Pathogenic gene causing eye-renal syndrome type 5, detection and application

Abstract

The invention provides a virulence gene causing eye-renal syndrome type 5, detection and application. Compared with the wild-type IQCB1 gene, the pathogenic gene has the advantages that the 691th basic group of the No.8 exon of the wild-type IQCB1 gene is mutated from the basic group C to the basic group T. The pathogenic gene mutation can be used as a biomarker for detecting the eye-renal syndrome type 5; by detecting whether a subject carries the mutation, the mutation is used for screening or diagnosing eye-renal syndrome type 5 virulence gene mutation patients so as to provide prenatal and postnatal care and treatment intervention guidance. An important foundation is laid for research on pathogenesis of the eye-renal syndrome type 5, and a brand new theoretical basis is provided for treatment of patients with the eye-renal syndrome type 5. And a possible drug target can be provided for treating the eye-renal syndrome type 5.

Description

Technical Field

[0001] The present invention relates to the field of detection reagents, and in particular to a pathogenic gene causing oculorenal syndrome type 5, and its detection and application. Background Art

[0002] Senior-Loken syndrome is oculorenal syndrome, or familial nephro-retinal dystrophy. It has two specific symptoms: one is the kidney symptom called nephronophthisis, and the other is the eye symptom of Leber congenital amaurosis. Nephronophthisis causes fluid-filled cysts to develop in the kidneys starting in childhood, impairing kidney function, initially causing polyuria, malaise and extreme fatigue, and leading to end-stage renal disease in late childhood or adolescence. Leber congenital amaurosis mainly affects the retina, causing vision problems, including photophobia, nystagmus and extreme farsightedness. Leber congenital amaurosis can be divided into infantile and juvenile forms: Infantile forms are blind at birth, and parents often find that their children cannot follow lights after a few months and seek medical attention. The eyeballs often appear sunken and have pendulum-like tremors. Children often have a special movement of pressing their eyeballs with their fists. There is no obvious abnormality in the fundus at the beginning. After a period of time, small white spots and pigment particles appear around the fundus, showing a salt-and-pepper appearance. Then, on the one hand, it gradually expands to the posterior pole, and on the other hand, the pigment particles continue to proliferate and fuse into osteocytes. The retinal blood vessels become thinner, the optic disc is waxy yellow, the pigment epithelium and the choroidal capillary layer atrophy, exposing the large blood vessels of the choroid, forming a diffuse choroidal atrophy-like fundus, and even forming an albinism-like fundus; the juvenile type has a serious decrease in vision at the age of 5 to 6, and is completely blind at around 30 years old. The fundus manifestations are extremely inconsistent. Most cases have salt-and-pepper changes in the periphery; in a few cases, even if they are completely blind, the fundus remains normal. Some cases of the above two types may be accompanied by mental and neurological symptoms, such as mental retardation, hearing impairment, epilepsy, etc., and some older people may develop keratoconus or spherical cornea. Common pathogenic genes include IQCB1, CEP290, GUCY2D, CRB1, RPE65, IMPDH1, AIPL1 and RPGRI P1. Gene mutation is an important genetic basis for the occurrence and development of the disease, and genetic diagnosis is an important genetic criterion for the diagnosis of Senior-Loken syndrome.

[0003] In the prior art, the genotype of gene mutation sites can be detected by other methods such as restriction fragment length polymorphism, single-stranded conformation polymorphism, allele-specific oligonucleotide hybridization, etc., but these detection methods cannot simultaneously meet the purpose of qualitative, quantitative and clear sequence of mutant genes. Summary of the invention

[0004] The main purpose of the present invention is to provide a pathogenic gene, detection and application that causes Senior-Loken syndrome type 5, so as to solve the technical problems of screening and detection of Senior-Loken syndrome type 5.

[0005] To achieve the above object, the present invention provides a pathogenic gene that causes Senior-Loken syndrome type 5, wherein compared with the wild-type IQCB1 gene, the 691st base in exon 8 of the wild-type IQCB1 gene mutates from base C to base T.

[0006] The present invention also provides a detection reagent for Senior-Loken syndrome type 5 caused by the above-mentioned pathogenic gene, and the detection reagent comprises primers or sequencing primers designed for the site of the gene mutation.

[0007] Furthermore, the primers include IQCB1-1F and IQCB1-1R, the nucleotide sequence of IQCB1-1F is shown as SEQ ID NO.1, and the nucleotide sequence of IQCB1-1R is shown as SEQ ID NO.2.

[0008] Furthermore, the sequencing primers include IQCB1-Seq1F and IQCB1-Seq1R, the nucleotide sequence of IQCB1-Seq1F is shown in SEQ ID NO.3, and the nucleotide sequence of IQCB1-Seq1R is shown in SEQ ID NO.4.

[0009] The present invention also provides a Senior-Loken syndrome type 5 detection kit, comprising the detection reagent as described above.

[0010] Furthermore, it also includes PCR amplification reagents; the PCR amplification reagents include dNTPs, 10×PCR buffer, magnesium ions and Taq polymerase.

[0011] The present invention also provides a use of the detection reagent as described in any one of the above items in a kit and its preparation.

[0012] Furthermore, the detection sample of the detection reagent includes blood and / or amniotic fluid.

[0013] The beneficial effects achieved by the present invention are:

[0014] The present invention uses exome sequencing technology to discover for the first time that the pathogenic gene that causes Senior-Loken syndrome type 5 is mutated from base C to base T in the 691st base of exon 8 of the wild-type IQCB1 gene compared to the wild-type IQCB1 gene. On the one hand, by detecting whether the subject carries the above mutation, it is used to screen or diagnose patients with Senior-Loken syndrome type 5 pathogenic gene mutations to provide guidance on eugenics and therapeutic intervention. On the other hand, the present invention lays an important foundation for the study of the pathogenesis of Senior-Loken syndrome type 5 and provides a new theoretical basis for the treatment of Senior-Loken syndrome type 5 patients. On the third aspect, the present invention can provide possible drug targets for the treatment of Senior-Loken syndrome type 5.

[0015] The kit provided by the present invention greatly improves the accuracy and reliability of the detection results by optimizing the primers and reaction system for detecting the mutation site of the pathogenic gene IQCB1, and can be used to quickly and effectively predict or diagnose Senior-Loken syndrome type 5. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the structures shown in these drawings without paying creative work.

[0017] Figure 1 This is the genetic map of the Senior-Loken syndrome family 1; Indicates male carriers, indicates female carrier, □ indicates normal male individual, ■ indicates male patient, ◇ indicates fetus, ↗ indicates proband;

[0018] Figure 2 This is the result of using Sanger sequencing to detect the genotype of the IQCB1:NM_001023571.3:exon8:c.691C>T:p.R231* site, in which the proband of family No. 1 is a homozygous mutation of "c.691C>T", and the proband's father, proband's mother, and fetus are heterozygous mutation carriers of "c.691C>T" (the arrow in the sequencing diagram indicates the mutation location);

[0019] Figure 3 This is the genetic map of the Senior-Loken syndrome family No. 2; □ represents a normal male individual, Indicates male carriers, indicates female carrier, ● indicates female patient, ↗ indicates proband;

[0020] Figure 4 This is the result of using the kit to detect the genotype of the IQCB1:NM_001023571.3:exon8:c.691C>T:p.R231* site in Family No. 2. The proband of Family No. 2 had the homozygous mutation of "c.691C>T", the proband's father and mother had the heterozygous mutation of "c.691C>T", and the proband's brother was wild type (the arrow in the sequencing image indicates the location of the mutation).

[0021] The realization of the purpose, functional features and advantages of the present invention will be further explained in conjunction with the embodiments and with reference to the accompanying drawings. DETAILED DESCRIPTION

[0022] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0023] Furthermore, the technical solutions between the various embodiments of the present invention may be combined with each other, but this must be based on the fact that they can be implemented by ordinary technicians in the field. When the combination of technical solutions is mutually contradictory or cannot be implemented, it should be deemed that such combination of technical solutions does not exist and is not within the scope of protection required by the present invention.

[0024] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the molecular genetics, nucleic acid chemistry and molecular biology related terms and laboratory operation procedures used herein are widely used terms and conventional procedures in the corresponding fields. At the same time, in order to better understand the present invention, the definitions and explanations of the relevant terms are provided below.

[0025] The term "autosomal recessive inheritance" in this article means that when both parents have disease-causing genes on their autosomes, one disease-causing gene will not cause the disease, but if the parents pass both disease-causing genes to their children, the children may become sick, regardless of gender.

[0026] The term "mutation" herein refers to a change in a wild-type polynucleotide sequence, and refers to the addition, deletion and / or substitution of one or several (e.g., several) bases in a gene sequence or DNA sequence, resulting in a variant, which may be naturally occurring or non-naturally occurring. When the term "mutation" is used to describe a product or protein encoded by a gene, "mutation" refers to the addition, deletion and / or substitution of one or several (e.g., several) amino acid residues in the protein or encoded product.

[0027] As used herein, the term "heterozygous mutation" refers to a mutation that is present in only one gene in a pair of alleles.

[0028] The term "homozygous mutation" in this article means that both alleles have the same mutation, that is, biallelic mutation, and each chromosome is mutated.

[0029] The term "nonsense mutation" in this article refers to the mutation of the codon representing a certain amino acid into a stop codon due to the change of a certain base, thereby terminating the peptide chain synthesis prematurely. That is, after the base replacement, the triplet codon encoding a certain amino acid becomes the stop codon UAA, UAG or UGA that does not encode any amino acid. Although nonsense mutations do not cause errors in amino acid coding, since the stop codon appears in the middle of an mRNA, the termination of the polypeptide chain during translation is terminated, forming an incomplete polypeptide chain.

[0030] The term "diagnosis" as used herein includes prediction of disease risk, diagnosis of whether a disease has occurred, and assessment of disease prognosis.

[0031] The term "prenatal diagnosis" in this article refers to the clear diagnosis of high-risk fetuses mainly through genetic testing and imaging examinations on the basis of genetic counseling, and the purpose of fetal selection is achieved through selective abortion of sick fetuses, thereby reducing the birth defect rate and improving the quality of eugenics and population quality.

[0032] In the present invention, "primer" refers to a polynucleotide fragment used to amplify a target nucleic acid in a PCR reaction, which is usually an oligonucleotide, for example, a polynucleotide fragment 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. The primer does not have to 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,

[0033] The term "specific amplification" means that the primer can amplify the target gene through PCR reaction without amplifying other genes. For example, specific amplification of IQCB1 gene means that in PCR reaction, the primer only amplifies IQCB1 gene without amplifying other genes.

[0034] In order to solve the technical problems of screening and detection of Senior-Loken syndrome type 5, the present invention provides a pathogenic gene that causes Senior-Loken syndrome type 5. Compared with the wild-type IQCB1 gene, the pathogenic gene mutates from base C to base T at the 691st base of exon 8 of the wild-type IQCB1 gene.

[0035] It should be noted that mutations in the IQCB1 gene (MIM 609237) can lead to the autosomal recessive disease Senio r-Loken syndrome type 5 (MIM 609254); the IQCB1 gene is located on chromosome 3q13.33, including 15 exons and 14 introns. The gene is 65.3kb long and encodes an IQCB1 protein with a 598 amino acid sequence, which contains a helical domain (340-373 amino acid residues) and two calmodulin IQ binding domains (294-323 and 387-416 amino acid residues). This protein is mainly expressed in the cilia connecting photoreceptor cells and the primary cilia of renal tubular epithelial cells, and is responsible for the integrity of epithelial cells. Impairments in the synthesis of this protein can lead to retinal degeneration and renal cyst formation. Nonsense mutations in the IQCB1 gene can introduce a stop codon prematurely, causing premature termination of mRNA transcription and producing an abnormal truncated protein. Since the truncated protein loses the complete functional domains of the original protein, such as the helical domain and the calmodulin IQ binding domain, it leads to protein function defects and thus causes disease.

[0036] Specifically, exon sequencing was first used to screen for pathogenic gene mutations that were highly correlated with Senior-Loken syndrome type 5. In order to avoid false positive results, Sanger sequencing was then used for verification, and finally the pathogenic gene mutation of Senior-Loken syndrome type 5 was obtained, IQCB1: NM_001023571.3: exon8: c.691C>T: p.R231*. The pathogenic gene mutations screened by the present invention can distinguish Senior-Loken syndrome type 5 patients from normal people. Therefore, the pathogenic gene mutations of the present invention can be used as biomarkers for diagnosing Senior-Loken syndrome type 5. The IQCB1:NM_001023571.3:exon8:c.691C>T:p.R231* mutation site of the present invention refers to the mutation of the 691st base C of the 8th exon of the two alleles of the IQCB1 gene to T, forming an IQCB1 gene mutant. The nucleotide sequence of the IQCB1 gene mutant is preferably as shown in SE Q ID NO.5( The letters in the boxes indicate the mutated bases.

[0037] Among them, compared with the protein encoded by the wild-type IQCB1 gene, the 231st amino acid of the IQCB1 mutant protein is mutated from arginine (R) to a stop codon (*), that is, the IQCB1 mutant protein contains a p.R231* mutation, which is caused by a nonsense mutation of c.691C>T; the amino acid sequence of the IQCB1 mutant protein is shown in SEQ ID NO.6 (RAMRLS*) (* is the stop codon after the mutation).

[0038] The present invention uses exome sequencing technology to discover for the first time that the pathogenic gene that causes Senior-Loken syndrome type 5 is mutated from base C to base T in the 691st base of exon 8 of the wild-type IQCB1 gene compared to the wild-type IQCB1 gene. On the one hand, by detecting whether the subject carries the above mutation, it is used to screen or diagnose patients with Senior-Loken syndrome type 5 pathogenic gene mutations to provide guidance on eugenics and therapeutic intervention. On the other hand, the present invention lays an important foundation for the study of the pathogenesis of Senior-Loken syndrome type 5 and provides a new theoretical basis for the treatment of Senior-Loken syndrome type 5 patients. On the third aspect, the present invention can provide possible drug targets for the treatment of Senior-Loken syndrome type 5.

[0039] The present invention also provides a detection reagent for Senior-Loken syndrome type 5 caused by the above pathogenic gene, and the detection reagent comprises primers or sequencing primers designed for the site of gene mutation.

[0040] Furthermore, the primers include IQCB1-1F and IQCB1-1R, the nucleotide sequence of IQCB1-1F is shown in SEQ ID NO.1, and the nucleotide sequence of IQCB1-1R is shown in SEQ ID NO.2.

[0041] Specifically, the nucleotide sequence of the primer for the c.691C>T mutation site is:

[0042] IQCB1-1F:5'-ACAGCAGCAGATGACAAGC-3'

[0043] IQCB1-1R:5'-GGAGATGAAGTGAAATAAAGG-3'

[0044] Furthermore, the sequencing primers include IQCB1-Seq1F and IQCB1-Seq1R, the nucleotide sequence of IQCB1-Seq1F is shown in SEQ ID NO.3, and the nucleotide sequence of IQCB1-Seq1R is shown in SEQ ID NO.4.

[0045] Specifically, the nucleotide sequence of the sequencing primer for the c.691C>T mutation site is:

[0046] IQCB1-Seq1F:5'-GTTGCTGGAGATAAATAGGC-3'

[0047] IQCB1-Seq1R:5'-GGGAGATTGTTCATCGTCA-3'

[0048] It should be noted that the present invention has no particular limitation on the sources of primers and sequencing primers, and any primer synthesis method known in the art may be used.

[0049] The present invention also provides a Senior-Loken syndrome type 5 detection kit, comprising the above detection reagent. The kit provided by the present invention greatly improves the accuracy and reliability of the detection results by optimizing the primers and reaction system for detecting the mutation site of the pathogenic gene IQCB1, and can be used to quickly and effectively predict or diagnose Senior-Loken syndrome type 5.

[0050] Furthermore, it also includes PCR amplification reagents; the PCR amplification reagents include dNTPs, 10×PCR buffer, magnesium ions and Taq polymerase.

[0051] Specifically, the 10×PCR buffer contains an aqueous solution of the following components: 500 mmol / L KCl, 100 mmol / L Tris-Cl at pH 8.3, and 15 mmol / L MgCl 2 .

[0052] The reaction system for amplifying the IQCB1 gene sequence is preferably 2.0 μL of 10×PCR buffer, 0.4 μL of 10 mmol / L dNTPs, 0.5 μL of 100 ng / μL IQCB1-1F, 0.5 μL of 100 ng / μL IQCB1-1R, 1.0 μL of 100 ng / μL extracted DNA, 0.2 μL of 5 U / μL Taq enzyme, and 15.4 μL of ddH2O. The reaction procedure for amplifying the IQCB1 gene sequence is preferably the first step: 95°C, 5 minutes; the second step: 30 cycles (95°C, 30 seconds → 54°C, 30 seconds → 72°C, 60 seconds); the third step: 72°C, 7 minutes; and the fourth step: 4°C insulation.

[0053] The present invention also provides a use of any of the above detection reagents in a kit and its preparation.

[0054] Specifically, the method for detecting the mutation site of the pathogenic gene IQCB1 that causes Senior-Loken syndrome type 5 preferably comprises the following steps:

[0055] Extracting genomic DNA from the sample to be tested;

[0056] Using the genomic DNA as a template, amplifying the IQCB1 gene sequence with the reagent;

[0057] DNA sequencing was performed on the amplified IQCB1 gene sequence;

[0058] The DNA sequencing results of the sample to be tested are compared with the normal human genome DNA sequence. If the results are completely consistent, it means that the IQCB1 gene in the sample to be tested has not mutated to the wild type; if the comparison results show that the 691st base C in exon 8 of the two alleles of the IQCB1 gene (NM_001023571.3) is mutated to T, the genotype is judged to be "c.691C>T homozygous mutation".

[0059] Specifically, the mutation site of gene IQCB1 can be IQCB1: NM_001023571.3: exon8: c.691C>T: p.R231*. The primers for detecting the mutation site of gene IQCB1 can be used in the preparation of an auxiliary diagnostic kit for Senior-Loken syndrome type 5. Specific primers are designed based on the upstream and downstream sequences of the mutation site of gene IQCB1, and the primers are used to amplify a DNA fragment containing the mutation site, and the genotype of the DNA fragment is used to determine whether there is a risk of Senior-Loken syndrome type 5.

[0060] In the present invention, the method for assisting diagnosis of Senior-Loken syndrome type 5 using the kit preferably comprises the following steps:

[0061] The kit is used to detect the genotype of the IQCB1 gene mutation site in the sample to diagnose whether the individual suffers from Senior-Loken syndrome type 5:

[0062] If the detected genotype is "c.691C>T homozygous mutation", the individual is diagnosed as Senior-Loken syndrome type 5;

[0063] If the detected genotype contains a single heterozygous mutation "c.691C>T heterozygous mutation", the individual is a carrier;

[0064] If the detected genotype is "wild type", the subject is diagnosed as a normal person.

[0065] Furthermore, the detection sample of the detection reagent includes blood and / or amniotic fluid.

[0066] The present invention is further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are generally carried out according to conventional conditions such as those described in Sambrook et al., Molecular Cloning A LABORATORY MANUAL 1 SECON D EDITION; New York: Cold Spring Harbor Laboratory Press, 2014, or according to the conditions recommended by the manufacturer.

[0067] Example 1

[0068] Sample acquisition

[0069] The inventors discovered a Senior-Loken syndrome type 5 pedigree (referred to as Family 1). The clinical information of some members of this pedigree is shown in Table 1. Figure 1 Family tree, including Indicates male carriers, indicates a female carrier, ■ indicates a male patient, ◇ indicates a fetus, and ↗ indicates a proband.

[0070] 1. Diagnostic criteria:

[0071] Please refer to the 2010 edition of Human Single Gene Inherited Diseases:

[0072] The disease can have two specific symptoms: one is a kidney symptom called nephronophthisis, and the other is Leber congenital amaurosis

[0073] Eye symptoms of Leber congenital amaurosis. Nephronophthisis causes fluid-filled cysts to develop in the kidneys beginning in childhood, impairing kidney function, initially causing polyuria, malaise and extreme fatigue, leading to end-stage renal disease in late childhood or adolescence. Leber congenital amaurosis primarily affects the retina, causing vision problems, including photophobia, nystagmus and extreme farsightedness. Clinical diagnostic criteria for Leber congenital amaurosis: 1) Children under 6 months of age with severe visual impairment or blindness, accompanied by symptoms such as fixation disorders, nystagmus or eye pressure; 2) Electroretinogram showing severe reduction of a and b waves, or even extinguished type; 3) Diffuse pigment granule deposition in the retina, presenting osteocyte-like or salt-and-pepper-like changes.

[0074] Table 1 Clinical information of the members of the pedigree No. 1 of Senior-Loken syndrome type 5

[0075]

[0076] like Figure 1As shown, the numbering is Ⅰ (first generation) and Ⅱ (second generation).

[0077] Peripheral blood DNA from family members Ⅰ1 (father of the proband), Ⅰ2 (mother of the proband), Ⅱ1 (proband) and amniotic fluid DNA from Ⅱ2 (fetus) were used for sequencing analysis.

[0078] Example 2

[0079] Exome sequencing

[0080] 1. Instruments and equipment are shown in Table 2.

[0081] Table 2 List of instruments and equipment

[0082]

[0083] 2. Reagents and consumables

[0084] Human whole exome sequencing kit (Agilent), DNA 1000 kit (Agilent), 96-well plate (Axygen), different types of pipette tips (Axygen), 200 μL centrifuge tubes (Eppendorf), 1.5 mL centrifuge tubes (Eppendorf), capillary electrophoresis buffer (Thermo), sequencing standards (Thermo), anhydrous ethanol (Thermo), BigDye Terminator V3.1 (Thermo), peripheral blood gDNA extraction kit (TIANGEN), agarose (TIANGEN), EB staining solution (Amresco).

[0085] 3. Reagent Formulation

[0086] 5×TBE electrophoresis buffer stock solution was prepared according to Table 3.

[0087] Table 3 5×TBE electrophoresis buffer formula

[0088] Reagents Tris Boric acid EDTA (pH 8.0, 0.5 mol / L) <![CDATA[ddH2O]]> Volume / Weight 5.4g 750mg 2mL 90mL

[0089] Adjust the final volume to 100 mL with ddH2O.

[0090] 0.5×TBE electrophoresis working solution can be diluted 10 times with ddH2O.

[0091] 10× red blood cell lysis buffer was prepared according to Table 4.

[0092] Table 4 10× red blood cell lysis buffer formula

[0093] Reagents <![CDATA[NH4Cl]]> <![CDATA[KHCO3]]> EDTA <![CDATA[Add ddH2O]]> Volume / Weight 82.9g 10g 0.37g Up to 1000mL

[0094] Autoclave and store at 4°C.

[0095] 1× cell nuclear lysis buffer was prepared according to Table 5.

[0096] Table 5 1× Cell Nuclear Lysis Solution Formula

[0097] Reagents 2M Tris-HCl, pH 8.2 4M NaCl 2mM EDTA Volume / Weight 0.5mL 10mL 0.4mL

[0098] 4. Experimental Procedure

[0099] After the informed consent was signed, 3-5 mL of peripheral blood and 10-20 mL of amniotic fluid were collected from members Ⅰ1 (father of the proband), Ⅰ2 (mother of the proband), and Ⅱ1 (proband) of family No. 1.

[0100] 4.1 Sample DNA extraction

[0101] 1) Place 3-5 mL of the sample into a 15 mL centrifuge tube, add 2-3 times the volume of 1× red blood cell lysis buffer, mix well, and place on ice for 30 minutes until the solution becomes transparent.

[0102] 2) Centrifuge at 4℃, 3000 rpm for 10 minutes, and carefully remove the supernatant. Add 1mL 1× nuclear lysis buffer to the precipitate, mix well, then add 2mL 1× nuclear lysis buffer and 150μL 20% SDS, shake well until it becomes viscous and transparent. Add 10μL 20mg / mL proteinase K, shake well. Digest at 37℃ for more than 6 hours or overnight.

[0103] 3) Add an equal volume of saturated phenol, shake gently to mix, and centrifuge at 3000 rpm for 10 minutes at room temperature.

[0104] 4) Carefully transfer the supernatant to another centrifuge tube, add an equal volume of phenol / chloroform (1:1 v / v) and mix well, and centrifuge at 3000 rpm for 10 minutes at room temperature.

[0105] 5) Carefully transfer the supernatant. If the supernatant is not clear and transparent, extract again with an equal volume of chloroform.

[0106] 6) Transfer the supernatant to another centrifuge tube, add two times the volume of anhydrous ethanol, shake well, and white flocculent DNA will be seen. Use a flame-sterilized glass hook to hook out the DNA, wash twice with 70% ethanol, dry at room temperature for 5 minutes, and then dissolve the DNA in 200 μL 1×TE and dissolve overnight in a drum. Measure the OD value under UV.

[0107] 7) DNA dissolved in TE can be stored at 4°C for one year. If long-term storage is required, add 2 times the volume of anhydrous ethanol and store at -70°C.

[0108] 4.2 Exome sequencing

[0109] Refer to the instructions of the Human Whole Exome Sequencing Kit (Agilent) and the operating instructions of the Molecular Cloning A LABORATORY MANUAL 1 SECOND EDITION; New York: Cold Spring Harbor Laboratory Press, 2014.

[0110] 1) Take 2 μg DNA, mechanically shear it to a fragment size of about 200 bp, and cut the gel to recover 150-250 bp fragments;

[0111] 2) DNA fragments are end-repaired and 3' end A is added;

[0112] 3) Connect the sequencing adapter, purify the connection product, perform PCR amplification, and purify the amplified product;

[0113] 4) The probe of the Agilent kit is added to the purified amplified product for hybridization capture. The hybridized product is eluted and recovered for PCR amplification. The final product is then recovered and a small sample is taken for agarose gel electrophoresis for quality control analysis.

[0114] 5) NextSeq500 sequencer sequencing and data analysis.

[0115] 4.3 Results

[0116] Finally, a homozygous mutation of the gene with pathogenic significance was obtained, IQCB1:NM_001023571.3:exon8:c.691C>T:p.R231*; the c.691C>T mutation was a mutation of the 691st base of exon 8 from G to T, resulting in a nonsense mutation. The genotype of the IQCB1:NM_001023571.3:exon8:c.691C>T:p.R231* site in the patient (proband) of family No. 1 was a homozygous mutation of "c.691C>T"; the genotype of the site in the carrier of family No. 1 was a heterozygous mutation of "c.691C>T".

[0117] Example 3

[0118] Sanger sequencing validation

[0119] The exome sequencing results were further verified by Sanger sequencing for the IQCB1: NM_001023571.3: exon8: c.691C>T: p.R231* site. The genotype of the IQCB1: NM_001023571.3: exon8: c.691C>T: p.R231* site was tested for 4 persons, including Ⅰ1 (father of the proband), Ⅰ2 (mother of the proband), Ⅱ1 (proband), and Ⅱ2 (fetus) in line 1 in Example 1 and 100 normal persons outside the family.

[0120] The specific steps are as follows:

[0121] 1. DNA extraction

[0122] The genomic DNA was extracted according to the method of Example 1.

[0123] 2. Design, verification and optimization of candidate primers

[0124] 2.1 Candidate primer design was based on the human genome sequence database hg19 / build36.3 (https: / / www.ncbi.nlm.nih.gov / genome, or http: / / genome.ucsc.edu / cgi-bin / hgGateway?redirect=manual&source=genome.ucsc.edu).

[0125] 2.2 19 pairs of candidate primers were designed for the c.691C>T site (see Table 6), and PCR experiments were used to verify and evaluate the advantages and disadvantages of each pair of candidate primers.

[0126] Table 6 Basic information of each pair of candidate primers and the results of verification experiments

[0127]

[0128]

[0129]

[0130] Note: Normal PCR amplification results only have one specific band after electrophoresis. If primer dimer bands or non-specific product bands appear, they are the result of abnormal primer reactions. Target primers should avoid such situations as much as possible. In addition, refer to the following principles to comprehensively evaluate and select the optimal primer pair:

[0131] ① The primer length is 15 to 30 nt, usually around 20 nt;

[0132] ② The G+C content should be 40% to 60%. Too little G+C will result in poor amplification, while too much G+C will result in non-specific bands. ATGC is best distributed randomly;

[0133] ③ Avoid references to strings of more than 5 purine or pyrimidine nucleotides;

[0134] ④ There should be no complementary sequence inside the primer;

[0135] ⑤ There should be no complementary sequences between the two primers, especially avoid complementary overlap at the 3' end;

[0136] ⑥ The homology between the primer and the sequence of the non-specific amplification region should not exceed 70%, and the 8 consecutive bases at the 3' end of the primer cannot have completely complementary sequences outside the region to be amplified, otherwise it will easily lead to non-specific amplification.

[0137] 2.3 Candidate Primer PCR Verification Reaction

[0138] Carry out PCR according to the reaction system in Table 7 and keep the reaction system on ice; set up 8 reaction test tubes for each pair of primers (sequence numbers 1 to 8 in Table 7).

[0139] Table 7 Primer detection PCR reaction system

[0140]

[0141]

[0142] Reaction conditions: Place the above test reaction tube into the PCR instrument and perform the following reaction program:

[0143] Step 1: 95°C, 5 minutes;

[0144] Step 2: 30 cycles (95°C, 30 seconds → Tm, 30 seconds → 72°C, 60 seconds); (PCR amplification parameters were set according to the Tm values ​​of the primers in Table 6. If double primers were used, the average Tm value was taken);

[0145] Step 3: 72°C, 7 minutes;

[0146] Step 4: 4°C until sampling.

[0147] 2.4 The PCR results of candidate primers were tested by agarose gel electrophoresis to evaluate the effectiveness and specificity of the primer reaction:

[0148] 1) Seal both ends of the cleaned and dried gel sampler with tape, place it on a flat surface, and place a comb about 1 cm from one end of the sampler.

[0149] 2) Weigh 2 g of agar powder into a conical flask, add 100 mL of 0.5×TBE electrophoresis buffer, shake well, and heat in a microwave oven or electric stove (with asbestos mesh). After boiling, take out and shake well, and heat again until the gel is completely melted, then take out and cool at room temperature.

[0150] 3) When the gel cools to about 50°C, pour it into the sealed gel sampler to make the thickness about 5mm.

[0151] 4) After the gel solidifies, remove the tape and place the gel and sampler into the electrophoresis tank.

[0152] 5) Add electrophoresis buffer to make the liquid level 1-2mm above the gel surface, and pull out the comb upwards; use a micropipette to mix the sample and DNA size standard with the loading solution respectively, and add them to each well. Since the specific gravity of sucrose in the loading solution is relatively large, the DNA sinks to the bottom of the well.

[0153] 6) Cover the electrophoresis tank, turn on the power, adjust to the appropriate voltage, and start electrophoresis. According to the indication of bromophenol blue in the sample loading solution, determine the approximate position of the sample and decide whether to terminate the electrophoresis.

[0154] 7) Turn off the power, take out the gel, and place it in a 0.5 g / mL EB aqueous solution for staining for 10-15 minutes.

[0155] 8) Place the gel under a transmission UV irradiator to observe the results at a wavelength of 254 nm, and take photos with a camera equipped with a red filter or use a gel scanning system to record the electrophoresis results.

[0156] 2.5 Result evaluation:

[0157] 1) If only one bright and clear target band appears in tube 7 and no other bands are present, it is judged that the primer pair and reaction system have good effectiveness and strong specificity;

[0158] 2) If no target band appears in tube 7, the primer pair and reaction system are considered invalid;

[0159] 3) If a primer dimer band other than the target band appears in tube 7, and also appears in some tubes 2, 3, 4, 5, and 6, it is judged that the effectiveness of the primer pair and reaction system is poor;

[0160] 4) If a non-specific band other than the target band appears in tube 7, and non-specific bands also appear in some tubes 5 and 6, it is judged that the primer pair and reaction system have poor specificity;

[0161] 5) If primer dimers and non-specific bands outside the target band appear in tube 7, and primer dimers and non-specific bands also appear in some tubes 2, 3, 4, 5, and 6, it is judged that the effectiveness and specificity of this pair of primers and reaction system are poor.

[0162] 2.6 According to the statistical results after the verification test in Table 6, the best pair (candidate primer pair No. 1 in Table 6) is selected as the primer for mutation family detection:

[0163] The PCR amplification primer sequences for the IQCB1:NM_001023571.3:exon8:c.691C>T:p.R231* site are as follows:

[0164] IQCB1-1F:5'-ACAGCAGCAGATGACAAGC-3' (SEQ ID NO.1);

[0165] IQCB1-1R: 5'-GGAGATGAAGTGAAATAAAGG-3' (SEQ ID NO. 2).

[0166] Other candidate primers were discarded because they had hairpin structures, primer dimers, or nonspecific binding amplification, resulting in poor PCR results.

[0167] 3. PCR amplification of mutation sites in family members No. 1 and 100 non-family members

[0168] Carry out PCR according to the reaction system in Table 8 and keep the reaction system on ice.

[0169] Table 8 Mutation site PCR reaction system

[0170] Reagents volume 10× PCR buffer 2.0μL 10mmol / L dNTPs 0.4μL 100ng / μL IQCB1-1F 0.5μL 100ng / μL IQCB1-1R 0.5μL 100ng / μL DNA extraction 1.0μL 5U / μL Taq enzyme 0.2μL <![CDATA[ddH2O]]> 15.4μL

[0171] Reaction conditions: Place the reaction system into the PCR instrument and perform the following reaction procedure:

[0172] The PCR amplification procedure for the IQCB1:NM_001023571.3:exon8:c.691C>T:p.R231* site is as follows:

[0173] Step 1: 95°C, 5 minutes;

[0174] Step 2: 30 cycles (95°C, 30 seconds → 54°C, 30 seconds → 72°C, 60 seconds);

[0175] Step 3: 72°C, 7 minutes;

[0176] Step 4: 4°C until sampling.

[0177] 4. Agarose gel electrophoresis detection

[0178] Refer to step 2.4 above.

[0179] 5. Enzymatic purification of PCR products: Add 0.5 μL of exonuclease I (Exo I) and 1 μL of alkaline phosphatase (AIP) to 5 μL of PCR product, digest at 37°C for 15 minutes, and inactivate the enzyme at 85°C for 15 minutes.

[0180] 6. BigDye reaction

[0181] The BigDye reaction system is shown in Table 9.

[0182] Table 9 BigDye reaction system

[0183] Reagents Dosage PCR product purified DNA 2.0μL 3.2pmol / μL sequencing primer 1.0μL BigDye 0.5μL 5×BigDye sequencing buffer 2.0μL <![CDATA[ddH2O]]> 4.5μL

[0184] Sequencing PCR cycling conditions:

[0185] Step 1: 96°C, 1 minute;

[0186] Step 2: 33 cycles (96°C, 30 sec → 55°C, 15 sec → 60°C, 4 min);

[0187] Step 3: 4°C until sampling.

[0188] 7. Purification of BigDye reaction products:

[0189] 1) Add 1 μL 125 mM EDTA (pH 8.0) to each tube until it reaches the bottom of the tube, then add 1 μL 3 mol / L NaAc (pH 5.2);

[0190] 2) Add 70 μL of 70% alcohol, shake and mix 4 times, and leave at room temperature for 15 minutes;

[0191] 3) Centrifuge at 3000g, 4°C for 30 minutes; immediately invert the 96-well plate and centrifuge at 185g for 1 minute;

[0192] 4) Leave at room temperature for 5 minutes to allow the residual alcohol to evaporate, add 10 μL Hi-Di formamide to dissolve the DNA, denature at 96°C for 4 minutes, quickly place on ice for 4 minutes, and sequence on the machine.

[0193] 8. Sequencing

[0194] The purified BigDye reaction product was subjected to DNA sequencing.

[0195] Sequencing primers are designed based on the above-mentioned preferred PCR primers as nested primers (the second set of primers is designed within the product sequence range amplified by the first set of primers) as sequencing primers:

[0196] The sequencing primer sequences for the IQCB1:NM_001023571.3:exon8:c.691C>T:p.R231* site are as follows:

[0197] IQCB1-Seq1F:5'-GTTGCTGGAGATAAATAGGC-3' (SEQ ID NO.3);

[0198] IQCB1-Seq1R: 5'-GGGAGATTGTTCATCGTCA-3' (SEQ ID NO. 4).

[0199] 9. Results Analysis

[0200] Figure 2 The Sanger sequencing results showed that the genotype of the IQCB1:NM_001023571.3:exon8:c.691C>T:p.R231* site of a person in family 1 was the homozygous mutation "c.691C>T". Figure 2 The position indicated by the arrow in the sequencing diagram shows that the genotype of the IQCB1:NM_001023571.3:exon8:c.691C>T:p.R231* site in the D layer is the homozygous mutation of "c.691C>T"; Figure 2 The position indicated by the arrow in the sequencing diagram shows that the genotype of the IQCB1:NM_001023571.3:exon8:c.691C>T:p.R231* site in the A, B and C layers is the "c.691C>T" heterozygous mutation.

[0201] Example 4

[0202] Senior-Loken syndrome type 5 diagnostic kit and its application

[0203] 1. Kit composition:

[0204] 1) Amplification primers (1.2 μg each): as shown in Example 3;

[0205] 2) Buffer (500 μL of 10× PCR buffer: 500 mmol / L KCl, 100 mmol / L Tris-Cl (pH 8.3), 15 mmol / L MgCl2);

[0206] 3) Taq enzyme (20 U);

[0207] 4) dNTPs (4 mM each of four dNTPs);

[0208] 5) IQCB1: c.691C>T positive mutation reference DNA, the positive reference is a double-stranded DNA, the specific sequence is as follows: (SEQ ID NO.7). The single underlined bases are the positions of the upstream and downstream primers for PCR amplification, the double underlined bases are the positions of the upstream and downstream primers for sequencing primers, the bases in the boxes are the mutation sites, the lowercase letters are the sequences of the intron region, and the uppercase letters are the sequences of the exon region.

[0209] 6) Sequencing primers: as shown in Example 3.

[0210] 2. Usage:

[0211] A total of 119 individuals from 31 families with eye diseases were screened and tested, and 19 patients and 38 carriers from 16 families were found again (see Table 10). This kit is now used to test patients from family No. 2 as an example (see Table 11).

[0212] Table 10. Screening status of Senior-Loken syndrome type 5

[0213]

[0214]

[0215] *: This family had a successful pregnancy after preimplantation genetic diagnosis and embryo transfer, and prenatal diagnosis confirmed that the fetus was a normal individual.

[0216] #: One of the allele mutations is a new mutation.

[0217] Table 11 Clinical information of the members of the second family with Senior-Loken syndrome type 5

[0218]

[0219]

[0220] like Figure 3 As shown, the numbering is Ⅰ (first generation) and Ⅱ (second generation).

[0221] The peripheral blood DNA of members Ⅰ1, Ⅰ2, Ⅱ1 and Ⅱ2 of family No. 2 was used for test kit detection.

[0222] 1) Genomic DNA extraction: Extract genomic DNA from samples.

[0223] 2) First, the PCR amplification reaction is performed using the above-mentioned PCR amplification primers, Taq enzyme, buffer, dNTPs, sample genomic DNA, etc., as in Example 3;

[0224] 3) Purify the PCR amplification product;

[0225] 4) using the above sequencing primers to perform BigDye reaction on the purified PCR product;

[0226] 5) Purification of BiyDye reaction products;

[0227] 6) Sequence the BiyDye reaction product and compare the sequence with the normal sequence.

[0228] Figure 4 The test results of the kit showed that the genotype of the proband of Family No. 2 at the IQCB1:NM_001023571.3:exon8:c.691C>T:p.R231* site was a homozygous mutation of "c.691C>T". Figure 4 The position indicated by the arrow in the sequencing diagram shows that the genotype of the IQCB1:NM_001023571.3:exon8:c.691C>T:p.R231* site in layer A is the homozygous mutation of "c.691C>T" (one mutation is inherited from his father and the other is inherited from his mother); Figure 4 The position indicated by the arrow in the sequencing diagram shows that the genotype of individuals in layers C and D is a heterozygous mutation of "c.691C>T", and the genotype of individuals in layer B is "wild type". The test results confirmed that the proband is a patient with Senior-Loken syndrome type 5, and his father and mother are carriers of the mutant gene; the probability of the proband's parents giving birth to the same patient again is 1 / 4, the probability of giving birth to a carrier is 1 / 2, and the probability of giving birth to a normal individual is 1 / 4.

[0229] Example 5

[0230] Gene mutation rating and interpretation (pathogenicity of mutations)

[0231] Mutation interpretation is based on our current understanding of Senior-Loken syndrome type 5 and the pathogenic gene IQCB1 (https: / / www.omim.org / entry / 609254), and the clinical phenotype association of the test results with the subjects. Mutations follow the HGVS guidelines for mutation nomenclature (http: / / www.hgvs.org / ) and are named according to the GenBank accession number (https: / / www.ncbi.nlm.nih.gov / genbank / ). The rules for interpreting genetic variation data refer to the relevant guidelines of the 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; and China's "Standards and Guidelines for the Classification of Genetic Variations": Wang Qiuju, Shen Yiping, Wu Lingqian, et al. Standards and Guidelines for the Classification of Genetic Variations. Science China: Life Sciences, 2017, 47: 668–688.

[0232] The classification of genetic variation in the "Genetic Variation Classification Standards and Guidelines" is based on typical data types (such as population data, computational data, functional data, and co-segregation data) to classify the variation into five levels: "pathogenic,

[0233] The following are the most common phenotypes: "likely pathogenic (P)", "likely pathogenic (LP)", "variant of uncertain significance (VUS)", "likely benign (LB)" and "benign (

[0234] B)”; the five-level classification is based on the comprehensive score after interpreting and analyzing each aspect / sub-item of the variation (Table 12).

[0235] Table 12 Criteria for determining pathogenicity of variants

[0236]

[0237] Before the five-level assessment, each aspect / sub-item of the mutation / variation needs to be analyzed / interpreted. Among them, the pathogenic variation criteria can be divided into: very strong (PVS1), strong (PS1~4), moderate (PM1~6), or supporting evidence (PP1~5; benign variation evidence can be divided into: stand-alone (BA1), strong (BS1~4), or supporting evidence (BP1~6) (see Table 13 below). For a given mutation / variation, it is first necessary to select the criteria in Table 13 based on the observed evidence to determine whether the various aspects / sub-items of the mutation / variation can meet the criteria. Which of the items in Table 13 is each rated as PVS1 / PS1~4 / PM1~6 / PP1~5 / BA1 / BS1~4 / BP1~6? Finally, the sub-item standards that can be met are combined according to the scoring rules in Table 12, and then a classification is selected from the five-level system according to the combination standards in Table 12; for example, if the aspects / sub-items of the mutation have PVS1, PM1, and PM2 after analysis, the mutation is rated as "pathogenic" [that is, it meets the criteria (i) + (b) in "Pathogenic (P)" in Table 12].

[0238] Table 13. Variant interpretation criteria and variant pathogenicity determination criteria

[0239]

[0240]

[0241]

[0242] The analysis / interpretation of each aspect / sub-item of mutation / variation needs to be based on the corresponding bioinformatics analysis tools (see Table 15) and a large number of available data (libraries) (see Table 16), including data obtained from existing cases and published data, such as public databases (such as ClinVar or site-specific databases) and laboratory-owned databases. When using various data (libraries) to analyze mutations / variations, the degree judgment evaluation criteria used are shown in Table 14.

[0243] Table 14 Evaluation criteria for degree judgment

[0244]

[0245]

[0246]

[0247] Table 15 Bioinformatics analysis tools

[0248]

[0249]

[0250] Table 16 Population databases, disease-specific databases and sequence databases

[0251]

[0252]

[0253] According to the above standards or criteria, the IQCB1 gene c.691C>T mutation in the present invention is evaluated as "pathogenic", and the judgment criteria and specific evidence are as shown in Table 17:

[0254] Table 17 Interpretation of pathogenicity of IQCB1 gene c.691C>T mutation

[0255]

[0256] AR: refers to autosomal recessive inheritance.

[0257] IQCB1:NM_001023571.3:exon8:c.691C>T:p.R231* Variant rating evidence is as follows:

[0258] 1. PVS1: The c.691C>T mutation in the IQCB1 gene is a nonsense mutation, which affects the function of the truncated protein (the mutation occurs in exon 8. The IQCB1 gene has 15 exons, which means that 7 exons from 9 to 15 are truncated);

[0259] 2. PS2: The variant was confirmed as a new variant in one family analysis after testing of the proband's parents and other family members (see Table 10);

[0260] 3. PS4: Based on the literature and this case, this variant was detected in multiple patients (19) (see Table 10);

[0261] 4. PM2: The c.691C>T variant of the IQCB1 gene was not found in the 1000 Genomes (1000G) of the reference population, the Exome Database (ExAC), and the GnomAD database;

[0262] 5. PM3: For recessive genetic diseases, the pathogenic variant has been detected in 20 patients at the trans position of the variant (on the other homologous chromosome) (i.e., homozygous mutation - the variant is detected on both chromosomes);

[0263] 6. PP3: Multiple computer software predicts that the variant will have a deleterious effect on the gene or gene product;

[0264] Therefore, the comprehensive evidence of this mutation / variation (PVS1+PS2+PS4+PM2+PM3+PP3) meets the "pathogenic (P)" judgment criteria (i) or (ii) in Table 12, and the IQCB1 gene c.691C>T variation is comprehensively judged to be "pathogenic".

[0265] Example 6

[0266] Follow-up and diagnostic kit performance analysis

[0267] All family members were followed up, and the IQCB1 gene targeted capture chip method was resequencing analysis and validation for all individuals (see Table 18).

[0268] Table 18 Performance analysis results of c.691C>T site detection

[0269] index formula result True positive variant sites (TP) / 61 True negative variant site (TN) / 10 False positive variant sites (FP) / 0 False negative variant site (FN) / 0 Sensitivity (N=58) TP / (TP+FN) 100% (95% CI: 99.03% ~ 100%) Specificity (N=10) 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%)

[0270] Note: This table contains follow-up data for Family 1; all variants detected in patients and carriers were positive.

[0271] Combining Table 1 and Table 10, it can be seen that positive patients (20 cases) and carriers (41 cases) were found when testing 17 families. The above positive site detection results were verified by the IQCB1 gene targeted capture chip method. According to the follow-up and verification results, a total of 61 true positive cases, 10 true negative cases, 0 false negative cases and 0 false positive cases were found. The sensitivity of the c.691C>T variant site marker for detection was 100.00%, and its 95% CI (95% confidence interval) was 99.03%~100%, and the specificity was 100%, and its 95% CI was 99.03%~100%. The above results show that this kit has good detection performance in clinical application.

[0272] It can be seen from the results of the above examples that the present invention has discovered a new IQCB1 gene mutant and confirmed that the new mutant is closely related to the onset of Senior-Loken syndrome type 5. The pathogenic mutant can be used for the molecular diagnosis of Senior-Loken syndrome type 5 and the differential diagnosis of related diseases.

[0273] The above technical solutions of the present invention are only preferred embodiments of the present invention, and the patent scope of the present invention is not limited thereto. All equivalent structural changes made by using the contents of the present invention specification and drawings under the technical concept of the present invention, or directly / indirectly applied in other related technical fields are included in the patent protection scope of the present invention.

Claims

1. A pathogenic gene for oculorenal syndrome type 5, characterized in that: Compared with the wild-type IQCB1 gene, the pathogenic gene has a mutation from base C to base T at base 691 in exon 8 of the wild-type IQCB1 gene.

2. A detection reagent for oculorenal syndrome type 5 caused by the pathogenic gene of claim 1, characterized in that: The detection reagent includes primers or sequencing primers designed for the site of the gene mutation.

3. The detection reagent according to claim 2, characterized in that: The primers include IQCB1-1F and IQCB1-1R. The nucleotide sequence of IQCB1-1F is shown in SEQ ID NO.1, and the nucleotide sequence of IQCB1-1R is shown in SEQ ID NO.

2.

4. The detection reagent according to claim 2, characterized in that The sequencing primers include IQCB1-Seq1F and IQCB1-Seq1R, the nucleotide sequence of IQCB1-Seq1F is shown in SEQ ID NO.3, and the nucleotide sequence of IQCB1-Seq1R is shown in SEQ ID NO.

4.

5. A detection kit for oculorenal syndrome type 5, characterized in that: Comprising the detection reagent of claim 3 or 4.

6. The detection kit according to claim 5, characterized in that: Also included are PCR amplification reagents; the PCR amplification reagents include dNTPs, 10×PCR buffer, magnesium ions and Taq polymerase.

7. Use of the detection reagent according to any one of claims 2 to 4 in a kit and its preparation.

8. The use according to claim 7, characterized in that: The detection sample of the detection reagent includes blood and / or amniotic fluid.

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