Patent Publication Number: US-2023152313-A1

Title: Antibody Tests for Identifying RO Negative Sjogren&#39;s Syndrome and Use as Biomarkers for Dysregulated B Cell Responses, B Cell Lymphoma, Tissue Fibrosis and Salivary Gland Dysfunction

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This application is a National Stage of International Application of PCT/US2021/016596, filed Feb. 4, 2021, which claims the benefit of U.S. Provisional Application No. 62/970,043, filed Feb. 4, 2020 and U.S. Provisional Application Serial No. 63/110,476 filed Nov. 6, 2020, the entire contents of which is incorporated herein by reference. 
    
    
     TECHNICAL FIELD OF THE INVENTION 
     The present invention relates in general to the field of novel antibody tests for identifying Ro negative Sjögren’s Syndrome and use as biomarkers for dysregulated B cell responses, B cell lymphoma, tissue fibrosis and salivary gland dysfunction. 
    
    
     STATEMENT OF FEDERALLY FUNDED RESEARCH 
     This invention was made with government support under 1R01AR074310-02 awarded by the National Institutes of Health. The government has certain rights in the invention. 
    
    
     BACKGROUND OF THE INVENTION 
     Without limiting the scope of the invention, its background is described in connection with Sjögren’s Syndrome. 
     Sjögren’s syndrome (SS) is a rheumatic autoimmune disease selectively targeting salivary and lacrimal glands, leading to painful dry mouth and eyes, oral infections, severe dental caries/tooth loss, fatigue, arthritis, nervous system involvement and malignant B cell lymphoma. Current internationally accepted disease classification criteria rely on either the presence of anti-Ro antibodies (these may target either the Ro60 antigen, Ro52 antigen or both) or the presence of focal lymphocytic infiltrates in a minor salivary gland lip biopsy for diagnosis (1, 2). 
     Among 475 individuals attending the Oklahoma Sjögren’s Research clinic and meeting classification criteria for primary SS, 38% lacked antibodies to Ro antigen. These individuals met classification criteria for SS due to a focus score ≥ 1 on examination of minor salivary gland lip biopsy. The antigen(s) driving the aberrant immune response in these individuals is unknown. 
     Novel methods are needed for identifying anti-Ro antibody negative patients with SS, but without the need to perform a salivary gland lip biopsy. 
     SUMMARY OF THE INVENTION 
     In one embodiment, the present invention includes a method of determining that a patient negative for Ro autoantibodies has Sjögren’s syndrome (SS) with or without performing a lip biopsy comprising: obtaining a biological sample from the patient; detecting if the biological sample has autoantibodies to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 proteins selected from their encoding gene names: RPAP3, ARFGAP1, C9orf78, CNDP2, FABP1, FXYD5, GRAMD1A, HNRNPAB, LIX1, MCCC2, PEAS/ND, POU6F1, PPIL3, SPSB2, SRPK2, WDR20, CCDC155, DDB1, MUM1L1, NFU1, RPS29, SOX5, TCP10, ZNF655, or RPAP3 wherein the level of autoantibody is ≥ 3 standard deviations (SD) above the mean of healthy controls. In one aspect, the method further comprises the step of detecting if the biological sample has autoantibodies to at least one of: CCDC155, DDB1, MUM1L1, NFU1, RPS29, SOX5, TCP10, ZNF655, or RPAP3 from a Ro positive or Ro negative patient and determining that the patient has SS without performing a lip biopsy. In another aspect, the autoantibody has a reactivity of ≥ 4SD above the mean of healthy controls to at least one of: RPAP3, MUM1L1, RPS29, SOX5 or RPAP3 to identify three-fourths of the Ro negative individuals with SS. In another aspect, the method further comprises the step of detecting if the biological sample has autoantibodies to at least one of: CCDC155, DDB1, MUM1L1, NFU1, RPS29, SOX5, TCP10, ZNF655, or RPAP3, identifies 93% of the Ro positive cases and 87% of the Ro negative cases. In another aspect, the autoantibodies to SOX5 also cross-react with at least one of SOX3, SOX4, SOX6, SOX9, SOX11, SOX13 and SOX30. In another aspect, the method further comprises the step of detecting if the liquid biological sample has autoantibodies to one or more proteins in Table 1, other than MUM1L1 and SOX5. In another aspect, the method further comprises the step of detecting if the liquid biological sample has autoantibodies to one or more proteins in Table 2 from patients with or without Ro autoantibodies. In another aspect, the autoantibodies are detected using an assay selected from at least one of: ELISA, flow cytometry, fluorimetry, microscopy, immunofluorescence, radioimmunoassay, immunoenzymatic assay, fluorescence activated cell sorting (FACS), differential display, representational difference analysis, microarray, Western blotting, immunohistochemical staining, immunocytochemical staining, dot blots, or surface plasmon resonance detection. In another aspect, the liquid biological sample selected from a saliva, a blood, a plasma, a serum, or a tear sample. In another aspect, the method further comprises the step of treating the patient negative for Ro autoantibodies with a therapy that treats or reduces the symptoms of SS. In another aspect, the biological sample is negative for autoantibodies to KCNAB1, KCNAB2, or as listed in Table 1, Table 2,  FIG.  11   , or  FIG.  16    in Ro negative cases are used to detect SS. 
     In another embodiment, the present invention includes an assay for detecting autoantibodies to RNA Polymerase II Associated Protein 3 (RPAP3) comprising: contacting a biological sample suspected of having autoantibodies with an RPAP3 protein under conditions in which the presence of autoantibodies to RNA Polymerase II Associated Protein 3 (RPAP3) are detected. In one aspect, the assay is an ELISA, flow cytometry, fluorimetry, microscopy, immunofluorescence, radioimmunoassay, immunoenzymatic assay, fluorescence activated cell sorting (FACS), differential display, representational difference analysis, microarray, Western blotting, immunohistochemical staining, and immunocytochemical staining, dot blots, or surface plasmon resonance detection. In another aspect, the biological sample is a liquid biological sample selected from a saliva, a blood, a serum, a plasma, or a tear sample. In another aspect, the assay further comprises detecting autoantibodies to at least 1, 2, 3, 4, 5, 6, 7 or 8 proteins selected from: CCDC155, DDB1, MUM1L1, NFU1, RPS29, SOX5, TCP10, or ZNF655 antigens. In another aspect, the autoantibody has a reactivity ≥ 2SD, ≥ 3SD, or ≥4SD, above the mean of healthy controls to at least one of: CCDC155, DDB1, MUM1L1, NFU1, RPS29, SOX5, TCP10, ZNF655 antigens in the Ro negative group to identify three-fourths of the Ro negative individuals. In another aspect, the autoantibodies to SOX5 also cross-react with at least one of SOX3, SOX4, SOX6, SOX9, SOX11, SOX13 and SOX30. In another aspect, the biological sample is negative for autoantibodies to KCNAB1, KCNAB2, or as listed in Table 1, Table 2,  FIG.  11   , or  FIG.  16    in Ro neg cases are used to detect SS. 
     In another embodiment, the present invention includes a kit comprising a synthetic or recombinant polypeptide covalently attached to a solid support, wherein the synthetic or recombinant polypeptide comprises 1, 2, 3, 4, 5, 6, 7, 8, or 9 proteins selected from: CCDC155, DDB1, MUM1L1, NFU1, RPS29, SOX5, TCP10, ZNF655, or RPAP3. In one aspect, the kit further comprises instructions for contacting the solid support with a biological sample from a patient suspected of having Sjögren’s syndrome. In one aspect, the solid support is selected from the group consisting of a multiwell plate, an enzyme-linked immunosorbent assay (ELISA) plate, a microarray, a bead, a porous strip, and a nitrocellulose filter. In another aspect, the kit is an assay selected from the group consisting of a Western blot, an ELISA, a radioimmunoassay (MA), an immunoprecipitation assay, an electrochemiluminescence assay, a chemiluminescence assay, a fluorescence assay, a microarray, a multiplex bead-based assay, a dot blot, or a surface plasmon resonance detection. In another aspect, the kit further comprises a secondary antibody labeled directly or indirectly with a detectable moiety. In another aspect, the one or more synthetic or recombinant polypeptides further comprise one or more proteins selected from the group consisting of 2, 3, 4, 5, 6, 7 or 8 proteins selected from: CCDC155, DDB1, MUM1L1, NFU1, RPS29, SOX5, TCP10, or ZNF655, or RPAP3. In another aspect, the kit further comprises reagents for detecting if the liquid biological sample has autoantibodies to one or more proteins in Table 1, Table 2, or Table 3, other than MUM1L1 and SOX5. 
     In another embodiment, the present invention includes a method of determining that a patient negative for Ro autoantibodies has Sjögren’s syndrome (SS) without performing a lip biopsy comprising: obtaining a liquid biological sample from the patient suspected of having SS; determining that the patient is negative for Ro autoantibodies; and detecting autoantibodies to 1, 2, 3, 4, 5, 6, 7, 8, or 9 proteins selected from: CCDC155, DDB1, MUM1L1, NFU1, RPS29, SOX5, TCP10, ZNF655, or RPAP3, in the liquid sample, wherein the present of autoantibodies indicates that the patient has SS. In one aspect, the method further comprises the step of detecting if the liquid biological sample has autoantibodies to 2, 3, 4, 5, 6, 7, 8, or 9 proteins selected from: CCDC155, DDB1, MUM1L1, NFU1, RPS29, SOX5, TCP10, ZNF655, or RPAP3. In one aspect, the autoantibody has a reactivity ≥ 2SD, ≥ 3SD, or ≥ 4SD above the mean of healthy controls to any one of any one of CCDC155, DDB1, MUM1L1, NFU1, RPS29, SOX5, TCP10, ZNF655 antigens in the Ro negative group to identify three-fourths of the Ro negative individuals. In another aspect, the autoantibodies to SOX5 also cross-react with at least one of SOX3, SOX4, SOX6, SOX9, SOX11, SOX13 and SOX30. In another aspect, the method further comprises the step of detecting if the liquid biological sample has autoantibodies to one or more proteins in Table 1, Table 2, or Table 3, other than MUM1L1 and SOX5. In another aspect, the step of detecting autoantibodies in an assay selected from the group consisting of a Western blot, an ELISA, a radioimmunoassay (MA), an immunoprecipitation assay, an electrochemiluminescence assay, a chemiluminescence assay, a fluorescence assay, a microarray, a multiplex bead-based assay, a dot blot, or a surface plasmon resonance detection. 
     In another embodiment, the present invention includes a method of determining that a patient negative for Ro autoantibodies has Sjögren’s syndrome (SS) without performing a lip biopsy comprising: obtaining a liquid biological sample from the patient suspected of having SS; determining that the patient is negative for Ro autoantibodies; and detecting autoantibodies 1, 2, 3, 4, 5, 6, 7, 8, or 9 proteins selected from: CCDC155, DDB1, MUM1L1, NFU1, RPS29, SOX5, TCP10, ZNF655, or RPAP3, wherein the present of autoantibodies to RPAP3 indicates that the patient has SS. 
     In another embodiment, the present invention includes a kit comprising a synthetic or recombinant polypeptide covalently attached to a solid support, wherein the synthetic or recombinant polypeptide comprises 1, 2, 3, 4, 5, 6, 7, 8, or 9 proteins selected from: CCDC155, DDB1, MUM1L1, NFU1, RPS29, SOX5, TCP10, ZNF655, or RPAP3, and reagents to detect autoantibodies to the 1, 2, 3, 4, 5, 6, 7, 8, or 9 proteins selected from: CCDC155, DDB1, MUM1L1, NFU1, RPS29, SOX5, TCP10, ZNF655, or RPAP3. In one aspect, the kit further comprises instructions for contacting the solid support with a biological sample from a patient suspected of having Sjögren’s syndrome. In one aspect, the solid support is selected from the group consisting of a multiwell plate, an enzyme-linked immunosorbent assay (ELISA) plate, a microarray, a bead, a porous strip, and a nitrocellulose filter. In another aspect, the kit is an assay selected from the group consisting of a Western blot, an ELISA, a radioimmunoassay (MA), an immunoprecipitation assay, an electrochemiluminescence assay, a chemiluminescence assay, a fluorescence assay, a microarray, a multiplex bead-based assay, a dot blot, or a surface plasmon resonance detection. In another aspect, the kit further comprises a secondary antibody labeled directly or indirectly with a detectable moiety. In another aspect, the kit further comprises reagents for detecting if the liquid biological sample has autoantibodies to one or more proteins in Table 1, other than MUM1L1 and SOX5. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       For a more complete understanding of the features and advantages of the present invention, reference is now made to the detailed description of the invention along with the accompanying figures and in which: 
         FIG.  1    shows the antigens identified on HuProt3.2 arrays that were bound by anti-Ro antibody positive SS patients (Ro Pos, n=15)) or anti-Ro antibody negative SS patients (Ro Neg, n=15) compared to matched healthy controls (n=15). Positive binding was defined as normalized and background-corrected intensity values ≥ 4SD above the mean of healthy controls. The antigens shown showed significant differences from the healthy control group by Fisher’s exact test. 
         FIG.  2    is a graph that shows binding of two versions of MUM1L1 protein by both anti-Ro negative SS cases (squares, left) and anti-Ro positive SS cases (triangles, right) but not matched healthy controls (circles, center). Red dashed lines indicate threshold of 4SD above the mean of the healthy control group. P-values using one-tailed Mann-Whitney U test compared to the healthy control group are shown. 
         FIG.  3    is a graph that shows binding of RPS29 protein by both anti-Ro negative SS cases (squares, left) and anti-Ro positive SS cases (triangles, right) but not matched healthy controls (circles, center). Red dashed line indicates threshold of 4SD above the mean of the healthy control group. P-values using one- tailed Mann-Whitney U test compared to the healthy control group are shown. 
         FIG.  4    is a graph that shows binding of two isoforms of SOX5 protein by both anti-Ro negative SS cases (squares, left) and anti-Ro positive SS cases (triangles, right) but not matched healthy controls (circles, center). Red dashed lines indicate threshold of 4SD above the mean of the healthy control group. P-values using one-tailed Mann-Whitney U test compared to the healthy control group are shown. 
         FIG.  5    is a graph that shows binding of RPAP3 protein by anti-Ro negative SS cases (squares, left) but not anti-Ro positive SS cases (triangles, right) or matched healthy controls (circles, center). Red dashed line indicates threshold of 4SD above the mean of the healthy control group. P-values using a one- tailed Mann-Whitney U test compared to the healthy control group are shown. 
         FIG.  6    is a graph that shows binding of two versions of MUM1L1 protein by the combined anti-Ro negative and anti-Ro positive patient groups (n=30, circles, left) but not matched healthy controls (n=15, squares, right). Red dashed lines indicate threshold of 4SD above the mean of the healthy control group. P-values using one-tailed Mann-Whitney U tests compared to the healthy control group are shown. 
         FIG.  7    is a graph that shows binding of the RPS29 protein by the combined anti-Ro negative and anti-Ro positive patient groups (n=30, circles, left) but not matched healthy controls (n=15, squares, right). Red dashed line indicates threshold of 4SD above the mean of the healthy control group. P-value using a one- tailed Mann-Whitney U test compared to the healthy control group is shown. 
         FIG.  8    is a graph that shows binding of two isoforms of SOX5 protein by the combined anti-Ro negative and anti-Ro positive patient groups (n=30, circles, left) but not matched healthy controls (n=15, squares, right). Red dashed lines indicate threshold of 4SD above the mean of the healthy control group. P-values using one-tailed Mann-Whitney U tests compared to the healthy control group are shown. 
         FIG.  9    is a table that shows that multiple SOX family members are bound by plasma IgG antibodies of individual SS cases in the anti-Ro negative and anti-Ro positive groups above the healthy controls. Positive binding is color-coded by the threshold of positivity used, where 2SD (green) is binding above the mean of the controls plus 2SD, 3SD (yellow) is binding above the mean of healthy controls plus 3SD, and 4SD (red) is binding above the mean of healthy controls plus 4SD. Each row represents binding data by an individual SS patient. 
         FIG.  10    shows the known and predicted protein-protein interactions involving SOX5 and Ro60 identified by the STRING database. Note close connections of SOX5 and SOX13, connections between SOX proteins, a collagen network, and a TGFbeta family member network. These networks are connected to an innate immune signaling network known to be dysregulated in SS. The latter network is linked to the canonical Ro60 antigen. 
         FIG.  11    is a table that shows the significant features reaching qValue&lt;0.05 differentiation threshold that discriminate between SS cases and healthy controls as assessed using the PAA Bioconductor package in R. A total of 9 proteins representing 13 separate features of the HuProt3.2 arrays significantly discriminated SS cases from healthy controls using a stringent significance threshold accounting for multiple comparisons. 
         FIG.  12    is a graph that shows binding of KCNAB 1 protein by healthy controls (circles, center) but not Ro negative SS cases (squares, left) or Ro positive SS cases (triangles, right). P-values using two-tailed Mann-Whitney U tests compared to the healthy control group are shown. 
         FIG.  13    is a graph that shows binding of KCNAB 1 protein by the combined Ro positive and Ro negative SS cases (circles, left) compared to healthy controls (squares, right). The p-value shown reflects a two- tailed Mann-Whitney U test. 
         FIG.  14    shows a subset of 45 proteins selected by glmnet from a panel of 126 proteins identified by the multivariate RF-RFE approach as useful for discriminating between SS patient groups and healthy controls. Data reflect patient plasma IgG antibody binding to proteins on HuProt3.2 arrays. Analysis conducted using the PAA Bioconductor package in R. 
         FIG.  15    shows one representative example showing how a subset of proteins identified by the PAA Bioconductor multivariate RF-RFE approach can distinguish Ro negative SS cases from Ro positive SS cases and healthy controls. 
         FIG.  16    is a Venn diagram that shows proteins bound in by plasma IgG among 15 anti-Ro antibody positive SS cases (“Ro Pos”) and/or 15 anti-Ro antibody negative SS cases (“Ro Neg”) above threshold of mean + 3SD of plasma IgG among 15 healthy controls. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     While the making and using of various embodiments of the present invention are discussed in detail below, it should be appreciated that the present invention provides many applicable inventive concepts that can be embodied in a wide variety of specific contexts. The specific embodiments discussed herein are merely illustrative of specific ways to make and use the invention and do not delimit the scope of the invention. 
     To facilitate the understanding of this invention, a number of terms are defined below. Terms defined herein have meanings as commonly understood by a person of ordinary skill in the areas relevant to the present invention. Terms such as “a”, “an” and “the” are not intended to refer to only a singular entity but include the general class of which a specific example may be used for illustration. The terminology herein is used to describe specific embodiments of the invention, but their usage does not limit the invention, except as outlined in the claims. 
     The present invention is used for the diagnosis of Ro antibody negative Sjögren’s syndrome (SS) without a minor salivary gland lip biopsy. Thus, the present invention allows for the first time the diagnosis of Ro antibody negative Sjögren’s syndrome without a minor salivary gland lip biopsy. 
     The present invention also teaches biomarker(s) of dysregulated B cell responses, B cell lymphoma, salivary gland fibrosis and/or salivary gland dysfunction. 
     Diagnosis of SS in individuals lacking antibodies to Ro antigen currently requires observation of focal lymphocytic infiltrates on salivary gland lip biopsy. The inventors have discovered antigens recognized by antibodies in “Ro negative” SS patients. This discovery permits diagnosis and/or treatment of SS without a lip biopsy. 
     SS is a rheumatic autoimmune disease selectively targeting salivary and lacrimal glands, leading to painful dry mouth and eyes, oral infections, severe dental caries/tooth loss, fatigue, arthritis, nervous system involvement and malignant B cell lymphoma. Current internationally accepted disease classification criteria rely on either the presence of anti-Ro antibodies (these may target either the Ro60 antigen, Ro52 antigen or both) or the presence of focal lymphocytic infiltrates in a salivary gland lip biopsy for diagnosis (1, 2). Either one of these features, in combination with one or more objective dryness measures are necessary for fulfillment of classification criteria for SS. 
     Among 475 individuals attending the Oklahoma Sjögren’s Research clinic and meeting classification criteria for primary SS, 38% lacked antibodies to Ro antigen. These individuals met classification criteria for SS due to a focus score ≥ 1 on examination of salivary gland lip biopsy. The antigen(s) driving the aberrant immune response in these individuals is unknown. The inventors’ prior published (3) and unpublished (Longobardi and Farris, unpublished data) work shows that many of the antigens recognized by monoclonal antibodies derived from salivary gland antibody-forming plasmablasts are also present in plasma, serum or saliva. However, what is needed are novel methods for detecting SS patients that are Ro antigen negative. 
     To discover novel autoantigens in Ro antibody negative SS cases, the inventors tested plasma from Ro negative SS cases (n=15), Ro positive SS cases (n=15) and healthy controls (n=15) for binding to more than 15,500 antigens using human proteome microarrays (HuProt3.2, CDI Laboratories). All groups were matched on age within 5 years, race and sex. The arrays were probed with 1:500 dilutions of plasma and signals detected using a fluorescently labeled anti- human IgG secondary antibody (Jackson ImmunoResearch AlexaFluor647 AffiniPure F(ab′)2 Fragment Goat Anti-Human IgG (H+L), cat#109-606-003 at 1:2000 dilution). After scanning fluorescent signals using a GenePix scanner, the arrays were probed with an anti-GST antibody, which captured signals for all proteins, as all proteins on the array carry a GST tag. Each array was manually aligned using GST signals. Manual analysis of the foreground/background ratio of GST signals of one representative array probed with anti-GST antibody alone was used to assess adequate presence of each protein in each set of duplicate spots. This analysis revealed no instance of protein being present on only one spot on the array. Using the Protein Array Analyzer (PAA) Bioconductor open source software in R, all 45 arrays were normalized to one another using robust linear model (rlm) normalization. Of the duplicate spots of each protein, the protein exhibiting the lower signal was selected for analysis. Appropriate normalization was verified by the observation of equalized positive immunoglobulin light chain spots present on each array, as these proteins should be identically detected using the fluorescently labeled anti-human IgG reagent employed. The PAA background correction feature was employed to correct for intra-array fluctuations in background. The normalized and background-corrected data were analyzed using two methods. 
     Using the first analysis method, the average ±4 SD of the healthy control group was computed for each protein. The patient groups were evaluated for antigens that: (i) bound greater than the average + 4 SD above the healthy control group and; (ii) exhibited significant differences between either anti-Ro antibody negative or anti-Ro antibody positive cases versus healthy controls by a Fisher’s exact test. Using these criteria, four novel antigens were significantly bound by the anti-Ro negative SS group, while 19 antigens were significantly bound by the anti-Ro positive SS group ( FIG.  1   ). Among these antigens, three novel antigens were bound by individuals in both patient groups but not the healthy control group. These antigens were listed on the CDI human proteome arrays as MUM1L1, RPS29 and SOX5. One novel antigen was uniquely bound by the anti-Ro negative group compared to healthy controls. This antigen was listed on the CDI human proteome arrays as RPAP3. Notably, reactivity ≥ 4SD above the mean of healthy controls to any one of the four novel antigens in the Ro negative group could identify nearly three-fourths of the Ro negative individuals (positive predictive value=73.3%, negative predictive value=100%). Antigens bound by antibodies in the anti-Ro positive group included the known canonical antigens Ro60 (identified on the HuProt3.2 arrays as TROVE2) and Ro52 (identified on the HuProt3.2 arrays as TRIM21 and as ND JHU00287.B2C16R4), validating the approach and analysis methods. Additional antigens identified in the anti-Ro positive group are also novel and are being explored as biomarkers for clinical and histological disease features. 
       FIG.  1    shows the antigens identified on HuProt3.2 arrays that were bound by anti-Ro positive SS patients (Ro Pos, n=15) or anti-Ro negative SS patients (Ro Neg, n=15) compared to matched healthy controls (n=15). Positive binding was defined as normalized and background-corrected intensity values ≥ 4SD above the mean of healthy controls. The antigens shown showed significant differences from the healthy control group by Fisher’s exact test. 
     To further demonstrate the magnitude of the responses to the novel antigens bound by the anti-Ro negative group, the normalized and background-corrected intensity values of the antigen binding in all three groups were plotted ( FIGS.  2 - 5   ). Two-way comparisons between each SS group and the healthy control group were made using the Mann-Whitney U test, and the resulting p-values are shown in each figure. 
       FIG.  2    is a graph that shows binding of two versions of MUM1L1 protein by both anti-Ro negative SS cases (squares, left) and anti-Ro positive SS cases (triangles, right) but not matched healthy controls (circles, center). Red dashed lines indicate the threshold of 4SD above the mean of the healthy control group. P-values using one-tailed Mann-Whitney U test compared to the healthy control group are shown. 
       FIG.  3    is a graph that shows binding of RPS29 protein by both anti-Ro negative SS cases (squares, left) and anti-Ro positive SS cases (triangles, right) but not matched healthy controls (circles, center). Red dashed line indicates the threshold of 4SD above the mean of the healthy control group. P-values using one- tailed Mann-Whitney U test compared to the healthy control group are shown. 
       FIG.  4    is a graph that shows binding of two isoforms of SOX5 protein by both anti-Ro negative SS cases (squares, left) and anti-Ro positive SS cases (triangles, right) but not matched healthy controls (circles, center). Red dashed lines indicate the threshold of 4SD above the mean of the healthy control group. P-values using one-tailed Mann-Whitney U test compared to the healthy control group are shown. 
       FIG.  5    is a graph that shows binding of RPAP3 protein by anti-Ro negative SS cases (squares, left) but not anti-Ro positive SS cases (triangles, right) or matched healthy controls (circles, center). Red dashed line indicates the threshold of 4SD above the mean of the healthy control group. P-values using a one- tailed Mann-Whitney U test compared to the healthy control group are shown. 
     As three of the novel antigens were bound by individuals in both patient groups, the inventors also evaluated the magnitude of binding by the combined patient groups compared to the healthy control group using the Mann-Whitney U test. These results are shown in  FIGS.  6 - 8   . 
       FIG.  6    is a graph that shows binding of two versions of MUM1L1 protein by the combined anti-Ro negative and anti-Ro positive patient groups (n=30, circles, left) but not matched healthy controls (n=15, squares, right). Red dashed lines indicate the threshold of 4SD above the mean of the healthy control group. P-values using one-tailed Mann-Whitney U tests compared to the healthy control group are shown. 
       FIG.  7    is a graph that shows binding of the RPS29 protein by the combined anti-Ro negative and anti-Ro positive patient groups (n=30, circles, left) but not matched healthy controls (n=15, squares, right). Red dashed line indicates threshold of 4SD above the mean of the healthy control group. P-value using a one- tailed Mann-Whitney U test compared to the healthy control group is shown. 
       FIG.  8    is a graph that shows binding of two isoforms of SOX5 protein by the combined anti-Ro negative and anti-Ro positive patient groups (n=30, circles, left) but not matched healthy controls (n=15, squares, right). Red dashed lines indicate threshold of 4SD above the mean of the healthy control group. P-values using one-tailed Mann-Whitney U tests compared to the healthy control group are shown. 
     As multiple SOX family members were targeted in the anti-Ro positive SS cases, the inventors evaluated binding to all SOX family members present on the proteome arrays using various thresholds of positivity, defined by the mean of healthy controls plus 2SD, 3SD or 4SD. The results showed that plasma IgG from Ro negative cases also bound to SOX3, SOX4, SOX6, SOX9, SOX11, SOX13 and SOX30, in addition to SOX5. Both anti-Ro negative and anti-Ro positive cases could bind to multiple SOX family members ( FIG.  9   ). As folding of protein on the proteome arrays may not be in completely native conformation, these data show that additional SOX family members may be useful for the application of diagnosing Ro negative SS cases. 
       FIG.  9    is a table that shows that multiple SOX family members are bound by plasma IgG antibodies of individual SS cases in the anti-Ro negative and anti-Ro positive groups above the healthy controls. Positive binding is color-coded by the threshold of positivity used, where 2SD (green) is binding above the mean of the controls plus 2SD, 3SD (yellow) is binding above the mean of healthy controls plus 3SD, and 4SD (red) is binding above the mean of healthy controls plus 4SD. Each row represents binding data by an individual SS patient. 
     SOX5 has been shown to be expressed in late stage B cell differentiation including atypical memory B cells, germinal center B cells and plasmablasts (4). As ectopic germinal centers are prevalent, and as plasmablasts are numerous in the salivary gland biopsy tissue of SS patients (5, 6), antibodies to SOX5 may be useful as a biomarker for dysregulated B cells occurring in salivary gland, blood or other tissues in SS patients. A biomarker for dysregulated B cell function in SS may be a useful biomarker for determining patient response to therapy, both in clinical trials and in patient care. SOX5 and other SOX family members have been shown to mediate epithelial to mesenchymal cell transition (EMT) (7, 8), a key pathway for promotion of tissue fibrosis. The inventors have shown that tissue fibrosis, as assessed by morphological criteria, is increased in the salivary gland biopsy tissue of SS cases (9). Therefore, antibodies to SOX5 in serum or saliva may be a useful biomarker for salivary gland tissue fibrosis, which could be useful for selecting patients for certain clinical trials or therapies. SOX5 is a marker of malignant B cells (10), and SS patients have a substantially increased risk of malignant B cell lymphoma (11, 12). Therefore, antibodies to SOX5 in SS patient samples may be useful as a biomarker for B cell lymphoma or risk for B cell lymphoma in SS patients. These are novel antigens in SS. 
     Evaluation of protein-protein interactions connecting the SOX5 protein and the canonical Ro60 antigen in SS using the Search Tool for Retrieval of Interacting Genes/Proteins (STRING) database revealed a close relationship between SOX5 and SOX13, which are among the most strongly bound SOX family members in SS ( FIG.  10   ). The analysis also revealed direct interactions between SOX5 and SOX13 to a network of TGFbeta receptor and TGF beta receptor family member signaling, and direct connections between SOX5 and multiple collagen proteins ( FIG.  10   ). TGFbeta signaling and collagen proteins are well known key mediators of tissue fibrosis. Moreover, the TGFbeta receptor signaling family member BMP proteins are involved in salivary gland differentiation and regeneration (13), and at least one BMP protein has been shown to be altered in SS and associated with salivary gland dysfunction in SS (14). Notably, the interactions above are also linked to a network of inflammatory signaling proteins known to be dysregulated in SS (15), as well as the canonical Ro antigens ( FIG.  10   ). These data provide additional evidence that antibodies to SOX family members may be useful as biomarkers for fibrosis and disease activity in SS. 
       FIG.  10    shows the known and predicted protein-protein interactions involving SOX5 and Ro60 identified by the STRING database. Note close connections of SOX5 and SOX13, connections between SOX proteins, a collagen network, and a TGFbeta family member network. These networks are connected to an innate immune signaling network known to be dysregulated in SS. The latter network is linked to the canonical Ro60 antigen. 
     A second analysis method available in the PAA Bioconductor R package was also employed to find novel SS antigens that can discriminate SS cases from healthy controls. Using this analysis antibody reactivity to 13 proteins were shown to significantly discriminate between SS cases and healthy controls after adjusting for multiple comparisons ( FIG.  11   ). 
       FIG.  11    is a table that shows the significant features reaching qValue&lt;0.05 differentiation threshold that discriminate between SS cases and healthy controls as assessed using the PAA Bioconductor package in R. A total of 9 proteins representing 13 separate features of the HuProt3.2 arrays significantly discriminated SS cases from healthy controls using a stringent significance threshold accounting for multiple comparisons. 
     Among these proteins, antibodies to KCNAB1 significantly discriminated between the Ro negative SS group and healthy controls, with antibody reactivity to KCNAB1 being significantly reduced in the SS cases. Evaluation of the magnitude of antibody binding to KCNAB1 in the three subject groups by Mann-Whitney U-test is shown in  FIG.  12   . 
       FIG.  12    is a graph that shows binding of KCNAB 1 protein by healthy controls (circles, center) but not Ro negative SS cases (squares, left) or Ro positive SS cases (triangles, right). P-values using two-tailed Mann-Whitney U tests compared to the healthy control group are shown. 
     As antibodies to KCNAB1 were reduced in both anti-Ro positive and anti-Ro negative SS cases, the two SS patient groups were combined and compared to the healthy controls. The results are shown in  FIG.  13   . 
       FIG.  13    is a graph that shows binding of KCNAB1 protein by the combined anti-Ro positive and anti-Ro negative SS cases (circles, left) compared to healthy controls (squares, right). The p-value shown reflects a two- tailed Mann-Whitney U test. 
     Using the PAA Bioconductor package in R, the multivariate RF-RFE approach selected 126 proteins that discriminated between SS cases and healthy controls. Of these, 45 that were sub-selected with glmnet are shown in  FIG.  14   . These include KCNAB2, MECR, LSM1, PARVA, MAFK, VEGF, ABHD8, ELFN2, KCNAB1, C15orf57, HDAC6, ESCO1, NDNF_FRAG, ABI2, ND, ISM2, BUD13, TPM1, TRIM21, AGTR1, CASC1, ARHGEF16, VWA5A, MUM1L1, HDAC6, TPM3, dsDNA, c9ORF61, HDAC6, PRRT2, AKR1B1, SPANXN2, PSMD4, TROVE2, ANKHD1, AVEN, MUM1L1, SKP1, PRKAR1B, NUPL2, KIR2DL1, PCMTD1, RAD23A, AQP5 and SMARCC1 (for proteins, see genecards.org for full names). 
       FIG.  14    shows a subset of 45 proteins selected by glmnet from a panel of 126 proteins identified by the multivariate RF-RFE approach as useful for discriminating between SS patient groups and healthy controls. Data reflect patient plasma IgG antibody binding to proteins on HuProt3.2 arrays. Analysis conducted using the PAA Bioconductor package in R. 
     The entire list of 126 proteins identified as being useful for discriminating anti-Ro positive SS cases, anti-Ro negative SS cases and healthy controls is shown in Table 1. 
     
       
         
          TABLE 1
           
               
               
               
               
               
             
               
                 List of 126 proteins (or gene names encoding the proteins) identified by the PAA Bioconductor Package as being useful for discriminating Ro positive SS, Ro negative SS, and healthy 
               
             
            
               
                 ABHD8 
                 EID3 
                 KCNAB1-1R49 
                 P2RX4 
                 SOX6 
               
               
                 ABI2 
                 ELFN2 
                 KIR2DL1 
                 PAIP1 
                 SPANXN2 
               
               
                 ACY1 
                 ERP27 
                 KIZ 
                 PARVA 
                 SRRT 
               
               
                 AGTR1 
                 ESCO1 
                 LCN1 
                 PBK 
                 STMN4 
               
               
                 AKR1B1 
                 FAF1 
                 LIPF 
                 PCMTD1 
                 TCOF1 
               
               
                 ANKHD1 
                 FAM131B 
                 LSM1 
                 PIK3CA 
                 TEX29 
               
               
                 ANKRD45 
                 FAM131C 
                 MAFK 
                 PPP1R3F 
                 TLK1 
               
               
                 AQP5 
                 FAM175B 
                 MAG 
                 PPP4R3A 
                 TMED8 
               
               
                 ARHGEF16-R40 
                 FAM53C 
                 MAPK8 
                 PRB1 
                 TMEM185B 
               
               
                 ARHGEF16-R32 
                 GAB1 
                 MECR-R5 
                 PRKAR1B 
                 TPM1-R43 
               
               
                 AVEN 
                 GAGE10 
                 MECR-R6 
                 PRRT2 
                 TPM1-R42 
               
               
                 BUD 13 
                 GAR1 
                 MTMR14 
                 PSMD4 
                 TPM3 
               
               
                 C14orf37 
                 GBP5 
                 MUC20 
                 PTP4A1 
                 TRIM21** 
               
               
                 C15orf57 
                 GCSH 
                 MUM1L1-R24 
                 RAB34 
                 TROVE2*** 
               
               
                 C9orf61 
                 GMPR 
                 MUM1L1-R4 
                 RAD23A-R21 
                 TSC22D3 
               
               
                 CAAP1 
                 GNAS 
                 NAF1 
                 RAD23A-R7 
                 TSSC4 
               
               
                 CASC1 
                 GORASP1 
                 NAT6 
                 RCC1 
                 VEGF 
               
               
                 CAST 
                 HDAC6-R14 
                 ND MIR16 
                 RTN4 
                 VWA5A 
               
               
                 CCDC155 
                 HDAC6-28R20 
                 ND SIAT7D 
                 SERPINB1 
                 WASF2 
               
               
                 CCER1 
                 HDAC6-20R20 
                 ND MTHFR 
                 SFT2D2 
                 ZBTB7C 
               
               
                 CYTH1 
                 HLCS 
                 ND C20orf58* 
                 SGIP1 
                 ZCCHC10 
               
               
                 DDX58 
                 HSPA13 
                 ND UBE3C 
                 SKP1-R26 
                 ZEB2 
               
               
                 DNAJC12 
                 ISM2 
                 ND PTHB 1 
                 SKP1-R6 
                   
               
               
                 dsDNA 
                 KBTBD7 
                 NDNF_frag 
                 SMARCC1 
                   
               
               
                 ECE1 
                 KCNAB1 
                 NFU1 
                 SMYD5_frag 
                   
               
               
                 EEF1D 
                 KCNAB2-16R49 
                 NUPL2 
                 SOX5 
                   
               
               
                 *ND C20orf68=FAM77A=NKAIN4; **TRIM21=Ro52 canonical SS antigen; ***TROVE2=Ro60 canonical SS antigen. 
               
            
           
         
       
     
     Combinations of smaller subsets of the 126 proteins selected with the PAA Bioconductor multivariate RF-RFE approach may be useful for diagnosing Ro negative SS, as well as distinguishing Ro negative SS from healthy controls and Ro positive SS. An illustration of this is an example in which only 3 proteins, KCNAB2, CREM and TRIM21, could distinguish all three subject categories tested. This example is shown in  FIG.  15   . 
       FIG.  15    shows one representative example showing how a subset of proteins identified by the PAA Bioconductor multivariate RF-RFE approach can distinguish anti-Ro negative SS cases from anti-Ro positive SS cases and healthy controls. 
     Sjögren’s Syndrome is a debilitating disease, and the autoantibodies identified in these analyses represent potential biomarkers to aid in diagnosis and care of these patients. Furthermore, it could reduce the number of lip biopsies necessary, especially for the nearly 40% of patients that do not develop autoantibodies to the canonical Ro antigens. 
     Novel shared antibody specificities in anti-Ro antibody negative Sjögren’s Syndrome. 
     Sjögren’s syndrome (SS) is a rheumatic autoimmune disease characterized by focal lymphocytic infiltrates in the lacrimal and salivary glands, severe dry mouth and eyes, pain and debilitation. Diagnosis requires autoantibodies to ubiquitous Ro antigens or a lip biopsy positive for focal lymphocytic infiltrates. In this study, human proteome arrays were used to identify novel antibodies in plasma from Ro positive and Ro antibody negative SS patients compared with healthy controls. 
     Anti-Ro positive (n=15) and anti-Ro negative (n=15) cases meeting 2016 ACR/EULAR classification criteria for SS were age, race, and sex matched with each other and healthy controls (n=15)(15-15-15). Plasma IgG binding to human proteome arrays containing &gt;19,500 recombinant human proteins representing &gt;80% of the human proteome (HuProt v3.2 arrays, CDI Laboratories) was assessed. Data were normalized by the Robust Linear Model using the PAA Bioconductor Package in R and log intensity values for each protein generated. Thresholds of mean + 3SD were established using the controls. Antigens bound by IgG in at least 4 cases compared to controls were considered significant (p&lt;0.05, one-tailed Fisher’s exact test). 
     IgG from anti-Ro positive SS cases significantly bound 42 proteins, including the canonical SS antigens Ro60, Ro52, and La, with an average of 15 specificities per individual. IgG from anti-Ro negative SS cases significantly bound 24 proteins compared to controls, with an average of 7 specificities per individual. Of the antigens identified, 8 were shared in both the Ro positive and Ro negative groups. Binding to at least one of these 8 proteins identified 93% of the Ro positive cases and 87% of the Ro negative cases. 
     A set of 8 novel antigens were bound by plasma IgG in both anti-Ro positive and anti-Ro negative cases. One or more of these antigens are used for diagnosing SS without a lip biopsy, including 1, 2, 3, 4, 5, 6, 7, or all 8 antigens or biomarkers.  
     
       
         
          TABLE 2
           
               
               
               
               
               
               
             
               
                 Proteins bound in 15-15-15 plasma above threshold of mean + 3SD of healthy controls. 
               
               
                 Ro Pos Name 
                 ID 
                 Ro Neg Name 
                 ID 
                 Shared Name 
                 ID 
               
             
            
               
                 AGTR1 
                 JHU16058.B8C25R46 
                 ARFGAP1 
                 JHU05382.B7C23R30 
                 CCDC155 
                 JHU09052.B9C16R21 
               
               
                 ATL2 
                 JHU07112.B7C25R49 
                 C9orf78 
                 JHU02218.B4C21R32 
                 DDB1 
                 JHU08569.B11C22R18 
               
               
                 CBX3 
                 JHU06273.B24C5R40 
                 CCDC155 
                 JHU09052.B9C16R21 
                 MUM1L1 
                 JHU15438.B17C15R24 
               
               
                 CCDC155 
                 JHU09052.B9C16R21 
                 CNDP2 
                 JHU02518.B1C4R41 
                 MUM1L1 
                 JHU15438.B18C27R4 
               
               
                 CPSF2 
                 JHU10483.B10C9R44 
                 DDB1 
                 JHU08569.B11C22R18 
                 NFU1 
                 JHU14404.B14C16R45 
               
               
                 DDB1 
                 JHU08569.B11C22R18 
                 FABP1 
                 JHU03967.B5C29R1 
                 RPS29 
                 JHU06790.B7C1R48 
               
               
                 dsDNA 
                 auto-antigen 
                 FXYD5 
                 JHU01290.B4C5R36 
                 SOX5 
                 JHU13672.B15C6R33 
               
               
                 EGFLAM 
                 JHU12185.B15C31R8 
                 GRAMD1A 
                 JHU07810.B9C8R2 
                 SOX5 
                 JHU06796.B8C19R46 
               
               
                 FAM118A 
                 JHU09047.B11C25R21 
                 HNRNPAB 
                 JHU08680.B9C25R18 
                 TCP10 
                 JHU10063.B12C3R42 
               
               
                 FUT3 
                 JHU11356.B9C24R57 
                 LIX1 
                 JHU03115.B21C10R42 
                 ZNF655 
                 JHU12825.B14C27R23 
               
               
                 FUT8 
                 JHU10594.B11C19R46 
                 MCCC2 
                 JHU04945.B8C31R14 
                   
                   
               
               
                 GAB1 
                 JHU09535.B23C26R51 
                 MUM1L1 
                 JHU15438.B17C15R24 
                   
                   
               
               
                 GMNN 
                 JHU01959.B4C2R36 
                 MUM1L1 
                 JHU15438.B18C27R4 
                   
                   
               
               
                 HSFX1 
                 JHU01774.B19C26R47 
                 NFU1 
                 JHU14404.B14C16R45 
                   
                   
               
               
                 KLHDC8A 
                 JHU05613.B8C24R28 
                 PEAS/ND 
                 JHU00407.B3C12R10 
                   
                   
               
               
                 KRR1 
                 JHU01681.B23C25R42 
                 POU6F1 
                 JHU14773.B15C1R49 
                   
                   
               
               
                 MAPRE1 
                 JHU11847.B16C31R1 
                 PPIL3 
                 JHU11100.B9C13R50 
                   
                   
               
               
                 MCFD2 
                 JHU15435.B18C14R6 
                 RPAP3 
                 JHU07034.B5C14R53 
                   
                   
               
               
                 MUM1L1 
                 JHU15438.B18C27R4 
                 RPS29 
                 JHU06790.B7C1R48 
                   
                   
               
               
                 MUM1L1 
                 JHU15438.B17C15R24 
                 SOX5 
                 JHU13672.B15C6R33 
                   
                   
               
               
                 NFU1 
                 JHU14404.B14C16R45 
                 SOX5 
                 JHU06796.B8C19R46 
                   
                   
               
               
                 NUP50 
                 JHU09563.B12C30R28 
                 SPSB2 
                 JHU04978.B7C10R14 
                   
                   
               
               
                 PCMTD1 
                 JHU09086.B9C6R22 
                 SRPK2 
                 JHU16239.B19C22R17 
                   
                   
               
               
                 PDPK1 
                 JHU08321.B12C6R12 
                 TCP10 
                 JHU10063.B12C3R42 
                   
                   
               
               
                 PLEKHA4 
                 JHU16370.B18C7R13 
                 WDR20 
                 JHU01727.B24C18R40 
                   
                   
               
               
                 PLTP 
                 JHU22252.B22C1R59 
                 ZNF655 
                 JHU12825.B14C27R23 
                   
                   
               
               
                 PML 
                 JHU17186.B17C1R33 
                   
                   
                   
                   
               
               
                 POLR3H 
                 JHU18583.B21C2R22 
                   
                   
                   
                   
               
               
                 POTEG 
                 JHU25886.B24C2R70 
                   
                   
                   
                   
               
               
                 PRRG2/ND 
                 JHU01414.B1C19R22 
                   
                   
                   
                   
               
               
                 PRRT2 
                 JHU07266.B6C10R53 
                   
                   
                   
                   
               
               
                 RAD23A 
                 JHU16026.B17C31R7 
                   
                   
                   
                   
               
               
                 RCAN3 
                 JHU18309.B24C29R16 
                   
                   
                   
                   
               
               
                 RO52/ND 
                 JHU00287.B2C16R4 
                   
                   
                   
                   
               
               
                 RPP38 
                 JHU26369.B24C3R70 
                   
                   
                   
                   
               
               
                 RPS29 
                 JHU06790.B7C1R48 
                   
                   
                   
                   
               
               
                 SEC23IP 
                 JHU09577.B9C21R29 
                   
                   
                   
                   
               
               
                 SKIL 
                 JHU07088.B19C25R40 
                   
                   
                   
                   
               
               
                 SOX13 
                 JHU10917.B11C27R52 
                   
                   
                   
                   
               
               
                 SOX5 
                 JHU13672.B15C6R33 
                   
                   
                   
                   
               
               
                 SOX5 
                 JHU06796.B8C19R46 
                   
                   
                   
                   
               
               
                 SOX6 
                 JHU06898.B6C31R48 
                   
                   
                   
                   
               
               
                 SOX6 
                 JHU06898.B22C25R37 
                   
                   
                   
                   
               
               
                 SSB 
                 JHU18504.B22C28R13 
                   
                   
                   
                   
               
               
                 TCP10 
                 JHU10063.B12C3R42 
                   
                   
                   
                   
               
               
                 TNRC6C 
                 JHU18133.B21C18R43 
                   
                   
                   
                   
               
               
                 TPI1 
                 JHU06136.B7C18R35 
                   
                   
                   
                   
               
               
                 TRAPPC8 
                 JHU06764.B7C24R46 
                   
                   
                   
                   
               
               
                 TRIM21 
                 JHU00287.B22C24R52 
                   
                   
                   
                   
               
               
                 TROVE2 
                 JHU06040.B5C11R35 
                   
                   
                   
                   
               
               
                 ZBTB16 
                 JHU19956.B23C12R65 
                   
                   
                   
                   
               
               
                 ZBTB46 
                 JHU12250.B15C28R10 
                   
                   
                   
                   
               
               
                 ZNF394 
                 JHU01440.B1C25R22 
                   
                   
                   
                   
               
               
                 ZNF655 
                 JHU12825.B14C27R23 
                   
                   
                   
                   
               
               
                 ZNF655_frag 
                 JHU12919.B14C25R20 
                   
                   
                   
                   
               
            
           
         
       
     
     The column on left are proteins bound in the Ro Positive group. The middle column are proteins bound in the Ro Negative group. Protein Names/IDs highlighted in pink are shared between two groups, which are also listed in the columns on right. 
       FIG.  16    is a Venn diagram that shows proteins bound in 15-15-15 plasma above threshold of mean + 3SD of healthy controls. Duplicates were removed to save space, i.e. MUM1L1, SOX 5, SOX6, Ro52/ND (TRIM21 present) and ZNF655_frag (ZNF655 present). Proteins bound in 15-15-15 (Anti-Ro positive (n=15), anti-Ro negative (n=15) cases meeting 2016 ACR/EULAR classification criteria for SS were age, race, and sex matched with each other and healthy controls (n=15)) plasma above threshold of mean + 3SD of healthy controls. *p&lt;0.05, Fisher’s Exact test, **bound with mean + 4SD threshold. 
     
       
         
          TABLE 3
           
               
               
             
               
                 Plasma biomarkers overlapping between anti-Ro negative and anti-Ro positive patients. 
               
               
                 Protein marker 
                 Full name, protein 
               
             
            
               
                 CCDC155 
                 Coiled-Coil Domain Containing 155, UniProtKB: Q8N6L0 
               
               
                 DDB1 
                 Damage Specific DNA Binding Protein 1, UniProtKB: Q16531 
               
               
                 MUM1L1** 
                 Melanoma Associated Antigen (Mutated) 1-Like 1, UniProtKB: Q5H9M0 
               
               
                 NFU1 
                 NFU1 Iron-Sulfur Cluster Scaffold, UniProtKB: Q9UMS0 
               
               
                 RPS29** 
                 Ribosomal Protein S29, UniProtKB: P62273 
               
               
                 SOX5** 
                 SRY-Box Transcription Factor 5, UniProtKB: P35711 
               
               
                 TCP10 
                 T-Complex 10 Like 3, Pseudogene, UniProtKB: Q12799 
               
               
                 ZNF655 
                 Zinc Finger Protein 655, UniProtKB: Q8N720 
               
               
                 ** bound with mean + 4SD threshold 
               
            
           
         
       
     
     It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method, kit, reagent, or composition of the invention, and vice versa. Furthermore, compositions of the invention can be used to achieve methods of the invention. 
     It will be understood that particular embodiments described herein are shown by way of illustration and not as limitations of the invention. The principal features of this invention can be employed in various embodiments without departing from the scope of the invention. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific procedures described herein. Such equivalents are considered to be within the scope of this invention and are covered by the claims. 
     All publications and patent applications mentioned in the specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. 
     The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and/or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.” The use of the term “or” in the claims is used to mean “and/or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and/or.” Throughout this application, the term “about” is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the study subjects. 
     As used in this specification and claim(s), the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited features, elements, components, groups, integers, and/or steps, but do not exclude the presence of other unstated features, elements, components, groups, integers and/or steps. In embodiments of any of the compositions and methods provided herein, “comprising” may be replaced with “consisting essentially of” or “consisting of”. As used herein, the term “consisting” is used to indicate the presence of the recited integer (e.g., a feature, an element, a characteristic, a property, a method/process step or a limitation) or group of integers (e.g., feature(s), element(s), characteristic(s), property(ies), method/process steps or limitation(s)) only. As used herein, the phrase “consisting essentially of” requires the specified features, elements, components, groups, integers, and/or steps, but do not exclude the presence of other unstated features, elements, components, groups, integers and/or steps as well as those that do not materially affect the basic and novel characteristic(s) and/or function of the claimed invention. 
     The term “or combinations thereof” as used herein refers to all permutations and combinations of the listed items preceding the term. For example, “A, B, C, or combinations thereof” is intended to include at least one of: A, B, C, AB, AC, BC, or ABC, and if order is important in a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. Continuing with this example, expressly included are combinations that contain repeats of one or more item or term, such as BB, AAA, AB, BBC, AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan will understand that typically there is no limit on the number of items or terms in any combination, unless otherwise apparent from the context. 
     As used herein, words of approximation such as, without limitation, “about”, “substantial” or “substantially” refers to a condition that when so modified is understood to not necessarily be absolute or perfect but would be considered close enough to those of ordinary skill in the art to warrant designating the condition as being present. The extent to which the description may vary will depend on how great a change can be instituted and still have one of ordinary skill in the art recognize the modified feature as still having the required characteristics and capabilities of the unmodified feature. In general, but subject to the preceding discussion, a numerical value herein that is modified by a word of approximation such as “about” may vary from the stated value by at least ±1, 2, 3, 4, 5, 6, 7, 10, 12 or 15%. 
     All of the compositions and/or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims. 
     To aid the Patent Office, and any readers of any patent issued on this application in interpreting the claims appended hereto, applicants wish to note that they do not intend any of the appended claims to invoke paragraph 6 of 35 U.S.C. § 112, U.S.C. § 112 paragraph (f), or equivalent, as it exists on the date of filing hereof unless the words “means for” or “step for” are explicitly used in the particular claim. 
     For each of the claims, each dependent claim can depend both from the independent claim and from each of the prior dependent claims for each and every claim so long as the prior claim provides a proper antecedent basis for a claim term or element. 
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