CN116804220A - Method for detecting linkage carrying of double-copy chromosomes of pathogenic genes - Google Patents

Abstract

The application relates to a method for detecting double-copy chromosome linkage carrying of a pathogenic gene, in particular to a method for detecting 2+0 carrying of a pathogenic gene. The method for detecting the 2+0 carrying of the pathogenic gene comprises the following steps: (1) Providing a genomic DNA sample of at least two generations of a family including a subject; (2) obtaining long fragment DNA of the target pathogenic gene; (3) Sequencing the long fragment DNA by using a long reading and long sequencing platform and constructing a haplotype; (4) And obtaining whether the pathogenic genes of the subject are carried by 2+0 through family haplotype analysis.

Description

Method for detecting linkage carrying of double-copy chromosomes of pathogenic genes

Technical Field

The application relates to a method for detecting double-copy chromosome linkage carrying of a pathogenic gene, in particular to a method for detecting 2+0 carrying of a pathogenic gene. In particular, the application relates to a method for judging whether a subject has pathogenic gene 2+0 carried by using two-generation family and haplotype construction.

Background

Spinal muscular atrophy (Spinal Muscular Atrophy, SMA) is the most common neuromuscular disease in children, and its main clinical features include muscle weakness and muscular atrophy caused by degeneration of alpha-motor neurons at the anterior horn of the spinal cord, the main type of which is 1 @ by motor neuron survival gene located at 5q13SMN1) 5q-SMA resulting from pathogenic variation on both chromosomes ^[1] . The crowd carrying rate of SMA is about 1/50, and the incidence rate is about 1/10000 ^[2] . Presence on 5q13SMN1Highly homologous genesSMN2。SMN2AndSMN1there was only a 5 base difference in which the C to T of c.840 in exon 7 was different, resulting in 90%SMN2Gene mRNA exon 7 is alternatively spliced, only 10% of whichSMN2Expression of functional SMN proteins ^[3] . Thus, the first and second substrates are bonded together,SMN1pathogenic variation determines the occurrence of the disease,SMN2affecting the severity and progression of the disease. In normal peopleSMN2From zero to four, there is a positive correlation between the copy number and the severity of the phenotype ^[4] . In general, patients of type I have severe symptoms, 1-3SMN2Copying; patients type II had 3SMN2Copying; patients of type III have 3 or 4SMN2Copying; patients of type IV have relatively mild symptoms, 4 in numberSMN2Copying ^[5] . The american society of gynaecology and obstetrics (American College of Obstetricians and Gynecologists, ACOG) in 2009 recommended SMA carrier screening for pre-pregnant and early pregnant women ^[7] . China also carries out large-queue research on the screening of the SMA carrier, and moves forward the SMA prevention and control window ^[8-9] . SMA carriers are mainly composed of three typesSMN1Variation causes about 90-95% of whichSMN1Copy number loss (1+0), i.e.1 on one chromosomeSMN1Copy, no other chromosomeSMN1Copying; 2% -4% isSMN1Point mutation (1+1) ^D ) I.e. 1 chromosome with normal functionSMN1Copying, 1 on the other chromosome with pathogenic mutationSMN1Copying; in addition 3-8% isSMN1Gene 2+0 carries, i.e. there are two on a chromosomeSMN1Copy, no other chromosomeSMN1Copying ^[10-12] 。

SMN1Gene 1+0 carrying can be carried out by qPCR ^[13] 、MLPA ^[14] 、NGS ^[15] Or single molecule long read length sequencing ^[16] 1+1 of the method of (2) ^D Carrying can be combined with nested PCR by long fragments ^[17] 、RT-PCR ^[18] Or single molecule long read length sequencing ^[16] But 2+0 carrying currently lacks an efficient method for detection.SMN1Gene 2+0 carryover is usually absent in the familySMN1The copy forerunner (patient) has two parentsSMN1After copying, the formula is inferred to be 2+0 carrying through genetic rules ^{[19, 20]} . In the absence of a prover, it is then necessary to infer 2+0 carrying by analysis of marker loci associated with the SMN1 gene using the third generation family ^[19] 。SMN1Gene 2+0 is carried in a specific population and is associated with a single or a set of SNPs (haplotypes), indicating that there may be a creator effect. In the De-line Utah and African Americans, a single SNP (g.27134T>G) And (3) withSMN1The gene 2+0 distribution has a strong association relationship ^[21] . Further studies have shown that in african americans, g.27134t is involved>G monomer type S1-9dSMN1Gene 2+0 distribution correlation ^[22] . But g.27134T>G and S1-9d andSMN1the 2+0 correlation is not found in other populations such as caucasians, ramericans and asians ^{[15, 21, 22]} This method is not universal. Method based on long read long sequencing to find partial 2+0 carriersSMN1Downstream of the gene isSMN2SNP of the gene, thus presumably carried 2+0 (patent No. ZL 202210234941.6), but the sensitivity of this method is only about 50% and is due to the normal 1 copySMN1Downstream of the gene may also haveSMN2SNP of a gene, which results in a low specificity of the method, is not suitable as a carrier screening method ^[16] . When the couple party is definitely by the traditional methodSMN1In the case of gene 1+0 or 1+1D carriers, it is important to detect whether the partner is a 2+0 carrier. The SMN1 group was foundThe 2+0 carrying method has the following limitations: (1) Relying on the presence of the patient to infer by genetic relationship; (2) By and with the third generation familySMN1Deducing a marker of gene association; (3) Specific SNPs or haplotypes, depending on the specific race, are not universal; (4) detection sensitivity and specificity are low. Thus, there is currently a lack of viable method implementationsSMN1Population screening of gene 2+0 carriers. And (3) withSMN1Modified genes with high homologySMN2Similarly, causative genes of congenital adrenocortical hyperplasiaCYP21A2Pseudogenes with high homologyCYP21A1PThere is also the possibility of 2+0 carrying ^[23] However, there is currently no good detection method.

Disclosure of Invention

In view of this, the present application provides a method for detecting 2+0 carrying of a pathogenic gene based on two generations of families and monomers. Specifically, for a subject whose 2 copies of a specific pathogenic gene need to be detected as 2+0 carrying or not, genomic DNA of the subject and parents or subjects, spouse and children is obtained, then haplotypes of the pathogenic genes of two-generation family members are obtained by a long-reading long sequencing method, and whether the subject is 2+0 carrying of the pathogenic genes is deduced by genetic conditions of the haplotypes.

Haplotype (haplotype) refers to a set of associated SNP alleles located at a region on a chromosome. SNPs refer to DNA sequence diversity at the genomic level caused by variation of a single nucleotide. SNPs as used herein include all sites that are not identical to the reference genome.

The method has high sensitivity and specificity, is feasible in clinical operation and high in repeatability, and is beneficial to realizing comprehensive carrier screening of the pathogenic genes. The present protocol does not look at the association of a specific SNP or haplotype with the 2+0 carrying of a pathogenic gene, but detects the 2+0 carrying of a pathogenic gene by simultaneously identifying the SNP or haplotype in a generation 2 human. Specifically, the genetic relationship of the pathogenic gene can be determined by simultaneously identifying SNP or haplotype of 2 generation human, and the pathogenic gene 2+0 carrying can be identified by genetic reasoning.

In this method, since association of SNP or haplotype with 2+0 is not required, it is not necessary to identify a specific SNP or haplotype, it is general, and there are no problems of low accuracy and creator effect compared to the prior art.

Accordingly, in a first aspect, the present application provides a method for detecting the 2+0 carrying of a pathogenic gene comprising the steps of:

(1) Providing a genomic DNA sample of at least two generations of a family including a subject;

(2) Obtaining long fragment DNA of target pathogenic gene;

(3) Sequencing the long fragment DNA by using a long reading and long sequencing platform and constructing a haplotype;

(4) And detecting whether the pathogenic genes of the subjects are carried by 2+0 through family haplotype analysis.

In one embodiment, the pathogenic gene is a pathogenic gene for which a modified gene or pseudogene is present that is highly homologous thereto.

In one embodiment, the pathogenic gene is selected from the group consisting of:SMN1、CYP21A2、GBA、IKBKGandIDS。

in one embodiment, the 2+0 carrying means that one chromosome contains 2 of the pathogenic genes and the other chromosome does not have the pathogenic genes.

In one embodiment, the subject has a copy number of 2 of the pathogenic genes.

In one embodiment, the two-generation family refers to the subject and its father, mother; or refers to a subject, his spouse, and child.

In one embodiment, the method of obtaining long fragment DNA of a pathogenic gene of interest is selected from the group consisting of: long fragment PCR amplification, obtaining pathogenic gene long fragment DNA using probe hybridization method or CRISPR/Cas9 cleavage method.

In one embodiment, the long fragment DNA is 500bp-200kb of DNA.

In one embodiment, the method further comprises detecting haplotypes located at a long fragment of the DNA molecule using a long read long sequencing platform.

In one embodiment, the family haplotype analysis comprises the steps of:

when the two-generation family refers to a subject and father and mother thereof, analyzing whether 2 pathogenic gene haplotypes of the subject are inherited from one or two parents, and when all 2 pathogenic gene haplotypes of the subject are inherited from one of the parents, carrying the pathogenic gene of the subject by 2+0, otherwise, carrying the pathogenic gene by not 2+0;

or when two-generation family is meant to include a subject, its spouse and a child, analyzing whether all, none or part of the haplotypes of 2 pathogenic genes of the subject are inherited to the child, and when none or all haplotypes of the subject are inherited by the pathogenic genes of the child of the subject, the pathogenic genes of the subject are carried by 2+0, whereas the pathogenic genes of the subject are not carried by 2+0.

In a second aspect, the application also provides a kit for carrying out the above method.

The excellent effect of the method for detecting the 2+0 carrying of the pathogenic gene is mainly that:

(1) The genetic relationship is not relied on to infer the 2+0 carrying of the pathogenic genes of family members after the occurrence of patients, so that the wedding guidance and the fertility selection can be carried out through carrier screening, and the birth of severe children is avoided.

(2) The method is independent of the presumption of the third generation family through the associated marker, and can be detected only by the two generation families through haplotypes, so that the clinical two generation family samples are easier to realize.

(3) The pathogenic gene 2+0 is detected independent of the creator effect (i.e., the association of a haplotype of a particular SNP or a particular SNP combination with 2+0. Any race and any similarity toSMN1、CYP21A2The 2+0 carrying genes possibly can be used for carrying and detecting the pathogenic genes 2+0 by the method of the application, and the method has the universality of race and pathogenic genes.

(4) The method for detecting the 2+0 carrying of the pathogenic gene by using the monomer type combined family obtained by long-reading and long-sequencing has high sensitivity and specificity and has the performance of screening large-scale crowd carriers.

Drawings

FIG. 1 subjectSMN1Gene 2+0 carries the detection flow chart.

Fig. 2A: long fragment sequencingSMN1Haplotypes of the genes, wherein the two haplotypes of the sample represent 2SMN1Copy of the gene. Fig. 2B: long fragment sequencingSMN1Haplotypes of the genes, wherein the three haplotypes of the sample represent 3SMN1Copy of the gene. The vertical lines in fig. 2A and 2B represent SNPs, and different haplotypes are distinguished by SNP combinations.

Fig. 3A: detecting whether the subject is a parent through the two-generation family of the subject and the parentSMN1Gene 2+0 is carried and is a family chart. Fig. 3B: detecting whether the subject is a parent through the two-generation family of the subject and the parentSMN1Gene 2+0 carries and the method of the application is used to detect whether a subject isSMN1Gene 2+0 is carried.

Fig. 4A: detecting whether a subject is by the two-generation family of the subject, spouse and childSMN1Gene 2+0 is carried and is a family chart. Fig. 4B: detecting whether a subject is by the two-generation family of the subject, spouse and childSMN1Gene 2+0 carries and the method of the application is used to detect whether a subject isSMN1Gene 2+0 is carried.

Fig. 5A: detecting whether the subject is a parent through the two-generation family of the subject and the parentSMN1Gene 2+0 is carried and is a family chart. Fig. 5B: detecting whether the subject is a parent through the two-generation family of the subject and the parentSMN1Gene 2+0 carries and the method of the application is used to detect whether a subject isSMN1The arrangement mode of two copies of the gene is 1+1, and the two copies are not carried by 2+0.

Fig. 6A: detecting whether a subject is by the two-generation family of the subject, spouse and childSMN1Gene 2+0 is carried and is a family chart. Fig. 6B: detecting whether a subject is by the two-generation family of the subject, spouse and childSMN1Gene 2+0 carries and the method of the application is used to detect whether a subject isSMN1The arrangement mode of two copies of the gene is 1+1, and the two copies are not carried by 2+0.

Detailed Description

The following detailed description of the preferred embodiments of the application, taken in conjunction with the accompanying drawings, is given by way of illustration and not limitation, and any other similar situations are intended to fall within the scope of the application.

Unless otherwise defined herein, scientific and technical terms used in connection with the present application will have the meanings commonly understood by one of ordinary skill in the art. Furthermore, unless the context requires otherwise, terms in the singular shall include the plural and terms in the plural shall include the singular. More specifically, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. In the present application, the use of "or" means "and/or" unless stated otherwise. Furthermore, the use of the term "include" and other forms (such as "include" and "contain") is not limiting. Furthermore, the scope provided in the specification and the appended claims includes all values between the endpoints and between the endpoints.

Example 1: the SMA pathogenic gene is obtained by the method of the applicationSMN1Monomer type of (2)

Step 1: a reaction system was prepared according to the following Table 1, and amplification was performed using genomic DNA as a templateSMN1The whole length of the gene.

Table 1:

step 2: PCR amplification was performed on a PCR apparatus under the conditions shown in Table 2 below.

Table 2:

step 3: a ligation reaction system was prepared and a PacBio library was constructed according to Table 3 below.

Table 3:

step 4: ligation reactions were performed on a PCR apparatus under the conditions shown in Table 4 below.

Table 4:

after completion of the reaction, 0.5 mL Exonuclease III (NEB, cat#m0206l) and 0.5 mL Exonuclease VII (NEB, cat#m0379L) were added and placed on a PCR machine to react for 1 hour at 37 ℃. The DNA was purified twice with 0.6XAmpure PB beads (PacBIO, cat# 100-265-900) according to the manufacturer's instructions, and finally eluted with 10uL of Elutation Buffer, and the obtained DNA was the PacBIO sequencing pre-library.

Step 5: pacBIO was sequenced on a machine.

The DNA concentration was determined on a Qubit 3 Flurometer (ThermoFisher, cat#Q 33216) with the Qubit dsDNA HS reagent (ThermoFisher, cat#Q 32851). When a plurality of samples need to be sequenced at the same time, equal amounts of pre-libraries with different Barcode can be mixed together to prepare a mixed pre-library. Based on the total and molar concentrations of the pre-library, the appropriate volumes of library were reacted with binding reagents (PacBIO, cat# 101-820-200) and primers (PacBIO, cat# 100-970-100) to prepare the final on-machine library. The library was sequenced on a PacBio sequence II sequencing platform for 30 hours.

Step 6:SMN1analysis of Gene haplotypes.

In the SMRT Link analysis system provided by PacBIO, the original sureads of PacBIO were subjected to consistency analysis to obtain high quality CCS reads, which were then aligned to the hg38 reference genome, by base discrimination at the c.840 position on exon 7SMN1AndSMN2is set in the CCS reads of (C). Will beSMN1CCS reads of the Gene were obtained by SNP information using the PacBio's pbampliconculatng analysis softwareSMN1Haplotype information of the gene. Representative results are shown in fig. 2A and 2B, wherein: the two haplotypes of the sample in FIG. 2A represent that the sample has 2SMN1Gene copy, FIG. 2BThree haplotypes of (a) represent 3 of the samplesSMN1Copy of the gene.

Example 2: detecting whether a subject is using the methods of the applicationSMN1Gene 2+0 carrying

Collecting 2 families of patients with SMA precursors, and 2 families of patients without SMA, wherein each memberSMN1The gene copy number was determined by MLPA assay. Family 1 includes third generation members, with precursor III-1 not presentSMN1Gene (i.e., 0+0), but its father II-1 has 2SMN1Genes, specifying these 2SMN1Genes are all on one chromosome and none on the otherSMN1Thus deducing its father as through genetic relationshipSMN1Gene 2+0 carries and then deduces its ancestor I-2SMN1The genes are in a 2+1 distribution (FIG. 3A). The method of the present application is now used to detect whether subject II-1 is or is not, without relying on a proverSMN1Gene 2+0 is carried. First, haplotypes of a subject and their parent were determined using the method in example 1. As shown in FIG. 3B, threeSMN1The number of gene haplotypes is consistent with the copy number of MLPA detection, which indicates that one gene copy number corresponding to each haplotype in the three samples can be found by haplotype analysis, and the two haplotypes of the subject are all inherited from the mother I-2 and different from the haplotype of the father I-1, which indicates that the two haplotypes are located on the same chromosome, thus the subjectSMN1The gene was 2+0 carrying (FIG. 3B), consistent with the result of the dependency precursor inferred by genetic relationship.

Family 2 includes two generations of members, with precursor II-2 not presentSMN1Gene (i.e., 0+0) and 2 of its mother I-2SMN1Genes, specifying these 2SMN1Genes are all on one chromosome and none on the otherSMN1Thus deducing its mother as by genetic relationshipSMN1Gene 2+0 carries (FIG. 4A). The method of the present application is now utilized to detect whether or not subject I-2 is, without relying on a proverSMN1Gene 2+0 is carried. First, the haplotypes of the subject, spouse I-1 and her daughter II-1 were determined using the method in example 1. As shown in FIG. 4B, threeSMN1Number of gene haplotypes and MThe copy numbers of LPA detection are consistent, which indicates that each haplotype in the three samples corresponds to a gene. As can be seen by haplotype analysis, both haplotypes of the subject were inherited by the daughter, indicating that both haplotypes are located on the same chromosome, and therefore the subjectSMN1The gene was 2+0 carrying (FIG. 4B), consistent with the result of the dependency precursor inferred by genetic relationship.

Family 3 comprises two generations of members, father I-1, mother I-2 and subject (son) II-1 all containing 2SMN1Copy of the gene (FIG. 5A). By haplotype analysis using the methods of the application, two subjects were testedSMN1Gene haplotypes were inherited from the father and mother, respectively, indicating that bothSMN1The genes were arranged in a 1+1 manner, not 2+0 (FIG. 5B). Family 4 comprises two generations of members, subject (father) I-1 has 2SMN1Gene copies, mother I-2 and son II-1 all have 1SMN1Copy of the gene (FIG. 6A). Analysis of two subjects by monomer using the methods of the applicationSMN1One of the gene haplotypes was inherited to the son, indicating two subjectsSMN1Genes were located on both chromosomes, i.e., arranged in a 1+1, not 2+0, pattern (FIG. 6B).

Thus, the subject can be accurately detected for the pathogenic gene 2+0 by the two-generation family using the method of the present application without relying on a forerunner.

It should be noted that the foregoing examples are illustrative only and are intended to illustrate some of the features of the application. The appended claims are intended to claim the broadest possible scope and the embodiments presented herein are merely illustrative of selected implementations based on combinations of all possible embodiments. Accordingly, it is not the intention of the applicant that the appended claims be limited by the choice of examples illustrating the features of the application. As used in the claims, the term "comprising" and its semantic variants also logically include different and varying terms such as, but not limited to, "consisting essentially of" or "consisting of. Where desired, numerical ranges are provided, and such ranges also include subranges therebetween. Variations in these ranges are also self-evident to those skilled in the art and should not be considered as being donated to the public, but rather should be construed as being covered by the appended claims where possible. Reagents, reaction conditions, etc., as involved in long fragment PCR reactions and in the construction of third generation sequencing libraries may be adapted and varied according to particular needs. But further advancements in technology will form possible equivalents or sub-substitutions not presently considered due to imprecision of language with such changes being construed where possible as covered by the appended claims.

Reference to the literature

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[12] Song, huang Shangzhi, beijing society of medical science and genetics, beijing society of rare disease diagnosis and treatment and guarantee. Spinal muscular atrophy genetic diagnostic specialists agree. Journal of chinese medicine, 11/3/2020, volume 100, 40.

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[16] 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.

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Claims (11)

1. A method for detecting 2+0 carrying of a pathogenic gene comprising the steps of:

(1) Providing a genomic DNA sample of at least two generations of a family including a subject;

(2) Obtaining long fragment DNA of target pathogenic gene;

(3) Sequencing the long fragment DNA by using a long reading and long sequencing platform and constructing a haplotype;

(4) And detecting whether the pathogenic genes of the subjects are carried by 2+0 through family haplotype analysis.

2. The method of claim 1, wherein the pathogenic gene is a pathogenic gene for which a modified gene or pseudogene is present that is highly homologous thereto.

3. The method of claim 1, wherein the pathogenic gene is selected from the group consisting of:SMN1、CYP21A2、GBA、IKBKGandIDS。

4. the method of claim 1, wherein the 2+0 carrying means that one chromosome contains 2 of the pathogenic genes and the other chromosome does not contain the pathogenic genes.

5. The method of claim 1, wherein the subject has a copy number of 2 of the pathogenic genes.

6. The method of claim 1, wherein the two-generation family is a subject and its father, mother; or refers to a subject, his spouse, and child.

7. The method of claim 1, wherein the method of obtaining long fragment DNA of a pathogenic gene of interest is selected from the group consisting of: long fragment PCR amplification, obtaining pathogenic gene long fragment DNA using probe hybridization method or CRISPR/Cas9 cleavage method.

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

9. The method of claim 1, wherein the method further comprises detecting haplotypes located in a long fragment of the DNA molecule using a long read long sequencing platform.

10. The method of claim 1, wherein the family haplotype analysis comprises the steps of:

when the two-generation family refers to a subject and father and mother thereof, analyzing whether 2 pathogenic gene haplotypes of the subject are inherited from one or two parents, and when all 2 pathogenic gene haplotypes of the subject are inherited from one of the parents, carrying the pathogenic gene of the subject by 2+0, otherwise, carrying the pathogenic gene by not 2+0;

or when two-generation family is meant to include a subject, its spouse and a child, analyzing whether all, none or part of the haplotypes of 2 pathogenic genes of the subject are inherited to the child, and when none or all haplotypes of the subject are inherited by the pathogenic genes of the child of the subject, the pathogenic genes of the subject are carried by 2+0, whereas the pathogenic genes of the subject are not carried by 2+0.

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