Method for the diagnosis of disorders caused by fetal alcohol syndrome

Abstract

The present invention provides a method for the diagnosis of disorders caused by foetal alcohol syndrome, said method comprising the assaying of PLGF (placental growth factor).

Claims

1. A method of treatment of foetal alcohol spectrum disorders (FASDs) in a subject believed to be suffering from an FASD comprising the following steps: a) measuring the amount of placental growth factor (PIGF) in a biological sample from said subject by measuring the amount of the polypeptide; b) comparing the amount of PIGF from step a) with a reference; c) verifying an FASD in said subject, and d) treating a subject verified to have an FASD.

2. The method of claim 1, characterised in that the reference is a measurement of the amount of PIGF in a healthy individual.

3. The method of claim 1, characterised in that an amount of FIGF from step a) lower than the reference indicates that the subject suffers from an FASD.

4. The method of claim 1, characterised in that an amount of PIGF from step a) lower than the reference indicates a brain vascular disorganisation in the subject.

5. The method of claim 1, characterised in that said biological sample is obtained from the placenta.

6. The method of claim 1, characterised in that the amount of PIGF is measured by a method selected from immunohistology, immunoprecipitation, Western blot, dot blot, ELISA or ELISPOT, ECLIA, protein arrays, antibody arrays, or tissue arrays coupled with immunohistochemistry, FRET or BRET techniques, microscopy or histochemistry methods, confocal microscopy and electron microscopy methods, methods based on the use of one or more excitation wavelengths and a suitable optical method, an electrochemical method (voltammetry and amperometry techniques), atomic force microscopy, and radio frequency methods, multipolar resonance spectroscopy, confocal and non-confocal, detection of fluorescence, luminescence, chemiluminescence, absorbance, reflectance, transmittance, and birefringence or refractive index, surface plasmon resonance, ellipsometry, a resonant mirror method, flow cytometry, radioisotope or magnetic resonance imaging, analysis by polyacrylamide gel electrophoresis (SDS-PAGE), HPLC-mass spectrophotometry and liquid chromatography-mass spectrophotometry/mass spectrometry (LC-MS/MS).

7. The method of claim 1, characterised in that the amount of PIGF is determined by a method selected from immunoprecipitation, immunohistology, Western blot, dot blot, ELISA or ELISPOT, ECLIA, protein arrays, antibody arrays, or tissue arrays coupled with immunohistochemistry.

8. The method of claim 1, characterised in that the amount of PIGF is determined by Western blot or by ELISA.

9. The method of claim 1, characterised in that the amount of PIGF is normalised relative to a control marker.

10. The method of claim 9, characterised in that the control marker is a gene selected from the group consisting of -2 microglobulin gene (B2M), transferrin receptor gene (TFRC), Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide (YWHAZ), ribosomal protein LO gene(RPLO), 18S ribosomal RNA, Beta-glucuronidase gene (GUSB), ubiquitin C gene (UBC), Tata Binding Protein gene (TBP), Glyceraldehyde -3-phosphate dehydrogenase gene (GAPDH), peptidylprolyl isomerase A gene (PPIA), DNA-directed RNA polymerase II subunit RPB1 gene (POLR2A), -actin gene (ACTB), Phosphoglycerate kinase 1 gene (PGK1), hypoxanthine-guanine phosphoribosyltransferase gene (HPRT1), Importin 8 gene (IPO8) and hydroxymethylbilane synthase gene (HMBS), or a polypeptide selected from the products of said genes.

11. The method of claim 1, characterized in that said biological sample is obtained from the cord blood.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1. Effects of in utero alcohol exposure on cortical angiogenesis in E20 mouse embryos. A, B: Effects of foetal alcohol exposure from GD15 to GD20 on cortical microvessel organisation in control animals (A) and in alcohol-exposed animals (B). Brain microvessels were visualised using anti-CD31 immunohistochemistry. The arrows indicate brain microvessels with a radial orientation in the Control group. Note a loss of the radial organisation in the Alcohol group. I-VI: Cortical layers; CC: Corpus callosum. C: Distribution of the orientation (angle classes) of cortical microvessels in the immature cortex of GD20 foetuses. Statistical analysis was performed using the x.sup.2 test. D: Quantification by Western blot of the effects of foetal alcohol exposure during the last week of gestation on the cortical expression of CD31 at GD20. ns vs the Control group using an unpaired t-test.

(2) FIG. 2. Effects of in utero alcohol exposure on the expression of VEGF/PlGF family members in E20 mouse embryos. A-E: Quantification by Western blot of VEGF-A (A), PlGF (B), sVEGF-R1 (C), mVEGF-R1 (D) and VEGF-R2 protein levels in the cortex of the Control and Alcohol groups. F: Comparison by Western blot of PlGF protein levels in the cortex and the placenta of E20 embryos of the Control group. ***p<0.001 vs the Control group using an unpaired t-test.

(3) FIG. 3. Effects of in utero alcohol exposure on the ultrastructural features of the placenta in GD20 mice. A: Observation by cresyl violet staining of the effect of alcohol exposure on the laminar structure of the placenta. The maternal side of the placenta is pointing up. Alcohol affects segregation of the junctional and labyrinth zones (dotted lines). B: Quantification by image analysis of the effects of alcohol on Reichert's membrane thickness. C, D: Observation at low magnification of the giant trophoblast layer in the Control (C) and Alcohol (D) groups. Giant trophoblasts are indicated by arrows. They have a typical rectangular shape in the placenta of the Control group, whereas in the Alcohol group they have a round shape. E-H: Images acquired by electron microscopy at medium (E, F) and high (G, H) magnification showing the cell morphology of giant trophoblasts and the presence of tight junctions (arrows) in the Control (E, G) and Alcohol (F, H) groups. Tight junctions (stars) are no longer visible in the alcohol-treated animals. The insets in E and F indicate the zone observed at higher magnification in G and H, respectively. D: maternal decidua; J: junctional zone; L: labyrinth zone; Tg: giant trophoblast layer. ***p<0.001 vs the Control group using an unpaired t-test.

(4) FIG. 4. Effects of in utero alcohol exposure on the expression of proteins involved in the placental barrier and in placental energy metabolism. A, B: Immunohistochemical observation of ZO-1 protein in the placental labyrinth zone of mice of the Control (A) and Alcohol (B) groups. ZO-1 protein appears as forming groups of dots (arrows) in the Control group whereas staining is diffuse in the Alcohol group. The trophoblast layers were revealed by immunoreactivity with the glucose transporter Glut-1. Nuclei were stained with Hoechst. C: Double staining with antibodies against monocarboxylate MCT-1 and glucose transporters in the labyrinth zone of a Control placenta. By contrast with Glut-1, the expression of MCT-1 is associated with the maternal layer of the syncytiotrophoblast. Nuclei were stained with Hoechst. D: Quantification by Western blot of ZO-1 and MCT-1 protein expression levels in placentas of the Control and Alcohol groups. *p<0.05, **p<0.01 vs the Control group using an unpaired t-test.

(5) FIG. 5. Effects of in utero alcohol exposure on the expression of VEGF/PlGF family members in murine placentas. A-F: Quantification by Western blot of the effects of alcohol exposure during the last week of gestation on the placental expression of VEGF-A (A), PlGF (B), sVEGF-R1 (C), mVEGF-R1 (D), VEGF-R2 (E) and CD31 (F) at GD20. G, H: Immunohistochemical staining showing the VEGF-R2 (G) distribution in placental syncytiotrophoblast layers labelled with Glut-1 (H). Nuclei were stained with Hoechst. *p<0.05 vs the Control group using an unpaired t-test.

(6) FIG. 6. Diffusion of Evans blue injected in utero from the placenta to the foetal brain. A, B: Time-course visualisation of Evans blue administered by microinjection into the placenta of pregnant mice at GD15. Fluorescence was detected by UV illumination (A) and is represented using a false-colour scale (B). C, D: Time-course visualisation of Evans blue fluorescence in the foetal brain after placental microinjection at GD15. Fluorescence was detected by UV illumination (C) and is represented using a false-colour scale (D). E, F: Time-course quantification by spectrophotometry of absorbance at 595 nm of the signal of the injected Evans blue in the placentas (E) and subsequently in the brains of the corresponding foetuses (F). G: ELISA quantification of human PlGF in foetal mouse brain 30 min after injection of hPlGF in the placentas of pregnant mice at GD15. *p<0.05 vs the Control group using an unpaired t-test.

(7) FIG. 7: Effect of repression of placental PlGF by in utero transfection on brain VEGF-R1 levels. A: Microphotography showing eGFP expression 48 hours after in utero transfection of a plasmid encoding eGFP in placentas of pregnant mice at GD15. B, C: Triple staining eGFP/Glut-1/Hoechst showing that eGFP fluorescence (B) is mainly associated with the foetal trophoblast layer labelled with Glut-1 (C; arrowheads). The maternal trophoblast layer, also labelled with Glut-1, is not transfected. The foetal trophoblast layer is identified by the presence of nucleated red blood cells characteristic of foetal circulation (arrow). D: Visualisation by Western blot of PlGF, GFP and actin proteins in the placentas of non-transfected (Sh.sup./GFP.sup.), GFP-transfected (Sh.sup./GFP.sup.+) and shPlGF/GFP-transfected (Sh.sup.+/GFP.sup.+) animals. E, F: Quantification by Western blot of PlGF (E) and GFP (F) expression levels in the placentas of non-transfected (Sh.sup./GFP.sup.), GFP-transfected (Sh.sup./GFP.sup.+) and shPlGF/GFP-transfected (Sh.sup.+/GFP.sup.+) animals. G: Quantification by Western blot of VEGF-R1 expression levels in the foetal brain from non-transfected (Sh.sup./GFP.sup.), GFP-transfected (Sh.sup./GFP.sup.+) and shPlGF/GFP-transfected (Sh.sup.+/GFP.sup.+) placentas. *p<0.05 vs the Sh.sup./GFP.sup. group using ANOVA followed by Tukey's HSD multiple comparison test.

(8) FIG. 8. Morphometric characterisation of the effects of in utero alcohol exposure on gestational week 20 to 25 human placenta. A, B: Anti-CD31 immunohistochemical staining and toluidine blue counterstaining to visualise the microvessels (brown) present in the placental villi (blue) of the Control (A) and FAS/pFAS (B) groups at 20 to <25 weeks of gestation (WG). C: Percentage of villi classified by size in placentas of the Control and FAS/pFAS groups at 20 to <25 WG. D: Vessel distribution by villus size in placentas of the Control and FAS/pFAS groups at 20 to <25 WG. E: Vascular surface area by villus size in placentas of the Control and FAS/pFAS groups at 20 to <25 WG. *p<0.05 vs the Control group using an unpaired t-test.

(9) FIG. 9. Morphometric characterisation of the effects of in utero alcohol exposure on gestational week 25 to 35 human placenta. A, B: Anti-CD31 immunohistochemical staining and toluidine blue staining to visualise the microvessels (brown) present in the placental villi (blue) of the Control (A) and FAS/pFAS (B) groups at 25 to <35 WG. C: Percentage of villi classified by size in placentas of the Control and FAS/pFAS groups at 25 to <35 WG. D: Vessel distribution by villus size in placentas of the Control and FAS/pFAS groups at 25 to <35 WG. E: Vascular surface area by villus size in placentas of the Control and FAS/pFAS groups at 25 to <35 WG. *p<0.05 vs the Control group using an unpaired t-test.

(10) FIG. 10. Morphometric characterisation of the effects of in utero alcohol exposure on gestational week 35 to 42 human placenta. A, B: Anti-CD31 immunohistochemical staining and toluidine blue staining to visualise the microvessels (brown) present in the placental villi (blue) of the Control (A) and FAS/pFAS (B) groups at 35 to <42 WG. The microvessel lumen area is greatly reduced in the FAS/pFAS group. C: Percentage of villi classified by size in placentas of the Control and FAS/pFAS groups at 35 to <42 WG. D: Vessel distribution by villus size in placentas of the Control and FAS/pFAS groups at 35 to <42 WG. E: Vascular surface area by villus size in placentas of the Control and FAS/pFAS groups at 35 to <42 WG. *p<0.05 vs the Control group using an unpaired t-test.

(11) FIG. 11. Time-course effects of in utero alcohol exposure on villus and vessel densities in human placentas and Western blot characterisation of pro-angiogenic proteins and energy metabolism. A: Changes in villus densities in placentas of the Control (A) and FAS/pFAS (B) groups at 20 to <25 WG, 25 to <35 WG, and 35 to <42 WG. B: Changes in vessel densities in placentas of the Control and FAS/pFAS groups at 20 to <25 WG, 25 to <35 WG, and 35 to <42 WG. .sup.#p<0.05, .sup.##p<0.01 vs the Control group as indicated on the graph. *p<0.05, ***p<0.001 for the Control vs Alcohol groups for a given gestational age group. C-H: Quantification by Western blot of ZO-1 (C), MCT-1 (D), PlGF (E), VEGF-A (F), VEGF-R1 (G) and VEGF-R2 (H) protein levels in placentas of the Control and FAS/pFAS groups. *p<0.05 vs the Control group using an unpaired t-test.

(12) FIG. 12. Comparison of cerebral and placental damage observed in human foetuses and induced by in utero alcohol exposure and statistical correlation. A-H: Vascular organisation in the brains (A, D) and the placentas (E, H) of patients of the Control group at 22 WG (A, E) and 31 WG (C, G) and vascular organisation in the brains (B, D) and the placentas (F, H) of patients of the FAS/pFAS group at 21 WG (B, F) and 33 WG (D, H). I, J: Statistical correlation between cortical vascular disorganisation and placental vascular density in patients of the Control (I) and FAS/pFAS (J) groups.

EXAMPLES

(13) Brain Angiogenesis Abnormalities Following In Utero Alcohol Exposure

(14) Effects of In Utero Alcohol Exposure on Brain Vasculature Development

(15) The present inventors previously showed that prenatal alcohol exposure induces brain vascular disorganisation. In particular, the effect of alcohol is associated with a significant decrease in the number of cortical vessels with a radial orientation and an increase in the number of microvessels with a random orientation (FIG. 1). In parallel with the study carried out in mice, analysis of brain microvasculature in humans showed that, as in mice, the cortical microvessels that have a radial orientation in the Control group are completely disorganised in the FAS/pFAS group (FIG. 12 and Jegou et al., 2012).

(16) Effects of In Utero Alcohol Exposure on the Expression of Genes Representative of the Vasculature in Mice

(17) Quantitative RT-PCR (mRNA) and Western blot (protein) studies revealed a marked dysregulation of the levels of VEGF-R1 and VEGF-R2 receptors which relay the pro-angiogenic effects of factors such as VEGF-A or PlGF. Brain vasculature abnormalities are thus associated with a dysregulation of the expression of brain pro-angiogenic receptors (FIG. 2 and Jegou et al., 2012).

(18) Abnormalities of Placental Angiogenesis Following In Utero Alcohol Exposure

(19) Various placental parameters were studied in mice (FIGS. 3-5) and in humans (FIGS. 8-10) by an immunohistochemical approach coupled with morphometric analysis comprising in particular placental villus density and size, vascular density and surface area, and proportion of vessels per villus. In humans, these parameters were measured and compared between 34 placentas from control individuals and 36 placentas from individuals exposed to alcohol in utero. The placentas were divided into three age groups comparable with those of the brain study (Jegou et al., 2012). The results concerning the age groups 20 to <25 WG, 25 to <35 WG, and 35 to <42 WG are presented in this document.

(20) In particular, morphometric analysis indicates that the distribution of placental vessels by villus size and the vascular surface area are significantly affected by alcohol exposure (FIG. 11). Moreover, longitudinal analysis of vascular density, taking into account the age factor, indicates that in the Controls group placental angiogenesis strongly increases between the age groups 20 to <25 WG and 25 to <35 WG. This high placental vascularization is explained by significant brain development during the third trimester of pregnancy requiring increased oxygen and nutrients. On the other hand, foetal alcohol induces a stagnation or a lowering of placental vascular density (FIG. 11).

(21) In conclusion, the present results indicate that there exists in the human placenta, as in the cerebral cortex, vascular abnormalities in the alcohol-exposed subjects. These results thus support the hypothesis of a correlate between brain disorders and impaired placental angiogenesis.

(22) Demonstration of a Correlation Between Placental and Brain Vascular Abnormalities

(23) The placental and brain vascular abnormalities observed in humans following in utero alcohol exposure may be the result of completely independent processes with no cause and effect relationship or, conversely, may be closely interlinked. The fact that the source of PlGF is unique and of placental origin speaks in favour of the second hypothesis. However, in order to show a link between cerebral and placental vascular defects, we carried out a correlation study in subjects of the Control group and another in individuals of the FAS/pFAS group (FIG. 12).

(24) The results show that in the Control group, the increase in placental vascularization does not affect the radial organisation of the cortical vessels (R.sup.2 0.4719). On the other hand, the lack of placental vascularization observed in the FAS/pFAS group is closely correlated with the random orientation of the cortical vessels (R.sup.2 0.9995). There is thus a highly significant interaction between placental and brain vascular alterations.

(25) Demonstration of a Functional Link Between Placental PlGF and the Brain Receptor Thereof

(26) In utero administration of a fluorescent molecule into the placenta of gestating (GD15) mice is found after 20-30 min in the foetal brain (FIG. 6). In addition, recombinant human PlGF injected into the placenta of mice is detected after 30 min by ELISA in the foetal brain (FIG. 6). These data indicate that placental molecules, and in particular PlGF, are able to reach the foetal brain.

(27) Invalidation by in utero placenta transfection for murine PlGF by shRNA results in a repression of placental PlGF protein levels after 48 hours (FIG. 7). This effect is associated at the cerebral level by a decrease in VEGF-R1 receptor protein levels (FIG. 7). These results indicate that i) the specific repression of placental PlGF directly effects the expression of the brain receptor, ii) the specific repression of placental PlGF mimics the effects of alcohol on brain VEGF-R1 expression (FIGS. 2 and 7).

(28) Identification of the Placental Factors that are Biomarkers of Brain Damage

(29) The correlation study above shows for the first time that the placental vascular defects induced by foetal alcohol exposure are directly linked with brain vascular defects. Consequently, placental factors whose role in angiogenesis is proven become candidate biomarkers of brain vascular defects.

(30) Expression levels of proteins known to be either factors of angiogenesis or specific proteins of the vasculature were quantified by Western blot. This work was carried out in animals (mice; placenta/brain) and in humans (placenta).

(31) In mice, quantification of placental VEGF-A and PlGF expression levels show a significant decrease in PlGF alone (for which the placenta is the only source in the organism; FIG. 5). In parallel, quantification of VEGF-A and PlGF receptors indicates that the expression of VEGF-R1 (the unique PlGF receptor) is decreased in both the placenta and the brain (FIGS. 2 and 5). This very marked reduction is on the order of 50%. VEGF-R2 expression in the brain, in turn, is not affected. Moreover, quantification of vascular ZO-1 protein, involved in establishing the placental and haematoencephalic barrier, is strongly decreased in the placenta (FIG. 4).

(32) In parallel to the work carried out in mice, analysis of protein expression was carried out on human placentas for which maternal alcohol exposure was proven and the children were living. We collected 7 Control placentas and 6 Alcohol placentas and quantified by Western blot the candidate markers identified in mice. The results indicate that in the Alcohol group PlGF expression and ZO-1 expression are very strongly decreased as in mice (FIG. 11). These data indicate that the effects of foetal alcohol observed in the placenta and in the brain are found in two different species, mice and humans.

(33) Evaluation of PlGF Concentrations in Umbilical Cord Blood, Placenta and Maternal Blood from Two Groups of Patients (Control vs Exposed to Alcohol In Utero)

(34) The main objective of this clinical study is to compare PlGF concentrations in the umbilical cord and the placenta between two groups of patients and to carry out a follow-up at 2 and 6 years of the neurodevelopment of both groups of patients. In the first group, the patients were exposed to alcohol in utero. The second group is a control group of patients who were not exposed to alcohol in utero.

(35) This clinical study has the following objectives: comparison of PlGF concentrations in the maternal blood; neurological clinical examination upon birth of the child; follow-up at 2 years of age in paediatric consultation to evaluate neurodevelopment, notably via an Ages and Stages Questionnaire (ASQ), and follow-up at 6 years of age in paediatric consultation to evaluate neurodevelopment, via a parental questionnaire and a neuropsychological assessment.

(36) In this clinical study, 30 women who consumed alcohol during their pregnancy and 30 abstinent pregnant women (control group) are monitored. All the women monitored are at least 18 years of age and signed a consent protocol.

(37) The documented alcohol consumption during pregnancy is chronic consumption of at least 30 g of alcohol per week or acute binge drinking-type consumption during pregnancy (with a unit of 10 g of pure alcohol corresponding to 25 cL of 4.5 beer, 10 cL of 12 wine, 3 cL of whisky, 7 cL of sherry, etc.).

(38) In the control group, no alcohol consumption during pregnancy is documented.

(39) Thirty patients in each group are needed to show a difference in PlGF level of 4.7 pg/dL with the power of the test being 80%.

(40) The assaying of PlGF in the umbilical cord blood and the placenta is carried out by electrochemiluminescence immunoassay (automated ECLIA analysis of PlGF (Cobas e411 Analyzer) made available by Roche Diagnostics) on samples of cord blood and placentas (control group vs alcohol-exposed group).

(41) Tissue samples of cord blood and placentas are taken and then frozen and stored at 80 C. Quantification on tissue extracts of blood and placental PlGF is then carried out.

(42) A clinical examination upon discharge from maternity (weight, height, head circumference, axial and peripheral tone, reactivity, primitive reflexes, postural adaptations, facial dysmorphism suggestive of FAS, possible malformations) is carried out.

(43) A follow-up of the children at 2 and 6 years of age is carried out by targeting cognitive development and behavioural disorders.

(44) During the consultation at 2 years of age, weight, height and head circumference (HC) are measured. An ASQ is filled out and a neurological examination (brain MRI to investigate brain malformations) and an assessment of signs of facial dysmorphism are carried out. Investigation of vascular rigidity of the retinal vessels by an ophthalmologist is also carried out.

(45) During the consultation at 6 years of age, weight, height and HC are measured and a neurological and neuropsychological examination using neurodevelopmental scales (WISC IV and NEPSY) is carried out. Conners parent and teacher questionnaires (for screening hyperactivity) and social communication questionnaires (SCQ) for parents (related to behaviour) are also filled out during this consultation.

(46) In both groups, at birth, at 2 years of age and at 6 years of age, clinical examinations (behaviour, eye pursuit-fixation, axial and peripheral tone, neuromotor assessment, stretch reflexes, complete physical examination to investigate malformations) and paraclinical examinations (fundus of the eye, brain MRI, parental ASQ, WISC IV and NEPSY developmental scales, Conners and SCQ questionnaires for parents and teachers) are carried out.

(47) The two groups of patients are compared using the Mann-Whitney nonparametric test. A significance threshold of 5% is set.

(48) The results obtained are consistent with that which was expected.

(49) Conclusion

(50) In the light of the various results obtained by the inventors in mice and in humans, it appears that i) foetal alcohol exposure affects brain angiogenesis and the organisation of the brain vasculature, ii) these brain alterations are correlated with placental vascular abnormalities, iii) a placental pro-angiogenic factor is able to reach the foetal brain, iv) the neurodevelopmental abnormalities of brain angiogenesis in FASD children are associated with a dysregulation of the placental PlGF/brain VEGF-R1 system, v) placental invalidation for PlGF reproduces the effects of foetal alcohol exposure on brain VEGF-R1, vi) a dysregulation of placental PlGF levels following foetal alcohol exposure makes it possible to predict brain damage, vii) a placental protein factor, PlGF, was identified as a biomarker of brain damage induced by in utero alcohol exposure.