CN115851898B

CN115851898B - Application of BPES syndrome pathogenic gene FOXL2 mutation site and diagnostic reagent thereof

Info

Publication number: CN115851898B
Application number: CN202210822994.XA
Authority: CN
Inventors: 曾桥; 刘鑫林; 薛斌
Original assignee: Hunan Jiahui Biotechnology Co Ltd
Current assignee: Hunan Jiahui Biotechnology Co Ltd
Priority date: 2022-07-13
Filing date: 2022-07-13
Publication date: 2024-04-05
Anticipated expiration: 2042-07-13
Also published as: CN115851898A

Abstract

The invention provides application of a BPES syndrome pathogenic gene FOXL2 mutation site and a diagnostic reagent thereof, and belongs to the technical field of gene diagnosis. The invention discovers for the first time that the mutation of FOXL2: NM_023067.3: exon1: c.316_321dupCACAAC: p.His106_Asn107dup site can cause BPES syndrome. The gene FOXL2 mutation site is applied to preparation of a BPES syndrome diagnostic reagent or preparation of a medicament for preventing and/or treating BPES syndrome target spots. The detection reagent aiming at the mutation site can be used for rapidly and effectively predicting or diagnosing the BPES syndrome, lays an important foundation for researching pathogenesis of the BPES syndrome, provides a brand-new theoretical basis for treating patients with the BPES syndrome, and also provides a possible drug target for treating the BPES syndrome.

Description

Application of BPES syndrome pathogenic gene FOXL2 mutation site and diagnostic reagent thereof

Technical Field

The invention belongs to the technical field of gene diagnosis, and particularly relates to application of a BPES syndrome pathogenic gene FOXL2 mutation site and a diagnostic reagent thereof.

Background

The narrow lid-ptosis-inverted inner canthus neoplasm Pi Zeng syndrome (BPES), also known as congenital small lid syndrome (Congenital blepharophimosis syndrome) or Komoto's syndrome, is an autosomal dominant inherited disease. The incidence rate is 1/5000, the incidence rate accounts for 3.5% of ptosis, and the ratio of men to women is 2:1.BPES syndrome is a frequently familial pathogenesis with 100% of the manifestation.

The clinical manifestation of BPES syndrome mainly includes bilateral complete severe ptosis, inverted inner canthus skin tag, the length of eyelid cleavage is generally less than 20mm (normal length 25-30 mm, width 7-12 mm), the inner canthus interval is obviously widened, nose bridge is flat, low ear and the like. Some of them have the combined symptoms of small eyeballs, nystagmus, internal and external eyelid, strabismus, etc., and if forming excessive coverage to pupils, the vision development can be affected, and the vision is often represented. The female suffering from the illness has primary or secondary amenorrhea, irregular menstruation, infertility and the like. BPES is divided into two types: type I and type II. Type I is common and is commonly transmitted by diseased men, with BPES female patients having an eyelid abnormal phenotype, along with ovarian failure; type II, both men and women suffering from the disease can inherit the offspring, and BPES patients only have eyelid defect phenotype. The main difference between the two types is mainly that the type I affected women are accompanied with infertility. To date, no reliable and effective method has been found clinically to treat infertility in BPES type i patients, and ptosis in BPES patients can be corrected only by reconstructive surgery to improve their appearance.

Mutations in the Forkhead box l2 (FOXL 2, MIM 605597) gene lead to BPES syndrome. Studies have shown that the FOXL2 gene is the most common, first causative candidate gene for BPES, and that mutations in the FOXL2 gene are found in both BPES type I and type II patients. The FOXL2 gene is a single exon gene located on chromosome 3q23, is 2.7kb in length, and comprises a unique forkhead DNA domain of 101 amino acids (located in the 54 th-148 th residue region) and a polyalanine peptide fragment separated therefrom, the function of which has not been elucidated. The FOXL2 gene is an autosomal gene that plays an important role in maintaining follicular development and normal ovarian function, and is mainly expressed in granulosa cells in the small follicular phase, and plays an important role in the proliferation and differentiation of follicular granulosa cells and the production of ovarian steroid hormones by inhibiting the transcriptional activity of promoters such as CYP11A1, CYP1941, CCND2, etc. which are downstream target genes. The gene mutation can influence the transcription inhibition of the FOXL2 protein on downstream regulatory genes CYP19A1 and CCND2 promoters, thereby causing abnormal functions of granulosa cells and steroid hormone generation and finally causing infertility.

Thus, gene mutation is an important genetic basis for the development of diseases, and gene diagnosis is an important genetic criterion for the diagnosis of BPES syndrome. There is a clinical need to establish corresponding detection techniques for different mutations and for clear etiology and disease diagnosis.

Disclosure of Invention

Accordingly, the present invention is directed to an application of mutation sites of the pathogenic gene FOXL2 of BPES syndrome and a diagnostic reagent thereof for distinguishing patients with BPES syndrome from normal human groups.

The invention provides an application of a BPES syndrome pathogenic gene FOXL2 mutation site FOXL2:NM_023067.3:exon1:c.316_321dupCACAAC:p.His106_Asn107dup in preparation of a BPES syndrome diagnostic reagent or preparation of a medicament for preventing and/or treating a BPES syndrome target.

The invention provides a reagent for diagnosing BPES syndrome genes, which is a primer for amplifying FOXL2 mutation sites FOXL2: NM_023067.3: exon1: c.316_321dupCACAAC: p.His106_Asn107dup, and comprises FOXL2-F with a nucleotide sequence shown as SEQ ID NO. 1 and FOXL2-R with a nucleotide sequence shown as SEQ ID NO. 2.

Preferably, the reagent comprises a sequencing primer comprising FOXL2-SeqF having a nucleotide sequence shown in SEQ ID NO. 3 and FOXL2-SeqR having a nucleotide sequence shown in SEQ ID NO. 4.

Preferably, the reagents further comprise PCR amplification reagents.

The invention provides application of the reagent in preparation of a BPES syndrome diagnosis kit.

Preferably, the diagnosis kit is used for detecting the genotype of a gene mutation site in a sample to diagnose whether an individual suffers from BPES syndrome, wherein the gene mutation site is FOXL2:NM_023067.3:exon1:c.316_321dupCACAAC:p.His106_Asn107dup, and the detection result is 'c.316_321 dupCACAAC heterozygote', and the existence of mutation in the FOXL2 gene is judged, so that the individual suffers from BPES syndrome; if the site has no mutation, the FOXL2 gene is judged to be wild type, and the individual is normal.

Preferably, the sample is blood.

The invention provides a BPES syndrome diagnosis kit, which comprises the reagent of claims 2-4.

The invention provides an application of a BPES syndrome pathogenic gene FOXL2 mutation site FOXL2:NM_023067.3:exon1:c.316_321dupCACAAC:p.His106_Asn107dup in preparation of a BPES syndrome diagnostic reagent or preparation of a medicament for preventing and/or treating a BPES syndrome target. The invention can lead to the onset of BPES syndrome through the first exact FOXL2: NM_023067.3: exon1: c.316_321dupCACAAC: p.His106_Asn107dup site mutation by the exome sequencing technology. Therefore, the FOXL2 mutation site is used as a molecular marker to diagnose BPES syndrome. In one aspect, the method is used for screening or diagnosis of genetic BPES syndrome by detecting whether a subject carries the mutation described above to guide treatment. In particular, the diagnostic kit provided by the invention can be used for rapidly and effectively predicting or diagnosing BPES syndrome. On the other hand, the invention lays an important foundation for researching pathogenesis of the BPES syndrome and provides a brand new theoretical basis for treating patients with the BPES syndrome. In a third aspect, the invention may provide a potential drug target for the treatment of BPES syndrome.

Drawings

FIG. 1 is a genetic map of the BPES syndrome family No. 1; wherein ∈r represents a normal male individual, ∈r represents a normal female, ■ represents a male patient, +.r represents a female patient, ↗ represents a forerunner.

FIG. 2 shows the results of detection of the genotype of family 1 FOXL 2:NM-023067.3:exo1:c.316_321dupCACAAC:p.His 106_Asn107dup locus by Sanger sequencing with the first and first father and first pair of siblings of family 1 as patients (the position of mutation is indicated by the arrow in the sequencing).

FIG. 3 shows a genetic map of family 2 of BPES syndrome; wherein ∈r represents a normal male individual, ∈r represents a normal female, ∈r represents a normal male individual, ∈r represents a suspected dead male individual, +.;

FIG. 4 shows a graph of the results of detection of the genotype of family 2 FOXL 2:NM-023067.3:exon1:c.316_321dupCACAAC:p.His 106_Asn107dup locus using Sanger sequencing, with the first-evidence being patient, the first-evidence husband and the first-evidence sister being wild-type (the position of mutation occurrence indicated by the arrow in the sequencing).

Detailed Description

In the invention, the mutant core nucleotide sequence of the pathogenic gene FOXL2 is shown in SEQ ID NO. 6 (ATCCGCCACAACCACAACCTCAGCC, the bold part is a mutation site repetitive sequence). Mutation site FOXL2 of the pathogenic gene FOXL2 of BPES syndrome, NM_023067.3, exon1, c.316_321dupCACAAC, p.His106_Asn107dup, indicates that the 106 th histidine and the 107 th asparagine in the protein coded by the FOXL2 gene are repeated, and the mutant core amino acid sequence of the pathogenic protein FOXL2 is shown as SEQ ID NO. 7 (IRHNHNLS).

The invention provides a reagent for diagnosing BPES syndrome genes, which is a primer for amplifying FOXL2 mutation sites FOXL2: NM_023067.3: exon1: c.316_321dupCACAAC: p.His106_Asn107dup, and comprises FOXL2-F with a nucleotide sequence shown as SEQ ID NO. 1 (CGGTCGCACAGTCAAGGAG) and FOXL2-R with a nucleotide sequence shown as SEQ ID NO. 2 (ATCTGGCAGGAGGCATAGGG).

In the present invention, the reagent preferably includes a sequencing primer, which preferably includes FOXL2-SeqF having a nucleotide sequence shown in SEQ ID NO. 3 (GCTCACGCTGTCCGGCATCT) and FOXL2-SeqR having a nucleotide sequence shown in SEQ ID NO. 4 (AGGCATAGGGCATGGGTGAGG). The reagents preferably also include PCR amplification reagents. The PCR amplification reagents include, but are not limited to dNTPs, PCR buffers, magnesium ions, and Tap polymerase.

In the invention, the diagnosis method preferably uses the diagnosis kit to detect the genotype of a gene mutation site in a sample to diagnose whether an individual has BPES syndrome, wherein the gene mutation site is FOXL2: NM_023067.3: exon1: c.316_321dupCACAAC: p.his106_Asn107dup, and the detection result is "c.316_321dupCACAAC heterozygote", and the existence of mutation in the FOXL2 gene is judged to be the individual has BPES syndrome; if the site has no mutation, the FOXL2 gene is judged to be wild type, and the individual is normal. The sample is preferably blood. The method for detecting the genotype of the gene mutation site in the sample by the diagnostic kit preferably extracts the genomic DNA of the sample, uses an amplification primer for PCR amplification, and uses a PCR product to carry out DNA sequencing, and according to the sequencing result, whether CACACAAC repetition exists in the interval 316_321 is judged, if the repetition exists and the hybridization exists, the genotype is 'c.316_321 dupCACAAC heterozygote', and if the repetition does not exist, the genotype is judged to be normal, and the genotype is judged to be normal.

The invention provides a BPES syndrome diagnosis kit, which comprises the reagent.

In the present invention, unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art. Also, the terms related to molecular genetics, nucleic acid chemistry and molecular biology and laboratory procedures used herein are all widely used terms and conventional procedures in the corresponding field. Meanwhile, in order to better understand the present invention, definitions and explanations of related terms are provided below.

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 "mutation" as used herein refers to the alteration of a wild-type polynucleotide sequence into a variant, which may be naturally occurring or non-naturally occurring.

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. As used herein,

the term "specifically amplify" refers to a primer that is capable of amplifying a gene of interest by a PCR reaction, but not other genes. For example, specifically amplifying the FOXL2 gene means that the primers amplify only the FOXL2 gene and not the other genes in a PCR reaction.

The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit 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, 1989) or as recommended by the manufacturer.

The following examples are presented to illustrate the application of the mutation site of the disease causing gene FOXL2 of BPES syndrome and the diagnostic reagent thereof in detail, but they should not be construed as limiting the scope of the present invention.

Example 1

The inventors have found a family of BPES syndromes (referred to as the FOXL2 family) and the clinical information of the members of the FOXL2 family are shown in Table 1. Fig. 1 shows a FOXL2 family map, wherein ∈Σ2 represents a normal male individual, ∈o represents a normal female, ■ represents a male patient, ∈c represents a female patient, ↗ represents a forerunner.

1. Diagnostic criteria:

reference may be made to "human monogenic genetic disease" 2010 edition:

1) Clinical characteristics:

the palpebral fissure syndrome is a congenital dysplasia, and the main manifestations in the eyes are four signs:

first, reverse direction type inner canthus skin tag: the inner canthus neoplasm skin is mainly the inner canthus neoplasm skin of the lower eyelid.

Second, ptosis: the upper eyelid covers more of the black eye bead.

Third, the lid split is relatively small: the eye appears to be relatively small.

Fourth, saddle nose: the nose is saddle-like and the canthus spacing is relatively wide.

Type i palpebral fissure syndrome can be manifested in women as premature ovarian failure, infertility.

2) Genetic characterization

BPES syndrome is an autosomal dominant inherited disease, most commonly caused by mutations in the FOXL2 gene.

TABLE 1 clinical information of BPES syndrome family members

As shown in FIG. 1, the numbers I (first generation) and II (second generation) are adopted.

Family members I1 (father), I2 (mother), II 2 (precursor), II 3 (sister) and II 5 (brother) peripheral blood DNA were used for sequencing analysis.

Example 2

Exon sequencing

1. The instrument is shown in table 2.

Table 2 list of instruments and devices

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 (Thermo), peripheral blood gDNA extraction kit (tengen), agarose (tengen), EB dye solution (amerco).

3. Reagent formulation

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

Table 3 5 XTBE electrophoresis liquid formula

Reagent(s)	Volume/weight
		Tris	5.4g
Boric acid	750mg
		EDTA(pH 8.0,0.5mol/L)	2mL
ddH ₂ O	90mL

With ddH ₂ O adjusts the final volume to 100mL.

0.5 XTBE working solution was run on ddH ₂ O is diluted by 10 times.

10 Xerythrocyte lysate was prepared according to Table 4.

TABLE 4 10 Xerythrocyte lysate formula

Reagent(s)	Volume/weight
		NH4Cl	82.9g
KHCO ₃	10g
		EDTA	0.37g
Adding dH2O	To 1000mL

Autoclaving and storing at 4deg.C.

1 Xnuclear lysate was prepared according to Table 5.

Table 5 1 XNuclear lysate formula

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

4. Experimental procedure

After signing informed consent, 3-5mL of peripheral blood of members of family I1 (father), I2 (mother), II 2 (forensic person), II 3 (sister), II 5 (brother) and the like are collected.

4.1 sample DNA extraction

1) If the sample is heparin anticoagulated peripheral blood sample, 3-5mL of peripheral blood is put into a 15mL centrifuge tube, and 2-3 times of 1 Xerythrocyte lysate is added, and the mixture is uniformly mixed and kept stand on ice for 30 minutes until the solution becomes transparent.

2) Centrifuge at 4℃for 10 min at 3000 rpm, 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 shaking, and centrifuging at room temperature of 3000 rpm for 10 min.

4) The supernatant was carefully transferred to another centrifuge tube, mixed with an equal volume of phenol/chloroform (1:1 v/v) and centrifuged at 3000 rpm 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 gel, and recovering 150-250bp 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, 1 pathogenic gene mutation FOXL2:NM_023067.3:exon1:c.316_321dupCACAAC:p.His106_Asn107dup is obtained, which causes duplication of histidine at position 106 and asparagine at position 107. The genotype of the FOXL2: NM-023067.3: exon1: c.316_321dupCACAAC: p.His106_Asn107dup site was a "c.316_321dupCACAAC heterozygous" mutation in individuals of the family patient and the genotype was a wild-type with no mutation in normal individuals of the FOXL2 family.

Example 3

Sanger sequencing validation

For the exome sequencing results, the FOXL2: NM-023067.3: exo1: c.316_321dupCACAAC: p.His106_Asn107dup site was further verified using Sanger sequencing. FOXL2: NM-023067.3: exon1: c.316_321dupCACAAC: p.His106Asn107dup site genotype was examined for family members such as I1, I2, II 3 and II 5 in example 1 and for 100 extrafamily normal persons, respectively.

The specific method comprises the following steps:

1. DNA extraction

Genomic DNA was extracted according to the method of example 1.

2. Candidate primer design, verification and preference

2.1 primer design reference human genome sequence database hg19/build36.3. Primer sequences were synthesized by Shanghai Biotechnology.

2.2 designing 18 pairs of candidate primers for the c316_321 dupCACAAC site, respectively (see Table 6), and verifying and evaluating the merits of each pair of candidate primers by PCR experiments

TABLE 6 list of candidate primer base conditions and validation experiment results for each pair

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 primer avoids such a situation as much as possible.

The optimal primer pairs were also comprehensively evaluated and selected with reference to the following principles:

(1) the length of the primer is 15-30nt, and is usually about 20 nt;

(2) the content of G+C is preferably 40-60%, too little G+C has poor amplification effect, and excessive G+C is easy to generate nonspecific bands. ATGC is preferably randomly distributed;

(3) avoiding a serial alignment of more than 5 purine or pyrimidine nucleotides;

(4) complementary sequences should not occur inside the primer;

(5) no complementary sequences should exist between the two primers, in particular to avoid complementary overlapping of the 3' ends;

(6) the homology of the primer and the sequence of the non-specific amplification region is not more than 70 percent, the continuous 8 bases at the 3' -end of the primer cannot have a complete complementary sequence outside the region to be amplified, otherwise, the non-specific amplification is easy to cause;

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: 95 ℃ for 5 minutes;

and a second step of: 30 cycles (95 ℃,30 seconds→tm,30 seconds→72 ℃,60 seconds); (the Tm value is calculated for each primer in Table 6 by setting PCR amplification parameters based on the Tm value of each primer).

And a third step of: 72 ℃,7 minutes;

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 1-2mm 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 only one bright and clear target strip appears in the tube No. 7 and no other strip exists, 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 verification test in Table 6, the optimal pair (pair No. 1 in Table 6) was selected as the primers for mutation family detection, and the primer sequences for FOXL2: NM-023067.3: exon1: c.316_321dupCACAAC: p.His106_Asn107dup sites are as follows:

5’-CGGTCGCACAGTCAAGGAG-3’(SEQ ID NO：1)

5’-CCATCTGGCAGGAGGCATAG-3’(SEQ ID NO：2)

3. PCR amplification of mutation sites in 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 site PCR reaction system

Reagent(s)	Volume of
		10 XPCR buffer	2.0μL
10mmol/L dNTPs	0.4μL
		100ng/μL FOXL2-F	0.5μL
100ng/μL FOXL2-R	0.5μL
		100 ng/. Mu.L of peripheral blood extract DNA	1.0μL
5 u/. Mu.L Taq enzyme	0.2μL
		ddH ₂ O	15.4μL

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

the first step: 95 ℃ for 5 minutes;

and a second step of: 30 cycles (95 ℃,30 seconds- > 60 ℃,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 15 minutes and inactivated at 85℃for 15 minutes.

6. BigDye reaction

The BigDye reaction system is shown in Table 9.

TABLE 9 BigDye reaction System

Reagent(s)	Dosage of
		DNA after purification of PCR product	2.0μL
3.2 pmol/. Mu.L sequencing primer	1.0μL
		BigDye	0.5μL
5 XBigDye sequencing buffer	2.0μL
		ddH ₂ O	4.5μL

Sequencing PCR cycling conditions:

the first step: 96℃for 1 minute;

and a second step of: 33 cycles (96 ℃,30 seconds- > 55 ℃,15 seconds- > 60 ℃,4 minutes);

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 mu L of 70% alcohol, shaking and mixing for 4 times, and standing at room temperature for 15 minutes;

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

4) After 5 minutes at room temperature, the residual alcohol was allowed to evaporate at room temperature, 10. Mu.L Hi-Di formamide was added to dissolve DNA, denatured at 96℃for 4 minutes, quickly placed on ice for 4 minutes, and sequenced on the machine.

8. Sequencing

DNA sequencing is carried out on the purified BigDye reaction product, and a nest primer (a second set of primers are designed within the range of the product sequence obtained by amplifying the first set of primers) is designed on the basis of the PCR preferred primers as a sequencing primer, wherein the sequence of the sequencing primer is as follows:

5’-GCTCACGCTGTCCGGCATCT-3’(SEQ ID NO：3)

5’-AGGCATAGGGCATGGGTGAGG-3’(SEQ ID NO：4)。

9. analysis of results

The Sanger sequencing results of FIG. 2 show that the genotype of the family 3 patients FOXL2: NM-023067.3: exon1: c.316_321dupCACAAC: p.His106_Asn107dup locus is "c.316_321dupCACAAC heterozygote". The position indicated by the arrow in the sequencing diagram of FIG. 2 shows that the FOXL 2:NM_023067.3:exo1:c.316_321dupCACAAC:p.His106_Asn 107dup locus genotype of A, C, E layers of BPES syndrome patient is the "c.316_321dupCACAAC heterozygote" mutation.

Example 4

FOXL2 gene c.316_321dupCACAAC mutation diagnosis kit and application

1. The kit comprises the following components:

1) Amplification primers: as shown in example 3

2) Buffer solution

3) Taq enzyme

4)dNTPs

5) FOXL2: c.316-321 dupCACAAC positive mutant reference DNA, which is a double-stranded DNA, the specific sequence is shown below:

(SEQ ID NO: 5) wherein the bolded bases represent the positions of the upstream and downstream primers for PCR amplification, respectively; underlined bases indicate mutation sites; double underlined bases represent upstream and downstream sequencing primers.

6) Sequencing primer: as shown in example 3.

2. The using method comprises the following steps:

the method is applied to the family 2 for gene mutation detection.

TABLE 10 clinical information of family members of BPES syndrome number 2

As shown in FIG. 3, the numbers I (first generation) and II (second generation) are used.

The peripheral blood DNA of family personnel II 1 (forerunner husband), II 2 (forerunner) and II 3 (forerunner sister) are used for detection and analysis of the kit.

1) Genomic DNA extraction: and extracting the genomic DNA of the sample.

2) Firstly, carrying out PCR amplification reaction by adopting the PCR amplification primer, taq enzyme, buffer solution, dNTPs, sample genome DNA and the like;

3) Purifying the PCR amplification product;

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

5) Purifying the BiyDye reaction product;

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

FIG. 4 shows a graph of the results of the detection of the genotype of the family 2 FOXL2: NM-023067.3: exon1: c.316_321dupCACAAC: p.His106_Asn107dup locus, the genotype of the first-evidence being c.316_321dupCACAAC heterozygote, the first-evidence husband and first-evidence sister being wild-type, using a kit; the detection result confirms that the first person is a BPES syndrome patient.

From the results of the above examples, it can be seen that the present invention has found a novel FOXL2 gene mutation and confirmed that the novel mutant is closely related to the onset of BPES syndrome, which can be used for molecular diagnosis of BPES syndrome and differential diagnosis of related diseases.

The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims

1. Application of positive mutation reference DNA containing BPES syndrome pathogenic gene FOXL2 mutation site FOXL2: NM_023067.3: exon1: c.316_321dupCACAAC: p.His106_Asn107dup in preparation of BPES syndrome diagnostic kit;

the positive mutation reference DNA is a double-stranded DNA;

the nucleotide sequence of the positive mutation reference DNA is shown as SEQ ID NO. 5.

2. The application of a reagent for BPES syndrome gene diagnosis in preparing a BPES syndrome diagnosis kit is characterized in that the reagent consists of an amplification primer and a sequencing primer for amplifying a FOXL2 mutation site FOXL 2:NM_023067.3:exo1:c.316_321dupCACAAC:p.His106_Asn 107 dup;

the amplification primer comprises FOXL2-F with a nucleotide sequence shown as SEQ ID NO. 1 and FOXL2-R with a nucleotide sequence shown as SEQ ID NO. 2;

the sequencing primer comprises FOXL2-SeqF with a nucleotide sequence shown in SEQ ID NO. 3 and FOXL2-SeqR with a nucleotide sequence shown in SEQ ID NO. 4.