CN101063678A - Method for forecasting pregnancy badness come-off generating risks - Google Patents
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Abstract
This invention relates to one predication method for imperfect pregnancy through personal prolinase carboxypeptidase gene E112D multi-position gene type, wherein when the gene is of 112EE pure wild type, the incidence is low; when the gene type is of 112ED mixture or 112DD pure burst type, the incident is high. This invention can predict the imperfect pregnancy risk to help doctor identify the danger to take actions in time for proper medicine process.
Description
Technical field
The present invention relates to utilize blood vessel and endothelial function to regulate the method for the external prediction pregnancy badness come-off generating risks of single nucleotide polymorphism (SNP) of the important enzyme gene on the path, comprise pregnancy badness come-offs such as threatened premature labor and premature labor, intrauterine growth retardation and LBW, pregnancy-hypertension syndrome and pre-eclampsia and eclampsia, belong to field of medicaments.
Background technology
The reproduction child-bearing health relates to the worldwide problem of baby's health, happy family life and population social sustainable development.Pregnancy duration generation threatened premature labor and premature labor (Preterm), fetal intrauterine growth retardation (Uterus GrowthRetardation, UGR) and LBW (Low Birth Weight, LBW) and pregnancy-hypertension syndrome (pregnancy induced hypertension syndrome, PIHs) and three big pregnancy badness come-offs such as the pre-eclampsia of its severe clinical manifestation and eclampsia, be infringement mother and sons' health perinatal period, influence the most common and most important reason on pregnant and lying-in women and neonate, ID and related disorders ground.Before pregnant, pregnant in the common and main healthy reproduction problem of early prevention, to providing the horizontal meaning of baby's health perinatal period very important.
Premature labor (Preterm delivery) refers to stop and childbirth person between pregnant 28-37 foot week (196-258 day).Threatened premature labor refers to occur just before giving birth omen between week at pregnant 28-37, flattens and dilatation of cervix with cervical canal.In a single day with the general difficult control of premature labor of show and premature rupture of fetal membranes, and dilatation of cervix occurs, neck tube flattens, and then often is developed to the premature labor childbirth.2002～2003 years premature's retrospective surveys in the whole nation show, among 1 year 6179 neonate of 77 policlinics, and the incidence of preterm birth 7.8% of obstetrics' birth, the premature accounts for 19.7% among the inpatient, and property was than 1.67: 1.Gestational age 32-36 week accounts for 63.5%.LBW (＜1500g) account for 32.3%.The high risk factor of premature labor is followed successively by mother miscarry history (36.8%), polyembryony (20.1%), premature rupture of fetal membranes (19.8%) and preeclampsia (12.6%).[paediatrics branch of Chinese Medical Association neonatology group, Chinese city epidemiology of prematurity preliminary investigation report, China's Contemporary paediatrics magazine, 2005,7 (1): 25-28] disease of the unusual and fetus itself of other clinical common factors that cause premature labors acute infectious diseases, genitals of also having mother.About 30% premature labor does not have obvious cause.
LBW refers to that neonatal weight born after the normal gestation period is lower than 2500 grams.Very low birth weight infant＜1500 grams.Premature labor and low birth weight infant are the main causes of enclosing newborn baby's death and disease.The low birth weight infant incidence of disease is 4.52% (476/10522), does not have significant change in the period of each, and wherein incidence of preterm birth reaches half (53.57%), and term birth and postmature delivery are respectively 36.13% and 10.29%.[Wang Gangqin, Yuan Shuyu, correlative factor analysis and final result that birth weight infants takes place, the modern gynecotokology magazine of China, 2006,3 (1): 14] clinical observation finds that the pathogenic factors of LBW is relevant with premature labor, premature rupture of fetal membranes, pregnancy-induced hypertension syndrome, multifetation etc.Previously the premature labor number of times is many more, and what premature labor took place this gestation may be big more.The puerpera of pregnancy and delivery neonatal weight≤1.5kg for the first time, the chance of II-para generation premature labor is 50%.
Gestational period women crowd hypertension is modal to be gestation hypertension syndrome (abbreviation preeclampsia).Essential hypertension person is arranged before pregnant, be defined as chronic hypertension and merge gestation.Blood pressure is normal before pregnant, and second trimester just begins elevation of blood pressure, is defined as pregnancy-hypertension syndrome.Preeclampsia mostly occurred in gestation 20 week backs to 2 weeks of postpartum, developed into the severe preeclampsia, then was pre-eclampsia (Preeclampsia) and eclampsia (Eclampsia).China is with reference to the suggestion criteria for classification of WHO, and nineteen eighty-three has been formulated existing classification (seeing Table 1) [chief editor such as remained shock ball, " practical hypertension " Science Press, second edition in 1998,609-23 page or leaf]:
The classification of table 1. pregnancy-hypertension syndrome
| Classification | Definition |
| Slight pregnancy-hypertension syndrome | Blood pressure is 130/90～140/90mmhg (17.3/12.0～18.7/13.3kpa), or, can accompany slight albuminuria and oedema than basic blood pressure rising 30/15mmhg (4.0/2.0kpa) |
| The moderate pregnancy-hypertension syndrome | Blood pressure＞140/90,＜160/110mmhg (＞17.3/12.0,＜21.3/14.7kpa), albuminuria is in "+" or with oedema and slight subjective symptoms such as giddy etc. |
| Severe pregnancy-hypertension syndrome (pre-eclampsia and eclampsia) | 1. pre-eclampsia: blood pressure 〉=160/110mmhg (21.3/14.7kpa), or albuminuria " ++ " → " ++ ++ ", 24 hours albuminuria quantitatively 〉=5g, subjective symptoms person's 2. eclampsias such as companion's oedema and headache: on the PIH basis, have a convulsion |
| Unfiled: 1. cyesedema 2. | Oedema prolong and huckle and above person pregnant before no albuminuria, gestational period albuminuria "+" and more than and recover normal person postpartum and comprise hyperpietic due to a variety of causes |
Annotate: blood pressure is not when meeting above standard, and then the high person with its systolic pressure or diastolic pressure is a standard, for example blood pressure be 150/110 or 170/100 all by the severe preeclampsia it.
Preeclampsia is the distinctive a kind of systemic disease of pregnant and lying-in women crowd, mainly shows as oedema, hypertension, albuminuria three big syndromes clinically.The patient is with headache, dim eyesight even tic, stupor.The overall incidence of disease of preeclampsia is 5-15%, and the incidence of disease of China is 9-10%.China preeclampsia scientific research cooperative groups census of population's nineties 3700000 shows, preeclampsia average attack rate 9.2%, the incidence of disease of severe pre-eclampsia and eclampsia is respectively 4.7%, 2.6%, 1.7% and 0.2% in light, it only is 0.2%[whole nation preeclampsia scientific research cooperative groups that chronic hypertension merges gestation, whole nation preeclampsia epidemiology survey, China's journal of obstetrics and gynecology, 1991,26:67-70].The gravid woman suffers from pregnancy-induced hypertension syndrome, particularly with chronic, make fetus intrauterine growth retardation, hypoxemia, acidosis, premature labor, neonate's LBW; Also can make mother eclampsia, cerebral hemorrhage, placental abruption, disseminated intravascular coagulation, hepatorrhagia, renal failure, pulmonary edema, apoplexy occur, even threat to life.Investigation shows that mild pre-eclampsia is with the people that diagnoses a disease, and early productive rate is significantly higher than the general population, reaches 13-54%.Faint from fear and the placental abruption incidence is respectively 0.2% and 1%, fetus or infant mortality rate are about 1%, growth retardation of fetus occurrence rate 5-13%.
The cause of disease deeper mechanisms of pregnancy badness come-off it be unclear that.Previously study knownly, the gravid woman suffers from pregnancy-induced hypertension syndrome, particularly with chronic, is one of key factor that causes premature labor, fetal intrauterine growth retardation and LBW.Think that at present the distinctive pathological change of tool of preeclampsia is the extensive little blood vessel endothelium injury of Utero-placenta unit hypoxic-ischemic and whole body.Utero-placenta ischemic may be preeclampsia and the initial link that pre-eclampsia takes place, the placental blood hypoperfusion causes its metabolism to change, discharge certain factor and enter the peripheral blood circulation, cause extensive vascular endothelial cell damage, cause vessel retraction spasm, blood coagulation system changes of function, body fluid to be shunted again then; May be preeclampsia clinical manifestation and multiple organ dysfunction thereof, and the basis that further develops the premature labor, fetal intrauterine growth retardation and the LBW related pathologies mechanism that cause multiple bad pregnancy outcome by preeclampsia.Report is arranged, and pre-eclampsia patient's HELLP syndrome (haemolysis, liver enzyme raise, platelet count descends) and gestation 34 all preceding mother and baby's case fatality rate obviously increase.Other has the research prompting, and gestational period abuse central nervous excitation agent cocaine causes the local anesthesia and the blood vessel function that contracts, can cause the placenta that exsomatizes and shrink strongly, strengthen a kind of slow effect that swashs the blood vessel thing that contracts that phthalein induces, can obviously reduce the fetal blood confession, and cause anoxic, cause fetal intrauterine growth retardation.The pregnancy duration life is overstretched or have serious life event to hit, and can cause premature labor.Pointed out thus, Utero-placenta ischemic and vascular endothelial cell damage may be organized the total important deep layer cause of disease mechanism clue of pregnancy badness come-off more.
Find and early prediction threatened premature labor and premature labor and pregnancy badness come-offs such as intrauterine growth retardation or LBW, the high-risk women crowd of early detection prevents and treats, still imperfectly understand under the situation in the cause of disease interior at present, EARLY RECOGNITION people at highest risk still depends on clinical more epiphytotics hazards at present.As: young unigravida, elderly primipara, preeclampsia or pre-eclampsia disease or threatened premature labor or LBW history, high body mass index person are arranged, family's hypertension, ephritis or diabetes medical history person, multifetation, hydramnion, vesicular mole patient, economic condition is poor, malnutrition, the anemia person; The gestational period, the psychology social pressures were big, and to fear of pregnancy, spiritual overstrain or irriate person, the living-hygienic situation is poor, with acute or chronic genital disease; This artificial low birth weight infant of mother, premature, or the unusual person of chronic hypertension medical history, diabetes, disturbance of blood coagulation and lipid metabolism is arranged.But these indexs are responsive and special not enough.The morbidity of pregnancy badness come-off also can increase when cold season, air pressure rising.
Previously research prompting, pregnancy badness come-offs such as preeclampsia, premature labor and LBW all are called the complex inheritance disease, are the complex disease features that the environment and heredity acting in conjunction causes, and the familial inheritance tendency is all arranged.There is the pregnant woman of preeclampsia family history higher 8 times, shows that the pregnant woman has hereditary capacity [Wang Zhijian, surplus bright red to the neurological susceptibility of preeclampsia than no family history person's incidence of disease.Pregnancy-hypertension syndrome merges the expression of the FGR placenta tissue vascular cell adhesion factor 1.China's perinatal medicine magazine, 2003; 6:75-7].
Pregnancy badness come-off all tends to multiple-factor inheritance, and definite hereditary basis is still not fully aware of.The more pathogenesis related genes of research has at present: the tumor susceptibility gene of 1, regulating blood pressure and body fluid volume: angiotensinogen gene (angiotensinogen, AGT), Ag-II 1 receptor gene (angiotensin I receptor, AT-1), the angiotensin converting enzyme gene (angiotensin-converting enzyme, ACE); 2, thrombotic tumor susceptibility gene: labile factor leiden sudden change, MTHFR (methylenetetrahydrofolate, MTHFR), factor gene, factor regulate albumen (thrombomodulin, TM); 3, the gene relevant with inner skin cell function: nitric oxide synthase gene (endothelial nitric oxide sythase gene, eNOS); 4, participate in the tumor susceptibility gene of lipid-metabolism: the lipoprotein lipase gene (lipoprotein lipase gene, LPL), apolipoprotein E gene (apolipoprotein E, apo E); 5, with Ia gene: humam leucocyte antigen (HLA) tumor susceptibility gene, TNF (tumor necrosis factor-α, TNF-α) gene and promoter; 6, the tumor susceptibility gene relevant: cytochrome C oxidase and long-chain-3-hydroxyl acetyl-coa dehydrogenase with mitochondria.7, imprinted genes.
The candidate genes polymorphism of disease association is the important science of heredity basis of disease occurrence risk individual difference.The DNA detection disease generation correlation candidate gene pleiomorphism means that functional genomics is based on.The hereditary feature of preeclampsia tumor susceptibility gene also shows as among the mononucleotide point variation formation crowd at gene pleiomorphism, i.e. single nucleotide variations feature on gene level between Different Individual.SNP sudden change on the important enzyme gene can be insignificant, recessive on the biology path, also may cause the active and whole biological function of important enzyme to change, even influence whole biology access function, and is directly relevant with clinical individual morbidity difference.It is estimated that a SNP can appear in average per 1000 pairs of gene bases, and nearly 3,000,000 SNP between two irrelevant individualities.The onset risk difference that difference between this individuality on the science of heredity causes can reach more than the hundreds of times.Detect as the single nucleotide polymorphism (SNP) of some diseases related gene is carried out SNP in ill crowd, can predict that the possible degree of a certain specified disease feature takes place the patient, thereby provide new auxiliary foundation for medical diagnosis on disease.In addition, also can help to be familiar with the generation and the controlling mechanism of disease or its certain symptom.
The hereditary feature of preeclampsia tumor susceptibility gene can also help to infer the chromosome segment relevant with preeclampsia.At present found that the chromosome segment relevant with preeclampsia comprises: No. 6 chromosomes; No. 17 chromosomes (angiotensin converting enzyme); No. 21 chromosomes (superoxide dismutase gene); No. 3 chromosomes (angiotensin I receptor) [Brought PF.What is the place of genetics in the pathogenesis of preeclampia.BiolNeonate, 1999,76:325-330].
In June, 2004, the patent of Granted publication on the 30th (was authorized publication number: CN 1155722C; Denomination of invention: gene warning diagnostic kit and detection method thereof before pregnant) so-called warning gene has comprised N5 in, N10-MTHFR and methionine synthetase reductase gene are used in pregnant preceding diagnosis filial generation and whether neural tube defects can take place.
Rely on clinical experience, sensitivity and the low deficiency of specific parameters in order to overcome the dlinial prediction pregnancy badness come-off, need utilize the genetic marker characteristic information of biological specimen, set up a kind of method of predicting the occurrence risk of pregnancy badness, help to improve forecasting efficiency.The people at highest risk that this will help more early stage effective protection pregnancy badness come-off reduces clinical poor prognosis risk, and the blindness of reduction medical therapy and expensive improves mother and baby's quality of life perinatal period.
Summary of the invention
Purpose of the present invention just provides a kind of early stage, safety, convenient, quick and sensitive, by measuring biological specimen and external prediction and pregnancy badness come-off, comprise threatened premature labor and premature labor, intrauterine growth retardation and LBW, and pregnancy-hypertension syndrome and eclampsia etc., pleomorphism site genotype on the common proline carboxypeptidase gene of being correlated with, can before pregnant, predict the method for mother's pregnancy badness come-off generating risks, reduce blindness and hysteresis quality on clinical prevention and the control pregnancy badness come-off, improve public health and clinical assistant diagnosis level, early detection individual difference and people at highest risk reduce the pregnancy badness come-off incidence of disease between pregnant and perinatal period, complication and case fatality rate.This method also can be used for developing the prediction kit of prediction pregnancy badness come-off generating risks.
For achieving the above object, take following technical scheme:
The method of prediction pregnancy badness come-off generating risks of the present invention, by measuring from proline carboxypeptidase gene (Prolylcarboxypeptidase in experimenter's the biological sample, PRCP) genotype of pleomorphism site E112D is predicted the occurrence risk of pregnancy badness come-off: when genotype was the 112EE homozygous wildtype, the pregnancy badness come-off incidence was lower; When genotype was 112ED heterozygous or 112DD homozygous mutation type, the pregnancy badness come-off incidence was higher.
Proline carboxypeptidase is that blood vessel and endothelial function are regulated the key enzyme on the path, and the generation of PRCP E112D (rs2298668) pleomorphism site and pregnancy badness come-off is relevant, has remarkable prediction effect.Concrete, when
When (1) described PRCP loci gene type was the 112EE homozygous wildtype, prediction pregnancy badness come-off incidence was lower;
When (2) described PRCP loci gene type was 112ED heterozygous or 112DD homozygous mutation type, prediction pregnancy badness come-off incidence was higher;
(3) when the gravid woman merges chronic hypertension and PRCP genotype and is the 112EE heterozygous, above-mentioned prediction effect is more obvious, predict that promptly the pregnancy badness come-off probability takes place the genotypic no hypertensive gravid woman of PRCP EE homozygous wildtype, be lower than gravid woman with chronic hypertension.
(4) prediction PRCP genotype is the gravid woman of 112ED heterozygous or 112DD homozygous mutation type, and during as if the merging chronic hypertension, the pregnancy badness come-off incidence is high in the genotypic gravid woman of other PRCP.
In said method of the present invention, the pregnancy badness come-off of described prediction comprises threatened premature labor and pregnancy badness come-offs such as premature labor, intrauterine growth retardation and LBW, pregnancy-hypertension syndrome and pre-eclampsia and eclampsia, the pregnancy badness come-off of these different phases may have the pathomechanism that common morbidity develops on Utero-placenta ischemic and vascular endothelial cell damage.
The genotype of the pleomorphism site E112D (rs2298668) of above-mentioned PRCP is except directly measuring, can also exist other pleomorphism site genotype of linkage disequilibrium to determine with it by measuring this site annex, such pleomorphism site comprises nonsense mutation site, missense mutation site and the pleomorphism site that is positioned at gene intron position, Gene regulation position.
In the present invention, can utilize the polymorphism parting oligonucleotide to detect the genotype of above-mentioned pleomorphism site.Preferably, described polymorphism parting oligonucleotide is: (1) allele-specific nucleic acid primer, the proline carboxypeptidase gene segment that is used to increase and contains described pleomorphism site, it can detect the pleomorphism site genotype of the key gene PRCP gene of blood vessel and endothelial function adjusting path, and/or (2) be used to detect the genotypic oligonucleotide probe of pleomorphism site that blood vessel and endothelial function are regulated the key gene PRCP gene of path, and it can be specifically regulates the nucleic acid hybridization of pleomorphism site of the key gene PRCP gene of path with blood vessel and endothelial function.Preferably, the length of described oligonucleotide probe is 17-50 nucleotide.
Thus, the present invention predicts the method for pregnancy badness come-off generating risks, specifically can may further comprise the steps: 1) utilize the pleomorphism site genotype of above-mentioned polymorphism parting oligonucleotide detection from key gene described in the sample of individuality; 2) foundation contains the genotypic forecast model of test sample PRCP pleomorphism site; 3), and merge and follow chronic hypertension person that the risk of pregnancy badness come-off takes place according to the risk of described polymorphism site genotype estimation pregnancy badness come-off.
In method of the present invention, can be used for predicting pregnancy badness come-off generating risks pleomorphism site comprise E112D (rs2298668) pleomorphism site of above-mentioned PRCP at least, can also further comprise other with the pleomorphism site that has linkage disequilibrium with it, comprise nonsense mutation site, missense mutation site and the pleomorphism site that is positioned at gene intron position, Gene regulation position.
In method of the present invention, measure the genotype of described pleomorphism site and can use the following difference foranalysis of nucleic acids technology that is selected from: polymerase chain reaction (PCR), polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), PCR-alleles-specific oligonucleotide probe method, PCR-sequence specific oligonucleotides method, sequencing, PCR-sequence specific primers method, the PCR-fluorescence method, the PCR finger-printing method, oligonucleotides connects to be analyzed, the detection method of fluorescent energy resonance transfer, biochip, nucleic acid chip, mass-spectrometric technique, genescan, single-strand conformation polymorphism, denaturing gradient gel electrophoresis, enzyme or chemical mispairing patterning method, the Taqman biological detecting method.Wherein preferred detection method is PCR, PCR-RFLP, Taqman technology, biochip, nucleic acid chip or kit.
PCR, PCR-RFLP, biochip, sequencing, Teqman genescan technology are the conventional methods of using of those skilled in the art.The Taqman technology is a kind of method of using fluorescent technique to carry out real-time quantitative PCR.Biochip is meant methods such as adopting the synthetic or micro-sampling of Hiroshima original position, with big molecular proportion of large number of biological such as nucleic acid fragment, peptide molecule even histotomy, biological samples such as cell solidify in holder in an orderly manner (as slide, silicon chip, polyacrylamide gel, carriers such as nylon membrane) surface, form intensive two-dimentional molecules align, then with the molecular hyridization that hits of the biological sample to be measured of mark, by specific instrument such as laser confocal scanning instrument or electric charge coupling photographic camera (CCD) intensity of hybridization signal is carried out fast, parallel, check and analysis efficiently, thereby the quantity of target molecule or quality in the judgement sample.
The present invention is not to be qualification for detection method for the explanation of the detection method of loci gene type, any those skilled in the art adopt conventional biological technique method to predict with the pregnancy badness come-off incidence to be that other risks of core all belong to content of the present invention by detecting pleomorphism site genotype of the present invention, can further include adopt the difference of conventional biological technique method by detecting genotypic transcription product of pleomorphism site functional form of the present invention and/or expression product indirectly the pleomorphism site that is associated of reflection predict the example of pregnancy badness come-off incidence.
In method of the present invention, wherein said biological sample from individuality is selected from: as the blood sample of periphery whole blood, peripheral blood cells, leucocyte, serum etc., humoral sample such as urine, saliva, tissue samples such as oral mucosa examination, hair, skin, biopsy are organized sample secretion, fecal matter sample, cultured cell, preferably, described sample is blood sample, and is optional, described sample can carry out purifying in advance, for example separates total nucleic acid.
Proline carboxypeptidase (PRCP) is the nervous plain II digestive enzyme of a kind of important vessel.Equaling nineteen sixty-eight by Yang HYT at first finds.PRCP equals at 5 o'clock at pH, has best enzymatic activity, the 20-50% when enzymatic activity only is left maximum when pH is elevated to 7.PRCP belongs to the serine stretch protein enzyme family, and activity can be suppressed by phenylmethylsulfonyl fluoride (PMSF).Studies show that in a large number PRCP can be at the outer film expression of endothelial cell.PRCP is the correctives between renin-angiotensin-aldosterone system (RAAS) and the kassinin kinin-bradykinin system (KKS), is the another kind of important regulating system of blood vessel and endothelial function.The PRCP Angiotensin II of degrading makes that having the vasoactive Angiotensin II that contracts strongly is transformed into the hypertensin 1-7 with vasodilator activity, participates in the adjusting of RAAS systemic-function.In addition, PRCP still is the plasmakinin activation of zymogen thing outside a kind of XIIa of being independent of, and can make prokininase activate and generate bradykinin.The Angiotensin II because PRCP can degrade, the generation that increases hypertensin 1-7 can promote the release of bradykinin again, two kinds of effects can both make the generation of NO increase, and then vasodilator, play the effect that brings high blood pressure down.
Proline carboxypeptidase (Prolylcarboxypeptidase, PRCP) gene was cloned in 1993, be positioned human No. 11 autosomal long-armed go up (11q14), a kind of lysosomal enzyme of encoding, effect is that enzyme is cut the C end amino acid that is connected with proline as peptide classes such as Angiotensin II, Angiotensin II I and bradykinins.Be positioned at the base mutation of an A-C on its extron, caused the 112nd amino acids (Glu) to become asparatate (Asp) by glutamic acid.We discover, the expression of this pleomorphism site and the mRNA of PRCP and activity etc. are relevant.In view of the important regulating action of PRCP to RAAS system and blood pressure, this functional mononucleotide polymorphism site probably can have influence on the drug effect of ACEI class medicine.But utilize the method for this site estimation pregnancy badness come-off and kit still not to have bibliographical information.
Common single nucleotide polymorphism (SNP) site can be positioned at extron position, introne position and the noncoding region position of gene, is preferably the extron position, especially can change the pleomorphism site of amino acid sequence coded.
What the present invention relates to discovers:
Find and early prediction threatened premature labor and premature labor and pregnancy badness come-offs such as intrauterine growth retardation or LBW, the high-risk women crowd of early detection prevents and treats, still imperfectly understand under the situation in the cause of disease interior, EARLY RECOGNITION people at highest risk still depends on clinical more epiphytotics hazards at present.But these indexs are responsive and special not enough.Previously research prompting, pregnancy badness come-offs such as preeclampsia, premature labor and LBW all are called the complex inheritance disease, be the complex disease feature that the environment and heredity acting in conjunction causes, all the pregnant woman has hereditary capacity familial inheritance tendency to the neurological susceptibility of pregnancy badness come-off.
The cause of disease deeper mechanisms of pregnancy badness come-off it be unclear that.Previously study knownly, the gravid woman suffers from pregnancy-induced hypertension syndrome, particularly with chronic hypertension, is one of key factor that causes premature labor, fetal intrauterine growth retardation and LBW.Think that at present the distinctive pathological change of tool of preeclampsia is the extensive little blood vessel endothelium injury of Utero-placenta unit hypoxic-ischemic and whole body.Utero-placenta ischemic may be preeclampsia and the initial link that pre-eclampsia takes place, the placental blood hypoperfusion causes its metabolism to change, discharge certain factor and enter the peripheral blood circulation, cause extensive vascular endothelial cell damage, cause vessel retraction spasm, blood coagulation system changes of function, body fluid to be shunted again then; May be preeclampsia clinical manifestation and multiple organ dysfunction thereof, and the basis that further develops the premature labor, fetal intrauterine growth retardation and the LBW related pathologies mechanism that cause multiple bad pregnancy outcome by preeclampsia.Report is arranged, and pre-eclampsia patient's HELLP syndrome (haemolysis, liver enzyme raise, platelet count descends) and gestation 34 all preceding mother and baby's case fatality rate obviously increase.Other has the research prompting, and gestational period abuse central nervous excitation agent cocaine causes the local anesthesia and the blood vessel function that contracts, can cause the placenta that exsomatizes and shrink strongly, strengthen a kind of slow effect that swashs the blood vessel thing that contracts that phthalein induces, can obviously reduce the fetal blood confession, and cause anoxic, cause fetal intrauterine growth retardation.The pregnancy duration life is overstretched or have serious life event to hit, and can cause premature labor.Pointed out thus, Utero-placenta ischemic and vascular endothelial cell damage may be organized the total important deep layer cause of disease mechanism clue of pregnancy badness come-off more.
The epidemiologic data prompting, pregnancy badness come-off has the familial inheritance tendency, there is the pregnant woman of preeclampsia family history higher 8 times than no family history person's incidence of disease, show that the pregnant woman has hereditary capacity to the neurological susceptibility of preeclampsia, but at present should disease definite hereditary basis is also not fully aware of, and the gene that can regulate blood pressure, body fluid volume, placenta growth, thrombosis, blood vessel double teeming, blood vessel inner skin cell function all may be the tumor susceptibility gene of preeclampsia.Therefore, foundation can be predicted the kit of pregnancy badness come-off generation correlated inheritance feature, may make the high-risk women's who discerns pregnancy badness come-off more morning more accurate, make from entering reproduction age gestation and can take preventive measures as early as possible and treatment measures to producing each phase, can reduce the incidence of disease, complication and case fatality rate to greatest extent, be the key of saving and treating pregnancy badness come-off.
Pregnancy badness come-off is one group of multigenic disease.(renin-angiotension system, RAAS) gene may be main profound cause of disease candidate gene on the pathogenesis of pregnancy badness come-off to renin-angiotensin-aldosterone system.RAAS is an a kind of hormone system, circulation RAAS is the feritin in kidney source, makes the synthetic proangiotensin of liver in peripheral blood, changes angiotensin I into, in the effect of lungs menses angiotensin conversion enzyme, be transformed into Angiotensin II (Ang II) again; Ang II is secreted in the blood,, combines with specific receptor on the target organ to far-end target organ (blood vessel, heart, brain, kidney, adrenal gland etc.) with blood flow, regulates the cardiovascular system water-electrolyte balance of unifying.There is the composition of local RAAS in a lot of periphery target organs (as blood vessel, heart, brain, kidney, adrenal gland, placenta, uterus, ovary, testis etc.).The principal ingredient of RAAS comprises feritin, Angiotensin-Converting, angiotensins proenzyme, angiotensin I, Angiotensin II and their acceptor, all is present in placenta.
Proline carboxypeptidase (PRCP) is the nervous plain II digestive enzyme of a kind of important vessel.Equaling nineteen sixty-eight by Yang HYT at first finds.PRCP equals at 5 o'clock at pH, has best enzymatic activity, the 20-50% when enzymatic activity only is left maximum when pH is elevated to 7.PRCP belongs to the serine stretch protein enzyme family, and activity can be suppressed by phenylmethylsulfonyl fluoride (PMSF).Studies show that in a large number PRCP can be at the outer film expression of endothelial cell.PRCP is the correctives between RAAS and the kassinin kinin-bradykinin system (KKS), is the another kind of important regulating system of blood vessel and endothelial function.The PRCP Angiotensin II of degrading makes that having the vasoactive Angiotensin II that contracts strongly is transformed into the hypertensin 1-7 with vasodilator activity, participates in the adjusting of RAAS systemic-function.In addition, PRCP still is the plasmakinin activation of zymogen thing outside a kind of XIIa of being independent of, and can make prokininase activate and generate bradykinin.The Angiotensin II because PRCP can degrade; the generation that increases hypertensin 1-7 can promote the release of bradykinin again; two kinds of effects can both make the generation of NO increase; and then vasodilator; play the effect that brings high blood pressure down and lower the vascular endothelial cell infringement; in addition, may cell in pregnant process in the blood supply and blood pressure regulating or protection of ecs of and mother's whole body local to placenta.
Proline carboxypeptidase (Prolylcarboxypeptidase, PRCP) gene was cloned in 1993, be positioned human No. 11 autosomal long-armed go up (11q14), a kind of lysosomal enzyme of encoding, effect is that enzyme is cut the C end amino acid that is connected with proline as peptide classes such as Angiotensin II, Angiotensin II I and bradykinins.Be positioned at the base mutation of an A-C on its extron, caused the 112nd amino acids to become asparatate (Asp) by glutamic acid (Glu).We discover, the expression of this pleomorphism site and the mRNA of PRCP and activity etc. are relevant.In view of the important regulating action of PRCP to RAAS system and blood pressure, this functional mononucleotide polymorphism site probably can have influence on the drug effect of ACEI class medicine.But still there is not bibliographical information with the relation and the pretest agent method thereof of preeclampsia, premature labor and intrauterine growth retardation and LBW.
The present invention finds successively and proves: on analysis-by-synthesis pregnancy badness come-off aspect, observe among the pregnancy and delivery women, the ratio (3%) of PRCP 112DD homozygous mutation type obviously increases (21%) than gravid woman's ratio of no pregnancy badness come-off in the pregnancy badness come-off patient.Adjusted factors such as conceptional age, pregnant age, multiparity number of times, smoking, BMI through overcorrection after, the OR value of finding PRCP 112DD homozygous mutation type genotype and the genotypic pregnancy badness come-off incidence of PRCP 112EE homozygous wildtype be 4 (95% CI:1-12, p=0.031).Results suggest PRCP gene can be predicted the generation of pregnancy badness come-off, and PRCP E112D pleomorphism site genotype can be predicted the generation of pregnancy badness come-off.
The present invention finds successively and proves: analyze on threatened premature labor and the premature labor aspect in each case, in the gravid woman, the ratio (3%) of PRCP 112DD homozygous mutation type gravid woman's ratio than absence of aura premature labor and premature labor in threatened premature labor and premature labor patient obviously increases (21%).Adjusted factors such as conceptional age, pregnant age, multiparity number of times, smoking, BMI through overcorrection after, the OR value of finding PRCP 112DD homozygous mutation type genotype and genotypic threatened premature labor of PRCP 112EE homozygous wildtype and incidence of preterm birth be 4 (95% CI:1-12, p=0.031).Results suggest PRCP gene can be predicted the generation of threatened premature labor and premature labor, and PRCP E112D pleomorphism site genotype can be predicted the generation of threatened premature labor and premature labor.
The present invention finds successively and proves: analyze on fetal intrauterine growth retardation and the LBW aspect in each case, in the gravid woman, the ratio (3%) of PRCP 112DD homozygous mutation type fetal intrauterine growth retardation and LBW ratio than absence of aura premature labor and premature labor in fetal intrauterine growth retardation and LBW patient obviously increases (21%).Adjusted factors such as conceptional age, pregnant age, multiparity number of times, smoking, BMI through overcorrection after, the OR value of finding PRCP 112DD homozygous mutation type genotype and genotypic fetal intrauterine growth retardation of PRCP 112EE homozygous wildtype and LBW incidence be 4 (95% CI:1-12, p=0.031).Results suggest PRCP gene can be predicted the generation of fetal intrauterine growth retardation and LBW, and PRCP E112D pleomorphism site genotype can be predicted the generation of fetal intrauterine growth retardation and LBW.
The present invention finds successively and proves: analyze on preeclampsia and the eclampsia aspect in each case, in the gravid woman, the ratio (3%) of PRCP 112DD homozygous mutation type obviously increases (21%) than gravid woman's ratio of no preeclampsia and eclampsia in preeclampsia and eclamptic patients.Adjusted factors such as conceptional age, pregnant age, multiparity number of times, smoking, BMI through overcorrection after, the OR value of finding PRCP 112DD homozygous mutation type genotype and the genotypic preeclampsia incidence of PRCP 112EE homozygous wildtype be 4 (95% CI:1-12, p=0.031).Results suggest PRCP gene can be predicted the generation of preeclampsia, and PRCP E112D pleomorphism site genotype can be predicted the generation of preeclampsia.
The present invention carries out further correlation analysis after the layering with chronic hypertension, find and confirmation: compare with the genotypic gravid woman of PRCP 112EE homozygous wildtype who does not merge chronic hypertension, chronic hypertension has increased by 11 times of risks of suffering from preeclampsia separately, and PRCP E112D heterozygous genotype gravid woman merges the risks that chronic hypertension has increased by 120 times of trouble preeclampsias.Results suggest PRCP E112D (rs2298668) pleomorphism site genotype can be predicted the generation (seeing Table 2) of gravid woman's preeclampsia, and is especially more obvious for the gravid woman's predicting function that merges chronic hypertension.
Use achievement of the present invention, select the predicted gene of PRCP, strengthened the women of child-bearing age that present tradition carries out, pregnant and lying-in women the gentle control ability of prevention of water at pregnancy badness come-off as pregnancy badness come-off.When being convenient to community health care of women consulting and/or the clinician to the first visit pregnant woman by measuring PRCP gene polymorphism sites genotype, for the consulting service that prescription on individual diagnosis person more scientifically provides prediction pregnancy badness come-off and hypertension prevention and control, find and the protection people at highest risk.
Description of drawings
Fig. 1 is to use the PCR-RFLP method to detect the genotypic gel electrophoresis figure of E112D pleomorphism site of PRCP gene.
Fig. 2 is to use the Taqman method to detect the genotypic fluorescence pattern of E112D pleomorphism site of PRCP gene.
Wherein:
1---the 167bp segment; 2---the 101bp segment; 3---the 66bp segment;
4---send the FAM fluorescence area; 5---send two kinds of fluorescence areas;
6---send the VIC fluorescence area.
Embodiment
Embodiment 1: measure the pleomorphism site genotype of PRCP gene and predict the generation of pregnancy badness come-off
(1) E112D (rs2298668) the pleomorphism site genotype of mensuration PRCP gene:
(1) genomic DNA of extraction host cell: according to the molecular biology method operation of routine.
(a) add the 30ml erythrocyte cracked liquid in whole blood, slowly shake up, room temperature left standstill 10 minutes, during, shake for several times, thoroughly splitting erythrocyte;
(b) in 4 ℃, 2000 leave the heart/minute, 10 minutes, remove supernatant, the leucocyte that will precipitate is broken up on the oscillator in rotation, adds proteinase 40 μ l, RNA enzyme 50 μ l, shakes up, and adds write cell lysis buffer and puts 15ml, 37 ℃ of water-baths of mixing were taken out after 20 minutes, put in the cold water;
(c) add cold albumen precipitation liquid 4ml, be placed on-20 ℃ of refrigerators 5 minutes behind the mixing, take out in 4 ℃, 3000 rev/mins centrifugal 10 minutes.Supernatant poured into slowly shake in the 50ml centrifuge tube that has added the 15ml isopropyl alcohol for several times, separate out to the DNA floccus;
(d) the DNA floccus of separating out is moved to another 1.5ml and has packed on the filter paper of 75% ethanol, make evaporate dried;
(e) add DNA hydrating fluid 1.5ml, put shaking table, shaken over night, standby;
(f) mensuration of DNA concentration adopts ultraviolet spectrophotometry, measures the OD value under two wavelength of 260nm and 280nm respectively, is DNA concentration with OD260nm * 50 income values.And with OD260nm/OD280nm ratio estimation DNA purity.
(2) use the Taqman method to detect Glu112Asp (E112D) the pleomorphism site genotype of PRCP gene
(a) with PCR instrument amplification PRCP functional gene polymorphic site and flanking sequence thereof, in 5 μ l PCR reaction systems, contain genomic DNA 10ng, 2.5 (composition comprises the Taqman 2X Universal PCR Master Mix NoAmpErase UNG of μ l: AmpliTaq Gold DNA Polymerase, dNTPs with Dutp, Passive Reference, with the damping fluid of optimizing), and the forward primer of 0.90 μ M, each 0.25 μ M. of allele-specific probe of the reverse primer of 0.90 μ M and two sections band fluorescence report groups
Primer sequence is
Forward primer: 5 ' GTTTGCCAAAAGGTTCAGTGACTT 3 ' (SEQ ID No.1)
Reverse primer: 5 ' TCTCCATAGTATCGATGTTCAGCAAAC 3 ' (SEQ ID No.2)
The sequence of allele-specific probe is:
VIC-5’CATAGCTTTCAGTTCCTCA 3’-NFQ(SEQ ID No.3)
Corresponding to " T (or Glu) " allele, carry VIC fluorescence report group.
FAM-5’ATAGCTTTCAGGTCCTCA 3’-NFQ(SEQ ID No.4)
Corresponding to " G (or Asp) " allele, carry FAM fluorescence report group.
The PCR reaction conditions:
95 ℃ of 10min, 1 circulation;
92℃ 15s，
60℃ 1min，
50 circulations.
(b) on ABI Primer 7900 type quantitative real time PCR Instruments, detect fluorescence information and carry out genotypic evaluation
The PCR plate of finishing the PCR reaction is put on the 7900 type quantitative real time PCR Instruments, is selected for use " Allelic Discrimination " program in SDS 2.1 softwares, scan judgement with the result:
The genotype of sending FAM fluorescence person is the Asp/Asp homozygote;
The genotype of sending VIC fluorescence person is the Glu/Glu homozygote;
The genotype of sending two kinds of fluorescence persons is the Asp/Glu heterozygote.
(3) use PCR and restriction fragment length polymorphism analysis method (PCR-RFLP) to detect the PRCPE112D pleomorphism site
According to PRCP E112D gene order design PCR Auele Specific Primer, comprise PCR forward primer and PCR reverse primer, carry out conventional pcr amplification by following condition.
Primer sequence:
Forward primer: 5 ' ATGGTTTGCCAAAAGGTTCA 3 ' (SEQ ID No.5)
Reverse primer: 5 ' TGTCACCAAAGGGGAGAGAC 3 ' (SEQ ID No.6)
The PCR reaction system:
Genomic DNA 15ng/ μ l, upstream and downstream primer 10pmol (20 μ mol/L), dNTPs 1.25mmol/L, 10 * buffer, 1.0 μ l, Gold Taq archaeal dna polymerase 3U, dH
2O supplies cumulative volume to 6.55 μ l.
The PCR reaction conditions:
Behind 94 ℃ of pre-sex change 3min; 94 ℃ of sex change 45sec, 62 ℃ of annealing 45sec, 72 ℃ are extended 1sec, 38 cycle periods; Last 72 ℃ are extended 7min. Obtain the amplified fragments of 167bp.
Enzyme tangent condition and system (15 μ l):
PRCP E112D site PCR product purpose fragment length is 167bp, total enzyme system of cutting is 15 μ l, PCR product 10ul wherein, 10 * NEBuffer#2,1.5 μ l, (its identification segment is G/GWCC to the AVaII restriction endonuclease, wherein W is A or T) 4U (0.4 μ l) and 3.1 μ l ddH2O, 37 ℃ are spent the night.
Genotype result judges:
Product point sample after the DNA enzyme cut after 37 ℃ of enzymes are cut and spent the night, reads glue figure and carries out genotyping on 2.5% agarose gel under uviol lamp.Idiotype is identified as follows:
Endonuclease bamhi is 167bp, and the PRCP genotype is 112EE (wild type);
Endonuclease bamhi is 167+101+66bp, and the PRCP genotype is 112ED (heterozygote);
Endonuclease bamhi is 101+66bp, and the PRCP genotype is 112DD (homozygote).
(2) prediction of generation pregnancy badness come-off risk
When the PRCP genotype was 112ED heterozygous or 112DD homozygous mutation type, prediction pregnancy badness come-off incidence was higher; Genotype is 112EE when homozygous, and prediction pregnancy badness come-off incidence is lower.
When the gravid woman merged chronic hypertension, above-mentioned prediction effect was more obvious.When the PRCP genotype that is the gravid woman of chronic hypertension was 112ED heterozygous or 112DD homozygous mutation type, prediction pregnancy badness come-off incidence was higher.
Above method is divided into three groups with the pregnancy badness come-off patient earlier: homozygous wildtype group, heterozygous group, homozygous mutation type group by clinical verification.Adopt clinical epidemiology case-control method, three groups of crowds are carried out PRCP pleomorphism site genotype and the total dangerous correlation analysis of pregnancy badness come-off.By whether following chronic hypertension chromatographic analysis pregnant and lying-in women that the preeclampsia situation takes place, the result shows (table 2): gestation before no chronic hypertension, carry among the genotypic gravid woman of PRCP gene DD homozygous mutation, pregnancy badness come-off generation ratio higher (52.6%), adjusted factors such as conceptional age, pregnant age, multiparity number of times, smoking, BMI through overcorrection after, find PRCP DD homozygous mutation type genotype, relative EE homozygous wildtype genotype, the OR value is 2.7,95%CI is 1.1-11.6, p=0.035.
The correlation analysis of table 2.PRCP gene E112D polymorphism genotype and pregnancy badness come-off danger
| Genotype | Gestation number (%) | Correlation analysis | Correction result § | |||
| Badness come-off is arranged | There is not bad final result | OR | 95％CI | OR | 95％CI | |
| EE ED DD | 301(34.3) 77(34.2) 11(52.6) | 577(65.7) 155(65.8) 8(48.4) | # 0.95 2.63 * | - (0.49，1.70) (1.03，9.84) | # 0.96 2.68 * | - (0.54，1.62) (1.10，11.61) |
*P＜0.05,
*P＜0.0001; The # control group; § has adjusted factors such as sex, conceptional age, pregnant age, multiparity number of times, smoking, diabetes, BMI, education degree.EE represents homozygous wildtype; ED represents heterozygous; DD represents homozygous mutation type (95% CI:95% credibility interval).
Embodiment 2: measure the pleomorphism site genotype of PRCP gene and predict threatened premature labor and the generation of premature labor
(1) E112D (rs2298668) the pleomorphism site genotype of mensuration PRCP gene:
(1) genomic DNA of extraction host cell: according to the molecular biology method operation of routine.(with embodiment 1)
(2) use the Taqman method to detect Glu112Asp (E112D) the pleomorphism site genotype (with embodiment 1) of PRCP gene
(3) use PCR and restriction fragment length polymorphism analysis method (PCR-RFLP) to detect PRCPE112D pleomorphism site (with embodiment 1)
(2) prediction of generation threatened premature labor and premature delivery risk
When the PRCP genotype was 112ED heterozygous or 112DD homozygous mutation type, prediction threatened premature labor and incidence of preterm birth were higher; Genotype is 112EE when homozygous, and prediction threatened premature labor and incidence of preterm birth are lower.
When the gravid woman merged chronic hypertension, above-mentioned prediction effect was more obvious.When the PRCP genotype that is the gravid woman of chronic hypertension was 112ED heterozygous or 112DD homozygous mutation type, prediction threatened premature labor and incidence of preterm birth were higher.
Above method is divided into three groups with threatened premature labor and premature labor patient earlier: homozygous wildtype group, heterozygous group, homozygous mutation type group by clinical verification.Adopt clinical epidemiology case-control method, three groups of crowds are carried out the total dangerous correlation analysis of PRCP pleomorphism site genotype and threatened premature labor and premature labor.By whether following chronic hypertension chromatographic analysis pregnant and lying-in women that the preeclampsia situation takes place, the result shows (table 3): gestation before no chronic hypertension, carry among the genotypic gravid woman of PRCP gene DD homozygous mutation, threatened premature labor and premature labor generation ratio higher (36.1%), adjusted factors such as conceptional age, pregnant age, multiparity number of times, smoking, BMI through overcorrection after, find PRCP DD homozygous mutation type genotype, relative EE homozygous wildtype genotype, the OR value is 1.6,95%CI is 1.1-10.6, p=0.041.
The dangerous correlation analysis of table 3.PRCP gene E112D polymorphism genotype and threatened premature labor and premature labor
| Genotype | Gestation number (%) | Correlation analysis | Correction result § | |||
| (tendency) premature labor is arranged | There is not (tendency) premature labor | OR | 95％CI | OR | 95％CI | |
| EE ED DD | 239(27.2) 45(19.7) 7(36.8) | 639(72.8) 183(80.3) 12(63.2) | # 0.75 1.56 * | - (0.21，1.57) (1.03，7.84) | # 0.73 1.59 * | - (0.22，1.63) (1.06，10.55) |
*P＜0.05,
*P＜0.0001; The # control group; § has adjusted factors such as sex, conceptional age, pregnant age, multiparity number of times, smoking, diabetes, BMI, education degree.EE represents homozygous wildtype; ED represents heterozygous; DD represents homozygous mutation type (95% CI:95% credibility interval).
Embodiment 3: measure the pleomorphism site genotype of PRCP gene and predict intrauterine growth retardation and the generation of LBW
(1) E112D (rs2298668) the pleomorphism site genotype of mensuration PRCP gene:
(1) genomic DNA of extraction host cell: according to the molecular biology method operation of routine.(with embodiment 1)
(2) use the Taqman method to detect Glu112Asp (E112D) the pleomorphism site genotype (with embodiment 1) of PRCP gene
(3) use PCR and restriction fragment length polymorphism analysis method (PCR-RFLP) to detect PRCPE112D pleomorphism site (with embodiment 1)
(2) prediction of generation intrauterine growth retardation and LBW risk
When the PRCP genotype was 112ED heterozygous or 112DD homozygous mutation type, prediction intrauterine growth retardation and LBW incidence were higher; Genotype is 112EE when homozygous, and prediction intrauterine growth retardation and LBW incidence are lower.
When the gravid woman merged chronic hypertension, above-mentioned prediction effect was more obvious.When the PRCP genotype that is the gravid woman of chronic hypertension was 112ED heterozygous or 112DD homozygous mutation type, prediction intrauterine growth retardation and LBW incidence were higher.
Above method is divided into three groups with intrauterine growth retardation and LBW patient earlier: homozygous wildtype group, heterozygous group, homozygous mutation type group by clinical verification.Adopt clinical epidemiology case-control method, three groups of crowds are carried out the total dangerous correlation analysis of PRCP pleomorphism site genotype and intrauterine growth retardation and LBW.By whether following chronic hypertension chromatographic analysis pregnant and lying-in women that the preeclampsia situation takes place, the result shows (table 4): gestation before no chronic hypertension, carry among the genotypic gravid woman of PRCP gene DD homozygous mutation, intrauterine growth retardation and LBW generation ratio higher (47.4%), adjusted factors such as conceptional age, pregnant age, multiparity number of times, smoking, BMI through overcorrection after, find PRCP DD homozygous mutation type genotype, relative EE homozygous wildtype genotype, the OR value is 1.7,95%CI is 1.2-11.2, p=0.036.
The dangerous correlation analysis of table 4.PRCP gene E112D polymorphism genotype and intrauterine growth retardation and LBW
| Genotype | Gestation number (%) | Correlation analysis | Correction result § | |||
| The hypoevolutism under-weight is arranged | There is not the slow under-weight of growth | OR | 95％CI | OR | 95％CI | |
| EE ED DD | 301(34.3) 60(36.3) 9(47.4) | 577(65.7) 168(69.3) 10(52.6) | # 0.68 1.73 * | - (0.11，1.43) (1.09，9.92) | # 0.69 1.72 * | - (0.12，1.55) (1.18，11.23) |
*P＜0.05,
*P＜0.0001; The # control group; § has adjusted factors such as sex, conceptional age, pregnant age, multiparity number of times, smoking, diabetes, BMI, education degree.EE represents homozygous wildtype; ED represents heterozygous; DD represents homozygous mutation type (95%CI:95% credibility interval).
Embodiment 4: measure the pleomorphism site genotype of PRCP gene and predict preeclampsia and the generation of eclampsia
(1) E112D (rs2298668) the pleomorphism site genotype of mensuration PRCP gene:
(1) genomic DNA of extraction host cell: according to the molecular biology method operation of routine.(with embodiment 1)
(2) use the Taqman method to detect Glu112Asp (E112D) the pleomorphism site genotype (with embodiment 1) of PRCP gene
(3) use PCR and restriction fragment length polymorphism analysis method (PCR-RFLP) to detect PRCPE112D pleomorphism site (with embodiment 1)
(2) prediction of generation preeclampsia and eclampsia risk
When the PRCP genotype was 112ED heterozygous or 112DD homozygous mutation type, prediction preeclampsia and eclampsia incidence were higher; Genotype is 112EE when homozygous, and prediction preeclampsia and eclampsia incidence are lower.
When the gravid woman merged chronic hypertension, above-mentioned prediction effect was more obvious.When the PRCP genotype that is the gravid woman of chronic hypertension was 112ED heterozygous or 112DD homozygous mutation type, prediction preeclampsia and eclampsia incidence were higher.
Above method is divided into three groups with preeclampsia and eclampsia patient earlier: homozygous wildtype group, heterozygous group, homozygous mutation type group by clinical verification.Adopt clinical epidemiology case-control method, three groups of crowds are carried out the total dangerous correlation analysis of PRCP pleomorphism site genotype and preeclampsia and eclampsia.By whether following chronic hypertension chromatographic analysis pregnant and lying-in women that the preeclampsia situation takes place, the result shows (table 5): gestation before no chronic hypertension, carry among the genotypic gravid woman of PRCP gene DD homozygous mutation, preeclampsia and eclampsia generation ratio higher (15.8%), adjusted factors such as conceptional age, pregnant age, multiparity number of times, smoking, BMI through overcorrection after, find PRCP DD homozygous mutation type genotype, relative EE homozygous wildtype genotype, the OR value is 2.9,95%CI is 1.2-10.1, p=0.035.
Carry the genotypic pregnant woman of PRCP gene EE, before the gestation with chronic hypertension person with compare without chronic hypertension person, the risk of suffering from preeclampsia and eclampsia significantly increases, the OR value is 8.8,95%CI is 3.6-12.7; And the pregnant and lying-in women that have PRCP ED heterozygous genes type simultaneously with chronic hypertension before the gestation, the risk of suffering from preeclampsia and eclampsia is than EE genotype and more remarkable without chronic hypertension person, and the OR value is 138..Above results suggest, PRCP E112D (rs2298668) pleomorphism site genotype and preeclampsia and eclampsia have very significant prediction incidence relation.
The dangerous correlation analysis of table 5.PRCP gene E112D polymorphism genotype and chronic hypertension and preeclampsia and eclampsia
| Whether merge chronic hypertension | Genotype | Gestation number (%) | Correlation analysis | Correction result § | |||
| Preeclampsia is arranged | No preeclampsia | OR | 95％CI | OR | 95％CI | ||
| Not | EE ED DD | 70(8.2) 15(7.1) 4(15.8) | 808(91.8) 213(92.9) 16(74.2) | # 0.81 2.89 * | - (0.51，1.58) (1.01，8.83) | # 0.89 2.92 * | - (0.56，1.50) (1.18，10.12) |
| Be | EE ED DD | 23(44.2) 12(100.0) 0 | 29(55.8) 0(0.0) 0 | 9.15 ** 138.5 ** - | (7.64，16.33) (22.62，∝) - | 8.82 ** - - | (3.56，12.72) - - |
*P＜0.05,
*P＜0.0001; The # control group; § has adjusted factors such as sex, conceptional age, pregnant age, multiparity number of times, smoking, diabetes, BMI, education degree.EE represents homozygous wildtype; ED represents heterozygous; DD represents homozygous mutation type (95%CI:95% credibility interval).
Sequence table (SEQUENCE LISTING)
＜110〉Anhui Biological Medical Inst
＜120〉a kind of method of predicting pregnancy badness come-off generating risks
<130> JSP060090
<160> 6
<170> PatentIn version 3.1
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Claims (10)
1. method of predicting pregnancy badness come-off generating risks, predict the occurrence risk of pregnancy badness come-off from the genotype of the pleomorphism site E112D of proline carboxypeptidase gene in experimenter's the biological sample by mensuration: when genotype was the 112EE homozygous wildtype, the pregnancy badness come-off incidence was lower; When genotype was 112ED heterozygous or 112DD homozygous mutation type, the pregnancy badness come-off incidence was higher.
2. the method for claim 1 is characterized in that, described experimenter merges the gravid woman for chronic hypertension, and when its genotype was the 112EE homozygous wildtype, the pregnancy badness come-off incidence was higher than no hypertensive gravid woman; When its genotype was 112ED heterozygous or 112DD homozygous mutation type, the pregnancy badness come-off incidence was apparently higher than no hypertensive gravid woman.
3. the method for claim 1 is characterized in that, described biological sample is selected from: blood sample, humoral sample, tissue sample, organize sample secretion, fecal matter sample, cultured cell.
4. method as claimed in claim 3 is characterized in that, described blood sample is selected from: periphery whole blood, peripheral blood cells, leucocyte, serum; Described humoral sample is selected from: urine sample, saliva; Described tissue sample is selected from: oral mucosa examination, hair, skin, biopsy.
5. the method for claim 1 is characterized in that, described pregnancy badness come-off comprises threatened premature labor, premature labor, intrauterine growth retardation, LBW, pregnancy-hypertension syndrome and pre-eclampsia and eclampsia.
6. the method for claim 1, it is characterized in that the genotypic method of wherein measuring the pleomorphism site E112D of proline carboxypeptidase gene is selected from following difference foranalysis of nucleic acids technology: polymerase chain reaction, the PCR-restriction fragment length polymorphism is analyzed, PCR-alleles-specific oligonucleotide probe method, PCR-sequence specific oligonucleotides method, sequencing, PCR-sequence specific primers method, the PCR-fluorescence method, the PCR finger-printing method, oligonucleotides connects to be analyzed, the detection method of fluorescent energy resonance transfer, biochip, nucleic acid chip, mass-spectrometric technique, genescan, single-strand conformation polymorphism, denaturing gradient gel electrophoresis, enzyme or chemical mispairing patterning method, and Taqman biological detecting method.
7. the method for claim 1, it is characterized in that, by utilizing the E112D pleomorphism site genotype of proline carboxypeptidase gene in the polymorphism parting oligonucleotide detection of biological sample, wherein, described polymorphism parting oligonucleotide is: (1) allele-specific nucleic acid primer, the proline carboxypeptidase gene segment that is used to increase and contains described pleomorphism site, and/or (2) be used to detect the genotypic oligonucleotide probe of pleomorphism site of described proline carboxypeptidase gene, and it can be specifically and the nucleic acid hybridization that contains described proline carboxypeptidase gene polymorphism sites.
8. method as claimed in claim 7 is characterized in that, the length of described oligonucleotide probe is 15-50 nucleotide.
9. the method for claim 1 is characterized in that, the genotype of the pleomorphism site E112D of described proline carboxypeptidase gene is to exist the genotype of other pleomorphism site of linkage disequilibrium to determine with it by this pleomorphism site annex.
10. method as claimed in claim 9 is characterized in that, described other pleomorphism site comprises nonsense mutation site, missense mutation site and the pleomorphism site that is positioned at gene intron position, Gene regulation position.
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| CN 200610011838 CN101063678A (en) | 2006-04-30 | 2006-04-30 | Method for forecasting pregnancy badness come-off generating risks |
| PCT/CN2007/001491 WO2007128244A1 (en) | 2006-04-30 | 2007-04-30 | Method, oligonucleotide probe and test kit of predicting risk of negative pregnancy outcomes, and use thereof |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112331340A (en) * | 2020-10-14 | 2021-02-05 | 国家卫生健康委科学技术研究所 | Intelligent prediction method and system for pregnancy probability of pregnant couple |
| CN113396332A (en) * | 2018-09-21 | 2021-09-14 | 斯坦福大学托管董事会 | Method for evaluating pregnancy progression and preterm birth miscarriage for clinical intervention and uses thereof |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113396332A (en) * | 2018-09-21 | 2021-09-14 | 斯坦福大学托管董事会 | Method for evaluating pregnancy progression and preterm birth miscarriage for clinical intervention and uses thereof |
| CN112331340A (en) * | 2020-10-14 | 2021-02-05 | 国家卫生健康委科学技术研究所 | Intelligent prediction method and system for pregnancy probability of pregnant couple |
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