DIAGNOSTIC MARKERS FOR CROHN'S DISEASE

Abstract

The invention relates to markers for Crohn's disease and to specific compounds for detecting said markers. The invention also relates to methods for detecting, diagnosing and monitoring the progression of Crohn's disease as well as to methods for evaluating the efficacy of a treatment for said disease, and to compositions and kits that can be used for implementing said methods.

Claims

1-8. (canceled)

9. A method for detecting the presence of a bacterium in a subject suspected of having Crohn's disease comprising detecting the expression of a peptide sequence comprising an amino acid sequence selected from SEQ ID NO: 2, SEQ ID NO: 1, SEQ ID NO: 3 and a 7 to 25 amino acid fragment thereof, or comprising a nucleic acid encoding said peptide sequence by a bacterium, or a portion of said bacterium in a sample comprising fecal matter, fecal water and/or intestinal epithelial tissue from a subject to be tested for Crohn's disease.

10. The method according to claim 9, characterized in that the fragment of the amino acid sequence comprises SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 or SEQ ID NO: 13.

11. The method according to claim 9, characterized in that the detection of said at least one bacterium or a portion thereof is carried out by targeted quantitative proteomics.

12. The method according to claim 9, characterized in that it comprises: contacting, in vitro or ex vivo, a sample comprising fecal matter, fecal water and/or intestinal epithelial tissue from the subject to be tested with a compound binding all or a portion of said at least one bacterium; and detecting the bond between said compound and all or a portion of said at least one bacterium when it is present in the sample.

13. The method according to claim 9, characterized in that the subject is an animal.

14. The method according to claim 13, wherein said animal is a human.

15. The method according to claim 14, wherein said human has a chronic digestive disorder, an unclassified chronic inflammatory bowel disease (IBD), or a family history of IBD.

16. A method for obtaining information useful in the diagnosis of Crohn's disease comprising implementing, in vitro or ex vivo, a detection method according to claim 9 on a sample comprising fecal matter, fecal water and/or intestinal epithelial tissue from a subject to be tested, characterized in that the absence of the bacterium or a portion thereof, or the detection of the bacteria or portions thereof, in a quantity below a control value within the sample, makes it possible to diagnose Crohn's disease in said subject, the presence within the sample of the bacteria or portions thereof in a quantity above said control value making it possible, conversely, to exclude the existence of Crohn's disease in said subject.

17. A method for evaluating, in a subject, the therapeutic efficacy of a treatment for Crohn's disease, said method comprising implementing, in vitro or ex vivo, a detection method according to claim 9 on a sample comprising fecal matter, fecal water and/or intestinal epithelial tissue from a subject treated for Crohn's disease, the appearance of or an increase in the quantity of bacteria or portions thereof revealing the efficacy of said treatment, and the disappearance of or the reduction in the quantity of bacteria or portions thereof revealing, conversely, the inefficacy of said treatment, when compared with a control value, for example when compared with the quantity of bacteria or portions thereof detected in said subject before said subject was treated for Crohn's disease.

Description

FIGURES

[0090] FIG. 1: Identifiers, sequences and functional and phylogenetic annotation of three proteins SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 (mass matching against the MetaHIT-2009 databank, reannotated 2014). The peptides in bold are those seen in label-free LC-MS/MS shotgun experiments. The peptides underlined and in bold indicate the specific peptides targeted in SRM. The vertical bars delimit the peptides seen in shotgun experiments or targeted in SRM.

[0091] FIG. 2: Sequence alignment of the three proteins having the sequences SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 (AlignUniprot—http://www.uniprot.org/blast/)

[0092] FIG. 3: Search for the protein domains of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 in the InterPro database (EMBL-EBI—http://www.ebi.ac.uk/Tools/pfa/iprscan5/)

EXAMPLES

[0093] The inventors discovered and validated, in patients in remission or having moderate flare-ups, a panel of bacterial and human protein signals associated with Crohn's disease (Juste et al., 2014). The bacterial proteins and a few human proteins adhering to the bacterial cells are extracted from the subjects' stools. Among the myriad of proteins present, it was shown, surprisingly, that some are remarkably less abundant in the patients compared with healthy controls, matched for age, sex and tobacco use.

[0094] Three microbial proteins were discovered as being more abundant in the healthy subjects compared with the ill by a label-free shotgun tandem mass spectrometry method developed by the inventors (Guillot et al., 2013). Identification of the proteins rests on the mass matching technique and on querying the MetaHIT metagenomics database (Qin et al., 2010) using the bioinformatics tool X!TandemPipeline (http://pappso.inra.fr/bioinfo/xtandempipeline/). Their identifiers in the MetaHIT 2009 database reannotated in February 2014, their sequences, and the peptides seen in mass spectrometry and having allowed their identification appear in FIG. 1.

[0095] Materials and Methods

[0096] Twelve intestinal microbiotas (all intestinal microbial populations) are extracted from 12 fresh stool samples (w=2 g) collected from 6 Crohn's patients in remission and 6 controls with no intestinal pathology. The extraction procedure is that described by the inventors (Juste et al., 2014). It is based on harvesting bacterial cells at their floating density within a continuous Nycodenz gradient, pre-formed by freezing/thawing. The stool sample weighed down in Nycodenz is deposited beneath the preformed gradient and the bacterial cells return to their floating density during low-speed centrifugation (14,567×g, 45 min, 4° C.). The anaerobic nature and the integrity of the bacteria are thus preserved throughout the extraction process. The bacterial pellets, washed in 20 mM Tris, 138 mM NaCl, 2.7 mM KCl, 0.03% (w/v) Na-deoxycholate, pH 7.4, are kept at −80° C. A 1.5 ml volume of lysis buffer (8.75 M urea, 2.5 M thiourea, 5% (w/v) CHAPS, 75 mM DTT, and 31.2 mM spermine dihydrate base) is deposited on each still-frozen pellet. The chemical lysis is performed at room temperature for 1 h during which the samples are vortexed vigorously every 10 min. The lysates are centrifuged at very high speed (245,419×g, 1 h, 18° C.) and the supernatants are collected and brought to neutral pH with concentrated HCl. The proteins are purified with the PlusOne SDS-PAGE Kit (GE Healthcare), then assayed with the 2-D Quant Kit (GE Healthcare). All the samples are brought to a protein concentration of 4 μg/μl with Laemmli denaturing buffer.

[0097] For the discovery step, we use a label-free shotgun proteomics method with prefractionation of the sample on SDS-PAGE gel for better coverage of the proteome. Briefly, 60 μg of proteins from a Crohn's patient and 60 μg of proteins from a healthy subject migrate for about 45 min in an SDS-PAGE gel (4-12% NuPAGE Novex gel, 200V, 110 mA) mounted on an XCell SureLock™ device (Invitrogen). After migration, the gel is rinsed with water, stained with Coomassie blue and scanned. Each of the two lanes is cut into 20 equivalent bands and each of the 40 pieces of gel is subjected to reduction, alkylation and tryptic digestion according to a standard protocol (Shevchenko et al., 2007). Tryptic peptides are extracted from the gel with 50% acetonitrile, 0.2% formic acid, then dried under vacuum (SpeedVac), taken up in 25 μl of HPLC buffer (0.1% formic acid, 2% acetonitrile) and analyzed individually (i.e., 40 analyses in total) by coupled LC-MS/MS (NanoLC Ultra System, Eksigent) connected to a mass spectrometer (Q Exactive, Thermo). To that end, 4 μl of peptide extract is loaded on a precolumn (stationary phase: PepMap 100 C18, 5 μm; column: 300 μm ID, 5 mm, Dionex) with a 7.5 μl/min flow rate. After 3 min, the precolumn is connected to a separation column (stationary phase: C18 Biosphere, 3 μm; column: 75 μm ID, 150 μm, NanoSeparations) and the peptides are eluted by using a linear gradient (5-35%) of buffer B (80% acetonitrile, 0.1% formic acid) in buffer A (2% acetonitrile, 0.1% formic acid) for 40 min, for a total acquisition time of 50 min per piece of gel. Ionization is carried out via a ‘PicoTip’ needle emitter (20 μm ID, 360 μm OD, New Objective). The peptide ions are detected automatically in data-dependent mode with the following parameters: “full scan” MS (m/z 400-1400 Th) to measure the m/z ratio of the peptides and fragmentation of the peptides observed with a normalized collision energy set to 30% in HCD, under the control of the Xcalibur software (version 2.1, Thermo). In this study, only the doubly and triply charged precursor ions were selected to be fragmented in MS/MS with an exclusion window of 40 sec while having activated the auto-calibration mode on the ion 445.12003 (dimethylcyclosiloxane).

[0098] We use the X!Tandem software to compare the measured masses of the fragmented peptides with the theoretical fragmentation masses generated from the MetaHIT database [3,299,822 complete or incomplete ORFs (Qin et al., 2010) reannotated for their function and their taxonomy in February 2014], combined with the UniProtKB/Swiss-Prot H. sapiens database and our own contaminant database. The search parameters and the validation thresholds for the peptides and the proteins are those described recently by the inventors (Juste et al., 2014). To finish, we use the X!TandemPipeline software (http://pappso.inra.fr/bioinfo/xtandempipeline/) to summarize the results in the form of tables giving the number of fragmented peptide ions attributed to each protein, as well as the sequence of these peptides. By inference, this number makes it possible to estimate the quantity of said protein in several samples. In the present invention, and by this method called “spectral counting”, the total number of fragmentation spectra attributed to each protein was summed for the 20 fractions of the “Crohn” sample on the one hand, and the 20 fractions of the “Healthy” sample on the other hand. The sum of the spectra attributed to the proteins SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 is remarkably low in the “Crohn” sample compared with the “Healthy” sample.

[0099] To confirm these results, the 6 “Crohn” samples and the 6 “Healthy” samples prepared above (100 μg of purified protein from each sample) were analyzed by the targeted proteomics method called selected reaction monitoring (SRM) with labeling, detailed by the inventors (Juste et al., 2014). Tryptic peptides specific for (not shared with other proteins within the MetaHIT database) the proteins SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 are identified. Among them, the peptides already identified in label-free shotgun experiments and having fragmentation spectra of very good quality are singled out. The selected peptides have an ideal length of 7 to 25 amino acids and contain neither miscleavage nor methionine. The selection of transitions, the carrying out of microLC-SRM assays with labeling, the data analysis, the quality controls and the statistical analyses are those detailed by the inventors (Juste et al., 2014). In the present invention, and by this SRM method with labeling, the low abundance of the peptide sequences SEQ ID NO: 4 to SEQ ID NO: 13 and, by inference, the low abundance of the protein sequences SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, are confirmed among the ill subjects relative to the controls.

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