CN117487817B

CN117487817B - IL1RAPL1 gene mutant, mutant protein, reagent, kit and application

Info

Publication number: CN117487817B
Application number: CN202311850243.XA
Authority: CN
Inventors: 曾桥; 刘亚宁; 徐霞
Original assignee: Hunan Jiahui Biotechnology Co Ltd
Current assignee: Hunan Jiahui Biotechnology Co Ltd
Priority date: 2023-12-29
Filing date: 2023-12-29
Publication date: 2024-04-23
Anticipated expiration: 2043-12-29
Also published as: CN117487817A

Abstract

The invention provides an IL1RAPL1 gene mutant, mutant protein, a reagent, a kit and application thereof, wherein the IL1RAPL1 gene mutant is compared with a wild type IL1RAPL1 gene, and the 109 th base of the 3 rd exon of the wild type IL1RAPL1 gene is mutated from a base G to a base A. The pathogenic gene mutation can be used as a biomarker for diagnosing mental retardation type 21. The invention can be used to screen or diagnose genetic diagnosis of mental retardation type 21 by detecting whether a subject carries the mutation described above to guide treatment. The detection kit provided by the invention can be used for rapidly and effectively predicting or diagnosing the mental retardation type 21.

Description

IL1RAPL1 gene mutant, mutant protein, reagent, kit and application

Technical Field

The invention relates to the field of detection reagents, in particular to an IL1RAPL1 gene mutant, mutant protein, a reagent, a kit and application.

Background

Mental disability (ID) affects 1-3% of the population, a genetically heterogeneous X-linked ID (XLID) disease caused by mutations in the X chromosome, with about 1.7 being affected in every 1000 men. Up to now, 120 or more have been found and described. Understanding the genetic cause of ID can provide valuable insight into cognitive development and function in humans and drug targets for rational development of effective therapies. The identification of new ID genes has become important due to extreme clinical and genetic heterogeneity.

Mental retardation type 21 (MIM 300143) is one of XLID and is characterized by recessive inheritance of the X chromosome, characterized by impaired or disabled cognitive neurological function, which can range from moderate dysnoesia to high functional autism, with male patients often severely affected and with some female carriers potentially exhibiting a milder phenotype.

The IL1RAPL1 gene (MIM 300206) is a pathogenic gene of mental retardation type 21, which is located on chromosome Xp21.3-p21.2, the total length of the gene is 1369.3kb, and comprises 11 exons and 10 introns, and codes for 696 amino acids IL1RAPL1. The IL1RAPL1 protein belongs to one of the IL-1/Toll receptor family members and comprises, in addition to 3 extracellular Ig-like domains and 1 intracellular TIR domain (the TIR domain is characteristic of the IL-1/Toll receptor family), a specific C-terminal domain consisting of 150 amino acids which has no significant homology to any known functional protein. IL1RAPL1 protein modulates Ca2+ -dependent exocytosis by its specific C-terminal domain interaction with neuronal calcium receptor-1 (neuronal calcium sensor-1, NCS-1); NCS-1 can indirectly regulate the activity of N-type voltage-gated calcium ion channel (N-type voltage-gated calcium channels, N-VGCC), indicating that IL1RAPL 1-related mental retardation is caused by disruption of N-VGCC and/or NCS-1 dependent synaptic and neuronal activity. The non-repeated deletion, inversion, mutation and the like of the IL1RAPL1 gene are main reasons for mental retardation, which suggest that the loss of function or abnormal expression of the IL1RAPL1 gene has correlation with mental retardation of human beings.

Gene mutation is an important genetic basis for the development of mental retardation type 21, and gene diagnosis is a gold standard for diagnosing mental retardation type 21. The clinical needs to establish corresponding detection technology aiming at different mutations and be used for clear etiology and disease diagnosis, the gene mutation site related to mental retardation type 21 is found, the screening and diagnosis of the mental retardation type 21 gene mutation are assisted, and the method has important significance for drug screening, drug effect evaluation and targeted treatment.

Disclosure of Invention

The invention mainly aims to provide a primer, a gene mutant, mutant protein, a reagent and application for detecting mental retardation type 21, so as to solve the technical problems of screening and diagnosis of mental retardation type 21.

To achieve the above object, the present invention provides an IL1RAPL1 gene mutant, wherein the IL1RAPL1 gene mutant is mutated from base G to base a at base 109 of exon 3 of the wild-type IL1RAPL1 gene, as compared with the wild-type IL1RAPL1 gene.

The invention provides an IL1RAPL1 mutant protein, compared with a wild IL1RAPL1 protein, the 37 th amino acid of the IL1RAPL1 mutant protein is mutated from aspartic acid to asparagine.

The invention provides an application of an IL1RAPL1 gene mutant as described above in preparing a mental retardation 21 type detection reagent or preparing a mental retardation 21 type detection kit as a detection target.

Further, the detection reagent and/or the detection kit comprises amplification primers comprising an upstream primer IL1RAPL1-1F and a downstream primer IL1RAPL1-1R; the upstream primer IL1RAPL1-1F comprises a nucleotide sequence shown as SEQ ID NO.1, and the downstream primer IL1RAPL1-1R comprises a nucleotide sequence shown as SEQ ID NO. 2.

Further, the detection reagent and/or the detection kit comprises a sequencing primer, wherein the sequencing primer comprises an upstream primer IL1RAPL1-Seq1F and a downstream primer IL1RAPL1-Seq1R; the upstream primer IL1RAPL1-Seq1F comprises a nucleotide sequence shown as SEQ ID NO. 3; the downstream primer IL1RAPL1-Seq1R comprises a nucleotide sequence shown as SEQ ID NO. 4.

The invention provides an application of IL1RAPL1 mutant protein as described above in preparing a detection reagent for mental retardation type 21 and/or preparing a detection kit for mental retardation type 21.

The invention provides a detection reagent for mental retardation type 21, and a detection target point of the detection reagent comprises the IL1RAPL1 gene mutant or the IL1RAPL1 mutant protein.

The invention provides a detection kit for mental retardation type 21, which comprises the detection reagent.

The application proposes the pathogenic gene causing mental retardation type 21, and can effectively distinguish mental retardation type 21 patients from normal human groups, so that the pathogenic gene mutation can be used as a biomarker for diagnosing mental retardation type 21. The invention can be used for genetic diagnosis, screening and prenatal and postnatal guidance of mental retardation type 21 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 mental retardation type 21. The invention lays a new foundation and a new path for researching pathogenesis of mental retardation type 21, provides a brand-new theoretical basis for treating mental retardation type 21 patients, and provides a possible medicine target for treating mental retardation type 21.

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 genetic map of phylogenetic retardation 21 type No. 1 family; wherein, it represents normal male individuals, it represents female carriers, ■ represents male patients, ↗ represents forerunner;

FIG. 2 shows a graph of the results of detection of genotype at the position IL1 RAPL1:NM-014271.4:exo3:c.109G > A:p.D37N of family 1 using Sanger sequencing; wherein, layer C: hemizygous mutation in family 1; layer B: genotype 1 family is heterozygous mutant; layer A: genotype in line 1 is wild type (position of mutation indicated by arrow in sequencing diagram);

FIG. 3 shows a genetic map of phylogenetic retardation 21 type No. 2 family; wherein, it represents normal male individuals, it represents female carriers, ■ represents male patients, ↗ represents forerunner;

FIG. 4 shows a graph of the results of detection of genotype at position 2 family IL1 RAPL1:NM-014271.4:exo3:c.109G > A:p.D37N locus using the kit; wherein, layer C: genotype in family 2 is hemizygous mutant patient; layer A: genotype in family No.2 is heterozygous mutation carrier; layer B: genotype in line No.2 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 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, the term "X-linked recessive inheritance" means that the pathogenic recessive mutant gene is on the X chromosome, the heterozygote is ill-resistant, and the carrier is ill-resistant only when the female is homozygous, and the hemizygous male is ill-resistant. Namely: when inherited in a recessive manner, since females have two X chromosomes, the trait or genetic disease controlled by the recessive gene is not revealed when the recessive pathogenic gene is in a heterozygous state (XAXa), such female phenotypically normal pathogenic gene carriers; it is only shown when both alleles on the X-chromosome are homozygous for the recessive pathogenic gene (XaXa). In male cells, there is only one X chromosome and the Y chromosome lacks a homologous segment, so that it occurs as long as there is a recessive pathogenic gene (XaY) on the X chromosome. Thus, only one gene of the paired alleles is present in the male cell, and is called a hemizygous.

In the present invention, the term "missense mutation" means that a codon encoding an amino acid is changed into a codon encoding another amino acid after base substitution, thereby changing the kind and sequence of the amino acid of the polypeptide chain.

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 "homozygous mutation" means that the same mutation occurs in all alleles, that is, a double allelic mutation, and each chromosome is mutated.

In the present invention, the term "hemizygous mutation" means that a gene is monovalent, exists on only one chromosome, has no corresponding allele, and is called hemizygous, and the gene is mutated, namely hemizygous mutation.

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 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.

In order to enrich the pathogenic mutation spectrum of mental retardation type 21, accurately judge mental retardation type 21, be used for genetic diagnosis of mental retardation type 21 to guide treatment, and genetic diagnosis and prenatal and postnatal care before embryo implantation, the invention provides IL1RAPL1 gene mutant, mutant protein, reagent, kit and application.

It should be understood that, in the present invention, the Genbank accession number of the wild type IL1RAPL1 gene is NM-014271.4, and the specific sequence is shown in SEQ ID NO. 46. The protein coded by the wild IL1RAPL1 gene has the ID number of NP-055086.1 and the specific sequence shown in SEQ ID No. 47.

The invention provides an IL1RAPL1 mutant protein, compared with a wild IL1RAPL1 protein, the 37 th amino acid of the IL1RAPL1 mutant protein is mutated from aspartic acid to asparagine, and the specific sequence is shown as SEQ ID NO. 48.

The invention also provides an IL1RAPL1 gene mutant, wherein the IL1RAPL1 gene mutant is compared with a wild type IL1RAPL1 gene, the 109 th base of the 3 rd exon of the wild type IL1RAPL1 gene is mutated from a base G to a base A, and the specific sequence is shown as SEQ ID NO. 49.

The IL1RAPL1 mutant gene of the invention comprises a gene shown in SEQ ID NO.5 (5' -TCTATC)ATATCA-3'), the nucleotide sequence shown (the letters in the box are bases after mutation). The accession number of the wild type IL1RAPL1 gene is NM_014271.4. The IL1RAPL1 mutant gene of the invention causes missense mutation, which leads to the mutation of the 37 th amino acid from aspartic acid (D) to asparagine (N) compared with the protein encoded by the wild IL1RAPL1 gene, wherein the sequence of the core amino acid generated by the mutation is shown as SEQ ID NO.6 (DWSI/>IKKY) (the letters in the box are mutated amino acids) influence normal IL1RAPL1 function, cause mental retardation type 21, have pathogenicity, and are specifically IL1 RAPL1:NM_014271.4:exo3:c.109G > A:p.D37N.

In order to avoid false positive results, the invention uses exon sequencing to screen pathogenic gene mutation highly related to mental retardation type 21, and verifies through Sanger sequencing that the IL1RAPL1 gene is related to mental retardation type 21 when c.109G > A exists for the first time, so that mental retardation type 21 can be detected by detecting whether the IL1RAPL1 gene exists c.109G > A or not. The IL1RAPL1 gene mutant provided by the invention can distinguish patients with mental retardation 21 from normal human groups, is a biomarker for diagnosing mental retardation 21, is beneficial to screening and diagnosing mental retardation 21 gene mutation, and provides a new technical support for drug screening, drug effect evaluation and targeted therapy.

The invention provides an application of the IL1RAPL1 gene mutant as described above as a detection target in preparation of a reagent or a preparation kit, wherein the reagent comprises a reagent for detecting mental retardation type 21;

The kit comprises one or more of a mental retardation prevention 21 type kit, a mental retardation diagnosis 21 type kit, a pre-pregnancy genetic disease screening kit, a pre-pregnancy genetic disease diagnosis kit and an auxiliary mental retardation 21 type kit.

The invention provides an amplification primer for detecting mental retardation type 21, which comprises an upstream primer IL1RAPL1-1F and a downstream primer IL1RAPL1-1R; the upstream primer IL1RAPL1-1F comprises a nucleotide sequence shown as SEQ ID NO.1, and the downstream primer IL1RAPL1-1R comprises a nucleotide sequence shown as SEQ ID NO. 2.

The nucleotide sequence of SEQ ID NO. 1-2 is specifically as follows:

SEQ ID NO.1：5'-ACTGTAGGAGCCAGGTGC-3'；

SEQ ID NO.2：5'- CCAGATGGTATCGCTTTTC-3'；

The invention provides a sequencing primer for detecting mental retardation type 21, which comprises an upstream primer IL1RAPL1-Seq1F and a downstream primer IL1RAPL1-Seq1R; the upstream primer IL1RAPL1-Seq1F comprises a nucleotide sequence shown as SEQ ID NO. 3; the downstream primer IL1RAPL1-Seq1R comprises a nucleotide sequence shown as SEQ ID NO. 4.

The nucleotide sequence of SEQ ID NO. 3-4 is specifically as follows:

SEQ ID NO.3：5'-TGTCTCACCAGTGCCCTCT-3'；

SEQ ID NO.4：5'-TTCTTTGCTCATTCTACTTCC-3'；

The invention provides a primer combination for detecting mental retardation type 21, comprising an amplification primer as described above and/or a sequencing primer as described above.

The primer combination can detect whether a mutation site of c.109G > A exists on the IL1RAPL1 gene, distinguish IL1RAPL1 gene mutant from wild type IL1RAPL1 gene, and specifically, the sequencing primer 1 can sequence the amplification product of the amplification primer 1 to judge whether the mutation site of c.109G > A exists in the IL1RAPL1 gene. The primer combination can be used for distinguishing patients with mental retardation type 21 from normal people, and can be used for rapidly and accurately diagnosing the mental retardation type 21.

The invention provides application of a primer combination in preparation of a reagent for detecting mental retardation type 21.

In some embodiments, the reagents may further comprise a primer combination as described in the above technical scheme, preferably further comprising one or more of dntps, PCR buffer, magnesium ion and Tap polymerase; the PCR buffer preferably comprises KCl 50mmol/L, tris-HCl 10mmol/L and MgCl ₂ 1.5.5 mmol/L, and the pH of Tris-HCl is preferably 8.3.

Further, the detection target of the mental retardation type 21 comprises an IL1RAPL1 gene mutant, wherein the IL1RAPL1 gene mutant is compared with a wild type IL1RAPL1 gene, and the 109 th base of the 3 rd exon of the wild type IL1RAPL1 gene is mutated from a base G to a base A.

The present invention provides an agent for detecting mental retardation type 21, which comprises the primer combination as described above.

The invention provides an application of the kit in preparation of the kit and the kit, wherein the kit comprises one or more of a mental retardation prevention 21-type kit, a mental retardation diagnosis 21-type kit, a pre-pregnancy genetic disease screening kit, a pre-pregnancy genetic disease diagnosis kit and an auxiliary mental retardation 21-type kit.

The invention also provides a kit for diagnosing mental retardation type 21, which comprises the reagent according to the technical scheme, and preferably further comprises c.109G > A site positive mutation reference DNA; the specific sequence of the positive reference of the mutation site of 109G > A is shown as SEQ ID NO. 7.

The kit of the invention diagnoses whether the individual suffers from mental retardation type 21 or not through the genotype of the IL1RAPL1 gene mutant in the detection sample of the male individual and/or the female individual; the test sample preferably comprises blood or amniotic fluid. The criteria for genotyping individuals with mental retardation type 21 are specifically:

When the genotype of male individual c.109G > A is "c.109G > A hemizygous mutation", then the patient is;

when the genotype of female individual c.109G > A is "c.109G > A heterozygote mutation", then the female individual c.109G > A is the carrier;

when the genotype of female individual c.109G > A is "c.109G > A homozygous mutation", then the patient is;

when the genotype of an individual is "wild type", the individual is a normal person.

The invention also preferably provides a method for identifying the genotype of the IL1RAPL1 gene mutant, which comprises the following steps:

taking the DNA of the sample to be detected as a template, and carrying out PCR amplification by using the amplification primer 1 to obtain an amplification product;

sequencing the amplified product by using the sequencing primer 1 to determine the genotype of the IL1RAPL1 gene mutant.

In the present invention, the reaction system for PCR amplification preferably comprises, in 20. Mu.L, 10 XPCR buffer 2. Mu. L, dNTPs 0.4.4. Mu. L, IL1RAPL1-1F or IL1RAPL1-2F 0.5. Mu. L, IL1RAPL1-1R or IL1RAPL1-2R 0.5. Mu.L, 100 ng/. Mu.L of template 1. Mu. L, taq enzyme 0.2. Mu.L and the balance ddH ₂ O.

In the present invention, when the primer pairs in the reaction system of the PCR amplification are IL1RAPL1-1F and IL1RAPL1-1R, the reaction progress of the PCR amplification preferably includes: pre-denaturation at 95 ℃ for 5min; denaturation at 95℃for 30s, annealing at 53℃for 30s, elongation at 72℃for 60s,30 cycles; the reaction was carried out at 72℃for 7min.

The invention can preferably determine the correlation between individuals providing the sample to be tested and the mental retardation type 21 according to the genotype of the IL1RAPL1 gene c.109G > A, and specifically comprises the following steps: the sequencing result and the positive reference DNA in the scheme indicate that c.109G > A mutation occurs if the sequence base of the sequencing result and the positive reference DNA are the same, and specific genotype diagnosis standards are as above and are not repeated herein.

In the specific implementation process, 298 individuals in 66 mental retardation families are screened and detected, wherein 14 patients with mental retardation type 21 are detected, 15 carriers are detected, and the individuals in 12 mental retardation type 21 families are found to be IL1RAPL1 gene c.109G > A mutation.

In order to further illustrate the present invention, the IL1RAPL1 mutant protein, IL1RAPL1 gene mutant, primer combinations, reagents and applications for detecting IL1RAPL1 gene mutant provided by the present invention will be described in detail with reference to the accompanying drawings and examples, and in order to illustrate the correlation between IL1RAPL1 mutant protein, IL1RAPL1 gene mutant and mental retardation type 21 of the present invention, 2 families selected and detected at random will be illustrated, but they should not be construed as limiting the scope of the present invention.

The experimental procedure, which does not address the specific conditions in the examples below, is generally followed by routine conditions such as Sambrook et al, molecular cloning: conditions described in the laboratory Manual (New York: cold Spring Harbor LaboratoryPress, 2014), or as recommended by the manufacturer.

Example 1

1. Diagnostic criteria:

Reference is made to the 2010 edition of human monogenic genetic diseases and the 2019 edition of the diagnosis and treatment guidelines for rare diseases.

Mutations in the IL1RAPL1 gene are associated with mental retardation type 21 and are characterized by impaired or disabled cognitive neurological function, ranging from moderate dysnoesia to high functional autism, often severely affected in male patients, and may appear in some female carriers with a milder phenotype.

Genetic counseling and prenatal diagnosis: as the X chromosome linked recessive genetic disease, the pathogenic genes are determined through gene detection, so that the risk of the patient for producing inhibitors can be judged, and the treatment is guided; the method can also identify the carrier in the family of the patient and provide basis for prenatal diagnosis. The probability of a female carrier transmitting a pathogenic mutation site to its male offspring is 50% except that some new mutations occur. When pathogenic mutation of a precursor is known, prenatal diagnosis of a male fetus can be performed, and wool membrane, amniotic fluid cell DNA sequencing, umbilical cord blood detection and the like can be performed. Prenatal diagnosis is mainly performed by using technologies such as amniocentesis and chorionic villus sampling, and genetic analysis is performed on amniotic fluid, amniotic fluid cells and chorionic villus to judge whether chromosomes or genes of fetuses and the like are normal. Different sampling methods are adopted in different pregnancy stages, and generally, fluff is taken in early pregnancy (7-9 weeks of pregnancy), amniotic fluid is taken through amniocentesis in the middle pregnancy (16-20 weeks of pregnancy), fetal specimens are taken through a fetal mirror (18-20 weeks of pregnancy), umbilical vein blood is taken directly through the abdominal wall (18 weeks of pregnancy), and the like.

2. Object of detection

1 Mental retardation 21 family (called 1 family for short) is taken as a tested object, clinical information of part members of the 1 family is shown in a table 1, and a family map is shown in figure 1.

TABLE 1 clinical information of mental retardation 21 st family member

Note that: i and II represent the first generation and the second generation in sequence, and the 1 st family personnel I: 1, I: 2, II: 1 peripheral blood DNA is used for sequencing.

Example 2

Exon sequencing

1. The instrument is shown in table 2.

Table 2 instrumentation

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 V3.1.1 (Thermo), peripheral blood gDNA extraction kit (TIANGEN), agarose (TIANGEN) and EB dye (amerco).

3. Reagent formulation

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

Table 35 XTBE electrophoresis liquid formula

2) The working solution of 5 XTBE was diluted 10 times with ddH ₂ O to the stock solution of 5 XTBE in Table 3.

3) 10 Xerythrocyte lysate was prepared according to Table 4.

TABLE 410 Xerythrocyte lysate formula

4) The 1 x nuclear lysate formulation was formulated according to table 5.

Table 51 XNuclear lysate formula

4. Experimental procedure

After signing the informed consent, collecting 3-5 mL of peripheral blood of members I1, I2 and II 1 in the family 1 as a study sample.

4.1 Sample DNA extraction

1) Filling 3-5 mL of a sample into a 15mL centrifuge tube, adding 2-3 times of 1 Xerythrocyte lysate, uniformly mixing, and standing on ice for 30 minutes until the solution becomes transparent; if the villus tissue is adopted, the step 2) is directly carried out.

2) Centrifuge at 3000rpm for 10min at 4℃and 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 light shaking, and centrifuging at 3000rpm for 10 minutes at room temperature.

4) The supernatant was carefully transferred to another centrifuge tube, and an equal volume of a phenol/chloroform mixture (phenol/chloroform volume ratio 1:1) was added and mixed well and centrifuged at 3000rpm 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 glue, and recovering 150-250 bp 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 obtaining the pathogenic gene mutation IL1RAPL1:NM_014271.4:exon3:c.109G > A:p.D37N; wherein a mutation of c.109G > A to base G at position 109 of exon3 of the IL1RAPL1 gene to A may result in a missense mutation.

The genotype of the IL1 RAPL1:NM_014271.4:exo3:c.109G > A:p.D37N locus is "c.109G > A hemizygous mutation" in male individuals of family 1, the genotype of this locus is "c.109G > A heterozygous mutation" in female carrier individuals of family 1, and the genotype of this locus is "wild type" in normal individuals.

Example 3

Sanger sequencing validation

The results of the family 1 exome sequencing were further verified for the IL1 RAPL1:NM-014271.4:exo3:c.109G > A:p.D37N sites using Sanger sequencing. Genotype detection was performed on the 4 persons of family 1 (forerunner, forerunner father, forerunner mother) and the 100 normal persons outside family in example 1, respectively, at the positions IL1 RAPL1:NM-014271.4:exo3:c.109G > A: p.D37N.

The specific method comprises the following steps:

DNA extraction

Genomic DNA was extracted according to the method of example 2.

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/HGGATEWAYREDIRECT = manual & source = genome. Ucsc. Edu).

2.2 20 Pairs of candidate primers were designed for mutation site c.109G > A (see Table 6), and the merits of each pair of candidate primers were verified and evaluated by PCR experiments.

TABLE 6 basic conditions and verification experiment results for c.109G > A site candidate primers

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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 primers avoid such primers as much as possible.

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: pre-denaturation at 95 ℃ for 5min;

and a second step of: 30 cycles (denaturation at 95℃for 30 sec. Fwdarw. Tm annealing for 30 sec. Fwdarw. 72℃for 60 sec); (PCR amplification parameters were set according to the Tm values of the primers in Table 6);

And a third step of: extending at 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 5mm.

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 an electrophoresis buffer solution to enable the liquid level to be 1-2 mm higher than the glue 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, and putting the gel 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 the tube No. 7 only has a bright band and no band, 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 partial tubes of 2,3, 4, 5 and 6, judging that the effectiveness of the pair of primers and the reaction system is poor;

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

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

2.6 Based on the results of the statistics after the validation test of Table 7, SEQ ID NO.1 and SEQ ID NO.2 of Table 6 were selected as amplification primers for the IL1 RAPL1:NM-014271.4:exon 3:c.109G > A:p.D37N sites.

IL1RAPL1-Seq1F：5-ACTGTAGGAGCCAGGTGC-3’（SEQ ID NO.1）

IL1RAPL1-Seq1R：5’-CCAGATGGTATCGCTTTTC-3’（SEQ ID NO.2）

3. PCR amplification of mutation sites by primers screened in step 2.6 for 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 point PCR reaction system

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

PCR amplification procedure for IL1RAPL1:NM_014271.4:exon3:c.109G > A:p.D37N sites 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 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 15min and inactivated at 85℃for 15min.

6. BigDye reaction

The BigDye reaction system is shown in Table 9.

Table 9 BigDye reaction System

Sequencing PCR cycling conditions:

The first step: pre-denaturation at 96℃for 1min;

And a second step of: 33 cycles (denaturation at 96℃for 30 sec. Fwdarw. Annealing at 55℃for 15 sec. Fwdarw. 60℃for 4 min);

and a third step of: 4℃until sampling.

7. And (3) purifying a BigDye reaction product:

1) mu.L of 125mM EDTA (pH 8.0) was added to each tube, and 1. Mu.L of 3mol/L NaAc (pH 5.2) was added to the bottom of the tube;

2) Adding 70 μL 70% alcohol, shaking and mixing for 4 times, and standing at room temperature for 15min;

3) 3000g, centrifuging at 4 ℃ for 30min; immediately inverting the 96-well plate, and centrifuging 185g for 1min;

4) Standing at room temperature for 5min, volatilizing residual alcohol at room temperature, adding 10 μl Hi-Di formamide to dissolve DNA, denaturing at 96 deg.C for 4min, rapidly placing on ice for 4min, and sequencing on machine.

8. Sequencing

And (3) carrying out DNA sequencing on the purified BigDye reaction product, wherein sequencing primers are designed on the basis of SEQ ID NO.1 and SEQ ID NO.2, and nest primers (the second set of primers are designed within the range of the product sequence obtained by amplifying the first set of primers) are used as sequencing primers, and the primer sequences are shown as follows.

The sequencing primer sequences for the IL1RAPL1:NM_014271.4:exon3:c.109G > A:p.D37N sites were as follows:

IL1RAPL1-Seq1F：5’-TGTCTCACCAGTGCCCTCT-3’（SEQ ID NO.3）

IL1RAPL1-Seq1R：5’-TTCTTTGCTCATTCTACTTCC-3’（SEQ ID NO.4）

9. Analysis of results

The sequencing results for the IL1RAPL1:NM_014271.4:exon3:c.109G > A:p.D37N sites are shown in FIG. 2. From FIG. 2, it can be seen that the genotype of the c.109G > A locus of 1 male patient in family 1 is a "c.109G > A" hemizygous mutation; heterozygous mutation of the genotype of the locus of 1 female patient in family 1 is 'c.109G > A'; the c.109G > A locus genotype of 1 normal individual in line 1 and 100 normal controls without blood relationship is "wild-type".

Example 4

Mental retardation 21-type diagnostic kit and application thereof

1. The kit comprises the following components:

The kit comprises: 1) Amplification primers: SEQ ID NO. 1-2 as in example 3; 2) PCR buffer (10 XPCR buffer, composed of 500mmol/L KCl,100mmol/L Tris-HCl (pH 8.3), 15mmol/L MgCl ₂ and the balance water); 3) Taq enzyme (20U); 4) dNTPs (4 mM each of the four dNTPs); 5) c.109G > A positive reference DNA, the reference is a double-stranded DNA, the specific sequence of the c.109G > A mutation site positive reference is shown as SEQ ID NO.7, the specific sequence is shown as SEQ ID NO.7 ：5'-actgtaggagccaggtgctgaagaaggcttgcacgctgtaagagcctgccaagtgagcacattgaaaataggaagcaaatccctttcttcctgaaatgtctcaccagtgccctctccccacaaagcttcagtgccagctagttaagaaaaacatatctcaaaggcccagatctatttttaccgagcaggcagaaaggtacatttagaactaagaggcaataaatcaataaccagcacactgatataaacaaataataaataacagcagaagcagactcagtgaagaatatattgtttgttagcagagtccactactttgcagggccattctcagtttgattcattgctatgtgctctgtgcctagaataaaatctgacccaatatggatgctcagtaaatatttgttggatgaatgaataatctaattgcttattttttttgcctctaatgtttttcctctctttctctgtctctttttttacgatagCCGATGGATGCACTGACTGGTCTATC ATATCAAGAAATATCAAGTTTTGGTGGGAGAGCCTGTTCGAATCAAATGTGCACTCTTTTATGGTTATATCAGAACAAATTACTCCCTTGCCCAAAGTGCTGGACTCAGTTTGATGTGGTACAAAAGTTCTGGTCCTGGAGACTTTGAAGAGCCAATAGCCTTTGACGGAAGTAGAATGAGCAAAGAAGAAGACTCCATTTGGTTCCGGCCAACATTGCTACAGGACAGTGGTCTCTACGCCTGTGTCATCAGgtatccctttaattctattactgctgaatcaaagaaagcacaggctgctttctcgtacttaaatgttgctgctatctcttttgcctaaagccgttgtctcaatatttgcatgtgttgctgctttctaaaatttagaatcaaaatcataaaaagatgggattggaatttatattagacattatttctagcttatggagaaaagcgataccatctgg-3'; Single underlined bases are positions of an upstream primer and a downstream primer of PCR amplification, bases in a square frame are point mutation sites, single underlined bolded italic bases are positions of an upstream sequencing primer and a downstream sequencing primer, lower case letters are sequences of an intron region, and upper case letters are sequences of an exon region; 6) Sequencing primer: the sequence is shown as SEQ ID NO. 3-4.

2. The using method comprises the following steps:

298 individuals out of 66 mental retardation families were screened and tested altogether, and 11 families consistent with the present invention were found again, of which mental retardation type 21 patients were 12 and carriers were 14 (see table 10); the application of the gene mutation detection kit is now described by taking a family No. 2 as an example. The clinical information of family 2 is shown in table 11, and the family 2 map is shown in fig. 3.

TABLE 10 mental retardation 21 screening cases List

: Through amniotic fluid puncture and prenatal diagnosis and detection, the second embryo is a sick boy, and the two couples select to terminate pregnancy; the third embryo was identified as a carrier girl by embryo implantation pre-genetic diagnosis and prenatal diagnosis, and the postnatal method results were the same for infants.

#: By amniotic fluid puncture and prenatal diagnosis and detection, the second fetus is a normal girl, and the postnatal method results are the same for infants.

And (3) the following steps: by amniotic fluid puncture and prenatal diagnosis and detection, the second fetus is a girl carrier, and the postnatal method results are the same.

And (2): is a new variant, not inherited from its parent.

TABLE 11 clinical information of mental retardation 21 # 2 family members

Note that: i and II sequentially represent a first generation and a second generation.

The peripheral blood DNA of family personnel No. 2I 1, I2 and II 1 is used for the detection of the kit, and the steps are as follows:

1) Genomic DNA extraction: extracting a sample genomic DNA according to the procedure of example 2;

2) Firstly, adopting PCR amplification primers, taq enzyme, buffer solution, dNTPs, sample genome DNA and the like in a kit to carry out PCR amplification reaction;

3) Purifying the PCR amplification product;

4) Performing BigDye reaction on the purified PCR product by using the sequencing primer in the kit;

5) Purifying BiyDye reaction products;

6) BiyDye reaction products were sequenced and the sequenced sequence was compared to the normal sequence.

The detection result of the kit for the family member No. 2 is shown in FIG. 4, wherein the arrow indicates the mutation occurrence position. As can be seen from fig. 4, the genotype of the c.109g > a locus of the precursor in line No. 2 is the "c.109g > a" hemizygous mutation (layer C), the precursor mother is heterozygous mutation (layer a); the detection result confirms that the first-evidence patient is a mental retardation 21-type patient, and the mother genotype of the first-evidence patient is heterozygous and is a carrier; the male parent genotype (layer B) of the first person is wild type and is normal. The probability of male child born after parents of the prior art is 25% for mental retardation 21 type patients, the probability of female child born for carriers is 25% and the probability of normal individuals born is 50%, and the male child born after parents of the prior art is recommended to continue to bear if necessary, and needs to go to the hospital for genetic diagnosis or prenatal diagnosis before embryo implantation.

Example 5

Gene mutation ranking and interpretation (pathogenicity of mutation)

Mutation interpretation is based on our current knowledge of mental retardation type 21 and pathogenic gene IL1RAPL1 (https:// www.omim.org/entry/300143), and the clinical phenotypic association of the test subjects. Mutations follow the HGVS guidelines for mutation nomenclature (http:// www.hgvs.org /) and are named according to GenBank accession numbers (https:// www.ncbi.nlm.nih.gov/GenBank /). The rules for interpretation of genetic variation data refer to guidelines ：Richards,S,et al.,Standards and guidelines for the interpretation of sequence variants:a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology.Genet Med,advance online publication 5 March 2015.doi:10.1038/gim.2015.30; associated with the american society for genetics and Genomics (ACMG), chinese classification standards and guidelines for genetic variation: wang Qiuju, shen Yiping, ling kilovolts, etc. Genetic variation classification criteria and guidelines. Chinese science: life sciences, 2017, 47:668-688.

The genetic variation classification in the "genetic variation classification criteria and guidelines" is to perform five-level classification on variations based on typical data types (such as crowd data, calculation data, functional data, co-segregation data), which are respectively: "pathogenic (P)", "potentially pathogenic (likely pathogenic, LP)", "ambiguous (variant of uncertain significance, VUS)", "potentially benign (likely benign, LB)", and "benign (benign, B)"; the five-level classification was determined based on the composite score after interpretation analysis of each side/sub-item of variation (table 12).

TABLE 12 determination criteria for pathogenicity of variation

Before a five-level assessment, the sides/sub-items of the mutation/variation need to be analyzed/interpreted. Among these, the pathogenic mutation criteria can be classified as: for a given mutation/mutation, first, the criteria in Table 13 need to be selected based on observed evidence, it is determined which side/sub-items of the mutation/mutation can meet in Table 13, each is evaluated as being PVS1/PS 1-4/PM 1-6/PM 1-5/BA 1/BS 1-4/BP 1-6, and finally, the sub-items of the mutation/mutation can be combined according to the scoring rules of Table 12, and then a classification is selected from the five-level system according to the combined criteria of Table 12, e.g., if the side/sub-items of the mutation/mutation meet in Table 13 after analysis of the side/sub-items of the mutation/mutation by the criteria [ i.e., P1 ] "(i.e., the comprehensive criteria of the" comprehensive "P1, P1" is satisfied by the comprehensive criteria of "in Table 12)".

TABLE 13 variant interpretation criteria and variant pathogenicity criterion

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Analysis/interpretation of the sides/sub-items of mutations/variations is based on the corresponding bioinformatic analysis tools (see table 15) and a number of available data (libraries) (see table 16), including data obtained from existing cases, as well as data obtained from existing publications, such as public databases (e.g., clinVar or site-specific databases) and laboratory owned databases. The degree judgment evaluation criteria used in the analysis of mutation/mutation using various data (libraries) are shown in table 14.

Table 14 degree judgment evaluation criteria

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Table 15 biological information analysis tool

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Table 16 crowd database, disease-specific database and sequence database

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According to the above criteria or guidelines, the mutation of the IL1RAPL1 gene c.109G > A of the present invention is assessed as "pathogenic", and the criteria and specific evidence are shown in Table 17 below:

TABLE 17 pathogenic interpretation of IL1RAPL1 Gene c.109G > A mutations

XLR: refers to X chromosome recessive inheritance.

IL1RAPL1:NM_014271.4:exon3:c.109G > A:p.D37N variation rating evidence is as follows:

1. PS 2: through family personnel detection such as parents of a forerunner, the variation is analyzed and verified in 1 family to be a new variation (see table 10), namely the variation is a new variation confirmed by the relationship;

2. PS4: combining literature and this case, this variation was detected in multiple patients (see table 10);

3. PM1: the mutation is located in the hotspot mutation region, in the key domain of the IL1RAPL1 protein (Ig-like C2-type 1 Domain);

4. PM2: IL1RAPL1 gene c.109G > A variation was not found in the reference human thousand genome (1000G), human exon database (ExAC) and human genome mutation frequencies database (gnomAD);

5. PP3: various computer software predicts that this variation will have deleterious effects on the gene or gene product;

Thus, the comprehensive evidence of this mutation/variation (ps2+ps4+pm1+pm2+pp3) meets the "pathogenicity (P)" criterion (ii) in table 12, where the IL1RAPL1 gene c.109g > a variation is comprehensively determined to be "pathogenicity".

Example 6

Follow-up and diagnostic kit detection performance analysis

All family members were followed and re-sequenced analysis was performed on all individuals using the IL1RAPL1 gene targeted capture chip method (see table 18).

TABLE 18 results of Performance analysis for detection of the 109G > A site

Note that: the table contains follow-up data for family 1; the detected variation was positive in both the patient and the carrier.

From table 10, it can be seen that positive patients (13 cases) and carriers (15 cases) were found when the 12 families were examined. The positive site detection results are verified by an IL1RAPL1 gene targeting capture chip method. According to the follow-up and verification results, 28 true positive cases, 23 true negative cases, 0 false negative cases and 0 false positive cases are found in total. The sensitivity of detecting the mutation site marker of c.109G > A is 100.00%, 95% CI is 99.03% -100%, the specificity is 100%, and 95% CI is 99.03% -100%. The results show that the kit has good detection performance in clinical application.

According to the above examples, it can be seen that the IL1RAPL1 mutant protein and the IL1RAPL1 gene mutant of the invention can be used as biomarkers for diagnosing mental retardation type 21, and can provide possible drug targets for treating mental retardation type 21.

Although the foregoing embodiments have been described in some, but not all, embodiments of the invention, it should be understood that other embodiments may be devised in accordance with the present embodiments without departing from the spirit and scope of the invention.

Claims

1. An IL1RAPL1 gene mutant, characterized in that said IL1RAPL1 gene mutant is mutated from base G to base a at base 109 of exon 3 of said wild-type IL1RAPL1 gene as compared to the wild-type IL1RAPL1 gene; the accession number of the wild type IL1RAPL1 gene is NM_014271.4.

2. An IL1RAPL1 mutant protein, characterized in that the amino acid 37 of said IL1RAPL1 mutant protein is mutated from aspartic acid to asparagine compared to the wild type IL1RAPL1 protein; the accession number of the coding gene of the wild type IL1RAPL1 protein is NM_014271.4.

3. The application of a detection reagent of the IL1RAPL1 gene mutant as a detection target in preparing a mental retardation 21 type detection reagent or preparing a mental retardation 21 type detection kit, wherein the detection reagent and the detection kit comprise a positive reference, and the sequence of the positive reference is shown as SEQ ID NO. 7.

4. The use according to claim 3, wherein the detection reagent and/or the detection kit comprises amplification primers comprising an upstream primer IL1RAPL1-1F and a downstream primer IL1RAPL1-1R; the upstream primer IL1RAPL1-1F comprises a nucleotide sequence shown as SEQ ID NO.1, and the downstream primer IL1RAPL1-1R comprises a nucleotide sequence shown as SEQ ID NO. 2.

5. Use according to claim 3, characterized in that the detection reagent and/or the detection kit comprises sequencing primers comprising an upstream primer IL1RAPL1-Seq1F and a downstream primer IL1RAPL1-Seq1R; the upstream primer IL1RAPL1-Seq1F comprises a nucleotide sequence shown as SEQ ID NO. 3; the downstream primer IL1RAPL1-Seq1R comprises a nucleotide sequence shown as SEQ ID NO. 4.

6. The application of a detection reagent of the IL1RAPL1 mutant protein as a detection target spot in preparing a detection reagent of mental retardation type 21 and/or preparing a detection kit of mental retardation type 21, wherein the detection reagent and the detection kit comprise positive references of nucleotides corresponding to the mutant protein, and the sequences of the positive references are shown as SEQ ID NO. 7.

7. A mental retardation type 21 detection reagent, wherein the detection target of the detection reagent comprises the IL1RAPL1 gene mutant according to claim 1 or the IL1RAPL1 mutant protein according to claim 2, and the detection reagent comprises a positive reference, and the sequence of the positive reference is shown as SEQ ID NO. 7.

8. A kit for detecting mental retardation type 21, comprising the detection reagent according to claim 7.