Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
  • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
  • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2310/00—Structure or type of the nucleic acid
  • C12N2310/10—Type of nucleic acid
  • C12N2310/14—Type of nucleic acid interfering N.A.
  • C12N2310/141—MicroRNAs, miRNAs
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2320/00—Applications; Uses
  • C12N2320/10—Applications; Uses in screening processes
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/118—Prognosis of disease development
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/158—Expression markers
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA

Definitions

• the present invention generally relates to the determination of cardiovascular risk in women after complicated pregnancy.
• the invention relates to molecular markers for the stratification of patients at risk of onset and development of cardiovascular and cerebrovascular diseases.
• the presence of aberrant expression profile of cardiovascular microRNAs in whole peripheral venous blood of the women after complicated pregnancy selects a risk population that may benefit from preventative measures to reduce or prevent the onset and development of cardiovascular and cerebrovascular diseases.
• Gestational hypertension GH
• preeclampsia PE
• FGR fetal growth restriction
• Fetal growth restriction is diagnosed if the fetus lags in growth with respect to the relevant gestational age (the estimated fetal weight is below the lOth percentile for the respective gestational period) and at the same time abnormal flow rates are present in the umbilical artery, uterine artery and/or signs of centralization of the fetal circulation are present (ACOG, 2013).
• late preeclampsia onset after 34th gestational week
• late FGR onset after 32nd gestational week
• secondary placental insufficiency due to dysfunction of the maternal cardiovascular system
• pregnancy-related complications are likely to be the first early manifestations of endothelial dysfunction, vascular and metabolic diseases (Kaaja et al., 2005), the American Heart Association recommends long-term follow-up of women with pregnancy-related complications such as preeclampsia, fetal growth restriction, gestational diabetes mellitus and/or preterm labor (A Guideline From the American Heart Association, 2011).
• Women who have been diagnosed with preeclampsia and/or eclampsia during pregnancy are at an increased risk of occurrence of hypertension, atherosclerosis, ischemic heart disease, myocardial infarction, stroke, deep venous thrombosis and metabolic syndrome (Bellamy et al., 2007; Haukkamaa et al., 2009; Lykke et al., 2009; Berks et al., 2013; Craici et al., 2008).
• This invention is based on an investigation of postpartal expression profile of microRNAs that play a role in the pathogenesis of cardiovascular and cerebrovascular diseases in women with prior occurence of pregnancy-related complications (gestational hypertension, preeclampsia and/or fetal growth restriction).
• microRNAs assayed within the framework of the present invention were miR-l-3p, miR-l6-5p, miR-l7-5p, miR-20a-5p, miR-20b-5p, miR-2l-5p, miR-23a-3p, miR-24-3p, miR-26a-5p, miR-29a-3p, miR-92a-3p, miR-l00-5p, miR-l03a-3p, miR-l25b-5p, miR-l26-3p, miR-l30b-3p, miR- l33a-3p, miR-l43-3p, miR-l45-5p, miR-l46a-5p, miR-l55-5p, miR-l8la-5p, miR-l95-5p, miR-l99a- 5p, miR-2lO-3p, miR-22l-3p, miR-342-3p, miR-499a-5p and miR-574-3p.
• MicroRNAs belong to a family of small non-coding RNA molecules of 18-25 nucleotides in length that regulate gene expression at the post-transcriptional level by degradation and/or blocking of the target rnRNA.
• a number of tissues affected by the pathological process have a characteristic microRNA expression profile that distinguishes the physiological tissue from the tissue affected by the pathology, which is successfully used in diagnostics.
• Recent studies have documented an aberrant expression profile of microRNAs in placental tissue, umbilical cord blood and maternal circulation in gestational hypertension, preeclampsia and fetal growth restriction.
• microRNAs in whole peripheral venous blood of women after a pregnancy-related complication (gestational hypertension, preeclampsia and/or fetal growth restriction) was described. We focused especially on those microRNAs that play a role in the pathogenesis of dyslipidemia, hypertension, vascular inflammation, insulin resistance and diabetes, atherosclerosis, angiogenesis, coronary arterial disease, myocardial infarction, heart failure, stroke, intracranial aneurysm, pulmonary arterial hypertension, and peripartal cardiomyopathy.
• Object of the present invention is to describe which microRNAs or their combination may be useful for screening and stratification of women with a history of pregnancy-related complications with regard to risk of cardiovascular diseases.
• a newly identified risk group of women based on the screening of microRNAs associated with cardiovascular and cerebrovascular diseases may benefit from a follow-up at out-patients departments of general practitioners and/or specialized physicians and from implementation of targeted preventive regimen measures aimed at reducing or preventing the onset and development of cardiovascular and cerebrovascular diseases.
• microRNAs show aberrant expression profile: miR-l-3p, miR-l7-5p, miR-20a-5p, miR-20b-5p, miR-29a-3p, miR-l00-5p, miR-l25b-5p, miR-l26-3p, miR-l30b-3p, miR-l33a-3p, miR-l43-3p, miR-l45-5p, miR-l46a-5p, miR-l8la-5p, miR-l99a-5p, miR-22l-3p, and miR-499a-5p.
• the present invention thus provides a method of predicting a risk of onset and development of cardiovascular and/or cerebrovascular disease for a woman with a history of pregnancy-related complications selected from gestational hypertension, preeclampsia and/or fetal growth restriction, said method comprising the steps of:
• said reference cut-off value is obtained by statistical analysis of samples from women with a history of pregnancy-related complications and samples from women without a history of pregnancy-related complications (i.e., women with a history of physiological (non- complicated) pregnancies);
• the risk of onset and development of cardiovascular or cerebrovascular disease means a higher risk of onset and development of cardiovascular disease for the tested woman than the risk of onset and development of cardiovascular or cerebrovascular disease for a healthy woman with a history of physiological pregnancy.
• the women in risk of onset and development of cardiovascular or cerebrovascular disease are subjected to preventative measures such as diet adjustment, appropriate physical activity, and/or pharmacological intervention (administration of cardiovascular or cerebrovascular preventative medication).
• preventative measures such as diet adjustment, appropriate physical activity, and/or pharmacological intervention (administration of cardiovascular or cerebrovascular preventative medication).
• the term garman with a history of pregnancy-related complications means a woman which had at least one pregnancy complicated by gestational hypertension, preeclampsia and/or fetal growth restriction.
• Healthy woman with a history of physiological (normal) pregnancy means a woman which had at least one pregnancy but the pregnancy was normal and not complicated by any of gestational hypertension, preeclampsia and/or fetal growth restriction.
• Normal pregnancies are defined as those without medical, obstetrical, or surgical complications, who subsequently delivered full term, singleton healthy infants weighing > 2500 g after 37 completed weeks of gestation.
• cardiovascular and/or cerebrovascular diseases include, in particular, dyslipidemia, hypertension, vascular inflammation, insulin resistance and diabetes, atherosclerosis, angiogenesis, coronary artery disease, myocardial infarction, heart failure, stroke, intracranial aneurysm, pulmonary arterial hypertension, and peripartum cardiomyopathy.
• MicroRNA marker is an individual microRNA. Combination of microRNAs is a combination of individual microRNAs. The statistical analysis assesses each microRNA separately in case of individual microRNA markers. In case of combination of microRNAs, individual microRNA markers may be assessed in one combined statistical test. When the combination of microRNAs is assayed, the expressions of individual microRNAs contained in the combination are determined, and the combination of their expressions is assessed in the statistical analysis with one cut-off value. To select the optimal combinations of microRNA biomarkers, logistic regression was used (MedCalc Software bvba, Ostend, Belgium) by the inventors. The logistic regression procedure allows to analyse the relationship between one dichotomous dependent variable and one or more independent variables.
• Determination of expression of microRNA markers can be performed using commercially available kits from various manufacturers, the procedure is described in manufacturer's instructions.
• microRNA markers is preferably normalized using a geometric mean of two selected endogenous controls (RNU58A and RNU38B).
• Pregnancy-related complications (gestational hypertension, preeclampsia and/or fetal growth restriction) induce epigenetic changes that are usually associated with the occurrence of cardiovascular and cerebrovascular diseases.
• These epigenetic changes can be detected in whole peripheral venous blood in women (mothers) in the long term (3-11 years after delivery).
• Postpartal screening of individual microRNAs associated with the occurrence of cardiovascular and cerebrovascular diseases and/or combinations of such microRNAs identifies women at increased cardiovascular risk.
• the group of mothers with an aberrant cardiovascular microRNA expression profile in whole peripheral venous blood represents a newly identified risk group of patients who may benefit from preventive measures to reduce or prevent the onset and development of cardiovascular and cerebrovascular diseases.
• Gestational hypertension leads to the induction of upregulation of several microRNAs that are associated with the occurrence of cardiovascular and cerebrovascular diseases (miR-20a-5p, miR-l43- 3p, miR-l46a-5p, miR-l8la-5p, miR-l99a-5p, miR-22l-3p, and miR-499a-5p).
• Severe preeclampsia also induces upregulation of miR-l30b-3p (10.39%) and upregulation of miR-l33a-3p (14.29%) in a proportion of women.
• miR-l33a-3p is also upregulated in 16.67% of women with early form of preeclampsia requiring termination of pregnancy before the 34th gestational week.
• Postpartal levels of some cardiovascular microRNAs detected in whole peripheral venous blood of women (mothers) also correlate with the severity of preeclampsia and/or FGR as assessed by Doppler ultrasound examination during pregnancy-related complications.
• RNA is isolated from homogenized cell lysates using the mirVana microRNA Isolation Kit (Ambion, Austin, USA, no: AM1560), followed by the process of enrichment of RNA for small RNA molecules (siRNAs, microRNAs) according to the manufacturer's recommendations.
• RNA was DNA-free.
• the ratio of absorbance values A(260/280) is optimally in the range of 1.8 - 2.0 (the obtained RNA is DNA-free).
• the ratio of absorbance values A(260/230) should be more than 1.6 (the RNA obtained is polysaccharide-free).
• Each microRNA is reverse transcribed into complementary DNA (cDNA) using a TaqMan MicroRNA Assay that contains a microRNA-specific stem-loop RT primer and a reverse transcription kit (TaqMan MicroRNA Reverse Transcription Kit, Applied Biosystems, Branchburg, USA, no: 4366597) in a total reaction volume of 10 pL as recommended by the manufacturer.
• Reverse transcription is performed on a cycler (7500 Real-Time PCR system, Applied Biosystems, Branchburg, USA) under the following conditions: 30 minutes at 16 °C, 30 minutes at 42 °C, 5 minutes at 85 °C and 4 °C without limitations.
• Relative quantification of microRNA using real-time PCR 3 pL cDNA are added to the reaction mixture containing specific primers and a TaqMan MGB probe (TaqMan MicroRNA Assay, Applied Biosystems, Branchburg, USA) and master mix ingredients (TaqMan Universal PCR Master Mix, Applied Biosystems, Branchburg, USA, no: 4318157) at total reaction volume 15 pL.
• Detection and quantification of the respective microRNA is performed on a cycler (7500 Real-Time PCR system, Applied Biosystems, Branchburg, USA) under standard conditions according to the manufacturer's recommendations. Detection is always performed in duplicates.
• NTC water for PCR applications instead of cDNA template
• NAC untranscribed RNA samples
• genomic DNA DNA isolated from equivalent samples
• RNA enriched for small RNAs isolated from the fetal part of one randomly selected placenta from normal pregnancy was used as a reference sample for relative quantification across assays.
• MicroRNA gene expression was compared between individual groups (more than two compared groups) by Kniskai -Wallis test followed by post-hoc analysis. The level of statistical significance was determined at a p-value of p ⁇ 0.05.
• Receivers operating characteristic (ROC) curves were constructed for the appropriate microRNAs associated with cardiovascular and cerebrovascular diseases within the framework of the postpartal screening in women with a history of pregnancy-related complications.
• the area under the curve, the sensitivity and specificity of individual microRNAs, and the optimal cut-off value were evaluated.
• the sensitivity of a given microRNA biomarker was determined at 90.0% specificity (information on the percentage of women having increased expression of a particular microRNA at 10.0% false positivity) (MedCalc Software bvba, Ostend, Belgium).
• combined statistical analysis logistic regression and ROC analysis was performed to select the optimal combination of microRNA biomarkers for a given situation. This application provides the following parameters: area under the curve, sensitivity, specificity, optimal cut-off value, sensitivity of the given combination of microRNA biomarkers at 90.0% specificity.
• This analysis identifies women who have due to a history of pregnancy-related complications an increased risk of onset and development of cardiovascular and cerebrovascular diseases. Based on the results of individual ROC analyses and combined ROC analyses, mothers after complicated pregnancy were differentiated into those without risk of onset and development of cardiovascular and cerebrovascular diseases and those with risk of onset and development of cardiovascular and cerebrovascular diseases.
• results of the experimental data are presented as box plot charts (Statistica software, version 9.0; StatSoft, Inc., USA).
• Each box plot chart shows a median of a logarithmic value of the normalized gene expression of the respective microRNA.
• the upper and lower limits of the boxes indicate the 75th and 25th percentiles, respectively.
• the upper and lower whiskers indicate maximum and minimum values that are no more than 1.5 times the span of the interquartile range (range of the values between the 25th and the 75th percentiles). Circles indicate outliers and asterisks indicate extreme values.
• MicroRNAs associated with cardiovascular and cerebrovascular diseases are dysregulated postpartally in whole peripheral venous blood of women (mothers) with a history of pregnancy-related complications
• microRNAs associated with cardiovascular and cerebrovascular diseases were studied postpartally in whole peripheral venous blood in women (mothers), 3 to 11 years after delivery. MicroRNA gene expression was compared between women with a history of pregnancy-related complications (gestational hypertension, preeclampsia and/or fetal growth restriction) and women with a history of normal pregnancy. Postpartal gene expression of microRNAs was also analyzed with regard to the severity of pregnancy-related complications according to clinical symptomatology (mild vs. severe preeclampsia, absence vs. presence of anhydramnion/oligohydramnion in fetus, absence vs.
• microRNA The percentage of women who are overexpressing a particular microRNA at 10.0% false positivity is also shown. Overexpression of individual microRNAs is observed in 8.0% - 18.0% of women with a history of gestational hypertension depending on the respective microRNA [miR-20a-5p (8.0%), miR- l43-3p (8.0%), miR-l46a-5p (12.0%), miR-l8la-5p (18.0%), miR-l99a-5p (12.0%), miR-22l-3p (14.0%) and miR-499a-5p (8.0%)].
• This analysis identifies women who have an increased risk of onset and development of cardiovascular and cerebrovascular diseases following pregnancy-related complications.
• AUC 0.626, p 0.018, 11.11% sensitivity
• Postpartal screening of a combination of microRNAs associated with cardiovascular and cerebrovascular diseases is more accurate in women with a history of gestational hypertension than screening of individual upregulated microRNA biomarkers, as it identifies more women at increased risk of the onset and development of cardiovascular and cerebrovascular diseases than individual microRNA biomarkers
• Postpartal screening based on a combination of 7 microRNAs with aberrant expression profile in women with a history of gestational hypertension showed a higher accuracy (AUC 0.680, p ⁇ 0.00l, 68.75% sensitivity, 62.92% specificity, cut off value >0.320869) than single microRNA biomarkers upregulated in women with a history of gestational hypertension.
• Postpartal screening based on the combination of miR-20a-5p, miR-l43-3p, miR-l46a-5p, miR-l8la-5p, miR-l99a-5p, miR-22l-3p and miR-499a-5p identified at 10.0% false positivity 29.17% of women with a history of gestational hypertension with an increased cardiovascular risk.
• Postpartal combined screening of all 5 microRNA biomarkers upregulated in women with a history of gestational hypertension and/or preeclampsia was also more accurate in identifying women with a history of gestational hypertension at increased cardiovascular risk (AUC 0.711, p ⁇ 0.00l, 58.33% sensitivity, 82.02% specificity, cut off value >0.365924) than individual microRNA biomarkers upregulated in women with a history of gestational hypertension and/or preeclampsia.
• Postpartal screening based on the combination of miR-l- 3p, miR-l7-5p, miR-20b-5p, miR-29a-3p and miR-l26-3p identified 33.33% of women with a history of gestational hypertension and with increased cardiovascular risk, at 10.0% false positivity.
• Postpartal screening of a combination of microRNAs associated with cardiovascular and cerebrovascular diseases is more accurate in women with a history of preeclampsia, regardless of the severity of the disease and gestational age of termination of pregnancy, than postpartal screening of individual upregulated microRNA biomarkers, because it identifies more women at increased risk of the onset and development of cardiovascular diseases than individual microRNA biomarkers
• Postpartal screening based on the combination of miR-l7-5p, miR-29a-3p and miR-l33a-3p was much more accurate in identifying women with a history of preeclampsia, regardless of disease severity and gestational age of termination of pregnancy, at increased cardiovascular risk than screening of individual microRNA biomarkers (miR-l7-5p, miR-29a-3p and miR-l33a-3p).
• Postpartal screening based on the combination of miR-l7-5p, miR-29a-3p and miR-l33a-3p identified 18.81% of women with a history of preeclampsia regardless of the severity of the disease and gestational age of termination of pregnancy at increased cardiovascular risk, at 10.0% false positivity.
• Postpartal screening of the combination of microRNAs associated with cardiovascular and cerebrovascular diseases is more accurate in women with a history of severe preeclampsia than postpartal screening of individual microRNA biomarkers, as it identifies more women at an increased risk of the onset and development of cardiovascular and cerebrovascular diseases than individual microRNA biomarkers
• Postpartal combined screening showed a greater accuracy in identifying women with a history of early form of preeclampsia at increased cardiovascular risk than individual microRNA biomarkers (miR-l-3p, miR-l7-5p and miR-l33a-3p).
• a single microRNA biomarker (miR-29a-3p) postpartal screening is more accurate in women with a history of late preeclampsia than a postpartal screening of a combination of microRNA biomarkers associated with cardiovascular and cerebrovascular diseases, as it identifies more women at increased risk of the onset and development of cardiovascular and cerebrovascular diseases than the microRNA biomarkers combination
• a single microRNA biomarker (miR-125b-5p) postpartal screening is more accurate in women with a history of preeclampsia and/or FGR with abnormal flow rates in the uterine artery during previous pregnancy than a postpartal screening of a combination of microRNAs associated with cardiovascular and cerebrovascular disease, as it identifies more women at the risk of the onset and development of cardiovascular and cerebrovascular diseases than a combination of microRNA biomarkers
• Postpartal combination screening based on the combination of miR-l33a-3p and miR-l45-5p, which showed the highest accuracy out of the various combinations of microRNA biomarkers, identified at 10.0% false positivity 14.29% of women with increased cardiovascular risk in patients with a history of preeclampsia and/or FGR with increased pulsatility index in the uterine artery in previous pregnancy.
• MiR-l-3p is generated from miR-l-l and miR-l-2 precursors whose genes are located on chromosomes 20ql3.3 and 18ql 1.2.
• MiR-l is highly expressed in heart muscle, especially in the myocardium, and in skeletal muscles. Circulating miR-1 levels are significantly elevated in acute myocardial infarction and correlate with circulating troponin T levels, a marker of cardiac damage.
• MiR-1 is a potential target of therapeutic intervention in cardiovascular diseases, cardiac ischemia and complications following myocardial infarction.
• Inhibition of miR-1 by oligonucleotides is cardioprotective, as it leads to a reduction in apoptosis, an increase in resistance to oxidative stress and a reduction in spontaneous arrhythmias.
• oligonucleotides have a history of gestational hypertension (16.0%) or early preeclampsia (11.11%) in their previous pregnancies, have an increased cardiovascular risk and should be monitored long-term.
• MiR-17-5p a member of the miR-17-92 cluster, has a gene located on chromosome 13q31, is highly expressed in endothelial cells and poorly expressed in vascular smooth muscle cells.
• MicroRNAs derived from the miR-17p-92 cluster play a critical role in heart development, as the hearts of miR- 17p-92 deficient mutant experimental mouse embryos had a ventricular septal defect.
• ischemic-reperfusion heart damage I/R- I. Upregulation of miR-17-5p promotes oxidative stress induced apoptosis in an in vivo I/R-I mouse model through regulation of Stat3 expression.
• miR-17-5p expression led to inhibition of cell death and induced cell survival in both in vivo I/R-I mouse model and in vitro cellular model of oxidative stress, and improved heart function following acute myocardial infarction by leading to suppression of endothelial cell apoptosis.
• MiR-17-5p expression is also activated during ischemia-reperfusion kidney damage in mice.
• circulating miR-17-5p represents one of major potential upregulated biomarkers of diffuse myocardial fibrosis in patients with hypertrophic cardiomyopathy, in patients with acute ischemic stroke, and it is also a biomarker of the severity of coronary atherosclerosis in patients with coronary artery disease.
• miR-17-5p is upregulated in a proportion of women with a history of gestational hypertension (20.0%), preeclampsia regardless of disease severity and gestational age of pregnancy termination (19.8%), severe preeclampsia (19.48%), early preeclampsia (16.67%) and late preeclampsia (21.54%) in previous pregnancy.
• miR-17-5p upregulation is associated with a higher cardiovascular risk.
• MiR-20a-5p also belongs to the miR-l7 family and is transcribed from the miR- 17-92 cluster.
• MiR- 20a plays a role in the inflammatory response in pulmonary hypertension.
• Intraperitoneal administration of antagomiR-20a significantly reduced miR-20a-5p expression and restored BMPR2 function in pulmonary arteries in an experimental mouse model of hypoxia-induced pulmonary hypertension.
• Previous studies have also shown increased plasma levels of miR-l7-5p and miR-20a-5p in patients with GDM between 16-19 gestational weeks. Our data support the role of miR-20a-5p in the pathogenesis of pathological conditions increasing the cardiovascular risk.
• MiR-20b-5p also belongs to the miR-17 family, but is transcribed from the miR-l06a-363 cluster. Recent studies have confirmed that increased plasma levels of miR-20b may represent a possible biomarker to monitor the efficacy of therapy and disease progression in hypertension-induced heart failure in an experimental animal model. In humans, elevated levels of miR-20b-5p were then detected in the serum of mothers with SGA fetuses during the second trimester of pregnancy. In our study, miR- 20b-5p represented a microRNA biomarker that is upregulated in a proportion of women with a history of gestational hypertension (14.0%), severe preeclampsia (18.18%), and late preeclampsia (18.46%) in previous pregnancy. We conclude that this group of women is at risk of an onset and development of cardiovascular disease and would benefit from preventive programs aimed at reducing cardiovascular risk.
• MiR-29a-3p a member of the miR-29 family, has a gene located on chromosome 7q32.3.
• Administration of antagomiR-29a significantly increased Mcl-2 protein expression, thereby significantly reduced the magnitude of myocardial infarction and apoptosis in an experimental model of ischemic -reperfusion injury to the heart.
• Upregulation of miR-29a-3p was also observed in cardiac cachexia, a frequent complication of heart failure, also in an experimental model.
• overexpression of mi-29a-3p was found in heart biopsy, which was associated with decreased expression of CACNA1C protein.
• Circulating miR-29a-3p also represents one of the potential upregulated biomarkers in diffuse myocardial fibrosis in patients with hypertrophic cardiomyopathy.
• Previous studies have shown increased plasma levels of miR-29a-3p in patients with GDM between 16-19 gestational weeks. Elevated serum levels of miR-29a-3p have also been observed in patients with a recent diagnosis of type 2 DM.
• MiR-100-5p is part of the MIR100HG cluster located on chromosome 11.
• MiR-lOO is upregulated in a failing heart, as well as in patients with idiopathic dilated cardiomyopathy and ischemic cardiomyopathy. Overexpression of miR-lOO results in suppression of the expression of genes associated with hypertension.
• Upregulation of miR-l00-5p reduces the levels of the NOX4 (NADPH oxidase 4) transcript, an oxidoreductase that catalyzes the conversion of oxygen to reactive oxygen species in dermal microvascular endothelial cells, and reduces H2O2 production.
• NOX4 NADPH oxidase 4
• Our study demonstrated upregulation of miR-l00-5p in a proportion of women with a history of preeclampsia and/or FGR with abnormal flow rates in the arteria cerebri media during previous pregnancy (10.81%), indicating poor perinatal prognosis. For these reasons, we conclude that the compensatory effect of miR-l00-5p occurs in this group of women to normalize cardiovascular function. Flowever, we consider the group of women with aberrant postpartal miR-l00-5p expression profile to be at cardiovascular risk. This group of women should be followed up and monitored in the long term.
• miR-l25b-l on chromosome l lq24.l and miR-125h-2 on chromosome 21 q21.1, both producing miR-125b-5p.
• miR-l25b-5p upregulated circulating microRNAs, including miR-l25b-5p, are associated with acute ischemic stroke and acute myocardial infarction. Upregulation of miR-l25b- 5p protects endothelial cells from apoptosis caused by oxidative stress due to negative regulation of SMAD4 (SMAD family member 4). Furthermore, miR-l25b-5p acts as a protector against ischemia- induced apoptosis of cardiomyocytes.
• Cardiomyocytes with upregulated miR-l25b-5p expression survive longer and protect the heart from acute myocardial infarction by repressing proapoptotic genes bakl and klf 13.
• Our study demonstrated upregulation of miR-l25b-5p in a proportion of women with a history of preeclampsia and/or FGR with abnormal flow rates in the arteria cerebri media and in uterine artery (increased resistance and presence of unilateral and/or bilateral notch).
• the increased uterine artery pulsatility index generally reflects a defect in maternal spiral arteries in placenta in pregnancies complicated by hypertension and fetal growth restriction.
• miR-l25b-5p may be a long-term consequence of severe pregnancy-related complications to protect endothelial cells and cardiomyocytes from apoptosis.
• postpartal upregulation of miR-l25b-5p in the circulation of women as a risk factor indicative of increased cardiovascular risk.
• MiR-l26 producing miR-126-3p, is encoded by a gene located in intron 7 of the EGFL7 (epidermal growth factor-like protein 7) gene on chromosome 9q34.3.
• MiR-l26 regulates endothelial expression of vascular cell adhesion molecule 1 (VCAM-l) and controls vascular inflammation. Upregulation of miR-l26 decreases VCAM-l expression and conversely, transfection of endothelial cells with miR-l26 antagomirs increases TNFa-stimulated VCAM-l expression.
• MiR-l26-3p is significantly downregulated in the serum of patients with acute myocardial infarction and in plasma of patients with type 2 DM.
• the MiR-l30b gene is a member of the miR-130 family located on chromosome 22ql l.2l. Circulating levels of miR-130b-3p reflect the level of obesity and can therefore be used in monitoring hypertriglyceridemia and metabolic syndrome. Elevated levels of circulating miR-l30b-3p were also observed in patients with type 2 DM and in patients with intracranial aneurysm. MiR-l30b is also abundantly expressed in large and small arteries in patients after ischemic stroke.
• MiR-133a-3p belongs to the miR-l33 family and is transcribed from a multiple copy gene on chromosome 18 (miR-l33a-l) and chromosome 20 (miR-l33a-2).
• MiR-l33 is expressed in cardiomyocytes and skeletal muscles and has an anti-apoptotic effect by suppressing expression of caspase 9.
• MiR-l33 also plays an important role in the development of the heart by influencing the expression of HCN2 and HCN4 genes.
• MiR-l33 is downregulated in heart hypertrophy, heart failure, and downregulation of miR-l33 also contributes to arrhythmogenesis in heart hypertrophy and heart failure.
• miR-l33 overexpression reduces cardiac hypertrophy and leads to correction of abnormalities in the transferring cardiac system. Furthermore, circulating miR-l33a-3p is one of the potential biomarkers of diffuse myocardial fibrosis in patients with hypertrophic cardiomyopathy and coronary artery calcification. Our study demonstrated upregulation of miR-l33a-3p in a proportion of women with a history of preeclampsia regardless of disease severity and gestational age of pregnancy termination (13.86%), severe preeclampsia (14.29%) and early preeclampsia (16.67%).
• MiR-143-3p and miR-145-5p have genes located on chromosome 5q33 approximately 1.3 kb apart. Both microRNAs are downregulated in most tumors and act as tumor suppressors.
• the target genes for miR-l43 are ERKS and KRAS, and for miR-l45 the c-myc and IRS-l genes.
• the miR- 143/145 cluster plays a crucial role in the differentiation of vascular smooth muscle cells (VSMCs) by regulating the expression of a network of transcription factors including Klf4, myocardine and Elk-l.
• miR-l43/l45 cluster expression is reduced under conditions associated with acute and chronic vascular stress such as aortic aneurysm and coronary arterial disease
• upregulation of miR-l43-3p and miR-l45-5p has been observed in VSMC pulmonary arteries and VSMC -derived exosomes and in lung tissue in patients with idiopathic and hereditary pulmonary hypertension.
• miR-143/miR- 145 overexpression was found in smooth muscle cells in the saphenous vein in patients with type 2 DM and in atherosclerotic plaques in patients with hypertension. Circulating miR-l43-3p was also validated as one of the upregulated biomarkers associated with acute ischemic stroke.
• MiR-l46 family contains 2 members with an almost identical sequence, miR-146a-5p and miR-14(Sh- 5p.
• the MIR146A gene is located within the gene encoding the long non-coding RNA, MIR3142HG, on chromosome 5q33.3.
• MiR-l46a-5p is actively involved in a variety of cancer processes such as immunosuppression, metastasis and angiogenesis.
• MiR-l46a-5p is an anti-inflammatory microRNA, negatively regulates inflammation by targeting the IRAK1 and TRAF6 genes, resulting in inhibition of NF-KB activation.
• miR-l46a is one of the microRNAs highly sensitive to hypoxia. Upregulation of miR-l46a appears to protect the myocardium from damage.
• MiR-l8la and miR-l8lb have genes located on chromosomes lq32.l and 9q33.3.
• MiR-l8la forms several mature microRNAs: miR-181a-5p, miR-l8la-3p and miR-l8la2-3p.
• the MiR-l8l family plays a central role in both acute and chronic inflammatory diseases such as atherosclerosis, DM type 2 and obesity.
• the decreased expression of miR-l8la observed in monocytes of obese patients was associated with metabolic syndrome and coronary arterial disease.
• MiR-199a-5p is encoded by 2 genes located on chromosomes lq24.3 and 19r13.2.
• MiR-l99a is essential for maintaining cardiomyocyte size and plays a role in the regulation of cardiac hypertrophy. Upregulation of miR-l99a in cardiomyocytes increases their size, and conversely, silencing the endogenous expression of miR-l99a decreases their size. MiR-l99a is upregulated 10-fold in hypertrophic heart in rats after abdominal aorta constriction even after 12 weeks. Upregulation of miR- l99a was also observed in a model of late post-infarction heart failure in rats. Furthermore, miR-l99a is very sensitive to hypoxia.
• MiR-l99a directly targets and inhibits the translation of HIF-la mRNA during normoxia, and downregulation of miR-l99a is required for upregulation of HIF-la during hypoxia.
• upregulation of miR-l99a-5p was found in a proportion of women with a history of gestational hypertension in previous pregnancy (12.0%). We conclude that the upregulation of miR- l99a-5p has a protective role rather than a deleterious effect in order to induce normoxia.
• MiR-22l/222 cluster containing genes for miR-221 and miR-222 located on Xpl l chromosome, is involved in the regulation of vascular smooth muscle and endothelial cells and myocardial function.
• MiR-221/222 expression is positively regulated by angiotensin II and contributes to several serious pathological conditions of the heart including myotonic dystrophy type 2 and hypertrophic cardiomyopathy.
• vascular expression of miR-221/222 is upregulated in the initial phase of atherosclerosis, in later stages the chronic inflammation results in downregulation of miR-221/222 in endothelial cells.
• Circulating miR-221 and/or miR-221-3p is one of the new diagnostic biomarkers of hypertension, obesity, metabolic syndrome, coronary arterial disease, carotid atherosclerosis, and acute myocardial infarction.
• Our study demonstrated the presence of upregulation of miR-22 l-3p in a proportion of women with a history of gestational hypertension in previous pregnancy (14.0%). We conclude that this group of women is at risk of developing cardiovascular disease in later stages of life and requires early implementation of prevention programs to reduce cardiovascular risk.
• MiR-499a-5p encoded by the miR-499a gene located on chromosome 20ql l.22, is strongly expressed in the heart under physiological conditions.
• MiR-499 is involved in inhibiting cardiomyocyte apoptosis by suppressing calcineurin-mediated dephosphorylation of the Drpl protein. It has also been reported that p53 downregulates the expression of miR-499.
• MiR-499 is also associated significantly with myocardial infarction, and together with miR-l33a and miR-208a, they represent potential early diagnostic biomarkers of myocardial infarction.
• miR-499 is significantly involved in the inflammatory signaling pathways of bronchial asthma.
• Fig. 1 Analysis results for microRNA markers combination according to Example 1
• Fig. 2 Analysis results for miR-20a-5p according to Example 1
• Fig. 14 Analysis results for miR-l26-3p according to Example 2
• Fig. 15 Analysis results for microRNA markers combination according to Example 3
• Fig. 25 Analysis results for miR-l33a-3p according to Example 4.
• Fig. 28 Analysis results for miR-l7-5p according to Example 5
• Fig. 30 Analysis results for miR-29a-3p according to Example 6
• Fig. 31 Analysis results for microRNA markers combination according to Example 6
• Fig. 32 Analysis results for miR-l7-5p according to Example 6
• Fig. 33 Analysis results for miR-20b-5p according to Example 6
• Fig. 34 Analysis results for miR-l25b-5p according to Example 7 (abnormal PI in arteria uterine)
• Fig. 35 Analysis results for microRNA markers combination according to Example 7
• Fig. 36 Analysis results for miR-l33a-3p according to Example 7 (abnormal PI in arteria uterine)
• Fig. 37 Analysis results for miR-l45-5p according to Example 7
• Fig. 38 Analysis results for miR-l25b-5p according to Example 7 (presence of unilateral and/or bilateral diastolic notch)
• Fig. 40 Analysis results for miR-l33a-3p according to Example 7 (presence of unilateral and/or bilateral diastolic notch)
• Fig. 41 Analysis results for miR-l25b-5p according to Example 8.
• Fig. 42 Analysis results for miR-l00-5p according to Example 9 Examples of carrying out the invention
• Example 1 Postpartal combined screening of miR-20a-5p, miR-143-3p, miR-146a-5p, miR-181a- 5p, miR-199a-5p, miR-221-3p and miR-499a-5p identifies a group of women with a history of gestational hypertension, who have an increased cardiovascular risk
• Postpartal screening based on a combination of 7 microRNAs with aberrant expression profile in women with a history of gestational hypertension showed a higher accuracy (AUC 0.680, p ⁇ 0.00l, 68.75% sensitivity, 62.92% specificity, cut off value >0.320869) than individual unique microRNA biomarkers upregulated in women with a history of gestational hypertension.
• Example 2 Postpartal combined screening of miR-l-3p, miR-17-5p, miR-20b-5p, miR-29a-3p and miR-126-3p identifies a group of women with a history of gestational hypertension, who have an increased cardiovascular risk
• Postpartal combined screening of 5 microRNA biomarkers upregulated in women with a history of gestational hypertension and/or preeclampsia was more accurate in identifying women with a history of gestational hypertension at increased cardiovascular risk (AUC 0.711, p ⁇ 0.00l, 58.33% sensitivity, 82.02% specificity, cut off value >0.365924) than individual microRNA biomarkers upregulated in women with a history of gestational hypertension and/or preeclampsia.
• Example 3 Postpartal combination screening of miR-17-5p, miR-29a-3p and miR-133a-3p identifies a group of women with a history of preeclampsia, regardless of the severity of the disease and gestational age of termination of pregnancy, who are at increased cardiovascular risk
• Postpartal screening based on a combination of miR-l7-5p, miR-29a-3p and miR-l33a-3p was much more accurate in identifying women with a history of preeclampsia, regardless of disease severity and gestational age of termination of pregnancy, at increased cardiovascular risk than screening of individual microRNA biomarkers upregulated in women with a history of preeclampsia regardless of disease severity and gestational age of termination of pregnancy (miR-l7-5p, miR-29a-3p and miR- l33a-3p).
• Postpartal screening based on a combination of miR-l7-5p, miR-29a-3p and miR-l33a-3p identified 18.81% of women with a history of preeclampsia regardless of the severity of the disease and gestational age of termination of pregnancy at increased cardiovascular risk, at 10.0% false positivity.
• Example 4 Postpartal combination screening of miR-17-5p, miR-20b-5p, miR-29a-3p, miR-126- 3p and miR-133a-3p identifies a group of women with a history of severe preeclampsia who are at increased cardiovascular risk
• Example 5 Postpartal combination screening of miR-l-3p, miR-17-5p, and miR-133a-3p identifies a group of women with a history of early preeclampsia who are at increased cardiovascular risk
• Example 7 Postpartal screening of miR-125b-5p identifies a group of women with a history of preeclampsia and/or FGR with abnormal flow rates in the uterine artery during a previous pregnancy who have an increased cardiovascular risk
• Postpartal combination screening based on the combination of miR-l33a-3p and miR-l45-5p, which showed the highest accuracy out of the various combinations of microRNA biomarkers, identified at 10.0% false positivity 14.29% of women with increased cardiovascular risk in the whole group of patients with a history of preeclampsia and/or FGR with increased pulsatility index in the uterine artery in previous pregnancy.
• Table 38 Analysis results for miR-125b-5p according to Example 7 (presence of unilateral and/or bilateral diastolic notch)
• Table 40 Analysis results for miR-133a-3p according to Example 7 (presence of unilateral and/or bilateral diastolic notch)
• Example 8 Postpartal screening of miR-125b-5p identifies a group of women with a history of preeclampsia and/or FGR with abnormal flow rates in the arteria cerebri media in previous pregnancy who have an increased cardiovascular risk
• Example 9 Postpartal screening of miR-100-5p identifies a group of women with a history of preeclampsia and/or FGR with abnormal values of cerebroplacental ratio in a previous pregnancy who have an increased cardiovascular risk
• Facca TA Mastroianni-Kirsztajn G, Sabino ARP, Passos MT, Dos Santos LF, Fama EAB,

Landscapes

• Chemical & Material Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Health & Medical Sciences (AREA)
• Organic Chemistry (AREA)
• Engineering & Computer Science (AREA)
• Genetics & Genomics (AREA)
• Zoology (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Wood Science & Technology (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Analytical Chemistry (AREA)
• General Engineering & Computer Science (AREA)
• Biotechnology (AREA)
• Molecular Biology (AREA)
• Biophysics (AREA)
• Physics & Mathematics (AREA)
• Microbiology (AREA)
• Biochemistry (AREA)
• General Health & Medical Sciences (AREA)
• Immunology (AREA)
• Biomedical Technology (AREA)
• Pathology (AREA)
• Plant Pathology (AREA)
• Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Applications Claiming Priority (2)

Publications (1)

Family

ID=69191784

Family Applications (1)

Country Status (2)

Cited By (3)

Families Citing this family (1)

Citations (3)

Family Cites Families (1)

• 2018
  • 2018-10-31 CZ CZ2018-597A patent/CZ308178B6/cs unknown
• 2019
  • 2019-10-30 WO PCT/CZ2019/050051 patent/WO2020088704A1/en active Application Filing

Patent Citations (3)

Non-Patent Citations (27)

Also Published As

Similar Documents

Legal Events