WO2023235773A1

WO2023235773A1 - Method to identify a patient with an increased likelihood of chronic spontaneous urticaria

Info

Publication number: WO2023235773A1
Application number: PCT/US2023/067722
Authority: WO
Inventors: Diana CHANG; Brian Louis YASPAN; David Fong CHOY
Original assignee: Genentech Inc
Current assignee: Genentech Inc
Priority date: 2022-06-01
Filing date: 2023-05-31
Publication date: 2023-12-07
Anticipated expiration: 2024-12-01

Abstract

Provided herein are, inter alia, methods, compositions and kits for diagnosing and preventing chronic spontaneous urticaria (CSU). Also provided are methods, compositions and kits for diagnosing and preventing autoimmune diseases.

Description

METHOD TO IDENTIFY A PATIENT WITH AN INCREASED LIKELIHOOD OF CHRONIC SPONTANEOUS URTICARIA

FIELD OF THE INVENTION

The present invention is directed to methods for identifying patients as having a genetic predisposition to autoimmune disorders and chronic spontaneous urticaria (CSU).

SEQUENCE LISTING

The instant application contains a Sequence Listing, which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on May 31, 2023 is named 048893-571001WO_SL_ST26.xml and is 3,586 bytes in size.

BACKGROUND OF THE INVENTION

Chronic spontaneous urticaria (CSU) is a dermatologic condition that is characterized by spontaneous, pruritic hives and/or angioedema that persist for six weeks or longer with no identifiable trigger. Though anti-histamines, as first line therapy for CSU patients, and second line therapy such as omalizumab are effective for some CSU patients, others remain symptomatic with significant impact on quality of life. CSU is a heritable disorder and uncovenng the genetics underlying CSU can highlight additional pathways involved in disease risk and patient heterogeneity. Omalizumab reduces free IgE which in turn leads to the destabilization and downregulation of FcsRI on the cell surface, thus desensitizing these cells to FcsRI-dependent stimuli¹⁴. While omalizumab has been proven to be efficacious and has a rapid onset of action for CSU patients, 38 to 44% of subjects on omalizumab did not achieve well controlled status in pivotal phase 3 studies¹⁵, indicating additional disease mediators are involved and can be targeted therapeutically.

Thus, there is a need for more effective means for determining which patients will respond to which treatment and for incorporating such determinations into more effective treatment regimens for patients with an antihistamine, a corticosteroid, an immunosuppressant molecule, or an antibody, whether used as single agents or combined with other agents

SUMMARY OF THE INVENTION

Provided herein are, inter alia, methods, compositions and kits for diagnosing and

1

SUBSTITUTE SHEET ( RULE 26 ) preventing chronic spontaneous urticaria (CSU). Also provided are methods, compositions and kits for diagnosing and preventing autoimmune diseases.

In aspects, provided herein are methods for identifying a subject as having a genetic predisposition to CSU, where the method comprises (a) providing a biological sample comprising a nucleic acid from the subject, (b) assaying the nucleic acid for the presence of human leukocyte antigen (HLA) alleles comprising one or more of DRBl*04:04 or DQAl*03:03, and (c) diagnosing the subject at risk of having a genetic predisposition to CSU upon presence of either HLA-DRBl*04:04 and/or HLA-DQAl*03:03. In other embodiments, the method for identifying a subject as having a genetic predisposition to chronic spontaneous urticaria (CSU) further includes step (d) of treating the subject with a therapy.

In other embodiments, the method for identifying a subject as having a genetic predisposition to CSU includes assaying, where the assaying comprises amplifying the HLA DRBl*04:04 allele and/or the HLA DQAl*03:03 allele in the sample and detecting the presence of the HLA DRBl*04:04 allele and/or the HLA DQAl*03:03 allele with an agent that specifically binds to the HLA DRBl*04:04 allele and/or the HLA DQAl*03:03 allele; or wherein the assaying comprises sequencing one or more HLA alleles in the sample, thereby identifying the presence of the HLA DRBl*04:04 allele and/or the HLA DQAl*03:03 allele.

In embodiments, the therapy comprises an antihistamine, a corticosteroid, an immunosuppressant molecule, or an antibody. For example, the therapy comprises an antihistamine (e.g., an Hi antihistamine). In other examples, the therapy comprises an antibody, e.g., Omalizumab.

In embodiments, the method further includes that the presence of DRB1 *04:04 or DQAl*03:03 is further associated with a high chronic urticaria (CU) index, wherein a high CU index is > 10.

In additional embodiments, the method further includes that the presence of DRBl*04:04 or DQA1 *03:03 is further associated with an autoimmune disorder. For example, the autoimmune disorder includes hypothyroidism, rheumatoid arthritis, type-1 diabetes, or vitiligo.

In other aspects, provided herein are methods of identifying a subject having CSU as likely to respond to a therapy, the method including (a) providing a biological sample comprising a nucleic acid from the subject, (b) assaying the nucleic acid for the presence of HLA alleles comprising DRBl*04:04 or DQAl*03:03, (c) identifying the subject as more likely to respond to the therapy, when the sample has the presence of either HLA-DRBl*04:04 and/or HLA- DQAl*03:03. In other embodiments, the method of identifying a subject having CSU as likely to respond to a therapy further includes step (d) of treating the subject with a therapy.

In other embodiments, the method for identifying a subject having CSU as likely to respond to therapy includes assaying, where the assaying comprises amplifying the HLA DRBl*04:04 allele and/or the HLA DQAl*03:03 allele in the sample and detecting the presence of the HLA DRBl*04:04 allele and/or the HLA DQAl*03:03 allele with an agent that specifically binds to the HLA DRBl*04:04 allele and/or the HLA DQAl*03:03 allele; or wherein the assaying comprises sequencing one or more HLA alleles in the sample, thereby identifying the presence of the HLA DRBl*04:04 allele and/or the HLA DQAl*03:03 allele.

In other aspects, provided herein are methods of identifying a subject as having a susceptibility to CSU, wherein the method includes determining that the subject bears an HLA DRBl*04:04 allele, or an HLA DQAl*03:03 allele, and classifying the subject as having susceptibility to CSU based upon the presence of the HLA DRBl*04:04 allele and/or the HLA DQAl*03:03 allele. In other embodiments, the method of identifying a subject as having a susceptibility to CSU further includes treating the subject with a therapy. In embodiments, the therapy comprises an antihistamine, a corticosteroid, an immunosuppressant molecule, or an antibody. For example, the therapy comprises an antihistamine (e.g., an Hi antihistamine). In other examples, the therapy comprises an antibody, e.g., Omalizumab.

In embodiments, the method for identifying of identifying a subject has having a susceptibility to CSU includes assaying, where the assaying comprises amplifying the HLA DRB 1*04: 04 allele and/or the HLA DQAl*03:03 allele in the sample and detecting the presence of the HLA DRBl*04:04 allele and/or the HLA DQAl*03:03 allele with an agent that specifically binds to the HLA DRBl*04:04 allele and/or the HLA DQAl*03:03 allele; or wherein the assaying comprises sequencing one or more HLA alleles in the sample, thereby identifying the presence of the HLA DRBl*04:04 allele and/or the HLA DQAl*03:03 allele.

In aspects, also provided herein are methods of monitoring the efficacy of a therapy in a subject having CSU, the method including: assaying a biological sample from the subject for the presence of HLA alleles comprising DRBl*04:04 or DQAl*03:03, and adapting the dosage of the therapy when the sample has the presence of the HLA DRB 1*04:04 allele and/or the HLA DQAl*03:03 allele.

In other embodiments, the method monitoring the efficacy of a therapy in a subject having CSU includes assaying, where the assaying comprises amplifying the HLA DRBl*04:04 allele and/or the HLA DQA1 *03:03 allele in the sample and detecting the presence of the HLA DRB 1*04: 04 allele and/or the HLA DQAl*03:03 allele with an agent that specifically binds to the HLA DRBl*04:04 allele and/or the HLA DQAl*03:03 allele; or wherein the assaying comprises sequencing one or more HLA alleles in the sample, thereby identifying the presence of the HLA DRBl*04:04 allele and/or the HLA DQAl*03:03 allele. In embodiments, the therapy comprises an antihistamine, a corticosteroid, an immunosuppressant molecule, or an antibody. For example, the therapy comprises an antihistamine (e.g., an Hi antihistamine). In other examples, the therapy comprises an antibody, e.g., Omalizumab.

In additional embodiments, the method further includes that the presence of DRB 1*04: 04 or DQA1 *03:03 is further associated with an autoimmune disorder. For example, the autoimmune disorder includes hypothyroidism, rheumatoid arthritis, type-1 diabetes, or vitiligo.

In further aspects, provided herein are methods for treating a subject having CSU, the method comprising: (a) selecting a subject with CSU having a HLA DRB1 *04:04 allele and/or a HLA DQA1*O3:O3 allele (b) administering a therapy to the subject. In embodiments, the therapy comprises an antihistamine, a corticosteroid, an immunosuppressant molecule, or an antibody. For example, the therapy comprises an antihistamine (e.g., an Hi antihistamine). In other examples, the therapy comprises an antibody, e.g., Omalizumab.

In embodiments, the method for treating a subject having CSU, wherein selecting the subject comprises (i) assaying a biological sample from the subject for the presence of human leukocyte antigen (HLA) alleles comprising DRBl*04:04 or DQAl*03:03; and (ii) identifying the subject as more likely to respond to a therapy when HLA DRBl*04:04 allele and/or the HLA DQAl*03:03 allele is detected in the sample.

In other embodiments, the method for treating a subject having CSU includes assaying, where the assaying comprises amplifying the HLA DRB1 *04:04 allele and/or the HLA DQAl*03:03 allele in the sample and detecting the presence of the HLA DRBl*04:04 allele and/or the HLA DQAl*03:03 allele with an agent that specifically binds to the HLA DRBl*04:04 allele and/or the HLA DQAl*03:03 allele; or wherein the assaying comprises sequencing one or more HLA alleles in the sample, thereby identifying the presence of the HLA DRBl*04:04 allele and/or the HLA DQAl*03:03 allele.

In aspects, provided herein is a method of identifying a subject as having a susceptibility to an autoimmune disease, where the method includes (a) providing a biological sample comprising a nucleic acid from the subject, (b) assaying the nucleic acid for the presence and/or expression level of inositol 1,4, 5 -trisphosphate kinase B (ITPKB and (c) diagnosing the subject as having a susceptibility to an autoimmune disease if the sample has the presence of and/or increased expression level of ITPKB. For example, if a subject has the presence of and/or increased expression level of ITPKB, the subject may have an increased susceptibility to an autoimmune disease.

In embodiments, the method further includes administering a therapy to the subject. In embodiments, the method includes assaying, where the assaying includes contacting the sample with an agent that binds ITPKB, thereby forming a complex between the agent and ITPKB, and thereby detecting the presence of and/or level of ITPKB. In embodiments, the biological sample comprises blood tissue.

In embodiments, the therapy comprises an antihistamine, a corticosteroid, an immunosuppressant molecule, or an antibody. For example, the therapy comprises an antihistamine (e.g., an Hi antihistamine). In other examples, the therapy comprises an antibody, e.g., Omalizumab. In other embodiments, the therapy includes an inhibitor of ITPKB expression and/or activity.

In embodiments, the level of ITPKB is increased 1.5-, 1.75-, 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 15-, 20-, 25-, 30-, 40-, 50-, 60-, 70-, 75-, 80-, 90-, or 100-fold compared to a reference level. For example, the level of ITPKB refers to the nucleic acid level (e.g., the mRNA level) or the protein level of ITPKB.

In further aspects, provided herein are methods of identifying a subject having an autoimmune disease as likely to respond to a therapy, the method including: (a) providing a biological sample comprising a nucleic acid from the subject, (b) assaying the nucleic acid for the presence and/or expression level of inositol 1,4, 5 -trisphosphate kinase B (ITPKB), and, (b) identifying the subject as more likely to respond to the therapy, when the sample has the presence of and/or increased expression level of ITPKB. In embodiments, the method further includes administering a therapy to the subject. For example, if a subject has the presence of and/or increased expression level of ITPKB, the subject having an autoimmune disease may be more likely to respond to therapy.

In embodiments, the method includes assaying, where the assaying includes contacting the sample with an agent that binds ITPKB, thereby forming a complex between the agent and ITPKB, and thereby detecting the presence of and/or level of ITPKB. In embodiments, the biological sample comprises blood tissue.

In embodiments, the therapy comprises an antihistamine, a corticosteroid, an immunosuppressant molecule, or an antibody. For example, the therapy comprises an antihistamine (e.g., an Hi antihistamine). In other examples, the therapy comprises an antibody, e.g., Omalizumab. In other embodiments, the therapy includes an inhibitor of ITPKB expression and/or activity. In embodiments, the level of ITPKB is increased 1.5-, 1.75-, 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 15-, 20-, 25-, 30-, 40-, 50-, 60-, 70-, 75-, 80-, 90-, or 100-fold compared to a reference level. For example, the level of ITPKB refers to the nucleic acid level (e.g., the mRNA level) or the protein level of ITPKB.

In aspects, provided herein are methods of identifying a subject as having an autoimmune disease, the method including: determining that the subject has the presence and/or increased expression level of inositol 1,4, 5 -trisphosphate kinase B (ITPKB), classifying the subject as having an autoimmune disease based upon the presence of and/or increased expression level of ITPKB. In embodiments, the method further includes administering a therapy to the subject. For example, if a subject has the presence of and/or increased expression level of ITPKB, the subject may be identified as having an autoimmune disease.

In further aspects, provided herein are methods of monitoring the efficacy of a therapy in a subject an autoimmune disease, the method including: (a) assaying a biological sample from the subject for the presence of and/or increased expression level of ITPKB, and (b) adapting the dosage of the therapy when the sample has the presence of and/or increased expression level of ITPKB. In embodiments, the method includes assaying, where the assaying includes contacting the sample with an agent that binds ITPKB, thereby forming a complex between the agent and ITPKB, and thereby detecting the presence of and/or level of ITPKB.

In embodiments, the biological sample comprises blood tissue.

In other aspects, provided herein are methods of treating a subject having an autoimmune disease, the method including: (a) identifying a subject having an increased expression level of ITPKB, and (b) administering a therapy to the subject. In embodiments, identifying the subject includies: (i) identifying a subject for the presence of and/or increased expression level of ITPKB, and (ii) identifying the subject as more likely to respond to a therapy when the presence of and/or increased expression level of ITPKB is detected in the sample.

In embodiments, the level of ITPKB is increased 1.5-, 1.75-, 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 15-, 20-, 25-, 30-, 40-, 50-, 60-, 70-, 75-, 80-, 90-, or 100-fold compared to a reference level. For example, the level of ITPKB refers to the nucleic acid level (e.g., the mRNA level) or the protein level of ITPKB. In some embodiments, a blood tissue level of ITPKB that is at or above a reference level, indicates that the patient may benefit from therapy for an autoimmune disease, is more likely to be responsive to the therapy for an autoimmune disease, or has increased likelihood of benefit from therapy for the autoimmune disease.

These and other embodiments are further described by the detailed description that follows.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. FIG. 1 is a graph showing CSU risk in a GWAS Manhattan plot. Two-sided p-values from comparing 679 CSU patients to 4,446 non-CSU controls are displayed. The black line represents the significance threshold after multiple testing correction (P=5xl0‘⁸).

FIG. 2 are data showing the association of polygenic risk scores for 15 disorders with CSU and CU-index risk. The log(odds-ratio) is plotted on the x-axis with bars indicating 95% confidence intervals. Associations passing multiple testing correction (P<1.67x10-3) are displayed as filled-in diamonds. Corresponding association statistics are available in Table 12. (CD=Crohn’s disease, HTH=hypothyroidism, MS=multiple sclerosis, PSOR=psoriasis, RA=rheumatoid arthritis, SLE=systemic lupus erythematosus, TlD=type-l diabetes, UC=ulcerative colitis, AD=atopic dermatitis, Allergic dis. =allergic diseases, CAD=coronary artery disease, COVID 19=hospitalized COVID 19, T2D=type-2 diabetes).

FIG. 3 is plot showing the regional association plot for rs34141382. The index SNP is indicated with a purple diamond. Correlation (measured by r²) between the index SNP and all other variants is indicated by the color of each point.

FIG. 4 is a plot showing the regional association plot for rs 1097296. The index SNP is indicated with a purple diamond. Correlation (measured by r²) between the index SNP and all other variants is indicated by the color of each point.

FIG. 5 is a plot showing a Manhattan plot comparing 455 CU-index low patients to 187 CU-index high patients; a CU-index GWAS Manhattan plot is shown. Two-sided p-values from comparing 455 CU-index low CSU to 187 CU-index high patients are displayed. The black line represents the significance threshold after multiple testing correction (P=5xl0‘⁸).

DETAILED DESCRIPTION OF THE INVENTION

I. Introduction

The invention provides methods for identifying patients having an increased (genetic predisposition to) susceptibility to CSU. The invention further provides methods for identifying patients as having an increased susceptibility to an autoimmune disease.

Chronic spontaneous urticaria (CSU) is a dermatologic condition with an overall prevalence of 0.5- 1 %^x that is characterized by spontaneous, pruritic hives and/or angioedema that persist for longer than six weeks despite antihistamine treatment²'⁴. CSU symptoms have a significant impact on quality of life in patients⁵. An autoimmune basis for CSU has been suggested. IgG autoantibodies to FCERI and IgE, and IgE with specificities to autoantigens are thought to elicit mast cell and basophil degranulation by autoantibody mediated FceRI/IgE crosslinking, releasing pruritogenic and angioedema causing mediators⁶. The presence and function of these autoantibodies in serum can be tested by a basophil histamine release assay, known as the Chronic Urticaria (CU)-index test, with up to 50% of CSU patients testing positive⁷.

Omalizumab is a recombinant monoclonal antibody with specificity to the FCERI binding region of IgE Fc that is an approved therapy for CSU⁸'¹³. Omalizumab reduces free IgE which in turn leads to the destabilization and downregulation of FCERI on the cell surface, thus desensitizing these cells to FcERI-dependent stimuli¹⁴. While omalizumab has been proven to be efficacious and has a rapid onset of action for CSU patients, 38 to 44% of subjects on omalizumab did not achieve well controlled status in pivotal phase 3 studies¹⁵, indicating additional disease mediators are involved and can be targeted therapeutically.

A familial study of CSU found that CSU occurred eight times more frequently in individuals with affected first degree relatives than expected, suggesting that genetic factors predispose individuals for the risk of developing CSU¹⁶. To date, only candidate single nucleotide polymorphism (SNP) analyses in small cohorts (n_Cases<200) with limited replication have been reported¹⁷'²¹. Herein, the first GWAS study of 679 CSU patients was performed who participated in the ASTERIA I (ClmicalTnals.gov NCT01287117), ASTERIA II (NCT01292473), GLACIAL (NCT01292473)ⁿ'¹³and SHASTA studies (NCT03693625).

II. Definitions

The following definitions are included for the purpose of understanding the present subject matter and for constructing the appended patent claims. The abbreviations used herein have their conventional meanings within the chemical and biological arts.

While various embodiments and aspects of the present invention are shown and described herein, it will be obvious to those skilled in the art that such embodiments and aspects are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention.

The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. All documents, or portions of documents, cited in the application including, without limitation, patents, patent applications, articles, books, manuals, and treatises are hereby expressly incorporated by reference in their entirety for any purpose.

Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by a person of ordinary skill in the art. See, e.g., Singleton et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY 2nd ed., J. Wiley & Sons (New York, NY 1994); Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, Cold Springs Harbor Press (Cold Springs Harbor, NY 1989). Any methods, devices and materials similar or equivalent to those described herein can be used in the practice of this invention. The following definitions are provided to facilitate understanding of certain terms used frequently herein and are not meant to limit the scope of the present disclosure.

The transitional term “comprising,” which is synonymous with “including,” “containing,” or “characterized by,” is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. By contrast, the transitional phrase “consisting of’ excludes any element, step, or ingredient not specified in the claim. The transitional phrase “consisting essentially of’ limits the scope of a claim to the specified materials or steps “and those that do not materially affect the basic and novel characteristic(s)” of the claimed invention.

In the descriptions herein and in the claims, phrases such as “at least one of’ or “one or more of’ may occur followed by a conjunctive list of elements or features. The term “and/or” may also occur in a list of two or more elements or features. Unless otherwise implicitly or explicitly contradicted by the context in which it is used, such a phrase is intended to mean any of the listed elements or features individually or any of the recited elements or features in combination with any of the other recited elements or features. For example, the phrases “at least one of A and B;” “one or more of A and B;” and “A and/or B” are each intended to mean “A alone, B alone, or A and B together.” A similar interpretation is also intended for lists including three or more items. For example, the phrases “at least one of A, B, and C;” “one or more of A, B, and C;” and “A, B, and/or C” are each intended to mean “A alone, B alone, C alone, A and B together, A and C together, B and C together, or A and B and C together.” In addition, use of the term “based on,” above and in the claims is intended to mean, “based at least in part on,” such that an unrecited feature or element is also permissible. A “control” sample or value refers to a sample that serves as a reference, usually a known reference, for comparison to a test sample. For example, a test sample can be taken from a test subject, e.g., a subject with an autoimmune disease or CSU, and compared to samples from known conditions, e.g., a subject (or subjects) that does not have an autoimmune disease or CSU (a negative or normal control), or a subject (or subjects) who does have an autoimmune disease or CSU (positive control). A control can also represent an average value gathered from a number of tests or results. One of skill in the art will recognize that controls can be designed for assessment of any number of parameters. One of skill in the art will understand which controls are valuable in a given situation and be able to analyze data based on comparisons to control values. Controls are also valuable for determining the significance of data. For example, if values for a given parameter are variable in controls, variation in test samples will not be considered as significant.

The term, “normal amount” with respect to a compound (e.g., a protein or mRNA) refers to a normal amount of the compound in an individual who does not have an autoimmune disease or CSU in a healthy or general population. The amount of a compound can be measured in a test sample and compared to the “normal control” level, utilizing techniques such as reference limits, discrimination limits, or risk defining thresholds to define cutoff points and abnormal values (e.g., for an autoimmune disease or CSU or a symptom thereof). The normal control level means the level of one or more compounds or combined compounds typically found in a subject known not suffering from an autoimmune disease or CSU. Such normal control levels and cutoff points may vary based on whether a compounds is used alone or in a formula combining with other compounds into an index. Alternatively, the normal control level can be a database of compounds patterns from previously tested subjects who did not develop an autoimmune disease or CSU or a particular symptom thereof over a clinically relevant time horizon.

The level that is determined may be the same as a control level or a cut off level or a threshold level, or may be increased or decreased relative to a control level or a cut off level or a threshold level. In some aspects, the control subject is a matched control of the same species, gender, ethnicity, age group, smoking status, body mass index (BMI), current therapeutic regimen status, medical history, or a combination thereof, but differs from the subject being diagnosed in that the control does not suffer from the disease (or a symptom thereof) in question or is not at risk for the disease. Relative to a control level, the level that is determined may an increased level. As used herein, the term “increased” with respect to level (e.g., protein or mRNA level) refers to any % increase above a control level. In various embodiments, the increased level may be at least or about a 5% increase, at least or about a 10% increase, at least or about a 15% increase, at least or about a 20% increase, at least or about a 25% increase, at least or about a 30% increase, at least or about a 35% increase, at least or about a 40% increase, at least or about a 45% increase, at least or about a 50% increase, at least or about a 55% increase, at least or about a 60% increase, at least or about a 65% increase, at least or about a 70% increase, at least or about a 75% increase, at least or about a 80% increase, at least or about a 85% increase, at least or about a 90% increase, at least or about a 95% increase, relative to a control level.

Relative to a control level, the level that is determined may a decreased level. As used herein, the term “decreased” with respect to level (e.g., protein or mRNA level) refers to any % decrease below a control level. In various embodiments, the decreased level may be at least or about a 5% decrease, at least or about a 10% decrease, at least or about a 15% decrease, at least or about a 20% decrease, at least or about a 25% decrease, at least or about a 30% decrease, at least or about a 35% decrease, at least or about a 40% decrease, at least or about a 45% decrease, at least or about a 50% decrease, at least or about a 55% decrease, at least or about a 60% decrease, at least or about a 65% decrease, at least or about a 70% decrease, at least or about a 75% decrease, at least or about a 80% decrease, at least or about a 85% decrease, at least or about a 90% decrease, at least or about a 95% decrease, relative to a control level.

In certain embodiments, the term “increase” or “above” refers to a level above the reference level or to an overall increase of 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 100% or greater, in a sample ITPKB level detected by the methods described herein, as compared to the ITPKB level from a reference sample. In certain embodiments, the term increase refers to the increase in blood tissue ITPKB level wherein, the increase is at least about 1.5-, 1.75-, 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 15-, 20-, 25-, 30-, 40-, 50-, 60-, 70-, 75-, 80-, 90-, or 100- fold higher as compared to the blood tissue ITPKB level e.g. predetermined from a reference sample. In one preferred embodiment the term increased level relates to a value at or above a reference level.

In certain embodiments, the term “decrease” or “below” herein refers to a level below the reference level or to an overall reduction of 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater, in ITPKB level detected by the methods described herein, as compared to the ITPKB level from a reference sample. In certain embodiments, the term decrease refers to the decrease in ITPKB level, wherein the decreased level is at most about 0.9-, 0.8-, 0.7-, 0.6-, 0.5-, 0.4-, 0.3-, 0.2-, 0.1-, 0.05-, or 0.01- fold the ITPKB level from the reference sample or lower.

In certain embodiments, the term “at a reference level” refers to a level that is the same as the ITPKB level detected by the methods described herein, from a reference sample.

In certain embodiments, the term “reference level” herein refers to a predetermined value. As the skilled artisan will appreciate the reference level is predetermined and set to meet the requirements in terms of e.g. specificity and/or sensitivity. These requirements can vary, e.g. from regulatory body to regulatory body. It may for example be that assay sensitivity or specificity, respectively, has to be set to certain limits, e.g. 80%, 90% or 95%. These requirements may also be defined in terms of positive or negative predictive values. Nonetheless, based on the teaching given in the present invention it will always be possible to arrive at the reference level meeting those requirements. In one embodiment, the reference level is determined in healthy individuals. The reference value in one embodiment has been predetermined in the disease entity to which the patient belongs. In certain embodiments the reference level can e.g. be set to any percentage between 25% and 75% of the overall distribution of the values in a disease entity investigated. In other embodiments the reference level can e.g. be set to the median, tertiles or quartiles as determined from the overall distribution of the values in a disease entity investigated. In one embodiment the reference level is set to the median value as determined from the overall distribution of the values in a disease entity investigated.

The phrase “substantially reduced,” or “substantially different,” as used herein, denotes a sufficiently high degree of difference between two numeric values (generally one associated with a molecule and the other associated with a reference/comparator molecule) such that one of skill in the art would consider the difference between the two values to be of statistical significance within the context of the biological characteristic measured by said values (e.g., Kd values). The difference between said two values is, for example, greater than about 10%, greater than about 20%, greater than about 30%, greater than about 40%, and/or greater than about 50% as a function of the value for the reference/comparator molecule.

A “disorder” or “disease” is any condition that would benefit from treatment with a substance/molecule or method of the invention. This includes chronic and acute disorders or diseases including those pathological conditions which predispose the mammal to the disorder in question. Non-limiting examples of disorders to be treated herein include CSU and autoimmune disorders, including hypothyroidism, type-1 diabetes and vitiligo. Other autoimmune disorders can include rheumatoid arthritis, lupus erythematosus, inflammatory bowel disease (TBD), multiple sclerosis (MS), Type 1 diabetes mellitus, Guillain-Barre syndrome, chronic inflammatory demyelinating polyneuropathy, psoriasis, Grave’s Disease, Hashimoto's thyroiditis, Myasthenia gravis, scleroderma, vasculitis, or other autoimmune-related disorders.

As used herein, “treatment” refers to clinical intervention in an attempt to alter the natural course of the individual or cell being treated, and can be performed either for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. In some embodiments, antibodies of the invention are used to delay development of a disease or disorder.

An “effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.

A “therapeutically effective amount” of a substance/molecule of the invention, an antihistamine, a corticosteroid, an immunosuppressant molecule, or an antibody. In other embodiments, the therapy includes an inhibitor of ITPKB expression and/or activity may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the substance/molecule, an antihistamine, a corticosteroid, an immunosuppressant molecule, or an antibody to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the substance/molecule, an antihistamine, a corticosteroid, an immunosuppressant molecule, or an antibody are outweighed by the therapeutically beneficial effects. The term “therapeutically effective amount” refers to an amount of an antibody, polypeptide or an antihistamine, a corticosteroid, an immunosuppressant molecule, or an antibody of this invention effective to “treat” a disease or disorder in a mammal (e.g., a patient).

A “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically but not necessarily, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount is less than the therapeutically effective amount.

An “an antihistamine” may refer to a molecule that binds to histamine Hi receptors in mast cells, smooth muscle, and endothelium in the body as well as in the tuberomammillary nucleus in the brain. Exemplary Hi antihistamines include Acrivastine, Amitriptyline (tricyclic antidepressant), Amoxapine, Azelastine, Bilastine, Bromodiphenhydramine, Brompheniramine, Buclizine, Carbinoxamine, Cetirizine (Zyrtec), Chlorodiphenhydramine, Chlorpheniramine, Chlorpromazine, Chlorprothixene, Chloropyramine, Cinnarizine, Clemastine, Clomipramine, Clozapine, Cyclizine, Cyproheptadine, Desloratadine, Dexbrompheniramine, Dexchlorpheniramine, Dimenhydrinate, Dimetindene, Diphenhydramine (Benadryl), Dosulepin, Doxepin, Doxylamine, Ebastine, Embramine, Fexofenadine (Allegra/Telfast), Hydroxyzine, Imipramine, Levocabastine (Livostin/Livocab), Levocetirizine (Xyzal), Levomepromazine, Loratadine (Claritin), Maprotiline, Meclizine, Mianserin, Mirtazapine, Olanzapine, Olopatadine, Orphenadrine, Periciazine, Phenindamine, Pheniramine, Phenyltoloxamine, Promethazine, Pyrilamine, Quetiapine, Rupatadine, Trazodone, Tripelennamine, or Triprolidine.

As used herein, the term “patient” refers to any single animal, more preferably a mammal (including such non-human animals as, for example, dogs, cats, horses, rabbits, zoo animals, cows, pigs, sheep, and non-human primates) for which treatment is desired. Most preferably, the patient herein is a human.

A “subject” herein is any single human subject, including a patient, eligible for treatment who is experiencing or has experienced one or more signs, symptoms, or other indicators of an autoimmune disorder or CSU. Intended to be included as a subject are any subjects involved in clinical research trials not showing any clinical sign of disease, or subjects involved in epidemiological studies, or subjects once used as controls. The subject may have been previously treated with an autoimmune therapy, or not so treated. The subject may be naive to an additional agent(s) being used when the treatment herein is started, i.e., the subject may not have been previously treated with, for example, an autoimmune therapy (i.e., at a set point in time before the administration of a first dose of an autoimmune therapy in the treatment method herein, such as the day of screening the subject before treatment is commenced). Such “naive” subjects are generally considered to be candidates for treatment with such additional agent(s). The term “pharmaceutical formulation” refers to a sterile preparation that is in such form as to permit the biological activity of the medicament to be effective, and which contains no additional components that are unacceptably toxic to a subject to which the formulation would be administered.

A “sterile” formulation is aseptic or free from all living microorganisms and their spores.

A “package insert” is used to refer to instructions customarily included in commercial packages of therapeutic products or medicaments, that contain information about the indications, usage, dosage, administration, contraindications, other therapeutic products to be combined with the packaged product, and/or warnings concerning the use of such therapeutic products or medicaments, etc.

A “kit” is any manufacture (e.g. a package or container) comprising at least one reagent, e.g., a medicament for treatment of an autoimmune disorder, or a probe for specifically detecting a biomarker gene or protein of the invention. The manufacture is preferably promoted, distributed, or sold as a unit for performing the methods of the present invention.

By “reducing the risk of a negative side effect” is meant reducing the risk of a side effect resulting from treatment with the autoimmune therapy herein to a lower extent than the risk observed resulting from treatment of the same patient or another patient with a previously administered medicament. Such side effects include those set forth above regarding toxicity or infection.

By “correlate” or “correlating” is meant comparing, in any way, the performance and/or results of a first analysis or protocol with the performance and/or results of a second analysis or protocol. For example, one may use the results of a first analysis or protocol in carrying out a second protocols and/or one may use the results of a first analysis or protocol to determine whether a second analysis or protocol should be performed. With respect to various embodiments herein, one may use the results of an analytical assay to determine whether a specific therapeutic regimen using an autoimmune therapy, such as an antihistamine, a corticosteroid, an immunosuppressant molecule, or an antibody. For example, the therapy comprises an antihistamine (e.g., an Hi antihistamine). In other examples, the therapy comprises an antibody, e.g., Omalizumab.

III. Methods

Chronic spontaneous urticaria (CSU) The present invention provides for methods for identifying a subject as having a genetic predisposition to chronic spontaneous urticaria (CSU), where the method comprises (a) providing a biological sample comprising a nucleic acid from the subject, (b) assaying the nucleic acid for the presence of human leukocyte antigen (HLA) alleles comprising one or more of DRBl*04:04 or DQAl*03:03, and (c) diagnosing the subject at risk of having a genetic predisposition to CSU upon presence of either HLA-DRBl*04:04 and/or HLA-DQAl*03:03. In other embodiments, the method for identifying a subject as having a genetic predisposition to chronic spontaneous urticaria (CSU) further includes step (d) of treating the subject with a therapy.

The invention further provides methods of identifying a subject having CSU as likely to respond to a therapy, the method including (a) providing a biological sample comprising a nucleic acid from the subject, (b) assaying the nucleic acid for the presence of HLA alleles comprising DRBl*04:04 or DQAl*03:03, (c) identifying the subject as more likely to respond to the therapy, when the sample has the presence of either HLA-DRBl*04:04 and/or HLA- DQAl*03:03. In other embodiments, the method of identifying a subject having CSU as likely to respond to a therapy further includes step (d) of treating the subject with a therapy.

In embodiments, the therapy comprises an antihistamine, a corticosteroid, an immunosuppressant molecule, or an antibody. For example, the therapy comprises an antihistamine (e.g., an Hi antihistamine). In embodiments, the therapy comprises a corticosteroid. In embodiments, the therapy comprises an immunosuppressant molecule. In other examples, the therapy comprises an antibody, e.g., Omalizumab.

The invention also provides methods of identifying a subject as having a susceptibility to CSU, wherein the method includes determining that the subject bears an HLA DRB1 *04:04 allele, or an HLA DQA1 *03:03 allele, and classifying the subject as having susceptibility to CSU based upon the presence of the HLA DRBl*04:04 allele and/or the HLA DQAl*03:03 allele. In other embodiments, the method of identifying a subject as having a susceptibility to CSU further includes treating the subject with a therapy. In embodiments, the therapy comprises an antihistamine, a corticosteroid, an immunosuppressant molecule, or an antibody. For example, the therapy comprises an antihistamine (e.g., an Hi antihistamine). In other examples, the therapy comprises an antibody, e.g., Omalizumab.

In embodiments, the nucleic acid is assayed for the presence of the DRBl*04:04 allele. In embodiments, the nucleic acid is assayed for the presence of the DQAl*03:03 allele. In embodiments, the nucleic acid is assayed for the presence of the DRB1 *04:04 allele and the DQAl*03:03 allele. In embodiments, the subject is diagnosed at risk of having a genetic predisposition to CSU upon presence of HLA-DRB 1*04:04. In embodiments, the subject is diagnosed at risk of having a genetic predisposition to CSU upon presence of HLA- DQAl*03:03. In embodiments, the subject is diagnosed at risk of having a genetic predisposition to CSU upon presence of HLA-DRB 1*04: 04 and HLA-DQAl*03:03. In embodiments, the subject is identified as more likely to respond to therapy upon presence of HLA-DRB 1*04: 04. In embodiments, the subject is identified as more likely to respond to therapy upon presence of HLA-DQAl*03:03. In embodiments, the subject is identified as more likely to respond to therapy upon presence of HLA-DRB 1*04: 04 and HLA-DQAl*03:03. In embodiments, the subject is classified as having susceptibility to CSU upon presence of HLA-DRB 1*04: 04. In embodiments, the subject is classified as having susceptibility to CSU upon presence of HLA - DQAl*03:03. In embodiments, the subject is classified as having susceptibility to CSU upon presence of HLA-DRB 1*04: 04 and HLA-DQA1 *03:03.

In aspects, also provided herein are methods of monitoring the efficacy of a therapy in a subject having CSU, the method including: assaying a biological sample from the subject for the presence of HLA alleles comprising DRBl*04:04 or DQAl*03:03, and adapting the dosage of the therapy when the sample has the presence of the HLA DRB 1*04:04 allele and/or the HLA DQAl*03:03 allele. In embodiments, the dosage of the therapy is adapted upon presence of HLA-DRBl*04:04. In embodiments, the dosage of the therapy is adapted upon presence of HLA-DQAl*03:03. In embodiments, the dosage of the therapy is adapted upon presence of HLA-DRB 1*04: 04 and HLA-DQA1 *03:03.

The invention also provides methods for treating a subject having CSU, the method comprising: (a) selecting a subject with CSU having a HLA DRB1 *04:04 allele and/or a HLA DQAl*03:03 allele (b) administering a therapy to the subject. In embodiments, a subject having a HLA-DRB 1*04: 04 allele is selected. In embodiments, t a subject having a HLA-DQAl*03:03 allele is selected. In embodiments, a subject having a HLA-DRB 1*04: 04 allele and a HLA- DQAl*03:03 allele is selected. In embodiments, the therapy comprises an antihistamine, a corticosteroid, an immunosuppressant molecule, or an antibody. For example, the therapy comprises an antihistamine (e.g., an Hi antihistamine). In other examples, the therapy comprises an antibody, e.g., Omalizumab.

The disclosed methods and assays provide for convenient, efficient, and potentially cost- effective means to obtain data and information useful in assessing appropriate or effective therapies for treating patients. For example, according to the methods of the invention, a patient could provide a sample before treatment with an autoimmune therapy comprising an antihistamine, a corticosteroid, an immunosuppressant molecule, or an antibody and the level of an HLA DRBl*04:04 allele, or an HLA DQAl*03:03 allele in the sample could be determined and compared to the level of an HLA DRBl*04:04 allele, or an HLA DQAl*03:03 allele in a reference sample or to a predetermined reference value, respectively. Patients with the presence of an HLA DRBl*04:04 allele, or an HLA DQAl*03:03 allele are identified as patients likely to respond to autoimmune therapy comprising an antihistamine, a corticosteroid, an immunosuppressant molecule, or an antibody. The methods may be conducted in a variety of assay formats, including assays detecting protein expression (such enzyme immunoassays) and biochemical assays detecting appropriate activity. Determination of expression or the presence of such biomarkers in the samples is predictive that the patient providing the sample will be sensitive to the biological effects of an autoimmune therapy. Typically an expression level of an HLA DRBl*04:04 allele, or an HLA DQAl*03:03 allele in a sample obtained from the patient at or above a reference level indicates that a patient will respond to or be sensitive to treatment with an autoimmune therapy.

One of skill in the medical arts, particularly pertaining to the application of diagnostic tests and treatment with therapeutics, will recognize that biological systems are somewhat variable and not always entirely predictable, and thus many good diagnostic tests or therapeutics are occasionally ineffective. Thus, it is ultimately up to the judgment of the attending physician to determine the most appropriate course of treatment for an individual patient, based upon test results, patient condition and history, and his or her own experience. There may even be occasions, for example, when a physician will choose to treat a patient with an autoimmune therapy even when a patient is not predicted to be particularly sensitive to an autoimmune therapy, based on data from diagnostic tests or from other criteria, particularly if all or most of the other obvious treatment options have failed, or if some synergy is anticipated when given with another treatment.

Some methods of the invention further comprise administering an autoimmune therapy to a patient with the presence of an HLA DRBl*04:04 allele, or an HLA DQAl*03:03 allele in a sample. A physician having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required, depending on such factors as the particular type of autoimmune therapy. For example, the physician could start with doses of such autoimmune therapy comprising an antihistamine, a corticosteroid, an immunosuppressant molecule, or an antibody, employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. The effectiveness of a given dose or treatment regimen of the an antihistamine, a corticosteroid, an immunosuppressant molecule, or an antibody can be determined, for example, by assessing signs and symptoms in the patient using standard methods.

In one embodiment, the autoimmune therapy is an antihistamine, a corticosteroid, an immunosuppressant molecule, or an antibody that is administered at a dose of about 100 or 400 mg every 1, 2, 3, or 4 weeks or is administered a dose of about 1, 3, 5, 7.5, 10, 15, or 20 mg/kg every 1, 2, 3, or 4 weeks. The dose may be administered as a single dose or as multiple doses (e.g., 2 or 3 doses), such as infusions.

In yet another aspect, the invention provides, after the diagnosis step, a method of determining whether to continue administering an autoimmune therapy is an antihistamine, a corticosteroid, an immunosuppressant molecule, or an antibody to a subject diagnosed with CSU, comparing imaging findings in the subject at the first time and at the second time, and continuing administration of the autoimmune disorder if there is no improvement.

In a still further embodiment, a step is included in the treatment method to test the subject’s response to treatment after the administration step to determine that the level of response is effective to treat CSU.

Autoimmune Disorders

The present invention provides methods for identifying a subject as having a susceptibility to an autoimmune disease, where the method includes (a) providing a biological sample comprising a nucleic acid from the subject, (b) assaying the nucleic acid for the presence and/or expression level of inositol 1,4,5-trisphosphate kinase B (ITPKB), and (c) diagnosing the subject as having a susceptibility to an autoimmune disease if the sample has the presence of and/or increased expression level of ITPKB. For example, if a subject has the presence of and/or increased expression level of ITPKB, the subject may have an increased susceptibility to an autoimmune disease.

The invention also provides methods of identifying a subject having an autoimmune disease as likely to respond to a therapy, the method including: (a) providing a biological sample comprising a nucleic acid from the subject, (b) assaying the nucleic acid for the presence and/or expression level of inositol 1,4, 5 -trisphosphate kinase B (ITPKB), and, (b) identifying the subject as more likely to respond to the therapy, when the sample has the presence of and/or increased expression level of ITPKB. In embodiments, the method further includes administering a therapy to the subject. For example, if a subject has the presence of and/or increased expression level of ITPKB, the subject having an autoimmune disease may be more likely to respond to therapy.

In embodiments, the presence of ITPKB is assayed. In embodiments, an expression level of ITPKB is assayed.

The invention further provides methods of identifying a subject as having an autoimmune disease, the method including: determining that the subject has the presence and/or increased expression level of inositol 1,4, 5 -trisphosphate kinase B (ITPKB), classifying the subject as having an autoimmune disease based upon the presence of and/or increased expression level of ITPKB. In embodiments, the method further includes administering a therapy to the subject. For example, if a subject has the presence of and/or increased expression level of ITPKB, the subject may be identified as having an autoimmune disease. In embodiments, the presence of ITPKB is determined. In embodiments, an expression level of ITPKB is determined.

The invention further provides methods of monitoring the efficacy of a therapy in a subject an autoimmune disease, the method including: (a) assaying a biological sample from the subject for the presence of and/or increased expression level of ITPKB, and (b) adapting the dosage of the therapy when the sample has the presence of and/or increased expression level of ITPKB. In embodiments, the method includes assaying, where the assaying includes contacting the sample with an agent that binds ITPKB, thereby forming a complex between the agent and ITPKB, and thereby detecting the presence of and/or level of ITPKB.

The invention also provides methods of treating a subject having an autoimmune disease, the method including: (a) identifying a subject having presence of and/or an increased expression level of ITPKB, and (b) administering a therapy to the subject. In embodiments, identifying the subject includes: (i) identifying a subject for the presence of and/or increased expression level of ITPKB, and (ii) identifying the subject as more likely to respond to a therapy when the presence of and/or increased expression level of ITPKB is detected in the sample. In embodiments, the subject is identified as more likely to respond to a therapy when the presence of ITPKB is detected in the sample. In embodiments, the subject is identified as more likely to respond to a therapy when an increased expression level of ITPKB is detected in the sample.

The disclosed methods and assays provide for convenient, efficient, and potentially cost- effective means to obtain data and information useful in assessing appropriate or effective therapies for treating patients. For example, according to the methods of the invention, a patient could provide a sample (e.g., blood tissue sample) before treatment with an autoimmune therapy comprising an antihistamine, a corticosteroid, an immunosuppressant molecule, or an antibody and the level of ITPKB in the sample could be determined and compared to the level of ITPKB in a reference sample or to a predetermined reference value, respectively. Patients with an increased level of ITPKB are identified as patients likely to respond to autoimmune therapy comprising an antihistamine, a corticosteroid, an immunosuppressant molecule, or an antibody. The methods may be conducted in a variety of assay formats, including assays detecting protein expression (such enzyme immunoassays) and biochemical assays detecting appropriate activity. Determination of expression or the presence of such biomarkers in the samples is predictive that the patient providing the sample will be sensitive to the biological effects of an autoimmune therapy. Typically an expression level of ITPKB in a sample obtained from the patient at or above a reference level indicates that a patient will respond to or be sensitive to treatment with an autoimmune therapy.

In certain embodiments, ITPKB is present when detected at a level above a reference level. In certain embodiments, expression of ITPKB is increased when the level is 5% or greater, in a sample ITPKB level detected by the methods described herein, as compared to the ITPKB level from a reference sample. In certain embodiments, expression of ITPKB is increased when the level is 10% or greater as compared to the ITPKB level from a reference sample. In certain embodiments, expression of ITPKB is increased when the level is 15% or greater as compared to the ITPKB level from a reference sample. In certain embodiments, expression of ITPKB is increased when the level is 20% or greater as compared to the ITPKB level from a reference sample. In certain embodiments, expression of ITPKB is increased when the level is 30% or greater as compared to the ITPKB level from a reference sample. In certain embodiments, expression of ITPKB is increased when the level is 40% or greater as compared to the ITPKB level from a reference sample. In certain embodiments, expression of ITPKB is increased when the level is 50% or greater as compared to the ITPKB level from a reference sample. In certain embodiments, expression of ITPKB is increased when the level is 60% or greater as compared to the ITPKB level from a reference sample. In certain embodiments, expression of ITPKB is increased when the level is 70% or greater as compared to the ITPKB level from a reference sample. In certain embodiments, expression of ITPKB is increased when the level is 80% or greater as compared to the ITPKB level from a reference sample. In certain embodiments, expression of ITPKB is increased when the level is 90% or greater as compared to the ITPKB level from a reference sample.

In certain embodiments, expression of ITPKB is increased when the increase is about 1.5-fold to about 500-fold higher as compared to the blood tissue ITPKB level e.g. predetermined from a reference sample. In embodiments, expression of ITPKB is increased when the increase is at least about 1.75-fold higher. In embodiments, expression of ITPKB is increased when the increase is at least about 2-fold higher. In embodiments, expression of ITPKB is increased when the increase is at least about 3 -fold higher. In embodiments, expression of ITPKB is increased when the increase is at least about 4-fold higher. In embodiments, expression of ITPKB is increased when the increase is at least about 5-fold higher. In embodiments, expression of ITPKB is increased when the increase is at least about 6-fold higher. In embodiments, expression of ITPKB is increased when the increase is at least about 7-fold higher. In embodiments, expression of ITPKB is increased when the increase is at least about 8- fold higher. In embodiments, expression of ITPKB is increased when the increase is at least about 9-fold higher. In embodiments, expression of ITPKB is increased when the increase is at least about 10-fold higher. In embodiments, expression of ITPKB is increased when the increase is at least about 15-fold higher. In embodiments, expression of ITPKB is increased when the increase is at least about 20-fold higher. In embodiments, expression of ITPKB is increased when the increase is at least about 25-fold higher. In embodiments, expression of ITPKB is increased when the increase is at least about 30-fold higher. In embodiments, expression of ITPKB is increased when the increase is at least about. In embodiments, expression of ITPKB is increased when the increase is at least about 40-fold higher. In embodiments, expression of ITPKB is increased when the increase is at least about 50-fold higher. In embodiments, expression of ITPKB is increased when the increase is at least about 60-fold higher. In embodiments, expression of ITPKB is increased when the increase is at least about 70-fold higher. In embodiments, expression of ITPKB is increased when the increase is at least about 75- fold higher. In embodiments, expression of ITPKB is increased when the increase is at least about 80-fold higher. In embodiments, expression of ITPKB is increased when the increase is at least about 90-fold higher. In embodiments, expression of ITPKB is increased when the increase is at least about 100-fold higher fold higher. In one preferred embodiment the term increased level relates to a value at or above a reference level.

Some methods of the invention further comprise administering an autoimmune therapy to a patient with an increased level of ITPKB compared to a reference sample. A physician having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required, depending on such factors as the particular type of autoimmune therapy. For example, the physician could start with doses of such autoimmune therapy comprising an antihistamine, a corticosteroid, an immunosuppressant molecule, or an antibody, employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. The effectiveness of a given dose or treatment regimen of the antihistamine, a corticosteroid, an immunosuppressant molecule, or an antibody can be determined, for example, by assessing signs and symptoms in the patient using standard m In one embodiment, the autoimmune therapy is an antihistamine, a corticosteroid, an immunosuppressant molecule, or an antibody that is administered at a dose of about 100 or 400 mg every 1, 2, 3, or 4 weeks or is administered a dose of about 1, 3, 5, 7.5, 10, 15, or 20 mg/kg every 1, 2, 3, or 4 weeks. The dose may be administered as a single dose or as multiple doses (e.g., 2 or 3 doses), such as infusions.

In yet another aspect, the invention provides, after the diagnosis step, a method of determining whether to continue administering an autoimmune therapy is an antihistamine, a corticosteroid, an immunosuppressant molecule, or an antibody to a subject diagnosed with an autoimmune disorder, comparing imaging findings in the subject at the first time and at the second time, and continuing administration of the autoimmune disorder if there is no improvement.

In a still further embodiment, a step is included in the treatment method to test the subject's response to treatment after the administration step to determine that the level of response is effective to treat the autoimmune disorder.

VI. Kits

For use in detection of HLA alleles DRBl*04:04 and DQAl*03:03, kits or articles of manufacture are also provided by the invention. For use in detection of ITPKB, kits or articles of manufacture are also provided by the invention. Such kits can be used to determine if a subject with an autoimmune disease or CSU will be effectively responsive to an autoimmune therapy such as an antihistamine, a corticosteroid, an immunosuppressant molecule, or an antibody. For example, the therapy comprises an antihistamine (e.g., an Hi antihistamine). In other examples, the therapy comprises an antibody, e.g., Omalizumab.

These kits may comprise a carrier means being compartmentalized to receive in close confinement one or more container means such as vials, tubes, and the like, each of the container means comprising one of the separate elements to be used in the method. For example, one of the container means may comprise a compound that specifically binds alleles HLA-DRBl*04:04 and/or HLA-DQAl*03:03. For example, one of the container means may comprise a compound that specifically binds ITPKB.

Such kit will typically comprise the container described above and one or more other containers comprising materials desirable from a commercial and user standpoint, including buffers, diluents, filters, needles, syringes, and package inserts with instructions for use. A label may be present on the container to indicate that the composition is used for a specific application, and may also indicate directions for either in vivo or in vitro use, such as those described above.

The kits of the invention have a number of embodiments. A typical embodiment is a kit comprising a container, a label on said container, and a composition contained within said container, wherein the composition includes compound(s) that specifically bind(s) alleles HLA- DRBl*04:04 and/or HLA-DQA1 *03:03, and the label on said container indicates that the composition can be used to detect alleles HLA-DRBl*04:04 and/or HLA-DQA1 *03:03, and wherein the kit includes instructions for using the compound(s) for detecting alleles HLA- DRBl*04:04 and/or HLA-DQA1 *03:03. The kit can further comprise a set of instructions and materials for preparing and using the compound(s).

The kits of the invention have a number of embodiments. A typical embodiment is a kit comprising a container, a label on said container, and a composition contained within said container, wherein the composition includes compound(s) that specifically bind(s) ITPKB, and the label on said container indicates that the composition can be used to detect ITPKB, and wherein the kit includes instructions for using the compound(s) for detecting ITPKB. The kit can further comprise a set of instructions and materials for preparing and using the compound(s).

Other optional components of the kit include one or more buffers (e.g., dilution buffer, etc.), other reagents such as carrier (e.g., dextran, albumin). Kits can also include instructions for interpreting the results obtained using the kit.

EXAMPLES

The following examples illustrate certain specific embodiments of the invention and are not meant to limit the scope of the invention.

Embodiments herein are further illustrated by the following examples and detailed protocols. However, the examples are merely intended to illustrate embodiments and are not to be construed to limit the scope herein. The contents of all references and published patents and patent applications cited throughout this application are hereby incorporated by reference.

Example 1: Two loci significantly associated with CSU

The association with CSU at 10,077,611 variants with minor allele frequency (MAF) > 0.5% that passed quality-control filters (Methods) was tested. A significant inflation of test statistics (AGC=1.02) was not observed. A linkage disequilibrium (LD)-score regression was assigned to this GWAS²³ and estimated the common- variant SNP heritability (h²) of CSU to be 0 160 (s.e. 0.114) assuming a prevalence of 1%². Two loci that were significantly associated with CSU (P<5xl0^-8) (FIG. 1, Table 1, Table 5) were identified. The SNP in the HLA (rs34141382, chr6: 32640701 :C:T) comprises the sequence (where the variant is bold and underlined): gaggcattgtttattattatatat (SEQ ID NO: 1).

Table 1. Sentinel SNPs for two loci significantly associated in this GWAS comparing 679 CSU cases to 4,446 non-CSU controls. Two-sided unadjusted p-values are reported. (CHR=chromosome, BP=GRCh38 coordinates, EA=effect allele, NEA=non-effect allele, OR=odds-ratio, P=p-value, RAF=isk allele frequency)

Table 2. HLA alleles significantly associated with CSU risk. Association statistics for alleles significantly associated with CSU risk are provided. Two-sided unadjusted p-values are provided. (OR=odds-ratio, Revalue)

The most significantly associated region was located in the HLA (rs34141382, ORc- aiieie=l .74, P=4.98xl0'¹³) (FIG. 3). Due to the heterogeneous nature of the controls, the reverse regression method²⁴ was applied to flag variants where diseases represented in the control samples may have contributed to the association signal. Though this method flagged a subset of the controls as contributing to the HLA signal, the association remained highly significant despite removing the flagged controls from the analysis (P=1.75xlO'¹⁰). Furthermore, the MAF of this variant in cases (0.22) was larger than the MAF in the study controls (0. 12) as well as the MAF in an external reference dataset such as gnomAD²⁵ (MAFnoii-Finiush-EUR⁼0. 16).

In other examples, the HLA comprises HLA-DQa, having an arginine residue at position 56. For example, the variable amino acid change at position 56 of HLA-DQa (P=1.69xl0'⁹) is associated with an increased risk of CSU (OR = 1.64), wherein the reference protein sequence comprises the following sequence (position 56 bolded/underlined): EDIVADHVASCGVNLYQFYGPSGQYTHEFDGDEEFYVDLERKETAWRWPEFSKFGG FDPQGALRNMAVAK (SEQ ID NO: 3).

28

SUBSTITUTE SHEET ( RULE 26 ) HLA alleles have previously been reported to be associated with CSU susceptibility¹⁷. To refine the observed HLA associations imputed HLA class I (A, B, C) and class II (DQA1, DQB1, DPB1, DRB1) alleles were inputted using HIBAG²⁶ and tested for association with CSU risk by comparing 679 CSU cases to 3,406 controls (controls flagged by revreg above were excluded from this analysis). After imputation, there were 131 HLA alleles with an allele frequency > 1% (Table 6). Four alleles (DQAl*03:03, DRBl*04:04, DQBl*03:02, and DQAl*03:01) were significantly associated with CSU risk after multiple testing correction (P<3.82xl0‘⁴) (Table 1). These associations replicate previous CSU risk candidate gene associations reported for DRB1*O4 (2-digit resolution) and DQBl*03:02 in a UK population²⁷. Given the extensive linkage disequilibrium between HLA alleles, the four alleles associated with CSU risk above were iteratively conditioned to identify the HLA alleles that were independently associated with CSU risk. Only DRBl*04:04 and DQAl*03:03 remained significantly associated with CSU risk after conditioning on significantly associated HLA alleles (Table 7). For both alleles, the allele frequency (AF) was higher in CSU cases (AFDRBI*04:04=0.11, AFDQAI*03:03 =0.17) as compared to study controls (AFDRBI*04:04=0.06, AFDQAI*03:03=0.09), and external controls from the Allele Frequency Net Database (AFDRBI*04:04=0.05, AFDQA1*03:03=0.07).

The second genome-wide significant signal mapped to an index SNP near ITPKB (rs!097296, P=1.57xl0'⁸, ORc -aiieie=1.44) (FIG. 4). Reverse regression did not flag any controls contributing to this signal, and conditioning on the index SNP, rs 1097296, did not reveal any independent associations with P<lxl0^-6. rs!097296 is cis-eQTL (expression quantitative trait loci) for several genes nearby, where the risk allele (C) is associated with increased expression of ITPKB, reduced expression of PSEN2, and increased expression of COQ8A in GTEx across several tissues (Table 8). To confirm shared causal variants between the CSU risk association and these eQTL associations, co-localization analysis was carried out. The risk association only co-localized with the ITPKB eQTL in GTEx whole-blood tissue (probability=0.997) (Table 9). The SNP near ITPKB (rs!097296, chrl : 226755787:C:T includes an exemplary sequence (variant bold and underlined): tgaaaaagcagcagcaagttatccagaag (SEQ ID NO: 2).

Example 2: CU-index stratified analyses highlight the autoimmune basis of CU-index

Roughly 90% of CSU patients with a CU index greater than or equal to 10 are symptomatic despite use of anti-histamines and leukotriene receptor agonists⁷. To investigate whether genetic differences underlie CU index, the CSU patients were stratified in the study by whether they had a high ( > 10) or low (<10) CU-index⁷ for 642 samples with available data, resulting in 455 CU-index low patients and 187 CU-index high patients. A logistic regression analysis comparing CU-index low to CU-index high patients did not identify any associations meeting genome-wide significance (FIG. 5). Of the loci significantly associated with CSU risk in the full cohort, the SNP near ITPKB (rsl097296) was not associated with CU-index (P=0.64) and had comparable risk allele frequencies between subgroups (RAFcu-indexiow⁼0.67, RAF cu-index iow=0.68), which was higher than the RAF in controls (0.59) and samples with similar ancestry from the external reference population gnomAD²⁵ (0.63) (Table 10). The HLA index SNP was associated with CU-index (P=6.02xl0‘⁶). In contrast to the association signal near ITPKB, the RAF in CU-index high patients was larger than in CU-index low patients (0.30 versus 0.19), though the RAF in both subgroups was larger than the RAF in controls and gnomAD (0.12 and 0.16, respectively), (Table 10). The HLA alleles DRBl*04:04 and DQAl*03:03 were significantly associated with CU-index (P<9.72xl0‘⁵) (Table 11). The allele frequency of both alleles was also higher in CU-index high patients (AFDRBI*04:04=0.16, AFDQAI*03:03 =0.25) as compared to CU-index low (AFDRBI*04:04=0.08, AFDQAI*03:03 =0.14) and external controls (AFDRB 1*04:04=0.05, AFDQAI*03:03=0.07). In summary, while the CSU risk association near ITPKB did not associate with CU-index, there is evidence of an HLA association with CU-index.

Several autoimmune disorders have been shown to be more frequent in CSU patients^{6, 28}. While the HLA is a well-known autoimmune locus²⁹, it was investigated whether pleiotropy between CSU and/or CU-index with autoimmune disorders extended beyond the HLA region. To do this, polygenic risk scores (PRS) was estimated for nine autoimmune disorders (five of which have been identified as having a higher prevalence in CSU patients: hypothyroidism, type-1 diabetes, rheumatoid arthritis, celiac disease, and systemic lupus erythematosus³⁰). PRS were first calculated with PRS-cs³¹for individuals in the study. It was then tested whether they were associated with CSU risk and CU-index (see Online Methods). Three allergic traits were included, and three additional “control” traits (coronary artery disease, idiopathic pulmonary fibrosis, and type-2 diabetes) for which there is little overlapping biology with CSU and for which no association is expected. Altogether 15 PRS were tested for association with CU-index and CSU risk. To minimize confounding from the control samples, the controls were excluded that were flagged earlier by the reverse regression analysis. To investigate the autoimmune pleiotropy beyond the HLA region associations results for PRS calculated without the HLA are presented, though w these results were compared to HLA-inclusive PRS as well.

CSU risk was significantly associated with four autoimmune PRS (hypothyroidism, rheumatoid arthritis, type-1 diabetes and vitiligo) and a composite PRS for allergic disease (hay fever, eczema, and asthma) (P<1.67xl0‘³, correcting for 15 PRS and two phenotypes) (Table 12). These risk association became more significant when the HLA was included into the PRS calculation (Table ). Three of the autoimmune PRS were also significantly associated with CU- index (hypothyroidism, type-1 diabetes and vitiligo) (FIG. 2, Table 12) with greater autoimmune risk associated with the CU-index high group. When including HLA in the PRS calculation, only the hypothyroidism and vitiligo association remained significant (Table 12). Of the three PRS associated with CU-index (hypothyroidism, type-1 diabetes and vitiligo), these autoimmune diseases share associations listed in the GWAS Catalog at two SNPs, rs2111485 and rs2476601, mapping to regions near IFIH1 and PTPN22, respectively. A moderate association of rs2476601 with CU-index (P=0.0025, ORA-aiieie=L72) was observed, but no association of rs2111485 (P=0.48). The association of rs2476601 with CU-index was consistent with the overall PRS association where the allele associated with the CU-index high subgroup was also associated with increased risk of T1D³², vitiligo³³ and hypothyroidism risk³⁴. Neither SNP was associated with CSU risk (P>0.1).

Lastly, considering that chronic inflammatory diseases are often associated with alterations of blood cell composition, and given the crucial role of various immune cells in CSU, it was tested whether CSU risk or CU-index may also share genetics with loci underlying cellcount measures. Employing the same method as above, it was estimated PRS for cell count measures (Methods) using a large-scale blood cell count GWAS³⁵. The eosinophil PRS was significantly associated with CSU risk (P=6.1xl0‘⁴) after multiple testing correction for 12 blood cell count measures and two CSU-related phenotypes, but did not observe associations of blood cell count PRS with CU-index (Table 12).

Discussion

This is the first systematic genome-wide association study of CSU. Two loci significantly associated with CSU risk with the most significant association mapping to the HLA region. This builds upon previously reported smaller candidate gene studies that show nominal significance for association of HLA alleles with CSU risk^{17, 18}. In this study, a genome- wide significant association of the HLA locus with CSU risk was shown, but also two HLA alleles (DRBl*04:04 and DQAl*03:03) that are independently associated with CSU risk were identified.

Furthermore, a novel locus that that is an eQTL for ITPKB, where the risk allele is associated with increased ITPKB expression in the blood was identified. Inositol 1,4,5- trisphosphate kinase B (ITPKB is part of a 3 member family of kinases which phosphorylates inositol (1,4,5) (IP3). In the context of the immune system, IP3 is a secondary messenger involved in the penultimate step of calcium signaling, driving a plethora of intracellular processes. IP3 binds to IP3 receptors (IP3R) in the endoplasmic reticulum (ER) membrane, causes Ca²⁺ release from ER stores resulting in a Ca²⁺ influx across the plasma membrane in a process called store-operated calcium entry (SOCE)³⁶. The function of itpkb has been studied extensively in murine genetic models which has highlighted the role of Itpkb in limiting neutrophil, NK, T cell function, and myelopoiesis³⁷ and inhibition of Itkb has been shown to be efficacious in preclinical models of autoimmune disease³⁸ and potentiates calcium-dependent secretory responses in mast cells³⁹. Pruritogenic mediators such as histamine directly activate receptors on peripheral sensory neurons in a SOCE dependent manner. Taken together, this highlighted a dual role of ITPKB in CSU manifestations via immune and neuronal cell types.

By stratifying the CSU patients by CU-index, shared genetics were identified between three autoimmune disorders and CU-index that extended beyond the HLA locus. In particular, moderate association of the PTPN22 locus with CU-index (index variant rs2476601) was observed, which is shared between hypothyroidism, type-1 diabetes, and vitiligo, with the CU- index high subgroup but no association with overall CSU risk. A recent candidate SNP study found nominal evidence supporting association of another PTPN22 variant (rs!310182) with CSU risk comparing 93 CSU cases to 100 healthy controls from Iran¹⁹ (P=0.007), but was unable to replicate this association in the study despite larger sample sizes (P=0.68). PTPN22 encodes lymphoid specific tyrosine phosphatase which is an inhibitor of T-cell activation and may highlight the greater role of T-cell mediated mast cell activation in CU-index high patients⁴⁰. These results provide a genetic basis for clinical observations that CSU patients are enriched for autoimmune comorbidities and highlight T-cell mediated pathology underlying patient heterogeneity highlighted by CU-index.

One limitation of this study is the lack of a replication cohort. Though there exists a validated ICD-based algorithm to define CSU²⁸- ⁴¹, application of this algorithm to the UK Biobank identified < 100 CSU patients. Assuming similar allele frequencies and odds-ratios as observed in this study <60% power was used to replicate the association near ITPKB at a significance threshold of 0.05. Furthermore, there would be no information on the CU-index status of these participants barring replication of the CU-index stratified findings. Therefore, full replication of the results is not yet possible until additional genetic data is available from other CSU cohorts.

In summary, this study is the first GWAS of a cohort of clinically well characterized CSU patients. Two loci significantly associated with CSU risk were identified. Leveraging the depth of phenotypic characterization of patients in the cohort, it was further examined the genetic underpinnings of CSU patients with or without functional evidence of autoantibodies present in serum (CU-index test) and identified a genetic basis for autoimmune comorbidities underlying CU-index high patients.

Tables

Table 3. CSU case and non-CSU control demographics

Table 4. Sample counts for non-CSU controls grouped by phenotype

SUBSTITUTE SHEET ( RULE 26 )

Attorney Docket No: 048893-571001WO

GNE: P37552-WO

Table 5. Most significantly associated genotyped and imputed variants for the two CSU risk loci

Table 6. HLA allele associations for all alleles with minor allele frequency > 1%

Table 7. Conditional analysis of HLA alleles

Table 8. GTEx eQTLs for rsl097296

Table 9. Co-localization results between locus around rs 1097296 and GTEx eQTLs

Table 10. Index variants from CSU risk loci stratified by CU-index

Table 11. HLA allele associations stratified by CU-index

Table 12. Association of polygenic risk scores with CSU risk and CU-index subgroup

Atorney Docket No: 048893-571001WO

GNE: P37552-WO

Table 13. Association statistics for previously reported CSU candidate gene associations

Table 14. List of input GWAS used to generate polygenic risk scores

Methods

Samples

Chronic spontaneous urticaria (CSU) cases in this genome-wide association study (GWAS) were obtained from CSU clinical trials participants from the following trials: ASTERIA I (ChmcalTrials.gov number: NCT01287117), ASTERIA II (NCTO 1292473), GLACIAL (NCTO 1264939) ³ and SHASTA studies (NCT03693625). Clinical characteristics and general demographics are available in Table 3. Controls were either participants in non-CSU clinical trials (n=4388) or healthy volunteers (n=58) (Table 4). All data used in this study were generated from clinical trial participants who signed informed consent forms approved by the ethics committee or IRB responsible for the country or site where the trials participants donated samples for research. Informed consent included use of these data for genetics research. Participants included in this study have signed informed consent that approves use of these data in genetic studies.

Samples were genotyped with the Infinium Global Screen Array (v2) by Illumina and imputed using BEAGLE (v5.1)⁴ and the 1000 Genomes reference panel (GRCh38 aligned version)⁵. All samples had genotyping rate > 0.98 and only samples with majority European ancestry (fraction > 0.7) as estimated using the supervised mode of ADMIXTURE^{6, 7} were retained for analysis. 17 related samples (Z0 > 0.4) were removed, 19 individuals with excess heterozygosity (> 6 standard deviations from the mean), and 58 samples that were excluded based upon iterative principal component analysis (PCA) outlier removal (default parameters as provided by EIGENSOFT⁸). Variants were excluded with imputation quality < 0.3, Hardy- Weinberg equilibrium p-value < IxlO'⁶, and minor allele frequency < 0.005. After the above quality control 10,077,611 variants were retained across 679 cases and 4,446 non-CSU controls for analysis. Though the full control set was used for the risk analysis, 3,406 controls were retained that were not flagged by the revreg method (see below) for the HLA allele and polygenic risk score analysis.

Statistical methods

Variant-level association analyses were performed in PLINK (vl.90b6.16)^{9, 10}. All association analyses corrected for genetic sex and the first five principal components. Genetic sex was estimated from X-chromosome heterozygosity via PLINK. Principal components were generated with EIGENSOFT (v6.1.4)⁸ on a set of linkage disequilibrium (LD)-pruned variants and regions of long-range LD excluded.

Due to the heterogeneous nature of the controls, revreg, was employed as a method that utilizes reverse regression and a Bayesian spike and slab prior to flag control groups that may contribute to association signals¹¹. Further details are provided in the Supplementary Note.

HLA alleles were imputed with HIBAG using provided reference panels¹². Alleles with a minimum frequency > 0.01 were retained for analysis. Association analysis was carried out in R using the MiDAS package¹³ assuming a dominant inheritance model. Independently associated alleles were determined by iterative conditional analyses on the most significant alleles until no further alleles are associated with Padjusted<0.05. Multiple testing correction was carried out via Bonferroni correction. All analyses corrected for genetic sex and the first five principal components. Reference HLA allele frequencies were obtained from The Allele Frequency Net Database^ using 307 samples from the “USA Caucasian Bethesda population”.

Heritability was estimated using LD-score regression as implemented in the LDSC tool (vl.0.1)¹⁵. The CSU risk GWAS summary statistics were harmonized and subset to variants overlapping HapMap3 variants using the munge sumstats.py function. To calculate h² on the liability scale a CSU prevalence of 1%¹⁶ was assumed. Pre-computed LD-scores were used from 1000 Genomes samples of European ancestry as linked to by the LDSC github (github.com/bulik/ldsc/wiki/LD-Score-Estimation-Tutorial).

Bayesian co-localization analysis was performed using the coloc package¹⁷. For each region, p-values were imputed and minor allele frequencies for variants within 1 megabase of most significantly associated variant and assumed default prior probabilities to estimate colocalization probabilities. Minor allele frequencies were estimated from the samples used in this GWAS. Summary statistics for eQTLs from GTEx (V8)¹⁸ were obtained.

Polygenic risk scores (PRS) were generated for 15 diseases and 12 blood cell type traits using PRS-cs¹⁹ (Table 12). Only variants in each GWAS overlapping variants in the study were used as input to PRS-cs. Furthermore, Two PRS were estimated for each trait/disease -one for which the HLA region was included, and another one in which variants within three megabases of the HLA were excluded. Association with CSU risk and CU-index subgroup were carried out in R using the glm function with the family =binomial parameter. Example 3: Assessing the impact of heterogeneous controls used in the CSU risk GW AS

In this genome-wide association study (GWAS) of chronic spontaneous urticaria (CSU) a set of controls were used that were genotyped on the same array (Illumina Infinium Global Screen Array v2). Though these controls included a small number of healthy volunteers, the majority of the control samples were participants of clinical trials of nine other diseases (Table 4). To investigate whether these disease samples contributed to an association signal, revreg¹ (Tom J et al. Enabling genome-wide association testing with multiple diseases and no healthy controls. Gene 2019; 684: 118-23) was applied to flag control groups and reran the analysis at that association signal after removing the flagged groups. The method is generally described below, but further details are provided in the original study (Tom et al 2019).

For each variant, the key input to the revreg method are the genotypes of samples at the locus and the disease status of all the samples in the original GWAS (diseases present in control samples are listed in Table 4). Here the genotype is the dependent variable and is regressed on an intercept term, 10 binary vectors indicating disease status for each disease present in the controls (nine vectors for diseases present in Table 4 plus an additional vector for the healthy control samples), and six additional covariates (here genetic sex and the first five principle components were included). To determine which diseases contributed to the original association signal, variable selection was carried out via the Bayesian spike and slab prior. The inclusion probability was estimated with MCMC sampling, with probabilities > 0.9 indicating a strong likelihood that a control disease was contributing to the original association signal. revreg was applied to the two genome-wide association signals observed in the study (Table 13) and observed ulcerative colitis (UC) was flagged with a high inclusion probability (0.9978) for the association locus near the HLA (index SNP= rs34141382). The control samples with ulcerative colitis were removed, and the association analysis at this locus was reran. The association signal remained statistically significant (P=1.75xlO'¹⁰).

Table 13: Ulcerative colitis (UC) was flagged with a high inclusion probability (0.9978) for the association locus near the HLA (index SNP= rs34141382)

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OTHER EMBODIMENTS

While the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

The patent and scientific literature referred to herein establishes the knowledge that is available to those with skill in the art. All references, e.g., U.S. patents, U.S. patent application publications, PCT patent applications designating the U.S.., published foreign patents and patent applications cited herein are incorporated herein by reference in their entireties. Genbank and NCBI submissions indicated by accession number cited herein are incorporated herein by reference. All other published references, documents, manuscripts and scientific literature cited herein are incorporated herein by reference. In the case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.

Claims

WHAT IS CLAIMED:

1. A method of identifying a subject as having a genetic predisposition to chronic spontaneous urticaria (CSU), the method comprising:

(a) providing a biological sample comprising a nucleic acid from the subject,

(b) assaying the nucleic acid for the presence of human leukocyte antigen (HLA) alleles comprising one or more of DRBl*04:04 or DQAl*03:03, and

(c) diagnosing the subject at risk of having a genetic predisposition to CSU upon presence of either HLA-DRB 1*04: 04 and/or HLA-DQAl*03:03.

2. A method of identifying a subject having CSU as likely to respond to a therapy, the method comprising:

(a) providing a biological sample comprising a nucleic acid from the subject,

(b) assaying the nucleic acid for the presence of HLA alleles comprising DRBl*04:04 or DQAl*03:03,

(c) identifying the subject as more likely to respond to the therapy, when the sample has the presence of either HLA-DRB 1*04: 04 and/or HLA-DQAl*03:03.

3. The method of claim 1 or 2, further comprising (d) treating the subject with a therapy.

4. A method of identifying a subject as having a susceptibility to CSU, the method comprising: determining that the subject bears an HLA DRBl*04:04 allele, or an HLA DQAl*03:03 allele, classifying the subject as having susceptibility to CSU based upon the presence of the HLA DRBl*04:04 allele and/or the HLA DQAl*03:03 allele.

5. The method of claim 4, further comprising treating the subject with a therapy.

6. A method of monitoring the efficacy of a therapy in a subject having CSU, the method comprising:

(a) assaying a biological sample from the subject for the presence of HLA alleles comprising DRBl*04:04 or DQAl*03:03, and (b) adapting the dosage of the therapy when the sample has the presence of the HLA DRBl*04:04 allele and/or the HLA DQAl*03:03 allele.

7. A method of treating a subject having CSU, the method comprising:

(a) selecting a subject with CSU having a HLA DRBl*04:04 allele and/or a HLA DQAl*03:03 allele

(b) administering a therapy to the subject.

8. The method of any one of claim 7, wherein selecting the subject comprises (i) assaying a biological sample from the subject for the presence of human leukocyte antigen (HLA) alleles comprising DRBl*04:04 or DQAl*03:03; and (ii) identifying the subject as more likely to respond to a therapy when HLA DRB1 *04:04 allele and/or the HLA DQA1 *03:03 allele is detected in the sample.

9. The method of any one of claims 1-6 or 8, wherein the assaying comprises amplifying the HLA DRBl*04:04 allele and/or the HLA DQAl*03:03 allele in the sample and detecting the presence of the HLA DRBl*04:04 allele and/or the HLA DQAl*03:03 allele with an agent that specifically binds to the HLA DRBl*04:04 allele and/or the HLA DQAl*03:03 allele; or wherein the assaying comprises sequencing one or more HLA alleles in the sample, thereby identifying the presence of the HLA DRBl*04:04 allele and/or the HLA DQAl*03:03 allele.

10. The method of any one of claims 1-9, wherein the therapy comprises an antihistamine, a corticosteroid, an immunosuppressant molecule, or an antibody.

11. The method of any one of claims 1-10, wherein the presence of DRBl*04:04 or DQAl*03:03 is further associated with a high chronic urticaria (CU) index, wherein a high CU index is > 10.

12. The method of any one of claims 1-11, wherein the presence of DRBl*04:04 or DQAl*03:03 is further associated with an autoimmune disorder, wherein the autoimmune disorder comprises hypothyroidism, rheumatoid arthritis, type-1 diabetes, or vitiligo.

13. A method of identifying a subject as having a susceptibility to an autoimmune disease, the method comprising:

(a) providing a biological sample comprising a nucleic acid from the subject,

(b) assaying the nucleic acid for the presence and/or expression level of inositol 1,4,5- trisphosphate kinase B (ITPKB), and

(c) diagnosing the subject as having a susceptibility to an autoimmune disease if the sample has the presence of and/or increased expression level of ITPKB.

14. A method of identifying a subject having an autoimmune disease as likely to respond to a therapy, the method comprising:

(a) providing a biological sample comprising a nucleic acid from the subject,

(b) assaying the nucleic acid for the presence and/or expression level of inositol 1,4,5- trisphosphate kinase B (ITPKB), and,

(b) identifying the subject as more likely to respond to the therapy, when the sample has the presence of and/or increased expression level of ITPKB.

15. A method of identifying a subject as having an autoimmune disease, the method comprising: determining that the subject has the presence and/or increased expression level of inositol 1,4,5- trisphosphate kinase B (ITPKB), classifying the subject as having an autoimmune disease based upon the presence of and/or increased expression level of ITPKB.

16. The method of any one of claims 13-15, further comprising administering a therapy to the subject.

17. A method of monitoring the efficacy of a therapy in a subject an autoimmune disease, the method comprising:

(a) assaying a biological sample from the subject for the presence of and/or increased expression level of ITPKB, and

(b) adapting the dosage of the therapy when the sample has the presence of and/or increased expression level of ITPKB.

18. A method of treating a subject having an autoimmune disease, the method comprising:

(a) identifying a subject having an increased expression level of ITPKB, and

(b) administering a therapy to the subject.

19. The method of claim 18, wherein identifying a subject comprises:

(i) identifying a subject for the presence of and/or increased expression level of ITPKB, and

(ii) identifying the subject as more likely to respond to a therapy when the presence of and/or increased expression level of ITPKB is detected in the sample.

20. The method of any one of claims 13-17 or 19, wherein the assaying comprises contacting the sample with an agent that binds ITPKB, thereby forming a complex between the agent and ITPKB, and thereby detecting the presence of and/or level of ITPKB.

21. The method of any one of claims 13-20, wherein the therapy comprises an antihistamine, a corticosteroid, an immunosuppressant molecule, or an antibody.

22. The method of any one of claims 13-21, wherein the therapy comprises an inhibitor of ITPKB expression and/or activity.

23. The method of any one of claims 13-22, wherein the biological sample comprises blood tissue.

24. The method of any one of claims 13-23, wherein the level of ITPKB is increased 1.5-, 1.75-, 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 15-, 20-, 25-, 30-, 40-, 50-, 60-, 70-, 75-, 80-, 90-, or 100-fold compared to a reference level.

25. The method of any one of the above claims, wherein the therapy comprises omalizumab.