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    • 专利摘要:
    The present invention provides a method for diagnosing disorders caused by fetal alcoholization, said method comprising assaying PLGF.
    公开号:FR3037657A1
    申请号:FR1555727
    申请日:2015-06-22
    公开日:2016-12-23
    发明作者:Bruno Jose Gonzales；Stephane Marret；Matthieu Jean Alexandre Lecuyer；Annie Laquerriere；Soumeya Bekri；Celine Lesueur；Sylvie Marguerite Alberte Jegou；Pascale Yvonne Josephine Marcorelles
    申请人:Univ Rouen Centre Hospitalier；Universite de Rouen；Institut National de la Sante et de la Recherche Medicale INSERM；Centre Hospitalier Universitaire de Rouen；
    IPC主号:
    专利说明:

    [0001] 1 INTRODUCTION Alcohol is a physical and behavioral teratogen. In humans, prenatal exposure to alcohol can lead to alterations in brain development. Thus, the consumption of alcohol during pregnancy (fetal alcoholization) is the leading cause of disability and in particular mental retardation of non-genetic origin in the world but also in France. The damage varies according to the period in which the fetus has been exposed, blood alcohol levels, genetic and environmental factors, the mode of consumption (chronic, binge drinking).
    [0002] Fetal Alcohol Syndrome (FAS) is the most extreme and disabling manifestation of fetal alcohol spectrum disorder (FASD). It combines physical abnormalities such as hypotrophy (growth retardation), cranio-facial dysmorphism and neurobehavioral abnormalities resulting in cognitive function disorders (attention deficit, motor skills, learning disorders, lock). The diagnosis of SAF children is relatively easy. On the basis of morphological abnormalities, it can be established in utero or at birth. In contrast, many FASD children do not have the morphological abnormalities of FAS children, which compromises early diagnosis. However, these children are not devoid of attacks. These disabilities / disabilities will be detected in the first years of life (hyperactivity, disorders of attention) while precious months of care could have been valued in the first year of life. These deficits are longer term associated with social, occupational and family inaptitudes. The future of these children and their professional integration are therefore seriously mortgaged. A diagnosis from birth would allow early management of these children essential to minimize the disabilities associated with fetal alcohol. To date, significant efforts have been made to identify fetal alcohol exposure biomarkers, that is, markers to answer the question: has the child been exposed to fetal alcohol exposure alcohol during his fetal life However, this important information is not able to improve on its own the care of infants and for several reasons. First of all, there is no threshold for alcohol toxicity. In other words, a proven exposure will not necessarily be associated with developmental disorders of the child. In return, an episodic exposure at a key moment in neurodevelopment will not be without consequences, and the concept of a window of vulnerability is now clearly admitted. In addition, consumption patterns have changed significantly. In teenagers, episodic consumption, such as week-end drunkenness, is clearly increasing in both girls and boys. Finally, since the biomarkers of exposure developed until now are most often targeted at chronic exposure, there is a real risk of false negatives. There is therefore a need to develop biomarkers that track the effects of alcohol abuse in utero. DESCRIPTION The present invention provides the opportunity to develop a placental biomarker for cerebral involvement of fetal alcohol. This type of biomarker has never been developed to date. Indeed, current biomarkers of fetal alcoholization are so-called exposure biomarkers that determine whether the mother consumed alcohol during pregnancy or if the child was exposed in utero. But, apart from the most severe cases (Fetal Alcohol Syndrome, FAS), a biomarker of exposure does not allow to conclude on a cerebral impact of alcohol in utero. To date, the majority of FASD children are not diagnosed early. Moreover, and for obvious economic reasons, it is not possible to take care of all children whose mothers have consumed alcohol during pregnancy.
    [0003] The present invention makes it possible, unlike biomarkers of the prior art, to monitor the effects of fetal alcoholization. Indeed, the inventors have shown that the PIGF assay can identify, in children exposed in utero to alcohol, those who have suffered brain damage. Notably, the PIGF level indicates which children have a disrupted cerebral vascular system resulting from impaired cerebral angiogenesis. But these children, to this day, escape an early diagnosis. The present invention thus overcomes the early diagnosis deficit observed for children with FASD, who in France represent 9 cases per 1000 births and whose clinical signs (hyperactivity, attention disorders, etc.) are not detected until late ( for example between 4 and 5 years old, during schooling). The present invention therefore makes it possible to implement early adapted care of these children. This management will notably include stimulating the motor, sensory and cognitive functions of the child at a period 5 (early childhood) when the brain plasticity is maximal. According to a first aspect, the subject of the invention is an in vitro method for diagnosing fetal alcohol spectrum disorder (FASD) in a subject, said method comprising steps of: a) measuring the amount of PIGF in a biological sample and b) determining a fetal alcohol disorder. The term "PIGF" or "Placental growth factor" or "placental growth factor" (all these terms are synonymous) means a protein of the family of vascular endothelial growth factors (VEGF). More particularly, PIGF within the meaning of the invention is a 149 amino acid protein highly similar to VEGF-A which is recognized by the same receptor as VEGF-R1. PIGF is strongly expressed by the placenta, but not by the fetal brain. N-terminally glycosylated PIGF is secreted and functions as a dimer to stimulate angiogenesis. The term "PIGF" refers in particular to all 4 isoforms PIGF1-4: PIGF-1 and PIGF-3 are isoforms that do not bind heparin while PIGF-2 and PIGF-4 contain additional domains. which make it possible to fix heparin. Even more preferentially, PIGF is understood to mean a murine protein whose sequence is available under the accession number NP_001258634 or a human protein whose sequence is available under the accession number NP_001193941.1.
    [0004] Fetal Alcohol Spectrum Disorder (FASD) is defined as "all disorders in children resulting from exposure to alcohol during pregnancy. This term includes, among other things, all of the behavioral disorders that will gradually become apparent with age. Children with these disorders are called FASD children. In their most severe version, the TACFs correspond to fetal alcohol syndrome (FAS). This results in craniofacial dysmorphia (including shortened palpebral fissures, a smooth, elongated, erased nasolabial fold, and a thin upper lip); nonspecific growth retardation (prenatal or postnatal height or weight or head circumference) or both; 3037657 4 and neurodevelopment disorders, sometimes expressed as mental retardation and more often as learning difficulties. Children with FAS are called FAS children. The inventors have shown that exposure to alcohol causes cerebral vascular damage. By "cerebrovascular disease" is meant here any alteration of the cerebrovascular system, including an alteration resulting in impaired or even defective operation of said system. A cerebrovascular disease in the sense of the invention may in particular be a disorganization of the cerebrovascular system. More particularly, fetal alcoholization induces a random orientation of the cerebral vessels. In a particular embodiment, the fetal alcohol disorder is related to cerebrovascular disease. Even more particularly, said fetal alcohol disorder is related to disorganization of the cerebrovascular system. The term "subject" according to the invention is understood to mean a human, and preferably an embryo, a fetus or a child. An "embryo" as it is understood here corresponds to a fertilized oocyte of less than three months old. "Fetus" means an individual taken before birth and whose gestational age is between 3 and 9 months. After childbirth, the subject becomes a child. By "child" is meant according to the invention an individual whose age is less than 3 years. Included in the category of children according to the invention are newborns, whose age is between 0 and 1 month, infants, who are between 1 month and 2 years old, and the children themselves, who are at least 2 years old. A "newborn", as we understand it here, may as well be born at term as it is premature. The term - a subject with fetal alcohol disorders "or - subject TCAF" as used herein refers to an embryo, fetus or subject, in particular human, which is exposed to in utero alcohol and who suffers from fetal alcohol impairment or who is at risk of developing maternal alcohol consumption as a condition related to fetal alcohol spectrum disorder, including the effects described above. In particular, a TCAF subject has a disorganized cerebral vascular network, said disorganization being notably related to a random orientation of the cerebral vessels. The method of the invention is particularly useful because it allows non-invasive prediction of cerebral deficits. It makes it possible to detect, from a biological sample, in particular a placental sample, the subjects who risk suffering from FASD, which makes it possible to take charge of them. The term "biological sample" according to the invention means any sample that can be taken from a subject. Alternatively, the biological sample is a sample from the placenta, including the umbilical cord. Indeed, PIGF is expressed by placental cells throughout pregnancy. This allows to dose the PIGF without compromising the integrity of the subject, especially when it is an embryo or a fetus. In general, the biological sample must allow the determination of the level of expression of the biological marker of the invention.
    [0005] The test sample may be used as obtained directly from the biological source or as a result of pretreatment to modify the character of the sample. For example, such pretreatment may include plasma preparation from blood, dilution of viscous fluids, and so on. Pre-treatment methods may also involve filtration, precipitation, dilution, distillation, mixing, concentration, inactivation of disrupting components, addition of reagents, lysis, and the like. In addition, it may be beneficial to modify a solid test sample to form a liquid medium or to release the analyte. PIGF protein is a secreted protein (DeFalco, Exp Mol Med 44 (1): 1-9, 2012).
    [0006] Preferred biological samples for determining the level of expression of said biomarkers include in particular blood, plasma, or lymph samples. Preferably, the biological sample is a blood sample. Even more preferably, the biological sample is a sample of placental blood or cord blood. This is usually collected during delivery. Blood from placental vessels can then be obtained to measure the level of PIGF in the blood. This allows a non-invasive diagnosis of a fetal alcohol disorder, including brain damage. In fact, the simple determination of PGIF in the blood makes it possible to determine whether the in utero exposure to alcohol has led to FASD, in particular because of cerebral vascular disorganization. The inventors have thus shown that the PIGF makes it possible to determine that cerebral involvement has occurred, unlike the biomarkers of the prior art which detected only the exposure of the fetus to alcohol. PIGF is therefore a reliable biomarker of FASD. By "biomarker" is meant within the meaning of the present application a feature that is objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes, or pharmacological responses to a therapeutic intervention. A biomarker therefore refers to a variety of different substances and parameters. For example, a biomarker may be a substance whose detection indicates a particular disease state (for example the presence of activated protein C as a marker of infection), or on the contrary a substance whose detection indicates a specific physiological state . The biomarker according to the invention is preferably a gene, the products of a gene such as its transcripts and the peptides derived from its transcripts, a lipid, a sugar or a metabolite. According to one embodiment of the present invention, the biomarker is a gene, the products of a gene such as transcripts or peptides, a lipid, a sugar or a metabolite whose expression changes, in particular level 15. of expression, correlate with a physiological state of the child resulting from in utero exposure to alcohol. In a particular embodiment, the biomarker is a peptide having growth factor activity. The candidate biomarker according to the invention is preferably a gene marker, a protein marker, a lipid marker or a metabolic marker. For each of these types of markers, many methods are available to those skilled in the art for measuring the expression of said biomarker and thus to identify a difference in expression between children exposed in utero to alcohol and children. healthy, that is, not exposed to alcohol. In a first embodiment, said marker is a gene marker or a protein marker. In this case, the method of the invention may comprise one or more intermediate steps between taking the sample of cutaneous cells and measuring the expression of PGLF, said steps corresponding to extraction from said placenta sample. a sample of mRNA (or the corresponding cDNA) or a protein sample. This can then be directly used to measure the expression of PGLF. Preparation or extraction of mRNA (as well as retrotranscription thereof into cDNA) or proteins from a cell sample are only routine procedures well known to those skilled in the art.
    [0007] Once a sample of mRNA (or corresponding cDNA) or protein is obtained, the expression of PGLF at either the mRNA level (i.e., all mRNAs or cDNAs present in the sample), or proteins (i.e., all of the proteins present in the sample), can be measured. The method used to do this depends on the type of transformation (mRNA, cDNA or protein) and the type of sample available. When the expression of PGLF is measured at the level of the mRNA (or corresponding cDNA), any technology usually used by those skilled in the art can be implemented. Such gene expression level analysis technologies, such as transcriptome analysis, include well-known methods such as PCR (Polymerase Chain Reaction, based on DNA), RT-PCR ( Reverse Transcription-PCR, starting from RNA) or quantitative RT-PCR or nucleic acid chips (including DNA chips and oligonucleotide chips) for higher throughput.
    [0008] "Nucleic acid chips" here means several different nucleic acid probes that are attached to a substrate, which may be a microchip, a glass slide, or a microsphere-sized bead. The microchip may consist of polymers, plastics, resins, polysaccharides, silica or a material based on silica, carbon, metals, inorganic glass, or nitrocellulose. The probes can be nucleic acids such as cDNAs ("cDNA chips"), mRNAs ("mRNA chips") or oligonucleotides ("oligonucleotide chips"), said oligonucleotides typically having a length of between about 25 and 60 nucleotides.
    [0009] To determine the expression profile of a particular gene, a nucleic acid corresponding to all or part of said gene is labeled, then brought into contact with the chip under hybridization conditions, leading to the formation of complexes between said acid. labeled target nucleic acid and probes attached to the surface of the chip that are complementary to that nucleic acid. The presence of labeled hybridized complexes is then detected. These technologies make it possible to monitor the level of expression of a particular gene or of several genes or even of all the genes of the genome (full genome or full transcriptome) in a biological sample (cells, tissues, etc.). These technologies are used routinely by those skilled in the art and therefore there is no need to detail them here. Examples of embodiments of the invention based on gene expression analysis (cDNA microarrays) and quantitative PCR are described in the experimental section. Alternatively, it is possible to use any current or future technology to determine gene expression based on the amount of mRNA in the sample. For example, one skilled in the art can measure the expression of a gene by hybridization with a labeled nucleic acid probe, such as, for example, Northern blot (for mRNA) or Southern blot (for cDNA). ), but also by techniques such as the method of serial analysis of gene expression (SAGE) and its derivatives, such as LongSAGE, SuperSAGE, DeepSAGE, etc. It is also possible to use fabric chips (also known as TMAs: - tissue microarrays "). The tests usually employed with tissue chips include immunohistochemistry and fluorescent in situ hybridization. For mRNA analysis, the tissue chips can be coupled with fluorescent in situ hybridization. Finally, it is possible to use bulk sequencing in parallel to obtain the amount of mRNA in the sample (RNA-Seq or Whole Transcriptome Shotgun Sequencing). For this purpose, several methods of bulk parallel sequencing are available. Such methods are described in, for example, US 4,882,127, U.S. 4,849,077; U.S. 7,556,922; U.S. 6,723,513; WO 03/066896; WO 2007/111924; US 2008/0020392; WO 2006/084132; US 2009/0186349; US 2009/0181860; US 2009/0181385; US 2006/0275782; EP-B11141399; Shendure And Ji, Nat Biotechnol., 26 (10): 1135-45. 2008; Pihlak et al., Nat Biotechnol., 26 (6): 676-684, 2008; Fuller et al., Nature Biotechnol., 27 (11): 1013-1023, 2009; Tuesdays, Genome Med., 1 (4): 40, 2009; Metzker, Nature Rev. Genet., 11 (1): 31-46, 2010. When marker expression is measured at the protein level, it is possible to employ specific antibodies, particularly in well-known technologies such as immunoprecipitation, immunohistology, western blot, dot blot, ELISA or ELISPOT, protein chips, antibody chips, or tissue chips coupled to immunohistochemistry. Other techniques that can be used include FRET or BRET techniques, microscopy or histochemistry methods, including confocal microscopy and electron microscopy methods, methods based on the use of one or more excitation wavelengths and a suitable optical method, such as an electrochemical method (voltammetry and annealing techniques), the atomic force microscope, and radiofrequency methods, such as multipolar resonance spectroscopy , confocal and non-confocal, fluorescence detection, luminescence, chemiluminescence, absorbance, reflectance, transmittance, and birefringence or refractive index (for example, by surface plasmon resonance, or - plasmon resonance surface in English, by ellipsometry, by resonant mirror method, tec.), flow cytonetry, radioisotopic or magnetic resonance imaging, polyacrylamide gel electrophoresis analysis (SDS-PAGE); by HPLC-Mass spectrophotometry, by liquid chromatography / mass spectrophotometry / mass spectrometry (LC-MS / MS). All these techniques are well known to those skilled in the art and it is not necessary to detail them here.
    [0010] Preferably, the expression of PIGF is measured at the protein level. More preferably, the expression of PIGF is measured using a test employing specific antibodies recognizing said biomarker, in particular in well-known technologies such as immunoprecipitation, immunohistology, western blot, dot blot. , ELISA or ELISPOT, protein chips, antibody chips, or tissue chips coupled to immunohistochemistry. Antibodies directed against PIGF are commercially available (see, for example, REtD Systems, Danta Cruz, Abcam, etc.) and may be used in the methods of the invention. Even more preferably, the expression of PIGF is measured by Western Blot or ELISA.
    [0011] In a preferred embodiment of the invention, it may be useful to compare the level of PIGF obtained in step a) of the method with a reference level. By - a level of reference expression of a biological marker "is meant within the meaning of the present application any level of expression of said marker used for reference. For example, a level of reference expression can be obtained by measuring the level of expression of the marker of interest in a biological sample of a healthy subject, for example a placenta of a healthy subject, that is, to say a subject who has not been exposed to alcohol in utero. In this case, a PIGF level of step a) below the reference level indicates a TCAF. In particular, the inventors have shown that a level of PIGF lower than that of a healthy subject signals a defective cerebral vascular organization. According to an advantageous embodiment of the present invention, the expression of the candidate marker is normalized with respect to the expression of a control marker.
    [0012] A "control marker" according to the present invention is a marker whose expression is identical regardless of the cell type considered and the age of the donor. In a particular embodiment, when the candidate biomarker is a gene marker or a protein marker, the control marker is a gene that is expressed in all cell types, regardless of the subject's age, or protein product. In a more particular embodiment, said control marker is a household gene or the protein product of said household gene. A household gene is a gene that is expressed in all cell types and provides a basic function that is necessary for the survival of the cell. For example, a list of human household genes can be found in Eisenberg et al. (Trends in Genetics 19: 362-365, 2003). A preferred household gene according to the invention is a gene selected from the group consisting of B2M, TFRC, YWHAZ, RPLO, 18S, GUSB, UBC, TBP, GAPDH, PPIA, POLR2A, ACTB, PGK1, HPRT1, IP08 and HMBS. The method of the invention is particularly useful because it allows for non-invasive diagnosis from an early age. Children who have been diagnosed with brain damage as a result of uterine exposure to alcohol can be cared for early and quickly. However, it has been shown that the earlier the management is, the better the functional and cognitive recovery (Toutain et al., Pychotropes, 13: 49-68, 2007).
    [0013] According to another aspect, the subject of the invention is a method for treating fetal alcohol disorders in a subject. The method comprises steps of: a) diagnosing FASD in said subject by any of the methods above; and b) treating said subject if step a) concludes that said subject has FASD. By "treatment" is meant any action to reduce or eradicate the symptoms or causes of FASD. Treatment within the meaning of the invention may include administration of a pharmacological substance and / or psychotherapeutic monitoring. The invention will be described more precisely by means of the examples below. Said examples are provided here by way of illustration and are not, unless otherwise indicated, intended to be limiting. LEGENDS OF FIGURES Figure 1. Effects of in utero alcohol exposure on cortical angiogenesis in mouse E20 embryos. A, B: Effects of fetal alcoholic exposure from 10 GD15 to GD20 on the organization of cortical microvessels in control (A) animals and exposed to alcohol (B). Microvessels of the brain were visualized by immunohistochemistry against CD31. The arrows indicate the microvessels of the brain with a radial orientation in the "control" group. It should be noted a loss of radial organization in the group - Alcohol ». I-VI: Cortical layers; CC: Corpus callosum. C: Distribution of orientation (angle categories) of cortical microvessels in the immature cortex of fetus GD20. Statistical analysis was performed using the x2 test. D: Western blot quantification of effects of fetal alcohol exposure during the last week of gestation on cortical expression of CD31 at GD20. vs the "control" group using an unpaired t-test. Figure 2. Effects of In utero Alcohol Exposure on Expression of VEGF / PIGF Family Members in Mouse E20 Embryos. A-E: Western blot quantification of protein levels of VEGFA (A), PIGF (B), sVEGF-R1 (C), mVEGF-R1 (D) and VEGF-R2 in the cortex of the groups - Control and Alcohol. F: Western blot comparison of PIGF protein levels in the cortex and placenta of E20 embryos of the - control group. *** p <0.001 vs group - control "using unpaired t-test. Figure 3. Effects of in utero alcohol exposure on the ultrastructural characteristics of the placenta in GD20 mice. A: Observation by Cresyl violet staining of the effect of alcoholic exposure on the laminar structure of the placenta. The maternal side of the placenta is upward. Alcohol affects the segregation of junction areas and labyrinth (dotted lines). B: 3037657 12 Quantification by image analysis of the effects of alcohol on the thickness of Reichert's membrane. C, D: Low magnification observation of the layer of giant trophoblasts in the groups - Control »(C) and - Alcohol» (D). The arrows indicate giant trophoblasts. These have a typical rectangular shape in the placenta of the group - "Witness", whereas in the group "Alcohol" they have a rounded shape. EH: Images acquired by medium (E, F) and strong (G, H) electron microscopy showing the cellular morphology of giant trophoblasts and the presence of zonula occludens (arrows) in the "control" (E, G) and - Alcohol "(F, H). Zonula occludens (stars) is no longer visible in the 10 animals treated with alcohol. The inserts in E and F indicate the area observed at higher magnification at G and H, respectively. D: maternal decidua; J: junction area; L: labyrinth area; Tg: layer of giant trophoblasts. *** p <0.001 vs group - control "using unpaired t-test. Figure 4. Effects of in utero alcohol exposure on the expression of proteins involved in the placental barrier and placental energy metabolism. A, B: Observation by innnnunohistochinnie ZO-1 protein in the maze area of the mouse placenta groups - Control "(A) and - Alcohol" (B). The ZO-1 protein appears to form groups of points (arrows) in the "control" group while the labeling is diffuse in the "alcohol" group. The trophoblast layers were demonstrated by immunoreactivity with the Glut-1 glucose transporter. The nuclei were marked at Hoechst. C: Double labeling with antibodies against MCT-1 monocarboxylate and glucose transporters in the labyrinth zone of a placenta - Control. In contrast to Glut-1, the expression of MCT-1 is associated with the maternal layer of the syncytiotrophoblast. The nuclei were marked at Hoechst. D: Quantification by western blot of expression levels of ZO-1 and MCT-1 proteins in the placentas of the "Control" and "Alcohol" groups. * p <0.05, ** p <0.01 vs the control group "using an unpaired t-test. Figure 5. Effects of in utero alcohol exposure on expression of VEGF / PIGF family members in murine placenta. AF: Western blot quantification of the effects of alcohol exposure during the last week of gestation on placental expression of VEGF-A (A), PIGF (B), sVEGF-R1 (C), nnVEGFR1 (D) ), VEGF-R2 (E) and CD31 (F) to GD20. G, H: Immunohistochemistry labeling showing the distribution of VEGF-R2 (G) in Glut-1 labeled (Gl) -labelled syncytiotrophoblast layers. The nuclei were marked at Hoechst. * p <0.05 vs the - control group "using an unpaired t-test. Figure 6. Diffusion of Evans blue injected in utero from the placenta into the fetal brain. A, B: Time-course visualization of Evans Blue administered by microinjection into the placenta of a GD15-gravid mouse. The fluorescence was detected by UV illumination (A) and is represented using a dummy color scale (B). C, D: Time visualization of Evans blue fluorescence in the fetal brain following GD15 placental microinjection. The fluorescence was detected by UV illumination (C) and is represented using a dummy color scale (D). E, F: Quantification over time by spectrophotometry of the 595 nm absorbance of the Evans blue signal injected into the placentas (E) and subsequently into the brains of the corresponding fetuses (F). G: ELISA quantification of human PIGF in mouse fetal brain 30 min after injection of hPIGF into placentas of GD15 pregnant mice. * p &lt; 0.05 vs control group, using unpaired t-test. Figure 7: Effect of Placental PIGF Repression by In utero Transfection on VEGF-R1 Levels of the Brain. A: Photomicrograph showing expression of eGFP 48 hours after in utero transfection of a plasmid encoding eGFP in GD15-pregnant mouse placenta. B, C: Triple labeling eGFP / Glut-1 / Hoechst 20 showing that the fluorescence of eGFP (B) is essentially associated with the fetal trophoblastic layer labeled with Glut-1 (C, arrowheads). The maternal trophoblastic layer that is also labeled with Glut-1 is not transfected. The fetal trophoblastic layer is identified by the presence of nucleated red blood cells characteristic of the fetal circulation (arrow). D: Western blot display of PIGF, GFP and actin proteins in non-transfected (sh- / GFP-) transfected animals transfected with shPIGF / GFP (sh- / GFP ±) ± / ± GFP). E, F: Western blot quantification of PIGF (E) and GFP (F) expression levels in non-transfected (sh- / GFP-) animals transfected with GFP (sh- / GFP) ±) and transfected with shPIGF / GFP (sh ± / GFP ±). G: Western blot quantification of VEGF-R1 expression levels in the fetal brain from non-transfected (sh- / GFP-) placentas transfected with GFP (sh- / GFP ±) and shPIGF / transfected GFP (sh ± / GFP ±). * p <0.05 vs the "sh- / GFP-" group using the ANOVA test followed by a Tukey multiple HSD comparison test.
    [0014] Figure 8. Morphometric characterization of the effects of in utero alcohol exposure on the human placenta from gestational weeks 20 to 25. A, B: Immunohistochemical labeling against CD31 and counterstaining with toluidine blue to visualize microvessels (chestnuts ) present in the placental villi (blue) groups - control "(A) and" FAS / pFAS "(B) at gestational ages [20-25 WG [. C: Percentage of villi classified by size in the placentas of the control groups "and" FAS / pFAS "at gestational ages [20-25 WG [. D: Distribution of vessels by size of villi in the placentas of the groups "Control" and "FAS / pFAS" at gestational ages [20-25 WG [. E: Vascular area by size of villi in placentas of the control groups and FAS / pFAS at gestational ages [20-25 WG [. * P <0.05 vs the control group] using an unpaired t-test. Figure 9. Morphometric characterization of the effects of in utero alcohol exposure on the human placenta of gestational weeks 25 to 35. A, B: Immunohistochemical labeling against CD31 and toluidine blue staining to visualize the microvessels (chestnuts) present in the placental villi (blue) of the groups - Control "(A) and" FAS / pFAS "(B) at gestational ages [25-35 WG [. C: Percentage of villi classified by size in the placentas of the "control" and "FAS / pFAS" groups at gestational ages [25-35 WG [. D: Villal size distribution of villi in the placentas of the "control" and "FAS / pFAS" groups at gestational ages [25-35 WG]. E: Vascular area by villous size in the placentas of the control groups "and" FAS / pFAS "at gestational ages [25-35 WG [. * p <0.05 vs the - control group "using an unpaired t-test. Figure 10. Morphometric characterization of the effects of in utero alcohol exposure on the human placenta of gestational weeks 35 to 42. A, B: Immunohistochemical labeling against CD31 and toluidine blue staining to visualize microvessels (chestnuts) present in placental villi (blue) of the groups - Control "(A) and" FAS / pFAS "(B) at gestational ages ranging from [35-42 WG [. The luminal region of the microvessels is greatly reduced in the "FAS / pFas" group. C: Percentage of villi classified by size in the placentas of the "control" and "FAS / pFAS" groups at gestational ages ranging from [35-42 WG [. D: Distribution of vessels by villous size in placentas of the "control" and "FAS / pFAS" groups at gestational ages ranging from [35-42 WG]. E: Vascular villous area size in the placenta of the control group and FAS / pFAS at gestational ages ranging from 35-42 WGVp <0.05 vs the control group using an unpaired t-test. Figure 11. Time-course effects of in utero alcohol exposure on villous and vessel densities in human placentas and western blot characterization of pro-angiogenic proteins and energy metabolism. A: Evolution of villous densities in the placentas of the control groups (A) and FAS / pFAS (B) at gestational ages [20-25 WG [, [25-35 WG [and [35-42 WG [. B: Evolution of vessel densities in the placentas of the groups "Control" and "FAS / pFAS" at gestational ages [20-25 WG [, [25-35 WG [and [35-42 WG [. #p <0.05, ep <0.01 vs the group - Indicator "as shown on the graph. * p <0.05, *** p <0.001 for the groups - control "vs - alcohol" for a given class of gestational age. CH: Quantification by western blot of protein levels of ZO-1 (C), MCT-1 (D), PIGF (E), VEGFA (F), VEGF-R1 (G) and VEGF-R2 (H) in placentas groups - Witness "and" FAS / pFAS ". * p <0.05 vs control group 15 using an unpaired t-test. Figure 12. Comparison of cerebral and placental damage observed in human fetuses and induced by in utero alcohol exposure and statistical correlation. AH: Vascular organization in the brains (A, D) and placentas (E, H) of patients in the group - Control WG22 (A, E) and WG31 (C, G) and vascular organization in the brains (B , D) and placentas (F, H) of patients from the group "FAS / pFAS" to WG21 (B, F) and WG33 (D, H). I, J: Statistical correlation between cortical vascular disorganization and placental vascular density in patients in the control groups "(I) and FAS / pFAS (J).
    [0015] EXAMPLES Abnormalities of cerebral angiogenesis following alcohol abuse in utero Effects of in utero exposure to alcohol on cerebrovascular network development The present inventors have previously demonstrated that prenatal alcohol alcoholization induces cerebral vascular disorganization. In particular, the effect of alcohol is associated with a significant decrease in the number of cortical vessels having a radial orientation in favor of the number of microvessels having a random orientation (Figure 1). In parallel with the study in the mouse, an analysis of the cerebral microvascular system in humans has shown that, as in the mouse, the cortical microvessels which have a radial orientation in the group "control" are totally disorganized in the group. SAF / pFAS "(Figure 12 and Jegou et al., 2012). Effects of in utero exposure to alcohol on the expression of representative genes of the vascular system in mice Quantitative RT-PCR (mRNA) and Western blot (protein) studies revealed a marked deregulation of VEGF-R1 and VEGF-R2 receptors that relay the pro-angiogenic effects of factors such as VEGFA or PIGF. The abnormalities of the cerebrovascular network are thus associated with a deregulation of the expression of pro-angiogenic cerebral receptors (Figure 2 and Jegou et al., 2012).
    [0016] Anomalies of placental angiogenesis following alcohol in utero Various placental parameters have been studied in mice (FIGS. 3-5) and in humans (FIGS. 8-10) by an immunohistochemical approach coupled with an nnorphonetric analysis comprising in particular density and size of placental villi, density and vascular area, or the proportion of vessels per villus. In humans, these parameters were measured and compared between 34 placentas of control individuals and 36 placentas from individuals exposed in utero to alcohol. The placentas were divided into three age classes comparable to those of the brain study (Jegou et al., 2012). This paper presents the results for the age groups [20-25GW [, [25-35GW [and [35-42GW [.
    [0017] In particular, morphometric analysis indicates that the distribution of placental vessels by villous sizes and vascular area are significantly impacted by alcoholization (Figure 11). In addition, a longitudinal analysis of the factor-age vascular density indicates that in the control group, placental angiogenesis strongly increases between the age groups [20-25GW [and [25-35GW [. This strong placental vascularization is explained by a significant development of the brain during the third trimester of pregnancy which requires increased oxygen and nutrient requirements. On the other hand, fetal alcoholization induces a stagnation or even a decrease in placental vascular density (FIG. 11). In conclusion, the present results indicate that there are vascular abnormalities in the human placenta as well as in the cerebral cortex in subjects who have been exposed to alcohol. These results therefore support the hypothesis of a correlation between brain disorders and placental deficits of angiogenesis. Demonstration of a correlation between placental and cerebral vascular abnormalities The placental and cerebral vascular abnormalities observed in humans following alcohol in utero may be the result of totally independent processes without cause-and-effect relationship or, conversely, closely entangled. The fact that the source of PIGF is unique and of placental origin argues in favor of the second hypothesis. However, in order to demonstrate a link between cerebrovascular and placental disorders, we carried out a correlation study on the one hand in the subjects of the "control" group and, on the other hand, in the individuals of the group - FAS / pFAS "(Figure 12). The results demonstrate that in the "control" group, the increase in placental vascularization does not impact the radial organization of cortical vessels (R2 0.4719). In contrast, the lack of placental vascularization observed in the FAS / pFAS group is closely correlated with the random orientation of the cortical vessels (R2 0.9995). There is therefore a very significant interaction between placental and cerebral vascular changes. Demonstration of a functional link between placental PIGF and its brain receptor In utero administration of a placental fluorescent molecule in pregnant mice (GD15) is found after 20-30 min in the fetal brain (FIG. ). In addition, human recombinant PIGF injected into the mouse at the placenta is detected after 30 min by ELISA in the fetal brain (FIG. 6). These data indicate that placental molecules and especially PIGF are able to reach the fetal brain.
    [0018] Invalidization by uterine placental uptake of murine PIGF by shRNA results in repression of placental PIGF protein levels after 48 hours (Figure 7). This effect is associated with the brain level by a drop in the protein levels of the VEGF-R1 receptor (Figure 7). These results indicate that i) specific repression of placental PIGF directly impacts the expression of the brain receptor, ii) specific repression of placental PIGF mimics the effects of alcohol on cerebral VEGF-R1 expression (FIGS. 7). Identification of placental factors biomarkers of cerebral involvement The above correlation study demonstrates for the first time that fetal alcohol-induced placental vascular involvement is directly related to cerebral vascular alterations. Therefore, placental factors whose role on angiogenesis is proven to become biomarkers candidates for cerebrovascular disorders. Expression levels of proteins known to be either actors of angiogenesis or proteins specific to the vascular system have been quantified by western blotting. This work was done in animals (mice, placenta / brain) and humans (placenta). In mice, the quantification of placental VEGFA and PIGF expression levels demonstrates a significant decrease only in PIGF (of which the placenta is the only source in the body, Figure 5). At the same time, quantification of VEGFA and PIGF receptors indicates that expression of VEGFR1 (the only receptor of PIGF) is decreased in both the placenta and the brain (Figures 2 and 5). This decrease, very marked, is of the order of 50%. The expression of VEGFR2 at the cerebral level is not affected. In addition, the quantification of vascular protein ZO-1 involved in placental and hematoencephalic barrier establishment is greatly diminished in the placenta (Figure 4). In parallel with work conducted in mice, protein expression analysis was conducted on human placentas with known maternal alcoholization and live children. We collected 7 placentas - controls "and 6 placentas alcohol" and quantified by western blot the candidate markers identified in the mouse. The results indicate that in the group - Alcohol »the expressions of PIGF 3037657 19 and ZO-1 are very strongly diminished as in the mouse (Figure 11). These data indicate that the fetal alcohol effects observed at the placental and cerebral levels are found in two different species, mice and humans.
    [0019] Conclusion In view of the different results obtained by the inventors in mice and humans, it appears that i) fetal alcoholization impacts cerebral angiogenesis and the organization of the cerebral vascular network, ii) these brain changes are correlated with placental vascular abnormalities, iii) a placental pro-angiogenic factor is able to reach the fetal brain, iv) neurodevelopmental abnormalities of cerebral angiogenesis in FASD children are associated with dysregulation of the placental PIGF system. Cerebral VEGF-R1 v) Placental invalidation of PIGF reproduces the effects of fetal alcoholization on cerebral VEGF-R1 vi) Deregulation of placental rates of PIGF following fetal alcoholization can predict cerebral involvement vii) a factor placental protein, PIGF has been identified as a biomarker of cerebral involvement induced by alcohol in uter o.
    权利要求:
    Claims (14)
    [0001]
    CLAIMS1) In vitro method for diagnosis of fetal alcohol disorder (FASD) in a subject comprising steps of: a) measuring the amount of Placental Growth Factor (PIGF) in a biological sample of said subject and; b) determining a FASD in said subject.
    [0002]
    2) The method of claim 1, characterized in that it comprises an additional step of comparing the amount of PIGE of step a) with a reference.
    [0003]
    3) The method of any one of claims 1 or 2, characterized in that the reference is a measure of the amount of PIGE in a healthy individual.
    [0004]
    4) The method of any one of claims 1 to 3, characterized in that a PIGF amount of step a) below the reference indicates that the subject is suffering from FASD.
    [0005]
    5) The method of any one of claims 1 to 4, characterized in that a quantity of PLU of step a) below the reference indicates cerebral vascular disorganization in the subject.
    [0006]
    6) The method of any one of claims 1 to 5, characterized in that said biological sample comes from the placenta, including cord blood.
    [0007]
    7) The method of any one of claims 1 to 6, characterized in that the amount of PIGE is determined by measuring the amount of PIGF nucleic acid.
    [0008]
    8) The method of any one of claims 1 to 7, characterized in that the amount of PIGE is measured by a method selected from Northern blot, Southern blot, PCR, RT-PCR, RT-PCR quantitative, SAGE and its derivatives, nucleic acid chips, including cDNA chips, oligonucleotide chips and mRNA chips, tissue chips and RNA-Seq.
    [0009]
    9) The method of any one of claims 1 to 6, characterized in that the amount of PIGF is determined by measuring the amount of the polypeptide. 3037657 21
    [0010]
    10) The method of claim 9, characterized in that the amount of PIGF is measured by a method selected from immunohistology, immunoprecipitation, western blot, dot blot, ELISA or ELISPOT, fleas proteins, antibody chips, or tissue chips coupled to immunohistochemistry, FRET or BRET techniques, microscopy or histochemistry methods, including confocal microscopy and electron microscopy methods, methods based on the use of one or more excitation wavelengths and a suitable optical method, such as an electrochemical method (voltammetry and amperometry techniques), the atomic force microscope, and radiofrequency methods, such as multipole, confocal and nonconfocal resonance spectroscopy, fluorescence detection, luminescence, chemiluminescence, absorbance, reflectance, transmittance, and birefringence or india x refraction (in particular by surface plasmon resonance, by ellipsometry or by resonant mirror method), flow cytometry, radioisotopic or magnetic resonance imaging, polyacrylamide gel electrophoresis (SDS-PAGE) analysis; by HPLC-Mass spectrophotometry, by liquid chromatography / mass spectrophotometry / mass spectrometry (LC-MS / MS).
    [0011]
    11) The method of any one of claims 9 or 10, characterized in that the amount of PIGF is determined by a method selected from immunoprecipitation, immunohistology, western blot, dot blot, ELISA or the ELISPOT, the protein chips, your antibody chips, or the tissue chips coupled to immunohistochemistry.
    [0012]
    12) The method of any one of claims 9 to 11, characterized in that the amount of PIGF is determined by Western blot or ELISA.
    [0013]
    13) The method of any one of claims 1 to 12, characterized in that the amount of PIGF is normalized with respect to a control marker.
    [0014]
    14) The method of claim 13, characterized in that the control marker is a gene selected from the group consisting of B2M, TFRC, YWHAZ, RPLO, 18S, GUSB, UBC, TBP, GAPDH, PPIA, POLR2A, ACTB, PGK1, HPRT1, IP08 and HMBS, or a polypeptide selected from the product of said genes.
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    同族专利:
    公开号 | 公开日
    US10793908B2|2020-10-06|
    CN108450002A|2018-08-24|
    AU2016282842A1|2018-01-25|
    EP3311174A1|2018-04-25|
    WO2016207253A1|2016-12-29|
    ES2758424T3|2020-05-05|
    PL3311174T3|2020-03-31|
    HRP20192085T1|2020-02-21|
    HK1253894B|2020-07-10|
    DK3311174T3|2019-11-18|
    JP6663936B2|2020-03-13|
    JP2018519819A|2018-07-26|
    CA2990304A1|2016-12-29|
    KR20180063036A|2018-06-11|
    PT3311174T|2019-11-22|
    EP3311174B1|2019-08-28|
    HUE047242T2|2020-04-28|
    US20180195124A1|2018-07-12|
    FR3037657B1|2017-06-23|
    BR112017027707A2|2018-09-11|
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    2018-06-15| CD| Change of name or company name|Owner name: UNIVERSITE DE ROUEN NORMANDIE, FR Effective date: 20180509 Owner name: INSTITUT NATIONAL DE LA SANTE ET DE LA RECHERC, FR Effective date: 20180509 Owner name: CENTRE HOSPITALIER UNIVERSITAIRE DE ROUEN, FR Effective date: 20180509 |
    2019-06-12| PLFP| Fee payment|Year of fee payment: 5 |
    2020-04-10| RM| Correction of a material error|Effective date: 20200303 |
    2021-03-12| ST| Notification of lapse|Effective date: 20210205 |
    优先权:
    申请号 | 申请日 | 专利标题
    FR1555727A|FR3037657B1|2015-06-22|2015-06-22|METHOD OF DIAGNOSING DISORDERS CAUSED BY FETAL ALCOHOLIZATION|FR1555727A| FR3037657B1|2015-06-22|2015-06-22|METHOD OF DIAGNOSING DISORDERS CAUSED BY FETAL ALCOHOLIZATION|
    PCT/EP2016/064480| WO2016207253A1|2015-06-22|2016-06-22|Method for the diagnosis of disorders caused by foetal alcohol syndrome|
    HUE16736402A| HUE047242T2|2015-06-22|2016-06-22|Method for the diagnosis of fetal acolhol spectrum disorder|
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    BR112017027707-7A| BR112017027707A2|2015-06-22|2016-06-22|in vitro method for the diagnosis of disorders|
    CN201680048286.8A| CN108450002A|2015-06-22|2016-06-22|Method for diagnosing the illness caused by fetal alcohol syndrome|
    PT167364025T| PT3311174T|2015-06-22|2016-06-22|Method for the diagnosis of fetal acolhol spectrum disorder|
    CA2990304A| CA2990304A1|2015-06-22|2016-06-22|Method for the diagnosis of disorders caused by foetal alcohol syndrome|
    EP16736402.5A| EP3311174B1|2015-06-22|2016-06-22|Method for the diagnosis of fetal acolhol spectrum disorder|
    PL16736402T| PL3311174T3|2015-06-22|2016-06-22|Method for the diagnosis of fetal acolhol spectrum disorder|
    KR1020187001742A| KR20180063036A|2015-06-22|2016-06-22|Diagnosis of disorders caused by Fetal Alcohol Syndrome|
    AU2016282842A| AU2016282842A1|2015-06-22|2016-06-22|Method for the diagnosis of disorders caused by foetal alcohol syndrome|
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    JP2017566398A| JP6663936B2|2015-06-22|2016-06-22|Methods for diagnosing disorders caused by fetal alcohol syndrome|
    US15/738,922| US10793908B2|2015-06-22|2016-06-22|Method for the diagnosis of disorders caused by fetal alcohol syndrome|
    HK18113035.0A| HK1253894B|2015-06-22|2018-10-11|Method for the diagnosis of fetal alcohol spectrum disorder|
    HRP20192085TT| HRP20192085T1|2015-06-22|2019-11-20|Method for the diagnosis of fetal alcohol spectrum disorder|
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