CN103602671A - SNP in KRT8 gene exon area and determination method thereof - Google Patents

Abstract

The invention discloses a SNP in KRT8 gene exon area and determination method thereof, and the SNP locus with G/T polymorphisms (GG) is at 12563 site of the eighth exon area of the KRT8 gene and has nucleotide sequences SEQ ID NO:1 and SEQ ID NO:2. The invention also provides a determination method of the SNP locus, and research and development of the polymorphisms can be applied to researches in the aspect of primary biliary cirrhosis, for example, control of primary biliary cirrhosis attack and hepatic cirrhosis degree or improvement of liver transplantation success rate.

Description

SNP site and definite method thereof in a kind of KRT8 gene extron subregion

Technical field

The present invention relates to the nucleotide sequence in SNP site in a kind of KRT8 gene extron subregion, and definite method of this nucleotide sequence, and it is applied to control primary biliary cirrhosis morbidity.

Background technology

Cytoskeleton is comprised of between the intermediate filament before both microfilament, microtubule and size.Keratin sulfate is the most important of intermediate filament family and the abundantest member of content, be divided into acidity (I type, K9-28) and alkaline (II type, K1-8, K71-80) two classes.All epithelial cells are at least expressed the Keratin sulfate monomer of a kind of I type and a kind of II type, and the two antiparallel formation dimer and then the composition tetramer, form the basic solubility unit of Keratin sulfate.Keratin sulfate is expressed in epidermis and epithelial cell and has tissue specificity, for example main expressing K 8 of liver cell and pancreatic acinar cell and K18, bile duct epithelial cell expressing K 7/K8/K18/K19, and a small amount of K20.Except maintain cell mechanicalness complete, Keratin sulfate also participates in being permitted cellulous important biomolecule and learns function, comprises transhipment, anti-stress and the anti-apoptosis capacity etc. of cell fission, motion, vesica.Mouse and the mankind, the sudden change of the Keratin sulfate of keratinocyte is the direct cause of disease of some skin and keratopathy, and the Keratin sulfate sudden change of nonkeratinocyte can increase the susceptibility of disease, as hepatic diseases.

The CK8 assignment of genes gene mapping is in No. 12 long-armed Shi San of karyomit(e) districts, Subcellular Localization: cytoplasm.It is to form one of cyto-architectural main component, and its sudden change can cause cryptogenic cirrhosis.KRT8, KRT18 often exists with composite form, is at first to find at non-tesselated epithelium layer, has the effect that protection liver is avoided machinery and toxic injury.

Animal model shows that Keratin sulfate has important provide protection to liver cell.In liver injury situation, the expression amount of mouse liver K8 and K18mRNA and albumen can raise nearly 3 times.K8-/-mouse has obvious liver phenotype, and the K8-of C57/B1 background/-mouse has the extensive hepatorrhagia of dead companion 94% embryonic stage; Although and the K8-of FVB/n background/-mouse 55% has normal lifetime, show as slight hepatitis when base state, the apoptosis susceptibility of Fas mediation obviously increased.The KRT8 gene that transgenic mice is crossed expression sudden change can make the fracture of mouse liver cell skeleton.Compare with crossing expression wild type gene, these mouse will be developed into chronic hepatitis, easily cause hepatic injury simultaneously.

In the recent decade, the human keratin mutational site relevant to liver injury and liver failure found gradually.For KRT8SNPs research majority, be positioned at exon 1, the research of non-translational region is few.In US and European white race crowd, the modal mutational site of K8 is R341H, and secondly common mutational site is G62.The R341H frequency of occurrences is 6.7% U.S. liver problem sufferer, and control group is 3.1%(p<0.01); German patient, be 3.5%, and in control group, do not detect (p<0.09).In acute hepatic failure case, compare K8R341H ratio in white people corresponding higher (p=0.01), and Afro American K8G435S more common (p=0.02) with control group.These results all point out Keratin sulfate sudden change to have racial difference.The report that China carries out systems analysis research to keratin gene variation and disease relationship is still few.Meanwhile, at present not about being positioned at the report of the upper site rs11554491 of gene KRT8.And the modal site rs11554495(pG61C of western countries) polymorphism and PBC are without obvious dependency.

Summary of the invention

The invention provides a kind of SNP site and definite method thereof in KRT8 gene extron subregion, method is simple, and is applied in the recent studies on for the treatment of or prevention primary biliary cirrhosis aspect.

The present invention realizes by following technical scheme:

A SNP site in KRT8 gene extron subregion, is the polymorphic SNP sites of 12563 G/T of KRT8 gene the 8th exon region, has the nucleotide sequence of SEQ ID NO:1 and SEQ ID NO:2.

The present invention also provides definite method in SNP site in this KRT8 gene extron subregion, is to realize by following step:

(1) extract the genomic templates DNA of host cell;

(2) described template DNA is carried out to pcr amplification, its primer is:

Upstream: 5 '-ACGTTGGATGTTCTTCACAACCACGGCCCT-3 ',

Downstream: 5 '-ACGTTGGATGTTCTTCACAACCACGGCCCT-3 ';

PCR product after amplification is carried out to purifying;

(3) the PCR product after purifying carries out PCR extension, and the primer of described extension is:

Upstream: 5 '-ACGTTGGATGTTCTTCACAACCACGGCCCT-3 ',

Downstream: 5 '-GGAGGAGCTGGTGCGG-3 ',

Finally carry out resin purification;

(4) after chip point sample, adopt mass spectrograph to detect, carry out data analysis, determine SNP loci gene type.

Beneficial effect of the present invention is: in the definite KRT8 gene extron subregion of the present invention, SNP site is relevant to primary biliary cirrhosis, degree of cirrhosis, liver transplantation success ratio etc.Therefore, by the research and development of this gene polymorphic be can be used for to primary biliary cirrhosis aspect, as controlled primary biliary cirrhosis morbidity, liver cirrhosis lesion degree or liver transplantation success ratio.

Embodiment

Below in conjunction with embodiment, the present invention will be further described.

(1) research object

1, candidate gene SNPs detects sample

According to U.S.'s clinical practice guideline: primary biliary cirrhosis (Poupon R.Primary biliary cirrhosis:a2010update.J Hepatol.2010; 52 (5): 745-58.) make a definite diagnosis patients with primary biliary cirrhosis sample 76 examples, patients' relatives in contrast, contrasts 215 examples.

2, selected SNPs somatotype sample

1) primary biliary cirrhosis group (PBC group)

From attached Xijing Gastroenterology dept. of hospital of The Fourth Military Medical University ward; totally 76 examples; mean age 51 ± 11(mean is added and subtracted standard deviation) year, primary biliary cirrhosis is diagnosed with reference to U.S.'s clinical practice guideline: primary biliary cirrhosis (Poupon R.Primary biliary cirrhosis:a2010update.J Hepatol.2010; 52 (5): the primary biliary cirrhosis Case definition of 745-58.) promulgating, all through transcutaneous aspiration biopsy of liver lesions, make a definite diagnosis.Get rid of the patients such as viral hepatitis, drug induced hepatitis.

2) normal healthy controls group (Control group)

From being the family members of patients with primary biliary cirrhosis after diagnosing, totally 215 examples, 51 ± 13 years old mean age, get rid of the possibility of autoimmune liver disease through autoantibody examination.

3, object fragment PCR amplification

1) PCR reaction system: adopt multiplex PCR amplification technique, each reaction cumulative volume 5 μ l, comprises template DNA 10 η g, Hotstar Taq0.5U, every 0.5pmol of amplimer, 25mM dNTP, 0.1 μ l.

2) PCR response procedures: reaction conditions be 94 ℃ 4 minutes; 94 ℃ 20 seconds, 56 ℃ 30 seconds, 72 ℃ 1 minute, 45 circulations; 72 ℃ 3 minutes; 4 ℃ of ∞.

4, PCR product purification

Purification system: PCR reaction product is used 0.5U SAP(shrimp alkaline phosphatase) process free dNTP in removal system.Reaction system 7 μ l, PCR product 5 μ l wherein, SAP mixed solution 2 μ l (SAP0.5U, buffer0.17 μ l).

Purification reaction: 37 ℃ of response procedures 20 minutes; 85 ℃ 5 minutes; 4 ℃ of ∞.

5, sequencing reaction (two-way direct Sequencing)

1) reaction system: adopt multiplex PCR amplification technique, each reaction cumulative volume 5 μ l, comprises template DNA 10 η g, Hotstar Taq0.5U, every 0.5pmol of amplimer, 25mMdNTP, 0.1 μ l.

2) pcr amplification reaction:

94 ℃ 4 minutes, 94 ℃ 20 seconds, 56 ℃ 30 seconds, 72 ℃ 1 minute, 72 ℃ 3 minutes, 4 ℃ of ∞.Wherein, 94 ℃ 20 seconds, 56 ℃ 30 seconds, 72 ℃ within 1 minute, carry out 45 times circulation.

3) ethanol precipitation.

4) sequencing reaction product polyacrylamide gel electrophoresis.

6, determine SNP site, as the nucleotide sequence of SEQ ID NO:1 and SEQ ID NO:2.

(2) SNP somatotype

MALDI-TOF mass spectrometric detection

1, design of primers: application MassARRAYTM Assay Design2.0 software design multiple PCR primer.

2, pcr amplification reaction

1) reaction system: each reaction cumulative volume 5 μ l, comprises template DNA 10 η g, Hotstar Taq0.5U, every 0.5pmol of amplimer, 25mM dNTP, 0.1 μ l.

2) response procedures: reaction conditions be 94 ℃ 4 minutes; 94 ℃ 20 seconds, 56 ℃ 30 seconds, 72 ℃ 1 minute, 45 circulations; 72 ℃ 3 minutes; 4 ℃ of ∞.

3, PCR product purification

1) reaction system: PCR reaction product is used 0.5U SAP(shrimp alkaline phosphatase) process free dNTP in removal system.Reaction system 7 μ l, PCR product 5 μ l wherein, SAP mixed solution 2 μ l (SAP0.5U, buffer0.17 μ l).

2) response procedures: 37 ℃ of response procedures 20 minutes; 85 ℃ 5 minutes; 4 ℃ of ∞.

4, extension

1) reaction system: cumulative volume 9 μ l reaction systems comprise SAP process after PCR product 7 μ l, each extension primer mixture 0.804 μ l wherein, iPLEX enzyme 0.041 μ l, extends mixture 0.2 μ l.

2) response procedures: response procedures be 94 ℃ 30 seconds; 94 ℃ 5 seconds; 52 ℃ 5 seconds, 80 ℃ of 5 circulations in 5 seconds; Return to 94 ℃ of totally 40 circulations in 5 seconds; 72 ℃ 3 minutes, 4 ℃ of ∞.

5, Resin resin purification: 6mg Clean Resin resin purification for each extension product.

6, chip point sample: purified product is moved on 384 hole SpectroCHIP (Sequenom) chips, and upper machine is measured.SpectroCHIP chip is used MALDI-TOF(matrix-assisted laser desorption/ionization time of flight) analysis of matrix assisted laser desorption ionization ionization time of flight mass spectrometry.

7, MassARRAYTM Analyzer mass spectrometric detection (SEQUENOM), detected result is used TYPER4.0 software (sequenom) somatotype Output rusults.

(3) statistical method

Application SPSS13.0for Windows software carries out data analysis and statistics: between group, mean relatively adopts t check; Between group, frequency distribution relatively adopts chi square test; Relative disease genotype relative risk adopts and represents than (odds ratio, OR) and 95% credibility interval (95%CI) than number; Between many groups, mean relatively adopts One-way ANOVA (One-way ANOVA).P<0.05 is for there being statistical significance.

Claims (2)

1. a SNP site in KRT8 gene extron subregion, it is characterized in that the polymorphic SNP site of 12563 G/T of KRT8 gene the 8th exon region, has the nucleotide sequence of SEQ ID NO:1 and SEQ ID NO:2.

2. definite method in SNP site in KRT8 gene extron subregion, it is characterized in that realizing by following step:

(1) extract the genomic templates DNA of host cell;

(2) described template DNA is carried out to pcr amplification, its primer is:

Upstream: 5 '-ACGTTGGATGTTCTTCACAACCACGGCCCT-3 ',

Downstream: 5 '-ACGTTGGATGTTCTTCACAACCACGGCCCT-3 ';

PCR product after amplification is carried out to purifying;

(3) the PCR product after purifying carries out PCR extension, and the primer of described extension is:

Downstream: 5 '-GGAGGAGCTGGTGCGG-3 ',

Finally carry out resin purification;

(4) after chip point sample, adopt mass spectrograph to detect, carry out data analysis, determine SNP loci gene type.