CN116814772A

CN116814772A - Primer combination, kit and method for detecting FANCA gene mutation

Info

Publication number: CN116814772A
Application number: CN202310829370.5A
Authority: CN
Inventors: 张毅; 杜娟; 胡晓; 林戈; 何文斌; 戴婧; 万振兴
Original assignee: Guang Xiu Gao Xin Life Science Co ltd Hunan; Reproductive and Genetic Hospital of CITIC Xiangya Co Ltd
Current assignee: Guang Xiu Gao Xin Life Science Co ltd Hunan; Reproductive and Genetic Hospital of CITIC Xiangya Co Ltd
Priority date: 2023-07-07
Filing date: 2023-07-07
Publication date: 2023-09-29

Abstract

The application relates to a primer combination, a kit and a method for detecting FANCA gene mutation, wherein the primer combination is selected from a plurality of primer pairs 1 to 105 with nucleotide sequences shown as SEQ ID No.1 to SEQ ID No. 210. The application provides a method for detecting FANCA mutation by embryo implantation pre-genetics, which has strong universality and high accuracy, can be used for detecting FANCA mutation at embryo level by high-throughput sequencing, and helps family fertility healthy offspring with the fertility history of infants suffering from Vanconi anemia.

Description

Primer combination, kit and method for detecting FANCA gene mutation

Technical Field

The application relates to the technical field of molecular biology, in particular to a primer combination, a kit and a method for detecting FANCA gene mutation.

Background

Fanconi Anemia (FA) is a rare monogenic genetic disease characterized primarily by progressive bone marrow hematopoietic failure, the most common of congenital hematopoietic failing diseases. Vanconi anemia, which can be caused by mutation of the FANCA gene, is an autosomal recessive inheritance, is a disease affecting multiple organs of the body, has high clinical heterogeneity, and is mainly clinically manifested by abnormal body, bone marrow failure and increased risk of malignant tumor.

Pre-embryo implantation genetic testing (Preimplantation genetic testing, PGT) is an effective means of preventing fanconi anemia birth defects. PGT refers to the step of performing genetic detection by biopsy of a plurality of cells when an embryo develops in vitro to a blastomeres stage or blastocyst stage, and finally selecting an embryo without risk of illness to implant into the mother uterus, thereby achieving the purpose of growing healthy offspring. PGT can effectively avoid physical and psychological injuries caused by termination of pregnancy due to pregnant genetics disease fetuses, and is becoming the first choice for high-risk fetuses couples with birth genetics disease. A common method for FANCA gene detection at the embryo level is multiplex nested PCR and Karyoming technology. The multiplex nested PCR method requires screening heterozygous Short Tandem Repeats (STRs) for FANCA gene carriers in advance, then establishing a multiplex PCR system containing mutation sites and heterozygous STR sites at lymphocyte level, evaluating amplification efficiency and allele release rate at each site at cell level, and being applicable to embryo detection after qualification. Because the STR number is limited and the heterozygosity frequency in the crowd is different, the design of the multiple nest PCR method is more personalized, the workload is large, the period is long, and the universality is lower. Compared with STR loci, the number of single nucleotide polymorphism loci (SNP) is widely distributed, the occurrence frequency in the crowd exceeds 1%, the total number of the SNP is 300 ten thousand, 1/3 Kb is averaged, and the SNP is easy to realize automatic analysis on a high-throughput sequencing platform. The Karyoming technique indirectly eliminates gene defects through SNP linkage analysis. The chip has 30 ten thousand SNP probes for linkage analysis of whole genome, and is a comprehensive universal single gene disease PGT technology. However, karyomopping is not able to detect gene mutations, so that either a pre-prover sample or a suitable family member sample is necessary. If there are not enough pedigree samples, additional mutation detection is required.

Therefore, there is an urgent need to develop a method for judging the genotype of an embryo FANCA mutation, which can more fully cover the SNP sites in and upstream and downstream of the FANCA gene.

Disclosure of Invention

Based on this, it is necessary to provide a primer combination, a kit and a method for detecting FANCA gene mutation, which are highly versatile and have a high diagnostic rate.

In a first aspect of the present application, there is provided a primer set for detecting a mutation of the FANCA gene, the primer set being selected from a plurality of primer pairs 1 to 105 having nucleotide sequences shown in SEQ ID No.1 to SEQ ID No. 210.

In one embodiment, the primer set includes primer pair 1 through primer pair 105 having nucleotide sequences shown in SEQ ID No.1 through SEQ ID No. 210.

In a second aspect of the present application, there is provided a kit for detecting a mutation of the FANCA gene, comprising the primer combination.

In one embodiment, the kit further comprises PCR reaction reagents.

In one embodiment, the PCR reaction reagents include one or more of PCR buffer, DNA polymerase, mg2+, and dNTPs.

In a third aspect of the present application, there is provided a method for detecting mutation of FANCA gene, comprising the steps of:

obtaining genomic DNA of a sample;

performing PCR amplification on the FANCA gene coding region of the genomic DNA of the sample and the upstream and downstream SNP loci thereof by using the primer combination or the kit; and analyzing the PCR amplification product, and determining the FANCA mutation type according to an analysis result.

In one embodiment, the FANCA gene coding region and its upstream and downstream SNP sites comprise: chr16: chr16: the chr16 r16 the-r 16 the-r 16-the-the.

In one embodiment, the method of analyzing the PCR amplification product comprises: and performing high-throughput sequencing on the PCR amplification product.

In one embodiment, the sample is selected from one or more of peripheral blood, semen, oral mucosal cells, and embryonic cells.

In a fourth aspect of the application, there is provided the use of said primer combination or said kit for the preparation of a product for detecting a mutation in the FANCA gene.

Compared with the traditional technology, the application also has the following beneficial effects:

the application provides a primer combination for detecting FANCA gene mutation, which can detect the haplotype and genotype of FANCA of a sample by a PCR amplification method of a FANCA gene coding region and upstream and downstream SNP loci thereof. The application adopts the primer combination to construct a genetic detection method before embryo implantation with strong universality and high diagnosis rate, omits a cell level pre-experiment, can be used for detecting FANCA mutation at the embryo level by high-throughput sequencing, and helps family fertility healthy offspring with the fertility history of Vanconi anemia infants. The advantages of the application mainly include the following points:

(1) The number of SNPs detected was large: the application can more comprehensively detect FANCA gene coding region and upstream and downstream SNP loci thereof, and can also detect unknown SNP mutation loci.

(2) The universality is high, the accuracy is high: the test was performed in 16 samples, and the detection pass rate of the samples was 100% by standard setting that the genes were not less than 2 effective SNPs on both sides.

(3) The cost is low: when the detection method is adopted for detection, the cost for analyzing the haplotype of FANCA is lower.

Detailed Description

In order that the application may be readily understood, a more particular description of the application will be rendered by reference to specific embodiments that are illustrated in the appended drawings. This application may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

Terminology

Unless otherwise indicated or contradicted, terms or phrases used herein have the following meanings:

in the present application, a selection range in reference to "and/or", "and/or" includes any one of two or more of the items listed in relation to each other, as well as any and all combinations of the items listed in relation to each other, including any two of the items listed in relation to each other, any more of the items listed in relation to each other, or all combinations of the items listed in relation to each other. It should be noted that when at least three items are connected by a combination of at least two conjunctions selected from the group consisting of "and/or", "and/or", it is to be understood that the technical solution undoubtedly includes technical solutions that are all connected by "logical and", and undoubtedly also includes technical solutions that are all connected by "logical or".

In the present application, reference to "optional", "optional" refers to the presence or absence of the "optional" or "optional" means either of the "with" or "without" side-by-side arrangements. If multiple "alternatives" occur in a technical solution, if no particular description exists and there is no contradiction or mutual constraint, then each "alternative" is independent.

In the present application, references to "preferred", "better", "preferred" are merely to describe embodiments or examples of better results, and it should be understood that they do not limit the scope of the present application.

In the present application, references to "further", "still further", "particularly" and the like are used for descriptive purposes and indicate that the application is not to be interpreted as limiting the scope of the application.

In the present application, the terms "first", "second", "third", "fourth", etc. are used for descriptive purposes only and are not to be construed as indicating or implying a relative importance or quantity, nor as implying an importance or quantity of a technical feature indicated. Moreover, the terms "first," "second," "third," "fourth," and the like are used for non-exhaustive list description purposes only, and are not to be construed as limiting the number of closed forms.

In the present application, a numerical range (i.e., a numerical range) is referred to, and optional numerical distributions are considered to be continuous within the numerical range and include two numerical endpoints (i.e., a minimum value and a maximum value) of the numerical range and each numerical value between the two numerical endpoints unless otherwise specified. Where a numerical range merely refers to integers within the numerical range, including both end integers of the numerical range, and each integer between the two ends, unless otherwise indicated, each integer is recited herein as being directly recited, such as t being an integer selected from 1 to 10, meaning t is an integer selected from any one of the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. Further, when a plurality of range description features or characteristics are provided, these ranges may be combined. In other words, unless otherwise indicated, the ranges disclosed herein are to be understood to include any and all subranges subsumed therein.

In the application, the technical characteristics described in an open mode comprise a closed technical scheme composed of the listed characteristics and also comprise an open technical scheme comprising the listed characteristics.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein in the description of the application is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. The term "and/or" as used herein includes any and all combinations of one or more of the associated listed items.

Reads refer to sequence fragments obtained by sequencing.

Single nucleotide polymorphism (single nucleotide polymorphism, SNP) refers to a DNA sequence polymorphism caused by variation of a single nucleotide at the genomic level.

Haplotypes (Haplotype), also known as haplotypes or haplotypes, refer to a group of interrelated single nucleotide polymorphisms located in a particular region of a chromosome and are prone to be inherited as a whole to offspring.

The sequencing depth refers to the ratio of the total number of bases sequenced to the size of the genome to be tested, and can be understood as the average number of repeated sequencing (in terms of number of bases) per measured base in the genome, in one embodiment of the application, the sequencing depth is 1000x, which means that the strip of specific PCR amplification product is sequenced 1000 times.

In order to solve the above-mentioned problems, in a first aspect of the present application, there is provided a primer combination for detecting a mutation of the FANCA gene, which is selected from a plurality of primer pairs 1 to 105 having nucleotide sequences shown in SEQ ID No.1 to SEQ ID No. 210.

Optionally, the primer combination comprises a primer pair 1 to a primer pair 105, wherein the nucleotide sequences of the primer pair 1 to the primer pair 105 are shown as SEQ ID No.1 to SEQ ID No. 210.

Optionally, the kit further comprises a PCR reaction reagent.

Optionally, the PCR reaction reagents include one or more of PCR buffer, DNA polymerase, mg2+, and dNTPs.

obtaining genomic DNA of a sample;

Optionally, the coding region of the FANCA gene and the SNP loci upstream and downstream thereof comprise: chr16: chr16: the chr16 r16 the-r 16 the-r 16-the-the.

Optionally, the method for analyzing the PCR amplification product comprises: and performing high-throughput sequencing on the PCR amplification product.

Alternatively, the high throughput sequencing platform is IlluminaMiseq.

Alternatively, the PCR amplification method is multiplex PCR amplification.

Optionally, the sample is selected from one or more of peripheral blood, semen, oral mucosal cells, and embryonic cells.

Further alternatively, the sample is an embryonic cell in the blastocyst stage or blastocyst stage.

Preferably, the DNA content in each DNA sample is greater than 500ng.

In a specific example, high throughput sequencing of the PCR amplification products comprises: comparing the sequencing result with the DNA sequences of the parents of the sample, and analyzing the haplotype of the embryo cell FANCA.

In a specific example, high throughput sequencing of the PCR amplification product further comprises: the sequencing results were aligned with human genome reference sequences and analyzed for fold coverage and genotype of SNPs.

In one embodiment, the detection method of the present application is to take a plurality of cells for genetic detection when fertilized eggs develop in vitro to the blastomeres or blastocysts, and it is understood that the method is not directly implemented by living human or animal bodies, and is not a disease diagnosis and treatment method.

The following examples are further offered to illustrate, but not to limit, the materials used in the examples are commercially available, and the equipment used is commercially available, and the processes involved are routinely selected by those skilled in the art without any specific description.

Example 1

SNP locus screening and primer design

Screening principle of SNP loci: high frequency SNPs were selected from the thousand person genome project database (http:// www.ncbi.nlm.nih.gov/variation/tools/1000genome /); high frequency SNP sites with minimum allele frequencies greater than 0.2; removing SNP sites with GC content of >70% in the polynucleotide (polyN) and 50bp sequences upstream and downstream of the sites; removing SNP sites with a plurality of positions from the upstream and downstream 50bp sequences (namely removing SNP sites with high homology) to human genome hg 19;

SNP selection range: SNP sites within the FANCA gene and within 1Mb upstream and downstream of the gene (or mutation site) are preferably selected, and if the number of SNPs within 1Mb is small, the range can be appropriately widened, for example, 2Mb upstream and downstream.

The FANCA gene coding region and the upstream and downstream SNP sites thereof were selected according to the above-described method.

2. Primer design: designing corresponding primers according to the screening principle of the SNP loci by an online platform (https:// www.ampliseq.com /); the primer sequence is shown as SEQ ID NO. 1-SEQ ID NO.210, the specificity of the primer is high, the primer has similar annealing temperature and the size of the PCR product fragment is in the range of 125bp-375 bp.

The SNP loci and the sequences of the primers are shown in Table 1, the sequence numbers 1-77 are SNP loci and primer pairs within 2M of the downstream of the FANCA gene, and the sequence numbers 77-105 are SNP loci and primer pairs within 2M of the upstream of the FANCA gene.

TABLE 1

Example 2

The results of gene detection suggest that the FANCA gene (NM-000135) of the infant has a complex heterozygous variation of c.3948delG (parent source) and c.2101A > G (p.Lys 701Glu) (parent source) for a pair of couples suspected of having Vanconi anemia infant fertility history. The couple applied for embryo implantation genetic test (PGT) against the pathogenic mutation mentioned above, and healthy offspring were bred. Since the genetic material of the child is insufficient, the blood samples of both parents of the couple are collected for genetic detection. The blood samples of parents of the couple are collected for genetic detection, and the result shows that the mutation carried by the female is inherited from the mother, the mutation carried by the male is inherited from the father, and the risk chromosome haplotype of the male and the female is defined through family information.

The couples are assisted by PGT to obtain 3 embryos (LYX-6, LYX-7 and LYX-8), the embryos develop to the blastula stage to carry out trophoblast biopsy, and the known variation and haplotype of FANCA genes are further detected after the whole genome of the biopsy cells are amplified. Of the 3 embryos, 2 were normal and 1 patient. The pair of couples transplanted a normal embryo, the ultrasonic diagnosis and the gene diagnosis before 16 weeks of pregnancy indicate that the fetus is normal in development, and the genetic detection indicates that the fetus FANCA gene does not have c.3948delG and c.2101A > G mutation, which are consistent with the PGT result. The specific detection steps are as follows:

1 multiplex PCR capture: mixing 105 pairs of primer pairs in Table 1 into two PCR reaction tubes, performing 105-fold reaction on the DNA of the biopsy cell sample in the two PCR reaction tubes, and performing multiplex PCR amplification on the SNP in the FANCA gene and upstream and downstream of the biopsy cell sample to obtain a whole genome amplified product of the biopsy cell sample. The DNA content of each sample was greater than 500ng.

2, library building and sequencing are carried out according to an Illumina standard library building flow.

And (3) carrying out library establishment on the whole genome amplified products of the biopsy cell sample to be detected according to an Illumina standard library establishment flow, and sequencing by using Miseq.

When the DNA molecule to be tested is from a plurality of samples to be tested, each sample may be tagged with a different tag sequence (barcode) for sample discrimination during sequencing, thereby allowing simultaneous sequencing of multiple samples.

3, data analysis: removing the linker sequence from the original data generated by the Illumina sequencer by using trimmabic software, aligning to the human hg19 reference genome by using BWA software, aligning the read with the reference genome sequence by BWA (Burrow-Wheeler-Aligner), and obtaining the position of the read on the reference genome. And finally, analyzing haplotype SNP coverage fold and genotype of the sample to be tested. The average sequencing depth reaches more than 100x, and the sequencing depth is regarded as qualified.

The detection results of haplotypes of the samples to be measured are shown in table 2. The quality control results of the sequencing are shown in Table 3, the sequencing depth is 100 times or more, the 30 times coverage is 70% or more, the 100 times coverage is 50% or more, and the quality control is qualified. Wherein F0 represents a male risk chromosome and F1 represents a male normal chromosome; m0 represents a female risk chromosome, and M1 represents a female normal chromosome.

TABLE 2

Name of name	Haplotype type	Pathogenic gene detection (PGT-M)
			Female party	M0/M1	Carrying about
Male prescription	F0/F1	Carrying about
			Male mother	F1/	Normal state
Women's mother	M0/	Carrying about
			LYX-6	M0/F0	Pathogenicity is caused by
LYX-7	M1/F1	Normal state
			LYX-8	M1/F1	Normal state

TABLE 3 Table 3

Table 4 shows the detection results of SNP mutations, and since the design scheme covers the entire coding region of FANCA gene, the gene mutations occurring in the coding region can be detected efficiently, and the sequencing depth is greater than 100×.

TABLE 4 Table 4

Note that: * Heterozygosity: hom indicates that the mutation site is homozygous, het indicates that the mutation site is heterozygous, hemi indicates that the mutation site is hemizygous, and Normal indicates that no mutation is detected at the site.

SNP haplotypes are shown in Table 5, wherein, "? "represents undetected, black bolded square frame sites as trip sites. F0 represents a male risk chromosome, and F1 represents a male normal chromosome; m0 represents a female risk chromosome, and M1 represents a female normal chromosome.

TABLE 5

/>

Samples of offspring embryos were verified using a generation of sequencing, the verification results are shown in table 6, consistent with PGT results.

TABLE 6

Name of name	First generation verification (c.3948delG)	First generation verification (c.2101A)>G)
			LYX-6	Het	Het
LYX-7	Normal	Normal
			LYX-8	Normal	Normal

Example 3

The test was carried out in 16 human samples by the method of example 2, and 16 FANCA carriers were detected, the mutation site covered 43 exons of FANCA pathogenic gene, and the pass rate of 16 subjects was 100% by standard setting that the genes had not less than 2 effective SNPs on both sides. The method for detecting FANCA gene mutation has higher universality and accuracy.

As the FANCA gene has a large number of single nucleotide polymorphism Sites (SNP) and a wide distribution, the occurrence frequency in the population exceeds 1%, the total number of the FANCA gene reaches 300 ten thousands, and the average number of the FANCA gene is 1/3 Kb. The application provides a genetic detection method before embryo implantation, which can analyze 210 SNP, and has the advantages of large number of detected SNP and high universality; the method detects 16 FANCA carriers, and sets that the two sides of a mutation site are respectively provided with not less than 2 effective SNP by the standard, and the passing rate of 16 testees is 100 percent, so that the universality is higher; the method has the advantages that the cell level pre-experiment is omitted, the automatic analysis can be easily realized on a high-throughput sequencing platform, different tag sequences are added on each sample based on the high-throughput sequencing technology, a large number of samples can be analyzed at one time, and the cost of FANCA haplotype analysis is reduced; the detection method can be used for detecting chromosome aneuploidy, chromosome structural abnormality and monogenic diseases, has high sequencing depth and average sequencing depth of not less than 100X, and overcomes the defect that chip detection is easily influenced by probes.

The technical features of the above-described embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above-described embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.

The above examples illustrate only a few embodiments of the application, which are described in detail and are not to be construed as limiting the scope of the application. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the application, which are all within the scope of the application. Accordingly, the scope of protection of the present application is to be determined by the appended claims.

Claims

1. A primer combination for detecting FANCA gene mutation is characterized in that the primer combination is selected from a plurality of primer pairs 1 to 105 with nucleotide sequences shown as SEQ ID No.1 to SEQ ID No. 210.

2. The primer combination of claim 1, wherein the primer combination comprises primer pair 1 to primer pair 105 having nucleotide sequences shown in SEQ ID No.1 to SEQ ID No. 210.

3. A kit for detecting a mutation in the FANCA gene comprising the primer combination of claim 1 or 2.

4. The kit of claim 3, further comprising PCR reaction reagents.

5. The kit of claim 4, wherein the PCR reaction reagents comprise PCR buffer, DNA polymerase, mg ²⁺ And one or more of dNTPs.

6. A method for detecting FANCA gene mutation, comprising the steps of:

obtaining genomic DNA of a sample;

performing PCR amplification of the FANCA gene coding region of genomic DNA of the sample and its upstream and downstream SNP sites using the primer combination of claim 1 or 2 or the kit of any one of claims 3 to 5; and analyzing the PCR amplification product, and determining the FANCA mutation type according to an analysis result.

7. The method according to claim 6, wherein the FANCA gene coding region and the SNP sites upstream and downstream thereof comprise: chr16: chr16: the chr16 r16 the-r 16 the-r 16-the-the.

8. The method according to claim 6, wherein the method for analyzing the PCR amplification product comprises: and performing high-throughput sequencing on the PCR amplification product.

9. The method of claim 6, wherein the sample is selected from one or more of peripheral blood, semen, oral mucosal cells, and embryonic cells.

10. Use of a primer combination according to claim 1 or 2 or a kit according to any one of claims 3 to 5 for the preparation of a product for detecting a mutation in the FANCA gene.