CN106282171B - STR locus of PKD2 gene and application thereof - Google Patents

Abstract

The invention discloses an STR locus of a PKD2 gene, which is selected from nucleic acid sequences shown in SEQ ID NO. 1-SEQ ID NO. 6. The invention also discloses application of the STR locus of the PKD2 gene, which is used for embryo pre-implantation diagnosis or prenatal diagnosis of the autosomal dominant hereditary polycystic kidney disease. The STR locus of the PKD2 gene has higher resolution, is used for linkage genetic analysis of the PKD2 gene, can obviously improve the typing and identifying efficiency and accuracy, and provides a basis for clinical embryo pre-implantation diagnosis of autosomal dominant hereditary polycystic kidney disease.

Description

STR locus of PKD2 gene and application thereof

Technical Field

The invention relates to the technical field of clinical molecular diagnosis, in particular to an STR locus of a PKD2 gene and application thereof.

Background

Autosomal Dominant Polycystic Kidney Disease (ADPKD) is one of the most common monogenic genetic diseases in humans, with a prevalence rate of about 0.1%. The main clinical feature is that bilateral kidneys form multiple liquid vesicles, and cysts grow progressively, which results in damage to kidney structure and function, and about 50% of patients develop terminal renal failure by the age of 60, accounting for about 10% of the causes of terminal renal failure. Patients with polycystic kidney disease can affect various organs of the whole body, such as liver, pancreas, spleen cyst, intracranial aneurysm, hypertension and the like, besides the change of the kidney. Other scholars believe that it is associated with pancreatic carcinogenesis. ADPKD is inherited in a delayed manner, and patients generally develop clinical symptoms after about 40 years of age, at which time most of the disease-causing genes are passed on to the next generation. Because no effective treatment method for ADPKD exists at present, the timely presymptomatic gene diagnosis of members at risk of disease in the ADPKD family, especially the prenatal diagnosis and the preimplantation genetic diagnosis of high-risk fetuses or embryos, is the key for controlling the occurrence and development of the ADPKD.

There is genetic heterogeneity in ADPKD, and there may be 3 mutant genes currently known to cause the disease, PKD2, PKD2 and PKD3, respectively. Of which about 15% of patients are caused by PKD2 mutation. The PKD2 gene is located in the region 2 to the region 3 of the long arm 2 of chromosome 4 (4q22-23), the length of the gene is 68kb, the gene consists of 15 exons, the transcription starting point is in exon 1, the transcribed mRNA is about 5.4kb, and the encoded protein is a transmembrane glycoprotein containing 968 amino acids. The PKD2 gene has more types of mutation, and no obvious mutation hot spot exists, which brings difficulty to the mutation detection of the PKD2 gene. At present, the following methods are mainly used for detecting the gene of PKD 2:

first, gene chip detection

The gene chip is prepared by fixing detection probes on the surface of a carrier according to a preset array matrix, hybridizing the detection probes with fluorescence-labeled sample nucleic acid molecules, and detecting the structure or expression condition of genes in a sample by detecting fluorescence signals displayed on the detection probes. The gene chip has the characteristics of large information amount, high sensitivity, parallel rapid detection and the like. In 2006, Wu faithful standard and the like establish a genotyping gene chip aiming at a common PKD2 gene mutation site in Chinese population, and the chip is used for detecting PKD2 gene mutation, so that certain achievements are obtained. However, the technique has the defects that the operation is complicated and tedious, and only specific mutations can be detected, so that the clinical application is greatly limited.

Two, single strand conformation polymorphism analysis (SSCP)

SSCP is a detection method in which the migration rate changes when non-denaturing polyacrylamide gel electrophoresis is performed, utilizing the difference in conformation of single strands of DNA due to the difference in the constituent bases. The SSCP technology is low in cost, simple, convenient and quick, is not limited by family linkage analysis, can detect mutation of a single patient, and has high mutation detection rate of a single locus. Deltas et al used this technique to detect PKD2 mutations with a total of at least 57 mutations detected, 30% being nonsense, 51% being insertion or deletion, 14% being shear and 15% being missense. However, the technology has a missing rate of 5-30%, and only the existence of the mutation can be detected, but the position of the mutation cannot be determined, and the defects seriously limit the popularization and application of the technology in clinic.

Modified high performance liquid chromatography (DHPLC)

DHPLC is used for carrying out PCR amplification on an amplification region, heating and denaturing a PCR product into a single-chain state, detecting the content of a base in the PCR product by a high-pressure liquid chromatography analyzer, and if the content of the base is changed, moving a peak value occurs, so that whether mutation occurs or not is detected. Mizoguchi et al first used this technique to detect 8 mutation sites from the partial coding region of PKD2 (exons 23-34) and the full coding region of PKD 2. Rossetti et al then performed PKD2 and PKD2 total coding region mutation assays on 45 ADPKD patients, respectively, using DHPLC technology. The DHPLC technology is rapid and simple, can judge whether mutation exists, but cannot determine the position and the type of SNP, and needs standard samples or combined sequencing verification. Only detecting heterozygous mutation is the main disadvantage of DHPLC, and the detection flux and sensitivity of point mutation screening are different from other detection technologies.

Denaturing Gradient Gel Electrophoresis (DGGE)

Denaturing gradient gel electrophoresis (Denaturing gradient gel electrophoresis) is a method for separating DNA fragments with the same fragment size and different base compositions by changing the electrophoretic mobility according to the difference of melting behaviors of DNA in denaturants with different concentrations. Specifically, a specific double-stranded DNA fragment is electrophoresed in a polyacrylamide gel containing a linear denaturant gradient from low to high, the DNA fragment migrates to a direction of a high-concentration denaturant along with the electrophoresis, when the DNA fragment reaches the minimum concentration denaturant required by denaturation, the double-stranded DNA forms a partial melting state, and the migration rate is slowed, and due to the sequence specificity of the denaturation, the DGGE can distinguish the DNA fragments with low to single base difference, and the DNA fragment is used in the field of ADPKD gene mutation detection. The defect of the technology is that the detection flux is low, the specific type of the mutation cannot be determined, and the application of the technology in clinical detection is limited.

Five, short tandem repeat Sequences (STR)

STRs are widely present in genomes, and are sequences formed by tandem repeat of units with 2-6 bases as cores, and are also called microsatellite DNA (micro satellite DNA). Genetic polymorphisms are present in the population due to variations in the number of repeats of the core sequence, but are highly conserved within the same family, inherited in a mendelian fashion. Thus, ADPKD patients can be screened in the same family using microsatellite DNA polymorphism linkage analysis. STRs currently used for ADPKD linkage analysis are basically (CA) n repeats, and microsatellite DNA linked with PKD2 includes D4S414, D4S423, D4S1542, D4S1563 and the like. Because of the complexity of the ADPKD gene and the uncertainty of the mutation site, linkage analysis remains the major means of ADPKD gene diagnosis at present. However, none of the STR loci currently used is an optimal choice, and some STR loci are difficult to genotype. For example, the dinucleotide slides during repeated PCR amplification, the detection is unstable, and misjudgment is easy to occur; D4S423 is far away from PKD2 gene, and misdiagnosis may be caused by gene recombination; some situations that the amplification of the STR locus fails in actual detection may affect the interpretation of the detection result, and the like. In order to solve the problems, more information STR loci which are close to the PKD2 gene, small in genetic distance, low in recombination rate and high in heterozygosity are required to be screened out to be used as genetic markers to carry out gene detection on autosomal dominant hereditary polycystic kidney disease, so that the detection efficiency is improved, and a basis is provided for clinical diagnosis and embryo preimplantation diagnosis of the disease.

Disclosure of Invention

The invention aims to solve the technical problem that the detection efficiency is not high due to the defects of long distance, high recombination rate, low heterozygosity and the like of the STR locus of the PKD2 gene at present, provides a new STR locus of the PKD2 gene with higher resolution, and the new STR locus is used for linkage genetic analysis of the PKD2 gene and can effectively improve the typing identification efficiency and accuracy.

In addition, it is also needed to provide a detection kit for PKD2 gene-linked genetic analysis.

In order to solve the technical problems, the invention is realized by the following technical scheme:

in one aspect of the invention, the STR locus of the PKD2 gene is provided and is selected from the nucleic acid sequences shown in SEQ ID NO. 1-SEQ ID NO. 6.

In another aspect of the invention, the invention also provides an application of the STR locus of the PKD2 gene, which is used for embryo pre-implantation diagnosis or prenatal diagnosis of the autosomal dominant polycystic kidney disease.

In another aspect of the invention, the invention also provides a detection kit for diagnosing autosomal dominant hereditary polycystic kidney disease, which comprises specific primer pairs respectively aiming at any more than two STR loci in SEQ ID NO. 1-SEQ ID NO. 6.

Preferably, the primer pair comprises specific primers aiming at STR loci shown in SEQ ID NO. 1-SEQ ID NO.6 respectively.

In a preferred embodiment of the present invention, the sequences of the specific primer pair for the STR locus shown in SEQ ID No.1 are SEQ ID No.28 and SEQ ID No.29, the sequences of the specific primer pair for the STR locus shown in SEQ ID No.2 are SEQ ID No.16 and SEQ ID No.17, the sequences of the specific primer pair for the STR locus shown in SEQ ID No.3 are SEQ ID No.12 and SEQ ID No.13, the sequences of the specific primer pair for the STR locus shown in SEQ ID No.4 are SEQ ID No.20 and SEQ ID No.21, the sequences of the specific primer pair for the STR locus shown in SEQ ID No.5 are SEQ ID No.18 and SEQ ID No.19, and the sequences of the specific primer pair for the STR locus shown in SEQ ID No.6 are SEQ ID No.30 and SEQ ID No. 31.

More preferably, the kit also comprises a specific primer pair aiming at more than one STR locus in SEQ ID NO. 7-SEQ ID NO. 11.

In another aspect of the present invention, there is also provided a multiplex amplification system for STR locus linkage genetic analysis of PKD2 genes, comprising: specific primer pairs respectively aiming at more than any three STR loci in SEQ ID NO. 1-SEQ ID NO. 11.

Preferably, the multiplex amplification system comprises: specific primer pairs respectively aiming at STR loci shown in SEQ ID NO. 1-SEQ ID NO. 10.

The 6 new STR loci shown in SEQ ID NO. 1-SEQ ID NO.6 of the invention do not have CNV (copy number variation) in the region where the loci are located, and the 6 loci have good compatibility, thereby being beneficial to forming an efficient composite amplification system with other STR locus loci.

The main advantages of the invention are as follows:

1. the newly screened STR locus is obtained by screening the existing latest human whole genome sequence information, and influence factors such as poor compatibility, CNV or high repetitive sequence region are eliminated.

2. The screened 6 related PKD2 gene STR new sites enrich the selection range of STR loci in the related application fields of prenatal diagnosis, embryo pre-implantation diagnosis and the like.

3. The new STR loci with higher resolution are provided, the new STR loci and the known STR loci form the same system for detecting related genetic diseases, and typing data can be obtained more efficiently and accurately.

Drawings

The present invention will be described in further detail with reference to the accompanying drawings and specific embodiments.

FIG. 1 is a raw peak plot of a plurality of STR loci according to example 2 of the present invention.

Detailed Description

The invention screens new STR locus loci with high resolution in PKD2 gene, and the new STR loci are combined with some existing STR loci to be applied to the fields of molecular research and molecular diagnosis of autosomal dominant hereditary polycystic kidney disease (ADPKD), thereby providing basis for clinical diagnosis and embryo pre-implantation diagnosis of the disease.

Example 1 selection of STR novel sites of 6 PKD2 genes

Prediction of STR

(1) Using a biological public database to consult and sort the STR information, (referring to a database with NCBI http:// www.ncbi.nlm.nih.gov/database before 11 months in 2011), and obtaining reported STR information of the NCBI;

(2) predicting STR by bioinformatics to obtain a plurality of loci with the repetition times of 2bp of a repeating unit being more than 10 times;

(3) comparing the repetition times of the STR loci in three genomes (refer, HuRef and Celera) in an NCBI database to obtain STR locus information with large difference of the repetition times;

(4) combining literature, biological databases and bioinformatics prediction, screening 6 bioinformatics predicted STRs according to the sequence characteristics of the actual STR, and respectively naming the STRs as T4S0005(SEQ ID NO.1), T4S0006(SEQ ID NO.2), T4S0008(SEQ ID NO.3), T4S0009(SEQ ID NO.4), T4S0012(SEQ ID NO.5) and T4S0014(SEQ ID NO. 6).

Detection of STR

All predicted STR sites are detected by a fluorescent labeling amplification product length polymorphism analysis method, primer3 software is adopted in primer design, and special sequences such as known SNP polymorphic sites and highly repetitive sequence regions in a database are avoided as much as possible in design. Diluting the PCR product, mixing a small amount of the diluted PCR product with an internal standard mark, directly applying ABI3130xl to perform capillary electrophoresis, and analyzing a data file by using GeneMapper4.0(applied biosystems); obtaining information such as Polymorphic Information Content (PIC) of the screened new STR loci.

Example 2STR New site sample validation

The newly screened 6 STR loci (T4S0005, T4S0006, T4S0008, T4S0009, T4S0012, T4S0014) were combined with 4 commonly used loci, D4S395(SEQ ID NO.7), D4S2929(SEQ ID NO.8), D4S414(SEQ ID NO.9), and Amelogenin (Amelo X: SEQ ID NO. 10; Amelo Y: SEQ ID NO.11), and 2 individual samples were tested using a two-step multiplex fluorescence PCR technique.

The specific experimental operation steps are as follows:

(1) DNA sample preparation

Collecting blood from 2 selected sample individuals in a hospital; DNA samples are obtained by extracting through a DNA extraction kit, 1 mul of 1% agarose electrophoresis is respectively taken to carry out quality inspection and concentration estimation on the samples, and then the samples are diluted to the working concentration of 5-10 ng/mul according to the estimated concentration.

(2) PCR reaction

1ul of each sample was taken and subjected to PCR reaction.

The total volume of the PCR reaction was 10. mu.l (1. mu.l of 10 XPCR buffer (Qiagen), 0.2. mu.l of 25mmol magnesium chloride, 1. mu.l of mixed primers for detecting multiple STR loci (primer sequences shown in Table 1), 0.8. mu.l of 2.5mmol dNTP, 0.04. mu.l of HotStarplus Taq enzyme (Qiagen), 5.96. mu.l of ddH₂O。

The PCR conditions were set as follows: 10 minutes at 95 ℃; 7 cycles of the Touchdown program (94 ℃ 20 seconds, 65 ℃ 1 ℃/40 seconds and 72 ℃ 2min),28 cycles of amplification (94 ℃ 20 seconds, 65 ℃ 30 seconds and 72 ℃ 2min), extension at 72 ℃ for 2min, extension at 60 ℃ for 60 min, 4 ℃ storage.

(3) Adding fluorescence reaction

1ul of each sample was taken and subjected to PCR reaction.

The total volume of the PCR reaction system was 10. mu.l (1. mu.l of 10 XPCR buffer (Qiagen), 0.2. mu.l of 25mmol magnesium chloride, 1. mu.l of mixed primers for detecting multiple STR loci, 0.8. mu.l of 2.5mmol dNTP, 0.04. mu.l of HotStarplusTaq enzyme (Qiagen), 5.96. mu.l of ddH₂O。

The PCR conditions were set as follows: 5 minutes at 95 ℃; 30 cycles (94 ℃ for 20 seconds, 56 ℃ for 40 seconds and 72 ℃ for 2min), extension at 72 ℃ for 2 minutes, and storage at 4 ℃.

TABLE 1PKD2 Gene 10 locus primer sequences

STR loci	Primer sequences	Fluorescent markers
			T4S0008	F:5’-ATGGCCAGCTCCAAGACAAAAA-3’(SEQ ID NO.12)	FAM

	R:5’-CTTTATGTGGATGAAGGTTGTCTGG-3’(SEQ ID NO.13)
			Amelo	F:5’-GCCCTGGGCTCTGTAAAGAATAGTG-3’(SEQ ID NO.14)	FAM
	R:5’-GTTTCTTGAGGCCAACCATCAGAGCTTA-3’(SEQ ID NO.15)
			T4S0006	F:5’-AGAGCCCCTAATCCATCCCAGT-3’(SEQ ID NO.16)	FAM
	R:5’-GGAGCAATTTTCCTCTGAACATCTTG-3’(SEQ ID NO.17)
			T4S0012	F:5-TTTTTGCATTTGGGCAGTAGCA-3’(SEQ ID NO.18)	VIC
	R:5-CGATGAATTGGAGTGGTTTTGTTG-3’(SEQ ID NO.19)
			T4S0009	F:5-TCACCATTTTCCCCAGTTGTCA-3’(SEQ ID NO.20)	VIC
	R:5-TTCCAGTCCAGCTTAGGCAACA-3’(SEQ ID NO.21)
			D4S2929	F:5-CAGAGTGGTCAATCCTGGCACT-3’(SEQ ID NO.22)	NED
	R:5-TTCTCACCCATTCCTAGGCTGTC-3’(SEQ ID NO.23)
			D4S395	F:5-GCCAGGATGATAGCAATGGTCA-3’(SEQ ID NO.24)	NED
	R:5-AACAGACGCACAGGCCAGGT-3’(SEQ ID NO.25)
			D4S414	F:5-CAGAGTTCTTACGCTGGTTCTTTCAG-3’(SEQ ID NO.26)	PET
	R:5-TTGCTATCATACCCCCCCTGTG-3’(SEQ ID NO.27)
			T4S0005	F:5-TTGCACAAGTGTTCACGCTGTC-3’(SEQ ID NO.28)	PET
	R:5-AGGATTTGGTGGCGTGAGAAAA-3’(SEQ ID NO.29)
			T4S0014	F:5-GGTTTCACCTTGTGCATGTGATG-3’(SEQ ID NO.30)	PET
	R:5-GTCCTGTTGAGCCTGCCAGATT-3’(SEQ ID NO.31)

After the PCR reaction is finished, diluting the PCR amplification product by 20 times, taking out 1ul, adding 8.9ul HIDI and 0.1ul LIZ, mixing uniformly, then carrying out 5min at 95 ℃, and carrying out capillary electrophoresis sample loading; because the STR locus detection primer has fluorescence, amplified fragments with different colors and different sizes are detected by capillary electrophoresis, and STR typing information (shown in table 2) is obtained by analyzing results by a fluorescence detection system.

The results are shown in FIG. 1 and tables 2 to 4. Table 2 shows the STR locus distribution of the PKD2 gene.

TABLE 2STR locus distribution of PKD2 gene

Fig. 1 is a graph of the raw peaks detected for multiple STR loci from two samples, sample 1 (top panel) and sample 2 (bottom panel), respectively. In FIG. 1, "A" is a FAM peak map, "B" is a VIC peak map, "C" is a NED peak map, and "D" is a PET peak map.

The information of each STR locus in fig. 1 is illustrated in table 3 below.

TABLE 3 PCR amplified fragment size for each STR locus of PKD2 gene

The STR measurements obtained from fig. 1 are shown in table 4 below.

TABLE 410 detection results for STR loci

As can be seen from fig. 1 and table 4, the newly screened 6 STR loci show higher discrimination on the results, and can be applied to biomedical detection such as embryo pre-implantation diagnosis, STR typing, paternity test, individual identification and the like, so that the typing identification efficiency and accuracy can be effectively improved, and the system has high individual identification rate and strong practicability on the linkage genetic analysis of the PKD2 gene.

The above-mentioned embodiments only express the embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims (6)

1. A detection kit for diagnosing autosomal dominant hereditary polycystic kidney disease is characterized by comprising specific primer pairs respectively aiming at STR loci of more than two PKD2 genes in SEQ ID NO. 1-SEQ ID NO. 6.

2. The detection kit according to claim 1, comprising specific primer pairs for STR loci shown in SEQ ID No.1 to SEQ ID No.6, respectively.

3. The detection kit according to claim 2, wherein the sequences of the specific primer pair for the STR locus shown in SEQ ID No.1 are SEQ ID No.28 and SEQ ID No.29, the sequences of the specific primer pair for the STR locus shown in SEQ ID No.2 are SEQ ID No.16 and SEQ ID No.17, the sequences of the specific primer pair for the STR locus shown in SEQ ID No.3 are SEQ ID No.12 and SEQ ID No.13, the sequences of the specific primer pair for the STR locus shown in SEQ ID No.4 are SEQ ID No.20 and SEQ ID No.21, the sequences of the specific primer pair for the STR locus shown in SEQ ID No.5 are SEQ ID No.18 and SEQ ID No.19, and the sequences of the specific primer pair for the STR locus shown in SEQ ID No.6 are SEQ ID No.30 and SEQ ID No. 31.

4. The detection kit according to any one of claims 1 to 3, further comprising a specific primer pair for at least one STR locus selected from SEQ ID No.7 to SEQ ID No. 11.

5. The detection kit according to claim 4, further comprising specific primer pairs for STR loci shown in SEQ ID No.7 to SEQ ID No.10, respectively.

6. The detection kit according to claim 5, wherein each primer pair has a fluorescein label of the same species or different species.

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