RU2815068C2 - Variants of protein related to interleukin-1 receptor and containing single immunoglobulin domain (sigirr), and use thereof - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology, specifically to biomarkers of inflammatory bowel disease, and can be used in medicine to detect inflammatory bowel disease with onset at an early age or to detect the risk of inflammatory bowel disease with onset at an early age. Inflammatory bowel disease (IBD) with onset at an early age is diagnosed when a human protein related to the interleukin-1 receptor and containing a single immunoglobulin domain (SIGIRR) is detected, which contains serine in position corresponding to position 186 in accordance with SEQ ID NO: 9 and is truncated in position corresponding to position 215 in accordance with SEQ ID NO: 9, or nucleic acid coding it.

EFFECT: invention makes it possible to facilitate diagnosis of IBD in children with inflammatory bowel disease with onset at an early age.

80 cl, 3 dwg, 5 ex

Description

Cross reference to related applications

This application claims priority to US Provisional Application No. 62/554857, filed September 6, 2017, which is incorporated herein by reference in its entirety.

Link to Sequence List

This application includes a Sequence Listing filed electronically as a text file entitled 18923800602SEQ, created on August 30, 2018, with a size of 56 kilobytes. The sequence listing is incorporated herein by reference.

Technical field of the invention

This disclosure relates generally to the field of genetics. In particular, this disclosure relates to gene changes and polypeptide variants of the Single Immunoglobulin Interleukin-1 Receptor Related (SIGIRR) protein that are associated, for example, with inflammatory bowel disease with onset at an early age (IBD-RV).

BACKGROUND OF THE INVENTION

Various references, including patents, patent applications, accession numbers, technical articles and scientific articles, are cited throughout the text of this specification. Each reference is incorporated herein by reference in its entirety and for all purposes.

Inflammatory bowel disease (IBD) is a genetically heterogeneous, chronic inflammatory disorder caused by an inappropriate immune response to commensal microbiota in the gastrointestinal tract, with a median age of onset of 30 years. Severe monogenic forms of IBD can occur with childhood onset (<18 years) and have been associated with rare, highly penetrant variants in about 50 “Mendelian” genes. However, the genetic architecture of early-onset inflammatory bowel disease (IBD-ER) is poorly understood and most patients remain genetically undiagnosed.

This disclosure provides variants of SIGIRR that will help understand the biology of SIGIRR and facilitate diagnosis and treatment of children with early-onset inflammatory bowel disease.

Brief description of the invention

This disclosure provides novel nucleic acid molecules (i.e., genomic DNA, mRNA, and cDNA) encoding variant SIGIRR polypeptides and variant SIGIRR polypeptides that have been demonstrated herein to be associated with inflammatory bowel disease, such as inflammatory bowel disease with onset at an early age.

In particular, this disclosure provides isolated nucleic acid molecules containing a nucleic acid sequence encoding a human SIGIRR protein, wherein the protein is truncated at position corresponding to position 215 in accordance with SEQ ID NO:9, or a complementary strand of the nucleic acid sequence.

This disclosure also provides genomic DNA molecules containing a nucleic acid sequence encoding at least a portion of the human SIGIRR protein, wherein the protein is truncated at position corresponding to position 215 in accordance with SEQ ID NO:9, or a complementary strand of the nucleic acid sequence.

This disclosure also provides cDNA molecules containing a nucleic acid sequence encoding at least a portion of the human SIGIRR protein, wherein the protein is truncated at position corresponding to position 215 in accordance with SEQ ID NO:9, or a complementary strand of the nucleic acid sequence.

This disclosure also provides mRNA molecules comprising a nucleic acid sequence encoding at least a portion of the human SIGIRR protein, wherein the protein is truncated at position corresponding to position 215 in accordance with SEQ ID NO:9, or a complementary strand of the nucleic acid sequence.

This disclosure also provides vectors containing any of the isolated nucleic acid molecules disclosed herein.

This disclosure also provides compositions comprising any of the isolated nucleic acid molecules or vectors disclosed herein and a carrier.

This disclosure also provides host cells containing any of the isolated nucleic acid molecules or vectors disclosed herein.

This disclosure also provides isolated recombinant polypeptides containing at least a portion of the human SIGIRR protein, wherein the protein is truncated at position corresponding to position 215 in accordance with SEQ ID NO:9.

This disclosure also provides compositions comprising any of the isolated or recombinant polypeptides disclosed herein and a carrier.

This disclosure also provides a probe or primer comprising a nucleic acid sequence of at least about 5 nucleotides that hybridizes to a nucleic acid sequence encoding the human SIGIRR protein, wherein the protein is truncated at a position corresponding to position 215 in accordance with SEQ ID NO :9, or which hybridizes to the complementary strand of a nucleic acid sequence encoding the human SIGIRR protein, wherein the protein is truncated at the position corresponding to position 215 in accordance with SEQ ID NO:9.

This disclosure also provides supports containing a substrate to which any of the probes disclosed herein can hybridize.

This disclosure also provides a change-specific probe or primer comprising a nucleic acid sequence that is complementary to a nucleic acid sequence encoding a SIGIRR protein truncated at position corresponding to position 215 in accordance with SEQ ID NO:9, wherein specific in regarding the change, the probe or primer contains a nucleic acid sequence that is complementary to a portion of the nucleic acid molecule encoding any of a number of positions corresponding to positions 186-209 or 211-215 in accordance with SEQ ID NO:9. In some embodiments, the change-specific probe or primer specifically hybridizes to a portion of the nucleic acid molecule encoding the position corresponding to position 186 of SEQ ID NO:9, or a complementary strand thereof. The change-specific probe or primer does not hybridize to a nucleic acid molecule having a nucleic acid sequence encoding a wild-type SIGIRR protein.

This disclosure also provides methods for identifying a human subject having inflammatory bowel disease or early-onset inflammatory bowel disease, or a risk of developing inflammatory bowel disease or early-onset inflammatory bowel disease, the method comprising detecting in a sample obtained from subject, presence or absence of truncated SIGIRR protein; and/or a nucleic acid molecule encoding a truncated SIGIRR protein; wherein the presence of a truncated SIGIRR protein and/or a nucleic acid molecule encoding a truncated SIGIRR protein indicates that the subject has inflammatory bowel disease or early-onset inflammatory bowel disease, or is at risk of developing inflammatory bowel disease or early-onset inflammatory bowel disease at an early age.

This disclosure also provides methods for identifying a human subject having inflammatory bowel disease or early-onset inflammatory bowel disease, or a risk of developing inflammatory bowel disease or early-onset inflammatory bowel disease, the method comprising detecting in a sample obtained from the subject, presence or absence of: SIGIRR protein truncated at position corresponding to position 215 in accordance with SEQ ID NO:9; and/or a nucleic acid molecule encoding a SIGIRR protein truncated at the position corresponding to position 215 in accordance with SEQ ID NO:9; wherein the presence of a truncated SIGIRR protein and/or a nucleic acid molecule encoding a truncated SIGIRR protein indicates that the subject has inflammatory bowel disease or early-onset inflammatory bowel disease, or is at risk of developing inflammatory bowel disease or early-onset inflammatory bowel disease at an early age.

This disclosure also provides methods for diagnosing inflammatory bowel disease or early-onset inflammatory bowel disease or identifying the risk of developing inflammatory bowel disease or early-onset inflammatory bowel disease in a human subject, comprising: detecting a change in a nucleic acid molecule encoding a SIGIRR protein obtained from a human subject, wherein the change encodes a truncated SIGIRR protein; and diagnosing a human subject with early-onset inflammatory bowel disease if the subject has one or more symptoms of inflammatory bowel disease or early-onset inflammatory bowel disease, or diagnosing the human subject as being at risk of developing inflammatory bowel disease or an inflammatory disease early-onset bowel disease, unless the subject has one or more symptoms of inflammatory bowel disease or early-onset inflammatory bowel disease.

This disclosure also provides methods for diagnosing inflammatory bowel disease or early-onset inflammatory bowel disease or identifying the risk of developing inflammatory bowel disease or early-onset inflammatory bowel disease in a human subject, comprising: detecting a change in a nucleic acid molecule encoding a SIGIRR protein obtained from a human subject, wherein the change encodes a SIGIRR protein truncated at position corresponding to position 215 in accordance with SEQ ID NO:9; and diagnosing a human subject with early-onset inflammatory bowel disease if the subject has one or more symptoms of inflammatory bowel disease or early-onset inflammatory bowel disease, or diagnosing the human subject as being at risk for developing inflammatory bowel disease or an inflammatory disease early-onset bowel disease, unless the subject has one or more symptoms of inflammatory bowel disease or early-onset inflammatory bowel disease.

Brief description of graphic materials

The accompanying figures, which are included in and constitute a part of this specification, illustrate several aspects and, together with the description, serve to explain the principles of the present invention.

In FIG. Figure 1, panels A, B, and C, illustrates the truncated variant in SIGIRR with dominant segregation in a family with Crohn's disease.

In FIG. Figure 2 illustrates the results from a panel of pro-inflammatory cytokines from a mesoscale primary study conducted on cell culture supernatants taken from LCL generated from cells from healthy donors, a patient with PF-SIGIRR, and 4 patients with IBD-RV without PF-SIGIRR, without or with stimulation treatment with 2 mg/ml LPS for 72 hours.

In FIG. Figure 3 illustrates the results from a panel of pro-inflammatory cytokines from a mesoscale primary study conducted on cell culture supernatants taken from LCL generated from cells from healthy donors, a patient with PF-SIGIRR, and 4 patients with IBD-RV without PF-SIGIRR, without or with stimulation treatment with 2 mg/ml anti-IgM/anti-CD40 for 16 hours.

Additional advantages of this disclosure will be set forth in part in the description that follows, and in part will be apparent from the description or may be learned by practice of the embodiments disclosed herein. The advantages of this disclosure will be realized and achieved by the elements and combinations expressly set forth in the appended claims. It should be understood that both the foregoing general description and the following detailed description are illustrative and explanatory only and are not limiting of the claimed embodiments.

Description

Various terms are used throughout the specification and claims to relate to aspects of the disclosure. Such terms should be assigned their ordinary meaning in the art unless otherwise noted. Other expressly defined terms should be construed in a manner consistent with the definition provided herein.

Unless expressly stated otherwise, it is not in any way intended that any method or aspect described herein should be construed as requiring its steps to be carried out in a particular order. Accordingly, unless a method-related claim or description expressly states that the steps are to be limited to a particular order, it is in no way intended that the order should be disturbed in any respect. This is true for any possible unstated basis for interpretation, including questions of logic regarding the order of steps or flowchart, obvious meaning implied by grammatical construction or punctuation, or the number or type of aspects described in the invention.

As used herein, singular forms include references to the plural unless the context clearly indicates otherwise.

In the context of this document, the terms “subject” and “patient” are used interchangeably. The subject may include any animal, including mammals. Mammals include, but are not limited to, farm animals ( eg , horses, cows, pigs), domestic animals ( eg , dogs, cats), laboratory animals ( eg , mice, rats, rabbits) and non-human primates. In some embodiments, the subject is a human.

As used herein, a “nucleic acid,” “nucleic acid molecule,” “nucleic acid sequence,” “polynucleotide,” or “oligonucleotide” may contain a polymeric form of nucleotides of any length, may contain DNA and/or RNA, and may be single-stranded, double-stranded, or multi-chain. One strand of a nucleic acid is also called its complementary strand.

As used herein, the expression "corresponds" or its grammatical variations when used in the context of numbering a given amino acid or nucleotide sequence or position, refers to the numbering of a particular reference sequence when the given amino acid or nucleotide sequence is compared with a reference sequence ( e.g. , in this document, reference sequence is a nucleic acid molecule or polypeptide (wild type or full length) SIGIRR). In other words, the residue number ( eg , amino acid or nucleotide) or residue position ( eg , amino acid or nucleotide) of a given polymer is designated relative to a reference sequence, and not according to the actual numerical position of the residue in a given amino acid or nucleotide sequence. For example, a given amino acid sequence can be aligned with a reference sequence by introducing gaps to optimize residue matches between the two sequences. In such cases, although gaps are present, the numbering of the residue in a given amino acid or nucleotide sequence is relative to the reference sequence to which it was aligned.

For example, the expression “SIGIRR protein truncated at position 215 in SEQ ID NO:9” (and similar expressions) means that if the amino acid sequence of the SIGIRR protein is aligned with the sequence of SEQ ID NO:9, the SIGIRR protein is truncated at the position , which corresponds to position 215 in SEQ ID NO:9 (eg, the terminal amino acid of the SIGIRR protein is the amino acid at position 215). Or, in other words, these expressions refer to a SIGIRR protein that is truncated at a position that is homologous to position 215 in SEQ ID NO:9. This protein is also referred to herein as the “truncated SIGIRR protein” or “variant p.K186fs*31.”

The SIGIRR protein truncated at position 215 in SEQ ID NO:9 can be readily identified by performing a sequence alignment between the specified SIGIRR protein and the amino acid sequence of SEQ ID NO:9. Likewise, a SIGIRR protein having a serine at a position corresponding to position 186 in SEQ ID NO:9 can be readily identified by performing a sequence alignment between a given SIGIRR protein and the amino acid sequence of SEQ ID NO:9. There are a number of computational algorithms that can be used to perform sequence alignments to identify a truncation at a position that corresponds to position 215 in SEQ ID NO:9, or to identify a serine at a position that corresponds to position 186 in SEQ ID NO:9. For example, using the NCBI BLAST algorithm (Altschul et al., 1997, Nucleic Acids Res., 25, 3389-3402) or CLUSTALW software (Sievers et al., 2014, Methods Mol. Biol., 1079, 105-116), sequence alignment can be performed. In this case, sequences can also be aligned manually.

According to the disclosure, certain variations in SIGIRR have been observed to be associated with the risk of developing early-onset inflammatory bowel disease. In general, the function of this protein is not fully understood, particularly in children under 18 years of age. None of the variants in the SIGIRR gene or protein are thought to have known associations with early-onset inflammatory bowel disease in humans. Additionally, none of the variants in the SIGIRR gene or protein are thought to have known associations with early-onset inflammatory bowel disease, particularly in children. Consistent with this disclosure, a rare variant in the SIGIRR gene was identified that cosegregates with an early-onset inflammatory bowel disease phenotype in affected members of the same family. For example, a genetic change that results in a deletion of adenine at position 557 of the human SIGIRR mRNA or cDNA (e.g., wild-type SEQ ID NO:3 and SEQ ID NO:5, respectively) has been observed to result in a frameshift yielding the SIGIRR protein, truncated at position corresponding to position 215 in accordance with SEQ ID NO:9 (eg, the terminal amino acid is located at position 215), indicates that a person having such a change may develop inflammatory bowel disease with early onset. Overall, the genetic analyzes described herein suggested that the SIGIRR gene, and in particular truncating or loss-of-function variants in the SIGIRR gene, are associated with increased susceptibility to the development of early-onset inflammatory bowel disease. Therefore, human subjects who have SIGIRR changes that are associated with early-onset inflammatory bowel disease can be treated to inhibit early-onset inflammatory bowel disease, reduce its symptoms, and/or suppress the development of symptoms. Accordingly, this disclosure provides isolated or recombinant variant SIGIRR genes, including cDNA and mRNA, as well as isolated or recombinant variant SIGIRR polypeptides. Additionally, this disclosure provides methods for leveraging the identification of such variants in subjects to identify or stratify the risk of such subjects developing inflammatory bowel disease or early-onset inflammatory bowel disease or for diagnosing subjects as having inflammatory bowel disease or early-onset inflammatory bowel disease. at an early age so that treatment can be given to subjects at risk or subjects with active disease.

The amino acid sequences of the two wild-type SIGIRR proteins are shown in SEQ ID NO:7 and SEQ ID NO:8. The wild type SIGIRR protein having SEQ ID NO:7 is 410 amino acids long, while the wild type SIGIRR protein having SEQ ID NO:8 is 504 amino acids long. In the case of both SEQ ID NO:7 and SEQ ID NO:8, positions 186-215 of the wild-type proteins contain the following amino acids in this order: Lys-Pro-Gln-Leu-Glu-Arg-Arg-Arg-Gly-Tyr -Lys-Leu-Phe-Leu-Asp-Asp-Arg-Asp-Leu-Leu-Pro-Arg-Ala-Glu-Pro-Ser-Ala-Asp-Leu-Leu (SEQ ID NO:10).

This disclosure provides nucleic acid molecules encoding variant SIGIRR proteins that are associated with inflammatory bowel disease or early-onset inflammatory bowel disease. In some embodiments, the nucleic acid molecules encode a truncated variant protein of SIGIRR. For example, this disclosure provides isolated nucleic acid molecules containing a nucleic acid sequence encoding a human SIGIRR protein, wherein the protein is truncated at position corresponding to position 215 in accordance with SEQ ID NO:9, or the complementary strand of the nucleic acid sequence.

In some embodiments, the isolated nucleic acid molecule contains or consists of a nucleic acid sequence that encodes a truncated SIGIRR protein containing a serine at the position corresponding to position 186 in accordance with SEQ ID NO:9.

In some embodiments, the isolated nucleic acid molecule contains or consists of a nucleic acid sequence that encodes a truncated SIGIRR protein comprising the amino acid sequence of SEQ ID NO:11 at positions corresponding to positions 186-215 of SEQ ID NO:9.

In some embodiments, the nucleic acid molecule contains or consists of a nucleic acid sequence that encodes a human SIGIRR protein having an amino acid sequence that is at least about 90%, at least about 91%, at least about 92%, at least at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO:9, or the complementary strand of the nucleic acid sequence. As used herein, when reference is made to percentage sequence identity, higher percentage sequence identity values are preferred to lower values.

In some embodiments, the isolated nucleic acid molecule contains or consists of a nucleic acid sequence that encodes a truncated SIGIRR protein, wherein the truncated SIGIRR protein comprises an amino acid sequence of SEQ ID NO:9 or an amino acid sequence that has at least 90% sequence identity with SEQ ID NO:9, and contains serine at the position corresponding to position 186 in accordance with SEQ ID NO:9.

The nucleic acid sequence of the wild type SIGIRR genomic DNA is shown in SEQ ID NO:1. The wild type SIGIRR genomic DNA containing SEQ ID NO:1 is 11,739 nucleotides in length. In the case of SEQ ID NO:1, position 9962 of wild type SIGIRR genomic DNA is represented by adenine.

This disclosure provides genomic DNA molecules encoding a variant SIGIRR protein. In some embodiments, the genomic DNA molecules encode a truncated SIGIRR protein. In some embodiments, the genomic DNA contains or consists of a nucleic acid sequence encoding a SIGIRR protein truncated at the position corresponding to position 215 in accordance with SEQ ID NO:9. In some embodiments, the genomic DNA contains or consists of a nucleic acid sequence encoding a SIGIRR protein truncated at a position corresponding to position 215 in accordance with SEQ ID NO:9, and containing a serine at a position corresponding to position 186 in accordance with SEQ ID NO: 9. In some embodiments, the genomic DNA contains or consists of a nucleic acid sequence encoding a SIGIRR protein truncated at a position corresponding to position 215 in accordance with SEQ ID NO:9, and containing the following amino acid sequence at positions corresponding to positions 186-215 in accordance with SEQ ID NO:9. Ser-Arg-Ser-Trp-Ser-Gly-Val-Gly-Ala-Thr-Ser-Ser-Ser-Trp-Thr-Thr-Ala-Thr-Ser-Cys-Arg-Ala-Leu-Ser-Pro- Pro-Pro-Thr-Ser-Trp (SEQ ID NO:11). In some embodiments, the genomic DNA contains or consists of a nucleic acid sequence that encodes a variant SIGIRR protein having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO:9. In some embodiments, the genomic DNA contains or consists of a nucleic acid sequence encoding a SIGIRR protein having SEQ ID NO:9. In some embodiments, the genomic DNA contains or consists of a nucleic acid sequence that encodes a truncated SIGIRR protein, wherein the truncated SIGIRR protein comprises the amino acid sequence of SEQ ID NO:9 or an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 9, and contains serine at the position corresponding to position 186 in accordance with SEQ ID NO:9.

In some embodiments, the genomic DNA contains or consists of a nucleic acid sequence containing guanine at the position corresponding to position 9962 in accordance with SEQ ID NO:2. In contrast, wild-type SIGIRR genomic DNA contains an adenine at the position corresponding to position 9962 according to SEQ ID NO:1. The change in the genomic DNA of the variant SIGIRR is due to the deletion of this adenine, which results in a frame shift of one nucleotide, thereby resulting in the appearance of a guanine at the position corresponding to position 9962 in SEQ ID NO:2. In some embodiments, the genomic DNA contains or consists of a nucleic acid sequence that is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94% , at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO:2. In some embodiments, the genomic DNA contains or consists of a nucleic acid sequence according to SEQ ID NO:2.

In some embodiments, the isolated nucleic acid molecules contain less than the complete genomic DNA sequence. In some embodiments, the isolated nucleic acid molecules contain or consist of at least about 15, at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, at least at least about 45, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about 100, at least about 200, at least about 300, at least about 400, at least about 500, at least about 600, at least about 700, at least about 800, at least about 900, at least about 1000, at least about 2000, at least about 3000, at least about 4000, at least about 5000, at least about 6000, at least about 7000, at least about 8000, at least about 9000, at least about 10000, at least at least about 11,000 or at least about 11,500 contiguous nucleotides of SEQ ID NO:2. In some embodiments, the isolated nucleic acid molecules contain or consist of from at least about 1000 to at least about 2000 contiguous nucleotides of SEQ ID NO:2.

In some embodiments, the isolated nucleic acid molecules contain or consist of at least about 15, at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, at least at least about 45, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about 100, at least about 200, at least about 300, at least about 400, at least about 500, at least about 600, at least about 700, at least about 800, at least about 900, at least about 1000, at least about 1000, at least about 1100, at least about 1200, at least about 1300, at least about 1400, at least about 1500, at least about 1600, at least about 1700, at least about 1800, at least at least about 1900, at least about 2000, at least about 2100, at least about 2200, at least about 2300, at least about 2400, or at least about 2500 contiguous nucleotides of SEQ ID NO:2. In some embodiments, such contiguous nucleotides can be combined with other nucleic acid molecules from contiguous nucleotides to produce the cDNA molecules described herein.

Such isolated nucleic acid molecules can be used, for example, to express variant SIGIRR mRNA and proteins or as exogenous donor sequences. It should be understood that gene sequences within a population may vary due to polymorphisms such as SNPs. The examples given herein represent only exemplary sequences, but other sequences are also possible.

In some embodiments, the isolated nucleic acid molecules comprise a variant SIGIRR minigene in which one or more nonessential coding segments of SEQ ID NO:9 have been deleted relative to the corresponding wild-type SIGIRR genomic DNA. In some embodiments, the deleted non-essential segments contain one or more intronic sequences. In some embodiments, the SIGIRR minigene has at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to a portion of SEQ ID NO:2, wherein the minigene comprises a nucleic acid sequence having a guanine at position , corresponding to position 9962 in accordance with SEQ ID NO:2.

The nucleic acid sequence of wild type SIGIRR mRNA is shown in SEQ ID NO:3. The wild type SIGIRR mRNA containing SEQ ID NO:3 is 1230 nucleotides in length. In the case of SEQ ID NO:3, position 557 of the wild type SIGIRR mRNA is represented by adenine.

This disclosure also provides mRNA molecules encoding a variant SIGIRR protein. In some embodiments, the mRNA molecules encode a truncated SIGIRR protein. In some embodiments, the mRNA contains or consists of a nucleic acid sequence encoding a SIGIRR protein truncated at the position corresponding to position 215 in accordance with SEQ ID NO:9. In some embodiments, the mRNA contains or consists of a nucleic acid sequence encoding a SIGIRR protein truncated at a position corresponding to position 215 in accordance with SEQ ID NO:9, and containing a serine at a position corresponding to position 186 in accordance with SEQ ID NO:9 . In some embodiments, the mRNA contains or consists of a nucleic acid sequence encoding a SIGIRR protein truncated at a position corresponding to position 215 in accordance with SEQ ID NO:9, and containing the following amino acid sequence at positions corresponding to positions 186-215 in accordance with SEQ ID NO:9. Ser-Arg-Ser-Trp-Ser-Gly-Val-Gly-Ala-Thr-Ser-Ser-Ser-Trp-Thr-Thr-Ala-Thr-Ser-Cys-Arg-Ala-Leu-Ser-Pro- Pro-Pro-Thr-Ser-Trp (SEQ ID NO:11). In some embodiments, the mRNA contains or consists of a nucleic acid sequence that encodes a SIGIRR variant protein having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO:9. In some embodiments, the mRNA contains or consists of a nucleic acid sequence encoding a SIGIRR protein having SEQ ID NO:9. In some embodiments, the mRNA contains or consists of a nucleic acid sequence that encodes a truncated SIGIRR protein, wherein the truncated SIGIRR protein comprises an amino acid sequence of SEQ ID NO:9 or an amino acid sequence that has at least 90% sequence identity to SEQ ID NO:9 , and contains a serine at the position corresponding to position 186 in accordance with SEQ ID NO:9.

In some embodiments, the mRNA contains or consists of a nucleic acid sequence containing guanine at the position corresponding to position 557 in accordance with SEQ ID NO:4. In contrast, wild-type SIGIRR mRNA contains an adenine at the position corresponding to position 557 according to SEQ ID NO:3. The change in the variant SIGIRR mRNA is due to the deletion of this adenine, which results in a frame shift of one nucleotide, thereby resulting in the appearance of a guanine at a position corresponding to position 557 in SEQ ID NO:4. In some embodiments, the mRNA contains or consists of a nucleic acid sequence containing codons CUA and AGC at positions corresponding to positions 553-555 and 556-558, respectively, in accordance with SEQ ID NO:4. In contrast, wild-type SIGIRR mRNA contains codons CUA and AAG at positions corresponding to positions 553-555 and 556-558, respectively, according to SEQ ID NO:3. In some embodiments, the mRNA contains or consists of a nucleic acid sequence that is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO:4. In some embodiments, the mRNA contains or consists of a nucleic acid sequence according to SEQ ID NO:4.

In some embodiments, the isolated nucleic acid molecule contains fewer nucleotides than the complete SIGIRR mRNA sequence. In some embodiments, the isolated nucleic acid molecules contain or consist of at least about 5, at least about 8, at least about 10, at least about 12, at least about 15, at least about 20, at least at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about 100, at least about 200, at least about 300, at least about 400, at least about 500, or at least about 600 contiguous nucleotides SEQ ID NO: 4. In some embodiments, the isolated nucleic acid molecules contain or consist of at least about 200 to at least about 500 contiguous nucleotides of SEQ ID NO:4. In this regard, longer mRNA molecules are preferable to shorter ones. In some embodiments, the isolated nucleic acid molecules contain or consist of at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about 100, at least at least about 200, at least about 300, at least about 400, or at least about 500 contiguous nucleotides of SEQ ID NO:4. In this regard, longer mRNA molecules are preferable to shorter ones. In some embodiments, such mRNA molecules contain a codon that encodes a serine at a position that corresponds to position 186 in accordance with SEQ ID NO:9. In some embodiments, such mRNA molecules contain guanine at a position corresponding to position 557 in accordance with SEQ ID NO:4. In some embodiments, such mRNA molecules contain codons CUA and AGC at positions corresponding to positions 553-555 and 556-558, respectively, in accordance with SEQ ID NO:4.

The nucleic acid sequence of the wild type SIGIRR cDNA is shown in SEQ ID NO:5. The wild type SIGIRR cDNA containing SEQ ID NO:5 is 1233 nucleotides in length, including the stop codon. In the case of SEQ ID NO:5, position 557 of the wild type SIGIRR cDNA is adenine.

This disclosure also provides cDNA molecules encoding a variant SIGIRR protein. In some embodiments, the cDNA molecules encode a truncated SIGIRR protein. In some embodiments, the cDNA contains or consists of a nucleic acid sequence encoding a SIGIRR protein truncated at the position corresponding to position 215 in accordance with SEQ ID NO:9. In some embodiments, the cDNA contains or consists of a nucleic acid sequence encoding a SIGIRR protein truncated at a position corresponding to position 215 in accordance with SEQ ID NO:9, and containing a serine at a position corresponding to position 186 in accordance with SEQ ID NO:9 . In some embodiments, the cDNA contains or consists of a nucleic acid sequence encoding a SIGIRR protein truncated at the position corresponding to position 215 in accordance with SEQ ID NO:9, and containing the following amino acid sequence at positions corresponding to positions 186-215 in accordance with SEQ ID NO:9. Ser-Arg-Ser-Trp-Ser-Gly-Val-Gly-Ala-Thr-Ser-Ser-Ser-Trp-Thr-Thr-Ala-Thr-Ser-Cys-Arg-Ala-Leu-Ser-Pro- Pro-Pro-Thr-Ser-Trp (SEQ ID NO:11). In some embodiments, the cDNA contains or consists of a nucleic acid sequence that encodes a variant SIGIRR protein having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO:9. In some embodiments, the cDNA contains or consists of a nucleic acid sequence encoding a SIGIRR protein having SEQ ID NO:9. In some embodiments, the cDNA contains or consists of a nucleic acid sequence that encodes a truncated SIGIRR protein, wherein the truncated SIGIRR protein comprises the amino acid sequence of SEQ ID NO:9 or an amino acid sequence that has at least 90% sequence identity to SEQ ID NO:9 , and contains a serine at the position corresponding to position 186 in accordance with SEQ ID NO:9.

In some embodiments, the cDNA contains or consists of a nucleic acid sequence containing guanine at the position corresponding to position 557 in accordance with SEQ ID NO:6. In contrast, the wild type SIGIRR cDNA contains an adenine at the position corresponding to position 557 according to SEQ ID NO:5. The change in the cDNA of the variant SIGIRR is due to the deletion of this adenine, which results in a frame shift of one nucleotide, thereby resulting in the appearance of a guanine at a position corresponding to position 557 in SEQ ID NO:6. In some embodiments, the cDNA contains or consists of a nucleic acid sequence containing codons CTA and AGC at positions corresponding to positions 553-555 and 556-558, respectively, in accordance with SEQ ID NO:6. In contrast, the wild-type SIGIRR cDNA contains codons CTA and AAG at positions corresponding to positions 553-555 and 556-558, respectively, according to SEQ ID NO:5. In some embodiments, the cDNA contains or consists of a nucleic acid sequence that is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO:6. In some embodiments, the cDNA contains or consists of a nucleic acid sequence according to SEQ ID NO:6.

In some embodiments, the cDNA molecules contain less than the entire sequence of the SIGIRR variant cDNA molecule. In some embodiments, the cDNA molecules contain or consist of at least about 5, at least about 8, at least about 10, at least about 12, at least about 15, at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about 100, at least about 200, at least about 300, at least about 400, at least about 500, or at least about 600 contiguous nucleotides of SEQ ID NO:6. In some embodiments, the cDNA molecules contain or consist of at least about 200 to at least about 500 contiguous nucleotides of SEQ ID NO:6. In this regard, longer cDNA molecules are preferable to shorter ones. In some embodiments, the cDNA molecules contain or consist of at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about 100, at least about 200, at least about 300, at least about 400, or at least about 500 contiguous nucleotides of SEQ ID NO:6. In this regard, longer cDNA molecules are preferable to shorter ones. In some embodiments, such cDNA molecules contain a codon that encodes a serine at a position that corresponds to position 186 in accordance with SEQ ID NO:9. In some embodiments, such cDNA molecules contain guanine at the position corresponding to position 557 in accordance with SEQ ID NO:6. In some embodiments, such cDNA molecules contain CTA and AGC codons at positions corresponding to positions 553-555 and 556-558, respectively, in accordance with SEQ ID NO:4.

This disclosure also provides isolated nucleic acid molecules that hybridize to variant SIGIRR genomic DNA (such as SEQ ID NO:2), variant SIGIRR minigenes, variant SIGIRR mRNA (such as SEQ ID NO:4) and/or variant SIGIRR cDNA ( such as SEQ ID NO:6). In some embodiments, such isolated nucleic acid molecules contain or consist of at least about 5, at least about 8, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 21, at least about 22, at least about 23, at least about 24, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, at least about 50 , at least about 55, at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, at least about 90, according to at least about 95, at least about 100, at least about 200, at least about 300, at least about 400, at least about 500, at least about 600, at least about 700, at least about 800, at least about 900, at least about 1000, at least about 2000, at least about 3000, at least about 4000, at least about 5000, at least about 6000, at least about 7000 , at least about 8000, at least about 9000, at least about 10000, at least about 11000, or at least about 11500 nucleotides. In some embodiments, the isolated nucleic acid molecule contains or consists of at least 15 nucleotides. In some embodiments, the isolated nucleic acid molecule contains or consists of from at least 15 nucleotides to at least about 35 nucleotides. In some embodiments, such isolated nucleic acid molecules hybridize to variant SIGIRR genomic DNA (such as SEQ ID NO:2), variant SIGIRR minigenes, variant SIGIRR mRNA (such as SEQ ID NO:4), and/or variant SIGIRR cDNA (such as SEQ ID NO:6) under harsh conditions. Such nucleic acid molecules can be used, for example, as probes, as primers, or as variation-specific probes or primers, as described and illustrated herein.

In some embodiments, the isolated nucleic acid molecules hybridize to at least about 15 contiguous nucleotides of the nucleic acid molecule that are at least about 70%, at least about 75%, at least about 80%, at least about about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99 % or 100% identical to variant SIGIRR genomic DNA (such as SEQ ID NO:2), variant SIGIRR minigenes, variant SIGIRR mRNA (such as SEQ ID NO:4) and/or variant SIGIRR cDNA (such as SEQ ID NO:6) . In some embodiments, the isolated nucleic acid molecules contain or consist of from about 15 to about 100 nucleotides, or from about 15 to about 35 nucleotides. In some embodiments, the isolated nucleic acid molecules contain or consist of from about 15 to about 100 nucleotides. In some embodiments, the isolated nucleic acid molecules contain or consist of from about 15 to about 35 nucleotides

In some embodiments, any of the nucleic acid molecules, genomic DNA molecules, cDNA molecules, or mRNA molecules disclosed herein may be purified, for example , are at least about 90% pure. In some embodiments, any of the nucleic acid molecules, genomic DNA molecules, cDNA molecules, or mRNA molecules disclosed herein may be purified, e.g. , are at least about 95% pure. In some embodiments, any of the nucleic acid molecules, genomic DNA molecules, cDNA molecules, or mRNA molecules disclosed herein may be purified, e.g. , are at least about 99% pure. Cleaning is performed by humans using human-designed cleaning techniques.

This disclosure also provides fragments of any of the isolated nucleic acid molecules, genomic DNA molecules, cDNA molecules, or mRNA molecules disclosed herein. In some embodiments, the fragments contain or consist of at least about 5, at least about 8, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14 , at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 21, at least about 22, according to at least about 23, at least about 24, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, at least about 55, at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, at least about 90, at least about 95 or at least about 100 contiguous residues of any of the nucleic acid sequences disclosed herein or any complementary strand thereof. In this regard, longer fragments are preferable to shorter ones. In some embodiments, the fragments contain or consist of at least about 5, at least about 8, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14 , at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 21, at least about 22, according to at least about 23, at least about 24, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45 or at least about 50 contiguous residues. In this regard, longer fragments are preferable to shorter ones. In some embodiments, the fragments contain or consist of at least about 20, at least about 25, at least about 30, or at least about 35 contiguous residues. In some embodiments, the fragments contain or consist of at least about 20 contiguous residues. In some embodiments, the fragments contain or consist of at least about 25 contiguous residues. In some embodiments, the fragments contain or consist of at least about 30 contiguous residues. In some embodiments, the fragments contain or consist of at least about 35 contiguous residues. The fragments are intended to contain or consist of a portion of a nucleic acid molecule that encodes a serine at a position corresponding to position 186 in accordance with SEQ ID NO:9, or encodes a position corresponding to positions 186-215 in accordance with SEQ ID NO:9. Such fragments can be used, for example, as probes, as primers, or as allele-specific primers, as described and illustrated herein.

This disclosure also provides probes and primers. A probe or primer according to this disclosure has a nucleic acid sequence that specifically hybridizes to any of the nucleic acid molecules disclosed herein or a complementary strand thereof. In some embodiments, the probe or primer specifically hybridizes to any of the nucleic acid molecules disclosed herein under stringent conditions. This disclosure also provides nucleic acid molecules having nucleic acid sequences that hybridize under moderate conditions with any of the nucleic acid molecules disclosed herein or a complementary strand thereof. The probe or primer according to the disclosure preferably includes a nucleic acid codon that encodes a serine at the position corresponding to position 186 in accordance with SEQ ID NO:9, or a complementary strand thereof. Thus, in a preferred embodiment, this disclosure provides variation-specific primers, which are defined in more detail above and below.

A probe in accordance with this disclosure can be used to detect a variant SIGIRR nucleic acid molecule (eg, genomic DNA, mRNA and/or cDNA) encoding a variant SIGIRR protein ( eg , in accordance with SEQ ID NO:9). In addition, a primer in accordance with this disclosure can be used to amplify a nucleic acid molecule encoding a variant SIGIRR protein, or a fragment thereof. This disclosure also provides a primer pair comprising one of the primers described above. For genomic polymerase chain reaction (PCR) amplification of a SIGIRR fragment containing a truncating frameshift variant, suitable primer sequences include, but are not limited to: forward primer (5' to 3'): TCAGTGGCTCTGAACTGCAC (SEQ ID NO: 12) and reverse primer (5' to 3'): GGTCCTGTTGAGCAGAGGAG (SEQ ID NO:13).

The nucleic acid molecules disclosed herein may contain a naturally occurring genomic DNA, cDNA, or SIGIRR mRNA transcript nucleic acid sequence, or may contain a non-naturally occurring sequence. In some embodiments, a naturally occurring sequence may differ from a non-naturally occurring sequence due to synonymous mutations or mutations that do not affect the encoded SIGIRR polypeptide. For example, the sequence may be identical except for synonymous mutations or mutations that do not affect the encoded SIGIRR polypeptide. A synonymous mutation or substitution is the replacement of one nucleotide with another in an exon of a protein-coding gene so that the resulting amino acid sequence remains unmodified. This is possible due to the degeneracy of the genetic code, when some amino acids are encoded by more than one codon of three base pairs. Synonymous substitutions are used, for example, in the process of codon optimization. The nucleic acid molecules disclosed herein may be codon optimized.

Also provided herein are functional polynucleotides that can interact with the disclosed nucleic acid molecules. Functional polynucleotides are nucleic acid molecules that have a specific function, such as binding a target molecule or catalyzing a specific reaction. Examples of functional polynucleotides include, but are not limited to, antisense molecules, aptamers, ribozymes, triple helix forming molecules and external guide sequences. Functional polynucleotides can act as effectors, inhibitors, modulators and stimulators of a particular activity possessed by the target molecule, or functional polynucleotides can have de novo activity independent of any other molecules.

Antisense molecules are designed to interact with a target nucleic acid molecule through canonical or non-canonical base pairing. The interaction of the antisense molecule and the target molecule is aimed at promoting the destruction of the target molecule through, for example, RNase-H-mediated degradation of the RNA-DNA hybrid. Alternatively, the antisense molecule is designed to interrupt a processing function that would normally occur with the target molecule, such as transcription or replication. Antisense molecules can be designed based on the sequence of the target molecule. There are numerous ways to optimize the effectiveness of antisense molecules by identifying the most accessible regions of the target molecule. Typical methods include, but are not limited to, in vitro selection experiments and DNA modification studies using DMS and DEPC. In general, antisense molecules bind the target molecule with a dissociation constant ( _kd ) of less than or equal to about 10 ^-6 , less than or equal to about 10 ^-8 , less than or equal to about 10 ^-10 , or less than or equal to about 10 ^-12 . Typical examples of methods and techniques that assist in the development and use of antisense molecules can be found in the following non-limiting list of US patents: 5135917; 5294533; 5627158; 5641754; 5691317; 5780607; 5786138; 5849903; 5856103; 5919772; 5955590; 5990088; 5994320; 5998602; 6005095; 6007995; 6013522; 6017898; 6018042; 6025198; 6033910; 6040296; 6046004; 6046319; and 6,057,437. Examples of antisense molecules include, but are not limited to, antisense RNAs, small interfering RNAs (siRNAs), and short hairpin RNAs (shRNAs).

The isolated nucleic acid molecules disclosed herein may include RNA, DNA, or both RNA and DNA. The isolated nucleic acid molecules can also be linked or fused to a heterologous nucleic acid sequence, such as a vector, or a heterologous tag. For example, the isolated nucleic acid molecules disclosed herein may be contained in a vector or exogenous donor sequence containing the isolated nucleic acid molecule and a heterologous nucleic acid sequence. The isolated nucleic acid molecules can also be associated or fused with a heterologous label, such as a fluorescent tag. Other examples of labels are disclosed elsewhere in this document.

The label may be suitable for direct detection ( eg a fluorophore) or indirect detection ( eg a hapten, enzyme or fluorophore quencher). Such labels can be detected by spectroscopic, photochemical, biochemical, immunochemical or chemical methods. Such tags include, for example, radioactive tags, which can be measured using radiation measuring devices; pigments, dyes or other chromogens that can be observed visually or measured using a spectrophotometer; spin labels, which can be measured using a spin label analyzer; and fluorescent tags ( eg fluorophores), in which the output signal is generated by excitation of a suitable molecular adduct and which can be visualized by excitation with light that is absorbed by the dye, or can be measured using standard fluorometers or imaging systems. The label may also be, for example, a chemiluminescent substance, in which case the output signal is generated by chemical modification of the signal compound; metal-containing substance; or an enzyme, in which case enzyme-dependent secondary signal generation occurs, such as the formation of a colored product from a colorless substrate. The term “tag” may also be referred to as a “tag” or hapten, which can selectively bind to a conjugated molecule so as to utilize the conjugated molecule when added sequentially along with a substrate to generate a detectable signal. For example, biotin can be used as a tag, and then use an avidin or streptavidin conjugate of horseradish peroxidase (HRP) to bind the tag, and then use a calorimetric substrate ( e.g. , tetramethylbenzidine (TMB)) or a fluorogenic substrate to detect the presence of HRP. Typical tags that can be used as tags to facilitate purification include, but are not limited to, myc, HA, FLAG or 3XFLAG, 6XHis or polyhistidine, glutathione S-transferase (GST), maltose binding protein, epitope tag or Fc moiety immunoglobulin. Numerous labels are known and include, for example, particles, fluorophores, haptens, enzymes and their calorimetric, fluorogenic and chemiluminescent substrates, as well as other labels.

The disclosed nucleic acid molecules may contain, for example, nucleotides or non-natural or modified nucleotides, such as nucleotide analogues or nucleotide substituents. Such nucleotides include a nucleotide that contains a modified base, sugar or phosphate group, or whose structure includes a non-natural compound. Examples of non-natural nucleotides include, but are not limited to, dideoxynucleotides, biotinylated, aminated, deaminated, alkylated, benzylated, or fluorophore-labeled nucleotides.

The nucleic acid molecules disclosed herein may also contain one or more nucleotide analogues or one or more substitutions. A nucleotide analog is a nucleotide that contains a modification in any of the base, sugar, or phosphate moieties. Modifications in the main fragment include, but are not limited to, natural and synthetic modifications A, C, G and T/U, as well as various purine or pyrimidine bases, such as, for example, pseudouridine, uracil-5-yl, hypoxanthine-9- yl (I) and 2-aminoadenin-9-yl. Modified bases include, but are not limited to, 5-methylcytosine.

(5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halogenuracil and cytosine,

5-propynyluracil and cytosine, 6-azouracil, cytosine and thymine, 5-uracil (pseudouracil),

4-thiouracil, 8-halogen, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halogen, in particular 5-bromo, 5-trifluoromethyl and other 5- substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine,

7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Certain nucleotide analogues, such as, for example, 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including, but not limited to, 2-aminopropyladenine,

5-propynyluracil, 5-propynylcytosine and 5-methylcytosine can increase the stability of double helix formation. Often base modifications can be combined, for example with a sugar modification such as 2'-O-methoxyethyl, to provide unique properties such as increased double helix stability.

Nucleotide analogues may also include modifications in the sugar moiety. Modifications to the sugar moiety include, but are not limited to, natural modifications of ribose and deoxyribose, as well as synthetic modifications. Sugar modifications include, but are not limited to, the following modifications at the 2' position: OH; F; O-, S- or N-alkyl; O-, S- or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein alkyl, alkenyl, and alkynyl may be substituted or unsubstituted C _1-10 alkyl or C _2-10 alkenyl and C _2-10 alkynyl. Typical 2' sugar modifications also include, but are not limited to, -O[(CH ₂ ) _n O] _m CH ₃ , -O(CH ₂ ) _n OCH ₃ , -O(CH ₂ ) _n NH ₂ , -O( CH ₂ ) _n CH ₃ , -O(CH ₂ ) _n -ONH ₂ and -O(CH ₂ ) _n ON[(CH ₂ ) _n CH ₃ )] ₂ , where n and m are from 1 to about 10.

Other modifications at the 2' position include, but are not limited to, C _1-10 alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH ₃ , OCN, Cl, Br, CN, CF ₃ , OCF ₃ , SOCH ₃ , SO ₂ CH ₃ , ONO ₂ , NO ₂ , N ₃ , NH ₂ , heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleaving group, reporter group, intercalator, group to improve pharmacokinetic properties oligonucleotide or a group to improve the pharmacodynamic properties of the oligonucleotide, and other substituents having similar properties. Similar modifications can be made at other positions in the sugar, in particular at the 3' position of the sugar in the 3' terminal nucleotide or in the 2'-5' linked oligonucleotides and at the 5' position of the 5' terminal nucleotide. Modified sugars also include those that contain modifications in the connecting oxygen of the ring, such as CH ₂ and S. Nucleotide sugar analogues may also contain sugar mimetics, such as cyclobutyl compounds, in place of the pentofuranosyl sugar.

Nucleotide analogues can also be modified in the phosphate moiety. Modified phosphate moieties include, but are not limited to, those that can be modified so that the linkage between two nucleotides contains a thiophosphate, chiral thiophosphate, dithiophosphate, phosphotriester, aminoalkyl phosphotriester, methyl and other alkyl phosphonates, including 3'-alkylene phosphonate and chiral phosphonates, phosphinates, phosphoramidates, including 3'-aminophosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphorotriesters and boranophosphates. This phosphate or modified phosphate bond between two nucleotides may be due to a 3'-5' bond or a 2'-5' bond and may involve inverted polarity such as from 3'-5' to 5'-3' or from 2'- 5' to 5'-2'. Also included are various forms of salts, mixed salts and free acids.

Nucleotide substitutions include molecules that have similar functional properties to nucleotides, but which do not contain a phosphate moiety, such as peptide nucleic acid (PNA). Nucleotide surrogates include molecules that recognize nucleic acids in a Watson-Crick or Hoogsten manner, but which are linked to each other through a moiety other than a phosphate moiety. Nucleotide substitutes are capable of adopting a double helix structure when interacting with the corresponding target nucleic acid.

Nucleotide substitutes also include nucleotides and nucleotide analogues with substituted phosphate moieties or sugar moieties. In some embodiments, the nucleotide replacements may not contain a standard phosphorus atom. The phosphate substitutes may be, for example, short-chain alkyl or cycloalkyl internucleoside linkages, complex heteroatom-alkyl or cycloalkyl internucleoside linkages, or one or more short-chain heteroatom or heterocyclic internucleoside linkages. They include components having morpholino bonds (derived in part from the sugar moiety of the nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone skeletons; formacetyl and thioformacetyl backbones; methylene-formacetyl and thioformacetyl backbones; alkene-containing backbones; sulfamate backbones; methylene imino and methylene hydrazine backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N-, O-, S- and CH ₂ components.

It should also be understood that in a nucleotide substitute, both the sugar and phosphate components of the nucleotide can be replaced, for example, by an amide-type (aminoethylglycine) (PNA) bond.

It is also possible to link other types of molecules (conjugates) with nucleotides or nucleotide analogues to enhance, for example, cellular uptake. Conjugates can be chemically linked to nucleotides or nucleotide analogues. Such conjugates include, for example, lipid moieties such as a cholesterol moiety, cholic acid, a thioester such as hexyl-S-tritylthiol, thiocholesterol, an aliphatic chain such as dodecanediol or undecyl units, a phospholipid such as dihexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate, polyamine or polyethylene glycol chain, adamantane acetic acid, palmityl moiety or octadecylamine moiety or hexylaminocarbonyloxycholesterol.

This disclosure also provides vectors containing any one or more of the nucleic acid molecules disclosed herein. In some embodiments, the vectors contain one or more of the nucleic acid molecules disclosed herein and a heterologous nucleic acid. Vectors may be viral or non-viral vectors capable of carrying a nucleic acid molecule. In some embodiments, the vector is a plasmid or cosmid ( eg , circular double-stranded DNA into which additional DNA segments can be ligated). In some embodiments, the vector is a viral vector in which additional DNA segments can be ligated into the viral genome. In some embodiments, the vector can autonomously replicate in the host cell into which it is introduced ( eg , bacterial vectors having a bacterial origin of replication and mammalian episomal vectors). In some embodiments, the vector ( eg , mammalian non-episomal vectors) may be integrated into the genome of a host cell upon entry into the host cell and thereby replicate along with the host genome. In addition, specific vectors can control the expression of genes to which they are functionally linked. Such vectors are referred to herein as “recombinant expression vectors” or “expression vectors”. Such vectors may also be targeting vectors (ie, exogenous donor sequences).

In some embodiments, proteins encoded by various genetic variants disclosed herein are expressed by inserting nucleic acid molecules encoding the disclosed genetic variants into expression vectors such that the genes are operably linked to expression control sequences, such as transcriptional and translational control sequences. Expression vectors include, but are not limited to, plasmids, cosmids, retroviruses, adenoviruses, adeno-associated viruses (AAV), plant viruses such as cauliflower mosaic virus and tobacco mosaic virus, yeast artificial chromosomes (YAC), Epstein-Barr virus episomes ( EBV) and other expression vectors known in the art. In some embodiments, nucleic acid molecules containing the disclosed genetic variants can be ligated into a vector such that the transcriptional and translational control sequences in the vectors perform their intended function of regulating the transcription and translation of the genetic variant. The expression vector and expression control sequences are selected to be compatible with the host cell used for expression. Nucleic acid sequences containing the disclosed genetic variants can be inserted into separate vectors or into a single expression vector as variant genetic information. The nucleic acid sequence containing the disclosed genetic variants can be inserted into an expression vector by standard methods ( for example , ligation of complementary restriction sites in the nucleic acid containing the disclosed genetic variants and the vector, or blunt-end ligation in the case of no restriction sites).

In addition to the nucleic acid sequence containing the disclosed genetic variants, recombinant expression vectors may carry regulatory sequences that direct expression of the genetic variant in a host cell. The design of the expression vector, including the choice of regulatory sequences, may depend on factors such as the choice of host cell to be transformed, the desired level of protein expression, etc. The necessary regulatory sequences for expression in mammalian host cells may include, for example, viral elements that mediate high levels of protein expression in mammalian cells, such as promoters and/or enhancers derived from retroviral LTRs, cytomegalovirus (CMV) (such as the CMV promoter/enhancer), simian virus 40 (SV40) (such as the SV40 promoter/enhancer ), adenovirus, ( eg adenovirus major late promoter (AdMLP)), polyomas, and strong mammalian promoters such as native immunoglobulin and actin promoters. Methods for expressing polypeptides in bacterial cells or fungal cells ( eg yeast cells) are also well known.

The promoter may be, for example, a constitutively active promoter, a conditional promoter, an inducible promoter, a temporally restricted promoter ( eg , a developmentally regulated promoter), or a spatially restricted promoter ( eg , a cell-specific or tissue-specific promoter). Examples of promoters can be found, for example, in WO 2013/176772.

Examples of inducible promoters include, for example, chemically regulated promoters and physically regulated promoters. Chemically regulated promoters include, for example, alcohol-regulated promoters ( e.g. , alcohol dehydrogenase (alcA) gene promoter), tetracycline-regulated promoters ( e.g. , tetracycline-responsive promoter, tetracycline operator sequence (tetO), tet-On promoter, or tet-Off promoter) , steroid-regulated promoters ( eg , rat glucocorticoid receptor, estrogen receptor promoter, or ecdysone receptor promoter) or metal-regulated promoters ( eg , metalloprotein promoter). Physically regulated promoters include, for example, temperature-regulated promoters ( eg , heat shock promoter) and light-regulated promoters ( eg , light-inducible promoter or light-repressible promoter).

Tissue-specific promoters may be, for example, neuron-specific promoters, glia-specific promoters, muscle cell-specific promoters, cardiac cell-specific promoters, kidney cell-specific promoters, bone cell-specific promoters, endothelial cell-specific promoters, or immune cell-specific promoters ( e.g. , B-cell promoter or T cell promoter).

Developmentally regulated promoters include, for example, promoters that are active only during the embryonic stage of development or only in the adult cell.

In addition to the nucleic acid sequence containing disclosed genetic variants and regulatory sequences, recombinant expression vectors may carry additional sequences, such as sequences that regulate the replication of the vector in host cells ( eg , origins of replication) and selectable marker genes. The selectable marker gene may facilitate selection of host cells into which the vector has been introduced ( see, for example , US Pat. Nos. 4,399,216; 4,634,665; and 5,179,017). For example, a selectable marker gene may confer resistance to drugs, such as G418, hygromycin, or methotrexate, to the host cell into which the vector has been introduced. Exemplary selectable marker genes include, but are not limited to, the dihydrofolate reductase (DHFR) gene (for use in dhfr host cells with methotrexate selection/amplification), the neo gene (for G418 selection), and the glutamate synthetase (GS) gene.

Additional vectors are described, for example, in US Provisional Application No. 62/367973, filed July 28, 2016, which is incorporated herein by reference in its entirety.

This disclosure also provides compositions containing any one or more of the isolated nucleic acid molecules, genomic DNA molecules, cDNA molecules, or mRNA molecules disclosed herein. In some embodiments, the composition is a pharmaceutical composition.

This disclosure also provides variant SIGIRR polypeptides. In some embodiments, the SIGIRR variant polypeptide is truncated. In some embodiments, the SIGIRR variant polypeptide is truncated at a position corresponding to position 215 in accordance with SEQ ID NO:9. In some embodiments, the SIGIRR variant polypeptide is truncated at position corresponding to position 215 in accordance with SEQ ID NO:9, and contains a serine at position corresponding to position 186 in accordance with SEQ ID NO:9. In some embodiments, the SIGIRR variant polypeptide is truncated at position corresponding to position 215 in accordance with SEQ ID NO:9, and contains a number of amino acids at positions corresponding to positions 186-215 in accordance with SEQ ID NO:9. In some embodiments, the SIGIRR variant polypeptide is truncated at position corresponding to position 215 in accordance with SEQ ID NO:9, and contains the following amino acid sequence at positions corresponding to positions 186-215 in accordance with SEQ ID NO:9. Ser-Arg-Ser-Trp-Ser-Gly-Val-Gly-Ala-Thr-Ser-Ser-Ser-Trp-Thr-Thr-Ala-Thr-Ser-Cys-Arg-Ala-Leu-Ser-Pro- Pro-Pro-Thr-Ser-Trp (SEQ ID NO:11). In some embodiments, the variant SIGIRR polypeptide has at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence according to SEQ ID NO:9. In some embodiments, the SIGIRR variant polypeptide contains or consists of an amino acid sequence according to SEQ ID NO:9. In some embodiments, the truncated SIGIRR protein contains or consists of an amino acid sequence of SEQ ID NO:9 or an amino acid sequence that has at least 90% sequence identity with SEQ ID NO:9, and contains a serine at a position corresponding to position 186 in accordance with SEQ ID NO:9.

This disclosure also provides fragments of any of the polypeptides disclosed herein. In some embodiments, the fragments comprise at least about 10, at least about 15, at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, at least at least about 45, at least about 50, at least about 55, at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, at least about 90, at least about 95, at least about 100, at least about 150, or at least about 200 contiguous amino acid residues of the encoded polypeptide (such as polypeptides having the amino acid sequence of SEQ ID NO:9) . In this regard, longer fragments are preferable to shorter ones. In some embodiments, the fragments comprise at least about 10, at least about 15, at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, at least at least about 45, at least about 50, at least about 55, at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, at least about 90, at least about 95, or at least about 100 contiguous amino acid residues of the encoded polypeptide. In this regard, longer fragments are preferable to shorter ones.

This disclosure also provides dimers comprising an isolated polypeptide comprising a variant SIGIRR polypeptide, the polypeptide being selected from any of the polypeptides disclosed herein.

In some embodiments, isolated polypeptides disclosed herein are linked or fused to heterologous polypeptides or heterologous molecules or tags, numerous examples of which are disclosed elsewhere herein. For example, proteins can be fused to a heterologous polypeptide to provide increased or decreased stability. The fusion domain of a heterologous polypeptide may be located at the N-terminus, C-terminus, or within the polypeptide. The fusion partner may, for example, help provide T helper epitopes (immunological fusion partner) or may promote expression of the protein (expression enhancer) at a higher yield than the native recombinant polypeptide. Certain fusion partners are both immunological and expression enhancing fusion partners. Other fusion partners may be selected to increase the solubility of the polypeptide or to facilitate targeting of the polypeptide to unnecessary intracellular compartments. Some fusion partners include affinity tags that facilitate purification of the polypeptide.

In some embodiments, the fusion protein is directly fused to a heterologous molecule or fused to a heterologous molecule through a linker, such as a peptide linker. Suitable peptide linker sequences can be selected, for example, based on the following factors: 1) ability to adopt a flexible elongated conformation; 2) resistance to the formation of a secondary structure that could interact with functional epitopes on the first and second polypeptides; and 3) the absence of hydrophobic or charged residues that can react with functional epitopes of the polypeptide. For example, peptide linker sequences may contain Gly, Asn and Ser residues. Other neutral amino acids such as Thr and Ala can also be used in the linker sequence. Amino acid sequences that can be used as linkers include, for example, those disclosed in Maratea et al. , Gene , 1985, 40, 39-46; Murphy et al. , Proc. Natl. Acad. Sci. USA , 1986, 83, 8258-8262; and US Patents 4,935,233 and 4,751,180. The length of the linker sequence can generally be, for example, from 1 to about 50 amino acids. Linker sequences are generally not needed when the first and second polypeptides have regions of non-essential N-terminal amino acids that can be used to separate functional domains and avoid steric interference.

In some embodiments, the polypeptides are operably linked to a cell entry domain. For example, the cell entry domain may be derived from the HIV-1 TAT protein, the TLM cell entry motif from human hepatitis B virus, MPG, Pep-1, VP22, the cell entry peptide from herpes simplex virus, or a polyarginine peptide sequence. See for example WO 2014/089290. The cell entry domain can be located at the N-terminus, C-terminus, or anywhere within the protein.

In some embodiments, the polypeptides are operably linked to a heterologous polypeptide to facilitate tracking or purification, such as a fluorescent protein, purification tag, or epitope tag. Examples of fluorescent proteins include, but are not limited to, green fluorescent proteins ( e.g. GFP, GFP-2, tagGFP, turboGFP, eGFP, Emerald, Azami Green, Monomeric Azami Green, CopGFP, AceGFP, ZsGreenl), yellow fluorescent proteins ( e.g. YFP, eYFP, Citrine, Venus, YPet, PhiYFP, ZsYellowl), blue fluorescent proteins ( e.g. eBFP, eBFP2, Azurite, mKalamal, GFPuv, Sapphire, T-sapphire), blue fluorescent proteins ( e.g. eCFP, Cerulean, CyPet, AmCyanl, Midoriishi-Cyan), red fluorescent proteins ( for example , mKate, mKate2, mPlum, DsRed monomer, mCherry, mRFP1, DsRed-Express, DsRed2, DsRed-Monomer, HcRed-Tandem, HcRedl, AsRed2, eqFP611, mRaspberry, mStrawberry, Jred), orange fluorescent proteins ( eg mOrange, mKO, Kusabira-Orange, Monomeric Kusabira-Orange, mTangerine, tdTomato) and any other suitable fluorescent protein. Examples of tags include, but are not limited to, glutathione S-transferase (GST), chitin binding protein (CBP), maltose binding protein, thioredoxin (TRX), poly(NANP), tandem affinity purification (TAP) tag, myc, AcV5 , AU1, AU5, E, ECS, E2, FLAG, hemagglutinin (HA), nus, Softag 1, Softag 3, Strep, SBP, Glu-Glu, HSV, KT3, S, S1, T7, V5, VSV-G, histidine (His), biotin and carboxyl carrier protein (BCCP) and calmodulin. In some embodiments, the heterologous molecule is an immunoglobulin Fc domain, a peptide tag, a transduction domain, a poly(ethylene glycol), a polysialic acid, or a glycolic acid.

In some embodiments, the isolated polypeptides contain unnatural or modified amino acids or peptide analogs. For example, there are numerous D-amino acids or amino acids that have a different functional substituent from naturally occurring amino acids. Opposite stereoisomers of naturally occurring peptides, as well as stereoisomers of peptide analogues, are disclosed. These amino acids can easily be incorporated into polypeptide chains by charging tRNA molecules with the amino acid of choice and designing genetic constructs that use, for example, amber codons to insert the amino acid analogue into the peptide chain in a site-specific manner.

In some embodiments, the isolated polypeptides are peptide mimetics, which can be designed to resemble peptides but which are not linked through a natural peptide bond. For example, bonds for amino acids and amino acid analogues include, but are not limited to, -CH ₂ NH-, -CH ₂ S-, -CH ₂ -, -CH=CH- (cis and trans), -COCH ₂ -, -CH (OH)CH ₂ - and -CHH ₂ SO-. Peptide analogues may have more than one atom between bonding atoms, such as beta-alanine, gamma-aminobutyric acid, and the like. Amino acid analogues and peptide analogues often have enhanced or desirable properties, such as more economical production, greater chemical stability, enhanced pharmacokinetic properties (half-life, absorption, potency, potency, etc.), altered specificity ( e.g. broad-spectrum biological activity ), reduced antigenicity and other necessary properties.

In some embodiments, the isolated polypeptides contain D-amino acids, which can be used to create more stable peptides since D-amino acids are not recognized by peptidases. Systematic replacement of one or more amino acids of the consensus sequence with a D-amino acid of the same type ( e.g. D-lysine instead of L-lysine) can be used to create more stable peptides. Cysteine residues can be used to cyclize or link two or more peptides together. This may be useful when restricting peptides to specific conformations ( see, for example , Rizo and Gierasch, Ann. Rev. Biochem ., 1992, 61, 387).

This disclosure also provides nucleic acid molecules encoding any of the polypeptides disclosed herein. This includes all degenerate sequences associated with a particular polypeptide sequence (all nucleic acids having a sequence that encodes one particular polypeptide sequence, as well as nucleic acids, including degenerate nucleic acids encoding disclosed variants and derivatives of protein sequences). Thus, although a specific nucleic acid sequence may not be set forth herein, in fact each sequence is disclosed and described herein by means of the disclosed polypeptide sequences.

The percentage identity (or percentage complementarity) between specific regions of nucleotide sequences in nucleic acids or amino acid sequences in polypeptides can be routinely determined using the BLAST (basic local alignment search tools) and PowerBLAST programs (Altschul et al. , J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656) or using the Gap program (Wisconsin Sequence Analysis Package, version 8 for Unix, Genetics Computer Group , University Research Park, Madison Wis.) with default settings, which uses the Smith and Waterman algorithm (Adv. Appl. Math., 1981, 2, 482-489). As used herein, when reference is made to percent sequence identity, higher percent sequence identity values are preferred to lower values.

This disclosure also provides compositions comprising any one or more of the nucleic acid molecules and/or any one or more of the polypeptides disclosed herein, and a carrier and/or excipient. In some embodiments, the carrier increases the stability of the nucleic acid molecule and/or polypeptide ( e.g. , prolongs the period under specified storage conditions ( e.g. , -20°C, 4°C, or ambient temperature) during which degradation products remain below a threshold value , for example, below 0.5% by weight of the original nucleic acid or protein; or increases stability in vivo ). Examples of carriers include, but are not limited to, poly(lactic acid) (PLA) microspheres, poly(D,L-lactic-co-glycolic acid) (PLGA) microspheres, liposomes, micelles, inverse micelles, lipid cochelates, and lipid microtubules. The carrier may contain a buffered saline solution such as PBS, SBS, etc.

This disclosure also provides methods for producing any of the polypeptides or fragments thereof disclosed herein. Such polypeptides or fragments thereof can be prepared by any suitable method. For example, polypeptides or fragments thereof can be obtained from host cells containing nucleic acid molecules ( eg , recombinant expression vectors) encoding such polypeptides or fragments thereof. Such methods may involve culturing a host cell containing a nucleic acid molecule ( eg , a recombinant expression vector) encoding a polypeptide or fragment thereof under conditions sufficient to produce the polypeptide or fragment thereof, thereby producing a polypeptide or fragment thereof. The nucleic acid can be operably linked to a promoter active in the host cell, and culture can be carried out under conditions in which the nucleic acid is expressed. Such methods may further include isolating the expressed polypeptide or fragment thereof. Isolation may further include purification of the polypeptide or fragment thereof.

Examples of suitable systems for protein expression include host cells, such as, for example, bacterial cell-based expression systems ( eg , Escherichia coli , Lactococcus lactis ), yeast cell-based expression systems ( eg , Saccharomyces cerevisiae , Pichia pastoris ), expression systems insect cell-based ( e.g. for baculovirus-mediated protein expression) and mammalian cell-based expression systems.

Examples of nucleic acid molecules encoding polypeptides or fragments thereof are disclosed in more detail elsewhere herein. In some embodiments, the nucleic acid molecules are codon optimized for expression in a host cell. In some embodiments, the nucleic acid molecules are operably linked to a promoter active in a host cell. The promoter may be a heterologous promoter (For example, a promoter that is not a naturally occurring promoter). Examples of promoters suitable forEscherichia coliinclude, but are not limited to, arabinose promoters,lac,tac, and T7. Examples of promoters suitable forLactococcus lactis, include, but are not limited to, the P170 and nisin promoters. Examples of promoters suitable forSaccharomyces cerevisiae, include, but are not limited to, constitutive promoters such as alcohol dehydrogenase (ADHI) or enolase (ENO) promoters, or inducible promoters such as PHO, CUP1, GAL1 and G10. Examples of promoters suitable forPichia pastoris, include, but are not limited to, the alcohol oxidase I (AOX I) promoter, the glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter, and the glutathione-dependent formaldehyde dehydrogenase (FLDI) promoter. An example of a promoter suitable for a baculovirus-mediated system is the late viral polyhedrin strong promoter.

In some embodiments, the nucleic acid molecules encode a tag in frame with a polypeptide or fragment thereof to facilitate protein purification. Examples of tags are disclosed elsewhere in this document. Such tags may, for example, bind to a partner ligand ( eg , immobilized on a resin) so that the tagged protein can be separated from all other proteins ( eg , host cell proteins). Affinity chromatography, high performance liquid chromatography (HPLC), and size exclusion chromatography (SEC) are examples of techniques that can be used to improve the purity of the expressed protein.

Other methods may also be used to obtain polypeptides or fragments thereof. For example, two or more peptides or polypeptides can be linked together using protein chemistry techniques. For example, peptides or polypeptides can be chemically synthesized using Fmoc (9-fluorenylmethyloxycarbonyl) or Boc ( tert -butyloxycarbonyl) chemistry. Such peptides or polypeptides can be synthesized using standard chemical reactions. For example, a peptide or polypeptide can be synthesized and not separated from the synthesis resin, while another fragment of the peptide or protein can be synthesized and subsequently separated from the resin, thereby exposing an end group that is operably blocked on the other fragment. Using peptide condensation reactions, these two fragments can be covalently joined by a peptide bond at the carboxyl and amino terminus, respectively. Alternatively, the peptide or polypeptide can be independently synthesized in vivo as described herein. Once isolated, these independent peptides or polypeptides can be linked to form a peptide or fragment thereof through similar peptide condensation reactions.

In some embodiments, enzymatic ligation of cloned or synthetic peptide segments allows relatively short peptide fragments to be joined to produce larger peptide fragments, polypeptides, or entire protein domains (Abrahmsen et al. , Biochemistry , 1991, 30, 4151). Alternatively, native chemical ligation of synthetic peptides can be used to synthetically construct large peptides or polypeptides from shorter peptide fragments. This method may consist of a two-step chemical reaction (Dawson et al. , Science , 1994, 266, 776-779). The first step may be a chemoselective reaction of an unprotected synthetic thioester peptide with another unprotected peptide segment containing an amino-terminal Cys residue to produce a thioester-linked intermediate as the starting covalent product. Without changing the reaction conditions, this intermediate can undergo a spontaneous, rapid intramolecular reaction to form a native peptide bond at the ligation site.

In some embodiments, the exposed peptide segments may be chemically linked, wherein the bond formed between the peptide segments by chemical ligation is a non-peptide bond (Schnolzer et al. , Science , 1992, 256, 221).

In some embodiments, polypeptides may have post-expression modifications, such as, for example, glycosylation, acetylation, and phosphorylation, as well as other modifications known in the art, both naturally occurring and non-naturally occurring. The polypeptide may be a whole protein or a subsequence thereof.

This disclosure also provides methods for producing any of the polypeptides disclosed herein, comprising culturing a host cell containing recombinant expression vectors containing nucleic acid molecules containing a polynucleotide capable of encoding one or more of the polypeptides disclosed herein, or a complementary strand thereof, producing , thus a polypeptide.

This disclosure also provides cells ( eg , recombinant host cells) containing any one or more nucleic acid molecules, including vectors containing nucleic acid molecules, and/or any one or more polypeptides disclosed herein. Cells can be in vitro , ex vivo or in vivo . Nucleic acid molecules can be linked to a promoter and other regulatory sequences so that they are expressed to produce the encoded protein. additional lines of such cells have been proposed.

In some embodiments, the cell is a totipotent cell or a pluripotent cell ( eg , an embryonic stem (ES) cell such as a rodent ES cell, a mouse ES cell, or a rat ES cell). Totipotent cells include undifferentiated cells that can develop into any cell type, and pluripotent cells include undifferentiated cells that have the ability to develop into more than one differentiated cell type. Such pluripotent and/or totipotent cells may be, for example, ES cells or ES-like cells, such as induced pluripotent stem (iPS) cells. ES cells include embryonic totipotent or pluripotent cells that are capable of contributing to any tissue of the developing embryo once introduced into the embryo. ES cells can be derived from the inner cell mass of the blastocyst and are capable of differentiating into cells of any of the three vertebrate germ layers (endoderm, ectoderm and mesoderm). In accordance with this disclosure, the embryonic stem cells may be non-human embryonic stem cells.

In some embodiments, the cell is a primary somatic cell or a cell that is not a primary somatic cell. Somatic cells can include any cell that is not a gamete, germ cell, gametocyte, or undifferentiated stem cell. In some embodiments, the cell may also be a primary cell. Primary cells include cells or cell cultures that have been directly isolated from an organism, organ, or tissue. Primary cells include cells that are neither transformed nor immortalized. Primary cells include any cell obtained from an organism, organ, or tissue that has not previously been passaged in tissue culture or that has been previously passaged in tissue culture but which cannot be passaged indefinitely in tissue culture. Such cells can be isolated by conventional methods and include, for example, somatic cells, hematopoietic cells, endothelial cells, epithelial cells, fibroblasts, mesenchymal cells, keratinocytes, melanocytes, monocytes, mononuclear cells, adipocytes, preadipocytes, neurons, glial cells, hepatocytes, skeletal myoblasts and smooth muscle cells. For example, primary cells can be obtained from connective tissues, muscle tissues, nervous system tissues, or epithelial tissues.

In some embodiments, cells typically cannot proliferate indefinitely, but due to mutation or change have escaped normal cellular aging and instead continue to divide. Such mutations or changes may occur naturally or be intentionally induced. Examples of immortalized cells include, but are not limited to, Chinese hamster ovary (CHO) cells, human embryonic kidney cells ( eg HEK 293 cells) and mouse embryonic fibroblasts ( eg 3T3 cells). Numerous types of immortalized cells are well known. Immortalized or primary cells include cells that are typically used for culture or for the expression of recombinant genes or proteins. In some embodiments, the cell is a differentiated cell, such as a liver cell ( eg , a human liver cell).

The cell can be obtained from any source. For example, the cell may be a eukaryotic cell, an animal cell, a plant cell, or a fungal cell ( such as yeast). Such cells may be fish cells or avian cells, or such cells may be mammalian cells, such as human cells, non-human mammalian cells, rodent cells, mouse cells, or rat cells. Mammals include, but are not limited to, humans, non-human primates, monkeys, monkeys, cats, dogs, horses, bovines, deer, bison, sheep, rodents ( e.g. mice, rats, hamsters, guinea pigs), farm animals ( e.g. bovine species such as cows, bulls, etc.; ovine species such as sheep, goats, etc.; and porcine species such as pigs and hogs). Birds include, but are not limited to, chickens, turkeys, ostriches, geese, ducks, etc. Pets and farm animals are also included. The term "non-human animal" excludes humans.

Additional host cells are described, for example, in US Patent Application Publication No. US2018/0030114, which is incorporated herein by reference in its entirety.

The nucleic acid molecules and polypeptides disclosed herein can be introduced into a cell by any means. Transfection protocols, as well as protocols for introducing nucleic acids or proteins, may vary. Non-limiting transfection methods include chemical transfection methods using liposomes, nanoparticles, calcium, dendrimers, and cationic polymers such as DEAE-dextran or polyethylenimine. Non-chemical methods include electroporation, sonoporation and optical transfection. Particle-based transfection includes the use of a gene gun or magnetic transfection. Viral methods can also be used for transfection.

Introduction of nucleic acids or proteins into a cell may also be mediated by electroporation, intracytoplasmic injection, viral infection, adenovirus, adeno-associated virus, lentivirus, retrovirus, transfection, lipid-mediated transfection, or nucleofection. Nucleofection is an improved electroporation technique that allows delivery of nucleic acid substrates not only into the cytoplasm, but also across the nuclear membrane and into the nucleus. In addition, the use of nucleofection in the methods disclosed herein typically requires fewer cells than conventional electroporation ( eg , only about 2 million compared to 7 million for conventional electroporation). In some embodiments, nucleofection is performed using the ^LONZA® NUCLEOFECTOR™ system.

The introduction of nucleic acids or proteins into a cell can also be accomplished through microinjection. Microinjection of mRNA is usually carried out into the cytoplasm (for example, to deliver mRNA directly to the translation apparatus), while microinjection of protein or DNA is usually carried out into the nucleus. Alternatively, microinjection can be performed by injecting into both the nucleus and the cytoplasm: the needle can first be inserted into the nucleus and inject a first amount, and when the needle is removed from the cell, inject a second amount into the cytoplasm. If a nuclease agent protein is introduced into the cytoplasm, the protein may contain a nuclear localization signal to ensure delivery to the nucleus/pronucleus.

Other methods of delivering nucleic acids or proteins into a cell may include, for example, vector delivery, particle-mediated delivery, exosome-mediated delivery, lipid nanoparticle-mediated delivery, cell-penetrating peptide-mediated delivery, or implantable device-mediated delivery. Methods for administering nucleic acids or proteins to a subject to modify cells in vivo are disclosed elsewhere herein. The introduction of nucleic acids and proteins into cells can also be achieved through hydrodynamic delivery (HDD).

Other methods of delivering nucleic acids or proteins into a cell may include, for example, vector delivery, particle-mediated delivery, exosome-mediated delivery, lipid nanoparticle-mediated delivery, cell-penetrating peptide-mediated delivery, or implantable device-mediated delivery. In some embodiments, the nucleic acid or protein can be delivered into a cell in a carrier such as a poly(lactic acid) (PMA) microsphere, a poly(D,L-lactic-co-glycolic acid) (PMGA) microsphere, a liposome , micelle, inverse micelle, lipid cochelate or lipid microtubule.

This disclosure also provides probes and primers. Examples of probes and primers are disclosed, for example, above. This disclosure provides probes and primers containing a nucleic acid sequence that specifically hybridizes to any of the nucleic acid molecules disclosed herein. For example, the probe or primer may contain a nucleic acid sequence that hybridizes to or that hybridizes to any of the nucleic acid molecules described herein that encode a variant SIGIRR protein that is truncated at position corresponding to position 215 in accordance with SEQ ID NO:9 with the complementary chain of a nucleic acid molecule. In some embodiments, the probe or primer comprises a nucleic acid sequence that hybridizes to a nucleic acid molecule encoding a variant SIGIRR protein according to SEQ ID NO:9, or that hybridizes to a complementary strand of that nucleic acid molecule. In some embodiments, the probe or primer comprises a nucleic acid sequence that hybridizes to a nucleic acid molecule encoding a variant SIGIRR polypeptide that is truncated at a position corresponding to position 215 in accordance with SEQ ID NO:9 and contains a serine at a position corresponding to position 186 in accordance with SEQ ID NO:9, or which hybridizes with the complementary strand of this nucleic acid molecule. In some embodiments, the probe or primer comprises a nucleic acid sequence that hybridizes to a nucleic acid molecule encoding a variant SIGIRR polypeptide that is truncated at position corresponding to position 215 in accordance with SEQ ID NO:9, and contains a number of amino acids at positions corresponding to positions 186-215 according to SEQ ID NO:9, or which hybridizes to the complementary strand of that nucleic acid molecule. In some embodiments, the probe or primer comprises a nucleic acid sequence that hybridizes to a nucleic acid molecule encoding a variant SIGIRR polypeptide that is truncated at position corresponding to position 215 in accordance with SEQ ID NO:9, and contains the following amino acid sequence at positions corresponding to positions 186-215 in accordance with SEQ ID NO:9: Ser-Arg-Ser-Trp-Ser-Gly-Val-Gly-Ala-Thr-Ser-Ser-Ser-Trp-Thr-Thr-Ala-Thr-Ser -Cys-Arg-Ala-Leu-Ser-Pro-Pro-Pro-Thr-Ser-Trp (SEQ ID NO:11), or which hybridizes to the complementary strand of that nucleic acid molecule. In some embodiments, the probe or primer comprises a nucleic acid sequence that hybridizes to a nucleic acid molecule encoding a variant SIGIRR polypeptide that is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence in accordance with SEQ ID NO:9, or which hybridizes with the complementary strand of this nucleic acid molecule. In some embodiments, the probe or primer comprises a nucleic acid sequence that hybridizes to a nucleic acid molecule encoding a variant SIGIRR polypeptide that contains or consists of an amino acid sequence according to SEQ ID NO:9, or that hybridizes to a complementary strand of that nucleic acid molecule . In some embodiments, the probe or primer specifically hybridizes to a portion of the nucleic acid molecule containing a codon that encodes a serine at the position corresponding to position 186 in accordance with SEQ ID NO:9.

The probe or primer may be of any suitable length, non-limiting examples of which include at least about 5, at least about 8, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 21, at least about 22, at least about 23, at least about 24, or at least about 25 nucleotides. In preferred embodiments, the probe or primer is at least about 18 nucleotides in length. The probe or primer may have a length of about 10 to about 35, about 10 to about 30, about 10 to about 25, about 12 to about 30, about 12 to about 28, about 12 to about 24, about 15 to about 30, about 15 to about 25, about 18 to about 30, about 18 to about 25, about 18 to about 24, or about 18 to about 22 nucleotides. In preferred embodiments, the probe or primer is between about 18 and about 30 nucleotides in length.

This disclosure also provides variation-specific probes and variation-specific primers. The variation-specific probe or variation-specific primer contains a nucleic acid sequence that is complementary to and/or hybridizes to, or specifically hybridizes to, a nucleic acid sequence encoding a variant SIGIRR protein that is truncated at the position corresponding to position 215 in accordance with SEQ ID NO:9, or its complementary chain. As used herein, “specifically hybridizes” means that the probe or primer ( eg , a variation-specific probe or variation-specific primer) does not hybridize to a nucleic acid molecule encoding a wild-type SIGIRR protein. In some embodiments, the change-specific probe specifically hybridizes to a nucleic acid codon that encodes a serine at the position corresponding to position 186 of SEQ ID NO:9, or a complementary strand thereof. In some embodiments, the variation-specific primer or pair of primers specifically hybridizes to the region(s) of a nucleic acid molecule encoding the SIGIRR variant protein such that a codon that encodes a serine at a position corresponding to position 186 in accordance with SEQ ID NO:9 , appears to be included in any transcript obtained from it. In some embodiments, the probe or primer specifically hybridizes to a portion of the nucleic acid molecule containing a codon that encodes a serine at the position corresponding to position 186 in accordance with SEQ ID NO:9.

In some embodiments, the variation-specific probe or variation-specific primer comprises a nucleic acid sequence that is complementary to and/or hybridizes to, or specifically hybridizes to, a nucleic acid sequence encoding a variant SIGIRR protein, wherein said protein comprises a truncation at a position corresponding to position 215 in accordance with SEQ ID NO:9, or its complementary chain.

In some embodiments, the variation-specific probe or variation-specific primer comprises a nucleic acid sequence that is complementary to and/or hybridizes to, or specifically hybridizes to, a genomic DNA molecule encoding a variant SIGIRR protein truncated at a position corresponding to position 215 according to with SEQ ID NO:9, or its complementary chain. In some embodiments, the variation-specific probe or variation-specific primer comprises a nucleic acid sequence that is complementary to and/or hybridizes to, or specifically hybridizes to, a genomic DNA molecule encoding a variant SIGIRR protein truncated at a position corresponding to position 215 according to with SEQ ID NO:9, and containing serine at the position corresponding to position 186 in accordance with SEQ ID NO:9. In some embodiments, the variation-specific probe or variation-specific primer comprises a nucleic acid sequence that is complementary to and/or hybridizes to, or specifically hybridizes to, a genomic DNA molecule encoding a variant SIGIRR protein truncated at a position corresponding to position 215 according to with SEQ ID NO:9, and containing the following amino acid sequence at positions corresponding to positions 186-215 in accordance with SEQ ID NO:9. Ser-Arg-Ser-Trp-Ser-Gly-Val-Gly-Ala-Thr-Ser-Ser-Ser-Trp-Thr-Thr-Ala-Thr-Ser-Cys-Arg-Ala-Leu-Ser-Pro- Pro-Pro-Thr-Ser-Trp (SEQ ID NO:11). In some embodiments, the variation-specific probe or variation-specific primer comprises a nucleic acid sequence that is complementary to and/or hybridizes or specifically hybridizes to a genomic DNA molecule encoding a variant SIGIRR protein having at least about 90% at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least at least about 98% or at least about 99% sequence identity to SEQ ID NO:9. In some embodiments, the variation-specific probe or variation-specific primer comprises a nucleic acid sequence that is complementary to and/or hybridizes or specifically hybridizes to a genomic DNA molecule encoding a variant SIGIRR protein having SEQ ID NO:9. In some embodiments, the probe or primer specifically hybridizes to a portion of genomic DNA containing a codon that encodes a serine at the position corresponding to position 186 in accordance with SEQ ID NO:9.

In some embodiments, the variation-specific probe or variation-specific primer comprises a nucleic acid sequence that is complementary to and/or hybridizes to, or specifically hybridizes to, a genomic DNA molecule that contains or consists of a nucleic acid sequence containing a guanine at the position corresponding to position 9962 in accordance with SEQ ID NO:2. In some embodiments, the variation-specific probe or variation-specific primer comprises a nucleic acid sequence that is complementary to and/or hybridizes, or specifically hybridizes, to a genomic DNA molecule that contains or consists of a nucleic acid sequence that has at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO:2. In some embodiments, the variation-specific probe or variation-specific primer comprises a nucleic acid sequence that is complementary to and/or hybridizes to, or specifically hybridizes to, a genomic DNA molecule that contains or consists of a nucleic acid sequence in accordance with SEQ ID NO :2.

In some embodiments, the variation-specific probe or variation-specific primer comprises a nucleic acid sequence that is complementary to and/or hybridizes to, or specifically hybridizes to, an mRNA molecule encoding a variant SIGIRR protein truncated at a position corresponding to position 215 according to SEQ ID NO:9. In some embodiments, the variation-specific probe or variation-specific primer comprises a nucleic acid sequence that is complementary to and/or hybridizes to, or specifically hybridizes to, an mRNA molecule encoding a variant SIGIRR protein truncated at a position corresponding to position 215 according to SEQ ID NO:9, and containing serine at the position corresponding to position 186 in accordance with SEQ ID NO:9. In some embodiments, the variation-specific probe or variation-specific primer comprises a nucleic acid sequence that is complementary to and/or hybridizes to, or specifically hybridizes to, an mRNA molecule encoding a variant SIGIRR protein truncated at a position corresponding to position 215 according to SEQ ID NO:9, and containing the following amino acid sequence at positions corresponding to positions 186-215 in accordance with SEQ ID NO:9. Ser-Arg-Ser-Trp-Ser-Gly-Val-Gly-Ala-Thr-Ser-Ser-Ser-Trp-Thr-Thr-Ala-Thr-Ser-Cys-Arg-Ala-Leu-Ser-Pro- Pro-Pro-Thr-Ser-Trp (SEQ ID NO:11). In some embodiments, the variation-specific probe or variation-specific primer comprises a nucleic acid sequence that is complementary to and/or hybridizes or specifically hybridizes to an mRNA molecule encoding a variant SIGIRR protein having at least about 90% of at least at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity to SEQ ID NO:9. In some embodiments, the variation-specific probe or variation-specific primer comprises a nucleic acid sequence that is complementary to and/or hybridizes or specifically hybridizes to an mRNA molecule encoding the variant SIGIRR protein having SEQ ID NO:9. In some embodiments, the probe or primer specifically hybridizes to a portion of the mRNA molecule containing a codon that encodes a serine at the position corresponding to position 186 in accordance with SEQ ID NO:9.

In some embodiments, the variation-specific probe or variation-specific primer comprises a nucleic acid sequence that is complementary to and/or hybridizes to, or specifically hybridizes to, an mRNA molecule that contains or consists of a nucleic acid sequence containing guanine at a position corresponding to position 557 in accordance with SEQ ID NO:4. In some embodiments, the variation-specific probe or variation-specific primer comprises a nucleic acid sequence that is complementary to and/or hybridizes to, or specifically hybridizes to, an mRNA molecule that contains codons CUA and AGC at positions corresponding to positions 553-555 and 556-558, respectively, in accordance with SEQ ID NO:4. In some embodiments, the variation-specific probe or variation-specific primer comprises a nucleic acid sequence that is complementary to and/or hybridizes to, or specifically hybridizes to, an mRNA molecule that contains or consists of a nucleic acid sequence that has at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97 %, at least about 98%, or at least about 99% sequence identity with SEQ ID NO:4. In some embodiments, the variation-specific probe or variation-specific primer comprises a nucleic acid sequence that is complementary to and/or hybridizes to, or specifically hybridizes to, an mRNA molecule that contains or consists of a nucleic acid sequence according to SEQ ID NO: 4.

In some embodiments, the variation-specific probe or variation-specific primer comprises a nucleic acid sequence that is complementary to and/or hybridizes to, or specifically hybridizes to, a cDNA molecule encoding a variant SIGIRR protein truncated at a position corresponding to position 215 in accordance with SEQ ID NO:9. In some embodiments, the variation-specific probe or variation-specific primer comprises a nucleic acid sequence that is complementary to and/or hybridizes to, or specifically hybridizes to, a cDNA molecule encoding a variant SIGIRR protein truncated at a position corresponding to position 215 in accordance with SEQ ID NO:9, and containing serine at the position corresponding to position 186 in accordance with SEQ ID NO:9. In some embodiments, the variation-specific probe or variation-specific primer comprises a nucleic acid sequence that is complementary to and/or hybridizes to, or specifically hybridizes to, a cDNA molecule encoding a variant SIGIRR protein truncated at a position corresponding to position 215 in accordance with SEQ ID NO:9, and containing the following amino acid sequence at positions corresponding to positions 186-215 in accordance with SEQ ID NO:9. Ser-Arg-Ser-Trp-Ser-Gly-Val-Gly-Ala-Thr-Ser-Ser-Ser-Trp-Thr-Thr-Ala-Thr-Ser-Cys-Arg-Ala-Leu-Ser-Pro- Pro-Pro-Thr-Ser-Trp (SEQ ID NO:11). In some embodiments, the variation-specific probe or variation-specific primer comprises a nucleic acid sequence that is complementary to and/or hybridizes or specifically hybridizes to a cDNA molecule encoding a variant SIGIRR protein having at least about 90% of at least at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity to SEQ ID NO:9. In some embodiments, the variation-specific probe or variation-specific primer comprises a nucleic acid sequence that is complementary to and/or hybridizes or specifically hybridizes to a cDNA molecule encoding a variant SIGIRR protein having SEQ ID NO:9. In some embodiments, the probe or primer specifically hybridizes to a portion of a cDNA molecule containing a codon that encodes a serine at the position corresponding to position 186 in accordance with SEQ ID NO:9.

In some embodiments, the variation-specific probe or variation-specific primer comprises a nucleic acid sequence that is complementary to and/or hybridizes to, or specifically hybridizes to, a cDNA molecule that contains or consists of a nucleic acid sequence containing guanine at a position corresponding to position 557 in accordance with SEQ ID NO:6. In some embodiments, the variation-specific probe or variation-specific primer comprises a nucleic acid sequence that is complementary to and/or hybridizes to, or specifically hybridizes to, a cDNA molecule that contains codons CUA and AGC at positions corresponding to positions 553-555 and 556-558, respectively, in accordance with SEQ ID NO:6. In some embodiments, the variation-specific probe or variation-specific primer comprises a nucleic acid sequence that is complementary to and/or hybridizes to, or specifically hybridizes to, a cDNA molecule that contains or consists of a nucleic acid sequence that has at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97 %, at least about 98%, or at least about 99% sequence identity with SEQ ID NO:6. In some embodiments, the variation-specific probe or variation-specific primer comprises a nucleic acid sequence that is complementary to and/or hybridizes to, or specifically hybridizes to, a cDNA molecule that contains or consists of a nucleic acid sequence according to SEQ ID NO: 6.

The length described above in connection with the probe or primer of the disclosure applies mutatis mutandis also to the variation-specific probe or variation-specific primer of the disclosure.

This disclosure also provides a pair of variation-specific primers comprising two of the variation-specific primers described above.

In some embodiments, the probe or primer ( eg , a variation-specific probe or variation-specific primer) comprises DNA. In some embodiments, the probe or primer ( eg , a variation-specific probe or a variation-specific primer) comprises RNA. In some embodiments, a probe or primer ( eg , a variation-specific probe or a variation-specific primer) hybridizes to a nucleic acid sequence encoding a variant SIGIRR protein under stringent conditions, such as high stringency conditions.

In some embodiments, the probe contains a label. In some embodiments, the label is a fluorescent label, a radioactive label, or biotin. In some embodiments, the length of the probe is described above. Alternatively, in some embodiments, the probe comprises or consists of at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, at least about 55, at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, at least at least about 90, at least about 95, or at least about 100 nucleotides. A probe ( eg , an allele-specific probe) can be used, for example, to detect any of the nucleic acid molecules disclosed herein. In preferred embodiments, the probe is at least about 18 nucleotides in length. The probe may have a length of from about 10 to about 35, from about 10 to about 30, from about 10 to about 25, from about 12 to about 30, from about 12 to about 28, from about 12 to about 24, from about 15 to about 30, about 15 to about 25, about 18 to about 30, about 18 to about 25, about 18 to about 24, or about 18 to about 22 nucleotides. In preferred embodiments, the probe has a length of from about 18 to about 30 nucleotides.

This disclosure also provides supports containing a substrate to which is attached any one or more of the probes disclosed herein. Solid supports are solid phase substrates or supports to which molecules, such as any probes disclosed herein, can bind. The form of the solid support is a matrix. Another form of solid substrate is an array detector. An array detector is a solid substrate to which a number of different probes have been coupled in the form of a matrix, lattice, or other arrangement.

Solid phase substrates for use in solid supports can include any solid material to which molecules can be bonded. This includes materials such as acrylamide, agarose, cellulose, nitrocellulose, glass, polystyrene, polyethylene vinyl acetate, polypropylene, polymethacrylate, polyethylene, polyethylene oxide, polysilicates, polycarbonates, Teflon, fluorocarbons, nylon, silicone rubber, polyanhydrides, polyglycolic acid, polylactic acid, complex polyorthoesters, polypropyl fumarate, collagen, glycosaminoglycans and polyamino acids. Solid phase substrates can be in any convenient form, including thin film, membrane, bottles, tablets, fibers, wool fibers, molded polymers, particles, granules, microparticles, or a combination thereof. Solid phase substrates and solid supports can be porous or non-porous. One form of solid phase substrate is a microtiter plate, for example the standard 96-well type. In some embodiments, a multi-well glass slide may be used, which typically contains one array per well. This allows for better control of assay reproducibility, improved throughput and sample handling, and facilitates automation. In some embodiments, the substrate is a microarray.

Any of the polypeptides disclosed herein may further contain one or more substitutions (such as conservative amino acid substitutions), insertions or deletions. Insertions include, for example, amino- or carboxy-terminal fusions, as well as insertions of one or more amino acid residues within a sequence. Methods for creating substitutions at predetermined sites in DNA having a known sequence are well known, for example, mutagenesis with primer M13 and PCR mutagenesis. Amino acid substitutions, as a rule, are carried out at one residue, but can be carried out simultaneously in a number of different locations; insertions typically range from 1 to 10 amino acid residues; and deletions range from 1 to 30 residues. Deletions or insertions can be made in adjacent pairs, i.e. deletion of 2 residues or insertion of 2 residues. Substitutions, deletions, insertions, or any combination thereof can be combined to produce the final construct. In some embodiments, the mutations do not displace the sequence out of reading frame or create complementary regions that may result in the formation of secondary structure of the mRNA.

This disclosure also provides kits for creating the compositions and using the methods described herein. The kits described herein may contain components for performing an assay or assays for detecting one or more genetic variants in a subject's sample.

In some embodiments, SIGIRR variant identification kits utilize the compositions and methods described above. In some embodiments, the base set may comprise a container containing at least one pair of oligonucleotide primers or probes, such as variation-specific probes or variation-specific primers, for a locus at any of the nucleic acid molecules disclosed herein (such as eg SEQ ID NO:2, SEQ ID NO:4 and/or SEQ ID NO:6). The kit may optionally contain instructions for use. The kit may also contain other optional kit components, such as, for example, one or more allelic ladders targeting each of the loci to be amplified, a sufficient amount of enzyme for amplification, an amplification buffer to facilitate amplification, a divalent cation solution to improve enzyme activity, dNTPs for chain extension during amplification, a loading solution for preparing amplified material for electrophoresis, genomic DNA as a template control, a size marker to ensure that materials move as expected in the separation media, and a protocol and guide to train the user and limit application errors . The amounts of different reagents in the kits can also be varied depending on a number of factors, such as the optimal sensitivity of the method. The scope of these principles includes the provision of test kits for use in manual applications or test kits for use with automatic sample preparation devices, reaction units, detectors and analyzers.

In some embodiments, the kits contain at least one pair of oligonucleotide primers ( e.g. , variation-specific primers) for amplification or at least one labeled oligonucleotide probe ( e.g. , variation-specific probe) for detecting a genomic DNA molecule encoding the variant protein SIGIRR, truncated at the position corresponding to position 215 in accordance with SEQ ID NO:9, or its complementary chain. In some embodiments, the kits contain at least one pair of oligonucleotide primers ( e.g. , variation-specific primers) for amplification or at least one labeled oligonucleotide probe ( e.g. , variation-specific probe) for detecting a genomic DNA molecule encoding the variant protein SIGIRR, truncated at position corresponding to position 215 in accordance with SEQ ID NO:9, and containing serine at position corresponding to position 186 in accordance with SEQ ID NO:9. In some embodiments, the kits contain at least one pair of oligonucleotide primers ( e.g. , variation-specific primers) for amplification or at least one labeled oligonucleotide probe ( e.g. , variation-specific probe) for detecting a genomic DNA molecule encoding the variant protein SIGIRR, truncated at the position corresponding to position 215 in accordance with SEQ ID NO:9, and containing the following amino acid position at positions corresponding to positions 186-215 in accordance with SEQ ID NO:9. Ser-Arg-Ser-Trp-Ser-Gly-Val-Gly-Ala-Thr-Ser-Ser-Ser-Trp-Thr-Thr-Ala-Thr-Ser-Cys-Arg-Ala-Leu-Ser-Pro- Pro-Pro-Thr-Ser-Trp (SEQ ID NO:11). In some embodiments, the kits contain at least one pair of oligonucleotide primers ( e.g. , variation-specific primers) for amplification or at least one labeled oligonucleotide probe ( e.g. , variation-specific probe) for detecting a genomic DNA molecule encoding the variant protein SIGIRR having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96 %, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO:9. In some embodiments, the kits contain at least one pair of oligonucleotide primers ( e.g. , variation-specific primers) for amplification or at least one labeled oligonucleotide probe ( e.g. , variation-specific probe) for detecting a genomic DNA molecule encoding the variant protein SIGIRR having SEQ ID NO:9.

In some embodiments, the kits contain at least one pair of oligonucleotide primers ( e.g. , variation-specific primers) for amplification or at least one labeled oligonucleotide probe ( e.g. , variation-specific probe) for detecting a genomic DNA molecule that contains or consists of a nucleic acid sequence containing guanine at the position corresponding to position 9962 in accordance with SEQ ID NO:2. In some embodiments, the kits contain at least one pair of oligonucleotide primers ( e.g. , variation-specific primers) for amplification or at least one labeled oligonucleotide probe ( e.g. , variation-specific probe) for detecting a genomic DNA molecule that contains or consists of a nucleic acid sequence that is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO:2. In some embodiments, the kits contain at least one pair of oligonucleotide primers ( e.g. , variation-specific primers) for amplification or at least one labeled oligonucleotide probe ( e.g. , variation-specific probe) for detecting a genomic DNA molecule that contains or consists of a nucleic acid sequence according to SEQ ID NO:2.

In some embodiments, the kits contain at least one pair of oligonucleotide primers ( e.g. , change-specific primers) for amplification or at least one labeled oligonucleotide probe ( e.g. , change-specific probe) for detecting an mRNA molecule encoding the variant SIGIRR protein truncated at the position corresponding to position 215 in accordance with SEQ ID NO:9. In some embodiments, the kits contain at least one pair of oligonucleotide primers ( e.g. , change-specific primers) for amplification or at least one labeled oligonucleotide probe ( e.g. , change-specific probe) for detecting an mRNA molecule encoding the variant SIGIRR protein truncated at position corresponding to position 215 in accordance with SEQ ID NO:9, and containing serine at a position corresponding to position 186 in accordance with SEQ ID NO:9. In some embodiments, the kits contain at least one pair of oligonucleotide primers ( e.g. , change-specific primers) for amplification or at least one labeled oligonucleotide probe ( e.g. , change-specific probe) for detecting an mRNA molecule encoding the variant SIGIRR protein truncated at the position corresponding to position 215 in accordance with SEQ ID NO:9, and containing the following amino acid position at positions corresponding to positions 186-215 in accordance with SEQ ID NO:9. Ser-Arg-Ser-Trp-Ser-Gly-Val-Gly-Ala-Thr-Ser-Ser-Ser-Trp-Thr-Thr-Ala-Thr-Ser-Cys-Arg-Ala-Leu-Ser-Pro- Pro-Pro-Thr-Ser-Trp (SEQ ID NO:11). In some embodiments, the kits contain at least one pair of oligonucleotide primers ( e.g. , change-specific primers) for amplification or at least one labeled oligonucleotide probe ( e.g. , change-specific probe) for detecting an mRNA molecule encoding the variant SIGIRR protein having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96% , at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO:9. In some embodiments, the kits contain at least one pair of oligonucleotide primers ( e.g. , change-specific primers) for amplification or at least one labeled oligonucleotide probe ( e.g. , change-specific probe) for detecting an mRNA molecule encoding the variant SIGIRR protein having SEQ ID NO:9.

In some embodiments, the kits contain at least one pair of oligonucleotide primers ( e.g. , change-specific primers) for amplification or at least one labeled oligonucleotide probe ( e.g. , change-specific probe) for detecting an mRNA molecule that contains or consists of from a nucleic acid sequence containing guanine at a position corresponding to position 557 in accordance with SEQ ID NO:4. In some embodiments, the kits contain at least one pair of oligonucleotide primers ( e.g. , change-specific primers) for amplification or at least one labeled oligonucleotide probe ( e.g. , change-specific probe) for detecting an mRNA molecule that contains CUA codons and AGC at positions corresponding to positions 553-555 and 556-558, respectively, in accordance with SEQ ID NO:4. In some embodiments, the kits contain at least one pair of oligonucleotide primers ( e.g. , change-specific primers) for amplification or at least one labeled oligonucleotide probe ( e.g. , change-specific probe) for detecting an mRNA molecule that contains or consists of of a nucleic acid sequence that is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO:4. In some embodiments, the kits contain at least one pair of oligonucleotide primers ( e.g. , change-specific primers) for amplification or at least one labeled oligonucleotide probe ( e.g. , change-specific probe) for detecting an mRNA molecule that contains or consists of from a nucleic acid sequence according to SEQ ID NO:4.

In some embodiments, the kits contain at least one pair of oligonucleotide primers ( e.g. , change-specific primers) for amplification or at least one labeled oligonucleotide probe ( e.g. , change-specific probe) for detecting a cDNA molecule encoding a variant SIGIRR protein truncated at the position corresponding to position 215 in accordance with SEQ ID NO:9. In some embodiments, the kits contain at least one pair of oligonucleotide primers ( e.g. , change-specific primers) for amplification or at least one labeled oligonucleotide probe ( e.g. , change-specific probe) for detecting a cDNA molecule encoding a variant SIGIRR protein truncated at position corresponding to position 215 in accordance with SEQ ID NO:9, and containing serine at a position corresponding to position 186 in accordance with SEQ ID NO:9. In some embodiments, the kits contain at least one pair of oligonucleotide primers ( e.g. , change-specific primers) for amplification or at least one labeled oligonucleotide probe ( e.g. , change-specific probe) for detecting a cDNA molecule encoding a variant SIGIRR protein truncated at the position corresponding to position 215 in accordance with SEQ ID NO:9, and containing the following amino acid position at positions corresponding to positions 186-215 in accordance with SEQ ID NO:9. Ser-Arg-Ser-Trp-Ser-Gly-Val-Gly-Ala-Thr-Ser-Ser-Ser-Trp-Thr-Thr-Ala-Thr-Ser-Cys-Arg-Ala-Leu-Ser-Pro- Pro-Pro-Thr-Ser-Trp (SEQ ID NO:11). In some embodiments, the kits contain at least one pair of oligonucleotide primers ( e.g. , change-specific primers) for amplification or at least one labeled oligonucleotide probe ( e.g. , change-specific probe) for detecting a cDNA molecule encoding a variant SIGIRR protein having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96% , at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO:9. In some embodiments, the kits contain at least one pair of oligonucleotide primers ( e.g. , change-specific primers) for amplification or at least one labeled oligonucleotide probe ( e.g. , change-specific probe) for detecting a cDNA molecule encoding a variant SIGIRR protein having SEQ ID NO:9.

In some embodiments, the kits contain at least one pair of oligonucleotide primers ( e.g. , variation-specific primers) for amplification, or at least one labeled oligonucleotide probe ( e.g. , variation-specific probe) for detecting a cDNA molecule that contains or consists of from a nucleic acid sequence containing guanine at a position corresponding to position 557 in accordance with SEQ ID NO:6. In some embodiments, the kits contain at least one pair of oligonucleotide primers ( e.g. , change-specific primers) for amplification or at least one labeled oligonucleotide probe ( e.g. , change-specific probe) for detecting a cDNA molecule that contains CTA codons and AGC at positions corresponding to positions 553-555 and 556-558, respectively, in accordance with SEQ ID NO:6. In some embodiments, the kits contain at least one pair of oligonucleotide primers ( e.g. , variation-specific primers) for amplification, or at least one labeled oligonucleotide probe ( e.g. , variation-specific probe) for detecting a cDNA molecule that contains or consists of of a nucleic acid sequence that is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO:6. In some embodiments, the kits contain at least one pair of oligonucleotide primers ( e.g. , variation-specific primers) for amplification, or at least one labeled oligonucleotide probe ( e.g. , variation-specific probe) for detecting a cDNA molecule that contains or consists of from a nucleic acid sequence according to SEQ ID NO:6.

In some embodiments, any of the kits disclosed herein may further comprise any one or more of: a nucleotide ladder, a protocol, an enzyme (such as an enzyme used for amplification such as polymerase chain reaction (PCR)), a dNTP, a buffer, a salt or salts and a nucleic acid control. In some embodiments, any of the kits disclosed herein may further comprise any one or more of a detectable label, products and reagents necessary to perform the annealing reaction, and instructions.

In some embodiments, kits disclosed herein may contain a primer or probe, or a variation-specific primer or variation-specific probe, containing a 3' terminal nucleotide that hybridizes directly to guanine at a position corresponding to position 9962 in SEQ ID NO: 2, or at the position corresponding to position 557 in SEQ ID NO:4 and/or SEQ ID NO:6.

Those skilled in the art will appreciate that the detection techniques employed are generally not limiting. More precisely, the disclosed methods and kits include a wide range of detection means, provided they are capable of detecting the presence or absence of an amplicon.

In some aspects, the kits may contain one or more primers or probes disclosed herein. For example, the kit may contain one or more probes that hybridize to one or more of the disclosed genetic variants.

In some aspects, the kit may contain one of the disclosed cells or cell lines. In some aspects, the kit may contain materials necessary to create a transgenic cell or cell line. For example, in some aspects, the kit may contain a cell and a vector containing a nucleic acid sequence containing one or more of the disclosed genetic variants. The kit may additionally contain cell culture medium.

This disclosure also provides methods for detecting the presence of genomic DNA, mRNA, cDNA and/or polypeptide of a variant SIGIRR in a biological sample from a human subject. It should be understood that gene sequences in a population, as well as the mRNA and proteins encoded by such genes, may vary due to polymorphism, such as single nucleotide polymorphism. The sequences given herein for genomic DNA, mRNA, cDNA and SIGIRR polypeptide are representative sequences only. Other genomic DNA, mRNA, cDNA and SIGIRR polypeptide sequences are also possible.

A biological sample can be obtained from any cell, tissue, or biological fluid from a subject. The sample may contain any clinically relevant tissue, such as a bone marrow sample, a tumor biopsy, a fine needle aspirate, or a body fluid sample such as blood, periodontal fluid, plasma, serum, lymph, ascites fluid, cystic fluid, or urine. In some cases, the sample contains a mouth swab. The sample used in the methods disclosed herein will vary depending on the assay format, the nature of the detection method, and the tissue, cells, or extracts that are used as the sample. A biological sample can be processed differently depending on the assay used. For example, when a SIGIRR variant nucleic acid molecule is detected, preprocessing may be used to isolate or enrich the sample for genomic DNA. A number of known techniques can be used for these purposes. Once the mRNA level of a variant SIGIRR is detected, different techniques can be used to enrich the biological sample for mRNA. Various methods can be used to detect the presence or level of mRNA or the presence of a particular variant locus in genomic DNA.

In some embodiments, this disclosure provides methods for detecting the presence or absence of a variant SIGIRR protein, comprising sequencing at least a portion of the protein in a biological sample to determine whether the protein contains an amino acid sequence encoding a truncated SIGIRR protein. In some embodiments, this disclosure provides methods for detecting the presence or absence of a variant SIGIRR protein, comprising sequencing at least a portion of the protein in a biological sample to determine whether the protein contains an amino acid sequence encoding a SIGIRR protein truncated at a position corresponding to position 215 in accordance with SEQ ID NO:9. In some embodiments, this disclosure provides methods for detecting the presence or absence of a variant SIGIRR protein, comprising sequencing at least a portion of the protein in a biological sample to determine whether the protein contains an amino acid sequence encoding a SIGIRR protein truncated at a position corresponding to position 215 in accordance with SEQ ID NO:9, and containing serine at the position corresponding to position 186 in accordance with SEQ ID NO:9.

In some embodiments, this disclosure provides methods for detecting the presence or absence of a variant SIGIRR nucleic acid molecule, comprising sequencing at least a portion of the nucleic acid in a biological sample to determine whether the nucleic acid contains a nucleic acid sequence encoding a truncated SIGIRR protein. In some embodiments, this disclosure provides methods for detecting the presence or absence of a variant SIGIRR nucleic acid molecule, comprising sequencing at least a portion of the nucleic acid in a biological sample to determine whether the nucleic acid contains a nucleic acid sequence encoding a SIGIRR protein truncated at a position corresponding to position 215 in accordance with SEQ ID NO:9. In some embodiments, this disclosure provides methods for detecting the presence or absence of a variant SIGIRR nucleic acid molecule, comprising sequencing at least a portion of the nucleic acid in a biological sample to determine whether the nucleic acid contains a nucleic acid sequence encoding a SIGIRR protein truncated at a position corresponding to position 215 in accordance with SEQ ID NO:9, and containing serine at a position corresponding to position 186 in accordance with SEQ ID NO:9. Any of the variant nucleic acid molecules disclosed herein can be detected using any of the probes and primers described herein.

In some embodiments, methods for detecting the presence or absence of an inflammatory bowel disease-associated variant SIGIRR nucleic acid molecule or an early-onset inflammatory bowel disease-associated variant SIGIRR nucleic acid molecule ( e.g. , genomic DNA, mRNA, or cDNA) in a subject, comprising: performing an assay on a biological sample obtained from a subject that determines whether a nucleic acid molecule in the biological sample contains any of the variant SIGIRR nucleic acid sequences disclosed herein ( e.g. , a nucleic acid molecule that encodes a truncated SIGIRR protein, a nucleic acid molecule that encodes a SIGIRR protein truncated at position 215 according to SEQ ID NO:9, a nucleic acid molecule that encodes a SIGIRR protein truncated at position 215 according to SEQ ID NO:9 and containing a serine at position , corresponding to position 186 in accordance with SEQ ID NO:9). In some embodiments, the biological sample comprises a cell or cell lysate. Such methods may further include, for example, obtaining a biological sample from a subject containing genomic DNA or SIGIRR mRNA, and, in the case of mRNA, optionally, reverse transcribing the mRNA into cDNA, and performing an analysis of the biological sample that determines whether the position encodes the genomic DNA , SIGIRR mRNA or cDNA truncated SIGIRR protein. Such methods may further include, for example, obtaining a biological sample from a subject containing genomic DNA or SIGIRR mRNA, and, in the case of mRNA, optionally, reverse transcribing the mRNA into cDNA, and performing an analysis of the biological sample that determines whether the position encodes the genomic DNA , SIGIRR mRNA, or cDNA SIGIRR protein truncated at position 215 of SEQ ID NO:9, performing an assay on a biological sample that determines whether a position in the genomic DNA, mRNA, or cDNA encodes a SIGIRR protein truncated at position corresponding to position 215 in accordance with SEQ ID NO:9 and containing serine at a position corresponding to position 186 in accordance with SEQ ID NO:9. Such assays may include, for example, determining the identity of these positions on a particular SIGIRR nucleic acid molecule. In some embodiments, the subject is a human.

In some embodiments, the analysis includes: sequencing at least a portion of the genomic DNA sequence of a SIGIRR nucleic acid molecule in a biological sample from the subject, wherein the portion to be sequenced includes a position corresponding to the position encoding serine at position 186 in the SIGIRR protein in accordance with SEQ ID NO:9 ; sequencing at least a portion of the SIGIRR mRNA sequence of a nucleic acid molecule in a biological sample from the subject, wherein the sequenced portion includes a position corresponding to the position encoding serine at position 186 in the SIGIRR protein in accordance with SEQ ID NO:9; or sequencing at least a portion of the SIGIRR cDNA sequence of a nucleic acid molecule in a biological sample from the subject, wherein the sequenced portion includes a position corresponding to the position encoding serine at position 186 in the SIGIRR protein in accordance with SEQ ID NO:9.

In some embodiments, the analysis includes: a) contacting the biological sample with a primer that hybridizes to: i) a portion of the SIGIRR genomic DNA sequence that is proximal to the position of the SIGIRR genomic DNA sequence at a position corresponding to the position encoding serine at position 186 in accordance with SEQ ID NO:9; ii) part of the SIGIRR mRNA sequence which is adjacent to the position of the SIGIRR mRNA sequence at a position corresponding to the position encoding serine at position 186 in accordance with SEQ ID NO:9; or iii) a portion of the SIGIRR cDNA sequence that is adjacent to a position of the SIGIRR cDNA sequence at a position corresponding to the position encoding serine at position 186 in accordance with SEQ ID NO:9; b) extending the primer through at least: i) a genomic DNA sequence position SIGIRR corresponding to nucleotide positions beyond the codon encoding the serine at the position corresponding to position 186 in accordance with SEQ ID NO:9; ii) a SIGIRR mRNA sequence position corresponding to nucleotide positions beyond the codon encoding serine at the position corresponding to position 186 according to SEQ ID NO:9; or iii) a SIGIRR cDNA sequence position corresponding to nucleotide positions beyond the codon encoding the serine at the position corresponding to position 186 in accordance with SEQ ID NO:9; and c) determining whether the primer extension product contains nucleotides encoding a serine at the position corresponding to position 186 in accordance with SEQ ID NO:9. In some embodiments, only SIGIRR genomic DNA is analyzed. In some embodiments, only SIGIRR mRNA is analyzed. In some embodiments, only the SIGIRR cDNA derived from the SIGIRR mRNA is analyzed.

In some embodiments, the analysis includes: a) contacting the biological sample with a variation-specific primer that hybridizes to i) a portion of the genomic DNA sequence SIGIRR containing nucleotides encoding a serine at a position corresponding to position 186 in accordance with SEQ ID NO:9 ; ii) part of the SIGIRR mRNA sequence containing nucleotides encoding serine at a position corresponding to position 186 in accordance with SEQ ID NO:9; or iii) part of the SIGIRR cDNA sequence containing nucleotides encoding serine at the position corresponding to position 186 in accordance with SEQ ID NO:9; b) primer extension using a variation-specific polymerase chain reaction method; and c) determining whether elongation has occurred. Variation-specific polymerase chain reaction techniques can be used to detect mutations such as deletions in a nucleic acid sequence. Variation-specific primers are used because DNA polymerase does not provide extension in the presence of a template mismatch. A number of variations on the basic variation-specific polymerase chain reaction method are available to those skilled in the art.

The change-specific primer may contain a nucleic acid sequence that is complementary to a nucleic acid sequence encoding a SIGIRR protein containing a serine at the position corresponding to position 186 in accordance with SEQ ID NO:9, or a complementary strand to the nucleic acid sequence. For example, the variation-specific primer may contain a nucleic acid sequence that is complementary to the nucleic acid sequence encoding SEQ ID NO:9, or to a complementary strand to that nucleic acid sequence. The variation-specific primer preferably specifically hybridizes to a nucleic acid sequence encoding the variant SIGIRR protein when the nucleic acid sequence encodes a serine at a position corresponding to position 186 in accordance with SEQ ID NO:9.

In some embodiments, the assay includes contacting a biological sample with a primer or probe that specifically hybridizes to a genomic DNA sequence, an mRNA sequence, or a cDNA sequence of a variant SIGIRR , but not to a corresponding wild-type SIGIRR sequence, under stringent conditions, and determining whether hybridization.

In some embodiments, the analysis includes RNA sequencing (RNA-seq). In some embodiments, the analysis also includes reverse transcribing the mRNA into cDNA via reverse transcriptase polymerase chain reaction (RT-PCR).

In some embodiments, the methods use probes and primers of sufficient nucleotide length to bind a target nucleic acid sequence and specifically detect and/or identify a polynucleotide comprising genomic DNA, mRNA, or cDNA of a SIGIRR variant. Hybridization conditions or reaction conditions can be determined by the operator to achieve this result. The nucleotide length can be any length sufficient for use in the selected detection method, including any assay described or illustrated herein. In general, for example, probes or primers having about 8, about 10, about 11, about 12, about 14, about 15, about 16, about 18, about 20, about 22, about 24, about 26, about 28 are used. , about 30, about 40, about 50, about 75, about 100, about 200, about 300, about 400, about 500, about 600 or about 700 nucleotides or more, or about 11 to about 20, about 20 to about 30, from about 30 to about 40, from about 40 to about 50, from about 50 to about 100, from about 100 to about 200, from about 200 to about 300, from about 300 to about 400, from about 400 to about 500 , from about 500 to about 600, from about 600 to about 700, or from about 700 to about 800 or more nucleotides in length. In preferred embodiments, the probe or primer is at least about 18 nucleotides in length. The probe or primer may have a length of about 10 to about 35, about 10 to about 30, about 10 to about 25, about 12 to about 30, about 12 to about 28, about 12 to about 24, about 15 to about 30, about 15 to about 25, about 18 to about 30, about 18 to about 25, about 18 to about 24, or about 18 to about 22 nucleotides. In preferred embodiments, the probe or primer is between about 18 and about 30 nucleotides in length.

Such probes and primers can hybridize specifically to the target sequence under high stringency hybridization conditions. Probes and primers may have complete nucleic acid sequence identity from contiguous nucleotides to the target sequence, although conventional methods can construct probes that differ from the target nucleic acid sequence but retain the ability to specifically detect and/or identify the target nucleic acid sequence. Accordingly, probes and primers may have about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% , about 99% or 100% sequence identity or complementarity with the target nucleic acid molecule.

In some embodiments, specific primers can be used to amplify the variant SIGIRR locus and/or variant SIGIRR mRNA or cDNA to produce an amplicon that can be used as a specific probe, or that can be detected on its own to identify the variant SIGIRR locus or to detect the level of specific SIGIRR mRNA or cDNA in a biological sample. The variant SIGIRR locus can be used to designate a genomic nucleic acid sequence comprising positions corresponding to the positions encoding serine at position 186 in accordance with SEQ ID NO:9. When a probe is hybridized to a nucleic acid molecule in a biological sample under conditions that allow the probe to bind to the nucleic acid molecule, this binding can be detected and provide an indication of the presence of a variant SIGIRR locus or the presence of a variant SIGIRR mRNA or cDNA level in the biological sample. Such identification of the associated probe has been described. The specific probe may contain a sequence that is at least about 80%, from about 80% to about 85%, from about 85% to about 90%, from about 90% to about 95%, and from about 95% to about 100 % identical (or complementary) to a specific region of the variant SIGIRR gene. The specific probe may contain a sequence that is at least about 80%, from about 80% to about 85%, from about 85% to about 90%, from about 90% to about 95%, and from about 95% to about 100 % identical (or complementary) to a specific region of the variant SIGIRR mRNA. The specific probe may contain a sequence that is at least about 80%, from about 80% to about 85%, from about 85% to about 90%, from about 90% to about 95%, and from about 95% to about 100 % identical (or complementary) to a specific cDNA region of a variant SIGIRR .

In some embodiments, to determine whether the complementary nucleic acid strand of a biological sample contains a nucleic acid sequence encoding a variant SIGIRR protein (e.g., a truncated SIGIRR protein or a variant SIGIRR protein containing a serine at the position corresponding to position 186 according to SEQ ID NO: 9), a biological sample can be subjected to nucleic acid amplification using a primer pair that contains a first primer derived from the 5' flanking sequence adjacent to the positions encoding the serine at the position corresponding to position 186 in accordance with SEQ ID NO:9, and a second a primer derived from the 3' flanking sequence adjacent to the positions encoding the serine at the position corresponding to position 186 in accordance with SEQ ID NO:9, to produce an amplicon that is diagnostic of the presence of nucleotides at the positions encoding the serine at the position corresponding to the position 186 according to SEQ ID NO:9. In some embodiments, the amplicon length may vary from the combined length of the primer pairs plus one nucleotide base pair to any amplicon length obtained using a DNA amplification protocol. This distance can vary from one nucleotide base pair to the limit of the amplification reaction, or up to about twenty thousand nucleotide base pairs. Optionally, the primer pair flanks a region including positions encoding serine at position 186 according to SEQ ID NO:9, and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides on each side of positions encoding serine at position 186 in accordance with SEQ ID NO:9. Similar amplicons can be generated from mRNA and/or cDNA sequences.

Typical methods for preparing and using probes and primers are described, for example, in Molecular Cloning: A Laboratory Manual, 2 ^nd Ed., Vol. 1-3, ed. Sambrook et al. , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY 1989 (hereinafter, “Sambrook et al. , 1989”); Current Protocols in Molecular Biology , ed. Ausubel et al. , Greene Publishing and Wiley-Interscience, New York, 1992 (with periodic updates) (hereinafter referred to as “Ausubel et al. , 1992”); and Innis et al. , PCR Protocols: A Guide to Methods and Applications, Academic Press: San Diego, 1990). PCR primer pairs can be generated from a known sequence, for example, using computer programs designed for this purpose, such as the Vector NTI PCR Primer Analysis Tool, version 10 (Informax Inc., Bethesda Md.); PrimerSelect (DNASTAR Inc., Madison, Wis.); and Primer3 (Version 0.4.0.COPYRGT., 1991, Whitehead Institute for Biomedical Research, Cambridge, Mass.). In addition, the sequence can be examined visually and primers can be identified manually using known guidelines.

Any hybridization or amplification or nucleic acid sequencing method can be used to specifically detect the presence of the SIGIRR variant gene locus and/or the level of mRNA or cDNA derived from the SIGIRR variant mRNA. In some embodiments, the nucleic acid molecule can be used as a primer to amplify a region of a SIGIRR nucleic acid, or the nucleic acid molecule can be used as a probe that specifically hybridizes, for example, under stringent conditions, to a nucleic acid molecule containing a variant SIGIRR gene locus. or a nucleic acid molecule containing mRNA or cDNA derived from a SIGIRR variant mRNA.

A number of techniques are available in the art, including, for example, nucleic acid sequencing, nucleic acid hybridization, and nucleic acid amplification. Illustrative examples of nucleic acid sequencing techniques include, but are not limited to, strand terminator (Sanger) sequencing and dye terminator sequencing.

Other methods include nucleic acid hybridization methods other than sequencing, including the use of labeled primers and probes directed against purified DNA, amplified DNA, and fixed cell preparations (fluorescence in situ hybridization (FISH)). In some methods, the target nucleic acid can be amplified prior to or simultaneously with detection. Illustrative examples of nucleic acid amplification techniques include, but are not limited to, polymerase chain reaction (PCR), ligase chain reaction (LCR), strand displacement amplification (CDA), and nucleic acid sequence-based amplification (NASA). Other methods include, but are not limited to, ligase chain reaction, strand displacement amplification, and thermophilic ADZ (tAZC).

Any method can be used to detect both unamplified and amplified polynucleotides, including, for example, hybridization protection analysis (HPA), real-time quantification of the amplification process, and determination of the amount of target sequence initially present in a non-amplified sample. in real time.

Also provided are methods for identifying nucleic acids that do not necessarily require sequence amplification and that are based, for example, on known Southern blot hybridization (DNA:DNA), in situ hybridization (ISH) and fluorescence in situ hybridization (FISH) methods of chromosomal material. Southern blotting can be used to detect specific nucleic acid sequences. In such methods, the nucleic acid that is extracted from the sample is fragmented, electrophoretically separated on a matrix gel, and transferred to a membrane filter. The filter-bound nucleic acid is hybridized with a labeled probe complementary to the sequence of interest. Detection of the hybridized probe associated with the filter is carried out. In any such method, the process may involve hybridization using any of the probes described or illustrated herein.

Hybridization methods can employ stringent conditions so that the probe or primer specifically hybridizes to its target. In some embodiments, the polynucleotide primer or probe will hybridize under stringent conditions to its target sequence ( e.g. , a variant SIGIRR gene locus, variant SIGIRR mRNA, or variant SIGIRR cDNA) to a markedly greater extent than to other sequences ( e.g. , the corresponding wild-type SIGIRR locus , wild-type mRNA or wild-type cDNA), for example, at least 2 times, at least 3 times, at least 4 times or more above background, including more than 10 times above background. In some embodiments, the polynucleotide primer or probe will hybridize under stringent conditions to its target sequence to a markedly greater extent than to other sequences, by at least a factor of 2. In some embodiments, the polynucleotide primer or probe will hybridize under stringent conditions to its target sequence to a markedly greater extent than to other sequences, by at least 3-fold. In some embodiments, the polynucleotide primer or probe will hybridize under stringent conditions to its target sequence to a markedly greater extent than to other sequences, by at least 4-fold. In some embodiments, the polynucleotide primer or probe will hybridize under stringent conditions to its target sequence to a markedly greater extent than to other sequences, greater than 10 times background. The stringency conditions depend on the sequence and will vary in different circumstances. By adjusting the hybridization stringency and/or washing conditions, target sequences that are 100% complementary to the probe can be identified (homologous probing). Alternatively, the stringency conditions can be adjusted to allow some sequence mismatch so that lesser degrees of identity can be detected (heterologous probing).

Appropriate stringency conditions that stimulate DNA hybridization, such as 6X sodium chloride/sodium citrate (SSC) at about 45°C followed by a 2X SSC wash at 50°C, are known and are described in Current Protocols in Molecular Biology , John Wiley & Sons , NY (1989), 6.3.1-6.3.6. Typically, stringent conditions for hybridization and detection are those where the salt concentration is less than about 1.5 M Na ions, typically a concentration of about 0.01 to 1.0 M Na ions (or other salts) at pH 7.0-8.3 and a temperature of at least 30°C for short probes ( eg , 10-50 nucleotides) and at least about 60°C for longer probes ( eg , more than 50 nucleotides). Harsh conditions can also be achieved by adding destabilizing agents such as formamide. Typical low stringency conditions include hybridization with a buffer solution of 30-35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulfate) at 37°C and wash in 1X - 2X SSC (20X SSC = 3.0 M NaCl/0, 3 M trisodium citrate) at 50-55°C. Typical moderate stringency conditions include hybridization in 40-45% formamide, 1.0 M NaCl, 1% SDS at 37°C and wash in 0.5X - 1X SSC at 55-60°C. Typical high stringency conditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37°C and wash in 0.1X SSC at 60-65°C. Optionally, wash buffers may contain from about 0.1% to about 1% SDS. The duration of hybridization is generally less than 24 hours, typically from about 4 to about 12 hours. The flushing period should be at least long enough to achieve equilibrium.

In hybridization reactions, specificity is generally a function of post-hybridization washes, with the ionic strength and temperature of the final wash solution being important factors. In the case of DNA-DNA hybrids, T _m can be approximated from the equation in Meinkoth and Wahl, Anal. Biochem ., 1984, 138, 267-284: T _m =81.5°C+16.6 (log M) + 0.41 (% GC) - 0.61 (% form.) - 500/L; where M is the molarity of monovalent cations, %GC is the percentage of guanosine and cytosine nucleotides in DNA, % forms. is the percentage of formamide in the hybridization solution, and L is the length of the hybrid in base pairs. T _m is the temperature (at a certain ionic strength and pH) at which 50% of the complementary target sequence hybridizes to a perfectly matching probe. T _m decreases by about 1°C for every 1% mismatch; thus, T _m , hybridization and/or washing conditions can be adjusted to hybridize sequences with the desired identity. For example, if sequences with ≥ 90% identity are being searched, T _m can be reduced by 10°C. In general, stringency conditions are chosen such that the temperature is about 5°C lower than the melting temperature ( _Tm ) for a particular sequence and its complementary chain at a particular ionic strength and pH. However, under conditions of very high stringency, hybridization and/or washing at a temperature of 1°C, 2°C, 3°C or 4°C lower than the melting temperature ( _Tm ) can be used; under moderate stringency conditions, hybridization and/or washing at 6°C, 7°C, 8°C, 9°C or 10°C lower than the melting temperature ( _Tm ) can be used; in low stringency conditions, hybridization and/or washing at temperatures 11°C, 12°C, 13°C, 14°C, 15°C or 20°C lower than the melting temperature (T _m ) can be used. Using the equation, the hybridization and wash compositions, and the required T _m , those skilled in the art will understand that variations in the stringency of the hybridization and/or wash solutions are inherently described here. If the required degree of mismatch results in a T _m less than 45°C (aqueous solution) or 32°C (formamide solution), it is optimal to increase the SSC concentration so that a higher temperature can be used.

Also provided are methods for detecting the presence or quantifying levels of a variant SIGIRR polypeptide in a biological sample, including, for example, protein sequencing and immunoassays. In some embodiments, a method of detecting the presence of a variant SIGIRR protein ( e.g. , SEQ D NO:9) in a human subject includes performing an assay on a biological sample from the human subject that detects the presence of a variant SIGIRR protein ( e.g. , SEQ D NO:4) in biological sample.

Illustrative non-limiting examples of protein sequencing techniques include, but are not limited to, mass spectrometry and Edman digestion. Illustrative examples of immunoassay methods include, but are not limited to, immunoprecipitation, Western blotting, immunohistochemistry, ELISA, immunocytochemistry, flow cytometry, and immuno-PCR. Polyclonal or monoclonal antibodies labeled for detection by various known techniques ( eg calorimetric, fluorescent, chemiluminescent or radioactive) are suitable for use in immunoassays. As for the immunoassay, the variant SIGIRR protein has a different size compared to the wild-type SIGIRR protein and therefore corresponds to a different molecular weight in the protein gel. Thus, using a single antibody, it is possible to distinguish wild-type SIGIRR protein from variant SIGIRR protein, for example, in a Western blot analysis.

This disclosure also provides methods for diagnosing inflammatory bowel disease or early-onset inflammatory bowel disease or identifying the risk of developing inflammatory bowel disease or early-onset inflammatory bowel disease in a human subject, comprising: detecting a nucleic acid molecule derived from a human subject, any of the changes in any of the SIGIRR nucleic acid molecules described herein; and diagnosing a human subject with inflammatory bowel disease or early-onset inflammatory bowel disease if the subject has one or more symptoms of inflammatory bowel disease or early-onset inflammatory bowel disease, or diagnosing the human subject as being at risk of developing an inflammatory disease inflammatory bowel disease or early-onset inflammatory bowel disease, unless the subject has one or more symptoms of inflammatory bowel disease or early-onset inflammatory bowel disease. In some embodiments, the human subject requires such a diagnosis. In some embodiments, the human subject may have relatives who have been diagnosed with inflammatory bowel disease or early-onset inflammatory bowel disease.

Symptoms of inflammatory bowel disease or early-onset inflammatory bowel disease include, but are not limited to, diarrhea, fever, weakness, abdominal pain, abdominal cramps, nausea, vomiting, blood in the stool, anemia, decreased appetite, and unintentional weight loss, or any combination thereof.

In some embodiments, the methods include detecting the presence of genomic DNA, mRNA, or mRNA-derived cDNA of a variant SIGIRR obtained from a biological sample obtained from the subject. It should be understood that gene sequences in a population, as well as the mRNA and proteins encoded by such genes, may vary due to polymorphisms such as single nucleotide polymorphisms (SNPs). The sequences given herein for genomic DNA, mRNA, cDNA, and SIGIRR polypeptide are representative sequences only, but other similar sequences are possible, including additional SIGIRR alleles.

In some embodiments, the discovery step includes sequencing at least a portion of a nucleic acid molecule that encodes the truncated SIGIRR protein. In some embodiments, the discovery step includes sequencing at least a portion of a nucleic acid molecule that encodes a truncated SIGIRR, wherein the nucleic acid molecule to be sequenced encodes an amino acid sequence that contains a position corresponding to position 186 in accordance with SEQ ID NO:9. Any nucleic acid molecules disclosed herein ( eg , genomic DNA, mRNA, or cDNA) may be sequenced. In some embodiments, the discovery step includes sequencing the entire nucleic acid molecule.

In some embodiments, the discovery step includes: amplifying at least a portion of a nucleic acid molecule that encodes a SIGIRR protein; labeling the nucleic acid molecule with a detectable label; bringing the labeled nucleic acid into contact with a support containing a probe, the probe comprising a nucleic acid sequence that hybridizes under stringent conditions to a nucleic acid sequence encoding a truncated SIGIRR protein; and detecting a detectable label. In some embodiments, the detection step includes: amplifying at least a portion of a nucleic acid molecule that encodes a SIGIRR protein, wherein the amplified nucleic acid molecule encodes an amino acid sequence that contains a position corresponding to position 186 in accordance with SEQ ID NO:9; labeling the nucleic acid molecule with a detectable label; bringing the labeled nucleic acid into contact with a support containing a probe, the probe comprising a nucleic acid sequence that hybridizes under stringent conditions to a nucleic acid sequence encoding aspartic acid at a position corresponding to position 186 in accordance with SEQ ID NO:9; and detecting a detectable label. Any nucleic acid molecules disclosed herein can be amplified. For example, any of the genomic DNA, cDNA, or mRNA molecules disclosed herein can be amplified. In some embodiments, the nucleic acid molecule is mRNA, and the method further includes reverse transcribing the mRNA into cDNA prior to the amplification step.

In some embodiments, the detection step includes: contacting a nucleic acid molecule that encodes a SIGIRR protein with a probe containing a detectable label, the probe comprising a nucleic acid sequence that hybridizes under stringent conditions to a nucleic acid sequence encoding a truncated SIGIRR protein , and detecting a detectable label. In some embodiments, the detection step includes: contacting a nucleic acid molecule that encodes a SIGIRR protein with a probe containing a detectable label, the probe comprising a nucleic acid sequence that hybridizes under stringent conditions to a nucleic acid sequence encoding an amino acid sequence, which contains a serine at the position corresponding to position 186 in accordance with SEQ ID NO:9, and detection of a detectable label. In some embodiments, the nucleic acid molecule is present in a cell obtained from a human subject and detection is carried out in accordance with an in situ hybridization method.

In some embodiments, the detection step includes contacting a nucleic acid molecule that encodes the SIGIRR protein with a variation-specific primer and amplifying the nucleic acid molecule using variation-specific PCR techniques. The variation-specific primer may be any such primer described herein and may be specific for variant SIGIRR proteins that encode a serine at the position corresponding to position 186 in accordance with SEQ ID NO:9.

Other assays that may be used in the methods disclosed herein include, for example, reverse transcription polymerase chain reaction (RT-PCR) or quantitative RT-PCR (qRT-PCR). Other analytical methods that can be used in the methods disclosed herein include, for example, RNA sequencing (RNA-seq) and then detecting the presence and amount of variant mRNA or cDNA in a biological sample.

This disclosure also provides methods for identifying a human subject having inflammatory bowel disease or early-onset inflammatory bowel disease, or a risk of developing inflammatory bowel disease or early-onset inflammatory bowel disease. these methods generally involve detecting the presence or absence of a variant SIGIRR protein in a sample obtained from a subject; and/or the presence or absence of any of the nucleic acid molecules encoding the SIGIRR variant protein described herein. The presence of a truncated SIGIRR protein indicates that the subject has inflammatory bowel disease or early-onset inflammatory bowel disease, or is at risk of developing inflammatory bowel disease or early-onset inflammatory bowel disease. The presence of guanine (due to deletion of adenine) at a position corresponding to position 9962 according to SEQ ID NO:2 ( for example , genomic DNA), or at a position corresponding to position 557 according to SEQ ID NO:4 ( for example , mRNA), or at position corresponding to position 557 of SEQ ID NO:6 ( e.g. , cDNA), which in each case results in a variant SIGIRR protein truncated at position corresponding to position 215 of SEQ ID NO:9 and containing serine at the position corresponding to position 186 in accordance with SEQ ID NO:9, indicates that the subject has inflammatory bowel disease or early-onset inflammatory bowel disease, or is at risk of developing inflammatory bowel disease or early-onset inflammatory bowel disease . This method can be carried out in vitro , in situ or in vivo .

This disclosure also provides methods for identifying a human subject having early-onset inflammatory bowel disease or a risk of developing early-onset inflammatory bowel disease, the method comprising detecting in a sample obtained from the subject the presence or absence of: SIGIRR protein containing serine at a position corresponding to position 186 in accordance with SEQ ID NO:9, and truncated at a position corresponding to position 215 in accordance with SEQ ID NO:9; and/or a nucleic acid molecule encoding a SIGIRR protein containing a serine at a position corresponding to position 186 in accordance with SEQ ID NO:9, and truncated at a position corresponding to position 215 in accordance with SEQ ID NO:9; wherein the presence of a truncated SIGIRR protein and/or a nucleic acid molecule encoding a truncated SIGIRR protein indicates that the subject has early-onset inflammatory bowel disease or is at risk of developing early-onset inflammatory bowel disease. In some embodiments, the truncated SIGIRR protein contains a different amino acid compared to the wild-type SIGIRR protein at any of the positions corresponding to positions 186-209 and 211-215 according to SEQ ID NO:9. In some embodiments, the truncated SIGIRR protein contains the amino acid sequence of SEQ ID NO:11 at positions corresponding to positions 186-215 of SEQ ID NO:9.

In some embodiments, detection of the presence or absence of truncated SIGIRR protein in samples is performed using an antibody specific for truncated SIGIRR. In some embodiments, an antibody that is specific for a truncated SIGIRR is specific for: i) a serine at the position corresponding to position 186 in accordance with SEQ ID NO:9; or ii) an epitope created in the SIGIRR protein due to a frameshift mutation that results in a serine at the position corresponding to position 186 in accordance with SEQ ID NO:9. In some embodiments, the detection further includes comparing the response of an antibody specific for the truncated SIGIRR with that of an antibody specific for the wild type SIGIRR. In some embodiments, detection of the presence or absence of a truncated SIGIRR protein in a specified sample is performed using an enzyme-linked immunosorbent assay (ELISA). In some embodiments, detecting the presence or absence of a specified nucleic acid molecule encoding a variant SIGIRR protein in a sample is accomplished by determining whether there is a frameshift mutation in the nucleic acid molecule creating a codon encoding a serine at a position corresponding to position 186 in accordance with SEQ ID NO:9. In some embodiments, the portion of the nucleic acid molecule to be sequenced contains a number of positions spanning the codon encoding the position corresponding to position 186 in accordance with SEQ ID NO:9.

In some embodiments of the method, the discovery step includes sequencing at least a portion of the nucleic acid molecule that encodes the SIGIRR protein. In some embodiments of the method, the discovery step includes sequencing at least a portion of the nucleic acid molecule that encodes the truncated SIGIRR protein. The nucleic acid molecule to be sequenced may encode an amino acid sequence that contains a position corresponding to position 186 in accordance with SEQ ID NO:9. The presence of guanine (due to deletion of adenine) at a position corresponding to position 9962 according to SEQ ID NO:2 ( for example , genomic DNA), or at a position corresponding to position 557 according to SEQ ID NO:4 ( for example , mRNA), or at position corresponding to position 557 of SEQ ID NO:6 ( e.g. , cDNA), which in each case results in a variant SIGIRR protein truncated at position corresponding to position 215 of SEQ ID NO:9 and containing serine at the position corresponding to position 186 in accordance with SEQ ID NO:9. The discovery step may involve sequencing the nucleic acid molecule encoding the entire SIGIRR protein.

In some embodiments of the method, the detection step includes amplifying at least a portion of a nucleic acid molecule that encodes a truncated SIGIRR protein, labeling the nucleic acid molecule with a detectable label, contacting the labeled nucleic acid molecule with a support containing a probe, wherein the probe contains a nucleic acid sequence an acid that specifically hybridizes, including, for example, under stringent conditions, with a nucleic acid sequence encoding a truncated SIGIRR protein, and detecting a detectable label. In some embodiments of the method, the detection step includes amplifying at least a portion of a nucleic acid molecule that encodes a SIGIRR protein, labeling the amplified nucleic acid molecule with a detectable label, contacting the labeled nucleic acid molecule with a support containing a probe, wherein the probe contains a nucleic acid sequence an acid that specifically hybridizes, including, for example, under stringent conditions, with a nucleic acid sequence encoding a serine at the position corresponding to position 186 in accordance with SEQ ID NO:9 (or with a nucleic acid sequence containing guanine (due to deletion of adenine ) at a position corresponding to position 9962 in accordance with SEQ ID NO:2 ( for example , genomic DNA), or at a position corresponding to position 557 in accordance with SEQ ID NO:4 ( for example , mRNA), or at a position corresponding to position 557 according to SEQ ID NO:6 ( e.g. cDNA), which in each case results in a variant SIGIRR protein truncated at the position corresponding to position 215 according to SEQ ID NO:9 and containing a serine at the position corresponding to position 186 in accordance with SEQ ID NO:9), and detecting a detectable label. The nucleic acid molecule to be amplified preferably encodes an amino acid sequence that contains the position corresponding to position 186 in accordance with SEQ ID NO:9. If the nucleic acid includes mRNA, the method may further include reverse transcribing the mRNA into cDNA before the amplification step. In some embodiments, the detection step includes contacting a nucleic acid molecule that encodes a SIGIRR protein with a probe containing a detectable label and detecting the detectable label. The probe preferably contains a nucleic acid sequence that specifically hybridizes, including, for example, under stringent conditions, with a nucleic acid sequence encoding an amino acid sequence that contains a serine at the position corresponding to position 186 in accordance with SEQ ID NO:9 (or a nucleic acid sequence containing guanine (due to deletion of adenine) at a position corresponding to position 9962 according to SEQ ID NO:2 ( for example , genomic DNA), or at a position corresponding to position 557 according to SEQ ID NO:4 ( for example , mRNA), or at the position corresponding to position 557 according to SEQ ID NO:6 ( for example , cDNA), which in each case results in a variant protein SIGIRR truncated at the position corresponding to position 215 according to SEQ ID NO:9 , and containing serine at the position corresponding to position 186 in accordance with SEQ ID NO:9). The nucleic acid molecule may be located within a cell obtained from a human subject.

In some embodiments, the detection step includes: amplifying at least a portion of a nucleic acid molecule that encodes a SIGIRR protein, wherein the amplified nucleic acid molecule includes a codon encoding an amino acid at a position corresponding to position 186 in accordance with SEQ ID NO:9; labeling the nucleic acid molecule with a detectable label; bringing a labeled nucleic acid molecule into contact with a support containing a probe, wherein the probe contains a nucleic acid sequence that specifically hybridizes under stringent conditions to a nucleic acid sequence containing a codon encoding a serine at a position corresponding to position 186 in accordance with SEQ ID NO:9 ; and detecting a detectable label.

In some embodiments, the detection step includes: contacting a nucleic acid molecule that encodes a SIGIRR protein with a probe containing a detectable label, wherein the probe contains a nucleic acid sequence that specifically hybridizes under stringent conditions to a nucleic acid sequence containing a codon, encoding a serine at a position corresponding to position 186 in accordance with SEQ ID NO:9, and detecting a detectable tag. In some embodiments, the human subject is under 18 years of age. In some embodiments, the human subject is identified as having Crohn's disease or at risk of developing Crohn's disease.

This disclosure also provides methods for diagnosing inflammatory bowel disease or early-onset inflammatory bowel disease or identifying the risk of developing inflammatory bowel disease or early-onset inflammatory bowel disease in a human subject, comprising: detecting a truncated SIGIRR protein in a sample obtained from a human subject; and diagnosing a human subject with inflammatory bowel disease or early-onset inflammatory bowel disease if the subject has one or more symptoms of inflammatory bowel disease or early-onset inflammatory bowel disease, or diagnosing the human subject as being at risk of developing an inflammatory disease inflammatory bowel disease or early-onset inflammatory bowel disease, unless the subject has one or more symptoms of inflammatory bowel disease or early-onset inflammatory bowel disease. In some embodiments, the human subject requires such a diagnosis. This disclosure also provides methods for diagnosing inflammatory bowel disease or early-onset inflammatory bowel disease or identifying the risk of developing inflammatory bowel disease or early-onset inflammatory bowel disease in a human subject, comprising: detecting a variant SIGIRR protein, such as containing SEQ ID NO:9, in a sample obtained from a human subject; and diagnosing a human subject with inflammatory bowel disease or early-onset inflammatory bowel disease if the subject has one or more symptoms of inflammatory bowel disease or early-onset inflammatory bowel disease, or diagnosing the human subject as being at risk of developing an inflammatory disease inflammatory bowel disease or early-onset inflammatory bowel disease, unless the subject has one or more symptoms of inflammatory bowel disease or early-onset inflammatory bowel disease. In some embodiments, the human subject requires such a diagnosis. In some embodiments, the human subject may have relatives who have been diagnosed with inflammatory bowel disease or early-onset inflammatory bowel disease.

This disclosure also provides methods for diagnosing early-onset inflammatory bowel disease or identifying the risk of early-onset inflammatory bowel disease in a human subject, comprising: detecting a nucleic acid molecule encoding the SIGIRR protein in a sample obtained from the human subject , wherein the SIGIRR protein contains a serine at a position corresponding to position 186 in accordance with SEQ ID NO:9, and is truncated at a position corresponding to position 215 in accordance with SEQ ID NO:9; and/or detection of a SIGIRR protein, in a sample obtained from a human subject, wherein the SIGIRR protein contains a serine at a position corresponding to position 186 in accordance with SEQ ID NO:9, and truncated at a position corresponding to position 215 in accordance with SEQ ID NO: :9; and diagnosing a human subject as having early-onset inflammatory bowel disease if the subject has one or more symptoms of early-onset inflammatory bowel disease, or diagnosing the human subject as being at risk for early-onset inflammatory bowel disease if the subject does not have one or more symptoms of early-onset inflammatory bowel disease. In some embodiments, the truncated SIGIRR protein contains a different amino acid compared to the wild-type SIGIRR protein at any of the positions corresponding to positions 186-209 and 211-215 according to SEQ ID NO:9. In some embodiments, the truncated SIGIRR protein contains the amino acid sequence of SEQ ID NO:13 at positions corresponding to positions 186-215 of SEQ ID NO:9. In some embodiments, detection of the truncated SIGIRR protein is performed using an antibody specific for the truncated SIGIRR. In some embodiments, an antibody that is specific for a truncated SIGIRR is specific for: i) a serine at the position corresponding to position 186 in accordance with SEQ ID NO:9; or ii) an epitope created in the SIGIRR protein due to a frameshift mutation that results in a serine at the position corresponding to position 186 in accordance with SEQ ID NO:9. In some embodiments, the detection further includes comparing the response of an antibody specific for the truncated SIGIRR with that of an antibody specific for the wild type SIGIRR. In some embodiments, detection of the truncated SIGIRR protein is performed using an enzyme-linked immunosorbent assay (ELISA). In some embodiments, detection of a nucleic acid molecule encoding said truncated SIGIRR protein is performed by detecting whether there is a frameshift mutation in said nucleic acid molecule creating a codon encoding a serine at a position corresponding to position 186 in accordance with SEQ ID NO:9 . In some embodiments, the portion of the nucleic acid molecule to be sequenced contains a number of positions spanning the codon encoding the position corresponding to position 186 in accordance with SEQ ID NO:9.

In some embodiments, the detection step includes: amplifying at least a portion of a nucleic acid molecule that encodes a SIGIRR protein, wherein the amplified nucleic acid molecule includes a codon encoding an amino acid at a position corresponding to position 186 in accordance with SEQ ID NO:9; labeling the nucleic acid molecule with a detectable label; bringing a labeled nucleic acid molecule into contact with a support containing a probe, the probe comprising a nucleic acid sequence that specifically hybridizes under stringent conditions to a nucleic acid sequence containing a codon encoding a serine at a position corresponding to position 186 in accordance with SEQ ID NO:9 ; and detecting a detectable label.

In some embodiments, the detection step includes: contacting a nucleic acid molecule that encodes a SIGIRR protein with a probe containing a detectable label, wherein the probe contains a nucleic acid sequence that specifically hybridizes under stringent conditions to a nucleic acid sequence containing a codon, encoding serine at a position corresponding to position 186 in accordance with SEQ ID NO:9; and detecting a detectable label.

In some embodiments, the human subject described herein from whom the sample is obtained ( eg , the human subject being diagnosed or treated) is not an adult. In some embodiments, the human subject described herein from which the sample is obtained is 18 years of age or younger. In some embodiments, the human subject described herein from which the sample is obtained is 15 years of age or younger. In some embodiments, the human subject described herein from which the sample is obtained is 13 years of age or younger. In some embodiments, the human subject described herein from which the sample is obtained is 10 years of age or less. In some embodiments, a human subject who is 6 years of age or less may have very early onset inflammatory bowel disease. In some embodiments, the human subject does not have necrotizing enterocolitis.

In some embodiments, any of the methods described herein may further comprise treating the subject with an agent effective for treating inflammatory bowel disease or early-onset inflammatory bowel disease. In some embodiments, the methods further comprise treating the subject with an agent effective for treating inflammatory bowel disease or early-onset inflammatory bowel disease when a change is detected in the subject and the subject is diagnosed with inflammatory bowel disease or early-onset inflammatory bowel disease.

This disclosure also provides the use of any genomic DNA molecules, mRNA, cDNA, variant SIGIRR polypeptides disclosed herein, and hybridization of nucleic acid molecules disclosed herein in diagnosing early-onset inflammatory bowel disease or diagnosing the risk of developing early-onset inflammatory bowel disease. in young years.

All patent documents, websites, other publications, accession numbers, etc. cited above or below are incorporated by reference in their entirety and for all purposes to the same extent as if each individual source had been specifically and separately indicated as being incorporated by reference. If different versions of a sequence have been associated with an accession number at different times, the version associated with the accession number at the time of the effective filing date of this application is assumed to be the version associated with the accession number. Effective filing date means the earlier of the actual filing date or the filing date of the priority application, referring to the accession number, if applicable. Likewise, if different versions of a publication, website, etc. published at different times, the most recent published version as of the effective date of application is implied, unless otherwise stated. Any feature, step, element, embodiment, or aspect of this disclosure may be used in combination with any other feature, step, element, embodiment, or aspect unless specifically stated otherwise. Although the present invention has been described in some detail by way of illustrations and examples for the purposes of clarity and understanding, it should be understood that certain changes and modifications may be practically made within the scope of the appended claims.

Nucleotide and amino acid sequences given herein are shown using standard letter abbreviations for nucleotide bases and single letter codes for amino acids. The nucleotide sequences follow the standard convention of starting at the 5' end of the sequence and continuing (ie from left to right in each line) to the 3' end. Only one strand of each nucleotide sequence is shown, but the complementary strand is intended to be included whenever the strand shown is mentioned. Amino acid sequences follow the standard convention of starting at the amino end of the sequence and continuing (i.e., from left to right in each line) to the carboxy end.

The following examples are provided to describe the embodiments in more detail. They are intended to illustrate, but not limit, the claimed embodiments.

Examples

The following examples are provided to provide those skilled in the art with a complete disclosure and description of how to prepare and evaluate the compounds, compositions, articles, devices and/or methods claimed herein and are intended to be illustrative only and not intended to be limiting. the scope of what the inventors consider to be the subject matter of the invention. Care has been taken to ensure accuracy in terms of numbers ( eg quantity, temperature, etc.), but the possibility of some errors and deviations should be taken into account. Unless otherwise noted, fractions are fractions by mass, temperature is in °C or equal to ambient temperature, and pressure is at or near atmospheric pressure.

Example 1: Patient Recruitment and Phenotyping

Whole genome sequencing and triple variant analysis were performed on a 13-year-old patient with IBD-RV, his mother with IBD, and his unaffected father. This family was identified for genetic evaluation of the patient's IBD-RV.

Example 2: Genomic samples

Genomic DNA was extracted from peripheral blood samples and transported to the Regeneron Genetics Center (RGC) for whole exome sequencing and stored in automated biobanks at -80°C. Fluorescence quantification was performed to ensure adequate quality and quantity of DNA for sequencing purposes.

1 μg of DNA was cut to an average fragment length of 150 bp (Covaris LE220) and prepared for exome capture using a custom reagent kit from Kapa Biosystems. Sample capture was performed using exome-specific NimbleGen SeqCap VCRome 2.1 or Integrated DNA Technologies xGen designs. Samples were barcoded, pooled, and multiplexed for sequencing using 75 bp. paired-end sequencing on Illumina HiSeq 2500 with v4 chemistry. Captured fragments were sequenced to achieve a minimum of 85% target base coverage with coverage of 20x or greater. After sequencing, the data were processed using a cloud-based workflow developed by RGC, which leverages DNAnexus and AWS to run standard tools for sample-level data acquisition and analysis. Briefly, sequencing data were generated and demultiplexed using Illumina's CASAVA software. Sequence reads were mapped and aligned to the reference human genome assembly GRCh37/hg19 using BWA-mem. After alignment, duplicate reads were marked and flagged using Picard tools, and indels were realigned using GATK to improve the quality of variant detection. SNP and INdel variants and genotypes were determined using GATK's HaplotypeCaller, and GATK's Variant Quality Score Recalibration (VQSR) was used to indicate overall variant quality scores. Sequencing statistics and data quality metrics were obtained for each sample to evaluate capture efficiency, alignment efficiency, and variant detection.

Example 3: Genomic Data Analysis

Standard quality control filters for minimum read depth (>10), genotype quality (>30) and allelic balance (>20%) were applied to certain variants. Passing variants were classified and annotated based on their potential functional effects (synonymous, nonsynonymous, splice variants, frameshift or non-frameshift variants) using an RGC-developed notation and analysis workflow. Familial relationships were verified using a descent identity metric from genetic data to determine relatedness and relationships within a group using PRIMUS (Staples et al ., Amer. J. Human Genet., 2014, 95, 553-564) and cross-referencing with reported origin for this family.

Ancestry-based variant analysis and segregation were performed to identify candidate disease genes with an autosomal dominant inheritance profile given known family history. Variants common to the affected proband and his affected mother, but not common to the unaffected father, were subsequently designated and filtered by observed frequency in population-based benchmark databases such as dbSNP, 1000 Genomes Project, NHLBI Exome Sequencing Project, Exome Aggregation Consortium Database ( ExAc), and internal RGC databases to filter common polymorphisms and high-frequency probable benign variants. Bioinformatic prediction algorithms for the functional effects of variants, such as LRT, Poly-phen2, SIFT, CADD, and Mutation Taster, along with alignment-based conservation estimates for multiple species (i.e., GERP, PhastCons, PhyloP) were included as part of the variant designation process and were used to communicate the potential harmfulness of identified candidate variants.

A rare truncation indel variant (SIGIRR: c.557delA; p.K186fs*31) has been identified in the SIGIRR gene, cosegregating with early-onset inflammatory bowel disease in a 13-year-old patient, which was inherited from his IBD mother . In FIG. Figure 1 (Panels A, B, and C) illustrates the identification of a truncation variant in the SIGIRR gene with dominant segregation in a family with Crohn's disease (CD). Panel A shows a table describing the truncating SIGIRR variant in c.557delA/p.K186fs*31 with maternal inheritance; the variant site has a neutral conservation score among species and is predicted to disrupt protein function; this variant has an alternating allele frequency of 0.000471 in the ExAC browser. Panel B shows the genealogy for an IBD-RV patient (Utah81427), his Crohn's disease-affected mother (Utah81428), and his unaffected father (Utah81429); colored symbols indicate individuals with CD, uncolored symbols indicate individuals without the disease; circles represent women and squares represent men. Panel C provides visual confirmation of the truncation-causing SIGIRR variant that segregates in CD-affected Utah81427 and his CD-affected mother, but is absent in his non-diseased father.

Example 4: Detection

The presence of a particular genetic variant in a subject may indicate that the subject is at increased risk of developing early-onset inflammatory bowel disease. A sample, such as a blood sample, may be obtained from the subject. Nucleic acids can be isolated from a sample using conventional nucleic acid extraction kits. After nucleic acid is isolated from a sample obtained from a subject, the nucleic acid is sequenced to determine whether it contains a genetic variant. The nucleic acid sequence can be compared to a control sequence (wild type sequence). Detection of a difference between the nucleic acid obtained from the sample obtained from the subject and the reference sequence indicates the presence of a genetic variant. These steps can be carried out as described in the examples above and throughout the text of this disclosure. The presence of one or more genetic variants indicates an increased risk for a subject to develop early-onset inflammatory bowel disease.

Example 5: Research

Materials and methods

Cell lines and culture conditions:

Epstein-Barr virus-transformed lymphoblastic cell lines (EBLs) were established from cells from pediatric IBD patients under IRB (Institutional Review Board) approval at the University of Utah Health Science Center. Healthy LCL were purchased from the American Type Culture Collection (ATCC; Manassas, VA). Cells were cultured in RPMI 1640 medium (Gibco product ID: 12633) supplemented with 10% fetal bovine serum (Gibco product ID: 10438026), 1X penicillin-streptomycin (Gibco product ID: 15140122) and 1X L-glutamine (Gibco product ID: 25030081 ).

Stimulation conditions:

LCLs derived from cells from healthy controls, a patient with SIGIRR loss of function, and LCLs from 4 IBD-RV patients without SIGIRR loss of function were stimulated with 2 mg/ml LPS (InvivoGen, San Diego, CA) for 72 hours or 2 mg/ml αIgM/αCD40 (Affymetrix, Santa Clara, CA) for 16 hours. Following stimulation, cell culture supernatants were collected and used to quantify pro-inflammatory cytokine secretion using the V-Plex Mesoscale Human Proinflammatory Cytokine Panel (K15049D) and quantified using the QuickPlex SQ 120 Mesoscale Assay and then complemented by RNA-seq.

Statistics:

Significance was determined using 2-way ANOVA, and PCA was calculated using results from 3 independent experiments using GraphPad PRISM (La Jolla, CA)

Results:

As illustrated in FIG. 2, LCLs generated from loss-of-function SIGIRR patient cells produced more IFN-γ, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, and TNF-α than healthy controls LCL or LCL from IBD-RV patients without SIGIRR with loss of function. Additionally, unstimulated LCL generated from loss-of-function SIGIRR patient cells secreted increased levels of IFN-γ, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70 and TNF-α compared with healthy LCL or some LCL from IBD-RV patients, indicating that SIGIRR loss-of-function LCL are constitutively active and resistant to LPS stimulation. In FIG. 2 blue bars indicate supernatants isolated from unstimulated cells; red bars indicate supernatants isolated from LPS-stimulated cells. “*” denotes p < 0.05 by two-way Anova; Error bars indicate PCA from 3 independent experiments.

As illustrated in FIG. 3, after stimulation with αIgM/αCD40, LCLs generated from SIGIRR loss-of-function patient cells produced more IFN-γ, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70 and TNF-α than healthy LCL or some LCL from IBD-RV patients without SIGIRR with loss of function. In addition, unstimulated LCL generated from SIGIRR loss-of-function patient cells secreted increased levels of IFN-γ, IL-2, IL-4, IL-6, IL-8, IL-10, and TNF-α compared to healthy controls. LCL or some LCL from IBD-RV patients, indicating that SIGIRR loss-of-function LCL are constitutively active and resistant to anti-IgM/anti-CD40 stimulation. In FIG. 3 blue bars indicate supernatants isolated from unstimulated cells; red bars indicate supernatants isolated from anti-IgM/anti-CD40 stimulated cells. “*” denotes p<0.05 by two-way Anova; Error bars indicate PCA from 3 independent experiments.

In unstimulated cells, up-regulation of key immune stimulators including IL-1β, IL-8 and IL-6 was observed in IBD-RV patient LCLs with a truncated (c.557delA; p.K186fs*31) SIGIRR compared to LCLs generated from healthy control cells, and with LCLs generated from cells from IBD-RV patients lacking SIGIRR loss-of-function (LF) variants. This observation supports a unique inflammatory signature in IBD patients harboring loss-of-function SIGIRR variants. Additionally, MCL from IBD-RV patients with SIGIRR have been observed to be resistant to IL-1β or TPR stimulation, suggesting constitutive activation of these proinflammatory pathways in the absence of SIGIRR.

--->

LIST OF SEQUENCES

<110> Regeneron Pharmaceuticals, Inc.

<120> INTERLEUKIN-1 RECEPTOR-RELATED PROTEIN VARIANTS

AND CONTAINING A SINGLE IMMUNOGLOBULIN DOMAIN (SIGIRR),

AND THEIR APPLICATION

<130> 189238.00602

<150> 62/554,857

<151> 2017-09-06

<160> 16

<170> PatentIn, version 3.5

<210> 1

<211> 11739

<212> DNA

<213> Homo sapiens

<220>

<223> wild-type SIGIRR genomic sequence

<400> 1

actgcggggc ggggccgcac ctgctgccgc tgccggggct acgccgccgc cggggctgtt 60

gccgctgcgc acctggctca ggtgagctgc cccgcccccg cccggcgcga gccccaggtc 120

ctggcagcag gtgagtggcg gtcgccatca gacccgcgaa ggtgagtgga ggtcgcagtc 180

ctcccagagc cgctgcaggc ctggagtcgg ggcccggggc gctgcttgtc cctgggcgcg 240

gtctctcagg aaatggctcg gggccgggga ggttcccgcg tcgcccggaa gcccgagctc 300

gcccgcgtcc tggccctttc cagggctggg ggaagggagg aaagtgggcg aagggggcgg 360

gggccaccct ttcctctgcc cgacgtggtc agcggctttt tggtgaccct cgcgcagaac 420

agagcggggc taggagcgcc gaggcgcgca gggatcccac ctcggtcgcc caagcctgga 480

tcccagctcg aggcttcctg aggcgccgag ggcggaccaa gagtcaggct ggggcccgag 540

ctcgcccggc gccgggtgga gcttccgcca tccgcgcgcc tctgccccgg ccgagcgccc 600

cgagccgcac ccccgtgccc gcctcccgcg cgcgtcctcg ttctccgccc tcctcacttg 660

gagggcgccc agggcgcagg tgggcgcggg gtaggggagc gaaccagagc ccctcggcct 720

gcggggccgc agccttttac ccacgttcct gcttcaggac aggcgctggg ggcccagcgc 780

gcacggggag cggaggggcg gcgcgtccga gctgagcggg agtcgcccgc agggtgctgg 840

gtgctgagcg tcgaacccac cgggcagggg cggccgcgga ccgaggacgc tggctgggtc 900

tgagggcggg aggagctggc gctttctaag aacctcaagg tgcccagcag agtgcggtgg 960

gcagtgcgag gcgaccaggg ccaggcttgg actgagattc tgggcgcctc ggaagagctg 1020

tgcgggcgag ggtcggagac tttgtgggtg ttcatgctca ccctggacgg tggacctggg 1080

ggctcggggg tgcaggacgt tagcagagag agggaaggag agggtgccaa gccagccctg 1140

agagatggtg acgctgagac cgggtgcaca gagcagccgc cgagccgggg cttcacagtt 1200

ggcggctgac cccagggcct gtgctggagg ccctgctcct gcttcctgcg ggggtgccct 1260

gggccctgct gtgcctgctt cctgacctgc agtgtgggga gaaccttctt gaggggcttc 1320

caaggaccca cttcaaggag gggctggtac caggctctgt gggggaaggg gtgctgtctt 1380

tgggaccctg agcccaaggc cccacatcac ccaaggctga gtaccgggac aaaggaaggt 1440

cctagcaggg ctgggactca gtcagacaag tacctgtaga aagcctgctg ctgagaggt 1500

gctcagccag ggccgggagg ctggatggca gccctccaca atgaagccct gcccccatgt 1560

ggctgagggc agggccaggg tccaaaggag acccttgtct gattctgtgg gcaataccag 1620

ccaccggtca ctgggtcccc agtcccaggt caacctgggg agtgtcagtt tggtgacaac 1680

aggagatgcc atctgcccct catctgctct gagccaggag cctcggccac ctgtgtttgc 1740

tccttatttg gaaaaggggg cgctgccctc ccaccccagc ctgaagggaa ggtccacaag 1800

atgccatagt cccttgtaac tgctcaccca tcgggggagc tatgtctggg agagggacgg 1860

ggagagggcg gaagagttct gctggagccc cattgttgac cgggagtttc tgaaggccct 1920

gttccccggg gtcagaggtc aaagacacat gggggagcaa accggcccct tggctcctga 1980

gtgtgagctc tagtagcgtc tggaaggcat gccgcgcggt ccccggtcct ccttgtatgt 2040

gtgcagggtg gccctggcgg gggtctcctg gctggaggac cccccatgca ttacccctgg 2100

aacctgcccc tttgagcacc ccccacccca caacacacac gcacacacac aaacgcacgc 2160

acacgcacat actgcacaca cattgcacac acgcagacac actgcacaca cacacacaca 2220

cacacacaca cacacacaca cacacacgct gcacagagga aagtgccgtg cctgggtggg 2280

ttggggcggg ctccagagga aggagccgct tgactcagag ctacaggaag agccggggca 2340

ggcggggtga gaaccaccaa ctgcccgtcc tcccgcccgc ctagggaagg ggtgggtgcc 2400

tgggtggggt ttagggttag gagaggatgt gactgcaagg cccaggaagct acagggaaag 2460

ttctgatcaa aatacacaaa gacacaagcc aggtccccac cgcgcttggc gatgcatcca 2520

tagcagcctt ggtgctgtgg acactccctg gggcccagcg aaggggagag tttgctccca 2580

aaggcgcacc aatgaccaac atttgccccc cggaggaaag aactggaacc aggtgactgc 2640

cttccccgct gctctgagtg agcctgtgtc agggcctggc tgagaggagg cccacggcgc 2700

ccaagaccca tatgcatgtg tgtgcatgtg tgtgtgagac agagcgaggg gcagcaggag 2760

gctgtgtcgc atcagcggcc aagggtgggt gtcaggtgca gtgtgtgtgc tgtggggtgc 2820

ctgtggtcct aggctctgtg acaagcgttg caatgggtgt tgtatatact caacagttgt 2880

gtgaggggtc tgcagcctaa gctgtgtgca gtggtaatgg gaggtgtgtg tggtaggaag 2940

tgtgctgtgt aggaggtgca agtgtgcaca cgggctgaga ctgaagctct gtgcaggtgc 3000

acatgggcca gagggctgtg tgtatgtgat gtgtgcaggg gaggaggggc agaggagagg 3060

gtgcagggga agttagggag gggagggggt acaaggaaag gtgtgcaggg gagagggtgc 3120

agggaaaggt ggggagggga gagggtgcag gggaaggtag gcagaggaga gggtgcaggg 3180

gagagggtgc aggggaaggt gtgcagggaa aggtggggag ggggagaggt gcaggggaag 3240

gtaggcagag gagagggtgc aggggaaggt gtgcagggga gagggtgcag ggaaaggtgg 3300

ggaggggaga gggtgcaggg gaaggtaggc agaggagagg gtgcagggga aggtgtgcag 3360

ggaaaggtgg ggagggagag ggtgcagggg aaggtaggca gaggagagag tgcaggggaa 3420

ggtgtgcagg ggagagggtg cagggaaagg tggggagggg agagggtgca ggggaaggta 3480

ggcagacgag agtacaggga aaggtaggga ggagagaagg tgcaggagga ggcaggcaga 3540

agagaggtg caggggaagg tggggagggg agaggtgca gggggaggca ggcagaggag 3600

agggttcagg ggagagggtg caggggaagg tgggcagggg aaggtgggga gtggagagcg 3660

tgcaggggga ggcaggcaga ggagagggtg caggggaagg tgcgtagggg agagggagca 3720

ggggagaggg tgcgggaaga gggtgcaggg gagggtttgc agggaggg tgcaggggag 3780

agggtgcaag gagaggcagg cagcggagag ggtgcagagg agggtttgca ggggagggca 3840

cctgctttct tgctgtgctc gtatcctggc agtctgccct tgacttccag aaggctctga 3900

ggctgctttt ggagggagga ctttgaggtc tgagcttggg gcggtcacgg aagcactgca 3960

ggagatgcca ggggttctgc tgggctggcg gtgtgacctc ccgggcttgc tgtgaggggt 4020

tggtttcctg ggaagtgtgt gctttaagga ccctcagtgg gtggttcccc tgggtcaagg 4080

actttccttg gagtgtcctc caccgcaaag gacttcttca aggaccccct ggccatcccc 4140

catgggtgtg tgcctcatgc tgtatcggtt cccgaggaaa ctgccaaaca gtcaaacctg 4200

gggcctctgg ctgtgcttca cccatggaga gggaataggt ggcaggctaa gaggactgtc 4260

cctgccactg cctctggggc cttagcgggg aggaatcctg agggagctgg ggtgcagaaa 4320

cttaggcttg atccccacat ccccacttcc actgtgtccc tgcagcaggg aggggagtag 4380

acagtcagtg cctgttggat aaacagggtg accctgtggc atccagtgag gagaggcccc 4440

tggggccctg ggctgccaag gaagaggctc tgggcttctc cctgcagcca cctcactgag 4500

gagctttggg agacttgagg tgtgtgtggc tgctgctacg ggctgggaaa ggacatggga 4560

cccgagacct ccatccccac cgggcgctgt tcaccctccg gcctcacaac cgcagggcga 4620

ggctaagctg agggttcccg aggtgtttcc cgaggtgttt cgaggtgtct gtgctcacgt 4680

gcactcctga ctgtaaccag acgtgggcca gagctgggca ccccccctcg actgcatcca 4740

gacatggtta gagctgggca cccccccccc ccgactgtat ccagacatgg tcagagctga 4800

gcacccctcc ccgactgtat ccagacatgt tcagagctgg gcaccccccc ccgactgtat 4860

ccagacatgt tcagagctgg gcaccccccc ccgactgtat ccagacatgg tcagagctgg 4920

gcacccccccc ccgactgtat ccagacatgg tcagagctgg gcaccccccg actgcatcca 4980

gacatggtca gagctgagca cccccccccg cccgactgta tccagacatg gtcagagctg 5040

ggcacccccc gactgcatcc agacatggtc agagctgggc acccccctcc ccgactgcat 5100

tcagacatgg tcagagctgg gcacccccgc cgactgcatc cagacatggt cagagttggg 5160

cacccccccc ccgactgtat ccagacatgg tcagagctgg gcaccccccg actgcatcca 5220

gacatggtca gagctgggca cccctccccg actgtatcca gacatggtca gagctgggca 5280

cccccccccg actgtatcca gacatggtca gagctgggca cctccccccg actgtatcca 5340

gacatggtca gagctgggca cctcccccga ctgcatccag acatggtcag agctgggcac 5400

cccccccgac tgcatccaga catggtcaga gctgggcacc cccccccccc actgtatcca 5460

gacatggtca gagctgggca cccccctccc cgactgcatc cagacatggt cagagctgag 5520

cacccccccc ccccgactgt atccagacat ggtcagagct gggcaccccc ctccccgact 5580

gtatccagac atggtcagag ctgagccccc cccccgcccg actgtatcca gacatggtca 5640

gagctgggca ccccccgact gcatccagac atggtcagag ctgagcaccc ctccccgact 5700

gcatccagac atggtcagag ctgggcaccc ccccccccca ccgactgtat ccagacatgg 5760

tcagagctgg gcaaccccca ccgactgtat ccagacatgg tcagagctgg gcaccccccg 5820

actgcatcca gacatggtca gagctgggca ccccccgccg actgtatcca gacatggtca 5880

gagctgggca cccccctccc cgactgcatc cagacatggt cagagctggg cacccccctc 5940

cccgactgca tccagacatg gtcagagctg ggcacccctc cccgactgta tccagacatg 6000

gtcagagctg ggcacccccc tccccgactg tatccagaca tggtcagagc tgggcacccc 6060

cgccgactgt atccagacat ggtcagagct gggcaccccc cgactgcatc cagacatggt 6120

cagagctggg cacccctccc cgactgtatc cagacatggt cagagctggg cacccccccc 6180

ccccgactgt atccagacat ggtcagagct gagcacccct ccccgactgt atccagacat 6240

gttcagagct gggcaccccc ccccccgact gtatccagac atggtcagag ctgggcaccc 6300

ccctccccga ctgcatccag acatggtcag agctgagcac cccccccgcc cgactgcatc 6360

cagacatggt cagagctggg caccccccga ctgcatccag acatggtcag agctgggcac 6420

ccccctcccc gactgcattc agacatggtc agagctgggc acccccctcc ccgactgcat 6480

ccagacatgg tcagagctgg gcacccccgc cgactgtatc cagacatggt cagagctggg 6540

cacccccctc cccgactgta tccagacatg gtcagagctg ggcacccccg ccgactgtat 6600

ccagacatgg tcagagctgg gcacccccct ccccgactgc atccagacat gttcagagct 6660

gggcaccccc ccccgactgt atccagacat ggtcagagct gggcaccccc cgactgcatc 6720

cagacatggt cagagctggg caaccccctc cctgactgca tccagacatg gtcagagctg 6780

ggcacccccc ccggctgtat ccagacatgg tcagagctgg gcaccccccc cccccgactg 6840

catccagaca tggtcagagc taagcaaagc cccccgccga ctgtatccag acatggtcag 6900

agctgggcac ccccacccca actgcatcca gacatggtca gagctgagca ccccccaccg 6960

actgtatcca gacatggtca gagctgggca tctcccatgg tgtaggttac atcactcaca 7020

cccctgcccc aaggccaggc tgtgtctctg gcactgcagc accccctgca gtggcagctc 7080

tgttgaaggg gacagccagg agtgaggcca actgccatgt cgggagacag ggtcccacct 7140

agtgttcgtc cactgcaccc tggggccgtc atggtggccc cagggccggt ggaggtgatc 7200

ctggacttga tggcccctcc ccgtggtcct gggctgcgtg aagtggtgcc cattctggcc 7260

acgtgtgagg ggctcaaaga gacaccctgc tggcctccga gaactctgcc agcatccatg 7320

cggctgctca gggtcgcatc tggtcagggc cagtgctgtc tgtggccggc agttactggt 7380

taactcatcc tgttcctgtt taacttgttc cctggggatg gcggcagcct ctgacctgtc 7440

caggtgccct gtccagctga ctgcaaggac agagaggagt cctgcccagc tcttggatca 7500

gtctgctggc cgaggagccc ggtggagcca ggggtgaccc tggagcccag cctgccccga 7560

ggaggccccg gctcagagcc atgccaggta ggcaggtcag agagggatgg gcttgaattc 7620

cccactccca cctcctgccc ctcaggctgg ctccacattg tgcatcctgc tcttatccca 7680

ggcactctgc ctggccacat gccgggggta caaaggcccc tgctcacccc tcccactggg 7740

cccttggccc tgaaaccagg gcgagtggtg aggatgagcc ctgcaccctc tcctgcccca 7800

gaccaggctc atgagggtca gtaaagccca ggaatgctag gagtgggcac tgctggggcc 7860

cacagcaggg tgtggccgat gtgagctggc aggagaccat accctactga gaagatggtg 7920

gtttgacctc ccgctagggc tcatcaccct gatgtaaaga tgaacaagtt caggcctcag 7980

gtggtgggga gggcttgcct gtggccagga gcagaggggcc ccaagacccc caaggcagga 8040

atgaggccaa ctgctccagg ctcccagctg tgcccatcca ggtggaaggt gctggcacac 8100

tgggcatctg ggtccccaaa cccactggtg cccacactct ctgcagattc cccagcagag 8160

gcaggccagg ctgaggggtg cagtgcccgg cccctcctat gagacaggca tcatggtgtc 8220

accaggggtg ggcaacaggt gcatctggga aatggggggc tggactcaca tcctgggtgg 8280

acaagcagcc agatggggct tgagcgtggg cctggggcag ctcaactgtg gccactgctc 8340

tctgtcccct cagcacaggg tgggcggaac acccaaagcc tggccactac tggggaaaga 8400

cgtgccaagt gctaagcctg tccccatggg actcgtggac agacatggtg tgactgccaa 8460

gccacacaga ggccttattt cttcctgcac cacggaccac tgtggaggtg gtggggccag 8520

gcaccctggc acagctgacc cactgtgtcc tctctcttca ggtgtctgtg atagggcccc 8580

tgacttcctc tccccgtctg aagaccaggt gctgaggcct gccttgggca gctcagtggc 8640

tctgaactgc acggcttggg tagtctctgg gccccactgc tccctgcctt cagtccagtg 8700

gctgaaagac gggcttccat tgggaattgg gggccactac agcctccacg agtactcctg 8760

gtaagagacc cgggtatcca gggtcagagt gacctctcag acatgccaag ggcagtggtc 8820

tccctgacct atgcctgggt ctgtaaccac ctggccagac atgtcacgag cagagtgcct 8880

ctgtggaccc agacggaggg tcagtaatgt cagggacaga cccttgggtc tgcatggccc 8940

ctgtaggcac acacctcgta actgcccagg gctacgggtg tcagcaaagt ctagggtctg 9000

aatgaacacc gaccagctgg ccaagtctgt gatcaaagga cctccaggcc ccagacccag 9060

gactgggacg gctgttctga aacaaatctg catcccctcc aggttcaggt cacttctctt 9120

ggcttaaggc ccaagaacca cccaggaggc ccaattctgc agcctccctg ggtgaaccca 9180

gacaggtggg gttcggggggt gtccactggt atccccagtc gaccctgacc ctggcttggt 9240

atccccaggg tcaaggccaa cctgtcagag gtgcttgtgt ccagtgtcct gggggtcaac 9300

gtgaccagca ctgaagtcta tggggccttc acctgctcca tccagaacat cagcttctcc 9360

tccttcactc ttcagagagc tggtgatggg ggttccccaa ggtggagggt agaaggggac 9420

ctagacctag tgaggcccag ggatcttgag gcttgggggc tgctggaggg gcagcggctc 9480

aggcaaccca tccgcaggcc ctacaagcca cgtggctgcg gtgctggcct ccctcctggt 9540

cctgctggcc ctgctgctgg ccgccctgct ctatgtcaag tgccgtctca acgtgctgct 9600

ctggtaccag gacgcgtatg gggaggtgga gataaacggt gcgtggggcc cgcgtgggcg 9660

cgaggagggg tcgccacggc ctccctgaga gtgcgctgct gagctcggct ttggaggcgt 9720

gcggcgggga ggggttagcc cccggggtgct ctgtgcggcc cggctggggt taaagtccgg 9780

ggcgggtctg cccctgtgca cgtgggagat ggttaggggt gttggggacc ccgagggctg 9840

gtgtgaggcc tcgggagcca tcggggtgac tggccgccgt ccgcagacgg gaagctctac 9900

gacgcctacg tctcctacag cgactgcccc gaggaccgca agttcgtgaa cttcatccta 9960

aagccgcagc tggagcggcg tcggggctac aagctcttcc tggacgaccg cgacctcctg 10020

ccgcgcgctg gtatcccggg ccccaccccg tgccccgccc acccggaggg cccgccccgc 10080

cccgccccat gctccgtccc acccggggcc ccacccccac catcgagctc cgcccccatc 10140

cccgcccacc cgaggccccg cctcaaaaac ccgcccaccc cgcaggcccc gcccctccct 10200

tagagctctg cgtccggcac cgcccctgag cctccggccc cgccctcccc cgcccccgcc 10260

cctgcgccgc cgacgcccgc cctcccgcag agccctccgc cgacctcttg gtgaacctga 10320

gccgctgccg acgcctcatc gtggtgcttt cggacgcctt cctgagccgg gcctggtgca 10380

gccacagctt ccggtgggtc ccgcgcgggg ttgggtgggc cccagcgtag cacccacccc 10440

cctgacggtc ccgccccgca gggagggcct gtgccggctg ctggagctca cccgcagacc 10500

catcttcatc accttcgagg gccagaggcg cgaccccgcg cacccggcgc tccgcctgct 10560

gcgccagcac cgccacctgg tgaccttgct gctctggagg cccggctccg tggtgcggag 10620

caggcgcggg agggtccggg gctagcggcg ggttagagat gggcggtgcc cgggctccag 10680

gctgggaccc ctccgtgggg agctctgcgg caccacgctt tgtgaatggg ccctgggggg 10740

aggttccgct gcctggggcc ccgatgcggg gagccgccct tgaggccccc ggagccacgg 10800

aatagctgtc gcagggcgtg gaacccgtgg gcagccgcag gtgtgctctt gggggccagg 10860

acgccagggg cttccgaggt gttcacacct gcaaaccgcc ccgacctggc ccccagactc 10920

cttcctccga tttttggaaa gaagtgcagc tggcgctgcc gcggaaggtg cagtacaggc 10980

ctgtggaagg agacccccag acgcagctgc aggacgacaa ggaccccatg ctgattcttc 11040

gaggccgagt ccctgagggc cgggccctgg actcagaggt ggacccggac cctgagggcg 11100

acctgggtat gcccgcccag ccccactccc caactggaga agctcagcac agggcggagt 11160

gggggcaggc acagggcaca gggcctggag gggctccagg tgttgaggac tcttcccggc 11220

accgggagcc cctgcacggc ctctgccctg gaggtgctcg gccctcggtc tgcctgggaa 11280

cttcctgggc ctcacaggcc atcacagcag ggggtgagca ggggcagccc ctggcagtgg 11340

gtctgggcca aggctgtggg tggccacctc aggcgtctcg gtctccccac cccaggtgtc 11400

cggggggcctg tctttggaga gccatcagct ccaccgcaca ccagtggggt ctcgctggga 11460

gagagccgga gcagcgaagt ggacgtctcg gatctcggct cgcgaaacta cagtgcccgc 11520

acagacttct actgcctggt gtccaaggat gatatgtagc tcccacccca gagtgcagga 11580

tcatagggac agcggggggcc agggcagcgg cgtcgctcct ctgctcaaca ggaccacaac 11640

ccctgccagc agccctggga ccctgccagc agccctggga aaaggctgtg gcctcagggc 11700

gcctcccagt gccagaaaat aaagtccttt tggattctg 11739

<210> 2

<211> 11738

<212> DNA

<213> Homo sapiens

<220>

<223> Genomic sequence of variant SIGIRR(c.557delA)

<400> 2

actgcggggc ggggccgcac ctgctgccgc tgccggggct acgccgccgc cggggctgtt 60

gccgctgcgc acctggctca ggtgagctgc cccgcccccg cccggcgcga gccccaggtc 120

ctggcagcag gtgagtggcg gtcgccatca gacccgcgaa ggtgagtgga ggtcgcagtc 180

ctcccagagc cgctgcaggc ctggagtcgg ggcccggggc gctgcttgtc cctgggcgcg 240

gtctctcagg aaatggctcg gggccgggga ggttcccgcg tcgcccggaa gcccgagctc 300

gcccgcgtcc tggccctttc cagggctggg ggaagggagg aaagtgggcg aagggggcgg 360

gggccaccct ttcctctgcc cgacgtggtc agcggctttt tggtgaccct cgcgcagaac 420

agagcggggc taggagcgcc gaggcgcgca gggatcccac ctcggtcgcc caagcctgga 480

tcccagctcg aggcttcctg aggcgccgag ggcggaccaa gagtcaggct ggggcccgag 540

ctcgcccggc gccgggtgga gcttccgcca tccgcgcgcc tctgccccgg ccgagcgccc 600

cgagccgcac ccccgtgccc gcctcccgcg cgcgtcctcg ttctccgccc tcctcacttg 660

gagggcgccc agggcgcagg tgggcgcggg gtaggggagc gaaccagagc ccctcggcct 720

gcggggccgc agccttttac ccacgttcct gcttcaggac aggcgctggg ggcccagcgc 780

gcacggggag cggaggggcg gcgcgtccga gctgagcggg agtcgcccgc agggtgctgg 840

gtgctgagcg tcgaacccac cgggcagggg cggccgcgga ccgaggacgc tggctgggtc 900

tgagggcggg aggagctggc gctttctaag aacctcaagg tgcccagcag agtgcggtgg 960

gcagtgcgag gcgaccaggg ccaggcttgg actgagattc tgggcgcctc ggaagagctg 1020

tgcgggcgag ggtcggagac tttgtgggtg ttcatgctca ccctggacgg tggacctggg 1080

ggctcggggg tgcaggacgt tagcagagag agggaaggag agggtgccaa gccagccctg 1140

agagatggtg acgctgagac cgggtgcaca gagcagccgc cgagccgggg cttcacagtt 1200

ggcggctgac cccagggcct gtgctggagg ccctgctcct gcttcctgcg ggggtgccct 1260

gggccctgct gtgcctgctt cctgacctgc agtgtgggga gaaccttctt gaggggcttc 1320

caaggaccca cttcaaggag gggctggtac caggctctgt gggggaaggg gtgctgtctt 1380

tgggaccctg agcccaaggc cccacatcac ccaaggctga gtaccgggac aaaggaaggt 1440

cctagcaggg ctgggactca gtcagacaag tacctgtaga aagcctgctg ctgagaggt 1500

gctcagccag ggccgggagg ctggatggca gccctccaca atgaagccct gcccccatgt 1560

ggctgagggc agggccaggg tccaaaggag acccttgtct gattctgtgg gcaataccag 1620

ccaccggtca ctgggtcccc agtcccaggt caacctgggg agtgtcagtt tggtgacaac 1680

aggagatgcc atctgcccct catctgctct gagccaggag cctcggccac ctgtgtttgc 1740

tccttatttg gaaaaggggg cgctgccctc ccaccccagc ctgaagggaa ggtccacaag 1800

atgccatagt cccttgtaac tgctcaccca tcgggggagc tatgtctggg agagggacgg 1860

ggagagggcg gaagagttct gctggagccc cattgttgac cgggagtttc tgaaggccct 1920

gttccccggg gtcagaggtc aaagacacat gggggagcaa accggcccct tggctcctga 1980

gtgtgagctc tagtagcgtc tggaaggcat gccgcgcggt ccccggtcct ccttgtatgt 2040

gtgcagggtg gccctggcgg gggtctcctg gctggaggac cccccatgca ttacccctgg 2100

aacctgcccc tttgagcacc ccccacccca caacacacac gcacacacac aaacgcacgc 2160

acacgcacat actgcacaca cattgcacac acgcagacac actgcacaca cacacacaca 2220

cacacacaca cacacacaca cacacacgct gcacagagga aagtgccgtg cctgggtggg 2280

ttggggcggg ctccagagga aggagccgct tgactcagag ctacaggaag agccggggca 2340

ggcggggtga gaaccaccaa ctgcccgtcc tcccgcccgc ctagggaagg ggtgggtgcc 2400

tgggtggggt ttagggttag gagaggatgt gactgcaagg cccaggaagct acagggaaag 2460

ttctgatcaa aatacacaaa gacacaagcc aggtccccac cgcgcttggc gatgcatcca 2520

tagcagcctt ggtgctgtgg acactccctg gggcccagcg aaggggagag tttgctccca 2580

aaggcgcacc aatgaccaac atttgccccc cggaggaaag aactggaacc aggtgactgc 2640

cttccccgct gctctgagtg agcctgtgtc agggcctggc tgagaggagg cccacggcgc 2700

ccaagaccca tatgcatgtg tgtgcatgtg tgtgtgagac agagcgaggg gcagcaggag 2760

gctgtgtcgc atcagcggcc aagggtgggt gtcaggtgca gtgtgtgtgc tgtggggtgc 2820

ctgtggtcct aggctctgtg acaagcgttg caatgggtgt tgtatatact caacagttgt 2880

gtgaggggtc tgcagcctaa gctgtgtgca gtggtaatgg gaggtgtgtg tggtaggaag 2940

tgtgctgtgt aggaggtgca agtgtgcaca cgggctgaga ctgaagctct gtgcaggtgc 3000

acatgggcca gagggctgtg tgtatgtgat gtgtgcaggg gaggaggggc agaggagagg 3060

gtgcagggga agttagggag gggagggggt acaaggaaag gtgtgcaggg gagagggtgc 3120

agggaaaggt ggggagggga gagggtgcag gggaaggtag gcagaggaga gggtgcaggg 3180

gagagggtgc aggggaaggt gtgcagggaa aggtggggag ggggagaggt gcaggggaag 3240

gtaggcagag gagagggtgc aggggaaggt gtgcagggga gagggtgcag ggaaaggtgg 3300

ggaggggaga gggtgcaggg gaaggtaggc agaggagagg gtgcagggga aggtgtgcag 3360

ggaaaggtgg ggagggagag ggtgcagggg aaggtaggca gaggagagag tgcaggggaa 3420

ggtgtgcagg ggagagggtg cagggaaagg tggggagggg agagggtgca ggggaaggta 3480

ggcagacgag agtacaggga aaggtaggga ggagagaagg tgcaggagga ggcaggcaga 3540

agagaggtg caggggaagg tggggagggg agaggtgca gggggaggca ggcagaggag 3600

agggttcagg ggagagggtg caggggaagg tgggcagggg aaggtgggga gtggagagcg 3660

tgcaggggga ggcaggcaga ggagagggtg caggggaagg tgcgtagggg agagggagca 3720

ggggagaggg tgcgggaaga gggtgcaggg gagggtttgc agggaggg tgcaggggag 3780

agggtgcaag gagaggcagg cagcggagag ggtgcagagg agggtttgca ggggagggca 3840

cctgctttct tgctgtgctc gtatcctggc agtctgccct tgacttccag aaggctctga 3900

ggctgctttt ggagggagga ctttgaggtc tgagcttggg gcggtcacgg aagcactgca 3960

ggagatgcca ggggttctgc tgggctggcg gtgtgacctc ccgggcttgc tgtgaggggt 4020

tggtttcctg ggaagtgtgt gctttaagga ccctcagtgg gtggttcccc tgggtcaagg 4080

actttccttg gagtgtcctc caccgcaaag gacttcttca aggaccccct ggccatcccc 4140

catgggtgtg tgcctcatgc tgtatcggtt cccgaggaaa ctgccaaaca gtcaaacctg 4200

gggcctctgg ctgtgcttca cccatggaga gggaataggt ggcaggctaa gaggactgtc 4260

cctgccactg cctctggggc cttagcgggg aggaatcctg agggagctgg ggtgcagaaa 4320

cttaggcttg atccccacat ccccacttcc actgtgtccc tgcagcaggg aggggagtag 4380

acagtcagtg cctgttggat aaacagggtg accctgtggc atccagtgag gagaggcccc 4440

tggggccctg ggctgccaag gaagaggctc tgggcttctc cctgcagcca cctcactgag 4500

gagctttggg agacttgagg tgtgtgtggc tgctgctacg ggctgggaaa ggacatggga 4560

cccgagacct ccatccccac cgggcgctgt tcaccctccg gcctcacaac cgcagggcga 4620

ggctaagctg agggttcccg aggtgtttcc cgaggtgttt cgaggtgtct gtgctcacgt 4680

gcactcctga ctgtaaccag acgtgggcca gagctgggca ccccccctcg actgcatcca 4740

gacatggtta gagctgggca cccccccccc ccgactgtat ccagacatgg tcagagctga 4800

gcacccctcc ccgactgtat ccagacatgt tcagagctgg gcaccccccc ccgactgtat 4860

ccagacatgt tcagagctgg gcaccccccc ccgactgtat ccagacatgg tcagagctgg 4920

gcacccccccc ccgactgtat ccagacatgg tcagagctgg gcaccccccg actgcatcca 4980

gacatggtca gagctgagca cccccccccg cccgactgta tccagacatg gtcagagctg 5040

ggcacccccc gactgcatcc agacatggtc agagctgggc acccccctcc ccgactgcat 5100

tcagacatgg tcagagctgg gcacccccgc cgactgcatc cagacatggt cagagttggg 5160

cacccccccc ccgactgtat ccagacatgg tcagagctgg gcaccccccg actgcatcca 5220

gacatggtca gagctgggca cccctccccg actgtatcca gacatggtca gagctgggca 5280

cccccccccg actgtatcca gacatggtca gagctgggca cctccccccg actgtatcca 5340

gacatggtca gagctgggca cctcccccga ctgcatccag acatggtcag agctgggcac 5400

cccccccgac tgcatccaga catggtcaga gctgggcacc cccccccccc actgtatcca 5460

gacatggtca gagctgggca cccccctccc cgactgcatc cagacatggt cagagctgag 5520

cacccccccc ccccgactgt atccagacat ggtcagagct gggcaccccc ctccccgact 5580

gtatccagac atggtcagag ctgagccccc cccccgcccg actgtatcca gacatggtca 5640

gagctgggca ccccccgact gcatccagac atggtcagag ctgagcaccc ctccccgact 5700

gcatccagac atggtcagag ctgggcaccc ccccccccca ccgactgtat ccagacatgg 5760

tcagagctgg gcaaccccca ccgactgtat ccagacatgg tcagagctgg gcaccccccg 5820

actgcatcca gacatggtca gagctgggca ccccccgccg actgtatcca gacatggtca 5880

gagctgggca cccccctccc cgactgcatc cagacatggt cagagctggg cacccccctc 5940

cccgactgca tccagacatg gtcagagctg ggcacccctc cccgactgta tccagacatg 6000

gtcagagctg ggcacccccc tccccgactg tatccagaca tggtcagagc tgggcacccc 6060

cgccgactgt atccagacat ggtcagagct gggcaccccc cgactgcatc cagacatggt 6120

cagagctggg cacccctccc cgactgtatc cagacatggt cagagctggg cacccccccc 6180

ccccgactgt atccagacat ggtcagagct gagcacccct ccccgactgt atccagacat 6240

gttcagagct gggcaccccc ccccccgact gtatccagac atggtcagag ctgggcaccc 6300

ccctccccga ctgcatccag acatggtcag agctgagcac cccccccgcc cgactgcatc 6360

cagacatggt cagagctggg caccccccga ctgcatccag acatggtcag agctgggcac 6420

ccccctcccc gactgcattc agacatggtc agagctgggc acccccctcc ccgactgcat 6480

ccagacatgg tcagagctgg gcacccccgc cgactgtatc cagacatggt cagagctggg 6540

cacccccctc cccgactgta tccagacatg gtcagagctg ggcacccccg ccgactgtat 6600

ccagacatgg tcagagctgg gcacccccct ccccgactgc atccagacat gttcagagct 6660

gggcaccccc ccccgactgt atccagacat ggtcagagct gggcaccccc cgactgcatc 6720

cagacatggt cagagctggg caaccccctc cctgactgca tccagacatg gtcagagctg 6780

ggcacccccc ccggctgtat ccagacatgg tcagagctgg gcaccccccc cccccgactg 6840

catccagaca tggtcagagc taagcaaagc cccccgccga ctgtatccag acatggtcag 6900

agctgggcac ccccacccca actgcatcca gacatggtca gagctgagca ccccccaccg 6960

actgtatcca gacatggtca gagctgggca tctcccatgg tgtaggttac atcactcaca 7020

cccctgcccc aaggccaggc tgtgtctctg gcactgcagc accccctgca gtggcagctc 7080

tgttgaaggg gacagccagg agtgaggcca actgccatgt cgggagacag ggtcccacct 7140

agtgttcgtc cactgcaccc tggggccgtc atggtggccc cagggccggt ggaggtgatc 7200

ctggacttga tggcccctcc ccgtggtcct gggctgcgtg aagtggtgcc cattctggcc 7260

acgtgtgagg ggctcaaaga gacaccctgc tggcctccga gaactctgcc agcatccatg 7320

cggctgctca gggtcgcatc tggtcagggc cagtgctgtc tgtggccggc agttactggt 7380

taactcatcc tgttcctgtt taacttgttc cctggggatg gcggcagcct ctgacctgtc 7440

caggtgccct gtccagctga ctgcaaggac agagaggagt cctgcccagc tcttggatca 7500

gtctgctggc cgaggagccc ggtggagcca ggggtgaccc tggagcccag cctgccccga 7560

ggaggccccg gctcagagcc atgccaggta ggcaggtcag agagggatgg gcttgaattc 7620

cccactccca cctcctgccc ctcaggctgg ctccacattg tgcatcctgc tcttatccca 7680

ggcactctgc ctggccacat gccgggggta caaaggcccc tgctcacccc tcccactggg 7740

cccttggccc tgaaaccagg gcgagtggtg aggatgagcc ctgcaccctc tcctgcccca 7800

gaccaggctc atgagggtca gtaaagccca ggaatgctag gagtgggcac tgctggggcc 7860

cacagcaggg tgtggccgat gtgagctggc aggagaccat accctactga gaagatggtg 7920

gtttgacctc ccgctagggc tcatcaccct gatgtaaaga tgaacaagtt caggcctcag 7980

gtggtgggga gggcttgcct gtggccagga gcagaggggcc ccaagacccc caaggcagga 8040

atgaggccaa ctgctccagg ctcccagctg tgcccatcca ggtggaaggt gctggcacac 8100

tgggcatctg ggtccccaaa cccactggtg cccacactct ctgcagattc cccagcagag 8160

gcaggccagg ctgaggggtg cagtgcccgg cccctcctat gagacaggca tcatggtgtc 8220

accaggggtg ggcaacaggt gcatctggga aatggggggc tggactcaca tcctgggtgg 8280

acaagcagcc agatggggct tgagcgtggg cctggggcag ctcaactgtg gccactgctc 8340

tctgtcccct cagcacaggg tgggcggaac acccaaagcc tggccactac tggggaaaga 8400

cgtgccaagt gctaagcctg tccccatggg actcgtggac agacatggtg tgactgccaa 8460

gccacacaga ggccttattt cttcctgcac cacggaccac tgtggaggtg gtggggccag 8520

gcaccctggc acagctgacc cactgtgtcc tctctcttca ggtgtctgtg atagggcccc 8580

tgacttcctc tccccgtctg aagaccaggt gctgaggcct gccttgggca gctcagtggc 8640

tctgaactgc acggcttggg tagtctctgg gccccactgc tccctgcctt cagtccagtg 8700

gctgaaagac gggcttccat tgggaattgg gggccactac agcctccacg agtactcctg 8760

gtaagagacc cgggtatcca gggtcagagt gacctctcag acatgccaag ggcagtggtc 8820

tccctgacct atgcctgggt ctgtaaccac ctggccagac atgtcacgag cagagtgcct 8880

ctgtggaccc agacggaggg tcagtaatgt cagggacaga cccttgggtc tgcatggccc 8940

ctgtaggcac acacctcgta actgcccagg gctacgggtg tcagcaaagt ctagggtctg 9000

aatgaacacc gaccagctgg ccaagtctgt gatcaaagga cctccaggcc ccagacccag 9060

gactgggacg gctgttctga aacaaatctg catcccctcc aggttcaggt cacttctctt 9120

ggcttaaggc ccaagaacca cccaggaggc ccaattctgc agcctccctg ggtgaaccca 9180

gacaggtggg gttcggggggt gtccactggt atccccagtc gaccctgacc ctggcttggt 9240

atccccaggg tcaaggccaa cctgtcagag gtgcttgtgt ccagtgtcct gggggtcaac 9300

gtgaccagca ctgaagtcta tggggccttc acctgctcca tccagaacat cagcttctcc 9360

tccttcactc ttcagagagc tggtgatggg ggttccccaa ggtggagggt agaaggggac 9420

ctagacctag tgaggcccag ggatcttgag gcttgggggc tgctggaggg gcagcggctc 9480

aggcaaccca tccgcaggcc ctacaagcca cgtggctgcg gtgctggcct ccctcctggt 9540

cctgctggcc ctgctgctgg ccgccctgct ctatgtcaag tgccgtctca acgtgctgct 9600

ctggtaccag gacgcgtatg gggaggtgga gataaacggt gcgtggggcc cgcgtgggcg 9660

cgaggagggg tcgccacggc ctccctgaga gtgcgctgct gagctcggct ttggaggcgt 9720

gcggcgggga ggggttagcc cccggggtgct ctgtgcggcc cggctggggt taaagtccgg 9780

ggcgggtctg cccctgtgca cgtgggagat ggttaggggt gttggggacc ccgagggctg 9840

gtgtgaggcc tcgggagcca tcggggtgac tggccgccgt ccgcagacgg gaagctctac 9900

gacgcctacg tctcctacag cgactgcccc gaggaccgca agttcgtgaa cttcatccta 9960

agccgcagct ggagcggcgt cggggctaca agctcttcct ggacgaccgc gacctcctgc 10020

cgcgcgctgg tatcccgggc cccaccccgt gccccgccca cccggagggc ccgccccgcc 10080

ccgccccatg ctccgtccca cccggggccc cacccccacc atcgagctcc gcccccatcc 10140

ccgcccaccc gaggccccgc ctcaaaaacc cgcccacccc gcaggccccg cccctccctt 10200

agagctctgc gtccggcacc gcccctgagc ctccggcccc gccctccccc gcccccgccc 10260

ctgcgccgcc gacgcccgcc ctcccgcaga gccctccgcc gacctcttgg tgaacctgag 10320

ccgctgccga cgcctcatcg tggtgctttc ggacgccttc ctgagccggg cctggtgcag 10380

ccacagcttc cggtgggtcc cgcgcggggt tgggtggggcc ccagcgtagc acccaccccc 10440

ctgacggtcc cgccccgcag ggagggcctg tgccggctgc tggagctcac ccgcagaccc 10500

atcttcatca ccttcgaggg ccagaggcgc gaccccgcgc acccggcgct ccgcctgctg 10560

cgccagcacc gccacctggt gaccttgctg ctctggaggc ccggctccgt ggtgcggagc 10620

aggcgcggga gggtccgggg ctagcggcgg gttagagatg ggcggtgccc gggctccagg 10680

ctgggacccc tccgtgggga gctctgcggc accacgcttt gtgaatgggc cctgggggga 10740

ggttccgctg cctggggccc cgatgcgggg agccgccctt gaggcccccg gagccacgga 10800

atagctgtcg cagggcgtgg aacccgtggg cagccgcagg tgtgctcttg ggggccagga 10860

cgccaggggc ttccgaggtg ttcacacctg caaaccgccc cgacctggcc cccagactcc 10920

ttcctccgat ttttggaaag aagtgcagct ggcgctgccg cggaaggtgc agtacaggcc 10980

tgtggaagga gacccccaga cgcagctgca ggacgacaag gaccccatgc tgattcttcg 11040

aggccgagtc cctgagggcc gggccctgga ctcagaggtg gacccggacc ctgaggcga 11100

cctgggtatg cccgcccagc cccactcccc aactggagaa gctcagcaca gggcggagtg 11160

ggggcaggca cagggcacag ggcctggagg ggctccaggt gttgaggact cttcccggca 11220

ccgggagccc ctgcacggcc tctgccctgg aggtgctcgg ccctcggtct gcctgggaac 11280

ttcctgggcc tcacaggcca tcacagcagg gggtgagcag gggcagcccc tggcagtggg 11340

tctgggccaa ggctgtgggt ggccacctca ggcgtctcgg tctccccacc ccaggtgtcc 11400

gggggcctgt ctttggagag ccatcagctc caccgcacac cagtggggtc tcgctgggag 11460

agagccggag cagcgaagtg gacgtctcgg atctcggctc gcgaaactac agtgcccgca 11520

cagacttcta ctgcctggtg tccaaggatg atatgtagct cccaccccag agtgcaggat 11580

catagggaca gcggggggcca gggcagcggc gtcgctcctc tgctcaacag gaccacaacc 11640

cctgccagca gccctgggac cctgccagca gccctgggaa aaggctgtgg cctcagggcg 11700

cctcccagtg ccagaaaata aagtcctttt ggattctg 11738

<210> 3

<211> 1230

<212> DNA

<213> Homo sapiens

<220>

<223> wild type SIGIRR mRNA

<400> 3

augccaggug ucugugauag ggccccugac uuccucuccc cgucugaaga ccaggugcug 60

aggccugccu ugggcagcuc aguggcucug aacugcacgg cuuggguagu cucugggccc 120

cacugcuccc ugccuucagu ccaguggcug aaagacgggc uuccauuggg aauugggggc 180

cacuacagcc uccacgagua cuccuggguc aaggccaacc ugucagaggu gcuugugucc 240

aguguccugg gggucaacgu gaccagcacu gaagucuaug gggccuucac cugcuccauc 300

cagaacauca gcuucuccuc cuucacucuu cagagagcug gcccuacaag ccacguggcu 360

gcggugcugg ccucccuccu gguccugcug gcccugcugc uggccgcccu gcucuauguc 420

aagugccguc ucaacgugcu gcucugguac caggacgcgu auggggaggu ggagauaaac 480

gacgggaagc ucuacgacgc cuacgucucc uacagcgacu gccccgagga ccgcaaguuc 540

gugaacuuca uccuaaagcc gcagcuggag cggcgucggg gcuacaagcu cuuccuggac 600

gaccgcgacc uccugccgcg cgcugagccc uccgccgacc ucuuggugaa ccugagccgc 660

ugccgacgcc ucaucguggu gcuuucggac gccuuccuga gccgggccug gugcagccac 720

agcuuccggg agggccugug ccggcugcug gagcucaccc gcagacccau cuucaucacc 780

uucgagggcc agaggcgcga ccccgcgcac ccggcgcucc gccugcugcg ccagcaccgc 840

caccugguga ccuugcugcu cuggaggccc ggcuccguga cuccuuccuc cgauuuuugg 900

aaagaagugc agcuggcgcu gccgcggaag gugcaguaca ggccugugga aggagacccc 960

cagacgcagc ugcaggacga caaggacccc augcugauuc uucgaggccg agucccugag 1020

ggccgggccc uggacucaga gguggacccg gacccugagg gcgaccuggg uguccggggg 1080

ccugucuuug gagagccauc agcuccaccg cacaccagug gggucucgcu gggagagagc 1140

cggagcagcg aaguggacgu cucggaucuc ggcucgcgaa acuacagugc ccgcacagac 1200

uucuacugcc ugguguccaa ggaugauaug 1230

<210> 4

<211> 645

<212> DNA

<213> Homo sapiens

<220>

<223> mRNA variant (c.557delA) SIGIRR

<400> 4

augccaggug ucugugauag ggccccugac uuccucuccc cgucugaaga ccaggugcug 60

aggccugccu ugggcagcuc aguggcucug aacugcacgg cuuggguagu cucugggccc 120

cacugcuccc ugccuucagu ccaguggcug aaagacgggc uuccauuggg aauugggggc 180

cacuacagcc uccacgagua cuccuggguc aaggccaacc ugucagaggu gcuugugucc 240

aguguccugg gggucaacgu gaccagcacu gaagucuaug gggccuucac cugcuccauc 300

cagaacauca gcuucuccuc cuucacucuu cagagagcug gcccuacaag ccacguggcu 360

gcggugcugg ccucccuccu gguccugcug gcccugcugc uggccgcccu gcucuauguc 420

aagugccguc ucaacgugcu gcucugguac caggacgcgu auggggaggu ggagauaaac 480

gacgggaagc ucuacgacgc cuacgucucc uacagcgacu gccccgagga ccgcaaguuc 540

gugaacuuca uccuaagccg cagcuggagc ggcgucgggg cuacaagcuc uuccuggacg 600

accgcgaccu ccugccgcgc gcugagcccu ccgccgaccu cuugg 645

<210> 5

<211> 1233

<212> DNA

<213> Artificial sequence

<220>

<223> wild-type SIGIRR cDNA

<400> 5

atgccaggtg tctgtgatag ggcccctgac ttcctctccc cgtctgaaga ccaggtgctg 60

aggcctgcct tgggcagctc agtggctctg aactgcacgg cttgggtagt ctctgggccc 120

cactgctccc tgccttcagt ccagtggctg aaagacgggc ttccattggg aattggggggc 180

cactacagcc tccacgagta ctcctgggtc aaggccaacc tgtcagaggt gcttgtgtcc 240

agtgtcctgg gggtcaacgt gaccagcact gaagtctatg gggccttcac ctgctccatc 300

cagaacatca gcttctcctc cttcactctt cagagagctg gccctacaag ccacgtggct 360

gcggtgctgg cctccctcct ggtcctgctg gccctgctgc tggccgccct gctctatgtc 420

aagtgccgtc tcaacgtgct gctctggtac caggacgcgt atggggaggt ggagataaac 480

gacgggaagc tctacgacgc ctacgtctcc tacagcgact gccccgagga ccgcaagttc 540

gtgaacttca tcctaaagcc gcagctggag cggcgtcggg gctacaagct cttcctggac 600

gaccgcgacc tcctgccgcg cgctgagccc tccgccgacc tcttggtgaa cctgagccgc 660

tgccgacgcc tcatcgtggt gctttcggac gccttcctga gccgggcctg gtgcagccac 720

agcttccggg agggcctgtg ccggctgctg gagctcaccc gcagacccat cttcatcacc 780

ttcgaggggcc agaggcgcga ccccgcgcac ccggcgctcc gcctgctgcg ccagcaccgc 840

cacctggtga ccttgctgct ctggaggccc ggctccgtga ctccttcctc cgatttttgg 900

aaagaagtgc agctggcgct gccgcggaag gtgcagtaca ggcctgtgga aggagacccc 960

cagacgcagc tgcaggacga caaggacccc atgctgattc ttcgaggccg agtccctgag 1020

ggccgggccc tggactcaga ggtggacccg gaccctgagg gcgacctggg tgtccggggg 1080

cctgtctttg gagagccatc agctccaccg cacaccagtg gggtctcgct gggagagagc 1140

cggagcagcg aagtggacgt ctcggatctc ggctcgcgaa actacagtgc ccgcacagac 1200

ttctactgcc tggtgtccaa ggatgatatg tag 1233

<210> 6

<211> 648

<212> DNA

<213> Artificial sequence

<220>

<223> cDNA variant (c.557delA) SIGIRR

<400> 6

atgccaggtg tctgtgatag ggcccctgac ttcctctccc cgtctgaaga ccaggtgctg 60

aggcctgcct tgggcagctc agtggctctg aactgcacgg cttgggtagt ctctgggccc 120

cactgctccc tgccttcagt ccagtggctg aaagacgggc ttccattggg aattggggggc 180

cactacagcc tccacgagta ctcctgggtc aaggccaacc tgtcagaggt gcttgtgtcc 240

agtgtcctgg gggtcaacgt gaccagcact gaagtctatg gggccttcac ctgctccatc 300

cagaacatca gcttctcctc cttcactctt cagagagctg gccctacaag ccacgtggct 360

gcggtgctgg cctccctcct ggtcctgctg gccctgctgc tggccgccct gctctatgtc 420

aagtgccgtc tcaacgtgct gctctggtac caggacgcgt atggggaggt ggagataaac 480

gacgggaagc tctacgacgc ctacgtctcc tacagcgact gccccgagga ccgcaagttc 540

gtgaacttca tcctaagccg cagctggagc ggcgtcgggg ctacaagctc ttcctggacg 600

accgcgacct cctgccgcgc gctgagccct ccgccgacct cttggtga 648

<210> 7

<211> 410

<212> Protein

<213> Homo sapiens

<220>

<223> wild type SIGIRR protein

<400> 7

Met Pro Gly Val Cys Asp Arg Ala Pro Asp Phe Leu Ser Pro Ser Glu

1 5 10 15

Asp Gln Val Leu Arg Pro Ala Leu Gly Ser Ser Val Ala Leu Asn Cys

20 25 30

Thr Ala Trp Val Val Ser Gly Pro His Cys Ser Leu Pro Ser Val Gln

35 40 45

Trp Leu Lys Asp Gly Leu Pro Leu Gly Ile Gly Gly His Tyr Ser Leu

50 55 60

His Glu Tyr Ser Trp Val Lys Ala Asn Leu Ser Glu Val Leu Val Ser

65 70 75 80

Ser Val Leu Gly Val Asn Val Thr Ser Thr Glu Val Tyr Gly Ala Phe

85 90 95

Thr Cys Ser Ile Gln Asn Ile Ser Phe Ser Ser Phe Thr Leu Gln Arg

100 105 110

Ala Gly Pro Thr Ser His Val Ala Ala Val Leu Ala Ser Leu Leu Val

115 120 125

Leu Leu Ala Leu Leu Leu Ala Ala Leu Leu Tyr Val Lys Cys Arg Leu

130 135 140

Asn Val Leu Leu Trp Tyr Gln Asp Ala Tyr Gly Glu Val Glu Ile Asn

145 150 155 160

Asp Gly Lys Leu Tyr Asp Ala Tyr Val Ser Tyr Ser Asp Cys Pro Glu

165 170 175

Asp Arg Lys Phe Val Asn Phe Ile Leu Lys Pro Gln Leu Glu Arg Arg

180 185 190

Arg Gly Tyr Lys Leu Phe Leu Asp Asp Arg Asp Leu Leu Pro Arg Ala

195 200 205

Glu Pro Ser Ala Asp Leu Leu Val Asn Leu Ser Arg Cys Arg Arg Leu

210 215 220

Ile Val Val Leu Ser Asp Ala Phe Leu Ser Arg Ala Trp Cys Ser His

225 230 235 240

Ser Phe Arg Glu Gly Leu Cys Arg Leu Leu Glu Leu Thr Arg Arg Pro

245 250 255

Ile Phe Ile Thr Phe Glu Gly Gln Arg Arg Asp Pro Ala His Pro Ala

260 265 270

Leu Arg Leu Leu Arg Gln His Arg His Leu Val Thr Leu Leu Leu Trp

275 280 285

Arg Pro Gly Ser Val Thr Pro Ser Ser Asp Phe Trp Lys Glu Val Gln

290 295 300

Leu Ala Leu Pro Arg Lys Val Gln Tyr Arg Pro Val Glu Gly Asp Pro

305 310 315 320

Gln Thr Gln Leu Gln Asp Asp Lys Asp Pro Met Leu Ile Leu Arg Gly

325 330 335

Arg Val Pro Glu Gly Arg Ala Leu Asp Ser Glu Val Asp Pro Asp Pro

340 345 350

Glu Gly Asp Leu Gly Val Arg Gly Pro Val Phe Gly Glu Pro Ser Ala

355 360 365

Pro Pro His Thr Ser Gly Val Ser Leu Gly Glu Ser Arg Ser Ser Glu

370 375 380

Val Asp Val Ser Asp Leu Gly Ser Arg Asn Tyr Ser Ala Arg Thr Asp

385 390 395 400

Phe Tyr Cys Leu Val Ser Lys Asp Asp Met

405 410

<210> 8

<211> 504

<212> Protein

<213> Homo sapiens

<220>

<223> wild-type SIGIRR protein isoform

<400> 8

Met Pro Gly Val Cys Asp Arg Ala Pro Asp Phe Leu Ser Pro Ser Glu

1 5 10 15

Asp Gln Val Leu Arg Pro Ala Leu Gly Ser Ser Val Ala Leu Asn Cys

20 25 30

Thr Ala Trp Val Val Ser Gly Pro His Cys Ser Leu Pro Ser Val Gln

35 40 45

Trp Leu Lys Asp Gly Leu Pro Leu Gly Ile Gly Gly His Tyr Ser Leu

50 55 60

His Glu Tyr Ser Trp Val Lys Ala Asn Leu Ser Glu Val Leu Val Ser

65 70 75 80

Ser Val Leu Gly Val Asn Val Thr Ser Thr Glu Val Tyr Gly Ala Phe

85 90 95

Thr Cys Ser Ile Gln Asn Ile Ser Phe Ser Ser Phe Thr Leu Gln Arg

100 105 110

Ala Gly Pro Thr Ser His Val Ala Ala Val Leu Ala Ser Leu Leu Val

115 120 125

Leu Leu Ala Leu Leu Leu Ala Ala Leu Leu Tyr Val Lys Cys Arg Leu

130 135 140

Asn Val Leu Leu Trp Tyr Gln Asp Ala Tyr Gly Glu Val Glu Ile Asn

145 150 155 160

Asp Gly Lys Leu Tyr Asp Ala Tyr Val Ser Tyr Ser Asp Cys Pro Glu

165 170 175

Asp Arg Lys Phe Val Asn Phe Ile Leu Lys Pro Gln Leu Glu Arg Arg

180 185 190

Arg Gly Tyr Lys Leu Phe Leu Asp Asp Arg Asp Leu Leu Pro Arg Ala

195 200 205

Glu Pro Ser Ala Asp Leu Leu Val Asn Leu Ser Arg Cys Arg Arg Leu

210 215 220

Ile Val Val Leu Ser Asp Ala Phe Leu Ser Arg Ala Trp Cys Ser His

225 230 235 240

Ser Phe Arg Glu Gly Leu Cys Arg Leu Leu Glu Leu Thr Arg Arg Pro

245 250 255

Ile Phe Ile Thr Phe Glu Gly Gln Arg Arg Asp Pro Ala His Pro Ala

260 265 270

Leu Arg Leu Leu Arg Gln His Arg His Leu Val Thr Leu Leu Leu Trp

275 280 285

Arg Pro Gly Ser Val Thr Pro Ser Ser Asp Phe Trp Lys Glu Val Gln

290 295 300

Leu Ala Leu Pro Arg Lys Val Gln Tyr Arg Pro Val Glu Gly Asp Pro

305 310 315 320

Gln Thr Gln Leu Gln Asp Asp Lys Asp Pro Met Leu Ile Leu Arg Gly

325 330 335

Arg Val Pro Glu Gly Arg Ala Leu Asp Ser Glu Val Asp Pro Asp Pro

340 345 350

Glu Gly Asp Leu Gly Met Pro Ala Gln Pro His Ser Pro Thr Gly Glu

355 360 365

Ala Gln His Arg Ala Glu Trp Gly Gln Ala Gln Gly Thr Gly Pro Gly

370 375 380

Gly Ala Leu Gly Val Glu Asp Ser Ser Arg His Arg Glu Pro Leu His

385 390 395 400

Gly Leu Cys Pro Gly Gly Ala Arg Pro Ser Val Cys Leu Gly Thr Ser

405 410 415

Trp Ala Ser Gln Ala Ile Thr Ala Gly Gly Glu Gln Gly Gln Pro Leu

420 425 430

Ala Val Gly Leu Gly Gln Gly Cys Gly Trp Pro Pro Gln Ala Ser Arg

435 440 445

Ser Pro His Pro Arg Cys Pro Gly Ala Cys Phe Trp Arg Ala Ile Ser

450 455 460

Ser Thr Ala His Gln Trp Gly Leu Ala Gly Arg Glu Pro Glu Gln Arg

465 470 475 480

Ser Gly Arg Leu Gly Ser Arg Leu Ala Lys Leu Gln Cys Pro His Arg

485 490 495

Leu Leu Leu Pro Gly Val Gln Gly

500

<210> 9

<211> 215

<212> Protein

<213> Homo sapiens

<220>

<223> Variant SIGIRR (c.557delA)

<400> 9

Met Pro Gly Val Cys Asp Arg Ala Pro Asp Phe Leu Ser Pro Ser Glu

1 5 10 15

Asp Gln Val Leu Arg Pro Ala Leu Gly Ser Ser Val Ala Leu Asn Cys

20 25 30

Thr Ala Trp Val Val Ser Gly Pro His Cys Ser Leu Pro Ser Val Gln

35 40 45

Trp Leu Lys Asp Gly Leu Pro Leu Gly Ile Gly Gly His Tyr Ser Leu

50 55 60

His Glu Tyr Ser Trp Val Lys Ala Asn Leu Ser Glu Val Leu Val Ser

65 70 75 80

Ser Val Leu Gly Val Asn Val Thr Ser Thr Glu Val Tyr Gly Ala Phe

85 90 95

Thr Cys Ser Ile Gln Asn Ile Ser Phe Ser Ser Phe Thr Leu Gln Arg

100 105 110

Ala Gly Pro Thr Ser His Val Ala Ala Val Leu Ala Ser Leu Leu Val

115 120 125

Leu Leu Ala Leu Leu Leu Ala Ala Leu Leu Tyr Val Lys Cys Arg Leu

130 135 140

Asn Val Leu Leu Trp Tyr Gln Asp Ala Tyr Gly Glu Val Glu Ile Asn

145 150 155 160

Asp Gly Lys Leu Tyr Asp Ala Tyr Val Ser Tyr Ser Asp Cys Pro Glu

165 170 175

Asp Arg Lys Phe Val Asn Phe Ile Leu Ser Arg Ser Trp Ser Gly Val

180 185 190

Gly Ala Thr Ser Ser Ser Trp Thr Thr Ala Thr Ser Cys Arg Ala Leu

195 200 205

Ser Pro Pro Pro Thr Ser Trp

210 215

<210> 10

<211> 30

<212> Protein

<213> Homo sapiens

<220>

<223> Amino acids 186–215 SIGIRR wild type

<400> 10

Lys Pro Gln Leu Glu Arg Arg Arg Gly Tyr Lys Leu Phe Leu Asp Asp

1 5 10 15

Arg Asp Leu Leu Pro Arg Ala Glu Pro Ser Ala Asp Leu Leu

20 25 30

<210> 11

<211> 30

<212> Protein

<213> Homo sapiens

<220>

<223> Amino acids 186–215 of variant SIGIRR

<400> 11

Ser Arg Ser Trp Ser Gly Val Gly Ala Thr Ser Ser Ser Trp Thr Thr

1 5 10 15

Ala Thr Ser Cys Arg Ala Leu Ser Pro Pro Pro Thr Ser Trp

20 25 30

<210> 12

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> forward PCR primer for SIGIRR

<400> 12

tcagtggctc tgaactgcac 20

<210> 13

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> reverse PCR primer for SIGIRR

<400> 13

ggtcctgttg agcagaggag 20

<210> 14

<211> 910

<212> Protein

<213> Artificial sequence

<220>

<223> rilonacept fusion protein

<400> 14

Met Val Leu Leu Trp Cys Val Val Ser Leu Tyr Phe Tyr Gly Ile Leu

1 5 10 15

Gln Ser Asp Ala Ser Glu Arg Cys Asp Asp Trp Gly Leu Asp Thr Met

20 25 30

Arg Gln Ile Gln Val Phe Glu Asp Glu Pro Ala Arg Ile Lys Cys Pro

35 40 45

Leu Phe Glu His Phe Leu Lys Phe Asn Tyr Ser Thr Ala His Ser Ala

50 55 60

Gly Leu Thr Leu Ile Trp Tyr Trp Thr Arg Gln Asp Arg Asp Leu Glu

65 70 75 80

Glu Pro Ile Asn Phe Arg Leu Pro Glu Asn Arg Ile Ser Lys Glu Lys

85 90 95

Asp Val Leu Trp Phe Arg Pro Thr Leu Leu Asn Asp Thr Gly Asn Tyr

100 105 110

Thr Cys Met Leu Arg Asn Thr Thr Tyr Cys Ser Lys Val Ala Phe Pro

115 120 125

Leu Glu Val Val Gln Lys Asp Ser Cys Phe Asn Ser Pro Met Lys Leu

130 135 140

Pro Val His Lys Leu Tyr Ile Glu Tyr Gly Ile Gln Arg Ile Thr Cys

145 150 155 160

Pro Asn Val Asp Gly Tyr Phe Pro Ser Ser Val Lys Pro Thr Ile Thr

165 170 175

Trp Tyr Met Gly Cys Tyr Lys Ile Gln Asn Phe Asn Asn Val Ile Pro

180 185 190

Glu Gly Met Asn Leu Ser Phe Leu Ile Ala Leu Ile Ser Asn Asn Gly

195 200 205

Asn Tyr Thr Cys Val Val Thr Tyr Pro Glu Asn Gly Arg Thr Phe His

210 215 220

Leu Thr Arg Thr Leu Thr Val Lys Val Val Gly Ser Pro Lys Asn Ala

225 230 235 240

Val Pro Pro Val Ile His Ser Pro Asn Asp His Val Val Tyr Glu Lys

245 250 255

Glu Pro Gly Glu Glu Leu Leu Ile Pro Cys Thr Val Tyr Phe Ser Phe

260 265 270

Leu Met Asp Ser Arg Asn Glu Val Trp Trp Thr Ile Asp Gly Lys Lys

275 280 285

Pro Asp Asp Ile Thr Ile Asp Val Thr Ile Asn Glu Ser Ile Ser His

290 295 300

Ser Arg Thr Glu Asp Glu Thr Arg Thr Gln Ile Leu Ser Ile Lys Lys

305 310 315 320

Val Thr Ser Glu Asp Leu Lys Arg Ser Tyr Val Cys His Ala Arg Ser

325 330 335

Ala Lys Gly Glu Val Ala Lys Ala Ala Lys Val Lys Gln Lys Val Pro

340 345 350

Ala Pro Arg Tyr Thr Val Ser Gly Gly Ala Pro Met Leu Ser Glu Ala

355 360 365

Asp Lys Cys Lys Glu Arg Glu Glu Lys Ile Ile Leu Val Ser Ser Ala

370 375 380

Asn Glu Ile Asp Val Arg Pro Cys Pro Leu Asn Pro Asn Glu His Lys

385 390 395 400

Gly Thr Ile Thr Trp Tyr Lys Asp Asp Ser Lys Thr Pro Val Ser Thr

405 410 415

Glu Gln Ala Ser Arg Ile His Gln His Lys Glu Lys Leu Trp Phe Val

420 425 430

Pro Ala Lys Val Glu Asp Ser Gly His Tyr Tyr Cys Val Val Arg Asn

435 440 445

Ser Ser Tyr Cys Leu Arg Ile Lys Ile Ser Ala Lys Phe Val Glu Asn

450 455 460

Glu Pro Asn Leu Cys Tyr Asn Ala Gln Ala Ile Phe Lys Gln Lys Leu

465 470 475 480

Pro Val Ala Gly Asp Gly Gly Leu Val Cys Pro Tyr Met Glu Phe Phe

485 490 495

Lys Asn Glu Asn Asn Glu Leu Pro Lys Leu Gln Trp Tyr Lys Asp Cys

500 505 510

Lys Pro Leu Leu Leu Asp Asn Ile His Phe Ser Gly Val Lys Asp Arg

515 520 525

Leu Ile Val Met Asn Val Ala Glu Lys His Arg Gly Asn Tyr Thr Cys

530 535 540

His Ala Ser Tyr Thr Tyr Leu Gly Lys Gln Tyr Pro Ile Thr Arg Val

545 550 555 560

Ile Glu Phe Ile Thr Leu Glu Glu Asn Lys Pro Thr Arg Pro Val Ile

565 570 575

Val Ser Pro Ala Asn Glu Thr Met Glu Val Asp Leu Gly Ser Gln Ile

580 585 590

Gln Leu Ile Cys Asn Val Thr Gly Gln Leu Ser Asp Ile Ala Tyr Trp

595 600 605

Lys Trp Asn Gly Ser Val Ile Asp Glu Asp Asp Pro Val Leu Gly Glu

610 615 620

Asp Tyr Tyr Ser Val Glu Asn Pro Ala Asn Lys Arg Arg Ser Thr Leu

625 630 635 640

Ile Thr Val Leu Asn Ile Ser Glu Ile Glu Ser Arg Phe Tyr Lys His

645 650 655

Pro Phe Thr Cys Phe Ala Lys Asn Thr His Gly Ile Asp Ala Ala Tyr

660 665 670

Ile Gln Leu Ile Tyr Pro Val Thr Asn Ser Gly Asp Lys Thr His Thr

675 680 685

Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe

690 695 700

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

705 710 715 720

Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val

725 730 735

Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

740 745 750

Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val

755 760 765

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

770 775 780

Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser

785 790 795 800

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

805 810 815

Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val

820 825 830

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

835 840 845

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

850 855 860

Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp

865 870 875 880

Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

885 890 895

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

900 905 910

<210> 15

<211> 116

<212> Protein

<213> Artificial sequence

<220>

<223> heavy chain variable region of an antibody targeting IL-6R

<400> 15

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Arg Phe Thr Phe Asp Asp Tyr

20 25 30

Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Gly Ile Ser Trp Asn Ser Gly Arg Ile Gly Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Glu Asn Ser Leu Phe

65 70 75 80

Leu Gln Met Asn Gly Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Ala Lys Gly Arg Asp Ser Phe Asp Ile Trp Gly Gln Gly Thr Met Val

100 105 110

Thr Val Ser Ser

115

<210> 16

<211> 107

<212> Protein

<213> Artificial sequence

<220>

<223> light chain variable region of an antibody targeting IL-6R

<400> 16

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Gly Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Ser Tyr Tyr Cys Gln Gln Ala Asn Ser Phe Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<---

Claims (109)

1. A method for identifying a human subject having early-onset inflammatory bowel disease or a risk of developing early-onset inflammatory bowel disease, the method comprising detecting in a sample obtained from the subject the presence or absence of: a SIGIRR protein containing a serine at a position corresponding to position 186 in accordance with SEQ ID NO:9, and truncated at a position corresponding to position 215 in accordance with SEQ ID NO:9; and/or a nucleic acid molecule encoding a SIGIRR protein containing a serine at a position corresponding to position 186 in accordance with SEQ ID NO:9, and truncated at a position corresponding to position 215 in accordance with SEQ ID NO:9; wherein the presence of said truncated SIGIRR protein and/or a nucleic acid molecule encoding said truncated SIGIRR protein indicates that the subject has early-onset inflammatory bowel disease, or is at risk of developing early-onset inflammatory bowel disease, wherein said truncated SIGIRR protein contains an amino acid sequence that has at least 90% sequence identity with SEQ ID NO:9. 2. The method of claim 1, wherein said truncated SIGIRR protein contains a different amino acid compared to the wild-type SIGIRR protein at any of the positions corresponding to positions 186-209 and 211-215 in accordance with SEQ ID NO:9. 3. The method according to claim 1 or 2, characterized in that said truncated SIGIRR protein contains the amino acid sequence of SEQ ID NO:11 at positions corresponding to positions 186-215 in accordance with SEQ ID NO:9. 4. The method of claim 1, wherein if a human subject exhibits one or more symptoms of early-onset inflammatory bowel disease, said human subject is identified as having early-onset inflammatory bowel disease. 5. The method according to claim 1, characterized in that said method is an in vitro method. 6. The method according to claim 1, characterized in that the detection of the presence or absence of the specified truncated SIGIRR protein in the specified sample is carried out using an antibody specific for the truncated SIGIRR. 7. The method according to claim 6, characterized in that said antibody specific for truncated SIGIRR is specific for: i) serine at a position corresponding to position 186 in accordance with SEQ ID NO:9; or ii) an epitope created in the SIGIRR protein due to a frameshift mutation that results in a serine at the position corresponding to position 186 in accordance with SEQ ID NO:9. 8. The method of claim 6 or 7, wherein the detection further comprises comparing the response of an antibody specific for truncated SIGIRR with that of an antibody specific for wild-type SIGIRR. 9. The method according to claim 1, characterized in that the detection of the presence or absence of the specified truncated SIGIRR protein in the specified sample is carried out using an enzyme-linked immunosorbent assay (ELISA). 10. The method according to claim 1, characterized in that the detection of the presence or absence of the specified nucleic acid molecule encoding the specified truncated SIGIRR protein in the specified sample is carried out by determining whether there is a frameshift mutation in the specified nucleic acid molecule creating a codon encoding serine at a position corresponding to position 186 in accordance with SEQ ID NO:9. 11. The method of claim 1, wherein the discovery step comprises sequencing at least a portion of a nucleic acid molecule that encodes a SIGIRR protein, wherein the sequenced nucleic acid molecule encodes a SIGIRR protein truncated at a position corresponding to position 215 in accordance with SEQ ID NO:9. 12. The method of claim 11, wherein the sequenced portion of the nucleic acid molecule contains a number of positions spanning the codon encoding the position corresponding to position 186 in accordance with SEQ ID NO:9. 13. The method according to claim 11 or 12, characterized in that the discovery step includes sequencing the entire nucleic acid molecule encoding the SIGIRR protein. 14. The method according to claim 1, characterized in that the detection step includes: amplifying at least a portion of a nucleic acid molecule that encodes a SIGIRR protein, wherein the nucleic acid molecule to be amplified includes a codon encoding an amino acid at a position corresponding to position 186 in accordance with SEQ ID NO:9. labeling the nucleic acid molecule with a detectable label; bringing a labeled nucleic acid molecule into contact with a support containing a probe, wherein the probe contains a nucleic acid sequence that specifically hybridizes under stringent conditions to a nucleic acid sequence containing a codon encoding a serine at a position corresponding to position 186 in accordance with SEQ ID NO:9 ; And detection of a detectable tag. 15. The method according to claim 14, wherein the nucleic acid molecule in the sample is mRNA, and the detection step further includes reverse transcribing the mRNA into cDNA before the amplification step. 16. The method according to claim 1, characterized in that the detection step includes: bringing a nucleic acid molecule that encodes the SIGIRR protein into contact with a probe containing a detectable tag, the probe comprising a nucleic acid sequence that specifically hybridizes under stringent conditions to a nucleic acid sequence containing a codon encoding a serine at a position corresponding to position 186 in accordance with SEQ ID NO:9, and detection of a detectable tag. 17. The method of claim 16, wherein the nucleic acid molecule is present in a cell obtained from a human subject. 18. The method according to claim 16 or 17, characterized in that said human subject is under 18 years of age. 19. The method of claim 1, wherein said human subject is identified as having Crohn's disease or at risk of developing Crohn's disease. 20. A method for diagnosing early-onset inflammatory bowel disease or identifying the risk of early-onset inflammatory bowel disease in a human subject, comprising: detecting a nucleic acid molecule encoding a SIGIRR protein obtained from a human subject, wherein the SIGIRR protein contains a serine at a position corresponding to position 186 in accordance with SEQ ID NO:9, and truncated at a position corresponding to position 215 in accordance with SEQ ID NO: 9; and/or detecting a SIGIRR protein obtained from a human subject, wherein the SIGIRR protein contains a serine at a position corresponding to position 186 in accordance with SEQ ID NO:9, and truncated at a position corresponding to position 215 in accordance with SEQ ID NO:9; And diagnosing a human subject with early-onset inflammatory bowel disease if the subject has one or more symptoms of early-onset inflammatory bowel disease, or diagnosing the human subject as at risk for early-onset inflammatory bowel disease if the subject does not has one or more symptoms of early-onset inflammatory bowel disease; wherein said truncated SIGIRR protein contains an amino acid sequence that has at least 90% sequence identity with SEQ ID NO:9. 21. The method of claim 20, wherein said truncated SIGIRR protein contains a different amino acid compared to the wild-type SIGIRR protein at any of the positions corresponding to positions 186-209 and 211-215 in accordance with SEQ ID NO:9. 22. The method of claim 20 or 21, wherein said truncated SIGIRR protein contains the amino acid sequence of SEQ ID NO:11 at positions corresponding to positions 186-215 of SEQ ID NO:9. 23. The method according to claim 20, characterized in that the detection of said truncated SIGIRR protein is carried out using an antibody specific for the truncated SIGIRR. 24. The method according to claim 23, characterized in that said antibody specific for truncated SIGIRR is specific for: i) serine at a position corresponding to position 186 in accordance with SEQ ID NO:9; or ii) an epitope created in the SIGIRR protein due to a frameshift mutation that results in a serine at the position corresponding to position 186 in accordance with SEQ ID NO:9. 25. The method of claim 23 or 24, wherein the detection further comprises comparing the response of an antibody specific for truncated SIGIRR with that of an antibody specific for wild-type SIGIRR. 26. The method according to claim 20, characterized in that the detection of said truncated SIGIRR protein is carried out using an enzyme-linked immunosorbent assay (ELISA). 27. The method of claim 20, wherein the detection of said nucleic acid molecule encoding said truncated SIGIRR protein is carried out by determining whether there is a frameshift mutation in said nucleic acid molecule creating a codon encoding a serine at a position corresponding to the position 186 according to SEQ ID NO:9. 28. The method of claim 20, wherein the discovery step comprises sequencing at least a portion of the nucleic acid molecule that encodes the SIGIRR protein. 29. The method of claim 28, wherein the sequenced portion of the nucleic acid molecule contains a number of positions spanning the codon encoding the position corresponding to position 186 in accordance with SEQ ID NO:9. 30. The method of claim 20, wherein the discovery step includes sequencing the entire nucleic acid molecule encoding the SIGIRR protein. 31. The method according to claim 20, characterized in that the detection step includes: amplifying at least a portion of a nucleic acid molecule that encodes a SIGIRR protein, wherein the amplified nucleic acid molecule spans a codon encoding an amino acid at a position corresponding to position 186 in accordance with SEQ ID NO:9; labeling the nucleic acid molecule with a detectable label; bringing a labeled nucleic acid molecule into contact with a support containing a probe, wherein the probe contains a nucleic acid sequence that specifically hybridizes under stringent conditions to a nucleic acid sequence containing a codon encoding a serine at a position corresponding to position 186 in accordance with SEQ ID NO:9 ; And detection of a detectable tag. 32. The method according to claim 31, characterized in that the nucleic acid molecule is mRNA, and said method further includes reverse transcribing the mRNA into cDNA before the amplification step. 33. The method according to claim 20, characterized in that the detection step includes: bringing a nucleic acid molecule that encodes the SIGIRR protein into contact with a probe containing a detectable tag, the probe comprising a nucleic acid sequence that specifically hybridizes under stringent conditions to a nucleic acid sequence containing a codon encoding a serine at a position corresponding to position 186 in accordance with SEQ ID NO:9; And detection of a detectable tag. 34. The method of claim 33, wherein the nucleic acid molecule is present in a cell obtained from a human subject. 35. The method according to claim 20, characterized in that said human subject is under 18 years of age. 36. The method of claim 20, wherein said human subject is identified as having Crohn's disease or at risk of developing Crohn's disease. 37. A method of treating early-onset inflammatory bowel disease or risk of early-onset inflammatory bowel disease in a subject, comprising: detecting a nucleic acid molecule encoding a SIGIRR protein obtained from a subject, wherein the SIGIRR protein contains a serine at a position corresponding to position 186 in accordance with SEQ ID NO:9, and truncated at a position corresponding to position 215 in accordance with SEQ ID NO:9; and/or detecting a SIGIRR protein obtained from a subject, wherein the SIGIRR protein contains a serine at a position corresponding to position 186 in accordance with SEQ ID NO:9, and truncated at a position corresponding to position 215 in accordance with SEQ ID NO:9; And diagnosing a subject with early-onset inflammatory bowel disease if the subject has one or more symptoms of early-onset inflammatory bowel disease, or diagnosing the subject as being at risk for early-onset inflammatory bowel disease if the subject does not have one or more symptoms of inflammatory bowel disease with early onset; And treating a subject with an agent effective for treating early-onset inflammatory bowel disease or at risk for inflammatory bowel disease; wherein said truncated SIGIRR protein contains an amino acid sequence that has at least 90% sequence identity with SEQ ID NO:9. 38. The method according to claim 20, characterized in that said human subject is under 18 years of age. 39. The method of claim 20, wherein said human subject is identified as having Crohn's disease or at risk of developing Crohn's disease. 40. An isolated nucleic acid molecule for introduction into a cell, wherein the isolated nucleic acid molecule contains a nucleic acid sequence encoding a human interleukin-1 receptor related protein containing a single immunoglobulin domain (SIGIRR), wherein said SIGIRR protein contains a serine at the position, corresponding to position 186 in accordance with SEQ ID NO:9, and truncated at the position corresponding to position 215 in accordance with SEQ ID NO:9, or a complementary nucleic acid strand encoding the specified SIGIRR protein, wherein detecting the presence of the nucleic acid in the sample obtained from a subject means that the subject is at risk of developing early-onset inflammatory bowel disease, wherein the truncated SIGIRR protein contains an amino acid sequence that has at least 90% sequence identity with SEQ ID NO:9. 41. The isolated nucleic acid molecule according to claim 40 or 41, characterized in that said nucleic acid molecule contains DNA. 42. The isolated nucleic acid molecule according to claim 40, characterized in that said nucleic acid molecule is a cDNA. 43. The isolated nucleic acid molecule according to claim 40, characterized in that said nucleic acid molecule contains RNA. 44. The isolated nucleic acid molecule of claim 40, wherein said nucleic acid molecule is genomic DNA and contains guanine at the position corresponding to position 9962 in accordance with SEQ ID NO:2. 45. The isolated nucleic acid molecule of claim 44, wherein said nucleic acid molecule contains SEQ ID NO:2. 46. The isolated nucleic acid molecule of claim 40, wherein said nucleic acid molecule is mRNA and contains guanine at the position corresponding to position 557 in accordance with SEQ ID NO:4. 47. The isolated nucleic acid molecule according to claim 40, characterized in that said nucleic acid molecule is mRNA and contains codons CUA and AGC at positions corresponding to positions 553-555 and 556-558, respectively, in accordance with SEQ ID NO: 4. 48. The isolated nucleic acid molecule of claim 46, wherein said nucleic acid molecule contains SEQ ID NO:4. 49. The isolated nucleic acid molecule of claim 40, wherein said truncated SIGIRR protein contains a different amino acid compared to the wild-type SIGIRR protein at any of the positions corresponding to positions 186-209 and 211-215 in accordance with SEQ ID NO: 9. 50. The isolated nucleic acid molecule of claim 40, wherein said truncated SIGIRR protein contains the amino acid sequence of SEQ ID NO:11 at positions corresponding to positions 186-215 of SEQ ID NO:9. 51. The isolated nucleic acid molecule of claim 40, wherein said truncated SIGIRR protein contains the amino acid sequence of SEQ ID NO:9. 52. An expression vector containing an isolated nucleic acid molecule according to any one of claims. 40-51. 53. The vector according to claim 52, characterized in that said vector is a plasmid. 54. The vector according to claim 52, characterized in that said vector is a virus. 55. cDNA transcribed from an mRNA nucleic acid molecule from a sample obtained from a subject, wherein the mRNA nucleic acid molecule contains a nucleic acid sequence encoding a SIGIRR protein, wherein said SIGIRR protein contains a serine at a position corresponding to position 186 in accordance with SEQ ID NO:9, and truncated at position corresponding to position 215 in accordance with SEQ ID NO:9, wherein the presence of said mRNA nucleic acid molecule capable of being transcribed into cDNA means that the subject is at risk of developing early-onset inflammatory bowel disease and wherein the truncated SIGIRR protein contains an amino acid sequence that has at least 90% sequence identity with SEQ ID NO:9. 56. The cDNA of claim 55, wherein said truncated SIGIRR protein contains a different amino acid from the wild-type SIGIRR protein at any of the positions corresponding to positions 186-209 and 211-215 according to SEQ ID NO:9. 57. The cDNA of claim 55, wherein said truncated SIGIRR protein contains the amino acid sequence of SEQ ID NO:11 at positions corresponding to positions 186-215 of SEQ ID NO:9. 58. The cDNA of claim 55, wherein said truncated SIGIRR protein contains the amino acid sequence of SEQ ID NO:9 or an amino acid sequence that has at least 90% sequence identity with SEQ ID NO:9, and contains a serine at the position corresponding to position 186 in accordance with SEQ ID NO:9. 59. The cDNA according to claim 55, wherein said cDNA contains guanine at a position corresponding to position 557 in accordance with SEQ ID NO:6. 60. The cDNA of claim 55, wherein said cDNA contains codons CTA and AGC at positions corresponding to positions 553-555 and 556-558, respectively, in accordance with SEQ ID NO:6. 61. The cDNA of claim 55, wherein said cDNA contains SEQ ID NO:6. 62. An expression vector containing cDNA according to any one of paragraphs. 55-61. 63. The vector according to claim 62, characterized in that said vector is a plasmid. 64. The vector according to claim 62, characterized in that said vector is a virus. 65. An isolated or recombinant polypeptide for diagnosing early-onset inflammatory bowel disease or identifying the risk of developing early-onset inflammatory bowel disease in a subject, comprising a truncated SIGIRR protein, characterized in that said SIGIRR protein contains a serine at a position corresponding to the position 186 in accordance with SEQ ID NO:9, and truncated at the position corresponding to position 215 in accordance with SEQ ID NO:9, wherein the presence of the specified nucleic acid molecule in a sample obtained from the subject means that the subject is at risk of developing an inflammatory disease intestine with early onset, wherein the truncated SIGIRR protein contains an amino acid sequence that has at least 90% sequence identity with SEQ ID NO:9, and wherein the truncated SIGIRR protein is a loss-of-function variant of the wild-type SIGIRR protein. 66. The isolated or recombinant polypeptide of claim 65, wherein said truncated SIGIRR protein contains a different amino acid compared to the wild-type SIGIRR protein at any of the positions corresponding to positions 186-209 and 211-215 in accordance with SEQ ID NO: 9. 67. The isolated or recombinant polypeptide of claim 65, wherein said truncated SIGIRR protein contains the amino acid sequence of SEQ ID NO:11 at positions corresponding to positions 186-215 of SEQ ID NO:9. 68. The isolated or recombinant polypeptide of claim 66, wherein said truncated SIGIRR protein contains a serine at a position corresponding to position 186 in accordance with SEQ ID NO:9. 69. The isolated or recombinant polypeptide of claim 65, characterized in that said truncated SIGIRR protein contains the amino acid sequence of SEQ ID NO:9. 70. An isolated or recombinant polypeptide according to claim 65, characterized in that said polypeptide is fused to a heterologous polypeptide. 71. The isolated or recombinant polypeptide of claim 70, wherein said heterologous polypeptide contains a purification peptide tag, a fluorescent protein, or both a purification peptide tag and a fluorescent protein. 72. The isolated or recombinant polypeptide of claim 65, characterized in that said polypeptide is associated with a detectable label. 73. The isolated or recombinant polypeptide according to claim 72, characterized in that said detectable label is a fluorescent label or a radioactive label. 74. A set of probes or primers for detecting early-onset inflammatory bowel disease or identifying the risk of early-onset inflammatory bowel disease, wherein the probe or primer comprises a nucleic acid sequence of at least about 15 nucleotides that specifically hybridizes to a nucleic acid molecule having a nucleic acid sequence encoding a human SIGIRR protein containing a serine at a position corresponding to position 186 in accordance with SEQ ID NO:9, and truncated at a position corresponding to position 215 in accordance with SEQ ID NO:9, or which specifically hybridizes to the complementary strand of the nucleic acid sequence encoding the specified truncated human SIGIRR protein. 75. The kit according to claim 74, characterized in that said probe or primer contains DNA. 76. The kit according to claim 74, characterized in that said probe or primer contains RNA. 77. Set according to any one of paragraphs. 74-76, characterized in that said probe or primer specifically hybridizes to a portion of a nucleic acid molecule containing a codon that encodes a serine at a position corresponding to position 186 in accordance with SEQ ID NO:9. 78. The kit according to claim 74, characterized in that said probe or primer specifically hybridizes to a nucleic acid sequence encoding a truncated SIGIRR protein, or its complementary strand, under stringent conditions. 79. The kit according to claim 74, characterized in that said probe or primer contains a label. 80. The kit according to claim 79, characterized in that the specified label is a fluorescent label, a radioactive label or biotin.

Images