CN116004789A

CN116004789A - Application of CEP152 mutation site of Seckel syndrome pathogenic gene and diagnostic reagent thereof

Info

Publication number: CN116004789A
Application number: CN202210915749.3A
Authority: CN
Inventors: 曾桥; 刘亚宁; 伊宁
Original assignee: Hunan Jiahui Biotechnology Co Ltd
Current assignee: Hunan Jiahui Biotechnology Co Ltd
Priority date: 2022-08-01
Filing date: 2022-08-01
Publication date: 2023-04-25
Anticipated expiration: 2042-08-01
Also published as: CN116004789B

Abstract

The invention provides an application of a CEP152 mutation site of a Seckel syndrome pathogenic gene and a diagnostic reagent thereof, belonging to the technical field of gene diagnosis. The invention confirms for the first time that the gene CEP152 is compounded with heterozygous mutation CEP152:NM_001194998.2:exon9:c.1060C > T:p.R354 and exon12:c.1414-14A > G; the composite heterozygous mutation leads to the onset of Seckel syndrome, and the CEP152 gene composite heterozygous mutation site is applied to the preparation of a Seckel syndrome diagnostic reagent or the preparation of a medicine for preventing and treating Seckel syndrome. The detection reagent aiming at the mutation site can be used for rapidly and effectively predicting or diagnosing Seckel syndrome, provides a brand-new theoretical basis for subsequent treatment, and also provides a possible drug target for treating the Seckel syndrome.

Description

Application of CEP152 mutation site of Seckel syndrome pathogenic gene and diagnostic reagent thereof

Technical Field

The invention belongs to the technical field of gene diagnosis, and particularly relates to application of a CEP152 mutation site of a Seckel syndrome pathogenic gene and a diagnostic reagent thereof.

Background

Seckel syndrome (MIM 613823) is also known as Seckel syndrome, bird head dwarfism, bird head-dwarfism, small head syndrome, virchow-Seckel syndrome, original small head malformation dwarfism and the like, and Seckel in 1950 is reported first on bird head dwarfism, so the Seckel syndrome is called. The disease is an autosomal recessive genetic disease, both sexes can be affected, the disease is symptomatic at birth, the weight is light at birth, the growth is slow, the main clinical characteristics of the disease are symmetrically dwarfing, premature senility, obvious beak-like nose and jaw (bird-like face), small head, mental retardation, sweating tendency, hip dislocation, rod-like feet, thumb deficiency, cloaca-like deformity of genitourinary tract and rectum, hair rarity and early white, palmoplegia increase, palm skin redundancy and wrinkles, subcutaneous tissue deficiency, low-order ear and auricle deformity, post-hairline low, eyelid sagging, cryptorchism, light sensitivity and increase of histidine level in urine.

Seckel syndrome is one of the primary microcephaly malformations, also known as true microcephaly malformations or autosomal stealth genetic microcephaly malformations (autosomal recessive primary microcephaly, MCPH), a neurological developmental disorder characterized primarily by decreased head circumference with some degree of non-progressive intellectual degradation. MCPH is currently widely recognized as a disease of polygenic stealth inheritance. Several MCPH-related gene loci have been found to date, such as CEP152, microcephalin, WDR62, CDK5RAP2, ASPM, CNPJ, STIL, etc.

Wherein CEP152 (MIM 613529) is the causative gene of Seckel syndrome. The CEP152 gene is located on chromosome 15q21.1, is 73.1kb in length, contains 27 exons and 26 introns, encodes a protein consisting of 1710 amino acids, human centrosome protein 152 (CEP 152), designated CEP152 because of its relative molecular weight of 152kD, which is involved in centrosome and flagellum formation. In cells, CEP152 is positioned in the pericentrosomal mass PCM, the C-terminal end of CEP152 is positioned in the centrosomal peripheral cartridge wall and the N-terminal end is radially outward, spanning the middle to the inner region of the PCM. CEP152 is an important regulator of centrosome formation and centrosome function, and is essential for centrosome formation and centrosome replication, CEP152 is involved in the initiation of centrosome replication. The role played by CEP152 in cilia formation is also highly conserved. The mutation of the gene causes abnormal or missing function of CEP152, thereby preventing centrosome replication and cell division, and premature closure of skull bone suture, so that the skull can not continue to grow, thereby pressing brain tissues, increasing intracranial pressure and affecting brain development.

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 Seckel syndrome. Establishing corresponding detection techniques for different mutations is a necessary means for the comprehensive diagnosis of Seckel syndrome.

Disclosure of Invention

Therefore, the invention aims to provide an application of novel Seckel syndrome pathogenic gene CEP152 composite heterozygous mutation c.1060C > T and c.1414-14A > G in preparation of Seckel syndrome diagnostic reagent or preparation of medicine for preventing and treating Seckel syndrome.

The invention also aims to provide a reagent for diagnosing the Seckel syndrome genes, which can accurately detect the genotype of mutation sites and provides an effective tool for diagnosing whether an individual suffers from the Seckel syndrome.

The invention provides an application of Seckel syndrome pathogenic gene CEP152 compound heterozygous mutation c.1060C > T and c.1414-14A > G in preparation of Seckel syndrome diagnostic reagent or preparation of medicine for preventing and treating Seckel syndrome.

The invention provides a reagent for diagnosing Seckel syndrome genes, which is a primer for amplifying a pathogenic gene CEP152 compound heterozygous mutation c.1060C > T and c.1414-14A > G, and comprises CEP152-1F and a nucleotide sequence shown as SEQ ID NO. 1, CEP152-1R and a nucleotide sequence shown as SEQ ID NO. 2, CEP152-2F and a nucleotide sequence shown as SEQ ID NO. 3 and CEP152-2R and a nucleotide sequence shown as SEQ ID NO. 4.

Preferably, the reagents include sequencing primers comprising CEP152-SEQ1F having a nucleotide sequence shown in SEQ ID NO. 5, CEP152-SEQ1R having a nucleotide sequence shown in SEQ ID NO. 6, CEP152-SEQ2F having a nucleotide sequence shown in SEQ ID NO. 7, and CEP152-SEQ2R having a nucleotide sequence shown in SEQ ID NO. 8.

Preferably, the reagents further comprise PCR amplification reagents.

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

Preferably, the diagnosis kit is used for detecting genotypes of gene mutation sites in a sample to diagnose whether an individual suffers from Seckel syndrome, wherein the gene mutation sites are c.1060C > T and c.1414-14A > G, and when the detection results are 'c.1060C > T heterozygous mutation' and 'c.1414-14A > G heterozygous mutation', the CEP152 gene is judged to exist, and when the individual is a patient, the individual is diagnosed to suffer from Seckel syndrome.

Preferably, the sample is blood and/or amniotic fluid.

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

The invention provides an application of Seckel syndrome pathogenic gene CEP152 compound heterozygous mutation c.1060C > T and c.1414-14A > G in preparation of Seckel syndrome diagnostic reagent or preparation of medicine for preventing and treating Seckel syndrome. The first gene CEP152 compound heterozygous mutation c.1060C > T and c.1414-14A > G can cause Seckel syndrome. The mutation site is thus used as a molecular marker for diagnosing Seckel syndrome. In one aspect, the method is used for screening or diagnosis of genetic Seckel syndrome to guide treatment by detecting whether a subject carries the mutation. In particular, the diagnostic kit provided by the invention can be used for rapidly and effectively predicting or diagnosing Seckel syndrome. On the other hand, the invention lays an important foundation for researching pathogenesis of Seckel syndrome and provides a brand new theoretical basis for treating Seckel syndrome patients. In a third aspect, the invention may provide a potential drug target for the treatment of Seckel syndrome.

The invention also provides a corresponding diagnosis kit for screening and diagnosing the power-assisted Seckel syndrome gene mutation, and provides a new technical support for drug screening, drug effect evaluation and targeted therapy.

Drawings

FIG. 1 is the Seckel syndrome No. 1 pedigree heritageAnd transmitting the map. Wherein, the utility model represents the male carrier,

representing female carriers, ■ for male patients, diamond-solid for diseased fetuses, ↗ for pre-evidence.

FIG. 2 is a graph showing the results of detecting the genotype of the CEP 152:NM-001194998.2:exo9:c.1060C > T:p.R354 locus of line 1 by Sanger sequencing, wherein the precursor, precursor mother and fetus of line 1 are heterozygous mutations of c.1060C > T (the arrows in the sequencing figures indicate the mutation occurrence positions).

FIG. 3 is a graph showing the results of detection of the genotype of the CEP 152:NM-001194998.2:exo12:c.1414-14A > G locus of family 1 by Sanger sequencing, showing that the ancestor, the male parent and the fetus of family 1 are heterozygous mutations of c.1414-14A > G (the position of the mutation is indicated by the arrow in the sequencing).

FIG. 4 is a genetic map of Seckel syndrome No. 2 family. Wherein, the utility model represents the male carrier,

representing female carriers, ■ male patients, +.female patients, ↗ forerunner.

FIG. 5 is a graph showing the results of the kit detection of genotype at position 2 line CEP 152:NM-001194998.2:exon 9:c.1060C > T:p.R354, with the first and second cases in line 2 both representing heterozygous mutations at position c.1060C > T (the position of the mutation indicated by the arrow in the sequencing).

FIG. 6 is a diagram showing the results of the detection of genotype at CEP 152:NM-001194998.2:exo12:c.1414-14A > G locus in line 2 by using the kit, wherein the first-evidence and the first-evidence are heterozygous mutations of c.1414-14A > G in line 2 (the position of the mutation is indicated by the arrow in the sequencing diagram).

Detailed Description

In the invention, the c.1060C > T mutation refers to that the 1060 th base of the 9 th exon of the wild CEP152 gene is mutated from C to T, so as to form a CEP152 gene mutant, and the nucleotide sequence of the CEP152 gene mutant is shown as SEQ ID NO. 11 (TTCAATGAGCT). Compared with the wild type CEP152 gene encoding protein, the CEP152 mutant protein provided by the invention has the advantages that the 354 th amino acid residue of the CEP152 protein is mutated from benzene arginine (R) to a stop codon (x), so that the truncated protein is 1357 amino acid residues less, namely, the CEP152 mutant protein contains p.R354 x mutation, and the mutation is caused by c.1060C > T mutation; the amino acid sequence of the CEP152 mutant protein is shown as SEQ ID NO. 12 (SESLQ).

The c.1414-14A > G mutation refers to that the base at the 11 th intron-14 which is adjacent to the 12 th exon of a wild CEP152 gene is mutated from A to G to form a CEP152 gene mutant, the nucleotide sequence of the CEP152 gene mutant is shown as SEQ ID NO. 13 (TAATAGTTTTG), and the mutation is positioned in the intron and can influence the intron cleavage, so that mRNA cleavage abnormality is caused.

The invention provides a reagent for diagnosing Seckel syndrome genes, which is a primer for amplifying a pathogenic gene CEP152 compound heterozygous mutation c.1060C > T and c.1414-14A > G, and comprises CEP152-1F and a nucleotide sequence shown as SEQ ID NO. 1, CEP152-1R and a nucleotide sequence shown as SEQ ID NO. 2, CEP152-2F and a nucleotide sequence shown as SEQ ID NO. 3 and CEP152-2R and a nucleotide sequence shown as SEQ ID NO. 4. The reagents preferably comprise sequencing primers comprising CEP152-SEQ1F having the nucleotide sequence shown in SEQ ID NO. 5, CEP152-SEQ1R having the nucleotide sequence shown in SEQ ID NO. 6, CEP152-SEQ2F having the nucleotide sequence shown in SEQ ID NO. 7 and CEP152-SEQ2R having the nucleotide sequence shown in SEQ ID NO. 8. The reagents preferably also include PCR amplification reagents. The PCR amplification reagents include, but are not limited to dNTPs, PCR buffers, magnesium ions, and Tap polymerase.

In the invention, the diagnosis method preferably uses the diagnosis kit to detect the genotype of a gene mutation site in a sample to diagnose whether an individual has Seckel syndrome, wherein the gene mutation site is c.1060C > T and c.1414-14A > G, and when the detection result is "c.1060C > T heterozygous mutation" and "c.1414-14A > G heterozygous mutation", the CEP152 gene is judged to have compound heterozygous mutation, and when the individual is a patient, the individual is diagnosed to have Seckel syndrome. The sample is preferably blood and/or amniotic fluid. 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 DNA sequencing to detect that the CT genotype exists at 1060 th base according to the sequencing result, and meanwhile, the genotype is 'CT heterozygous genotype' when the AG genotype exists at 1414 th base, if the genotype is 'c.1060C > T' or 'c.1414-14A > G', the genotype is 'CT heterozygous genotype', the genotype belongs to the wild type.

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

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 CEP152 gene means that the primer amplifies only the CEP152 gene and not the other genes in the PCR reaction.

The application of the CEP152 mutation site of Seckel syndrome pathogenic gene and its diagnostic reagents provided in the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.

Example 1

Sample acquisition

Applicants have found a Seckel syndrome family (abbreviated as family 1) with clinical information for members of the family 1 portion shown in Table 1. FIG. 1 shows a family pattern of accession number 1, wherein,

representing a male carrier, is->

1. Diagnostic criteria:

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

clinical manifestations: both sexuality can be affected, symptoms exist at birth, growth is retarded, dwarfism is obvious, and the nose and the jaw, the small head and the intelligence are low; palm folds are increased, palm skin is superfluous and wrinkled, and subcutaneous tissues are deficient; low ear and auricle deformity, low posterior hairline, etc.

X-ray examination: the distance between two eyes is widened, the upper jaw and the lower jaw are underdeveloped, the craniocerebral suture is early closed, and the teeth fall off or shrink; scoliosis and kyphosis; sternal deformity; dislocation of the small head of the radius, inward bending of distal phalanges and malformation of the phalanges; hip defect, tibiofibular joint defect and short fibula.

Histopathology: the skin showed slight irregular atrophy of the epidermis, shortening of the epithelial ridge and thinning of the stratum spinosum. The corner layers are in the shape of a basket. Basal layer has focal cell vacuole formation and cell layer structure destruction. The boundary between the epidermis and the dermis in some areas is blurred, and the dermis elastic tissue is denatured.

TABLE 1 clinical information of Seckel syndrome No. 1 family members

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

Family 1 personnel I1 (father), I2 (mother), II 1 (forerunner) peripheral blood and II 2 (fetus) amniotic fluid 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

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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 35 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
		NH ₄ Cl	82.9g
KHCO ₃	10g
		EDTA	0.37g
Adding dH ₂ O	To 1000mL

Autoclaving and storing at 4deg.C.

1 Xnuclear lysate was prepared according to Table 5.

Table 51 XNuclear lysate formula

Reagent(s)	Volume/weight
		2M Tris-HCl，pH 8.2	0.5mL
4M NaCl	10mL
		2mM EDTA	0.4mL

4. Experimental procedure

After signing the informed consent, 3-5mL of peripheral blood and 5-10 mL of amniotic fluid of I1 (father), I2 (mother), II 1 (forerunner) and other members in family 1 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. The amniotic fluid sample directly enters the next step.

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, 2 pathogenic gene mutations CEP152:NM_001194998.2:exon9:c.1060C > T:p.R354 and exon12:c.1414-14A > G are obtained; c.0C > T mutation to nonsense mutation, resulting in the 354 th arginine to stop codon, resulting in a truncated protein with 1357 amino acid residues less; the mutation 1414-14A > G is located at the junction of an intron and an exon, and can influence intron cleavage to cause mRNA cleavage abnormality. The genotypes of CEP 152:NM-001194998.2:exo9:c.1060C > T: p.R354 and exon12:c.1414-14A > G sites were the "c.1060C > T and c.1414-14A > G" composite heterozygous mutations in the patient individuals of family 1, and the genotypes were the "c.1060C > T" or "c.1414-14A > G" single heterozygous mutations in the carrier individuals of family 1.

Example 3

Sanger sequencing validation

The exome sequencing results were further verified using Sanger sequencing to verify CEP152: NM-001194998.2: exon9: c.1060C > T: p.R354 and exon12: c.1414-14A > G sites. CEP 152:NM_001194998.2:exo9:c.1060C > T:p.R354 and exon12:c.1414-14A > G loci genotype test was performed on line 1 personnel such as I1, I2, II 1, II 2, etc. and 100 extraline normal persons, respectively, in example 1.

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 candidate primer design reference human genome sequence database hg19/build36.3.

2.2 design 15 pairs of candidate primers for the c.1060C > T and c.1414-14A > G sites, respectively (see Table 6), and use PCR experiments to verify and evaluate the merits of each pair of candidate primers

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

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Note that: after electrophoresis, the normal PCR amplification result has only one specific band, and if the primer dimer band and the non-specific product band are all the results of abnormal reaction of the primer; the target primer avoids such a situation as much as possible. The optimal primer pairs were also comprehensively evaluated and selected with reference to the following principles:

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

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

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

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

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

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

2.3 candidate primer PCR verification reaction

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

TABLE 7 primer detection PCR reaction System

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

the first step: 95 ℃ for 5min;

and a second step of: 30 cycles (95 ℃,30sec→tm,30sec→72 ℃,60 sec); (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 ℃ for 7min;

fourth step: 4℃until sampling.

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

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

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

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

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

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

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

7) Cutting off the power supply, taking out the gel, putting the gel into an EB water solution with the concentration of 0.5g/ml, and dyeing for 10-15 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 according to the results of statistics after the verification test of Table 6, an optimal pair of primers (two mutation site No. 1 primers in Table 6) was selected as primers for detecting the mutant family, and the sequences of the primers are as follows:

the PCR primer sequences for CEP152: NM-001194998.2: exon9: c.1060C > T: p.R354. Site are as follows:

5’-CCATGATGAGCGGAGAC-3’(SEQ ID NO：1)

5’-GGGATTACAGGCGTGAG-3’(SEQ ID NO：2)

the PCR primer sequences for CEP152: NM-001194998.2: exon12: c.1414-14A > G sites are shown below:

5’-TAGTAAGCCAGAAAGCG-3’(SEQ ID NO：3)

5’-AATACCCAAATCCACAT-3’(SEQ ID NO：4)

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 CEP152-F	0.5μL
100ng/μL CEP152-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 placed in a PCR instrument.

The following reaction procedure was performed for CEP152:nm_001194998.2:exon9:c.1060c > t:p.r354:

the first step: 95 ℃ for 5 minutes;

and a second step of: 30 cycles (95 ℃,30 seconds- > 51 ℃,30 seconds- > 72 ℃,60 seconds);

and a third step of: 72 ℃,7 minutes;

fourth step: 4℃until sampling.

The following reaction procedure was performed for CEP 152:NM-001194998.2:exon 12:c.1414-14A > G:

the first step: 95 ℃ for 5 minutes;

and a second step of: 30 cycles (95 ℃,30 seconds- > 46 ℃,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/LNaAc (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 sequencing primer is used as a sequencing primer by designing a nest primer (a second primer set is designed within the range of the product sequence obtained by amplifying the first primer set) on the basis of the PCR preferred primer.

The primer sequences for CEP152:nm_001194998.2:exon9:c.1060c > t:p.r354 sites are as follows:

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

5’-TCATCCCAACCTGTTCTTTA-3’(SEQ ID NO：6)。

DNA sequencing of the purified BigDye reaction product was performed, and the primer sequences for CEP152: NM-001194998.2: exon12: c.1414-14A > G sites were as follows:

5’-TTTTGCCTCCTGTTGAT-3’(SEQ ID NO：7)

5’-TACCCAAATCCACATACGAT-3’(SEQ ID NO：8)。

9. analysis of results

The Sanger sequencing results of fig. 2 show that the genotype of the cen 152:nm_001194998.2:exon9:c.1060c > t:p.r354 locus of 3 persons of family 1 is a "c.1060c > T" heterozygous mutation. The position indicated by the arrow in the sequencing diagram of FIG. 2 shows B, C and D layers CEP152: NM-001194998.2: exon9: c.1060C > T: p.R354. Site genotype is "c.1060C > T" heterozygous mutation, in which the gene site of A layer is wild type.

The Sanger sequencing results of FIG. 3 show that the CEP 152:NM-001194998.2:exon 12:c.1414-14A > G site genotype of 3 persons of family 1 is a "c.1414-14A > G" heterozygous mutation. The position indicated by the arrow in the sequencing diagram of FIG. 3 shows A, C and the D layer individuals CEP152: NM-001194998.2: exo12: c.1414-14A > G site genotype is a "c.1414-14A > G" heterozygous mutation, and the B layer of the site genotype is wild type.

And judging that the first-evidence patient and the fetus are Seckel syndrome patients according to the results.

Example 4

CEP152 gene c.1060C > T, c.1414-14A > G composite heterozygous mutation diagnostic kit

1. The kit comprises the following components:

1) Amplification primers: as shown in example 3

2) Buffer solution

3) Taq enzyme

4)dNTPs

5) CEP152, c.1060C > T, c.1414-14A > G positive mutation reference DNA the reference is a double-stranded DNA, and the specific sequence of the c.1060C > T positive mutation reference is as follows:

wherein, single underlined base is the position of the primer at the upstream and downstream of PCR amplification, the base in the square frame is the point mutation site, and double underlined base is the position of the primer at the upstream and downstream of sequencing.

The specific sequences of c.1414-14A > G positive mutant references are shown below:

wherein, single underlined base is the position of the primer at the upstream and downstream of PCR amplification, base in the square frame is point mutation site, double underlined base is the position of the primer at the upstream and downstream sequencing, and wavy underlined base is the overlapping region of the primer at the downstream sequencing and the primer at the PCR.

6) Sequencing primer: as shown in example 3

2. The using method comprises the following steps:

the method is applied to family 2 patient detection.

TABLE 10 clinical information of Seckel syndrome No. 2 family members

As shown in FIG. 4, the numbers I (first generation), II (second generation) and III (third generation) are used.

The peripheral blood DNA of family members III 1, III 2 (precursor) was used for the detection 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.

The detection result of the kit in fig. 5 shows that the genotype of the position 2 of family 2 patients with CEP152:nm_001194998.2:exon9:c.1060c > t:p.r354 is the heterozygous mutation of "c.1060c > T". The position indicated by the arrow in the sequencing diagram of FIG. 5 shows that the A and B layers CEP 152:NM-001194998.2:exon 9:c.1060C > T:p.R354. Site genotypes are "c.1060C > T" heterozygous mutations.

The detection result of the kit of FIG. 6 shows that the genotype of CEP 152:NM_001194998.2:exo12:c.1414-14A > G site of 2 patients of family 2 is a heterozygous mutation of 'c.1414-14A > G'. The position indicated by the arrow in the sequencing diagram of FIG. 6 shows that the CEP 152:NM-001194998.2:exon 12:c.1414-14A > G site genotypes of individuals of the A and B layers are "c.1414-14A > G" heterozygous mutations. And confirming that the first evidence and the first evidence are Seckel syndrome patients according to the detection result.

From the results of the above examples, it can be seen that the present invention has found a novel CEP152 gene mutant, and confirmed that the novel mutant is closely related to the onset of sechel syndrome, which can be used for molecular diagnosis of sechel syndrome and differential diagnosis of related diseases.

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

Claims

1. Application of Seckel syndrome pathogenic gene CEP152 compound heterozygous mutation c.1060C > T and c.1414-14A > G in preparation of Seckel syndrome diagnostic reagent or preparation of medicine for preventing and treating Seckel syndrome.

2. A reagent for diagnosing Seckel syndrome gene is characterized in that the reagent is a primer for amplifying a pathogenic gene CEP152 composite heterozygous mutation c.1060C > T and c.1414-14A > G, and comprises CEP152-1F and a nucleotide sequence shown as SEQ ID NO. 1, CEP152-1R and a nucleotide sequence shown as SEQ ID NO. 2, CEP152-2F and a nucleotide sequence shown as SEQ ID NO. 3 and CEP152-2R and a nucleotide sequence shown as SEQ ID NO. 4.

3. The reagent of claim 2, wherein the reagent comprises a sequencing primer comprising CEP152-SEQ1F having the nucleotide sequence shown in SEQ ID No. 5, CEP152-SEQ1R having the nucleotide sequence shown in SEQ ID No. 6, CEP152-SEQ2F having the nucleotide sequence shown in SEQ ID No. 7, and CEP152-SEQ2R having the nucleotide sequence shown in SEQ ID No. 8.

4. A reagent according to claim 2 or 3, wherein the reagent further comprises a PCR amplification reagent.

5. Use of the reagent according to any one of claims 2 to 4 for the preparation of a diagnostic kit for Seckel syndrome.

6. The use of claim 5, wherein the diagnosis kit is used to detect the genotype of a mutation site of a gene in a sample, wherein the mutation site of a gene is c.1060C > T and c.1414-14A > G, and wherein when the detection result is "c.1060C > T heterozygous mutation" and "c.1414-14A > G heterozygous mutation", the presence of a complex heterozygous mutation in the CEP152 gene is determined, and wherein the individual is a patient, the individual is diagnosed as suffering from Seckel syndrome.

7. The use according to claim 6, wherein the sample is blood and/or amniotic fluid.

8. A diagnostic kit for sechel syndrome, comprising the reagent of claims 2 to 4.