Method for detecting linked carrying of pathogenic gene in two-copy chromosome

Abstract

The present invention relates to a method for detecting the linked carrying of a pathogenic gene in a two-copy chromosome, and in particular to a method for detecting the carrying of a pathogenic gene 2+0. The method for detecting the carrying of a pathogenic gene 2+0 comprises the following steps: (1) providing genomic DNA samples of at least two generations of families including a subject; (2) acquiring long-fragment DNA of a target pathogenic gene; (3) using a long-read sequencing platform to sequence the long-fragment DNA and constructing haplotypes; and (4) analyzing the haplotypes of the families to obtain whether the subject carries a pathogenic gene 2+0.

Description

A method for detecting double-copy chromosome linkage of pathogenic genes Technical Field The present invention relates to a method for detecting double-copy chromosome linkage of a pathogenic gene, and in particular to a method for detecting 2+0 carriers of a pathogenic gene. Specifically, the present invention relates to a method for determining whether a subject has 2+0 carriers of a pathogenic gene by using two-generation pedigrees and haplotype construction. Background Art Spinal muscular atrophy (SMA) is the most common neuromuscular disease in children. Its main clinical features include muscle weakness and atrophy caused by degeneration of α-motor neurons in the anterior horn of the spinal cord. The main type is 5q-SMA caused by pathogenic mutations in the survival motor neuron 1 (SMN1) gene located at 5q13 on both chromosomes. [1] The carrier rate of SMA in the population is about 1 / 50, and the incidence rate is about 1 / 10000. [2] There is a gene SMN2 on 5q13 that is highly homologous to SMN1. There are only 5 base differences between SMN2 and SMN1, including a C to T difference at c.840 in exon 7, which results in 90% of SMN2 gene mRNA exon 7 being selectively spliced, and only 10% of SMN2 expresses functional SMN protein. [3] . Therefore, SMN1 pathogenic variants determine the occurrence of the disease, and SMN2 affects the severity and progression of the disease. The number of copies of SMN2 in the normal population ranges from zero to four, and there is a positive correlation between its copy number and the severity of the phenotype. [4] Typically, people with type I have severe symptoms and have 1-3 copies of SMN2; people with type II have 3 copies of SMN2; people with type III have 3 or 4 copies of SMN2; and people with type IV have milder symptoms and have 4 copies of SMN2. [5] In 2009, the American College of Obstetricians and Gynecologists (ACOG) recommended that women should be screened for SMA carriers before and during early pregnancy. [7] my country has also conducted a large cohort study on SMA carrier screening, moving the prevention and control window of SMA forward. [8-9] SMA carriers are mainly caused by three types of SMN1 mutations, of which about 90-95% are SMN1 copy number deletions (1+0). That is, there is one copy of SMN1 on one chromosome and no copy of SMN1 on the other chromosome; 2%-4% are SMN1 point mutations (1+1 D), that is, there is one SMN1 copy with normal function on one chromosome and one SMN1 copy with pathogenic mutation on the other chromosome; another 3-8% are SMN1 gene 2+0 carriers, that is, there are two SMN1 copies on one chromosome and no SMN1 copy on the other chromosome [10-12] . SMN1 gene 1+0 carrier can be detected by qPCR

[0013] MLPA

[0014] , NGS

[0015] or single-molecule long-read sequencing

[0016] The method is used for detection, 1+1 D Long fragment binding nested PCR

[0017] , RT-PCR

[0018] or single-molecule long-read sequencing

[0016] However, there is no effective method to detect 2+0 carriers. SMN1 gene 2+0 carriers are usually diagnosed when a proband (patient) without SMN1 copies appears in the family and one of their parents has two SMN1 copies. The genetic law is used to infer that the parent is a 2+0 carrier. [19,20] In the absence of a proband, a three-generation pedigree is needed to infer the 2+0 carrier by analyzing marker loci associated with the SMN1 gene.

[0019] The SMN1 gene 2+0 carrier is associated with a single or a group of SNPs (haplotypes) in a specific population, indicating a possible founder effect. In Ashkenazi Jews and African Americans, a single SNP (g.27134T>G) has a strong association with the SMN1 gene 2+0 distribution.

[0021] Further studies have shown that in African Americans, the haplotype S1-9d containing g.27134T>G is associated with a 2+0 distribution of the SMN1 gene.

[0022] However, the correlation between g.27134T>G and S1-9d and SMN1 gene 2+0 was not found in other populations such as Caucasians, Hispanics, and Asians. [15,21,22] , indicating that this method is not universal. The method based on long-read sequencing found that some 2+0 carriers have SMN2 gene SNPs downstream of the SMN1 gene, from which it can be inferred that 2+0 carriers (patent number ZL202210234941.6), but the sensitivity of this method is only about 50%, and because the normal 1 copy of the SMN1 gene downstream may also have SMN2 gene SNPs, the specificity of this method is very low, and it is not suitable as a carrier screening method.

[0016] When one spouse is confirmed to be a 1+0 or 1+1D carrier of the SMN1 gene through traditional methods, it is crucial to detect whether the spouse is a 2+0 carrier. The above methods for discovering SMN1 gene 2+0 carriers have the following limitations: (1) They rely on the appearance of patients and thus infer through genetic relationships; (2) They rely on three-generation pedigrees and infer through markers associated with the SMN1 gene; (3) They rely on specific The specific SNP or haplotype of a certain race is not universal; (4) The detection sensitivity and specificity are low. Therefore, there is currently a lack of feasible methods to achieve population screening for SMN1 gene 2+0 carriers. Similar to SMN1, which has a highly homologous modifier gene SMN2, the pathogenic gene CYP21A2 of congenital adrenal hyperplasia has a highly homologous pseudogene CYP21A1P, which also has the possibility of 2+0 carriers.

[0023] , but there is currently no good detection method. Summary of the invention In view of this, the present invention provides a method for detecting 2+0 carriers of pathogenic genes based on two-generation pedigrees and haplotypes. Specifically, for subjects who clearly have two copies of the pathogenic gene and need to be tested for 2+0 carriers, genomic DNA of the subject and parents or the subject, spouse and children is obtained, and then the haplotypes of the pathogenic genes of two generations of family members are obtained by long-read sequencing, and the genetic situation of the haplotype is used to infer whether the subject is a 2+0 carrier of the pathogenic gene. Haplotype refers to a group of associated SNP alleles located in a certain region on a chromosome. SNP refers to the DNA sequence diversity caused by the variation of a single nucleotide at the genome level. As used herein, SNP includes all sites that are different from the reference genome. The method of the present invention has high sensitivity and specificity, is feasible in clinical operation and has strong repeatability, and is conducive to realizing comprehensive carrier screening of such pathogenic genes. This scheme does not focus on the correlation between a specific SNP or haplotype and the 2+0 carrier of the pathogenic gene, but detects the 2+0 carrier of the pathogenic gene by simultaneously identifying the SNP or haplotype of two generations of people. Specifically, the genetic relationship of the pathogenic gene can be determined by simultaneously identifying the SNP or haplotype of two generations of people, and the 2+0 carrier of the pathogenic gene can be identified through genetic reasoning. In this method, since the association of SNPs or haplotypes with 2+0 is not required, there is no need to identify specific SNPs or haplotypes, and this method is universal and does not have the problems of low accuracy and founder effect compared to existing technologies. Therefore, in the first aspect, the present invention provides a method for detecting the 2+0 carrier of a pathogenic gene, comprising the following steps: (1) Providing genomic DNA samples of at least two generations of the family including the subject; (2) Obtaining long DNA fragments of target pathogenic genes; (3) Sequencing long DNA fragments and constructing haplotypes using a long-read sequencing platform; (4) Family haplotype analysis was used to detect whether the subject was a 2+0 carrier of the pathogenic gene. In one embodiment, the pathogenic gene is a pathogenic gene for which there is a highly homologous modifying gene or pseudogene. In one embodiment, the causative gene is selected from the group consisting of: SMN1, CYP21A2, GBA, IKBKG and IDS. In one embodiment, the 2+0 carrier refers to the presence of two pathogenic genes on one chromosome and no pathogenic gene on the other chromosome. In one embodiment, the subject has two copies of the disease-causing gene. In one embodiment, the two-generation pedigree refers to the subject and his / her father, mother; or refers to the subject, his / her spouse and children. In one embodiment, the method for obtaining the long-fragment DNA of the target pathogenic gene is selected from: long-fragment PCR amplification, a method using probe hybridization, or a CRISPR / Cas9 cutting method to obtain the long-fragment DNA of the pathogenic gene. In one embodiment, the long fragment DNA is 500 bp-200 kb DNA. In one embodiment, the method further comprises detecting a haplotype located in a long DNA molecule using a long read sequencing platform. In one embodiment, the pedigree haplotype analysis comprises the following steps: When the two-generation family refers to the subject and his / her father and mother, the subject's two pathogenic gene haplotypes are analyzed to see whether they are inherited from one or both parents. If the subject's two pathogenic gene haplotypes are all inherited from one parent, the subject is a 2+0 carrier of the pathogenic gene, otherwise it is not a 2+0 carrier. Or when a two-generation pedigree refers to a family including the subject, his / her spouse and children, the two pathogenic gene haplotypes of the subject are analyzed to see whether they are completely, not or partially inherited to the children. When the pathogenic gene of the subject's children is not inherited to the subject's haplotype, or only inherits all of the subject's haplotypes, the subject is a 2+0 carrier of the pathogenic gene, otherwise the subject is not a 2+0 carrier. In a second aspect, the present invention also provides a kit for implementing the above method. The excellent effects of the method for detecting 2+0 carriers of pathogenic genes described in the present invention are mainly in the following aspects: (1) It does not rely on the genetic relationship after the patient appears to infer that family members are carriers of the 2+0 pathogenic gene. Therefore, carrier screening can be used to provide marriage and childbearing guidance and fertility selection to avoid the birth of severely ill children. (2) It does not rely on three generations of family members to infer through associated markers, but only two generations of family members can be tested through haplotypes, which is easier to achieve clinically. (3) It does not rely on the founder effect, that is, the association between a specific SNP or a specific SNP combination haplotype and 2+0 to detect the pathogenic gene 2+0. Any race and any gene that may carry 2+0, such as SMN1 and CYP21A2, can be used to detect the pathogenic gene 2+0 carrier using the method of the present invention, which has the universality of race and pathogenic gene. (4) The method of using the haplotype obtained by long-read sequencing combined with family pedigree to detect 2+0 carriers of the pathogenic gene has high sensitivity and specificity and is capable of screening carriers in large populations. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1. Flow chart of SMN1 gene 2+0 carrier detection in subjects. Figure 2A: The haplotypes of the SMN1 gene obtained by long-fragment sequencing, where two haplotypes of the sample represent two copies of the SMN1 gene. Figure 2B: The haplotypes of the SMN1 gene obtained by long-fragment sequencing, where three haplotypes of the sample represent three copies of the SMN1 gene. The vertical lines in Figures 2A and 2B represent SNPs, and different haplotypes are distinguished by SNP combinations. Figure 3A: A pedigree diagram showing whether a subject is a 2+0 carrier of the SMN1 gene by testing the subject and his parents for two generations. Figure 3B: A pedigree diagram showing whether a subject is a 2+0 carrier of the SMN1 gene by testing the subject and his parents for two generations. The method of the present invention is used to detect whether a subject is a 2+0 carrier of the SMN1 gene. Figure 4A: A pedigree chart showing whether the subject is a 2+0 carrier of the SMN1 gene through the two generations of the subject, spouse, and children. Figure 4B: A pedigree chart showing whether the subject is a 2+0 carrier of the SMN1 gene through the two generations of the subject, spouse, and children. The two-generation family tests whether the subject is a 2+0 carrier of the SMN1 gene, and the method of the present invention is used to detect whether the subject is a 2+0 carrier of the SMN1 gene. Figure 5A: A pedigree diagram showing whether the subject is a 2+0 carrier of the SMN1 gene through the two generations of the subject and the parents. Figure 5B: A pedigree diagram showing whether the subject is a 2+0 carrier of the SMN1 gene through the two generations of the subject and the parents. The method of the present invention is used to detect whether the arrangement of the two copies of the SMN1 gene in the subject is 1+1, not 2+0. Figure 6A: A pedigree diagram showing whether the subject is a 2+0 carrier of the SMN1 gene through the pedigree of the subject, spouse and children. Figure 6B: A pedigree diagram showing whether the subject is a 2+0 carrier of the SMN1 gene through the pedigree of the subject, spouse and children. The method of the present invention is used to detect whether the arrangement of the two copies of the SMN1 gene in the subject is 1+1, not 2+0. DETAILED DESCRIPTION The preferred embodiments of the present invention are further described in detail below in conjunction with the accompanying drawings of the specification. The following description is exemplary and not limiting of the present invention. Any other similar situations also fall within the protection scope of the present invention. Unless otherwise defined herein, scientific and technical terms used in conjunction with the present invention will have the meanings commonly understood by those of ordinary skill in the art. In addition, unless the context otherwise requires, terms in the singular should include plural forms, and terms in the plural should include singular forms. More specifically, as used in this specification and the appended claims, unless the context otherwise clearly indicates, the singular forms "a", "an" and "the" include plural indicators. In this application, unless otherwise stated, the use of "or" means "and / or". In addition, the use of the term "comprising" and other forms (such as "including" and "containing") is not restrictive. In addition, the ranges provided in the specification and the appended claims include endpoints and all values ​​between the endpoints. Example 1: Obtaining the haplotype of the SMA pathogenic gene SMN1 using the method of the present invention Step 1: Prepare the reaction system according to Table 1 below and amplify using genomic DNA as template Full-length SMN1 gene. Table 1: Step 2: Perform PCR amplification on a PCR instrument according to the conditions shown in Table 2 below. Table 2: Step 3: Prepare the ligation reaction system according to Table 3 below to construct the PacBio library. Table 3: Step 4: On a PCR instrument, perform a ligation reaction according to the conditions shown in Table 4 below. Table 4: After the reaction is completed, add 0.5 mL of Exonuclease III (NEB, Cat#M0206L) and 0.5 mL of Exonuclease VII (NEB, Cat#M0379L), and place in a PCR instrument at 37°C for 1 hour. Purify twice with 0.6x Ampure PB magnetic beads (PacBio, Cat#100-265-900) according to the manufacturer's instructions, and finally elute with 10uL of Elution Buffer. The resulting DNA is the PacBio sequencing pre-library. Step 5: Sequencing on PacBio machine. The DNA concentration was determined using Qubit dsDNA HS reagent (ThermoFisher, Cat#Q32851) on a Qubit 3 Flurometer (ThermoFisher, Cat#Q33216). When multiple samples need to be sequenced simultaneously, equal amounts of pre-libraries with different barcodes can be mixed together to prepare a mixed pre-library. According to the total concentration and molar concentration of the pre-library, an appropriate volume of the library was reacted with the binding reagent (PacBio, Cat#101-820-200) and primers (PacBio, Cat#100-970-100) to prepare the final machine-ready library. The machine-ready library was sequenced on the PacBio Sequel II sequencing platform for 30 hours. Step 6: SMN1 genotype analysis. In the SMRT Link analysis system provided by PacBio, the original subreads downloaded from PacBio were analyzed for consistency to obtain high-quality CCS reads, which were then aligned to the hg38 reference genome. The CCS reads of SMN1 and SMN2 were distinguished by the base at the c.840 site on exon 7. The CCS reads of the SMN1 gene were analyzed using PacBio's pbampliconclustering analysis software to obtain the haplotype information of the SMN1 gene through SNP information. Representative results are shown in Figures 2A and 2B, where: the two haplotypes of the sample in Figure 2A represent that the sample has two copies of the SMN1 gene, and the three haplotypes of the sample in Figure 2B represent that the sample has three copies of the SMN1 gene. Example 2: Using the method of the present invention to detect whether a subject is a 2+0 carrier of the SMN1 gene Two families with SMA probands (patients) and two families without SMA patients were collected, and the SMN1 gene copy number of each member was determined by MLPA detection. Family 1 includes three generations of members, proband III-1 has no SMN1 gene (i.e., 0+0), and its father II-1 has 2 SMN1 genes, indicating that the 2 SMN1 genes are on one chromosome, and there is no SMN1 on the other chromosome. Therefore, it is inferred through genetic relationship that its father is a 2+0 carrier of the SMN1 gene, and then it is inferred that the SMN1 gene of its grandmother I-2 is 2+1 distribution (Figure 3A). Without relying on the proband, the method of the present invention is now used to detect whether the subject II-1 is a 2+0 carrier of the SMN1 gene. First, the method in Example 1 is used to determine the haplotype of the subject and his parents. As shown in Figure 3B, the number of SMN1 gene haplotypes in the three subjects was consistent with the copy number detected by MLPA, indicating that each haplotype in the three samples corresponds to one gene copy number. Haplotype analysis showed that the two haplotypes of the subject were all inherited from the mother I-2 and were different from the haplotype of the father I-1, indicating that the two haplotypes were located on the same chromosome. Therefore, the SMN1 gene of the subject was 2+0 carrier (Figure 3B), which was consistent with the result inferred by genetic relationship relying on the proband. Family 2 includes two generations. The proband II-2 has no SMN1 gene (i.e., 0+0), while her mother I-2 has two SMN1 genes, indicating that the two SMN1 genes are on the same chromosome. On the chromosome, there is no SMN1 on the other chromosome, so it is inferred that the mother is a 2+0 carrier of the SMN1 gene through genetic relationship (Fig. 4A). Without relying on the proband, the method of the present invention is now used to detect whether the subject I-2 is a 2+0 carrier of the SMN1 gene. First, the method in Example 1 is used to determine the haplotypes of the subject, spouse I-1 and his daughter II-1. As shown in Fig. 4B, the number of SMN1 gene haplotypes of the three is consistent with the copy number detected by MLPA, indicating that each haplotype in these three samples corresponds to a gene by. It can be found through haplotype analysis that the two haplotypes of the subject are all inherited to the daughter, indicating that the two haplotypes are located on the same chromosome, so the SMN1 gene of the subject is 2+0 carrier (Fig. 4B), which is consistent with the result of relying on the proband to infer through genetic relationship. Family 3 includes two generations of members, and the father I-1, the mother I-2 and the subject (son) II-1 all have 2 copies of the SMN1 gene (Figure 5A). Using the method of the present invention, through haplotype analysis, the two SMN1 gene haplotypes of the subject are inherited from the father and mother respectively, indicating that the arrangement of the two SMN1 genes is 1+1, not 2+0 carriers (Figure 5B). Family 4 includes two generations of members, the subject (father) I-1 has 2 copies of the SMN1 gene, and the mother I-2 and the son II-1 both have 1 copy of the SMN1 gene (Figure 6A). Using the method of the present invention, through haplotype analysis, one of the two SMN1 gene haplotypes of the subject is inherited to the son, indicating that the two SMN1 genes of the subject are located on two chromosomes, that is, the arrangement is 1+1, not 2+0 carriers (Figure 6B). Therefore, without relying on a proband, the method of the present invention can accurately detect 2+0 pathogenic genes in subjects through two generations of pedigrees. It should be noted that the aforementioned examples are merely illustrative and are used to explain some of the features of the present application. The attached claims are intended to claim the widest possible scope that can be imagined, and the embodiments presented herein are merely illustrative of selected implementation methods based on the combination of all possible embodiments. Therefore, the applicant's intention is that the attached claims are not limited by the selection of examples illustrating the features of the present application. As used in the claims, the term "comprising" and its semantic variants logically also include different and varying terms, such as but not limited to "essentially consisting of" or "consisting of". When necessary, some numerical values ​​are provided. range, and these ranges also include sub-ranges therebetween. Changes in these ranges are also self-evident to those skilled in the art and should not be considered to be donated to the public, and these changes should also be interpreted as being covered by the attached claims where possible. For example, the reaction reagents, reaction conditions, etc. involved in long-fragment PCR reactions and third-generation sequencing library construction can be adjusted and changed accordingly according to specific needs. Moreover, progress in science and technology will form possible equivalents or sub-substitutions that are not currently considered due to inaccurate language expression, and these changes should also be interpreted as being covered by the attached claims where possible. References [1]Eugenio Mercuri, Charlotte J Summer, Francesco Muntoni, et al. Spinal muscular atrophy. Nat Rev Dis Primers. 2022Aug 4;8(1):52.doi:10.1038 / s41572-022-00380-8. [2]Ingrid E C Verhaart,Agata Robertson,Ian J Wilson,et al.Prevalence,incidence nd carrier frequency of 5q-linked spinal muscular atrophy-a literature review.Orphanet J Rare Dis.2017 Jul 4;12(1):124.doi:10.1186 / s13023-017-0671-8. [3]Christian L Lorson,Eric Hahnen,Elliot J Androphy,et al.A single nucleotide in the SMN gene regulates splicing and is responsible for spinal muscular atrophy.Proc Natl Acad Sci U S A.1999 May 25;96(11):6307-11.doi:10.1073 / pnas.96.11.6307. 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[9]Sumin Zhao,Wangyang Wang,Yaoshen Wang,et al.NGS-based spinal muscular atrophy carrier screening of 10,585 diverse couples in China:a pan-ethnic study.Eur J Hum Genet.2021 Jan;29(1):194-204.doi:10.1038 / s41431-020-00714-8.

[0010] Melanie Smith,Vanessa Calabro,Belinda Chong,et al.Population screening and cascade testing for carriers of SMA.Eur J Hum Genet.2007 Jul;15(7):759-66.doi:10.1038 / sj.ejhg.5201821.

[0011] BC Hendrickson, C Donohoe, VR Akmaev, et al. Differences in SMN1 allele frequencies among ethnic groups within North America. J Med Genet. 2009Sep; 46(9):641-4.doi:10.1136 / jmg.2009.066969.

[0012] Song Fang, Huang Shangzhi, Medical Genetics Branch of Beijing Medical Association, Beijing Rare Disease Diagnosis and Treatment and Protection Society. Expert consensus on genetic diagnosis of spinal muscular atrophy. Chinese Medical Journal, Vol. 100, No. 40, November 3, 2020. Markus Feldkotter, Verana Schwarzer, Radu Wirth, et al. Quantitative analyzes of SMN1 and SMN2 based on real-time lightCycler PCR: fast and highly reliable carrier testing and prediction of severity of spinal muscular atrophy. Am J Hum Genet. 2002 Feb;70(2):358-68.doi:10.1086 / 338627.

[0014] Eva L Arkbald, Niklas Darin, Kerstin Berg, et al. Multiplex ligation-dependent probe amplification improves diagnostics in spinal muscular atrophy. Neuromuscul Disord. 2006 Dec; 16 (12): 830-8. doi: 10.1016 / j.nmd.2006.08.011.

[0015] Yanming Feng,Xiaoyan Ge,Linyan Meng,et al.The next generation of population-based spinal muscular atrophy carrier screening:comprehensive pan-ethnic SMN1 copy-number and sequence variant analysis by massively parallel sequencing.Genet Med.2017 Aug;19(8):936-944.doi:10.1038 / gim.2016.215.

[0016] Shuyuan Li,Xu Han,Yan Xu,et al.Comprehensive Analysis of Spinal Muscular Atrophy:SMN1 Copy Number,Intragenic Mutation,and 2+0 Carrier Analysis by Third-Generation Sequencing.J Mol Diagn.2022 Sep;24(9):1009-1020.doi:10.1016 / j.jmoldx.2022.05.001.

[0017] Yan Xu,Bing Xiao,Yu Liu,et al.Identification of novel SMN1 subtle mutations using an allelic-specific RT-PCR.Neuromuscul Disord. 2020 Mar;30(3):219-226.doi:10.1016 / j.nmd.2019.11.010.

[0018] Yuji Kubo,Hisahide Nishio,Kayobo Saito.A new method for SMN1 and hybrid SMN gene analysis in spinal muscular atrophy using long-range PCR followed by sequencing.J Hum Genet.2015 May;60(5):233-9.doi:10.1038 / jhg.2015.16.

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Claims

1. A method for detecting the presence of a pathogenic gene 2+0, comprising the following steps: (1) Providing genomic DNA samples of at least two generations of the family including the subject; (2) Obtaining long DNA fragments of target pathogenic genes; (3) Sequencing long DNA fragments and constructing haplotypes using a long-read sequencing platform; (4) Family haplotype analysis was used to detect whether the subject was a 2+0 carrier of the pathogenic gene.

2. The method according to claim 1, wherein: The pathogenic gene is a pathogenic gene having a highly homologous modifying gene or pseudogene.

3. The method according to claim 1, wherein: The pathogenic gene is selected from the group consisting of: SMN1, CYP21A2, GBA, IKBKG and IDS.

4. The method according to claim 1, wherein: The 2+0 carrier means that one chromosome contains 2 pathogenic genes, while the other chromosome does not contain the pathogenic gene.

5. The method according to claim 1, wherein: The subject has 2 copies of the pathogenic gene.

6. The method according to claim 1, wherein: The two-generation pedigree refers to the subject and his / her father, mother; or refers to the subject, his / her spouse and children.

7. The method according to claim 1, wherein: The method for obtaining the long-fragment DNA of the target pathogenic gene is selected from: long-fragment PCR amplification, a method using probe hybridization, or a CRISPR / Cas9 cutting method to obtain the long-fragment DNA of the pathogenic gene.

8. The method according to claim 1, wherein: The long DNA fragment is 500bp-200kb DNA.

9. The method according to claim 1, wherein: The method also includes using a long-read sequencing platform to detect a haplotype located in a long DNA molecule.

10. The method according to claim 1, wherein: The family haplotype analysis comprises the following steps: When the two-generation pedigree refers to the subject and his / her father and mother, the two pathogenic gene haplotypes of the subject are analyzed to see whether they are inherited from one or both parents. If all haplotypes of the pathogenic gene are inherited from one parent, the subject is a 2+0 carrier of the pathogenic gene, otherwise it is not a 2+0 carrier; Or when a two-generation pedigree refers to a family including the subject, his / her spouse and children, the two pathogenic gene haplotypes of the subject are analyzed to see whether they are completely, not or partially inherited to the children. When the pathogenic gene of the subject's children is not inherited to the subject's haplotype, or only inherits all of the subject's haplotypes, the subject is a 2+0 carrier of the pathogenic gene, otherwise the subject is not a 2+0 carrier.

11. A kit for carrying out the method according to any one of claims 1 to 10.

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