CN115851898B - Application of BPES syndrome pathogenic gene FOXL2 mutation site and diagnostic reagent thereof - Google Patents
Application of BPES syndrome pathogenic gene FOXL2 mutation site and diagnostic reagent thereof Download PDFInfo
- Publication number
- CN115851898B CN115851898B CN202210822994.XA CN202210822994A CN115851898B CN 115851898 B CN115851898 B CN 115851898B CN 202210822994 A CN202210822994 A CN 202210822994A CN 115851898 B CN115851898 B CN 115851898B
- Authority
- CN
- China
- Prior art keywords
- bpes
- syndrome
- foxl2
- gene
- mutation
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 102100035137 Forkhead box protein L2 Human genes 0.000 title claims abstract description 70
- 208000033931 Blepharophimosis-ptosis-epicanthus inversus syndrome Diseases 0.000 title claims abstract description 67
- 101001023356 Homo sapiens Forkhead box protein L2 Proteins 0.000 title claims abstract description 67
- 208000011580 syndromic disease Diseases 0.000 title claims abstract description 65
- 108010010285 Forkhead Box Protein L2 Proteins 0.000 title claims abstract description 43
- 230000035772 mutation Effects 0.000 title claims abstract description 43
- 239000003153 chemical reaction reagent Substances 0.000 title claims abstract description 33
- 230000001717 pathogenic effect Effects 0.000 title claims abstract description 13
- 238000003745 diagnosis Methods 0.000 claims abstract description 19
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 17
- 238000002360 preparation method Methods 0.000 claims abstract description 11
- 238000012163 sequencing technique Methods 0.000 claims description 24
- 108020004414 DNA Proteins 0.000 claims description 23
- 239000002773 nucleotide Substances 0.000 claims description 14
- 125000003729 nucleotide group Chemical group 0.000 claims description 14
- 230000003321 amplification Effects 0.000 claims description 8
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 8
- KIAPWMKFHIKQOZ-UHFFFAOYSA-N 2-[[(4-fluorophenyl)-oxomethyl]amino]benzoic acid methyl ester Chemical compound COC(=O)C1=CC=CC=C1NC(=O)C1=CC=C(F)C=C1 KIAPWMKFHIKQOZ-UHFFFAOYSA-N 0.000 claims description 4
- 101100333985 Drosophila melanogaster tos gene Proteins 0.000 claims description 4
- 101150112849 EXO1 gene Proteins 0.000 claims description 4
- 238000009007 Diagnostic Kit Methods 0.000 claims description 3
- 102000053602 DNA Human genes 0.000 claims description 2
- 238000001514 detection method Methods 0.000 abstract description 14
- 239000003814 drug Substances 0.000 abstract description 4
- 230000008506 pathogenesis Effects 0.000 abstract description 3
- 239000003596 drug target Substances 0.000 abstract description 2
- 238000006243 chemical reaction Methods 0.000 description 29
- 102000015784 Forkhead Box Protein L2 Human genes 0.000 description 27
- 239000000523 sample Substances 0.000 description 19
- 101150072550 FOXL2 gene Proteins 0.000 description 14
- 238000012408 PCR amplification Methods 0.000 description 13
- 238000000034 method Methods 0.000 description 13
- 239000000499 gel Substances 0.000 description 12
- 239000000047 product Substances 0.000 description 12
- 206010064571 Gene mutation Diseases 0.000 description 10
- 238000001962 electrophoresis Methods 0.000 description 10
- 201000010099 disease Diseases 0.000 description 8
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 8
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 7
- 239000006166 lysate Substances 0.000 description 7
- 210000005259 peripheral blood Anatomy 0.000 description 7
- 239000011886 peripheral blood Substances 0.000 description 7
- 230000000295 complement effect Effects 0.000 description 6
- 210000000744 eyelid Anatomy 0.000 description 6
- 230000002068 genetic effect Effects 0.000 description 6
- 239000007788 liquid Substances 0.000 description 6
- 206010015995 Eyelid ptosis Diseases 0.000 description 5
- 239000000872 buffer Substances 0.000 description 5
- 239000000539 dimer Substances 0.000 description 5
- 208000000509 infertility Diseases 0.000 description 5
- 230000036512 infertility Effects 0.000 description 5
- 231100000535 infertility Toxicity 0.000 description 5
- 201000003004 ptosis Diseases 0.000 description 5
- 238000007480 sanger sequencing Methods 0.000 description 5
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 4
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 4
- 239000007795 chemical reaction product Substances 0.000 description 4
- 239000012634 fragment Substances 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- 206010010356 Congenital anomaly Diseases 0.000 description 3
- 238000007400 DNA extraction Methods 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 3
- 238000000246 agarose gel electrophoresis Methods 0.000 description 3
- 239000007853 buffer solution Substances 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 210000002503 granulosa cell Anatomy 0.000 description 3
- 238000009396 hybridization Methods 0.000 description 3
- 238000011068 loading method Methods 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- 238000005070 sampling Methods 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 238000012795 verification Methods 0.000 description 3
- KDCGOANMDULRCW-UHFFFAOYSA-N 7H-purine Chemical compound N1=CNC2=NC=NC2=C1 KDCGOANMDULRCW-UHFFFAOYSA-N 0.000 description 2
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 2
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 2
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 description 2
- 238000001712 DNA sequencing Methods 0.000 description 2
- ZHNUHDYFZUAESO-UHFFFAOYSA-N Formamide Chemical compound NC=O ZHNUHDYFZUAESO-UHFFFAOYSA-N 0.000 description 2
- 102100024185 G1/S-specific cyclin-D2 Human genes 0.000 description 2
- 208000028782 Hereditary disease Diseases 0.000 description 2
- 101000980741 Homo sapiens G1/S-specific cyclin-D2 Proteins 0.000 description 2
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 description 2
- 208000024556 Mendelian disease Diseases 0.000 description 2
- 206010029098 Neoplasm skin Diseases 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 230000002159 abnormal effect Effects 0.000 description 2
- 239000002390 adhesive tape Substances 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 2
- 229960001230 asparagine Drugs 0.000 description 2
- 235000009582 asparagine Nutrition 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 239000000284 extract Substances 0.000 description 2
- 210000001508 eye Anatomy 0.000 description 2
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 2
- 102000039446 nucleic acids Human genes 0.000 description 2
- 108020004707 nucleic acids Proteins 0.000 description 2
- 150000007523 nucleic acids Chemical class 0.000 description 2
- 210000004940 nucleus Anatomy 0.000 description 2
- 230000002611 ovarian Effects 0.000 description 2
- 230000036961 partial effect Effects 0.000 description 2
- 102000040430 polynucleotide Human genes 0.000 description 2
- 108091033319 polynucleotide Proteins 0.000 description 2
- 239000002157 polynucleotide Substances 0.000 description 2
- 238000004321 preservation Methods 0.000 description 2
- 201000000195 skin tag Diseases 0.000 description 2
- 239000003270 steroid hormone Substances 0.000 description 2
- 238000011144 upstream manufacturing Methods 0.000 description 2
- 238000007482 whole exome sequencing Methods 0.000 description 2
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 229920000936 Agarose Polymers 0.000 description 1
- 201000000736 Amenorrhea Diseases 0.000 description 1
- 206010001928 Amenorrhoea Diseases 0.000 description 1
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 1
- 102100029361 Aromatase Human genes 0.000 description 1
- 206010005155 Blepharophimosis Diseases 0.000 description 1
- -1 CYP1941 Proteins 0.000 description 1
- 108010084976 Cholesterol Side-Chain Cleavage Enzyme Proteins 0.000 description 1
- 102100027516 Cholesterol side-chain cleavage enzyme, mitochondrial Human genes 0.000 description 1
- 235000002673 Dioscorea communis Nutrition 0.000 description 1
- 241000544230 Dioscorea communis Species 0.000 description 1
- 206010058314 Dysplasia Diseases 0.000 description 1
- 108010067770 Endopeptidase K Proteins 0.000 description 1
- 108010007577 Exodeoxyribonuclease I Proteins 0.000 description 1
- 102100029075 Exonuclease 1 Human genes 0.000 description 1
- 108090000852 Forkhead Transcription Factors Proteins 0.000 description 1
- 108091092584 GDNA Proteins 0.000 description 1
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 description 1
- 101000919395 Homo sapiens Aromatase Proteins 0.000 description 1
- 208000026350 Inborn Genetic disease Diseases 0.000 description 1
- JLVVSXFLKOJNIY-UHFFFAOYSA-N Magnesium ion Chemical compound [Mg+2] JLVVSXFLKOJNIY-UHFFFAOYSA-N 0.000 description 1
- 208000037093 Menstruation Disturbances Diseases 0.000 description 1
- 206010027339 Menstruation irregular Diseases 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 108091034117 Oligonucleotide Proteins 0.000 description 1
- 206010033165 Ovarian failure Diseases 0.000 description 1
- 108010033276 Peptide Fragments Proteins 0.000 description 1
- 102000007079 Peptide Fragments Human genes 0.000 description 1
- 208000035753 Periorbital contusion Diseases 0.000 description 1
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 1
- 208000002500 Primary Ovarian Insufficiency Diseases 0.000 description 1
- 108700005075 Regulator Genes Proteins 0.000 description 1
- 208000004350 Strabismus Diseases 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- 230000007488 abnormal function Effects 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 231100000540 amenorrhea Toxicity 0.000 description 1
- 235000001014 amino acid Nutrition 0.000 description 1
- 229940024606 amino acid Drugs 0.000 description 1
- 235000019270 ammonium chloride Nutrition 0.000 description 1
- 239000010425 asbestos Substances 0.000 description 1
- 125000000613 asparagine group Chemical group N[C@@H](CC(N)=O)C(=O)* 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- 230000002146 bilateral effect Effects 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 description 1
- 239000004327 boric acid Substances 0.000 description 1
- UDSAIICHUKSCKT-UHFFFAOYSA-N bromophenol blue Chemical compound C1=C(Br)C(O)=C(Br)C=C1C1(C=2C=C(Br)C(O)=C(Br)C=2)C2=CC=CC=C2S(=O)(=O)O1 UDSAIICHUKSCKT-UHFFFAOYSA-N 0.000 description 1
- 210000005252 bulbus oculi Anatomy 0.000 description 1
- 208000035269 cancer or benign tumor Diseases 0.000 description 1
- 238000005251 capillar electrophoresis Methods 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 210000000349 chromosome Anatomy 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 230000001351 cycling effect Effects 0.000 description 1
- 238000007405 data analysis Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000003748 differential diagnosis Methods 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 229920001971 elastomer Polymers 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000012467 final product Substances 0.000 description 1
- 230000003325 follicular Effects 0.000 description 1
- 230000008217 follicular development Effects 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 208000016361 genetic disease Diseases 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 230000005484 gravity Effects 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 229960002897 heparin Drugs 0.000 description 1
- 229920000669 heparin Polymers 0.000 description 1
- 125000000487 histidyl group Chemical group [H]N([H])C(C(=O)O*)C([H])([H])C1=C([H])N([H])C([H])=N1 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 229910001425 magnesium ion Inorganic materials 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 239000003147 molecular marker Substances 0.000 description 1
- 206010029864 nystagmus Diseases 0.000 description 1
- 201000004535 ovarian dysfunction Diseases 0.000 description 1
- 231100000539 ovarian failure Toxicity 0.000 description 1
- 230000011599 ovarian follicle development Effects 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 150000002989 phenols Chemical class 0.000 description 1
- 108010054442 polyalanine Proteins 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 206010036601 premature menopause Diseases 0.000 description 1
- 208000017942 premature ovarian failure 1 Diseases 0.000 description 1
- 238000004393 prognosis Methods 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 235000018102 proteins Nutrition 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 210000001747 pupil Anatomy 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 239000002213 purine nucleotide Substances 0.000 description 1
- 239000002719 pyrimidine nucleotide Substances 0.000 description 1
- 150000003230 pyrimidines Chemical class 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- 238000002278 reconstructive surgery Methods 0.000 description 1
- 238000003303 reheating Methods 0.000 description 1
- 230000008439 repair process Effects 0.000 description 1
- 230000003252 repetitive effect Effects 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 229910052895 riebeckite Inorganic materials 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000011550 stock solution Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 230000002103 transcriptional effect Effects 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 238000010200 validation analysis Methods 0.000 description 1
- 238000012418 validation experiment Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
- 239000012224 working solution Substances 0.000 description 1
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
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
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
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.
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.
The invention provides application of the reagent in preparation of a BPES syndrome diagnosis kit.
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
/>
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 (2)
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.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202210822994.XA CN115851898B (en) | 2022-07-13 | 2022-07-13 | Application of BPES syndrome pathogenic gene FOXL2 mutation site and diagnostic reagent thereof |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202210822994.XA CN115851898B (en) | 2022-07-13 | 2022-07-13 | Application of BPES syndrome pathogenic gene FOXL2 mutation site and diagnostic reagent thereof |
Publications (2)
Publication Number | Publication Date |
---|---|
CN115851898A CN115851898A (en) | 2023-03-28 |
CN115851898B true CN115851898B (en) | 2024-04-05 |
Family
ID=85660307
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202210822994.XA Active CN115851898B (en) | 2022-07-13 | 2022-07-13 | Application of BPES syndrome pathogenic gene FOXL2 mutation site and diagnostic reagent thereof |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN115851898B (en) |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN107406874A (en) * | 2014-12-19 | 2017-11-28 | 表观基因组股份有限公司 | The method for methylating and diagnosing cancer for detecting CpG |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20110195070A1 (en) * | 2008-10-09 | 2011-08-11 | British Columbia Cancer Agency Branch | Detection of granulosa-cell tumors |
-
2022
- 2022-07-13 CN CN202210822994.XA patent/CN115851898B/en active Active
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN107406874A (en) * | 2014-12-19 | 2017-11-28 | 表观基因组股份有限公司 | The method for methylating and diagnosing cancer for detecting CpG |
Non-Patent Citations (2)
Title |
---|
Foxl 2基因的结构特点和生物学功能概述;罗小云等;生物学教学;第45卷(第8期);第63-64页 * |
Identification of 34 novel and 56 known FOXL2 mutations in patients with blepharophimosis syndrome;Diane Beysen等;Human Mutation;第29卷(第11期);E205-E219 * |
Also Published As
Publication number | Publication date |
---|---|
CN115851898A (en) | 2023-03-28 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN116240280A (en) | Pathogenic gene for leading to Rubin-Taybi syndrome, detection and application | |
CN116121365B (en) | Pathogenic gene causing COFS syndrome, detection and application | |
CN115772214B (en) | F8 mutant protein, F8 gene mutant, primer combination for detecting F8 gene mutant, reagent and application | |
CN115141837B (en) | Novel SLC9A6 mutant gene and diagnostic reagent thereof | |
CN116287213A (en) | Pathogenic gene causing MRD7 type mental disorder, detection and application | |
CN116179686A (en) | Pathogenic gene for Malan syndrome, detection and application | |
CN115725716B (en) | PKD1 pathogenic mutant gene and application thereof in preparation of polycystic kidney disease diagnosis kit | |
CN116377054A (en) | Pathogenic gene causing Snijders Blok type intellectual disability, detection and application | |
CN115927356B (en) | SLC45A2 pathogenic mutant gene, pathogenic mutant and application thereof in preparation of eye skin albinism IV type diagnostic kit | |
CN116426630A (en) | Pathogenic gene for causing Joubert syndrome, detection and application | |
CN115851898B (en) | Application of BPES syndrome pathogenic gene FOXL2 mutation site and diagnostic reagent thereof | |
CN116083556A (en) | Application of pathogenic gene ASH1L mutation site for causing MRD52 type dysnoesia, detection reagent and application | |
CN116656801A (en) | Application of Cowcdock syndrome pathogenic gene AIFM1 mutation site, detection reagent and application thereof | |
CN115992212A (en) | UBE3A mutant protein, UBE3A gene mutant, amplification primer, detection reagent and application | |
CN115216536A (en) | Novel NIPBL mutant gene and diagnostic reagent thereof | |
CN115948530B (en) | Turner type X-linked syndrome pathogenic gene and primer pair for mental retardation and application thereof | |
CN115851918B (en) | Application and detection reagent of pathogenic gene CFAP410 mutation site causing retinal dystrophy | |
CN117467761B (en) | FKTN gene mutant, mutant protein, reagent, kit and application | |
CN117487923B (en) | Application of HABP2 gene mutant as detection target, detection reagent and/or detection kit with HABP2 gene mutant | |
CN115873938B (en) | LRPPRC gene composite mutant for causing French-Canadian Leigh syndrome | |
CN116377055A (en) | Pathogenic gene causing gill-ear-kidney syndrome, detection and application | |
CN117487906B (en) | GAMT gene mutant, reagent, kit and application | |
CN116144753A (en) | Application and detection reagent of pathogenic gene CHD1 mutation site | |
CN116287214A (en) | Pathogenic gene for Dysferlin myopathy, detection and application | |
CN115992224A (en) | Application of pathogenic gene CHD7 mutation site for causing CHRGE syndrome and detection reagent |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
GR01 | Patent grant | ||
GR01 | Patent grant |