CN117683872A

CN117683872A - Application, reagent and kit of NR5A1 mutant gene as detection target

Info

Publication number: CN117683872A
Application number: CN202311665346.9A
Authority: CN
Inventors: 曾桥; 徐霞; 刘亚宁; 范园; 何亮波
Original assignee: Hunan Jiahui Biotechnology Co Ltd
Current assignee: Hunan Jiahui Biotechnology Co Ltd
Priority date: 2023-12-06
Filing date: 2023-12-06
Publication date: 2024-03-12

Abstract

The invention provides application, a reagent and a kit of NR5A1 mutant gene as a detection target. The invention provides application, reagent and kit of NR5A1 mutant gene as detection target spot, enriches detection method for detecting 46, XY reverse 3 type, provides basis for treating 46, XY reverse 3 type, shortens detection time, facilitates doctor or patient to know illness state in time, and carries out subsequent treatment in a targeted manner.

Description

Application, reagent and kit of NR5A1 mutant gene as detection target

Technical Field

The invention relates to the technical field of biological detection, in particular to application, a reagent and a kit of NR5A1 mutant gene as a detection target.

Background

Sexual dysplasia (disorders of sex development, DSD) is a generic term for a large group of genetically heterogeneous diseases in which chromosome karyotypes, gonadal phenotypes, and gonadal anatomy are inconsistent. The main clinical manifestation is that the internal and external genital organs are blurred, which can be combined with the amphoteric characteristics of men and women and even the sex is blurred and difficult to determine. It is reported that the prevalence of genital fuzzy infants in live infants is close to 1/1000. DSD is broadly divided into: sex chromosomes DSD, 46, XYDSD and 46, XX DSD. Patients with xy DSD have a normal structure and number of 46, xy karyotypes, with reduced androgen secretion, manifested by mild hypourethral cleavages to complete internal and external female genitals. 46, xy DSD is mainly associated with androgen synthesis/dysfunction and abnormal testis differentiation. 46, xy DSD mainly comprises: (1) gonadal dysplasia: complete Gonadal Dysplasia (CGD), partial Gonadal Dysplasia (PGD), ovarian dysplasia, testicular dysplasia, and the like. 46, xy CGD is characterized by a complete female external genitalia, a distinct Miaole structure, and the gonad consists of a series of fibrous tissues. The external genitalia is female due to the absence of testosterone and the production of anti-Miaole hormone, which results in the presence of uterus, fallopian tubes, miaole tubes. 46, xy PGD is characterized by normal development of part of the testes, with or without well-developed yolk ducts and Miaole ducts, different degrees of maleation of the external genitalia; (2) androgen synthesis/dysfunction: luteinizing Hormone (LH) receptor abnormality, 5 a-reductase type 2 abnormality, steroid synthesis acute regulatory protein abnormality, congenital Adrenocortical Hyperplasia (CAH), complete Androgen Insensitivity Syndrome (CAIS), partial Androgen Insensitivity Syndrome (PAIS) and the like; (3) external male genital organs are obscured. It has now been found that over 40 genes are associated with 46, xy DSD, such as SRY, CBX2, MAP3K1, WWOX, NR5A1, etc., where mutations in the NR5A1 gene can result in 46, xy reversed form 3 (MIM 612965) leading to autosomal dominant inheritance, with the main clinical manifestations of sex reversal, scrotum-type hypourethral cleavage, vulvar gender, uterine dysplasia, gonadal hypoplasia, premature ovarian failure, occult sperm disease, etc.

NR5A1 gene (MIM 184757) is a 46, XY inverted 3 pathogenic gene, which is located on chromosome 9q33.3, the gene is 26.2kb in length, contains 7 exons and 6 introns, encodes 461 amino acid sterogenic factor 1 (SF 1/NR5A 1), SF1 is a solitary nucleus receptor, and consists of a DNA Binding Domain (DBD) containing two zinc finger structures and an FTZ-F1 cassette, an auxiliary hinge region, and a Ligand Binding Domain (LBD) two function activating domains AF-1 and AF-2. SF1 acts as a transcription factor that binds directly to DNA in a sequence-specific manner and recruits cofactors and/or ligands to enable transcriptional activation or inhibition of downstream target genes. SF1 is expressed in adrenal glands and bipotent gonads of developing embryos, SF1 is a key regulator of steroid synthesis and reproductive development, controls an important process of adrenal gland and gonadal development, and during 46, XY embryo development, it is first expressed in the urogenital ridge and promotes testis differentiation along with the SRY, SOX9 and GATA4 genes. Mice deleted for the NR5A1 gene show adrenal and gonadal complete dysplasia; in XY mice, the reproductive phenotype includes female external genitalia and persistent Miaole structure. The NR5A1 mutations are associated with sexual dysplasia, while NR5A1 is associated with fertility, these symptoms including gonadal and testicular dysplasia with or without a Miaole residual structure, genital ambiguity, mild and severe hypourethral fissures, male dysplasia of varying severity: such as penile, cryptorchidism, no testosterone, male infertility. The missense mutation of NR5A1 is most common, other mutation types include frameshift mutation, nonsense mutation, non-frameshift mutation, splicing mutation and the like, and the mutation is distributed in a gene coding sequence without obvious mutation hot areas. The NR5A1 gene mutation may cause 46, XY-reversed type 3 in 46, XY individuals, 46, XX-reversed type 4 (MIM 617480), premature ovarian failure type 7 (MIM 612964) in 46, XX individuals, and the like.

The NR5A1 gene mutation is an important genetic basis for the development of 46, XY reversed type 3, and the genetic diagnosis is a gold standard for diagnosing 46, XY reversed type 3. The clinical needs to establish corresponding detection technology aiming at different mutations and be used for clear etiology and disease diagnosis, the gene mutation sites related to 46, XY inversion type 3 are found, the screening and diagnosis of the gene mutation of the 46, XY inversion type 3 are assisted, and the method has important significance for drug screening, drug effect evaluation and targeted treatment. In the prior art, the genotype of a mutation site of a gene can be detected by adopting other methods such as restriction enzyme fragment length polymorphism, single-strand conformation polymorphism, allele-specific oligonucleotide hybridization and the like, but the detection methods cannot meet the purposes of qualitative, quantitative and definite mutation gene sequence at the same time, so that a specific amplification primer and a sequencing primer aiming at the specific mutation site are needed, the mutant gene is further sequenced by the primers, and the detection method is applied to treatment of 46, XY reverse 3 type.

In view of the above, there is an urgent need for an application, a reagent and a kit of NR5A1 mutant gene as a detection target.

Disclosure of Invention

The invention mainly aims to provide an application, a reagent and a kit of an NR5A1 mutant gene as a detection target spot, so as to solve the technical problem that qualitative, quantitative and definite mutant gene sequences cannot be simultaneously satisfied in the prior art.

In order to achieve the above purpose, the invention provides an application of an NR5A1 mutant gene serving as a detection target in preparing a reagent for detecting 46, XY reversed 3 type and/or a kit for detecting 46, XY reversed 3 type, wherein the sequence of the NR5A1 mutant gene is shown as SEQ ID NO.45, and the 870+1 base of the 4 th exon is A.

The NR5A1 mutant gene is used as a detection target spot for preparing a reagent or a preparation kit, so that the variety of detection means for 46, XY reverse 3 type is enriched, 46, XY reverse 3 type caused by the NR5A1 mutant gene can be directly detected through the reagent or the kit, the detection time is shortened, a doctor or a patient can know the condition in time conveniently, and the subsequent treatment can be carried out pertinently.

Preferably, the reagent and/or kit comprises amplification primers comprising an upstream primer NR5A1-1F with a nucleotide sequence shown as SEQ ID NO.1 and a downstream primer NR5A1-1R with a nucleotide sequence shown as SEQ ID NO. 2.

The amplification primer provided by the invention can specifically amplify NR5A1 mutant genes and wild NR5A1 genes, can be matched with other sequencing primers to realize the detection of the nucleotide sequence of the target genes, and is used for detecting 46, XY inversion type 3.

Preferably, the reagent and/or kit comprises a sequencing primer, wherein the sequencing primer comprises an upstream primer NR5A1-SEQ1F with a nucleotide sequence shown as SEQ ID NO.3 and a downstream primer NR5A1-SEQ1R with a nucleotide sequence shown as SEQ ID NO. 4.

The sequencing primer provided by the invention can specifically detect NR5A1 mutant genes, thereby detecting whether 46, XY inversion type 3 exists.

The invention also provides a reagent for detecting 46, XY reversed 3 type, the reagent comprises an NR5A1 mutant gene, the sequence of the NR5A1 mutant gene is shown as SEQ ID NO.45, wherein the 870+1 base of the 4 th exon is A.

The invention also provides a kit for detecting 46, XY reversed type 3, which comprises the reagent.

Preferably, the kit comprises one or more of a preventive 46, xy reversed 3 type kit, a diagnostic or auxiliary diagnostic 46, xy reversed 3 type kit, a pre-pregnancy and/or pre-prenatal genetic disease screening kit, a pre-pregnancy and/or pre-prenatal genetic disease diagnostic kit, and an auxiliary therapeutic 46, xy reversed 3 type kit.

The 46, XY reversed 3 type caused by the gene mutation can be interfered and treated in different disease periods through different types of kits.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to the structures shown in these drawings without inventive effort for a person skilled in the art.

FIG. 1 is a genetic map of line 1, type 3 with XY inverted polarity, according to an embodiment of the invention, 46; wherein ∈r represents a normal male individual, ∈r represents a normal female individual, ∈r represents a patient, ∈r represents a fetus, and ↗ represents a forerunner;

FIG. 2 is a graph showing the results of detection of genotype of the No.1 family NR5A1: NM-004959.5: exon4: c.870+1G > A locus using Sanger sequencing in an embodiment of the present invention; wherein, layer C: heterozygote mutation in family 1; A. b and D layers: genotype in line 1 is wild type (position of mutation indicated by arrow in sequencing diagram);

FIG. 3 is a genetic map of line 2, type 3 with XY inverted polarity, 46, according to one embodiment of the invention; wherein ∈Σ represents a normal male individual, ∈o represents a normal female individual, +.t represents a patient, ↗ represents a forerunner;

FIG. 4 is a diagram showing the result of detecting genotype of the NM-004959.5:exo n4:c.870+1G>A locus of family No.2 NR5A1 by using the kit according to an embodiment of the present invention; wherein, B: genotype in family 2 is heterozygous mutant patient; layers a and C: genotype in line No.2 is wild type (position of mutation indicated by arrow in sequencing).

The achievement of the object, functional features and advantages of the present invention will be further described with reference to the drawings in connection with the embodiments.

Detailed Description

The following description of the embodiments of the present invention will be made clearly and fully with reference to the accompanying drawings, in which it is evident that the embodiments described are only some, but not all embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the present invention without making any inventive effort, are intended to fall within the scope of the present invention.

It should be noted that all directional indicators (such as upper and lower … …) in the embodiments of the present invention are merely used to explain the relative positional relationship, movement conditions, etc. between the components in a specific posture, and if the specific posture is changed, the directional indicators are changed accordingly.

Furthermore, descriptions such as those referred to as "first," "second," and the like, are provided for descriptive purposes only and are not to be construed as indicating or implying a relative importance or implying an order of magnitude of the indicated technical features in the present disclosure. Thus, a feature defining "a first" or "a second" may explicitly or implicitly include at least one such feature.

Moreover, the technical solutions of the embodiments of the present invention may be combined with each other, but it is necessary to be based on the fact that those skilled in the art can implement the embodiments, and when the technical solutions are contradictory or cannot be implemented, it should be considered that the combination of the technical solutions does not exist, and is not within the scope of protection claimed by the present invention.

In the present invention, the term "autosomal dominant inheritance" means that whenever a pathogenic gene is present on one of parents' autosomes and transmitted to children, whether or not another gene of children is normal, it may cause a disease regardless of sex. The patients with family history of genetic diseases need to be checked before pregnancy to avoid the influence of the genetic diseases on the next generation.

In the present invention, the term "heterozygous mutation" means that the mutation exists in only one gene of a pair of alleles.

In the present invention, the term "homozygous mutation" means that the same mutation occurs in all alleles, that is, a double allelic mutation, and each chromosome is mutated.

In the present invention, the term "splice mutation" refers to a mutation occurring in a conserved sequence flanking a splice donor, acceptor recognition site, or both, such that the normal splice site disappears or the splice site is newly created, and the manner of splicing the RNA precursor is altered such that the resulting mature RNA contains introns or deleted exon sequences.

The term "diagnosis" herein includes prediction of disease risk, diagnosis of the onset or absence of a disease, and also the assessment of disease prognosis.

The term "prenatal diagnosis" herein refers to definitive diagnosis of a high-risk fetus based on genetic counseling, mainly through genetic detection and imaging examination, and achieves the purpose of fetal selection through selective abortion of a diseased fetus, thereby reducing birth defect rate and improving prenatal quality and population quality.

In the present invention, a "primer" refers to a polynucleotide fragment, typically an oligonucleotide, containing at least 5 bases, such as 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more bases, for amplifying a target nucleic acid in a PCR reaction. The primer need not be completely complementary to the target gene to be amplified or its complementary strand, as long as it can specifically amplify the target gene.

The invention provides application of an NR5A1 mutant gene serving as a detection target in preparation of a reagent for detecting 46, XY inversion type 3 and/or in preparation of a kit for detecting 46, XY inversion type 3, wherein the sequence of the NR5A1 mutant gene is shown as SEQ ID NO.45, and the 870+1 base of the 4 th exon is A. The NR5A1 mutant gene comprises a sequence shown as SEQ ID NO.5 # -, and the like) The nucleotide sequences shown (the letters in the boxes are the bases after mutation, the capital letters are the exon sequences, and the lowercase letters are the intron sequences). The wild NR5A1 gene has an ID number of NM_004959.5 and a sequence shown as SEQ ID No. 43. The NR5A1 mutant gene causes shearing abnormality, the ID number of the original wild type NR5A1 gene protein is NP-004950.2, the amino acid sequence is shown as SEQ ID No.44, the mutated NR5A1 mutant gene protein is shown as SEQ ID No.46, the normal expression of normal NR5A1 is influenced, 46 is caused, XY inversion type 3 is caused, and the mutant has pathogenicity, specifically, NR5A1: NM-004959.5: exon4: c.870+1G>A。

The NR5A1 mutant gene provided by the invention is a 46, XY reverse 3-type pathogenic gene, and whether the mutant gene has 46, XY reverse 3-type disease can be judged by detecting the NR5A1 mutant gene, so that the existing detection means for detecting whether the mutant gene has 46, XY reverse 3-type disease is enriched, and early intervention and treatment, genetic diagnosis before embryo implantation and prenatal and postnatal care are conveniently and timely carried out.

In some embodiments, the reagents and/or kits comprise amplification primers comprising an upstream primer NR5A1-1F having a nucleotide sequence shown as SEQ ID NO.1 and a downstream primer NR5A1-1R having a nucleotide sequence shown as SEQ ID NO. 2;

the nucleotide sequences of SEQ ID NO. 1-2 are specifically as follows:

SEQ ID NO.1：5'-TGGCTGGCTACCTCTACC-3'；

SEQ ID NO.2：5'-CCCCAAACTCTAAAACACTAAG-3'。

In some embodiments, the reagents and/or kits comprise sequencing primers comprising an upstream primer NR5A1-SEQ1F having a nucleotide sequence shown as SEQ ID NO.3 and a downstream primer NR5A1-SEQ1R having a nucleotide sequence shown as SEQ ID NO. 4;

the nucleotide sequences of SEQ ID NO. 3-4 are specifically as follows:

SEQ ID NO.3：5'-CCGTGCCATCAAGTCTGA-3'；

SEQ ID NO.4：5'-GCTGTGATAAGTGGAAATGCT-3'。

The invention also provides a reagent for detecting 46, XY reversed 3 type, the reagent comprises an NR5A1 mutant gene, the sequence of the NR5A1 mutant gene is shown as SEQ ID NO.45, wherein the 870+1 base of the 4 th exon is A. The reagent comprises the amplification primer and the sequencing primer, and further comprises dNTP, PCR buffer solution, magnesium ions and Tap polymerase; the PCR buffer comprises KCl (preferably at a concentration of 50 mmol/L), tris-HCl (preferably at a concentration of 10mmol/L, pH preferably at 8.3) and MgCl ₂ (the concentration is preferably 1.5 mmol/L);

the invention also provides a method for identifying the NR5A1 mutant gene, which comprises the following steps:

taking the DNA of the sample to be detected as a template, and carrying out PCR amplification by using the amplification primer to obtain an amplification product; in the present invention, the PCR amplification reactionThe system preferably comprises, in 20. Mu.L of 10 XPCR buffer 2. Mu. L, dNTPs 0.4, 0.4. Mu. L, NR5A 1-1F0.5. Mu. L, NR5A1-1R 0.5. Mu.L, 100 ng/. Mu.L of template (sample DNA to be tested) 1. Mu. L, taq enzyme 0.2. Mu.L and the balance ddH ₂ O; in the present invention, when the amplification primers in the reaction system of the PCR amplification are the upstream primer NR5A1-1F and the downstream primer NR5A1-1R, the reaction progress of the PCR amplification preferably comprises: pre-denaturation at 95 ℃ for 5min;30 cycles (denaturation at 95℃for 30s, annealing at 53℃for 30s, extension at 72℃for 60 s); the reaction was carried out at 72℃for 7min.

Sequencing the amplified product by using the sequencing primer to determine the genotype of the NR5A1 mutant gene.

The invention also provides a kit for detecting 46, XY reversed 3 type, the kit comprising the above-described reagents, the kit comprising one or more of a preventive 46, XY reversed 3 type kit, a diagnostic or auxiliary diagnostic 46, an XY reversed 3 type kit, a pre-pregnancy and/or pre-prenatal genetic disease screening kit, a pre-pregnancy and/or pre-prenatal genetic disease diagnostic kit, and an auxiliary therapeutic 46, XY reversed 3 type kit.

In some embodiments, the kit comprises a positive mutation reference DNA with a nucleotide sequence shown as SEQ ID NO.6, and the sequencing result of the kit is compared with the positive mutation reference DNA, and if the sequencing result is identical with the sequence base of the positive mutation reference DNA, the sequence base is used for indicating that the c.870+1G > A mutation occurs.

The kit of the invention diagnoses whether the individual suffers from 46, XY inversion type 3 or not through the genotype of NR5A1 gene in the detection sample of the male individual and/or the female individual; the test sample preferably comprises blood or tissue. The criteria for genotyping individuals with 46, xy reversed type 3, are specifically:

when 46, the genotype of the XY individual NR5A1: c.870+1G > A is "c.870+1G > A heterozygote mutation", the patient is;

when 46, the genotype of the XY individual NR5A1: c.870+1G > A is "c.870+1G > A homozygote mutation", the patient is;

when 46, the genotype of XY subject NR5A1 is "wild type", the subject is normal.

The invention prepares the reagent containing the amplification primer and the sequencing primer on the basis of the amplification primer and the sequencing primer, and successfully prepares different types of kits by using the reagent, including one or more of a 46-preventive, XY reversed 3-type kit, a 46-diagnostic or auxiliary-diagnostic, a 46-XY reversed 3-type kit, a pre-pregnancy and/or pre-prenatal genetic disease screening kit, a pre-pregnancy and/or pre-prenatal genetic disease diagnosis kit and an auxiliary-therapeutic 46, and the 46-XY reversed 3-type kit caused by the mutation of the gene can be intervened and treated in different disease periods through the different types of kits.

In the specific implementation process, 207 individuals in 55 personality dysplasia families are screened and detected, wherein 17 individuals of 46, XY inversion type 3 patients are 'c.870+1G > A mutation'.

For further explanation of the present invention, the detection of NR5A1 mutant gene of type 46, XY inverted 3, primers, reagents and applications provided by the present invention will be described in detail with reference to the accompanying drawings and examples, and for explanation of the correlation of NR5A1 mutant gene of the present invention with type 46, XY inverted 3, 2 families for random selection screening and detection will be described, but they should not be construed as limiting the scope of the present invention.

The experimental procedure, which does not address the specific conditions in the examples below, is generally followed by routine conditions such as Sambrook et al, molecular cloning: conditions described in the laboratory Manual (New York: cold Spring Harbor LaboratoryPress, 2014), or as recommended by the manufacturer.

Example 1

1. Diagnostic criteria:

reference is made to the 2010 edition of human monogenic genetic diseases and the 2019 edition of the diagnosis and treatment guidelines for rare diseases.

The NR5A1 gene mutation is related to X linkage inheritance 46, XY sex reversal type 3, clinical manifestations are complex and various, external genitalia can be expressed as complete feminization to complete maleinization, wherein vulva is difficult to distinguish most frequently, female external genitalia accompanied with clitoral hypertrophy, complete feminization of external genitalia, complete maleinization of hypourethrosis and the like also occur. Some patients do not have Miaole tube structure, some patients have gonads which are testes, most of them are bilateral, and histoscopy often shows dysplastic testes, lack of spermatogenic cells or fibrosis and the like.

2. Object of detection

The detection is carried out by taking 1 family (called 1 family for short) with the XY reversed sex of type 3 as a tested object, the clinical information of the members of the family part 1 is shown in a table 1, and the family map is shown in figure 1.

TABLE 1 clinical information of XY Reversal No.3 family members

Note that: i and II represent the first generation and the second generation in sequence, and the 1 st family personnel I:1, I: 2, II: 1 peripheral blood DNA and II: 2 amniotic fluid DNA are used for sequencing.

Example 2

Exon sequencing

1. The instrument is shown in table 2.

Table 2 instrumentation

2. Reagent consumable

Human whole exon sequencing kit (Agilent), DNA 1000 kit (Agilent), 96 well plate (Axygen), different model tips (Axygen), 200 μl centrifuge tube (Eppendorf), 1.5mL centrifuge tube (Eppendorf), capillary electrophoresis buffer (Thermo), sequencing standard (Thermo), absolute ethanol (Thermo), bigDye Terminator V3.1.3.1 (Thermo), peripheral blood gDNA extraction kit (TIANGEN), agarose (TIANGEN) and EB dye solution (amerco).

3. Reagent formulation

1) A5 XTBE stock solution of electrophoresis liquid was prepared in accordance with Table 3.

Table 3 5 XTBE electrophoresis liquid formula

2) 0.5 XTBE working solution was run on ddH ₂ O dilution of the 5 XTBE stock solution in Table 3 was performed 10 times.

3) 10 Xerythrocyte lysate was prepared according to Table 4.

TABLE 4 10 Xerythrocyte lysate formula

4) The 1 x nuclear lysate formulation was formulated according to table 5.

Table 5 1 XNuclear lysate formula

Reagent(s)	2M Tris-HCl，pH8.2	4M NaCl	2mM EDTA
				Volume/weight	0.5mL	10mL	0.4mL

4. Experimental procedure

After signing informed consent, collecting 3-5 mL of peripheral blood of I1, I2 and II 1 in the family 1 and 10-20mL of amniotic fluid of II 2 members as study samples.

4.1 sample DNA extraction

1) Filling 3-5 mL of sample into a 15mL centrifuge tube, adding 2-3 times of 1 Xerythrocyte lysate, uniformly mixing, and standing on ice for 30 minutes until the solution becomes transparent; if the villus tissue is adopted, the step 2) is directly carried out.

2) Centrifuge at 3000rpm for 10 min at 4℃and carefully remove the supernatant. 1mL of 1 Xcell nucleus lysate was added to the pellet, mixed well, and 2mL of 1 Xcell nucleus lysate and 150. Mu.L of 20% SDS were added thereto, and shaken well until a viscous transparent state appeared. Add 10. Mu.L of 20mg/mL proteinase K and shake well. Digestion is performed at 37℃for more than 6 hours or overnight.

3) Adding saturated phenol with equal volume, mixing by light shaking, and centrifuging at 3000rpm for 10 minutes at room temperature.

4) The supernatant was carefully transferred to another centrifuge tube, and an equal volume of a phenol/chloroform mixture (phenol/chloroform volume ratio 1:1) was added and mixed well and centrifuged at 3000rpm for 10 minutes at room temperature.

5) The supernatant was carefully removed and if not clear, extracted once more with an equal volume of chloroform.

6) Transferring the supernatant into another centrifuge tube, adding diploid absolute ethanol, shaking, and obtaining white flocculent DNA. The DNA was hooked with a flame sterilized glass crochet, washed twice with 70% ethanol, dried at room temperature for 5 minutes, and then dissolved in 200. Mu.L of 1 XTE and drum-dissolved overnight. OD was measured by uv.

7) The TE-dissolved DNA can be preserved for one year at 4deg.C, and if long-term preservation is required, 2 times volume of absolute ethanol is added for preservation at-70deg.C.

4.2 exon sequencing

1) Taking 2 mug DNA, mechanically breaking to ensure that the fragment size is about 200bp, cutting glue, and recovering 150-250 bp fragments;

2) DNA fragment is used for terminal repair and A is added to the 3' -terminal;

3) Connecting sequencing joints, purifying the connection products, performing PCR amplification, and purifying the amplified products;

4) Adding the purified amplification product into an Agilent kit probe for hybridization capture, eluting and recovering the hybridization product, performing PCR amplification, recovering the final product, and performing quality control analysis by agarose gel electrophoresis on a small sample;

5) NextSeq500 sequencer sequencing and data analysis.

4.3 results

Finally obtaining the gene mutation NR5A1 with pathogenic significance, NM_004959.5:exon4:c.870+1G > A; wherein c.870+1G > A is represented by mutation of the 870+1 th base near the 4 th exon (1 st base in the 4 th intron) of the NR5A1 gene from G to A, resulting in a shearing abnormality.

Genotype of the NR5A1: NM-004959.5: exon4: c.870+1G > A locus in line 46, XY reversed 3 patient individuals is "c.870+1G > A heterozygous mutation", and the locus genotype is "wild type" in normal line 1 individuals.

Example 3

Sanger sequencing validation

The results of the family 1 exome sequencing were further verified for the NR5A1: NM-004959.5: exon4: c.870+1G > A sites using Sanger sequencing. Genotype detection was performed on4 persons (forerunner, forerunner father, forerunner mother, fetus) from line 1 and 100 normal persons outside line in example 1, respectively, at the NR5A1: NM-004959.5: exon4: c.870+1G > A locus.

The specific method comprises the following steps:

DNA extraction

Genomic DNA was extracted according to the method of example 2.

2. Candidate primer design, verification and preference

2.1 candidate primer design references the human genome sequence database hg19/build36.3 (https:// www.ncbi.nlm.nih.gov/genome, or http:// genome. Ucsc. Edu/cgi-bin/hgGateway.

2.2 designing 19 pairs of candidate primers for mutation site c870+1G > A (see Table 6), and verifying and evaluating the merits of each pair of candidate primers by PCR experiments

TABLE 6 basic conditions and verification experiment results for c870+1G > A site candidate primers

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Note that: after electrophoresis, the normal PCR amplification result has only one specific band, and if the primer dimer band and the non-specific product band are all the results of abnormal reaction of the primer; the target primers avoid such primers as much as possible.

2.3 candidate primer PCR verification reaction

PCR was performed according to the reaction system in Table 7 and the reaction system was kept on ice; each pair of primers was provided with 8 reaction test tubes (SEQ ID NOS 1 to 8 in Table 7).

TABLE 7 primer detection PCR reaction System

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Reaction conditions: the test reaction tube was placed in a PCR instrument and the following reaction procedure was performed:

the first step: pre-denaturation at 95 ℃ for 5min;

and a second step of: 30 cycles (denaturation at 95℃for 30 sec. Fwdarw. Tm annealing for 30 sec. Fwdarw. 72℃for 60 sec); (PCR amplification parameters were set according to the Tm values of the primers in Table 6).

And a third step of: extending at 72 ℃ for 7min;

fourth step: 4℃until sampling.

2.4 candidate primer PCR results agarose gel electrophoresis detection was performed to evaluate the effectiveness, specificity of the primer reactions:

1) Sealing the two ends of the gel sampler with adhesive tape, placing on a horizontal table, and placing a comb at about 1cm position at one end of the sampler.

2) Weighing 2g of agar powder in a conical flask, adding 100mL of 0.5 XTBE electrophoresis buffer, shaking uniformly, heating on a microwave oven or an electric furnace (adding asbestos gauze), taking out after boiling, shaking uniformly, reheating until the gel is completely melted, taking out and cooling at room temperature.

3) After the gel is cooled to about 50 ℃, pouring the gel into a sealed gel sampler to enable the thickness to be about 5 mm.

4) Gel is solidified and the adhesive tape is removed, and the gel and the sampler are put into an electrophoresis tank together.

5) Adding electrophoresis buffer solution to make the liquid level 1-2 mm higher than the rubber surface, and pulling out the comb upwards; and (3) uniformly mixing the sample and the DNA size standard substance with the sample loading liquid by using a micropipette, and adding the mixture into each sample loading hole, wherein the DNA is sunk into the hole bottom due to the fact that the sucrose in the sample loading liquid has a larger specific gravity.

6) And (5) covering an electrophoresis tank, switching on a power supply, adjusting to a proper voltage, and starting electrophoresis. And judging the approximate position of the sample according to the indication of bromophenol blue in the sample carrying liquid, and determining whether to terminate electrophoresis.

7) The power supply is cut off, the gel is taken out, and the gel is put into an EB water solution with the concentration of 0.5g/mL for dyeing for 10 to 15 minutes.

8) The gel was observed under a transmissive ultraviolet irradiator at 254nm and the electrophoresis results were recorded either with a camera with a red filter or with a gel scanning system.

2.5 evaluation of results:

1) If the tube No. 7 only has a bright band and no band, judging that the pair of primers and a reaction system are good in effectiveness and strong in specificity;

2) If no target band appears in the tube 7, judging that the pair of primers and the reaction system are invalid;

3) If the No. 7 tube has a primer dimer band outside the target band and also has a primer dimer band in the No.2, 3, 4, 5 and 6 partial tubes, judging that the effectiveness of the pair of primers and the reaction system is poor;

4) If the No. 7 tube has a nonspecific band outside the target band and also has a nonspecific band in the No.5 and 6 partial tubes, judging that the specificity of the pair of primers and the reaction system is poor;

5) If primer dimer and non-specific band outside the target band appear in the tube No. 7, and primer dimer and non-specific band also appear in the tube No.2, 3, 4, 5, 6, the effectiveness and specificity of the pair of primers and the reaction system are judged to be poor.

2.6 based on the results of the statistics after the validation test of Table 7, SEQ ID NO.1 and SEQ ID NO.2 of Table 6 were selected as amplification primers for the NR5A1: NM-004959.5: exon4: c.870+1G > A site.

NR5A1-1F：5’-TGGCTGGCTACCTCTACC-3’(SEQ ID NO.1)

NR5A1-1R：5’-CCCCAAACTCTAAAACACTAAG-3’(SEQ ID NO.2)

3. PCR amplification of mutation sites by primers screened in step 2.6 for family 1 personnel and 100 off-family personnel

PCR was performed according to the reaction system in Table 8 and the reaction system was kept on ice.

TABLE 8 mutation point PCR reaction system

Reaction conditions: the reaction system was put into a PCR instrument, and the following reaction procedure was performed:

PCR amplification procedure for NR5A1: NM-004959.5: exon4: c.870+1G > A site was as follows: the first step: 95 ℃ for 5 minutes; and a second step of: 30 cycles (95 ℃,30 seconds- > 53 ℃,30 seconds- > 72 ℃,60 seconds); and a third step of: 72 ℃,7 minutes; fourth step: 4℃until sampling.

4. Agarose gel electrophoresis detection

Refer to step 2.4 above.

5. Purifying a PCR product by an enzymolysis method: to 5. Mu.L of the PCR product, 0.5. Mu.L of exonuclease I (Exo I), 1. Mu.L of alkaline phosphatase (AIP) was added, and the mixture was digested at 37℃for 15min and inactivated at 85℃for 15min.

6. BigDye reaction

The BigDye reaction system is shown in Table 9.

TABLE 9 BigDye reaction System

Sequencing PCR cycling conditions:

the first step: pre-denaturation at 96℃for 1min;

and a second step of: 33 cycles (denaturation at 96℃for 30 sec. Fwdarw. Annealing at 55℃for 15 sec. Fwdarw. 60℃for 4 min);

and a third step of: 4℃until sampling.

7. And (3) purifying a BigDye reaction product:

1) mu.L of 125mM EDTA (pH 8.0) was added to each tube, and 1. Mu.L of 3mol/L NaAc (pH 5.2) was added to the bottom of the tube;

2) Adding 70 μL 70% alcohol, shaking and mixing for 4 times, and standing at room temperature for 15min;

3) 3000g, centrifuging at 4 ℃ for 30min; immediately inverting the 96-well plate, and centrifuging 185g for 1min;

4) Standing at room temperature for 5min, volatilizing residual alcohol at room temperature, adding 10 μl Hi-Di formamide to dissolve DNA, denaturing at 96 deg.C for 4min, rapidly placing on ice for 4min, and sequencing on machine.

8. Sequencing

And (3) carrying out DNA sequencing on the purified BigDye reaction product, wherein sequencing primers are designed on the basis of SEQ ID NO.1 and SEQ ID NO.2, and nest primers (the second set of primers are designed within the range of the product sequence obtained by amplifying the first set of primers) are used as sequencing primers, and the primer sequences are shown as follows.

Sequencing primer sequences for the NR5A1: NM-004959.5: exon4: c.870+1G > A site are as follows:

NR5A1-Seq1F：5’-CCGTGCCATCAAGTCTGA-3’(SEQ ID NO.3)

NR5A1-Seq1R：5’-GCTGTGATAAGTGGAAATGCT-3’(SEQ ID NO.4)

9. analysis of results

The sequencing results for the NR5A1: NM-004959.5: exon4: c.870+1G > A sites are shown in FIG. 2. From FIG. 2, it can be seen that the genotype of the c.870+1G > A locus of the individual patient of type 3 with XY polarity inversion in line 1 is a "c.870+1G > A" heterozygous mutation; the genotype of the c.870+1G > A locus of normal individuals in line 1 and 100 non-blood related normal controls is "wild type".

Example 4

46, XY reverse 3 type diagnostic kit and application

1. The kit comprises the following components:

the kit comprises: 1) Amplification primers: SEQ ID NOS.1 to 2 as in example 3; 2) PCR buffer (10 XPCR buffer, consisting of 500mmol/L KCl,100mmol/L Tris-HCl (pH 8.3), 15mmol/L MgCl) ₂ And the balance water); 3) Taq enzyme (20U); 4) dNTPs (4 mM each of the four dNTPs); 5) c.870+1G>A positive mutant reference DNA, which is a double-stranded DNA, c.870+1G>The specific sequence of the A positive mutation reference is shown in SEQ ID NO.6, and specifically comprises the following steps:

single underlined bases are positions of an upstream primer and a downstream primer of PCR amplification, bases in a square frame are mutation sites, double underlined bases are positions of an upstream sequencing primer and a downstream sequencing primer, capital letters are exon sequences, and lowercase letters are intron sequences; 6) Sequencing primer: as shown in SEQ ID NO. 3-4.

2. The using method comprises the following steps:

207 individuals in 55 personally dysplastic families were screened and detected altogether, and 16 families (16 patients with XY reversed 3 type, see Table 10) conforming to the invention were found again, and the application of the gene mutation detection kit is now described by taking family No.2 as an example. The clinical information of family 2 is shown in table 11, and the family 2 map is shown in fig. 3.

Table 10, 46, XY reverse 3 screening Profile List

TABLE 11 clinical information of XY Reversal No.3 family members

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Note that: i and II sequentially represent a first generation and a second generation.

The peripheral blood DNA of family personnel No. 2I 1, I2 and II 1 is used for the detection of the kit, and the steps are as follows:

1) Genomic DNA extraction: sample genomic DNA was extracted according to the procedure of example 2.

2) Firstly, adopting PCR amplification primers, taq enzyme, buffer solution, dNTPs, sample genome DNA and the like in a kit to carry out PCR amplification reaction;

3) Purifying the PCR amplification product;

4) Performing BigDye reaction on the purified PCR product by using the sequencing primer in the kit;

5) Purifying the BiyDye reaction product;

6) The biydiye reaction products were sequenced and the sequenced sequences were compared to the normal sequences.

The detection result of the kit for the family member No.2 is shown in FIG. 4, wherein the arrow indicates the mutation occurrence position. As can be seen from FIG. 4, the genotype of the c.870+1G > A locus of the precursor in the family No.2 is "c.870+1G > A" heterozygous mutation (B layer), and the detection result confirms that the precursor is 46, the XY reversed 3 type patient, the parent genotype of the precursor (A and C layers) is wild type, and the normal individual. Considering that the parents of the pre-identified individuals will later develop 46, the probability of xy reversed type 3 patients is low, but it is still recommended that later birth be continued if necessary, and prenatal diagnosis be performed in the hospital.

Example 5

Gene mutation ranking and interpretation (pathogenicity of mutation)

Mutation interpretation is based on our current understanding of 46, XY reversed 3 type and pathogenic gene NR5A1 (https:// www.omim.org/entry/612965), and the clinical phenotypic association of the test subjects. Mutations follow the HGVS guidelines for mutation nomenclature (http:// www.hgvs.org /), and are named according to GenBank accession numbers (https:// www.ncbi.nlm.nih.gov/GenBank /). The rules for interpretation of genetic variation data refer to guidelines associated with the american society of medical genetics and genomics (American College of Medical Genetics and Genomics, ACMG): richards, 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. Genet Med, advance online publication 5 March 2015.doi:10.1038/gim.2015.30; chinese classification standards and guidelines for genetic variation: wang Qiuju, shen Yiping, ling KV, et al, classification standards and guidelines for genetic variation: life sciences, 2017, 47:668-688.

the genetic variation classification in the "genetic variation classification criteria and guidelines" is to perform five-level classification on variations based on typical data types (such as crowd data, calculation data, functional data, co-segregation data), which are respectively: "pathogenic (P)", "potentially pathogenic (likely pathogenic, LP)", "ambiguous (variant of uncertain significance, VUS)", "potentially benign (LB)", and "benign (B)"; the five-level classification was determined based on the composite score after interpretation analysis of each side/sub-item of variation (table 12).

TABLE 12 determination criteria for pathogenicity of variation

Before a five-level assessment, the sides/sub-items of the mutation/variation need to be analyzed/interpreted. Among these, the pathogenic mutation criteria can be classified as: for a given mutation/mutation, first it is necessary to select the criteria in Table 13 based on observed evidence, determine which of Table 13 can be met by the sides/sub-items of the mutation/mutation, each of which is rated as PVS1/PS 1-4/PM 1-6/PP 1-5/BA 1/BS 1-4/BP 1-6, and finally combine the sub-item criteria that can be met according to the rating rules of Table 12, then select a classification from the five-level system according to the combined criteria of Table 12, for example if the sides/sub-items of the mutation/mutation are analyzed to have the criteria of PVS1, PM 1) +PbP 1.2 in Table 13, then it is rated as PM 1-4/PM 1-6/PP 1-5/BA 1/BS 1-4/BP 1-6, and finally it is rated as PM (i.e.p) according to the combined criteria of Table 12.

TABLE 13 variant interpretation criteria and variant pathogenicity criterion

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Analysis/interpretation of the sides/sub-items of mutations/variations needs to be based on the corresponding bioinformatic analysis tools (see table 15) and a number of available data (libraries) (see table 16), including data obtained from existing cases, as well as data that has been published, such as public databases (e.g., clinVar or site-specific databases) and laboratory owned databases. The degree judgment evaluation criteria used in the analysis of mutation/mutation using various data (libraries) are shown in table 14.

Table 14 degree judgment evaluation criteria

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Table 15 biological information analysis tool

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Table 16 crowd database, disease-specific database and sequence database

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According to the above criteria or guidelines, the NR5A1 gene c.870+1G > A mutation in the present invention is assessed as "pathogenic", judging criteria and specific evidence are shown in Table 17 below:

TABLE 17 pathogenic interpretation of NR5A1 gene c.870+1g > A mutation

AD: refers to autosomal dominant inheritance.

The evidence of variation ratings for NR5A1: NM-004959.5: exon4: c.870+1G > A is as follows:

1. PVS1: the mutation of the NR5A1 gene c.870+1G > A occurs in the splicing region, namely, the nucleotide position G of the 870+1 intron region is mutated into A, the site is a classical cleavage site, and the mutation affects the function of the protein;

2. PS2: through family personnel detection such as parents of a forerunner, the variation is a new variation (see table 10) verified by 17 family analysis, namely the variation is a new variation confirmed by the relationship;

3. PS4: combining literature and this case, this variation was detected in multiple patients (see table 10);

4. PM2: the NR5A1 gene c.870+1g > a variation was not found in the reference human thousand genome (1000G), the human exon database (ExAC) and the human genome mutation frequency database (gnomAD);

5. PP3: various computer software predicts that this variation will have deleterious effects on the gene or gene product;

thus, the comprehensive evidence of this mutation/variation (pvs1+ps2+ps4+pm2+pp3) meets the "pathogenicity (P)" criteria (i) in (a) and (c), or (ii) in table 12, where the NR5A1 gene c.870+1g > a variation is comprehensively determined to be "pathogenicity".

Example 6

Follow-up and diagnostic kit detection performance analysis

All family members were followed and re-sequenced analysis was performed on all individuals using the NR5A1 gene targeted capture chip method (see Table 18).

TABLE 18 c.870+1G > A site detection Performance analysis results

Index (I)	Formula (VI)	Results
			True positive mutation site (TP)	/	17
True negative mutation site (TN)	/	45
			False positive mutation site (FP)	/	0
False negative mutation site (FN)	/	0
			Sensitivity (n=17)	TP/(TP+FN)	100％(95％CI:99.03％～100％)
Specificity (N=45)	TN/(TN+FP)	100％(95％CI:99.03％～100％)
			Positive predictive value	TP/(TP+FP)	100％(95％CI:99.03％～100％)
Negative predictive value	TN/(TN+FN)	100％(95％CI:99.03％～100％)

Note that: the table contains follow-up data for family 1.

From tables 1 and 10, it can be seen that positive patients (17 cases) were found when 17 families were examined. The positive site detection results are verified by an NR5A1 gene targeting capture chip method. According to the follow-up and verification results, 17 true positive cases, 45 true negative cases, 0 false negative cases and 0 false positive cases are found in total. The sensitivity of detection of the 870+1G > A mutation site marker is 100.00%, the 95% CI (namely 95% confidence interval) is 99.03% -100%, the specificity is 100%, and the 95% CI is 99.03% -100%. The results show that the kit has good detection performance in clinical application.

From the above examples, it can be seen that the NR5A1 mutant gene of the present invention can be used as a biomarker for diagnosing 46, XY reversed 3 type, and provides a possible drug target for treating 46, XY reversed 3 type.

In the above technical solution of the present invention, the above is only a preferred embodiment of the present invention, and therefore, the patent scope of the present invention is not limited thereto, and all the equivalent structural changes made by the description of the present invention and the content of the accompanying drawings or the direct/indirect application in other related technical fields are included in the patent protection scope of the present invention.

Claims

1. The application of the NR5A1 mutant gene serving as a detection target in preparing a reagent for detecting 46, XY reversed 3 type and/or a kit for detecting 46, XY reversed 3 type is characterized in that the sequence of the NR5A1 mutant gene is shown as SEQ ID NO.45, wherein the 870+1 base of the 4 th exon is A.

2. The use according to claim 1, wherein the reagents and/or kits comprise amplification primers comprising an upstream primer NR5A1-1F having the nucleotide sequence shown in SEQ ID NO.1 and a downstream primer NR5A1-1R having the nucleotide sequence shown in SEQ ID NO. 2.

3. The use according to claim 1, wherein the reagent and/or kit comprises a sequencing primer comprising an upstream primer NR5A1-SEQ1F having the nucleotide sequence shown in SEQ ID NO.3 and a downstream primer NR5A1-SEQ1R having the nucleotide sequence shown in SEQ ID NO. 4.

4. A reagent for detecting 46, XY reversed type 3, characterized in that the reagent comprises an NR5A1 mutant gene, the sequence of the NR5A1 mutant gene is shown as SEQ ID NO.45, wherein the 870+1 base of the 4 th exon is A.

5. A kit for detecting 46, xy reversal type 3, characterized in that the kit comprises the reagent of claim 4.