CN101798578A - ATP 8 B1 gene hotspot linked mutation and application - Google Patents
ATP 8 B1 gene hotspot linked mutation and application Download PDFInfo
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- CN101798578A CN101798578A CN200910046069A CN200910046069A CN101798578A CN 101798578 A CN101798578 A CN 101798578A CN 200910046069 A CN200910046069 A CN 200910046069A CN 200910046069 A CN200910046069 A CN 200910046069A CN 101798578 A CN101798578 A CN 101798578A
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Abstract
The invention belongs to the field of genomics and relates to the gene diagnosis of hereditary diseases, in particular to an ATP 8 B1 gene hotspot linked mutation and application. The ATP 8 B1 gene hotspot linked mutation is hotspot linked mutational site 625C>A (P209T)/IVS6+5G>T in Chinese children with PFIC1 (Progressive Familial Intrahepatic Cholestasis 1), and the sequence of the ATP 8 B1 gene hotspot linked mutation has the structure of the sequence 1. In the invention, the ATP 8 B1 gene hotspot linked mutation can be used for linked mutational site pre-detection on children with the PFIC, negative common pathogenesis and continuously low gamma-GT, which is, beneficial for the elimination or diagnosis of the PFIC1. The invention can adopt the ATP 8 B1 gene hotspot linked mutation to prepare diagnosis probes and reagent kits so that the PFIC1 can be conveniently and rapidly diagnosed. Therefore, the invention provides a favorable chance for the influences of protein structures and/or functions for the discussion on the pathogenesis of the PFIC1.
Description
Technical field
The invention belongs to genomics field, relate to the gene diagnosis of heredopathia, be specifically related to ATP8B1 gene hot linked mutation and purposes.
Background technology
Studies show that infancy, the hepatopathy overwhelming majority showed as onset in neonatal period or infantile intrahepatic cholestasis, clinical baby's hepatopathy syndrome that claims again.Foreign literature report life birth baby incidence about 1: 2500-5000, the corresponding epidemiologic data of domestic shortage, but clinical practice generally believes that sickness rate is apparently higher than western countries.The harm of cholestasis maximum be cholate in liver cell, accumulate can inducing hepatocyte accent die and/or downright bad, finally cause liver cirrhosis and liver failure.Wherein, most of infant prognosis bona be liver cirrhosis or liver failure but also there is the minority infant to make progress, and PFIC1 is exactly one of reason that causes this part infant deterioration of liver function.
Clinical study confirms that PFIC1 is a kind of recessive hereditary disease, finds in Amish descendant that at first some clinical manifestations are subsequently similarly distributed disease with it and also are classified as this disease.PFIC1 be everlasting neonatal period or infancy onset, clinical jaundice, itch, the growth retardation etc. of mainly showing as of first few months.Along with hepatosplenomegaly, malabsorption can appear in the progress infant of the state of an illness, the infant that has also shows pancreatitis, hearing disability, cough etc.The state of an illness continues progress, and infant is everlasting and was developed into liver cirrhosis and liver failure in 20 years old in the past.Blood biochemistry show gpt (ALT), glutamic-oxal(o)acetic transaminase (AST), 5 '-phosphonuclease (5 '-NT), alkaline phosphatase (ALP), bile acide raise, but gamma glutamyltransferase (γ-GT) not high all the time.Hepatic pathology mainly shows as intrahepatic cholestasis, along with hepatic fibrosis and liver cirrhosis can appear in the progress of the state of an illness.This disease difficult and some other disease of hanging down γ-GT that shows as in clinical manifestation differentiates mutually, as: carrying out property familial intrahepatic cholestasis disease 2 types (ProgressiveFamilial Introhepatic Cholestasis type 2, PFIC2).Gene diagnosis at present is unique means of effectively making a definite diagnosis.
Studies show that, PFIC1 (claims the FIC1 gene again by the ATP8B1 gene, GeneID:5205) sudden change causes, this gene is positioned at human chromosomal 18q21-q22, totally 85382 base pairs, wherein comprise 27 exons that participate in encoding histone, the phosphoramidate translocase (FIC1 albumen) that coding is made up of 1251 amino acid, this albumen all has expression at a plurality of organs such as liver, small intestine and pancreas of the mankind.At present the mechanism of the albumen intrahepatic cholestasis that defective causes that causes for transgenation is not clear and definite as yet.More at western countries' report for the ATP8B1 gene mutation site that causes PFIC1 to take place, TaiWan, China also has the minority report, but there is no the focus sudden change.Because the sequence of ATP8B1 gene is very long, therefore rely on gene studies to diagnose this disease to have very big difficulty, so the relevant researchist in this area gives bigger concern to finding the focus sudden change, expectation can significantly reduce the workload of gene diagnosis thus.
Find content
The purpose of this invention is to provide ATP8B1 gene hot linked mutation and purposes.Especially ATP8B1 gene hot linked mutation is in carrying out property familial intrahepatic cholestasis disease 1 type (Progressive Familial Introhepatic Cholestasis type 1, the purposes in genetics diagnosis PFIC1).
ATP8B1 gene hot linked mutation provided by the invention is focus linked mutation site 625C＞A (P209T)/IVS6+5G＞T in the Chinese PFIC1 infant.Wherein missense mutation 625C＞A is positioned at first base site of last codon of No. 6 exon of ATP8B1 gene, sports VITAMIN B4 (A) by cytosine(Cyt) (C), causes the proline(Pro) (P) of the 209th in FIC1 albumen to change Threonine (T) into; IVS6+5G＞T is positioned at the 5th base site of No. 6 intron, sports thymus pyrimidine (T) by guanine (G), 6 Nucleotide in interval between these two sites.Its place sequence has the structure of sequence 1.Be specially
GTAA 
GAACAAATTCTGCTTCTCTTGTTCCTTGTTCTCCCCCA GCCCCTGTGCTCGGCTTTTCCCTCAGGGTCTCGGTGTTATTTGTGTGTTTTGTTTT GGA CAG,Wherein, black matrix is the full sequence of No. 6 exon, and underscore partly is the full sequence of No. 6 intron, and italic is 2 mutational sites.
ATP881 gene hot linked mutation of the present invention obtains by following step,
1) collect specimen: collect the peripheral vein hemocyte of doubtful carrying out property familial intrahepatic cholestasis disease 1 type infant, blood plasma is used for clinical diagnosis (after the approval family members of Ethics Committee informed consent);
2) extracting of genomic dna: operate by poba gene group DNA extraction agent box operation instructions, test kit is given birth to worker's biotechnology company limited by Shanghai and is provided;
3) sequential analysis
(1) design and synthesize primer exon and the contiguous sequence that participates in encoding histone increased,
Wherein, all primers are to crossing over the adjacent introne 1 00-300 base in corresponding exon two ends;
4) pcr amplification
System is 50ul, contains genomic dna 100ng, two-way primer 2 00～800nM, 10 * buffer, 5 μ l, Mg
2+1.5～2mM, dNTPs 200nM, Taq Gold 1.25u～2.5u;
Amplification condition is: 95 ℃ of pre-sex change 15min, and 94 ℃ of sex change 30s, 55 ℃ of annealing 30s, 72 ℃ are extended 30s, and 35 circulations are extended 4min for back 72 ℃ eventually.
5) the PCR product is served and is directly checked order after worker's biotechnology company limited purifying is given birth in the sea;
6) ocr software interpreting blueprints of sequencer map employing Chromas gene and artificial nucleus are right, compare the back to mutant nucleotide sequence place exon repetition PCR and backward sequencing with the standard sequence (GeneID:5205) in the gene pool.
Among the present invention, can adopt ATP8B1 gene hot linked mutation to be progressive intrahepatic cholestasis, common disease and detect in advance, help to get rid of or diagnosis PFIC1 because of equal negative and not high all the time infants of γ-GT carries out the linked mutation site to clinical manifestation.
Among the present invention, can adopt ATP8B1 gene hot linked mutation to prepare diagnostic probe, carrying out property of diagnosis that can be more simple and efficient familial intrahepatic cholestasis disease 1 type disease.
Further, among the present invention, preparation contains the test kit of said mutation site 625C＞A (P209T)/IVS6+5G＞T according to a conventional method.
The discovery of focus linked mutation of the present invention has brought significant facility to the gene diagnosis work of relevant PFIC1.Can provide good opportunity by studying this linked mutation to the pathogenesis of inquiring into PFIC1 to the influence of relevant protein structure and/or function.
Description of drawings
Fig. 1 is a heterozygous mutant, and arrow is depicted as the site of undergoing mutation.
Fig. 2 is a homozygous mutation, and arrow is depicted as the site of undergoing mutation.
Fig. 3 is normal sequencer map, and arrow is depicted as the site of undergoing mutation.
Embodiment
Embodiment 1 clinical study experiment
1. selection research object, concrete standard is:
(1). neonatal period or infancy onset, show as and continue or the jaundice (it is main that conjugative bilirubin raises) or the serious itch of outbreak repeatedly;
(2). SBA raises, and γ-GT is all the time in normal range;
(3). to the common factor that can cause baby's intrahepatic cholestasis carry out examination with except the cause of disease such as infection, metabolic disturbance, medicine and surgical disease;
(4). except have in congenital malformation and the multiple organ dysfunction person taken place.
2. collect specimen: collect infant peripheral vein hemocyte 0.5～1.0ml after the approval family members of Ethics Committee informed consent, blood plasma is used for clinical diagnosis.
3. extracting genomic dna: operate by poba gene group DNA extraction agent box operation instructions, test kit is given birth to worker's biotechnology company limited by Shanghai and is provided.
4. sequential analysis
(1) designing and synthesizing 25 pairs of primers increases to 27 exons and the contiguous sequence that participates in encoding histone.All primers are to crossing over the adjacent introne 1 00-300 base in corresponding exon two ends;
(2) pcr amplification: system is 50ul, contains genomic dna 100ng, two-way primer 2 00～800nM, 10 * buffer, 5 μ l, Mg
2+1.5～2mM, dNTPs 200nM, Taq Gold1.25u～2.5u;
Amplification condition is: 95 ℃ of pre-sex change 15min, and 94 ℃ of sex change 30s, 55 ℃ of annealing 30s, 72 ℃ are extended 30s, and 35 circulations are extended 4min for back 72 ℃ eventually;
(3) the PCR product is served and is directly checked order after worker's biotechnology company limited purifying is given birth in the sea;
(4) sequencer map adopt the interpreting blueprints of Chromas gene ocr software and the artificial nucleus is right and gene pool in standard sequence (GeneID:5205) back of comparing mutant nucleotide sequence place exon is repeated PCR and backward sequencing.
5. adopt the ATP8B1 gene hot linked mutation that makes to prepare diagnostic probe, carrying out property of diagnosis familial intrahepatic cholestasis disease 1 type disease:
The result shows, 9 examples exist in the infant of ATP8B1 transgenation has 4 examples to have linked mutation 625C＞A (P209T)/IVS6+5G＞T, accounts for 44.4%, wherein each 2 example of heterozygosis and homozygous mutation infant.
In view of 625C＞A (P209T)/IVS6+5G＞T linked mutation is a focus mutation type in the Chinese PFIC1 infant.ATP8B1 gene hot linked mutation provided by the present invention can be used for to Chinese clinical manifestation be progressive intrahepatic cholestasis, common disease because of the infant that all negative and γ-GT is not high all the time can carry out the detection in this chain site earlier, can help get rid of or diagnosis PFIC1.This focus linked mutation has brought remarkable facility to the gene diagnosis work of PFIC1.
ATP8B1 gene hot linked mutation and purposes sequence
SEQUENCE?LISTING
＜110〉Children's Hospital, Fudan University
＜120〉ATP8B1 gene hot linked mutation and purposes
<130>11
<160>1
<170>PatentIn?version?3.1
<210>1
<211>179
<212>DNA
＜213〉people
<400>1
gttcaaagtt?gctaagtgga?aagaaattca?agttggagac?gtcattcgtc?tgaaaaaaaa 60
tgattttgtt?ccagtaagtg?aacaaattct?gcttctcttg?ttccttgttc?tcccccagcc 120
cctgtgctcg?gcttttccct?cagggtctcg?gtgttatttg?tgtgttttgt?tttggacag 179
Claims (6)
1.ATP8B1 the gene hot linked mutation is characterized in that containing focus linked mutation site 625C＞A (P209T)/IVS6+5G＞T.
2. by the described ATP8B1 gene hot of claim 1 linked mutation, it is characterized in that described linked mutation site 625C＞A (P209T)/IVS6+5G＞T, wherein, missense mutation 625C＞A is positioned at first base site of last codon of No. 6 exon of ATP8B1 gene, sport VITAMIN B4 (A) by cytosine(Cyt) (C), cause the proline(Pro) (P) of the 209th in FIC1 albumen to change Threonine (T) into; IVS6+5G＞T is positioned at the 5th base site of No. 6 intron, sports thymus pyrimidine (T) by guanine (G), 6 Nucleotide in interval between described two sites.
3. by the described ATP8B1 gene hot of claim 1 linked mutation, it is characterized in that described linked mutation site 625C＞A (P209T)/IVS6+5G＞T, its place sequence has the structure of sequence 1.
4. a diagnostic probe is characterized in that containing the described focus linked mutation site 625C＞A of claim 1 (P209T)/IVS6+5G＞T.
5. a test kit is characterized in that containing the described focus linked mutation site 625C＞A of claim 1 (P209T)/IVS6+5G＞T.
6. the described ATP8B1 gene hot of claim 1 linked mutation is preparing the purposes of diagnosing in the sick diagnostic reagent of carrying out property familial intrahepatic cholestasis disease 1 type.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104313698A (en) * | 2014-10-29 | 2015-01-28 | 华中科技大学同济医学院附属同济医院 | DNA (Deoxyribonucleic Acid) library for detecting cholestatic jaundice pathogenic genes and application thereof |
| CN105463083A (en) * | 2015-12-11 | 2016-04-06 | 上海新培晶医学检验所有限公司 | Kit for detecting gene mutation of progressive familial intrahepatic choleatasia (PFIC) and detection method of kit |
| CN115404273A (en) * | 2022-06-30 | 2022-11-29 | 湖南家辉生物技术有限公司 | ATP8B1 gene mutant causing progressive familial intrahepatic cholestasis type I, protein and application |
-
2009
- 2009-02-10 CN CN200910046069A patent/CN101798578A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104313698A (en) * | 2014-10-29 | 2015-01-28 | 华中科技大学同济医学院附属同济医院 | DNA (Deoxyribonucleic Acid) library for detecting cholestatic jaundice pathogenic genes and application thereof |
| CN104313698B (en) * | 2014-10-29 | 2020-05-12 | 华中科技大学同济医学院附属同济医院 | DNA library for detecting cholestatic jaundice pathogenic gene and application thereof |
| CN105463083A (en) * | 2015-12-11 | 2016-04-06 | 上海新培晶医学检验所有限公司 | Kit for detecting gene mutation of progressive familial intrahepatic choleatasia (PFIC) and detection method of kit |
| CN105463083B (en) * | 2015-12-11 | 2019-02-12 | 上海新培晶医学检验所有限公司 | A kind of kit and its detection method of the siltation disease gene mutation of detection progressive familial hepatic bile |
| CN115404273A (en) * | 2022-06-30 | 2022-11-29 | 湖南家辉生物技术有限公司 | ATP8B1 gene mutant causing progressive familial intrahepatic cholestasis type I, protein and application |
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Application publication date: 20100811 |