; If the target gene is moderately associated with the disease, the evidence for loss-of-function pathogenicity will be downgraded by one level; If the target gene is weakly associated with the disease, the loss-of-function pathogenicity evidence is downgraded to the second level.

26. A device for determining evidence of loss-of-function pathogenicity, characterized in that: include: The category acquisition module is used to obtain the category of the target variant based on its impact on transcription; The impact judgment module is used to judge the impact of the target variant on the target gene function based on the category of the target variant; The evidence judgment module is used to determine whether the target variant is evidence of loss-of-function pathogenicity based on the impact of the target variant on the target gene function.

27. A device for determining the strength of evidence for loss-of-function pathogenicity, characterized in that include: A strength determination module is used to determine the strength of the loss-of-function pathogenicity evidence obtained by the device of claim 26 based on the degree of impact of the target variant on the target gene function.

28. A device for correcting evidence of loss-of-function pathogenicity, characterized in that include: A correction module is used to correct the strength of the evidence of loss-of-function pathogenicity determined by the device of claim 27.

29. A device for determining evidence of loss-of-function pathogenicity, characterized in that: include: Memory, used to store programs; A processor, configured to implement the method according to claims 1 to 7 by executing a program stored in a memory.

30. A device for determining the strength of evidence for loss-of-function pathogenicity, characterized in that include: Memory, used to store programs; A processor, configured to implement the method according to claims 8 to 22 by executing a program stored in a memory.

31. A device for correcting the strength of loss-of-function evidence, characterized in that: include: Memory, used to store programs; A processor, configured to implement the method according to claims 23 to 25 by executing a program stored in a memory.

32. A computer-readable storage medium, characterized in that The medium stores a program, and the program can be executed by a processor to implement the method according to any one of claims 1 to 25.

Images