EP1776480A2 - Methoden und marker zum nachweis pränataler chromosomaler abnormalitäten - Google Patents

Methoden und marker zum nachweis pränataler chromosomaler abnormalitäten

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Publication number
EP1776480A2
EP1776480A2 EP05795957A EP05795957A EP1776480A2 EP 1776480 A2 EP1776480 A2 EP 1776480A2 EP 05795957 A EP05795957 A EP 05795957A EP 05795957 A EP05795957 A EP 05795957A EP 1776480 A2 EP1776480 A2 EP 1776480A2
Authority
EP
European Patent Office
Prior art keywords
hnp
markers
peaks
syndrome
defensins
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP05795957A
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English (en)
French (fr)
Inventor
Nathalie Leporrier
Michel Herrou
Sophie Feldblum
Pierre-Yves Simonin
Jean-François Cornuel
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Tycoon R & D Ltd
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Tycoon R & D Ltd
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Publication date
Application filed by Tycoon R & D Ltd filed Critical Tycoon R & D Ltd
Priority to EP05795957A priority Critical patent/EP1776480A2/de
Publication of EP1776480A2 publication Critical patent/EP1776480A2/de
Withdrawn legal-status Critical Current

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    • 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
    • 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

Definitions

  • the present invention relates to prenatal screening for chromosomal abnormalities. More particularly, the invention is based on the identification of novel markers of chromosomal abnormalities, in particular trisomy 21, whose assay from a biological fluid sample of the pregnant woman allows, alone or in combination with other assays, to determine the probability that the fetus is suffering from a chromosomal abnormality, with greater accuracy than the tests currently used.
  • Trisomy 21 or Down syndrome is the most common of the viable chromosome abnormalities. At the origin of psychomotor handicaps, it affects one birth in 800, and knowing that about 800 000 births are observed in France, the incidence is 1000 children with Down Syndrome per year.
  • the risk of trisomy increases with maternal age, especially after age 35 (exponential growth). The risk is also increased if one of the parents carries a balanced translocation or if the couple has already given birth to a child with a chromosomal abnormality. Fetal abnormalities detected on echography may also lead to the prescription of a fetal karyotype.
  • Antenatal screening for Trisomy 21 consists of studying the fetal karyotype, either on amniotic cells, on trophoblastic cells, or on cord blood. The sample is at risk of miscarriage.
  • the caiyotype is a long examination that is performed in a specialized laboratory and authorized by the Ministry of Health. It is therefore reserved for a population of women called "at high risk of chromosomal abnormalities".
  • Ultrasound call signs demonstrated morphological abnormalities of the fetus, internal or external, proven intrauterine growth retardation, amniotic fluid quantity abnormalities.
  • AFP alpha-fetoprotein
  • hCG chorionic gonadotropin hormone
  • ThCG ThCG
  • AFP unconjugated estriol
  • uE3 unconjugated estriol
  • ⁇ subunit of hCG ⁇ subunit of hCG.
  • the maternal serum markers hCG, uE3 and AFP
  • the next test is B145 or 39 euros 15.
  • a blood sample between the 15th and 18th week of gestation, for the determination of at least two of these markers is carried out, allowing a calculation of risk taking into account the risk a priori of the age of the mother modulated according to the observed values of these markers.
  • the threshold risk considered as decision-making for prenatal screening is arbitrarily set at 1: 250.
  • the determination of serum markers alone does not establish the diagnosis of trisomy 21. It is a screening test that makes it possible to estimate the risk of the presence of a child with Down Syndrome.
  • Screening sensitivity is approximately 60% if the risk threshold chosen for access to amniocentesis is 1/250. This leads to an amnio ⁇ centesis for about 5% of pregnant women.
  • the positive predictive value (speci ⁇ city) is of the order of 1 to 2%.
  • the sensitivity of the screening is better, with equal amniocentesis rate, if the markers are more discriminating and combined with each other.
  • the most commonly used combinations are the triple test (hCG + AFP + uE3) and the double tests, hCG + AFP or hCG + uE3, the latter being the best.
  • False positives of screening are amniocentesis with normal karyotype. The number of false positives should be reduced as much as possible because of the risk of fetal loss after amniocentesis (about 1%). Moreover, they can generate great anxiety in women while waiting for karyotype results.
  • the false negatives of screening are trisomy 21 cases not detected. They aggravate the emotional shock at the time of birth. In fact, couples who have benefited from a serum marker assay do not understand the notion of residual risk and often think that the test is a diagnostic tool. The failure of screening is then strongly felt. This situation is unsatisfactory in that it allows 30 to 40% of pregnancies with trisomy 21 to be missed. It is necessary to find a technique aimed at increasing sensitivity and specificity, reducing the number of useless procedures. invasive (false positives) and especially reduce the number of false negatives (30 to 40%).
  • serum markers in the first trimester of pregnancy are the PAPP-A protein, the hCG free beta subunit, and the nuchal translucency measurement at 12 weeks of amenorrhea. Some teams are also trying to develop the detection of fetal cells or free fetal DNA in maternal blood without effective screening.
  • the performance of the serum markers depends on the chosen strategy: risk threshold beyond which an amniocentesis is proposed, nature of the markers used, and type of association between them. Some combinations of markers perform better than others (WaId et al, Lancet, 2003, 8, 361, 855-6).
  • the inventors conducted studies from 280 frozen sera of women. pregnant with trisomic fetuses 21 (including those that were detected and those not detected by serum markers in the second trimester), and more than 280 control sera of women without fetuses trisomic 21 whose outcomes are known.
  • the placenta is a barrier between the mother and the fetus, but there is evidence that some molecules of small molecular weight pass this barrier.
  • the inventors have therefore chosen to apply the proteomic technique in the serum of pregnant women, in order to determine a differential expression profile making it possible to almost certainly distinguish those with trisomic fetuses 21 by comparison. her with sera of normal pregnant women. This approach, illustrated in the experimental part below, allowed them to identify several markers allowing to virtually distinguish serums of women with fetuses with trisomic 21 compared with sera of pregnant women with normal fetuses.
  • the present invention thus relates, in the first place, to the use of one or more marker (s) chosen from (i) the human neutrophil defensins HNP-2, HNP-I and HNP-3, and / or (ii) detectable molecular species in mass spectrometry with anion exchange type separation at the following molecular weights: 4461.60 Da; 4570.90 Da; 4630.70 Da; 4714.30 Da; 4859.10 Da; 8195.65 Da; 8905.85 Da; 9121.00 Da; 9700.50 Da, and / or (iii) molecular species detectable by mass spectrometry with a hydrophobic type separation, at the following molecular weights: 2625.47 Da; 4068.5 Da; 4075.95 Da; 4084.3 Da; 4084.7 Da; 4093.13 Da; 4320.79 Da; 4642.66 Da; 4658.47 Da; 6419.80 Da; 8107 Da; 8124.5 Da; 8140.5 Da; 8155 Da;
  • molecular species refers here, inter alia, to any protein, glycosylated or otherwise, phosphorylated or otherwise, any protein complex, any product of metabolism, or any fragment of any of these entities.
  • the molecular weights reported herein were obtained under experimental conditions specified below. Some technologies, different from those used by the inventors, usable to implement the invention, are cited below. It is clear that some variation is likely to occur when using different technologies and / or equipment. Nevertheless, it is also very clear that the person skilled in the art is able to transpose the results described in the experimental section below to other technologies, with a satisfactory recovery rate, and to easily identify the signals. (peaks,%) Corresponding to the markers highlighted by the inventors.
  • the molecular weights listed above must be heard with an accuracy of ⁇ 20%, preferably ⁇ 10%, even ⁇ 5%, and in good conditions ⁇ 1%. It should be noted that under the conditions described in the experimental section below, the measurement of the molecular weights of the different markers is reproducible, with a coefficient of variation that is very much less than 10%, typically of the order of 0.3. at 0.5%, for molecular weight markers less than 12000 Da. An accuracy of ⁇ 0.1% is even obtained up to 6000 Da. Due to the calibrators used, this coefficient of variation is of the order of 10 to 25% for molecular weight markers greater than 12000 Da.
  • the choice of the calibrator does not influence the values of the intensities of the peaks observed.
  • the use of different calibrators from those used by the inventors may possibly lead to a variation in the mass measured for the marker, the peaks corresponding to each of the markers nevertheless remaining always identifiable.
  • the invention also relates to an in vitro screening method for determining whether a pregnant woman is carrying a fetus suffering from an abnormality, comprising a detection or assaying step, in a biological sample of said pregnant woman, of at least one marker selected from any one of the groups (i) to (iii) defined above.
  • the word "assay” refers to a quantitative or semi-quantitative measurement in the broad sense, for example a relative quantification of a compound, with respect to the same compound in another sample, or with respect to another composed in the same sample.
  • a comparison of profiles, obtained for example by mass spectrometry is considered a "dosage”.
  • the method of the invention comprises a step of assaying at least two or three markers, at least one of which is selected from groups (i) and / or (ii) and / or or (iii) above, and the other one or two others are (are) selected from either the same groups or from the markers already characterized and used today, here constituting group (iv), including second-trimester markers - chorionic gonadotropin hormone (hCG) or its free ⁇ subunit, unconjugated estriol (uE3), p ⁇ -fetoprotein (AFP) and / or inhibin A - or first trimester - under -unit ⁇ free of hCG and / or protein Plasma associated with pregnancy (PAPP-A).
  • group (iv) including second-trimester markers - chorionic gonadotropin hormone (hCG) or its free ⁇ subunit, unconjugated estriol (uE3), p ⁇ -fetoprotein (AFP) and / or inhibin A -
  • an "improved triple test”, based on the hCG assay, uE3, and one of the markers cited above, is an integral part of the invention.
  • the assay of a marker of groups (i) to (iii) above may be combined with one or more other known test (s), such as for example the examination of the nuchal translucency.
  • the method of the invention involves a dosage step from a biological sample from the pregnant woman, which may be any body fluid, such as a sample of blood, serum, plasma, urine. ..
  • the number of markers selected from the molecules identified by the inventors may be equal to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or more, whether markers selected from groups (i), (ii), (iii) or (iv) (at least one of the markers being selected from one of groups (i) to (iii)).
  • markers selected from groups (i), (ii), (iii) or (iv) at least one of the markers being selected from one of groups (i) to (iii)).
  • Those skilled in the art will determine the number of markers to be assayed according to efficiency constraints (search for the most sensitive and selective test possible), and economic constraints.
  • these methods also comprise, for each measured marker, a step of comparing the measured concentration of this marker in the biological sample of the pregnant woman, with values of reference to the concentration of this marker in pregnant women with normal fetuses, and in pregnant women with fetuses with a known chromosomal and / or genetic abnormality, the comparison being indicative of the risk that the pregnant woman has a fetus. a chromosomal and / or genetic anomaly.
  • These references will be chosen by those skilled in the art so as to correspond to the gestational age of the sample taken in the pregnant woman.
  • the assay of at least one marker is carried out by mass spectrometry, for example by low-resolution or high-resolution mass spectrometry.
  • mass spectrometry for example by low-resolution or high-resolution mass spectrometry.
  • other separation techniques, purification, or dosing can be used in the context of the invention.
  • two types of technology can be envisaged to develop a diagnostic device according to the invention: the comparison of profiles, or the spot dosage of the markers.
  • this method comprises a step of analyzing at least one spectrum obtained from a biological sample from a pregnant woman; according to this variant, the signal of at least 2 peaks selected from (i) the peaks corresponding to the human neutrophil defenders HNP-2, HNP-I and HNP-3, and / or (ii) the observable peaks is measured.
  • the term “spectrum” here designates any type of spectrum (mass, MALDI, MALDI-TOF, SELDI, SELDI-TOF, plasmon resonance surface, liquid chromatography, ICAT, ESI, ESI-TOF, etc.), the person skilled in the art knowing how to transpose the results presented hereinafter in mass spectrometry to another technology and find the relevant peaks.
  • the term “signal” refers to any observable (statistical variable) or observable ratio, such as intensity, signal on noise, Tof ⁇ time offlighi area), half-height Tof ⁇ Tof Width), resolution, etc.
  • the markers characterized by the inventors three have been identified as the human neutrophil defensins HNP-2, HNP-I and HNP-3 (see experimental part, D-9). These defensins are overexpressed or expressed normally in controls, and under-expressed in trisomy 21 ( Figure 15).
  • at least one of the markers for which the assay is carried out is selected from HNP -2, HNP-I and HNP-3.
  • a high rate of the marker in question is indicative of a healthy fetus, while a low rate is indicative of a high probability of trisomy 21.
  • the inventors have found that the ratios between these defensins HNP-2, HNP-I and HNP-3 vary depending on whether the fetus has Down syndrome or not.
  • the longitudinal ratios HNP-1 / HNP-3 and HNP-2 / HNP-3 are statistically higher in case of trisomy 21 than when the pregnant woman is a carrier of a non-trisomic fetus.
  • These ratios which are particularly indicative, also have the advantage of being independent of the calibration of the measuring apparatus.
  • the inventors have furthermore found that the expression levels of the defensins HNP2, HNP1 and HNP3, as well as the ratios between these different levels are not correlated with the age of the pregnant woman, nor with the pregnancy week, nor with any markers currently used (AFP, uE3 and HCG in particular), nor with any of the other markers identified in the studies. reported here.
  • the ratio between the concentrations of HNP-3 and HNP-I and / or HNP -2 in the sample is measured.
  • these ratios can be measured from a spectrum obtained according to any of the techniques mentioned above. It is also possible to measure the expression ratios of defensins by a nucleic acid amplification technique, for example by quantitative or semi-quantitative RT-PCR, from the biological sample, or from RNA. extracted from this sample. Primers that can be used for this purpose are, for example, described in the article by Linzmeier, RM and T. Ganz. Human defensin gene copy number polymorphisms: Comprehensive analysis of independent variation in alpha- and beta-defensin regions at 8p22-p23 Genomics 2005 ).
  • the first detected marker is the sum of the defensins HNP-2, HNP-I and HNP-3.
  • This detection can in particular be carried out by immunological techniques, for example by ELISA or PCR.
  • HNP-I human neutrophil defensins selected from HNP-I, HNP-2 and HNP-3
  • HND-I, HND-2 and HND-3 also denoted HND-I, HND-2 and HND-3
  • Antibodies can be produced against the markers of the invention, by any technique, including monoclonal antibodies, chemical synthesis, biological production (animal route [example Bioprotein etc.], vegetal route [example Lemna-Biolex-Bayer] , Meristems Therapeutics also etc ...]). They can then be used, either to directly perform an immunoassay of the corresponding markers, or to purify the markers prior to their assay.
  • Immuno Assay Immuno Assay, or IA Assay: Different technologies, known to those skilled in the art, may be envisaged, such as the Sandwich Immunoassay (Sandwich IA), the surface interaction (Surface Plasma Resonance). , or SPR), or immunoprecipitation (eg by the Atto-Lab developed Q-MAP technology).
  • Sandwich Immunoassay Sandwich IA
  • surface interaction Surface Plasma Resonance
  • SPR Surface Plasma Resonance
  • immunoprecipitation eg by the Atto-Lab developed Q-MAP technology.
  • the Sandwich IA approach is based on the double recognition of the marker - or antigen - by specific antibodies: the so-called “capture” antibodies immobilized on the chosen support (micro-titration plate, magnetic ball, etc.), and the so-called “detection” antibodies, in solution and coupled to a reactive molecule, responsible for the emission of the binding signal (directly or indirectly).
  • This set of non-overlapping specific antibodies constitutes the reagents common to the various current detection systems.
  • antigen recognition can be accomplished using aptamers, which are single-stranded oligonucleotide (DNA or RNA) sequences selected in vitro for their binding capacity to a given molecule; this selection can be done, for example, by the SELEX method (Systematic Evolution ofLigands by Exponential Enrichment) as described in US Pat. No. 5,270,163.
  • a functionalized aptamer may, for example, be used in place of a secondary antibody in a Sandwich ELISA immunoassay, as described by Sumedha Jayasena (Clinical Chemistry 1999, 45 (9): 1628-50, FIG. 6).
  • Aptamers can be optimized by different modifications (Sumedha Jayasena, supra, Fig. 2).
  • aptans it is possible to modify the aptans so that activation by UV after binding of their target ensures the covalent bonding of this target to the support (photoaptamers).
  • the different components of a diagnostic device of the immuno-reaction type also depend on the detection system used. Some reagents and supports that can be used to implement the invention are summarized in Table 1 below, as well as examples of suitable instrumentation.
  • Protein chips can be designed to determine selected protein expression patterns involving the desired markers. This technology is presented in the review paper by Sydor and Nock (Proteome Science, 2003, June 10; 1 (1): 3). Of course, those skilled in the art can improve the techno ⁇ logies presented in this article by using his general knowledge in this field.
  • MAP technology Luminex Corp
  • Bio Bar Codes Nanotechnologies Inc.
  • LumiPhos markers 530 and 480 Lumigen
  • RCA Rolling-circle Amplification
  • MAP technology Luminex Corp combines flow cytometry and microparticles in an IFA (Immunofuorescent Assay) type format.
  • IFA Immunofuorescent Assay
  • This technology logie proposes a double fluorescence analysis.
  • the first detection system makes it possible to capture the fluorescence emitted by the capture beads of the markers (one ball corresponds to one marker and one fluorescence); the second makes it possible to measure the fluorescence associated with the detection antibodies, that is to say with the quantification of the markers.
  • Bio Bar Codes (Nanotechnologies Inc.) Combine Nanoparticles and Scanning (Silver), Introducing a New Detection System Based on Hybridization of Unique Nucleic Acid Probes (Marker Identification)
  • LumiPhos 530 and 480 (Lumigen) markers combine chemiluminescent and fluorescent detection systems, thereby ensuring signal optimization.
  • RCA rolling-circle amplification
  • This technolo ⁇ gy described by Schweitzer et al involves the polymerization of a long chain of single-stranded DNA attached to the analyte (eg, an antibody) from a circular DNA template. This technology minimizes sample volumes and the use of expensive materials (monoclonal antibodies and enzymes).
  • signal amplification systems as another category of reagent in particular in the context of IFA and CLIA (eg biotin / avidin).
  • the markers can also be detected by a nucleic acid amplification technique by polymerase chain reaction (PCR or other technique).
  • PCR polymerase chain reaction
  • the invention makes it possible to detect chromosomal abnormalities, in particular Down syndrome (trisomy 21), Edwards syndrome (trisomy 18), Patau syndrome (Trisomy 13), Turner syndrome, Klinefelter syndrome, monosomy X, fragile X syndrome, and penta X syndrome and a deletion of the long arm of chromosome 7, but also other congenital anomalies, currently detected by ultrasound, for example Spina Bifida.
  • the invention can be applied to the detection of genetic or infectious (viral, bacterial, or parasitic) or metabolic fetal diseases, or to the search for a specific phenotype and in particular the sex of the fetus.
  • the invention is particularly suitable for the prenatal diagnosis of Down syndrome.
  • the present invention is applicable to prenatal screening during the second trimester, or earlier, during the first trimester of pregnancy.
  • the screening methods according to the invention may also include an initial step of preparing the sample.
  • this step may comprise fractionation as a function of the mass of the proteins, and / or a stage of depletion of albumin or other transport proteins, and / or a step of purifying the proteins on an anion-type preparative surface and / or hydrophobe.
  • the present invention also relates to a prenatal diagnostic kit for chromosomal abnormalities, comprising means suitable for carrying out one of the methods described above.
  • a prenatal diagnostic kit may for example comprise specific antibodies and / or aptamers of one or more markers mentioned above and / or amplification primers as well as probes.
  • each antibody, aptamer or primer is specific for a single marker.
  • each solution is preferably a specific single marker, or a group of associated markers (for example, defensins HND-I, HND-2 and HND-3).
  • anti-HND antibodies such as those marketed by Tebu-Bio SA (39 rue de Houdan, F-78612 Le Perray en Yvelines ) under the reference 038NCL-DEFENSIN (Anti-Neutrophil Defensin Mouse IgG1 Clone D21, 1 ml).
  • a diagnostic kit comprises, for at least one of the markers of the invention, a set of non-overlapping specific antibodies, that is to say at least two antibodies capable of simultaneously recognizing the marker by question, which makes it possible to carry out sandwich-type immuno-reaction tests.
  • the antibodies in particular the so-called “capture” antibodies, can be immobilized on a support, and the so-called “detection” antibodies coupled to a molecule allowing the detection.
  • supports and molecules for detection usable in the context of the invention, are mentioned above.
  • the invention also comprises other arrangements, which will emerge from the description which follows, which refers to examples of implementation of the method which is the subject of the present invention and to the appended drawings, in which which:
  • FIG. 1 illustrates the experimental strategy used
  • FIG. 2 illustrates a schematic preparation of the samples
  • FIG. 3 summarizes the different recording conditions, as a function of the molecular mass of the proteins to be detected
  • FIG. 4 illustrates the experimental scheme used in studies E1, E2, E3, E4 and E6;
  • FIG. 5 represents the spectra obtained on the spectral zone
  • FIGS 6-8 illustrate the spectra centered on the 4000 Dalton area (FIG. 6: spectra in global views, FIG. 7: pseudo-gels, FIG. 8: spectra in staggered views);
  • FIGS. 9-11 illustrate the spectra centered on the area 5000-6000 Daltons (FIG. 9: spectra in global views; FIG. 10: pseudo-gels; FIG.
  • Figures 12 to 14 illustrate the spectra centered on the spectral zone 3000-10000 Daltons (Figure 12: spectra in global views, Figure 13: pseudo-gels, Figure 14: views offset); FIG. 15 illustrates the spectra centered on the area 3000-4000
  • FIGS. 16 to 18 illustrate the spectra respectively centered on the areas 4000-5000 Daltons, 5000-7000 Daltons and 7000-10000 Daltons (spectra in global views);
  • FIG. 19 illustrates the spectra in global, standardized and superimposed views by clinical category, for the control group, the T21 group, and the Tl 8 group, on the 3000-10000 Dalton spectral zone (study E6);
  • FIGS. 20 and 21 show, for the same spectral zone as that of FIG. 19, the pseudo-gel views (FIG. 20) and the staggered views (FIG. 21) only for the control group and the T21 group;
  • FIGS. 22-24 show spectra on the spectral zone 3000-2000 Daltons, respectively in global views (standardized and superimposed spectra by clinical category) for the control groups, T21 and Tl8, in pseudo-gel views for the control groups and T21, and in staggered views for the same groups (study E6);
  • - Figure 25 shows the spectra centered on the 6500 zone, after normalization with respect to the intensity of the PM6622 cluster (studies E4 and E5). This figure shows the superposition of the spectra for the control and T21 categories (study E6);
  • FIG. 26 shows the spectra centered on the 6500 zone after normalization on the PM6622 peak (standardized and superimposed spectra by clinical category), for the T21, control, and Tl groups.
  • the peak referenced N corresponds to PM6622 cluster and is used to standardize spectra;
  • FIGS. 27 to 30 show examples of distribution statistics of the intensities of different clusters, for the control and T21 groups:
  • FIG. 27 peak (cluster) PM3371, anionic separation phase, any pH> 4.
  • FIG. 28 PM3442 peak, anionic separation phase, all pH> 4.
  • FIG. 29 PM3486 peak, anionic separation phase, all pH> 4.
  • FIG. 30 PM6423 peak, hydrophobic separation phase, normali ⁇ tion on the intensity of the PM6620 peak (PM is represented by MW in this figure);
  • FIG. 31 shows the ROC curves for the molecular weight markers 3371, 3442, 3486, and 6423 Da (MW is represented by MW in this figure);
  • FIG. 32 shows the result of a build with the Genecluster Software Version 1999 (Eisen M.) and Treeview Version 1.60 (Eisen M.) (Hierarchical classification on Data Set of the E6 study (95% CI) , Anionic Surface Q10);
  • Fig. 33 illustrates the principal component analysis for defensins (anion exchange surface Q10)
  • FIG. 34 illustrates the procedure for identifying the clusters PM6423, PM6439, PM6620 and PM6636 (PM is represented by MW in this figure).
  • FIG. 35 shows mass spectra acquired under conditions Q10, pH4, crude serum, -80 ° C., for the zone corresponding to the HNPs, for a control group and a group of sera of women carrying fetuses carrying trisomy 21. .
  • FIG. 36 presents the statistical data on the HNP, in signal on noise, in the form of "box plots" (box plots).
  • A signal on noise of each peak.
  • B longitudinal ratios of the signals / noise.
  • FIGS. 37 and 38 show the binormal ROC distributions and curves corresponding to each HNP (Fig. 37A: HNP1;
  • FIGS. 39 and 40 show mass spectra acquired under the same conditions as the spectrum of FIG. 35, centered on different zones.
  • FIG. 41 shows the distributions and ROC curves bi-normals associated with peaks other than PST identified as discriminants in QlO condi ⁇ tions, pH4, crude serum -8O 0 C.
  • FIG. 42 summarizes the data (in signal / noise) of the relevant peaks (other than the HNP) of the NS502 study, in the form of boxplots (box plots). Isolated points represent abnormal (outliers) values that fall outside the confidence interval.
  • FIGS. 43 to 48 show spectra acquired under purification conditions on H50, for certain zones identified as relevant in the studies NS526c and NS526d.
  • Figures 49 and 50 present the data for each peak (from NS526c and NS526d, respectively) in the form of distribution curves and ROC curves.
  • FIG. 51 and 52 show the results, in the form of box plots, for the markers identified in NS526c and NS526d.
  • Week of pregnancy adjusted to the number of days of the week at harvest number of days of gestation
  • Sex of the fetus, - Biological markers: hCG in IU / ml, uE3 in pg / ml, ⁇ -fetoprotein in IU / ml, the 3 converted into a multiple of the median (MoM measured value / median value),
  • Fetal karyotype outcome (conventional techniques) Outcome of pregnancy (birth, IMG, MFIU fetal death in utero, possibly histopathological findings).
  • the samples are partially depleted of abundant proteins such as albumin or immunoglobulins. Enrichment of Proteins Associated with Transport Molecules The transport proteins and the molecules associated with them are purified, thereby increasing the relative concentration of the molecules transported in the fraction of the sample analyzed. Serums fractionated according to native mass
  • Raw or partially depleted or enriched samples are fractionated under native conditions by separation as a function of protein mass. Synthesis of sample preparation
  • biochemical properties specific to each protein are used in order to separate them from each other according to their apparent surface charges (ionic properties) and their hydrophobicity. In parallel, their relative concentration within the sample and / or the analyzed fraction of the sample is studied. Four different approaches are used: Normal phase chromatography
  • a positively charged functional group e.g., quaternary amines
  • anionic character negatively charged
  • Two variable parameters are generally explored: the pH at which the incubation-association phase takes place and the degree of stringency of the eluent used.
  • a negatively charged functional group eg, carboxylate group
  • a known cationic character positively charged
  • various experimental combinations are applied to the association and elution phases. Hydrophobic interactions Hydrophobic interactions release structured water surrounding hydrophobic sectors of biomolecules. This has the effect of increasing the entropy, making the thermodynamically favorable interactions.
  • a chromato ⁇ graphy using a carbon chain for example: C 18 ) can be used. The association and elution conditions depend on the degree of hydrophobicity of the eluent used. Targeted purification
  • Two experimental schemes were used for a targeted approach to protein purification.
  • the biological characteristics of the proteins to be purified reveal the capacity of association with a metal ligand and / or chemical.
  • the identity of the protein to be isolated is known, allows to use an immune strategy, thus using an antibody specific for the targeted protein.
  • Laser desorption mass spectrometry Proteins are deposited on an active surface (SELDI mass spectrometry - Surface Enhanced Laser Desorption Ionisation), or passive (MALDI mass spectrometry - Matrix Assisted Laser Desorption Ionisation), associated with a chemical matrix allowing, under vacuum, their ionization by a laser beam. Once ionized, they are accelerated and directed under electrostatic stress to a detector placed at the end of a vacuum tube. The speed at which they reach the detector (TOF - Time of Flighf) is proportional to the square root of their mass. y Electrospray mass spectrometry
  • the proteins are nebulized and then ionized under the action of an inert gas, passing through a very fine, highly charged needle.
  • the analyzer is most often a "TOF" of the same type as for laser desorption mass spectrometry.
  • the recordings and the analysis of the spectra are carried out according to different protocols, which depend on the surface and the apparatus (laser intensity, mass sensitivity of the detector, gain of the detector, adjustment of the deflector). These parameters are optimized according to the zone of the mass range to be analyzed.
  • the standardization factors used are mainly Total Current Ion (TIC), some markers having a specific standardization.
  • Figure 3 summarizes the different recording conditions, depending on the molecular weight of the proteins to be detected.
  • the spectra and the associated clusters are annotated according to two complementary procedures, one automatic, the other manual pertaining to the art of signal processing. The two procedures are then confronted with each other.
  • the samples were either analyzed native unfractionated or analyzed fractionated according to the mass of proteins.
  • the eluates were used either native or analyzed after albumin depletion or other transport proteins, and then separated and purified in two modes, one anionic, the other hydrophobic.
  • An analyzer of the SELDI-TOF mass spectrometer type was used to acquire the spectral information. Interest, Associativity and Purification on anionic surface
  • the spectra presented are obtained on anionic separating surface.
  • the peaks of interest mentioned are present at pH 4 and at any pH value greater than 4, insofar as no protein aggregation or precipitation phenomenon occurs at basic pH.
  • FIG. 4 shows the experimental scheme that was used.
  • Protein chips were prepared according to the following procedure:
  • the protein chips are inserted into the mass spectrometer or any other appropriate reading or sequencing apparatus.
  • the different separation and purification conditions used and their preparation protocols are summarized in Table 2 above.
  • the protein chips are inserted into the mass spectrometer for reading according to the different intensities described above and in FIG. 3, or any other suitable reading device, optimized and adjusted accordingly.
  • the skilled person will choose the intensity of the laser to allow the proteins to fly, depending on their mass, some of their physicochemical properties, and any interactions they establish with partners.
  • the antibodies used have been described above.
  • Calibrators used on Normal Phase / Silicon Oxide and Maldi mode are as described in Table 3.
  • Fraction A molecular weight ⁇ 10 kDa
  • Fraction B 10 kDa ⁇ molecular weight ⁇ 50 kDa
  • Fraction C 50 kDa ⁇ molecular weight ⁇ 100 IcDa
  • Fraction D molecular weight> 100 kDa
  • Mass fractionation is intended to increase the relative protein concentration of a fraction relative to the total protein concentration without altering the absolute concentration of the separated proteins.
  • albumin is normally found as a major element, and can be specifically deleted if necessary. This deletion was not performed in the experiments described here; the removal of albumin will detect an additional peak corresponding to ⁇ -fetoprotein, which is currently masked by the albumin peak.
  • Vivaspin 500 from Vivascience with 3 distinct cutoffs: 10, 50 and 100 kDa.
  • the separation by filtration on membrane takes place in PBS IX, pH 7.4, in non-denaturing conditions. All that passes through the membrane is of smaller mass than the cutoff threshold, all that remains in the dead volume above the membrane is of mass greater than the cutoff threshold.
  • the proteins remaining in the upper part of Vivaspin 500 with a cut-off threshold of 100 kDa therefore have a native mass> 100 kDa. This is also true of heterogeneous multimeric complexes as well as homogeneous ones.
  • C - 3 Instrumentation (Example: Study E6 - Anionic Purification) Acquisition Parameters
  • the Total Ion Currenf (TIC) standardization method allows for each spectrum to average the intensities, and adjusts the average intensities of each spectrum so that the data is normalized.
  • the normalization parameters used corresponded to a "Total Ion Current” normalization starting at 3000, and ending at 20000, with subtraction of the baseline, a normalization coefficient of 0.2691 12, and without normalization to ground.
  • the calibration is made according to the mass range observed. In zone 3 at 20 kDa, three calibrations are used, depending on whether the calibrator is: Cytochrome C bovine insulin, bovine ⁇ -lactoglobulin-A, bovine
  • the calibration is made on insulin peaks (bovine) (5733.58 + 1H), and bovine insulin 2H + (2866.79 + 1H).
  • the calibration equation used is quadratic: m / z
  • the clustering consists in grouping the value of the intensity of the peaks corresponding to this protein for all the recorded spectra.
  • a cluster is therefore defined as a table returning the value of the intensity of the peak considered for each spectrum studied.
  • Controls Control
  • DS Down Syndrome
  • ES Edward Syndrome
  • the peaks are manually annotated.
  • the clusters are then completed by searching within a window of ⁇ 0.3% of the M + H value of a signal such as signal / noise - 2, otherwise the local background noise is used.
  • the viewing area is between 3 000 Da and 20 000 Da
  • cytochrome C (bovine) is used to calibrate.
  • ⁇ -lactoglobulin A (bovine) is used to calibrate.
  • the choice of the calibrator does not influence the intensity values in any way. Their values do not vary according to the calibration used.
  • the calibration makes it possible to increase the accuracy of the values of the masses of the peaks recorded, over a restricted spectral zone.
  • other calibrators may be used. Table 12 below presents a list of calibrators that can be used, as well as their molecular weight.
  • Tables 13 and 14 in the data obtained from the spectra obtained (one spectrum per sample of the populations of the three groups Control, DS (T21) and ES (Tl 8), out of group -80 ° C, except for the special case of the samples A7 and Al 5, recorded in triplicate during the study E6).
  • the number of manually annotated clusters is reduced according to the criterion "present in at least 30% of the spectral population".
  • Table 16 indicates for a few clusters observed during the experiments of the E6 study, the flight time (TOF), the p value of the cluster and for each group (Control, T21, Tl 8 ), the mean, minimum and maximum intensities as well as the standard intensity deviation.
  • FIG. 5 shows the spectra obtained in the spectral zone 3000-2000 Daltons, in global views, for the control group, the T21 group and the Tl 8 group.
  • the standardized spectra are superimposed thereon by clinical category.
  • Figures 6, 7 and 8 show, respectively, the spectral views (normalized and superimposed spectra by clinical category), the pseudo-gel views, and the shifted view spectra (study E6), centered on the area 4000 Da.
  • Figures 9, 10 and 11 show, respectively, the spectral views (normalized and superimposed spectra by clinical category), the pseudo-gel views, and the offset-view spectra (study E6), centered on the 5000-6000 Da area.
  • FIGS. 12 to 18 show the spectra centered on the spectral area 3000-10000 Daltons, respectively in global views (standardized and superimposed spectra by clinical category), pseudo-gel view, and offset views (El to E5).
  • Figure 15 shows the spectra centered on the spectral area 3000-4000 Daltons (E1 to E5).
  • Figures 16 to 18 show the spectra, normalized and superimposed by clinical category, respectively centered on the areas 4000-5000 Daltons, 5000-7000 Daltons, and 7000-10000 Daltons (studies E1 to E5). Profiles obtained by separation according to the hydrophobicity of the H50 proteins (study E6)
  • Figure 32 shows the result of a build with Genecluster Version 1999 (Eisen M.) and Treeview Version 1.60 (Eisen M.) software (Hierarchical Classifi cation on Data Set of Study E6 (95% CI), Anionic surface Q10).
  • Construction method Data adjustment by logarithmic transforma ⁇ tion then normalization in order, according to the spectra then the clusters. Then hierarchical clustering simultaneously on Spearman's clusters and correlation spectra.
  • the software used is Stata SE 8 software and Ciphergen Express Version 1.0 software. Both software confirm the results.
  • the eigenvalues confirm the presence and the molecular weight of the peaks identified as defensins whose masses are: PM3371, PM3442 and PM3486.
  • results obtained for the peaks in the 3000 Dalton zone, on anionic surface Q10 are shown in Table 17. These three peaks are under-expressed or absent in the T21 group, reflecting a significant decrease in immunity, and expressed normally. or over-expressed in the control group. Further study of the results will determine the detection threshold for these three peaks, which are correlated and dependent, such that at least one negative sign among the three or one positive sign among the three signifies the disease or absence of disease, respectively.
  • the results presented in Table 17 show variability for these three peaks, mainly related to the fact that they were obtained with samples stored at -2O 0 C, showing some degradation.
  • Table 18 presents the results and applications for the peaks in the 4000 - 9000 Dalton area, on anionic surface Q10.
  • Table 19 shows the results and applications for peaks in the 4000 -17500 Dalton area, on H50 hydrophobic surface.
  • HNPs Human Neutrophil Defensins
  • peptides have correlated biophysicochemical properties. They are present in a native fraction of the samples such that the mass of the products is between 50 and 100 kDa as well as in a fraction of native mass greater than 100 IcDa. They are thus peptides associated with several distinct proteins, such that the mass of the complexes thus constituted is between 50 and 100 kDa, or greater than 100 kDa. Considerations about mass deviations and possible identification
  • PM6636 - PM6439 197 Da +/- IDa valine + proline (196 Da) or Proline + Threonine (198 Da)
  • PM6636 - PM6423 181 Da +/- IDa ⁇ No combination of amino acids
  • PM6620 - PM6439 181 Da + / - IDa ⁇ No amino acid combination
  • PM6620 - PM6423 197 Da +/- IDa -> valine + proline (196 Da) or proline + threonine (198 Da)
  • PM6439 - PM6423 16 Da Possible hydrolysis of a proline in hydroxyproline
  • the mass peaks PM 3371, PM3442, PM 3486, identified as defensins are under-expressed in the T21 groups and overexpressed or normally expressed in the controls.
  • the mass peaks PM 6423, PM 6439, PM6620, PM6636 are identified in pairs by hydrolysis of a proline to hydroxyproline (+16 Da).
  • the inventors have calculated the longitudinal ratios ⁇ le., Between 2 peaks of the same spectrum) between the intensities of the peaks corresponding to the different HNPs. Remarkably, they observed, only in native serum condition, a significant difference in these ratios, depending on whether it is a sample of a woman carrying a fetus with trisomy 21 or a woman with a healthy fetus.
  • the results of three of these studies are presented below, as well as in Figures 35 to 38.
  • the studies whose results are presented in detail were made from three datasets. They are identified as follows:
  • IC or CI confidence interval
  • Dataset dataset
  • Obs. number of samples observed
  • GC or CG control group
  • Down Down syndrome
  • Err. Std. standard error
  • SN signal on noise
  • Avg. average
  • AUC Area under curve, or area under the curve
  • var AUC variance of AUC
  • pAUC (8%) Partial Area Under Curve, a false positive rate (FP) of 8% was imposed on the Dataset.
  • Cluster 3 Dataset
  • Cluster 4 Cluster 5 (Longitudinal Ratio HNPx / HNPy)
  • Cluster Cluster 3 Cluster 4
  • Cluster 5 Longitudinal Ratio HNPx / HNPy
  • the peak intensity ratios of HNP-1 / HNP-3, and HNP-2 / HNP-3 are particularly discriminatory markers for detecting pregnant women who may carry a fetus with Down syndrome.
  • the peaks at 4859.10 Da and 8195.65 Da respectively correspond to the peaks at 4860 Da and 8206 Da identified in the preliminary studies (see Table 14 above).
  • the peaks at approximately 4068.5 Da, 4320.79 Da, 4619.80 Da, 8107 Da, 8269.10 Da and 8610.30 Da respectively correspond to the peaks at 4067 Da, 4317 Da, 6423 Da, 8112 Da, 8263 Da, and 8627 Da identified in the preliminary studies (see Table 14 above), and the doublet at 4642.66 Da and 4658.47 Da corresponds to the peak at 4650 Da identified initially.
  • Peaks 36, 37 and 38 of the NS526c study represent multicharged proteins corresponding to the proteins revealed by the peaks 8, 9 and 11 of this same study. Similarly, peaks 59, 60 and 61 of study NS526d correspond to peaks 20, 21 and 24.
  • Figures 43 to 48 show spectra on which some of the above-mentioned peaks occur in H50 purification cases.
  • Figures 49 and 50 present the data for each peak (from NS526c and NS526d respectively) as distribution curves and ROC curves.
  • Figures 51 and 52 show the results for these new markers, in the form of box plots. The points outside the boxes represent the aberrant points.
  • MassStart 880
  • MassRange: MassEnd 25118.9
  • CutOffMass 900
  • Table 42 shows the performances (sensitivity, specificity, etc.) of a combination of two ratios between the HNP defensins, and a combination of these same ratios with the hcg, on the dataset. NS502.
  • Other associations can be made between the different markers identified by the inventors.

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WO2021004811A1 (en) 2019-07-05 2021-01-14 Agfa-Gevaert Nv A separator for alkaline water electrolysis
EP3933069A1 (de) 2020-07-03 2022-01-05 Agfa-Gevaert Nv Separator für alkalische wasserelektrolyse
WO2022002999A1 (en) 2020-07-03 2022-01-06 Agfa-Gevaert Nv A separator for water electrolysis
WO2023280600A1 (en) 2021-07-08 2023-01-12 Agfa-Gevaert Nv A separator for alkaline water electrolysis
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Publication number Priority date Publication date Assignee Title
WO2021004811A1 (en) 2019-07-05 2021-01-14 Agfa-Gevaert Nv A separator for alkaline water electrolysis
EP3933069A1 (de) 2020-07-03 2022-01-05 Agfa-Gevaert Nv Separator für alkalische wasserelektrolyse
WO2022002999A1 (en) 2020-07-03 2022-01-06 Agfa-Gevaert Nv A separator for water electrolysis
WO2022002904A1 (en) 2020-07-03 2022-01-06 Agfa-Gevaert Nv A separator for alkaline water electrolysis
WO2023280600A1 (en) 2021-07-08 2023-01-12 Agfa-Gevaert Nv A separator for alkaline water electrolysis
WO2023280760A1 (en) 2021-07-08 2023-01-12 Agfa-Gevaert Nv A separator for alkaline water electrolysis
WO2023280598A1 (en) 2021-07-08 2023-01-12 Agfa-Gevaert Nv A separator for alkaline water electrolysis

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