CN116121365A

CN116121365A - Pathogenic gene causing COFS syndrome, detection and application

Info

Publication number: CN116121365A
Application number: CN202310300995.2A
Authority: CN
Inventors: 曾桥; 伊宁; 薛斌; 范园
Original assignee: Hunan Jiahui Biotechnology Co Ltd
Current assignee: Hunan Jiahui Biotechnology Co Ltd
Priority date: 2023-03-27
Filing date: 2023-03-27
Publication date: 2023-05-16
Anticipated expiration: 2043-03-27
Also published as: CN116121365B

Abstract

The invention provides a pathogenic gene, detection and application for causing COFS syndrome; the pathogenic gene is mutated from base G to base A at the last base of the 17 th intron of the wild type ERCC2 gene immediately adjacent to the 18 th exon and/or mutated from base G to base C at the 1922 nd base of the 21 st exon of the wild type ERCC2 gene compared to the wild type ERCC2 gene. The pathogenic gene mutation can be used as a biomarker for diagnosing COFS syndrome; the invention can be used for genetic diagnosis, screening and prenatal and postnatal care guidance of the COFS syndrome by detecting whether a subject carries the mutation; the detection kit provided by the invention can be used for rapidly and effectively predicting or diagnosing the COFS syndrome.

Description

Pathogenic gene causing COFS syndrome, detection and application

Technical Field

The invention relates to the field of detection reagents, in particular to a pathogenic gene for causing COFS syndrome, detection and application.

Background

COFS syndrome, also known as brain-eye-face-bone (COFS) syndrome, is a rare autosomal recessive genetic disorder formally named by Pena and shekeir in 1974, and its main clinical features include small head deformity, ocular abnormalities, large auricles, small mandible, joint flexion and physical retardation in addition to severe mental retardation. COFS syndrome belongs to one of the diseases of nucleotide transcription coupled repair pathway disorder. Mutations in pathogenic genes are classified into 4 types according to their differences: COFS syndrome type 1 is caused by ERCC6 (CSB, MIM 126340) gene mutation, type 2 (MIM 610756) is caused by ERCC2 (XPD) gene mutation, type 3 is caused by ERCC5 (XPG) gene mutation, and type 4 is caused by ERCC1 gene mutation. ERCC2 gene mutations can cause dysplasia of the hair-thio type (TTD, MIM 601675) and xeroderma pigmentosum (MIM 278730) in addition to the type 2 brain-eye-face-bone syndrome (COFS 2, MIM 610756) that leads to autosomal recessive inheritance. COFS2 (MIM 610756) is a progressive neurodegenerative disease characterized by small head deformity, congenital cataracts, severe mental retardation, facial deformity, joint softening, and the like; TTD is commonly manifested clinically as skin, nerve and growth abnormalities, other clinical features being ichthyosis, intellectual/developmental disorders, reduced fertility, ocular abnormalities, short stature, etc.; pigment dry skin disease is clinically characterized by skin cancer in places where the body is exposed to sunlight.

The ERCC2 gene (MIM 126340) is located on chromosome 19q13.32, comprises 23 exons and 22 introns, is 20.8kb in length, and encodes 760 amino acid sequences of ERCC2 protein. The nucleotide excision repair pathway is a mechanism for repairing DNA damage, and the protein coded by the gene participates in transcription coupling nucleotide excision repair and is an indispensible member of a basic transcription factor btf/tfiih complex. The gene product has ATP dependent DNA helicase activity and belongs to RAD3/XPD subfamily of helicase. Mutation or inactivation of the ERCC2 gene may lead to diseases including: cancer, xeroderma pigmentosum, hair sulfur malnutrition, COFS syndrome, etc.

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 COFS syndrome. The invention discovers a novel compound heterozygous mutation of ERCC2 for the first time, which can lead to COFS syndrome, develops a corresponding diagnosis kit according to the novel compound heterozygous mutation, assists in screening and diagnosing COFS syndrome gene mutation, and provides a novel technical support for drug screening, drug effect evaluation and targeted therapy.

Disclosure of Invention

The invention mainly aims to provide a pathogenic gene, detection and application for causing COFS syndrome, so as to solve the technical problems of screening and diagnosis of the COFS syndrome.

To achieve the above object, the present invention provides a pathogenic gene causing COFS syndrome, which causes a base G mutation to a base a at the last base of the 17 th intron of the wild-type ERCC2 gene immediately adjacent to the 18 th exon, as compared to the wild-type ERCC2 gene;

and/or, the pathogenic gene is mutated from basic G to base C at base 1922 of exon21 of the wild-type ERCC2 gene as compared to the wild-type ERCC2 gene.

The invention also provides a detection reagent for the COFS syndrome triggered by the pathogenic gene, wherein the detection reagent comprises a specific amplification primer designed for a site where the pathogenic gene is mutated.

Further, the specific amplification primer comprises ERCC2-1F, ERCC2-1R, ERCC2-2F, ERCC2-2R, the nucleotide sequence of the ERCC2-1F is shown as SEQ ID NO.1, the nucleotide sequence of the ERCC2-1R is shown as SEQ ID NO.2, the nucleotide sequence of the ERCC2-2F is shown as SEQ ID NO.3, and the nucleotide sequence of the ERCC2-2R is shown as SEQ ID NO. 4.

The invention also provides a detection kit for the COFS syndrome, which comprises the detection reagent.

Further, reagents for PCR amplification reactions, and/or reagents and sequencing primers required for DNA sequencing are included.

Further, the sequencing primer comprises ERCC2-Seq1F, ERCC2-Seq1R, ERCC2-Seq2F and ERCC2-Seq2R, wherein the nucleotide sequence of the ERCC2-Seq1F is shown as SEQ ID No.5, the nucleotide sequence of the ERCC2-Seq1R is shown as SEQ ID No.6, the nucleotide sequence of the ERCC2-Seq2F is shown as SEQ ID No.7, and the nucleotide sequence of the ERCC2-Seq2R is shown as SEQ ID No. 8.

The invention also provides the use of a detection reagent as described in any of the above or a detection kit as described in any of the above in a method of detecting COFS syndrome.

Further, the test sample of the test reagent comprises blood and/or amniotic fluid.

The application proposes the pathogenic gene causing the COFS syndrome, and can effectively distinguish COFS syndrome patients from normal people, so that the pathogenic gene mutation can be used as a biomarker for diagnosing the COFS syndrome. The invention can be used for genetic diagnosis, screening and instruction of prenatal and postnatal care of COFS syndrome by detecting whether a subject carries the mutation. The detection kit provided by the invention can be used for rapidly and effectively predicting or diagnosing the COFS syndrome. The invention lays a new foundation and a new path for researching pathogenesis of the COFS syndrome and provides a brand new theoretical basis for treating COFS syndrome patients. The invention can provide a possible drug target for treating COFS syndrome.

Drawings

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

FIG. 1 shows a family genetic map of COFS syndrome No. 1; wherein,,

representing a male carrier, is->

Representing female carriers, ■ male patients, ↗ forerunner.

FIG. 2 shows a graph of the results of detection of ERCC2: NM-000400.4: exo18: c.1666-1G > A locus genotype using Sanger sequencing, wherein ancestor No.1, ancestor father "c.1666-1G > A heterozygous mutation", ancestor mother wild type (the position of mutation indicated by the arrow in the sequencing diagram).

FIG. 3 shows a graph of the results of detection of ERCC2: NM-000400.4: exon21: c.1922G > C: p.R641P locus genotype using Sanger sequencing, wherein ancestor 1, ancestor mother "c.1922G > C heterozygous mutation" and ancestor father wild type (the position of mutation indicated by the arrow in the sequencing).

FIG. 4 shows a family genetic map of COFS syndrome No. 2; wherein,,

representing a male carrier, is->

Representing a female carrier, +. representing a female patient, < > representing a fetus, ↗ representing a forerunner.

FIG. 5 shows a graph of the results of the detection of genotype at position A of the ERCC2: NM-000400.4: exo18: c.1666-1G > using a kit, wherein the ancestor of the No.2 family, the father, is the "c.1666-1G > A heterozygous mutation", the male parent, the fetus, are wild type (the position of the mutation is indicated by the arrow in the sequencing diagram).

FIG. 6 shows the results of the detection of genotype at position 2, line ERCC2: NM-000400.4: exo21: c.1922G > C: p.R641P using the kit, wherein the ancestor, mother, fetus of line 2 is "c.1922G > C heterozygous mutation", the ancestor mother is wild type (the position of the mutation is indicated by the arrow in the sequencing diagram).

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

Detailed Description

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

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

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

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

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

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

In the present invention, the term "complex heterozygous mutation" means a heterozygous mutation in which 1 or more parts of alleles occur, that is, a double allelic mutation, each chromosome being mutated.

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

In the present invention, a "primer" refers to a polynucleotide fragment, typically an oligonucleotide, containing at least 5 bases, such as 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more bases, for amplifying a target nucleic acid in a PCR reaction. The primer need not be completely complementary to the target gene to be amplified or its complementary strand, as long as it can specifically amplify the target gene. The term "specifically amplify" as used herein refers to the ability of a primer to amplify a gene of interest by a PCR reaction without amplifying other genes. For example, specifically amplifying the ERCC2 gene means that the primer amplifies only the ERCC2 gene and not the other genes in the PCR reaction.

The research thought of the invention is as follows: the pathogenic gene mutation highly related to the COFS syndrome is firstly screened by utilizing exon sequencing, in order to avoid false positive results, and then verified by Sanger sequencing, and finally the pathogenic gene mutation of the COFS syndrome is obtained, wherein ERCC2: NM-000400.4: exo18: c.1666-1G > A and exon21: c.1922G > C: p.R641P.

The selected pathogenic gene composite heterozygous mutation can distinguish COFS syndrome patients, carriers and normal people, so that the pathogenic gene composite heterozygous mutation can be used as a biomarker for diagnosing COFS syndrome.

Specifically, the invention provides a gene composite heterozygous mutation related to COFS syndrome, ERCC2: NM_000400.4: exo18: c.1666-1G > A and exon21: c.1922G > C: p.R641P.

c.1666-1G according to the invention>The A mutation refers to the mutation of the last base G of the 17 th intron of the wild type ERCC2 gene, which is adjacent to the 18 th exon, to A to form an ERCC2 gene mutant, and the nucleotide sequence of the ERCC2 gene mutant is preferably shown as SEQ ID NO.11 (cccaaGGGAT) (underlined in bold is the base after mutation, lowercase is the intron sequence, uppercase is the exon sequence).

c.1922G of the invention>The C mutation refers to the 192st alkali of the 21 st exon of the wild ERCC2 geneThe group G is mutated into C to form ERCC2 gene mutant, and the nucleotide sequence of the ERCC2 gene mutant is preferably shown as SEQ ID NO.12 (TACCTGC)GGGACCAG) (underlined letters are added to the base after mutation).

Compared with the protein encoded by the wild ERCC2 gene, the ERCC2 mutant protein of the invention has the 641 st position mutated from arginine (R) to proline (P), namely the error sense mutation occurs, namely the ERCC2 mutant protein contains the mutation of p.R641P, and the mutation is due to c.1922G>Missense mutation of C; the amino acid sequence of ERCC2 mutant protein is shown as SEQ ID NO.13 (YLPDQ) (underlined letters are bolded as mutated amino acids).

Based on the above discussion, the present invention screens out and provides a pathogenic gene causing COFS syndrome, which causes a base G mutation to a base a at the last base of the 17 th intron of the wild-type ERCC2 gene immediately adjacent to the 18 th exon, compared to the wild-type ERCC2 gene;

The invention also provides a detection reagent for the COFS syndrome triggered by any of the pathogenic genes, which comprises a specific amplification primer designed for a site where the pathogenic gene is mutated. The detection reagent may be a gene mutation site for detecting the therapeutic gene.

The specific amplification primer comprises ERCC2-1F, ERCC2-1R, ERCC2-2F, ERCC2-2R, the nucleotide sequence of ERC 2-1F is shown as SEQ ID NO.1, the nucleotide sequence of ERCC2-1R is shown as SEQ ID NO.2, the nucleotide sequence of ERCC2-2F is shown as SEQ ID NO.3, and the nucleotide sequence of ERCC2-2R is shown as SEQ ID NO. 4.

Specifically, the base sequence of PCR primer aiming at c.1666-1G > A locus is as follows:

ERCC2-1F:GGGGTCAAGGTTCATTTTC-3’(SEQ ID NO.1)

ERCC2-1R:GGAGGAAGAGCCCAGTC-3’(SEQ ID NO.2)

the base sequence of PCR primer aiming at c.1922G > C site is as follows:

ERCC2-2F:TAACTTGCGTCTCGGTCTC-3’(SEQ ID NO.3)

ERCC2-2R:GAAGGGCTGTGCCATCT-3’(SEQ ID NO.4)

the invention also provides a detection kit for the COFS syndrome, which comprises any detection reagent. In addition to specific amplification primers, the detection kit may also include conventional reagents for PCR amplification reactions, and/or reagents and sequencing primers required for DNA sequencing. The detection kit diagnoses whether an individual suffers from COFS syndrome by detecting the genotype of a mutation site in a sample.

The sequencing primer comprises ERCC2-Seq1F, ERCC2-Seq1R, ERCC2-Seq2F and ERCC2-Seq2R, wherein the nucleotide sequence of the ERCC2-Seq1F is shown as SEQ ID NO.5, the nucleotide sequence of the ERCC2-Seq1R is shown as SEQ ID NO.6, the nucleotide sequence of the ERCC2-Seq2F is shown as SEQ ID NO.7, and the nucleotide sequence of the ERCC2-Seq2R is shown as SEQ ID NO. 8.

Specifically, the base sequence of the sequencing primer aiming at the c.1666-1G > A locus is as follows:

ERCC2-Seq1F:GCCTGCCCCACTTATCC-3’(SEQ ID NO.5)

ERCC2-Seq1R:GGAGACCGAGACGCAAG-3’(SEQ ID NO.6)

the sequencing primer base sequence of the primer for the c.1922G > C site is as follows:

ERCC2-Seq2F:GCCGTCATCATGTTTGG-3’(SEQ ID NO.7)

ERCC2-Seq2R:AGCCTGGTTCTTGGAGC-3’(SEQ ID NO.8)

other conventional reagents in the PCR amplification reaction include, but are not limited to dNTPs, PCR buffers, magnesium ions, tap polymerase, and the like. The PCR buffer is 10 XPCR buffer: 500mmol/LKCl,100mmol/LTris-Cl (pH 8.3), 15mmol/LMgCl ₂ 。

The detection reagent and the detection kit belong to the application of the disease curing gene and the gene mutation site thereof, and the invention can design a specific amplification primer or a specific detection probe according to the upstream and downstream sequences of the gene mutation site.

In a specific application, the test sample of the test reagent may be blood and/or amniotic fluid.

In a specific application process, the invention also provides a diagnosis kit for the COFS syndrome, which comprises a reagent for detecting the mutation site of the gene.

In a specific application process, the method for detecting whether the ERCC2 gene has the gene mutation comprises the following steps:

1) Extracting sample genome DNA;

2) Amplifying ERCC2 gene sequence;

3) Sequencing DNA;

4) Comparing the DNA sequencing result of the sample to be detected with the genome DNA sequence of a normal person:

when the genotype of the c.1666-1g > a site is wild-type (i.e., no c.1666-1g > a mutation has occurred), the genotype of the c.1922g > C site is wild-type (i.e., no c.1922g > C mutation has occurred), the individual providing the test sample is a normal individual;

when the genotype of the c.1666-1G > A site is a c.1666-1G > A heterozygous mutation (one gene is mutated c.1666-1G > A and its allele is not mutated c.1666-1G > A), the genotype of the c.1922G > C site is a c.1922G > C heterozygous mutation (one gene is mutated c.1922G > C and its allele is not mutated c.1922G > C), and the two mutated sites are on both chromosomes,

or the genotype of the c.1666-1G > A site is a c.1666-1G > A homozygous mutation, the genotype of the c.1922G > C site is a c.1922G > C heterozygous mutation,

or the genotype of the c.1666-1G > A site is a c.1666-1G > A homozygous mutation, the genotype of the c.1922G > C site is a wild type,

or the genotype of the c.1666-1G > A site is wild type, the genotype of the c.1922G > C site is a c.1922G > C homozygous mutation,

or the genotype of the c.1666-1G > A site is a c.1666-1G > A heterozygous mutation, the genotype of the c.1922G > C site is a c.1922G > C homozygous mutation,

or when the genotype of the c.1666-1G > A locus is a c.1666-1G > A homozygous mutation and the genotype of the c.1922G > C locus is a c.1922G > C homozygous mutation, the individual providing the sample to be tested is a COFS syndrome patient;

when the genotype of the c.1666-1G > A locus is c.1666-1G > A heterozygous mutation, the genotype of the c.1922G > C locus is c.1922G > C heterozygous mutation, and the two mutated loci are on the same chromosome,

or when the genotype of the c.1666-1G > A site is wild type and the genotype of the c.1922G > C site is a c.1922G > C heterozygous mutation,

or the genotype of the c.1666-1G > A site is a c.1666-1G > A heterozygous mutation, and the genotype of the c.1922G > C site is wild type, the individual providing the test sample is the carrier of COFS syndrome.

The invention has the advantages that: according to the invention, through an exome sequencing technology, it is discovered for the first time that the complex heterozygous mutation of ERCC2: NM_000400.4: exo18: c.1666-1G > A and exon21: c.1922G > C: p.R641P site can cause the onset of COFS syndrome. In one aspect, the genetic diagnosis for screening or diagnosing COFS syndrome is used to guide treatment by detecting whether a subject carries the mutation described above. In particular, the diagnostic kit provided by the invention can be used for rapidly and effectively predicting or diagnosing the COFS syndrome. On the other hand, the invention lays an important foundation for research on pathogenesis of the COFS syndrome and provides a brand new theoretical basis for treatment of COFS syndrome patients. In a third aspect, the invention may provide a potential drug target for the treatment of COFS syndrome.

The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental procedures, which do not address the specific conditions in the examples below, are generally followed by conventional conditions such as those described in Sambrook et al, molecular cloning, A laboratory Manual (Molecular Cloning A LABORATORY MANUAL 1SECOND EDITION;NewYork:Cold Spring Harbor LaboratoryPress,2014), or by the manufacturer's recommendations.

Example 1 sample acquisition

The inventors found 1 family of COFS syndrome (abbreviated as family 1), and the clinical information of part members of this family is shown in table 1. FIG. 1 is a family chart, wherein,

representing a male carrier, is->

Representing female carriers, ■ male patients, ↗ forerunner.

1. Diagnostic criteria:

reference may be made to the 2010 version of the human monogenic genetic disease.

Diagnostic criteria for COFS syndrome: typical clinical manifestations (microcephaly, congenital cataract and/or microcephaly, prominent nasal root, prominent upper lip, joint flexion, severe developmental lag) and genetic analysis. Wherein the COFS2 is characterized by small head deformity, congenital cataract, severe mental retardation, facial deformity, joint softening, etc.

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

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

The peripheral blood DNA of family 1 personnel I1 (male parent of the first-wittedly), I2 (mother of the first-wittedly) and II 1 (mother of the first-wittedly) were used for sequencing analysis.

Example 2 exon sequencing

1. The instrument is shown in table 2.

Table 2 list of instruments and devices

2. Reagent consumable

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

3. Reagent formulation

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

Table 3 5 XTBE electrophoresis liquid formula

Reagent(s)	Tris	Boric acid	EDTA(pH8.0,0.5mol/L)	ddH ₂ O
					Volume/weight	5.4g	750mg	2mL	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)	NH ₄ Cl	KHCO ₃	EDTA	Adding ddH ₂ O
					Volume/weight	82.9g	10g	0.37g	To 1000mL

Autoclaving and storing at 4deg.C.

1 Xnuclear lysate was prepared according to Table 5.

Table 51 XNuclear lysate formula

Reagent(s)	2MTris-HCl，pH8.2	4MNaCl	2mMEDTA
				Volume/weight	0.5mL	10mL	0.4mL

4. Experimental procedure

After signing the informed consent, 3-5mL of peripheral blood of I1 (forerunner father), I2 (forerunner mother) and II 1 (forerunner) in family 1 were collected.

4.1 sample DNA extraction

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

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

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

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

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

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

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

4.2 exon sequencing

Reference is made to the manual of the human whole exon sequencing kit (Agilent) and the manual of the molecular cloning laboratory (third edition; molecular CloningALABORATORYMANUAL 1SECOND EDITION;NewYork:Cold Spring Harbor LaboratoryPress,2014) for instructions.

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

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

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

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

5) NextSeq500 sequencer sequencing and data analysis.

4.3 results

Finally, 1 gene composite heterozygous mutation ERCC2 with pathogenic significance is obtained, wherein NM_000400.4 is exo18 is c.1666-1G > A, exo21 is c.1922G > C is p.R641P; 1666-1G > A mutation to the last base G of intron 17 immediately adjacent to exon18 to A, which affects cleavage; the c.1922G > C mutation is a mutation of the base G at 1922 of the 21 st exon to C, resulting in a mutation of the 641 st arginine (R) to proline (P), i.e. a missense mutation. The genotypes of ERCC 2:NM-000400.4:exo18:c.1666-1G > A and exon21:c.1922G > C:p.R641P sites in family 1 patient (precursor) are "c.1666-1G > A+c.1922G > C" complex heterozygous mutations; the genotype of this site in line 1 carrier is the "c.1666-1G > A" heterozygous mutation or the "c.1922G > C" heterozygous mutation.

Example 3Sanger sequencing validation

The ERCC2: NM-000400.4: exo18: c.1666-1G > A and exon21: c.1922G > C: p.R641P sites were further verified using Sanger sequencing for exome sequencing results. Genotype test was performed on 3 persons such as I1 (male parent of the forerunner), I2 (mother of the forerunner), II 1 (mother of the forerunner) and the like in line 1 and 100 normal persons outside the line in example 1, respectively, with ER CC2: NM-000400.4: exo18: c.1666-1G > A and exon21: c.1922G > C: p.R641P locus.

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

2.2 18 pairs of candidate primers were designed for each of the c.1666-1G > A and c.1922G > C sites (see Table 6), and the merits of each pair of candidate primers were verified and evaluated by PCR experiments.

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

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

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

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

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

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

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

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

2.3 candidate primer PCR verification reaction

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

TABLE 7 primer detection PCR reaction System

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

the first step: 95 ℃ for 5 minutes;

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

And a third step of: 72 ℃,7 minutes;

fourth step: 4℃until sampling.

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

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

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

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

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

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

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

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

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

2.5 evaluation of results:

1) If only one bright and clear target strip appears in the tube No.7 and no other strip exists, judging that the pair of primers and a reaction system are good in effectiveness and strong in specificity;

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

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

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

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

2.6 based on the results of statistics after the verification test in Table 6, the optimal pair (No. 1 in Table 6) was selected as the primers for mutation family detection.

The PCR primer sequences for ERCC2: NM-000400.4: exo18: c.1666-1G > A sites are shown below:

ERCC2-1F：5’-GGGGTCAAGGTTCATTTT-3’(SEQ ID NO.1)

ERCC2-1R：5’-GGAGGAAGAGCCCAGTC-3’(SEQ ID NO.2)

primer sequences for ERCC 2:NM-000400.4:exo21:c.1922G > C:p.R641P sites are shown below:

ERCC2-2F：5’-TAACTTGCGTCTCGGTCTC-3’(SEQ ID NO.3)

ERCC2-2R：5’-GAAGGGCTGTGCCATCT-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

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

for ERCC 2:NM-000400.4:exo18:c.1666-1G > A site reaction procedure is as follows:

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.

For ERCC 2:NM-000400.4:exo21:c.1922G > C:p.R641P site reaction procedure was as follows:

the first step: 95 ℃ for 5 minutes;

and a second step of: 30 cycles (95 ℃,30 seconds- > 53 ℃,30 seconds- > 72 ℃,60 seconds);

and a third step of: 72 ℃,7 minutes;

fourth step: 4℃until sampling.

4. Agarose gel electrophoresis detection

Refer to step 2.4 above.

5. Purifying a PCR product by an enzymolysis method: to the 5. Mu.LPCR 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 9BigDye reaction System

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 the purified BigDye reaction product, wherein sequencing primers are designed on the basis of the PCR preferred primers (the second set of primers are designed within the range of the product sequence obtained by amplifying the first set of primers) as sequencing primers, and the sequencing primer sequences for ERCC2: NM_000400.4: exo18: c.1666-1G > A sites are as follows:

ERCC2-Seq1F:5’-GCCTGCCCCACTTATCC-3’(SEQ ID NO.5)

ERCC2-Seq1R:5’-GGAGACCGAGACGCAAG-3’(SEQ ID NO.6)

the sequencing primer sequences for ERCC 2:NM-000400.4:exo21:c.1922G > C:p.R641P sites are as follows:

ERCC2-Seq2F:5’-GCCGTCATCATGTTTGG-3’(SEQ ID NO.7)

ERCC2-Seq2R:5’-AGCCTGGTTCTTGGAGC-3’(SEQ ID NO.8)

9. analysis of results

The Sanger sequencing results of FIG. 2 show that the 2 persons in family 1 ERCC2: NM-000400.4: exo18: c.1666-1G > A locus genotype is "c.1666-1G > A heterozygote". The position indicated by the arrow in the sequencing diagram of FIG. 2 shows that the B, C layer ERCC 2:NM_000400.4:exo18:c.1666-1G > A locus genotype is a "c.1666-1G > A heterozygote" mutation; the position indicated by the arrow in the sequencing diagram of FIG. 2 shows that the A-layer individual genotype is wild type.

The Sanger sequencing results of FIG. 3 show that the genotype of 2 members of family 1 ERCC2: NM-000400.4: exo21: c.1922G > C: p.R641P locus is "c.1666-1G > A heterozygote". The position indicated by the arrow in the sequencing diagram of FIG. 3 shows that the genotype of the P locus of p.R641 is the "c.1922G > C heterozygote" mutation in A, C-layer individuals ERCC 2:NM-000400.4:exo21:c.1922G > C; the position indicated by the arrow in the sequencing diagram of FIG. 3 shows that the B-layer individual genotype is wild type.

Based on the detection results, the ERCC2 genotype of the precursor is c.1666-1G > A and c.1922G > C composite heterozygous mutation. And prompting the patient with the first syndrome to be the COFS syndrome patient according to the detection result.

Example 4 ERCC2 Gene c.1666-1G>A、c.1922G>C mutation diagnosis kit and application

1. The kit comprises the following components:

1) Amplification primers (1.2. Mu.g per primer): as shown in example 3

2) Buffer (500 μl of 10 XPCR buffer: 500mmol/LKCl,100mmol/LTris-Cl (pH 8.3), 15mmol/LMgCl 2)

3) Taq enzyme (20U)

4) dNTPs (four kinds of dNTPs 4mM each)

5) ERCC2: c.1666-1G > A, c.1922G > C positive mutant reference DNA the reference is a double-stranded DNA, the specific sequence of the c.1666-1G > A positive mutant reference DNA is as follows:

the specific sequence of c.1922G > C positive mutant reference DNA is as follows:

wherein, single underlined bases are positions of the upstream and downstream primers of PCR amplification, boxes are mutation occurrence sites, and double underlined bases are positions of the upstream and downstream sequencing primers.

6) Sequencing primer: as shown in example 3

2. The using method comprises the following steps:

98 individuals in 23 families with retarded language development after growth and development are screened and detected together, the families conforming to the invention are found again, and the application of the gene mutation detection kit is illustrated by taking the family number 2 as an example.

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

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

Family members No.2, I1 (father), I2 (mother), II 1 (forerunner) peripheral blood DNA and II 2 (fetus) amniotic fluid DNA were used for the kit detection.

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

2) Firstly, carrying out PCR amplification reaction by using the PCR amplification primer, taq enzyme, buffer solution, dNTPs, sample genome DNA and the like, as in the example 3;

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 A locus of the No.2 family father and forensic ERCC 2:NM_000400.4:exo18:c.1666-1G > A is a "c.1666-1G > A heterozygote". The position indicated by the arrow in the sequencing diagram of FIG. 5 shows that the B and C layers ERC 2: NM-000400.4: exo18: c.1666-1G > A locus genotype is a "c.1666-1G > A heterozygote" mutation; the position indicated by the arrow in the sequencing diagram of FIG. 5 shows that the A and D layers of the idiotypes are wild type. The detection results of the kit of FIG. 6 show that the genotype of the No.2 family mother, fetus and precursor ERCC2: NM-000400.4: exo21: c.1922G > C: p.R641P locus is "c.1922G > C heterozygote". FIG. 6 shows that the position indicated by the arrow in the sequencing diagram shows A, C and D layer ERCC2: NM-000400.4: exo21: c.1922G > C: p.R641P locus genotype is the "c.1922G > C heterozygote" mutation; the position indicated by the arrow in the sequencing diagram of FIG. 6 shows that the B-layer individual genotype is wild-type. The detection result confirms that the first evidence is a COFS syndrome patient, and the mother, father and fetus are mutant gene carriers; genetic counseling opinion is to continue pregnancy and monitor pregnancy. Fetal birth follow-up did not see CO FS syndrome related phenotypes.

From the results of the above examples, it can be seen that the present invention has found a novel ERCC2 gene mutant, and confirmed that the novel mutant is closely related to the onset of COFS syndrome, which can be used for molecular diagnosis of COFS syndrome and differential diagnosis of related diseases.

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

Claims

1. A pathogenic gene causing COFS syndrome, characterized in that the pathogenic gene is mutated from base G to base a at the last base of the 17 th intron of the wild-type ERCC2 gene immediately adjacent to exon18, compared to the wild-type ERCC2 gene;

2. A test agent for COFS syndrome triggered by the pathogenic gene of claim 1, comprising a specific amplification primer designed for the site of mutation of the pathogenic gene.

3. The detection reagent according to claim 2, wherein the specific amplification primer comprises ERCC2-1F, ERCC2-1R, ERCC2-2F, ERCC2-2R, the nucleotide sequence of ERCC2-1F is shown in SEQ ID NO.1, the nucleotide sequence of ERCC2-1R is shown in SEQ ID NO.2, the nucleotide sequence of ERCC2-2F is shown in SEQ ID NO.3, and the nucleotide sequence of ERCC2-2R is shown in SEQ ID NO. 4.

4. A test kit for COFS syndrome, comprising the test reagent according to claim 2 or 3.

5. The kit of claim 4, further comprising reagents for PCR amplification reaction, and/or reagents and sequencing primers required for DNA sequencing.

6. The detection kit according to claim 5, wherein the sequencing primer comprises ERCC2-Seq1F, ERCC2-Seq1R, ERCC2-Seq2F and ERCC2-Seq2R, the nucleotide sequence of ERCC2-Seq1F is shown in SEQ ID NO.5, the nucleotide sequence of ERCC2-Seq1R is shown in SEQ ID NO.6, the nucleotide sequence of ERCC2-Seq2F is shown in SEQ ID NO.7, and the nucleotide sequence of ERCC2-Seq2R is shown in SEQ ID NO. 8.

7. Use of the detection reagent of claim 2 or 3 or the detection kit of any one of claims 4 to 6 in a method for the detection of COFS syndrome.

8. The use according to claim 7, wherein the test sample of the test reagent comprises blood and/or amniotic fluid.