CN116287213A

CN116287213A - Pathogenic gene causing MRD7 type mental disorder, detection and application

Info

Publication number: CN116287213A
Application number: CN202310301796.3A
Authority: CN
Inventors: 曾桥; 刘亚宁; 刘鑫林; 李婵艺
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: 2023-06-23

Abstract

The invention discloses a pathogenic gene for causing MRD7 type intellectual disability, which is characterized in that heterozygous mutation occurs at a locus corresponding to 827 th base of 8 th exon of a wild DYRK1A gene compared with a wild DYRK1A gene. The research result of the invention can be used for screening or diagnosing MRD7 type dysnoesia pathogenic gene mutant or patient to guide patient treatment and help for prenatal and postnatal care, provides a new basis and path for research of pathogenesis of MRD7 type dysnoesia, provides a new theoretical basis for treatment of MRD7 type dysnoesia, and provides a possible medicine target for treatment of MRD7 type dysnoesia.

Description

Pathogenic gene causing MRD7 type mental disorder, detection and application

Technical Field

The invention relates to the field of detection reagents, in particular to a pathogenic gene causing MRD7 type dysnoesia, detection and application.

Background

According to the definition of the united states mental disorder association (American Association on Mental Retardation) for mental disorders (mental retardation, MR), congenital mental disorders are complex diseases mainly caused by central nervous system dysplasia and possibly accompanied by symptoms such as metabolic disorders, and patients often show significant defects in terms of mental and behavioral before 18 years of age. According to statistics, the mental disorder patients account for 1% -3% of the general population, and the proportion of men and women is 1.4-1.6:1. Intellectual disability is a major group of neurological developmental disorders with high clinical and genetic heterogeneity, whose etiology is complex, involving multiple factors such as genetics and environment. Wherein, the genetic factors account for 50 percent, especially in patients with moderate and severe mental disorder, and the genetic factors comprise chromosome number and structure abnormality, monogenic disease, mitochondrial disease, polygenic and/or epigenetic abnormality, etc. Currently, in the human Mendelian genetic database (OMIM), there are hundreds of genes associated with mental disorders, with autosomal dominant inheritance accounting for 13-20% of mental disorders; in addition, congenital metabolic defect diseases are mostly inherited by autosomal recessive inheritance, and account for 1-55 of mental disorder; the X-linked dysnoesia accounts for 10-12% of the children suffering from male dysnoesia.

The autosomal dominant inherited neurodevelopmental retardation type 7 (Intellectual developmental disorder, autosomal dominant, MRD 7) or MRD7 dysnoesia (MIM 614104), also known as DYRK 1A-related dysnoesia, is an autosomal dominant inherited disease; the main manifestations are: developmental retardation, mental retardation, febrile convulsion, language retardation, ataxia, abnormal gait, autism, etc.

MRD7 type intellectual disturbance pathogenic gene DYRK1A (MIM 600855) is located on chromosome 21q22.13, the gene is 160.3kb in total length, contains 13 exons and 12 introns, encodes a double-substrate specific tyrosine phosphorylation regulating kinase A consisting of 584 amino acids, belongs to DYRK family, is an important protein kinase closely related to Down syndrome occurrence and highly conserved in evolution, and is located in a critical region of Down syndrome. DYRK1A protein comprises a protein kinase domain as well as some other specific structures, mainly including Poly-Ser, poly-His, ser/Thr-rich. DYRK1A has very important and critical roles in the developmental processes of the nervous system, including neuronal proliferation, neurogenesis, neuronal differentiation, cell necrosis and synaptic plasticity. Mutations in the DYRK1A gene can lead to neurological dysplasia and dysnoesia.

Gene mutation is an important genetic basis for the development of MRD7 type dysnoesia, and gene diagnosis is a gold standard for diagnosing MRD7 type dysnoesia. In the prior art, the detection of the genotype of a gene mutation site can be realized by adopting other methods such as restriction enzyme fragment length polymorphism, single-chain conformation polymorphism, allele specific oligonucleotide hybridization and the like, but the detection methods can not simultaneously meet the purposes of qualitative, quantitative and definite mutant gene sequence, so that primers are required to be designed for the specific mutation site, and the detection of the gene sequence is realized by combining with Sanger sequencing technology. The invention discovers a novel heterozygous mutation of DYRK1A for the first time, can cause MRD7 type dysnoesia, develops a corresponding diagnosis kit according to the novel heterozygous mutation, assists screening and diagnosis of MRD7 type dysnoesia gene mutation, and provides a novel technical support for drug screening, drug effect evaluation and targeted therapy.

Disclosure of Invention

The main purpose of the invention is to provide a pathogenic gene, detection and application for causing MRD7 type dysnoesia, so as to solve the technical problems of screening and diagnosis of MRD7 type dysnoesia.

To achieve the above object, the present invention provides a pathogenic gene causing MRD7 type dysnoesia, which causes heterozygous mutation at a position corresponding to base 827 of exon8 of a wild-type DYRK1A gene as compared with the wild-type DYRK1A gene.

The present invention provides a reagent for detecting MRD7 type dysnoesia caused by pathogenic gene as described above, which comprises a specific amplification primer designed for the locus of the gene mutation.

Further, the specific amplification primer comprises DYRK1A-1F, DYRK A-1R, the nucleotide sequence of the DYRK1A-1F is shown as SEQ ID NO.1, and the nucleotide sequence of the DYRK1A-1R is shown as SEQ ID NO. 2.

The invention provides a kit for detecting MRD7 type dysnoesia, 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 DYRK1A-Seq1F and DYRK1A-Seq1R, the nucleotide sequence of the DYRK1A-Se q1F is shown as SEQ ID NO.3, and the nucleotide sequence of the DYRK1A-Seq1R is shown as SEQ ID NO. 4.

The invention also provides application of the detection kit in preparing a reagent for detecting MRD7 type dysnoesia.

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

Compared with the prior art, the invention at least comprises the following advantages: the pathogenic gene which leads to MRD7 type dysnoesia proposed by the application can effectively distinguish MRD7 type dysnoesia patients from normal people, therefore, the pathogenic gene mutation of the invention can be used as a biomarker for diagnosing MRD7 type dysnoesia. The invention can be used to screen or diagnose genetic diagnosis of MRD type 7 dysnoesia by detecting whether the subject carries the mutation described above. The detection kit provided by the invention can be used for rapidly and effectively predicting or diagnosing MRD7 type intellectual impairment. The invention lays an important foundation for researching pathogenesis of MRD7 type dysnoesia and provides a brand new theoretical basis for treating MRD7 type dysnoesia patients. The invention can provide a possible medicine target for treating MRD7 type dysnoesia.

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 MRD 7-type intellectual disability No.1 family genetic map; wherein, it is ≡indicates normal male individual, ≡o indicates normal female, ■ indicates male patient, ↗ indicates forerunner.

FIG. 2 shows a graph of the results of detection of genotypes at the R locus of the family 1 DYRK1A: NM-101395.2: exo8: c.827T > G: p.L276 using Sanger sequencing, wherein layer A: heterozygote mutation in family 1; b and C layers: genotype in line 1 is wild type (position of mutation indicated by arrow in sequencing).

FIG. 3 shows the MRD7 type intellectual disability # 2 family genetic map; wherein ∈r represents a normal individual in men, ∈r represents a normal female, ∈r represents a female patient, ∈r represents a fetus, and ↗ represents a forerunner.

FIG. 4 shows a graph of the results of detection of genotypes at the R locus of the family No.2 DYRK1A: NM-101395.2: exo18: c.827T > G: p.L276R using Sanger sequencing, wherein layer B: heterozygote mutation in family 3; A. layers C and D: genotype in line 3 is wild type (position of mutation indicated by arrow in sequencing).

FIG. 5 shows a MRD 7-type intellectual disability No.3 family genetic map; wherein, it is ≡indicates normal male individual, ≡o indicates normal female, ■ indicates male patient, ↗ indicates forerunner.

FIG. 6 shows a graph of the results of detection of genotypes at the R locus of the line 3 DYRK1 A:NM-101395.2:exo8:c.827T > G:p.L276 using Sanger sequencing, wherein layer A: heterozygote mutation in family 3; b and C layers: genotype in line 3 is wild type (position of mutation indicated by arrow in sequencing).

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 in detail and with reference to the accompanying drawings, wherein it is apparent that the embodiments described are only some, but not all embodiments of the present invention. All other embodiments, based on the embodiments of the invention, which are apparent to those of ordinary skill in the art without inventive faculty, are intended to be within the scope of the 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.

Where numerical ranges are provided in the examples, it is understood that unless otherwise stated herein, both endpoints of each numerical range and any number between the two endpoints are significant both in the numerical range. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs and to which this invention belongs, and any method, apparatus, or material of the prior art similar or equivalent to the methods, apparatus, or materials described in the examples of this invention may be used to practice the invention.

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. 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 DYRK1A gene means that the primer amplifies only the DYRK1A gene and not the other genes in the PCR reaction.

The research thought of the invention is as follows: firstly, screening pathogenic gene mutation highly related to MRD7 type dysnoesia by utilizing exon sequencing, and finally obtaining the pathogenic gene mutation of MRD7 type dysnoesia by verifying through Sanger sequencing in order to avoid false positive results, wherein DYRK1A is NM_101395.2:exo8:c.827T > G:p.L276R.

The heterozygous mutation of the pathogenic gene screened by the invention can distinguish MRD7 type dysnoesia patients from normal people, so that the heterozygous mutation of the pathogenic gene can be used as a biomarker for diagnosing MRD7 type dysnoesia.

A pathogenic gene causing MRD7 type dysnoesia, said pathogenic gene heterozygous mutated at the locus corresponding to base 827 of exon8 of the wild-type DYRK1A gene compared to the wild-type DYRK1A gene.

Specifically, the invention provides a gene heterozygous mutation related to MRD7 type intellectual impairment, DYRK1A: NM_101395.2: exo18: c.827T > G: p.L276R.

c.827T of the invention>The G mutation refers to mutation of 827 th base T of 8 th exon of wild DYRK1A gene into G to form DYRK1A gene mutant, and the nucleotide sequence of the DYRK1A gene mutant is preferably shown as SEQ ID NO.6 (CTTTTCCGTGCGACT) (underlined letter in bold is base after mutation).

Compared with the protein encoded by the wild DYRK1A gene, the DYRK1A mutant protein of the invention has the 276 th amino acid mutated from leucine (L) to arginine (R), namely the DYRK1A mutant protein contains the mutation of p.L276R, the mutation is due to c.827T>Missense of GMutation-induced; the amino acid sequence of DYRK1A mutant protein is shown as SEQ ID NO.7 (LFRAT) (underlined letter is the amino acid after mutation).

The invention also provides a reagent for detecting MRD7 type dysnoesia caused by the pathogenic gene, which comprises a specific amplification primer designed for the locus of the gene mutation.

In some embodiments, the specific amplification primers comprise DYRK1A-1F, DYRK1A-1R, the DYRK1A-1F having a nucleotide sequence shown in SEQ ID NO.1 and the DYRK1A-1R having a nucleotide sequence shown in SEQ ID NO. 2.

Specific: DYRK1A-1F:5'-GAGGGCTGCCAACTACA-3' (SEQ ID NO. 1)

DYRK1A-1R:5’-CTGACAAGAACTGCCAAAG-3’(SEQ ID NO.2)

The invention also provides a kit for detecting MRD7 type dysnoesia, which comprises the detection reagent as described above.

In some embodiments, reagents for PCR amplification reactions, and/or reagents and sequencing primers required for DNA sequencing are also included.

In other embodiments, the sequencing primer comprises DYRK1A-Seq1F and DYRK1A-Seq1R, wherein the nucleotide sequence of DYRK1A-Seq1F is shown in SEQ ID NO.3 and the nucleotide sequence of DYRK1A-Seq1R is shown in SEQ ID NO. 4.

Specific: DYRK1A-Seq1F 5'-GTTTGTCCATTTGGTCCTT-3' (SEQ ID NO. 3)

DYRK1A-Seq1R:5’-AACTGCCAAAGTCAACTATCT-3’(SEQ ID NO.4)

Other conventional reagents in the PCR amplification reaction include, but are not limited to dNTPs, PCR buffers, magnesium ions, ta p polymerase, and the like. The PCR buffer is 10 XPCR buffer: 500mmol/L KCl,100mmol/L Tris-Cl (pH 8.3), 15mmol/L MgCl ₂ 。

According to the invention, a specific amplification primer or a specific detection probe can be designed according to the upstream and downstream sequences of the gene mutation site.

The invention also provides application of the detection kit in preparation of a reagent for detecting MRD7 type dysnoesia.

In some embodiments, the test sample of the test reagent comprises blood and/or amniotic fluid.

The invention also provides a method for detecting the presence or absence of a gene mutation in the DYRK1A gene, the method comprising the steps of:

1) Extracting sample genome DNA;

2) Amplifying DYRK1A 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 human, and if the genotype of the DYRK1A: NM_101395.2: exo18: c.827T > G: p.L276R locus of the individual is found to be 'c.827T > G heterozygote mutation', the sample is the patient; if the genotype of the site is "wild-type", the individual is normal.

The present invention also provides a method of diagnosing MRD type 7 dysnoesia, the method comprising the steps of: detecting the genotype of DY RK1A: NM_101395.2: exo18: c.827T > G: p.L276R site, and diagnosing the subject as MRD 7-type intellectual impairment if the genotype of the individual DY RK1A: NM_101395.2: exo8: c.827T > G: p.L276R site is "c.827T > G heterozygous mutation".

The invention has the advantages that: the invention discovers that DYRK1A: NM_101395.2: exon8: c.827T > G: p.L276R site mutation can cause the onset of MRD7 type intellectual disability for the first time through an exome sequencing technology. In one aspect, the method is used to screen or diagnose a mutant or patient of the mental disorder causing gene of MRD7 type by detecting whether the subject has the mutation described above to provide instruction for eugenic and therapeutic intervention. In particular, the diagnostic kit provided by the invention can be used for rapidly and effectively predicting or diagnosing MRD7 type dysnoesia. On the other hand, the invention lays an important foundation for researching pathogenesis of MRD7 type dysnoesia and provides a brand-new theoretical basis for treating MRD7 type dysnoesia patients. In a third aspect, the invention may provide a potential drug target for the treatment of MRD 7-type dysnoesia.

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 (New York: cold Spring Harbor LaboratoryPress, 2014), or by the manufacturer's recommendations.

Example 1 sample acquisition

The inventors found 1MRD7 family of mental disorders (abbreviated as family 1), and the clinical information of the family members is shown in Table 1. FIG. 1 shows DYRK1A gene mutation family map, wherein ∈s represents normal male individuals, ∈s represents normal female, ■ represents male patients, ↗ represents forerunner.

1. Diagnostic criteria:

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

The MRD7 type intellectual disability is mainly manifested by: developmental retardation, mental retardation, febrile convulsion, language retardation, ataxia, gait abnormalities, autism and the like (small head deformity, abnormal facial features, especially significant overall developmental lag/mental disorder after language development).

TABLE 1 clinical information of MRD7 type intellectual disability # 1 family Member

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, I2 and II 1 were used for sequencing.

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), DNA1000 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 V3.1.3.1 (Thermo), peripheral blood gDNA extraction kit (TIANGEN), agarose (TIANGE N), EB dye solution (amerco).

3. Reagent formulation

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

Table 3 5 XTBE electrophoresis liquid formula

Reagent(s)	Tris	Boric acid	EDTA(pH 8.0,0.5mol/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 410 Xerythrocyte lysate formula

Autoclaving and storing at 4deg.C.

1 Xnuclear lysate was prepared according to Table 5.

Table 51 XNuclear lysate formula

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

5. Experimental procedure

After signing informed consent, 3-5mL of peripheral blood of members I1, I2 and II 1 in family 1 were collected as study samples.

5.1 sample DNA extraction

1) 3-5mL of the sample is put into a 15mL centrifuge tube, and 2-3 times of volume of 1 Xerythrocyte lysate is added, and the mixture is uniformly mixed, and the mixture is 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.

5.2 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.

5.3 results

Finally, the pathogenic gene mutation DYRK1A is obtained, wherein the gene mutation DYRK1A is NM_101395.2, exo18, c.827T > G, p.L276R; wherein the mutation of exo 8: c.827T > G to p.L276R is located at base 827 of exon8, the mutation resulting in the mutation of amino acid 276 from leucine (L) to arginine (R), i.e., missense mutation. The genotype of DYRK1A: NM-101395.2: exo8: c.827T > G: p.L276R site in patient # 1 family patient is "c.827T > G heterozygous mutation", and the genotype of this site in normal individuals is "wild-type".

Example 3Sanger sequencing validation

The DYRK1A: NM-101395.2: exo8: c.827T > G: p.L276R sites were further verified using Sanger sequencing for exome sequencing results. DYRK1A: NM-101395.2: exo8: c.827T > G: p.L276R locus genotype detection was performed on 3 persons (forensic, forensic mother, forensic father) of family 1 and 100 normal persons outside the family, 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 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 design 18 pairs of candidate primers for mutation site c.827T > G (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 5 minutes;

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

And a third step of: 72 ℃,7 minutes;

fourth step: 4℃until sampling.

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

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

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

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

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

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

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

7) The power supply is cut off, the gel is taken out, and the gel is put into an EB water solution with the concentration of 0.5g/mL for dyeing for 10-15 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 (two-site No.1 pair of candidate primers in Table 6) was selected as the primers for mutation family detection.

The PCR primers for DYRK1A: NM-101395.2: exon8: c.827T > G: p.L276R sites are:

DYRK1A-1F:5’-GAGGGCTGCCAACTACA-3’(SEQ ID NO.1)

DYRK1A-1R:5’-CTGACAAGAACTGCCAAAG-3’(SEQ ID NO.2)

3. PCR amplification of mutation sites in family 1 personnel and 100 off-family personnel

PCR was performed according to the reaction system in Table 8 and the reaction system was kept on ice.

TABLE 8 mutation site PCR reaction system

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

for DYRK1A: NM-101395.2: exo18: c.827T > G: p.L276R sites, the PCR 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.

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 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 the bottom of the tube, followed by 1. Mu.L of 3mol/L NaAc (pH 5).

2)；

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

The purified BigDye reaction product was subjected to DNA sequencing.

Sequencing primers nested primers (the second set of primers is designed within the range of the product sequence amplified from the first set of primers) were designed as sequencing primers based on the preferred primers for PCR described above.

Sequencing primers for DYRK1A: NM-101395.2: exon8: c.827T > G: p.L276R sites are:

DYRK1A-Seq1F:5’-GTTTGTCCATTTGGTCCTT-3’(SEQ ID NO.3)

DYRK1A-Seq1R:5’-AACTGCCAAAGTCAACTATCT-3’(SEQ ID NO.4)

9. analysis of results

Sanger sequencing results show that DYRK1A: NM_101395.2: exo18: c.827T > G: p.L276R site genotype of 1 patient in family 1 is a "c.827T > G" heterozygous mutation; the DYRK1A: NM-101395.2: exo8: c.827T > G: p.L276R loci of normal individuals in the family and 100 non-blood related normal controls are "wild type". The position A indicated by the arrow in the sequencing diagram of FIG. 2 shows that the genotype of DYRK1A: NM_101395.2: exo18: c.827T > G: p.L276R locus is a heterozygous mutation of "c.827T > G", and that the locus of a normal individual in the family is wild type in the B and C layers of FIG. 2.

Example 4MRD7 type mental disorder 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/L KCl,100mmol/L Tris-Cl (pH 8.3), 15mmol/L MgCl 2)

3) Taq enzyme (20U)

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

5) DYRK1A: c.827T > G positive mutation reference DNA, the reference is a double-stranded DNA, and the specific sequence is as follows:

wherein, single underlined bases are positions of the primer for PCR amplification, mutation sites are in the box, and lower case letters are positions of the primer for sequencing.

6) Sequencing primer: as shown in example 3

2. The using method comprises the following steps:

104 individuals in 23 intellectual disability families are screened and detected, the following families are found again, and the kit is applied to the gene mutation detection of the families No.2 and No. 3.

TABLE 10 clinical information of MRD7 family members with dysnoesia No.2

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As shown in fig. 3 and 5, numbers i (first generation) and ii (second generation) are used. Wherein ∈r represents a normal male individual, ∈r represents a normal female individual, ∈ ■ represents a male patient, +.r represents a female patient, +.r represents a fetus, and ↗ represents a forerunner.

No.2 family members I1, I2, II 1 peripheral blood DNA and II 2 amniotic fluid DNA were used for the detection of the kit.

The peripheral blood DNA of family 3 personnel I1, I2 and II 1 are used for 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 shows that the genotype of the pro-evidence DYRK1A: NM_101395.2: exo18: c.827T > G: p.L276R site in the No.2 family is a heterozygote mutation of 'c.827T > G'. The position indicated by the arrow on layer B in the sequencing diagram of FIG. 4 shows that the pro-evidence DYRK1A: NM-101395.2: exon8: c.827T > G: p.L276R locus genotype is a "c.827T > G" heterozygous mutation in the family; the parent and fetus of the forerunner are wild type; the detection result confirms that the first-person is MRD7 type mental disorder patient, the parent and the fetus of the first-person are normal individuals, and the pregnancy is recommended to be continued, and the pregnancy detection is finished. Infants were not seen with the phenotype associated with MRD type 7 dysnoesia following postnatal follow-up.

The detection result of the kit shows that the genotype of the first-evidence DYRK1A: NM_101395.2: exo18: c.827T > G: p.L276R site in the No.3 family is a heterozygote mutation of 'c.827T > G'. FIG. 6 shows, in the sequencing diagram, that the position indicated by the arrow at the A-layer shows that the genotype of the first-evidence DYRK1 A:NM-101395.2:exo8:c.827T > G:p.L276R locus in the family is a "c.827T > G" heterozygous mutation; the parent site of the forensic is wild-type; the detection result confirms that the first person is MRD7 type mental disorder patient, and the parent of the first person is normal individual; considering that new mutations are possible, the risk of parents reproducing the infant of the same class is low, but it is still recommended to regress later in gestation for prenatal diagnosis in hospitals.

As can be seen from the results of the above examples, the present invention has found a novel DYRK1A gene mutant and confirmed that the novel mutant is closely related to the onset of MRD 7-type mental disorder, which can be used for molecular diagnosis of MRD 7-type mental disorder 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 MRD7 type dysnoesia, characterized in that said pathogenic gene is heterozygous mutated at the locus corresponding to base 827 of exon8 of the wild type DYRK1A gene compared to the wild type DYRK1A gene.

2. A reagent for detecting a mental disorder of MRD7 type caused by a pathogenic gene according to claim 1, wherein the reagent comprises a specific amplification primer designed for a site of heterozygous mutation of the gene.

3. The detection reagent according to claim 2, wherein the specific amplification primer comprises DYRK1A-1F, DYRK A-1R, the nucleotide sequence of DYRK1A-1F is shown in SEQ ID NO.1, and the nucleotide sequence of DYRK1A-1R is shown in SEQ ID NO. 2.

4. A kit for the detection of mental disorders of the MRD7 type, characterized in that it comprises said detection 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 of claim 5, wherein the sequencing primer comprises DYRK1A-Seq1F and DYRK1A-Seq1R, the nucleotide sequence of DYRK1A-Seq1F is shown in SEQ ID NO.3, and the nucleotide sequence of DYR K1A-Seq1R is shown in SEQ ID NO. 4.

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 MRD 7-type intellectual impairment.

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