Compositions and methods for detecting non-celiac gluten sensitivity

Abstract

To provide compositions and methods for detecting proteins associated with non-celiac gluten sensitivity (NCGS). Such markers may be useful for individuals susceptible to NCGS to manage their food intake to avoid symptoms and further progression of the disease. In one embodiment, the method includes obtaining a sample from an individual and measuring the amount of at least one of IL-8, IL-10, TNF-α, or total IgE protein in the sample.

Description

[Technical field]

[0001] Related Applications This application claims priority to U.S. Provisional Patent Application No. 62 / 505,378, filed May 12, 2017, the contents of which are incorporated by reference in their entirety herein.

[0002] Field of Disclosure The present disclosure relates to methods and compositions for diagnosing non-celiac gluten sensitivity. [Background technology]

[0003] background Celiac disease is an autoimmune disorder with genetic, environmental and immunological components. Symptoms of celiac disease can be triggered by oral ingestion of wheat gluten and certain associated proteins of rye and barley. Such symptoms can include inflammation of the small intestine, villous atrophy and crypt hyperplasia. However, there are certain individuals who do not have the characteristic serological or histological evidence of celiac disease but experience a constellation of symptoms in response to oral ingestion of wheat. The terms non-celiac gluten sensitivity (NCGS) and non-celiac wheat sensitivity (NCWS) are commonly used to refer to this condition.

[0004] The biological mechanisms behind NCGS are unknown, and few, if any, biomarkers provide reliable indications of the condition. Nevertheless, adjusting the diet to avoid triggering the onset of symptoms and / or promoting further progression of the disease would help individuals susceptible to NCGS. Thus, there is a need to develop and evaluate biomarkers for NCGS. Summary of the Invention [Problem to be solved by the invention]

[0005] Abstract The present disclosure can be embodied in a variety of ways.

[0006] In one embodiment, a method is disclosed for detecting biomarkers associated with non-celiac gluten sensitivity (NCGS) in an individual, comprising obtaining a sample from the individual; and measuring the amount of at least one of IL-8, IL-10, TNF-α or total IgE protein in the sample.

[0007] Other features, objects, and advantages of the disclosure herein will be apparent in the detailed description, drawings, and claims that follow. It should be understood, however, that the detailed description, drawings, and claims, while indicating embodiments of the disclosed methods, compositions, and systems, are provided by way of illustration only and not by way of limitation. Various changes and modifications within the scope of the invention will become apparent to those skilled in the art.

[0008] drawing The present disclosure may be better understood in consideration of the following non-limiting drawings. [Brief description of the drawings]

[0009] [Figure 1] Figure 1 shows an example of a multi-node interaction network that identifies markers associated with NCGS. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] Detailed Description Terms and Definitions In order that this disclosure may be more readily understood, certain terms are first defined. Further definitions of the following terms, as well as other terms, are set forth throughout the specification.

[0011] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the present disclosure are approximations, the numerical values ​​set forth in the specific examples are reported as precisely as possible. However, numerical values ​​inherently contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements. Moreover, all ranges disclosed herein are to be understood to encompass any and all subranges subsumed therein. For example, a stated range of "1 to 10" should be considered to include any and all subranges between the minimum value of 1 and the maximum value of 10 (including both maximum and minimum values); that is, all subranges begin with a minimum value of 1 or more (e.g., 1 to 6.1) and end with a maximum value of 10 or less (e.g., 5.5 to 10). Moreover, any reference referred to as "incorporated herein" should be understood to be incorporated in its entirety.

[0012] It is further noted that, as used herein, the singular forms "a," "an," and "the" include plural referents unless expressly and unambiguously limited to one referent. The term "and / or" is typically used to refer to at least one or the other. In some instances, the term "and / or" is used synonymously with the term "or." The term "including" is used herein to mean, and is used synonymously with, the phrase "including but not limited to." The term "such as" is used herein to mean, and is used synonymously with, the phrase "such as but not limited to."

[0013] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Experts are directed to Current Protocols in Molecular Biology (Ausubel), in particular, for definitions and terms in the art.

[0014] Similarly, as used herein, "at least one" contemplates a number from 1 to the entire group. For example, with respect to a list of four markers, the phrase "at least one" is understood to mean 1, 2, 3, or 4 markers.

[0015] Similarly, as used herein, "comprising" more specifically includes embodiments defined using the term "consisting of."

[0016] Antibody: As used herein, the term "antibody" refers to a polypeptide consisting of one or more polypeptides substantially encoding immunoglobulin genes or fragments of immunoglobulin genes. Recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as various immunoglobulin variable region genes. Light chains are generally classified as either kappa or lambda. Heavy chains are generally classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes IgG, IgM, IgA, IgD, and IgE, respectively. A typical immunoglobulin (antibody) structural unit is known to comprise a tetramer. Each tetramer consists of two identical pairs of polypeptide chains, each pair having one "light" chain (about 25 kD) and one "heavy" chain (about 50-70 kD). The N-terminus of each chain defines a variable region of about 100-110 or more amino acids primarily responsible for antigen recognition. The terms "light chain variable chain" (VL) and "heavy chain variable chain" (VH) refer to these light and heavy chains, respectively. An antibody can be specific for a particular antigen. The antibody or its antigen can be either an analyte or a binding partner. Antibodies exist as intact immunoglobulins or as a number of well-characterized fragments produced by digestion with various peptidases. Thus, for example, pepsin digests an antibody under the disulfide bond of the hinge region to produce F(ab)'2, which is a dimer of Fab, itself a light chain linked to VH-CH1 by a disulfide bond. F(ab)'2 can be reduced under mild conditions to cleave the disulfide bond of the hinge region, thereby converting the (Fab')2 dimer into a Fab' monomer. The Fab' monomer is essentially an Fab with a portion of the hinge region (for a more detailed description of other antibody fragments, see Fundamental Immunology, WE Paul, ed., Raven Press, NY (1993)). While various antibody fragments have been defined in terms of the digestion of an intact antibody, one of skill in the art will appreciate that such Fab' fragments can be synthesized de novo either chemically or by using recombinant DNA methodology.Thus, the term "antibody" as used herein also includes antibody fragments produced by modification of whole antibodies or synthesized de novo using recombinant DNA methods. In some embodiments, the antibody is a single chain antibody, e.g., a single chain Fv (scFv) antibody in which the heavy and light variable chains are linked together (directly or via a peptide linker) to form a contiguous polypeptide. Single chain Fv ("scFv") polypeptides are covalently linked VH::VL heterodimers and can be expressed from nucleic acids that contain VH- and VL-encoding sequences that are directly linked or linked by a peptide-encoding linker. (See, e.g., Huston et al., (1988) Proc. Nat. Acad. Sci. USA, 85:5879-5883, the entire contents of which are incorporated herein by reference). Several structures exist for converting the naturally aggregated but chemically separated light and heavy polypeptide chains from antibody V regions into scFv molecules that fold into a three-dimensional structure substantially similar to the structure of the antigen-binding site. See, for example, U.S. Patent Nos. 5,091,513, 5,132,405, and 4,956,778.

[0017] The term "antibody" includes monoclonal antibodies, polyclonal antibodies, synthetic antibodies and chimeric antibodies, such as those generated by combinatorial mutagenesis and phage display. The term "antibody" also includes antibody mimetics or peptidomimetics. Peptidomimetics are compounds based on or derived from peptides and proteins. The peptidomimetics of the present disclosure can be obtained by structural modification of known peptide sequences, generally using non-natural amino acids, conformational constraints, isosteric substitutions, etc.

[0018] Allele: As used herein, the term "allele" refers to different versions of the nucleotide sequence at the same genetic locus (eg, gene).

[0019] Allele-specific primer extension (ASPE): As used herein, the term "allele-specific primer extension (ASPE)" refers to a mutation detection method using a primer that hybridizes to a corresponding DNA sequence and is extended upon successful hybridization of the 3'-terminal nucleotide of such primer. In general, an extension primer having a 3'-terminal nucleotide that forms a perfect match with the target sequence is extended to form an extension product. Modified nucleotides can be incorporated into the extension product, and such nucleotides can effectively label the extension product for detection purposes. Alternatively, the extension primer may instead contain a 3'-terminal nucleotide that forms a mismatch with the target sequence. In this case, primer extension will not occur unless the polymerase used for extension inadvertently has exonuclease activity.

[0020] Amplification: As used herein, the term "amplification" refers to methods known in the art for copying a target nucleic acid, thereby increasing the number of copies of a selected nucleic acid sequence. Amplification may be exponential or linear. The target nucleic acid may be either DNA or RNA. Generally, the sequence amplified in this manner comprises an "amplicon." Amplification may be accomplished in a variety of ways, including, but not limited to, polymerase chain reaction ("PCR"), transcription-based amplification, isothermal amplification, rolling circle amplification, and the like. Amplification may be performed with relatively similar amounts of each primer in a primer pair to generate a double-stranded amplicon. However, asymmetric PCR may be used to amplify primarily or exclusively single-stranded products as is well known in the art (e.g., Poddar et al., Molec. And Cell. Probes 14:25-32 (2000)). This may be achieved by using each pair of primers to greatly reduce the concentration of one primer in the pair compared to the other primer (e.g., 100-fold difference). Amplification by asymmetric PCR is usually linear. Those skilled in the art will appreciate that different amplification methods may be used together.

[0021] Animal: As used herein, the term "animal" refers to any member of the animal kingdom. In some embodiments, "animal" refers to humans at any stage of development. In some embodiments, "animal" refers to non-human animals at any stage of development. In certain embodiments, the non-human animals are mammals (e.g., rodents, mice, rats, rabbits, monkeys, dogs, cats, sheep, cows, primates, and / or pigs). In some embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, insects, and / or worms. In some embodiments, animals may be transgenic animals, genetically modified animals, and / or clones.

[0022] Approximately: As used herein, the term "approximately" or "about" as applied to one or more values ​​of interest refers to a value similar to the stated reference value. In certain embodiments, the term "approximately" or "about" refers to a range of values ​​that falls within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater or smaller) of the stated reference value, unless otherwise stated or otherwise clear from the context (unless such number exceeds 100% of possible values). Also, throughout this application, the term "about" is used to indicate that the value includes the inherent variation of error of the device, the method used to determine the value, or the variation that exists between samples.

[0023] Associated with a condition or disease of interest: As used herein, "associated with a condition or disease of interest" means that the variant is found more frequently in patients with the condition or disease of interest than in non-symptomatic or non-disease controls. Generally, the statistical significance of such association can be determined by assaying multiple patients.

[0024] Biological samples and samples: As used herein, the term "biological sample" encompasses any sample obtained from a biological source. Biological samples may include, by way of non-limiting example, blood, serum, plasma, tissue biopsy, cell-free DNA, amniotic fluid, serum, urine, feces, epithelial samples, skin samples, buccal swabs, sperm, amniotic fluid, cultured cells, bone marrow samples, and / or chorionic villi. Convenient biological samples may be obtained, for example, by scraping cells from the surfaces of the buccal cavity. The term biological sample encompasses samples that have been processed to release or make available nucleic acids or proteins for detection as described herein. For example, biological samples may include cDNA obtained by reverse transcription of RNA from cells in the biological sample. Biological samples may be obtained from stages of life, such as fetuses, young adults, adults, etc. Fixed or frozen tissues may also be used.

[0025] Biomarker: As used herein, the term "biomarker" refers to one or more nucleic acids, polypeptides, and / or other biological molecules (e.g., cholesterol, lipids) that can be used alone or in combination with other biomarkers to diagnose or aid in the diagnosis or prognosis of a disease or condition of interest; monitor the progression of a disease or condition of interest; and / or monitor the effectiveness of a treatment for a condition or disease of interest.

[0026] Binder: As used herein, the term "binder" refers to a molecule that can specifically and selectively bind to a second (i.e., different) molecule of interest. The interaction may be non-covalent, e.g., as a result of hydrogen bonding, van der Waals interactions, or electrostatic or hydrophobic interactions, or it may be covalent. The term "soluble binder" refers to a binder that is not bound (i.e., covalently or non-covalently bound) to a solid support.

[0027] Carrier: The term "carrier" refers to a person who has no symptoms but carries a mutation that can be passed on to their children. Generally, for autosomal recessive genetic disorders, a carrier has one allele that contains the disease causing mutation and a second allele that is normal or not associated with the disease.

[0028] Coding and non-coding sequences: As used herein, the term "coding sequence" refers to a sequence of a nucleic acid or its complement, or a portion thereof, that can be transcribed and / or translated to produce an mRNA of a polypeptide or a fragment thereof and / or a polypeptide or a fragment thereof. A coding sequence comprises exons in genomic DNA or in the premature primary RNA transcript, which are joined together by the biochemical machinery of the cell to produce a mature mRNA. The antisense strand is the complement of such a nucleic acid, from which the encoded sequence can be deduced. As used herein, the term "non-coding sequence" refers to a sequence or a portion thereof of a nucleic acid or its complement that is not transcribed in vivo into amino acids, or that does not interact or attempt to position an amino acid with a tRNA. Non-coding sequences include intron sequences, both in genomic DNA or in the premature primary RNA transcript, and gene associated sequences, such as promoters, enhancers, silencers, etc.

[0029] Complement: As used herein, the terms "complement," "complementary," and "complementarity" refer to the pairing of nucleotide sequences according to the Watson / Crick pairing rules. For example, the sequence 5'-GCGGTCCCA-3' has a complementary sequence of 5'-TGGGACCGC-3'. A complementary sequence can also be a sequence of RNA that is complementary to a DNA sequence. Certain bases not commonly found in natural nucleic acids may be included in complementary nucleic acids, including, but not limited to, inosine, 7-deazaguanine, locked nucleic acids (LNA) and peptide nucleic acids (PNA). Complementarity need not be perfect; a stable duplex may contain mismatched base pairs, degenerate or unmatched bases. Those skilled in the art of nucleic acid technology can empirically determine duplex stability, taking into account numerous variables, including, for example, the length of the oligonucleotide, the base composition and sequence of the oligonucleotide, ionic strength, and the incidence of mismatched base pairs.

[0030] Conserved: As used herein, the term "conserved residue" refers to amino acids that are the same among multiple proteins with the same structure and / or function. Regions of conserved residues may be important to the structure or function of a protein. Thus, adjacent conserved residues identified in a three-dimensional protein may be important to the structure or function of the protein. To find conserved residues, or conserved regions of 3D structure, comparisons of sequences of the same or similar proteins from different species or from individuals of the same species can be made.

[0031] Control: As used herein, the term "control" has its art-understood meaning of being a standard against which results are compared. In general, controls are used to increase the consistency of an experiment by isolating a variable in order to draw conclusions about such variable. In some embodiments, a control is a reaction or assay that is performed simultaneously with a test reaction or assay to obtain a basis for comparison. In one experiment, the "test" (i.e., the variable being tested) is applied. In a second experiment, the "control" (i.e., the variable being tested) is not applied. In some embodiments, a control is a historical control (i.e., a test or assay previously performed, or a control of a previously known amount or outcome). In some embodiments, a control is or includes a stored record, printed or otherwise. A control may be a positive or negative control.

[0032] A "control" or "predetermined standard" of a biomarker refers to the level of expression of the biomarker in a healthy subject, or the expression level of said biomarker in non-diseased or non-symptomatic tissue from the same subject. A control or predetermined standard expression level or protein amount of a given biomarker can be established by prospective and / or retrospective statistical studies using only routine experimentation. Such predetermined standard expression level and / or protein level (amount) can be determined by a person skilled in the art using well-known methods.

[0033] Crude: As used herein, the term "crude" when used in reference to a biological sample refers to a sample that is in a substantially unpurified state. For example, a crude sample can be a cell lysate or a biopsy tissue sample. A crude sample can be present in a dissolved state or as a dry preparation.

[0034] Deletion: As used herein, the term "deletion" includes a mutation that removes one or more nucleotides from a naturally occurring nucleic acid.

[0035] Disease or condition of interest: As used herein, the disease or condition of interest is NCGS. As used herein, NCGS includes non-celiac wheat sensitivity (NCWS).

[0036] Detection: As used herein, the terms "detection," "detected," or "detecting" include "measurement," "measured," or "measuring," and vice versa.

[0037] Detectable moiety: As used herein, the term "detectable moiety" or "detectable biomolecule" or "reporter" refers to a molecule that can be measured in a quantitative assay. For example, a detectable moiety may include an enzyme that can be used to convert a substrate into a product that can be measured (e.g., a visible product). Alternatively, the detectable moiety may be a radioisotope that can be quantified. Alternatively, the detectable moiety may be a fluorophore. Alternatively, the detectable moiety may be a luminescent molecule. Alternatively, other detectable molecules may be used.

[0038] Epigenetic: As used herein, epigenetic elements can change gene expression by mechanisms other than changes in the basic DNA sequence. Such elements can include paramutation, imprinting, gene silencing, X-chromosome inactivation, position effect, reprogramming, transvection, maternal effect, histone modification, and elements that regulate heterochromatin.

[0039] Epitope: As used herein, the term "epitope" refers to a fragment or portion of a molecule or molecular compound (eg, a polypeptide or protein complex) that contacts a particular antibody or antibody-like protein.

[0040] Exon: As used herein, an exon is a nucleic acid sequence found in mature or processed RNA after other parts of the RNA (e.g., intervening regions known as introns) have been removed by RNA splicing. Thus, an exon sequence usually codes for a protein or a portion of a protein. An intron is a portion of an RNA that is removed from the surrounding exon sequence by RNA splicing.

[0041] Expression and expressed RNA: As used herein, expressed RNA is RNA that codes for a protein or polypeptide ("coding RNA"), or any other RNA that is transcribed but not translated ("non-coding RNA"). The term "expression" is used herein to mean the process by which a polypeptide is produced from DNA. This process involves the transcription of a gene into mRNA and the translation of this mRNA into a polypeptide. Depending on the context in which it is used, "expression" can refer to the production of RNA, protein, or both.

[0042] Measurement of protein quantity and / or expression of the biomarkers of the present disclosure may be assessed by any of a wide variety of well-known methods for detecting expression of a transcribed molecule or its corresponding protein. Non-limiting examples of such methods include immunological methods for detection of secreted proteins, protein purification methods, protein function or activity assays, nucleic acid hybridization methods, nucleic acid reverse transcription methods, and nucleic acid amplification methods. In certain embodiments, expression of a marker gene is assessed using an antibody (e.g., a radiolabeled, chromophore-labeled, fluorophore-labeled, or enzyme-labeled antibody) that specifically binds to a protein corresponding to the marker gene, such as a protein encoded by an open reading frame corresponding to the marker gene or a protein that has undergone all or some of its normal post-translational modifications, an antibody derivative (e.g., an antibody conjugate bound to a substrate or to a protein or ligand of a protein-ligand pair {e.g., biotin-streptavidin}), or an antibody fragment (e.g., a single chain antibody, an isolated antibody hypervariable domain, etc.). In certain embodiments, the reagent may be directly or indirectly labeled with a detectable substance. The detectable substance may, for example, be selected from the group consisting of radioisotopes, fluorescent compounds, enzymes, and enzyme co-factors. Methods of labeling antibodies are well known in the art.

[0043] The expression of the marker gene is evaluated by preparing mRNA / cDNA (i.e., transcribed polynucleotide) from cells in the sample and hybridizing the mRNA / cDNA with a reference polynucleotide that is the complement of the polynucleotide containing the marker gene and fragments thereof. The cDNA can be amplified, if desired, using any of a variety of polymerase chain reaction methods prior to hybridization with the reference polynucleotide; preferably, it is not amplified.

[0044] Family history: As used herein, the term "family history" generally refers to the occurrence of events (e.g., disease-related disorders or mutation carriers) related to an individual's immediate family, including parents and siblings. Family history may also include grandparents and other relatives.

[0045] Flanking: As used herein, the term "flanking" means that the primer hybridizes to the target nucleic acid adjacent to the region of interest to be amplified on the target. Those skilled in the art will understand that the preferred primers are a pair of primers, one to each strand of the target double-stranded DNA molecule, hybridizing 3' from the region of interest, so that nucleotides are added to the 3' end of the primer by a suitable DNA polymerase. For example, a primer flanking a mutant sequence does not actually anneal to the mutant sequence, but rather to a sequence adjacent to the mutant sequence. In some cases, primers flanking an exon are usually designed to anneal to a sequence adjacent to the exon (e.g., an intron sequence) rather than to the exon sequence. However, in some cases, the amplification primers may be designed to anneal to an exon sequence.

[0046] Gene: As used herein, a gene is a unit of heredity. Generally, a gene is a portion of DNA that codes for a protein or functional RNA. A gene is a locatable region of a genomic sequence that corresponds to a unit of heredity. A gene may be associated with regulatory regions, transcriptional regions, and other functional sequence regions.

[0047] Genotype: As used herein, the term "genotype" refers to the genetic makeup of an organism. More specifically, the term refers to the identity of the alleles present in an individual. "Genotyping" an individual or DNA sample refers to determining the nature, in terms of nucleotide bases, of the two alleles that an individual possesses at known polymorphic sites.

[0048] Genetic regulatory element: As used herein, a genetic regulatory element or regulatory sequence is a segment of DNA to which regulatory proteins, such as transcription factors, bind to regulate gene expression. Such regulatory regions are often located upstream of the gene that they regulate.

[0049] Healthy individual: As used herein, the term "healthy individual" or "control" refers to a subject who has not been diagnosed with the condition and / or disease of interest.

[0050] Heterozygous: As used herein, the term "heterozygous" or "HET" refers to an individual who carries two different alleles of the same gene. As used herein, the term "heterozygous" encompasses "compound heterozygous" or "compound heterozygous mutant". As used herein, the term "compound heterozygous" refers to an individual who carries two different alleles. As used herein, the term "compound heterozygous mutant" refers to an individual who carries two different copies of an allele, and such alleles are characterized as mutant forms of the gene.

[0051] Homozygous: As used herein, the term "homozygous" refers to an individual who carries two copies of the same allele. As used herein, the term "homozygous mutant" refers to an individual who carries two copies of the same allele, such alleles being characterized as mutant forms of the gene.

[0052] Hybridize: As used herein, the term "hybridize" or "hybridization" refers to the process in which two complementary nucleic acid strands anneal to each other under appropriately stringent conditions. Oligonucleotides or probes suitable for hybridization generally contain 10-100 nucleotides in length (e.g., 18-50, 12-70, 10-30, 10-24, 18-36 nucleotides in length). Nucleic acid hybridization techniques are well known in the art. Those skilled in the art know how to estimate and adjust the stringency of hybridization conditions so that sequences with at least a desired level of complementarity will stably hybridize, while sequences with lower complementarity will not. For examples of hybridization conditions and parameters, see, for example, Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Press, Plainview, NY; Ausubel, FM et al., 1994, Current Protocols in Molecular See Biology. John Wiley & Sons, Secaucus, NJ.

[0053] Identity or percent identity: As used herein, the term "identity" or "percent identity" refers to the sequence identity between two amino acid sequences or between two nucleic acid sequences. Percent identity can be determined by aligning two sequences and refers to the number of identical residues (i.e., amino acids or nucleotides) at positions shared by the sequences being compared. Sequence alignment and comparison can be performed using standard algorithms in the art (e.g., Smith and Waterman, 1981, Adv. Appl. Math. 2:482; Needleman and Wunsch, 1970, J. Mol. Biol. 48:443; Pearson and Lipman, 1988, Proc. Natl. Acad. Sci., USA, 85:2444), or by computerized versions of these algorithms published as BLAST and FASTA (Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive, Madison, WI). Also, ENTREZ, available through the National Institutes of Health, Bethesda, Maryland, may be used for sequence comparison. In other examples, commercially available software, such as GenomeQuest, may be used to determine percent identity. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., BLASTN; available at the Internet site of the National Center for Biotechnology Information) may be used. In one embodiment, the percent identity of two sequences may be determined using GCG with a gap weight of 1, such that each amino acid gap is weighted as if it were a single amino acid mismatch between the two sequences. Alternatively, the ALIGN program (version 2.0), which is part of the GCG (Accelrys, San Diego, Calif.) sequence alignment software package, may be used.

[0054] As used herein, the term at least 90% identical thereto includes sequences that are in the range of 90-100% identity with the indicated sequence, including all ranges therebetween. Thus, the term at least 90% identical thereto includes sequences that are 91, 91.5, 92, 92.5, 93, 93.5, 94, 94.5, 95, 95.5, 96, 96.5, 97, 97.5, 98, 98.5, 99, 99.5 percent identical to the indicated sequence. Similarly, the term "at least 70% identical" includes sequences that are in the range of 70-100% identity, including all ranges therebetween. The determination of percent identity is determined using the algorithm described herein.

[0055] Insertion or Addition: As used herein, the term "insertion" or "addition" refers to a change in an amino acid or nucleotide sequence that results in the addition of one or more amino acid residues or nucleotides, respectively, as compared to the naturally occurring molecule.

[0056] In vitro: As used herein, the term "in vitro" refers to events that take place not within a multicellular organism, but in an artificial environment, such as a test tube or reaction vessel, cell culture, and the like.

[0057] In vivo: As used herein, the term "in vivo" refers to events that occur within a multicellular organism, such as a non-human animal.

[0058] Isolated: As used herein, the term "isolated" refers to (1) a substance and / or entity that is separated from at least some of the elements (whether in nature and / or in an experimental setting) with which it is associated when originally created, and / or (2) a substance and / or entity that is generated, prepared, and / or manufactured by the hand of man. An isolated substance and / or entity may be separated from at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98%, about 99%, substantially 100%, or 100% of other elements with which it is originally associated. In some embodiments, an isolated agent is greater than about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, substantially 100%, or 100% pure. As used herein, a substance is "pure" if it is substantially free of other elements. As used herein, the term "isolated cell" refers to a cell that is not contained in a multicellular organism.

[0059] Labeled: The terms "labeled" and "labeled with a detectable agent or moiety" are used interchangeably herein to specify that an entity (e.g., a nucleic acid probe, an antibody, etc.) can be measured, for example, by detection of the label (e.g., visual detection, such as radioactivity) after binding to another entity (e.g., a nucleic acid, a polypeptide, etc.). The detectable agent or moiety may be selected to generate a signal that can be measured and whose intensity is related (e.g., proportional) to the amount of bound entity. A wide variety of systems for labeling and / or detecting proteins and peptides are known in the art. Labeled proteins and peptides can be prepared by incorporation or conjugation of a label that is detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical, chemical or other means. A label or labeling moiety may be directly detectable (i.e., it does not require further reaction or manipulation to become detectable, e.g., a fluorophore is directly detectable) or indirectly detectable (i.e., it becomes detectable by reaction or binding with another entity that is detectable, e.g., a hapten is detectable by immunostaining after reaction with an appropriate antibody that contains a reporter such as a fluorophore). Suitable detectable agents include, but are not limited to, radionucleotides, fluorophores, chemiluminescent agents, microparticles, enzymes, colorimetric labels, magnetic labels, haptens, molecular beacons, aptamer beacons, and the like.

[0060] MicroRNA: As used herein, microRNA (miRNA) is a short (20-24 nucleotide) non-coding RNA involved in the post-transcriptional regulation of gene expression. MicroRNAs can affect both mRNA stability and translation. For example, microRNAs can bind to complementary sequences in the 3'UTR of target mRNAs, causing gene silencing. miRNAs are transcribed by RNA polymerase II as part of a capped and polyadenylated primary transcript (pri-miRNA), which may be either protein-coding or non-coding. The primary transcript can be cleaved by the Drosha ribonuclease III enzyme to generate an approximately 70 nucleotide stem-loop precursor miRNA (pre-miRNA), which is further cleaved by the cytoplasmic Dicer ribonuclease to generate mature miRNAs and antisense miRNA star (miRNA). * Mature miRNAs can be incorporated into an RNA-induced silencing complex (RISC), which can recognize target mRNAs through imperfect base pairing with the miRNA, most commonly resulting in translational inhibition or destabilization of the target mRNA.

[0061] Multiplex PCR: As used herein, the term "multiplex PCR" refers to the simultaneous amplification of two or more regions, each primed with a separate primer pair.

[0062] Multiplex ASPE: As used herein, the term "multiplex ASPE" refers to an assay that combines multiplex PCR with allele-specific primer extension (ASPE) to detect genetic polymorphisms. In general, multiplex PCR is used to first amplify regions of DNA that serve as target sequences for ASPE primers. See the definition of allele-specific primer extension.

[0063] Mutation and / or variant: In this specification, the terms mutation and variant are used interchangeably to describe the change in nucleic acid or protein sequence.The term "mutant" in this specification refers to a mutated or potentially non-functional form of gene.This term includes any mutation that makes gene non-functional, from point mutation to large chromosomal rearrangement, as known in the art.

[0064] Nucleic acid: As used herein, a "nucleic acid" is a polynucleotide such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). The term is used to include single-stranded nucleic acids, double-stranded nucleic acids, and RNA and DNA made from nucleotide or nucleoside analogs.

[0065] Polypeptide or protein: As used herein, the terms "polypeptide" and / or "protein" refer to a polymer of amino acids, not a specific length. Thus, peptides, oligopeptides and proteins are included within the definition of a polypeptide and / or protein. "Polypeptide" and "protein" are used interchangeably herein to describe protein molecules that may include partial or full-length proteins. The term "peptide" is used to indicate less than full-length proteins or very short proteins, unless the context indicates otherwise.

[0066] As known in the art, "proteins," "peptides," "polypeptides," and "oligopeptides" are chains of amino acids (generally L-amino acids) whose alpha carbons are linked by peptide bonds formed by a condensation reaction between the carboxyl group on the alpha carbon of one amino acid and the amino group on the alpha carbon of another amino acid. Generally, the amino acids constituting a protein are numbered sequentially, starting with the amino terminal residue and increasing toward the carboxy terminal residue of the protein. Amino acid residue abbreviations are the standard three-letter and / or one-letter codes used in the art to refer to one of the 20 common L-amino acids.

[0067] As used herein, a "domain" of a polypeptide or protein includes a region along a polypeptide or protein that comprises an independent unit. A domain may be defined in terms of structure, sequence and / or biological activity. In one embodiment, a polypeptide domain may include a region of a protein that folds in a manner that is substantially independent from the rest of the protein. Domains may be identified using domain databases such as, but not limited to, PFAM, PRODOM, PROSITE, BLOCKS, PRINTS, SBASE, ISREC PROFILES, SAMRT, and PROCLASS.

[0068] Primer: As used herein, the term "primer" refers to a short, single-stranded oligonucleotide capable of hybridizing to a complementary sequence in a nucleic acid sample. Generally, a primer serves as the initiation point for template-dependent DNA synthesis. Deoxyribonucleotides can be added to a primer by DNA polymerase. In some embodiments, such addition of deoxyribonucleotides to a primer is also known as primer extension. As used herein, the term primer includes all forms of primers that can be synthesized, including peptide nucleic acid primers, locked nucleic acid primers, phosphorothioate modified primers, labeled primers, and the like. A "primer pair" or "primer set" for a PCR reaction generally refers to a set of primers that includes a "forward primer" and a "reverse primer." As used herein, a "forward primer" refers to a primer that anneals to the antisense strand of dsDNA. A "reverse primer" anneals to the sense strand of dsDNA.

[0069] Genetic polymorphism: As used herein, the term "genetic polymorphism" refers to the coexistence of two or more forms of a gene or portions thereof.

[0070] Portion and Fragment: As used herein, the terms "portion" and "fragment" are used interchangeably to refer to a polypeptide, nucleic acid, or other molecular construct.

[0071] Sense and antisense strands: As used herein, the term "sense strand" refers to the strand of double-stranded DNA (dsDNA) that contains at least a portion of the coding sequence of a functional protein. As used herein, the term "antisense strand" refers to the strand of dsDNA that is the reverse complement of the sense strand.

[0072] Significant difference: As used herein, the term "significant difference" is well within the knowledge of a person skilled in the art and is empirically determined with reference to each particular biomarker. For example, a significant difference in the expression of a biomarker in a subject with a disease or condition group of interest compared to a healthy subject is a statistically significant difference in protein abundance.

[0073] Analogous or Homologous: As used herein, the term "analogous" or "homologous" when referring to an amino acid or nucleotide sequence refers to a polypeptide that has a degree of homology or identity with a wild-type amino acid sequence. Homology comparisons can be performed by eye, or more commonly, with the aid of readily available sequence comparison programs. These commercially available computer programs can calculate the percent homology between two or more sequences (e.g., Wilbur, WJ and Lipman, DJ, 1983, Proc. Natl. Acad. Sci. USA, 80:726-730). For example, homologous sequences can be construed to include amino acid sequences that are at least 70% identical, 75% identical, 80% identical, 85% identical, 90% identical, 95% identical, 97% identical, or 98% identical to each other in alternative embodiments.

[0074] Specific: As used herein, the term "specific" refers to an oligonucleotide or primer that is capable of hybridizing to a target of interest under suitable hybridization or washing conditions and does not substantially hybridize to nucleic acids that are not of interest. Higher levels of sequence identity are preferred, including at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity. In some embodiments, a specific oligonucleotide or primer comprises at least 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 35, 40, 45, 50, 55, 60, 65, 70 or more bases of sequence identity with the portion of the nucleic acid that it hybridizes or amplifies when the oligonucleotide and the nucleic acid are aligned.

[0075] As is known in the art, conditions for hybridizing nucleic acid sequences to each other can be described as ranging from low stringency to high stringency. Generally, highly stringent hybridization conditions refer to washing the hybrids with a low salt buffer at high temperature. Hybridization is carried out in 0.5M NaHPO 4 Filter the bound DNA at 65 °C using a hybridization solution standard in the art, such as 7% sodium dodecyl sulfate (SDS) and 0.25 M NaHPO. 4 , 3.5% SDS, followed by a wash in 0.1×SSC / 0.1% SDS at temperatures ranging from room temperature to 68° C., depending on the length of the probe (see, e.g., Ausubel, FM et al., Short Protocols in Molecular Biology, 4 th (See, Ed., Chapter 2, John Wiley & Sons, NY). For example, high stringency washes include washing in 6×SSC / 0.05% sodium pyrophosphate at 37° C. for 14-base oligonucleotide probes, or 48° C. for 17-base oligonucleotide probes, or 55° C. for 20-base oligonucleotide probes, or 60° C. for 25-base oligonucleotide probes, or 65° C. for nucleotide probes of about 250 nucleotides in length. The nucleic acid probe may be, for example, [γ- 32 with radioactive nucleotides by end-labeling with [P]ATP or by random primer labeling with [α- 32 Alternatively, the probe may be labeled by incorporation of biotinylated or fluorescein-labeled nucleotides and the probe may be detected using streptavidin or an anti-fluorescein antibody.

[0076] siRNA: As used herein, siRNA (small inhibitory RNA) is an essentially double-stranded RNA molecule consisting of about 20 complementary nucleotides. siRNA is generated by degradation of a larger double-stranded (ds) RNA molecule. siRNA can suppress gene expression by essentially splitting its corresponding mRNA into two by interaction of siRNA with mRNA, causing degradation of the mRNA. siRNA can also interact with DNA to promote chromatin silencing and heterochromatin expansion.

[0077] Subject: As used herein, the term "subject" refers to a human or any non-human animal. A subject may be a patient, which refers to a human who visits a health care provider for diagnosis or treatment of a disease. Humans include prenatal and postnatal forms. Also, as used herein, the terms "individual," "subject," or "patient" include all warm-blooded animals. In one embodiment, the subject is a human. In one embodiment, the individual is a subject who has NCGS or is at increased risk of developing NCGS.

[0078] Substantially: As used herein, the term "substantially" refers to a qualitative condition that indicates the entire or nearly entire extent or degree of a feature or characteristic of interest. Those skilled in the art of biology will understand that biological and chemical phenomena rarely, if ever, go to completion and / or proceed perfectly or achieve or avoid absolute results. Thus, the term "substantially" is used herein to capture the potential lack of completeness that is inherent in many biological and chemical phenomena.

[0079] Substantially complementary: As used herein, the term "substantially complementary" refers to two sequences that are capable of hybridizing under stringent hybridization conditions. One of skill in the art will understand that substantially complementary sequences need not hybridize over their entire length. In some embodiments, "stringent hybridization conditions" refers to hybridization conditions that are at least as stringent as the following: 50% formamide, 5X SSC, 50 mM NaH 2 PO 4 , pH 6.8, 0.5% SDS, 0.1 mg / mL sonicated salmon sperm DNA, and 5X Denhart's solution overnight at 42° C.; a wash with 2X SSC, 0.1% SDS at 45° C.; and a wash with 0.2X SSC, 0.1% SDS at 45° C. In some embodiments, stringent hybridization conditions should not allow hybridization of two nucleic acids that differ by more than two bases within a span of 20 contiguous nucleotides.

[0080] Substitution: As used herein, the term "substitution" refers to the replacement of one or more amino acids or nucleotides by different amino acids or nucleotides, respectively, as compared to a naturally occurring molecule.

[0081] Suffering from: An individual "suffering from" a disease, disorder, and / or condition has been diagnosed with or is exhibiting one or more symptoms of the disease, disorder, and / or condition.

[0082] Susceptible: An individual who is "susceptible" to a disease, disorder, and / or condition has not been diagnosed with the disease, disorder, and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition may not be exhibiting symptoms of the disease, disorder, and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition will develop the disease, disorder, and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition will not develop the disease, disorder, and / or condition.

[0083] Solid support: The term "solid support" or "support" refers to a structure that provides a substrate onto which a biomolecule can be bound. For example, a solid support can be a well of an assay (i.e., a microtiter plate, etc.), or a solid support can be a location on an array, or a mobile support such as a bead.

[0084] Upstream and downstream: As used herein, the term "upstream" refers to the residue that is N-terminal to the second residue if the molecule is a protein, or 5' to the second residue if the molecule is a nucleic acid. Also, as used herein, the term "downstream" refers to the residue that is C-terminal to the second residue if the molecule is a protein, or 3' to the second residue if the molecule is a nucleic acid. All protein, polypeptide and peptide sequences disclosed herein are written from the N-terminal amino acid to the C-terminal acid, and all nucleic acid sequences disclosed herein are written from the 5' end of the molecule to the 3' end of the molecule.

[0085] overview The disclosure herein provides novel mutations identified in genes for diseases and / or conditions of interest that can be used to more accurately diagnose disorders associated with the genes and / or conditions of interest.

[0086] In some embodiments, the sample contains nucleic acid. In some embodiments, the testing step includes sequencing. In some embodiments, the testing step includes hybridization. In some embodiments, the hybridization is performed using one or more oligonucleotide probes specific to the region of the biomarker of interest. In some embodiments, for detection of mutations, the hybridization is performed under sufficiently stringent conditions to not allow a single nucleotide mismatch. In some embodiments, the hybridization is performed using a microarray. In some embodiments, the testing step includes restriction enzyme digestion. In some embodiments, the testing step includes PCR amplification. In some embodiments, the PCR amplification is digital PCR amplification. In some embodiments, the testing step includes primer extension. In some embodiments, the primer extension is single-base primer extension. In some embodiments, the testing step includes performing multiplex allele-specific primer extension (ASPE).

[0087] In some embodiments, the sample contains proteins. In some embodiments, the testing step includes amino acid sequencing. In some embodiments, the testing step includes performing an immunoassay using one or more antibodies that specifically recognize the biomarker of interest. In some embodiments, the testing step includes protease digestion (e.g., trypsin digestion). In some embodiments, the testing step further includes performing 2D gel electrophoresis.

[0088] In some embodiments, the testing step includes determining the presence of one or more biomarkers using mass spectrometry. In some embodiments, the mass spectrometry format is selected from among matrix-assisted laser desorption / ionization, time-of-flight (MALDI-TOF), electrospray (ES), IR-MALDI, ion cyclotron resonance (ICR), Fourier transform, and combinations thereof.

[0089] In some embodiments, the sample is obtained from cells, tissues, whole blood, mouthwash, plasma, serum, urine, stool, saliva, umbilical cord blood, chorionic villus sample, chorionic villus sample culture, amniotic fluid, amniotic fluid culture, transcervical washing, or combinations thereof. In further embodiments, the sample is obtained from blood or a blood product (e.g., plasma or serum) from a pregnant woman and / or fetal DNA.

[0090] In some embodiments, the testing step comprises determining the identity of the nucleotide and / or amino acid at a predetermined position of the biomarker, hi some embodiments, the presence of a mutation is determined by comparing the identity of the nucleotide and / or amino acid at the predetermined position with a control.

[0091] In embodiments, the method may include performing an assay (eg, sequencing) on ​​multiple individuals to determine the statistical significance of the association.

[0092] In another aspect, the disclosure provides reagents for detecting a biomarker of interest, such as, but not limited to, a nucleic acid probe that specifically binds to the biomarker (e.g., a mutation in a DNA sequence) or an array containing one or more probes that specifically bind to the biomarker. In some embodiments, the disclosure provides an antibody that specifically binds to the biomarker. In some embodiments, the disclosure provides a kit comprising one or more such reagents. In some embodiments, the one or more reagents are provided in the form of a microarray. In some embodiments, the kit further comprises a reagent for primer extension. In some embodiments, the kit further comprises a control representative of a healthy individual. In some embodiments, the kit further comprises instructions on how to determine whether an individual has a symptom or disease of interest based on the biomarker of interest.

[0093] In some examples, the amount of one or more biomarkers may be detected in certain embodiments by: (a) detecting the amount of a polypeptide or protein that is regulated by said one or more biomarkers; (b) detecting the amount of a polypeptide or protein that regulates said biomarkers; or (c) detecting the amount of a metabolite of the biomarker.

[0094] In yet another aspect, the disclosure herein provides a computer readable medium encoding information corresponding to detection of a biomarker.

[0095] Methods and Compositions for Diagnosing NCGS Embodiments of the present disclosure include methods and compositions for diagnosing the presence of, or increased risk of developing, Non-Celiac Gluten Sensitivity (NCGS). The methods and compositions of the present disclosure can be used to obtain or provide genetic information from a subject to objectively diagnose the presence of, or increased risk of developing, NCGS for that subject or other subjects. The methods and compositions can be embodied in a variety of ways.

[0096] In one embodiment, a method is disclosed for detecting biomarkers associated with non-celiac gluten sensitivity (NCGS) in an individual, comprising obtaining a sample from the individual; and measuring the amount of at least one of IL-8, IL-10, TNF-α or total IgE protein in the sample. In some examples, the measuring may further comprise measuring the expression of one of IP-10, CD4 or CD45. Additionally and / or alternatively, the method may include measuring at least one normalizing (e.g., housekeeping) gene. In one non-limiting embodiment, the housekeeping gene may be glyceraldehyde 3-phosphate dehydrogenase. Alternatively, other housekeeping genes may be used. Additionally and / or alternatively, other biomarkers may be measured.

[0097] As disclosed herein, a variety of methods can be used to measure the biomarker of interest. In one embodiment, the measurement includes measuring a peptide or polypeptide biomarker. For example, in one embodiment, the measurement includes an immunoassay. Alternatively, the measurement can include flow cytometry. Alternatively, nucleic acid methods can be used, as discussed in detail herein.

[0098] A variety of sample types may be used, in certain embodiments, the sample comprises blood, serum, plasma or a tissue biopsy.

[0099] In certain embodiments, the present disclosure provides a method of identifying markers associated with NCGS in an individual, which may include identifying at least one marker that is increased or decreased in expression in NCGS but not in celiac disease compared to a control individual or population.

[0100] In other embodiments, the disclosure provides a method for detecting the presence or susceptibility of non-celiac gluten sensitivity (NCGS) in an individual. The method includes obtaining a sample from the individual; measuring the amount of at least one of IL-8, IL-10, TNF-α or total IgE protein in the sample; and comparing the expression of at least one of the IL-8, IL-10, TNF-α or total IgE in the sample to a control value of each of the IL-8, IL-10, TNF-α or total IgE. In some examples, the measuring may further include measuring the expression of at least one of IP-10, CD4 or CD45 and comparing the level of these markers to the level of the control value. In one embodiment, the control value is derived from a healthy individual or an individual with no or no detectable gastrointestinal pathology. In some embodiments, the control is a disease control. Such disease controls may include individuals with celiac disease (stratified according to whether the individual is on a gluten-free diet) and subjects with other GI diseases, such as inflammatory bowel disease (IBD), hepatitis, small intestinal bacterial overgrowth, and other diseases.

[0101] Additionally and / or alternatively, the method may include measuring at least one normalization (e.g., housekeeping) gene. In one non-limiting embodiment, the housekeeping gene may be glyceraldehyde 3-phosphate dehydrogenase. Alternatively, other housekeeping genes may be used. Additionally and / or alternatively, other biomarkers may be measured.

[0102] As disclosed herein, a variety of methods can be used to measure the biomarker of interest. In one embodiment, the measurement includes measuring a peptide or polypeptide biomarker. For example, in one embodiment, the measurement includes an immunoassay. Alternatively, the measurement can include flow cytometry. Alternatively, nucleic acid methods can be used, as discussed in detail herein.

[0103] A variety of sample types may be used, in certain embodiments, the sample comprises blood, serum, plasma or a tissue biopsy.

[0104] Still other embodiments include compositions for detecting biomarkers associated with non-celiac gluten sensitivity (NCGS) in an individual. In certain embodiments, the compositions include reagents for quantifying the level of at least one of the proteins IL-8, IL-10, TNF-α, or total IgE in a biological sample. In some examples, the compositions may further include reagents for measuring the expression of at least one of IP-10, CD4, or CD45.

[0105] For example, the composition may include reagents for measuring peptide or polypeptide biomarkers, as described in detail herein. In one embodiment, the composition may include reagents for performing an immunoassay. Or, the composition may include reagents for performing flow cytometry. Or, the composition may include reagents for determining the presence of a particular sequence and / or expression level of a nucleic acid, as described in detail herein.

[0106] Other embodiments include kits containing at least a portion of the compositions disclosed herein and / or reagents for carrying out the methods disclosed herein. Such kits may include control biological samples from healthy individuals or individuals with no or no detectable gastrointestinal pathology. In some embodiments, the control is a disease control. Such disease controls may include individuals with celiac disease (stratified according to whether the individual is on a gluten-free diet) and subjects with other GI diseases, such as inflammatory bowel disease (IBD), hepatitis, small intestinal bacterial overgrowth, and other diseases. Such kits may include instructions and / or computer readable media containing instructions and / or other information for carrying out the methods. Such instructions may include the control values ​​described herein.

[0107] Other embodiments include computer readable media containing instructions and / or other information for practicing the method that is separate from the kit or the reagents therein.

[0108] Peptide, Polypeptide and Protein Assays In certain embodiments, the biomarker of interest is detected at the protein (or peptide or polypeptide level), i.e., the gene product is analyzed. For example, the protein or fragments thereof can be analyzed by amino acid sequencing or immunoassay using one or more antibodies that specifically recognize one or more epitopes present on the biomarker of interest or, in some cases, specific for the mutation of interest. The protein can be analyzed by protease digestion (e.g., trypsin digestion), and in some embodiments, the digested protein product can be further analyzed by 2D gel electrophoresis.

[0109] Antibody detection Specific antibodies that bind to a biomarker of interest can be used in any of a variety of ways known in the art. Antibodies against a particular epitope, polypeptide, and / or protein can be generated using any of a variety of ways known in the art. For example, the epitope, polypeptide, or protein against which an antibody is desired can be produced and injected into an animal, typically a mammal (e.g., donkey, mouse, rabbit, horse, chicken, etc.), and the antibodies produced by the animal can be collected from the animal. Monoclonal antibodies can also be generated by generating hybridomas that express the antibody of interest in an immortalized cell line.

[0110] In some embodiments, the antibody is labeled with a detectable moiety described herein.

[0111] Antibody detection methods are well known in the art and include, but are not limited to, enzyme-linked immunosorbent assay (ELISA) and Western blot. Some such methods are suitable for implementation in an array format.

[0112] For example, in some embodiments, the biomarker of interest is detected using a first antibody (or antibody fragment) that specifically recognizes the biomarker. The antibody may be labeled with a detectable moiety (e.g., a chemiluminescent molecule), an enzyme, or a second binding agent (e.g., streptavidin). Alternatively, the first antibody may be detected using a second antibody as known in the art.

[0113] In certain embodiments, the method may further include adding a capture support, which includes at least one capture support binder that recognizes and binds the biomarker so as to immobilize the biomarker on the capture support. In certain embodiments, the method may further include adding a second binder that can specifically recognize and bind at least a portion of the plurality of binder molecules on the capture support. In one embodiment, the binder that can specifically recognize and bind at least a portion of the plurality of binder molecules on the capture support is a soluble binder (e.g., a second antibody). The second binder may be labeled (e.g., with an enzyme) so that binding of the biomarker of interest is measured by adding a substrate for the enzyme and quantifying the amount of product formed.

[0114] In one embodiment, the capture solid support can be an assay well (i.e., a microtiter plate, etc.). Alternatively, the capture solid support can be a location on an array, or a mobile support, such as a bead, or the capture support can be a filter.

[0115] In some examples, the biomarker can be complexed with a first binding agent (e.g., a primary antibody specific for the biomarker and labeled with a detectable moiety) and a second binding agent (e.g., a secondary antibody or a second primary antibody that recognizes the primary antibody), which forms a complex with a third binding agent (e.g., biotin), which can then interact with a capture support (e.g., magnetic beads) that has a reagent (e.g., streptavidin) that recognizes the third binding agent linked to the capture support. The complex (labeled primary antibody:biomarker:second primary antibody-biotin:streptavidin-beads) is then captured using a magnet (e.g., a magnetic probe) to measure the amount of the complex.

[0116] A variety of binding agents may be used in the disclosed methods. For example, the binding agent attached to the capture support or the second antibody may be either an antibody or an antibody fragment that recognizes the biomarker. Alternatively, the binding agent may include a protein that binds to a non-protein target (i.e., a protein that specifically binds to a small molecule biomarker, or a receptor that binds to a protein, etc.).

[0117] In certain embodiments, the solid support may be treated with a passivation agent. For example, in certain embodiments, the biomarker of interest may be captured on a passivated surface (i.e., a surface that has been treated to reduce non-specific binding). One such passivation agent is BSA. Additionally and / or alternatively, if the binding agent used is an antibody, the solid support may be coated with Protein A, Protein G, Protein A / G, Protein L, or another agent that binds with high affinity to the binding agent (e.g., an antibody). These proteins bind to the Fc domain of the antibody and thus can direct the binding of the antibody that recognizes one or more proteins of interest.

[0118] Nucleic Acid Assays In certain embodiments, the biomarkers disclosed herein are detected at the nucleic acid level. In one embodiment, the present disclosure includes a method for diagnosing the presence of or increased risk of developing a condition or disease of interest (e.g., NCGS) in a subject. The method may include obtaining nucleic acid from a tissue or bodily fluid sample of the subject and performing an assay to identify whether the subject's nucleic acid has a variant sequence (i.e., a mutation). In certain embodiments, the method may include comparing the variant to a known variant associated with the condition or disease of interest and determining whether the variant is a variant previously identified as being associated with the condition or disease of interest. Alternatively, the method may include identifying the variant as a new variant that has not been previously characterized. If the variant is a new variant, the method may further include performing an analysis to determine whether the mutation is expected to be deleterious to the expression of the gene and / or the function of the protein encoded by the gene. The method may further include using the variant profile (i.e., a compilation of mutations identified in the subject) to diagnose the presence of, or an increased risk of developing, the syndrome or disease of interest.

[0119] Nucleic acid analysis can be performed on genomic DNA, messenger RNA, and / or cDNA. In various embodiments, the nucleic acid includes genes, RNA, exons, introns, gene regulatory elements, expressed RNA, siRNA, or epigenetic elements. Regulatory elements including splice sites, transcription factor binding, AI editing sites, microRNA binding sites, and functional RNA structure sites can also be evaluated for mutations (i.e., variants). Thus, for each of the disclosed methods and compositions, the variants can include nucleic acid sequences that encompass at least one of the following: (1) A-to-I editing sites; (2) splice sites; (3) conserved functional RNA structures; (4) effective transcription factor binding sites (TFBS); (5) microRNA (miRNA) binding sites; (6) polyadenylation sites; (7) known regulatory elements; (8) miRNA genes; (9) ROI-encoded small nucleolar RNA genes; and / or (10) ultraconserved elements across placental mammals.

[0120] In many embodiments, the nucleic acid is extracted from a biological sample. In some embodiments, the nucleic acid is cell-free DNA. In some embodiments, the nucleic acid is analyzed without being amplified. In some embodiments, the nucleic acid is amplified using techniques known in the art, such as polymerase chain reaction (PCR), and the amplified nucleic acid is used for subsequent analysis. Multiplex PCR may be used, in which multiple sets of primer pairs are used to amplify several amplicons (e.g., from different genomic regions) at once. For example, the nucleic acid may be analyzed by sequencing, hybridization, PCR amplification, restriction enzyme digestion, primer extension, such as single-base primer extension or multiplex allele-specific primer extension (ASPE), or DNA sequencing. In some embodiments, the nucleic acid is amplified such that the size of the amplification product of the wild-type allele is different from the size of the mutant allele. Thus, the presence or absence of a particular mutant allele can be determined by detecting the difference in size of the amplification product, for example on an electrophoretic gel. For example, deletions or insertions of gene regions may be particularly suitable for use with size-based approaches.

[0121] Certain exemplary nucleic acid analysis methods are described in detail below.

[0122] Allele-specific amplification In some embodiments, for example when the biomarker for a disease and / or condition group of interest is a mutation, the biomarker is detected using an allele-specific amplification assay. This approach includes PCR amplification of specific alleles (PASA) (Sarkar et al., 1990 Anal. Biochem. 186:64-68), allele-specific amplification (ASA) (Okayama et al., 1989 J. Lab. Clin. Med. 114:105-113), allele-specific PCR (ASPCR) (Wu et al., 1989 Proc. Natl. Acad. Sci. USA. 86:2757-2760), and amplification-refractory mutation system (ARMS) (Newton et al., 1989 Nucleic Acids Res. 17:2503-2516). The entire contents of each of these references are incorporated herein. This method is applicable to single base substitutions as well as microdeletions / insertions.

[0123] For example, for PCR-based amplification methods, amplification primers can be designed to be able to distinguish between different alleles (e.g., between wild-type and mutant alleles). Thus, the presence or absence of an amplification product can be used to determine whether a genetic mutation is present in a given nucleic acid sample. In some embodiments, allele-specific primers can be designed such that the presence of an amplification product indicates a genetic mutation. In some embodiments, allele-specific primers can be designed such that the absence of an amplification product indicates a genetic mutation.

[0124] In some embodiments, two complementary reactions are used. One reaction uses a primer specific for the wild-type allele (the "wild-type specific reaction") and the other reaction uses a primer for the mutant allele (the "mutation specific reaction"). The two reactions may use a common second primer. PCR primers specific for a particular allele (e.g., the wild-type allele or the mutant allele) usually perfectly match one allelic variant of the target but not the other allelic variant (e.g., the mutant allele or the wild-type allele). The mismatch may be located at / near the 3' end of the primer, leading to preferential amplification of the perfectly matched allele. Whether an amplification product can be detected from one or both reactions indicates the presence or absence of the mutant allele. Detection of an amplification product only from the wild-type specific reaction indicates the presence of only the wild-type allele (e.g., homozygosity for the wild-type allele). Detection of an amplification product only from the mutation specific reaction indicates the presence of only the mutant allele (e.g., homozygosity for the mutant allele). Detection of amplification products from both reactions indicates (e.g., a heterozygote). Herein, this approach is also referred to as "allele-specific amplification (ASA)."

[0125] Allele-specific amplification can also be used to detect duplications, insertions, or rearrangements by using primers that partially hybridize across the junction. The extent of junction overlap can be varied to allow specific amplification.

[0126] The amplification products can be examined by methods known in the art, including visualizing (e.g., with one or more dyes) bands of nucleic acid that have migrated (e.g., by electrophoresis) through a gel to separate the nucleic acids by size.

[0127] Allele-specific primer extension In some embodiments, an allele-specific primer extension (ASPE) approach is used to detect genetic mutations. ASPE uses allele-specific primers in the extension reaction that can distinguish between alleles (e.g., mutant and wild-type alleles), so that extension products are obtained only in the presence of a particular allele (e.g., mutant or wild-type alleles). The extension products can be detectable or can be made detectable, for example, by using labeled deoxyribonucleotides in the extension reaction. Any of a variety of labels are suitable for use in these methods, including, but not limited to, radioactive labels, fluorescent labels, chemiluminescent labels, enzyme labels, and the like. The nucleotides are labeled with an entity that can then be bound (directly or indirectly) by a biotin molecule that can be bound to a detectable label, for example, a streptavidin-linked fluorescent dye. In some embodiments, the reaction is performed multiplexed, for example, using many allele-specific primers in the same extension reaction.

[0128] In some embodiments, the extension products are hybridized to a solid or semi-solid support, such as beads, matrices, gels, etc., among others. For example, the extension products may be tagged with a specific nucleic acid sequence (e.g., included as part of an allele-specific primer), and the solid support may be attached with an "anti-tag" (e.g., a nucleic acid sequence complementary to the tag in the extension product). The extension products may be captured and detected on the solid support. For example, the beads may be sorted and detected. One such system that can be used in this way is the LUMlNEX™ MAP system, which can be adapted for cystic fibrosis mutation detection by TM Bioscience and is commercially available as Universal Bead Array (TAG-IT™).

[0129] Single nucleotide primer extension In some embodiments, single nucleotide primer extension (SNuPE) assays are used, in which the primer is designed to extend only one nucleotide. In such methods, the identity of the nucleotide immediately downstream of the 3' end of the primer is known, and the mutant allele is different compared to the wild-type allele. SNuPE can be performed using an extension reaction in which only a specific type of deoxynucleotide is labeled (e.g., labeled dATP, labeled dCTP, labeled dGTP, or labeled dTTP). Thus, the presence of a detectable extension product can be used as an indication of the identity of the nucleotide at the position of interest (e.g., the position immediately downstream of the 3' end of the primer), and therefore, can be used as an indication of the presence or absence of a mutation at that position. SNuPE can be performed as described in U.S. Pat. No. 5,888,819; U.S. Pat. No. 5,846,710; U.S. Pat. No. 6,280,947; U.S. Pat. No. 6,482,595; U.S. Pat. No. 6,503,718; U.S. Pat. No. 6,919,174; Piggee, C. et al., Journal of Chromatography A 781 (1997), p. 367-375 ("Capillary Electrophoresis for the Detection of Known Point Mutations by Single-Nucleotide Primer Extension and Laser-Induced Fluorescence Detection"); Hoogendoom, B. et al., Human Genetics (1999) 104:89-93, ("Genotyping Single Nucleotide Polymorphism by Primer Extension and High Performance Liquid Chromatography"), the entire contents of each of which are incorporated herein by reference.

[0130] In some embodiments, primer extension can be combined with mass spectrometry to accurately and quickly detect the presence or absence of mutation.See U.S. Patent No. 5,885,775 to Haff et al. (analysis of single nucleotide polymorphism analysis by mass spectrometry); U.S. Patent No. 7,501,251 to Koster (DNA diagnosis based on mass spectrometry); the teachings of both are incorporated herein by reference.Suitable mass spectrometry formats include, but are not limited to, matrix-assisted laser desorption / ionization, time-of-flight (MALDI-TOF), electrospray (ES), IR-MALDI, ion cyclotron resonance (ICR), Fourier transform, and combinations thereof.

[0131] Oligonucleotide ligation assay In some embodiments, oligonucleotide ligation assay ("OLA" or "OL") is used. OLA uses two oligonucleotides designed to hybridize to adjacent sequences of a single strand of a target molecule. Typically, one oligonucleotide is biotinylated and the other is detectably labeled, for example, with a streptavidin-linked fluorescent moiety. When the exact complementary sequence is found in the target molecule, the oligonucleotides hybridize with their ends adjacent, creating a ligation substrate that can be captured and detected. See, for example, Nickerson et al. (1990) Proc. Natl. Acad. Sci. USA 87:8923-8927, Landegren, U. et al. (1988) Science 241:1077-1080 and U.S. Patent No. 4,998,617, the entire contents of which are incorporated herein by reference in their entirety.

[0132] Hybridization approach In some embodiments, nucleic acid is analyzed by hybridization using one or more oligonucleotide probes specific to the biomarker of interest, under sufficiently stringent conditions that do not allow single nucleotide mismatch.In certain embodiments, suitable nucleic acid probes can distinguish between normal and mutant genes.Therefore, for example, those skilled in the art can use the probe of the present invention to determine whether an individual is homozygous or heterozygous for a particular allele.

[0133] Nucleic acid hybridization techniques are well known in the art. Those skilled in the art understand how to estimate and adjust the stringency of hybridization conditions so that sequences with at least desired levels of complementarity will stably hybridize, while sequences with lower complementarity will not. For examples of hybridization conditions and parameters, see, for example, Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Press, Plainview, NY; Ausubel, FM et al., 1994, Current Protocols in Molecular Biology. John Wiley & Sons, Secaucus, NJ.

[0134] In some embodiments, probe molecules that hybridize to mutant or wild-type sequences can be used to detect such sequences in the amplification products by liquid phase or, more preferably, solid phase hybridization, which can be accomplished, for example, by attaching the probe to a microchip.

[0135] Nucleic acid probes may include ribonucleic acid and / or deoxyribonucleic acid. In some embodiments, the nucleic acid probes provided are oligonucleotides (i.e., "oligonucleotide probes"). Generally, oligonucleotide probes are long enough to specifically bind to homologous regions of a gene of interest, but short enough that a one nucleotide difference between the probe and the nucleic acid sample being tested disrupts hybridization. Generally, the size of an oligonucleotide probe ranges from approximately 10 to 100 nucleotides. In some embodiments, the oligonucleotide probes are 15-90, 15-80, 15-70, 15-60, 15-50, 15-40, 15-35, 15-30, 18-30, or 18-26 nucleotides in length. As will be appreciated by those of skill in the art, the optimal length of an oligonucleotide probe may depend on the particular method and / or conditions in which the oligonucleotide probe may be used.

[0136] In some embodiments, the nucleic acid probe is useful as a primer, for example, for nucleic acid amplification and / or extension reaction. For example, in certain embodiments, the gene sequence evaluated for variants includes exon sequences. In certain embodiments, exon sequences and additional flanking sequences (e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 or more nucleotides of UTR and / or intron sequences) are analyzed in the assay. Alternatively, intron sequences or other non-coding regions can be evaluated for potentially harmful mutations. Alternatively, parts of these sequences can be used. Such variant gene sequences can include sequences that have at least one of the mutations described herein.

[0137] Other embodiments of the present disclosure provide isolated gene sequences containing mutations associated with symptoms and / or diseases of interest. Such gene sequences can be used to objectively diagnose the presence of NCGS or increased risk of developing it for a subject. In certain embodiments, the isolated nucleic acid can contain non-variant sequences or variant sequences of any one or combination thereof. For example, in certain embodiments, the gene sequence includes exon sequences. In certain embodiments, exon sequences and additional flanking sequences (e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 or more nucleotides of UTR and / or intron sequences) are analyzed in the assay. Alternatively, intron sequences or other non-coding regions may be used. Or, portions of these sequences may be used. In certain embodiments, the gene sequence includes exon sequences from at least one of the biomarker genes disclosed herein.

[0138] In some embodiments, the nucleic acid probe is labeled with a detectable moiety as described herein.

[0139] array Various methods mentioned herein can be adapted for use with arrays, which allows a set of biomarkers to be analyzed and / or detected in a single experiment.For example, multiple mutations involving biomarkers can be analyzed simultaneously.In particular, methods involving the use of nucleic acid reagents (e.g., probes, primers, oligonucleotides, etc.) are particularly suitable for adaptation to array-based platforms (e.g., microarrays).In some embodiments, the array contains one or more probes that are specific for detecting mutations in biomarkers of interest.

[0140] DNA sequencing In certain embodiments, diagnosis of a biomarker of interest is performed by detecting variations in the sequence, genomic location or configuration, and / or genomic copy number of a nucleic acid or panel of nucleic acids by sequencing.

[0141] In some embodiments, the method may include obtaining nucleic acid from a tissue or bodily fluid sample from the subject and sequencing at least a portion of the nucleic acid to obtain a sample nucleic acid sequence of at least one gene. In certain embodiments, the method may include comparing the variant to known variants associated with NCGS to determine whether the variant is a variant previously identified as associated with NCGS. Alternatively, the method may include identifying the variant as a new variant that has not been previously characterized. If the variant is a new variant, or in some cases a previously characterized (i.e., identified) variant, the method may further include performing an analysis to determine whether the mutation is predicted to be deleterious to the expression of the gene and / or the function of the protein encoded by the gene. The method may further include using the variant profile (i.e., a compilation of variants identified in the subject) to diagnose the presence of NCGS or an increased risk of developing NCGS.

[0142] For example, in certain embodiments, next-generation (massively parallel sequencing) may be used. Alternatively, Sanger sequencing may be used. Alternatively, a combination of next-generation (massively parallel sequencing) and Sanger sequencing may be used. Additionally and / or alternatively, sequencing includes at least one single molecule sequencing-by-synthesis method. Thus, in certain embodiments, multiple DNA samples are analyzed in pools to identify samples that exhibit variation. Additionally and / or alternatively, in certain embodiments, multiple DNA samples are analyzed in pools to identify individual samples that exhibit the same variation in at least two pools.

[0143] One traditional method of performing sequencing is by chain termination and gel separation, as described in Sanger et al., 1977, Proc Natl Acad Sci USA, 74:5463-67. Another traditional sequencing method involves chemical degradation of nucleic acid fragments. See Maxam et al., 1977, Proc. Natl. Acad. Sci., 74:560-564. Methods based on sequencing by hybridization have also been developed. See, e.g., Harris et al., U.S. Patent Publication No. 20090156412. Each of these references is incorporated herein by reference in its entirety.

[0144] In other embodiments, nucleic acid sequencing is achieved by massively parallel sequencing (also known as "next-generation sequencing") of single molecule or a group of nearly identical molecules derived from a single molecule by amplification by methods such as PCR. Massively parallel sequencing is described, for example, in Lapidus et al., U.S. Patent No. 7,169,560; Quake et al., U.S. Patent No. 6,818,395; Harris, U.S. Patent No. 7,282,337; and Braslavsky et al., PNAS (USA), 100:3960-3964 (2003), the contents of each of which are incorporated herein by reference.

[0145] In next generation sequencing, PCR or whole genome amplification may be performed on the nucleic acid to obtain sufficient amounts of nucleic acid for analysis. In some forms of next generation sequencing, amplification is not required, as the method is capable of evaluating DNA sequence from unamplified DNA. Once determined, the sequence and / or genomic arrangement and / or genomic copy number of the nucleic acid from the test sample is compared to a standard reference obtained from one or more individuals who were not known to be affected by NCGS at the time the sample was collected. Any difference between the sequence and / or genomic arrangement and / or genomic arrangement and / or copy number of the nucleic acid from the test sample and the standard reference is considered a variant.

[0146] In the next generation (massively parallel sequencing), all regions of interest are sequenced together and the origin of each read sequence is determined by comparison (alignment) with a reference sequence. Regions of interest can be enriched together in one reaction or enriched separately and then combined before sequencing. In a particular embodiment, and as described in more detail in the examples herein, DNA sequences derived from coding exons of genes included in the assay are enriched by bulk hybridization of randomly fragmented genomic DNA to specific RNA probes. The same adapter sequence binds to the ends of all fragments, allowing enrichment of fragments captured in all hybridizations by PCR with one primer pair in one reaction. Regions that were not captured very efficiently by hybridization are amplified by PCR with specific primers. In addition, PCR with specific primers can be used to amplify exons where similar sequences ("pseudo-exons") are present elsewhere in the genome.

[0147] In certain embodiments where massively parallel sequencing is used, PCR products are ligated to form long stretches of DNA that are sheared (e.g., by acoustic energy) into short fragments. This step ensures that the ends of the fragments are distributed throughout the region of interest. A stretch of dA nucleotides is then added to the 3' end of each fragment, which allows the fragments to be bound to a flat surface coated with oligo(dT) primers ("flow cell"). Each fragment can then be sequenced by extending the oligo(dT) primers with fluorescently labeled nucleotides. During each sequencing cycle, only one type of nucleotide (A, G, T, or C) is added, and only one nucleotide is incorporated with a chain-terminating nucleotide. For example, during the first sequencing cycle, fluorescently labeled dCTP can be added. This nucleotide is incorporated only into the growing complementary DNA strand that requires a C as the next nucleotide. After each sequencing cycle, an image of the flow cell is taken to determine which fragments have been extended. DNA strands that have incorporated C emit light, while those that have not incorporated C appear dark. Chain termination is reversed, allowing the growing DNA strand to extend again, and the process is repeated for a total of 120 cycles.

[0148] The images are converted into strings of bases, commonly called "reads," that replicate the 25-60 bases at the 3' end of each fragment. The reads are then compared to a reference sequence for the analyzed DNA. Because any string of 25 bases typically occurs only once in the human genome, most reads can be "aligned" to one specific location in the human genome. Finally, a consensus sequence for each genomic region is constructed from the available reads and can be compared to the exact sequence of the reference at that location. Differences between the consensus sequence and the reference are called sequence variants.

[0149] Detectable Part In certain embodiments, certain molecules used in accordance with and / or provided by the present invention (e.g., nucleic acid probes, antibodies, etc.) contain one or more detectable entities or moieties, i.e., such molecules are "labeled" with such entities or moieties.

[0150] Any of a wide variety of detectable agents can be used in the practice of the present disclosure. Suitable detectable agents include, but are not limited to: various ligands, radionuclides; fluorescent dyes; chemiluminescent agents (e.g., acridinium esters, stabilized dioxetanes, etc.); bioluminescent agents; spectrally resolvable inorganic fluorescent semiconductor nanocrystals (i.e., quantum dots); microparticles; metal nanoparticles (e.g., gold, silver, copper, platinum, etc.); nanoclusters; paramagnetic metal ions; enzymes; colorimetric labels (e.g., dyes, colloidal gold, etc.); biotin; dioxygenin; haptens; and proteins for which antisera or monoclonal antibodies are available.

[0151] In some embodiments, the detectable moiety is biotin, which can be bound to avidin (such as streptavidin), which is generally bound (directly or indirectly) to other moieties that are themselves detectable (e.g., fluorescent moieties).

[0152] Below are some non-limiting examples of some detectable moieties that can be used.

[0153] Fluorescent dyes In certain embodiments, the detectable moiety is a fluorescent dye. A large number of known fluorescent dyes with a wide variety of chemical structures and physical properties are suitable for use in the practice of the present disclosure. The fluorescent detectable moiety is stimulated by a laser and the emitted light is captured by a detector. The detector can be a charge-coupled device (CCD) or a confocal microscope that records its intensity.

[0154] Suitable fluorescent dyes include, but are not limited to, fluorescein and fluorescein dyes (e.g., fluorescein isothiocyanine or FITC, naphthofluorescein, 4',5'-dichloro-2',7'-dimethoxyfluorescein, 6-carboxyfluorescein or FAM, etc.), carbocyanines, merocyanines, styryl dyes, oxonol dyes, phycoerythrin, erythrosine, eosin, rhodamine dyes (e.g., carboxytetramethylrhodamine or TAMRA, carboxyrhodamine 6G, carboxy-X-rhodamine (ROX), Lissamine rhodamine B, rhodamine 6G, rhodamine green, rhodamine red, tetramethylrhodamine (TMR), etc.), coumarin and coumarin dyes (e.g., methoxycoumarin, dialkylaminocoumarin, hydroxycoumarin, aminomethylcoumarin (AMCA), etc.), Q-DOTS. Oregon Green dyes (e.g., Oregon Green 488, Oregon Green 500, Oregon Green 514, etc.), Texas Red, Texas Red-X, SPECTRUM RED™, SPECTRUM GREENT™, cyanine dyes (e.g., CY-3™, CY-5™, CY-3.5™, CY-5.5™, etc.), ALEXA FLUOR™ dyes (e.g., ALEXA FLUOR™ 350, ALEXA FLUOR™ 488, ALEXA FLUOR™ 532, ALEXA FLUOR™ 546, ALEXA FLUOR™ 568, ALEXA FLUOR™ 594, ALEXA FLUOR™ 633, ALEXA FLUOR™ 660, ALEXA FLUOR™ 680, etc.), BODIPY™ dyes (e.g., BODIPY™ FL, BODIPY™ R6G, BODIPY™ TMR, BODIPY™ TR, BODIPY™ 530 / 550, BODIPY™ 558 / 568, BODIPY™ 564 / 570, BODIPY™ 576 / 589, BODIPY™ 581 / 591, BODIPY™ 630 / 650, BODIPY™ 650 / 665, etc.), IRDyes (e.g., IRD40, IRD 700, IRD 800, etc.), etc.For more examples of suitable fluorescent dyes and methods for conjugating fluorescent dyes to other chemical entities such as proteins and peptides, see, for example, "The Handbook of. See, "Fluorescent Probes and Research Products," 9th Edition, Molecular Probes, Inc., Eugene, Oreg. Favorable properties of fluorescent labeling agents include high molar absorption coefficients, high fluorescence quantum yields, and photostability. In some embodiments, the labeling fluorophores exhibit absorption and emission wavelengths in the visible range (i.e., between 400-750 nm) rather than the ultraviolet range (i.e., below 400 nm) of the spectrum.

[0155] The detectable moiety may include multiple chemical entities, such as those in fluorescence resonance energy transfer (FRET). Resonance transfer results in an overall increase in emission intensity. See, for example, Ju et al. (1995) Proc. Nat'l, the entire contents of which are incorporated herein by reference. See Acad. Sci. (USA) 92:4347. To achieve resonance energy transfer, a first fluorescent molecule (the "donor" fluorophore) absorbs light and transfers it to a second fluorescent molecule (the "acceptor" fluorophore) through the resonance of excited electrons. In one approach, both donor and acceptor dyes can be linked together and attached to an oligo primer. Methods for linking donor and acceptor dyes to nucleic acids are described, for example, in U.S. Pat. No. 5,945,526 to Lee et al., the entire contents of which are incorporated herein by reference. Donor / acceptor dye pairs that can be used include, for example, fluorescein / tetramethylrhodamine, IAEDANS / fluorescein, EDANS / DABCYL, fluorescein / fluorescein, BODIPY FL / BODIPY FL, and fluorescein / QSY7 dye. See, for example, U.S. Pat. No. 5,945,526 to Lee et al. Many of these dyes are also commercially available, for example from Molecular Probes Inc. (Eugene, Oreg.) Suitable donor fluorophores include 6-carboxyfluorescein (FAM), tetrachloro-6-carboxyfluorescein (TET), 2'-chloro-7'-phenyl-1,4-dichloro-6-carboxyfluorescein (VIC), and the like.

[0156] enzyme In certain embodiments, the detectable moiety is an enzyme. Examples of suitable enzymes include, but are not limited to, enzymes used in ELISA, such as horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase, etc. Other examples include β-glucuronidase, β-D-glucosidase, urease, glucose oxidase, etc. Enzymes can be attached to molecules using linker groups such as carbodiimides, diisocyanates, glutaraldehyde, etc.

[0157] radioactive isotope In certain embodiments, the detectable moiety is a radioisotope. For example, the molecule may be isotopically labeled (i.e., may contain one or more atoms replaced with an atom having an atomic mass or mass number different from that normally found in nature), or the isotope may be attached to the molecule. Non-limiting examples of isotopes that can be incorporated into the molecules include isotopes of hydrogen, carbon, fluorine, phosphorus, copper, gallium, yttrium, technetium, indium, iodine, rhenium, thallium, bismuth, astatine, samarium, and lutetium (i.e., 3H, 13C, 14C, 18F, 19F, 32P, 35S, 64Cu, 67Cu, 67Ga, 90Y, 99mTc, 111In, 125I, 123I, 129I, 131I, 135I, 186Re, 187Re, 201T1, 212Bi, 213Bi, 211At, 153Sm, 177Lu).

[0158] In some embodiments, signal amplification is achieved using labeled dendrimers as detectable moieties (see, e.g., Physiol Genomics 3:93-99, 2000), the entire contents of which are incorporated herein by reference in their entirety. Fluorescently labeled dendrimers are available from Genisphere (Montvale, NJ). These can be chemically attached to oligonucleotide primers by methods known in the art.

[0159] kit In certain embodiments, the present disclosure provides a kit for use according to the methods and compositions disclosed herein.Generally, the kit comprises one or more reagents for detecting biomarkers of interest.Suitable reagents can include nucleic acid probes and / or antibodies or fragments thereof.In some embodiments, suitable reagents are provided in the form of arrays such as microarrays or mutation panels.

[0160] In some embodiments, the provided kits further comprise reagents for carrying out the various detection methods described herein (e.g., sequencing, hybridization, primer extension, multiplex ASPE, immunoassays, etc.). For example, the kits may contain buffers, enzymes, and / or reagents for use in the methods described herein, as needed, for example, for amplifying nucleic acids via primer-directed amplification, for carrying out ELISA experiments, etc.

[0161] In some embodiments, the provided kit further comprises a control representative of a healthy individual, for example, a nucleic acid and / or protein sample from an individual who does not have the disease and / or symptoms of interest. In one embodiment, the control is derived from a healthy individual or an individual who has no or no detectable gastrointestinal pathology. In some embodiments, the control is a disease control. Such disease controls may include individuals with celiac disease (stratified according to whether the individual follows a gluten-free diet) and subjects with other GI diseases, such as inflammatory bowel disease (IBD), hepatitis, small intestinal bacterial overgrowth, and other diseases.

[0162] The kit may also include instructions on how to determine whether an individual has or is at risk for developing a disease and / or condition of interest.

[0163] In some embodiments, computer readable media is provided that encodes information corresponding to a biomarker of interest. Such computer readable media may be included in a kit of the invention.

[0164] How to Identify NCGS Markers Data Mining In certain embodiments of the present disclosure, biomarkers are identified using data mining approaches. For example, in some instances, public databases (e.g., PubMed) can be searched for genes that have been shown to be associated (directly or indirectly) with a particular disease. Such genes can then be evaluated as biomarkers. In one embodiment, the results of such an analysis identify IP-10, IL-8, IgE, TNF-α, IL-10, CD4, and CD45 as potential markers of interest. Figure 1 shows an example of a multi-node interaction network that identifies markers associated with NCGS, as described in detail in U.S. Provisional Patent Application No. 62 / 505,536, filed May 12, 2017, and U.S. Provisional Patent Application No. 62 / 523,382, filed June 22, 2017, which are incorporated herein by reference in their entireties. In the figure, circled markers include more relevant disease markers.

[0165] molecule In certain embodiments, the present disclosure includes methods for identifying biomarkers (i.e., variants in nucleic acid sequences that are associated with NCGS in a statistically significant manner) for a condition or disease of interest. Genes and / or genomic regions assayed for new markers may be selected based on their importance in biochemical pathways that indicate genetic linkage and / or biological causation to the condition and / or disease of interest. Alternatively, genes and / or genomic regions assayed for markers may be selected based on genetic linkage with DNA regions that are genetically related to the inheritance of NCGS in families. Alternatively, genes and / or genomic regions assayed for markers may be systematically evaluated to cover specific regions of chromosomes that have not yet been evaluated.

[0166] In other embodiments, the gene or genomic region evaluated for new markers may be part of a biochemical pathway that may be related to the onset of the condition and / or disease of interest (e.g., NCGS). Variants and / or combinations of variants may be evaluated for their clinical significance based on one or more of the following methods. If a variant and / or combination of variants is reported or known to occur more frequently in the nucleic acid from a subject with the condition and / or disease of interest than in a subject without the condition, it is considered to be at least potentially susceptible to the condition and / or disease of interest. If a variant and / or combination of variants is reported or known to be exclusively or preferentially transmitted to individuals with the condition and / or disease of interest, it is considered to be at least potentially susceptible to the condition and / or disease of interest. Conversely, if a variant is found at a similar frequency in both populations, it is unlikely to be related to the onset of the condition and / or disease of interest.

[0167] If a variant or combination of variants is reported or known to have a global detrimental effect on the function of this protein or biological system in an experimental model system suitable for measuring the function of the protein or biological system, and if this variant or combination of variants affects one or more genes known to be related to the condition and / or disease of interest, it is considered to be at least potentially susceptible to the condition and / or disease of interest.For example, if a variant or combination of variants is predicted based on the predicted effect on the sequence and / or structure of protein or nucleic acid to have a global detrimental effect on protein or gene expression (i.e., to cause nonsense mutation, frameshift mutation, or splice site mutation, or even missense mutation), and if this variant or combination of variants affects one or more genes known to be related to the condition and / or disease of interest, it is considered to be at least potentially susceptible to the condition and / or disease of interest.

[0168] Also, in certain embodiments, the total number of variants may be important.When one or several variants are detected in test sample, which are evaluated as possibly being related to at least symptoms and / or disease of interest, either individually or in combination, the individual who has this variant or these variants detected in their genetic material can be diagnosed as suffering from or at high risk of developing symptoms and / or disease of interest.

[0169] For example, the disclosure herein provides a method for diagnosing the presence of or increased risk of developing NCGS in a subject. Such a method may include obtaining nucleic acid from a tissue or bodily fluid sample. The method may further include sequencing the nucleic acid or determining the genomic location or copy number of the nucleic acid to detect whether there are one or more variants in the nucleic acid sequence or genomic location or copy number. The method may further include evaluating the clinical significance of the one or more variants. Such analysis may include evaluating the degree of association of the variant sequence in a diseased population (i.e., subjects with the disease). Such analysis may also include analyzing the degree of effect that the mutation may have on gene expression and / or protein function. The method may also include diagnosing the presence of or increased risk of developing NCGS based on the evaluation.

[0170] The following examples serve to illustrate certain aspects of the present disclosure and are not intended to be limiting in any way. EXAMPLES

[0171] Tumor necrosis factor alpha (TNF-α) Tumor necrosis factor alpha (TNF-α), also known as cachectin, and TNFSF1A are the prototype ligands of the TNF superfamily. TNF-α plays a central role in inflammation, immune system development, apoptosis, and lipid metabolism. TNF-α has also been implicated in a number of disease states, including asthma, Crohn's disease, rheumatoid arthritis, neuropathic pain, obesity, type 2 diabetes, septic shock, autoimmunity, and cancer.

[0172] TNF-α may be measured using the Quantikine assay. The Quantikine TNF-α Immunoassay is a 4.0-hour solid-phase ELISA designed to measure human TNF-α in serum and plasma. The assay contains recombinant human TNF-α from E. coli and an antibody raised against the recombinant factor, and uses a quantitative sandwich enzyme immunoassay technique. A monoclonal antibody specific for human TNF-α is precoated onto a microplate. Standards and samples are pipetted into the wells, and any TNF-α present binds to the immobilized antibody. After washing away unbound material, a biotinylated polyclonal antibody specific for human TNF-α is added to the wells, the wells are washed to remove unbound antibody-biotin reagent, and enzyme-linked streptavidin is added to the wells. After washing, a substrate solution (hydrogen peroxide and tetramethylbenzidine) is added to the wells, resulting in color development proportional to the amount of TNF-α bound in the initial step. Color development is stopped and the color intensity is measured at 450 nm and the measurements at 540 nm and 570 nm are subtracted.

[0173] The sample may be serum or plasma. For serum, serum separator tubes (SST) are used and the sample is allowed to clot at room temperature for 30 minutes, followed by centrifugation at 1000xg for 15 minutes. The serum is removed and assayed. The sample is used immediately or divided into aliquots and stored at -20°C or below. Plasma is collected using EDTA or heparin as the anticoagulant. The sample is centrifuged at 1000xg for 15 minutes within 30 minutes of collection, and the sample is either assayed immediately or divided into aliquots and stored at -20°C or below. For both serum and plasma samples, repeated freeze-thaw cycles should be avoided. In general, severely hemolyzed samples, high albumin samples, and citrated plasma should not be used.

[0174] Interleukin 10 (IL-10) Interleukin 10 (IL-10), originally named cytokine synthesis inhibitory factor (CSIF), is a pleiotropic cytokine that can exert either immunosuppressive or immunostimulatory effects on diverse cell types. IL-10 is a potent modulator of monocyte / macrophage function. As a downregulator of cellular immune responses, IL-10 can suppress the production of prostaglandin E2 and numerous inflammatory cytokines, including TNF-α, IL-1, IL-6, and IL-8, by monocytes after activation. IL-10 also promotes the release of soluble TNF receptors and inhibits the expression of surface ICAM-1 and B. IL-10 has been reported to suppress the synthesis of superoxide anions and reactive oxygen intermediates (ROIs), and to inhibit or promote NO synthesis depending on the time of exposure to activated macrophages. IL-10 also has a pronounced effect on B cells. For example, it induces IgA synthesis in CD40-activated cells and selects for the secretion of IgG1 and IgG3. IL-10 also has demonstrated activity on endothelial cells (mimicking IL-4), and on thymocytes and mast cells (acting as a growth co-stimulator).

[0175] IL-10 may be measured using the Quantikine assay. The Quantikine IL-10 immunoassay is a 3.5-4.5 hour solid-phase ELISA designed to measure IL-10 in cell culture supernatants, serum and plasma. It contains Sf21-expressed recombinant human IL-10 and an antibody raised against the recombinant factor. The assay uses a quantitative sandwich enzyme immunoassay technique. A monoclonal antibody specific for IL-10 is precoated onto a microplate. Standards and samples are pipetted into the wells and any IL-10 present binds to the immobilized antibody. After washing away unbound material, an enzyme-linked monoclonal antibody specific for IL-10 is added to the wells. After washing, a substrate solution (hydrogen peroxide and tetramethylbenzidine) is added to the wells, resulting in color development proportional to the amount of TNF-α bound in the initial step. Color development is stopped and the color intensity is measured at 450 nm and measurements at 540 nm and 570 nm are subtracted.

[0176] For cell culture supernatants, particulates are removed by centrifugation. Samples are assayed immediately or divided into aliquots and stored at -20°C or below. Repeated freeze-thaw cycles should be avoided. For serum, serum separator tubes (SSTs) are used and samples are allowed to clot for 30 minutes at room temperature, then centrifuged at 1000xg for 15 minutes. Serum is removed and assayed. Samples are used immediately or divided into aliquots and stored at -20°C or below. Plasma is collected using EDTA or heparin as an anticoagulant. Samples are centrifuged at 1000xg for 15 minutes within 30 minutes of collection, and samples are assayed immediately or divided into aliquots and stored at -20°C or below. Repeated freeze-thaw cycles should be avoided for both serum and plasma samples. In general, severely hemolyzed and high albumin samples should not be used.

[0177] Interleukin 8 (IL-8) Interleukin 8 (IL-8), a member of the neutrophil-specific CXC subfamily of chemokines, is a potent neutrophil chemotactic and activating factor. It is the major proinflammatory cytokine produced by many cells (including monocytes / macrophages, T cells, neutrophils, fibroblasts, endothelial cells, keratinocytes, hepatocytes, astrocytes and chondrocytes) in response to proinflammatory stimuli such as IL-1, TNF, LPS and viruses. Its function is, in part, to attract neutrophils to the site of inflammation and activate them. IL-8 binds to two seven-transmembrane G protein-coupled receptors, CXCR1 and CXCR2, as well as to the non-signaling Duffy antigen on red blood cells. The Duffy antigen may play a role in regulating IL-8 activity on functional receptors.

[0178] IL-8 can be measured using the Quantikine assay. The Quantikine IL-8 immunoassay is a 3.5-hour solid-phase ELISA designed to measure human IL-8 in cell culture supernatants, serum and plasma. It is based on an antibody raised against a 72 amino acid variant of human IL-8 from E. coli. It is calibrated with the same recombinant factor. The assay uses a quantitative sandwich enzyme immunoassay technique. A monoclonal antibody specific for IL-8 is precoated on a microplate. Standards and samples are pipetted into the wells and any IL-8 present binds to the immobilized antibody. After washing away unbound material, an enzyme-linked monoclonal antibody specific for IL-8 is added to the wells. After washing, a substrate solution (hydrogen peroxide and tetramethylbenzidine) is added to the wells, resulting in color development proportional to the amount of TNF-α bound in the initial step. The color development is stopped and the color intensity is measured at 450 nm and the measurements at 540 nm and 570 nm are subtracted.

[0179] For cell culture supernatants, particulates are removed by centrifugation. Samples are assayed immediately or divided into aliquots and stored at -20°C or below. Repeated freeze-thaw cycles should be avoided. For serum, serum separator tubes (SSTs) are used and samples are allowed to clot for 30 minutes at room temperature, then centrifuged at 1000xg for 15 minutes. Serum is removed and assayed. Samples are used immediately or divided into aliquots and stored at -20°C or below. Plasma is collected using EDTA or heparin as an anticoagulant. Samples are centrifuged at 1000xg for 15 minutes within 30 minutes of collection, and samples are assayed immediately or divided into aliquots and stored at -20°C or below. Repeated freeze-thaw cycles should be avoided for both serum and plasma samples. In general, severely hemolyzed and highly albuminized samples should not be used.

[0180] Total IgE Immunoglobulin E (IgE) plays an important role in immunological protection against parasitic infections and allergies (type 1 hypersensitivity). For example, binding of allergens to sensitized mast or basophil cells results in cross-linking of IgE on the cell membrane, leading to cell degranulation and release of factors (e.g., histamine), which cause the typical symptoms of type 1 hypersensitivity. IgE concentrations in serum are usually very low (less than 0.001% of total serum immunoglobulins). IgE concentrations are generally age-dependent, with the lowest value being that measured at birth. The concentration gradually increases and becomes stable between 5 and 7 years of age, although IgE values ​​can vary greatly within certain age groups. In infants and small children with recurrent airway diseases, the measurement of IgE may be relevant later. As IgE is important in allergies, elevated IgE concentrations can be found in patients with allergic diseases such as hay fever, atopic bronchitis and dermatitis. Elevated serum IgE concentrations can also occur in nonallergic diseases, such as bronchopulmonary aspergillosis, Wiskott-Aldrich syndrome, hyper-IgE syndrome, IgE myeloma, and parasitic infections.

[0181] The IgE II assay (Elecsys) uses a monoclonal antibody made specifically against human IgE. The Elecsys assay is a sandwich immunoassay. During the first incubation, IgE in the sample (10 μL) is mixed with a biotinylated monoclonal IgE-specific antibody, which is labeled with a ruthenium complex / complex (tris(2,2'-bipyridyl)ruthenium(II)-complex / complex (Ru(bpy)2+) to form a sandwich complex / complex. After adding streptavidin-coated microparticles, the complex binds to the solid phase via biotin-streptavidin interaction. The reaction mixture is then drawn into the measuring cell, where the microparticles are captured by magnetism on the surface of the electrode. Unbound material is then removed by the ProCell. A voltage is applied to the electrode, inducing chemiluminescence that is measured by a photomultiplier tube. The results are determined by a calibration curve generated instrument-specifically by two-point calibration and a master curve provided via the reagent barcode.

[0182] Alternatively, an assay based on ImmunoCAP (Phadia US, Inc.,) may be used. In this assay, anti-IgE covalently bound to ImmunoCAP reacts with total IgE in the patient sample. After washing, an enzyme-labeled antibody against IgE is added to form a complex. After incubation, unbound enzyme-anti-IgE is washed away and the bound complex is incubated with a developer. After stopping the reaction, the fluorescence of the eluate is measured. Fluorescence is directly proportional to the concentration of IgE in the sample. The higher the response, the more IgE is present in the sample. To evaluate the test results, the responses of the patient samples are converted to concentrations using a standard curve.

[0183] A variety of patient samples may be used. Serum may be sampled using standard sampling tubes or separation gels, Li + -Heparin, Sodium + -Heparin, K +-EDTA, and sodium citrate plasma may be collected using tubes containing If sodium citrate is used, results must be corrected by +10%.

[0184] References and citations to other documents, such as patents, patent applications, patent publications, journals, books, papers, web content, etc., have been made throughout this disclosure. All such documents are incorporated herein by reference in their entirety for all purposes. Various modifications and equivalents of what is described herein will be apparent to those skilled in the art from the entire contents of this document, including references to the scientific and patent literature cited herein. The subject matter of this specification, in its various embodiments and equivalents thereof, contains information, examples, and guidance that can be adapted to the practice of the present disclosure. In certain embodiments, for example, the following items are provided: (Item 1) 1. A method for detecting a biomarker associated with non-celiac gluten sensitivity (NCGS) in an individual, comprising: obtaining a sample from the individual; measuring the amount of at least one of IL-8, IL-10, TNF-α or total IgE protein in said sample; The method includes: (Item 2) 2. The method of claim 1, wherein the measuring step comprises an immunoassay. (Item 3) 2. The method of claim 1, wherein the measuring step comprises flow cytometry. (Item 4) The method of claim 1, further comprising measuring the expression of at least one of IP-10, CD4 or CD45. (Item 5) 5. The method according to any one of items 1 to 4, wherein the sample comprises blood, serum, plasma or a tissue biopsy. (Item 6) A method for identifying markers associated with NCGS in an individual, the method comprising identifying at least one marker whose expression is increased or decreased in NCGS but not in celiac disease compared to normal controls. (Item 7) A method for detecting the presence or susceptibility to non-celiac gluten sensitivity (NCGS) in an individual, obtaining a sample from said individual; measuring the amount of at least one of IL-8, IL-10, TNF-α, or total IgE protein in said sample; comparing the expression of at least one of said IL-8, IL-10, TNF-α or total IgE in said sample to a control value for each of said IL-8, IL-10, TNF-α or total IgE; The method includes: (Item 8) 8. The method of claim 7, wherein the measuring comprises an immunoassay. (Item 9) 8. The method of claim 7, wherein the measuring comprises flow cytometry. (Item 10) The method of item 7, further comprising measuring the expression of at least one of IP-10, CD4 or CD45. (Item 11) 11. The method according to any one of items 7 to 10, wherein the sample comprises blood, serum, plasma or a tissue biopsy. (Item 12) A composition for detecting a biomarker associated with non-celiac gluten sensitivity (NCGS) in an individual, comprising a reagent for quantifying the level of at least one of the proteins IL-8, IL-10, TNF-α or total IgE in a biological sample. (Item 13) 13. The composition of claim 12, wherein the protein is detected by immunoassay. (Item 14) 14. A kit comprising the composition according to item 12 or 13.

Claims

1. 1. A method for determining whether the amount of at least one protein in a sample obtained from an individual is indicative of non-celiac gluten sensitivity (NCGS) in said individual, said method comprising: measuring the amount of total immunoglobulin E (IgE) protein, the amount of IL-8 and IL-10 protein, the amount of IL-8 and TNF-α protein, the amount of IL-8 and total IgE protein, the amount of IL-10 and total IgE protein, or the amount of TNF-α and total IgE protein in said sample; and Comparing the measured protein amounts with corresponding control values. wherein the corresponding control value is based on the amount of total immunoglobulin E (IgE) protein, the amount of IL-8 and IL-10 protein, the amount of IL-8 and TNF-α protein, the amount of IL-8 and total IgE protein, the amount of IL-10 and total IgE protein, or the amount of TNF-α and total IgE protein in a sample obtained from a healthy individual.

2. The method of claim 1 , wherein the measuring step comprises performing an immunoassay.

3. The method of claim 1 or 2, further comprising measuring the expression of at least one of IP-10, CD4 or CD45 in the sample.

4. The method of claim 3 , wherein the step of measuring expression comprises performing flow cytometry.

5. The method of any one of claims 1 to 4, wherein the sample comprises blood, serum, plasma or a tissue biopsy.

6. 1. A method for determining the expression level of at least one protein marker in a sample obtained from an individual as an indication of non-celiac gluten sensitivity (NCGS) in said individual, said method comprising: Measuring the expression level of total immunoglobulin E (IgE), the expression levels of IL-8 and IL-10, the expression levels of IL-8 and TNF-α, the expression levels of IL-8 and total IgE, the expression levels of IL-10 and total IgE, or the expression levels of TNF-α and total IgE in the sample; and Comparing the measured expression levels with corresponding control values. wherein the corresponding control value is based on the expression level of total immunoglobulin E (IgE), the expression level of IL-8 and IL-10, the expression level of IL-8 and TNF-α, the expression level of IL-8 and total IgE, the expression level of IL-10 and total IgE, or the expression level of TNF-α and total IgE in a sample obtained from a healthy individual.

7. The method of claim 6 , wherein the measuring step comprises performing an immunoassay.

8. The method of claim 6 or 7, wherein the measuring step comprises performing flow cytometry.

9. The method of any one of claims 6 to 8, further comprising the step of measuring the expression of at least one of IP-10, CD4 or CD45 in the sample.

10. The method of any one of claims 6 to 9, wherein the sample comprises blood, serum, plasma or a tissue biopsy.

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