CN115948530B

CN115948530B - Turner type X-linked syndrome pathogenic gene and primer pair for mental retardation and application thereof

Info

Publication number: CN115948530B
Application number: CN202210914615.XA
Authority: CN
Inventors: 曾桥; 伊宁; 罗娇娇
Original assignee: Hunan Jiahui Biotechnology Co Ltd
Current assignee: Hunan Jiahui Biotechnology Co Ltd
Priority date: 2022-08-01
Filing date: 2022-08-01
Publication date: 2024-04-23
Anticipated expiration: 2042-08-01
Also published as: CN115948530A

Abstract

The invention relates to the technical field of gene diagnosis, in particular to a Turner type X linkage syndrome pathogenic gene and primer pair with low intelligence and application thereof. The invention provides a Turner type X linkage syndrome pathogenic gene with mental retardation, which has G > T mutation at 9994 th position of 67 th exon of gene with accession number NM_ 031407.7. The pathogenic gene can be used for screening and diagnosing Turner type X-linked complex mental retardation people, and can specifically distinguish Turner type X-linked complex mental retardation patients from normal people; provides a new basis and a new approach for researching pathogenesis of Turner type X linked syndrome mental retardation, provides a new theoretical basis for treating the Turner type X linked syndrome mental retardation, and can provide a possible drug target for treating the Turner type X linked syndrome mental retardation.

Description

Turner type X-linked syndrome pathogenic gene and primer pair for mental retardation and application thereof

Technical Field

The invention relates to the technical field of gene diagnosis, in particular to a Turner type X linkage syndrome pathogenic gene and primer pair with low intelligence and application thereof.

Background

X chromosome-linked dysnoesia (X-1inked intellectual disability,XLID) is a congenital dysnoesia caused by mutation of genes located on X chromosome, and the related congenital dysnoesia accounts for about 15% of all congenital dysnoesia. XLID fall into two categories depending on whether there are other physiological defects besides mental retardation: S-XLID (syndromic forms) and NS-XLID (non-syndromic forms), S-XLID are manifested by abnormalities or defects in metabolic aspects, neurological features or other signs such as bones, craniofacial aspects, in addition to mental disorders.

According to the definition of the united states mental disorder association (AmericanAssociation 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 ratio of men and women is (1.4:1) - (1.6:1). To date, studies have found that a loss of function of 102 genes can lead to 81S-XLID syndromes and 50 more families of NS-XLID, and another 30S-XLID syndromes and 48 families carrying NS-XLID are associated with a specific region of the X chromosome. Factors causing congenital mental disorder include changes in gene copy number, deletion or insertion of small nucleotide fragments, abnormal functions of regulatory elements, epigenetic changes, etc., 10% -15% of mental disorder is related to X chromosome linkage.

Turner type X-linked complex mental retardation is one of X-linked mental disorders. Turner type X-linked syndrome mental retardation (MIM 309590), or Turner type X-linked syndrome mental retardation (Turner-type X-linked syndromic intellectual developmental disorder, MRXST), also known as Juberg-marsidi syndrome, is a small-headed syndrome and X-linked mental retardation Turner type, which is a neurological disorder with a highly variable clinical phenotype, some patients exhibit X-linked recessive inheritance, female carriers are unaffected, in other families, men are severely affected, female mutation carriers exhibit milder cognitive abnormalities or malformation characteristics. Furthermore, despite the selective inactivation of the X chromosome, female patients with new mutations still exhibit a complete phenotype. The affected individuals show overall retardation from the beginning of infancy, varying mental development, poor or absent speech ability, often accompanied by retardation of walking. Deformity characteristics are common and may include head size, small head, eye depth, strabismus, blepharoptosis, dysplasia, large or low ear, long face, narrowing of the temple, high bow waist, thin upper lip, scoliosis, or mild distal skeletal abnormalities, such as short limbs or tapered fingers. Other features, such as hypotonia, seizures, and bone age delays, are more individual to individual. Gene localization and cloning indicated that one important gene associated with the disease was HUWE a 1.

The HUWE gene (MIM 300697) is 154.6kb long and is located on chromosome xp11.22, comprising 84 exons and 83 introns, which encode a HUWE protein that yields 4374 amino acids. HUWE1 protein is E3 ubiquitination ligase, has three functional domains of HECT, UBA and WWE, and has abnormal functions, which can cause ubiquitination abnormality of cancer-inhibiting factor P53, further cause imbalance of nerve cell fate regulation and abnormal development of a nervous system, influence organs such as eyes, bones and prostate, cause serious mental disability and delay skeletal maturation.

Thus, gene mutation is an important genetic basis for the development of diseases, and gene diagnosis is an important genetic criterion for diagnosing Turner type X-linked syndrome mental retardation. However, no diagnostic reagent has been reported to specifically distinguish patients suffering from Turner-type X-linked syndrome type mental retardation from mutation, carriers and normal individuals.

Disclosure of Invention

In order to solve the problems, the invention provides a Turner type X linkage syndrome pathogenic gene and primer pair with low intelligence and application thereof. The pathogenic gene can be used for screening and diagnosing Turner type X-linked syndrome mental retardation people, and can specifically distinguish Turner type X-linked syndrome mental retardation patients from normal people.

In order to achieve the above object, the present invention provides the following technical solutions:

The invention provides a Turner type X linkage syndrome pathogenic gene with mental retardation, wherein G > T mutation exists at 9994 th position of 67 th exon of a gene with accession number NM_ 031407.7.

The invention provides a primer pair for the pathogenic gene, wherein the primer pair comprises HUWE-F and HUWE-R;

The nucleotide sequence of HUWE-F is shown as SEQ ID NO. 1; the nucleotide sequence of HUWE-R is shown as SEQ ID NO. 2.

The invention provides application of the pathogenic gene as a target gene in preparing a reagent for diagnosing Turner type X-linked syndrome mental retardation.

The invention provides a reagent for diagnosing Turner type X linkage syndrome mental retardation, which comprises the primer pair.

Preferably, the reagent further comprises a sequencing primer pair; the sequencing primer pair comprises HUWE-SeqF and HUWE1-SeqR;

The nucleotide sequence of HUWE to SeqF is shown as SEQ ID NO. 3; the nucleotide sequence of HUWE-SeqR is shown as SEQ ID NO. 4.

The invention provides application of the pathogenic gene serving as a target gene or the reagent in preparation of a kit for diagnosing Turner type X linkage syndrome mental retardation.

The invention provides a kit for diagnosing Turner type X linkage syndrome mental retardation, which comprises the reagent.

Preferably, the kit further comprises a positive mutant reference DNA; the nucleotide sequence of the positive mutation reference DNA is shown as SEQ ID NO. 5.

The invention provides a method for identifying the genotype of HUWE < 1 > NM_031407.7:exo67:c.9994 sites, 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 primer pair to obtain an amplification product;

The amplified product was sequenced and the genotype of HUWE 1:NM-031407.7:exo67:c.9994 sites was determined.

Preferably, the sample to be tested comprises blood.

The beneficial effects are that:

The invention provides a Turner type X linkage syndrome pathogenic gene with low intelligence, wherein G > T mutation exists at 9994 th position of 67 th exon of a gene with accession number NM_031407.7 to form the pathogenic gene. The invention firstly utilizes the exon sequencing technology to screen pathogenic gene mutation highly related to Turner type X linkage syndrome mental retardation by utilizing the exon sequencing technology, and in order to avoid false positive results, the pathogenic gene mutation sites of the Turner type X linkage syndrome mental retardation are obtained by verification through Sanger sequencing, and the pathogenic gene mutation sites of the Turner type X linkage syndrome mental retardation are specifically: HUWE 1:NM-031407.7:exo67:c.9994G > T:p.V3332F, and found that HUWE 1:NM-031407.7:exo67:c.9994G > T:p.V3332F site mutations can lead to the onset of Turner type X-linked syndrome mental retardation. Thus, by detecting whether a subject carries the aforementioned pathogenic genes, a genetic diagnosis for screening or diagnosing Turner type X-linked syndrome mental retardation is used to guide the treatment. The invention lays an important foundation for researching pathogenesis of Turner type X linkage syndrome mental retardation, and provides a brand-new theoretical basis for treating Turner type X linkage syndrome mental retardation patients; the invention can provide a possible medicine target for treating Turner type X linkage syndrome mental retardation. Meanwhile, the pathogenic gene provided by the invention can be used for genetic diagnosis and prenatal and postnatal guidance of Turner type X linkage syndrome mental retardation.

Furthermore, the reagent and the diagnostic kit provided by the invention can be used for rapidly and effectively predicting or diagnosing whether a patient is Turner type X-linked syndrome type X-linked mental retardation.

Drawings

FIG. 1 shows a Turner type X linkage syndrome, mental retardation, family 1 genetic map; 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 HUWE of the genotype at the locus NM-031407.7:exo67:c.9994G > T:p.V3332F using Sanger sequencing, with the first evidence in line 1 being the patient (the position of the mutation occurrence indicated by the arrow in the sequencing);

FIG. 3 shows a Turner type X linkage syndrome, mental retardation, family 2 genetic map; wherein, it represents normal male individuals, it represents female carriers, ■ represents male patients, ↗ represents forerunner;

FIG. 4 shows a graph of the results of the detection HUWE of the genotype at the site NM-031407.7:exo67:c.9994G > T:p.V3332F using the kit, with the first-evidence and first-evidence brother being patients in family 2 (the position of mutation occurrence indicated by the arrow in the sequencing diagram).

Detailed Description

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

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

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

In the present invention, the term "hemizygous mutation" means that a mutation exists in a sex chromosome, and since a male has only one X chromosome, the gene is monovalent and there is no corresponding allele, and the mutation is called "hemizygous mutation".

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, 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, without amplifying other genes. For example, specifically amplifying HUWE genes means that the primers amplify only HUWE genes and not other genes in the PCR reaction.

The invention provides a Turner type X linkage syndrome pathogenic gene with low intelligence, wherein G > T mutation exists at 9994 th position of 67 th exon of a gene with accession number NM_031407.7 to form the pathogenic gene. In the present invention, when the 9994 th base of the 67 th exon of the gene of accession number HUWE 1:1 NM-031407.7 is mutated from G to T, it results in the 3332 th amino acid being changed from valine to phenylalanine.

The c.9994G > T mutation refers to that the 9994 th base G of a wild HUWE gene is mutated into T to form a HUWE gene mutant, and the nucleotide sequence of the HUWE gene mutant is preferably shown as SEQ ID NO.34 (particularly: CTGTTTTCTGC). Compared with the protein coded by the wild HUWE gene, the HUWE mutant protein of the invention has the 3332 th amino acid mutated from valine (V) to phenylalanine (F), namely the HUWE mutant protein contains p.V3332F mutation, and the mutation is caused by missense mutation of c.9994G > T; the amino acid sequence of HUWE mutant protein is shown as SEQ ID NO.35 (specifically: APVFCRH).

By detecting whether or not the subject carries the above-mentioned pathogenic gene, it is possible to diagnose whether or not the subject is a patient, and thus, it is possible to use the genetic diagnosis for screening or diagnosing Turner type X-linked syndrome mental retardation.

The invention also provides a primer pair for amplifying the pathogenic genes, wherein the primer pair comprises HUWE-F and HUWE-R; the nucleotide sequence of HUWE-F is shown as SEQ ID NO. 1; the nucleotide sequence of HUWE-R is shown as SEQ ID NO. 2. The primer provided by the invention has good specificity, and can not generate non-specific products during PCR amplification; 3 or more continuous complementary bases do not appear in the primer and among the primers, so that primer dimer is avoided; the content of the primer G+C is moderate, the amplification effect of PCR is not hindered, and the amplification effect is optimal; the primer has proper length, can meet the requirement of specific 'grabbing' of a DNA template of a PCR primer, and has moderate annealing temperature (Tm), the primer length is generally proportional to the Tm, and too long and too short primers can cause too large or too small Tm values.

The pathogenic gene provided by the invention can specifically distinguish Turner type X linkage syndrome patients with mental retardation and normal people. Therefore, the pathogenic gene disclosed by the invention can be used for preparing a reagent or a kit for diagnosing Turner type X linkage syndrome mental retardation.

The invention also provides application of the pathogenic gene serving as a target gene in preparation of a reagent for diagnosing Turner type X linkage syndrome mental retardation. The reagent of the invention can accurately diagnose whether the patient is a Turner type X-linked syndrome mental retardation patient.

The invention also provides a reagent for diagnosing Turner type X linkage syndrome mental retardation, which comprises a primer pair for amplifying the pathogenic genes. The present invention has been described in detail above for this primer pair, and thus, any description thereof will not be made here.

The invention also provides a kit for diagnosing Turner type X linkage syndrome mental retardation, which comprises the reagent and the sequencing primer pair. The reagents of the present invention have been described in detail above, and thus are not described in any detail herein. In the present invention, the kit preferably further comprises a positive mutation reference DNA; the nucleotide sequence of the positive mutation reference DNA is preferably shown in SEQ ID NO. 5.

The invention also provides a method for identifying the genotype of HUWE 1:NM_031407.7:exo67:c.9994 locus, which comprises the following steps:

sequencing the amplified product to determine the genotype of the pathogenic gene.

In the present invention, the reaction procedure 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.

In the present invention, the reaction system for PCR amplification preferably comprises, in 20. Mu.L, 2. Mu.L of 10 XPCR buffer, 10mmol/LdNTPs 0.4.4. Mu.L, 100 ng/. Mu. L HUWE 1-F0.5. Mu.L, 100 ng/. Mu. L HUWE 1-R0.5. Mu.L, 1. Mu.L of template, 0.2. Mu.L of 5 u/. Mu. LTaq enzyme and the balance ddH ₂ O.

In the present invention, the sample preferably includes blood. The blood is not particularly limited in the present invention, and preferably includes fresh blood, old blood, blood spots, blood marks, or the like, that is, blood containing DNA as long as it contains DNA.

In the present invention, when the sample is derived from a male, genotypes of HUWE 1:NM-031407.7:exo67:c.9994 sites are specifically two of "c.9994G > T hemizygous" and "c.9994G hemizygous"; when HUWE < 1 > NM_031407.7:exo67:c.9994 locus has genotype of "c.9994G > T hemizygous", the mutation of HUWE1 gene is judged, and male individuals are patients; when HUWE < 1 > NM_031407.7:exo67:c.9994 locus genotype is "c.9994G hemizygous", judging that HUWE gene has no mutation, and the male individual is normal;

When the sample is derived from a female, the genotypes of HUWE 1:NM-031407.7:exo67:c.9994 locus have three cases of "c.9994T/T homozygote", "c.9994G > T heterozygote" and "c.9994G/G homozygote"; when HUWE < 1 > NM_031407.7:exo67:c.9994 locus genotype is "c.9994T/T homozygote", determining that the HUWE1 gene has homozygote mutation, and female individuals are patients; when HUWE < 1 > NM_031407.7:exo67:c.9994 locus genotype is "c.9994G > T heterozygote", judging that the HUWE1 gene has heterozygote mutation, and the female individual is a carrier; when the genotype of HUWE 1:NM_031407.7:exo67:c.9994 locus is "c.9994G/G homozygote", the HUWE1 gene is judged to have no mutation, and the female individual is normal.

For further explanation of the present invention, a Turner type X-linked syndrome causative gene, primer pair and application thereof provided by the present invention are described in detail below with reference to the accompanying drawings and examples, but they should not be construed as limiting the scope of the present invention.

Example 1

Sample acquisition

The inventor discovers a Turner type X linkage syndrome mental retardation family (abbreviated as family 1), and clinical information of part members of the family 1 is shown in table 1. FIG. 1 shows a HUWE family map in which ∈Μ represents normal male individuals, "∈12 represents female carriers, ■ represents male patients, and ↗ represents forerunner.

1. Diagnostic criteria:

Reference may be made to the "human monogenic genetic disease" 2010 edition and the "diagnosis of childhood dysnoesia or general developmental delay etiology diagnosis policy expert consensus" 2018 edition:

Clinical characteristics: developmental retardation, different mental development, poor speech ability or loss, often accompanied by walking retardation; malformed features may include large head, small head, deep eye, strabismus, small eyelid fissures, dysplasia, large or low ear, long face, narrowing of the temple, high arch waist, thin upper lip, scoliosis, or mild distal skeletal abnormalities, such as short limbs or tapered fingers; men often have cryptorchism; other features such as hypotonia, seizures, and delayed bone age.

TABLE 1 Turner clinical information of members of family 1 for X-linked syndrome type mental retardation

The family spectrogram of No. 1 is shown in FIG. 1, and the numbers are I (first generation) and II (second generation).

The peripheral blood DNA of family 1 personnel I: 1 (father), I: 2 (mother), II: 2 (forensic) was used for sequencing analysis.

Exon sequencing

2. The instrument is shown in table 2.

Table 2 list of instruments and devices

3. Reagent consumable

Human whole exon sequencing kit (Agilent), DNA 1000 kit (Agilent), 96 well plate (Axygen), different model tips (Axygen), 200 μl centrifuge tube (Eppendorf), 1.5mL centrifuge tube (Eppendorf), capillary electrophoresis buffer (Thermo), sequencing standard (Thermo), absolute ethanol (Thermo), bigDye terminator v3.1 (Thermo), peripheral blood gDNA extraction kit (TIANGEN), agarose (TIANGEN) and EB dye solution (amerco).

4. Reagent formulation

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

Table 35 XTBE electrophoresis liquid formula

Reagent(s)	Volume/weight
		Tris	5.4g
Boric acid	750mg
		EDTA(pH8.0，0.5mol/L)	2mL
ddH₂O	90mL

The final volume was adjusted to 100mL with ddH ₂ O.

The working solution of the 0.5 XTBE electrophoresis solution was diluted 10-fold with ddH ₂ O.

10 Xerythrocyte lysate was prepared according to Table 4.

TABLE 410 Xerythrocyte lysate formula

Reagent(s)	Volume/weight
		NH4Cl	82.9g
KHCO₃	10g
		EDTA	0.37g
Adding ddH ₂ O	To 1000mL

Autoclaving and storing at 4deg.C.

1 Xnuclear lysate was prepared according to Table 5.

Table 51 XNuclear lysate formula

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

5. Experimental procedure

After signing the informed consent, 3-5 mL of peripheral blood of members of family such as I: 1 (father), I: 2 (mother), II: 2 (forerunner) and the like are collected.

5.1 Sample DNA extraction

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

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

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

4) The supernatant was carefully transferred to another centrifuge tube, and a mixed solution of phenol and chloroform (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

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

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.

5.3 Results

Finally, 1 pathogenic gene mutation HUWE 1:NM_031407.7:exo67:c.9994G > T:p.V3332F is obtained; mutation of base 9994 from G to T results in amino acid 3332 from valine to phenylalanine. The genotype of 2 male patients (forerunner and forerunner brother) HUWE 1:nm_031407.7:exo67:c.9994 g > t:p.v3332f sites in family 1 is a "c.9994g > T hemizygous" mutation; genotype of HUWE 1:NM-031407.7:exo67:c.9994G > T:p.V3332F site in individuals of female carrier in family 1 is a "c.9994G > T heterozygote" mutation; the genotype of 1 male normal individual in family 1 was a wild type without mutation.

Example 2

Sanger sequencing validation

The results of the exome sequencing were further verified using Sanger sequencing to verify HUWE 1:NM-031407.7:exo67:c.9994G > T:p.V3332F sites. Genotype detection was performed on 4 family members such as I1, I2, II 1, II 2 and the like and 100 normal persons outside the family in example 1 at HUWE:NM-031407.7:exo67:c.9994G > T:p.V3332F locus, respectively.

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

2.2 Designing 15 pairs of candidate primers for the c.9994G > T site, respectively (see Table 6), and verifying and evaluating the merits of each pair of candidate primers by PCR experiments

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

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

① The length of the primer is 15-30 nt, 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%, and the continuous 8 bases at the 3' -end of the primer cannot have a complete complementary sequence outside the region to be amplified, otherwise, the non-specific amplification is easy to cause.

2.3 Candidate primer PCR verification reaction

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

TABLE 7 primer detection PCR reaction System

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

The first step: 95 ℃ for 5min;

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

And a third step of: 72 ℃ for 7min;

fourth step: 4℃until sampling.

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

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

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

3) After the gel is cooled to about 50 ℃, pouring the gel into a sealed gel sampler to enable the thickness to be about 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 electrophoresis buffer solution to make the liquid level 1-2 mm higher than the rubber surface, and pulling out the comb upwards; and (3) uniformly mixing the sample and the DNA size standard substance with the sample loading liquid by using a micropipette, and adding the mixture into each sample loading hole, wherein the DNA is sunk into the hole bottom due to the fact that the sucrose in the sample loading liquid has a larger specific gravity.

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

7) Cutting off the power supply, taking out the gel, putting the gel into an EB water solution with the concentration of 0.5g/ml, and dyeing for 10-15 minutes.

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

2.5 Evaluation of results:

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

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

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

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

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

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

The primer sequences for HUWE 1:NM-031407.7:exo67:c.9994G > T:p.V3332F sites are as follows:

5’-TGAAACACCCAAACTCACTAC-3’(SEQ ID NO.1)；

5’-CAATACAGGGCTCAACACTT-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

Reagent(s)	Volume of
		10 XPCR buffer	2.0μL
10mmol/L dNTPs	0.4μL
		100ng/μL HUWE1-F	0.5μL
100ng/μL HUWE1-R	0.5μL
		100 Ng/. Mu.L of peripheral blood extract DNA	1.0μL
5U/. Mu.L Taq enzyme	0.2μL
		ddH₂O	15.4μL

Reaction conditions: the reaction system was put into 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- > 53 ℃,30 seconds- > 72 ℃,60 seconds);

And a third step of: 72 ℃,7 minutes;

fourth step: 4℃until sampling.

4. Agarose gel electrophoresis detection

Refer to step 2.4 above.

5. Purifying a PCR product by an enzymolysis method: to the 5. Mu.LPCR product, 0.5. Mu.L of exonuclease I (Exo I), 1. Mu.L of alkaline phosphatase (AIP) was added, and the mixture was digested at 37℃for 15 minutes and inactivated at 85℃for 15 minutes.

6. BigDye reaction

The BigDye reaction system is shown in Table 9.

Table 9 BigDye reaction System

Reagent(s)	Dosage of
		DNA after purification of PCR product	2.0μL
3.2 Pmol/. Mu.L sequencing primer	1.0μL
		BigDye	0.5μL
5 XBigDye sequencing buffer	2.0μL
		ddH₂O	4.5μL

Sequencing PCR cycling conditions:

the first step: 96℃for 1 minute;

And a second step of: 33 cycles (96 ℃,30 seconds- > 55 ℃,15 seconds- > 60 ℃,4 minutes);

and a third step of: 4℃until sampling.

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

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

2) Adding 70 mu L of 70% alcohol, shaking and mixing for 4 times, and standing at room temperature for 15 minutes;

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

4) After 5 minutes at room temperature, the residual alcohol was allowed to evaporate at room temperature, 10. Mu.L Hi-Di formamide was added to dissolve DNA, denatured at 96℃for 4 minutes, quickly placed on ice for 4 minutes, and sequenced on the machine.

8. Sequencing

DNA sequencing is carried out on the purified BigDye reaction product, and a nest primer (a second set of primers are designed within the range of the product sequence obtained by amplifying the first set of primers) is designed on the basis of the PCR preferred primers as a sequencing primer, wherein the sequence of the sequencing primer is as follows:

5’-GGGCGTAAACATACCGAGAA-3’(SEQ ID NO.3)；

5’-GCTGCCACCCTTCTTCCA-3’(SEQ ID NO.4)。

9. Analysis of results

The Sanger sequencing results of FIG. 2 show that the genotype of line 1 male patient HUWE 1:NM-031407.7:exo67:c.9994G > T:p.V3332F locus is "c.9994G > T hemizygous". FIG. 2 shows, in the position indicated by the arrow in the sequencing diagram, that the A-and D-tier Turner-type X-linked complex mental retardation patient HUWE 1:NM-031407.7:exo67:c.9994G > T and p.V3332F site genotypes are "c.9994G > T hemizygous" mutations; the position indicated by the arrow in the sequencing diagram of FIG. 2 shows that the B-layer female carrier HUWE 1:NM-031407.7:exo67:c.9994G > T:p.V3332F locus genotype is a "c.9994G > T heterozygote" mutation, and the C-layer individual genotype is a wild type.

Example 3

HUWE 1.9994G > T mutation (also called HUWE 1:NM_031407.7:exo67:c.9994G > T:p.V3332F) diagnostic kit and application

1. The kit comprises the following components:

1) Amplification primers (1.2. Mu.g per primer): as shown in example 2;

2) Buffer (500. Mu.L of 10 XPCR buffer, 500mmol/LKCl,100mmol/L Tris.Cl (pH 8.3), 15mmol/LMgCl ₂);

3) Taq enzyme (20U);

4) dNTPs (4 mM each of the four dNTPs);

5) HUWE 1:1 c.9994G > T positive mutation reference DNA the reference is a double-stranded DNA, the specific sequence is as follows:

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

6) Sequencing primer: as shown in example 2;

2. The using method comprises the following steps:

the method is applied to the detection of family 2 patients, and the details are shown in Table 10.

Table 10 Turner clinical information of members of family 2 for X-linked syndrome type mental retardation

The family spectrogram No. 2 is shown in FIG. 3, and the numbers are I (first generation) and II (second generation).

The peripheral blood DNA of family 2 personnel I: 1 (father), I: 2 (mother), II: 2 (patient) were used for sequencing analysis.

1) Genomic DNA extraction: sample genomic DNA was extracted as in example 1;

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

3) Purifying the PCR amplification product;

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

5) Purifying BiyDye reaction products;

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

As shown in FIG. 4, the genotype of genotype 1 male patient HUWE of family 2, NM-031407.7, exo67, c.9994G > T, p.V3332F locus was "c.9994G > T hemizygous". FIG. 4 shows, in the sequencing diagram, that the locus of the locus genotype of C-layer Turner type X-linked complex mental retardation patient HUWE1: NM-031407.7: exon67: c.9994G > T: p.V3332F is a "c.9994G > T hemizygous" mutation; the position indicated by the arrow in the sequencing diagram of FIG. 4 shows that the B-layer female carrier HUWE 1:NM-031407.7:exo67:c.9994G > T:p.V3332F locus genotype is a "c.9994G > T heterozygote" mutation, and the A-layer individual genotype is a wild type.

In summary, the invention discovers a novel HUWE gene mutant, confirms that the novel mutant is closely related to the incidence of Turner type X linked syndrome mental retardation, and is used for screening or diagnosing the genetic diagnosis of the Turner type X linked syndrome mental retardation by detecting whether a subject carries the pathogenic gene, and simultaneously, the reagent and the diagnostic kit provided by the invention can be used for rapidly and effectively predicting or diagnosing whether a patient is the Turner type X linked syndrome mental retardation.

While the invention has been described in terms of preferred embodiments, it is not intended to be limited thereto, but rather to enable any person skilled in the art to make various changes and modifications without departing from the spirit and scope of the present invention, which is therefore to be limited only by the appended claims.

Claims

1. A Turner type X linkage syndrome mental retardation pathogenic gene, wherein the pathogenic gene is characterized in that a 9994 th base is mutated from G to T in a coding region of a nucleotide sequence shown in accession No. nm_031407.7 on NCBI.

2. The use of the pathogenic gene of claim 1 as a detection target in the preparation of a reagent for diagnosing Turner type X-linked syndrome mental retardation.

3. A reagent for diagnosing Turner type X-linked complex mental retardation, comprising a primer pair and a sequencing primer pair for amplifying the pathogenic gene of claim 1;

The primer pair for amplifying the pathogenic gene of claim 1 consists of HUWE-F and HUWE-R;

The nucleotide sequence of HUWE-F is shown as SEQ ID NO. 1; the nucleotide sequence of HUWE-R is shown as SEQ ID NO. 2;

The sequencing primer pair consists of HUWE-SeqF and HUWE 1-SeqR;

4. Use of the pathogenic gene of claim 1 or the agent of claim 3 for the preparation of a kit for diagnosing Turner type X-linked syndrome mental retardation.

5. A kit for diagnosing Turner type X-linked complex mental retardation, comprising the agent of claim 3.

6. The kit of claim 5, further comprising a positive mutant reference DNA; the nucleotide sequence of the positive mutation reference DNA is shown as SEQ ID NO. 5.